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Purpose: Cancer registries offer an avenue to identify cancer clusters across large populations and efficiently examine potential environmental harms affecting cancer. The role of known metal carcinogens (i.e., cadmium, arsenic, nickel, chromium(VI)) in breast and colorectal carcinogenesis is largely unknown. Historically marginalized communities are disproportionately exposed to metals, which could explain cancer disparities. We examined area-based metal exposures and odds of residing in breast and colorectal cancer hotspots utilizing state tumor registry data and described the characteristics of those living in heavy metal-associated cancer hotspots., Methods: Breast and colorectal cancer hotspots were mapped across Kentucky, and area-based ambient metal exposure to cadmium, arsenic, nickel, and chromium(VI) were extracted from the 2014 National Air Toxics Assessment for Kentucky census tracts. Among colorectal cancer (n = 56,598) and female breast cancer (n = 77,637) diagnoses in Kentucky, we used logistic regression models to estimate Odds Ratios (ORs) and 95% Confidence Intervals to examine the association between ambient metal concentrations and odds of residing in cancer hotspots, independent of individual-level and neighborhood risk factors., Results: Higher ambient metal exposures were associated with higher odds of residing in breast and colorectal cancer hotspots. Populations in breast and colorectal cancer hotspots were disproportionately Black and had markers of lower socioeconomic status. Furthermore, adjusting for age, race, tobacco and neighborhood factors did not significantly change cancer hotspot ORs for ambient metal exposures analyzed., Conclusion: Ambient metal exposures contribute to higher cancer rates in certain geographic areas that are largely composed of marginalized populations. Individual-level assessments of metal exposures and cancer disparities are needed., (© 2024. The Author(s).)

Ubiquitin Proteasomal System (UPS) and autophagy dysregulation initiate cancer. These pathways are regulated by zinc finger proteins. Trivalent inorganic arsenic (iAs) displaces zinc from zinc finger proteins disrupting functions of important cellular proteins. The effect of chronic environmental iAs exposure (100 nM) on UPS has not been studied. We tested the hypothesis that environmental iAs exposure suppresses UPS, activating autophagy as a compensatory mechanism. We exposed skin (HaCaT and Ker-CT; independent quadruplicates) and lung (BEAS-2B; independent triplicates) cell cultures to 0 or 100 nM iAs for 7 or 8 weeks. We quantified ER stress (XBP1 splicing employing Reverse Transcriptase -Polymerase Chain Reaction), proteasomal degradation (immunoblots), and initiation and completion of autophagy (immunoblots). We demonstrate that chronic iAs exposure suppresses UPS, initiates autophagy, but suppresses autophagic protein degradation in skin and lung cell lines. Our data suggest that chronic iAs exposure inhibits autophagy which subsequently suppresses UPS., Competing Interests: Declaration of Competing Interest The authors declare they have no actual or potential competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biological processes are inherently stochastic, i.e., are partially driven by hard to predict random probabilistic processes. Carcinogenesis is driven both by stochastic and deterministic (predictable non-random) changes. However, very few studies systematically examine the contribution of stochastic events leading to cancer development. In differential gene expression studies, the established data analysis paradigms incentivize expression changes that are uniformly different across the experimental versus control groups, introducing preferential inclusion of deterministic changes at the expense of stochastic processes that might also play a crucial role in the process of carcinogenesis. In this study, we applied simple computational techniques to quantify: (i) The impact of chronic arsenic (iAs) exposure as well as passaging time on stochastic gene expression and (ii) Which genes were expressed deterministically and which were expressed stochastically at each of the three stages of cancer development. Using biological coefficient of variation as an empirical measure of stochasticity we demonstrate that chronic iAs exposure consistently suppressed passaging related stochastic gene expression at multiple time points tested, selecting for a homogenous cell population that undergo transformation. Employing multiple balanced removal of outlier data, we show that chronic iAs exposure induced deterministic and stochastic changes in the expression of unique set of genes, that populate largely unique biological pathways. Together, our data unequivocally demonstrate that both deterministic and stochastic changes in transcriptome-wide expression are critical in driving biological processes, pathways and networks towards clonal selection, carcinogenesis, and tumor heterogeneity., Competing Interests: Declaration of competing interest The authors declare that they have no competing conflict of interests regarding this work other than acknowledged grants supporting the work., (Copyright © 2024. Published by Elsevier Inc.)

Chronic arsenic exposure through drinking water is a global health issue, affecting >200 million people. Arsenic is a group I human carcinogen and causes chromosomal instability (CIN). Arsenic exposure is the second most common cause of skin cancer after UV radiation. hsa-miR-186 is overexpressed in arsenic-induced squamous cell carcinoma relative to premalignant hyperkeratosis. Among predicted targets of hsa-miR-186 are cell cycle regulators including regulators of mitotic progression. Disruption of mitotic progression can contribute to CIN. Thus, we hypothesized that hsa-miR-186 overexpression contributes to malignant transformation of arsenic exposed HaCaT cells by induction of CIN. Stable clones of HaCaT cells transfected with pEP-hsa-miR-186 expression vector or empty vector were maintained under puromycin selection and exposed to 0 or 100 nM NaAsO 2 and cultured for 29 weeks. HaCaT clones overexpressing hsa-miR-186 and exposed to NaAsO 2 showed increased CIN and anchorage independent growth at 29 weeks in a stochastic manner, in contrast to unexposed empty vector transfected clones. These results suggest that clonal variability mediates arsenic-induced carcinogenesis in hsa-miR-186 overexpressing human keratinocytes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J. Christopher States reports financial support was provided by National Institute of Environmental Health Sciences., (Copyright © 2023. Published by Elsevier Inc.)

Background: Liver retransplantation (reLT) has historically had inferior survival relative to primary liver transplant (LT). To improve outcomes after reLT, researchers have identified factors predicting overall (OS) and/or graft survival (GS) after reLT. This systematic review and random effects meta-analysis sought to summarize this literature to elucidate the strongest independent predictors of post-reLT., Methods: A systematic review was conducted to identify manuscripts reporting factors affecting survival in multivariable Cox proportional hazards analyses. Papers with overlapping cohorts were excluded., Results: All 25 included studies were retrospective, and 15 (60%) were single-center studies. Patients on pre-transplant ventilation (HR, 3.11; 95% CI, 1.56-6.20; p  = 0.001) and with high serum creatinine (HR, 1.46; 95% CI, 1.15-1.87; p  = 0.002) had the highest mortality risk after reLT. Recipient age, Model for End-Stage Liver Disease score, donor age, and cold ischemia time >12 h also conferred a significant risk of post-reLT death (all p  < 0.05). Factors affecting GS included donor age and retransplant interval (the time between LT and reLT; both p  < 0.05). OS is significantly higher when the retransplant interval is ≤7 days relative to 8-30 days ( p  = 0.04)., Conclusions: The meta-analysis was complicated by papers utilizing non-standardized cut-off values to group variables, which made between-study comparisons difficult. However, it did identify 7 variables that significantly impact survival after reLT, which could stimulate future research into improving post-reLT outcomes., Competing Interests: RMG is a member of the advisory board for TransMedics and has received stock in the company. The author RMG declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Brombosz, Moore, Mobley, Kodali, Saharia, Hobeika, Connor, Victor, Cheah, Simon, Gaber and Ghobrial.)

Chronic inorganic arsenic (iAs) exposure in drinking water is a global issue affecting >225 million people. Skin is a major target organ for iAs. miRNA dysregulation and chromosomal instability (CIN) are proposed mechanisms of iAs-induced carcinogenesis. CIN is a cancer hallmark and tetraploid cells can better tolerate increase in chromosome number and aberration, contributing to the evolution of CIN. miR-186 is overexpressed in iAs-induced squamous cell carcinoma relative to iAs-induced hyperkeratosis. Bioinformatic analysis indicated that miR-186 targets mRNAs of important cell cycle regulators including mitotic checkpoint serine/threonine kinase B (BUB1) and cell division cycle 27 (CDC27). We hypothesized that miR-186 overexpression contributes to iAs-induced transformation of keratinocytes by targeting mitotic regulators leading to induction of CIN. Ker-CT cells, a near diploid human keratinocyte cell line, were transduced with miR-186 overexpressing or scrambled control lentivirus. Stable clones were isolated after puromycin selection. Clones transduced with lentivirus expressing either a scrambled control miRNA or miR-186 were maintained with 0 or 100 nM iAs for 4 weeks. Unexposed scrambled control clones were considered as passage matched controls. Chronic iAs exposure increased miR-186 expression in miR-186 clones. miR-186 overexpression significantly reduced CDC27 levels irrespective of iAs exposure. The percentage of tetraploid or aneuploid cells was increased in iAs exposed miR-186 clones. Aneuploidy can arise from a tetraploid intermediate. Suppression of CDC27 by miR-186 may lead to impairment of mitotic checkpoint complex formation and its ability to maintain cell cycle arrest leading to chromosome misalignment. As a result, cells overexpressing miR-186 and chronically exposed to iAs may have incorrect chromosome segregation and CIN. These data suggest that dysregulation of miRNA by iAs mediates tetraploidy, aneuploidy and chromosomal instability contributing to iAs-induced carcinogenesis., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J. Christopher States reports financial support was provided by National Institute of Environmental Health Sciences. Alexandra Nail reports financial support was provided by National Institute of Environmental Health Sciences. J. Christopher States reports a relationship with Society of Toxicology that includes: board membership and travel reimbursement. J. Christopher States has patent #“Compounds For Treating Cancer, For Administering, And For Pharmaceutical Compositions” US Patent # 10,849,863 issued to University of Louisville., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Environmental factors affecting health and vulnerability far outweigh genetics in accounting for disparities in health status and longevity in US communities. The concept of the exposome, the totality of exposure from conception onwards, provides a paradigm for researchers to investigate the complex role of the environment on the health of individuals. We propose a complementary framework, community-level exposomics, for population-level exposome assessment. The goal is to bring the exposome paradigm to research and practice on the health of populations, defined by various axes including geographic, social, and occupational. This framework includes the integration of community-level measures of the built, natural and social environments, environmental pollution-derived from conventional and community science approaches, internal markers of exposure that can be measured at the population-level and early responses associated with health status that can be tracked using population-based monitoring. Primary challenges to the implementation of the proposed framework include needed advancements in population-level measurement, lack of existing models with the capability to produce interpretable and actionable evidence and the ethical considerations of labeling geographically-bound populations by exposomic profiles. To address these challenges, we propose a set of recommendations that begin with greater engagement with and empowerment of affected communities and targeted investment in community-based solutions. Applications to urban settings and disaster epidemiology are discussed as examples for implementation., Competing Interests: Conflict of interest statement None declared.

Arsenic-induced carcinogenesis is a worldwide health problem. Identifying the molecular mechanisms responsible for the induction of arsenic-induced cancers is important for developing treatment strategies. MicroRNA (miRNA) dysregulation is known to affect development and progression of human cancer. Several studies have identified an association between altered miRNA expression in cancers from individuals chronically exposed to arsenic and in cell models for arsenic-induced carcinogenesis. This chapter provides a comprehensive review for miRNA dysregulation in arsenic-induced cancer., Competing Interests: Conflict of interest statement The authors declare they have no financial conflicts of interest other than grants R01ES02778, R21ES030334, P30ES030283, T32ES011564 and R25CA134283 that supported the authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Disrupted cell cycle progression underlies the molecular pathogenesis of multiple diseases. Chronic exposure to inorganic arsenic (iAs) is a global health issue leading to multi-organ cancerous and non-cancerous diseases. Exposure to supratherapeutic concentrations of iAs causes cellular accumulation in G2 or M phase of the cell cycle in multiple cell lines by inducing cyclin B1 expression. It is not clear if iAs exposure at doses corresponding to serum levels of chronically exposed populations (∼100 nM) has any effect on cell cycle distribution. In the present study we investigated if environmentally relevant iAs exposure induced cell cycle disruption and mechanisms thereof employing two human keratinocyte cell lines (HaCaT and Ker-CT), flow cytometry, immunoblots and quantitative real-time PCR (qRT-PCR). iAs exposure (100 nM; 24 h) led to mitotic accumulation of cells in both cell lines, along with the stabilization of ANAPC11 ubiquitination targets cyclin B1 and securin, without affecting their steady state mRNA levels. This result suggested that induction of cyclin B1 and securin is modulated at the level of protein degradation. Moreover, zinc supplementation successfully prevented iAs-induced mitotic accumulation and stabilization of cyclin B1 and securin without affecting their mRNA levels. Together, these data suggest that environmentally relevant iAs exposure leads to mitotic accumulation possibly by displacing zinc from the RING finger subunit of anaphase promoting complex/cyclosome (ANAPC11), the cell cycle regulating E3 ubiquitin ligase. This early cell cycle disruptive effect of environmentally relevant iAs concentration could underpin the molecular pathogenesis of multiple diseases associated with chronic iAs exposure., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Environmentally relevant (100 nM) inorganic arsenic (iAs) exposure displaces zinc from zinc fingers of upstream splice regulator ZRANB2 disrupting the splicing of its target TRA2B. Excess zinc displaced iAs from ZRANB2 zinc fingers in cell free system. Thus, the hypothesis that zinc supplementation could prevent iAs-mediated disruption of ZRANB2 splice function in human keratinocytes was tested. The data show that zinc supplementation prevented iAs-induced dysregulation of TRA2B splicing by ZRANB2 as well as the induction of ZRANB2 protein expression. These results provide additional support for the hypothesis that zinc supplementation could prevent iAs-mediated disease in iAs-exposed populations., (Copyright © 2022 Elsevier B.V. All rights reserved.)

An estimated 220 million people worldwide are chronically exposed to inorganic arsenic (iAs) primarily as a result of drinking iAs-contaminated water. Chronic iAs exposure is associated with a plethora of human diseases including skin lesions and multi-organ cancers. iAs is a known clastogen, inducing DNA double strand breaks (DSBs) in both exposed human populations and in vitro. However, iAs does not directly interact with DNA, suggesting that other mechanisms, such as inhibition of DNA repair and DNA Damage Response (DDR) signaling, may be responsible for iAs-induced clastogenesis. Recent RNA-sequencing data from human keratinocytes (HaCaT cells) indicate that mRNAs for phosphatases important for resolution of DDR signaling are induced as a result of chronic iAs exposure prior to epithelial to mesenchymal transition. Here, we report that phosphorylation of ataxia telengectasia mutated (ATM) protein at a critical site (pSer1981) important for DDR signaling, and downstream CHEK2 activation, are significantly reduced in two human keratinocyte lines as a result of chronic iAs exposure. Moreover, RAD50 expression is reduced in both of these lines, suggesting that suppression of the MRE11-RAD50-NBS1 (MRN) complex may be responsible for reduced ATM activation. Lastly, we demonstrate that DNA double strand break accumulation and DNA damage is significantly higher in human keratinocytes with low dose iAs exposure. Thus, inhibition of the MRN complex in iAs-exposed cells may be responsible for reduced ATM activation and reduced DSB repair by homologous recombination (HR). As a result, cells may favor error-prone DSB repair pathways to fix damaged DNA, predisposing them to chromosomal instability (CIN) and eventual carcinogenesis often seen resulting from chronic iAs exposure., (Copyright © 2021. Published by Elsevier Inc.)

Cutaneous squamous cell carcinoma (cSCC) is a major deleterious health effect of chronic arsenic (iAs) exposure. The molecular mechanism of arsenic-induced cSCC remains poorly understood. We recently demonstrated that chronic iAs exposure leads to temporally regulated genome-wide changes in profiles of differentially expressed mRNAs and miRNAs at each stage of carcinogenesis (7, 19, and 28 weeks) employing a well-established passage-matched HaCaT cell line model of arsenic-induced cSCC. Here, we performed longitudinal differential expression analysis (miRNA and mRNA) between the different time points (7 vs 19 weeks and 19 vs 28 weeks) within unexposed and exposed groups, coupled to expression pairing and pathway analyses to differentiate the relative effects of long-term passaging and chronic iAs exposure. Data showed that 66-105 miRNA [p < .05; log2(fold change) > I1I] and 2826-4079 mRNA [p < .001; log2(fold change) > I1I] molecules were differentially expressed depending on the longitudinal comparison. Several mRNA molecules differentially expressed as a function of time, independent of iAs exposure were being targeted by miRNA molecules which were also differentially expressed in a time-dependent manner. Distinct pathways were predicted to be modulated as a function of time or iAs exposure. Some pathways were also modulated both by time and exposure. Thus, the HaCaT model can distinguish between the effects of passaging and chronic iAs exposure individually and corroborate our previously published data on effects of iAs exposure compared with unexposed passage matched HaCaT cells. In addition, this work provides a template for cell line-based longitudinal chronic exposure studies to follow for optimal efficacy., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Chronic arsenic exposure via drinking water is associated with an increased risk of developing cancer and noncancer chronic diseases. Pre-mRNAs are often subject to alternative splicing, generating mRNA isoforms encoding functionally distinct protein isoforms. The resulting imbalance in isoform species can result in pathogenic changes in critical signaling pathways. Alternative splicing as a mechanism of arsenic-induced toxicity and carcinogenicity is understudied., Objective: This study aimed to accurately profile differential alternative splicing events in human keratinocytes induced by chronic arsenic exposure that might play a role in carcinogenesis., Methods: Independent quadruplicate cultures of immortalized human keratinocytes (HaCaT) were maintained continuously for 28 wk with 0 or 100  nM sodium arsenite. RNA-sequencing (RNA-Seq) was performed with poly(A) RNA isolated from cells harvested at 7, 19, and 28 wk with subsequent replicate multivariate analysis of transcript splicing (rMATS) analysis to detect and quantify differential alternative splicing events. Reverse transcriptase-polymerase chain reaction (RT-PCR) for selected alternative splicing events was performed to validate RNA-Seq predictions. Functional enrichment was performed by gene ontology (GO) analysis of the differential alternative splicing event data set at each time point., Results: At least 600 differential alternative splicing events were detected at each time point tested, comprising all the five main types of alternative splicing and occurring in both open reading frames (ORFs) and untranslated regions (UTRs). Based on functional relevance ELK4 , SHC1 , and XRRA1 were selected for validation of predicted alternative splicing events at 7 wk by RT-PCR. Densitometric analysis of RT-PCR data corroborated the rMATS predicted alternative splicing for all three events. Protein expression validation of the selected alternative splicing events was challenging given that very few isoform-specific antibodies are available. GO analysis demonstrated that the enriched terms in differential alternatively spliced mRNAs changed dynamically with the time of exposure. Notably, RNA metabolism and splicing regulation pathways were enriched at the 7-wk time point, when the greatest number of differentially alternatively spliced mRNAs are detected. Our preliminary proteomic analysis demonstrated that the expression of the canonical isoforms of the splice regulators DDX42, RMB25, and SRRM2 were induced upon chronic arsenic exposure, corroborating the splicing predictions., Discussion: These results using cultures of HaCaT cells suggest that arsenic exposure disrupted an alternative splice factor network and induced time-dependent genome-wide differential alternative splicing that likely contributed to the changing proteomic landscape in arsenic-induced carcinogenesis. However, significant challenges remain in corroborating alternative splicing data at the proteomic level. https://doi.org/10.1289/EHP9676.

Genomic instability consists of a range of genetic alterations within the genome that contributes to tumor heterogeneity and drug resistance. It is a well-established characteristic of most cancer cells. Genome instability induction results from defects in DNA damage surveillance mechanisms, mitotic checkpoints and DNA repair machinery. Accumulation of genetic alterations ultimately sets cells towards malignant transformation. Recent studies suggest that miRNAs are key players in mediating genome instability. miRNAs are a class of small RNAs expressed in most somatic tissues and are part of the epigenome. Importantly, in many cancers, miRNA expression is dysregulated. Consequently, this review examines the role of miRNA dysregulation as a causal step for induction of genome instability and subsequent carcinogenesis. We focus specifically on mechanistic studies assessing miRNA(s) and specific subtypes of genome instability or known modes of genome instability. In addition, we provide insight on the existing knowledge gaps within the field and possible ways to address them., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chronic arsenic exposure causes skin cancer, although the underlying molecular mechanisms are not well defined. Altered microRNA and mRNA expression likely play a pivotal role in carcinogenesis. Changes in genome-wide differential expression of miRNA and mRNA at 3 strategic time points upon chronic sodium arsenite (As 3+ ) exposure were investigated in a well-validated HaCaT cell line model of arsenic-induced cutaneous squamous cell carcinoma (cSCC). Quadruplicate independent HaCaT cell cultures were exposed to 0 or 100 nM As 3+ for up to 28-weeks (wk). Cell growth was monitored throughout the course of exposure and epithelial-mesenchymal transition (EMT) was examined employing immunoblot. Differentially expressed miRNA and mRNA profiles were generated at 7, 19, and 28-wk by RNA-seq, followed by identification of differentially expressed mRNA targets of differentially expressed miRNAs through expression pairing at each time point. Pathway analyses were performed for total differentially expressed mRNAs and for the miRNA targeted mRNAs at each time point. RNA-seq predictions were validated by immunoblot of selected target proteins. While the As 3+ -exposed cells grew slower initially, growth was equal to that of unexposed cells by 19-wk (transformation initiation), and exposed cells subsequently grew faster than passage-matched unexposed cells. As 3+ -exposed cells had undergone EMT at 28-wk. Pathway analyses demonstrate dysregulation of carcinogenesis-related pathways and networks in a complex coordinated manner at each time point. Immunoblot data largely corroborate RNA-seq predictions in the endoplasmic reticulum stress (ER stress) pathway. This study provides a detailed molecular picture of changes occurring during the arsenic-induced transformation of human keratinocytes.

Introduction: Liver transplantation (LT) is an effective treatment for hepatocellular carcinoma (HCC), the most common type of primary liver cancer. Patients with small HCC (<5 cm) are given priority over others for transplantation due to clinical allocation policies based on tumor size. Attempting to shift from the prevalent paradigm that successful transplantation and longer disease-free survival can only be achieved in patients with small HCC to expanding the transplantation option to patients with HCC of the highest tumor burden (>5 cm), we developed a convergent artificial intelligence (AI) model that combines transient clinical data with quantitative histologic and radiomic features for more objective risk assessment of liver transplantation for HCC patients., Methods: Patients who received a LT for HCC between 2008-2019 were eligible for inclusion in the analysis. All patients with post-LT recurrence were included, and those without recurrence were randomly selected for inclusion in the deep learning model. Pre- and post-transplant magnetic resonance imaging (MRI) scans and reports were compressed using CapsNet networks and natural language processing, respectively, as input for a multiple feature radial basis function network. We applied a histological image analysis algorithm to detect pathologic areas of interest from explant tissue of patients who recurred. The multilayer perceptron was designed as a feed-forward, supervised neural network topology, with the final assessment of recurrence risk. We used area under the curve (AUC) and F-1 score to assess the predictability of different network combinations., Results: A total of 109 patients were included (87 in the training group, 22 in the testing group), of which 20 were positive for cancer recurrence. Seven models (AUC; F-1 score) were generated, including clinical features only (0.55; 0.52), magnetic resonance imaging (MRI) only (0.64; 0.61), pathological images only (0.64; 0.61), MRI plus pathology (0.68; 0.65), MRI plus clinical (0.78, 0.75), pathology plus clinical (0.77; 0.73), and a combination of clinical, MRI, and pathology features (0.87; 0.84). The final combined model showed 80 % recall and 89 % precision. The total accuracy of the implemented model was 82 %., Conclusion: We validated that the deep learning model combining clinical features and multi-scale histopathologic and radiomic image features can be used to discover risk factors for recurrence beyond tumor size and biomarker analysis. Such a predictive, convergent AI model has the potential to alter the LT allocation system for HCC patients and expand the transplantation treatment option to patients with HCC of the highest tumor burden., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Exposure to alkylanilines found in tobacco smoke and indoor air is associated with risk of bladder cancer. Genetic factors significantly influence the metabolism of arylamine carcinogens and the toxicological outcomes that result from exposure. We utilized nucleotide excision repair (NER)-deficient immortalized human fibroblasts to examine the effects of human N-acetyltransferase 1 (NAT1), CYP1A2, and common rapid (NAT2*4) and slow (NAT2*5B or NAT2*7B) acetylator human N-acetyltransferase 2 (NAT2) haplotypes on environmental arylamine and alkylaniline metabolism. We constructed SV40-transformed human fibroblast cells that stably express human NAT2 alleles (NAT2*4, NAT2*5B, or NAT2*7B) and human CYP1A2. Human NAT1 and NAT2 apparent kinetic constants were determined following recombinant expression of human NAT1 and NAT2 in yeast for the arylamines benzidine, 4-aminobiphenyl (ABP), and 2-aminofluorene (2-AF), and the alkylanilines 2,5-dimethylaniline (DMA), 3,4-DMA, 3,5-DMA, 2-6-DMA, and 3-ethylaniline (EA) compared with those of the prototype NAT1-selective substrate p-aminobenzoic acid and NAT2-selective substrate sulfamethazine. Benzidine, 3,4-DMA, and 2-AF were preferential human NAT1 substrates, while 3,5-DMA, 2,5-DMA, 3-EA, and ABP were preferential human NAT2 substrates. Neither recombinant human NAT1 or NAT2 catalyzed the N-acetylation of 2,6-DMA. Among the alkylanilines, N-acetylation of 3,5-DMA was substantially higher in human fibroblasts stably expressing NAT2*4 versus NAT2*5B and NAT2*7B. The results provide important insight into the role of the NAT2 acetylator polymorphism (in the presence of competing NAT1 and CYP1A2-catalyzed N-acetylation and N-hydroxylation) on the metabolism of putative alkyaniline carcinogens. The N-acetylation of two alkylanilines associated with urinary bladder cancer (3-EA and 3,5-DMA) was modified by NAT2 acetylator polymorphism.

miRNAs (miRNA) are essential players regulating gene expression affecting cellular processes contributing to disease development. Dysregulated miRNA expression has been observed in numerous diseases including hepatitis, cardiovascular diseases and cancers. In cardiovascular diseases, several miRNAs function as mediators of pathogenic stress-related signaling pathways that may lead to an excessive extracellular matrix production and collagen deposition causing cardiac stress resulting in fibrosis. In cancers, many miRNAs function as oncogenes or tumor suppressors facilitating tumor growth, invasion and angiogenesis. Furthermore, the association between distinct miRNA profile and tumor development, progression and treatment response has identified miRNAs as potential biomarkers for disease diagnosis and prognosis. Growing evidence demonstrates changes in miRNA expression levels in experimental settings or observational studies associated with environmental chemical exposures such as arsenic. Arsenic is one of the most well-known human carcinogens. Long-term exposure through drinking water increases risk of developing skin, lung and urinary bladder cancers, as well as cardiovascular disease. The mechanism(s) by which arsenic causes disease remains elusive. Proposed mechanisms include miRNA dysregulation. Epidemiological studies identified differential miRNA expression between arsenic-exposed and non-exposed individuals from India, Bangladesh, China and Mexico. In vivo and in vitro studies have shown that miRNAs are critically involved in arsenic-induced malignant transformation. Few studies analyzed miRNAs in other diseases associated with arsenic exposure. Importantly, there is no consensus on a consistent miRNA profile for arsenic-induced cancers because most studies analyze only particular miRNAs. Identifying miRNA expression changes common among humans, rodents and cell lines might guide future miRNA investigations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The impacts of acute arsenic exposure (i.e. vomiting, diarrhea, and renal failure) are distinct from those brought about by sustained, low level exposure from environmental sources or drinking of contaminated well water. Chronic arsenic exposure is a risk factor for the development of pulmonary diseases, including lung cancer. How arsenic exposure leads to pulmonary disease is not fully understood. Both acute versus chronic arsenic exposure increase EGFR expression, but do so via distinct molecular mechanisms. BEAS-2B cells were exposed to either acute sodium arsenite (5 μM for 24 h) or chronic sodium arsenite (100 nM for 24 weeks). Cells treated with acute arsenic exhibited a decrease in viability, changes in morphology, and increased mRNA level of BTC. In contrast, during 24 weeks of arsenic exposure, the cells had increased EGFR expression and activity, and increased mRNA and protein levels of TGFα. Further, chronic arsenic treatment caused an increase in cell migration in the absence of exogenous ligand. Elevated TGFα and EGFR expression are features of many non-small cell lung cancers. We propose that lung epithelial cells chronically exposed to low level arsenic increases EGFR signaling via TGFα production to enhance ligand-independent cell migration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Exposure to arsenic, a class I carcinogen, affects 200 million people globally. Skin is the major target organ, but the molecular etiology of arsenic-induced skin carcinogenesis remains unclear. Arsenite (As 3+ )-induced disruption of alternative splicing could be involved, but the mechanism is unknown. Zinc finger proteins play key roles in alternative splicing. As 3+ can displace zinc (Zn 2+ ) from C3H1 and C4 zinc finger motifs (zfm's), affecting protein function. ZRANB2, an alternative splicing regulator with two C4 zfm's integral to its structure and splicing function, was chosen as a candidate for this study. We hypothesized that As 3+ could displace Zn 2+ from ZRANB2, altering its structure, expression, and splicing function. As 3+ /Zn 2+ binding and mutual displacement experiments were performed with synthetic apo-peptides corresponding to each ZRANB2 zfm, employing a combination of intrinsic fluorescence, ultraviolet spectrophotometry, zinc colorimetric assay, and liquid chromatography-tandem mass spectrometry. ZRANB2 expression in HaCaT cells acutely exposed to As 3+ (0 or 5 μM, 0-72 h; or 0-5 μM, 6 h) was examined by RT-qPCR and immunoblotting. ZRANB2-dependent splicing of TRA2B mRNA, a known ZRANB2 target, was monitored by reverse transcription-polymerase chain reaction. As 3+ bound to, as well as displaced Zn 2+ from, each zfm. Also, Zn 2+ displaced As 3+ from As 3+ -bound zfm's acutely, albeit transiently. As 3+ exposure induced ZRANB2 protein expression between 3 and 24 h and at all exposures tested but not ZRANB2 mRNA expression. ZRANB2-directed TRA2B splicing was impaired between 3 and 24 h post-exposure. Furthermore, ZRANB2 splicing function was also compromised at all As 3+ exposures, starting at 100 nm. We conclude that As 3+ exposure displaces Zn 2+ from ZRANB2 zfm's, changing its structure and compromising splicing of its targets, and increases ZRANB2 protein expression as a homeostatic response both at environmental/toxicological exposures and therapeutically relevant doses.

Epidemiological evidence suggests that cadmium (Cd) is one of the causative factors of prostate cancer, but the effect of Cd on benign prostatic hyperplasia (BPH) remains unclear. This study aimed to determine whether Cd exposure could malignantly transform BPH1 cells and, if so, to dissect the mechanism of action. We deciphered the molecular signaling responsible for BPH1 transformation via RNA-sequencing and determined that Cd induced the expression of zinc finger of the cerebellum 2 (ZIC2) in BPH1 cells. We noted Cd exposure increased ZIC2 expression in the Cd-transformed BPH1 cells that in turn promoted anchorage-independent spheroids and increased expression of stem cell drivers, indicating their role in stem cell renewal. Subsequent silencing of ZIC2 expression in transformed cells inhibited spheroid formation, stem cell marker expression, and tumor growth in nude mice. At the molecular level, ZIC2 interacts with the glioma-associated oncogene family (GLI) zinc finger 1 (GLI1), which activates prosurvival factors (nuclear factor NFκB, B-cell lymphoma-2 (Bcl2), as well as an X-linked inhibitor of apoptosis protein (XIAP)) signaling in Cd-exposed BPH1 cells. Conversely, overexpression of ZIC2 in BPH1 cells caused spheroid formation confirming the oncogenic function of ZIC2. ZIC2 activation and GLI1 signaling induction by Cd exposure in primary BPH cells confirmed the clinical significance of this oncogenic function. Finally, human BPH specimens had increased ZIC2 versus adjacent healthy tissues. Thus, we report direct evidence that Cd exposure induces malignant transformation of BPH via activation of ZIC2 and GLI1 signaling.

Carcinogenic aromatic amines such as 4-aminobiphenyl (ABP) and 2-aminofluorene (AF) require metabolic activation to form electrophilic intermediates that mutate DNA leading to carcinogenesis. Bioactivation of these carcinogens includes N-hydroxylation catalyzed by CYP1A2 followed by O-acetylation catalyzed by arylamine N-acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in ABP- and AF-induced mutagenesis and DNA damage, nucleotide excision repair-deficient (UV5) Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. ABP and AF both caused significantly (P < 0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the UV5/CYP1A2/NAT2*4 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT2*5B cell lines. ABP- and AF-induced hprt mutant cDNAs were sequenced and over 80% of the single-base substitutions were at G:C base pairs. DNA damage also was quantified by γH2AX in-cell western assays and by identification and quantification of the two predominant DNA adducts, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) by liquid chromatography-mass spectrometry. DNA damage and adduct levels were dose-dependent, correlated highly with levels of hprt mutants, and were significantly (P < 0.0001) greater in the UV5/CYP1A2/NAT2*4 rapid acetylator cell line following treatment with ABP or AF as compared to all other cell lines. Our findings provide further clarity on the importance of O-acetylation in CHO mutagenesis assays for aromatic amines. They provide evidence that NAT2 genetic polymorphism modifies aromatic amine-induced DNA damage and mutagenesis that should be considered in human risk assessments following aromatic amine exposures. Environ. Mol. Mutagen. 61:235-245, 2020. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

Exposure to the environmental toxicant cadmium (Cd) contributes to the development of obesity-associated diseases. Obesity is a risk factor for a spectrum of unhealthy conditions including systemic metabolic dyshomeostasis. In the present study, the effects of whole-life exposure to environmentally-relevant concentrations of Cd on systemic essential metal distribution in adult mice fed a high-fat diet (HFD) were examined. For these studies, male and female mice were exposed to Cd-containing drinking water for >2 weeks before breeding. Pregnant mice and dams with offspring were exposed to Cd-containing drinking water. After weaning, offspring were continuously exposed to the same Cd concentration as their parents, and divided into HFD and normal (low) fat diet (LFD) groups. At 10 and 24 weeks, mice were sacrificed and blood, liver, kidney and heart harvested for metal analyses. There were significant concentration dependent increases in Cd levels in offspring with kidney > liver > heart. Sex significantly affected Cd levels in kidney and liver, with female animals accumulating more metal than males. Mice fed the HFD showed > 2-fold increase in Cd levels in the three organs compared to similarly treated LFD mice. Cadmium significantly affected essential metals levels in blood, kidney and liver. Additionally, HFD affected essential metal levels in these three organs. These findings suggest that Cd interacts with HFD to affect essential metal homeostasis, a phenomenon that may contribute to the underlying mechanism responsible for the development of obesity-associated pathologies.

The mechanism of arsenic-induced skin carcinogenesis is not yet fully understood. Chromosomal instability contributes to aneuploidy and is a driving force in carcinogenesis. Arsenic causes mitotic arrest and induces aneuploidy. hsa-miR-186 overexpression is associated with metastatic cancers as well as arsenic-induced squamous cell carcinoma and is reported to target several mitotic regulators. Decreased levels of these proteins can dysregulate chromatid segregation contributing to aneuploidy. This work investigates the potential aneuploidogenic role of hsa-miR-186 in arsenic carcinogenesis. Clones of immortalized human keratinocytes (HaCaT) stably transfected with a hsa-miR-186 expression or empty vector were isolated. Three clones with high and low hsa-miR-186 expression determined by RT-qPCR were selected for further analysis and cultured with 0 or 100 nM NaAsO 2 for 8 weeks. Analysis of mitoses revealed that chromosome number and structural abnormalities increased in cells overexpressing hsa-miR-186 and were further increased by arsenite exposure. Double minutes were the dominant structural aberrations. The peak number of chromosomes also increased. Cells with >220 to >270 chromosomes appeared after 2 months in hsa-miR-186 overexpressing cells, indicating multiple rounds of endomitosis had occurred. The fraction of cells with increased chromosome number or structural abnormalities did not increase in passage matched control cells. Levels of selected target proteins were determined by western blot. Expression of BUB1, a predicted hsa-miR-186 target was suppressed in hsa-miR-186 overexpressing clones, but increased with arsenite exposure. CDC27 remained constant under all conditions. These results suggest that overexpression of miR-186 in arsenic exposed tissues likely induces aneuploidy contributing to arsenic-induced carcinogenesis., (Copyright © 2019. Published by Elsevier Inc.)

Exposure to environmental stressors during susceptible windows of development can result in negative health outcomes later in life, a concept known as the Developmental Origins of Health and Disease (DOHaD). There is a growing body of evidence that exposures to metals early in life (in utero and postnatal) increase the risk of developing adult diseases such as cancer, cardiovascular disease, non-alcoholic fatty liver disease, and diabetes. Of particular concern is exposure to the metalloid arsenic, a drinking water contaminant and worldwide health concern. Epidemiological studies of areas with high levels of arsenic in the drinking water, such as some regions in Chile and Bangladesh, indicate an association between in utero arsenic exposure and the development of adult diseases. Therefore, the need for experimental models to address the mechanism underlining early onset of adult diseases have emerged including the in utero and whole-life exposure models. This review will highlight the epidemiological events and subsequent novel experimental models implemented to study the impact of early life exposure to arsenic on the development of adult diseases. In addition, current research using these models will be discussed as well as possible underlying mechanism for the early onset of disease. Abbreviations: ALT: alanine aminotransferase; AMI: acute myocardial infarction; AST: aspartate aminotransferase; ATSDR: Agency for Toxic Substances and Disease Registry; CVD: cardiovascular disease; DMA: dimethylarsinate; DOHaD: Developmental Origins of Health and Disease; EPA: U.S. Environmental Protection Agency; ER-α: estrogen receptor alpha; HDL: high-density lipoprotein; HOMA-IR: homeostatic model assessment of insulin resistance; iAs: inorganic arsenic; LDL: low-density lipoprotein; MetS: metabolic syndrome; MMA: monomethylarsonate; NAFLD: non-alcoholic fatty liver disease; PND: postnatal day; ppb: parts per billion; ppm: parts per million; SAM: S-adenosylmethionine; USFDA: United States Food and Drug Administration.

Arsenic is a toxic metalloid widely present in the earth's crust, and is a proven human carcinogen. Chronic arsenic exposure mainly through drinking water causes skin, lung, and urinary bladder cancers, and is associated with liver, prostate, and kidney cancers, cardiovascular and neurological disorders, and diabetes. Several modes of action have been suggested in arsenic carcinogenesis. However, the molecular etiology of arsenic-induced cancer remains unclear. Recent evidence clearly indicates that gene expression modifications induced by arsenic may involve epigenetic alterations, including miRNA dysregulation. Many miRNAs have been implicated in different human cancers as a consequence of losses and or gains of miRNA function that contribute to cancer development. Progress in identifying miRNA dysregulation induced by arsenic has been made using different approaches and models. The present review discusses the recent data regarding dysregulated expression of miRNA in arsenic-induced malignant transformation in vitro, gaps in current understanding and deficiencies in current models for arsenic-induced carcinogenesis, and future directions of research that would improve our knowledge regarding the mechanisms involved in arsenic-induced carcinogenesis.

Arsenic, a naturally occurring element, contaminates the drinking water of over 200 million people globally. Chronic arsenic exposure causes multiple cancers including those originating from skin, lung and bladder, and is associated with liver, kidney, and prostate cancers. Skin is the primary target organ for arsenic toxicity; chronic toxicity initially manifests as non-malignant hyperkeratoses (HK) and subsequently advances to malignant lesions, including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). In this study, we evaluate the miRNA expression profiles of premalignant (3 HK) and malignant (3 BCC and 3 SCC) skin lesions from individuals chronically exposed to high levels of arsenic (59-172 ppb) in their drinking water in West Bengal, India. The lesions were histologically complex requiring histopathologic identification of keratinocytes to be isolated for RNA analyses. Keratinocytes were harvested using Laser Capture Microdissection and miRNA expression profiles were determined using TaqMan® Array Human MiRNA A Card v2.0. Thirty-five miRNAs were differentially expressed among the three lesion types analyzed. Two miRNAs (miR-425-5p and miR-433) were induced in both BCC and SCC relative to HK indicating their association with malignancy. Two other miRNAs (miR-184 and miR-576-3p) were induced in SCC relative to both BCC and HK suggesting selective induction in tumors capable of metastasis. Six miRNAs (miR-29c, miR-381, miR-452, miR-487b, miR-494 and miR-590-5p) were selectively suppressed in BCC relative to both SCC and HK. In conclusion, the differential miRNA expression was both phenotype- and stage-related. These miRNAs are potential biomarkers and may serve as therapy targets for arsenic-induced internal tumors., Competing Interests: The authors have declared that no competing interests exist.

N-acetyltransferase 1 (NAT1) is an enzyme that metabolizes carcinogens, which suggests a potential role in breast carcinogenesis. High NAT1 expression in breast tumors is associated with estrogen receptor α (ER α +) and the luminal subtype. We report that NAT1 mRNA transcript, protein, and enzyme activity were higher in human breast tumors with high expression of ER α / ESR1 compared with normal breast tissue. There was a strong correlation between NATb promoter and NAT1 protein expression/enzyme activity. High NAT1 expression in tumors was not the result of adipocytes, as evidenced by low perilipin ( PLIN) expression. ESR1 , NAT1 , and XBP1 expression were associated in tumor biopsies. Direct regulation of NAT1 transcription by estradiol (E 2 ) was investigated in ER α (+) MCF-7 and T47D breast cancer cells. E 2 did not increase NAT1 transcript expression but increased progesterone receptor expression in a dose-dependent manner. Likewise, NAT1 transcript levels were not increased by dihydrotestosterone (DHT) or 5 α -androstane-3 β , (3 β -adiol) 17 β -diol. Dithiothreitol increased levels of the activated, spliced XBP1 in ER α (+) MCF-7 and T47D breast cancer cells but did not affect NAT1 or ESR1 expression. We conclude that NAT1 expression is not directly regulated by E 2 , DHT, 3 β -adiol, or dithiothreitol despite high NAT1 and ESR1 expression in luminal A breast cancer cells, suggesting that ESR1 , XBP1, and NAT1 expression may share a common transcriptional network arising from the luminal epithelium associated with better survival in breast cancer. Clusters of high-expression genes, including NAT1, in breast tumors might serve as potential targets for novel therapeutic drug development., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer., (© 2018 Wiley Periodicals, Inc.)

Arsenic is a widely distributed toxic natural element. Chronic arsenic ingestion causes several cancers, especially skin cancer. Arsenic-induced cancer mechanisms are not well defined, but several studies indicate that mutation is not the driving force and that microRNA expression changes play a role. Chronic low arsenite exposure malignantly transforms immortalized human keratinocytes (HaCaT), serving as a model for arsenic-induced skin carcinogenesis. Early changes in miRNA expression in HaCaT cells chronically exposed to arsenite will reveal early steps in transformation. HaCaT cells were maintained with 0/100 nM NaAsO2 for 3 and 7 weeks. Total RNA was purified. miRNA and mRNA expression was assayed using Affymetrix microarrays. Targets of differentially expressed miRNAs were collected from TargetScan 6.2, intersected with differentially expressed mRNAs using Partek Genomic Suite software, and mapped to their pathways using MetaCore software. MDM2, HMGB1 and TP53 mRNA, and protein levels were assayed by RT-qPCR and Western blot. Numerous miRNAs and mRNAs involved in carcinogenesis pathways in other systems were differentially expressed at 3 and 7 weeks. A TP53 regulatory network including MDM2 and HMGB1 was predicted by the miRNA and mRNA networks. Total TP53 and TP53-S15-phosphorylation were induced. However, TP53-K382-hypoacetylation suggested that the induced TP53 is inactive in arsenic exposed cells. Our data provide strong evidence that early changes in miRNAs and target mRNAs may contribute to arsenic-induced carcinogenesis.

We report a case of a 46-year-old Vietnamese man who developed widespread, numerous and concurrent cutaneous squamous cell carcinomas (SCCs) in non-sun exposed skin areas after taking a traditional medicine (TM) formulation for chronic plaque psoriasis. The SCC lesions began to develop within 12-15 months after beginning the arsenic-containing TM. The patient experienced both acute and chronic symptoms consistent with arsenic exposure. Laboratory investigation of a collected hair sample showed a significant arsenic level. The TM formulation used by the patient was tested and demonstrated an extremely high concentration of arsenic., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: Arsenic is naturally prevalent in the earth's crust and widely distributed in air and water. Chronic low arsenic exposure is associated with several cancers in vivo, including skin cancer, and with transformation in vitro of cell lines including immortalized human keratinocytes (HaCaT). Arsenic also is associated with cell cycle dysregulation at different exposure levels in multiple cell lines. In this work, we analyzed gene expression in HaCaT cells to gain an understanding of gene expression changes contributing to transformation at an early time point., Methods: HaCaT cells were exposed to 0 or 100nM NaAsO 2 for 7weeks. Total RNA was purified and analyzed by microarray hybridization. Differential expression with fold change≥|1.5| and p-value≤0.05 was determined using Partek Genomic Suite™ and pathway and network analyses using MetaCore™ software (FDR≤0.05). Cell cycle analysis was performed using flow cytometry., Results: 644 mRNAs were differentially expressed. Cell cycle/cell cycle regulation pathways predominated in the list of dysregulated pathways. Genes involved in replication origin licensing were enriched in the network. Cell cycle assay analysis showed an increase in G2/M compartment in arsenite-exposed cells., Conclusions: Arsenite exposure induced differential gene expression indicating dysregulation of cell cycle control, which was confirmed by cell cycle analysis. The results suggest that cell cycle dysregulation is an early event in transformation manifested in cells unable to transit G2/M efficiently. Further study at later time points will reveal additional changes in gene expression related to transformation processes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Recent investigations suggest role(s) of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer. Rat NAT2 is orthologous to human NAT1 and the gene products are functional homologs. We conducted in vivo studies using F344.WKY-Nat2 rapid/slow rats, congenic at rat Nat2 for high (rapid) and low (slow) arylamine N-acetyltransferase activity, to assess a possible role for rat NAT2 in mammary tumor susceptibility., Methods: Mammary carcinogens, methylnitrosourea (MNU) and 7,12-dimethylbenzanthracene (DMBA) neither of which is metabolized by N-acetyltransferase, were administered to assess mammary tumors. MNU was administered at 3 or 8 weeks of age. DMBA was administered at 8 weeks of age. NAT2 enzymatic activity and endogenous acetyl-coenzyme A (AcCoA) levels were measured in tissue samples and embryonic fibroblasts isolated from the congenic rats., Results: Tumor latency was shorter in rapid NAT2 rats compared to slow NAT2 rats, with statistical significance for MNU administered at 3 and 8 weeks of age (p = 0.009 and 0.050, respectively). Tumor multiplicity and incidence were higher in rapid NAT2 rats compared to slow NAT2 rats administered MNU or DMBA at 8 weeks of age (MNU, p = 0.050 and 0.035; DMBA, p = 0.004 and 0.027, respectively). Recombinant rat rapid-NAT2, as well as tissue samples and embryonic fibroblasts derived from rapid NAT2 rats, catalyzed p-aminobenzoic acid N-acetyl transfer and folate-dependent acetyl-coenzyme A (AcCoA) hydrolysis at higher rates than those derived from rat slow-NAT2. Embryonic fibroblasts isolated from rapid NAT2 rats displayed lower levels of cellular AcCoA than slow NAT2 rats (p < 0.01)., Conclusions: A novel role for rat NAT2 in mammary cancer was discovered unrelated to carcinogen metabolism, suggesting a role for human NAT1 in breast cancer.

Introduction: Human arylamine N -acetyltransferase 1 (NAT1) is a phase II xenobiotic metabolizing enzyme found in almost all tissues. Expression of NAT1 is elevated in several cancers including breast cancer. However, the exact mechanism by which NAT1 expression affects cancer risk and progression remains unclear., Objective: This study explored polar metabolome differences between MDA-MB-231 breast cancer cells expressing varying levels of NAT1 activity using an untargeted approach., Methods: Three MDA-MB-231 breast adenocarcinoma cell lines that stably express wild-type, increased, and decreased levels of human NAT1 were investigated for differences in polar metabolic profile using a comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF MS) system., Results: Increased levels of human NAT1 in the transformed cell lines resulted in a statistically significant decreased abundance of the metabolite palmitoleic acid (q = 0.0006), when compared to normal and decreased levels of human NAT1. The fatty acid synthesis pathway utilizes acetyl coenzyme A (acetyl-CoA) in the first two reactions of the pathway and eventually leads to the synthesis of palmitoleic acid., Conclusion: These data suggest a link between increased levels of NAT1 activity and decreased flux of acetyl-CoA through this portion of the fatty acid synthesis pathway., Competing Interests: The authors declare that they have no conflict of interest.

Polychlorinated biphenyls (PCBs) are environmental pollutants associated with non-alcoholic-steatohepatitis (NASH), diabetes, and obesity. We previously demonstrated that the PCB mixture, Aroclor 1260, induced steatohepatitis and activated nuclear receptors in a diet-induced obesity mouse model. This study aims to evaluate PCB interactions with the pregnane-xenobiotic receptor (Pxr: Nr1i2) and constitutive androstane receptor (Car: Nr1i3) in NASH. Wild type C57Bl/6 (WT), Pxr(-/-) and Car(-/-) mice were fed the high fat diet (42% milk fat) and exposed to a single dose of Aroclor 1260 (20 mg/kg) in this 12-week study. Metabolic phenotyping and analysis of serum, liver, and adipose was performed. Steatohepatitis was pathologically similar in all Aroclor-exposed groups, while Pxr(-/-) mice displayed higher basal pro-inflammatory cytokine levels. Pxr repressed Car expression as evident by increased basal Car/Cyp2b10 expression in Pxr(-/-) mice. Both Pxr(-/-) and Car(-/-) mice showed decreased basal respiratory exchange rate (RER) consistent with preferential lipid metabolism. Aroclor increased RER and carbohydrate metabolism, associated with increased light cycle activity in both knockouts, and decreased food consumption in the Car(-/-) mice. Aroclor exposure improved insulin sensitivity in WT mice but not glucose tolerance. The Aroclor-exposed, Pxr(-/-) mice displayed increased gluconeogenic gene expression. Lipid-oxidative gene expression was higher in WT and Pxr(-/-) mice although RER was not changed, suggesting PCB-mediated mitochondrial dysfunction. Therefore, Pxr and Car regulated inflammation, behavior, and energy metabolism in PCB-mediated NASH. Future studies should address the 'off-target' effects of PCBs in steatohepatitis., (Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.)

Arsenic is a Class I human carcinogen and is widespread in the environment. Chronic arsenic exposure causes cancer in skin, lung and bladder, as well as in other organs. Paradoxically, arsenic also is a potent chemotherapeutic against acute promyelocytic leukemia and can potentiate the cytotoxic effects of DNA damaging chemotherapeutics, such as cisplatin, in vitro. Arsenic has long been implicated in DNA repair inhibition, cell cycle disruption, and ubiquitination dysregulation, all negatively impacting the DNA damage response and potentially contributing to both the carcinogenic and chemotherapeutic potential of arsenic. Recent studies have provided mechanistic insights into how arsenic interferes with these processes including disruption of zinc fingers and suppression of gene expression. This review discusses these effects of arsenic with a view toward understanding the impact on the DNA damage response.

Arylamine N -acetyltransferases (NATs) are drug and xenobiotic metabolizing enzymes that catalyze the N-acetylation of arylamines and hydrazines and the O-acetylation of N-hydroxy-arylamines. Recently, studies report that human NAT1 and mouse Nat2 hydrolyze acetyl-coenzyme A (AcCoA) into acetate and coenzyme A in a folate-dependent fashion, a previously unknown function. In this study, our goal was to confirm these findings and determine the apparent Michaelis-Menten kinetic constants (Vmax and Km) of the folate-dependent AcCoA hydrolysis for human NAT1/NAT2, and the rodent analogs rat Nat1/Nat2, mouse Nat1/Nat2, and hamster Nat1/Nat2. We also compared apparent Vmax values for AcCoA hydrolysis and N-acetylation of the substrate para-aminobenzoic acid (PABA). Human NAT1 and its rodent analogs rat Nat2, mouse Nat2 and hamster Nat2 catalyzed AcCoA hydrolysis in a folate-dependent manner. Rates of AcCoA hydrolysis were between 0.25 - 1% of the rates for N-acetylation of PABA catalyzed by human NAT1 and its rodent orthologs. In contrast to human NAT1, human NAT2 and its rodent analogs rat Nat1, mouse Nat1, and hamster Nat1 did not hydrolyze AcCoA in a folate-dependent manner. These results are consistent with the possibility that human NAT1 and its rodent analogs regulate endogenous AcCoA levels.

Arsenic is an enigmatic xenobiotic that causes a multitude of chronic diseases including cancer and also is a therapeutic with promise in cancer treatment. Arsenic causes mitotic delay and induces aneuploidy in diploid human cells. In contrast, arsenic causes mitotic arrest followed by an apoptotic death in a multitude of virally transformed cells and cancer cells. We have explored the hypothesis that these differential effects of arsenic exposure are related by arsenic disruption of mitosis and are differentiated by the target cell's ability to regulate or modify cell cycle checkpoints. Functional p53/CDKN1A axis has been shown to mitigate the mitotic block and to be essential to induction of aneuploidy. More recent preliminary data suggest that microRNA modulation of chromatid cohesion also may play a role in escape from mitotic block and in generation of chromosomal instability. Other recent studies suggest that arsenic may be useful in treatment of solid tumors when used in combination with other cytotoxic agents such as cisplatin.