The hydroxysteroid sulfotransferases (HSTs) are a family of enzymes that catalyze the sulfation of hydroxysteroids, aliphatic alcohols and other xenobiotics to polar products. In addition, the HSTs activate polycyclic aromatic hydrocarbon (PAH) derivatives to ultimate carcinogens. HST sulfation of 5-hydroxymethylchrysene and 7,12- dihydroxymethylbenz(a)anthracene results in the formation of highly reactive sulfate esters. Loss of the sulfate group generates potent mutagenic and carcinogenic intermediates which damage DNA. Three major HSTs have been isolated from rat liver cytosol. Previous studies implicate gonadal, adrenal and pituitary hormones in HST enzyme regulation. In addition, metabolic studies show that rat liver HST enzyme activity increases with aging. However, to date, there is no information on HST gene regulation and limited information on HST gene expression. Studies conducted in this laboratory examined HST gene expression using an oligonucleotide probe complementary to published rat liver HST cDNA (HST-a). Northern and slot blot analyses revealed that rat liver HST-a-mRNA was differentially expressed with respect to age and gender. Hepatic HST-a mRNA levels which were approximately 4 to 6- fold higher in female rats relative to males, increased dramatically with aging in both sexes. Site-specific alterations in cytosine methylation profile has been linked to mammalian tissue-specific and developmental gene activation. The hypothesis to be examined in this proposal is that developmental expression of the HST genes is a result of transcriptional activation regulated by altered cytosine methylation profile.
The specific aims of this research are: 1.) To isolate and purify the HST proteins from rat liver and prepare HST mono-specific antibodies, 2.) To use HST mono-specific antibodies in immuno-blots and for cDNA library screening, 3.) To examine age- and gender- related HST gene expression in rat liver using HST cDNAs, oligonucleotide probes, or PCR fragment probes 4.) To define HST age- and gender-related transcriptional activation in rat liver using nuclear run on transcription assays, and 5). To examine age-and gender-related modulation of HST gene methylation. Activation of PAH derivatives by HSTs results in the formation of mutagenic and carcinogenic intermediates and constitutes a key pathway in the biotransformation of xenobiotics. These studies will be the first to characterize the developmental expression of the HST genes. Information of HST expression and developmental regulation is critical to understanding the impact of these important enzymes on the processes of cellular toxicity and chemical carcinogenesis.
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