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2022 Annual Meeting Report: Across a Life Span and Beyond—Lessons (and Key Terms!) from Wednesday’s Session on Persistent Epigenetic Alterations in Toxicology

By Sarah Carratt posted 04-05-2022 14:23

  

Symposium Session: “Role and Mechanisms of Persistent Epigenetic Alterations in Toxicological Responses”

Epigenetic changes are essential to normal cellular processes, modulating gene expression without changing the underlying DNA sequence. Environmental factors, from the food you eat to the chemicals to which you are exposed, can influence your epigenome. In this session, speakers from academia, government, and industry presented data showing how persistent epigenetic changes shape outcomes across the life span and multiple generations.

Brian Chorley (US EPA): “Genetic and Epigenetic Alterations Associated with Latent Liver Carcinogenesis due to Early-Life Dichloroacetic Acid Exposure in Mice”

In the first talk of the session, Dr. Chorley discussed how his team was able to leverage modern sequencing technology to retroactively assess archived frozen and paraformaldehyde-preserved samples—dating as far back as the 1990s!

In the historical study, mice had an increased incidence of liver cancer 84 weeks after exposure to dichloroacetic acid (DCA). To determine if the early-life exposure left a persistent epigenetic footprint responsible for the latent carcinogenicity, Dr. Chorley et al. assessed DNA methylation and gene expression at 78 weeks. While both a 10-week exposure to DCA and chronic DCA exposures led to an increase in liver cancer incidence, it turns out that carcinogenesis occurred through two unique mechanisms of action. Ten-week early-life exposures had a completely distinct, different genomic and epigenetic signature, activating fibrosis and biosynthesis gene expression pathways, while continuous exposure activated cancer pathways. Gene expression analysis at 10 weeks showed insulin dysregulation followed by dysregulation of extracellular membrane pathways.

Dr. Chorley’s presentation highlights the advantages of incorporating epigenetic analyses into toxicology assessments, and the potential for using DNA methylation patterns as biomarkers of exposure.

Kamin Johnson (Corteva Agriscience): “Epigenomic Point-of-Departure Modeling for Developmental Toxicity Safety Assessment”

Once we have identified epigenomic biomarkers, how do we leverage these data? In his talk, Dr. Johnson outlined how we can apply epigenomic (miRNA) and transcriptomic (mRNA) data to predict toxicity and identify safe chemical exposure limits.

Using ketoconazole (an anti-fungal agent) as a model developmental toxicant, Dr. Johnson performed an in vivo dose-response study and compared the utility of apical endpoints (histopathology, functional changes) with molecular endpoints. Because molecular changes precede apical changes, he predicted this would be a sensitive method. While using miRNA to estimate apical points of departure, this is a unique application in the context of developmental risk assessment. Because toxicant exposures during embryofetal development can have severe consequences, establishing reliable methods for predicting safe chemical exposure limits is critical.

Dr. Johnson found that point-of-departure (POD) calculations based on apical and molecular endpoints were similar, meaning that using a molecular-based POD would be protective. A downside of molecular endpoints seems to be that there are likely organ and cell-specific changes in gene expression following exposures. Without knowing the likely site of toxicity, how would you know which tissues to evaluate for molecular changes? And for that matter, which time point do you use? What happens if you miss the window where the greatest gene expression changes are occurring? There is certainly potential for molecular endpoints in POD modeling, but it’s difficult to understand how bulk miRNA and mRNA analyses will be useful for a chemical with unknown sites of toxicity.

Jodi Flaws (University of Illinois): “The Transgenerational Effects of Phthalates on Female Reproduction”

Phthalates have been called “everywhere chemicals,” an apt name due to their widespread use in everyday products—from toys to flooring to personal care products. Women and children have high exposure from increased contact with personal care products and toys. In her talk, Dr. Flaws discusses her lab’s efforts to understand how these ubiquitous endocrine-disrupting chemicals alter ovarian follicle growth, steroidogenesis, and female fertility.

One unique aspect of Dr. Flaws’s work on phthalates is that her lab is using a mixture containing six phthalates. She based the ratios of phthalates in this mixture on an epidemiology study (iKids), using ratios actually found in pregnant women. Both the doses and ratios were environmentally relevant and equivalent to human exposures.

What are the effects of this mixture on female reproduction? One of the most striking effects of phthalate exposure was on the ovaries, which—even to an untrained eye—were greatly enlarged and cystic. This effect was observed out to the F3 generation, which was not directly exposed. (The generations directly exposed were F0, F1, and F2.)

Dr. Flaws found that prenatal phthalate exposure altered female reproduction through F4. It is interesting and alarming to see that prenatal exposure to phthalates may accelerate several biomarkers of reproductive aging in a multi- and transgenerational manner in female mice.

Tracie Baker (University of Florida): “Zebrafish as a Model for Induction of Transgenerational Inheritance of Male Infertility due to Environmental Contaminants”

Closing out the session, Dr. Baker presented her lab’s research on the genetic and epigenetic regulation triggered by exposure to dioxin, an endocrine-disrupting chemical. To do this, she utilizes a zebrafish model. Why zebrafish? One of the advantages of this model is that it can be used to study adverse transgenerational outcomes and the fetal basis of adult disease. Additionally, zebrafish share with humans 99% of the genes that are essential for embryonic development.

As an endocrine-disrupting chemical, dioxin can mimic or interfere with the body’s hormones. Dr. Baker was therefore interested in understanding if exposures to dioxin in a parental cohort can affect subsequent generations of zebrafish. And in fact, she found that dioxin exposure leads to altered spermatogenesis, steroidogenesis, lipid metabolism, and xenobiotic metabolism pathway responses across multiple generations.

There are many ways by which parental exposures can affect outcomes in unexposed offspring, each of which involves changes in parental germ line cells (eggs and sperm). For example, exposure to some DNA-damaging agents can cause heritable DNA mutations. Dioxin and other endocrine-disrupting chemicals that Dr. Baker studies do not cause DNA damage. Dr. Baker explains that, in her transgenerational models, she is looking at a different kind of heritable change: the epigenetic process of DNA methylation.

Following dioxin exposure early in the development of a parental generation, she found that multiple genes involved in reproductive processes or epigenetic modifications were differentially methylated, with a uniquely high number of differentially methylated sites and genes in the F1 generation. She has linked this DNA methylation with transgenerational gene expression and phenotype changes, including decreased reproductive capacity mediated through the male germ line that persists for multiple, unexposed generations.

Key Term
Abbreviation(s)
Definition

2,3,7,8-Tetrachlorodibenzodioxin

TCDD

The chemical name for dioxin

Adverse outcome pathways

AOP

A model that identifies the sequence of molecular and cellular events required to produce a toxic effect when an organism is exposed to a substance

Agrochemicals

Chemical products composed of fertilizers, plant-protection chemicals or pesticides, and plant-growth hormones used in agriculture

Developmental and reproductive toxicity

DART

Developmental toxicity: adverse effects to the embryo or fetus

Reproductive toxicity: overlaps with developmental toxicity but also refers to the adverse effects on sexual function and fertility in adult males and females

Di(2-ethylhexyl)phthalate

DEHP

A manufactured chemical that is commonly added to plastics to make them flexible; best-studied model endocrine-disrupting chemical

Dichloroacetic Acid

DCA

A chlorinated acetic acid that has been reported to be a mouse liver carcinogen

Diisononyl Phthalate

DINP

A manufactured chemical that is commonly added to plastics to make them flexible; endocrine-disrupting chemical used to replace DEHP

Dioxins

TCDD, PCDDs, PCDFs,

(a) 2,3,7,8- tetrachlorodibenzo para dioxin (TCDD)

(b) A group of toxic chemical compounds that share certain chemical structures and biological characteristics, including polychlorinated dibenzo para dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs)

DNA methylation

An epigenetic process by which gene expression is regulated through the addition of methyl groups to the DNA molecule; heritable

Endocrine-disrupting chemicals

EDCs

Chemicals that mimic or interfere with the body’s hormones

Epigenetics

The study of changes in gene activity caused by mechanisms other than DNA sequence changes: DNA methylation, DNA-protein interactions, chromatin accessibility, histone modifications

Epigenetic silencing

Non-mutational modification that leads to inactivation of a gene, often due to changes in levels of DNA methylation or modification to histone proteins (and chromatin accessibility)

Gene expression

The process by which the information encoded in a gene is used to direct the assembly of a protein molecule; quantified by assessing levels of transcript mRNA (RT-qPCR, RNA-seq) or protein (western blot)

Genome

The complete set of genes or genetic material present in a cell or an organism

Heritable

Transmissible from parent to offspring

Ketoconazole

An antiandrogen and antifungal medication

Mechanism of action

MOA

The specific biochemical and/or molecular-level changes from the exposure of a living organism to a substance

MicroRNA

miRNA

A small single-stranded non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression

Mode of action

MoA

A functional or anatomical change, or a change at the cellular level, resulting from the exposure of a living organism to a substance

Multigenerational toxicity

Describes adverse effects when multiple generations have direct exposure to a toxicant (examples: when a gestating female mouse is exposed to a toxicant, the offspring also are exposed in utero; a female mouse co-housed with offspring are exposed to a toxicant)

Persistent organic pollutants

POPs

“Forever chemicals”; organic compounds that are resistant to environmental degradation through chemical, biological, and photolytic processes

Phthalates

“The everywhere chemical”; man-made chemical compounds used in the manufacture of plastics, solvents, and personal care products

Plasticizers

A substance that is added during the production of plastics to increase flexibility and durability

Point-of-Departure Modeling

POD

The point on a toxicological dose-response curve corresponding to an estimated low-effect level or no-effect level; highest dose producing no effect (at a specific response magnitude)

Spermatogenesis

The development of the sperm cells within the male reproductive organs, the testes

Steroidogenesis

The formation of steroids, as by the adrenal cortex, testes, and ovaries

TempO-seq

Multiplexed molecular profiling platform: targeted RNA expression or DNA variant assays designed to monitor tens to tens of thousands of genes or markers

Teratogens

An agent or factor that causes malformation of an embryo

Toxicoepigenomics

Research examining exposure-induced changes to the epigenome

Transcription factor

A protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence

Transcriptome

The sum total of all the messenger RNA molecules expressed from the genes of an organism

Transgenerational toxicity

Describes adverse effects of parental exposure that extend to offspring and future generations of offspring (example: exposure of a male mouse before fathering offspring leads to toxicity in offspring)

Whole-genome bisulfite sequencing

WGBS

A next-generation sequencing technology used to detect DNA methylation patterns

Xenobiotic

A chemical that is foreign to the body or to an ecological system (drugs, pesticides, cosmetics, food additives, pollutants, etc.)

Educational figures created with BioRender.com.

This blog was prepared by an SOT Reporter and represents the views of the author. SOT Reporters are SOT members who volunteer to write about sessions and events in which they participate during the SOT Annual Meeting and ToxExpo. SOT does not propose or endorse any position by posting this article. If you are interested in participating in the SOT Reporter program in the future, please email Giuliana Macaluso.

On-demand recordings of all Featured and Scientific Sessions delivered during the 2022 SOT Annual Meeting and ToxExpo will be available to meeting registrants in the SOT Event App and Online Planner after their conclusion, through July 31, 2022.


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