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.

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.

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.

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.

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.

The blood cell phosphatidylinositol glycan class A (PIG-A) gene mutation assay has been extensively researched in rodents for in vivo mutagenicity testing and is now being investigated in humans. The PIG-A gene is involved in glycosyl phosphatidylinositol (GPI)-anchor biosynthesis. A single mutation in this X-linked gene leads to loss of membrane bound GPI-anchors, which can be enumerated using flow cytometry. With many studies published to date measuring this mutation in erythrocytes, there is remarkable consistency across research groups. Moreover, with the low background level of mutant erythrocytes in healthy subjects (2.9 - 5.56 x 10-6 mutants), induction of mutation post genotoxic exposure can be detected. Cigarette smoking, radiotherapy and occupational exposures including lead, have been shown to increase mutant levels. Identification of harmful agents can allow us to recommend new exposure limits, minimising individual risk. Conversely, protective agents such as aspirin and healthy diets could mitigate these effects, reducing baseline somatic mutation levels and such behaviours can be encouraged. This mutational monitoring approach may also provide information on individuals at higher risk of cancer development. Patients with inflammatory bowel disease, oesophageal adenocarcinoma and pancreatic cancer have elevated numbers of PIG-A mutant erythrocytes compared to age-matched controls. With further technological progress including protocol standardisation and the development of cryopreservation methods to improve GPI-anchor stability, this assay can be widely employed in rural and low-income countries. Here we review the current literature on PIG-A mutation in human subjects and discuss the potential role of this assay in human biomonitoring and disease.

Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also requires understanding how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel RTPCR based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and the pathogenesis of autoimmune disease and cancer.

A document by Elena Esina. Click on the document to view its contents.

The understanding of radiation-induced non-cancer effects on the central nervous system (CNS) is essential for the medical setting (e.g., radiotherapy), and occupational exposures, such as nuclear workers or astronauts. Herein, the adverse outcome pathway (AOP) approach was used to consolidate relevant studies in the area of cognitive decline for identification of research gaps, countermeasure development, and for eventual use in risk assessments. AOPs are an analytical construct describing critical events to an adverse outcome (AO) in a simplified form beginning with a molecular initiating event (MIE). An AOP was constructed utilizing mechanistic information to build empirical support for the key event relationships (KERs) between the MIE of deposition of energy to the AO of learning and memory impairment through multiple key events (KEs). The evidence for the AOP was developed through a scoping review of the literature. In this AOP, the MIE is connected to the AO via six KEs of increased oxidative stress, increased deoxyribonucleic acid (DNA) strand breaks, altered signaling pathways, tissue resident cell activation, increased pro-inflammatory mediators and neural remodeling. Deposition of energy directly leads to oxidative stress, increased DNA strand breaks, an increase of pro-inflammatory mediators and tissue resident cell activation. These KEs, which are themselves interconnected, converge through increased DNA strand breaks, altered signaling pathways and pro-inflammatory routes and directly lead to neural remodeling. Broadly, it is envisioned that the outcome of these efforts could be applied to other cognitive disorders and support ongoing work by international authorities to review the system of radiological protection.

The genotoxic and clastogenic/aneugeneic potentials of four α, ß-unsaturated aldehydes, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal, 2-methyl-2-pentenal and p-methoxy cinnamaldehyde, which are used as fragrance materials, were assessed in avian fetal livers using the Chicken Egg Genotoxicity Assay (CEGA) and the Hen’s egg micronucleus (HET-MN) assay, respectively. Selection of materials was based on their chemical structures and the results of their assessment in the regulatory in vitro and/or in vivo genotoxicity test battery. Three tested materials, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal and 2-methyl-2-pentenal, were negative in both, CEGA and HET-MN assays. These findings were congruent with the results of regulatory in vivo genotoxicity assays. In contrast, p-methoxy cinnamaldehyde, which was also negative in the in vivo genotoxicity assays, produced evidence of DNA damage, including DNA strand breaks and DNA adducts in CEGA, however, no increase in the micronucleus formation in blood was reported in the HET-MN study. Pretreatment with a glutathione precursor, N-acetyl cysteine, negated positive outcomes produced by p-methoxy cinnamaldehyde in CEGA, indicating that difference in response observed in the egg and rodent models can be attributed to rapid glutathione depletion. Additionally, the dosing protocols for both HET-MN and CEGA assays are different, which can also be an important contributing factor. Overall, our findings support the conclusion that CEGA and/or HET-MN can be considered as a potential alternative to animal testing as follow-up strategies for assessment of genotoxic potential of fragrance materials with evidence of genotoxicity in vitro.

Bone loss, commonly seen in osteoporosis, is a condition that entails a progressive decline of bone mineral density and microarchitecture, often seen in post-menopausal women. Bone loss has been widely reported in astronauts exposed to a plethora of stressors and in patients with osteoporosis following radiotherapy for cancer. Studies on mechanisms are well documented but the causal connectivity of events to bone loss development remains incompletely understood. Herein, the adverse outcome pathway (AOP) framework was used to organize data and develop a qualitative AOP beginning from deposition of energy (the molecular initiating event) to bone loss (the adverse outcome). A literature review was conducted to compile and evaluate the state of knowledge based on the modified Bradford Hill criteria. Following review of 1865 studies, an empirically supported AOP was developed, showing the progression to bone loss through many factors affecting the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. The structural, functional, and quantitative basis of each proposed relationship was defined, for inference of causal changes between key events. Current knowledge and its gaps relating to dose-, time- and incidence-concordance across the key events were identified, as well as modulating factors that influence linkages. The new priorities for research informed by the AOP highlight areas for improvement to enable development of a quantitative AOP used to support risk assessment strategies for space travel or cancer radiotherapy.

At the 8th International Workshop on Genotoxicity Testing (IWGT) meeting in Ottawa, August 2022, a plenary session was dedicated to the genotoxicity risk evaluation of gene therapies, including insertional oncogenesis and off-target genome editing. This brief communication summarizes the topics of discussion and the main insights from the speakers. Common themes included recommendations to conduct tailored risk assessments based on a weight of evidence approach, to promote data sharing, transparency, and cooperation between stakeholders, and to develop state-of-the-art validated tests relevant to clinical scenarios.

Cardiovascular diseases (CVDs) are complex, encompassing many types of heart pathophysiologies and associated etiologies. It has been shown that fractionated radiation exposure at high doses (3-17 Gy) to the heart increases the incidence of CVD, as evident from radiotherapy studies. However, the effects of low doses of radiation on the cardiovascular system or the effects from space travel, where radiation and microgravity are important contributors to damage, are not clearly understood. Herein, the adverse outcome pathway (AOP) framework was applied to develop an AOP to vascular remodeling from the deposition of energy. Following the creation of a preliminary pathway with the guidance of field experts and authoritative reviews, a scoping review was conducted which informed final key event (KE) selection and facilitated evaluation Bradford Hill criteria of the key event relationships (KERs). The AOP begins with a molecular initiating event of deposition of energy; ionization events increase oxidative stress, which concurrently causes the release of pro-inflammatory mediators and alters signaling pathways. These KEs alter nitric oxide levels leading to endothelial dysfunction and subsequent vascular remodeling (the adverse outcome). The work identifies evidence needed to strengthen understanding of the causal associations for the KERs, emphasizing where there are knowledge gaps and uncertainties in both qualitative and quantitative understanding. The AOP is anticipated to direct future research to better understand the effects of space on the human body and potentially develop countermeasures to better protect future space travelers.