The research mission of the Inorganic Toxicology (IT) Group is to characterize responses to carcinogenic inorganics to elucidate mechanisms under a single project titled Mechanisms of Inorganic Toxicology. The major focus is on arsenic (As) with a smaller Cd project. Inorganic carcinogens are major human health hazards and defining mechanisms is key to defining risk. The development of rodent cancer models for As is a recent advent in which the IT Group members played a major role. We also extensively use target-relevant cell models. 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 inorganic As 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 and kidney are considered likely targets. Tens of 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. Members of IT Group developed a mouse transplacental cancer model in which multiple studies show As exposure in utero causes or facilitates tumors in adulthood at various sites, including known human targets such as the 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 in humans. Human data now link early life drinking water As exposure and lung, liver and kidney cancer. The population studied comes from Chile and was exposed to high natural levels of drinking water As from 1958 until its remediation in 1970. The results of these studies are so stunning it prompted Dr. Allen Smith, a prominent University of California at Berkeley epidemiologist to say that this As exposure in the drinking water had resulted in the greatest increases in cancer mortality in adults ever associated with early-life environmental exposure. There were also high dose pulse exposures in humans in early life that caused cancer in adulthood. In this case inorganic As-contaminated powdered milk caused a mass poisoning event of infants in the mid-1950s in Japan and is now linked to cancers in the survivors, including liver cancer. Using a whole life exposure model, which more reasonably duplicates typical human environmental exposure, we recently showed that mice exposed to low levels (50 ppb) of As in their 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 of inorganic As 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 inorganic As exposure. The early life period, including in utero and neonatal life, is also a time of high stem/progenitor cell activity due to organogenesis, global proliferative growth, etc. Since inorganic As as a cancer chemotherapeutic is known to impact stem cell (SC) programming as part of its therapeutic mode of action, this lead us to hypothesize early on that in 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 putative CSC overproduction. A major issue is how As can specifically target SCs and what the molecular manifestations of this targeting are. We are using specific, target-relevant SC cell lines (prostate, skin, kidney, liver) to look into these important questions. Recent 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. This fortifies the notion that a quiescent, long-lived cell (i.e. SCs) passes damage along for years. With chronic As exposure, cells adapt in various ways such as via altered methyl metabolism, oxidant stress response or enhanced export. Inorganic As undergoes enzymatic biomethylation (BML) by a specific methyltransferase (AS3MT). As BML was thought to be adaptive but many target cells of As carcinogenesis do not BML As. The role of As BML and ODD generation during malignant transformation has been tested in multiple models. ODD occurred in BML-capable cells prior to transformation but BML-deficient cells showed no ODD despite exposure past transformation. This indicates that multiple mechanisms can be involved during As carcinogenesis. Direct exposure to the BML product, methylarsonous acid (MMA3+) indicates that BML-capable and BML-deficient cells show similar patterns and levels of ODD which is very illuminating with regards to early steps in methylation and oxidative stress. Thus, As BML is obligatory for ODD, and hastens acquired cancer phenotype, but cells can acquire a cancer phenotype without ODD, again implicating multiple mechanisms. Furthermore, MMA3+ needs no further methylation to produce ODD. 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. This could indicate a unique sensitivity to MMA3+ in lung cells. We have a human lung cell line which we are using to study this important question. Besides methylation, As has other epigenetic mechanisms behind its cancer-inducing properties. The oncogene, KRAS, is highly up-regulated during As-induced malignant transformation of human prostate epithelial cells. We found no evidence of DNA damage during this process and this cell line is BML-deficient suggesting up-regulation of KRAS is not the result of mutation or methylation, but due to other 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. We are using these transformants to determine the effects of a miRNA-regulated RAS suppression on reversal of malignant phenotype. These studies will help further determine the role of miRNAs in this process and will be important to understanding the underlying molecular/epigenetic mechanisms of chemical transformation. The prostate is a potential human target of Cd. In contrast to As, which selects for SC accumulation, Cd early on selectively kills SCs. Cd caused 95% cytolethality in our prostate SC line exposed to a non-toxic, but transforming, level for the heterogeneous parental epithelial line. Though depleted, remaining SCs rapidly re-emerge and undergo transformation. We are determining if Cd has transformed these SCs and observing these SCs and observing the mature cell line for selection of hyper-resistant SCs. We are also developing liver and kidney SC models of cadmium transformation. We have developed a human lung epithelial cell transformant with Cd from a line that would produce adenocarcinoma, the tumors linked to Cd in humans and rodents. Cd appears to work, at least in part, by epigenetic mechanisms, including modification of DNA methylation.
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