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Nitro group containing xenobiotics include drugs, cancer chemotherapeutic agents, carcinogens (e.g., nitroarenes and aristolochic acid) and explosives. The nitro group undergoes a six-electron reduction to form sequentially the nitroso

The history of long-term environmental exposure to heavy metals can be recorded in the genome as sporadic and specific mutations. Variable environments introduce diverse and adaptive mutations to organisms. To reveal the information hidden in genomes about environmental exposure to heavy metals, we performed long-term mutation accumulation (MA) experiments with

Cancer is the uncontrolled proliferation of cells that involves accumulation of genetic mutations by different types of mutagens including physical, chemical, and biological. Conse-quently, normal cell cycles get interrupted. Immunological assays, histopathological tests, poly-merase chain reaction, computed tomography, magnetic resonance, and radiation therapy are some conventional techniques for cancer diagnostics. However, these techniques are not only expensive, time-consuming, tedious but also toxic to healthy cells. Therefore, these limitations are overcome by nanodevices that show high sensitivity, selectivity, rapidity, and cost-effectiveness in the detec-tion of cancer biomarkers. Electrochemical biosensors are more efficient in the early diagnosis of cancers that help in patients' effective and timely treatment. Distinct types of nanotools viz. inor-ganic, organic, and polymeric nanomaterials are used in cancer therapeutics. Nano approaches have shown many advantages: they are site-specific, require meager amounts of drugs, limited toxicity, avoid drug resistance, and are more efficient, sensitive, and reliable. Therefore, future research should focus on developing highly inventive nanotools for the diagnosis and therapeutics of cancers.

3-Methylthymine (m3T) is a long-known chemically stable but strongly mutagenic DNA base adduct; however, the sequencing method to determine m3T is lacking so far. Two of the main obstacles include the capacity of m3T to stall DNA elongation and its low abundance. To address the challenges, we report an unnatural base pairing strategy in this paper. A new m3T-TPT3 base pair was developed with a Vmax/Km value 82-fold higher than that of the m3T-A pair. The TPT3 nucleobase can be specifically incorporated opposite the m3T base, and the resulting m3T-TPT3 base pair can be effectively extended to give full-length products in the presence of commercial DNA polymerases. Importantly, the feature of the m3T-TPT3 pair enables a replacement PCR amplification to transfer m3T lesions at the exact positions into unnatural base pairs, which permits Sanger sequencings as well as biotin-streptavidin-based enrichments of m3T lesions. Taken together, this work offers a simple, convenient, and practical method for amplification, enrichment, and sequencing of m3T for the first time.

Plants, being sessile, are prone to genotoxin-induced macromolecule damage. Among the inevitable damaging agents are reactive carbonyls that induce glycation of DNA, RNA and proteins to result in the build-up of advanced glycated end-products. However, it is unclear how plants repair glycated macromolecules. DJ-1/PARK7 members are a highly conserved family of moonlighting proteins having double domains in higher plants and single domains in other phyla. Here we show that Arabidopsis DJ-1D offers robust tolerance to endogenous and exogenous stresses through its ability to repair glycated DNA, RNA and proteins. DJ-1D also reduced the formation of reactive carbonyls through its efficient methylglyoxalase activity. Strikingly, full-length double domain-containing DJ-1D suppressed the formation of advanced glycated end-products in yeast and plants. DJ-1D also efficiently repaired glycated nucleic acids and nucleotides in vitro and mitochondrial DNA in vivo under stress, indicating the existence of a new DNA repair pathway in plants. We propose that multi-stress responding plant DJ-1 members, often present in multiple copies among plants, probably contributed to the adaptation to a variety of endogenous and exogenous stresses.

Molnupiravir (MOV) is used to treat COVID-19. In cells, MOV is converted to the ribonucleoside analog N4-hydroxycytidine (NHC) and incorporated into the SARS-CoV-2 RNA genome during its replication, resulting in RNA mutations. The widespread accumulation of such mutations inhibits SARS-CoV-2 propagation. Although safety assessments by many regulatory agencies across the world have concluded that the genotoxic risks associated with the clinical use of MOV are low, concerns remain that it could induce DNA mutations in patients, particularly because numerous in vitro studies have shown that NHC is a DNA mutagen. In this study, we used HiFi sequencing, a technique that can detect ultralow-frequency substitution mutations in whole genomes, to evaluate the mutagenic effects of MOV in E. coli and of MOV and NHC in mouse lymphoma L5178Y cells and human lymphoblastoid TK6 cells. In all models, exposure to these compounds increased genome-wide mutation frequencies in a dose-dependent manner, and these increases were mainly composed of A:T → G:C transitions. The NHC exposure concentrations used for mammalian cells were comparable to those observed in the plasma of humans who received clinical doses of MOV.

Bacterial genotoxins are peptide or protein virulence factors produced by several pathogens, which make single-strand breaks (SSBs) and/or double-strand DNA breaks (DSBs) in the target host cells. If host DNA inflictions are not resolved on time, host cell apoptosis, cell senescence, and/or even bacterial pathogen-related cancer may occur. Two multi-protein AB toxins, cytolethal distending toxin (CDT) produced by over 30 bacterial pathogens and typhoid toxin from Salmonella Typhi, as well as small polyketide-peptides named colibactin that causes the DNA interstrand cross-linking and subsequent DSBs is the most well-characterized bacterial genotoxins. Using these three examples, this review discusses the mechanisms by which these toxins deliver themselves into the nucleus of the target host cells and exert their genotoxic functions at the structural and functional levels.

In animal studies, heterocyclic amines (HCAs) are recognized as having strong carcinogenicity, therefore we have hypothesized that HCAs might be associated with the risk of colorectal adenoma (CRA) and cancer (CRC).We used the Keywords of "Heterocyclic amines and colorectal cancer" to search, there were showing published articles (n=200). After reviews of titles, abstracts, and full articles, seven prospective cohort studies were included in the pooled analysis. Exposures to HCAs 2-amino-3,8-dimethylimidazo(4,5-j)quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), 2-amino-3,4,8-trimethylimidazo(4,5-f)quinoxaline (DiMeIQx), meat-derived mutagenicity (MDM), and the risk of CRA and CRC were examined. The estimated HCA intake as ng/day and ng/1,000 kcal/day by participants and by studies were examined. The ln(HR) and se(ln(HR)) were estimated from the multivariable-adjusted HR, 95%CI derived from seven published prospective studies in the US and EPIC. The random pooled multivariable-adjusted HR, 95%CI was analyzed using ln(HR) and se(ln(HR)) by STATA-10.For CRC and HCA intake, the null association was observed for MDM, the random pooled multivariable-adjusted HR, (95%CI): 1.11, (1.00, 1.23); for PhIP: 1.00, (0.91, 1.09); and for DiMeIQx: 1.03, (0.87, 1.22). A significant positive association was seen for MeIQx, the random pooled multivariable-adjusted HR, (95%CI):1.12, (1.03, 1.22). For CRA and HCA intake, the null association was observed for MDM, randomly pooled multivariable-adjusted HR, (95%CI): 1.15, (0.99, 1.34), and for DiMeIQx: 1.09, (0.97, 1.23). A significant positive association was seen for PhIP, the random pooled multivariable-adjusted HR, (95%CI): 1.19, (1.02, 1.39), and for MeIQx: 1.17, (1.01, 1.35). The major instances of HCAs were contributed by chicken (54%-74%) for PhIP and by red meat (83%-92%) for MeIQx. However, the estimated PhIP intake (ng/1,000 kcal/day) was remarkably different between studies.We observed a positive association between exposures to MeIQx and the risk of both CRC and CRA which supports the hypothesis of the role of HCAs in developing CRA and CRC. Improving the quality of the estimated HCA intake would be highly concerned for further investigation.

Although the health benefits of swimming are well-documented, health effects such as asthma and bladder cancer are linked to disinfection by-products (DBPs) in pool water. DBPs are formed from the reaction of disinfectants such as chlorine (Cl) or bromine (Br) with organics in the water. Our previous study (Daiber et al., Environ. Sci. Technol. 50, 6652; 2016) found correlations between the concentrations of classes of DBPs and the mutagenic potencies of waters from chlorinated or brominated swimming pools and spas. We extended this study by identifying significantly different concentrations of 21 individual DBPs in brominated or chlorinated pool and spa waters as well as identifying which DBPs and additional DBP classes were most associated with the mutagenicity of these waters. Using data from our previous study, we found that among 21 DBPs analyzed in 21 pool and spa waters, the concentration of bromoacetic acid was significantly higher in Br-waters versus Cl-waters, whereas the concentration of trichloroacetic acid was significantly higher in Cl-waters. Five Br-DBPs (tribromomethane, dibromochloroacetic acid, dibromoacetonitrile, bromoacetic acid, and tribromoacetic acid) had significantly higher concentrations in Br-spa versus Cl-spa waters. Cl-pools had significantly higher concentrations of Cl-DBPs (trichloroacetaldehyde, trichloromethane, dichloroacetic acid, and chloroacetic acid), whereas Br-pools had significantly higher concentrations of Br-DBPs (tribromomethane, dibromoacetic acid, dibromoacetonitrile, and tribromoacetic acid). The concentrations of the sum of all 4 trihalomethanes, all 11 Br-DBPs, and all 5 nitrogen-containing DBPs were each significantly higher in brominated than in chlorinated pools and spas. The 8 Br-DBPs were the only DBPs whose individual concentrations were significantly correlated with the mutagenic potencies of the pool and spa waters. These results, along with those from our earlier study, highlight the importance of Br-DBPs in the mutagenicity of these recreational waters.

DNA damage is an established initiating event in the mutagenicity and carcinogenicity of genotoxic chemicals. Accordingly, assessment of this endpoint is critical for chemicals which are being developed for use in humans. To assess the ability of the Chicken Egg Genotoxicity Assay (CEGA) to detect genotoxic pharmaceuticals, a set of 23 compounds with different pharmacological and reported genotoxic effects was tested for the potential to produce nuclear DNA adducts and strand breaks in the embryo-fetal livers using the 32P-nucleotide postlabeling (NPL) and comet assays, respectively. Due to high toxicity, two aneugens, colchicine and vinblastine, and an autophagy inhibitor, hydroxychloroquine, could not be evaluated. Out of the 20 remaining pharmaceuticals, 10 including estrogen modulators, diethylstilbestrol and tamoxifen, antineoplastics cyclophosphamide, etoposide, and mitomycin C, antifungal griseofulvin, local anesthetics lidocaine and prilocaine, and antihistamines diphenhydramine and doxylamine, yielded clear positive outcomes in at least one of the assays. The antihypertensive vasodilator hydralazine and antineoplastics streptozotocin and teniposide, produced only DNA strand breaks, which were not dose-dependent, and thus, the results with these 3 pharmaceuticals were considered equivocal. No DNA damage was detected for 7 compounds, including the purine antagonist 6-thioguanine, antipyretic analgesics acetaminophen and phenacetin, antibiotic ciprofloxacin, antilipidemic clofibrate, anti-inflammatory ibuprofen, and sedative phenobarbital. However, low solubility of these compounds limited dosages tested in CEGA. Overall, results in CEGA were largely in concordance with the outcomes in other systems in vitro and in vivo, indicating that CEGA provides reliable detection of DNA damaging activity of genotoxic compounds. Further evaluations with a broader set of compounds would support this conclusion.

Lowest observable adverse effects level (LOAEL) is a standard point-of-departure dose in toxicology. However, first observable adverse effects level (FOAEL) was recently reported and is used, in this study, as one criterion to detect a mutagenic stimulus in a live imager. Fluorescence ubiquitinated cell cycle indicator (FUCCI) embryonic stem cells (ESC) are green in the S-G2-M phase of the cell cycle and not green in G1-phase. Standard media change here is a mild stress that delays G1-phase and media change increases green 2.5- to 5-fold. Since stress is mild, media change rapidly increases green cell number, but higher stresses of environmental toxicants and positive control hyperosmotic stress suppress increased green after media change. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) previously suppressed progression of nongreen to green cell cycle progression. Here, bisphenol A (BPA), cortisol, and positive control hyperosmotic sorbitol also suppress green fluorescence, but benzo(a)pyrene (BaP) at high doses (10 μM) increases green fluorescence throughout the 74-h exposure. Since any stress can affect many cell cycle phases, messenger RNA (mRNA) markers are best interpreted in ratios as dose-dependent mutagens increase in G2/G1 and nonmutagens increase G1/G2. After 74-h exposure, RNAseq detects G1 and G2 markers and increasing BaP doses increase G2/G1 ratios but increasing hyperosmotic sorbitol and PFOA doses increase G1/G2 marker ratios. BaP causes rapid green increase in FOAEL at 2 h of stimulus, whereas retinoic acid caused significant green fluorescence increases only late in culture. Using a live imager to establish FOAEL and G2 delay with FUCCI ESC is a new method to allow commercial and basic developmental biologists to detect drugs and environmental stimuli that are mutagenic. Furthermore, it can be used to test compounds that prevent mutations. In longitudinal studies, uniquely provided by this viable reporter and live imager protocol, follow-up can be done to test whether the preventative compound itself causes harm.

Funder: Canada Research Chairs; doi: http://dx.doi.org/10.13039/501100001804, Risk assessments are increasingly reliant on information from in vitro assays. The in vitro micronucleus test (MNvit) is a genotoxicity test that detects chromosomal abnormalities, including chromosome breakage (clastogenicity) and/or whole chromosome loss (aneugenicity). In this study, MNvit datasets for 292 chemicals, generated by the US EPA's ToxCast program, were evaluated using a decision tree-based pipeline for hazard identification. Chemicals were tested with 19 concentrations (n = 1) up to 200 µM, in the presence and absence of Aroclor 1254-induced rat liver S9. To identify clastogenic chemicals, %MN values at each concentration were compared to a distribution of batch-specific solvent controls; this was followed by cytotoxicity assessment and benchmark concentration (BMC) analyses. The approach classified 157 substances as positives, 25 as negatives, and 110 as inconclusive. Using the approach described in Bryce et al. (Environ Mol Mutagen 52:280-286, 2011), we identified 15 (5%) aneugens. IVIVE (in vitro to in vivo extrapolation) was employed to convert BMCs into administered equivalent doses (AEDs). Where possible, AEDs were compared to points of departure (PODs) for traditional genotoxicity endpoints; AEDs were generally lower than PODs based on in vivo endpoints. To facilitate interpretation of in vitro MN assay concentration-response data for risk assessment, exposure estimates were utilized to calculate bioactivity exposure ratio (BER) values. BERs for 50 clastogens and two aneugens had AEDs that approached exposure estimates (i.e., BER < 100); these chemicals might be considered priorities for additional testing. This work provides a framework for the use of high-throughput in vitro genotoxicity testing for priority setting and chemical risk assessment.

The “totality” of the human exposure is conceived to encompass life-associated endogenous and exogenous aggregate exposures. Process-related contaminants (PRCs) are not only formed in foods by heat processing, but also occur endogenously in the organism as physiological components of energy metabolism, potentially also generated by the human microbiome. To arrive at a comprehensive risk assessment, it is necessary to understand the contribution of in vivo background occurrence as compared to the ingestion from exogenous sources. Hence, this review provides an overview of the knowledge on the contribution of endogenous exposure to the overall exposure to putative genotoxic food contaminants, namely ethanol, acetaldehyde, formaldehyde, acrylamide, acrolein, α,β-unsaturated alkenals, glycation compounds, N-nitroso compounds, ethylene oxide, furans, 2- and 3-MCPD, and glycidyl esters. The evidence discussed herein allows to conclude that endogenous formation of some contaminants appears to contribute substantially to the exposome. This is of critical importance for risk assessment in the cases where endogenous exposure is suspected to outweigh the exogenous one (e.g. formaldehyde and acrolein).

Bioassays have been used to complement the chemical characterization of aquatic mutagenicity, but the tests sometimes are done only with water liquid phase (LP). Particle-bound mutagens are important because they can be ingested by filtering organisms. Our objective was to evaluate the mutagenicity of organic extracts of the LP and the water suspended particulate matter (SPM) from 13 sites along Danube River with the Salmonella/microsome microsuspension assay using TA98, YG1041, TA1538, and YG5185 strains. A high incidence of mutagenicity was detected, 84% for LP and 92% for SPM samples. The contribution of SPM to the mutagenicity was relatively small when compared with LP however, for five sites SPM was responsible for the whole mutagenicity, highlighting the importance of analyzing SPM when assessing water mutagenicity. YG1041 was the most sensitive strain and should be considered in future water mutagenicity monitoring programs, but it will depend on the main pollution sources.

Studies during the past 50 years have characterized the carcinogenicity and mutagenicity of extractable organic material (EOM) of particulate matter (PM) in ambient air and from combustion emissions. We have summarized conclusions from these studies and present data supporting those conclusions for 50 combustion emissions, including carcinogenic potencies on mouse skin (papillomas/mouse/mg EOM), mutagenic potencies (revertants/μg EOM) in the Salmonella (Ames) mutagenicity assay, and mutagenicity emission factors (revertants/kg fuel or revertants/MJ

Octamethylcyclotetrasiloxane (D4), a highly lipophilic, volatile compound with low water solubility, is metabolized to lower molecular weight, linear silanols. Toxicity has been documented in several tissues in animals following mixed vapor/aerosol exposures by inhalation at near saturating vapor concentrations or with gavage dosing in vegetable oil vehicles. These results, together with more mechanism-based studies and detailed pharmacokinetic information, were used to assess likely modes of action (MOAs) and the tissue dose measures of D4 and metabolites that would serve as key events leading to these biological responses. This MOA analysis indicates that pulmonary effects arise from direct epithelial contact with mixed vapor/aerosol atmospheres of D4; liver hypertrophy and hepatocyte proliferation arise from adaptive, rodent-specific actions of D4 with nuclear receptor signaling pathways; and, nephropathy results from a combination of chronic progresive nephropathy and silanol metabolites binding with alpha-2u globulin (a male rat specific protein). At this time, the MOAs of other liver effects - pigment accumulation and bile duct hyperplasia (BDH) preferentially observed in Sprague-Dawley (SD) rats- are not known. Hypothalamic actions of D4 delaying the rat mid-cycle gonadotrophin releasing hormone (GnRH) surge that result in reproductive effects and subsequent vaginal/uterine/ovarian tissue responses, including small increases in incidence of benign endometrial adenomas, are associated with prolongation of endogenous estrogen exposures due to delays in ovulation. Human reproduction is not controlled by a mid-cycle GnRH surge. Since the rodent-specific reproductive and the vaginal/uterine/ovarian tissue responses are not relevant for risk assessments in human populations, D4 should neither be classified as a CMR (i.e., carcinogenic, mutagenic, or toxic for reproduction) substance nor be regarded as an endocrine disruptor. Bile duct hyperplasia (BDH) and pigment accumulation in liver seen in SD rats are endpoints that could serve to define a Benchmark Dose (BMD) or No-Observed-Effect-Level (NOEL) for D4 although their human relevance remains uncertain.

Aneuploidy is characterized by the presence of an abnormal number of chromosomes and is a common hallmark of cancer. However, exposure to aneugenic compounds does not necessarily lead to cancer. Aneugenic compounds are mainly identified using the in vitro micronucleus assay but this assay cannot standardly discriminate between aneugens and clastogens and cannot be used to identify the exact mode-of-action (MOA) of aneugens; tubulin stabilization, tubulin destabilization, or inhibition of mitotic kinases. To improve the classification of aneugenic substances and determine their MOA, we developed and validated the TubulinTracker assay that uses a green fluorescent protein-tagged tubulin reporter cell line to study microtubule stability using flow cytometry. Combining the assay with a DNA stain also enables cell cycle analysis. Substances whose exposure resulted in an accumulation of cells in G2/M phase, combined with increased or decreased tubulin levels, were classified as tubulin poisons. All known tubulin poisons included were classified correctly. Moreover, we correctly classified compounds, including aneugens that did not affect microtubule levels. However, the MOA of aneugens not affecting tubulin stability, such as Aurora kinase inhibitors, could not be identified. Here, we show that the TubulinTracker assay can be used to classify microtubule stabilizing and destabilizing compounds in living cells. This insight into the MOA of aneugenic agents is important, eg, to support a weight-of-evidence approach for risk assessment, and the classification as an aneugen as opposed to a clastogen or mutagen, has a big impact on the assessment.

Azo dyes are the most common colorants in consumer products, including clothing and cosmetics. Some azo dyes and their products from reductive degradation are known to be mutagenic, so dermal exposure to these species has been studied extensively. In contrast, oxidative degradation of azo dyes in consumer products has not been studied so thoroughly. In the indoor environment, ozone is ubiquitous, so reactive uptake of ozone to azo dyes could lead to dermal exposure to other classes of degradation products. Here, we report the first measurements of the reactive uptake of ozone to thin films of three widely used commercial azo dyes: sunset yellow, amaranth, and tartrazine. Steady-state uptake was observed for all three dyes, under all conditions investigated, even at the lowest relative humidity (RH) of 0%. The uptake coefficients increased with RH. For sunset yellow at 100 ppb of ozone, the value at 80% RH, (2.0 ± 0.5) × 10

Autophagy is a highly conserved process of cellular self-digestion that involves the formation of autophagosomes for the delivery of intracellular components and dysfunctional organelles to lysosomes. This process is induced by different signals including starvation, mitochondrial dysfunction, and DNA damage. The molecular link between autophagy and DNA damage is not well understood yet. Importantly, tumor cells utilize the mechanism of autophagy to cope with genotoxic anti-cancer drug therapy. Another mechanism of drug resistance is provided to cancer cells via the execution of the EMT program. One of the critical transcription factors of EMT is Zeb1. Here we demonstrate that Zeb1 is involved in the regulation of autophagy in several breast cancer cell models. On the molecular level, Zeb1 likely facilitates autophagy through the regulation of autophagic genes, resulting in increased LC3-II levels, augmented staining with Lysotracker, and increased resistance to several genotoxic drugs. The attenuation of Zeb1 expression in TNBC cells led to the opposite effect. Consequently, we propose that Zeb1 augments the resistance of breast cancer cells to genotoxic drugs, at least partially, via autophagy. Collectively, we have uncovered a novel function of Zeb1 in the regulation of autophagy in breast cancer cells.

BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been utilized at the Research Institute for Fragrance Materials in a safety assessment program as a screening tool to prioritize fragrance materials for higher-tier testing, as supporting evidence when using a read-across approach, and as evidence to adjust the threshold of toxicological concern. Predictive values for the BlueScreen HC assay were evaluated based on the ability of the assay to predict the outcome of in vitro and in vivo mutagenicity and chromosomal damage genotoxicity assays. A set of 371 fragrance materials was assessed in the BlueScreen HC assay along with existing or newly generated in vitro and in vivo genotoxicity data. Based on a weight-of-evidence approach, the majority of materials in the data set were deemed negative and concluded not to have the potential to be genotoxic, while only a small proportion of materials were determined to show genotoxic effects in these assays. Analysis of the data set showed a combination of high positive agreement but low negative agreement between BlueScreen HC results, in vitro regulatory genotoxicity assays, and higher-tier test results. The BlueScreen HC assay did not generate any false negatives, thereby providing robustness when utilizing it as a high-throughput screening tool to evaluate the large inventory of fragrance materials. From the perspective of protecting public health, it is desirable to have no or minimal false negatives, as a false-negative result may incorrectly indicate the lack of a genotoxicity hazard. However, the assay did have a high percentage of false-positive results, resulting in poor positive predictivity of the in vitro genotoxicity test battery outcome. Overall, the assay generated 100% negative predictivity and 3.9% positive predictivity. In addition to the data set of 371 fragrance materials, 30 natural complex substances were evaluated for BlueScreen HC, Ames, and in vitro micronucleus assay, and a good correlation in all three assays was observed. Overall, while a positive result may have to be further investigated, these findings suggest that the BlueScreen HC assay can be a valuable screening tool to detect the genotoxic potential of fragrance materials and mixtures.

Maintenance of genome stability is an essential requirement for all living organisms. Formaldehyde and UV-B irradiation cause DNA damage and affect genome stability, growth and development, but the interplay between these two genotoxic factors is poorly understood in plants. We show that Arabidopsis adh2/gsnor1 mutant, which lacks alcohol dehydrogenase 2/S-nitrosoglutathione reductase 1 (ADH2/GSNOR1), are hypersensitive to low fluence UV-B irradiation or UV-B irradiation-mimetic chemicals. Although the ADH2/GSNOR1 enzyme can act on different substrates, notably on S-hydroxymethylglutathione (HMG) and S-nitrosoglutathione (GSNO), our study provides several lines of evidence that the sensitivity of gsnor1 to UV-B is caused mainly by UV-B-induced formaldehyde accumulation rather than other factors such as alteration of the GSNO concentration. Our results demonstrate an interplay between formaldehyde and UV-B that exacerbates genome instability, leading to severe DNA damage and impaired growth and development in Arabidopsis, and show that ADH2/GSNOR1 is a key player in combating these effects.

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA–protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.

In order to evaluate the utility of the 3D reconstructed skin micronucleus assay (3DRSMN) to assess clastogenic/aneugenic potential of the fragrance chemicals, a set of 22 fragrance materials were evaluated in 3DRSMN assay. These materials evaluated were also evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per Organisation for Economic Co-operation and Development guidelines. The results of the RSMN assay were in 100% agreement with the in vivo micronucleus assay results. From this dataset, 18 materials were positive in an in vitro micronucleus assay but were negative in an in vivo micronucleus assay. All these 18 materials were also concluded to be negative in 3DRSMN assay, stressing the importance of the assay to help minimize misleading positive outcomes from the in vitro assay. Since the highest exposure for fragrances is through the dermal route, the RSMN assay fits the applicability domain for testing. Thus, RSMN assay is an important alternative to animal testing for characterization of the genotoxicity potential of fragrance materials.

Chlorothalonil is a fungicide widely used in agriculture as well as an active ingredient in antifouling paints. Although it causes toxic effects on non-target organisms and can accumulate in fish tissues, little is known about its sublethal effects. Thus, genotoxic and mutagenic effects of intraperitoneal injected chlorothalonil in Micropogonias furnieri, an estuarine fish of frequent human consumption and a promising test-organism for ecotoxicological assays, were assessed. Chlorothalonil showed to be genotoxic (DNA damage by comet assay) and mutagenic (micronuclei, nuclear buds, apoptotic fragments, and bilobed cells) even at the lowest dose tested (0.35 μg g−1) and in a dose-dependent manner (0.35 and 3.5 μg g−1) for micronuclei, apoptotic fragments, and bilobed cells. As genomic instability may lead to carcinogenesis, the present evidence can assist decision-makers in banning this compound since any benefit toward food production is outweighed by the hazard to aquatic ecosystems and human health.

Thermal treatment of protein-rich food can lead to the formation of biologically active heterocyclic aromatic amines (HAAs). One of the methods to learn how to reduce the content as well as the influence of these compounds on heath is the study of factors inhibiting their synthesis. In the current investigation, the effect of onion and garlic on the formation of six possibly carcinogenic non-polar HAAs (α-, γ- and δ-carbolines) and two co-mutagenic β-carbolines (harmane and norharmane) was evaluated by comparing their contents in meat and gravy samples obtained from pan-fried pork dishes prepared in the presence and absence of these vegetables. Carbolines were isolated from food samples by solid phase extraction. The quantitative analysis was performed by high-performance liquid chromatography with fluorescence detection. The concentrations of individual compounds in dishes prepared without added vegetables ranged from 0.02 ng g-1 (3-amino-1,4-dimethyl-5 H-pyrido(4,3-b)indole; Trp-P-1) to 10.1 ng g-1 of meat (2-amino-9 H-pyrido[2,3-b]indole; AαC). Onion (30 g/100 g of meat) and garlic (15 g/100 g of meat) lowered the total content (in meat and gravy) of the α-, δ- and γ-carbolines in the range from 52% to 87%. In contrast, onion caused an increase in the norharmane concentration both in meat and gravy. The percentage of carbolines in the gravies (assuming that their total content in meat and gravy is 100%) was higher in dishes prepared with onion and garlic than in dishes without these seasonings.

Acinetobacter baumannii strain 17978 is an opportunistic pathogen with a unique DNA damage repair response that lacks the LexA repressor but induces ~150 genes after DNA damage. It uses the UmuD homolog UmuDAb and the small protein DdrR, unique to Acinetobacter, to repress multiple horizontally acquirediumuDC/ierror-prone polymerase genes through an unknown mechanism. We used reverse transcription-quantitative PCR and immunoblotting to elucidate UmuDAb regulatory requirements and DdrR contributions to the corepression of this specialized regulon. Mutations in the putative UmuDAb helix-turn-helix (HTH) domain could not repress the expression of the UmuDAb/DdrR regulon. AiddrR/iinsertion mutation in these HTH mutant backgrounds produced even greater derepression of the regulon, suggesting that DdrR exerts an additional level of control over this mutagenic response. TheseiddrR/iHTH mutant A. baumannii cells overexpressed UmuDAb, cleaving it after treatment with the DNA-damaging agent mitomycin C. This showed that DdrR was not required for UmuDAb self-cleavage and that UmuDAb repression and self-cleavage actions were independent. An uncleavableiumuDAb/imutant with an A-to-Y change at position 83 (A83Y) could neither induce the UmuDAb/DdrR regulon nor conduct SOS mutagenesis. However, a prophage-encodediumuDrumB/ioperon was still partially induced after DNA damage in this mutant. Surprisingly, that prophage's putative repressor was dispensable for prophage-encodediumuDrumB/iinduction, implying another repressor's involvement. This study revealed that UmuDAb behaves like LexA, requiring an N-terminal HTH motif for repression and C-terminal self-cleavage for gene induction and subsequent SOS mutagenesis, and DdrR cooperates with it to exert an additional level of repressive control on this pathogen's mutagenic response to DNA damage.bIMPORTANCE/bAcinetobacter baumannii is a nosocomial pathogen that acquires antibiotic resistance genes through conjugative transfer and carries out a robust mutagenic DNA damage response. After exposure to conditions typically encountered in health care settings, such as antibiotics, UV light, and desiccation, this species induces error-prone UmuD'sub2/subC polymerases. This mutagenic capability increases A. baumannii survival and virulence and is regulated by the UmuDAb/DdrR corepressor system unique to the Acinetobacter genus. Our study has revealed that the DdrR protein provides an additional layer of control in preventing mutagenic polymerase expression by enhancing UmuDAb repression actions. Understanding these repressors could lead to new drug targets, as multidrug resistance in hospital-acquired infections has decreased treatment options, with limited new drugs being developed.

The increasing use of yttrium oxide (Y2O3) nanoparticles (NPs) entails an improved understanding of their potential impact on the environmental and human health. In the present study, the acute oral toxicity of Y2O3 NPs and their microparticles (MPs) was carried out in female albino Wistar rats with 250, 500 and 1000 mg kg−1 body weight doses. Before the genotoxicity evaluation, characterization of the particles by transmission electron microscopy, dynamic light scattering and laser Doppler velocimetry was performed. The genotoxicity studies were conducted using micronucleus and comet assays. Results showed that Y2O3 NP-induced significant DNA damage at higher dose (1000 mg kg−1 body weight) in peripheral blood leukocytes and liver cells, micronucleus formation in bone marrow and peripheral blood cells. The findings from biochemical assays depicted significant alterations in aspartate transaminase, alanine transaminase, alkaline phosphatase, malondialdehyde, superoxide dismutase, reduced glutathione, catalase and lactate dehydrogenase levels in serum, liver and kidneys at the higher dose only. Furthermore, tissue biodistribution of both particles was analyzed by inductively coupled plasma optical emission spectrometry. Bioaccumulation of yttrium (Y) in all tissues was significant and dose-, time- and organ-dependent. Moreover, Y2O3 NP-treated rats exhibited higher tissue distribution along with greater clearance through urine whereas Y2O3 MP-dosed animals depicted the maximum amount of Y in the feces. Hence, the results indicated that bioaccumulation of Y2O3 NPs via its Y ions may induce genotoxic effects.

Graphical abstract The etiology of the majority of human cancers is associated with a myriad of environmental causes, including physical, chemical, and biological factors. DNA damage induced by such mutagens is the initial step in the process of carcinogenesis resulting in the accumulation of mutations. Mutational events are considered the major triggers for introducing genetic and epigenetic insults such as DNA crosslinks, single- and double-strand DNA breaks, formation of DNA adducts, mismatched bases, modification in histones, DNA methylation, and microRNA alterations. However, DNA repair mechanisms are devoted to protect the DNA to ensure genetic stability, any aberrations in these calibrated mechanisms provoke cancer occurrence. Comprehensive knowledge of the type of mutagens and carcinogens and the influence of these agents in DNA damage and cancer induction is crucial to develop rational anticancer strategies. This review delineated the molecular mechanism of DNA damage and the repair pathways to provide a deep understanding of the molecular basis of mutagenicity and carcinogenicity. A relationship between DNA adduct formation and cancer incidence has also been summarized. The mechanistic basis of inflammatory response and oxidative damage triggered by mutagens in tumorigenesis has also been highlighted. We elucidated the interesting interplay between DNA damage response and immune system mechanisms. We addressed the current understanding of DNA repair targeted therapies and DNA damaging chemotherapeutic agents for cancer treatment and discussed how antiviral agents, anti-inflammatory drugs, and immunotherapeutic agents combined with traditional approaches lay the foundations for future cancer therapies.

Benzene is a recognized hematotoxin and leukemogen; however, its mechanism of action in humans remain unclear. To provide insight into the processes underlying benzene hematotoxicity, we performed high-resolution metabolomic profiling of plasma collected from a cross-sectional study of 33 healthy workers exposed to benzene (median 8-h time-weighted average exposure; 20 ppma), and 25 unexposed controls in Shanghai, China. Metabolic features associated with benzene were identified using a metabolome-wide association study (MWAS) that tested for the relationship between feature intensity and benzene exposure. MWAS identified 478 mass spectral features associated with benzene exposure at false discovery rate < 20%. Comparison to a list of 13 known benzene metabolites and metabolites predicted using a multi-component biotransformation algorithm showed five metabolites were detected, which included the known metabolites phenol and benzene diolepoxide. Metabolic pathway enrichment identified 41 pathways associated with benzene exposure, with altered pathways including carnitine shuttle, fatty acid metabolism, sulfur amino acid metabolism, glycolysis, gluconeogenesis and branched chain amino acid metabolism. These results suggest disruption to fatty acid uptake, energy metabolism and increased oxidative stress, and point towards pathways related to mitochondrial dysfunction, which has previously been linked to benzene exposure in animal models and human studies. Taken together, these results suggest benzene exposure is associated with disruption of mitochondrial pathways, and provide promising, systems biology biomarkers for risk assessment of benzene-induced hematotoxicity in humans.

Nucleotide excision repair (NER) is one of the main pathways for genome protection against structural DNA damage caused by sunlight, which in turn is extensively related to skin cancer development. The mutation spectra induced by UVB were investigated by whole-exome sequencing of randomly selected clones of NER proficient and XP-C deficient human skin fibroblasts. As a model, a cell line unable to recognize and remove lesions (XP-C) was used and compared to the complemented isogenic control (COMP). As expected, a significant increase of mutagenesis was observed in irradiated XP-C cells, mainly C>T transitions, but also CC>TT and C>A base substitutions. Remarkably, the C>T mutations occur mainly at the second base of dipyrimidine sites in pyrimidine-rich sequence contexts, with 5´TC sequence the most mutated. Although T>N mutations were also significantly increased, they were not directly related to pyrimidine dimers. Moreover, the large-scale study of a single UVB irradiation on XP-C cells allowed recovering the typical mutation spectrum found in human skin cancer tumors. Eventually, the data may be used for comparison with the mutational profiles of skin tumors obtained from XP-C patients and may help to understand the mutational process in non-affected individuals.

Nucleotide excision repair (NER) is a repair mechanism that removes DNA lesions induced by UV radiation, environmental mutagens and carcinogens. There exists sufficient evidence against acetaldehyde suggesting it to cause a variety of DNA lesions and be carcinogenic to humans. Previously, we found that acetaldehyde induces reversible intra-strand GG crosslinks in DNA similar to those induced by cis-diammineplatinum(II) that is subsequently repaired by NER. In this study, we analysed the repairability by NER mechanism and the mutagenesis of acetaldehyde. In an in vitro reaction setup with NER-proficient and NER-deficient xeroderma pigmentosum group A (XPA) cell extracts, NER reactions were observed in the presence of XPA recombinant proteins in acetaldehyde-treated plasmids. Using an in vivo assay with living XPA cells and XPA-correcting XPA cells, the repair reactions were also observed. Additionally, it was observed that DNA polymerase eta inserted dATP opposite guanine in acetaldehyde-treated oligonucleotides, suggesting that acetaldehyde-induced GG-to-TT transversions. These findings show that acetaldehyde induces NER repairable mutagenic DNA lesions.

The main bactericidal components of cold atmospheric plasma (CAP) are thought to be reactive oxygen and nitrogen species (RONS) and UV-radiation, both of which have the capacity to cause DNA damage and mutations. Here, the mutagenic effects of CAP on Escherichia coli were assessed in comparison to X- and UV-irradiation. DNA damage and mutagenesis were screened for using a diffusion-based DNA fragmentation assay and modified Ames test, respectively. Mutant colonies obtained from the latter were quantitated and sequenced. CAP was found to elicit a similar mutation spectrum to X-irradiation, which did not resemble that for UV implying that CAP-produced RONS are more likely the mutagenic component of CAP. CAP treatment was also shown to promote resistance to the antibiotic ciprofloxacin. Our data suggest that CAP treatment has mutagenic effects that may have important phenotypic consequences.

Lignin and lignin-based materials have received considerable attention in various fields due to their promise as sustainable feedstocks. Guaiacol (G) and syringol (S) are two primary monolignols that occur in different ratios for different plant species. As methoxyphenols, G and S have been targeted as atmospheric pollutants and their acute toxicity examined. However, there is a rare understanding of the toxicological properties on other endpoints and mixture effects of these monolignols. To fill this knowledge gap, our study investigated the impact of different S/G ratios (0.5, 1, and 2) and three lignin depolymerization samples from poplar, pine, and miscanthus species on mutagenicity and developmental toxicity. A multitiered method consisted of in silico simulation, in vitro Ames test, and in vivo chicken embryonic assay was employed. In the Ames test, syringol showed a sign of mutagenicity, whereas guaiacol did not, which agreed with the T.E.S.T. simulation. For three S and G mixture and lignin monomers, mutagenic activity was related to the proportion of syringol. In addition, both S and G showed developmental toxicity in the chicken embryonic assay and T.E.S.T. simulation, and guaiacol had a severe effect on lipid peroxidation. A similar trend and comparable developmental toxicity levels were detected for S and G mixtures and the three lignin depolymerized monomers. This study provides data and insights on the differential toxicity of varying S/G ratios for some important building blocks for bio-based materials.

Environmental mutagens are chemical and physical substances in the environment that has a potential to induce a wide range of mutations and generate multiple physiological, biochemical, and genetic modifications in humans. Most mutagens are having genotoxic effects on the following generation through germ cells. The influence of germinal mutations on health will be determined by their frequency, nature, and the mechanisms that keep a specific mutation in the population. Early prenatal lethal mutations have less public health consequences than genetic illnesses linked with long-term medical and social difficulties. Physical and chemical mutagens are common mutagens found in the environment. These two environmental mutagens have been associated with multiple neurological disorders and carcinogenesis in humans. Thus in this study, we aim to unravel the molecular mechanism of physical mutagens (UV rays, X-rays, gamma rays), chemical mutagens (dimethyl sulfate (DMS), bisphenol A (BPA), polycyclic aromatic hydrocarbons (PAHs), 5-chlorocytosine (5ClC)), and several heavy metals (Ar, Pb, Al, Hg, Cd, Cr) implicated in DNA damage, carcinogenesis, chromosomal abnormalities, and oxidative stress which leads to multiple disorders and impacting human health. Biological tests for mutagen detection are crucial; therefore, we also discuss several approaches (Ames test and Mutatox test) to estimate mutagenic factors in the environment. The potential risks of environmental mutagens impacting humans require a deeper basic knowledge of human genetics as well as ongoing research on humans, animals, and their tissues and fluids.

Epidemiology studies link cigarillos and shisha tobacco (delivered through a hookah waterpipe) to increased risk for cardiopulmonary diseases. Here we performed a comparative chemical constituent analysis between 3 cigarettes, 3 cigarillos, and 8 shisha tobacco products. The potency for genotoxicity and oxidative stress of each product’s generated total particulate matter (TPM) was also assessed using immortalized oral, lung, and cardiac cell lines to represent target tissues. Levels of the carcinogenic carbonyl formaldehyde were 32- to 95-fold greater, while acrolein was similar across the shisha aerosols generated by charcoal heating compared to cigarettes and cigarillos. Electric-mediated aerosol generation dramatically increased acrolein to levels exceeding those in cigarettes and cigarillos by up to 43-fold. Equivalent cytotoxic-mediated cell death and dose response for genotoxicity through induction of mutagenicity and DNA strand breaks was seen between cigarettes and cigarillos, while minimal to no effect was observed with shisha tobacco products. In contrast, increased potency of TPM from cigarillos compared to cigarettes for inducing oxidative stress via reactive oxygen radicals and lipid peroxidation across cell lines was evident, while positivity was seen for shisha tobacco products albeit at much lower levels. Together, these studies provide new insight into the potential harmful effects of cigarillos for causing tobacco-associated diseases. The high level of carbonyls in shisha products, that in turn is impacted by the heating mechanism, reside largely in the gas phase which will distribute throughout the respiratory tract and systemic circulation to likely increase genotoxic stress.

Our aim is to investigate the effect of the Mesenchymal stem cell (MSC) administration on the release of Mammalian Target of Rapamycin (mTOR) and Phosphorylated- mTOR(p-mTOR) in Cyclophosphomide (CTX) induced ovarian damage. Rats divided into three groups. The first group was categorized as the control(C group;n = 6), the second group as CTX-administered group (CTX group;n = 6), and the third group as CTX and MSC-administered group (CTX + SC group;n = 6). CTX was injected intraperitoneally at 50 mg/kg on the first day and at 8 mg/kg during the following 13 days. In Group 3, adipose-derived MSCs (5 × 104) were injected locally into the ovary. Both ovaries were removed at the end of the 8th week. The follicle count was made. The expression of mTOR and p-mTOR was analyzed immunohistochemically. The follicles in the ovary of Group C were observed in normal structures. Degeneration was evident in the CTX group. In the CTX + SC group, the degenerative appearance monitored in the CTX group vanished in most areas, and fibrosis was greatly reduced. The number of follicles in the CTX group was lower than that of both C and CTX + SC groups (p < 005). In the C group, mTOR showed strong positive staining while mTOR and p-mTOR expression was negative in all follicles in the CTX group. Both mTOR and p-mTOR revealed moderate positive expression in the CTX + SC group. MSC therapy rescued the damage ovarian function created by CTX, reducing follicle loss. MSCs were shown to inhibit the loss of mTOR and p-mTOR signaling, which is key to meiosis in oocytes.

Microbiota-derived metabolites that elicit DNA damage can contribute to colorectal cancer (CRC). However, the full spectrum of genotoxic chemicals produced by indigenous gut microbes remains to be defined. We established a pipeline to systematically evaluate the genotoxicity of an extensive collection of gut commensals from inflammatory bowel disease patients. We identified isolates from divergent phylogenies whose metabolites caused DNA damage and discovered a distinctive family of genotoxins—termed the indolimines—produced by the CRC-associated species Morganella morganii. A non–indolimine-producing M. morganii mutant lacked genotoxicity and failed to exacerbate colon tumorigenesis in mice. These studies reveal the existence of a previously unexplored universe of genotoxic small molecules from the microbiome that may affect host biology in homeostasis and disease.

Unexpected members of the gut microbiota produce diverse host cell genotoxins

Endosulfan is an extensively used organochlorine pesticide around the world, which was classified as a persistent organic pollutant (POP) in 2009. Although previous studies have documented the reproductive toxicity of endosulfan in a variety of organisms, little is known about the influence of endosulfan on the genome stability of germ cells and nonexposed progeny. Here we applied whole-genome sequencing to explore the germ cell mutagenicity of α-endosulfan in

We had previously developed an improved Ames module to directly determine the mutagenicity of gaseous formaldehyde (HCHO) and toluene without liquid extraction. This study further evaluated the suitability and sensitivity of this module on whole and real polluted air samples. For this, two common brands of stick incense (A and B) and cigarettes (A and B) were harvested, and various types of incense smoke (IS) and sidestream cigarette smoke (SCS) samples were generated by lighting 3, 6, 12, 24, 30, or 36 incense sticks, and by lighting 1, 2, or 3 cigarettes, respectively, in an acrylic box. CO

The WEEL guide for dimethyl ether (DME) was originally established in 1996 and updated in 2010. Literature searches to identify new toxicity information were performed in November 2020 and evaluated by the WEEL Revisions Subcommittee. No new studies or data relevant to the WEEL guide were identified. DME has very low acute inhalation toxicity with a 4 h LC

The dispersion of SARS-CoV-2 in aquatic environments via the discharge of domestic and hospital sewage has been confirmed in different locations. Thus, we aimed to evaluate the possible impacts of zebrafish (Danio rerio) exposure to SARS-CoV-2 peptide fragments (PSPD-2001, 2002, and 2003) alone and combined with a mix of emerging pollutants. Our data did not reveal the induction of behavioral, biometric, or mutagenic changes. But we noticed an organ-dependent biochemical response. While nitric oxide and malondialdehyde production in the brain, gills, and muscle did not differ between groups, superoxide dismutase activity was reduced in the "PSPD", "Mix", and "Mix+PSPD" groups. An increase in catalase activity and a reduction in DPPH radical scavenging activity were observed in the brains of animals exposed to the treatments. However, the "Mix+PSPD" group had a higher IBRv2 value, with NO levels (brain), the reduction of acetylcholinesterase activity (muscles), and the DPPH radical scavenging activity (brain and muscles), the most discriminant factors for this group. The principal component analysis (PCA) and hierarchical clustering analysis indicated a clear separation of the "Mix+PSPD" group from the others. Thus, we conclude that exposure to viral fragments, associated with the mix of pollutants, induced more significant toxicity in zebrafish adults than in others.

A forward mutagenesis assay using the supF gene has been widely employed for the last several decades in studies addressing mutation frequencies and mutation spectra associated with various intrinsic and environmental mutagens. In this study, by using a supF shuttle vector and non-SOS-induced Escherichia coli with short-read next-generation sequencing (NGS) technology, we present an advanced method for the study of mutations, which is simple, versatile, and cost-effective. We demonstrate the performance of our newly developed assay via pilot experiments with ultraviolet (UV) irradiation, the results from which emerge more relevant than expected. The NGS data obtained from samples of the indicator E. coli grown on titer plates provides mutation frequency and spectrum data, and uncovers obscure mutations that cannot be detected by a conventional supF assay. Furthermore, a very small amount of NGS data from selection plates reveals the almost full spectrum of mutations in each specimen and offers us a novel insight into the mechanisms of mutagenesis, despite them being considered already well known. We believe that the method presented here will contribute to future opportunities for research on mutagenesis, DNA repair, and cancer.

Background Environmental contamination from synthetic plastics and their additives is a widespread problem. Phthalate esters are a class of refractory synthetic organic compounds which are widely used in plastics, coatings, and for several industrial applications such as packaging, pharmaceuticals, and/or paints. They are released into the environment during production, use and disposal, and some of them are potential mutagens and carcinogens. Isophthalate (1,3-benzenedicarboxylic acid) is a synthetic chemical that is globally produced at a million-ton scale for industrial applications and is considered a priority pollutant. Here we describe the biochemical characterization of an enzyme involved in anaerobic degradation of isophthalate by the syntrophically fermenting bacterium Syntrophorhabdus aromaticivorans strain UI that activate isophthalate to isophthalyl-CoA followed by its decarboxylation to benzoyl-CoA. Results Isophthalate:Coenzyme A ligase (IPCL, AMP-forming) that activates isophthalate to isophthalyl-CoA was heterologously expressed in E. coli (49.6 kDa) for biochemical characterization. IPCL is homologous to phenylacetate-CoA ligase that belongs to the family of ligases that form carbon-sulfur bonds. In the presence of coenzyme A, Mg2+ and ATP, IPCL converts isophthalate to isophthalyl-CoA, AMP and pyrophosphate (PPi). The enzyme was specifically induced after anaerobic growth of S. aromaticivorans in a medium containing isophthalate as the sole carbon source. Therefore, IPCL exhibited high substrate specificity and affinity towards isophthalate. Only substrates that are structurally related to isophthalate, such as glutarate and 3-hydroxybenzoate, could be partially converted to the respective coenzyme A esters. Notably, no activity could be measured with substrates such as phthalate, terephthalate and benzoate. Acetyl-CoA or succinyl-CoA did not serve as CoA donors. The enzyme has a theoretical pI of 6.8 and exhibited optimal activity between pH 7.0 to 7.5. The optimal temperature was between 25 °C and 37 °C. Denaturation temperature (Tm) of IPCL was found to be at about 63 °C. The apparent KM values for isophthalate, CoA, and ATP were 409 μM, 642 μM, and 3580 μM, respectively. Although S. aromaticivorans is a strictly anaerobic bacterium, the enzyme was found to be oxygen-insensitive and catalysed isophthalyl-CoA formation under both anoxic and oxic conditions. Conclusion We have successfully cloned the ipcl gene, expressed and characterized the corresponding IPCL enzyme, which plays a key role in isophthalate activation that initiates its activation and further degradation by S. aromaticivorans. Its biochemical characterization represents an important step in the elucidation of the complete degradation pathway of isophthalate.

Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men worldwide. The treatment of advanced cases is based on chemotherapy, which lacks specificity and efficacy, due to severe side effects and resistance to the traditional drugs. Copper complexes have shown antitumoral efficacy and low toxicity, being considered a promising class of metal-based drugs for the treatment of malignant neoplasms. Thus, the present study aimed to evaluate the cellular effects of a copper(II) complex with 4-fluorophenoxyacetic acid hydrazide and 1,10-phenanthroline (1) on PCa cell lines, as well as the mutagenic/recombinogenic and anticarcinogenic potential of 1 in Drosophila melanogaster. PNT-2 (non-tumorigenic), LNCaP (hormone-responsive PCa) and PC-3 (androgen-independent PCa) cells were cultured, and cytotoxicity was assessed using the MTT assay. The expression levels of the proliferation markers Ki-67 and Cyclin D1 were analyzed by flow cytometry. Furthermore, the Somatic Mutation and Recombination Test (SMART) and the Epithelial Tumor Test (ETT) were performed. Complex 1 was selective to LNCaP cells, significantly reducing Ki-67 and Cyclin D1 expression levels. Sub-toxic concentrations of complex 1 were defined by the toxicity test in D. melanogaster, and no mutagenic/recombinogenic/carcinogenic effects were observed. Anticarcinogenic potential was observed in D. melanogaster, suggesting modulating activity of the complex 1 against Doxorubicin, a drug used as control by its carcinogenic properties. Therefore, complex 1 is a possible starting point for the development of new antitumor agents for the treatment of PCa.

29 p.2 fig.-3 tab.-1 graph.abst.+Sup. Inf. 4 p._4 tab., The Ames mutagenicity test constitutes the most frequently used assay to estimate the mutagenic potential of drug candidates. While this test employs experimental results using various strains of Salmonella typhimurium, the vast majority of the published in silico models for predicting mutagenicity do not take into account the test results of the individual experiments conducted for each strain. Instead, such QSAR models are generally trained employing overall labels (i.e., mutagenic and nonmutagenic). Recently, neural-based models combined with multitask learning strategies have yielded interesting results in different domains, given their capabilities to model multitarget functions. In this scenario, we propose a novel neural-based QSAR model to predict mutagenicity that leverages experimental results from different strains involved in the Ames test by means of a multitask learning approach. To the best of our knowledge, the modeling strategy hereby proposed has not been applied to model Ames mutagenicity previously. The results yielded by our model surpass those obtained by single-task modeling strategies, such as models that predict the overall Ames label or ensemble models built from individual strains. For reproducibility and accessibility purposes, all source code and datasets used in our experiments are publicly available., This work was partially supported by the Argentinean National Council of Scientific and Technological Research (CONICET for its acronym in Spanish) [Grant No.PIP 112-2017-0100829], by the National Agency for the Promotion of Research, Technological Development and Innovation of Argentina (AGENCIA I+D+i in Spanish),through the Fund for Scientific and Technological Research (FONCyT for its acronym in Spanish) [Grant No. PICT 2019-03350], by the Universidad Nacional del Sur (UNS), Bahía Blanca, Argentina [Grant No. PGI 24/N042], Ministerio de Economía, Industria y Competitividad, Gobierno de España, under Grant No. RTI2018-096100B-100, and by a Google Latin America Research Award 2021−2022.

The comet assay is an effective method for identifying DNA breaks and alkali-labile sites induced by genotoxins. Performed as a single-cell electrophoresis, this assay is especially simplistic, and the results are easily reproducible. DNA breakage can be quantitatively assessed by the induced comet tail regions, which can be measured using a variety of comet software. This protocol will finish within approximately two hours with adequate preparation, and digitized images can be taken using a confocal or standard fluorescence microscopes after staining the cell nucleus with a DNA dye.

(E)-1,2-Difluoroethylene (HFO-1132E) is a colorless gas used as a refrigerant. HFO-1132E has low acute toxicity following inhalation exposure in rats and dogs. HFO-1132E caused neither cardiac sensitization in dogs nor reproductive or developmental toxicity in rats following repeated inhalation exposure. Repeated inhalation exposure for up to 13 weeks in rats resulted in degeneration of the vomeronasal organ (VNO) at all exposure levels. However, the VNO is poorly developed or absent in humans. HFO-1132E was not genotoxic in either an in vitro chromosome aberration study or in vivo (rat) or in vitro micronucleus assays although the substance was mutagenic in 4 of 5