Workshop Report 30

APPENDIX 4: Background document

Background document on epigenetics in relation to the workshop on ‘The role of Epigenetics in reproductive Toxicity’

Authors: Miriam Jacobs and John Greally

Current OECD human health related TGs and potential for epigenetic additions in relation to reproductive / developmental chemical safety

All human health TGs cited are publically available from the OECD website (http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-4-health-effects_20745788).

Table 1: OECD Test Guidelines that could potentially be adapted for epigenomic studies of effects of endocrine disruptors (ED) and reproductive toxicity

Type of study Test Guidelines (TG) Description
Alternative models integrating multiple mechanisms of action TG 236 Zebrafish Embryo Acute Toxicity Assay (see discussion in Table 2)

Xenopus Embryo Thyroid Assay (XETA assay) in validation

General exposure studies TG 451

TG 452

TG 453

Carcinogenicity Studies

Chronic Toxicity Studies

Combined Chronic Toxicity / Carcinogenicity Studies

Post-mitotic cell studies TG 424 Neurotoxicity Study in Rodents
Prenatal effects TG 414

TG 426

Prenatal Development Toxicity Study

Developmental Neurotoxicity Study

Reproductive effects TG 415, TG 416

TG 421

TG 422
TG 443

One- and Two-Generation Reproduction Toxicity

(Revised) Reproduction / Developmental Toxicity Screening Test

(Revised) Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test

Extended One-Generation Reproductive Toxicity Study

Genotoxicity tests TG 483
TG 488
(Revised 2015) Mammalian Spermatogonial Chromosome Aberration Test

Transgenic Rodent Somatic and Germ Cell Gene Test

Potentially relevant tests to be used in combination TG 473 In vitro Mammalian Chromosomal Aberration Test

 

Table 2: Updated OECD Endocrine Disruptor Testing Conceptual Framework combined with potential epigenetic tests and preliminary reference chemicals

Level Mammalian and non-mammalian Toxicology Epigenetic test information Potential prototype chemicals to determine the sensitivity and specificity of model systems
1

Existing Data and
Non-Test Information

Physical and chemical properties, e.g. MW reactivity, volatility, biodegradability Epigenetic literature review information
1 All available (eco)toxicological data from standardised or
non-standardised tests
Epigenetic literature review information
1 Read across, chemical categories, QSARs and other in silico predictions, and ADME model predictions e.g. literature-derived information about DNA methylation, RNA and miRNA expression studies and chromatin structure and modification data, with analyses to identify biomarker s for detection of compounds with epigenetic ED activity
2

In vitro assays providing data about selected endocrine mechanism(s) / pathways

Oestrogen or androgen receptor binding affinity Combine with TG 473 but leave out the use of metaphase-arresting substances in exposed cells, this could then be used to screen for potential downstream epigenetic effects Positive: for ER

17β-Oestradiol

Positives: for ER and epigenetic effects

Diethylstilbestrol (DES), BPA, Genistein, Equol (includes metabolism)

Positives: for AR

Testosterone

Positives: for AR and epigenetic effects

Vinclozolin, Flutamide,
Hydroxyflutamide (metabolite)

Negatives: for ER effects

Corticosterone, Spironolactone, Atrazine, Linuron

2 Oestrogen receptor transcriptional activation (TG 455) Relevant endpoints:

– DNA modifications (cytosine methylation)

– miRNA and RNA expression studies

– Studies of chromatin components and structure

Positives: for ED and epigenetic effects

DES, Bisphenol A (BPA), Genistein, Equol (includes metabolism), OH Tamoxifen

2 Androgen or thyroid transcriptional activation (if/when TGs are available) Relevant endpoints:

– DNA modifications (cytosine methylation)

– miRNA and RNA expression studies

Positives: for ER and epigenetic effects

DES, BPA, Genistein, Equol (includes metabolism)

Androgen Receptor STTA TG (lead Japan) expected 2016, validation work completed – Studies of chromatin components and structure Positives: for AR and epigenetic effects

Vinclozolin, flutamide, hydroxyflutamide (metabolite)

2 Steroidogenesis in vitro (TG 456) Relevant endpoints:

– DNA modifications (cytosine methylation)

– miRNA and RNA expression studies

– Studies of chromatin components and structure

– Multivariate / systems analysis to identify key regulatory factors mediating variability of steroidogenesis on a chemical specific basis

Positives: for ED

Prochloraz, Forskolin, Atrazine, Aminoglutethimide, BPA, Dibutyl phthalate (DBP)

Negative: for ED

Human chorionic gonadotropin (HCG)

2 MCF-7 cell proliferation assays (ER ant/agonist) Relevant endpoints:

– DNA modifications (cytosine methylation)

– miRNA and RNA expression studies

– Studies of chromatin components and structure

– Multivariate / systems analysis to identify key what is mediating variability of cell proliferation

As for ER / transactivation assays, plus substances acting through oestrogenic but not receptor pathways (e.g. through non genomic pathways, sulphotransferases etc)

DBP

2 Zebrafish embryo epigenetic assay adaptation of zebrafish embryo toxicity test (zFET). TG 236 and EASZY assay (a zebrafish embryo-based assay for Endocrine Active Substances) Relevant endpoints:

– DNA modifications (cytosine methylation)

– Studies of chromatin components and structure

EASZY assay (in validation): Epigenetic effects upon thyroid

2 Xenopus Embryo Thyroid Assay (XETA assay) (in validation) Relevant endpoints:

Re: thyroid hormone pathway physiology and epigenetic modification particularly in trans- and multigenerational pathways

– DNA modifications (cytosine methylation)

– Studies of chromatin components and structure

Key epigenetic targets identified in the Zebrafish screens above could be examined cross species on the same genes in Xenopus embryos

2 Possible additional examples

1. Casa assay (sperm cell toxicant)

2. Comet assay (sperm cell mutagen)

Relevant endpoints:

– DNA modifications (cytosine methylation)

– miRNA and RNA expression studies

– Luminometric methylation analysis (LUMA) for global methylation analyses

– Studies of chromatin components and structure

 

1. Valproic acid, DES, lindane, carbenazim, nonylphenol

2. DES, lindane, carbenazim, nonylphenol di-2-(ethylhexyl) phthalate (DEHP), DBP

3. Sertoli cell assay

4. Leydig cell assay (cross ref with steroidogenesis assay TG 456)

5. Oogenesis, follicular culture

6. Mouse embryonic stem D3 cell assay (Kleinstreuer et al, 2011)

7. Human embryonic stem cells

8. Rat whole embryo culture toxicity assay

– Multivariate / systems biology / reverse engineering analyses for the identification of gene modules critically involved in transcript abundance during development, to elucidate relevant regulation factors and pathways 3. BPA and as above

4. DES, carbenazim, nonylphenol, taxol, ketoconazole

5. DES, genistein, carbenazim, nonylphenol, ketoconazole

3

In vivo assays providing data about selected endocrine mechanism(s) / pathway(s)

Uterotrophic assay (TG 440) Less relevant endpoint: correlation changes in uterine tissue with molecular changes (epigenomic assays)
3 Hershberger assay (TG 441) No end organ present, not appropriate for testing
4

In vivo assays providing data on adverse effects on endocrine relevant end-points

Repeated dose 28-day study (TG 407)

TG 422

Relevant endpoints:

– DNA modifications (cytosine methylation)

– miRNA and RNA expression studies

– Studies of chromatin components and structure

e.g. Testicular histopathology combined with epigenomic dysregulation assays

With tissues of interest available, need to consider issues of sample collection and preservation, cellular heterogeneity etc, as discussed in Greally and Jacobs, 2013

4 Repeated dose 90-day study (TG 408)
4 1-generation assay (TG 415) 1. Combination with TGs 451 (Carcinogenicity Studies), 452 (Chronic Toxicity Studies) and 453 (Combined Chronic Toxicity / Carcinogenicity Studies) with focus on hormonally-responsive tissues: combination with epigenomic assays

2. The rat model of IUGR and quantified cytosine methylation throughout the genome in beta islet cells from the pancreas of young adult rats, results indicate a distinct pattern of methylation discriminating the animals that had undergone IUGR, at loci already implicated in glucose metabolism or type 2 diabetes mellitus (Thompson, Fazzari et al, 2010). BPA studies all showed changes in cytosine methylation associated with exposure, some changes occurring at loci that were found to be transcriptionally altered.

Valproic acid (male: reduction of spermatogenesis, testicular atrophy, degeneration of seminiferous tubules; female: polycystic ovaries high serum testosterone and menstrual disorders. Teratogenic)
4 Prenatal Development Toxicity Study (TG 414)
4 Chronic toxicity and carcinogenicity studies (TG 451-3)
4 Reproductive screening test (TG 421 from July 2015: enhanced) Oestrus cycles, follicle counts, oocyte maturation, ovarian integrity; thyroid integrity, spermatogenesis combination with epigenomic assays including RNA analysis, toxicogenomics and multivariate data analyses
4 Combined 28-day / reproductive screening assay (TG 422 from July 2015: enhanced)

Developmental Neurotoxicity (TG 426)

Prenatal effects are potentially studied using TG 414 (Prenatal Development Toxicity Study) which involves the exposure to animals of agents during pregnancy, testing the foetus at term for abnormalities, while TG 426 (Developmental Neurotoxicity Study) allows the offspring to be born and to develop, testing specifically for neurological consequences. Tissues harvested at both time-points could shed light on epigenetic effects of agents used for exposure
5

In vivo assays providing more comprehensive data on adverse effects on endocrine relevant endpoints over more extensive parts of the life cycle of the organism

Extended one-generation reproductive Toxicity Study (TG 443) Necropsy and neurological studies of the tests for TG 426, 414, 424 etc Valproic acid, DES, lindane, carbenazim, nonylphenol

BPA, DBP, DEHP

Taxol, ketoconazole, genistein, vinclozolin, methoxychlor

5 2-generation assay (TG 416 most recent update) TG (416) could allow multiple tissues to be sampled in offspring of parents exposed to the agent of interest, allowing screening for inherited epimutations DES, lindane, carbenazim, nonylphenol

BPA

Taxol, ketoconazole, genistein, vinclozolin, methoxychlor and as above

Italicised tests are not in OECD TG workplan.

 

 

«
»