The carcinogenic potential of chemicals such as 2- acetylaminofluorene, safrole, dimethylbenz[a]anthracene, and cyproterone acetate is dependent on the sulfotransferase-mediated bioactivation of these compounds into mutagens and carcinogens. The goal of the present proposal is to determine which rat and human sulfotransferase enzymes are responsible for the activation of these four chemicals into genotoxic carcinogens. Both rats and humans are known to have seven major sulfotransferases. Rats have four phenol-sulfotransferases and three hydroxysteroid sulfotransferases, whereas humans have six phenol sulfotransferases and one hydroxysteroid sulfotransferase. We postulate that in both rats and humans specific sulfotransferase isoforms will be responsible for the bioactivation of 2- acetylaminofluorene, safrole, dimethylbenz[a]-anthracene, and cyproterone acetate.
In Aim 1, we will characterize the ability of the seven recombinant rat sulfotransferases to sulfurylate the P-450 metabolites of these four procarcinogens; determine which sulfotransferase isoform bioactivates these compounds to be mutagenic in the Ames Salmonella assay; and because the seven rat sulfotransferase isoforms are known to be differentially regulated by age, sex (e.g., estradiol and testosterone), pituitary (e.g., growth hormone), and adrenal hormones (e.g., corticosterone), we will exploit this differential regulation and use isolated hepatic cytosol to implicate individual sulfotransferses in the mutagenicity of these four procarcinogens.
In Aim 2, antibodies to each rat sulfotransferase will be raised to determine (1) the amount of each sulfotransferase in the liver of normal and physiologically manipulated rats, as well as (2) to immunotype the ability of each sulfotransferase to activate each promutagen to the mutagen.
In Aim 3, the investigators will determine the in vivo ability of male and female rats of different ages and with or without hypophysectomy or adrenalectomy to activate each of the four procarcinogens to covalently bind DNA. This will enable the investigators to determine whether the predicted in vitro activity of the sulfotransferase isoforms reflects their activity in vivo. If the in vitro data does not predict the in vivo data, it might be that the in vitro conditions do not contain steroid sulfatase, a microsomal enzyme that degrades sulfate metabolites. Therefore in Aim 4, regulation of the sulfatase enzyme will be determined under the conditions that are known to after the regulation of the various sulfotransferase isoforms.
In Aim 5, the ability of the seven expressed human sulfotransferases to activate the four procarcinogens to mutagens will be determined. Therefore, this proposal will determine which sulfotransferase isoenzyme in both rats and humans is responsible for activating each of the four procarcinogens to mutagens, a major unknown in understanding their mechanism of carcinogenicity.
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