The mission of the Stem Cell Toxicology (SCT) Group is to characterize responses to toxicants to elucidate mechanisms and identify the role of stem cells (SCs) in disease manifestation. While stem cells/cancer stem cells and potential mechanisms of action in inorganic carcinogenesis has been the major focus, research efforts are also extending beyond cancer to include those diseases and conditions associated with exposure to NTP chemicals. Arsenic (As) and cadmium (Cd) are inorganic carcinogens that are major human health hazards and defining mechanisms is key to defining risk. We use target-relevant cell models of both mature differentiated cells and SCs. Accepted human targets for iAs are the lung, skin and urinary bladder and suspected targets are the prostate, liver and kidney. Cd clearly targets the human lung but the prostate, liver, and kidney are considered likely targets. Millions of people worldwide are exposed to unhealthy As levels in drinking water but questions remain about health issues of low-level exposures, making elucidation of mechanisms all the more important. With Cd, environmental exposure may play an important role in cancer. Recently, members of SCT Group developed a mouse transplacental cancer model in which multiple studies show As exposure in utero causes or facilitates adulthood tumors at various sites, including known human targets. These studies stimulated work on early life human As exposure from the drinking water and its association with adulthood cancers. Human data from populations in Chile and Japan, both exposed to high levels of As during early life, link this As exposure to cancers later in life in both populations. Using a whole life exposure model, which more reasonably duplicates typical human exposure, we recently showed that mice exposed to low levels (50 ppb) of As in drinking water develop lung cancer as adults. This is the first study to show tumor development in animals exposed to very low levels of As, similar to which humans might be exposed. Exposure to even lower concentrations during gestation or early life is needed to help define molecular changes that result in disease manifestation later in life. Human and rodent evidence indicate early life is a time of sensitivity to iAs exposure. The early life period, including in utero and neonatal life, is also a time of high SC activity due to organogenesis, global proliferative growth, etc. iAs as a cancer chemotherapeutic can impact SC programming as part of its therapeutic mode of action, which led us to hypothesize that during early life SCs could be a key target population of As carcinogenesis. Perinatal As exposure, which induces or predisposes mice to lung, skin, urinary bladder, liver or kidney tumors as adults, also causes an over-abundance putative cancer SCs (CSCs) in many of these same tumors. We also find superior innate and acquired As resistance in human and rodent SC lines, involving general and As-specific adaptation. Malignant transformation of a heterogenous mature prostate line with As causes a stunning CSC overproduction. A major issue is how As can specifically target SCs and the molecular manifestations of this targeting. We are using target-relevant SC cell lines (prostate, skin, kidney, liver, lung) to look into these important questions. Human data indicate that where elevated As exposure is remediated, despite long-term exposure cessation, cancer risk remains elevated in lung and bladder for at least 40 years, fortifying the notion that a quiescent, long-lived cell (i.e. SC) passes damage along for years. In the lung, iAs can induce both squamous cell carcinoma and adenocarcinoma, the former predominantly associated with ingestion and the latter with inhalation. We used human peripheral lung cells to investigate the effects and mechanisms involved in As-induced lung adenocarcinoma. We find that low-level exposure induces a cancer phenotype in these cells. We can continue to use this human lung model to further define mechanisms and the role of lung SCs in the process of As lung carcinogenesis. We previously found that methylarsonous acid (MMA3+), a biomethylation product of iAs, does not need further methylation to produce oxidative DNA damage. This may be important in the lung, as a genetic predisposition to poorly methylate As past MMA was recently linked to lung cancer in humans, possibly indicating a unique sensitivity to MMA3+ in lung cells. Our lung cell model will allow us to investigate this important question. Neither As nor Cd appears to be directly mutagenic, suggesting they have other mechanisms of action. Epigenetic modifications are heritable changes in gene transcription that are not caused by changes in DNA sequence. Epigenetic modifications can play a role in cancers induced by both As and Cd. Using next generation sequencing we identified multiple dysregulated genes in an As-transformed cell model. Five of the most highly dysregulated genes were chosen for more in-depth analysis of DNA methylation in As and Cd transformants. Four of the five genes showed differential methylation in the transformants when compared to control cells. Methylation was inversely related to gene expression in the transformed cells. These results further demonstrate that epigenetic factors, specifically DNA methylation in this case, play a role in As and Cd carcinogenesis. The KRAS oncogene is highly up-regulated during As and Cd induced malignant transformation. We found no evidence of DNA damage during this process and this cell line is As biomethylation-deficient suggesting increased KRAS is not due to mutation or As methylation, but likely epigenetic factors. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at a post-transcriptional level. We find that As exposure dysregulates miRNA expression that appears to control RAS activation during malignant transformation of human prostate epithelial and SCs. Knockdown of RAS partially mitigates this malignant phenotype. These data suggest miRNA-regulated RAS expression is a putative driver in As-induced malignant transformation. Indeed, restoration of specific miRNAs mitigates As-induced malignant phenotype. Similarly, depleting KRAS in Cd-transformed cells also reverses cancer phenotype, although this did not appear to be miRNA-regulated. These studies will help determine the role of miRNAs and underlying epigenetic mechanisms involved in As and Cd carcinogenesis. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants that have a wide range of toxicities including cancer, reproductive and developmental effects, immunotoxicity. Together with the Toxicology Branch of DNTP, we are characterizing the toxicity of a broad range of individual PAHs, defined PAH mixtures, and complex environmental mixtures containing PAHs using various cell lines. Induced pluripotent SCs offer the ability to study effects of environmental toxicants on embryogenesis and reproductive toxicology. Together with the Biomolecular Screening Branch (NTP) and the Epigenetics and Stem Cell Biology Lab (DIR) we are developing assays to screen chemicals on various forms of pluripotent SCs to help discover and possibly better predict developmental toxicants and teratogens. The prostate is a human target of Cd. Unlike As, Cd initially selectively kills SCs causing 90% cytolethality during exposure to a non-toxic, but transforming, level for the heterogeneous parental line. The remaining SCs rapidly re-emerge and undergo transformation. We are determining if Cd has transformed these SCs and observing these SCs and the mature cell line for selection of hyper-resistant SCs. We are also developing liver and kidney SC models of As and Cd transformation and defining the metabolic profiles of these cells during the carcinogenic process.

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Ngalame, Ntube N O; Luz, Anthony L; Makia, Ngome et al. (2018) Arsenic Alters Exosome Quantity and Cargo to Mediate Stem Cell Recruitment Into a Cancer Stem Cell-Like Phenotype. Toxicol Sci 165:40-49
Luz, Anthony L; Tokar, Erik J (2018) Pluripotent Stem Cells in Developmental Toxicity Testing: A Review of Methodological Advances. Toxicol Sci 165:31-39
Ren, Natalie S X; Ji, Ming; Tokar, Erik J et al. (2017) Haploinsufficiency of SIRT1 Enhances Glutamine Metabolism and Promotes Cancer Development. Curr Biol 27:483-494
Ganini, Douglas; Leinisch, Fabian; Kumar, Ashutosh et al. (2017) Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells. Redox Biol 12:462-468
Ngalame, Ntube N O; Makia, Ngome L; Waalkes, Michael P et al. (2016) Mitigation of arsenic-induced acquired cancer phenotype in prostate cancer stem cells by miR-143 restoration. Toxicol Appl Pharmacol 312:11-18
Bailey, Kathryn A; Smith, Allan H; Tokar, Erik J et al. (2016) Mechanisms Underlying Latent Disease Risk Associated with Early-Life Arsenic Exposure: Current Research Trends and Scientific Gaps. Environ Health Perspect 124:170-5
Ngalame, Ntube N O; Waalkes, Michael P; Tokar, Erik J (2016) Silencing KRAS Overexpression in Cadmium-Transformed Prostate Epithelial Cells Mitigates Malignant Phenotype. Chem Res Toxicol 29:1458-67
Kumar, Ashutosh; Ehrenshaft, Marilyn; Tokar, Erik J et al. (2016) Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells. Biochim Biophys Acta 1860:1519-27
Carlin, Danielle J; Naujokas, Marisa F; Bradham, Karen D et al. (2016) Arsenic and Environmental Health: State of the Science and Future Research Opportunities. Environ Health Perspect 124:890-9
Tokar, Erik J (2016) MicroRNAs: small molecules with big effects. Toxicol Appl Pharmacol 312:1-2

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