Genotoxicity assessment is a critical component in the development and evaluation of chemicals. Traditional genotoxicity assays (i.e., mutagenicity, clastogenicity, aneugenicity) have been limited to dichotomous hazard classification, while other toxicity endpoints are assessed through quantitative determination of points-of-departure (PODs) for setting exposure limits. The more recent higher-throughput in vitro genotoxicity assays, many of which also provide mechanistic information, offer a powerful approach for determining high-precision PODs for potency ranking and risk assessment. In order to obtain relevant human dose context from the in vitro assays, in vitro to in vivo extrapolation (IVIVE) models are required to determine what dose would elicit a concentration in the body demonstrated to be genotoxic using in vitro assays. Previous work has demonstrated that application of IVIVE models to in vitro bioactivity data can provide PODs that are protective of human health, but there has been no evaluation of how these models perform with in vitro genotoxicity data. Thus, the Genetic Toxicology Technical Committee, under the Health and Environmental Sciences Institute, conducted a case study on 31 reference chemicals to evaluate the performance of IVIVE application to genotoxicity data. The results demonstrate that for most chemicals (20/31), the PODs derived from in vitro data and IVIVE are highly health protective relative to in vivo PODs from animal studies. PODs were also protective by individual assay type: mutations (8/13 chemicals), micronuclei (9/12) and aneugenicity markers (4/4). It is envisioned that this novel testing strategy could enhance prioritization, rapid screening, and risk assessment of genotoxic chemicals.
Quantitative risk assessments of chemicals are routinely performed in rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.
Inorganic Arsenic (iAs) is one of the largest toxic exposures to impact humanity worldwide. Exposure to iAs during pregnancy may disrupt the proper remodeling of the epigenome of F1 developing offspring and potentially their F2 grand-offspring via disruption of fetal primordial germ cells (PGCs). There is a limited understanding between the correlation of disease phenotype and methylation profile within offspring of both generations and whether it persists to adulthood. Our study aims to understand the intergenerational effects of in utero iAs exposure on the epigenetic profile and onset of disease phenotypes within F1 and F2 adult offspring, despite the life-long absence of direct arsenic exposure within these generations. We exposed F0 female mice (C57BL6/J) to the following doses of iAs in drinking water 2 weeks before pregnancy until the birth of the F1 offspring: 1 ppb, 10 ppb, 245 ppb, and 2300 ppb. We found sex- and dose-specific changes in weight and body composition that persist from early time to adulthood within both generations. Fasting blood glucose challenge suggests iAs exposure causes dysregulation of glucose metabolism, revealing generational, exposure, and sex specific differences. Toward understanding the mechanism, genome-wide DNA methylation data highlights exposure-specific patterns in liver, finding dysregulation within genes associated with cancer, T2D, and obesity. We also identified regions containing persistently differentially methylated CpG sites between F1 and F2 generations. Our results indicate F1 developing embryos and F2 PGCs retain epigenetic damage established during the prenatal period and are associated with adult metabolic dysfunction.
Consumption of foods contaminated with aflatoxin B1 (AFB1) is a recognized risk factor for developing hepatocellular carcinomas (HCCs). The mutational signature of AFB1 is characterized by high frequency G > T transversions in a limited subset of trinucleotide sequences. The 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) has been implicated as the primary DNA lesion responsible for AFB1-induced mutations. This study evaluated the mutagenic potential of AFB1-FapyGua in four contexts, including hot- and cold-spot sequences as apparent in the mutational signature. Vectors containing AFB1-FapyGua were replicated in primate cells and the products of replication were isolated and sequenced. Regardless of the sequence context, AFB1-FapyGua caused base substitutions at frequencies of ~ 80-90%, with G > T transversions being most common. Spectra of mutations were only slightly modulated by the sequence context. These data suggest that mechanism(s) defining sequence context-dependent distribution of AFB1-induced mutations likely operates prior to replication.
DNA damage occurs throughout life from a variety of sources, and it is imperative to repair damage in a timely manner to maintain genome stability. Thus, DNA repair mechanisms are a fundamental part of life. Nucleotide Excision Repair (NER) plays an important role in the removal of bulky DNA adducts, such as cyclobutane pyrimidine dimers (CPDs) from ultraviolet (UV) light or DNA crosslinking damage from platinum-based chemotherapeutics, such as cisplatin. A main component for the NER pathway is transcription factor IIH (TFIIH), a multifunctional, 10-subunit protein complex with crucial roles in both transcription and NER. In transcription, TFIIH is a component of the pre-initiation complex (PIC) and is important for promoter opening and the phosphorylation of RNA Polymerase II (RNA Pol II). During repair, TFIIH is important for DNA unwinding, recruitment of downstream repair factors, and verification of the bulky lesion. Several different disease states can arise from mutations within subunits of the TFIIH complex. Most strikingly are Xeroderma Pigmentosum (XP), XP combined with Cockayne Syndrome (CS), and Trichothiodystrophy (TTD). Here, we summarize the recruitment and functions of TFIIH in the two NER subpathways, global genomic (GG-NER) and transcription-coupled NER (TC-NER). We will also discuss how TFIIH’s roles in the two subpathways lead to different genetic disorders.
We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip for DNA damage and the micronucleus (MN) assay as a follow up for in vitro positive genotoxic response as alternatives to in vivo genotoxicity testing.. Naphthalene is genotoxic with rat liver S9 in human TK6 cells inducing a nonlinear dose-response for the induction of micronuclei in the presence of rat liver S9. To follow up this response, we used metabolically competent HepaRG™ cells as a New Approach Methodology (NAM) alternative to animals for genotoxicity assessment of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using 12 concentrations of naphthalene with the top dose used for assessment of genotoxicity of 1.7 mM corresponding to 45% cell survival. In contrast to human TK6 cell with S9, Naphthalene was not genotoxic in either the HepaRG™ MN Assay or the Comet Assay using CometChip. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. This data indicates that rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity BMD calculations when compared to hepatocytes. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents.