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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.

Educational figures created with BioRender.com.

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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.