• Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant oncogene expression

      Zhao, Christopher Qinchun; Waalkes, Michael P.; Squibb, Katherine S. (1997)
      Arsenic compounds are known human carcinogens but have proven equivocal in animal testing. The methylation of arsenic is a key aspect of its metabolism and consumption of the cellular methyl group donor, S-adenosyl-methionine (SAM), occurs concurrently with this metabolism. The fact that DNA methyltransferases (MeTases) require this same methyl group donor suggests a role for DNA hypomethylation in arsenic carcinogenesis. We tested the hypothesis that arsenic-induced cell transformation results from DNA hypomethylation caused by continuous methyl depletion due to chronic exposure to arsenic, and aberrant gene expression facilitated by the inheritable DNA methylation status. The hypothesis was tested by first inducing transformation in a rat liver epithelial cell line by chronic exposure to low-levels of arsenic, as confirmed by the development of highly aggressive, malignant tumors after inoculation of cells into Nude mice. Global DNA hypomethylation occurred concurrently with malignant transformation and in the presence of depressed levels of SAM. Arsenic-induced DNA hypomethylation was a function of dose and exposure duration, and remained constant even after withdrawal of arsenic. Consistent with hypomethylation as the means of transformation, activation of the oncogene c-myc was detected. Activation of c-myc often occurs during hepatocarcinogenesis and can induce transformation without mutation by simple overexpression. Hyperexpressibility of the MT gene, a gene for which expression is clearly controlled by DNA methylation, was also detected in transformed cells. Acute arsenic or arsenic at non-transforming levels did not induce global hypomethylation of DNA or activate c-myc. While transcription of DNA MeTase was elevated, the MeTase enzymatic activity was reduced with arsenic transformation. Taken together, these results indicate chronic, low levels of arsenic can act as a carcinogen by inducing genomic DNA hypomethylation, which facilitates aberrant oncogene expression, and they constitute the first tenable theory of mechanism in arsenic carcinogenesis.
    • Molecular mechanisms of cadmium carcinogenesis: The potential roles of lipid peroxidation, oncogene activation and metallothionein expression

      Abshire, Michael Krystian; Waalkes, Michael P. (1995)
      Cadmium is a potent metallic toxin and carcinogen. The metallothionein gene (MT) is highly metal-inducible and encodes for a metal-binding protein, metallothionein (MT), which can protect against the toxic effects of cadmium by sequestration of the metal. The molecular processes involved in cadmium carcinogenesis were examined. Levels of oxidative damage (lipid peroxidation) and the acute activation of proto-oncogenes c-myc and c-jun by cadmium in vitro and the effect of MT gene preactivation on these effects were studied. A low level of cadmium was able to induce expression of the c-myc and c-jun oncogenes and subsequently transform cultured cells. Cadmium caused strain-dependent oxidative damage in vivo in the liver, kidney and testes of mice. Levels of lipid peroxidation were higher in the testes of NFS mice, a strain known to be more susceptible to cadmium toxicity. MT protein preinduction by zinc prevented cadmium-induced lipid peroxidation. This was more marked in BALB/c mice, a strain resistant to the toxic effects of cadmium. Acute in vitro cadmium exposure was an effective inducer of expression of the c-myc and c-jun proto-oncogenes in rat L6 myoblasts; preexposure to zinc effectively repressed their transcriptional induction by cadmium. Chronic low dose cadmium exposure of L6 cells was a potent initiator of transformation. Low dose cadmium-transformed L6 cells, when inoculated into nude mice, produced tumors that were more malignant and had significantly higher growth rates and sizes than untreated control cells. Transformation induced by cadmium was associated with a chronic downregulation of c-myc and c-jun. However, lipid peroxidation was not associated with the increased expression of these oncogenes. Cadmium was an effective initiator of oxidative damage, proto-oncogene activation and L6 transformation. MT expression was associated with protection from acute cadmium toxicity in vitro and in vivo, and prevention of cadmium-induced oncogene activation. MT may thus be intimately involved in the molecular mechanisms of cadmium carcinogenesis.