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Created at: 2019-11-08 12:37

AOP ID and Title:

AOP 212: Histone deacetylase inhibition leading to testicular toxicity
Short Title: Histone deacetylase inhibition leading to testicular toxicity

Graphical Representation

Authors

Shihori Tanabe, Akihiko Hirose, Takashi Yamada

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


Status

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 Do not cite EAGMST Under Review 1.52 Included in OECD Work Plan

Abstract

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



Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

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

Key Event Relationships

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
Histone deacetylase inhibition adjacent Histone acetylation, increase High Moderate
Histone acetylation, increase adjacent p21 (CDKN1A) expression, increase Moderate Moderate
p21 (CDKN1A) expression, increase adjacent Cell cycle, disrupted High Moderate
Cell cycle, disrupted adjacent Apoptosis Moderate Moderate
Apoptosis adjacent spermatocyte depletion High Not Specified
spermatocyte depletion adjacent testicular toxicity High Not Specified
Histone deacetylase inhibition non-adjacent Cell cycle, disrupted High Moderate
Histone deacetylase inhibition non-adjacent Apoptosis Moderate Moderate
Histone deacetylase inhibition non-adjacent testicular toxicity Moderate Low

Stressors


Name Evidence
Methoxyacetic acid High
Butyrate High
Trichostatin A High

Overall Assessment of the AOP

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. (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

Life Stage Applicability
Life Stage Evidence
Adult, reproductively mature High
Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Moderate NCBI
mouse Mus musculus Moderate NCBI
rat Rattus norvegicus High NCBI
Sex Applicability
Sex Evidence
Male High

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

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].

Weight of Evidence Summary

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].

 

      NF-kappaB pathway

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].

 

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 Consideration

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)

  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

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


Appendix 1

List of MIEs in this AOP

Event: 1502: Histone deacetylase inhibition

Short Name: Histone deacetylase inhibition

Key Event Component

Process Object Action
enzyme inhibitor activity histone deacetylase 1 decreased

Stressors

Name
Methoxyacetic acid
Butyrate
Trichostatin A
Valproic acid
Suberoylanilide hydroxamic acid
MS-275
Apicidin

Biological Context

Level of Biological Organization
Molecular

Cell term

Cell term
cell

Organ term

Organ term
organ

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

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 (IC50 value of ~0.2 mM and ~8 mM, respectively), but no effect for HDAC8 (IC50 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].



Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
rat Rattus norvegicus High NCBI
human Homo sapiens High NCBI
mouse Mus musculus High NCBI
Life Stage Applicability
Life Stage Evidence
All life stages Moderate
Sex Applicability
Sex Evidence
Male High

The inhibition of HDAC by HDIs is well conserved between species from lower organism to mammals.

  • HDIs reduced lethality in Drosophila model and the HDAC activity was inhibited with HDIs in rat PC12 cells [Steffan, 2001].
  • 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].
  • HDIs were approved as drugs for multiple myeloma and T-cell lymphoma by FDA [Ansari, 2016].
  • HDIs inhibited cell growth in human non-small cell lung cancer cell lines [Miyanaga, 2008].
  • HDAC acetylation level was increased by HDIs in MRL-lpr/lpr murine model of lupus splenocytes [Mishra, 2003].
  • SAHA increased histone acetylation in brain and spleen of mice [Hockly, 2003].
  • MAA inhibits HDAC activity in HeLa cells and spleens from C57BL/6 mice [Jansen, 2004].
  • It is also reported that MAA inhibits HDAC activity in testis cytosolic and nuclear extract of juvenile rats (27 days old) [Wade, 2008].
  • VPA and TSA inhibit HDAC enzymatic activity in mouse embryo and human HeLa cell nuclear extract [Di Renzo, 2007].
  • 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].

Key Event Description

Site of action: The site of action for the molecular initiating event is a cell.

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].

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].

 

Description from EU-ToxRisk deliverable:

Eukaryotic histone deacetylases (HDACs) are grouped, according to phylogeny, into classes 1 through 4 (Gregoretti et al., 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+, and are considered impervious to most known HDAC inhibitors (Drummond et al., 2005).

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 recent 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 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 exert 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.


How it is Measured or Detected

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].

 

Description from EU-ToxRisk deliverable:

HDAC inhibition can be followed by several different approaches:

-Western blots applying antibodies targeting specific acetylated proteins.

-Commercial fluorimetric and colorimetric kits can be applied to assay HDAC activity from various biological extracts.


References

Damaskos C. et al. (2017) Histone deacetylase inhibitors: an attractive therapeutic strategy against breast cancer. Anticancer Research 37: 35-46.

Taunton J. et al. (1996) A mammalian histone deacetylase related to the Yeast transcriptional regulator Rpd3p. Science 272:408-411.

Lee SC. et al. (2016) Essential role of insulin-like growth factor 2 in resistance to histone deacetylase inhibitor. Oncogene 35:5515-5526.

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.

Weichert W. (2009) HDAC expression and clinical prognosis in human malignancies. Cancer Letters 280:168-176

Barneda-Zahonero B and Parra M (2012) Histone deacetylases and cancer. Mol Oncol 6:579-589

Bell EL et al. (2014) SirT1 is required in the male germ cell for differentiation and fecundity in mice. Development 141:3495-3504

Richon VM et al. (2004) Histone deacetylase inhibitors: assays to assess effectiveness in vitro and in vivo. Methods Enzymol. 376:199-205

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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

Johnson DS et al. (2007) Genome-wide mapping of in vivo protein-DNA interactions. Science 316:1497-1502

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

Steffan JS et al. (2001) Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature 413:739-743

Desjardins D et al. (2016) Histone deacetylase inhibition restores retinal pigment epithelium function in hyperglycemia. PLoS ONE 11: e0162596

Ansari J et al. (2016) Epigenetics in non-small cell lung cancer: from basics to therapeutics. Transl Lung Cancer Res 5:155-171

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

Mishra N et al. (2003) Histone deacetylase inhibitors modulate renal disease in the MRL-lpr/lpr mouse. J Clin Invest 111: 539-552

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

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

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

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

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Villae-Garea A and Esteller M. (2004) Histone deacetylase inhibitors: understanding a new wave of anticancer agents. Int J Cancer 112:171-178

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List of Key Events in the AOP

Event: 1503: Histone acetylation, increase

Short Name: Histone acetylation, increase

Key Event Component

Process Object Action
regulation of protein modification process histone increased

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
cell

Organ term

Organ term
organ

Domain of Applicability


Life Stage Applicability
Life Stage Evidence
Not Otherwise Specified Moderate
Sex Applicability
Sex Evidence
Unspecific High

The histone acetylation increase by HDIs is well conserved between species from lower organism to mammals.

・MAA, a HDAC inhibitor, induces acetylation of histones H3 and H4 in Sprague-Dawley (Rattus norvegicus) [Wade, 2008].

・It is also reported that MAA promotes acetylation of H4 in HeLa cells (Homo sapiens) and spleens from C57BL/6 mice (Mus musculus) treated with MAA [Jansen, 2014].

・VPA, a HDAC inhibitor, induces hyperacetylation of histone H4 in protein extract of mouse embryos (Mus musculus) exposed in utero for 1h to VPA [Di Renzo, 2007a].

・Apicidin, MS-275 and sodium butyrate, HDAC inhibitors, induce hyperacetylation of histone H4 in homogenates from mouse embryos (Mus musculus) after the treatments [Di Renzo, 2007b].

・MAA acetylates histones H3K9 and H4K12 in limbs of CD1 mice (Mus musculus) [Dayan, 2014].


Key Event Description

Gene transcription is regulated with the balance between acetylation and deacetylation. The acetylation and deacetylation are modulated on the NH3+ 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].

 

Description from EU-ToxRisk Deliverable:

The inhibition of HDACs result in an accumulation of acetylated proteins such as tubulin or histones.


How it is Measured or Detected

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 [3H] acetate [Gunjan, 2001].

 

Description from EU-ToxRisk Deliverable:

  1. Semi-quantitative: Western blot usining antibodies agains acetylated tubulin or histones

  2. Quantitative: enzyme assays using acetylated peptides and purified HDAC enzyme


References

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. (2004) Histone deacetylase inhibitors: assays to assess effectiveness in vitro and in vivo. Methods Enzymol 376:199-205

Gunjan A et al. (2001) Core histone acetylation is regulated by linker histone stoichiometry in vivo. J Biol Chem 276:3635-3640

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

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

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

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

 


Event: 1504: p21 (CDKN1A) expression, increase

Short Name: p21 (CDKN1A) expression, increase

Key Event Component

Process Object Action
positive regulation of gene expression cyclin-dependent kinase inhibitor increased

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:212 - Histone deacetylase inhibition leading to testicular toxicity KeyEvent

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
cell

Organ term

Organ term
organ

Domain of Applicability


Life Stage Applicability
Life Stage Evidence
Not Otherwise Specified Moderate
Sex Applicability
Sex Evidence
Unspecific High

The p21 up-regulation by HDIs is well conserved between species from lower organism to mammals.

・FK228, a HDAC inhibitor, up-regulated p21 level in human esophageal cancer TE2 cells (Homo sapiens) [Hoshino, 2007].

・MAA, a HDAC inhibitor, induced p21 up-regulation in human prostate cancer cell lines (Homo sapiens) [Parajuli, 2014].

・MAA increases p21 expression in human bladder carcinoma cells, T24 (Homo sapiens) [Glaser, 2003].

・MAA up-regulated p21 expression in limbs of CD1 embryonic mice (Mus musculus) [Dayan, 2014].


Key Event Description

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 G1 phase. p21 is important for cell cycle regulation.


How it is Measured or Detected

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].


References

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

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

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

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


Event: 1505: Cell cycle, disrupted

Short Name: Cell cycle, disrupted

Key Event Component

Process Object Action
regulation of cell cycle cell cycle-related cyclin disrupted

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:212 - Histone deacetylase inhibition leading to testicular toxicity KeyEvent

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
cell

Organ term

Organ term
organ

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Homo sapiens Homo sapiens High NCBI
Mus musculus Mus musculus High NCBI
Life Stage Applicability
Life Stage Evidence
Not Otherwise Specified Moderate
Sex Applicability
Sex Evidence
Unspecific High

The relationship between disrupted cell cycle and apoptosis is likely well conserved between species.

・The change in the amounts of cells in G1 phase and S phase of cell cycle was detected in mouse HDAC1 knock out fibroblast lines (Mus musculus) [Zupkovitz, 2010].

・The histone gene expression alters in each phase of cell cycle in human HeLa cell (Homo sapiens) [Heintz, 1982].


Key Event Description

The dysregulation of cell cycle leads to the decreases in cell number. The cell cycle consists of G1, S, G2, M, and G0 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].


How it is Measured or Detected

The percentage of cells at G1, G0, S, and G2/M phases was determined by flow cytometry analysis using DNA content frequency histogram deconvolution software [Li, 2013].

Cell cycle distribution was analyzed by fluorescence-activated cell sorter (FACS) analysis with a Partec PAS-II sorter [Zupkovitz, 2010].

The four cell cycle phases in living cells can be measured with four-color fluorescent proteins using live cell imaging [Bajar, 2016].

The incorporation of [3H]deoxycytidine or [3H]thymidine into cell DNA during S phase can be monitored as DNA synthesis [Heintz, 1982].


References

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

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

Child ES and Mann DJ. (2006) The intricacies of p21 phosphorylation. Cell Cycle 5:1313-1319

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.

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

Bajar BT et al. (2016) Fluorescent indicators for simultaneous reporting of all four cell cycle phases. Nat Methods 13: 993-996 


Event: 1262: Apoptosis

Short Name: Apoptosis

Key Event Component

Process Object Action
apoptotic process increased

Biological Context

Level of Biological Organization
Cellular

Cell term

Cell term
cell

Organ term

Organ term
organ

Domain of Applicability


Life Stage Applicability
Life Stage Evidence
Not Otherwise Specified High
Sex Applicability
Sex Evidence
Unspecific High

・The apoptosis and proliferation inhibition induced by MAA, a HDAC inhibitor,  was measured in human prostate cancer cell lines (Homo sapiens) [Parajuli, 2014].

・The cell viability inhibition induced by SAHA or TSA , which are HDAC inhibitors, was observed in NHDFs (Homo sapiens) [Glaser, 2003].

・The proliferation of the HDAC-/- ES cells was inhibited compared to HDAC+/+ ES cells (Homo sapiens) [Zupkovitz, 2010].


Key Event Description

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-/- 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].


How it is Measured or Detected

・The apoptosis is detected with the expression alteration of procaspases 7 and 3 by Western blotting using antibodies [Parajuli, 2014].

・The apoptosis is measured with down-regulation of anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, or cIAP1) [Parajuli, 2014].

・Apoptotic nucleosomes were detected using Cell Death Detection ELISA kit, which were calculated as absorbance subtraction at 405 nm and 490 nm [Parajuli, 2014].

・Cell viability was measured with live cell number changes using the CellTiter-Glo Luminescent Cell Viability Assay [Parajuli, 2014].

・Cleavage of PARP was detected with Western blotting [Parajuli, 2014].

・The proliferation/viability of NHDFs was measured with Alamar-Blue [modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] [Glaser, 2003].

・Proliferation of the HDAC-/- ES cells was determined with crystal violet and measurement of absorbance at 595 nm [Zupkovitz, 2010].

・Caspase-3 and caspase-9 activity is measured with the enzyme-catalyzed release of p-nitroanilide (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].

・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].


References

Susan E. (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35: 495-516

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

Pucci B et al. (2000) Cell cycle and apoptosis. Neoplasia 2:291-299

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

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

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

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

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

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


Event: 1515: spermatocyte depletion

Short Name: spermatocyte depletion

Key Event Component

Process Object Action
spermatocyte decreased

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:212 - Histone deacetylase inhibition leading to testicular toxicity KeyEvent

Biological Context

Level of Biological Organization
Tissue

Organ term

Organ term
testis

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Rattus norvegicus Rattus norvegicus Moderate NCBI
Life Stage Applicability
Life Stage Evidence
Adult Moderate
Sex Applicability
Sex Evidence
Male High

There are evidences of spermatocyte depletion.

・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 (Mus musculus) [Zindy, 2001].

・The sperm counts in the cauda epidydimis of rats exposed to butylparaben were significantly decreased (Rattus norvegicus) [Oishi, 2001].

・MAA treatment induced spermatocyte death in Sprague-Dawley rats (Rattus norvegicus) [Wade, 2008].


Key Event Description

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].


How it is Measured or Detected

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].

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].

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].

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].


References

Rooij DG. (2001) Proliferation and differentiation of spermatogonial stem cells. Reproduction 121:347-354

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

Oishi S. (2001) Effects of butylparaben on the male reproductive system in rats. Toxicol Indust Health 17:31-39

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

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


List of Adverse Outcomes in this AOP

Event: 1506: testicular toxicity

Short Name: testicular toxicity

Key Event Component

Process Object Action
testicular atrophy Testis decreased

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:212 - Histone deacetylase inhibition leading to testicular toxicity AdverseOutcome

Biological Context

Level of Biological Organization
Organ

Organ term

Organ term
testis

Domain of Applicability


Taxonomic Applicability
Term Scientific Term Evidence Links
Rattus norvegicus Rattus norvegicus Moderate NCBI
Life Stage Applicability
Life Stage Evidence
Adult, reproductively mature Moderate
Sex Applicability
Sex Evidence
Male High

There are some evidences on testicular toxicity induced by HDAC inhibitors.

・EGME or MAA treatment induced the testicular damage in rat (Rattus norvegicus) [Foster, 1983].

・EGME were shown to deplete the spermatocytes in CD-1 mice (Mus musculus) and CD rats (Rattus norvegicus), principally pachytene cells, but with other stages affected with increasing dose [Anderson, 1987].

・The testicular lesions induced by 2-methoxyethanol were observed in rats (Rattus norvegicus) and guinea pigs (Cavia porcellus), which are different in onset, characteristics and severity [Ku, 1984].

・EGME has effects in disruption of spermatogenesis in rabbits (Oryctolagus cuniculus) [Foote, 1995].

・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 (Rattus norvegicus).

 


Key Event Description

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 7th edition].


How it is Measured or Detected

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].

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].


Regulatory Significance of the AO

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.


References

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

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

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

Moss EJ et al. (1985) The role of metabolism in 2-methoxyethanol-induced testicular toxicity. Toxicol Appl Pharmacol 79:480-489

Casarett & Doull’s Toxicology, the Basic Science of Poisons, 7th Edition, Edited by Curtis D. Klaassen, Chapter 20 Toxic responses of the reproductive system

Foster PM et al. (1983) Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rats. Toxicol Appl Pharmacol 69:385-399

McDowell EM and Trump BF. (1976) Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100:405-414

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

Foote RH et al. (1995) Ethylene glycol monomethyl ether effects on health and reproduction in male rabbits. Reprod Toxicol 9:527-539

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

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


Appendix 2

List of Key Event Relationships in the AOP