A multigenerational study can detect the evolutionary response to BaP exposure in the non-biting freshwater midge Chironomus riparius.

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment is posing a threat to ecosystems and human health. Benzo(a)pyrene (BaP) is considered a biomarker of PAH exposure and is classified as a Group 1 carcinogen. However, it was not known whether BaP is mutagenic, i.e. induces inherited germline mutations. In this study, we used a recently established method, which combines short-term mutation accumulation lines (MAL) with whole genome sequencing (WGS) to assess mutagenicity in the non-biting midge Chironomus riparius. The mutagenicity analysis was supplemented by an evaluation of the development of population fitness in three successive generations in the case of chronic exposure to BaP at a high concentration (100 µg/L). In addition, the level of ROS-induced oxidative stress was examined in vivo. Exposure to the higher BaP concentration led to an increase in germline mutations relative to the control, while the lower concentration showed no mentionable effect. Against expectations, BaP exposure decreased ROS-level compared to the control and is thus probably not responsible for the increased mutation rate. Likewise, the higher BaP concentration decreased fitness measured as population growth rate per day (PGR) significantly over all generations, without signs of rapid evolutionary adaptations. Our results thus highlighted that high BaP exposure may influence the evolutionary trajectory of organisms.

Evaluation of genotoxic effect via expression of DNA damage responsive gene induced by ivermectin on MDBK cell line.

Ivermectin (IVM) is an anti-parasitic drug which is used for treating parasitic infestations. It has been used in humans for treating intestinal strongyloidiasis and onchocerciasis however, currently researchers are investigating its potential for treating coronavirus SARS-CoV-2. Due to its broad-spectrum activities, IVM is being used excessively in animals which has generated an interest for researchers to investigate its toxic effects. Cytotoxic and genotoxic effects have been reported in animals due to excessive usage of IVM. Therefore, this study aims to evaluate the cytotoxic and genotoxic effects of IVM on the Madin-Darby-Bovine-Kidney (MDBK) cell line by examining the expression of a DNA damage-responsive gene (OGG1). Cytotoxicity of IVM was tested using an assay (MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), whereas the genotoxicity was evaluated using comet assay along with micronucleus assay. Moreover, the gene expression of DNA damage response gene (OGG1) was measured by qRT-PCR, after extraction of RNA from the MDBK cell line using the TRIzol method and its conversion to cDNA by reverse-transcriptase PCR. During the experiment, cell viability percentage was measured at different doses of IVM i.e., 25%, 50%, 75%, along with LC50/2, LC50 and LC50*2. It was observed that the gene expression of OGG1 increased as the concentration of IVM increased. It was concluded that IVM has both cytotoxic and genotoxic effects on the MDBK cell line. Furthermore, it is recommended that studies related to the toxic effects of IVM at molecular level and on other model organisms should be conducted to combat its hazardous effects.

Assessment of the genotoxicity of tert-butyl alcohol in an in vivo thyroid comet assay.

Chronic exposure to high (20,000 ppm) concentrations of tert-butyl alcohol (TBA) in drinking water, equivalent to ~2100 mg/kg bodyweight per day, is associated with slight increases in the incidence of thyroid follicular cell adenomas and carcinomas in mice, with no other indications of carcinogenicity. In a recent toxicological review of TBA, the U.S. EPA determined that the genotoxic potential of TBA was inconclusive, largely based on non-standard studies such as in vitro comet assays. As such, the potential role of genotoxicity in the mode of action of thyroid tumors and therefore human relevance was considered uncertain. To address the potential role of genotoxicity in TBA-associated thyroid tumor formation, CD-1 mice were exposed up to a maximum tolerated dose of 1500 mg/kg-day via oral gavage for two consecutive days and DNA damage was assessed with the comet assay in the thyroid. Blood TBA levels were analyzed by headspace GC-MS to confirm systemic tissue exposure. At study termination, no significant increases (DNA breakage) or decreases (DNA crosslinks) in %DNA tail were observed in TBA exposed mice. In contrast, oral gavage of the positive control ethyl methanesulfonate significantly increased %DNA tail in the thyroid. These findings are consistent with most genotoxicity studies on TBA and provide mechanistic support for non-linear, threshold toxicity criteria for TBA. While the mode of action for the thyroid tumors remains unclear, linear low dose extrapolation methods for TBA appear more a matter of policy than science.

Assessment of genotoxicity biomarkers in gasoline station attendants due to occupational exposure.

Gasoline station attendants are exposed to numerous chemicals that might have genotoxic and carcinogenic potential, such as benzene in fuel vapor and particulate matter and polycyclic aromatic hydrocarbons in vehicle exhaust emission. According to IARC, benzene and diesel particulates are Group 1 human carcinogens, and gasoline has been classified as Group 2A "possibly carcinogenic to humans." At gas stations, self-service is not implemented in Turkey; fuel-filling service is provided entirely by employees, and therefore they are exposed to those chemicals in the workplace during all working hours. Genetic monitoring of workers with occupational exposure to possible genotoxic agents allows early detection of cancer. We aimed to investigate the genotoxic damage due to exposures in gasoline station attendants in Turkey. Genotoxicity was evaluated by the Comet, chromosomal aberration, and cytokinesis-block micronucleus assays in peripheral blood lymphocytes. Gasoline station attendants (n = 53) had higher tail length, tail intensity, and tail moment values than controls (n = 61). In gasoline station attendants (n = 46), the frequencies of chromatid gaps, chromosome gaps, and total aberrations were higher compared with controls (n = 59). Increased frequencies of micronuclei and nucleoplasmic bridges were determined in gasoline station attendants (n = 47) compared with controls (n = 40). Factors such as age, duration of working, and smoking did not have any significant impact on genotoxic endpoints. Only exposure increased genotoxic damage in gasoline station attendants independently from demographic and clinical characteristics. Occupational exposure-related genotoxicity risk may increase in gasoline station attendants who are chronically exposed to gasoline and various chemicals in vehicle exhaust emissions.

Computational framework for identifying and evaluating mutagenic and xenoestrogenic potential of food additives.

Food additives are chemicals incorporated in food to enhance its flavor, color and prevent spoilage. Some of these are associated with substantial health hazards, including developmental disorders, increase cancer risk, and hormone disruption. Hence, this study aimed to comprehend the in-silico toxicology framework for evaluating mutagenic and xenoestrogenic potential of food additives and their association with breast cancer. A total of 2885 food additives were screened for toxicity based on Threshold of Toxicological Concern (TTC), mutagenicity endpoint prediction, and mutagenic structural alerts/toxicophores identification. Ten food additives were identified as having mutagenic potential based on toxicity screening. Furthermore, Protein-Protein Interaction (PPI) analysis identified ESR1, as a key hub gene in breast cancer. KEGG pathway analysis verified that ESR1 plays a significant role in breast cancer pathogenesis. Additionally, competitive interaction studies of the predicted potential mutagenic food additives with the estrogen receptor-α were evaluated at agonist and antagonist binding sites. Indole, Dichloromethane, Trichloroethylene, Quinoline, 6-methyl quinoline, Ethyl nitrite, and 4-methyl quinoline could act as agonists, and Paraldehyde, Azodicarbonamide, and 2-acetylfuranmay as antagonists. The systematic risk assessment framework reported in this study enables the exploration of mutagenic and xenoestrogenic potential associated with food additives for hazard identification and management.

Identification of Novel Induced Mutations in Seed and Vegetatively Propagated Plants from Reduced Representation or Whole Genome Sequencing Data.

Treatment of plants with chemical mutagens results primarily in the production of novel single nucleotide variants. Mutagenesis is a mostly random process and as such plants derived from mutagenesis of different seeds or in vitro material are expected to accumulate different mutations. An important step in the creation of a mutant population for forward or reverse genetics is the choice of treatment conditions (e.g., dosage) such that sufficient mutations accumulate while not adversely affecting propagation of the plant. DNA sequencing provides a quick method to evaluate the effect of different treatment conditions and their effect on the density and spectrum of accumulated mutations. Whole genome sequencing or reduced representation sequencing is carried out followed by mapping to a reference genome and production of a Variant Call Format (VCF) file. We provide here a method for generating a multi-sample VCF from mutagenized plants and describe a new tool to streamline the process of recovering unique induced mutations and determining their possible effect on gene function.

Within-laboratory reproducibility of Ames test results: Are repeat tests necessary?

The Ames test is required by regulatory agencies worldwide for assessing the mutagenic and carcinogenic potential of chemical compounds. This test uses several strains of bacteria to evaluate mutation induction: positive results in the assay are predictive of rodent carcinogenicity. As an initial step to understanding how well the assay may detect mutagens present as constituents of complex mixtures such as botanical extracts, a cross-sector working group examined the within-laboratory reproducibility of the Ames test using the extensive, publicly available National Toxicology Program (NTP) Ames test database comprising more than 3000 distinct test articles, most of which are individual chemicals. This study focused primarily on NTP tests conducted using the standard Organization for Economic Co-operation and Development Test Guideline 471 preincubation test protocol with 10% rat liver S9 for metabolic activation, although 30% rat S9 and 10 and 30% hamster liver S9 were also evaluated. The reproducibility of initial negative responses in all strains with and without 10% S9, was quite high, ranging from 95% to 99% with few exceptions. The within-laboratory reproducibility of initial positive responses for strains TA98 and TA100 with and without 10% rat liver S9 was ≥90%. Similar results were seen with hamster S9. As expected, the reproducibility of initial equivocal responses was lower, <50%. These results will provide context for determining the optimal design of recommended test protocols for use in screening both individual chemicals and complex mixtures, including botanicals.

DNA adduct formation in Saccharomyces cerevisiae following exposure to environmental pollutants, as in vivo model for molecular toxicity studies.

DNA adduction in the model yeast Saccharomyces cerevisiae was investigated after exposure to the fungicide penconazole and the reference genotoxic compound benzo(a)pyrene, for validating yeasts as a tool for molecular toxicity studies, particularly of environmental pollution. The effect of the toxicants on the yeast's growth kinetics was determined as an indicator of cytotoxicity. Fermentative cultures of S. cerevisiae were exposed to 2 ppm of Penconazole during different phases of growth; while 0.2 and 2 ppm of benzo(a)pyrene were applied to the culture medium before inoculation and on exponential cultures. Exponential respiratory cultures were also exposed to 0.2 ppm of B(a)P for comparison of both metabolisms. Penconazole induced DNA adducts formation in the exponential phase test; DNA adducts showed a peak of 54.93 adducts/109 nucleotides. Benzo(a)pyrene induced the formation of DNA adducts in all the tests carried out; the highest amount of 46.7 adducts/109 nucleotides was obtained in the fermentative cultures after the exponential phase exposure to 0.2 ppm; whereas in the respiratory cultures, 14.6 adducts/109 nucleotides were detected. No cytotoxicity was obtained in any experiment. Our study showed that yeast could be used to analyse DNA adducts as biomarkers of exposure to environmental toxicants.

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing.

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

Evaluation of imidacloprid (Confidor OD®) genotoxicity in Chrysoperla externa eggs (Neuroptera: Chrysopidae) through comet assay.

The comet assay allows the analysis of DNA damage caused by different genotoxins. This assay has recently gained interest because of its ease of studying the interactions of xenobiotics with different organisms. Chrysoperla externa (Hagen, 1861) is a species of great economic relevance because it is a predator of major agricultural pests during its larval stage. Neonicotinoids are the most important chemical class of insecticides introduced into markets. A previous imidacloprid toxicity assessment on C. externa showed that this neonicotinoid insecticide reduced the egg viability. The objective of this study was to analyze the genotoxicity of Confidor OD® (imidacloprid 20% a.i., LS, Bayer CropScience) on the biological control agent C. externa at DNA level using the comet assay as an ecotoxicological biomarker. A comet assay protocol has been developed for this species at first time. For the bioassays, the commercial product formulated Confidor OD® was used at two concentrations: 100 and 180 mg/l of the active ingredient. Selected eggs were dipped in a Confidor OD® solution for 15 s. Descriptors evaluated in the comet assay were damage index, % DNA damage, and tail length. The damage index did not show any significant differences between the different concentrations evaluated, but differences were observed for tail length, because at higher concentrations of Confidor OD®, there were greater DNA breaks. The DNA of the cells from treated eggs analyzed at 48 h and 96 h of development showed the same % DNA damage; that is, they had no recovery capacity. Application of Confidor OD® to C. externa eggs produced irreparable breaks at the DNA level. The technique adjusted for C. externa can be used in other beneficial insects to study pesticide genotoxicity using a comet assay.

Strategy proposal using QSAR models to approach mutagenicity assessment of non intentionally added substances in recycled plastic resins.

CONTEXT: Transition to the use of recycled plastics raises an issue concerning safety assessment of Non Intentionally Added Substances (NIAS). To assess the mutagenic potential of the recycled polyethylene impurities and to evaluate the need to perform in vitro assays on recycled resins, this study lies in identifying existing NIAS associated with recycled Low/High Density Polyethylene and assessing the mutagenicity data-gaps by employing in silico tools. METHODS: Quantitative Structure-Activity Relationship (QSAR) models predicting Ames mutagenicity were selected from literature, then NIAS were run to 1/evaluate performances of each model, 2/apply a QSAR strategy on the NIAS molecular space and address data-gaps. RESULTS: Among the 165 NIAS identified, experimental Ames results were not found for 50 substances while the substances with experimental data were predominantly negatives. No individual model was able to predict all NIAS due to applicability domain limitations. Taking into account 1/calculated performances, 2/availability of applicability domain, 3/description of the Training Set, an Integrated Strategy was founded including Sarpy, Consensus and Protox to extend the applicability domain. CONCLUSION & PERSPECTIVES: Existing data and predictions generated by this strategy suggest a low mutagenic potential of NIAS. Further investigation is needed to explore other genotoxicity mechanisms.

The evolutionary safety of mutagenic drugs should be assessed before drug approval.

Some drugs increase the mutation rate of their target pathogen, a potentially concerning mechanism as the pathogen might evolve faster toward an undesired phenotype. We suggest a four-step assessment of evolutionary safety for the approval of such treatments.

Metabolism-dependent mutagenicity of two structurally similar tobacco-specific nitrosamines (N-nitrosonornicotine and N-nitrosoanabasine) in human cells, partially different CYPs being activating enzymes.

N-nitrosonornicotine (NNN) and N-nitrosoanabasine (NAB) are both tobacco-specific nitrosamines bearing two heterocyclic amino groups, NAB bearing an extra -CH2- group (conferring a hexa- rather than penta-membered cycle) but with significantly decreased carcinogenicity. However, their activating enzymes and related mutagenicity remain unclear. In this study, the chemical-CYP interaction was analyzed by molecular docking, thus the binding energies and conformations of NNN for human CYP2A6, 2A13, 2B6, 2E1 and 3A4 appeared appropriate as a substrate, so did NAB for human CYP1B1, 2A6, 2A13 and 2E1. The micronucleus test in human hepatoma (HepG2) cells with each compound (62.5-1000 µM) exposing for 48 h (two-cell cycle) was negative, however, pretreatment with bisphenol AF (0.1-100 nM, CYPs inducer) and ethanol (0.2% v:v, CYP2E1 inducer) potentiated micronucleus formation by both compounds, while CITCO (1 µM, CYP2B6 inducer) selectively potentiated that by NNN. In C3A cells (endogenous CYPs enhanced over HepG2) both compounds induced micronucleus, which was abolished by 1-aminobenzotriazole (60 µM, CYPs inhibitor) while unaffected by 8-methoxypsoralen (1 µM, CYP2A inhibitor). Consistently, NNN and NAB induced micronucleus in V79-derived recombinant cell lines expressing human CYP2B6/2E1 and CYP1B1/2E1, respectively, while negative in those expressing other CYPs. By immunofluorescent assay both compounds selectively induced centromere-free micronucleus in C3A cells. In PIG-A assays in HepG2 cells NNN and NAB were weakly positive and simply negative, respectively; however, in C3A cells both compounds significantly induced gene mutations, NNN being slight more potent. Conclusively, both NNN and NAB are mutagenic and clastogenic, depending on metabolic activation by partially different CYP enzymes.

Urinary mutagenicity and bladder cancer risk in northern New England.

The etiology of bladder cancer among never smokers without occupational or environmental exposure to established urothelial carcinogens remains unclear. Urinary mutagenicity is an integrative measure that reflects recent exposure to genotoxic agents. Here, we investigated its potential association with bladder cancer in rural northern New England. We analyzed 156 bladder cancer cases and 247 cancer-free controls from a large population-based case-control study conducted in Maine, New Hampshire, and Vermont. Overnight urine samples were deconjugated enzymatically and the extracted organics were assessed for mutagenicity using the plate-incorporation Ames assay with the Salmonella frameshift strain YG1041 + S9. Logistic regression was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) of bladder cancer in relation to having mutagenic versus nonmutagenic urine, adjusted for age, sex, and state, and stratified by smoking status (never, former, and current). We found evidence for an association between having mutagenic urine and increased bladder cancer risk among never smokers (OR = 3.8, 95% CI: 1.3-11.2) but not among former or current smokers. Risk could not be estimated among current smokers because nearly all cases and controls had mutagenic urine. Urinary mutagenicity among never-smoking controls could not be explained by recent exposure to established occupational and environmental mutagenic bladder carcinogens evaluated in our study. Our findings suggest that among never smokers, urinary mutagenicity potentially reflects genotoxic exposure profiles relevant to bladder carcinogenesis. Future studies are needed to replicate our findings and identify compounds and their sources that influence bladder cancer risk.

Discerning the stability behaviour of mavacamten availing liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy: In silico toxicity and mutagenicity prediction of degradation products.

The present study aimed to separate, identify, and characterise the degradation products formed when mavacamten is exposed to stress degradation as well as the stability of the drug in various environments and also to understand its degradation chemistry. Prediction of in silico toxicity and mutagenicity was aimed at the observed degradation products. Stress degradation along with stability studies and degradation kinetics were performed on mavacamten, and separation of degradation products was carried out by high-performance liquid chromatography. Tandem mass spectrometry studies were executed to characterise the structures of degradation products using product ion fragments. Orthogonally, nuclear magnetic resonance experiments were conducted to elucidate the structures having ambiguity in characterising them. Deductive Estimation of Risk from Existing Knowledge and Structure Activity Relationship Analysis using Hypotheses software were used to establish in silico toxicity and mutagenic profiles of mavacamten and its degradation products. Two degradation products of mavacamten found in acidic hydrolytic stress conditions were separated, identified, characterised, and proposed as 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione and 1-phenylethanamine. Mavacamten was found to be stable under different pH and gastrointestinal conditions. The degradation kinetics of mavacamten under 1 N acidic condition followed zero-order kinetics, and it was degraded completely within 6 h. In silico toxicity and mutagenicity studies revealed that 1-phenylethanamine can be a skin sensitiser. A high-performance liquid chromatography method was developed for the separation of degradation products of mavacamten and characterised by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance. During the manufacturing and storage of drug product, precautions need to be taken when dealing with acidic solutions as the drug is prone to hydrolysis in acidic conditions. The formation of 1-phenylethanamine under these conditions is to be monitored as it is a skin sensitiser.

Effect of cell treatment procedures on in vitro genotoxicity assessment.

So far, the majority of in vitro toxicological experiments are conducted after an acute 24 h treatment that does not represent a realistic human chemical exposure. Recently, new in vitro approaches have been proposed to study the chemical toxicological effect over several days in order to be more predictive of a representative exposure scenario. In this study, we investigated the genotoxic potential of chemicals (direct or bioactived clastogen, aneugen and apoptotic inducer) with the γH2AX and pH3 biomarkers, in the human liver-derived HepaRP cell line. We used different treatment durations, with or without a three-day recovery stage (release period), before genotoxicity measurement. Data were analysed with the Benchmark Dose approach. We observed that the detection of clastogenic compounds (notably for DNA damaging agents) was more sensitive after three days of repeated treatment compared to one or three treatments over 24 h. In contrast, aneugenic chemicals were detected as genotoxic in a similar manner whether after a 24 h exposure or a three-day repeated treatment. Globally, the release period decreases the genotoxicity measurement substantially. For DNA damaging agents, after high concentration treatments, γH2AX induction was always observed after a three-day release period. In contrast, for DNA topoisomerase inhibitors, no effect could be observed after the release period. In conclusion, in the HepaRP cell line, there are some important differences between a one-day acute and a three-day repeated treatment protocol, indicating that different cell treatment procedures may differentiate chemical genotoxic mechanisms of action more efficiently.

Safety assessment of high fructose corn syrup and fructose used as sweeteners in foods.

High Fructose Corn Syrup (HFCS) and Fructose (FR) are widely used sweeteners in many foods and beverages. This study aimed at investigating the cytotoxic effects of HFCS (5%-30%) and FR (62.5-2000 µg/mL) using MTT assay in Human Hepatocellular Carcinoma (HepG2) cells, and genotoxic effects of using Chromosome Aberrations (CAs), Sister Chromatid Exchanges (SCEs), Micronuclei (MN) and comet assays in human lymphocytes. HFCS significantly reduced the cell viability in HepG2 cells at between 7.5% and 30% for 24 and 48 h. 30% HFCS caused a very significant toxic effect. FR had a cytotoxic effect in HepG2 cells at all treatments. However, as fructose concentration decreased, the cell viability decreased. HFCS (10%-20%) and FR (250-2000 µg/mL) decreased the mitotic index at higher concentrations. IC50 value was found to be a 15% for 48 h. IC50 value of FR was detected as 62.5 µg/mL for 24 h and 48 h. HFCS significantly increased CAs frequency at 15% and 20%. FR significantly increased the frequency of CAs at 250, 1000, and 2000 µg/mL for 48 h. Both sweeteners increased the frequency of SCEs at all concentrations. HFCS (15% and 20%) and FR (250, 1000, and 2000 µg/mL) induced MN frequency at higher concentrations. HFCS caused DNA damage in comet assay at 10% -30%. FR increased tail intensity and moment at 125-2000 µg/mL and tail length at 62.5, 250 and 500 µg/mL. Therefore, HFCS and FR are clearly seen to be cytotoxic and genotoxic, especially at higher concentrations.

HFCS and FR exhibited cytotoxic effect at HepG2 and human lymphocytes at higher concentrations.Both sweeteners increased the frequencies of CAs and SCEs at higher concentrations.HFCS caused DNA damage at 10% -30% concentrations.HFCS (15% and 20%) and FR (250, 1000, and 2000 µg/mL) induced MN frequency.

Comparative analysis of micronucleus induction and DNA damage biomarkers in TK6 and A375 cells using flow cytometry.

Previously, we introduced an alternative adherent A375 cell line for clastogenicity and aneugenicity testing using a high content imaging platform. To further characterize the performance of A375 cells, we investigated the sensitivity and specificity of A375 and TK6 cells by directly comparing micronucleus (MN) induction, cytotoxicity (relative cell counts, viability, and apoptosis), clastogenicity (γH2AX), and aneuploidy markers (pH 3, MPM-2, and polyploidy) using flow cytometric methods. We evaluated 14 compounds across different mechanisms (non-genotoxic apoptosis inducers, clastogens, and aneugens with either tubulin binding or aurora kinase inhibiting phenotypes) at 4-h and 24-h post treatment. Both aneugens and clastogens tested positive for micronucleus induction in both cell lines. Apoptosis continued to be a confounding factor for flow cytometry-based micronuclei assessment in TK6 cells as evidenced by positive responses by the three cytotoxicants. Conversely, A375 cells were not affected by apoptosis-related false positive signals and did not produce a positive response in the in vitro micronucleus assay. Benchmark dose response (BMD) analysis showed that the induction of micronuclei and biomarkers occurred at similar concentrations in both cell lines for clastogens and aneugens. By showing that A375 cells have similar sensitivity to TK6 cells but a greater specificity, these results provide additional support for A375 cells to be used as an alternative adherent cell line for in vitro genetic toxicology assessment.

Are hydroxyapatite-based biomaterials free of genotoxicity? A systematic review.

Hydroxyapatite (HA) is a biomaterial widely used in clinical applications and pharmaceuticals. The literature on HA-based materials studies is focused on chemical characterization and biocompatibility. Generally, biocompatibility is analyzed through adhesion, proliferation, and differentiation assays. Fewer studies are looking for genotoxic events. Thus, although HA-based biomaterials are widely used as biomedical devices, there is a lack of literature regarding their genotoxicity. This systematic review was carried out following the PRISMA statement. Specific search strategies were developed and performed in four electronic databases (PubMed, Science Direct, Scopus, and Web of Science). The search used "Hydroxyapatite OR Calcium Hydroxyapatite OR durapatite AND genotoxicity OR genotoxic OR DNA damage" and "Hydroxyapatite OR Calcium Hydroxyapatite OR durapatite AND mutagenicity OR mutagenic OR DNA damage" as keywords and articles published from 2000 to 2022, after removing duplicate studies and apply include and exclusion criteria, 53 articles were identified and submitted to a qualitative descriptive analysis. Most of the assays were in vitro and most of the studies did not show genotoxicity. In fact, a protective effect was observed for hydroxyapatites. Only 20 out of 71 tests performed were positive for genotoxicity. However, no point mutation-related mutagenicity was observed. As the genotoxicity of HA-based biomaterials observed was correlated with its nanostructured forms as needles or rods, it is important to follow their effect in chronic exposure to guarantee safe usage in humans.

Application of duplex sequencing to evaluate mutagenicity of aristolochic acid and methapyrilene in Fisher 344 rats.

Duplex sequencing (DS) is an error-corrected next-generation sequencing (NGS) method that can overcome notorious high error rate from the process of NGS and detect ultralow-frequency mutations. In this study, we evaluated the mutagenicity of aristolochic acid, a known genotoxic carcinogen, and methapyrilene, a known nongenotoxic carcinogen using DS. Four male Fisher 344 rats were treated with aristolochic acid, methapyrilene, or the vehicle control for 6 weeks, liver tissues were collected one day after the treatment, and the DNA was isolated for analysis. The mutation frequency for the aristolochic acid-treated group was significantly increased over the vehicle control (44-fold), whereas no significant difference in the mutation frequency was observed between the methapyrilene-treated and the control groups. The primary type of mutation induced by aristolochic acid was A:T > T:A transversion, which occurred frequently at ApT sites, whereas the major type of mutation in the control and methapyrilene-treated groups was G:C > A:T transition, which occurred frequently at CpG sites. These findings are consistent with previously published data obtained with other in vivo mutation assays. Thus, our results suggest that the DS mutation assay is a promising technology for assessing mutagenicity of chemicals in vivo.