The research mission of the Stem Cells Toxicology (SCT) Group is to characterize responses to toxicants to elucidate mechanisms and identify the role of stem cells (SCs) in disease manifestation. The major focus has been on arsenic (As) with a smaller Cd project. Arsenic and cadmium are inorganic carcinogens that are major human health hazards and defining mechanisms is key to defining risk. We primarily use target-relevant cell models of both mature differentiated cells and SCs. Cadmium (Cd) is a well-defined human and rodent carcinogen, so cell models are used to define mechanisms in established/suspected human targets. Accepted human targets for iAs (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. In a recent advent, 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 (e.g. lung, liver, skin and urinary bladder). These mouse studies stimulated work on early life human As exposure from the drinking water and its association with adulthood cancers. Human data from a population in Chil that was exposed in early life to high natural levels of As in drinking water from 1958 to 1970 link As exposure to lung, liver and kidney cancer. These stunning results prompted Dr. Allen Smith, a prominent epidemiologist to say that this As exposure had resulted in the greatest increases in cancer mortality in adults ever associated with early-life environmental exposure. Similarly, a high-dose pulse exposure in early life caused by iAs-contaminated powdered milk in infants in the 1950s is now being linked to cancer in survivors, including liver cancer. 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 are 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 iAs carcinogenesis in the lung. We have previously found that methylarsonous acid (MMA3+), a biomethylation product of iAs, does not need to be further methylated to produce oxidative DNA damage. This may be important for 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 oncogene, KRAS, is highly up-regulated during As-induced malignant transformation. We found no evidence of DNA damage during this process and this cell line is biomethylation-deficient suggesting up-regulation of KRAS is not the result of mutation or methylation, but likely epigenetic factors. MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression at a post-transcriptional level. We find that As exposure causes a dysregulation of miRNA expression that appears to control RAS activation during malignant transformation of human prostate epithelial and SCs. Knockdown of RAS expression partially mitigates malignant phenotype of As-transformed epithelial cells and SCs. These data suggest miRNA-regulated RAS expression is a putative driver in As-induced malignant transformation. These studies will help determine the role of miRNAs and underlying epigenetic mechanisms involved in As 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 helping characterize the toxicity of a broad range of individual PAHs, defined PAH mixtures, and complex environmental mixtures containing PAHs using various cell lines. The prostate is a human target of Cd. Unlike As, which selects for SC accumulation, Cd early on selectively kills SCs causing 90% cytolethality in our prostate SC line exposed 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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