Styrene has been shown to induce lung tumors in mice, but not in rats. The current study investigated the potential role of genotoxicity as an initial key event in the mode of action for styrene-induced lung tumors in mice. Transgenic male B6C3F1 Big Blue® mice were treated by oral gavage for 28 consecutive days with 0 (corn oil), 75, 150, or 300 mg/kg/day of styrene. The 300 mg/kg/day represented the tumorigenic dose in the oral gavage carcinogenicity study conducted in B6C3F1 mice. Following a 28-day expression period, mutant frequencies were assessed at the cII locus of the transgene in the tumor target (lung) and non-target tissues (liver, glandular stomach, and duodenum). Mice treated with N-ethyl-N-nitrosourea (40 mg/kg/day) by oral gavage on Days 1, 2, and 3 of the study and sacrificed on Day 56 served as the positive control group. Genomic DNA was extracted from the selected tissues, processed for the recovery of the transgene into infectious phage, plated onto Escherichia coli strain G1250, and incubated at 37°C for titer determination or at 24°C for the selection of mutant plaques. There were no treatment-related increases in mutant frequency in any of the tissues. The positive control group had a significant increase in the frequency of cII mutants assuring the adequacy of the experimental conditions to detect induced mutations. To conclude, mutagenicity is not considered a plausible initial key event in the mode of action for styrene-induced mouse lung tumors as these data support that styrene is not an in vivo mutagen.

Inhalation of nanosized metal oxides may occur at the workplace. Thus, information on potential hazardous effects is needed for risk assessment. We report an investigation of the genotoxic potential of different metal oxide nanomaterials. Acellular and intracellular reactive oxygen species (ROS) production were determined for all the studied nanomaterials. Moreover, mice were exposed by intratracheal instillation to copper oxide (CuO) at 2, 6, and 12 μg/mouse, tin oxide (SnO) at 54 and 162 μg/mouse, aluminum oxide (AlO) at 18 and 54 μg/mouse, zinc oxide (ZnO) at 0.7 and 2 μg/mouse, titanium dioxide (TiO) and the benchmark carbon black at 162 μg/mouse. The doses were selected based on pilot studies. Post-exposure time points were 1 or 28 days. Genotoxicity, assessed as DNA strand breaks by the comet assay, was measured in lung and liver tissue. The acellular and intracellular ROS measurements were fairly consistent. The CuO and the carbon black bench mark particle were potent ROS generators in both assays, followed by TiO. AlO, ZnO, and SnO generated low levels of ROS. We detected no increased genotoxicity in this study using occupationally relevant dose levels of metal oxide nanomaterials after pulmonary exposure in mice, except for a slight increase in DNA damage in liver tissue at the highest dose of CuO. The present data add to the body of evidence for risk assessment of these metal oxides.

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 also 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). This qualitative AOP was developed in collaboration with bone loss research experts to aggregate relevant findings, supporting ongoing efforts to understand and mitigate human system risks associated with radiation exposures. A literature review was conducted to compile and evaluate the state of knowledge based on the modified Bradford Hill criteria. Following review of 2029 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.

We previously reported that certain sub-regions of the thyA gene of Escherichia coli are more mutable than others when many different mutagens and mutators are analyzed (Mashiach et al., Mutation Research Fundamental Molecular Mechansims of Mutagenesis, 821: 111702, 2021). In this study, we focus on a single mutagen, cisplatin and verify that mutations occur preferentially at specific 3 bp sequences, but only when they appear in certain subregions of the gene. Moreover, we show that hotspots for some premutational lesions are camouflaged by the preferential repair effected by the uvrA,B,C-encoded excision repair system, even when they appear on the same strand. We do this by using a novel reporter gene in E. coli, the tdk gene that codes for thymidine deoxykinase, and we describe some of the advantages of utilizing this detection system.

Cardiovascular diseases (CVDs) are complex, encompassing many types of heart pathophysiologies and associated etiologies. Radiotherapy studies have shown that fractionated radiation exposure at high doses (3-17 Gy) to the heart increases the incidence of CVD. 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 abnormal 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 that informed final key event (KE) selection and evaluation of the Bradford Hill criteria for the KE relationships (KERs). The AOP begins with a molecular initiating event of deposition of energy; ionization events increase oxidative stress, which when persistent concurrently causes the release of pro-inflammatory mediators, suppresses anti-inflammatory mechanisms and alters stress response signaling pathways. These KEs alter nitric oxide levels leading to endothelial dysfunction, and subsequent abnormal 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.

The human NEIL1 DNA glycosylase is one of 11 mammalian glycosylases that initiate base excision repair. While substrate preference, catalytic mechanism, and structural information of NEIL1's ordered residues are available, limited information on its subcellular localization, compounded by relatively low endogenous expression levels, have impeded our understanding of NEIL1. Here, we employed a previously developed computational framework to optimize the mitochondrial localization signal of NEIL1, enabling the visualization of its specific targeting to the mitochondrion via confocal microscopy. While we observed clear mitochondrial localization and increased glycosylase/lyase activity in mitochondrial extracts from low-moderate NEIL1 expression, high NEIL1 mitochondrial expression levels proved harmful, potentially leading to cell death.

Chlorothalonil (CTL) is a pesticide widely used in Brazil, yet its mutagenic potential is not fully determined. Thus, we assessed the mutagenicity of CTL and its bioactivation metabolites using the somatic mutation and recombination test (SMART) in Drosophila melanogaster, by exposing individuals, with basal and high bioactivation capacities (standard and high bioactivation cross offspring, respectively), from third instar larval to early adult fly stages, to CTL-contaminated substrate (0.25, 1, 10 or 20 μM). This substrate served as food and as physical medium. Increased frequency of large single spots in standard cross flies' wings exposed to 0.25 μM indicates that, if CTL is genotoxic, it may affect Drosophila at early life stages. Since the total spot frequency did not change, CTL cannot be considered mutagenic in SMART. The same long-term exposure design was performed to test whether CTL induces oxidative imbalance in flies with basal (wild-type, WT) or high bioactivation (ORR strain) levels. CTL did not alter reactive oxygen species and antioxidant capacity against peroxyl radicals levels in adult flies. However, lipid peroxidation (LPO) levels were increased in WT male flies exposed to 1 μM CTL. SMART and LPO alterations were observed only in flies with basal bioactivation levels, pointing to direct CTL toxicity to DNA and lipids. Survival, emergence and locomotor behavior were not affected, indicating no bias due to lethality, developmental and behavioral impairment. We suggest that, if related to CTL exposure, DNA and lipid damages may be residual damage of earlier life stages of D. melanogaster.

The genotoxic and clastogenic/aneugeneic potentials of four α,β-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 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 previous 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. Such variation in responses between the CEGA and HET-MN assay can be attributed to differences in the dosing protocols. 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 chicken egg and rodent models can be attributed to rapid glutathione depletion. 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.

Understanding radiation-induced non-cancer effects on the central nervous system (CNS) is essential for the risk assessment of medical (e.g., radiotherapy) and occupational (e.g., nuclear workers and astronauts) exposures. 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 acquired through a documented scoping review of the literature. In this AOP, the MIE is connected to the AO via six KEs: increased oxidative stress, increased deoxyribonucleic acid (DNA) strand breaks, altered stress response signaling, tissue resident cell activation, increased pro-inflammatory mediators, and abnormal neural remodeling that encompasses atypical structural and functional alterations of neural cells and surrounding environment. 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, can lead to abnormal neural remodeling impacting learning and memory processes. Identified knowledge gaps include improving quantitative understanding of the AOP across several KERs and additional testing of proposed modulating factors through experimental work. Broadly, it is envisioned that the outcome of these efforts could be extended to other cognitive disorders and complement ongoing work by international radiation governing bodies in their review of the system of radiological protection.

Reporting any uncommon or untapped changes in bacterial genetics or physiology would be of great importance to support the drug development process. We studied 120 Mycobacterium tuberculosis clinical isolates with different geographical origin within India and their resistance profile and found a significant number of isolates (109) harboring the polymorphism at nucleotide positions 61 and 284 of the gyrA gene. Bioinformatics analysis of these changes for drug binding suggested no significant change in the binding of the drug but have lower binding energies as compared with the wild-type proteins. Although functionally silent for the gyrA gene, these changes are indicating a silent geographical and evolutionary change that needs to be further studied for drug discovery and bacterial fitness.

Regulatory genetic toxicology testing is essential for identifying potentially mutagenic hazards. Duplex Sequencing (DS) is an error-corrected next-generation sequencing technology that provides substantial advantages for mutation analysis over conventional mutagenicity assays including: improved accuracy of mutation detection, ability to measure changes in mutation spectrum, and applicability across diverse biological models. To apply DS for regulatory toxicology testing, power analyses are required to determine suitable sample sizes and study designs. In this study, we explored study designs to achieve sufficient power for various effect sizes in chemical mutagenicity assessment. We collected data from MutaMouse bone marrow and liver samples that were analyzed by DS using TwinStrand's Mouse Mutagenesis Panel. Average duplex reads achieved in two separates studies on liver and bone marrow were 8.4 × 10 (± 7.4 × 10) and 9.5 × 10 (± 1.0 × 10), respectively. Baseline mean mutation frequencies (MF) were 4.6 × 10 (± 6.7 × 10) and 4.6 × 10 (± 1.1 × 10), with estimated standard deviations for the animal-to-animal random effect of 0.15 and 0.20, for liver and bone marrow, respectively. We conducted simulation analyses based on these empirically derived parameters. We found that a sample size of four animals per group is sufficient to obtain over 80% power to detect a two-fold change in MF relative to baseline. In addition, we estimated the minimal total number of informative duplex bases sequenced with different sample sizes required to retain power for various effect sizes. Our work provides foundational data for establishing suitable study designs for mutagenicity testing using DS.

ADP-ribosylation is a reversible post-translational modification that plays a role as a signaling mechanism in various cellular processes. This modification is characterized by its structural diversity, highly dynamic nature, and short half-life. Hence, it is tightly regulated at many levels by cellular factors that fine-tune its formation, downstream signaling, and degradation that together impacts cellular outcomes. Poly-ADP-ribosylation is an essential signaling mechanism in the DNA damage response that mediates the recruitment of DNA repair factors to sites of DNA damage via their poly-ADP-ribose (PAR)-binding domains (PBDs). PAR readers, encoding PBDs, convey the PAR signal to mediate cellular outcomes that in some cases can be dictated by PAR structural diversity. Several PBD families have been identified, each with variable PAR-binding affinity and specificity, that also recognize and bind to distinct parts of the PAR chain. PARylation signaling has emerged as an attractive target for the treatment of specific cancer types, as the inhibition of PAR formation or degradation can selectively eliminate cancer cells with specific DNA repair defects and can enhance radiation or chemotherapy response. In this review, we summarize the key players of poly-ADP-ribosylation and its regulation and highlight PBDs as tools for studying PARylation dynamics and the expanding potential to target PARylation signaling in cancer treatment.

Regulatory genetic toxicology focuses on DNA damage and subsequent gene mutations. However, genotoxic agents can also affect epigenetic marks, and incorporation of epigenetic data into the regulatory framework may thus enhance the accuracy of risk assessment. Additionally, epigenetic alterations may identify non-genotoxic carcinogens that are not captured with the current battery of tests. Epigenetic alterations could also explain long-term consequences and potential transgenerational effects in the absence of DNA mutations. Therefore, at the 2022 International Workshops on Genotoxicity Testing (IWGT) in Ottawa (Ontario, Canada), an expert workgroup explored whether including epigenetic endpoints would improve regulatory genetic toxicology. Here we summarize the presentations and the discussions on technical advancements in assessing epigenetics, how the assessment of epigenetics can enhance strategies for detecting genotoxic and non-genotoxic carcinogens and the correlation between epigenetic alterations with other relevant apical endpoints.

At the 8th International Workshop on Genotoxicity Testing meeting in Ottawa, in 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.

Methyl tertiary-butyl ether (MTBE) is used as a component of motor vehicle fuel to enhance combustion efficiency and to reduce emissions of carbon monoxide and nitrogen oxides. Although MTBE was largely negative in the in vitro and in vivo genotoxicity studies, isolated reports of positive findings along with the observation of tumors in the rat cancer bioassays raised concern for its in vivo mutagenic potential. To investigate this, transgenic male Big Blue Fischer 344 rats were exposed to 0 (negative control), 400, 1000, and 3000 ppm MTBE via whole body inhalation for 28 consecutive days, 6 h/day. Mutant frequencies (MF) at the cII locus of the transgene in the nasal epithelium (portal of entry tissue), liver (site of primary metabolism), bone marrow (rapidly proliferating tissue), and kidney (tumor target) were analyzed (5 rats/exposure group) following a 3-day post-exposure manifestation period. MTBE did not induce a mutagenic response in any of the tissues investigated. The adequacy of the experimental conditions to detect induced mutations was confirmed by utilizing tissue samples from animals treated with the known mutagen ethyl nitrosourea. These data provide support to the conclusion that MTBE is not an in vivo mutagen and male rat kidney tumors are not likely the result of a mutagenic mode of action.

The detection of N-nitrosamines in drug products has raised global regulatory interest in recent years due to the carcinogenic potential of some nitrosamines in animals and a need to identify a testing strategy has emerged. Ideally, methods used would allow for the use of quantitative analysis of dose-response data from in vivo genotoxicity assays to determine a compound-specific acceptable intake for novel nitrosamines without sufficient carcinogenicity data. In a previous study we compared the dose-response relationships of N-nitrosodiethylamine (NDEA) in three in vivo genotoxicity endpoints in rats. Here we report a comparison of NDEA's genotoxicity profile in mice. Big Blue® mice were administered NDEA at doses of 0.001, 0.01, 0.1, 1 and 3 mg/kg/day by oral gavage for 28 days followed by 3 days of expression. Statistically significant increases in the NDEA induced mutations were detected by both the transgenic rodent mutation assay (TGR) using the cII endpoint and by duplex sequencing in the liver but not bone marrow of mice. In addition, administration of NDEA for two consecutive days in male C57BL/6N mice caused elevated DNA damage levels in the liver as measured by % tail DNA in comet assay. The benchmark dose (BMD) analysis shows a BMDL of 0.03, 0.04 and 0.72 mg/kg/day for TGR, duplex sequencing and comet endpoints, respectively. Overall, this study demonstrated a similar genotoxicity profile of NDEA between mice and rats and provides a reference that can be used to compare the potential potency of other novel nitrosamines for the induction of gene mutations.

Evaluation and mitigation of the potential carcinogenic risks associated with nitrosamines in marketed pharmaceutical products are areas of interest for pharmaceutical companies and health authorities alike. Significant progress has been made to establish acceptable intake (AI) levels for N-nitrosamine drug substance-related impurities (NDSRIs) using SAR, however some compounds require experimental data to support derivation of a recommended AI. Many angiotensin-converting enzyme inhibitors, identified by the suffix "pril," have secondary amines that can potentially react to form nitrosamines. Here we consider a structural assessment and metabolism data, coupled with comprehensive in vitro and in vivo (mouse) genotoxicity testing to evaluate this particular class of nitrosamines. N-nitroso ramipril and N-nitroso quinapril, both of which are predicted to have inhibited nitrosamine bioactivation due to steric hinderance and branching at the α-position were non-genotoxic in the in vivo liver comet assay and non-mutagenic in the in vivo Big Blue® mutation and duplex sequencing assays. Predicted metabolism along with in vitro metabolism data and quantum chemical calculations related to DNA interactions offer a molecular basis for the negative results observed in both in vitro and in vivo testing. These nitrosamines are concluded to be non-mutagenic and non-carcinogenic; therefore, they should be controlled according to ICH Q3B guidance. Furthermore, these results for N-nitroso ramipril and N-nitroso quinapril should be considered when evaluating the appropriate AI and control strategy for other structurally similar "pril" NDSRIs.

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

Annotating genomic sequence alterations is sometimes a difficult decision, particularly in missense variants with uncertain pathogenic significance and also in those presumed as germline pathogenic variants. We here suggest that mutation spectrum may also be useful for judging them. From the public databases, 982 BRCA1/1861 BRCA2 germline missense variants and 294 BRCA1/420 BRCA2 somatic missense variants were obtained. We then compared their mutation spectra, i.e., the frequencies of two transition- and four transversion-type mutations, in each category. Intriguingly, in BRCA1 variants, A:T to C:G transversion, which was relatively frequent in the germline, was extremely rare in somatic, particularly breast cancer, cells (p = .03). Conversely, A:T to T:A transversion was most infrequent in the germline, but not rare in somatic cells. Thus, BRCA1 variants with A:T to T:A transversion may be suspected as somatic, and those with A:T to C:G as being in the germline. These tendencies of mutation spectrum may also suggest the biological and chemical origins of the base alterations. On the other hand, unfortunately, variants of uncertain significance (VUS) were not distinguishable by mutation spectrum. Our findings warrant further and more detailed studies.

As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a method reliably assessing DNA ligation capacity in protein extracts from murine tissues was aimed to establish. To optimize applicability, the use of radioactively labeled substrates was avoided and replaced by fluorescently labeled oligonucleotides. Briefly, tissue extracts were incubated with those complementary oligonucleotides so that in an ensuing gel electrophoresis ligated strands could be separated from unconnected molecules. Originally, the method was intended for use in cerebellum tissue to further elucidate possible mechanisms of neurodegenerative diseases. However, due to its inhomogeneous anatomy, DNA ligation efficiency varied strongly between different cerebellar areas, illuminating the established assay to be suitable only for homogenous organs. Thus, for murine liver tissue sufficient intra- and interday repeatability was shown during validation. In further experiments, the established assay was applied to an animal study comprising young and old (24 and 110 weeks) mice which showed that DNA ligation efficiency was affected by neither sex nor age. Finally, the impact of in vitro addition of the trace elements copper, iron, and zinc on DNA ligation in tissue extracts was investigated. While all three metals inhibited DNA ligation, variations in their potency became evident. In conclusion, the established method can be reliably used for investigation of DNA ligation efficiency in homogenous murine tissues.