The goal of the proposed study is to provide rigorous biochemical evidence that cytotoxicity and altered maintenance methylation (formation of 5-methylcytosine in DNA shortly after replication) in the rodent liver are mechanistically related and causative in cancer induced by environmental agents. The results would be applicable to the interpretation of cancer bioassays in which increased incidences of tumors appear only at exposure levels which cause cytotoxicity in the target tissue. The hypothesis proposed for experimental investigation is: cytotoxicity has a causative role in liver carcinogenesis by altering maintenance methylation patterns and/or levels, thus compromising differentiation of new daughter cells during compensatory regeneration. The experiments will use tools of both analytical chemistry and molecular biology to measure quantitating the extent to which 5-methylcytosine levels and patterns in newly synthesized DNA are altered and whether there are specific patterns of altered maintenance methylation related to cytotoxicity. The dose-response relationships for the cytotoxicity and altered maintenance methylation induced by hydrazine and other carcinogens will be determined. Chronic exposure to hamster of hydrazine in the drinking water provides a model in which the appearance of hepatocellular carcinomas is clearly associated with the accumulation of DNA adducts and the development of mixed necrosis and hyperplasia; this model will be used to investigate the association of altered maintenance methylation with cytotoxicity and carcinogenesis.
The specific aims of the proposed research are: 1) determine the dose- response relationship for the hypermethylation (excess of 5- methylcytosine) of genomic liver DNA and of defined sequences in liver DNA by hydrazine and other carcinogens; 2) develop a method for the analysis of liver DNA for changes in the amount and position of 5-methylcytosine using hybridization of treated DNA with specific probes to examine particular defined DNA sequences; 3) determine the persistence of the chemically-induced hypermethylation of liver DNA (5-methylcytosine); 4) determine the relationship and sequence of events for the formation of methylguanines, alteration of 5-methylcytosine patterns in liver DNA and development of liver necrosis in hamsters given three dose levels of hydrazine daily in the drinking water for two years, and 5) determine the structures of the DNA adducts formed by phenylhydrazine and hydralazine.
