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Aop: 212

AOP Title

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Histone deacetylase inhibition leading to testicular toxicity

Short name:

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Histone deacetylase inhibition leading to testicular toxicity

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Authors

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Shihori Tanabe, Akihiko Hirose, Takashi Yamada

Division of Risk Assessment, Biological Safety Research Center, National Institute of Health Sciences

Point of Contact

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Takashi Yamada   (email point of contact)

Contributors

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  • Takashi Yamada
  • Shihori Tanabe

Status

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Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite Under Development 1.52 Included in OECD Work Plan


This AOP was last modified on June 06, 2018 05:45

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Revision dates for related pages

Page Revision Date/Time
Histone deacetylase inhibition June 10, 2018 22:43
Histone acetylation, increase June 06, 2018 06:46
p21 (CDKN1A) expression, increase June 10, 2018 22:45
cell cycle, disrupted June 10, 2018 22:47
Apoptosis June 10, 2018 22:52
testicular toxicity June 06, 2018 06:56
spermatocyte depletion June 06, 2018 06:54
Histone deacetylase inhibition leads to Histone acetylation, increase June 10, 2018 22:08
Histone acetylation, increase leads to p21 (CDKN1A) expression, increase June 10, 2018 22:21
p21 (CDKN1A) expression, increase leads to cell cycle, disrupted June 10, 2018 22:34
cell cycle, disrupted leads to Apoptosis June 10, 2018 22:36
Histone deacetylase inhibition leads to p21 (CDKN1A) expression, increase June 10, 2018 23:04
Histone deacetylase inhibition leads to cell cycle, disrupted June 10, 2018 23:05
Apoptosis leads to spermatocyte depletion June 10, 2018 23:00
Histone deacetylase inhibition leads to Apoptosis June 06, 2018 06:33
spermatocyte depletion leads to testicular toxicity June 01, 2018 04:16
Histone deacetylase inhibition leads to testicular toxicity June 06, 2018 06:39
Methoxyacetic acid January 21, 2018 20:38
Butyrate January 21, 2018 20:39
Trichostatin A January 21, 2018 20:39

Abstract

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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 short chain 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 in vivo. 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.

 

Abbreviation: HDAC: histone deacetylase, HDI: HDAC inhibitor, SAHA: syberooylanilide hydroxamic acid, TSA: trichostatin A, MAA: methoxyacetic acid, MIE: molecular initiating event, KE: key event, AOP: adverse outcome pathway


Background (optional)

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Summary of the AOP

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Events: Molecular Initiating Events (MIE)

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Key Events (KE)

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Adverse Outcomes (AO)

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Sequence Type Event ID Title Short name
1 MIE 1502 Histone deacetylase inhibition Histone deacetylase inhibition
2 KE 1503 Histone acetylation, increase Histone acetylation, increase
3 KE 1504 p21 (CDKN1A) expression, increase p21 (CDKN1A) expression, increase
4 KE 1505 cell cycle, disrupted cell cycle, disrupted
5 KE 1262 Apoptosis Apoptosis
6 KE 1515 spermatocyte depletion spermatocyte depletion
7 AO 1506 testicular toxicity testicular toxicity

Relationships Between Two Key Events
(Including MIEs and AOs)

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Title Adjacency Evidence Quantitative Understanding
Histone deacetylase inhibition leads to Histone acetylation, increase adjacent High High
Histone acetylation, increase leads to p21 (CDKN1A) expression, increase adjacent Moderate Moderate
p21 (CDKN1A) expression, increase leads to cell cycle, disrupted adjacent High Moderate
cell cycle, disrupted leads to Apoptosis adjacent High Moderate
Apoptosis leads to spermatocyte depletion adjacent High Not Specified
spermatocyte depletion leads to testicular toxicity adjacent High Not Specified
Histone deacetylase inhibition leads to p21 (CDKN1A) expression, increase non-adjacent High High
Histone deacetylase inhibition leads to cell cycle, disrupted non-adjacent High High
Histone deacetylase inhibition leads to Apoptosis non-adjacent High High
Histone deacetylase inhibition leads to testicular toxicity non-adjacent High High

Network View

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Stressors

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Name Evidence Term
Methoxyacetic acid High
Butyrate High
Trichostatin A High

Life Stage Applicability

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Life stage Evidence
Adult, reproductively mature High

Taxonomic Applicability

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Term Scientific Term Evidence Link
human Homo sapiens Moderate NCBI
mouse Mus musculus Moderate NCBI
rat Rattus norvegicus High NCBI

Sex Applicability

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Sex Evidence
Male High

Overall Assessment of the AOP

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Attached file: Overallassessmentaop212

1. Support for Biological Plausibility of KERs

MIE => KE1: Histone deacetylase inhibition leads to histone acetylation increase

Biological Plausibility of the MIE => KE1 is high.
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. Activity of histone acetyltransferase (HAT) in testis nuclear protein was increased with MAA addition (Wade, 2008).

KE1 => KE2: Histone acetylation, increase leads to p21 (CDKN1A) expression, increase

Biological Plausibility of the KE1 => KE2 is moderate.
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).

KE2 => KE3: p21 (CDKN1A) expression, increase leads to cell cycle, disrupted

Biological Plausibility of the KE2 => KE3 is high.
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).

KE3 => KE4: Cell cycle disrupted leads to apoptosis

Biological Plausibility of the KE3 => KE4 is high.
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 G1 arrest (Li, 2012), and deregulated CCND2 is implicated in cell proliferation inhibition (Wu, 2016, Mermelstein, 2005, Dong, 2010).

KE4 => KE5: Apoptosis leads to spermatocyte depletion

Biological Plausibility of the KE4 => KE5 is high.
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).

KE5 => AO: Spermatocyte depletion leads to testicular toxicity

Biological Plausibility of the KE5 => AO is high.
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].

2. Support for essentiality of KEs

KE4: Apoptosis

Essentiality of the KE4 is moderate.
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).

3. Empirical support for KERs

MIE => KE1: Histone deacetylase inhibition leads to histone acetylation increase

Empirical Support of the MIE => KE1 is high.
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 in silico determination of modified sites on any protein on the basis of the primary sequence (Gnad, 2011).

KE1 => KE2: Histone acetylation, increase leads to p21 (CDKN1A) expression, increase

Empirical Support of the KE1 => KE2 is moderate.
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).

KE2 => KE3: p21 (CDKN1A) expression, increase leads to cell cycle, disrupted

Empirical Support of the KE2 => KE3 is high.
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).

KE3 => KE4: Cell cycle disrupted leads to apoptosis

Empirical Support of the KE3 => KE4 is high.
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).

KE4 => KE5: Apoptosis leads to spermatocyte depletion

Empirical Support of the KE4 => KE5 is high.
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).

KE5 => AO: Spermatocyte depletion leads to testicular toxicity

Empirical Support of the KE5 => AO is high.
Rationale: 2-methoxyethanol (ME) or its major metabolite, MAA induced spermatocyte depletion and testicular atrophy [Beattie, 1984].

Domain of Applicability

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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.


Essentiality of the Key Events

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Key Event

Direct/Indirect Evidence

MIE: histone deacetylase inhibition

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].

KE1: Histone acetylation, increase

The HDAC inhibition induced cell death in spermatocytes in both rat and human seminiferous tubules [Li, 1996].

KE2: p21 (CDKN1A) expression, increase

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).

KE3: Cell cycle, disrupted

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].

KE4: Apoptosis

HDAC inhibition leads to cell death through the apoptotic pathways (Falkenberg, 2014).

KE5: spermatocyte depletion

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].


Evidence Assessment

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Biological plausibility, coherence, and consistency of the experimental evidence

The available data supporting the AOP are logic, coherent and consistent with established biological knowledge, whereas there are possibilities for alternative pathways.

 

Alternative mechanism(s) that logically present themselves and the extent to which they may distract from the postulated AOP

 

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.

 

p53 pathway

The study in which in vivo 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 MAA induced up-regulation of p21 expression is mediated through histone hyperacetylation and independent of p53/p63/p73 (Parajuli, 2014).

 

     NFkB pathway

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).

 

Communication with Sertoli cells

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).

 

Decrease in deoxynucleotide pool by MAA

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-CH2-THF production by MAA may decreases deoxynucleotide pool in spermatocytes (Yamazoe, 2015).


Quantitative Understanding

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Concordance of dose-response relationships

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.

Temporal concordance among the key events and adverse outcome

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.

Strength, consistency, and specificity of association of adverse outcome and initiating event

The scientific evidence on the linkage between MIE and AO has been described.

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).

 


Considerations for Potential Applications of the AOP (optional)

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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.


References

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Wade MG et al. (2008) Methoxyacetic acid-induced spermatocyte death is associated with histone hyperacetylation in rats. Biol Reprod 78:822-831

Richon VM et al. (2000) Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. Proc Natl Acad Sci 97:10014-10019        

Struhl K. (1998) Histone acetylation and transcriptional regulatory mechanisms. Gene Dev 12:599-606

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           

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     

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  

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           

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

Kerr JFR et al. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257

Miller RR et al. (1982) Toxicity of methoxyacetic acid in rats. Fundam Appl Toxicol 2: 158-160

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

Lagger G et al. (2002) Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J 21:2672-2681                                       

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

Choudhary C et al. (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325:834-840

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

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

Gnad F et al. (2011) PHOSIDA 2011: the posttranslational modification database. Nucl Acids Res 39:D253-D260

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

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

Richon VM et al. (2000) Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation. Proc Natl Acad Sci 97:10014-10019

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

Gartel AL and Tyner AL (2002) The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther 1: 639-649

Chen J et al (1996) Cyclin-binding motifs are essential for the function of p21CIP1. Mol Cell Biol 16: 4673-4682                        

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

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

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     

Niu Z et al. (2011) microRNA-21 regulates the self-renewal of mouse spermatogonial stem cells. Proc Natl Acad Sci 108: 12740-12745

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

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

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

Fenic I et al. (2008) In vivo application of histone deacetylase inhibitor trichostatin-A impairs murine male meiosis. J Andro 29: 172-185

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

Wang C et al. (2017) CD147 regulates extrinsic apoptosis in spermatocytes by modulating NFkB signaling pathways. Oncotarget 8: 3132-3143

Chen S and Liu Y. (2015) Regulation of spermatogonial stem cell self-renewal and spermatocyte meiosis by Sertoli cell signaling. Reproduction 149: R159-R167

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

Foster PM et al. (1983) Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat. Toxicol Appl Pharmacol 69:385-39