Overview of the “Epigenetic End Points in Toxicologic Pathology and Relevance to Human Health” Session of the 2014 Society of Toxicologic Pathology Annual Symposium

The 1st presentation, by Dr. Jay Goodman from Michigan State University (East Lansing, MI), was an overview of epigenetics as heritable mechanisms superimposed on DNA that influence gene expression, but without an underlying change in the DNA sequence (Watson and Goodman 2002). Dr. Goodman described how factors influencing epigenetic alterations are integrated and interact together in a coordinated fashion to influence transcription, including methylation, chromatin remodeling, and noncoding RNAs. He emphasized that these alterations are important in toxicologic responses to chemicals (Moggs et al. 2004). He went on to discuss new alterations in methylation that have been recently discovered and that these changes are not completely understood. There is increasing interest in the effects of epigenetics on the mechanisms of toxicity and disease, and how these effects may play a causative role or serve as an underlying susceptibility factor in development of disease. He is particularly interested in the role of epigenetics in adverse transgenerational effects, that is, epigenetic alterations that are heritable and passed down through multiple generations. This would suggest that epigenetic changes from previous generations can influence disease in distant generations of offspring. Dr. Goodman went on to describe the potential effects of nutrition, inflammation, metabolism, and DNA damage on the control of epigenetic alterations. In addition, rationales for determining whether or not to consider analysis of epigenetic end points in preclinical studies with regard to mechanism of action and safety assessment were discussed. For example, given the wide array of changes that are influenced by multiple factors, and in turn influence gene expression in various ways, it is important to consider fundamental properties of toxicology when considering epigenetic analysis, including dose–response, determination of the maximum tolerated dose, consideration of normal intra-animal biologic variability, potential beneficial effects of epigenetic modifications, and importantly, translation of epigenetic changes and their effects from rodents to humans, and relevance of those changes (Goodman et al. 2010).

The 2nd presentation, by Dr. Reza Rasoulpour from The Dow Chemical Company (Midland, MI), provided an overview of the investigation of the role of epigenetics in product safety assessment. The field of epigenetics adds another layer of complexity to the interpretation of how the epigenome, genome, transcriptome, and exposome interact to cause disease. There is increasing concern regarding whether the current preclinical testing system accounts for epigenetic mechanisms, and if the current testing strategy needs to incorporate these end points (Alyea, Gollapudi, and Rasoulpour 2014). Dr. Rasoulpour stressed that as epigenetic mechanisms are more defined in terms of associations with toxicologic responses in the context of exposures, it becomes increasingly difficult to anchor those responses with changes in transcription or apical end points such as histopathology or clinical pathology. How are epigenetic changes interpreted in the space of toxicologic responses, tissue changes, and genomic alterations? How is the dose–response interpreted in terms of these changes? To address these issues, and to determine whether epigenetic end points could play a role in safety assessment, Dr. Rasoulpour gave examples from the literature as well as his own data. His laboratory has performed experiments involving exposure of multiple species to estrogenic and nongenotoxic molecules, followed by measurement and correlation of epigenetic, molecular, and apical end points (Alyea et al. 2012). He concluded that epigenetic changes could be correlated with current points of departure based on the no adverse effect level (NOAEL). However, since the epigenome is constantly changing based on development, aging, and nutritional status, determination of the causality of these changes in the development of toxicity and disease is very challenging. He stressed that a better understanding of how epigenetic changes influence dose–response in the context of apical end points is needed, and recommended that further epigenetic studies be explored to evaluate the correlation between epigenetic changes and apical end points, and to determine the meaning of dose–response related to epigenetic end points, and how they relate to those of apical end points (Alyea, Gollapudi, and Rasoulpour 2014).

The 3rd presentation was given by Dr. Stephen Baylin from the Sydney Kimmel Comprehensive Cancer Center at Johns Hopkins University (Baltimore, MD). He focused on epigenetic changes that occur in cancer, methodologies to detect them, and potential therapies involving targeting such changes. Chronic inflammation (reactive oxygen species [ROS] or hydrogen peroxide [H₂O₂]) with alterations in extracellular scaffolding proteins and their signaling have been identified as predisposing to epigenetic alterations. Recent work with cancer cell lines and freshly isolated neoplasms has identified a large number of epigenetic alterations in cancer cells. Dr. Baylin explained that, in normal cells, active transcription sites are acted upon by transcription factor proteins that provide fine levels of transcriptional control. Cell stress (such as oxidative damage or local inflammation) is associated with movement of these transcription controller proteins, so that they cover the transcription start sites to prevent damage. Long-term stress is associated with more permanent epigenetic modifications such as DNA methylation. Many of the genes that are suppressed are checkpoint genes, tumor suppressor genes, or immune tolerance molecules (Wee et al. 2014). Dr. Baylin discussed the fact that this altered gene expression profile either allows increased cell division with less checkpoint efficiency or helps to minimize the immune surveillance of the neoplasm. Many of these changes may occur more frequently in certain populations such as stem cells, where important proliferation genes are either expressed or in a poised state (Easwaran, Tsai, and Baylin 2014). Current investigations suggest use of low-dose epigenetic therapies such as azacytidine, a DNA methyltransferase (DNMT) inhibitor, may help remove these suppressive epigenetic marks in neoplastic cells, resulting in reexpression of tumor suppressor genes (Li et al. 2014). Indeed, priming with epigenetic therapies before administration of cancer chemotherapy has shown increased efficacy in some resistant cancers (Ahuja, Easwaran, and Baylin 2014). Further understanding of the molecular events will help provide rationale for enhanced efficacy while minimizing unintended toxicities of the priming agent as well as the chemotherapeutic agent.

The 4th presentation, by Dr. Michael Boyle from Amgen (Thousand Oaks, CA), provided an interesting discussion regarding chromatin remodeling as an epigenetic mechanism influencing gene transcription, its role in both normal development and disease, and potential applications for its use in target identification. The formation of chromatin involves wrapping of DNA around nucleosomes and assembly of these nucleosomes into secondary and tertiary structures in order to compress the approximately 1.7 m of DNA present within a normal cell into the nucleus (Ho and Crabtree 2010). In order for transcription to occur, portions of the genome that are compacted in this manner require temporary accessibility to cellular machinery, which requires elongation of the DNA sequence. Chromatin remodeling complexes facilitate this elongation through hydrolysis of ATP in order to allow transcriptional machinery access to the genome (Trotter and Archer 2004). Dr. Boyle further discussed the function of Brahma-related gene 1 (Brg1), a chromatin remodeling factor, in transcriptional control, and its protective role in animals exposed to doxorubicin, a cancer chemotherapeutic with cardiotoxic properties. Animals with induced deletion of Brg1 had increased incidence and severity of heart lesions consistent with cardiotoxicity.

The 5th presentation of the session, by Dr. Theresea Alenghat from the University of Pennsylvania (Philadelphia, PA), focused on the role of the microbiome in health and disease and epigenetic cross talk between the mammalian host and the microbiome. Dr. Alenghat discussed the role of the gut microflora as important environmental influences on normal physiologic homeostasis and disease development. Histone deacetylases (HDACs) are epigenetic enzymes that alter gene expression and may be influenced by host or environmental factors, including bacteria-derived signals (Chen et al. 2012). Dr. Alenghat went on to describe the interplay between mammalian HDACs and the microbiome. For example, alterations in gut microflora and increased susceptibility to intestinal disease occurred in mice lacking intestinal epithelial expression of HDAC3 (Alenghat et al. 2013). In addition, the commensal-derived metabolite, butyrate, can regulate the immune system through HDACs.

This session provided the membership with new and useful information about the use of epigenetic studies in toxicologic pathology and translational studies. As the field of epigenetics continues to expand into the toxicologic pathology space, pathologists will play an increasingly critical role in the study of these alterations. How these changes influence host disease and how they may be targeted for drug development in humans will continue to be an important consideration in translational science.