The interplay of DNA damage and repair, gene expression, and mutagenesis in mammalian cells during oxidative stress.

We investigated the interplay among oxidative DNA damage and repair, expression of genes encoding major base excision repair (BER) enzymes and bypass DNA polymerases, and mutagenesis in mammalian cells. Primary mouse embryonic fibroblasts were challenged with oxidative stress induced by methylene blue plus visible light, and formation and repair of DNA damage, changes in gene expression, and mutagenesis were determined at increasing intervals post-treatment (0 - 192 hours). Significant formation of oxidative DNA damage together with upregulation of Ogg1, Polß, and Polκ, and no changes in Mutyh and Nudt1 expression were found in treated cells. There was a distinct interconnection between Ogg1 and Polß expression and DNA damage formation and repair whereby changes in expression of these two genes were proportionate to the levels of oxidative DNA damage, once a 3-plus hour lag time passed (P < 0.05). Equally notable was the matching pattern of Polκ expression and kinetics of oxidative DNA damage and repair (P < 0.05). The DNA damage and gene expression data were remarkably consistent with mutagenicity data in the treated cells; the induced mutation spectrum is indicative of erroneous bypass of oxidized DNA bases and incorporation of oxidized deoxynucleoside triphosphates during replication of the genomic DNA. Our findings support follow-up functional studies to elucidate how oxidation of DNA bases and the nucleotide pool, overexpression of Polκ, delayed upregulation of Ogg1 and Polß, and inadequate expression of Nudt1 and Mutyh collectively affect mutagenesis consequent to oxidative stress.

Vaping Dose, Device Type, and E-Liquid Flavor are Determinants of DNA Damage in Electronic Cigarette Users.

INTRODUCTION: Despite the widespread use of electronic cigarettes, the long-term health consequences of vaping are largely unknown. AIMS AND METHODS: We investigated the DNA-damaging effects of vaping as compared to smoking in healthy adults, including "exclusive" vapers (never smokers), cigarette smokers only, and nonusers, matched for age, gender, and race (N = 72). Following biochemical verification of vaping or smoking status, we quantified DNA damage in oral epithelial cells of our study subjects, using a long-amplicon quantitative polymerase chain reaction assay. RESULTS: We detected significantly increased levels of DNA damage in both vapers and smokers as compared to nonusers (p = .005 and p = .020, respectively). While the mean levels of DNA damage did not differ significantly between vapers and smokers (p = .522), damage levels increased dose-dependently, from light users to heavy users, in both vapers and smokers as compared to nonusers. Among vapers, pod users followed by mod users, and those who used sweet-, mint or menthol-, and fruit-flavored e-liquids, respectively, showed the highest levels of DNA damage. The nicotine content of e-liquid was not a predictor of DNA damage in vapers. CONCLUSIONS: This is the first demonstration of a dose-dependent formation of DNA damage in vapers who had never smoked cigarettes. Our data support a role for product characteristics, specifically device type and e-liquid flavor, in the induction of DNA damage in vapers. Given the popularity of pod and mod devices and the preferability of sweet-, mint or menthol-, and fruit-flavored e-liquids by both adult- and youth vapers, our findings can have significant implications for public health and tobacco products regulation. IMPLICATIONS: We demonstrate a dose-dependent formation of DNA damage in oral cells from vapers who had never smoked tobacco cigarettes as well as exclusive cigarette smokers. Device type and e-liquid flavor determine the extent of DNA damage detected in vapers. Users of pod devices followed by mod users, and those who use sweet-, mint or menthol-, and fruit-flavored e-liquids, respectively, show the highest levels of DNA damage when compared to nonusers. Given the popularity of pod and mod devices and the preferability of these same flavors of e-liquid by both adult- and youth vapers, our findings can have significant implications for public health and tobacco products regulation.

DNA Hydroxymethylation in Smoking-Associated Cancers.

5-hydroxymethylcytosine (5-hmC) was first detected in mammalian DNA five decades ago. However, it did not take center stage in the field of epigenetics until 2009, when ten-eleven translocation 1 (TET1) was found to oxidize 5-methylcytosine to 5-hmC, thus offering a long-awaited mechanism for active DNA demethylation. Since then, a remarkable body of research has implicated DNA hydroxymethylation in pluripotency, differentiation, neural system development, aging, and pathogenesis of numerous diseases, especially cancer. Here, we focus on DNA hydroxymethylation in smoking-associated carcinogenesis to highlight the diagnostic, therapeutic, and prognostic potentials of this epigenetic mark. We describe the significance of 5-hmC in DNA demethylation, the importance of substrates and cofactors in TET-mediated DNA hydroxymethylation, the regulation of TETs and related genes (isocitrate dehydrogenases, fumarate hydratase, and succinate dehydrogenase), the cell-type dependency and genomic distribution of 5-hmC, and the functional role of 5-hmC in the epigenetic regulation of transcription. We showcase examples of studies on three major smoking-associated cancers, including lung, bladder, and colorectal cancers, to summarize the current state of knowledge, outstanding questions, and future direction in the field.

Secondhand smoke affects reproductive functions by altering the mouse testis transcriptome, and leads to select intron retention in Pde1a.

BACKGROUND: Human exposure to secondhand smoke (SHS) is known to result in adverse effects in multiple organ systems. However, the impact of SHS on the male reproductive system, particularly on the regulation of genes and molecular pathways that govern sperm production, maturation, and functions remains largely understudied. OBJECTIVE: We investigated the effects of SHS on the testis transcriptome in a validated mouse model. METHODS: Adult male mice were exposed to SHS (5 h/day, 5 days/week for 4 months) as compared to controls (clean air-exposed). RNA-seq analysis was performed on the testis of SHS-exposed mice and controls. Variant discovery and plink association analyses were also conducted to detect exposure-related transcript variants in SHS-treated mice. RESULTS: Exposure of mice to SHS resulted in the aberrant expression of 131 testicular genes. Whilst approximately two thirds of the differentially expressed genes were protein-coding, the remaining (30.5%) comprised noncoding elements, mostly lncRNAs (19.1%). Variant discovery analysis identified a homozygous frameshift variant that is statistically significantly associated with SHS exposure (P = 7.744e-06) and is generated by retention of a short intron within Pde1a, a key regulator of spermatogenesis. Notably, this SHS-associated intron variant harbors an evolutionarily conserved, premature termination codon (PTC) that disrupts the open reading frame of Pde1a, presumably leading to its degradation via nonsense-mediated decay. DISCUSSION: SHS alters the expression of genes involved in molecular pathways that are crucial for normal testis development and function. Preferential targeting of lncRNAs in the testis of SHS-exposed mice is especially significant considering their crucial role in the spatial and temporal modulation of spermatogenesis. Equally important is our discovery of a novel homozygous frameshift variant that is exclusively and significantly associated with SHS-exposure and is likely to represent a safeguard mechanism to regulate transcription of Pde1a and preserve normal testis function during harmful exposure to environmental agents.

A novel role for vaping in mitochondrial gene dysregulation and inflammation fundamental to disease development.

We constructed and analyzed the whole transcriptome in leukocytes of healthy adult vapers (with/without a history of smoking), 'exclusive' cigarette smokers, and controls (non-users of any tobacco products). Furthermore, we performed single-gene validation of expression data, and biochemical validation of vaping/smoking status by plasma cotinine measurement. Computational modeling, combining primary analysis (age- and sex-adjusted limmaVoom) and sensitivity analysis (cumulative e-liquid- and pack-year modeling), revealed that 'current' vaping, but not 'past' smoking, is significantly associated with gene dysregulation in vapers. Comparative analysis of the gene networks and canonical pathways dysregulated in vapers and smokers showed strikingly similar patterns in the two groups, although the extent of transcriptomic changes was more pronounced in smokers than vapers. Of significance is the preferential targeting of mitochondrial genes in both vapers and smokers, concurrent with impaired functional networks, which drive mitochondrial DNA-related disorders. Equally significant is the dysregulation of immune response genes in vapers and smokers, modulated by upstream cytokines, including members of the interleukin and interferon family, which play a crucial role in inflammation. Our findings accord with the growing evidence on the central role of mitochondria as signaling organelles involved in immunity and inflammatory response, which are fundamental to disease development.

Hydroxychloroquine induces oxidative DNA damage and mutation in mammalian cells.

Since the early stages of the pandemic, hydroxychloroquine (HCQ), a widely used drug with good safety profile in clinic, has come to the forefront of research on drug repurposing for COVID-19 treatment/prevention. Despite the decades-long use of HCQ in the treatment of diseases, such as malaria and autoimmune disorders, the exact mechanisms of action of this drug are only beginning to be understood. To date, no data are available on the genotoxic potential of HCQ in vitro or in vivo. The present study is the first investigation of the DNA damaging- and mutagenic effects of HCQ in mammalian cells in vitro, at concentrations that are comparable to clinically achievable doses in patient populations. We demonstrate significant induction of a representative oxidative DNA damage (8-oxodG) in primary mouse embryonic fibroblasts (MEFs) treated with HCQ at 5 and 25 µM concentrations (P = 0.020 and P = 0.029, respectively), as determined by enzyme-linked immunosorbent assay. Furthermore, we show significant mutagenicity of HCQ, manifest as 2.2- and 1.8-fold increases in relative cII mutant frequency in primary and spontaneously immortalized Big Blue® MEFs, respectively, treated with 25 µM dose of this drug (P = 0.005 and P = 0.012, respectively). The observed genotoxic effects of HCQ in vitro, achievable at clinically relevant doses, are novel and important, and may have significant implications for safety monitoring in patient populations. Given the substantial number of the world's population receiving HCQ for the treatment of various chronic diseases or in the context of clinical trials for COVID-19, our findings warrant further investigations into the biological consequences of therapeutic/preventive use of this drug.

Relationships among smoking, oxidative stress, inflammation, macromolecular damage, and cancer.

Smoking is a major risk factor for a variety of diseases, including cancer and immune-mediated inflammatory diseases. Tobacco smoke contains a mixture of chemicals, including a host of reactive oxygen- and nitrogen species (ROS and RNS), among others, that can damage cellular and sub-cellular targets, such as lipids, proteins, and nucleic acids. A growing body of evidence supports a key role for smoking-induced ROS and the resulting oxidative stress in inflammation and carcinogenesis. This comprehensive and up-to-date review covers four interrelated topics, including 'smoking', 'oxidative stress', 'inflammation', and 'cancer'. The review discusses each of the four topics, while exploring the intersections among the topics by highlighting the macromolecular damage attributable to ROS. Specifically, oxidative damage to macromolecular targets, such as lipid peroxidation, post-translational modification of proteins, and DNA adduction, as well as enzymatic and non-enzymatic antioxidant defense mechanisms, and the multi-faceted repair pathways of oxidized lesions are described. Also discussed are the biological consequences of oxidative damage to macromolecules if they evade the defense mechanisms and/or are not repaired properly or in time. Emphasis is placed on the genetic- and epigenetic alterations that may lead to transcriptional deregulation of functionally-important genes and disruption of regulatory elements. Smoking-associated oxidative stress also activates the inflammatory response pathway, which triggers a cascade of events of which ROS production is an initial yet indispensable step. The release of ROS at the site of damage and inflammation helps combat foreign pathogens and restores the injured tissue, while simultaneously increasing the burden of oxidative stress. This creates a vicious cycle in which smoking-related oxidative stress causes inflammation, which in turn, results in further generation of ROS, and potentially increased oxidative damage to macromolecular targets that may lead to cancer initiation and/or progression.

The consequential impact of JUUL on youth vaping and the landscape of tobacco products: The state of play in the COVID-19 era.

JUUL is a groundbreaking electronic cigarette (e-cig) and the preeminent vaping product on the market. We present an overview of the rapid and spectacular rise of JUUL and its remarkable fall within the timespan of 2015 - 2020. We highlight JUUL's entering the market in June 2015, becoming the industry leader in mid 2017, and experiencing a litany of setbacks by late 2019 through to early 2020. We address the role played by JUUL in the ongoing epidemic of youth vaping. We also feature competing views on the public health impact of JUUL use (in particular), and e-cig vaping (in general). We further highlight the latest trends in youth vaping and sales records for JUUL and tobacco cigarettes. In view of the ongoing pandemic of COVID-19, we briefly summarize the existing evidence on the relationship between vaping and smoking and the prevalence, disease course, and clinical outcomes of COVID-19.

Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization.

To investigate the role of oxidative stress-induced DNA damage and mutagenesis in cellular senescence and immortalization, here we profiled spontaneous and methylene blue plus light-induced mutations in the cII gene from λ phage in transgenic mouse embryonic fibroblasts during the transition from primary culture through senescence and immortalization. Consistent with detection of characteristic oxidized guanine lesions (8-oxodG) in the treated cells, we observed significantly increased relative cII mutant frequency in the treated pre-senescent cells which was augmented in their immortalized counterparts. The predominant mutation type in the treated pre-senescent cells was G:C→T:A transversion, whose frequency was intensified in the treated immortalized cells. Conversely, the prevailing mutation type in the treated immortalized cells was A:T→C:G transversion, with a unique sequence-context specificity, i.e. flanking purines at the 5' end of the mutated nucleotide. This mutation type was also enriched in the treated pre-senescent cells, although to a lower extent. The signature mutation of G:C→T:A transversions in the treated cells accorded with the well-established translesion synthesis bypass caused by 8-oxodG, and the hallmark A:T→C:G transversions conformed to the known replication errors because of oxidized guanine nucleosides (8-OHdGTPs). The distinctive features of photosensitization-induced mutagenesis in the immortalized cells, which were present at attenuated levels, in spontaneously immortalized cells provide insights into the role of oxidative stress in senescence bypass and immortalization. Our results have important implications for cancer biology because oxidized purines in the nucleoside pool can significantly contribute to genetic instability in DNA mismatch repair-defective human tumors.

Vaping epidemic: challenges and opportunities.

This article is a timely, concise, and unbiased analysis of the national and international responses to the spate of vaping-related lung illnesses and deaths and the epidemic of teen vaping. In view of the recent outbreak of vaping-related lung injuries and deaths in the USA and the epidemic of teen vaping, the viewpoints and recommendations presented in this article have immediate policy implications in the USA and around the world. The perspectives and recommendations are expected to assist medical communities, public health professionals, and regulatory authorities in addressing complex issues related to vaping regulation, which are intertwined with public health, economy, and politics of nations, worldwide.

Secondhand Smoke Induces Liver Steatosis through Deregulation of Genes Involved in Hepatic Lipid Metabolism.

We investigated the role of secondhand smoke (SHS) exposure, independently of diet, in the development of chronic liver disease. Standard diet-fed mice were exposed to SHS (5 h/day, 5 days/week for 4 months). Genome-wide gene expression analysis, together with molecular pathways and gene network analyses, and histological examination for lipid accumulation, inflammation, fibrosis, and glycogen deposition were performed on the liver of SHS-exposed mice and controls, upon termination of exposure and after one-month recovery in clean air. Aberrantly expressed transcripts were found in the liver of SHS-exposed mice both pre- and post-recovery in clean air (n = 473 vs. 222). The persistent deregulated transcripts (n = 210) predominantly affected genes and functional networks involved in lipid metabolism as well as in the regulation of the endoplasmic reticulum where manufacturing of lipids occurs. Significant hepatic fat accumulation (steatosis) was observed in the SHS-exposed mice, which progressively increased as the animals underwent recovery in clean air. Moderate increases in lobular inflammation infiltrates and collagen deposition as well as loss of glycogen were also detectable in the liver of SHS-exposed mice. A more pronounced phenotype, manifested as a disrupted cord-like architecture with foci of necrosis, apoptosis, inflammation, and macrovesicular steatosis, was observed in the liver of SHS-exposed mice post-recovery. The progressive accumulation of hepatic fat and other adverse histological changes in the SHS-exposed mice are highly consistent with the perturbation of key lipid genes and associated pathways in the corresponding animals. Our data support a role for SHS in the genesis and progression of metabolic liver disease through deregulation of genes and molecular pathways and functional networks involved in lipid homeostasis.

Hypomethylation of LINE-1 repeat elements and global loss of DNA hydroxymethylation in vapers and smokers.

The outbreak of vaping-related severe lung injuries and deaths and the epidemic of teen vaping in the U.S. underscore the urgent need for determining the biological consequences of electronic cigarette (e-cig) use. We have investigated the association between vaping and epigenetic changes by quantifying DNA methylation levels in Long Interspersed Nucleotide Element 1 (LINE-1) and global DNA hydroxymethylation (5-hmC) levels and measuring the expression level of enzymes catalysing the respective processes in peripheral blood of exclusive vapers, smokers, and controls, matched for age, gender, and race (n = 45). Both vapers and smokers showed significant loss of methylation in LINE-1 repeat elements in comparison to controls (P = 0.00854 and P = 0.03078, respectively). Similarly, vapers and smokers had significant reductions in 5-hmC levels relative to controls (P = 0.04884 and P = 0.0035, respectively). Neither the LINE-1 methylation levels nor the global 5-hmC levels were different between vapers and smokers. There was a direct correlation between methylation levels in the LINE-1 elements and global 5-hmC levels in the study subjects (r = 0.31696, P = 0.03389). Inverse and statistically significant correlations were found between both the LINE-1 methylation levels and the global 5-hmC levels and various vaping/smoking metrics in the study subjects. There were modest but not statistically significant changes in transcription of DNA methyltransferases and ten-eleven translocation enzymes in both vapers and smokers relative to controls. Our findings support follow-up genome-wide investigations into the epigenetic effects of vaping, which may further clarify the health consequences of e-cig use. ABBREVIATIONS: 5-mC: 5-methylcytosine; 5-hmC: 5-hydroxymethylcytosine; 8-OHdG: 8-hydroxy-2'-deoxyguanosine; ACTIN: actin beta; ANOVA: Analysis of Variance; BER: base excision repair; BMI: body mass index; CO: carbon monoxide; COHb: carboxyhaemoglobin; COBRA: combined bisulphite restriction analysis; COPD: chronic obstructive pulmonary disease; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase 3A; DNMT3B: DNA methyltransferase 3B; e-cigs: electronic cigarettes; ELISA: enzyme-linked immunosorbent assay; ENDS: electronic nicotine delivery systems; FDA: Food and Drug Administration; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HPLC: high-performance liquid chromatography; LINE-1: Long Interspersed Nucleotide Element 1; PBS: phosphate-buffered saline; RFU: relative fluorescence units; RT-qPCR: quantitative reverse-transcription polymerase chain reaction; ROS: reactive oxygen species; SAM, S-adenosylmethionine; SE: standard error; TET1: ten-eleven translocation 1; TET2: ten-eleven translocation 2; TET3: ten-eleven translocation 3.

DNA Hydroxymethylation at the Interface of the Environment and Nonalcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent forms of chronic liver disorders among adults, children, and adolescents, and a growing epidemic, worldwide. Notwithstanding the known susceptibility factors for NAFLD, i.e., obesity and metabolic syndrome, the exact cause(s) of this disease and the underlying mechanisms of its initiation and progression are not fully elucidated. NAFLD is a multi-faceted disease with metabolic, genetic, epigenetic, and environmental determinants. Accumulating evidence shows that exposure to environmental toxicants contributes to the development of NAFLD by promoting mitochondrial dysfunction and generating reactive oxygen species in the liver. Imbalances in the redox state of the cells are known to cause alterations in the patterns of 5-hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC), thereby influencing gene regulation. The 5hmC-mediated deregulation of genes involved in hepatic metabolism is an emerging area of research in NAFLD. This review summarizes our current knowledge on the interactive role of xenobiotic exposure and DNA hydroxymethylation in the pathogenesis of fatty liver disease. Increasing the mechanistic knowledge of NAFLD initiation and progression is crucial for the development of new and effective strategies for prevention and treatment of this disease.

Deregulation of Biologically Significant Genes and Associated Molecular Pathways in the Oral Epithelium of Electronic Cigarette Users.

We have investigated the regulation of genes and associated molecular pathways, genome-wide, in oral cells of electronic cigarette (e-cigs) users and cigarette smokers as compared to non-smokers. Interrogation of the oral transcriptome by RNA-sequencing (RNA-seq) analysis showed significant number of aberrantly expressed transcripts in both e-cig users (vapers) and smokers relative to non-smokers; however, smokers had ~50% more differentially expressed transcripts than vapers (1726 versus 1152). Whereas the deregulated transcripts in smokers were predominately from protein-coding genes (79% versus 53% in vapers), nearly 28% of the aberrantly expressed transcripts in vapers (versus 8% in smokers) belonged to regulatory non-coding RNAs, including long intergenic non-coding, antisense, small nucleolar and misc RNA (P < 0.0001). Molecular pathway and functional network analyses revealed that "cancer" was the top disease associated with the deregulated genes in both e-cig users and smokers (~62% versus 79%). Examination of the canonical pathways and networks modulated in either e-cig users or smokers identified the "Wnt/Ca⁺ pathway" in vapers and the "integrin signaling pathway" in smokers as the most affected pathways. Amongst the overlapping functional pathways impacted in both e-cig users and smokers, the "Rho family GTPases signaling pathway" was the top disrupted pathway, although the number of affected targets was three times higher in smokers than vapers. In conclusion, we observed deregulation of critically important genes and associated molecular pathways in the oral epithelium of vapers that bears both resemblances and differences with that of smokers. Our findings have significant implications for public health and tobacco regulatory science.

The Lambda Select cII Mutation Detection System.

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

Mutation Analysis in Cultured Cells of Transgenic Rodents.

To comply with guiding principles for the ethical use of animals for experimental research, the field of mutation research has witnessed a shift of interest from large-scale in vivo animal experiments to small-sized in vitro studies. Mutation assays in cultured cells of transgenic rodents constitute, in many ways, viable alternatives to in vivo mutagenicity experiments in the corresponding animals. A variety of transgenic rodent cell culture models and mutation detection systems have been developed for mutagenicity testing of carcinogens. Of these, transgenic Big Blue® (Stratagene Corp., La Jolla, CA, USA, acquired by Agilent Technologies Inc., Santa Clara, CA, USA, BioReliance/Sigma-Aldrich Corp., Darmstadt, Germany) mouse embryonic fibroblasts and the λ Select cII Mutation Detection System have been used by many research groups to investigate the mutagenic effects of a wide range of chemical and/or physical carcinogens. Here, we review techniques and principles involved in preparation and culturing of Big Blue® mouse embryonic fibroblasts, treatment in vitro with chemical/physical agent(s) of interest, determination of the cII mutant frequency by the λ Select cII assay and establishment of the mutation spectrum by DNA sequencing. We describe various approaches for data analysis and interpretation of the results. Furthermore, we highlight representative studies in which the Big Blue® mouse cell culture model and the λ Select cII assay have been used for mutagenicity testing of diverse carcinogens. We delineate the advantages of this approach and discuss its limitations, while underscoring auxiliary methods, where applicable.

A Versatile Assay for Detection of Aberrant DNA Methylation in Bladder Cancer.

Urothelial carcinoma of the bladder is one of the most common malignancies in the industrialized world, mainly caused by smoking and occupational exposure to chemicals. The favorable prognosis of early stage bladder cancer underscores the importance of early detection for the treatment of this disease. The high recurrence rate of this malignancy also highlights the need for close post-diagnosis monitoring of bladder cancer patients. As for other malignancies, aberrant DNA methylation has been shown to play a crucial role in the initiation and progression of bladder cancer, and thus holds great promise as a diagnostic and prognostic biological marker. Here, we describe a protocol for a versatile DNA methylation enrichment method, the Methylated CpG Island Recovery Assay (MIRA), which enables analysis of the DNA methylation status in individual genes or across the entire genome. MIRA is based on the ability of the methyl-binding domain (MBD) proteins, the MBD2B/MBD3L1 complex, to specifically bind methylated CpG dinucleotides. This easy-to-perform method can be used to analyze the methylome of bladder cancer or urothelial cells shed in the urine to elucidate the evolution of bladder carcinogenesis and/or identify epigenetic signatures of chemicals known to cause this malignancy.

Limited mutagenicity of electronic cigarettes in mouse or human cells in vitro.

OBJECTIVES: Electronic cigarettes (e-cig), which are promoted as safe alternatives to tobacco cigarettes or as aides to smoking cessation, are becoming increasingly popular among adult chronic smokers and adolescents experimenting with tobacco products. Despite the known presence of toxicants and carcinogens in e-cig liquid and vapor, the possible carcinogenic effects of e-cig use in humans are unknown. MATERIALS AND METHODS: We have utilized two validated in vitro model systems to investigate whether e-cig vapor induces mutation in mouse or human cells. We have exposed transgenic mouse fibroblasts in vitro to e-cig vapor extracts prepared from three popular brands, and determined the induction of mutagenesis in a reporter gene, the cII transgene. Furthermore, we have treated the pSP189 plasmid with e-cig vapor extract, transfected human fibroblast cells with the e-cig-treated plasmid, and screened for the induced mutations in the supF gene. RESULTS AND CONCLUSION: We observed no statistically significant increases in relative mutant frequency in the cII transgene or supF gene in the e-cig treated mouse or human cells, respectively. Our data indicate that e-cig vapor extracts from the selected brands and at concentrations tested in this study have limited mutagenicity in both mouse and human cells in vitro.

An opportune and unique research to evaluate the public health impact of electronic cigarettes.

In response to the growing public health concern regarding the risks or benefits of electronic cigarettes (e-cig) use relative to smoking, the National Institute on Drug Abuse (NIDA) has recently introduced the first standardized- and well- characterized e-cig device to the research community (see, https://www.drugabuse.gov/funding/supplemental-information-nida-e-cig ). E-cig are promoted as safe alternatives to conventional tobacco cigarettes and/or as aides to smoking cessation. E-cig are highly popular among cigarette smokers who are unable/unwilling to quit but are willing to switch to putatively less-harmful tobacco substitutes. E-cig are also becoming increasingly popular among youth who have never experimented with combustible cigarettes. However, chemical analyses of e-cig juices (both in liquid form and after being heated into vapor) have shown that many carcinogens present in cigarette smoke are also found in a range of e-cig products. To date, the cancer-causing potential of e-cig has not been investigated in e-cig users (i.e., vapers). Use of e-cig without a prior history of smoking is currently a rare phenomenon in adults, but is increasingly common among youth. Consequently, investigating the carcinogenic potential of e-cig in nonsmoking youth provides a unique opportunity to verify the health impact of e-cig use, without the confounding effects of cigarette smoking. Within this context, the availability of the NIDA Standard Research e-cig offers a unique research opportunity with tremendous public health implications. Comparing and contrasting the cancer-causing potentials of standard vaping and smoking in youth will help determine the health risks or benefits of e-cig use relative to cigarette smoking. This information will be instrumental in making scientifically based decisions on the development and evaluation of policies and regulations on e-cig manufacture, marketing, and distribution. Ultimately, evidence-based guidelines and legislations on e-cig will help reduce the burden of tobacco-related diseases, particularly on minors and vulnerable populations.