Genetic toxicology tests are used to categorize substances as genotoxic and potentially carcinogenic. In general, test results are designated as mutagenic, not mutagenic, or inconclusive and, depending on its potential use and applicable regulations, a mutagenic result can restrict or remove a substance from further development, or assign limits to its use. In these tests, mutation responses form a continuum without a clear delineation between an increase over the background, untreated, mutant frequency and a frequency that would define the test substance as a mutagen and a potential carcinogenic hazard. This situation is illustrated using the Salmonella mutagenicity (Ames) test which is the initial, and often only, test used to characterize substances as mutagenic or nonmutagenic. It has its widest use by industry and regulatory authorities to identify potential carcinogens among chemicals in development. The OECD Test Guideline No. 471 has been adopted by regulatory agencies internationally, and describes the minimum requirements for a negative response, but does not provide a specific approach for evaluating the test data. The most widely used criterion for making yes-or-no mutagenicity decisions is a 2- or 3-fold increase over the background (solvent) mutant frequency. Other approaches rely on formal statistics and/or expert judgment. These approaches and recently proposed modifications are evaluated here. Recommendations are made that are in conformity with the OECD guideline and are based on biological relevance and the biology of the mutagenic response rather than on arbitrary decision points (e.g., ≥2-fold increase or p ≤ .05).
Glyphosate, the most heavily used herbicide world-wide, is applied to plants in complex formulations that promote absorption. The National Toxicology Program reported in 1992 that glyphosate, administered to rats and mice at doses up to 50,000 ppm in feed for 13 weeks, showed little evidence of toxicity, and no induction of micronuclei was observed in the mice in this study. Subsequently, mechanistic studies of glyphosate and glyphosate-based formulations (GBFs) that have focused on DNA damage and oxidative stress suggest that glyphosate may have genotoxic potential. However, few of these studies directly compared glyphosate to GBFs, or effects among GBFs. To address these data gaps, we tested glyphosate, glyphosate isopropylamine (IPA), and (aminomethyl)phosphonic acid (AMPA, a microbial metabolite of glyphosate), 9 high-use agricultural GBFs, 4 residential-use GBFs, and additional herbicides (metolachlor, mesotrione, and diquat dibromide) present in some of the GBFs in bacterial mutagenicity tests, and in human TK6 cells using a micronucleus assay and a multiplexed DNA damage assay. Our results showed no genotoxicity or notable cytotoxicity for glyphosate or AMPA at concentrations up to 10 mM, while all GBFs and herbicides other than glyphosate were cytotoxic, and some showed genotoxic activity. An in vitro to in vivo extrapolation of results for glyphosate suggests that it is of low toxicological concern for humans. In conclusion, these results demonstrate a lack of genotoxicity for glyphosate, consistent with observations in the NTP in vivo study, and suggest that toxicity associated with GBFs may be related to other components of these formulations.
Ethyl tertiary-butyl ether (ETBE) is a fuel oxygenate used for the efficiency of motor vehicle fuels and their octane ratings. ETBE has been reported to induce liver adenomas in male rats in a 2-year bioassay at the highest inhalation concentration tested of 5000 ppm. To investigate the potential mutagenicity of ETBE in the liver, male Big Blue Fischer 344 rats were exposed for 28 consecutive days (6 h/day) to 0, 500, 1500, and 5000 ppm ETBE. The treated rats were sacrificed 3 days post-exposure and the frequencies of cII mutants were evaluated in the liver and bone marrow tissues. The mutant frequency (MF) of the liver in the negative control group was 36.3 × 10−6 and this value was not significantly different in ETBE-exposed animals (39.4, 37.3, and 45.9 × 10−6 in 500, 1500, and 5000 ppm groups, respectively). In the bone marrow, the mean MF in the negative control was 32.9 × 10−6 which was not different from the means of the exposed groups (33.8, 22.6, and 32.0 × 10−6 for groups exposed to 500, 1500 and 5000 ppm, respectively). These data, along with consistent negative response reported in the literature for other apical genotoxicity endpoints informs that mutagenicity is not likely the initial key event in the mode of action for ETBE-induced hepatocarcinogenesis in the rat.
Hydroxyurea is approved for treating children and adults with sickle cell anemia (SCA). Despite its proven efficacy, concerns remain about its mutagenic and carcinogenic potential that hamper its widespread use. Cell culture- and animal-based investigations indicate that hydroxyurea's genotoxic effects are due to indirect clastogenicity in select cell types when high dose and time thresholds are exceeded (reviewed by Ware & Dertinger, 2021). The current study extends these preclinical observations to pediatric patients receiving hydroxyurea for treatment of SCA. First, proof-of-principle experiments with testicular cancer patients exposed to a cisplatin-based regimen validated the ability of flow cytometric blood-based micronucleated reticulocyte (MN-RET) and PIG-A mutant reticulocyte (MUT RET) assays to detect clastogenicity and gene mutations, respectively. Second, these biomarkers were measured in a cross-sectional study with 26 SCA patients receiving hydroxyurea and 13 SCA patients without exposure. Finally, a prospective study was conducted with 10 SCA patients using pretreatment blood samples and after 6 or 12 months of therapy. Cancer patients exposed to cisplatin exhibited increased MN-RET within days of exposure, while the MUT RET endpoint required more time to reach maximal levels. In SCA patients, hydroxyurea induced MN-RET in both the cross-sectional and prospective studies. However, no evidence of PIG-A gene mutation was found in hydroxyurea-treated children, despite the fact that the two assays use the same rapidly-dividing, highly-exposed cell type. Collectively, these results reinforce the complementary nature of MN-RET and MUT RET biomarkers, and indicate that hydroxyurea can be clastogenic but was not mutagenic in young patients with SCA.
To determine the utility of the ToxTracker assay in animal alternative testing strategies, the genotoxic potential of four fragrance materials (2-octen-4-one, lauric aldehyde, veratraldehyde, and p-methoxy cinnamaldehyde) were tested in the ToxTracker assay. These materials have been previously evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per OECD guidelines. In addition to these studies, reconstructed human skin micronucleus studies were conducted on all four materials. All four materials were positive in an in vitro micronucleus assay but were negative in both in vivo and 3D skin micronucleus assays. The ToxTracker assay, in combination with in silico methods to predict metabolism was used to identify mechanisms for the misleading positive outcomes observed in the in vitro micronucleus assays. The results show that the ToxTracker assay, in conjunction with in silico predictions, can provide the information needed to aid in the identification of an appropriate animal alternative follow-up assay, for substances with positive results in the standard in vitro test battery. Thus, the ToxTracker assay is a valuable tool to identify the genotoxic potential of fragrance materials and can aid with replacing animal-based follow-up testing with appropriate animal alternative assay(s).
Diesel engine exhaust (DEE) is an established lung carcinogen, but the biological mechanisms of diesel-induced lung carcinogenesis are not well understood. MicroRNAs (miRNAs) are small noncoding RNAs that play a potentially important role in regulating gene expression related to lung cancer. We conducted a cross-sectional molecular epidemiology study to evaluate whether serum levels of miRNAs are altered in healthy workers occupationally exposed to DEE compared to unexposed controls. We conducted a two-stage study, first measuring 405 miRNAs in a pilot study of six DEE-exposed workers exposed and six controls. In the second stage, 44 selected miRNAs were measured using the Fireplex circulating miRNA assay that profiles miRNAs directly from biofluids of 45 workers exposed to a range of DEE (Elemental Carbon (EC), median, range: 47.7, 6.1–79.7 μg/m3) and 46 controls. The relationship between exposure to DEE and EC with miRNA levels was analyzed using linear regression adjusted for potential confounders. Serum levels of four miRNAs were significantly lower (miR-191-5p, miR-93-5p, miR-423-3p, miR-122-5p) and one miRNA was significantly higher (miR-92a-3p) in DEE exposed workers compared to controls. Of these miRNAs, miR-191-5p (ptrend = .001, FDR = 0.04) and miR-93-5p (ptrend = .009, FDR = 0.18) showed evidence of an inverse exposure–response with increasing EC levels. Our findings suggest that occupational exposure to DEE may affect circulating miRNAs implicated in biological processes related to carcinogenesis, including immune function.
In our country, great concern exists about diffuse pollution cause by the great use of pesticides in rural environments. A thorough analysis is needed to generate information, know the real situation and thus, be able to make decisions with the purpose of reducing environmental pollution. In situ bioassays have been carried out using Cnesterodon decemmaculatus within limnocorrals located in a surface natural water system that receives rainfall excess flowing from an agricultural basin with a typical crop rotation, including corn, wheat and soy. Specimens were taken from the limnocorrals 72 h after a probed natural runoff event toward the water body, and the gill cells were used to evaluate the DNA damage (comet assay, CA), catalase enzyme activity (CAT), and lipid peroxidation (LPO). In addition, the physicochemical analysis of the water (pH, temperature) and the presence and concentration of pesticides were carried out. The results showed significant differences on DNA damage and oxidative stress on the gill cells of the exposed fish compared to controls, being the combination of the rain regime and the mixtures of pesticides used in corn and soy more toxic than in wheat. These results highlight the necessity to understand detrimental processes caused by pesticides used in extensive systems of primary production, in order to prevent and minimize diffuse contamination, contributing to environmental recovery and sustainability.
In March 2020, the Italian government imposed a national lockdown which was almost completely removed in June 2020. Due to the abrupt stop of human activities, emissions of air pollutants decreased. Air pollution is an environmental risk factor for noncommunicable disease and mortality. Emerging evidence also suggests a role in male infertility. In this study, we compared sperm DNA fragmentation (sDF) levels and conventional semen parameters between subjects undergoing sDF determination and routine semen analysis in a single Italian centre, during about 6 months before (N = 119) and after lockdown (N = 105). After lockdown, we found an improvement of sperm progressive motility (48.00[38.50–58.00]% vs. 42.00[33.00–53.00]%) and sDF levels (as total: 24.79[18.33–33.97]% vs. 35.02[25.04–45.73]%, p < .001; brighter: 14.02[10.69–17.93]% vs 18.54[13.58–25.82]%, p < .001 and dimmer sDF: 9.24[5.64–15.78]% vs. 12.24[8.08–19.10]%, p < .01), mirrored by a decrease of leukocyte semen concentration (p < .01). The improvement of sperm motility and DNA quality was maintained after adjusting for leukocyte concentration and several conditions known to affect sperm motility and/or sDF levels. With a significant decrease in air pollution observed in Tuscany during and after lockdown, associated improvement in sperm motility and DNA quality in patients referred to the infertility clinic is suggestive of the potential role of air pollution in male infertility.
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