<td>Under development: Not open for comment. Do not cite</td>
<td>EAGMST Under Review</td>
<td>Open for citation & comment</td>
<td>WPHA/WNT Endorsed</td>
<td>1.52</td>
<td>Included in OECD Work Plan</td>
</tr>
</tbody>
</table>
</div>
</div>
<!-- Abstract Section, text as generated by author -->
<div id="abstract">
<h2>Abstract</h2>
<hr>
<p>Testicular toxicity is of interest for human health risk assessment especially in terms of reproductive and developmental toxicity, however, the testicular toxicity has not fully elucidated. HDIs are approved as anti-cancer drugs since HDIs have apoptotic effect in cancer cells. HDIs includes the shortchain fatty acids (e.g., butyrate, valproate ,MAA), hydroxamic acids (e.g., SAHA, TSA), cyclic tetrapeptides (e.g., FK-228), benzamides (e.g., N-acetyldinaline and MS-275) and epoxides (depeudecin, trapoxin A), of which MAA especially focused on have the testicular toxicity such as testis atrophy <em>in vivo</em>. The intracellular mechanisms of induction of the spermatocyte apoptosis by HDIs are suggested as HDAC inhibition as MIE, histone acetylation increase, p21 expression increase, disrupted cell cycle, apoptosis, and spermatocyte depletion as KEs. Adverse outcome includes testicular toxicity. The HDIs inhibit deacetylation of the histone, leading to the increase in histone acetylation, followed by increase in p21 gene expression. The apoptosis induced by disrupted cell cycle leads to spermatocyte depletion and testis atrophy. We propose new AOP for HDAC inhibition leading to testicular toxicity. This AOP may be one of the pathways induced by HDIs, which suggests the networks of the pathways with hyperacetylations of cellular proteins other than histones.</p>
<p> </p>
<h2>Abstract</h2>
<p>Testicular toxicity is of interest for human health risk assessment especially in terms of reproductive and developmental toxicity, however, the testicular toxicity has not been fully elucidated. Histone deacetylase inhibitors (HDIs) are approved as anti-cancer drugs since HDIs have apoptotic effects in cancer cells. HDIs include short-chain fatty acids, hydroxamic acids, benzamides, and epoxides. The intracellular mechanisms of induction of the spermatocyte apoptosis by HDIs are suggested as histone deacetylase (HDAC) inhibition as MIE, histone acetylation increase, disrupted cell cycle, apoptosis, and spermatocyte depletion as KEs. The adverse outcome has been defined as testicular atrophy. The HDIs inhibit deacetylation of the histone, leading to an increase in histone acetylation. The apoptosis induced by the disrupted cell cycle leads to spermatocyte depletion and testis atrophy. This AOP may be one of the pathways induced by HDIs, which suggests the pathway networks of protein hyperacetylations.</p>
<p>Biological Plausibility of the MIE => KE1 is high.<br />
Rationale: Upon the inhibition of HDAC by HDIs, the acetylation of lysine in histone remains and it leads to transcriptional activation or repression, changes in DNA replication and DNA damage repair. (Wade, 2008).</p>
Rationale: Upon the inhibition of HDAC by HDIs, the acetylation of lysine in histone remains and it leads to transcriptional activation or repression, changes in DNA replication, and DNA damage repair. The activity of histone acetyltransferase (HAT) in testis nuclear protein was increased with MAA addition [Wade et al., 2008].</p>
<p>Biological Plausibility of the KE1 => KE2 is moderate.<br />
Rationale: HDIs induce histone acetylation increase and p21 expression increase leading to the cell cycle arrest, which suggests the close correlation between histone acetylation increase and p21. In the models proposed for the relationship between histone acetylation and transcription, histone acetylation can be untargeted and occur at both promoter and nonpromoter regions, targeted generally to promoter regions, or targeted to specific promoters by gene-specific activator proteins (Richon, 2000, Struhl, 1998). Since several results supported a model in which increased histone acetylation is targeted to specific genes and occurs throughout the entire gene, not just the promoter regions, histone acetylation may leads to gene transcription of p21 (Richon, 2000).</p>
<p>Biological Plausibility of the KE2 => KE3 is high.<br />
Rationale: The study using the p21 deficient lungs showed that p21 is essential for the survival under hyperoxia and protects the lung from oxidative stress (O’Reilly, 2001). Hyperoxia inhibits DNA replication through p21 and histone H3 expression (O’Reilly, 2001). Hyperoxia decreased proliferation in p21 wild-type lungs but not in p21-deficient mice, which suggests that p21 is crucial for cell cycle regulation (O’Reilly, 2001).</p>
Rationale: Gene transcription is regulated by histone acetylation [Struhl, 1998]. Acetylation of histones neutralizes the positive charge of the histones. Thus, less compacted DNA can be bound more easily by transcription factors and transcribed. In the models proposed for the relationship between histone acetylation and transcription, histone acetylation can be untargeted and occur at both promoter and non-promoter regions, targeted generally to promoter regions, or targeted to specific promoters by gene-specific activator proteins [Richon et al., 2000; Struhl, 1998].</p>
</td>
</tr>
<tr>
<td style="height:101px; width:120px">
<p>KE3 => KE4: Cell cycle disrupted leads to apoptosis</p>
<td style="height:101px; width:143px">
<p>KE2 => KE3: Cell cycle, disrupted leads to apoptosis</p>
</td>
<td style="height:101px; width:423px">
<p>Biological Plausibility of the KE3 => KE4 is high.<br />
Rationale: microRNA-497, potentially targeting Bcl2 and Cyclin D2 (CCND2), induced apoptosis via the Bcl-2/Bax - caspase 9 - caspase 3 pathway and CCND2 protein in human umbilical vein endothelial cells (HUVECs) (Wu, 2016). The microRNA-497 activated caspases 9 and 3, and decreased Bcl2 and CCND2 (Wu, 2016). CCND2 is an important cell cycle gene that induces G<sub>1</sub> arrest (Li, 2012), and deregulated CCND2 is implicated in cell proliferation inhibition (Wu, 2016, Mermelstein, 2005, Dong, 2010).</p>
<td style="height:101px; width:425px">
<p>Biological Plausibility of the KE2 => KE3 is moderate.<br />
Rationale: Prolonged cell cycle arrest will lead to either senescence or apoptosis. Especially for fast-dividing and still differentiating cells, such an arrest will most certainly induce apoptosis as the normal cellular program cannot be followed.</p>
</td>
</tr>
<tr>
<td style="height:93px; width:120px">
<p>KE4 => KE5: Apoptosis leads to spermatocyte depletion</p>
<td style="height:93px; width:143px">
<p>KE3 => KE4: Apoptosis leads to spermatocyte depletion</p>
</td>
<td style="height:93px; width:423px">
<p>Biological Plausibility of the KE4 => KE5 is high.<br />
Rationale: Apoptosis is a basic biological phenomenon in which the cells are controlled in the atrophy of various tissues and organs through the deletion and turnover, as well as in tumor regression (Kerr, 1972).</p>
<td style="height:93px; width:425px">
<p>Biological Plausibility of the KE3 => KE4 is moderate.<br />
Rationale: During development and in tissue homeostasis, apoptosis is needed to control organ size. If apoptosis is induced at a higher rate, one can assume it leading to atrophy of the target organ. Especially when target organ/target cells are fast replicating, abnormal levels of apoptosis will lead to depletion.</p>
</td>
</tr>
<tr>
<td style="height:93px; width:120px">
<p>KE5 => AO: Spermatocyte depletion leads to testicular toxicity</p>
<td style="height:28px; width:143px">
<p>KE4 => AO: Spermatocyte depletion leads to testicular atrophy</p>
</td>
<td style="height:93px; width:423px">
<p>Biological Plausibility of the KE5 => AO is high.<br />
Rationale: HDAC inhibition induced testicular toxicity including testis atrophy [Miller, 1982]. HDAC inhibition in cell culture resulted in the testicular toxicity including germ cell apoptosis and cell morphology change [Li, 1996].</p>
<td style="height:28px; width:425px">
<p>Biological Plausibility of the KE4 => AO is moderate.<br />
Rationale: Spermatocyte depletion is one of the main characteristics of testicular atrophy.</p>
</td>
</tr>
<tr>
<td colspan="2" style="height:41px; width:543px">
<p>2. Support for essentiality of KEs</p>
<td colspan="2" style="height:27px; width:568px">
<p>2. Support for Essentiality of KEs</p>
</td>
</tr>
<tr>
<td style="height:89px; width:120px">
<p>KE4: Apoptosis</p>
<td style="height:89px; width:143px">
<p>KE2: Cell cycle, disrupted</p>
</td>
<td style="height:89px; width:423px">
<p>Essentiality of the KE4 is moderate.<br />
Rationale for Essentiality of KEs in the AOP: HDAC1-defecient embyonic stem cells showed reduced proliferation rates, which correlates with decreased cyclin-associated kinase activities and elevated levels of the cyclin-dependent kinase inhibitor p21 (Lagger, 2002). Loss of HDAC1 leads to significantly reduced overall deacetylase activity, hyperacetylation of a subset of histones H3 and H4 (Lagger, 2002).</p>
<td style="height:89px; width:425px">
<p>The essentiality of the KE2 is moderate.<br />
The rationale for the Essentiality of KEs in the AOP: HDAC1-deficient embryonic stem cells showed reduced proliferation rates, which correlates with decreased cyclin-associated kinase activities and elevated levels of the cyclin-dependent kinase inhibitor 1A, a cell cycle regulator p21 [Lagger et al., 2002]. Loss of HDAC1 leads to significantly reduced overall deacetylase activity, hyperacetylation of a subset of histones H3 and H4 [Lagger et al., 2002].</p>
<p>Empirical Support of the MIE => KE1 is high.<br />
Rationale: HDIs increase histone acetylation in brain (Schroeder, 2013). The HDI selectivity exists, in which SAHA is a more potent inducer of histone acetylation than MS-275, and more acetylation sites on the histones H3 and H4 are responsible to SAHA than MS-275 (Choudhary, 2009). HDI AR-42 induces acetylation of histone H3 in dose-response manner in human pancreatic cancer cell lines (Henderson 2016). To quantify acetylation by HDAC, stable isotope labeling with amino acids in cell culture (SILAC) method is used (Choudhary, 2009). SAHA and MS-275 increased acetylation of specific lysines on histones more than twofold (Choudhary, 2009). Acetylation of the variant histone H2AZ-a mark for DNA damage sites- was upregulated almost 20-fold by SAHA, whereas a number of sites on the core histones H3 and H4 are several times more highly regulated in response to SAHA than by MS-275 (Choudhary, 2009). TSA (100 ng/ml) accumulated the acetylated histones in a variety of mammalian cell lines, and the partially purified HDAC from wild-type FM3A cells was effectively inhibited by TSA (Ki = 3.4 nM) (Yoshida, 1990). To predict the degree of acetylation of lysine, a public database called Phosida (www.phosida.com), which is a member of ProteinEx-change and provides detailed information about each acetylation site is available (Choudhary, 2009, Gnad, 2011). The database contains high-accuracy species-specific phosphorylation and acetylation site predictors and allow the <em>in silico</em> determination of modified sites on any protein on the basis of the primary sequence (Gnad, 2011).</p>
Rationale: HDAC inhibitors increase histone acetylation in the brain [Schroeder et al., 2013]. The major empirical evidence came from the incubation of cell culture cells with small molecule compounds that inhibit HDAC enzymes followed by western blots or acid urea gel analysis. The first evidence was shown by Riggs et al. who showed that incubation of HeLa cells with <em>n</em>-butyrate leads to an accumulation of acetylated histone proteins [Riggs et al., 1977]. Later, it was shown that <em>n</em>-butyrate specifically increases the acetylation of histone by the incorporation of radioactive [<sup>3</sup>H]acetate and analysis of the histones on acid urea gels that allow the detection of acetylated histones [Cousens et al., 1979]. TSA was shown to be an HDAC inhibitor by acid urea gel analysis in 1990 [Yoshida et al., 1990] and good evidence for VPA as an HDAC inhibitor <em>in vitro</em> and <em>in vivo</em> was shown using acetyl-specific antibodies and western blot [Gottlicher et al., 2001].</p>
<p>Empirical Support of the KE1 => KE2 is moderate.<br />
Rationale: Histone acetylation regulates the gene transcriptional mechanism (Struhl, 1998). Histones, which may inhibit RNA synthesis, are acetylated and the acetylation of histones promote the RNA polymerase reaction (Allfrey, 1964, Pogo, 1966). HDIs accumulated acetylation of histones and induced p21 protein and mRNA expression (Richon, 2000, Wu, 2001). TSA (0.3 uM) induced p21 mRNA expression in 1 hr after stimulation and the induction is returned to the basal level in 24 hrs (Wu, 2001). Sodium butyrate (5 mM) and repetitive doses of TSA (0.3 uM, every 8 hrs) induced the p21 mRNA level in 24 hrs in HT-29 cells (Wu, 2001). Time course for histone H4 hyperacetylation in response to in repeated doses of TSA every 8hrs showed that histone hyperacetylation was peaked in 12 hrs in 8-fold increase and showed 5-fold increase in 24 hrs compared to control (Wu, 2001).</p>
Rationale: Increase in histone acetylation by HDAC inhibition induces the cell cycle regulator expression and inhibits progression through the cell cycle. Histone acetylation regulates the gene transcriptional mechanism [Struhl, 1998]. Acetylation of histones promotes the RNA polymerase reaction [Allfrey et al., 1964; Pogo et al., 1966]. Since several results supported a model in which increased histone acetylation is targeted to a specific gene and occurs throughout the entire genome, not just the promoter regions, histone acetylation may lead to gene transcription of the cell cycle regulator [Richon et al., 2000].</p>
<p>KE2 => KE3: Cell cycle, disrupted leads to apoptosis</p>
</td>
<td style="height:122px; width:423px">
<p>Empirical Support of the KE2 => KE3 is high.<br />
Rationale: HDIs induce p53-independent expression of p21 via Sp1 binding sites in the p21 promoter (Gartel, 2002). TSA induces p21 expression leading to cell cycle arrest (Gartel, 2002). Butyrate induced p21 and apoptosis in human colon tumor cell lines, whereas the absence of p21 increased the apoptosis in HCT116 colon carcinoma cell line, which indicates that p21 has a repressive effect for butyrate-induced apoptosis and protects the cells from butyrate-induced cell death (Gartel, 2002). SAHA induced p53-independent p21 expression and apoptosis in myelomonocytic leukemia cells (Gartel, 2002). The SAHA-related lethality was increased by anti-sense p21, which indicates a protective role of p21 against SAHA-induced apoptosis (Gartel, 2002). The peptide containing cyclin-binding domain of p21 in N-terminus inhibited the kinase activity of cyclin E-Cdk2 with concentration of inhibitor which inhibits kinase activity to 50% of activity (Ki) of 296 nM (Chen, 1996). The Ki was more than 300,000 nM for inhibition of the kinase activity of cyclin D1-Cdk4, and 220 nM for inhibition of the kinase activity of cyclin A-Cdk2 (Chen, 1996). The peptide containing cyclin-binding domain of p21 in C-terminus showed 32,000, 800, or >300,000 nM of Ki for inhibition of the kinase activity of cyclin E-Cdk2, cyclin A-Cdk2 or cyclin D1-Cdk4, respectively (Chen, 1996). GST fusion proteins of p21 without amino acids 17-24 (cyclin binding site in N-terminus of p21) showed 4.3, 0.4, or >850 nM of Ki for inhibition of the kinase activity of cyclin E-Cdk2, cyclin A-Cdk2, or cyclin D1-Cdk4, respectively (Chen, 1996). Deletion of either cyclin binding site in N-terminus or C-terminus of p21, or CDK binding domain was sufficient for the kinase activity inhibition (Chen, 1996).</p>
<td style="height:129px; width:425px">
<p>Empirical Support of the KE2 => KE3 is moderate.<br />
Rationale: Cell cycle arrests such as G<sub>1 </sub>arrest and G<sub>1</sub>/S arrest are observed in apoptosis [Li et al., 2012; Dong et al., 2010]. microRNA-1 and microRNA-206 repress CCND2, while microRNA-29 represses CCND2 and induces G<sub>1</sub> arrest and apoptosis in rhabdomyosarcoma [Li et al., 2012].</p>
</td>
</tr>
<tr>
<td style="height:129px; width:120px">
<p>KE3 => KE4: Cell cycle disrupted leads to apoptosis</p>
<td style="height:128px; width:143px">
<p>KE3 => KE4: Apoptosis leads to spermatocyte depletion</p>
</td>
<td style="height:129px; width:423px">
<td style="height:128px; width:425px">
<p>Empirical Support of the KE3 => KE4 is high.<br />
Rationale: Cell cycle arrest such as G1 arrest and G1/S arrest are observed in apoptosis (Li, 2012, Dong, 2010). microRNA-1 and microRNA-206 represses CCND2, while microRNA-29 represses CCND2 and induces G1 arrest and apoptosis in rhabdomyosarcoma (Li, 2012). Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of pNA and quantified at 405 nm (Wu, 2016). Apoptosis is measured with Annexin V-FITC probes, and the relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry (Wu, 2016).</p>
Rationale: microRNA-21 regulates the spermatogonial stem cell homeostasis, in which suppression of microRNA-21 with anti-miR-21 oligonucleotides led to apoptosis of spermatogonial stem cell-enriched germ cell cultures and the decrease in the number of spermatogonial stem cells [Niu et al., 2011].</p>
</td>
</tr>
<tr>
<td style="height:124px; width:120px">
<p>KE4 => KE5: Apoptosis leads to spermatocyte depletion</p>
<td style="height:114px; width:143px">
<p>KE4 => AO: Spermatocyte depletion leads to testicular atrophy</p>
</td>
<td style="height:124px; width:423px">
<p>Empirical Support of the KE4 => KE5 is high.<br />
Rationale: MicroRNA-21 regulates the spermatogonial stem cell homeostasis, in which suppression of microRNA-21 with anti-miR-21 oligonucleotides led to apoptosis of spermatogonial stem cell-enriched germ cell cultures and the decrease in the number of spermatogonial stem cells (Niu, 2011).</p>
</td>
</tr>
<tr>
<td style="height:124px; width:120px">
<p>KE5 => AO: Spermatocyte depletion leads to testicular toxicity</p>
</td>
<td style="height:124px; width:423px">
<p>Empirical Support of the KE5 => AO is high.<br />
Rationale: 2-methoxyethanol (ME) or its major metabolite, MAA induced spermatocyte depletion and testicular atrophy [Beattie, 1984].</p>
<td style="height:114px; width:425px">
<p>Empirical Support of the KE4 => AO is high.<br />
Rationale: The testicular atrophy seen in 2-methoxyethanol (2-ME), or its major metabolite MAA, treated rats <em>in vivo</em> and in human, and rat <em>in vitro</em> culture was associated with spermatocyte depletion [Beattie et al., 1984].</p>
<p>The AOP is applicable to the reproductively mature males in rats, mice and humans. The administration route or doses of HDAC inhibitors may affect the intensity of the toxicity.</p>
<p>The AOP is applicable to the reproductively mature males in rats, mice and humans. The administration route or doses of HDAC inhibitors may affect the intensity of the toxicity.</p>
<p>HDAC inhibition induced testicular toxicity including testis atrophy [Miller, 1982]. HDAC inhibition in cell culture resulted in the testicular toxicity including germ cell apoptosis and cell morphology change [Li, 1996].</p>
<td style="width:573px">HDAC inhibition induced testicular toxicity including testis atrophy [Miller et al., 1982]. HDAC inhibition in cell culture resulted in testicular toxicity including germ cell apoptosis and cell morphology change [Li et al., 1996].</td>
</tr>
<tr>
<td style="width:147px">
<p>KE2: p21 (CDKN1A) expression, increase</p>
</td>
<td style="width:408px">
<p>Loss of HDAC1 in mouse embryonic stem (ES) cells has demonstrated the acetylation of histones H3 and H4, up-regulation of cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1 and inhibition of proliferation (Lagger, 2002).</p>
<td style="width:573px">The HDAC inhibition induced cell death in spermatocytes in both rat and human seminiferous tubules [Li et al., 1996].</td>
</tr>
<tr>
<td style="width:147px">
<p>KE3: Cell cycle, disrupted</p>
</td>
<td style="width:408px">
<p>In HDAC1-/- fibroblast lines, increase in the amount of cells in G1 phase and decrease in the amount of cells in S phase were observed, which indicates the importance of HDAC inhibition in cell cycle regulation [Zupkovitz, 2010].</p>
<td style="width:573px">In HDAC1-/- fibroblast lines, an increase in the number of cells in the G<sub>1</sub> phase and a decrease in the number of cells in the S phase were observed, which indicates the importance of HDAC inhibition in cell cycle regulation [Zupkovitz et al., 2010].</td>
</tr>
<tr>
<td style="width:147px">
<p>KE4: Apoptosis</p>
</td>
<td style="width:408px">
<p>HDAC inhibition leads to cell death through the apoptotic pathways (Falkenberg, 2014).</p>
<td style="width:573px">HDAC inhibition leads to cell death through the apoptotic pathways [Falkenberg et al., 2014].</td>
</tr>
<tr>
<td style="width:147px">
<p>KE5: spermatocyte depletion</p>
</td>
<td style="width:408px">
<p>The HDAC inhibition induced cell death in spermatocytes in both rat and human seminiferous tubules [Li, 1996]. The HDAC inhibitor treatment resulted in degeneration in spermatocytes in rat seminiferous tubules [Li, 1996]. The HDAC inhibition induced the germ cell apoptosis in human testicular tissues [Li, 1996].</p>
<td style="width:573px">The HDAC inhibition induced cell death in spermatocytes in both rat and human seminiferous tubules [Li et al., 1996]. The HDAC inhibitor treatment resulted in degeneration in spermatocytes in rat seminiferous tubules [Li et al., 1996]. The HDAC inhibition induced germ cell apoptosis in human testicular tissues [Li et al., 1996].</td>
</tr>
</tbody>
</table>
<h3>Weight of Evidence Summary</h3>
<p style="margin-left:18.0pt"><em>Biological plausibility, coherence, and consistency of the experimental evidence</em></p>
<h3>Weight of Evidence Summary</h3>
<p style="margin-left:18.0pt"><em>Biological plausibility, coherence, and consistency of the experimental evidence</em></p>
<p style="margin-left:18.0pt">The available data supporting the AOP are logic, coherent and consistent with established biological knowledge, whereas there are possibilities for alternative pathways.</p>
<p style="margin-left:18.0pt">The available data supporting the AOP are logical, coherent, and consistent with established biological knowledge, whereas there are possibilities for alternative pathways.</p>
<p style="margin-left:18.0pt"> </p>
<p style="margin-left:18.0pt"><em>Alternative mechanism(s) that logically present themselves and the extent to which they may distract from the postulated AOP</em></p>
<p> </p>
<p style="margin-left:18.0pt">There are some other important apoptotic pathways that are involved in cell death, as well as other important spermatocyte signaling or mechanism influences testicular toxicity.</p>
<p style="margin-left:18.0pt"> </p>
<p style="margin-left:18.0pt">p53 pathway</p>
<ul>
<li>p53 pathway</li>
</ul>
<p style="margin-left:18.0pt">The study in which <em>in vivo</em> administration of trichostatin A (TSA), a HDI, in mice resulted in male meiosis impairment showed the involvement of p53-noxa-caspase-3 apoptotic pathway in TSA-induced spermatocyte apoptosis [Fenic, 2008]. Other study showed that MAAinduced up-regulation of p21 expression is mediated through histone hyperacetylation and independent of p53/p63/p73 [Parajuli, 2014].</p>
<p style="margin-left:18.0pt">The study in which <em>in vivo</em> administration of trichostatin A (TSA), an HDI, in mice resulted in male meiosis impairment showed the involvement of p53-noxa-caspase-3 apoptotic pathway in TSA-induced spermatocyte apoptosis [Fenic et al., 2008]. Another study showed that MAA-induced up-regulation of p21 expression is mediated through histone hyperacetylation and independent of p53/p63/p73 [Parajuli et al., 2014].</p>
<p style="margin-left:18.0pt">The present AOP focuses on p21 pathway leading to apoptosis, however, the alternative pathway such as NF-kappaB signaling pathways may be involved in apoptosis of spermatocytes [Wang, 2017].</p>
<p style="margin-left:18.0pt">The present AOP focuses on the p21 pathway leading to apoptosis, however, alternative pathways such as NF-kappaB signaling pathways may be involved in the apoptosis of spermatocytes [Wang et al., 2017].</p>
<p style="margin-left:18.0pt"> </p>
<p style="margin-left:18.0pt">Communication with Sertoli cells</p>
<ul>
<li>Communication with Sertoli cells</li>
</ul>
<p style="margin-left:18.0pt">The present AOP focuses on testicular atrophy by HDAC inhibition-induced apoptosis in spermatocytes, however, the signaling in Setoli cells may be involved in testicular atrophy. Sertoli cell secretes GDNF, FGF2, CXCL12 or Ccl9 molecules, which results in the activation of RET, FGFR, CXCR4 or CCR1 signaling in spermatogonial stem cells, respectively [Chen, 2015].</p>
<p style="margin-left:18.0pt">The present AOP focuses on testicular atrophy by HDAC inhibition-induced apoptosis in spermatocytes, however, the signaling in Sertoli cells may be involved in testicular atrophy. Sertoli cell secretes GDNF, FGF2, CXCL12, or Ccl9 molecules, which results in the activation of RET, FGFR, CXCR4, or CCR1 signaling in spermatogonial stem cells, respectively [Chen and Liu, 2015].</p>
<p style="margin-left:18.0pt"> </p>
<p style="margin-left:18.0pt">Decrease in deoxynucleotide pool by MAA</p>
<ul>
<li>Decrease in deoxynucleotide pool by MAA</li>
</ul>
<p style="margin-left:18.0pt">MAA induces decrease in deoxynucleotide pool, resulting apoptosis, which may be an alternative pathway other than p21-mediated pathway [Yamazoe, 2015]. Inhibition of 5,10-CH<sub>2</sub>-THF production by MAA may decreases deoxynucleotide pool in spermatocytes [Yamazoe, 2015].</p>
<p style="margin-left:18.0pt">MAA induces a decrease in the deoxynucleotide pool, resulting in apoptosis, which may be an alternative pathway other than the p21-mediated pathway [Yamazoe et al., 2015]. Inhibition of 5,10-CH<sub>2</sub>-THF production by MAA may decrease deoxynucleotide pool in spermatocytes [Yamazoe et al., 2015].</p>
<p style="margin-left:18.0pt"> </p>
<ul>
<li>Spermatocyte depletion by necrosis</li>
</ul>
<p style="margin-left:18.0pt">Spermatocyte may be decreased by necrosis. Cell death mechanisms other than apoptosis, such as necrosis, may be considered for spermatocyte depletion.</p>
<h3>Quantitative Consideration</h3>
<p style="margin-left:18.0pt"><em>Concordance of dose-response relationships</em></p>
<h3>Quantitative Consideration</h3>
<p style="margin-left:18.0pt"><em>Concordance of dose-response relationships</em></p>
<p style="margin-left:18.0pt">This is a quantitative description on dose-response relationships from MIE to AOP. But some KE relationships individually are not fully supported with dose-response relationships, while there is empirical evidence to support that a change in KEup leads to an appropriate change in the respective KEdown.</p>
<p style="margin-left:18.0pt">This is a quantitative description of dose-response relationships from MIE to AOP. But some KE relationships individually are not fully supported with dose-response relationships, while there is empirical evidence to support that a change in KEup leads to an appropriate change in the respective KEdown.</p>
<p style="margin-left:18.0pt"> </p>
<p style="margin-left:18.0pt"><em>Temporal concordance among the key events and adverse outcome</em></p>
<p style="margin-left:18.0pt">Temporal concordance between MIE and AOP has been described with in vivo experimental data. Empirical evidences show temporal concordance between MIE and the individual KEs, however, the temporal concordance among the individual KEs and AO is not fully elucidated.</p>
<p style="margin-left:18.0pt">Temporal concordance between MIE and AOP has been described with <em>in vivo</em> experimental data. Empirical evidence shows temporal concordance between MIE and the individual KEs, however, the temporal concordance among the individual KEs and AO is not fully elucidated.</p>
<p> </p>
<p style="margin-left:18.0pt"><em>Strength, consistency, and specificity of association of adverse outcome and initiating event</em></p>
<p style="margin-left:18.0pt">The scientific evidence on the linkage between MIE and AO has been described.</p>
<p> </p>
<p style="margin-left:18.0pt">The quantitative understanding of the AOP in terms of indirect relations between HDAC inhibition and testicular atrophy was examined in in vivo experiments [Foster, 1983, Miller, 1982].</p>
<p style="margin-left:18.0pt">The quantitative understanding of the AOP in terms of indirect relations between HDAC inhibition and testicular atrophy was examined in <em>in vivo</em> experiments [Foster et al., 1983; Miller et al., 1982].</p>
<p style="margin-left:18.0pt"> </p>
</div>
<!-- potential consierations, text as entered by author -->
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<hr>
<p> The present AOP can be used in risk assessment of HDAC inhibitors for the anti-cancer drugs in terms of testicular toxicity. HDAC inhibitors nowadays have been utilized as therapeutics for cancer or neurology disease, and the adverse effects of HDAC inhibitors should be evaluated. This AOP elucidating the pathway from HDAC inhibition through testicular toxicity may provides important insights for potential toxicity of HDAC inhibitors. It also provides a basis for the HDAC inhibition-induced epigenetic alteration and cell death. HDAC inhibitors such as rocilinostat are clinically evaluated as anti-cancer drugs in clinical trial.</p>
<h2>Considerations for Potential Applications of the AOP (optional)</h2>
<p>The AOP may be useful in the risk assessment on several types of HDI molecules including anti-cancer drugs, as well as other types of chemicals, biocides, or pesticides. HDAC inhibitors nowadays have been utilized as therapeutics for cancer or neurology disease, and the adverse effects of HDAC inhibitors should be evaluated. This AOP elucidating the pathway from HDAC inhibition to testicular atrophy may provide important insights into the potential toxicity of HDAC inhibitors. It also provides a basis for the HDAC inhibition-induced epigenetic alteration and cell death. HDAC inhibitors such as rocilinostat/ricolinostat are clinically evaluated as anti-cancer drugs in clinical trials [Yee et al., 2016]. The AOP may be useful for the risk assessment of chemi<span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">cals, since possible applications of HDAC inhibitors include the enhancement of salinity tolerance to increase agricultural sustainability. Other potential applications of the AOP include the risk assessment of biocides or pesticides, considering that HDAC inhibitors are being investigated as insecticides or amoebicides [Bagnall et al., 2017; Lee et al., 2020].</span></span> </p>
</div>
<!-- reference section, text as of right now but should be changed to be handled as table -->
<div id="references">
<h2>References</h2>
<hr>
<p style="margin-left:49.55pt">Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831</p>
<p style="margin-left:49.55pt">Richon VM et al. (2000) Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. Proc Natl Acad Sci 97:10014-10019 </p>
<p style="margin-left:49.55pt">Struhl K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Gene Dev 12:599-606</p>
<p style="margin-left:49.55pt">O’Reilly MA et al (2001) The cyclin-dependent kinase inhibitor p21 protects the lung from oxidative stress. Am J Respir Cell Mol Biol 24: 703-710 </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Allfrey, V. et al. (1964), "Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis", Proc Natl Acad Sci 51:786-794</span></span></p>
<p style="margin-left:49.55pt">Wu R et al. (2016) microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs. PLoS One 11: e0167052 </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Bagnall, N.H. et al. (2017), "Insecticidal activities of histone deacetylase inhibitors against a dipteran parasite of sheep, Lucilia cuprina", Int J Parasitology: Drugs Drug Resistance <em>7</em>(1):51–60 https://doi.org/10.1016/j.ijpddr.2017.01.001</span></span></p>
<p style="margin-left:49.55pt">Li L et al. (2012) Downregulation of microRNAs miR-1, -206 and -29 stabilizes PAX3 and CCND2 expression in rhabdomyosarcoma. Lab Invest 92: 571-583 </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Beattie, P.J. et al. (1984), "The effect of 2-methoxyethanol and methoxyacetic acid on Sertoli cell lactate production and protein synthesis in vitro", Toxicol Appl Pharmacol 76:56-61</span></span></p>
<p style="margin-left:49.55pt">Mermelshtein A et al. (2005) Expression of F-type cyclins in colon cancer and in cell lines from colon carcinomas. Br J Cancer 93: 33 </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Chen, S. and Liu, Y. (2015), "Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling", Reproduction 149:R159-R167</span></span></p>
<p style="margin-left:49.55pt">Dong Q et al. (2010) microRNA let-7a inhibits proliferation of human prostate cancer cells in vitro and in vivo by targeting E2F2 and CCND2. PLoS One 5: e10147</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Cousens, L.S., et al. (1979), "Different accessibilities in chromatin to histone acetylase", J Biol Chem 254:1716-1723</span></span></p>
<p style="margin-left:49.55pt">Kerr JFR et al. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Dong, Q. et al. (2010), "microRNA let-7a inhibits proliferation of human prostate cancer cells in vitro and in vivo by targeting E2F2 and CCND2", PLoS One 5:e10147</span></span></p>
<p style="margin-left:49.55pt">Miller RR et al. (1982) Toxicity of methoxyacetic acid in rats. Fundam Appl Toxicol 2: 158-160</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Fenic, I. et al. (2008), "In vivo application of histone deacetylase inhibitor trichostatin-A impairs murine male meiosis", J Andro 29:172-185</span></span></p>
<p style="margin-left:49.55pt">Li LH et al. (1996) 2-Methoxyacetic acid (MAA)-induced spermatocyte apoptosis in human and rat testes: an in vitro comparison. J Androl 17: 538-549</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Foster, P.M. et al. (1983), "Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat", Toxicol Appl Pharmacol 69:385-39</span></span></p>
<p style="margin-left:49.55pt">Lagger G et al. (2002) Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J 21:2672-2681 </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Gottlicher, M. et al. (2001), "Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells", EMBO J 20:6969-6978</span></span></p>
<p style="margin-left:49.55pt">Schroeder FA et al. (2013) A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests. PLoS One 8:e71323</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Lagger, G. et al. (2002), "Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression", EMBO J 21:2672-2681 </span></span></p>
<p style="margin-left:49.55pt">Choudhary C et al. (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834-840</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Lee, H-A. et al. (2020), "Application of histone deacetylase inhibitors MPK472 and KSK64 as a potential treatment option for Acanthamoeba keratitis" Antimicrob Agents Chemother 64:e01506-20 https://doi.org/10.1128/AAC.01506-20 </span></span></p>
<p style="margin-left:49.55pt">Henderson SE et al. (2016) Suppression of tumor growth and muscle wasting in a transgenic mouse model of pancreatic cancer by the novel histone deacetylase inhibitor AR-42. Neoplasia 18:765-774</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Li, L. et al. (2012), "Downregulation of microRNAs miR-1, -206 and -29 stabilizes PAX3 and CCND2 expression in rhabdomyosarcoma", Lab Invest 92:571-583 </span></span></p>
<p style="margin-left:49.55pt">Yoshida M et al. (1990) Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro trichostatin A. J Biol Chem 265:17174-17179</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Li, L.H. et al. (1996), "2-Methoxyacetic acid (MAA)-induced spermatocyte apoptosis in human and rat testes: an in vitro comparison", J Androl 17:538-549</span></span></p>
<p style="margin-left:49.55pt">Gnad F et al. (2011) PHOSIDA 2011: the posttranslational modification database. Nucl Acids Res 39:D253-D260</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Miller, R.R. et al. (1982), "Toxicity of methoxyacetic acid in rats", Fundam Appl Toxicol 2:158-160</span></span></p>
<p style="margin-left:49.55pt">Allfrey V et al (1964) Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Natl Acad Sci 51: 786-794</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Niu, Z. et al. (2011), "microRNA-21 regulates the self-renewal of mouse spermatogonial stem cells", Proc Natl Acad Sci 108:12740-12745</span></span></p>
<p style="margin-left:49.55pt">Pogo B et al (1966) RNA synthesis and histone acetylation during the course of gene activation in lymphocytes. Proc Natl Acad Sci 55: 805-812</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-312</span></span></p>
<p style="margin-left:49.55pt">Richon VM et al. (2000) Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. Proc Natl Acad Sci 97:10014-10019</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Pogo, B. et al. (1966), "RNA synthesis and histone acetylation during the course of gene activation in lymphocytes", Proc Natl Acad Sci 55:805-812</span></span></p>
<p style="margin-left:49.55pt">Wu JT et al. (2001) Transient vs prolonged histone hyper acetylation: effects on colon cancer cell growth, differentiation, and apoptosis. Am J Physiol Gastrointest Liver Physiol 280:G482-G490</p>
<p style="margin-left:49.55pt">Gartel AL and Tyner AL (2002) The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther 1: 639-649</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Riggs, M.G. et al. (1977), "N-butyrate causes histone modification in HeLa and friend erythroleukaemia cells", Nature 268:462-464</span></span></p>
<p style="margin-left:49.55pt">Chen J et al (1996) Cyclin-binding motifs are essential for the function of p21CIP1. Mol Cell Biol 16: 4673-4682 </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Schroeder, F.A. et al. (2013), "A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests", PLoS One 8:e71323</span></span></p>
<p style="margin-left:49.55pt">Li L et al. (2012) Downregulation of microRNAs miR-1, -206 and -29 stabilizes PAX3 and CCND2 expression in rhabdomyosarcoma. Lab Invest 92: 571-583</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Struhl, K. (1998), "Histone acetylation and transcriptional regulatory mechanisms", Gene Dev 12:599-606 </span></span></p>
<p style="margin-left:49.55pt">Dong Q et al. (2010) microRNA let-7a inhibits proliferation of human prostate cancer cells in vitro and in vivo by targeting E2F2 and CCND2. PLoS One 5: e10147</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</span></span></p>
<p style="margin-left:49.55pt">Wu R et al. (2016) microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs. PLoS One 11: e0167052 </p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143</span></span></p>
<p style="margin-left:49.55pt">Niu Z et al. (2011) microRNA-21 regulates the self-renewal of mouse spermatogonial stem cells. Proc Natl Acad Sci 108: 12740-12745</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Yamazoe, Y. et al. (2015), "Embryo- and testicular-toxicities of methoxyacetate and the related: a review on possible roles of one-carbon transfer and histone modification", Food Safety 3:92-107</span></span></p>
<p style="margin-left:49.55pt">Beattie PJ, et al. (1984) The effect of 2-methoxyethanol and methoxyacetic acid on Sertoli cell lactate production and protein synthesis in vitro. Toxicol Appl Pharmacol 76: 56-61</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Yee, A.J. et al. (2016), "Ricolinostat plus lenalidomide, and dexamethasone in relapsed or refractory multiple myeloma: a multicentre phase 1b trial", Lancet Oncol 17(11):1569-1578 https://doi.org/<a href="https://scifinder-n.cas.org/navigate/?answersPerPage=1&appId=8390a8c4-deb9-4044-b98f-91567683d716&externalLink=http%253A%252F%252Fdx.doi.org%252F10.1016%252Fs1470-2045(16)30375-8&fullTextOption=716&fullTextPresentedOptions=%5B%7B%22type%22%3A716%2C%22value%22%3Atrue%7D%2C%7B%22type%22%3A702%2C%22value%22%3Afalse%7D%5D&resultType=reference&state=externalLinks&uiContext=697&uriForDetails=document%2Fpt%2Fdocument%2F47951405" style="color:blue; text-decoration:underline" target="_blank">10.1016/s1470-2045(16)30375-8</a></span></span></p>
<p style="margin-left:49.55pt">Zupkovitz G et al. (2010) The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation. Mol Cell Biol 30:1171-1181</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Yoshida, M. et al. (1990), "Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro trichostatin A", J Biol Chem 265:17174-17179</span></span></p>
<p style="margin-left:49.55pt">Falkenberg KJ and Johnstone RW. (2014) Histone deacetylases and their inhibitors in cancer, neurological disease and immune disorders. Nat Rev Drug Discov 13:673-691</p>
<p style="margin-left:49.55pt">Fenic I et al. (2008) In vivo application of histone deacetylase inhibitor trichostatin-A impairs murine male meiosis. J Andro 29: 172-185</p>
<p style="margin-left:49.55pt">Parajuli KR et al. (2014) Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis. Am J Clin Exp Urol 2:300-312</p>
<p style="margin-left:49.55pt">Wang C et al. (2017) CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways. Oncotarget 8: 3132-3143</p>
<p style="margin-left:49.55pt">Chen S and Liu Y. (2015) Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling. Reproduction 149: R159-R167</p>
<p style="margin-left:49.55pt">Yamazoe Y. et al. (2015) Embryo- and testicular-toxicities of methoxyacetate and the related: a review on possible roles of one-carbon transfer and histone modification. Food Safety 3:92-107</p>
<p style="margin-left:49.55pt">Foster PM et al. (1983) Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat. Toxicol Appl Pharmacol 69:385-39</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181</span></span></p>
<!-- Evidence for Perturbation of This Event by Stressors -->
<h3>Evidence for Perturbation by Stressor</h3>
<hr>
<h4>Overview for Molecular Initiating Event</h4>
<p>HDIs are classified according to chemical nature and mode of mechanism: the short chain fatty acids (e.g., butyrate, valproate), hydroxamic acids (e.g., suberoylanilide hydroxamic acid or SAHA, Trichostatin A or TSA), cyclic tetrapeptides (e.g., FK-228), benzamides (e.g., N-acetyldinaline and MS-275) and epoxides (depeudecin, trapoxin A) [Richon, 2004, Ropero, 2007, Villar-Garea, 2004]. There is a report showing that TSA and butyrate competitively inhibits HDAC activity [Sekhavat, 2007]. HDIs inhibit preferentially HDACs with some selectiveness [Hu, 2003]. TSA inhibits HDAC1, HDAC3 and HDAC8, whereas MS-27-275 has inhibitory effect for HDAC1 and HDAC3 (IC<sub>50</sub> value of ~0.2 mM and ~8 mM, respectively), but no effect for HDAC8 (IC<sub>50</sub> value >10 mM) [Hu, 2003]. TSA inhibits HDAC1, 2, 3 of class I HDACs. HDAC 1, 4, 6 are related to tumor size [Damaskos, 2016]. MAA (2 or 5 mM) inhibited HDAC activity in dose-response manner in rat testis cytosolic and nuclear extracts [Wade, 2008].</p>
<br>
<br>
<!-- end Evidence for Perturbation of This Event by Stressors -->
<h4>Domain of Applicability</h4>
<br>
<!-- loop to find taxonomic applicability under event -->
<p>The inhibition of HDAC by HDIs is well conserved between species from lower organism to mammals.</p>
<ul>
<li>HDIs reduced lethality in <em>Drosophila</em> model and the HDAC activity was inhibited with HDIs in rat PC12 cells [Steffan, 2001].</li>
<li>HDIs inhibited restores the rate of resorption of subretinal blebs in hyper glycemia in brown Norway rat and HDAC activity was inhibited with HDIs in human ARPE19 cells [Desjardins, 2016].</li>
<li>HDIs were approved as drugs for multiple myeloma and T-cell lymphoma by FDA [Ansari, 2016].</li>
<li>HDIs inhibited cell growth in human non-small cell lung cancer cell lines [Miyanaga, 2008].</li>
<li>HDAC acetylation level was increased by HDIs in MRL-lpr/lpr murine model of lupus splenocytes [Mishra, 2003].</li>
<li>SAHA increased histone acetylation in brain and spleen of mice [Hockly, 2003].</li>
<li>MAA inhibits HDAC activity in HeLa cells and spleens from C57BL/6 mice [Jansen, 2004].</li>
<li>It is also reported that MAA inhibits HDAC activity in testis cytosolic and nuclear extract of juvenile rats (27 days old) [Wade, 2008].</li>
<li>VPA and TSA inhibit HDAC enzymatic activity in mouse embryo and human HeLa cell nuclear extract [Di Renzo, 2007].</li>
</ul>
<p>The inhibition of HDAC by HDIs is well conserved between species from lower organisms to mammals.</p>
<ul>
<li>HDAC inhibitors, phenylbutyrate (PB) (2 mM) and TSA (200 nM) acetylate histones H3 and H4 in synovial cells from rats with adjuvant arthritis [Chung, 2003].</li>
<li>HDAC inhibition restores the rate of resorption of subretinal blebs in hyperglycemia in brown Norway rat and HDAC activity was inhibited with HDIs in human ARPE19 cells [Desjardins et al., 2016].</li>
<li>Treatment of HDIs inducing HDAC inhibition showed anti-tumor effects in human non-small cell lung cancer cells [Ansari et al., 2016; Miyanaga et al., 2008].</li>
<li>HDAC acetylation level was increased by HDIs in the MRL-lpr/lpr murine model of lupus splenocytes [Mishra et al., 2003].</li>
<li>SAHA increased histone acetylation in the brain and spleen of mice [Hockly et al., 2003].</li>
<li>MAA inhibits HDAC activity in HeLa cells and spleens from C57BL/6 mice [Jansen et al., 2004].</li>
<li>It is also reported that MAA inhibits HDAC activity in testis cytosolic and nuclear extract of juvenile rats (27 days old) [Wade et al., 2008].</li>
<li>VPA and TSA inhibit HDAC enzymatic activity in the mouse embryo and human HeLa cell nuclear extract [Di Renzo et al., 2007].</li>
<li>The treatment with HDAC inhibitors, phenylbutyrate (PB) (2 mM) and TSA (200 nM), inhibits HDAC in adjuvant arthritis synovial cells derived from rats, causing higher acetylated histone [Chung et al., 2003].</li>
</ul>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p>Site of action: The site of action for the molecular initiating event is a cell.</p>
<p>The nucleosome consists of core histones having classes of H2A, H2B, H3 and H4) [Damaskos, 2017]. DNA strand (about 200 bp) wound around the core histones, where histone deacetylase (HDAC) effects on the lysine residue of the histone to hydrolyze the acetyl residue [Damaskos, 2017]. Histone deacetylase inhibitor (HDI) inhibits HDAC and acetylate the histones and release the DNA strand to induce the binding of transcription factors [Taunton, 1996]. HDIs have potentials as anti-cancer pharmaceuticals since HDIs induce the transcriptional restoration of epigenetically silenced tumor suppressor genes by regulating acetylation of histones and non-histone proteins [Lee, 2016] [Minucci, 2006].</p>
<p>It is known that 18 HDAC isoforms are classified into four classes: class I HDACs (isoforms 1, 2, 3, 8), class II isoforms (4, 5, 6, 7, 9, 10) and class III HDACs (the sirtuins) and HDAC11 [Weichert, 2009, Barneda-Zahonero, 2012]. HDACs 1, 2 and 3 are ubiquitously expressed, whereas HDAC8 is predominantly expressed in cells with smooth muscle/myoepithelial differentiation [Weichert, 2009]. HDAC6 is not observed to express in lymphocytes, stromal cells and vascular endothelial cells [Weichert, 2009]. Class III HDACs sirtuins are widely expressed and localized in different cellular compartments [Barneda-Zahonero, 2012]. SirT1 is highly expressed in testis, thymus and multiple types of germ cells [Bell, 2014]. HDAC11 expression is enriched in kidney, brain, testis, heart and skeletal muscle [Barneda-Zahonero, 2012].</p>
<p> </p>
<p>Description from EU-ToxRisk deliverable:</p>
<p>Eukaryotic histone deacetylases (HDACs) are grouped, according to phylogeny, into classes 1 through 4 (Gregoretti <em>et al.</em>, 2004). The members of groups 1,2 and 4 are dependent on a zinc ion and a water molecule at their active sites, for their deacetylase function. The Sirtuins of class 3 depend on NAD<sup>+</sup>, and are considered impervious to most known HDAC inhibitors (Drummond <em>et al.</em>, 2005).</p>
<p>Several structurally distinct groups of compounds have been found to inhibit HDACs of class 1, 2 and 4, among others short-chain fatty acids (e.g. butyrate and VPA), hydroxamic acids (e.g. TSA and SAHA) and epoxyketones (e.g. Trapoxin) (Drummond <em>et al.</em>, 2005). The hydroxamic acids seem to exert their inhibitory action by mimicking the acetyl-lysine target of HDACs, chelating the zinc ion in the active site and displacing the water molecule (Finnin <em>et al.</em>, 1999). Several recent high resolution crystal structures support this mode of inhibition (Decroos <em>et al.</em>, 2015; Luckhurst <em>et al.</em>, 2016). The mode of inhibition of epoxyketones seems to function the formation of a stable transition state analog with the zinc ion in the active site (Porter and Christianson, 2017). The inhibitory actions of the short-chain fatty acids are less well defined, but it has been speculated that VPA blocks access to the binding pocket (Göttlicher <em>et al.</em>, 2001). It has been shown that VPA exert similar gene regulatory effects to TSA, on a panel of migration related transcripts in neural crest cells (Dreser <em>et al.</em>, 2015) supporting a mode of action similar to hydroxamic acid type HDAC inhibitors.</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>The measurement of HDAC inhibition monitors the decrease in histone acetylation. The measurement methods include the immunological detection of histone acetylation with anti-acetylated histone antibodies [Richon, 2004]. The histones are isolated from pellets of cells treated with HDIs, followed by acid-urea-triton gel electrophoresis, western blotting, and immunohistochemistry [Richon, 2004]. Epigenetic modifications including the histone acetylation are measured using chromatin immunoprecipitation-microarray hybridization (ChIP-chip) [ENCODE Project Consortium, 2004, Ren, 2004]. ChIP detects physical interaction between transcription factors or cofactors and the chromosome [Johnson, 2007]. The HDAC activity is measured directly with ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) by calculating the ratio of deacetylated peptide and acetylated peptide [Zwick, 2016].</p>
<p> </p>
<p>Description from EU-ToxRisk deliverable:</p>
<p>HDAC inhibition can be followed by several different approaches:</p>
<h4>Key Event Description</h4>
<p>Nucleosomes consist of eight core histones, two of each histone H2A, H2B, H3, and H4 [Damaskos et al., 2017]. DNA strands (about 200 bp) wind around the core histones, which can be modified on their N-terminal ends. One possible modification is the acetylation of lysine residues, which decreases the binding strength between DNA and the core histone. Histone deacetylases (HDACs) hydrolyze the acetyl residues [Damaskos et al., 2017]. HDACs remove the acetyl groups from the lysine residues leading to the formation of a condensed and transcriptionally silenced chromatin. Thus, the inhibition of HDAC blocks this action and can result in hyperacetylation of histones associated mostly with increases in transcriptional activation. Histone deacetylase inhibitor (HDI) inhibits HDAC, causing increased acetylation of the histones and thereby facilitating binding of transcription factors [Taunton et al., 1996].</p>
<p>-Western blots applying antibodies targeting specific acetylated proteins.</p>
<p>It is known that eukaryotic HDAC isoforms are classified into four classes: class I HDACs (isoforms 1, 2, 3, 8), class II HDACs (isoforms 4, 5, 6, 7, 9, 10), class III HDACs (the sirtuins), and HDAC11 [Gregoretti et al., 2004; Weichert, 2009; Barneda-Zahonero and Parra, 2012]. HDACs 1, 2, and 3 are ubiquitously expressed, whereas HDAC8 is predominantly expressed in cells with smooth muscle/myoepithelial differentiation [Weichert, 2009]. HDAC6 is not observed to be expressed in lymphocytes, stromal cells, and vascular endothelial cells [Weichert, 2009]. Class III HDACs, sirtuins, are widely expressed and localized in different cellular compartments [Barneda-Zahonero and Parra, 2012]. SirT1 is highly expressed in testis, thymus, and multiple types of germ cells [Bell et al., 2014]. HDAC11 expression is enriched in the kidney, brain, testis, heart, and skeletal muscle [Barneda-Zahonero and Parra, 2012]. The members of classes 1, 2, and 4 are dependent on a zinc ion and a water molecule at their active sites, for their deacetylase function. The Sirtuins of class 3 depend on NAD<sup>+</sup> and are considered impervious to most known HDAC inhibitors [Drummond et al., 2005].</p>
<p>-Commercial fluorimetric and colorimetric kits can be applied to assay HDAC activity from various biological extracts.</p>
<p>Several structurally distinct groups of compounds have been found to inhibit HDACs of class 1, 2, and 4, among others short-chain fatty acids (e.g. butyrate and VPA), hydroxamic acids (e.g. TSA and SAHA), and epoxyketones (e.g. Trapoxin) [Drummond et al., 2005]. The hydroxamic acids seem to exert their inhibitory action by mimicking the acetyl-lysine target of HDACs, chelating the zinc ion in the active site, and displacing the water molecule [Finnin et al., 1999]. Several high-resolution crystal structures support this mode of inhibition [Decroos et al., 2015; Luckhurst et al., 2016]. The mode of inhibition of epoxyketones seems to function in the formation of a stable transition state analog with the zinc ion in the active site [Porter and Christianson, 2017]. The inhibitory actions of the short-chain fatty acids are less well defined, but it has been speculated that VPA blocks access to the binding pocket [Göttlicher et al., 2001]. It has been shown that VPA exerts similar gene regulatory effects to TSA, on a panel of migration-related transcripts in neural crest cells [Dreser et al., 2015], supporting a mode of action similar to hydroxamic-acid type HDAC inhibitors. Some <em>in silico</em> methods including molecular modeling, virtual screening, and molecular dynamics are used to find the common HDAC inhibitor structures [Huang et al., 2016; Yanuar et al. 2016].</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>The measurement of HDAC inhibition monitors changes in histone acetylation. HDAC inhibition can be detected directly by the measurement of HDAC activity using commercially available colorimetric or fluorimetric kits or indirectly by the increase of histone acetylation as the detection of global histone acetylation changes by Western blot or mass spectrometry (MS)-based proteomics methods or as detection of site-specific histone acetylation changes using chromatin immunoprecipitation (ChIP) or ChIP-on-Chip. The measurement methods include the immunological detection of histone acetylation with anti-acetylated histone antibodies [Richon et al., 2004]. The histones are isolated from pellets of cells treated with HDIs, followed by acid-urea-triton gel electrophoresis, western blotting, and immunohistochemistry [Richon et al., 2003]. The HDAC activity is measured directly with ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) by calculating the ratio of deacetylated peptide and acetylated peptide [Zwick et al., 2016]. <span style="color:#2980b9">HDAC inhibition can be predicted by perturbations in gene expression patterns as well; an 81-gene transcriptomic biomarker of HDAC inhibition, called TGx-HDACi, has shown to accurately predict HDAC inhibition after 4 hour exposures to HDI in TK6 human lymphoblastoid cells [Cho et al., 2021]. </span> </p>
<h4>References</h4>
<p>Damaskos C. et al. (2017) Histone deacetylase inhibitors: an attractive therapeutic strategy against breast cancer. Anticancer Research 37: 35-46.</p>
<h4>References</h4>
<p>Ansari, J. et al. (2016), "Epigenetics in non-small cell lung cancer: from basics to therapeutics", Transl Lung Cancer Res 5:155-171</p>
<p>Taunton J. et al. (1996) A mammalian histone deacetylase related to the Yeast transcriptional regulator Rpd3p. Science 272:408-411.</p>
<p>Barneda-Zahonero, B. and Parra, M. (2012), "Histone deacetylases and cancer", Mol Oncol 6:579-589</p>
<p>Lee SC. et al. (2016) Essential role of insulin-like growth factor 2 in resistance to histone deacetylase inhibitor. Oncogene 35:5515-5526.</p>
<p>Bell, E.L. et al. (2014), "SirT1 is required in the male germ cell for differentiation and fecundity in mice", Development 141:3495-3504</p>
<p>Minucci S, Pelicci PG. (2006) Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer. Jan;6(1):38-51.</p>
<p><span style="color:#2980b9">Cho, E. et al. (2021), "Development and validation of the TGx-HDACi transcriptomic biomarker to detect histone deacetylase inhibitors in human TK6 cells", Arch Toxicol 95:1631–1645</span></p>
<p>Weichert W. (2009) HDAC expression and clinical prognosis in human malignancies. Cancer Letters 280:168-176</p>
<p>Chung, Y.L. et al. (2003), "A therapeutic strategy uses histone deacetylase inhibitors to modulate the expression of genes involved in the pathogenesis of rheumatoid arthritis", Mol Ther 8:707-717</p>
<p>Barneda-Zahonero B and Parra M (2012) Histone deacetylases and cancer. Mol Oncol 6:579-589</p>
<p>Damaskos, C. et al. (2016), "Histone deacetylase inhibitors: a novel therapeutic weapon against medullary thyroid cancer?", Anticancer Res 36:5019-5024</p>
<p>Bell EL et al. (2014) SirT1 is required in the male germ cell for differentiation and fecundity in mice. Development 141:3495-3504</p>
<p>Damaskos, C. et al. (2017), "Histone deacetylase inhibitors: an attractive therapeutic strategy against breast cancer", Anticancer Research 37:35-46</p>
<p>Richon VM et al. (2004) Histone deacetylase inhibitors: assays to assess effectiveness in vitro and in vivo. Methods Enzymol. 376:199-205</p>
<p>Decroos, C. et al. (2015), "Biochemical and structural characterization of HDAC8 mutants associated with cornelia de lange syndrome spectrum disorders", Biochemistry 54:6501–6513</p>
<p>The ENCODE Project Consortium. (2004) The ENCODE (ENCyclopedia Of DNA Elements) Project. Science 306:636-640</p>
<p>Desjardins, D. et al. (2016), "Histone deacetylase inhibition restores retinal pigment epithelium function in hyperglycemia", PLoS ONE 11:e0162596</p>
<p>Ren B and Dynlacht D. (2004) Use of chromatin immunoprecipitation assays in genome-wide location analysis of mammalian transcription factors. Methods Enzymol. 376:304-315</p>
<p>Di Renzo, F. et al. (2007), "Boric acid inhibits embryonic histone deacetylases: A suggested mechanism to explain boric acid-related teratogenicity", Toxicol and Appl Pharmacol 220:178-185</p>
<p>Johnson DS et al. (2007) Genome-wide mapping of in vivo protein-DNA interactions. Science 316:1497-1502</p>
<p>Dreser, N. et al. (2015), "Grouping of histone deacetylase inhibitors and other toxicants disturbing neural crest migration by transcriptional profiling", Neurotoxicology 50:56–70</p>
<p>Zwick V et al. (2016) Cell-based multi-substrate assay coupled to UHPLC-ESI-MS/MS for a quick identification of class-specific HDAC inhibitors. J Enzyme Inhib Med Chem 31: 209-214</p>
<p>Drummond, D.C. et al. (2005), "Clinical development of histone deacetylase inhibitors as anticancer agents", Annu Rev Pharmacol Toxicol 45:495–528</p>
<p>Steffan JS et al. (2001) Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature 413:739-743</p>
<p>Finnin, M.S. et al. (1999), "Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors", Nature 401:188–193</p>
<p>Desjardins D et al. (2016) Histone deacetylase inhibition restores retinal pigment epithelium function in hyperglycemia. PLoS ONE 11: e0162596</p>
<p>Göttlicher, M. et al. (2001), "Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells", EMBO J 20:6969–6978</p>
<p>Ansari J et al. (2016) Epigenetics in non-small cell lung cancer: from basics to therapeutics. Transl Lung Cancer Res 5:155-171</p>
<p>Gregoretti, I.V. et al. (2004), "Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis", J Mol Biol 338:17–31</p>
<p>Miyanaga A et al. (2008) Antitumor activity of histone deacetylase inhibitors in non-small cell lung cancer cells: development of a molecular predictive model. Mol Cancer Ther 7:1923-1930</p>
<p>Hockly, E. et al. (2003), "Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington’s disease", Proc Nat Acad Sci 100:2041-2046</p>
<p>Mishra N et al. (2003) Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse. J Clin Invest 111: 539-552</p>
<p>Hu, E. et al. (2003), "Identification of novel isoform-selective inhibitors within class I histone deacetylases", J Pharmacol Exp Ther 307:720-728</p>
<p>Hockly E et al. (2003) Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington’s disease. Proc Nat Acad Sci 100:2041-2046</p>
<p>Huang, Y.X. et al. (2016), "Virtual screening and experimental validation of novel histone deacetylase inhibitors", BMC Pharmacol Toxicol 17(1):32</p>
<p>Jansen MS et al. (2014)Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition Proc Natl Acad Sci USA 101:7199-7204</p>
<p>Jansen, M.S. et al. (2014), "Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition", Proc Natl Acad Sci USA 101(18):7199-7204</p>
<p>Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831</p>
<p>Luckhurst, C.A. et al. (2016), "Potent, Selective, and CNS-Penetrant Tetrasubstituted Cyclopropane Class IIa Histone Deacetylase (HDAC) Inhibitors", ACS Med Chem Lett 7:34–39</p>
<p>Di Renzo F et al. (2007) Boric acid inhibits embryonic histone deacetylases: A suggested mechanism to explain boric acid-related teratogenicity. Toxicol and Appl Pharmacol 220:178-185</p>
<p>Mishra, N. et al. (2003), "Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse", J Clin Invest 111:539-552</p>
<p>Chung YL et al. (2003) A therapeutic strategy uses histone deacetylase inhibitors to modulate the expression of genes involved in the pathogenesis of rheumatoid arthritis. Mol Ther 8:707-717</p>
<p>Miyanaga, A. et al. (2008), "Antitumor activity of histone deacetylase inhibitors in non-small cell lung cancer cells: development of a molecular predictive model", Mol Cancer Ther 7:1923-1930</p>
<p>Ropero S and Esteller M. (2007) The role of histone deacetylases (HDACs) in human cancer. Mol Oncol 1:19-25</p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:16px">Ooi, J.Y.Y., et al. (2015), “HDAC inhibition attenuates cardiac hypertrophy by acetylation and deacetylation of target genes”, Epigenetics 10:418-430</span></span></p>
<p>Villae-Garea A and Esteller M. (2004) Histone deacetylase inhibitors: understanding a new wave of anticancer agents. Int J Cancer 112:171-178</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Park M.J. and Sohrabi F. (2016), “The histone deacetylase inhibitor, sodium butyrate, exhibits neuroprotective effects for ischemic stroke in middle-aged female rats”, J Neuroinflammation 13:300</span></span></p>
<p>Sekhavat A et al. (2007) Competitive inhibition of histone deacetylase activity by trichostatin A and butyrate. Biochemistry and Cell Biology 85:751-758</p>
<p>Porter, N.J., and Christianson, D.W. (2017), "Binding of the microbial cyclic tetrapeptide trapoxin A to the Class I histone deacetylase HDAC8", ACS Chem Biol 12:2281–2286</p>
<p>Hu E et al. (2003) Identification of novel isoform-selective inhibitors within class I histone deacetylases. J Pharmacol Exp Ther 307:720-728</p>
<p>Richon, V.M. et al. (2003), "Histone deacetylase inhibitors: assays to assess effectiveness in vitro and in vivo", Methods Enzymol. 376:199-205</p>
<p>Damaskos C et al. (2016) Histone deacetylase inhibitors: a novel therapeutic weapon against medullary thyroid cancer? Anticancer Res 36:5019-5024</p>
<p>Ropero, S. and Esteller, M. (2007), "The role of histone deacetylases (HDACs) in human cancer", Mol Oncol 1:19-25</p>
<p>Sekhavat, A. et al. (2007), "Competitive inhibition of histone deacetylase activity by trichostatin A and butyrate", Biochemistry and Cell Biology 85:751-758</p>
<p>Dreser, N., Zimmer, B., Dietz, C., Sügis, E., Pallocca, G., Nyffeler, J., et al. (2015), Neurotoxicology 50: 56–70.</p>
<p>Taunton, J. et al. (1996), "A mammalian histone deacetylase related to the Yeast transcriptional regulator Rpd3p", Science 272:408-411</p>
<p>Drummond, D.C., Noble, C.O., Kirpotin, D.B., Guo, Z., Scott, G.K., and Benz, C.C. (2005), Annu Rev Pharmacol Toxicol 45: 495–528.</p>
<p>Villar-Garea, A. and Esteller, M. (2004), "Histone deacetylase inhibitors: understanding a new wave of anticancer agents", Int J Cancer 112:171-178</p>
<p>Finnin, M.S., Donigian, J.R., Cohen, a, Richon, V.M., Rifkind, R. a, Marks, P. a, et al. (1999), Nature 401: 188–193.</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p>Göttlicher, M., Minucci, S., Zhu, P., Krämer, O.H., Schimpf, A., Giavara, S., et al. (2001), EMBO J 20: 6969–6978.</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wagner F.F. et al. (2015), “Kinetically selective inhibitors of histone deacetylase 2 (HDAC2) as cognition enhances”, Chem Sci 6:804</span></span></p>
<p>Gregoretti, I. V., Lee, Y.M., and Goodson, H. V. (2004), J Mol Biol 338: 17–31.</p>
<p>Weichert, W. (2009) "HDAC expression and clinical prognosis in human malignancies", Cancer Letters 280:168-176</p>
<p>Luckhurst, C.A., Breccia, P., Stott, A.J., Aziz, O., Birch, H.L., Bürli, R.W., et al. (2016), ACS Med Chem Lett 7: 34–39.</p>
<p>Yanuar, A. et al. (2016), "In silico approach to finding new active compounds from histone deacetylase (HDAC) family", Curr Pharm Des 22:3488-3497</p>
<p>Zwick, V. et al. (2016), "Cell-based multi-substrate assay coupled to UHPLC-ESI-MS/MS for a quick identification of class-specific HDAC inhibitors", J Enzyme Inhib Med Chem 31:209-214</p>
<p>The histone acetylation increase by HDIs is well conserved between species from lower organism to mammals.</p>
<p>・MAA, a HDAC inhibitor, induces acetylation of histones H3 and H4 in Sprague-Dawley (<em>Rattus norvegicus)</em> [Wade, 2008].</p>
<p>・It is also reported that MAA promotes acetylation of H4 in HeLa cells (<em>Homo sapiens</em>) and spleens from C57BL/6 mice (<em>Mus musculus</em>) treated with MAA [Jansen, 2014].</p>
<p>・VPA, a HDAC inhibitor, induces hyperacetylation of histone H4 in protein extract of mouse embryos (<em>Mus musculus</em>) exposed <em>in utero</em> for 1h to VPA [Di Renzo, 2007a].</p>
<p>・Apicidin, MS-275 and sodium butyrate, HDAC inhibitors, induce hyperacetylation of histone H4 in homogenates from mouse embryos (<em>Mus musculus</em>) after the treatments [Di Renzo, 2007b].</p>
<p>・MAA acetylates histones H3K9 and H4K12 in limbs of CD1 mice (<em>Mus musculus</em>) [Dayan, 2014].</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p>Gene transcription is regulated with the balance between acetylation and deacetylation. The acetylation and deacetylation are modulated on the NH<sub>3</sub><sup>+</sup> groups of lysine amino acid residues in histones. DNA in acetylated histones is more accessible for transcription factors, leading to increase in gene expression. HDAC inhibition promotes the hyperacetylation by inhibiting deacetylation of histones with classes of H2A, H2B, H3 and H4 in nucleosomes. [Wade, 2008].</p>
<p>The histone acetylation increase by HDIs is well conserved between species from lower organisms to mammals.</p>
<p> </p>
<p>・MAA, an HDAC inhibitor, induces acetylation of histones H3 and H4 in Sprague-Dawley rats (<em>Rattus norvegicus)</em> [Wade et al., 2008].</p>
<p>Description from EU-ToxRisk Deliverable:</p>
<p>・It is also reported that MAA promotes acetylation of H4 in HeLa cells (<em>Homo sapiens</em>) and spleens from C57BL/6 mice (<em>Mus musculus</em>) treated with MAA [Jansen et al., 2014].</p>
<p>The inhibition of HDACs result in an accumulation of acetylated proteins such as tubulin or histones.</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>Histone acetylation is measured by the immunological detection of histone acetylation with anti-acetylated histone antibodies [Richon, 2004]. Histone acetylation on chromatin can be measured using labeling method with sodium [<sup>3</sup>H] acetate [Gunjan, 2001].</p>
<p>・VPA, an HDAC inhibitor, induces hyperacetylation of histone H4 in protein extract of mouse embryos (<em>Mus musculus</em>) exposed <em>in utero</em> for 1 hr to VPA [Di Renzo et al., 2007a].</p>
<p> </p>
<p>Description from EU-ToxRisk Deliverable:</p>
<p>・Apicidin, MS-275 and sodium butyrate, HDAC inhibitors, induce hyperacetylation of histone H4 in homogenates from mouse embryos (<em>Mus musculus</em>) after the treatments [Di Renzo et al., 2007b].</p>
<ol>
<li>
<p>Semi-quantitative: Western blot usining antibodies agains acetylated tubulin or histones</p>
</li>
<li>
<p>Quantitative: enzyme assays using acetylated peptides and purified HDAC enzyme</p>
</li>
</ol>
<p>・MAA acetylates histones H3K9 and H4K12 in limbs of CD1 mice (<em>Mus musculus</em>) [Dayan and Hales, 2014].</p>
<br>
<h4>Key Event Description</h4>
<p>Gene transcription is regulated with the balance between acetylation and deacetylation. A dynamic balance of histone acetylation and histone deacetylation is typically catalyzed by enzymes with histone acetyltransferase (HAT) and HDAC activities. Histone acetylation relaxes chromatin and makes it accessible to transcription factors, whereas deacetylation is associated with gene silencing. The balance can be disturbed also by targeting HAT, not only HDACs. At least theoretically, an activation of HAT might lead to an increase in histone acetylation. The acetylation and deacetylation are modulated on the NH<sub>3</sub><sup>+</sup> groups of lysine amino acid residues in histones. HDAC inhibition promotes hyperacetylation by inhibiting the deacetylation of histones with classes of H2A, H2B, H3, and H4 in nucleosomes. [Wade et al., 2008]. The inhibition of HDACs results in an accumulation of acetylated proteins such as tubulin or histones.</p>
<h4>How it is Measured or Detected</h4>
<p>Histone acetylation is measured by the immunological detection of histone acetylation with anti-acetylated histone antibodies [Richon et al., 2004]. Histone acetylation on chromatin can be measured using the labeling method with sodium [<sup>3</sup>H]acetate [Gunjan et al., 2001]. The histone acetylation increase is detected as global histone acetylation changes by Western blot or mass spectrometry (MS)-based proteomics methods or as site-specific histone acetylation changes using chromatin immunoprecipitation (ChIP) or ChIP-on-Chip.</p>
<h4>References</h4>
<p style="margin-left:49.55pt">Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831</p>
<p style="margin-left:49.55pt">Richon VM et al. (2004) Histone deacetylase inhibitors: assays to assess effectiveness in vitro and in vivo. Methods Enzymol 376:199-205</p>
<h4>References</h4>
<p>Dayan, C. and Hales, B.F. (2014), "Effects of ethylene glycol monomethyl ether and its metabolite, 2-methoxyacetic acid, on organogenesis stage mouse limbs in vitro", Birth Defects Res (Part B) 101:254-261</p>
<p style="margin-left:49.55pt">Gunjan A et al. (2001) Core histone acetylation is regulated by linker histone stoichiometry <em>in vivo</em>. J Biol Chem 276:3635-3640</p>
<p>Di Renzo, F. et al. (2007a), "Boric acid inhibits embryonic histone deacetylases: A suggested mechanism to explain boric acid-related teratogenicity", Toxicol and Appl Pharmacol 220:178-185</p>
<p style="margin-left:49.55pt">Jansen MS et al. (2014) Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition Proc Natl Acad Sci USA 101:7199-7204</p>
<p>Di Renzo, F. et al. (2007b), "Relationship between embryonic histonic hyperacetylation and axial skeletal defects in mouse exposed to the three HDAC inhibitors apicidin, MS-275, and sodium butyrate", Toxicol Sci 98:582-588</p>
<p style="margin-left:49.55pt">Di Renzo F et al. (2007a) Boric acid inhibits embryonic histone deacetylases: A suggested mechanism to explain boric acid-related teratogenicity. Toxicol and Appl Pharmacol 220:178-185</p>
<p>Gunjan, A. et al. (2001), "Core histone acetylation is regulated by linker histone stoichiometry in vivo", J Biol Chem 276:3635-3640</p>
<p style="margin-left:49.55pt">Di Renzo F et al. (2007b) Relationship between embryonic histonic hyperacetylation and axial skeletal defects in mouse exposed to the three HDAC inhibitors apicidin, MS-275, and sodium butyrate. Toxicol Sci 98:582-588</p>
<p>Jansen, M.S. et al. (2014), "Short-chain fatty acids enhance nuclear receptor activity through mitogen-activated protein kinase activation and histone deacetylase inhibition", Proc Natl Acad Sci USA 101:7199-7204</p>
<p style="margin-left:49.55pt">Dayan C and Hales BF. (2014) Effects of ethylene glycol monomethyl ether and its metabolite, 2-methoxyacetic acid, on organogenesis stage mouse limbs in vitro. Birth Defects Res (Part B) 101:254-261</p>
<p>Richon, V.M. et al. (2004), "Histone deacetylase inhibitors: assays to assess effectiveness in vitro and in vivo", Methods Enzymol 376:199-205</p>
<p> </p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<td><a href="/aops/212">Aop:212 - Histone deacetylase inhibition leading to testicular atrophy</a></td>
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<td><a href="/aops/393">Aop:393 - AOP for thyroid disorder caused by triphenyl phosphate via TRβ activation</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/396">Aop:396 - Deposition of ionizing energy leads to population decline via impaired meiosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/331">Aop:331 - Excessive reactive oxygen species leading to growth inhibition via oxidative DNA damage and reduced cell proliferation</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/332">Aop:332 - Excessive reactive oxygen species leading to growth inhibition via lipid peroxidation and reduced cell proliferation</a></td>
<p>The p21 up-regulation by HDIs is well conserved between species from lower organism to mammals.</p>
<p>・FK228, a HDAC inhibitor, up-regulated p21 level in human esophageal cancer TE2 cells (<em>Homo sapiens</em>) [Hoshino, 2007].</p>
<p>・MAA, a HDAC inhibitor, induced p21 up-regulation in human prostate cancer cell lines (<em>Homo sapiens</em>) [Parajuli, 2014].</p>
<p>・MAA increases p21 expression in human bladder carcinoma cells, T24 (<em>Homo sapiens</em>) [Glaser, 2003].</p>
<p>・MAA up-regulated p21 expression in limbs of CD1 embryonic mice (<em>Mus musculus</em>) [Dayan, 2014].</p>
</div>
</div>
</div>
<br>
</div>
<p>The histone gene expression alters in each phase of the cell cycle in human HeLa cells (<em>Homo sapiens</em>) [Heintz et al., 1982].</p>
<!-- event text -->
<h4>Key Event Description</h4>
<p>p21 (CDKN1A; cyclin dependent kinase inhibitor 1A) binds to and inhibits the activity of cyclin-dependent kinase 2 or cyclin-dependent kinase 4 complexes, and regulates cell cycle progression in G<sub>1</sub> phase. p21 is important for cell cycle regulation.</p>
<h4>Key Event Description</h4>
<p>The disruption of the cell cycle leads to a decrease in cell number. The cell cycle consists of G<sub>1</sub>, S, G<sub>2</sub>, M, and G<sub>0</sub> phases. The cell cycle regulation is disrupted by the cell cycle arrest in certain cell cycle phases. The histone gene expression is regulated in cell cycle phases [Heintz et al., 1983].</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>The percentage of cells at G<sub>1</sub>, G<sub>0</sub>, S, and G<sub>2</sub>/M phases can be detected by flow cytometry [Li et al., 2013]. Cell cycle distribution was analyzed by fluorescence-activated cell sorter (FACS) analysis with a Partec PAS-II sorter [Zupkovitz et al., 2010]. The four cell-cycle phases in living cells can be measured with four-color fluorescent proteins using live-cell imaging [Bajar et al., 2016]. The incorporation of [<sup>3</sup>H]deoxycytidine or [<sup>3</sup>H]thymidine into cell DNA during the S phase can be monitored as DNA synthesis [Heintz et al., 1982].</p>
<h4>How it is Measured or Detected</h4>
<p>The p21 mRNA is measured with real-time RT-PCR technique using primers for p21 [Dayan, 2014]. Gene expression of p21 is measured with microarray technique using gene chips after cDNA preparation from total RNA extracted from the samples [Glaser, 2003, Hoshino, 2007]. Protein level of p21 is measured with Western blot analysis using anti-p21 antibody [Parajuli, 2014, Glaser, 2003].</p>
<br>
<h4>References</h4>
<p style="margin-left:49.55pt">Dayan C and Hales BF. (2014) Effects of ethylene glycol monomethyl ether and its metabolite, 2-methoxyacetic acid, on organogenesis stage mouse limbs in vitro. Birth Defects Res (Part B) 101:254-261</p>
<h4>References</h4>
<p>Bajar, B.T. et al. (2016), "Fluorescent indicators for simultaneous reporting of all four cell cycle phases", Nat Methods 13:993-996 </p>
<p style="margin-left:49.55pt">Glaser KB et al. (2003) Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines. Mol Cancer Ther 2:151-163</p>
<p>Heintz, N. et al. (1983), "Regulation of human histone gene expression: Kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle", Molecular and Cellular Biology 3:539-550</p>
<p style="margin-left:49.55pt">Hoshino I et al. (2007) Gene expression profiling induced by histone deacetylase inhibitor, FK228, in human esophageal squamous cancer cells. Oncol Rep 18:585-592</p>
<p style="margin-left:49.55pt">Parajuli KR et al. (2014) Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis. Am J Clin Exp Urol 2:300-313</p>
<p>Li, Q. et al. (2013), "Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis", Drug Des Devel Ther 7:635-643</p>
<td><a href="/aops/500">Aop:500 - Activation of MEK-ERK1/2 leads to deficits in learning and cognition via ROS and apoptosis</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/502">Aop:502 - Decrease, cholesterol synthesis leads to orofacial clefting</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/441">Aop:441 - Ionizing radiation-induced DNA damage leads to microcephaly via apoptosis and premature cell differentiation</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/535">Aop:535 - Binding and activation of GPER leading to learning and memory impairments</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/540">Aop:540 - Oxidative Stress in the Fish Ovary Leads to Reproductive Impairment via Reduced Vitellogenin Production</a></td>
<td>KeyEvent</td>
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<td><a href="/aops/563">Aop:563 - Aryl hydrocarbon Receptor (AhR) activation causes Premature Ovarian Insufficiency leading to Reproductive Failure</a></td>
<p>The relationship between disrupted cell cycle and apoptosis is likely well conserved between species.</p>
<p>・Apoptosis is induced in human prostate cancer cell lines (<em>Homo sapiens</em>) [Parajuli et al., 2014].</p>
<p>・The change in the amounts of cells in G<sub>1</sub> phase and S phase of cell cycle was detected in mouse HDAC1 knock out fibroblast lines (<em>Mus musculus</em>) [Zupkovitz, 2010].</p>
<p>・Apoptosis occurs in B6C3F1 mouse (<em>Mus musculus</em>) [Elmore, 2007].</p>
<p>・The histone gene expression alters in each phase of cell cycle in human HeLa cell (<em>Homo sapiens</em>) [Heintz, 1982].</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p>The dysregulation of cell cycle leads to the decreases in cell number. The cell cycle consists of G<sub>1</sub>, S, G<sub>2</sub>, M, and G<sub>0</sub> phase. The cell cyle regulation is disrupted by the cell cycle arrest in certain cell cycle phase. The histone gene expression is regulated in cell cyle phases [Heintz, 1983]. The phosphorylation of p21 (CDKN1A; cyclin dependent kinase inhibitor 1A) regulates its function [Moussa, 2015, Child, 2006]. The up-regulation of p21 level in iron-chelated cancer cells was observed [Moussa, 2015].</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>The percentage of cells at G<sub>1</sub>, G<sub>0</sub>, S, and G<sub>2</sub>/M phases was determined by flow cytometry analysis using DNA content frequency histogram deconvolution software [Li, 2013].</p>
<p>・Apoptosis occurs in Sprague-Dawley rat (<em>Rattus norvegicus</em>) [Elmore, 2007].</p>
<p>Cell cycle distribution was analyzed by fluorescence-activated cell sorter (FACS) analysis with a Partec PAS-II sorter [Zupkovitz, 2010].</p>
<p>・Apoptosis occurs in the nematode (<em>Caenorhabditis elegans</em>) [Elmore, 2007].</p>
<ul>
<li>Apoptosis occurs in breast cancer cells, human and mouse (Parton)</li>
</ul>
<p>The four cell cycle phases in living cells can be measured with four-color fluorescent proteins using live cell imaging [Bajar, 2016].</p>
<p> </p>
<p>The incorporation of [3H]deoxycytidine or [3H]thymidine into cell DNA during S phase can be monitored as DNA synthesis [Heintz, 1982].</p>
<br>
<p> </p>
<h4>Key Event Description</h4>
<p>Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Elmore, 2007]. Apoptosis, also called “physiological cell death”, is involved in cell turnover, physiological involution, and atrophy of various tissues and organs [Kerr et al., 1972]. The formation of apoptotic bodies involves marked condensation of both nucleus and cytoplasm, nuclear fragmentation, and separation of protuberances [Kerr et al., 1972]. Apoptosis is characterized by DNA ladder and chromatin condensation. Several stimuli such as hypoxia, nucleotides deprivation, chemotherapeutical drugs, DNA damage, and mitotic spindle damage induce p53 activation, leading to p21 activation and cell cycle arrest [Pucci et al., 2000]. The SAHA or TSA treatment on neonatal human dermal fibroblasts (NHDFs) for 24 or 72 hrs inhibited proliferation of the NHDF cells [Glaser et al., 2003]. Considering that the acetylation of histone H4 was increased by the treatment of SAHA for 4 hrs, histone deacetylase inhibition may be involved in the inhibition of the cell proliferation [Glaser et al., 2003]. The impaired proliferation was observed in HDAC1<sup>-/-</sup> ES cells, which was rescued with the reintroduction of HDAC1 [Zupkovitz et al., 2010]. An AOP focuses existes on p21 pathway leading to apoptosis, however, alternative pathways such as NF-kappaB signaling pathways may be involved in the apoptosis of spermatocytes [Wang et al., 2017].</p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">Apoptosis is defined as a </span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">programmed cell death</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">. </span></span></span><span style="font-family:"Times New Roman",serif"><span style="color:black"> A decrease in apoptosis or a resistance to cell death is noted is described as a hallmark of cancer by Hanahan et al. It is widely admitted as an essential step in tumor proliferation (Adams, Lowe). </span></span></span></span><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">Apoptosis occurs after activation of a number of intrinsic and extrinsic signals which activate the protease caspase system which in turn activates the destruction of the cell. </span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">The Bcl-2 is a protein family suppressing apoptosis by <span style="background-color:white">binding and inhibiting</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> two proapoptotic proteins (Bax and Bak)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> and transferring them to the mitochondrial outer membrane. In the absence of inhibition by Bcl2, Bax and Bak destroy the mitochondrial membrane and releases </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">proapoptotic signaling proteins, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">such as</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> cytochrome </span></span></span><em>c</em><em> </em><em><span style="background-color:white"><span style="color:black">which activated the caspase system. </span></span></em><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">An increased</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> expression of </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">these </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">antiapoptotic </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">proteins</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> (Bcl-2, Bcl-x</span></span></span><sub>L</sub>) <em><span style="background-color:white"><span style="color:black">occurs in cancer (Hanahan, Adams, Lowe). Several others pathways such as the l</span></span></em><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">oss of TP53 tumor suppressor function,</span></span></span> or <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">the increase </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">of survival signals (Igf1/2), </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">or decrease of</span></span></span> <span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">proapoptotic factors (Bax, Bim, Puma)</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black"> can also increase tumor growth <em>(Hanahan, Juntilla).</em></span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="font-family:"Times New Roman",serif"><span style="color:black">In breast cancer a decrease in apoptosis and a resistance to cell death has been described thoroughly, especially using a dysregulation of the Bcl2 system or TP53 (Parton, </span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Williams</span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">, </span></span></span><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Shahbandi</span></span></span><span style="font-family:"Times New Roman",serif"><span style="color:black">).</span></span></span></span></p>
<h4>References</h4>
<p style="margin-left:49.55pt">Heintz N et al. (1983) Regulation of human histone gene expression: Kinetics of accumulation and changes in the rate of synthesis and in the half-lives of individual histone mRNAs during the HeLa cell cycle. Molecular and Cellular Biology 3:539-550</p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Apoptosis is characterized by many morphological and biochemical changes <span style="color:black">such as homogenous condensation of chromatin to one side or the periphery of the nuclei, membrane blebbing and formation of apoptotic bodies with fragmented nuclei, DNA fragmentation, enzymatic activation of pro-caspases, or phosphatidylserine translocation that can be measured using electron and cytochemical optical microscopy, proteomic and genomic methods, and spectroscopic techniques [Archana et al., 2013; Martinez et al., 2010; Taatjes et al., 2008; Yasuhara et al., 2003].</span></span></span></p>
<p style="margin-left:49.55pt">Moussa RS et al. (2015) Differential targeting of the cyclin-dependent kinase inhibitor, p21CIP/WAF1, by chelators with anti-proliferative activity in a range of tumor cell-types. Oncotarget 6:29694-29711</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・<span style="color:black">DNA fragmentation can be quantified with comet assay using electrophoresis, where the tail length, head size, tail intensity, and head intensity of the comet are measured [Yasuhara et al., 2003].</span></span></span></p>
<p style="margin-left:49.55pt">Child ES and Mann DJ. (2006) The intricacies of p21 phosphorylation. Cell Cycle 5:1313-1319</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p style="margin-left:49.55pt">Li Q, Lambrechts MJ, Zhang Q, Liu S, Ge D, Yin R, Xi M and You Z. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis. Drug Des Devel Ther 2013; 7: 635-643.</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p style="margin-left:49.55pt">Zupkovitz G et al. (2010) The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation. Mol Cell Biol 30:1171-1181</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Apoptotic nucleosomes are detected using Cell Death Detection ELISA kit, which was calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p style="margin-left:49.55pt">Bajar BT et al. (2016) Fluorescent indicators for simultaneous reporting of all four cell cycle phases. Nat Methods 13: 993-996 </p>
<p>・The apoptosis and proliferation inhibition induced by MAA, a HDAC inhibitor, was measured in human prostate cancer cell lines (<em>Homo sapiens</em>) [Parajuli, 2014].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Cleavage of PARP is detected with Western blotting [Parajuli<span style="color:black"> et al.</span>, 2014].</span></span></p>
<p>・The cell viability inhibition induced by SAHA or TSA , which are HDAC inhibitors, was observed in NHDFs (<em>Homo sapiens</em>) [Glaser, 2003].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm [Wu<span style="color:black"> et al.</span>, 2016].</span></span></p>
<p>・The proliferation of the HDAC<sup>-/-</sup> ES cells was inhibited compared to HDAC<sup>+/+</sup> ES cells (<em>Homo sapiens</em>) [Zupkovitz, 2010].</p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p style="margin-left:18.0pt">Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Susan, 2007]. Apoptosis, also called as “physiological cell death”, is involved in cell turnover, physiological involution and atrophy of various tissues and organs [Kerr, 1972]. The formation of apoptotic bodies involves marked condensation of both nucleus and cytoplasm, nuclear fragmentation, and separation of protuberances [Kerr, 1972]. Apoptosis is characterized by DNA ladder and chromatin condensation. Several stimuli such as hypoxia, nucleotides deprivation, chemotherapeutical drugs, DNA damage, and mitotic spindle damage induce p53 activation, leading to p21 activation and cell cycle arrest [Pucci, 2000]. The SAHA or TSA treatment on neonatal human dermal fibroblasts (NHDFs) for 24 or 72 hrs inhibited proliferation of the NHDF cells [Glaser, 2003]. Considering that the acetylation of histone H4 was increased by the treatment of SAHA for 4 hrs, histone deacetylase inhibition may be involved in the inhibition of the cell proliferation [Glaser, 2003]. The impaired proliferation was observed in HDAC1<sup>-/-</sup> ES cells, which was rescued with the reintroduction of HDAC1 [Zupkovitz, 2010]. The present AOP focuses on p21 pathway leading to apoptosis, however, the alternative pathway such as NF-kB signaling pathways may be involved in apoptosis of spermatocytes [Wang, 2017].</p>
<br>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Apoptosis is measured with Annexin V-FITC probes, and the relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry [Wu et al., 2016].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・Apoptosis is detected with the Terminal dUTP Nick End-Labeling (TUNEL) method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks [Kressel and Groscurth, 1994].</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">・For the detection of apoptosis, the testes are fixed in neutral buffered formalin and embedded in paraffin. Germ cell death is visualized in testis sections by Terminal dUTP Nick End-Labeling (TUNEL) staining method [Wade et al., 2008]. The incidence of TUNEL-positive cells is expressed as the number of positive cells per tubule examined for one entire testis section per animal [Wade et al., 2008]</span></span></p>
<h4>How it is Measured or Detected</h4>
<p style="margin-left:18.0pt">・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli, 2014].</p>
<h4>References</h4>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Archana, M. et al. (2013), "Various methods available for detection of apoptotic cells", Indian J Cancer 50:274-283</span></span></p>
<p style="margin-left:18.0pt">・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli, 2014].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Elmore, S. (2007), "Apoptosis: a review of programmed cell death", Toxicol Pathol 35:495-516</span></span></p>
<p style="margin-left:18.0pt">・Apoptotic nucleosomes were detected using Cell Death Detection ELISA kit, which were calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli, 2014].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163</span></span></p>
<p style="margin-left:18.0pt">・Cell viability was measured with live cell number changes using the CellTiter-Glo Luminescent Cell Viability Assay [Parajuli, 2014].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Kerr, J.F.R. et al. (1972), "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer 26:239-257</span></span></p>
<p style="margin-left:18.0pt">・Cleavage of PARP was detected with Western blotting [Parajuli, 2014].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Kressel, M. and Groscurth, P. (1994), "Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA", Cell Tissue Res 278:549-556</span></span></p>
<p style="margin-left:18.0pt">・The proliferation/viability of NHDFs was measured with Alamar-Blue [modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] [Glaser, 2003].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Martinez, M.M. et al. (2010), "Detection of apoptosis: A review of conventioinal and novel techniques", Anal Methods 2:996-1004</span></span></p>
<p style="margin-left:18.0pt">・Proliferation of the HDAC<sup>-/-</sup> ES cells was determined with crystal violet and measurement of absorbance at 595 nm [Zupkovitz, 2010].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313</span></span></p>
<p style="margin-left:18.0pt">・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (pNA) and quantified at 405 nm [Wu, 2016].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Pucci, B. et al. (2000), "Cell cycle and apoptosis", Neoplasia 2:291-299</span></span></p>
<p style="margin-left:18.0pt">・Apoptosis is measured with Annexin V-FITC probes, and the relative percentage of Annexin V-FITC-positive/PI-negative cells is analyzed by flow cytometry [Wu, 2016].</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Taatjes, D.J. et al. (2008), "Morphological and cytochemical determination of cell death by apoptosis", Histochem Cell Biol 129:33-43</span></span></p>
<p style="margin-left:18.0pt">・Apoptosis is detected with the Terminal dUTP Nick End-Labeling (TUNEL) method to assay the endonuclease cleavage products by enzymatically end-labeling the DNA strand breaks [Kressel, 1994].</p>
<br>
<h4>References</h4>
<p style="margin-left:49.55pt">Susan E. (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35: 495-516</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</span></span></p>
<p style="margin-left:49.55pt">Kerr JFR et al. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143</span></span></p>
<p style="margin-left:49.55pt">Pucci B et al. (2000) Cell cycle and apoptosis. Neoplasia 2:291-299</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Wu, R. et al. (2016), "microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs", PLoS One 11:e0167052</span></span></p>
<p style="margin-left:49.55pt">Glaser KB et al. (2003) Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines. Mol Cancer Ther 2:151-163</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">Yasuhara, S. et al. (2003), </span>"<span style="color:black">Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis</span>"<span style="color:black">, J Histochem Cytochem 51:873-885</span></span></span></p>
<p style="margin-left:49.55pt">Zupkovitz G et al. (2010)The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation. Mol Cell Biol 30:1171-1181</p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181</span></span></p>
<p style="margin-left:49.55pt">Wang C et al. (2017) CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways. Oncotarget 8: 3132-3143</p>
<p> </p>
<p style="margin-left:49.55pt">Parajuli KR et al. (2014) Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis. Am J Clin Exp Urol 2: 300-313</p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230</span></span></span></span></span></p>
<p style="margin-left:49.55pt">Wu R et al. (2016) microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs. PLoS One 11: e0167052</p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981.</span></span></span></span></span></p>
<p style="margin-left:49.55pt">Kressel M and Groscurth P (1994) Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA. Cell Tissue Res 278: 549-556</p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. </span></span></span><em>Nature</em> <strong>432</strong>, 307–315 (2004). <a href="https://doi.org/10.1038/nature03098" style="color:#467886; text-decoration:underline"><span style="color:black">https://doi.org/10.1038/nature03098</span></a></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene. 2007 Feb 26;26(9):1324-37. doi: 10.1038/sj.onc.1210220. PMID: 17322918; PMCID: PMC2930981.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Lowe, S., Cepero, E. & Evan, G. Intrinsic tumour suppression. </span></span></span><em>Nature</em> <strong>432</strong>, 307–315 (2004). <a href="https://doi.org/10.1038/nature03098" style="color:#467886; text-decoration:underline"><span style="color:black">https://doi.org/10.1038/nature03098</span></a></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Junttila MR, Evan GI. p53--a Jack of all trades but master of none. Nat Rev Cancer. 2009 Nov;9(11):821-9. doi: 10.1038/nrc2728. Epub 2009 Sep 24. PMID: 19776747.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Williams MM, Cook RS. Bcl-2 family proteins in breast development and cancer: could Mcl-1 targeting overcome therapeutic resistance? Oncotarget. 2015 Feb 28;6(6):3519-30. doi: 10.18632/oncotarget.2792. PMID: 25784482; PMCID: PMC4414133.</span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Aptos,sans-serif"><span style="background-color:white"><span style="font-family:"Times New Roman",serif"><span style="color:black">Shahbandi A, Nguyen HD, Jackson JG. TP53 Mutations and Outcomes in Breast Cancer: Reading beyond the Headlines. Trends Cancer. 2020 Feb;6(2):98-110. doi: 10.1016/j.trecan.2020.01.007. Epub 2020 Feb 5. PMID: 32061310; PMCID: PMC7931175.</span></span></span></span></span></p>
<p style="text-align:justify"> </p>
<p>Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. BMJ. 2001 Jun 23;322(7301):1528-32. doi: 10.1136/bmj.322.7301.1528. PMID: 11420276; PMCID: PMC1120573.</p>
<p>There are evidences of spermatocyte depletion.</p>
<p><span style="font-size:14px">There are pieces of evidence of spermatocyte depletion in different species.</span></p>
<p>・It has been reported that mice lacking cyclin D-dependent kinase inhibitor proteins produced few mature sperm, and the residual spermatozoa had reduced motility and decreased viability (<em>Mus musculus</em>) [Zindy, 2001].</p>
<p><span style="font-size:14px">・Mature sperm counts were decreased and the residual spermatozoa had reduced motility and decreased viability (<em>Mus musculus)</em> [Zindy et al., 2001].</span></p>
<p>・The sperm counts in the cauda epidydimis of rats exposed to butylparaben were significantly decreased (<em>Rattus norvegicus</em>) [Oishi, 2001].</p>
<p><span style="font-size:14px">・The sperm counts in the cauda epididymis of rats were significantly decreased (<em>Rattus norvegicus</em>) [Oishi, 2001].</span></p>
<p>・MAA treatment induced spermatocyte death in Sprague-Dawley rats (<em>Rattus norvegicus</em>) [Wade, 2008].</p>
<p><span style="font-size:14px">・Spermatocyte death can be induced in Sprague-Dawley rats (<em>Rattus norvegicus</em>) [Wade et al., 2008].</span></p>
<br>
</div>
<h4>Key Event Description</h4>
<p><span style="font-size:14px">Spermatocytes are differentiated from spermatogonial stem cells <em>via</em> random proliferation, differentiation, and synchronized mitoses with several stages [Rooij, 2001]. In each step of the spermatogonial differentiation, different molecular mechanisms are activated in the testis [Rooij, 2001; de Kretser</span> et al.<span style="font-size:14px">, 2016]. The stem cell factor (SCF) genes are involved in differentiation into A1 spermatogonia. The expression of cyclin D2 is regulated in the epithelial stage VIII when the aligned spermatogonia differentiate into A1 spermatogonia [Rooij, 2001]. Upon the apoptosis of spermatogonia, overexpression of the apoptosis-inhibiting proteins Bcl-2 and Bcl-xL and deficiency of the apoptosis-inducing protein Bax have been shown to cause an accumulation of spermatogonia in the testis, leading to apoptosis of all cells [Rooij, 2001].</span></p>
<!-- event text -->
<h4>Key Event Description</h4>
<h2><span style="font-size:14px">The apoptosis of the cells lead to spermatocyte depletion. Spermatocytes are differentiated from spermatogonial stem cells via random proliferation, differentiation and synchronized mitoses with several stages [Rooij, 2001]. </span></h2>
<br>
<h4>How it is Measured or Detected</h4>
<p>The sperm-containing fluid was squeezed out of the cauda, and suspended in medium containing HEPEs buffer and bovine serum albumin, and incubated at 37C for 20 min. The number of spermatozoa was determined by hematocytometer [Zindy, 2001].</p>
<h4>How it is Measured or Detected</h4>
<p><span style="font-size:14px">Traditional spermatocytes assessment includes sperm count and concentration (haemocytometer, automated image-based system), morphology and motility (microscope, automated image-based system) and viability (for example propidium iodide staining of necrotic cells, TUNEL assay staining apoptotic cells). In addition, there are functional tests such as assays for genetic integrity (e.g. <em>via</em> measurement of DNA fragmentation/integrity -Halosperm kit or reactive oxygen species) and fertilization defects (through various measures of sperm-zona pellucida (ZP) interaction, such as measurement of ZP-receptor binding).</span></p>
<p>Testicular sperm counts and daily sperm production were determined by counting the total number of spermatids per testis, and divided by the testicular weight to give the results in spermatids per gram of testis [Oishi, 2001].</p>
<p><span style="font-size:14px">The sperm-containing fluid was squeezed out of the cauda, and suspended in medium containing HEPES buffer and bovine serum albumin, and incubated at 37ºC for 20 min. The number of spermatozoa was determined by a haematocytometer [Zindy et al., 2001].</span></p>
<p>For the detection of apoptosis, the testes were fixed in neutral buffered formalin, and embedded in paraffin. Germ cell death was visualized in testis sections by Terminal dUTP Nick End-Labeling (TUNEL) staining memthod [Wade, 2008]. The incidence of TUNEL-positive cells was expressed as the number of positive cells per tubule examined for one entire testis section per animal [Wade, 2008].</p>
<p><span style="font-size:14px">Testicular sperm counts and daily sperm production were determined by counting the total number of spermatids per testis and divided by the testicular weight to give the results in spermatids per gram of testis [Oishi, 2001].</span></p>
<p>For the testis cell analysis, fresh testes were dispersed using a two-stage enzymatic digestion and incubated in BSA containing collagenase and DNase I [Wade, 2006]. The seminiferous tubules were further digested and cells were fixed in ice-cold 70% ethanol [Wade, 2006]. Relative proportions of spermatogenic cell populations were assessed in fixed cells using a flow cytometeric method [Wade, 2006]. The principle of the test is that spermatogenic cells, as they differentiate from normal diploid spermatogonial stem cells through to mature spermatozoa with a highly condensed haploid complement of DNA, progress through various intermediate stages with differing nuclear DNA content and cellular content of mitochondria. Relative proportions of cells in each population were calculated with WinList software [Wade, 2006].</p>
<br>
<p><span style="font-size:14px">For the testis cell analysis, fresh testes were dispersed using two-stage enzymatic digestion and incubated in BSA containing collagenase and DNase I [Wade et al., 2006]. The seminiferous tubules were further digested and cells were fixed in ice-cold 70% ethanol [Wade et al., 2006]. Relative proportions of spermatogenic cell populations were assessed in fixed cells using a flow cytometric method [Wade et al., 2006]. The principle of the test is that spermatogenic cells, as they differentiate from normal diploid spermatogonial stem cells through to mature spermatozoa with a highly condensed haploid complement of DNA, progress through various intermediate stages with differing nuclear DNA content and cellular content of mitochondria. Relative proportions of cells in each population were calculated with WinList software [Wade et al., 2006].</span></p>
<h4>References</h4>
<p style="margin-left:49.55pt">Rooij DG. (2001) Proliferation and differentiation of spermatogonial stem cells. Reproduction 121:347-354</p>
<h4>References</h4>
<p><span style="font-size:14px">de Kretser, D.M. et al. (2016), "Endocrinology: Adult and Pediatric (Seventh Edition)", W.B. Saunders, Chapter 136-Spermatogenesis, pages 2325-2353.e9, Editors: J. Larry Jameson, Leslie J De Groot, David M. de Kretser, Linda C. Giudice, Ashley B. Grossman, Shlomo Melmed, John T. Potts, Gordon C. Weir</span></p>
<p style="margin-left:49.55pt">Zindy F et al. (2001) Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18<sup>Ink4c</sup> and p19<sup>Ink4d</sup>. Mol Cell Biol 21:3244-3255</p>
<p><span style="font-size:14px">Oishi, S. (2001), "Effects of butylparaben on the male reproductive system in rats", Toxicol Indust Health 17:31-39</span></p>
<p style="margin-left:49.55pt">Oishi S. (2001) Effects of butylparaben on the male reproductive system in rats. Toxicol Indust Health 17:31-39</p>
<p><span style="font-size:14px">Rooij, D.G. (2001), "Proliferation and differentiation of spermatogonial stem cells", Reproduction 121:347-354</span></p>
<p style="margin-left:49.55pt">Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831</p>
<p><span style="font-size:14px">Wade, M.G. et al. (2006), "Testicular toxicity of candidate fuel additive 1,6-dimethoxyhexane: comparison with several similar aliphatic ethers", Toxicol Sci 89:304-313</span></p>
<p style="margin-left:49.55pt">Wade MG et al. (2006) Testicular toxicity of candidate fuel additive 1,6-dimethoxyhexane: comparison with several similar aliphatic ethers. Toxicol Sci 89:304-313</p>
<br>
<!-- end event text -->
</div>
<p><span style="font-size:14px">Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</span></p>
<p><span style="font-size:14px">Zindy, F. et al. (2001), "Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18Ink4c and p19Ink4d", Mol Cell Biol 21:3244-3255</span></p>
<p>There are some evidences on testicular toxicity induced by HDAC inhibitors.</p>
<p>・EGME or MAA treatment induced the testicular damage in rat (<em>Rattus norvegicus</em>) [Foster, 1983].</p>
<p>・EGME were shown to deplete the spermatocytes in CD-1 mice (<em>Mus musculus</em>) and CD rats (<em>Rattus norvegicus</em>), principally pachytene cells, but with other stages affected with increasing dose [Anderson, 1987].</p>
<p>・The testicular lesions induced by 2-methoxyethanol were observed in rats (<em>Rattus norvegicus</em>) and guinea pigs (<em>Cavia porcellus</em>), which are different in onset, characteristics and severity [Ku, 1984].</p>
<p>・EGME has effects in disruption of spermatogenesis in rabbits (<em>Oryctolagus cuniculus</em>) [Foote, 1995].</p>
<p>・Dimethoxyhexane (DMH) induces testicular toxicity such as spermatocyte death in seminiferous tubule stages I-IV and stages XII-XIV and MAA increase in urine in Sprague-Dawley rats (<em>Rattus norvegicus</em>).</p>
<p> </p>
<br>
</div>
<!-- event text -->
<h4>Key Event Description</h4>
<p>It is hypothesized that the testicular effects of 1,6-dimethoxyhexane (DMH) are caused by its metabolism to MAA [Wade, 2006, Poon, 2004]. MAA produces testicular and thymic atrophy such as the decrease in size [Miller, 1982, Moss, 1985]. The spermatogenic stages in which the toxicity of MAA is induced are on the patchytene spermatocytes immediately before and during meiotic division, which are Stages XII-XIV of the cycle in the rat and the early pachytene spermatocytes at stages I-IV of the cycle. Dead germ cells can be seen as soon as 12 hours after the treatment of MAA [Casarett & Doull’s 7<sup>th</sup> edition].</p>
<br>
<h4>How it is Measured or Detected</h4>
<p>The weights of testes of MAA-treated rats were measured to detect the testicular atrophy [Foster, 1983]. Since zinc concentration has been shown to play an important role in the production of testicular injury by compounds, the effects of EGME and MAA on urinary zinc excretion and testicular zinc content was examined [Foster, 1983]. Testis were fixed for observations for light microscopy or transmission electron microscopy [McDowell, 1976, Mercantepe, 2018]. The testicular tissue structure was observed whether there are normal germinal epithelial cells and Leydig cells [Mercantepe, 2018]. Changes in sperm were measured by computer-assisted sperm analysis [Foote, 1995].</p>
<p>For the assessment of sperm morphology, eosin-stained sperm collected from the cauda epididymis were smeared onto two glass slides per sample, air-dried, and cover-slipped. At least 200 sperm on each slide were examined for the proportion of sperm with abnormal head (overhooked, blunt hook, banana-shaped, amorphous, or extremely oversized) or tail (twisted, bent, corkscrew, double. multiple) by one individual unaware of animal number or treatment [Wade, 2006]. For the measurement of the total number of condensed spermatids per testis, a weighed portion of the parenchyma from the left testis, as representative of the whole organ as possible, was homogenized [Wade, 2006]. For the measurement of the total number of sperm in the cauda epididymis, whole cauda and associated sperm suspension in DPBS were thawed on ice and homogenized [Wade, 2006]. Sperm or homogenization-resistant spermatid nuclei densities were calculated from the average number of nuclei and were expressed as total or as per gram of epididymis or testis weight [Wade, 2006]. For the determination of total LDH and LDH-X in supernatant of the homogenized testis fragment, enzyme activity was measured by monitoring extinction of NAD absorbance [Wade, 2006].</p>
<ul>
<li>The decrease in testis weight associated with testicular cell damage was induced by ethylene glycol monomethyl ether (EGME) or MAA treatment in rats (<em>Rattus norvegicus</em>) [Foster et al., 1983].</li>
<li>The number of spermatocytes, principally pachytene cells, is decreased by EGME treatment in CD-1 mice (<em>Mus musculus</em>) and CD rats (<em>Rattus norvegicus</em>) [Anderson et al., 1987].</li>
<li>The testicular lesions induced by 2-methoxyethanol (or EGME) were observed in rats (<em>Rattus norvegicus</em>) and guinea pigs (<em>Cavia porcellus</em>), which are different in onset, characteristics, and severity [Ku et al., 1984].</li>
<li>Spermatogenesis was disrupted by EGME treatment in rabbits (<em>Oryctolagus cuniculus</em>) [Foote et al., 1995].</li>
<li>Testicular toxicity such as spermatocyte death in seminiferous tubule stages I-IV and stages XII-XIV was induced by dimethoxyhexane (DMH) treatment in Sprague-Dawley rats (<em>Rattus norvegicus</em>) [Wade et al., 2006].</li>
</ul>
<br>
<h4>Key Event Description</h4>
<p>It is hypothesized that the testicular effects of 1,6-dimethoxyhexane (DMH) are caused by its metabolism to methoxyacetic acid (MAA) [Wade et al., 2006; Poon et al., 2004]. MAA produces testicular and thymic atrophy such as the decrease in size [Miller et al., 1982; Moss et al., 1985]. The spermatogenic stages in which the toxicity of MAA is induced are on the patchytene spermatocytes immediately before and during meiotic division, which are Stages XII-XIV of the cycle in the rat and the early pachytene spermatocytes at stages I-IV of the cycle. Dead germ cells can be seen as soon as 12 hours after the treatment of MAA [Casarett & Doull’s, 7<sup>th</sup> edition].</p>
<h4>Regulatory Significance of the AO</h4>
<p style="margin-left:.05pt">The testicular toxicity assessment is important for assessing the side effects of the medicines such as anti-cancer drugs. The unexpected effects may be predicted with this AO.</p>
<h4>How it is Measured or Detected</h4>
<ul>
<li>Testicular atrophy can be assessed by testicular volume measurement using an orchidometer, rulers, calipers, and ultrasonography or by testis weighing and histopathologic examination.</li>
<li>The testis weight is measured to detect testicular atrophy [Foster et al., 1983].</li>
<li>The urinary zinc excretion and testicular zinc content are examined since zinc concentration has been shown to play an important role in the production of testicular injury [Foster et al., 1983].</li>
<li>The testicular tissue structure is observed whether there are normal germinal epithelial cells and Leydig cells [Mercantepe et al., 2018]. The testis is fixed for observations by light microscopy or transmission electron microscopy [McDowell and Trump, 1976; Mercantepe et al., 2018].</li>
<li>Changes in sperm are measured by computer-assisted sperm analysis [Foote et al., 1995].</li>
<li>For the assessment of sperm morphology, eosin-stained sperm collected from the cauda epididymis is observed. At least 200 sperm on each slide were examined for the proportion of sperm with abnormal head (overhooked, blunt hook, banana-shaped, amorphous, or extremely oversized) or tail (twisted, bent, corkscrew, double/multiple) [Wade et al., 2006].</li>
<li>For the measurement of the total number of condensed spermatids per testis, a weighed portion of the parenchyma from the left testis was homogenized [Wade et al., 2006]. Sperm or homogenization-resistant spermatid nuclei densities were calculated from the average number of nuclei and were expressed as total or as per gram of epididymis or testis weight [Wade et al., 2006].</li>
<li>For the determination of total LDH and LDH-X in the supernatant of the homogenized testis fragment, enzyme activity was measured by monitoring the extinction of NAD absorbance [Wade et al., 2006].</li>
</ul>
<br>
<h4>Regulatory Significance of the AO</h4>
<p style="margin-left:.05pt">The testicular atrophy assessment is important for assessing the side effects of the medicines such as anti-cancer drugs, as well as the hazard and risk of chemicals. The testicular atrophy including a decrease in testis weight and sperm count, fertility, decrease in morphology and function of the sperm, can become one of the main endpoints as the adverse effects of the therapeutics. The unexpected effects of the therapeutics may be predicted with this Adverse Outcome (AO). In terms of chemical risk assessment, the AO may be related to the health effects caused by the usage of pesticides or biocides.</p>
<h4>References</h4>
<p style="margin-left:49.55pt">Wade MG et al. (2006) Testicular toxicity of candidate fuel additive 1,6-dimethoxyhexane: comparison with several similar aliphatic ethers. Toxicol Sci 89:304-313</p>
<h4>References</h4>
<p>Anderson, D. et al. (1987), "Effect of ethylene glycol monomethyl ether on spermatogenesis, dominant lethality, and F1 abnormalities in the rat and the mouse after treatment of F0 males", Teratog Carcinog Mutagen 7:141-158</p>
<p style="margin-left:49.55pt">Poon R et al. (2004) Short-term oral toxicity of pentyl ether, 1,4-diethoxybutane, and 1,6-dimethoxyhexane in male rats. Toxicol Sci 77:142-150</p>
<p>Casarett & Doull’s Toxicology, the Basic Science of Poisons, 7th Edition, Edited by Curtis D. Klaassen, Chapter 20 Toxic responses of the reproductive system</p>
<p style="margin-left:49.55pt">Miller R et al. (1982) Toxicity of methoxyacetic acid in rats. Fundam Appl Toxicol 2:158-160</p>
<p>Foote, R.H. et al. (1995), "Ethylene glycol monomethyl ether effects on health and reproduction in male rabbits", Reprod Toxicol 9:527-539</p>
<p style="margin-left:49.55pt">Moss EJ et al. (1985) The role of metabolism in 2-methoxyethanol-induced testicular toxicity. Toxicol Appl Pharmacol 79:480-489</p>
<p>Foster, P.M. et al. (1983), "Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rats", Toxicol Appl Pharmacol 69:385-399</p>
<p style="margin-left:49.55pt">Casarett & Doull’s Toxicology, the Basic Science of Poisons, 7<sup>th</sup> Edition, Edited by Curtis D. Klaassen, Chapter 20 Toxic responses of the reproductive system</p>
<p>Ku, W.W. et al. (1994), "Comparison of the testicular effects of 2-methoxyethanol (ME) in rats and guinea pigs", Exp Mol Pathol 61:119-133</p>
<p style="margin-left:49.55pt">Foster PM et al. (1983) Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rats. Toxicol Appl Pharmacol 69:385-399</p>
<p>McDowell, E.M. and Trump, B.F. (1976), "Histologic fixatives suitable for diagnostic light and electron microscopy", Arch Pathol Lab Med 100:405-414</p>
<p style="margin-left:49.55pt">McDowell EM and Trump BF. (1976) Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100:405-414</p>
<p>Mercantepe, T. et al. (2018), "Protective effects of amifostine, curcumin and caffeic acid phenethyl ester against cisplatin-induced testis tissue damage in rats", Exp Ther Med 15:3404-3412</p>
<p style="margin-left:49.55pt">Mercantepe T et al. (2018) Protective effects of amifostine, curcumin and caffeic acid phenethyl ester against cisplatin-induced testis tissue damage in rats. Exp Ther Med 15:3404-3412</p>
<p>Miller, R. et al. (1982), "Toxicity of methoxyacetic acid in rats", Fundam Appl Toxicol 2:158-160</p>
<p style="margin-left:49.55pt">Foote RH et al. (1995) Ethylene glycol monomethyl ether effects on health and reproduction in male rabbits. Reprod Toxicol 9:527-539</p>
<p>Moss, E.J. et al. (1985), "The role of metabolism in 2-methoxyethanol-induced testicular toxicity", Toxicol Appl Pharmacol 79:480-489</p>
<p style="margin-left:49.55pt">Anderson D et al. (1987) Effect of ethylene glycol monomethyl ether on spermatogenesis, dominant lethality, and F1 abnormalities in the rat and the mouse after treatment of F0 males. Teratog Carcinog Mutagen 7:141-158</p>
<p>Poon, R. et al. (2004), "Short-term oral toxicity of pentyl ether, 1,4-diethoxybutane, and 1,6-dimethoxyhexane in male rats", Toxicol Sci 77:142-150</p>
<p style="margin-left:49.55pt">Ku WW et al. (1994) Comparison of the testicular effects of 2-methoxyethanol (ME) in rats and guinea pigs. Exp Mol Pathol 61:119-133</p>
<p>Wade, M.G. et al. (2006), "Testicular toxicity of candidate fuel additive 1,6-dimethoxyhexane: comparison with several similar aliphatic ethers", Toxicol Sci 89:304-313</p>
<br>
<!-- end event text -->
</div>
<h2>Appendix 2</h2>
<h2>List of Key Event Relationships in the AOP</h2>
<!-- Evidence for relationship links section, this lists the relationships and then supports them -->
<p style="margin-left:18.0pt">The relationship between HDAC inhibition and hyperacetylation is likely well conserved between species from lower organisms to mammals.</p>
</div>
<p style="margin-left:18.0pt">The relationship between HDAC inhibition and increase in histone acetylation is conceivably well conserved among various species including mammals.</p>
<ul>
<li>Hyperacetylation by HDIs such as SAHA and Cpd-60 are observed in mouse (<em>Mus musculus</em>) [Schroeder, 2013].</li>
<li>TSA induces acetylation of histone H4 in time-dependent manner in mouse cell lines (<em>Mus musculus</em>) [Alberts, 1998].</li>
<li>AR-42, a novel HDI, induces hyperacetylation in human pancreatic cancer cells (<em>Homo sapiens</em>) [Henderson, 2016].</li>
<li>SAHA and MS-275 hyperacetylates lysine of histones in human cell lines of epithelial (A549) and lymphoid origin (Jurkat) (<em>Homo sapiens</em>) [Choudhary, 2009].</li>
<li>SAHA treatment induces the H3 and H4 histone acetylation in human corneal fibroblasts and conjunctiva from rabbits after glaucoma filtration surgery (<em>Homo sapiens</em>, <em>Oryctolagus cuniculus</em>) [Sharma, 2016].</li>
<li>TSA induces the acetylation of histones H3 and H4 in <em>Brassica napus</em> microspore cultures (<em>Brassica napu</em>) [Li, 2014].</li>
<li>Hyperacetylation by HDIs such as SAHA and Cpd-60 are observed in mice (<em>Mus musculus</em>) [Schroeder et al., 2013].</li>
<li>TSA induces acetylation of histone H4 in a time-dependent manner in mouse cell lines (<em>Mus musculus</em>) [Alberts et al., 1998].</li>
<li>AR-42, a novel HDI, induces hyperacetylation in human pancreatic cancer cells (<em>Homo sapiens</em>) [Henderson et al., 2016].</li>
<li>SAHA and MS-275 lead to the hyperacetylation of lysine residues of histones in human cell lines of epithelial (A549) and lymphoid origin (Jurkat) (<em>Homo sapiens</em>) [Choudhary et al., 2009].</li>
<li>SAHA treatment induces the H3 and H4 histone acetylation in human corneal fibroblasts and conjunctiva from rabbits after glaucoma filtration surgery (<em>Homo sapiens</em>, <em>Oryctolagus cuniculus</em>) [Sharma et al., 2016].</li>
<li>TSA induces the acetylation of histones H3 and H4 in <em>Brassica napus</em> microspore cultures (<em>Brassica napu</em>) [Li et al., 2014].</li>
</ul>
<h4>Key Event Relationship Description</h4>
<p>The HDAC inhibitors (HDIs) inhibit deacetylation of the histone, leading to the increase in histone acetylation and gene transcription. HDACs deacetylate acetylated histone in epigenetic regulation [Falkenberg, 2014].</p>
<h4>Key Event Relationship Description</h4>
<p>The HDAC inhibitors (HDIs) inhibit deacetylation of the histone, leading to the increase in histone acetylation and gene transcription. HDACs deacetylate acetylated histone in epigenetic regulation [Falkenberg and Johnstone, 2014].</p>
<p>Description from EU-ToxRisk Deliverable:</p>
<p>Histone acetylation is one of the major epigenetic mechanisms and belongs to the posttranslational modifications of histones. Histone acetyltransferases are setting the mark and deacetylases (HDAC) are responsible for removing the acetyl group from specific lysin residues of the histones. It has been shown that the inhibition of HDACs leads to a hyperacetylation of histones and in general to an imbalance of other histone modifications.</p>
<p>Histone acetylation is one of the major epigenetic mechanisms and belongs to the posttranslational modifications of histones. Histone acetyltransferase is setting the mark, and deacetylase (HDAC) is responsible for removing the acetyl group from specific lysine residues of the histones. It has been shown that the inhibition of HDACs leads to a hyperacetylation of histones and in general to an imbalance of other histone modifications.</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>HDACs are important proteins in the epigenetic regulation of gene transcription. Upon the inhibition of HDAC by HDIs, lysine in histone remains acetylated which leads to transcriptional activation or repression, changes in DNA replication, and DNA damage repair [Wade et al., 2008].</p>
<p>In all eukaryotes, the DNA containing the genetic information of an organism is organized in chromatin. The basic unit of chromatin is the nucleosome around which the naked DNA is wrapped. A nucleosome consists of two copies of each of the core histones H2A, H2B, H3, and H4 [Luger et al., 1997]. In order to dynamically regulate this highly complex structure several mechanisms are involved, including the posttranslational modifications of histones (reviewed in [Bannister and Kouzarides, 2011; Kouzarides, 2007]. For a long time, it is known that histones get acetylated and that this reaction is catalyzed by histone acetyltransferases (HAT) whereas the acetyl groups are removed by histone deacetylases (HDAC). Inhibition of HDACs by small-molecule compounds leads to hyperacetylation of the histones as the homeostasis of acetylation and deacetylation is disturbed (reviewed in [Gallinari et al., 2007]).</p>
<strong>Biological Plausibility</strong>
<p>HDACs are important proteins in epigenetic regulation of gene transcription. Upon the inhibition of HDAC by HDIs, the acetylation of lysine in histone remains and it leads to transcriptional activation or repression, changes in DNA replication and DNA damage repair. The treatment by HDIs increased histone acetylation [Wade, 2008].</p>
<strong>Empirical Evidence</strong>
<p>The major empirical evidence came from the incubation of cell culture cells with small molecule compounds that inhibit HDAC enzymes followed by western blots or acid urea gel analysis. The first evidence was shown by Riggs et al. who showed that incubation of HeLa cells with <em>n</em>-butyrate leads to an accumulation of acetylated histone proteins [Riggs et al., 1977]. Later, it was shown that <em>n</em>-butyrate specifically increases the acetylation of histone by the incorporation of radioactive [<sup>3</sup>H]acetate and analysis of the histones on acid urea gels that allow the detection of acetylated histones [Cousens et al., 1979]. TSA was shown to be an HDAC inhibitor by acid urea gel analysis in 1990 [Yoshida et al., 1990] and good evidence for VPA as an HDAC inhibitor <em>in vitro</em> and <em>in vivo</em> was shown using acetyl-specific antibodies and western blot [Gottlicher et al., 2001].</p>
<p>Description from EU-ToxRisk Deliverable:</p>
<p>There exist several pieces of evidence showing the link between histone deacetylase inhibition and increase in histone acetylation as follows:</p>
<p>In all eukaryotes the DNA containing the genetic information of an organism, is organized in chromatin. The basic unit of chromatin is the nucleosome around which the naked DNA is wrapped. A nucleosome consists of two copies of each of the core histones H2A, H2B, H3 and H4 (Luger et al., 1997). In order to dynamically regulate this highly complex structure severeal mechanism are involved, including the posttranslational modifications of histones (reviewed in (Bannister and Kouzarides, 2011; Kouzarides, 2007). For long time it is known that histones get acetylated and that this reaction is catalyzed by histone acetyl transferases (HAT) and the acetyl groups are removed by histone deacetylases (HDAC). Inhibition of HDACs by small molecule compounds lead to hyperacetylation of the histones as the homeostasis of acetylation and deacetylation is disturbed (reviewed in (Gallinari et al., 2007)).</p>
<strong>Empirical Evidence</strong>
<ul>
<li>HDAC inhibition by HDIs leads to hyperacetylation of histone and a large number of cellular proteins such as NF-kB [Falkenberg, 2014, Chen, 2018].</li>
<li>The concentrations of half-maximum inhibitory effect (IC<sub>50</sub>) for HDAC enzyme inhibition of 20 valproic acid derivatives correlated with teratogenic potential of the compounds, and HDAC inhibitory compounds showed hyperacetylation of core histone 4 in treated F9 cells [Eikel, 2006].</li>
<li>HDIs increase histone acetylation in brain [Schroeder, 2013].</li>
<li>The HDI selectivity exists, in which SAHA is a more potent inducer of histone acetylation than MS-275, and more acetylation sites on the histones H3 and H4 are responsible to SAHA than MS-275 [Choudhary, 2009].</li>
<li>HDI AR-42 induces acetylation of histone H3 in dose-response manner in human pancreatic cancer cell lines [Henderson, 2016].</li>
<li>MAA treatment in rats (650 mg/kg, for 4, 8, 12, and 24 hrs) induced hyperacetylation in histones H3 and H4 of testis nuclei [Wade, 2008].</li>
<li>HDAC inhibition induced by valproic acid (VPA) leads to histone hyperacetylation in mouse teratocarcinoma cell line F9 [Eikel, 2006].</li>
<li>Hyperacetylation of histone H3 was observed in HDAC1-deficient ES cells [Lagger, 2002].</li>
<li>The treatment of MAA induced histone acetylation in H3K9Ac and H4K12Ac, as well as p53K379Ac [Dayan, 2014].</li>
<ul>
<li>Exposure of mouse embryos <em>in utero</em> to VPA and TSA (two well-known HDAC inhibitors) showed an increased histone acetylation level in whole embryo extracts and was also shown <em>in situ</em> immuno-stainings [Menegola et al., 2005].</li>
<li>HDAC inhibition by HDIs leads to hyperacetylation of histone and a large number of cellular proteins such as NF-kappaB [Falkenberg and Johnstone, 2014; Chen et al., 2018].</li>
<li>The concentrations of half-maximum inhibitory effect (IC<sub>50</sub>) for HDAC enzyme inhibition of 20 valproic acid derivatives correlated with teratogenic potential of the compounds, and HDAC inhibitory compounds showed hyperacetylation of core histone 4 in treated F9 cells [Eikel et al., 2006].</li>
<li>HDIs increase histone acetylation in the brain [Schroeder et al., 2013].</li>
<li>More acetylation sites on the histones H3 and H4 are responsive to SAHA than to MS-275 indicating that an HDI selectivity exists [Choudhary et al., 2009].</li>
<li>HDI AR-42 induces acetylation of histone H3 in a dose-response manner in human pancreatic cancer cell lines [Henderson et al., 2016].</li>
<li>MAA treatment in rats (650 mg/kg, for 4, 8, 12, and 24 hrs) induced hyperacetylation in histones H3 and H4 of testis nuclei [Wade et al., 2008].</li>
<li>HDAC inhibition induced by valproic acid (VPA) leads to histone hyperacetylation in mouse teratocarcinoma cell line F9 [Eikel et al., 2006].</li>
<li>Hyperacetylation of histone H3 was observed in HDAC1-deficient ES cells [Lagger et al., 2002].</li>
<li>The treatment of MAA induced histone acetylation in H3K9Ac and H4K12Ac, as well as p53K379Ac [Dayan and Hales, 2014].</li>
</ul>
<p> </p>
<p>Description from EU-ToxRisk Deliverable:</p>
<p>The major empirical evidence came from the incubation of cell culture cells with small molecule compounds that inhibit HDAC enzymes followed by western blots or acid urea gel analysis.</p>
<p>The first evidence was shown by Riggs et al. who showed that incubation of HeLa cells with n-butyrate leads to an accumulation of acetylated histone proteins (Riggs et al., 1977)</p>
<p>Later, it was shown that n-butyrate specifically increases the acetylation of histone by the incorporation of radioactive [H<sup>3</sup>] acetate and analysis of the histones on acid urea gels that allow the detection of acetylated histones (Cousens et al., 1979)</p>
<p>TSA was shown to be an HDAC inhibitor by acid urea gel analysis in 1990 (Yoshida et al., 1990) and good evidence for VPA as an HDAC inhibitor in vitro and in vivo was shown using acetyl-specific antibodies and western blot (Gottlicher et al., 2001).</p>
<p>Exposure of mouse embryos in utero to VPA and TSA (two well-known HDAC inhibitors) showed an increased histone acetylation level in whole embryo extracts and was also shown in situ immuno stainings (Menegola et al., 2005).</p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="margin-left:18.0pt">HDACs affect a large number of cellular proteins including histones, which reminds us the HDAC inhibition by HDIs hyperacetylates cellular proteins other than histones and exhibit biological effects. It is also noted that HDAC functions as the catalytic subunits of large protein complex, which suggests that the inhibition of HDAC by HDIs affect the function of the large multiprotein complexes of HDAC [Falkenberg, 2014].</p>
<p style="margin-left:18.0pt"> </p>
<p>Description from EU-ToxRisk Deliverable:</p>
<p>All above mentioned analysis are indirect or in purified systems. Therefore a direct cause-consequence relation is difficult to obtain.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>To quantify acetylation by HDAC, stable isotope labeling with amino acids in cell culture (SILAC) method is used [Choudhary, 2009].</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>HDACs affect a large number of cellular proteins including histones, which reminds us the HDAC inhibition by HDIs hyperacetylates cellular proteins other than histones and exhibit additional biological effects. It is also noted that HDAC functions as the catalytic subunits of the large protein complex, which suggests that the inhibition of HDAC by HDIs affects the function of the large multiprotein complexes of HDAC [Falkenberg and Johnstone, 2014]. Related-analysis are usually indirect or in purified systems, therefore a direct cause-consequence relation is difficult to obtain.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>To quantify acetylation by HDAC, stable isotope labeling with amino acids in cell culture (SILAC) method is used [Choudhary et al., 2009].</p>
<strong>Response-response relationship</strong>
<p style="margin-left:18.0pt">SAHA and MS-275 increased acetylation of specific lysines on histones more than twofold [Choudhary, 2009]. Acetylation of the variant histone H2AZ-a mark for DNA damage sites- was upregulated almost 20-fold by SAHA, whereas a number of sites on the core histones H3 and H4 are several times more highly regulated in response to SAHA than by MS-275 [Choudhary, 2009].</p>
<strong>Response-response relationship</strong>
<p style="margin-left:18pt">SAHA or MS-275 treatment leads to an increase in acetylation of specific lysine residues on histones more than two-fold [Choudhary et al., 2009]. Acetylation of the variant histone H2AZ-a mark for DNA damage sites- was upregulated almost 20-fold by SAHA, whereas a number of sites on the core histones H3 and H4 are several times more highly regulated in response to SAHA than by MS-275 [Choudhary et al., 2009].</p>
<p style="margin-left:18.0pt">TSA (100 ng/ml) accumulated the acetylated histones in a variety of mammalian cell lines, and the partially purified HDAC from wild-type FM3A cells was effectively inhibited by TSA (<em>K<sub>i</sub></em> = 3.4 nM) [Yoshida, 1990].</p>
<p style="margin-left:18pt">TSA (100 ng/ml) treatment leads to accumulation of the acetylated histones in a variety of mammalian cell lines, and the partially purified HDAC from wild-type FM3A cells was effectively inhibited by TSA (<em>K<sub>i</sub></em> = 3.4 nM) [Yoshida et al., 1990].</p>
<p>Falkenberg KJ and Johnstone RW. (2014) Histone deacetylases and their inhibitors in cancer, neurological disease and immune disorders. Nat Rev Drug Discov 13:673-691Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831</p>
<p>Chen S et al. (2018) Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-kB pathway dependent of HDAC3. J Neuroinflammation 15:150</p>
<h4>References</h4>
<p>Alberts, A.S. et al. (1998), "Activation of SRF-regulated chromosomal templates by Rho-family GTPases requires a signal that also induces H4 hyperacetylation", Cell 92:475-487</p>
<p>Eikel D et al. (2006) Teratogenic effects mediated by inhibition of histone deacetylases: evidence from quantitative structure activity relationships of 20 valproic acid derivatives. Chem Res Toxicol 19:272-278</p>
<p>Bannister, A. J. and Kouzarides, T. (2011), "Regulation of chromatin by histone modifications", Cell Res 21:381-395</p>
<p>Schroeder FA et al. (2013) A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests. PLoS One 8:e71323</p>
<p>Chen, S. et al. (2018), "Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-kB pathway dependent of HDAC3", J Neuroinflammation 15:150</p>
<p>Choudhary C et al. (2009)Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834-840</p>
<p>Choudhary, C. et al. (2009), "Lysine acetylation targets protein complexes and co-regulates major cellular functions", Science 325:834-840</p>
<p>Henderson SE et al. (2016) Suppression of tumor growth and muscle wasting in a transgenic mouse model of pancreatic cancer by the novel histone deacetylase inhibitor AR-42. Neoplasia 18:765-774</p>
<p>Cousens, L. S. et al. (1979), "Different accessibilities in chromatin to histone acetylase", J Biol Chem 254:1716-1723</p>
<p>Lagger G et al. (2002) Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J 21:2672-2681</p>
<p>Dayan, C. and Hales, B.F. (2014), "Effects of ethylene glycol monomethyl ether and its metabolite, 2-methoxyacetic acid, on organogenesis stage mouse limbs in vitro", Birth Defects Res (Part B) 101:254-261</p>
<p>Dayan C and Hales BF. (2014) Effects of ethylene glycol monomethyl ether and its metabolite, 2-methoxyacetic acid, on organogenesis stage mouse limbs in vitro. Birth Defects Res (Part B) 101:254-261</p>
<p>Eikel, D. et al. (2006), "Teratogenic effects mediated by inhibition of histone deacetylases: evidence from quantitative structure activity relationships of 20 valproic acid derivatives", Chem Res Toxicol 19:272-278</p>
<p>Yoshida M et al. (1990) Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro trichostatin A. J Biol Chem 265:17174-17179</p>
<p>Falkenberg, K.J. and Johnstone, R.W. (2014), "Histone deacetylases and their inhibitors in cancer, neurological disease and immune disorders", Nat Rev Drug Discov 13:673-691</p>
<p>Alberts AS et al. (1998) Activation of SRF-regulated chromosomal templates by Rho-family GTPases requires a signal that also induces H4 hyperacetylation. Cell 92:475-487</p>
<p>Gallinari, P. et al. (2007), "HDACs, histone deacetylation and gene transcription: From molecular biology to cancer therapeutics", Cell Res 17:195-211</p>
<p>Sharma A et al. (2016) Epigenetic modification prevents excessive wound healing and scar formation after glaucoma filtration surgery. Invest Ophthalmol Vis Sci 57:3381-3389</p>
<p>Gottlicher, M. et al. (2001), "Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells", EMBO J 20:6969-6978</p>
<p>Li H et al. (2014) The histone deacetylase inhibitor trichostatin A promotes totipotentcy in the male gametophyte. Plant Cell 26:195-209</p>
<p>Henderson, S.E. et al. (2016), "Suppression of tumor growth and muscle wasting in a transgenic mouse model of pancreatic cancer by the novel histone deacetylase inhibitor AR-42", Neoplasia 18:765-774</p>
<p>Bannister, A. J. and Kouzarides, T. (2011). Regulation of chromatin by histone modifications. <em>Cell Res 21</em>, 381-395. doi:10.1038/cr.2011.22</p>
<p>Kouzarides, T. (2007), "Chromatin modifications and their function", Cell 128:693-705</p>
<p>Cousens, L. S., Gallwitz, D. and Alberts, B. M. (1979). Different accessibilities in chromatin to histone acetylase. <em>J Biol Chem 254</em>, 1716-1723.</p>
<p>Lagger, G. et al. (2002), "Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression", EMBO J 21:2672-2681</p>
<p>Gallinari, P., Di Marco, S., Jones, P. et al. (2007). Hdacs, histone deacetylation and gene transcription: From molecular biology to cancer therapeutics. <em>Cell Res 17</em>, 195-211. doi:7310149 [pii]</p>
<p>Li, H. et al. (2014), "The histone deacetylase inhibitor trichostatin A promotes totipotentcy in the male gametophyte", Plant Cell 26:195-209</p>
<p>10.1038/sj.cr.7310149</p>
<p>Luger, K. et al. (1997), "Crystal structure of the nucleosome core particle at 2.8 a resolution", Nature 389:251-260</p>
<p>Gottlicher, M., Minucci, S., Zhu, P. et al. (2001). Valproic acid defines a novel class of hdac inhibitors inducing differentiation of transformed cells. <em>Embo J 20</em>, 6969-6978. doi:10.1093/emboj/20.24.6969</p>
<p>Menegola, E. et al. (2005), "Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity", Birth Defects Res B Dev Reprod Toxicol 74:392-398</p>
<p>Kouzarides, T. (2007). Chromatin modifications and their function. <em>Cell 128</em>, 693-705.</p>
<p>Riggs, M.G. et al. (1977), "N-butyrate causes histone modification in HeLa and friend erythroleukaemia cells", Nature 268:462-464</p>
<p>Luger, K., Mader, A. W., Richmond, R. K. et al. (1997). Crystal structure of the nucleosome core particle at 2.8 a resolution. <em>Nature 389</em>, 251-260. doi:10.1038/38444</p>
<p>Schroeder, F.A. et al. (2013), "A selective HDAC 1/2 inhibitor modulates chromatin and gene expression in brain and alters mouse behavior in two mood-related tests", PLoS One 8:e71323</p>
<p>Menegola, E., Di Renzo, F., Broccia, M. L. et al. (2005). Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity. Birth Defects Res B Dev Reprod Toxicol 74, 392-398. doi:10.1002/bdrb.20053</p>
<p>Sharma, A. et al. (2016), "Epigenetic modification prevents excessive wound healing and scar formation after glaucoma filtration surgery", Invest Ophthalmol Vis Sci 57:3381-3389</p>
<p>Riggs, M. G., Whittaker, R. G., Neumann, J. R. et al. (1977). N-butyrate causes histone modification in hela and friend erythroleukaemia cells. <em>Nature 268</em>, 462-464.</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p>Yoshida, M., Kijima, M., Akita, M. et al. (1990). Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin a. <em>J Biol Chem 265</em>, 17174-17179.</p>
<p>Yoshida, M. et al. (1990), "Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A", J Biol Chem 265:17174-17179</p>
<p style="margin-left:18.0pt">The relationship between increased histone acetylation and p21 expression increase is likely well conserved between species.</p>
</div>
<p style="margin-left:18.0pt"><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">The relationship between increased histone acetylation and cell cycle disruption is likely well conserved between species. The present KER focuses on the pathway of p21, a cell-cycle regulator, leading to apoptosis. The examples are only given for mammals:</span></span></p>
<ul>
<li>TSA and sodium butyrate induced p21 mRNA expression in HT-29 human colon carcinoma cells (<em>Homo sapiens</em>) [Wu, 2001].</li>
<li>VPA increased acetylation of histone H3 from 3 hrs to 72 hrs after the treatment, and increased p21 expression in 24 hrs after the treatment in K562 cells (<em>Homo sapiens</em>) [Gurvich, 2004].</li>
<li>Scriptaid, a HDI, up-regulated p21 mRNA expression in mouse embryonic kidney cells (<em>Mus musculus</em>) [Chen, 2011].</li>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Chidamide induced histone acetylation and cell cycle arrest in RPMI8226 and U266 human myeloma cells (<em>Homo sapiens</em>) [Yuan et al., 2019].</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">TSA and sodium butyrate induced cell cycle regulator p21 mRNA expression in HT-29 human colon carcinoma cells (<em>Homo sapiens</em>) [Wu et al., 2001].</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">VPA increased acetylation of histone H3 from 3 hrs to 72 hrs after the treatment and increased p21 expression in 24 hrs after the treatment in K562 cells (<em>Homo sapiens</em>) [Gurvich et al., 2004].</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Scriptaid, an HDI, up-regulated p21 mRNA expression in mouse embryonic kidney cells (<em>Mus musculus</em>) [Chen et al., 2011].</span></span></li>
</ul>
<h4>Key Event Relationship Description</h4>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Upon histone acetylation increase, cell cycle regulation is disrupted, where acetylation in the promoter region of the coding genes has a close correlation [Gurvich et al., 2004]. Transient histone hyperacetylation was sufficient for the activation of molecules involving cell cycle regulation such as inducing p21 gene expression [Wu et al., 2001]. Histone hyperacetylating agents butyrate and TSA induced mRNA expression of cell cycle regulator gene [Archer et al., 1998]. SAHA induced the accumulation of acetylated histones in the chromatin of the gene regulating cell cycle [Richon et al., 2000]. </span></span></p>
<h4>Key Event Relationship Description</h4>
<p style="margin-left:18.0pt">Upon histone acetylation increase, p21 transcription and protein level are increased. Acetylation of p21 promoter and p21 mRNA level have a close correlation [Gurvich, 2004]. Transient histone hyperacetylation was sufficient for the activation of p21 [Wu, 2001]. Histone hyperacetylating agents butyrate and TSA induced p21 mRNA expression [Archer, 1998]. SAHA induced the accumulation of acetylated histones in the chromatin of the p21<sup>WAF1</sup> gene and this increase was associated with an increase in p21<sup>WAF1</sup> expression [Richon, 2000].</p>
<p style="margin-left:18.0pt"> </p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>HDIs induce histone hyperacetylation and p21 activation leading to the cell cycle arrest, which suggests the close correlation between histone hyperacetylation and p21. In the models proposed for the relationship between histone acetylation and transcription, histone acetylation can be untargeted and occur at both promoter and nonpromoter regions, targeted generally to promoter regions, or targeted to specific promoters by gene-specific activator proteins [Richon, 2000, Struhl, 1998]. Since several results supported a model in which increased histone acetylation is targeted to specific genes and occurs throughout the entire gene, not just the promoter regions, histone acetylation may leads to gene transcription of p21 [Richon, 2000].</p>
<strong>Empirical Evidence</strong>
<ul>
<li>MAA induced histone acetylation of H4 in prostate cancer cells including LNCaP, C4-2B, PC-3 and DU-145 parallel with p21 mRNA level increase [Parajuli, 2014].</li>
<li>HDIs accumulated acetylation of histones and induced p21 protein and mRNA expression [Richon, 2000, Wu, 2001].</li>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Histone deacetylase inhibitors induce histone hyperacetylation and the activation of downstream molecules leading to the cell cycle arrest, which suggests the close correlation between histone hyperacetylation and cell cycle arrest [Yuan et al., 2019]. The histone acetylation regulates the gene transcription through the promoter region of the coding gene, which may lead to the overexpression of cell cycle regulators [Richon et al., 2000; Struhl, 1998]. Histone deacetylase inhibition leads to acetylation of histone, inducing the expression of cyclin-dependent kinase inhibitors, followed by a cell-cycle arrest [Li and Seto, 2016].</span></span></p>
<strong>Empirical Evidence</strong>
<ul>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">MAA induced histone acetylation of H4 in prostate cancer cells including LNCaP, C4-2B, PC-3, and DU-145 parallel with cyclin-dependent kinase inhibitor p21, a cell cycle regulator, mRNA level increase [Parajuli et al., 2014].</span></span></li>
<li><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">HDIs accumulated acetylation of histones and induced cell cycle regulator p21 protein and mRNA expression [Richon et al., 2000; Wu et al., 2001].</span></span></li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">The histone acetylation causes cell cycle disruption in several pathways, in which the specific molecule involvement remains uncertain. </span></span></p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="margin-left:18.0pt">There are several pathways to activate p21 promoter by HDI. A HDI, apicidin, induced p21<sup>WAF1/Cip1</sup> mRNA independent of the <em>de novo</em> protein synthesis and activated the p21<sup>WAF1/Cip1</sup> promoter through Sp1 sites [Han, 2001]. Pretreatment with selective PKC inhibitors calphostin A and rottlerin suppressed the promoter activity of p21WAF1/Cip1 activated by apicidin [Han, 2001]. Furthermore, apicidin-induced translocation of PKCe from cytosolic to particulate fraction was reversed by pretreatment with calphostin C, which suggests the PKCe involvement in apicidin-induced p21<sup>WAF1/Cip1</sup> transcription [Han, 2001]. The p21 promoter activation through Sp1 sites induced by apicidin is thought to be independent of histone hyperacetylation [Han, 2001]. The apicidin is suggested to histone hyperacetylation leading to the antiproliferative activity [Han, 2000]. These results indicate the inconclusive discussion in the linkage between histone acetylation and p21 activation.</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>Histone H4 acetylation is induced with in 4 hrs and returned to basal level after 0.3 uM of trichostatin A (TSA) treatment [Wu JT].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Histone acetylation occurs in a dose-dependent manner with the treatment of chidamide for 48 hrs [Yuan et al., 2019]. The expression of proteins related to G<sub>0</sub>/G<sub>1</sub> cell cycle arrest, p21, and phosphorylated p53 is increased in a dose-dependent manner [Yuan et al., 2019]. </span></span></p>
<strong>Response-response relationship</strong>
<p>Dose-response of valproic acid (VPA) showed that 5, 10, and 20 mM of VPA inhibited HDAC6 and HDAC7 activity in 293T cells, and 0.1-2 mM of VPA induced acetylation of lysine in H3 in U937 cells [Gurvich 2004]. The p21 protein level was induced with the treatment of 0.25-2 mM of VPA in U937 cells [Gurvich 2004].</p>
<strong>Response-response relationship</strong>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Dose-response of histone acetylation and expression of p21 and phosphorylated p53 showed that treatment with 0.5, 1, or 2 microM of chidamide for 48hrs induced histone acetylation in RPMI8226 myeloma cells, while 2, 4, or 8 microM of chidamide for 48 hrs induced histone acetylation in U266 myeloma cells [Yuan et al., 2019]. Chidamide treatment in 0.5, 1, or 2 microM in RPMI8226 or 2, 4, or 8 microM in U266 induced G<sub>0</sub>/G<sub>1</sub> arrest in the myeloma cells [Yuan et al., 2019]. Dose-response of valproic acid (VPA) showed that 5, 10, and 20 mM of VPA inhibited HDAC6 and HDAC7 activity in 293T cells, and 0.1-2 mM of VPA induced acetylation of lysine in H3 in U937 cells [Gurvich et al., 2004]. The p21 protein level was induced with the treatment of 0.25-2 mM of VPA in U937 cells [Gurvich et al., 2004].</span></span></p>
<strong>Time-scale</strong>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Time course for histone H4 hyperacetylation in response to repeated doses of TSA every 8 hrs showed that histone hyperacetylation was peaked in 12 hrs in an 8-fold increase and showed a 5-fold increase in 24 hrs compared to control [Wu et al., 2001]. TSA (0.3 microM) induced cell cycle regulator p21 mRNA expression in 1 hr after stimulation and the induction is returned to the basal level in 24 hrs [Wu et al., 2001]. Sodium butyrate (5 mM) and repetitive doses of TSA (0.3 microM, every 8 hrs) induced the p21 mRNA level in 24 hrs in HT-29 cells [Wu et al., 2001]. Acetylation of p21 promoter and p21 mRNA induction were correlated in the treatment of valproic acid and analogs [Gurvich et al., 2004]. MAA-induced acetylation increases in histones H3 and H4 was occurred in 4, 8, 12 hrs and returned to basal level in 24 hrs after the treatment in rat testis [Wade et al., 2008].</span></span></p>
<strong>Time-scale</strong>
<p>Time course for histone H4 hyperacetylation in response to in repeated doses of TSA every 8 hrs showed that histone hyperacetylation was peaked in 12 hrs in 8-fold increase and showed 5-fold increase in 24 hrs compared to control [Wu JT]. TSA (0.3 uM) induced p21 mRNA expression in 1 hr after stimulation and the induction is returned to the basal level in 24 hrs [Wu JT]. Sodium butyrate (5 mM) and repetitive doses of TSA (0.3 uM, every 8 hrs) induced the p21 mRNA level in 24 hrs in HT-29 cells [Wu JT]. Acetylation of p21 promoter and p21 mRNA induction were correlated in treatment of valproic acid and analogs [Gurvich 2004]. MAA-induced acetylation increase in histones H3 and H4 was occurred in 4, 8, 12 hrs and returned to basal level in 24 hrs after the treatment in rat testis [Wade 2008].</p>
<p style="margin-left:49.55pt">Gurvich N et al. (2004) Histone deacetylase is a target of valproic acid-mediated cellular differentiation. Cancer Res 64:1079-1086</p>
<h4>References</h4>
<p>Archer, S.Y. et al. (1998), "p21WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells", Proc Natl Acad Sci USA 95:6791-6796</p>
<p style="margin-left:49.55pt">Wu JT et al. (2001) Transient vs prolonged histone hyper acetylation: effects on colon cancer cell growth, differentiation, and apoptosis. Am J Physiol Gastrointest Liver Physiol 280:G482-G490</p>
<p>Chen, S. et al. (2011), "Histone deacetylase (HDAC) activity for embryonic kidney gene expression, growth, and differentiation", J Biol Chem 286:32775-32789</p>
<p style="margin-left:49.55pt">Archer SY et al. (1998) p21<sup>WAF1</sup> is required for butyrate-mediated growth inhibition of human colon cancer cells. Proc Natl Acad Sci USA 95:6791-6796</p>
<p>Gurvich, N. et al. (2004), "Histone deacetylase is a target of valproic acid-mediated cellular differentiation", Cancer Res 64:1079-1086</p>
<p style="margin-left:49.55pt">Richon VM et al. (2000) Histone deacetylase inhibitor selectively induces p21<sup>WAF1</sup> expression and gene-associated histone acetylation. Proc Natl Acad Sci 97:10014-10019</p>
<p>Li, Y. and Seto, E. (2016), "HDACs and HDAC inhibitors in cancer development and therapy", Cold Spring Harb Perspect Med 6:a026831</p>
<p style="margin-left:49.55pt">Struhl K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Gene Dev 12:599-606</p>
<p>Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313</p>
<p style="margin-left:49.55pt">Parajuli KR et al. (2014) Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis. Am J Clin Exp Urol 2:300-313</p>
<p style="margin-left:49.55pt">Han JW et al. (2001) Activation of p21<sup>WAF1/Cip1</sup> transcription through Sp1 sites by histone deacetylase inhibitor apicidin: involvement of protein kinase C. J Biol Chem 276:42084-42090</p>
<p>Struhl, K. (1998), "Histone acetylation and transcriptional regulatory mechanisms", Gene Dev 12:599-606</p>
<p style="margin-left:49.55pt">Han JW et al. (2000) Apidin, a histone deacetylase inhibitor, inhibits proliferation of tumor cells via induction of p21<sup>WAF1/Cip1</sup> and gelsolin. Cancer Res 60:6068-6074</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p style="margin-left:49.55pt">Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831</p>
<p>Wu, J.T. et al. (2001), "Transient vs prolonged histone hyperacetylation: effects on colon cancer cell growth, differentiation, and apoptosis", Am J Physiol Gastrointest Liver Physiol 280:G482-G490</p>
<p style="margin-left:49.55pt">Chen S et al (2011) Histone deacetylase (HDAC) activity for embryonic kidney gene expression, growth, and differentiation. J Biol Chem 286: 32775-32789</p>
<p>Yuan, X. et al. (2019), "Chidamide, a histone deacetylase inhibitor, induces growth arrest and apoptosis in multiple myeloma cells in a caspase-dependent manner", Oncol Let 18:411-419</p>
<p>DNA replication in <em>Xenopus</em> was suppressed by the GST fusion protein of p21 without amino acids 17-24 or the peptide containing cyclin binding site in N-terminus of p21 protein [Chen, 1996]. P21 regulates the E2F transcriptional activity to control cell cycle in human U2OS osteosarcoma cells (<em>Homo sapiens</em>) [Delavaine, 1999]. Cell cycle is regulated by p21 through cyclins and CDKs in mice (<em>Mus musculus</em>) [Sherr CJ, 2004]. </p>
<h4>Key Event Relationship Description</h4>
<p>Cell cycle regulation through p21 (cyclin dependent kinase inhibitor 1A; CDKN1A) activation is demonstrated by the interactions of p21 with cyclins [Dotto, 2000]. p21 interacts directly with cyclins through a conserved region in close to its N-terminus (amino acids 17-24; Cy1) [Dotto, 2000]. The cyclin dependent kinase inhibitor, p21 has the secondary weak cyclin binding domain near its C-terminus region (amino acids 153-159), which overlaps with its proliferating cell nuclear antigen (PCNA) binding domain [Dotto, 2000]. Kinase activity of cyclin-dependent kinase (Cdk) was inhibited by Cy1 site of p21 that is important for the interaction of p21 with cyclin-Cdk complexes [Chen, 1996]. The p21 inhibits Cdk complexes such as cyclin A/E-Cdk2 or cyclin D-Cdk4 complexes, leading to the cell cycle disruption as G<sub>1</sub>/S arrest [Chen, 1996].</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="margin-left:18.0pt">p21 has a separate cyclin-dependent kinase 2 (CDK2) binding site in its N-terminus region (amino acids 53-58) and optimal cyclin/CDK inhibition requires binding by this site as well as one of the cyclin binding sites [Dotto, 2000]. The peptide containing Cy1 site inhibited the kinase activity of cyclin E-Cdk2 and cyclin A-Cdk2 [Chen, 1996]. The p21<em><sup>WAF1/CIP1/sdi1</sup></em> gene product inhibits the cyclin D/cdk4/6 and the cyclin E/cdk2 complexes in response to DNA-damage, resulting in G<sub>1</sub>/S arrest [Moussa, 2015, Ogryzko, 1997]. p21 inhibits cyclin-dependent kinases and regulates cell cycle to promote cell cycle arrest. Deletion of either cyclin binding site in N-terminus or C-terminus of p21, or CDK binding domain was sufficient for the kinase activity inhibition [Chen, 1996].</p>
<strong>Empirical Evidence</strong>
<ul>
<li>TSA induces p21 expression leading to cell cycle arrest [Gartel, 2002].</li>
<li>The up-regulation of p21 signaling and in testicular germ cells was observed in diabetes [Kilarkaje, 2015].</li>
<li>A study investing the effects of miR-6734 that has a sequence homology with a specific region of p21<sup>WAF1/CIP1</sup> promoter on HCT-116 colon cancer cell growth indicated that miR-6734 up-regulated p21 gene expression and induced cell cycle arrest [Kang, 2016]. This result suggests that the direct enhancement of p21 gene expression is related to the alteration of the cell cycle distribution [Kang, 2016].</li>
<li>The study of postnatal telomere indicated that dysfunction of premature telomere induces cell-cycle arrest through p21 activation in mammalian cardiomyocytes [Aix, 2016].</li>
<li>The p21<em><sup>WAF1/CIP1/sdi1</sup></em> gene product inhibits the cyclin D/cdk4/6 and the cyclin E/cdk2 complexes in response to DNA-damage, resulting in G<sub>1</sub>/S arrest [Moussa, 2015, Ogryzko, 1997].</li>
</div>
<p style="margin-left:18.0pt">The relationship between disrupted cell cycle and apoptosis is likely well conserved between species. The examples are only given for mammals:</p>
<ul>
<li>MicroRNA let-7a induced cell cycle arrest and inhibited CCND2 and proliferation of human prostate cancer cells (<em>Homo sapiens</em>) [Dong et al., 2010].</li>
<li>The microRNA-497 down-regulated CCND2 and induced apoptosis <em>via</em> the Bcl-2/Bax-caspase 9- caspase 3 pathway in HUVECs (<em>Homo sapiens</em>) [Wu et al., 2016].</li>
<li>The microRNA-26a regulated p53-mediated apoptosis and CCND2 and CCNE2 in mouse hepatocyte (<em>Mus musculus</em>) [Zhou et al., 2016].</li>
</ul>
<h4>Key Event Relationship Description</h4>
<p>Cell cycle dysregulation may lead to apoptosis. Cell cycles characterized by the DNA content changes regulate cell death and cell proliferation [Lynch et al., 1986].</p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="margin-left:18.0pt">TSA promotes apoptosis via HDAC inhibition and p53 signaling pathway activation [Deng, 2016a]. It is suggested that furazolidone induces reactive oxygen species leading to suppression of p-AKT and p21, andinduction of apoptosis [Deng, 2016b]. The dual roles of p21 in cell cycle arrest and anti-apoptotic effect in the testicular germ cells of diabetic rats are suggested [Kilarkaje, 2015]. The anti-apoptotic effect of p21 is mediated by caspase-3 inhibition, which demonstrates the possibility of cell-cycle independent effect on apoptosis [Deng, 2016b]. It has been demonstrated that p21 induces apoptosis in human cervical cancer cell lines [Tsao, 1999], whereas p21 is implicated in apoptosis inhibition by blocking activation of caspase-3 or interacting with ASK1 [Gartel, 2002, Zhan, 2007]. Up-regulation of p21 is implicated in the activation of DNA damage pathways, and deletion of p21 improved stem cell function and lifespan without accelerating chromosomal instability, which indicates that p21-dependent checkpoint induction affects the longevity limit [Choudhury, 2007].</p>
<h4>Evidence Supporting this KER</h4>
<p>The microRNA-497, potentially targeting Bcl-2 and cyclin D2 (CCND2), activated caspases 9/3, and induced apoptosis <em>via</em> the Bcl-2/Bax - caspase 9 - caspase 3 pathway and CCND2 protein in human umbilical vein endothelial cells (HUVECs) [Wu, 2016]. CCND2 is an important cell cycle gene, of which a decrease in expression induces G<sub>1</sub> arrest [Li et al., 2012], and dysregulated CCND2 is implicated in cell proliferation inhibition [Wu et al., 2016; Mermelstein et al., 2005; Dong et al., 2010].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<strong>Response-response relationship</strong>
<p style="margin-left:18.0pt">The peptide containing cyclin-binding domain of p21 in N-terminus inhibited the kinase activity of cyclin E-Cdk2 with 296 nM of the concentration in which kinase activity is inhibited in 50% (Ki) [Chen, 1996].</p>
<strong>Biological Plausibility</strong>
<p>The incidence of apoptosis was increased in vincristine-treated cells, in which metaphases were arrested, compared to untreated cells, which indicates that cell cycle dysregulation leads to apoptosis [Sarraf and Bowen, 1986]. Cell gain and loss are balanced with mitosis and apoptosis [Cree et al., 1987]. Apoptosis is mediated by caspase activation [Porter and Janicke, 1999]. Caspase-3 is activated in programmed cell death, and the pathways to caspase-3 activation include caspase-9 and mitochondrial cytochrome c release [Porter and Janicke, 1999]. The activation of caspase-3 leads to apoptotic chromatin condensation and DNA fragmentation [Porter and Janicke, 1999]. Sinularin, a marine natural compound, exhibited DNA damage and induced G<sub>2</sub>/M cell cycle arrest, followed by apoptosis in human hepatocellular carcinoma HepG2 cells [Chung et al., 2017]. Sinularin induced caspases 8, 9, and 3, and pro-apoptotic protein Bax, whereas it decreases the anti-apoptotic Bcl-2 protein expression level [Chung et al., 2017].</p>
<p style="margin-left:18.0pt">The peptide containing cyclin-binding domain of p21 in C-terminus showed 32,000, 800, or >300,000 nM of Ki for inhibition of the kinase activity of cyclin E-Cdk2, cyclin A-Cdk2 or cyclin D1-Cdk4, respectively [Chen, 1996].</p>
<p style="margin-left:18.0pt"> </p>
<strong>Empirical Evidence</strong>
<ul>
<li>Cell cycle arrests such as G<sub>1</sub> arrest and G<sub>1</sub>/S arrest are observed in apoptosis [Li et al., 2012; Dong et al., 2010].</li>
<li>microRNA-1 and microRNA-206 repress CCND2, while microRNA-29 represses CCND2 and induces G<sub>1</sub> arrest and apoptosis in rhabdomyosarcoma [Li et al., 2012].</li>
<li>The blockade of G<sub>1</sub>/S transition of cell cycle and reduction of CDK4 and CDK2, and apoptosis have occurred in HDAC inhibitor treatment [Parajuli et al., 2014].</li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p>MAA induces CDK4 and CDK2 decreases, cell cycle arrest, and apoptosis, which may be regulated by several pathways [Parajuli et al., 2014].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>Cell proliferation which was determined at daily intervals after a 24-hr pulse of [<sup>3</sup>H]thymidine changed as the amount of DNA in the cultures changed. Cell death which was measured by lactic dehydrogenase (LDH) activity in the medium changed in parallel with the changes in cell proliferation [Lynch et al., 1986].</p>
<strong>Response-response relationship</strong>
<p>Treatment with sinularin, a natural product isolated from cultured soft coral possessing antineoplastic activity, at 12.5, 25, 50 microM resulted in cell cycle disruption and apoptosis in a dose-dependent manner in hepatocellular carcinoma cells [Chun et al., 2017]. The cell cycle disruption and apoptosis are induced by 30 microM curcumin, a major component extracted from turmeric plants that have an anti-cancer effect [Liu et al., 2018].</p>
<p>MAA (5 mM) decreases CDK4, CDK2 expression 48 hrs after the treatment, which indicates the G<sub>1</sub> arrest [Parajuli et al., 2014]. MAA (5 mM) decreases the protein expression of procaspase 7 and 3 in 24 to 72 hrs after the treatment, indicating the activation of caspases 7 and 3 [Parajuli et al., 2014].</p>
<h4>References</h4>
<p style="margin-left:49.55pt">Dotto GP (2000) p21<sup>WAF1/Cip1</sup>: more than a break to the cell cycle? Biochim Biophys Acta 1471: M43-M56</p>
<h4>References</h4>
<p>Chung, T.W. et al. (2017), "Sinularin induces DNA damage, G2/M phase arrest, and apoptosis in human hepatocellular carcinoma cells", BMC Complement Altern Med 17:62</p>
<p style="margin-left:49.55pt">Chen J et al (1996) Cyclin-binding motifs are essential for the function of p21<em><sup>CIP1</sup></em>. Mol Cell Biol 16: 4673-4682</p>
<p>Cree, I.A. et al. (1987), "Cell death in granulomata: the role of apoptosis", J Clin Pathol 40:1314-1319</p>
<p style="margin-left:49.55pt">Moussa RS et al. (2015) Differential targeting of the cyclin-dependent kinase inhibitor, p21CIP/WAF1, by chelators with anti-proliferative activity in a range of tumor cell-types. Oncotarget 6:29694-29711</p>
<p>Dong, Q. et al. (2010), "microRNA let-7a inhibits proliferation of human prostate cancer cells in vitro and in vivo by targeting E2F2 and CCND2", PLoS One 5:e10147</p>
<p style="margin-left:49.55pt">Ogryzko VV et al. (1997) WAF1 retards S-phase progression primarily by inhibition of cyclin-dependent kinases. Mol Cell Biol 17:4877-4882</p>
<p>Kerr, J.F.R. et al. (1972), "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer 26:239-257</p>
<p style="margin-left:49.55pt">Gartel AL and Tyner AL (2002) The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther 1: 639-649</p>
<p>Li, L. et al. (2012), "Downregulation of microRNAs miR-1, -206 and -29 stabilizes PAX3 and CCND2 expression in rhabdomyosarcoma", Lab Invest 92:571-583</p>
<p style="margin-left:49.55pt">Kilarkaje N and Al-Bader MM. (2015) Diabetes-Induced Oxidative DNA Damage Alters p53-p21<sup>CIP1/Waf1</sup> Signaling in the Rat Testis. <em>Reproductive Sciences </em>22: 102–112</p>
<p>Liu, W. et al. (2018), "Curcumin suppresses gastric cancer biological activity by regulation of miRNA-21: an in vitro study", Int J Clin Exp Pathol 11:5820-5289</p>
<p style="margin-left:49.55pt">Kang MR et al (2016) miR-6734 up-regulates p21 gene expression and induces cell cycle arrest and apoptosis in colon cancer cells. PLoS One 11: e0160961</p>
<p>Lynch, M.P. et al. (1986), "Evidence for soluble factors regulating cell death and cell proliferation in primary cultures of rabbit endometrial cells grown on collagen", Proc Natl Acad Sci USA 83:4784-4788</p>
<p style="margin-left:49.55pt">Aix E et al (2016) Postnatal telomere dysfunction induces cardiomyocyte cell-cycle arrest through p21 activation. J Cell Biol 213: 571-583</p>
<p>Mermelshtein, A. et al. (2005), "Expression of F-type cyclins in colon cancer and in cell lines from colon carcinomas", Br J Cancer 93:338-345</p>
<p style="margin-left:49.55pt">Deng Z et al. (2016a) Histone deacetylase inhibitor trichostatin A promotes the apoptosis of osteosarcoma cells through p53 signaling pathway activation. Int J Biol Sci 12:1298-1308</p>
<p>Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313</p>
<p style="margin-left:49.55pt">Deng S et al (2016b) P21<sup>Waf1/Cip1</sup> plays a critical role in furazolidone-induced apoptosis in HepG2 cells through influencing the caspase-3 activation and ROS generation. Food Chem Toxicol 88: 1-12</p>
<p>Porter, A.G. and Janicke, R.U. (1999), "Emerging roles of caspase-3 in apoptosis", Cell Death Differ 6:99-104</p>
<p style="margin-left:49.55pt">Tsao YP et al (1999) Adenovirus-mediated p21<sup>WAF1/SDII/CIP1</sup> gene transfer induces apoptosis of human cervical cancer cell lines. J Virology 73: 4983-4990</p>
<p>Sarraf, C.E. and Bowen, I.D. (1986), "Kinetic studies on a murine sarcoma and an analysis of apoptosis", Br J Cancer 54:989-998</p>
<p style="margin-left:49.55pt">Zhan J et al (2007) Negative regulation of ASK1 by p21Cip1 involves a small domain that includes serine 98 that is phosphorylated by ASK1 in vivo. Mol Cell Biol 27: 3530-3541</p>
<p>Wu, R. et al. (2016), "microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs", PLoS One 11:e0167052</p>
<p style="margin-left:49.55pt">Choudhury AR et al (2007) Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation. Nat Genet 39: 99-105</p>
<p style="margin-left:49.55pt">Delavaine L and La Thangue NB (1999) Control of E2F activity by p21Waf1/Cip1. Oncogene 18: 5381-5392</p>
<p style="margin-left:49.55pt">Sherr CJ and Roberts JM (2004) Living with or without cyclins and cyclin-dependent kinases. Gene Dev 18: 2699-2711</p>
<p>Zhou, J. et al. (2016), "miR-26a regulates mouse hepatocyte proliferation via directly targeting the 3’ untranslated region of CCND2 and CCNE2", Hepatobiliary Pancreat Dis Int 15:65-72</p>
</div>
<br>
<div>
<h4><a href="/relationships/1712">Relationship: 1712: Cell cycle, disrupted leads to Apoptosis</a></h4>
<div>
<h4><a href="/relationships/1735">Relationship: 1735: Apoptosis leads to Spermatocyte depletion</a></h4>
<p style="margin-left:18.0pt">The relationship between disrupted cell cycle and apoptosis is likely well conserved between species.</p>
</div>
<p>The apoptosis of the cells leads to spermatocyte depletion. The relationship between apoptosis and spermatocyte depletion is likely well conserved between species. The examples are only given for mammals:</p>
<ul>
<li>MicroRNA let-7a induced cell cycle arrest, inhibited CCND2 and proliferation of human prostate cancer cells (<em>Homo sapiens</em>) [Dong, 2010].</li>
<li>microRNA-497 down-regulated CCND2 and induced apoptosis via the Bcl-2/Bax-caspase 9- caspase 3 pathway in HUVECs (<em>Homo sapiens</em>) [Wu, 2016].</li>
<li>micoRNA-26a regulated p53-mediated apoptosis and CCND2 and CCNE2 in mouse hepatocyte (<em>Mus musculus</em>) [Zhou, 2016].</li>
</ul>
<h4>Key Event Relationship Description</h4>
<p style="margin-left:18.0pt">Cell cycle dysregulation leads to apoptosis. Cell cycles characterized by the DNA content changes regulate cell death and cell proliferation [Lynch, 1986].</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<p>microRNA-497, potentially targeting Bcl2 and Cyclin D2 (CCND2), induced apoptosis via the Bcl-2/Bax - caspase 9 - caspase 3 pathway and CCND2 protein in human umbilical vein endothelial cells (HUVECs) [Wu, 2016]. The microRNA-497 activated caspases 9 and 3, and decreased Bcl2 and CCND2 [Wu, 2016]. CCND2 is an important cell cycle gene that induces G<sub>1</sub> arrest [Li, 2012], and deregulated CCND2 is implicated in cell proliferation inhibition [Wu, 2016, Mermelstein, 2005, Dong, 2010].</p>
<strong>Biological Plausibility</strong>
<p style="margin-left:18.0pt">The incidence of apoptosis was increased in vincristine-treated cells, in which metaphases were arrested, compared to untreated cells, which indicates that cell cycle dysregulation leads to apoptosis [Sarraf, 1986]. Cell gain and loss are balanced with mitosis and apoptosis [Cree, 1987]. Apoptosis is mediated by caspase activation [Porter, 1999]. Caspase-3 is activated in the programmed cell death, and the pathways to caspase-3 activation include caspase-9 and mitochondrial cytochrome c release [Porter, 1999]. The activation of caspase-3 leads to apoptotic chromatin condensation and DNA fragmentation [Porter, 1999]. Sinularin, a marine natural compound, exhibited DNA damage and induced G<sub>2</sub>/M cell cycle arrest, followed by apoptosis in human hepatocellular carcinoma HepG2 cells [Chung, 2017]. Sinularin induced caspases 8, 9, and 3, and pro-apoptotic protein Bax, whereas it decrease the anti-apoptotic Bcl-2 protein expression level [Chung, 2017].</p>
<p style="margin-left:18.0pt"> </p>
<strong>Empirical Evidence</strong>
<ul>
<li>Cell cycle arrest such as G<sub>1</sub> arrest and G<sub>1</sub>/S arrest are observed in apoptosis [Li, 2012, Dong, 2010].</li>
<li>microRNA-1 and microRNA-206 represses CCND2, while microRNA-29 represses CCND2 and induces G<sub>1</sub> arrest and apoptosis in rhabdomyosarcoma [Li, 2012].</li>
<li>The treatment with HDAC inhibitor, methoxyacetic acid (MAA) in prostate cancer cells induced growth arrest and apoptosis [Parajuli, 2014]. MAA blocks G<sub>1</sub>/S transition of cell cycle [Parajuli, 2014]. MAA reduces CDK4 and CDK2, and decreases protein expression of BIRC2 and activates caspase 7 and 3 [Parajuli, 2014].</li>
<li>Spermatogenesis was inhibited by the knockdown of Sucla2, a β subunit of succinyl coenzyme A synthase, <em>via</em> apoptosis in the mouse spermatocyte (<em>Mus musculus</em>) [Huang et al., 2016].</li>
<li>The suppression of microRNA-21 led to apoptosis of spermatogonial stem cell-enriched germ cell cultures and the decrease in the number of spermatogonial stem cells in mice (<em>Mus musculus</em>) [Niu et al., 2011].</li>
<li>MAA induced apoptosis and depletion of spermatocytes in adult rats (<em>Rattus norvegicus</em>) [Brinkworth et al., 1995].</li>
<li>
<p>The apoptosis and proliferation inhibition induced by MAA, an HDAC inhibitor, was measured in human prostate cancer cell lines (<em>Homo sapiens</em>) [Parajuli et al., 2014].</p>
</li>
<li>
<p>The cell viability inhibition induced by SAHA or TSA, which are HDAC inhibitors, was observed in NHDFs (<em>Homo sapiens</em>) [Glaser et al., 2003].</p>
</li>
<li>
<p>The proliferation of the HDAC<sup>-/-</sup> ES cells was inhibited compared to HDAC<sup>+/+</sup> ES cells (<em>Homo sapiens</em>) [Zupkovitz et al., 2010].</p>
</li>
<li>
<p>It has been reported that the mice lacking both <em>Ink4c</em> and <em>Ink4d</em>, cyclin D-dependent kinase inhibitors, produced few mature sperm, and the residual spermatozoa had reduced motility and decreased viability (<em>Mus musculus</em>) [Zindy et al., 2001].</p>
</li>
<li>
<p>The sperm counts in the cauda epididymis of rats exposed to butylparaben were significantly decreased (<em>Rattus norvegicus</em>) [Oishi, 2001].</p>
</li>
<li>
<p>MAA treatment-induced spermatocyte death in Sprague-Dawley rats (<em>Rattus norvegicus</em>) [Wade et al., 2008].</p>
</li>
</ul>
<h4>Key Event Relationship Description</h4>
<p>Apoptosis results in spermatocyte depletion <em>via</em> cell death. Apoptosis and spermatocyte depletion is correlated, where spermatocyte depletion <em>via</em> apoptosis is a general mechanism [Brinkworth et al., 1995].</p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="margin-left:18.0pt">MAA induces apoptosis, however, the MAA-induced changes of BCL2, BAX, BCL2L1, BAD, BID, MCL1, and CFLAR, pro-apoptotic and anti-apoptotic genes were not observed [Parajuli, 2014]. microRNA-497 induce activation of caspase-9 and -3, followed by apoptosis, however, the caspase-9 and -3 protein levels were repressed by the ectopic expression of microRNA-497, which remains uncertain [Wu, 2016].</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>Induced apoptosis during the development of germ cells results in the progressive depletion of spermatocytes [Brinkworth et al., 1995]. An HDAC inhibitor, MAA, induced apoptosis and spermatocyte depletion at stages IX-II [Brinkworth et al., 1995].</p>
<strong>Empirical Evidence</strong>
<p>In the mouse spermatocyte, spermatogenesis is inhibited by knockdown of Sucla2, a beta subunit of succinyl coenzyme A synthase, which is located in mitochondria and catalyzes the reversible synthesis of succinate and adenosine triphosphate in the tricarboxylic acid cycle [Huang et al., 2016]. The knockdown of Sucla2 induces apoptosis of mouse spermatocytes [Huang et al., 2016]. The prolonged cryptorchidism leads to germs cell apoptosis and testicular sperm count decrease [Barqawi et al., 2004]. CD147 was reported to regulate apoptosis in mouse testis and spermatocyte cell line (GC-2 cells) <em>via</em> NFκB pathway [Wang et al., 2017]. The microRNA-21 regulates the spermatogonial stem cell homeostasis, in which suppression of microRNA-21 with anti-miR-21 oligonucleotides led to apoptosis of spermatogonial stem cell-enriched germ cell cultures and the decrease in the number of spermatogonial stem cells [Niu et al., 2011].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>Cell proliferation which was determined at daily intervals agter a 24-hr pulse of [3H]thymidine changed as the amount of DNA in the cultures changed. Cell death which was measured by lactic dehydrogenase (LDH) activity in the medium changed in parallel with the changes in cell proliferation [Lynch, 1986]. The decrease in total DNA was measured, the increase in cell death was observed [Lynch, 1986].</p>
<strong>Uncertainties and Inconsistencies</strong>
<p>The process of apoptosis is necessary for the meiosis of the stem cell differentiation in the testis, which remains in question for the regulation of spermatocyte deletion and testis atrophy/weight loss [Dym, 1994].</p>
<strong>Time-scale</strong>
<p style="margin-left:18.0pt">MAA (5 mM) decreases CDK4, CDK2 expression in 48 hrs after the treatment, which indicates the G<sub>1</sub> arrest [Parajuli, 2014]. MAA (5 mM) decreases the protein expression of procaspase 7 and 3 in 24 to 72 hrs after the treatment, indicating the activation of caspases 7 and 3 [Parajuli, 2014].</p>
<p style="margin-left:49.55pt">Lynch MP et al. (1986) Evidence for soluble factors regulating cell death and cell proliferation in primary cultures of rabbit endometrial cells grown on collagen. Proc Natl Acad Sci USA 83: 4784-4788</p>
<p style="margin-left:49.55pt">Wu R et al. (2016) microRNA-497 induces apoptosis and suppressed proliferation via the Bcl-2/Bax-caspase9-caspase 3 pathway and cyclin D2 protein in HUVECs. PLoS One 11: e0167052</p>
<h4>References</h4>
<p>Barqawi, A. et al. (2004), "Effect of prolonged cryptorchidism on germ cell apoptosis and testicular sperm count", Asian J Androl 6:47-51</p>
<p style="margin-left:49.55pt">Li L et al. (2012) Downregulation of microRNAs miR-1, -206 and -29 stabilizes PAX3 and CCND2 expression in rhabdomyosarcoma. Lab Invest 92: 571-583</p>
<p>Bose, R. et al. (2017), "Ubiquitin ligase Huwe1 modulates spermatogenesis by regulating spermatogonial differentiation and entry into meiosis", Sci Rep 7:17759</p>
<p style="margin-left:49.55pt">Mermelshtein A et al. (2005) Expression of F-type cyclins in colon cancer and in cell lines from colon carcinomas. Br J Cancer 93: 338-345</p>
<p>Brinkworth, M. et al. (1995), "Identification of male germ cells undergoing apoptosis in adult rats", J Reprod Fertil 105:25-33</p>
<p style="margin-left:49.55pt">Dong Q et al. (2010) microRNA let-7a inhibits proliferation of human prostate cancer cells in vitro and in vivo by targeting E2F2 and CCND2. PLoS One 5: e10147</p>
<p>Dym, M. (1994), "Spermatogonial stem cells of the testis", Proc Natl Acad Sci USA 91:11287-11289</p>
<p style="margin-left:49.55pt">Kerr JFR et al. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257</p>
<p>Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163</p>
<p style="margin-left:49.55pt">Sarraf CE and Bowen ID (1986) Kinetic studies on a murine sarcoma and an analysis of apoptosis. Br J Cancer 54: 989-998</p>
<p>Huang, S. et al. (2016), "Knockdown of Sucla2 decreases the viability of mouse spermatocytes by inducing apoptosis through injury of the mitochondrial function of cells", Folia Histochem Cytobiol 54:134-142</p>
<p style="margin-left:49.55pt">Cree IA et al. (1987) Cell death in granulomata: the role of apoptosis J Clin Pathol 40: 1314-1319</p>
<p>Niu, Z. et al. (2011), "microRNA-21 regulates the self-renewal of mouse spermatogonial stem cells", Proc Natl Acad Sci 108:12740-12745</p>
<p style="margin-left:49.55pt">Porter AG and Janicke RU. (1999) Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6: 99-104</p>
<p>Oishi, S. (2001), "Effects of butylparaben on the male reproductive system in rats", Toxicol Indust Health 17:31-39</p>
<p style="margin-left:49.55pt">Chung TW et al. (2017) Sinularin induces DNA damage, G2/M phase arrest, and apoptosis in human hepatocellular carcinoma cells. BMC Complement Altern Med 17: 62</p>
<p>Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-313</p>
<p style="margin-left:49.55pt">Parajuli KR et al. (2014) Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis. Am J Clin Exp Urol 2:300-313</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p style="margin-left:49.55pt">Zhou J et al. (2016) miR-26a regulates mouse hepatocyte proliferation via directly targeting the 3’ untranslated region of CCND2 and CCNE2. Hepatobiliary Pancreat Dis Int 15: 65-72</p>
<p>Wang, C. et al. (2017), "CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways", Oncotarget 8:3132-3143</p>
<p> </p>
<p>Zindy, F. et al. (2001), "Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18Ink4c and p19Ink4d", Mol Cell Biol 21:3244-3255</p>
<p> </p>
<p>Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181</p>
</div>
<br>
<div>
<h4><a href="/relationships/1735">Relationship: 1735: Apoptosis leads to spermatocyte depletion</a></h4>
<div>
<h4><a href="/relationships/1734">Relationship: 1734: Spermatocyte depletion leads to Testicular atrophy</a></h4>
<p style="margin-left:18.0pt">The relationship between apoptosis and spermatocyte depletion is likely well conserved between species.</p>
</div>
<p style="margin-left:18.0pt">The relationship between spermatocyte depletion and testicular toxicity is likely well conserved between species.</p>
<ul>
<li>Spermatogenesis was inhibited by knockdown of Sucla2, a β subunit of succinyl coenzyme A synthase, via apoptosis in the mouse spermatocyte (<em>Mus musculus</em>) [Huang, 2016].</li>
<li>The suppression of microRNA-21 led to apoptosis of spermatogonial stem cell-enriched germ cell cultures and the decrease in the number of spermatogonial stem cells in mice (<em>Mus musculus</em>) [Niu Z, 2011].</li>
<li>MAA induced apoptosis and depletion of spermatocytes in adult rats (<em>Rattus norvegicus</em>) [Brinkworth, 1995].</li>
<li>
<p>The apoptosis and proliferation inhibition induced by MAA, a HDAC inhibitor, was measured in human prostate cancer cell lines (<em>Homo sapiens</em>) [Parajuli, 2014].</p>
</li>
<li>
<p>The cell viability inhibition induced by SAHA or TSA , which are HDAC inhibitors, was observed in NHDFs (<em>Homo sapiens</em>) [Glaser, 2003].</p>
</li>
<li>
<p>The proliferation of the HDAC<sup>-/-</sup> ES cells was inhibited compared to HDAC<sup>+/+</sup> ES cells (<em>Homo sapiens</em>) [Zupkovitz, 2010].</p>
</li>
<li>
<p>It has been reported that mice lacking both <em>Ink4c</em> and <em>Ink4d</em> ,cyclin D-dependent kinase inhibitors, produced few mature sperm, and the residual spermatozoa had reduced motility and decreased viability (<em>Mus musculus</em>) [Zindy, 2001].</p>
</li>
<li>
<p>The sperm counts in the cauda epidydimis of rats exposed to butylparaben were significantly decreased (<em>Rattus norvegicus</em>) [Oishi, 2001].</p>
</li>
<li>
<p>MAA treatment induced spermatocyte death in Sprague-Dawley rats (<em>Rattus norvegicus</em>) [Wade, 2008].</p>
</li>
<li>ME and MAA induced spermatocyte depletion and testicular atrophy in rats (<em>Rattus norvegicus</em>) [Beattie et al., 1984].</li>
<li>Ethylene glycol monomethyl ether induced depletion of late spermatocytes and testicular atrophy in F344 rat (<em>Rattus norvegicus</em>) [Chapin et al., 1984].</li>
<li>The epididymal tubules of rats with testicular degeneration had low sperm density (<em>Rattus norvegicus</em>) [Lee et al., 1993].</li>
<li>Hydroxyurea induced spermatocyte reduction and testicular atrophy (<em>Mus musculus</em>) [Wiger et al., 1995].</li>
</ul>
<h4>Key Event Relationship Description</h4>
<p>Spermatocyte depletion leads to testicular atrophy with a decrease in size. The spermatocyte depletion is involved in testicular atrophy and testicular toxicity [Chapin et al., 1984]. There are different insults that can induce spermatocyte depletion and consequently testicular atrophy.</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>Spermatocyte depletion caused by apoptosis leads to testicular atrophy. Apoptosis is a basic biological phenomenon in which the cells are controlled through the deletion and turnover in the atrophy of various tissues and organs as well as in tumor regression [Kerr et al., 1972].</p>
<h4>Key Event Relationship Description</h4>
<p style="margin-left:18.0pt">Apoptosis results in spermatocyte depletion via cell death. HDAC inhibitor, MAA, induced apoptosis and spermatocyte depletion at stages IX-II [Brinkworwth, 1995]. Induced apoptosis during development of germ cells results in progressive depletion of spermatocyte.</p>
<strong>Empirical Evidence</strong>
<p>2-methoxyethanol (ME) or its major metabolite, methoxyacetic acid (MAA), an HDAC inhibitor, induced spermatocyte depletion and testicular atrophy [Beattie et al., 1984].</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Uncertainties and Inconsistencies</strong>
<p>Spermatogonial stem cell self-renewal and spermatocyte meiosis are regulated by Sertoli cell signaling, which suggests that various pathways in spermatocytes or spermatogonia are involved in the spermatocyte deletion and testis atrophy/weight loss [Chen et al., 2015].</p>
<strong>Biological Plausibility</strong>
<p style="margin-left:18.0pt">In the mouse spermatocyte, spermatogenesis was inhibited by knockdown of Sucla2, a b subunit of succinyl coenzyme A synthase, via apoptosis [Huang, 2016]. The prolonged cryptorchidism leads to germs cell apoptosis and testicular sperm count decrease [Barqawi, 2004]. CD147 was reported to regulate apoptosis in mouse testis and spermatocyte cell line (GC-2 cells) via NFκB pathway [Wang, 2017].</p>
<strong>Empirical Evidence</strong>
<p style="margin-left:18.0pt">MicroRNA-21 regulates the spermatogonial stem cell homeostasis, in which suppression of microRNA-21 with anti-miR-21 oligonucleotides led to apoptosis of spermatogonial stem cell-enriched germ cell cultures and the decrease in the number of spermatogonial stem cells [Niu, 2011].</p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="margin-left:18.0pt">The process of apoptosis is necessary for the meitosis of the stem cell differentiation in the testis, which remains in question for the regulation of spermatocyte deletion and testis atrophy/weight loss [Dym, 1994].</p>
<p style="margin-left:49.55pt">Brinkworth M et al. (1995) Identification of male germ cells undergoing apoptosis in adult rats. J Reprod Fertil 105: 25-33</p>
<p style="margin-left:49.55pt">Huang S et al. (2016) Knockdown of Sucla2 decreases the viability of mouse spermatocytes by inducing apoptosis through injury of the mitochondrial function of cells. Folia Histochem Cytobiol 54: 134-142</p>
<p style="margin-left:49.55pt">Barqawi A et al. (2004) Effect of prolonged cryptorchidism on germ cell apoptosis and testicular sperm count. Asian J Androl 6: 47-51.</p>
<p style="margin-left:49.55pt">Wang C et al. (2017) CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways. Oncotarget 8: 3132-3143</p>
<p style="margin-left:49.55pt">Niu Z et al. (2011) microRNA-21 regulates the self-renewal of mouse spermatogonial stem cells. Proc Natl Acad Sci 108: 12740-12745</p>
<h4>References</h4>
<p>Abedi, N. et al. (2017), "Short and long term effects of different doses of paracetamol on sperm parameters and DNA integrity in mice", Middle East Fertility Society Journal 22:323-328</p>
<p style="margin-left:49.55pt">Dym M. (1994) Spermatogonial stem cells of the testis. Proc Natl Acad Sci USA 91: 11287-11289</p>
<p>Beattie, P.J. et al. (1984), "The effect of 2-methoxyethanol and methoxyacetic acid on Sertoli cell lactate production and protein synthesis in vitro", Toxicol Appl Pharmacol 76:56-61</p>
<p style="margin-left:49.55pt">Bose R et al. (2017) Ubiquitin ligase Huwe1 modulates spermatogenesis by regulating spermatogonial differentiation and entry into meiosis. Sci Rep 7: 17759</p>
<p>Chapin, R.E. et al. (1984), "The effects of ethylene glycol monomethyl ether on testicular histology in F344 rats", J Andro 5:369-380</p>
<p style="margin-left:49.55pt">Parajuli KR et al. (2014) Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis. Am J Clin Exp Urol 2: 300-313</p>
<p>Chen, S. and Liu, Y. (2015), "Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling", Reproduction 149:R159-R167</p>
<p style="margin-left:49.55pt">Glaser KB et al. (2003) Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines. Mol Cancer Ther 2:151-163</p>
<p>de Rooij, D.G. et al. (2001), "Proliferation and differentiation of spermatogonial stem cells", Reproduction 121:347-354</p>
<p style="margin-left:49.55pt">Zupkovitz G et al. (2010) The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation. Mol Cell Biol 30:1171-1181</p>
<p>de Rooij, D.G. (1998), "Stem cells in the testis", Int J Exp Path 79:67-80</p>
<p style="margin-left:49.55pt">Zindy F et al. (2001) Control of spermatogenesis in mice by the cyclin D-dependent kinase inhibitors p18<sup>Ink4c</sup> and p19<sup>Ink4d</sup>. Mol Cell Biol 21:3244-3255</p>
<p>Kerr, J.F.R. et al. (1972), "Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics", Br J Cancer 26:239-257</p>
<p style="margin-left:49.55pt">Oishi S. (2001) Effects of butylparaben on the male reproductive system in rats. Toxicol Indust Health 17:31-39</p>
<p>Lee, K.P. et al. (1993), "Testicular degeneration and spermatid retention in young male rats", Toxicol Pathol 21:292-302</p>
<p style="margin-left:49.55pt">Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831</p>
<p>Wiger, R. et al. (1995), "Effects of acetaminophen and hydroxyurea on spermatogenesis and sperm chromatin structure in laboratory mice", Reprod Toxicol 9:21-33</p>
</div>
<br>
<div>
<h4><a href="/relationships/1734">Relationship: 1734: spermatocyte depletion leads to testicular toxicity</a></h4>
<h3>List of Non Adjacent Key Event Relationships</h3>
<p style="margin-left:18.0pt">The relationship between spermatocyte depletion and testicular toxicity is likely well conserved between species.</p>
</div>
<p style="margin-left:18.0pt">MAA induced G<sub>1</sub> cell cycle arrest in human prostate cancer cells (<em>Homo sapiens</em>) [Parajuli et al., 2014].</p>
<ul>
<li>ME and MAA induced spermatocyte depletion and testicular atrophy in rat (<em>Rattus norvegicus</em>) [Beattie, 1984].</li>
<li>Ethylene glycol monomethyl ether induced depletion of late spermatocytes and testicular atrophy in F344 rat (<em>Rattus norvegicus</em>) [Chapin, 1984].</li>
<li>The epididymal tubules of rats with testicular degeneration had low sperm density (<em>Rattus norvegicus</em>) [Lee, 1993].</li>
<p style="margin-left:18.0pt">Spermatocyte depletion leads to testicular toxicity such as testicular atrophy with decrease in size. The spermatocyte depletion is involved in testicular atrophy and testicular toxicity [Chapin, 1984].</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>Spermatocyte depletion caused by apoptosis leads to the testicular toxicity. Apoptosis is a basic biological phenomenon in which the cells are controlled in the atrophy of various tissues and organs through the deletion and turnover, as well as in tumor regression [Kerr, 1972].</p>
<strong>Empirical Evidence</strong>
<p style="margin-left:18.0pt">2-methoxyethanol (ME) or its major metabolite, methoxyacetic acid (MAA), HDAC inhibitor, induced spermatocyte depletion and testicular atrophy [Beattie, 1984].</p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="margin-left:18.0pt">Spermatogonial stem cell self-renewal and spermatocyte meiosis are regulated by Sertoli cell signaling, which suggests us that various pathways in spermatocytes or spermatogonia are involved in the spermatocyte deletion and testis atrophy/weight loss [Chen, 2015].</p>
<p style="margin-left:18.0pt">Apicidin induced G<sub>1</sub> cell cycle arrest in HeLa cells (<em>Homo sapiens</em>) [Han et al., 2000].</p>
<p style="margin-left:18.0pt">The change in the amounts of cells in the G<sub>1</sub> phase and S phase of the cell cycle was detected in mouse HDAC1 knock-out fibroblast lines <em>(Mus musculus)</em> [Zupkovitz et al., 2010].</p>
<p style="margin-left:18.0pt">Loss of HDAC1 in mouse embryonic stem (ES) cells results in the acetylation of histones H3 and H4, up-regulation of cyclin-dependent kinase inhibitors p21<sup>WAF1/CIP1</sup> and p27<sup>KIP1</sup>, and inhibition of proliferation <em>(Mus musculus) </em>[Lagger et al., 2002].</p>
<h4>Key Event Relationship Description</h4>
<p>HDAC inhibition leads to cell cycle arrest including G<sub>1</sub>/S phase arrest [Falkenberg and Johnstone, 2014]. The HDAC inhibition-induced cell cycle arrest is mediated by transcriptional changes of the CDK inhibitors such as p21 [Falkenberg and Johnstone, 2014].</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The knockdown of HDACs may induce antitumor effects such as cell cycle arrest and inhibition of proliferation [Falkenberg and Johnstone, 2014]. In leukemia, an oncogenic fusion protein recruits a variety of proteins including HDACs to repress cell cycle inhibitors, which suggests that HDAC inhibition leads to cell cycle dysregulation [Falkenberg and Johnstone, 2014].</p>
<strong>Empirical Evidence</strong>
<ul>
<li>HDAC inhibition with SAHA, TSA, and MS-27-275 induced acetylation of histone H4, up-regulation of cyclin-dependent kinase inhibitor p21, and inhibition of proliferation in human bladder carcinoma cells [Glaser et al., 2003].</li>
<li>Apicidin [cyclo(<em>N</em>-<em>O</em>-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)], a fungal metabolite HDI, inhibits proliferation of tumor cells <em>via</em> p21 induction [Han et al., 2000]. Apicidin induced hyperacetylation of histone H4, up-regulation of p21, and G<sub>0</sub>/G<sub>1</sub> cell cycle arrest in HeLa cells [Han et al., 2000].</li>
<li>Falkenberg and Johnstone (2014) nicely reviewed that HDAC inhibition leads to cell cycle arrest in which G<sub>1</sub>/S phase arrest occurs <em>via</em> up-regulation of p21.</li>
<li>Loss of HDAC1 in mouse embryonic stem (ES) cells has demonstrated the acetylation of histones H3 and H4, up-regulation of cyclin-dependent kinase inhibitors p21<sup>WAF1/CIP1</sup> and p27<sup>KIP1</sup>, and inhibition of proliferation [Lagger et al., 2002].</li>
<li>G<sub>1</sub>/S transition blockade was observed in methoxyacetic acid (MAA)-treated prostate cancer cells [Parajuli et al., 2014].</li>
<li>The change in the amounts of cells in the G<sub>1</sub> phase and S phase of the cell cycle was detected in mouse HDAC1 knock-out fibroblast lines [Zupkovitz et al., 2010]. </li>
<li>MAA, an HDI, induced cell cycle arrest and up-regulation of p21 expression and inhibited prostate cancer cell growth [Parajuli et al., 2014].</li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p>The involvement of p53/p63/p73 in up-regulation of p21 induced by HDAC inhibition is not fully elucidated, where time course of the p21 and p53/p63/p73 mRNA expression has demonstrated the cell-line specific differences in the responses in 4 human prostate cancer cell lines LNCaP, C4-2B, PC-3 and DU-145 [Parajuli et al., 2014].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>MAA (20 mM) induced G<sub>1</sub> cell cycle arrest upon the treatment for 24 hrs in LNCaP, C4-2B, PC-3, and DU-145 human prostate cancer cell lines [Parajuli et al., 2014]. Almost 80% of the cells were arrested in the G<sub>1</sub> phase upon stimulation of MAA, whereas approximately 40 to 60 % of the cells were in the G<sub>1</sub> phase without MAA treatment [Parajuli et al., 2014].</p>
<p style="margin-left:18.0pt">MAA (5 mM) induced p21 up-regulation in 12 to 72 hrs in LNCaP, C4-2B, PC-3, and DU-145 human prostate cancer cell lines [Parajuli et al., 2014].</p>
<h4>References</h4>
<p style="margin-left:49.55pt">Chapin RE et al. (1984) The effects of ethylene glycol monomethyl ether on testicular histology in F344 rats. J Andro 5: 369-380</p>
<p style="margin-left:49.55pt">Kerr JFR et al. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257</p>
<h4>References</h4>
<p>Falkenberg, K.J. and Johnstone, R.W. (2014), "Histone deacetylases and their inhibitors in cancer, neurological disease and immune disorders", Nat Rev Drug Discov 13:673-691</p>
<p style="margin-left:49.55pt">Beattie PJ, et al. (1984) The effect of 2-methoxyethanol and methoxyacetic acid on Sertoli cell lactate production and protein synthesis in vitro. Toxicol Appl Pharmacol 76: 56-61</p>
<p>Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163</p>
<p style="margin-left:49.55pt">Chen S and Liu Y. (2015) Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling. Reproduction 149: R159-R167</p>
<p>Han, J.W. et al. (2000), "Apicidin, a histone deacetylase inhibitor, inhibits proliferation of tumor cells via induction of p21WAF1/Cip1 and gelsolin", Cancer Res 60:6068-6074</p>
<p style="margin-left:49.55pt">Abedi N et al. (2017) Short and long term effects of different doses of paracetamol on sperm parameters and DNA integrity in mice. Middle East Fertility Society Journal 22: 323-328</p>
<p>Lagger, G. et al. (2002), "Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression", EMBO J 21:2672-2681</p>
<p style="margin-left:49.55pt">Wiger R et al. (1995) Effects of acetaminophen and hydroxyurea on spermatogenesis and sperm chromatin structure in laboratory mice. Reprod Toxicol 9: 21-33</p>
<p>Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-312</p>
<p style="margin-left:49.55pt">De Rooij DG et al. (2001) Proliferation and differentiation of spermatogonial stem cells. Reproduction 121: 347-354</p>
<p style="margin-left:49.55pt">De Rooij DG. (1998) Stem cells in the testis. Int J Exp Path 79: 67-80</p>
<p style="margin-left:49.55pt">Lee KP et al. (1993) Testicular degeneration and spermatid retention in young male rats. Toxicol Pathol 21: 292-302</p>
<p>Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181</p>
</div>
<br>
<h3>List of Non Adjacent Key Event Relationships</h3>
<p style="margin-left:18.0pt">MAA induced G<sub>1</sub> cell cycle arrest in human prostate cancer cells (<em>Homo sapiens</em>) [Parajuli, 2014]. Apicidin induced G<sub>1</sub> cell cycle arrest in HeLa cells (<em>Homo sapiens</em>) [Han, 2000].</p>
<h4>Key Event Relationship Description</h4>
<p style="margin-left:18.0pt">HDAC inhibition leads to cell cycle arrest including G<sub>1</sub>/S phase arrest [Falkenberg, 2014]. The HDAC inhibition-induced cell cycle arrest is the mediated by transcriptional changes of the CDK inhibitors such as p21 [Falkenberg, 2014].</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
</div>
<p>・AR-42 inhibited proliferation of human pancreatic cancer cells (<em>Homo sapiens</em>) [Henderson et al., 2016].</p>
<p>・MAA induced apoptosis in human prostate cancer cell lines. The apoptosis and proliferation inhibition induced by MAA, an HDAC inhibitor, was measured in human prostate cancer cell lines (<em>Homo sapiens</em>) [Parajuli et al., 2014].</p>
<p>・SAHA or TSA, which are HDAC inhibitors, reduced cell viability in NHDFs (<em>Homo sapiens</em>) [Glaser et al., 2003].</p>
<p>・The proliferation of the HDAC<sup>-/-</sup> ES cells was inhibited compared to HDAC<sup>+/+</sup> ES cells (<em>Homo sapiens</em>) [Zupkovitz et al., 2010].</p>
<strong>Biological Plausibility</strong>
<p style="margin-left:18.0pt">The knockdown of HDACs may induce antitumor effects such as cell cycle arrest and inhibition of proliferation [Falkenberg, 2014]. In leukemia, acute myloid leukaemia 1-ETO, oncogenic fusion protein, recruits the variety of the proteins including HDACs to form multiprotein complexes to repress the cell cycle inhibitors, which suggests that the HDAC inhibition leads to cell cycle dysregulation [Falkenberg, 2014].</p>
<h4>Key Event Relationship Description</h4>
<p>HDAC inhibition leads to cell death through the apoptotic pathways [Falkenberg and Johnstone, 2014]. The intrinsic apoptosis pathway requires BH3-only proteins, and BCL-2 protein overexpression inhibits apoptosis [Falkenberg and Johnstone, 2014]. Administration of methoxyacetic acid (MAA), an HDAC inhibitor, causes apoptosis with DNA ladder in male germ cells [Brinkworth et al., 1995]. MAA induces the apoptosis of spermatocytes at spermatogenic cycle stage IX-II [Brinkworth et al., 1995].</p>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>HDAC inhibition in cancer results in apoptosis with the up-regulation of pro-apoptotic B cell lymphoma 2 (BCL-2) family genes and down-regulation of pro-survival BCL-2 genes [Falkenberg, 2014]. The antitumor effect of HDAC inhibition includes cell death and apoptosis [Falkenberg and Johnstone, 2014].</p>
<strong>Empirical Evidence</strong>
<ul>
<li>HDAC inhibition with SAHA, TSA and MS-27-275 induced acetylation of histone H4, up-regulation of cyclin-dependent kinase inhibitor p21, and inhibition of proliferation in human bladder carcinoma cells [Glaser, 2003].</li>
<li>Apicidin [cyclo(<em>N</em>-<em>O</em>-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)], a fungal metabolite HDI, inhibits proliferation of tumor cells via p21 induction [Han, 2000]. Apicidin induced hyperacetylation of histone H4, up-regulation of p21, and G<sub>0</sub>/G<sub>1</sub> cell cycle arrest in HeLa cells [Han, 2000].</li>
<li>HDAC inhibition leads to cell cycle arrest, where G<sub>1</sub>/S phase arrest occurs via up-regulation of p21 [Falkenberg, 2014].</li>
<li>Loss of HDAC1 in mouse embryonic stem (ES) cells has demonstrated the acetylation of histones H3 and H4, up-regulation of cyclin-dependent kinase inhibitors p21<sup>WAF1/CIP1</sup> and p27<sup>KIP1</sup> and inhibition of proliferation [Lagger, 2002].</li>
<li>G<sub>1</sub>/S transition blockade was observed in MAA-treated prostate cancer cells [Parajuli, 2014].</li>
<strong>Empirical Evidence</strong>
<ul>
<li>MAA-induced spermatocyte death is associated with histone acetylation increase [Wade et al., 2008].</li>
<li>The HDAC inhibition induced apoptosis markers such as BAK overexpression and suppression of phosphorylated AKT [Henderson et al., 2016].</li>
<li>The administration of MAA can cause apoptosis in the germ cells of adult male rats [Brinkworth et al., 1995].</li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p>It is uncertain through which pathway the HDAC inhibition induces apoptosis.</p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="margin-left:18.0pt">MAA, a HDI, induced cell cycle arrest and up-regulation of p21 expression, and inhibited prostate cancer cell growth [Parajuli, 2014]. The involvement of p53/p63/p73 in up-regulation of p21 induced by HDAC inhibition is not fully elucidated, where time course of the p21 and p53/p63/p73 mRNA expression has demonstrated the cell-line specific differences in the responses in 4 human prostate cancer cell lines LNCaP, C4-2B, PC-3 and DU-145 [Parajuli, 2014].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>MAA (20 mM) induced G<sub>1</sub> cell cycle arrest upon the treatment for 24 hrs in LNCaP, C4-2B, PC-3 and DU-145 human prostate cancer cell lines [Parajuli, 2014]. Almost 80% of the cells were arrested in G<sub>1</sub> phase upon stimulation of MAA, whereas approximately 40 to 60 % of the cells were in G<sub>1</sub> phase without MAA treatment [Parajuli, 2014].</p>
<strong>Time-scale</strong>
<p style="margin-left:18.0pt">MAA (5 mM) induced p21 up-regulation in 12 to 72 hrs in LNCaP, C4-2B, PC-3 and DU-145 human prostate cancer cell lines [Parajuli, 2014].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>MAA (5 mM) induced apoptosis in prostate cancer cell lines, LNCaP, C4-2B, PC-3, and DU-145, in which apoptotic nucleosomes were calculated as absorbance at 405 nm – absorbance at 490 nm [Parajuli et al., 2014].</p>
<strong>Time-scale</strong>
<p style="margin-left:18.0pt">MAA (5 mM) decreased protein expression of BIRC2 and activated caspases 7 and 3 within 72 hrs [Parajuli et al., 2014].</p>
<p style="margin-left:49.55pt">Falkenberg KJ and Johnstone RW. (2014) Histone deacetylases and their inhibitors in cancer, neurological disease and immune disorders. Nat Rev Drug Discov 13:673-691</p>
<h4>References</h4>
<p>Brinkworth, M.H. et al. (1995), "Identification of male germ cells undergoing apoptosis in adult rats", J Reprod Fertil 105:25-33</p>
<p style="margin-left:49.55pt">Glaser KB et al. (2003) Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines. Mol Cancer Ther 2:151-163</p>
<p>Falkenberg, K.J. and Johnstone, R.W. (2014), "Histone deacetylases and their inhibitors in cancer, neurological disease and immune disorders", Nat Rev Drug Discov 13:673-691</p>
<p style="margin-left:49.55pt">Han JW et al. (2000) Apicidin, a histone deacetylase inhibitor, inhibits proliferation of tumor cells via induction of p21<sup>WAF1/Cip1</sup> and gelsolin. Cancer Res 60:6068-6074</p>
<p>Glaser, K.B. et al. (2003), "Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines", Mol Cancer Ther 2:151-163</p>
<p style="margin-left:49.55pt">Lagger G et al. (2002) Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J 21:2672-2681</p>
<p>Henderson, S.E. et al. (2016), "Suppression of tumor growth and muscle wasting in a transgenic mouse model of pancreatic cancer by the novel histone deacetylase inhibitor AR-42", Neoplasia 18:765-774</p>
<p style="margin-left:49.55pt">Parajuli KR et al. (2014)Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis. Am J Clin Exp Urol 2:300-312</p>
<p>Parajuli, K.R. et al. (2014), "Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis", Am J Clin Exp Urol 2:300-312</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p>Zupkovitz, G. et al. (2010), "The cyclin-dependent kinase inhibitor p21 is a crucial target for histone deacetylase 1 as a regulator of cellular proliferation", Mol Cell Biol 30:1171-1181</p>
</div>
<br>
<div>
<h4><a href="/relationships/1716">Relationship: 1716: Histone deacetylase inhibition leads to Apoptosis</a></h4>
<div>
<h4><a href="/relationships/2010">Relationship: 2010: Histone deacetylase inhibition leads to Spermatocyte depletion</a></h4>
<p style="margin-left:18.0pt">AR-42 inhibited proliferation of human pancreatic cancer cells (<em>Homo sapiens</em>) [Henderson, 2016]. SAHA inhibited proliferation of NHDF (<em>Homo sapiens</em>) [Glaser, 2003]. MAA induced apoptosis in human prostate cancer cell lines (<em>Homo sapiens</em>) [Parajuli, 2014]. HDAC-deficient mouse ES cells showed decrease in proliferation (<em>Mus musculus</em>) [Lagger, 2002].</p>
<h4>Key Event Relationship Description</h4>
<p style="margin-left:18.0pt">HDAC inhibition leads to cell death through the apoptotic pathways [Falkenberg, 2014]. Intrinsic apoptosis pathway requires BH3-only proteins, and BCL-2 protein overexpression inhibits apoptosis [Falkenberg, 2014].</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p style="margin-left:18.0pt">HDAC inhibition in cancer results in apoptosis with the up-regulation of pro-apoptotic B cell lymphoma 2 (BCL-2) family genes and down- regulation of pro-survival BCL-2 genes [Falkenberg, 2014]. The antitumor effect of HDAC inhibition includes cell death and apoptosis [Falkenberg, 2014].</p>
<strong>Empirical Evidence</strong>
<ul>
<li>HDAC-deficient mouse embryonic stem (ES) cells showed reduced proliferation rates with up-regulation of cyclin-dependent kinase inhibitors p21 and p27 [Lagger, 2002]. HDAC-null embryoid bodies showed a reduced inner cell mass and reduced colony formation [Lagger, 2002].</li>
<li>HDAC inhibition by suberoylanilide hydroxamic acid (SAHA) inhibited proliferation of normal human dermal fibroblasts (NHDF) [Glaser, 2003].</li>
<li>MAA-induced spermatocyte death is associated with histone acetylation increase [Wade, 2008].</li>
<li>The HDAC inhibition induced p21 up-regulation, histone acetylation increase, and apoptosis markers such as BAK overexpression and suppression of phosphorylated AKT [Henderson, 2016].</li>
<li>The administration of methoxyacetic acid can cause apoptosis in the germ cells of adult male rats [Brinkworth, 1995].</li>
</ul>
<h4>Quantitative Understanding of the Linkage</h4>
<p>MAA (5 mM) induced apoptosis in prostate cancer cell lines, LNCaP, C4-2B, PC-3 and DU-145, in which apoptotic nucleosomes were calculated as absorbance at 405 nm – absorbance at 490 nm [Parajuli, 2014].</p>
</div>
<p>・Histone deacetylase inhibition by histone deacetylase inhibitors caused spermatocyte death in rats. MAA treatment induced spermatocyte death in Sprague-Dawley rats (<em>Rattus norvegicus</em>) [Wade et al., 2008].</p>
<p>・VPA exposure caused a decrease in sperm count in humans (<em>Homo sapiens</em>) [Yerby and McCoy, 1999; Kose-Ozlece et al., 2015].</p>
<p> </p>
<h4>Key Event Relationship Description</h4>
<p>Histone deacetylase inhibition triggered by histone deacetylase inhibitors such as methoxyacetic acid (MAA) leads to spermatocyte death causing spermatocyte depletion [Wade et al., 2008]. Histone deacetylase inhibition leads to an increase in histone acetylation, leading to spermatocyte apoptosis.</p>
<strong>Time-scale</strong>
<p style="margin-left:18.0pt">MAA (5 mM) decreased protein expression of BIRC2 and activated caspases 7 and 3 within 72 hrs [Parajuli, 2014].</p>
<h4>Evidence Supporting this KER</h4>
<p>MAA administration induces spermatocyte deaths, which has been revealed by section staining of the germ cell death [Wade et al., 2008].</p>
<strong>Biological Plausibility</strong>
<p>Histone deacetylase inhibition causes histone acetylation, which increases the gene expression of cell-cyle-related proteins, followed by spermatocyte apoptosis in testis.</p>
<strong>Empirical Evidence</strong>
<p>Administration of MAA in rats, a histone deacetylase inhibitor (HDI), demonstrated the emergence of TUNEL-positive spermatocytes, which indicates spermatocyte apoptosis [Wade et al., 2008]. Treatment of valproate (VPA) resulted in a decline in the sperm count [Yerby and McCoy, 1999; Kose-Ozlece et al., 2015].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>The administration of MAA in rats induced spermatocyte depletion which was confirmed with TUNEL-staining of the germ cells [Wade et al., 2008].</p>
<p>TUNEL-positive germ cells were increased after 8, 12, and 24 hrs of MAA exposure (650 mg/kg i.p.) in the rats [Wade et al., 2008]. TUNEL-positive zygotene spermatocytes have emerged after 12 hrs of MAA exposure in the rats, which was confirmed by the section staining [Wade et al., 2008]. </p>
<h4>References</h4>
<p>Falkenberg KJ and Johnstone RW. (2014) Histone deacetylases and their inhibitors in cancer, neurological disease and immune disorders. Nat Rev Drug Discov 13:673-691</p>
<p>Lagger G et al. (2002) Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J 21:2672-2681</p>
<h4>References</h4>
<p>Kose-Ozlece, H. et al. (2015), "Alterations in semen parameters in men wıth epilepsy treated with valproate", Iran J Neurol 14:164-167</p>
<p>Glaser KB et al. (2003) Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines. Mol Cancer Ther 2:151-163</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p>Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831</p>
<p>Yerby, M.S. and McCoy, G.B. (1999), "Male infertility: Possible association with valproate exposure", Epilepsia 40:520-521</p>
<p>Henderson SE et al. (2016) Suppression of tumor growth and muscle wasting in a transgenic mouse model of pancreatic cancer by the novel histone deacetylase inhibitor AR-42. Neoplasia 18:765-774</p>
<p> </p>
<p>Brinkworth MH et al. (1995) Identification of male germ cells undergoing apoptosis in adult rats. J Reprod Fertil 105:25-33.</p>
<p> </p>
<p>Parajuli KR et al. (2014) Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis. Am J Clin Exp Urol 2:300-312</p>
<p> </p>
<p> </p>
</div>
<br>
<div>
<h4><a href="/relationships/1717">Relationship: 1717: Histone deacetylase inhibition leads to testicular toxicity</a></h4>
<div>
<h4><a href="/relationships/1717">Relationship: 1717: Histone deacetylase inhibition leads to Testicular atrophy</a></h4>
<p style="margin-left:18.0pt">The administration of di(2-ethylhexyl)-phthalate induced testis atrophy in rats (<em>Rattus norvegicus</em>) [Oishi, 1994]. The administration of butylparaben resulted in decrease in sperm counts in rats (<em>Rattus norvegicus</em>) [Oishi, 2001]. MAA induced spermatocyte apoptosis in human testes (<em>Homo sapiens</em>) [Li, 1996].</p>
<h4>Key Event Relationship Description</h4>
<p style="margin-left:18.0pt">HDAC inhibition induced testicular toxicity including testis atrophy such as the decrease in size [Miller, 1982]. HDAC inhibition in cell culture resulted in the testicular toxicity including germ cell apoptosis and cell morphology change [Li, 1996]. Valproic acid, a HDAC inhibitor, caused a reduced testicular weight in the offspring in rats [Kallen, 2004].</p>
<!-- if nothing shows up in any of these fields, then evidence supporting this KER will not be displayed -->
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The HDAC inhibition induced cell death in spermatocytes in both rat and human seminiferous tubules [Li, 1996]. The HDAC inhibitor treatment resulted in degeneration in spermatocytes in rat seminiferous tubules [Li, 1996]. The HDAC inhibition induced the germ cell apoptosis in human testicular tissues [Li, 1996].</p>
</div>
<p>MAA induced spermatocyte apoptosis and cell morphology change in human testes (<em>Homo sapiens</em>) [Li et al., 1996].</p>
<p>Valproic acid caused the decrease in rat testicular weight (<em>Rattus norvegicus</em>) [Kallen, 2004].</p>
<p style="margin-left:18.0pt"> </p>
<h4>Key Event Relationship Description</h4>
<p>HDAC inhibition induced testicular toxicity including testis atrophy such as the decrease in size [Miller et al., 1982]. HDAC inhibition in cell culture resulted in testicular toxicity including germ cell apoptosis and cell morphology change [Li et al., 1996]. Valproic acid, an HDAC inhibitor, caused a reduced testicular weight in the offspring in rats [Kallen, 2004].</p>
<strong>Empirical Evidence</strong>
<ul>
<li>HDAC inhibitor, methoxyacetic acid (MAA), (300 mg/kg) induced testicular toxicity measured with testis weight loss [Miller, 1982].</li>
<li>MAA induced apoptosis and degeneration in spermatocytes in human testicular tissue and 25-day rat seminiferous tubule cultures [Li, 1996].</li>
<li>MAA-induced spermatocyte death with an association of histone acetylation increase [Wade, 2008].</li>
<li>Doxorubicin, which has a testicular toxicity, induced caspase 3 activation and g-H2AX induction, apoptosis markers, in human lung cancer A549 cells [El-Awady, 2015, Yamazoe, 2015].</li>
<li>Doxorubicin-resistant A549 cells showed reduced expression of HDAC1, 3 and 4 compared to A549 cells [El-Awady, 2015].</li>
<li>MAA-induced apoptosis in male germ cells was modulated by Sertoli cells via P/Q type voltage-operated calcium channels [Barone, 2005].</li>
<li>The <em>p.o.</em> administration of ethylene glycol monomethyl (500 mg/kg/day) in rats induced the testis or liver organ weight loss on 2, 4, 7 and 11 days or 24 hrs and 2, 4 and 7 days after treatment, respectively [Foster, 1983].</li>
<li>The investigation of 2-methoxyethanol (2-ME)-induced testicular toxicity has revealed that the conversion of 2-ME to MAA is required in 2-ME-induced testicular toxicity [Moss, 1985].</li>
<li>The exposure of MAA induced morphological changes on embryonic forelimbs [Dayan, 2014].</li>
<h4>Evidence Supporting this KER</h4>
<strong>Biological Plausibility</strong>
<p>The HDAC inhibition induced cell death in spermatocytes in both rat and human seminiferous tubules [Li et al., 1996]. The HDAC inhibitor treatment resulted in degeneration in spermatocytes in rat seminiferous tubules [Li et al., 1996]. The HDAC inhibition induced germ cell apoptosis in human testicular tissues [Li et al., 1996].</p>
<strong>Empirical Evidence</strong>
<ul>
<li>HDAC inhibitor, methoxyacetic acid (MAA), (300 mg/kg) induced testicular toxicity measured with testis weight loss [Miller et al., 1982].</li>
<li>MAA induced apoptosis and degeneration in spermatocytes in human testicular tissue and 25-day rat seminiferous tubule cultures [Li et al., 1996].</li>
<li>MAA-induced spermatocyte death with an association of histone acetylation increase [Wade et al., 2008].</li>
<li>MAA-induced apoptosis in male germ cells was modulated by Sertoli cells <em>via</em> P/Q type voltage-operated calcium channels [Barone et al., 2005].</li>
<li>The <em>p.o.</em> administration of ethylene glycol monomethyl (500 mg/kg/day) in rats induced the testis or liver organ weight loss on 2, 4, 7, and 11 days or 24 hrs and 2, 4, and 7 days after treatment, respectively [Foster et al., 1983].</li>
<li>The investigation of 2-methoxyethanol (2-ME)-induced testicular toxicity has revealed that the conversion of 2-ME to MAA is required in 2-ME-induced testicular toxicity [Moss et al., 1985].</li>
</ul>
<strong>Uncertainties and Inconsistencies</strong>
<p>It is reported that HDAC inhibition leads to teratogenic toxicity, whereas the correlation between testicular toxicity and teratogenic toxicity by HDAC inhibition is not fully understood [Menegola et al., 2006]. The oral administration of vorinostat (SAHA), an HDAC inhibitor, in Sprague-Dawley rats showed no indication of reproductive toxicity in drug-treated male rats, which suggested the involvement of some compensation mechanisms or digestion [Wise et al., 2008]. Some studies have demonstrated that the decrease in histone acetylation in spermatids is associated with impaired spermatogenesis corresponding with the well-known reduction of protamine expression in the cells [Sonnack et al., 2002; Li et al., 2014]. It has also been reported that the histological examination of sections revealed no difference between wild-type and HDAC6-deficient testes [Zhang et al., 2008].</p>
<strong>Uncertainties and Inconsistencies</strong>
<p style="margin-left:18.0pt">Methyl and ethyl esters of <em>p</em>-hydroxybenzoic acid did not show spermatotoxic effects in rats (<em>Rattus norvegicus</em>) [Oishi, 2004]. It is reported that HDAC inhibition leads to teratogenic toxicity, whereas the correlation with testicular toxicity and teratogenic toxicity by HDAC inhibition is not fully understood [Menegola, 2006]. The oral administration of vorinostat (SAHA), a HDAC inhibitor, in Sprague-Dawley rats showed no indication of reproductive toxicity in drug-treated male rats, which suggested the involvement of some compensation mechanisms or digestion [Wise, 2008].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p style="margin-left:18.0pt">MAA administration (592 mg/kg/day) for 4 days showed testis weight loss in which the relative organ weights were 0.773 ± 0.022 g/100 g body weight, compared to 0.985 ± 0.028 g/100g body weight in control treated with water [Foster, 1984].</p>
<h4>Quantitative Understanding of the Linkage</h4>
<p>MAA administration (592 mg/kg/day) for 4 days showed testis weight loss in which the relative organ weights were 0.773 ± 0.022 g/100 g body weight, compared to 0.985 ± 0.028 g/100g body weight in control treated with water [Foster et al., 1984].</p>
<strong>Time-scale</strong>
<p style="margin-left:18.0pt">The relative testicular weight was decreased at day 2 after the treatment of 500 mg/kg/day treatment of ethylene glycol monomethyl ether [Foster et al., 1984]. The treatment of 5 mM MAA for 5 hrs induced the pachytene spermatocyte death in early-stage tubules in 19 hrs [Li et al., 1996]. The degeneration in late spermatocytes was observed in late-stage tubules in 19 hrs after 5 mM MAA treatment for 5 hrs [Li et al., 1996].</p>
<strong>Time-scale</strong>
<p style="margin-left:18.0pt">The relative testicular weight was decreased at day 2 after the treatment of 500 mg/kg/day treatment of ethylene glycol monomethyl ether [Foster, 1984]. The treatment of 5 mM MAA for 5 hrs induced the pachytene spermatocyte death in early stage tubules in 19 hrs [Li, 1996]. The degeneration in late spermatocytes was observed in late-stage tubules in 19 hrs after 5 mM MAA treatment for 5 hrs [Li, 1996].</p>
<p style="margin-left:49.55pt">Miller RR et al. (1982) Toxicity of methoxyacetic acid in rats. Fundam Appl Toxicol 2: 158-160</p>
<p style="margin-left:49.55pt">Li LH et al. (1996) 2-Methoxyacetic acid (MAA)-induced spermatocyte apoptosis in human and rat testes: an in vitro comparison. J Androl 17: 538-549</p>
<h4>References</h4>
<p>Barone, F. et al. (2005), "Modulation of MAA-induced apoptosis in male germ cells: role of Sertoli cell P/Q-type calcium channels", Reprod Biol Endocrinol 3:13</p>
<p style="margin-left:49.55pt">Kallen B (2004) Valproic acid is known to cause hypospadias in man but does not reduce anogenital distance or causes hypospadias in rats. Basic Clin Pharmacol Toxicol 94: 51-54</p>
<p>Foster, P.M. et al. (1983), "Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat", Toxicol Appl Pharmacol 69:385-399</p>
<p style="margin-left:49.55pt">Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831</p>
<p>Foster, P.M. et al. (1984), "Testicular toxicity produced by ethylene glycol monomethyl and monoethyl esters in the rat", Environ Health Perspect 57:207-217</p>
<p style="margin-left:49.55pt">El-Awady RA et al. (2015) Epigenetics and miRNA as predictive markers and targets for lung cancer chemotherapy. Cancer Biol Ther 16: 1056-1070</p>
<p>Kallen, B. (2004), "Valproic acid is known to cause hypospadias in man but does not reduce anogenital distance or causes hypospadias in rats", Basic Clin Pharmacol Toxicol 94:51-54</p>
<p style="margin-left:49.55pt">Yamazoe Y. et al. (2015) Embryo- and testicular-toxicities of methoxyacetate and the related: a review on possible roles of one-carbon transfer and histone modification. Food Safety 3:92-107</p>
<p>Li, L.H. et al. (1996), "2-Methoxyacetic acid (MAA)-induced spermatocyte apoptosis in human and rat testes: an in vitro comparison", J Androl 17:538-549</p>
<p style="margin-left:49.55pt">Barone F. et al. (2005) Modulation of MAA-induced apoptosis in male germ cells: role of Sertoli cell P/Q-type calcium channels. Reprod Biol Endocrinol 3:13</p>
<p>Li, W. et al. (2014), "Chd5 orchestrates chromatin remodeling during sperm development", Nat Commun 5:3812</p>
<p style="margin-left:49.55pt">Foster PM et al. (1983) Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat. Toxicol Appl Pharmacol 69:385-399</p>
<p>Menegola, E. et al. (2006), "Inhibition of histone deacetylase as a new mechanism of teratogensis", Birth Defects Res 78:345-353</p>
<p style="margin-left:49.55pt">Moss EJ et al. (1985) The role of metabolism in 2-methoxyethanol-induced testicular toxicity. Toxicol Appl Pharmacol 79:480-489</p>
<p>Miller, R.R. et al. (1982), "Toxicity of methoxyacetic acid in rats", Fundam Appl Toxicol 2:158-160</p>
<p style="margin-left:49.55pt">Dayan C and Hales BF. (2014) Effects of ethylene glycol monomethyl ether and its metabolite, 2-methoxyacetic acid, on organogenesis stage mouse limbs in vitro. Birth Defects Res (Part B) 101:254-261</p>
<p>Moss, E.J. et al. (1985), "The role of metabolism in 2-methoxyethanol-induced testicular toxicity", Toxicol Appl Pharmacol 79:480-489</p>
<p style="margin-left:49.55pt">Oishi S. (2004) Lack of spermatotoxic effects of methyl and ethyl esters of p-hydroxybenzoic acid in rats. Food Chem Tox 42: 1845-1849</p>
<p>Sonnack, V. et al. (2002), "Expression of hyperacetylated histone H4 during normal and impaired human spermatogenesis", Andrologia. 34:384-390</p>
<p style="margin-left:49.55pt">Menegola E et al. (2006) Inhibition of histone deacetylase as a new mechanism of teratogensis. Birth Defects Res 78: 345-353</p>
<p>Wade, M.G. et al. (2008), "Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats", Biol Reprod 78:822-831</p>
<p style="margin-left:49.55pt">Wise LD et al. (2008)Assessment of female and male fertileity in Sprague-Dawley rats administered vorinostat, a histone deacetylase inhibitor. Birth Defects Res B Dev Reprod Toxicol 83:19-26</p>
<p>Wise, L.D. et al. (2008), "Assessment of female and male fertility in Sprague-Dawley rats administered vorinostat, a histone deacetylase inhibitor", Birth Defects Res B Dev Reprod Toxicol 83:19-26</p>
<p style="margin-left:49.55pt">Foster PM et al. (1984) Testicular toxicity produced by ethylene glycol monomethyl and monoethyl esters in the rat. Environ Health Perspect 57: 207-217</p>
<p style="margin-left:49.55pt">Oishi S. (1994) Prevention of Di(2-ethylhexyl)phthalate-induced testicular atrophy in rats by co-administration of the vitamin B12 derivative denosylcobalamin. Arch Environ Contam Toxicol 26: 497-503</p>
<p style="margin-left:49.55pt">Oishi S. (2001) Effects of butylparaben on the male reproductive system in rats. Tox Industr Health 17: 31-39</p>
<p>Zhang, Y. et al. (2008), "Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally", Mol Cel Biol 28:1688-1701</p>