Aopwiki

SNAPSHOT

Created at: 2020-11-04 14:43

AOP ID and Title:

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

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 Do not cite EAGMST Under Review 1.52 Included in OECD Work Plan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. Histone deacetylase inhibitors (HDIs) are approved as anti-cancer drugs, since HDIs have apoptotic effect in cancer cells. HDIs includes the 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. Adverse outcome has been defined as testicular atrophy. The HDIs inhibit deacetylation of the histone, leading to the increase in histone acetylation. The apoptosis induced by 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.

Abbreviation: AOP: adverse outcome pathway, HDAC: histone deacetylase, HDI: HDAC inhibitor, KE: key event, MIE: molecular initiating event



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 1505 Cell cycle, disrupted Cell cycle, disrupted
4 KE 1262 Apoptosis Apoptosis
5 KE 1515 Spermatocyte depletion Spermatocyte depletion
6 AO 1506 Testicular atrophy Testicular atrophy

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 Cell cycle, disrupted Moderate Moderate
Cell cycle, disrupted adjacent Apoptosis Moderate Moderate
Apoptosis adjacent Spermatocyte depletion High Not Specified
Spermatocyte depletion adjacent Testicular atrophy 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 Spermatocyte depletion Moderate Moderate
Histone deacetylase inhibition non-adjacent Testicular atrophy Moderate Moderate

Stressors


Name Evidence
Methoxyacetic acid High
Butyrate High
Trichostatin A High
Valproate Moderate

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. Activity of histone acetyltransferase (HAT) in testis nuclear protein was increased with MAA addition [Wade et al., 2008].

KE1 => KE2: Histone acetylation, increase leads to cell cycle, disrupted

Biological Plausibility of the KE1 => KE2 is moderate.
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 nonpromoter regions, targeted generally to promoter regions, or targeted to specific promoters by gene-specific activator proteins [Richon et al., 2000; Struhl, 1998].

KE2 => KE3: Cell cycle, disrupted leads to apoptosis

Biological Plausibility of the KE2 => KE3 is moderate.
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.

KE3 => KE4: Apoptosis leads to spermatocyte depletion

Biological Plausibility of the KE3 => KE4 is moderate.
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.

KE4 => AO: Spermatocyte depletion leads to testicular atrophy

Biological Plausibility of the KE4 => AO is moderate.
Rationale: Spermatocyte depletion is one of the main characteristics of testicular atrophy.

2. Support for essentiality of KEs

KE2: Cell cycle, disrupted

Essentiality of the KE2 is moderate.
Rationale for Essentiality of KEs in the AOP: HDAC1-defecient 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].

3. Empirical support for KERs

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

Empirical Support of the MIE => KE1 is high.
Rationale: HDAC inhibitors increase histone acetylation in 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 n-butyrate leads to an accumulation of acetylated histone proteins [Riggs et al., 1977]. Later, it was shown that n-butyrate specifically increases the acetylation of histone by the incorporation of radioactive [3H]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 in vitro and in vivo was shown using acetyl-specific antibodies and western blot [Gottlicher et al., 2001].

KE1 => KE2: Histone acetylation, increase leads to cell cycle, disrupted

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

KE2 => KE3: Cell cycle, disrupted leads to apoptosis

Empirical Support of the KE2 => KE3 is moderate.
Rationale: Cell cycle arrest such as G1 arrest and G1/S arrest are observed in apoptosis [Li et al., 2012; Dong et al., 2010]. microRNA-1 and microRNA-206 represses CCND2, while microRNA-29 represses CCND2 and induces G1 arrest and apoptosis in rhabdomyosarcoma [Li et al., 2012].

KE3 => KE4: Apoptosis leads to spermatocyte depletion

Empirical Support of the KE3 => KE4 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 et al., 2011].

KE4 => AO: Spermatocyte depletion leads to testicular atrophy

Empirical Support of the KE4 => AO is high.
Rationale: The testicular atrophy seen in 2-methoxyethanol (2-ME), or its major metabolite MAA, treated rats in vivo and in human, and rat in vitro culture was associated with spermatocyte depletion [Beattie et al., 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 et al., 1982]. HDAC inhibition in cell culture resulted in the testicular toxicity including germ cell apoptosis and cell morphology change [Li et al., 1996].
KE1: Histone acetylation, increase The HDAC inhibition induced cell death in spermatocytes in both rat and human seminiferous tubules [Li et al., 1996].
KE2: 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 et al., 2010].
KE3: Apoptosis HDAC inhibition leads to cell death through the apoptotic pathways [Falkenberg et al., 2014].
KE4: Spermatocyte depletion 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 the germ cell apoptosis in human testicular tissues [Li et al., 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 et al., 2008]. Other 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].

 

      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 et al., 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 and Liu, 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 et al., 2015]. Inhibition of 5,10-CH2-THF production by MAA may decreases deoxynucleotide pool in spermatocytes [Yamazoe et al., 2015].

Spermatocyte depletion by necrosis

Spermatocyte may be decreased by necrosis. Cell death mechanism other than apoptosis, such as necrosis, may be considered for spermatocyte depletion.

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 et al., 1983; Miller et al., 1982].

 

Considerations for Potential Applications of the AOP (optional)

The present AOP can be used in risk assessment of several types of the molecules including HDAC inhibitors as the anti-cancer drugs, as well as another types of the chemicals, 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 to testicular atrophy 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

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

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

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

Cousens, L.S., et al. (1979), "Different accessibilities in chromatin to histone acetylase", J Biol Chem 254:1716-1723

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

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

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

Gottlicher, M. et al. (2001), "Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells", EMBO J 20:6969-6978

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

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  

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

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

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

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

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

Riggs, M.G. et al. (1977), "N-butyrate causes histone modification in HeLa and friend erythroleukaemia cells", Nature 268:462-464

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

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

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

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

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

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

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


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 et al., 2003; Ropero and Esteller, 2007; Villar-Garea et al., 2004]. There is a report showing that TSA and butyrate competitively inhibits HDAC activity [Sekhavat et al., 2007]. HDIs inhibit preferentially HDACs with some selectiveness [Hu et al., 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 et al., 2003]. TSA inhibits HDAC1, 2, 3 of class I HDACs. [Damaskos et al., 2016].



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
Unspecific High

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

  • HDAC inhibition 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 et al., 2016].
  • 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].
  • HDAC acetylation level was increased by HDIs in MRL-lpr/lpr murine model of lupus splenocytes [Mishra et al., 2003].
  • SAHA increased histone acetylation in brain and spleen of mice [Hockly et al., 2003].
  • MAA inhibits HDAC activity in HeLa cells and spleens from C57BL/6 mice [Jansen et al., 2004].
  • 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].
  • VPA and TSA inhibit HDAC enzymatic activity in mouse embryo and human HeLa cell nuclear extract [Di Renzo et al., 2007].
  • 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].

Key Event Description

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].

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 kidney, brain, testis, heart and skeletal muscle [Barneda-Zahonero and Parra, 2012]. 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 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 increase of histone acetylation as the detection of global histone acetylation changes by Western blot or mass spectrometry (MS)-based proteomic 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]. ome in silico methods including molecular modelling, virtual screening and molecular dynamics are used to find the common HDAC inhibitor structures [Huang et al., 2016; Yanuar et al. 2016].

 


References

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

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

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

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

Damaskos, C. et al. (2016), "Histone deacetylase inhibitors: a novel therapeutic weapon against medullary thyroid cancer?", Anticancer Res 36:5019-5024

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

Decroos, C. et al. (2015), "Biochemical and structural characterization of HDAC8 mutants associated with cornelia de lange syndrome spectrum disorders", Biochemistry 54: 6501–6513.

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

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

Dreser, N. et al. (2015), "Grouping of histone deacetylase inhibitors and other toxicants disturbing neural crest migration by transcriptional profiling", Neurotoxicology 50: 56–70

Drummond, D.C. et al. (2005), "Clinical development of histone deacetylase inhibitors as anticancer agents", Annu Rev Pharmacol Toxicol 45: 495–528

Finnin, M.S. et al. (1999), "Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors", Nature 401: 188–193

Göttlicher, M. et al. (2001), "Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells", EMBO J 20: 6969–6978

Gregoretti, I.V. et al. (2004), "Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis", J Mol Biol 338: 17–31

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

Hu, E. et al. (2003), "Identification of novel isoform-selective inhibitors within class I histone deacetylases", J Pharmacol Exp Ther 307:720-728

Huang, Y.X. et al. (2016), "Virtual screening and experimental validation of novel histone deacetylase inhibitors", BMC Pharmacol Toxicol 17(1):32

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

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

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

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

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

Richon, V.M. et al. (2003), "Histone deacetylase inhibitors: assays to assess effectiveness in vitro and in vivo", Methods Enzymol. 376:199-205

Ropero, S. and Esteller, M. (2007), "The role of histone deacetylases (HDACs) in human cancer", Mol Oncol 1:19-25

Sekhavat, A. et al. (2007), "Competitive inhibition of histone deacetylase activity by trichostatin A and butyrate", Biochemistry and Cell Biology 85:751-758

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

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

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

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

Yanuar, A. et al. (2016), "In silico approach to finding new active compounds from histone deacetylase (HDAC) family", Curr Pharm Des 22: 3488-3497

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


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 et al., 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 et al., 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 et al., 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 et al., 2007b].

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


Key Event Description

Gene transcription is regulated with the balance between acetylation and deacetylation. A dynamic balance of histone acetylation and histone deacetylation are 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 lease theoretically, an activation of HAT might lead to increase in histone acetylation. 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 et al., 2008]. 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 et al., 2004]. Histone acetylation on chromatin can be measured using labeling method with sodium [3H]acetate [Gunjan et al., 2001]. The histone acetylation increase is detected as global histone acetylation changes by Western blot or mass spectrometry (MS)-based proteomic methods or as site specific histone acetylation changes using chromatin immunoprecipitation (ChIP) or ChIP-on-Chip.


References

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

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

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

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

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

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


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


Key Event Description

The disruption of the cell cycle leads to a decrease in the cell number. The cell cycle consists of G1, S, G2, M, and G0 phase. The cell cycle regulation is disrupted by the cell cycle arrest in certain cell cycle phase. The histone gene expression is regulated in cell cycle phases [Heintz et al., 1983].


How it is Measured or Detected

The percentage of cells at G1, G0, S, and G2/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 [3H]deoxycytidine or [3H]thymidine into cell DNA during S phase can be monitored as DNA synthesis [Heintz et al., 1982].


References

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

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

Li, Q. et al. (2013), "Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis", Drug Des Devel Ther 7:635-643


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

・Apoptosis is induced in human prostate cancer cell lines (Homo sapiens) [Parajuli et al., 2014].

・Apoptosis occurs in B6C3F1 mouse (Mus musculus) [Elmore, 2007].

・Apoptosis occurs in Sprague-Dawley rat (Rattus norvegicus) [Elmore, 2007].

・Apoptosis occurs in nematode (Caenorhabditis elegans) [Elmore, 2007].

 

 


Key Event Description

Apoptosis, the process of programmed cell death, is characterized by distinct morphology with DNA fragmentation and energy dependency [Elmore, 2007]. Apoptosis, also called as “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-/- ES cells, which was rescued with the reintroduction of HDAC1 [Zupkovitz et al., 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 et al., 2017].


How it is Measured or Detected

Apoptosis is characterized by many morphological and biochemical changes such as cell shrinkage and pyknosis, cell fragments into apoptotic bodies, DNA fragmentation, enzymatic activation of pro-caspases or phosphatidylserine translocation that can be measured using electron and cytochemical optical microscopy, proteoic and genomic methods, spectroscopic techniques [Martines, 2010, Taatjes, 2008, Archana, 2013]. Some of the methods are described as follows:

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

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

・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 et al., 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 and Groscurth, 1994].

・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].


References

Archana, M. et al. (2013), "Various methods available for detection of apoptotic cells", Indian J Cancer 50:274-283

Elmore, S. (2007), "Apoptosis: a review of programmed cell death", Toxicol Pathol 35:495-516

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

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

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

Martinez, M.M. et al. (2010), "Detection of apoptosis: A review of conventioinal and novel techniques", Anal Methods 2:996-1004

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

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

Taatjes, D.J. et al. (2008), "Morphological and cytochemical determination of cell death by apoptosis", Histochem Cell Biol 129:33-43

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

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

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

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


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 atrophy 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 in different species.

・Mature sperm counts were decreased and the residual spermatozoa had reduced motility and decreased viability (Mus musculus) [Zindy et al., 2001].

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

・Spermatocyte death can be induced in Sprague-Dawley rats (Rattus norvegicus) [Wade et al., 2008].


Key Event Description

Spermatocytes are differentiated from spermatogonial stem cells via random proliferation, differentiation and synchronized mitoses with several stages [Rooij, 2001]. In several steps of the spermatogonial differentiation, different molecular mechanism is activated in testis [Rooij, 2001, de Kretser et al., 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].


How it is Measured or Detected

Traditional spermatocytes assessment includes sperm count and concentration (haemocytometer, automated imaged-based syste), morphology and motility (microscope, automated imaged-based system) and viability (for example propidium iodide staining of necrotic cells, TUNEL assay staining apoptotic cells). In additioin, there are functional tests such as assays for genetic integrity (e.g. via measurement of DNA fragmentation/integrity -Halosperm kit or reactive oxygen species) and fertilization defets (through various measures of sperm-zona pellucida (ZP) interaction, such as measurement of ZP-receptor binding).

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 et al., 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 testis cell analysis, fresh testes were dispersed using a 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 cytometeric 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].


References

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

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

Rooij, D.G. (2001), "Proliferation and differentiation of spermatogonial stem cells", Reproduction 121:347-354

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

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

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


List of Adverse Outcomes in this AOP

Event: 1506: Testicular atrophy

Short Name: Testicular atrophy

Key Event Component

Process Object Action
Testicular atrophy Testis increased

AOPs Including This Key Event

AOP ID and Name Event Type
Aop:212 - Histone deacetylase inhibition leading to testicular atrophy 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
  • The derease in testis weight associated with testicular cell damage was induced by EGME or MAA treatment in rats (Rattus norvegicus) [Foster et al., 1983].
  • The number of spermatocytes, principally pachytene cells, is decreased by EGME treatment in CD-1 mice (Mus musculus) and CD rats (Rattus norvegicus) [Anderson et al., 1987].
  • The testicular lesions induced by 2-methoxyethanol (ethylene glycol monomethyl ether; EGME) were observed in rats (Rattus norvegicus) and guinea pigs (Cavia porcellus), which are different in onset, characteristics and severity [Ku et al., 1984].
  • Spermatogenesis was disrupted by EGME treatment in rabbits (Oryctolagus cuniculus) [Foote et al., 1995].
  • 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 (Rattus norvegicus) [Wade et al., 2006].

Key Event Description

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, 7th edition].


How it is Measured or Detected

  • Testicular atrophy can be assessed by testicular volume measurment using orchidometer, rulers, calipers, and ultrasonography or by testis weighing and histopathologic examination.
  • The testis weight is measured to detect the testicular atrophy [Foster et al., 1983].
  • 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].
  • The testicular tissue structure is observed whether there are normal germinal epithelial cells and Leydig cells [Mercantepe et al., 2018]. Testis is fixed for observations by light microscopy or transmission electron microscopy [McDowell and Trump, 1976; Mercantepe et al., 2018].
  • Changes in sperm are measured by computer-assisted sperm analysis [Foote et al., 1995].
  • 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) by one individual unaware of animal number or treatment [Wade et al., 2006].
  • 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].
  • 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 et al., 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, as well as hazard and risk of chemicals. The testicular atrophy including 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.


References

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

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

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

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

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

McDowell, E.M. and Trump, B.F. (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

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

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

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

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


Appendix 2

List of Key Event Relationships in the AOP