Forward Mutation Research Articles

HIV-1 Reverse Transcriptase Error Rates and Transcriptional Thresholds Based on Single-strand Consensus Sequencing of Target RNA Derived From In Vitro-transcription and HIV-infected Cells

Nucleotide incorporation and lacZ-based forward mutation assays have been widely used to determine the accuracy of reverse transcriptases (RTs) in RNA-dependent DNA polymerization reactions. However, they involve quite complex and laborious procedures, and cannot provide accurate error rates. Recently, NGS-based methods using barcodes opened the possibility of detecting all errors introduced by the RT, although their widespread use is limited by cost, due to the large size of libraries to be sequenced. In this study, we describe a novel and relatively simple NGS assay based on single-strand consensus sequencing that provides robust results with a relatively small number of raw sequences (around 60 Mb). The method has been validated by determining the error rate of HIV-1 (BH10 strain) RT using the HIV-1 protease-coding sequence as target. HIV-1 reverse transcription error rates in standard conditions (37 °C/3 mM Mg2+) using an in vitro-transcribed RNA were around 7.3 × 10−5. In agreement with previous reports, an 8-fold increase in RT’s accuracy was observed after reducing Mg2+ concentration to 0.5 mM. The fidelity of HIV-1 RT was also higher at 50 °C than at 37 °C (error rate 1.5 × 10−5). Interestingly, error rates obtained with HIV-1 RNA from infected cells as template of the reverse transcription at 3 mM Mg2+ (7.4 × 10−5) were similar to those determined with the in vitro-transcribed RNA, and were reduced to 1.8 × 10−5 in the presence of 0.5 mM Mg2+. Values obtained at low magnesium concentrations were modestly higher than the transcription error rates calculated for human cells, thereby suggesting a realistic transcriptional threshold for our NGS-based error rate determinations.

Molecular Mechanism of Cell Signaling Pathways of Cancer Cells in Forward and Reverse Mutation: A Perspective Review

Molecular Mechanism of Cell Signaling Pathways of Cancer Cells in Forward and Reverse Mutation: A Perspective Review

Acute exposure to dihydroxyacetone promotes genotoxicity and chromosomal instability in lung, cardiac, and liver cell models.

Inhalation exposures to dihydroxyacetone (DHA) occur through spray tanning and e-cigarette aerosols. Several studies in skin models have demonstrated that millimolar doses of DHA are cytotoxic, yet the genotoxicity was unclear. We examined the genotoxicity of DHA in cell models relevant to inhalation exposures. Human bronchial epithelial cells BEAS-2B, lung carcinoma cells A549, cardiomyocyte Ac16, and hepatocellular carcinoma HepG3 were exposed to DHA, and low millimolar doses of DHA were cytotoxic. IC90 DHA doses induced cell cycle arrest in all cells except the Ac16. We examined DHA's genotoxicity using strand break markers, DNA adduct detection by Repair Assisted Damage Detection (RADD), metaphase spreads, and a forward mutation assay for mutagenesis. Similar to results for skin, DHA did not induce significant levels of strand breaks. However, RADD revealed DNA adducts were induced 24 h after DHA exposure, with BEAS-2B and Ac16 showing oxidative lesions and A549 and HepG3 showing crosslink-type lesions. Yet, only low levels of reactive oxygen species or advanced glycation end products were detected after DHA exposure. Metaphase spreads revealed significant increases in chromosomal aberrations in the BEAS-2B and HepG3 with corresponding changes in ploidy. Finally, we confirmed the mutagenesis observed using the supF reporter plasmid. DHA increased the mutation frequency, consistent with methylmethane sulfonate, a mutagen and clastogen. These data demonstrate DHA is a clastogen, inducing cell-specific genotoxicity and chromosomal instability. The specific genotoxicity measured in the BEAS-2B in this study suggests that inhalation exposures pose health risks to vapers, requiring further investigation.

OmeDDG: Improved Protein Mutation Stability Prediction Based on Predicted 3D Structures.

Determining changes in the protein's thermal stability following mutations is critical in protein engineering and understanding pathogenic missense mutations. Despite the development of various computational methods to predict the effects of single-point mutations, their accuracy remains limited. In this study, we propose a new computational method, OmeDDG, that more accurately predicts mutation-induced Gibbs free energy changes in protein folding (ΔΔG). OmeDDG takes the sequences of wild-type and mutant proteins as input, utilizes OmegaFold to obtain the 3D structure, employs a convolutional neural network to extract structural features, and combines them with protein mutation features and pretraining features to predict the stability of single-point mutations in proteins. We performed a comprehensive comparison between OmeDDG and other available prediction methods on four blind test datasets, confirming that OmeDDG can effectively enhance protein mutation prediction performance. Notably, on the antisymmetric dataset Ssym, OmeDDG achieves the best performance, demonstrating favorable antisymmetry with PCC = 0.79 and RMSE = 0.96 for forward mutations and PCC = 0.77 and RMSE = 0.97 for reverse mutant types.

DDMut: predicting effects of mutations on protein stability using deep learning.

Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite significant efforts, community assessments of predictive tools have highlighted ongoing limitations, including computational time, low predictive power, and biased predictions towards destabilising mutations. To fill this gap, we developed DDMut, a fast and accurate siamese network to predict changes in Gibbs Free Energy upon single and multiple point mutations, leveraging both forward and hypothetical reverse mutations to account for model anti-symmetry. Deep learning models were built by integrating graph-based representations of the localised 3D environment, with convolutional layers and transformer encoders. This combination better captured the distance patterns between atoms by extracting both short-range and long-range interactions. DDMut achieved Pearson's correlations of up to 0.70 (RMSE: 1.37 kcal/mol) on single point mutations, and 0.70 (RMSE: 1.84 kcal/mol) on double/triple mutants, outperforming most available methods across non-redundant blind test sets. Importantly, DDMut was highly scalable and demonstrated anti-symmetric performance on both destabilising and stabilising mutations. We believe DDMut will be a useful platform to better understand the functional consequences of mutations, and guide rational protein engineering. DDMut is freely available as a web server and API at https://biosig.lab.uq.edu.au/ddmut.

50 Human Hazard Assessment of Nanomaterials: Supporting Risk Decision Making Through Interlaboratory Trial Data

Abstract The prevalent application of engineered nanomaterials (ENMs) has led to an extensive effort to develop tools facilitating their risk assessment and management. This interlaboratory trial (encompassing two laboratories) sought to investigate the genotoxic impact of ENMs using the hypoxanthine phosphoribosyltransferase (HPRT) forward mutation assay and in vitro cytokinesis-blocked micronucleus (CBMN) assay, utilising harmonised protocols. In the CBMN assay, human lymphoblast (TK6) cells were exposed to zinc oxide (ZnO), titanium dioxide (TiO2) and tungsten carbide-cobalt (WC/Co) for 24-hours after which 1000 binucleated cells were scored for micronuclei (n=2). For the HPRT assay, TK6 and mouse fibroblast (V79) cells were exposed to ZnO, TiO2, WC/Co, CuO and Nanocyl-CNTs for 24-hours after which 600 wells were scored for point mutations (n=2). Significant cytotoxicity and micronuclei frequency was reported following ZnO exposure (20µg/ml). Significant micronuclei induction was reported following WC/Co exposures (2-fold over control, 100µg/ml). No significant mutagenicity was detected with ZnO or TiO2; both laboratories observed significant cytotoxicity following exposure to ZnO (20µg/ml; 37% reduction in cell viability). In the second interlaboratory trial CuO induced significant cytotoxicity (36% reduction in viability: 0.5µg/ml), and a 32-fold increase in mutagenicity. The Nanocyl induced a 6-fold increase in mutagenicity. The data generated here has shown good statistical concordance between laboratories and has highlighted a promising route forward in supporting risk decision making for ENMs. The authors would like to acknowledge this research has received funding from the European Union’s Horizon 2020 research and innovation program for the RiskGONE project, grant agreement #814425.

Spontaneous deamination of cytosine to uracil is biased to the non-transcribed DNA strand in yeast

Transcription in Saccharomyces cerevisiae is associated with elevated mutation and this partially reflects enhanced damage of the corresponding DNA. Spontaneous deamination of cytosine to uracil leads to CG>TA mutations that provide a strand-specific read-out of damage in strains that lack the ability to remove uracil from DNA. Using the CAN1 forward mutation reporter, we found that C>T and G>A mutations, which reflect deamination of the non-transcribed and transcribed DNA strands, respectively, occurred at similar rates under low-transcription conditions. By contrast, the rate of C>T mutations was 3-fold higher than G>A mutations under high-transcription conditions, demonstrating biased deamination of the non-transcribed strand (NTS). The NTS is transiently single-stranded within the ∼15 bp transcription bubble, or a more extensive region of the NTS can be exposed as part of an R-loop that can form behind RNA polymerase. Neither the deletion of genes whose products restrain R-loop formation nor the over-expression of RNase H1, which degrades R-loops, reduced the biased deamination of the NTS, and no transcription-associated R-loop formation at CAN1 was detected. These results suggest that the NTS within the transcription bubble is a target for spontaneous deamination and likely other types of DNA damage.

Dynamics of drug on-drug off models with mutations in morbidostat — Dedicated to the seventieth birthday of Professor Gail Wolkowicz

<abstract><p>The morbidostat is a bacteria culture device that progressively increases antibiotic drug concentration. It is used to study the evolutionary pathway. In this article, we construct mathematical models for the morbidostat. First we consider the case of no mutations, we study limiting systems and obtain criteria for the large time behavior of the solutions. From the theoretical results and numerical simulations, we conclude that there are two competitive exclusion states of either wild type or mutant type as the threshold parameter $ U $ varies. There are three cases, wild type bacteria excludes all mutants; a mutant dominates in the competition; oscillation between the above two states.</p> <p>Next we study the systems of forward mutations and forward-backward mutations. Then we apply a result of pertubation for globally stable state.</p></abstract>

In vitro and in vivo assessments of the genotoxic potential of 3-chloroallyl alcohol.

3-Chloroallyl alcohol (3-CAA) can be found in the environment following the application of plant protection products. 3-CAA is formed in groundwater following the injection of 1,3-dichloropropene, a fumigant used to control nematodes. 3-CAA is also formed, in leafy crops, as a glycoside conjugate following application of the herbicide, clethodim. Human exposure may occur from groundwater used as drinking water or through dietary consumption. To characterize 3-CAA's potential to cause genotoxicity in mammals, in vitro and in vivo studies were conducted. 3-CAA was negative in an Ames test and positive in a mouse lymphoma forward mutation assay. 3-CAA was negative in an acute in vivo CD-1 mouse bone marrow micronucleus assay when administered up to a dose level of 125 mg/kg/day for two consecutive days. In a combined gene mutation assay and erythrocyte micronucleus assay, using transgenic Big Blue® Fischer 344 rats, 3-CAA was administered via drinking water at targeted dose levels of 0, 10, 30, and 100 mg/kg/day for 29 days. Peripheral blood samples, collected at the end of treatment, were analyzed for micronucleus induction in reticulocytes using flow cytometry. Liver and bone marrow samples, collected 2 days after the termination of the treatment, were analyzed for the induction of mutations at the cII locus. 3-CAA did not induce an increase in mutant frequency or micronuclei under the experimental conditions. In conclusion, the mutagenic response observed in the in vitro mouse lymphoma assay is not confirmed in the whole animal. 3-CAA is not considered to pose a mutagenic risk.

Physicochemical characterization and oxidative potential of size fractionated Particulate Matter: Uptake, genotoxicity and mutagenicity in V-79 cells

For many years, the impact of Particulate Matter (PM) in the ambient air has been one of the major concerns for the environment and human health. The consideration of the heterogeneity and complexity of different size fractions is notably important for the assessment of PM toxicological effects. The aim of the study was to present a comprehensive size-composition-morphology characterization and to assess the oxidative potential, genotoxicity, and mutagenicity of the atmospheric PM fractions, collected by using MOUDI near a busy roadside in Lucknow, India. Physicochemical characterization of ambient coarse particles (1.8–10 µm), fine particles (0.32–1.8 µm), quasi-ultrafine (0.1–0.32 µm) and ultrafine particles (≤0.1 µm) along with SRM 1649b was done using TEM, SEM, DLS, NTA, ICP-MS, and IC in parallel with the estimation of exogenous Reactive Oxygen Species (ROS) by acellular assays. In this study, two different acellular assays, dithiothreitol (DTT) and the CM-H2DCFDA assay, indicated stronger mass-normalized bioactivity for different size ranges. Enrichment factor analysis indicated that the different size fractions were highly enriched with elements of anthropogenic origin as compared to elements of crustal origin. The endotoxin concentration in different size fractions was also estimated. Cellular studies demonstrated significant uptake, cytotoxicity, ultrastructural changes, cellular ROS generation, and changes in the different phases of the cell cycle (Sub G1, G1, S, G2/M) exposed to different size fractions. The Comet assay and the Micronucleus assay were used to estimate genotoxicity. Mutagenic potential was revealed by the HGPRT gene forward mutation assay in V-97 cells. Conclusively, our results clearly indicate that the genotoxic and mutagenic potential of the coarse PM was greater than the other fractions, and interestingly, the ultrafine PM has higher bioactivity as compared to quasi-ultrafine PM.

PSP-GNM: Predicting Protein Stability Changes upon Point Mutations with a Gaussian Network Model.

Understanding the effects of missense mutations on protein stability is a widely acknowledged significant biological problem. Genomic missense mutations may alter one or more amino acids, leading to increased or decreased stability of the encoded proteins. In this study, we describe a novel approach—Protein Stability Prediction with a Gaussian Network Model (PSP-GNM)—to measure the unfolding Gibbs free energy change (ΔΔG) and evaluate the effects of single amino acid substitutions on protein stability. Specifically, PSP-GNM employs a coarse-grained Gaussian Network Model (GNM) that has interactions between amino acids weighted by the Miyazawa–Jernigan statistical potential. We used PSP-GNM to simulate partial unfolding of the wildtype and mutant protein structures, and then used the difference in the energies and entropies of the unfolded wildtype and mutant proteins to calculate ΔΔG. The extent of the agreement between the ΔΔG calculated by PSP-GNM and the experimental ΔΔG was evaluated on three benchmark datasets: 350 forward mutations (S350 dataset), 669 forward and reverse mutations (S669 dataset) and 611 forward and reverse mutations (S611 dataset). We observed a Pearson correlation coefficient as high as 0.61, which is comparable to many of the existing state-of-the-art methods. The agreement with experimental ΔΔG further increased when we considered only those measurements made close to 25 °C and neutral pH, suggesting dependence on experimental conditions. We also assessed for the antisymmetry (ΔΔGreverse = −ΔΔGforward) between the forward and reverse mutations on the Ssym+ dataset, which has 352 forward and reverse mutations. While most available methods do not display significant antisymmetry, PSP-GNM demonstrated near-perfect antisymmetry, with a Pearson correlation of −0.97. PSP-GNM is written in Python and can be downloaded as a stand-alone code.

Variation in G-quadruplex sequence and topology differentially impacts human DNA polymerase fidelity

G-quadruplexes (G4s), a type of non-B DNA, play important roles in a wide range of molecular processes, including replication, transcription, and translation. Genome integrity relies on efficient and accurate DNA synthesis, and is compromised by various stressors, to which non-B DNA structures such as G4s can be particularly vulnerable. However, the impact of G4 structures on DNA polymerase fidelity is largely unknown. Using an in vitro forward mutation assay, we investigated the fidelity of human DNA polymerases delta (δ4, four-subunit), eta (η), and kappa (κ) during synthesis of G4 motifs representing those in the human genome. The motifs differ in sequence, topology, and stability, features that may affect DNA polymerase errors. Polymerase error rate hierarchy (δ4 < κ < η) is largely maintained during G4 synthesis. Importantly, we observed unique polymerase error signatures during synthesis of VEGF G4 motifs, stable G4s which form parallel topologies. These statistically significant errors occurred within, immediately flanking, and encompassing the G4 motif. For pol δ4, the errors were deletions, insertions and complex errors within the G4 or encompassing the G4 motif and surrounding sequence. For pol η, the errors occurred in 3’ sequences flanking the G4 motif. For pol κ, the errors were frameshift mutations within G-tracts of the G4. Because these error signatures were not observed during synthesis of an antiparallel G4 and, to a lesser extent, a hybrid G4, we suggest that G4 topology and/or stability could influence polymerase fidelity. Using in silico analyses, we show that most polymerase errors are predicted to have minimal effects on predicted G4 stability. Our results provide a unique view of G4s not previously elucidated, showing that G4 motif heterogeneity differentially influences polymerase fidelity within the motif and flanking sequences. Thus, our study advances the understanding of how DNA polymerase errors contribute to G4 mutagenesis.

Forward mutations in sars-cov-2 cause waves of covid-19

Time space between waves of Covid-19 is linked to selection of most appropriate virus mutant(s)/variant(s), in the Spike (S) gene, generated during massive and error prone replication of the virus in the human populations that are either naive or partially immune due to infection immunity or vaccinal immunity, having higher transmissibility with immune-escape feature and increased pathogenicity. The mutations in the S gene have been found to be progressive to resist prevalent virus neutralizing antibodies in the hosts. The first mutation of significance was D (Asp) to G (Gly) at the position 614 of the Spike protein, over the original Wuhan strain/virus. Subsequently, with in a short time span of less than 18 months since December 2019, many Variants/mutants of SARS-CoV-2, viz., B.1.1.7, B.1.427, B.1.1.28.2, B.1.429, B.1.617, B.1.351, and P.1 have been found associated with the current waves of the Covid-19 pandemic in different countries. The latest significant S variants identified in the second wave in India and elsewhere are B.1.617 and its Kappa (k), Delta (8) and Delta plus (8+; Lys to Asn at position 417) strains, in the order of appearance. The lambda (X) variant is prevalent in South America. The P.2 variant B.1.1.28.2 was antigenically dominant over B.1 D614G virus. The k and 8 variants of the S gene were partially resistant to neutralization by existing vaccinal antibodies, and the 8 variant has been feared to ignite third wave in many countries of Americas, Asia and Europe. The SARS-CoV-2 possibly has ‘Quasispecies’ structure, like the transboundary animal pathogen Foot and Mouth Disease (FMD) virus, and continuous forward mutations in the S gene has been the cause of the fact that "previous immunity acquired during the course of the natural infection (or vaccination) is not fully competent to protect against subsequent mutants selected (in the nature) that leads to new waves of the disease". The waves of Covid-19 have been guided by emergence of sequential antigenic variants, e.g. B.1 D614G virus ^B.1 8 mutant ^B.1 8+ mutant. Intermittent/inter-wave drop in incidence of the infection between the Covid-19 waves could be attributed to very short term (less than 3 months?) infection/vaccination immunity that is breached by the successive antigenic mutant (s) selected. Vaccination and virus circulation happening simultaneously, can facilitate faster selection of neutralization-escape mutants, which is now evident and may lead to new waves of the disease/infection at short intervals of less than 6 months. All variants described here are antigenically dissimilar, whereas the current Covid-19 vaccines are monovalent. Therefore, it may be essential and beneficial to have multivalent vaccines or with higher antigenic mass in monovalent vaccines. Further, it is required that nasal swabs of a certain percentage of vaccinates be collected at the time of vaccination and preserved for retrospect analysis of their infection status, that in turn will reveal the infection status of the population in villages/cities/districts/State.

Identification of Schizosaccharomyces pombe ird Mutants Resistant to Glucose Suppression and Oxidative Stress

Glucose is both the favourite carbon and energy source and acts as a hormone that plays a regulating role in many biological processes. Calorie restriction extends the lifespan in many organisms, including Schizosaccharomyces pombe, while uptake of high glucose leads to undesired results, such as diabetes and aging. In this study, sequence analysis of Schizosaccharomyces pombe ird5 and ird11 mutants was performed using next-generation sequencing techniques and a total of 20 different mutations were detected. ird11 is resistant to oxidative stress without calorie restriction, whereas ird5 displays an adaptive response against oxidative stress. We selected nine candidate mutations located in the non-coding (6) and coding (3) region among a total of 20 different mutations. The nine candidate mutations, which are thought to be responsible for ird5 and ird11 mutant phenotypes, were investigated via forward and backward mutations by using various cloning techniques. The results of this study provide report-like information that will contribute to understanding the relationship between glucose sensing/signalling and oxidative stress response components.

Stochastic investigation of HIV infection and the emergence of drug resistance.

Drug-resistant HIV-1 has caused a growing concern in clinic and public health. Although combination antiretroviral therapy can contribute massively to the suppression of viral loads in patients with HIV-1, it cannot lead to viral eradication. Continuing viral replication during sub-optimal therapy (due to poor adherence or other reasons) may lead to the accumulation of drug resistance mutations, resulting in an increased risk of disease progression. Many studies also suggest that events occurring during the early stage of HIV-1 infection (i.e., the first few hours to days following HIV exposure) may determine whether the infection can be successfully established. However, the numbers of infected cells and viruses during the early stage are extremely low and stochasticity may play a critical role in dictating the fate of infection. In this paper, we use stochastic models to investigate viral infection and the emergence of drug resistance of HIV-1. The stochastic model is formulated by a continuous-time Markov chain (CTMC), which is derived based on an ordinary differential equation model proposed by Kitayimbwa et al. that includes both forward and backward mutations. An analytic estimate of the probability of the clearance of HIV infection of the CTMC model near the infection-free equilibrium is obtained by a multitype branching process approximation. The analytical predictions are validated by numerical simulations. Unlike the deterministic dynamics where the basic reproduction number R0 serves as a sharp threshold parameter (i.e., the disease dies out if R0<1 and persists if R0>1), the stochastic models indicate that there is always a positive probability for HIV infection to be eradicated in patients. In the presence of antiretroviral therapy, our results show that the chance of clearance of the infection tends to increase although drug resistance is likely to emerge.

Identification of a Mycobacterium tuberculosis-specific gene marker for diagnosis of tuberculosis using semi-nested melt-MAMA qPCR (lprM-MAMA)

Tuberculosis (TB) is the deadliest of infectious diseases. TB diagnosis, based on sputum microscopy, culture, and nucleic acid amplification tests (NAATs) to identify its main causative agent, Mycobacterium tuberculosis (MTB), remains challenging. The current available NAATs, endorsed by World Health Organization (WHO), can differentiate MTB from some MTB complex (MTBC) members. Using bioinformatics, we identified a single nucleotide polymorphism (SNP) in lprM (Rv1970) gene that differentiate MTB from other MTBC members. A forward mismatch amplification mutation assay (MAMA) primer was designed for the targeted mutation and was used in a semi-nested melt-MAMA qPCR (lprM-MAMA). Using the optimized protocol, lprM-MAMA was positive with all MTB reference and clinical strains, and negative with other MTBC members, non-tuberculous mycobacteria (NTM) and other non-mycobacterial (NM) reference strains. The limit of detection (LOD) of lprM-MAMA was 76.29 fg. Xpert® MTB/RIF (Xpert)-positive sputum samples were also positive by lprM-MAMA, except for samples classified as having “very low” bacterial load by Xpert. Xpert-negative sputum samples were also negative by lprM-MAMA. In conclusion, lprM-MAMA demonstrated to be a useful tool for specific MTB diagnosis. Further evaluation with higher number of reference strains, including NTM and NM; and sputum samples are required to determine its potential for clinical application.

New indicator Escherichia coli strain for rapid and accurate detection of supF mutations

BackgroundThe supF gene of Escherichia coli is useful for forward mutation analysis in bacterial and mammalian cells used in mutagenesis and DNA repair studies. Indicator E. coli strains, such as KS40/pOF105, have been used to analyze supF mutations. However, KS40/pOF105 is not enough to select supF mutants on nutrient-rich agar plates. Therefore, in this study, a new indicator E. coli strain for rapid and accurate detection of supF mutations was developed.ResultsThe gyrA and rpsL genes with an amber mutation were integrated into the chromosomal DNA of E. coli KS40 to produce a new indicator strain, RF01. RF01 cells transformed by the wild-type supF gene were sensitive to nalidixic acid and streptomycin on LB agar plates. supF mutant frequencies and mutation spectra in RF01 were similar to those in KS40/pOF105. In addition, some mutations in supF were only detected in RF01.ConclusionRF01 is a new and useful indicator E. coli strain for analyzing supF mutations.

Effects of melatonin to arecoline-induced reactive oxygen species production and DNA damage in oral squamous cell carcinoma

Arecoline, the major alkaloid of areca nut, is known to induce reactive oxygen species (ROS) and DNA damage during oral cancer progression. This study aim to evaluate whether melatonin, an antioxidant, supported or repressed the arecoline-induced carcinogenesis phenotypes in oral squamous cell carcinoma (OSCC). The cytotoxicity of arecoline or melatonin treatment alone and their co-treatment in the OSCC cell line OEC-M1 were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell cycle, cell death, and total ROS production were analyzed using flow cytometer. The protein expression was determined using western blot analysis. The genotoxicity and mutation rate were determined using micronucleus assay and hypoxanthine phosphoribosyl transferase (HPRT) forward mutation assay, respectively, in CHO-K1 cells. The ataxia telangiectasia mutated (ATM) promoter activity and DNA repair ability were determined through reporter assay. The result showed that both the arecoline and melatonin induced ROS production and antioxidant enzymes expression. Melatonin treatment enhanced arecoline-induced ROS production, cytotoxicity, G2/M phase arrest, and cell apoptosis in OSCC cells. On the other hand, melatonin treatment activated DNA repair activity to reverse arecoline-induced DNA damage and mutation. These results indicated that melatonin is a potential chemopreventive agent for betel quid chewers to prevent OSCC initiation and progression.

Mutagenicity assessment downstream of oil and gas produced water discharges intended for agricultural beneficial reuse.

Produced water is the largest waste stream associated with oil and gas operations. This complex fluid contains petroleum hydrocarbons, heavy metals, salts, naturally occurring radioactive materials and any remaining chemical additives. In the United States, west of the 98th meridian, the federal National Pollutant Discharge Elimination System (NPDES) exemption allows release of produced water for agricultural beneficial reuse. The goal of this study was to quantify mutagenicity of a produced water NPDES release and discharge stream. We used four mutation assays in budding yeast cells that provide rate estimates for copy number variation (CNV) duplications and deletions, as well as forward and reversion point mutations. Higher mutation rates were observed at the discharge and decreased with distance downstream, which correlated with the concentrations of known carcinogens detected in the stream (e.g., benzene, radium), described in a companion study. Mutation rate increases were most prominent for CNV duplications and were higher than mutations observed in mixtures of known toxic compounds. Additionally, the samples were evaluated for acute toxicity in Daphnia magna and developmental toxicity in zebrafish. Acute toxicity was minimal, and no developmental toxicity was observed. This study illustrates that chemical analysis alone (McLaughlin et al., 2020) is insufficient for characterizing the risk of produced water NPDES releases and that a thorough evaluation of chronic toxicity is necessary to fully assess produced water for beneficial reuse.

Timing of appearance of new mutations during yeast meiosis and their association with recombination.

Mutations in budding yeast occur in meiosis at higher frequencies than in cells grown vegetatively. In contrast to mutations that occur in somatic cells, meiotic mutations have a special, long-range impact on evolution, because they are transferred to the following generations through the gametes. Understanding the mechanistic basis of meiotic mutagenicity is still lacking, however. Here, we report studies of mutagenicity in the reporter gene CAN1, in which forward mutation events in meiosis are sevenfold higher than in mitotic cells, as determined by fluctuation analysis. Meiotic mutations appear approximately at the same time as heteroallelic-recombination products and as meiotic DSBs. Recombination-associated timing of meiotic mutagenicity is further augmented by the absence of meiotic mutations in cells arrested after pre-meiotic DNA synthesis. More than 40% of the mutations generated in meiosis in CAN1 are found on chromosomes that have recombined in the 2.2kb covering the reporter, implying that the mutations have resulted from recombination events and that meiotic recombination is mutagenic. The induced expression in yeast meiosis of low-fidelity DNA polymerases coded by the genes REV1, REV3, RAD30, and POL4 makes them attractive candidates for introducing mutations. However, in our extensive experiments with polymerase-deleted strains, these polymerases do not appear to be the major source of meiotic mutagenicity. From the connection between meiotic mutagenicity and recombination, one may conclude that meiotic recombination has another diversification role, of introducing new mutations at the DNA sequence level, in addition to reshuffling of existing variation. The new, rare meiotic mutations may contribute to long-range evolutionary processes and enhance adaptation to challenging environments.