Dihydrodiol dehydrogenase (DD;EC 1.3.1.20) has been implicated in the detoxication of polycyclic aromatic hydrocarbons (PAH) which are human carcinogens. DD catalyzes the NAD(P)+ dependent oxidation of PAH-trans- dihydrodiols to non-carcinogenic catechols and can suppress the formation of the ultimate carcinogens (anti-diol-epoxides). The rat liver enzyme has been cloned and sequenced, and is identical to 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD). Data indicates that enzyme activity is regulated by estrogens (E2) and growth hormone (GH) and this occurs by mechanisms independent of the Ah receptor. To understand these mechanisms the regulation of rat liver 3alpha-HSD/DD gene expression will be studied. E2, GH and insulin-like growth factor-1 (IGF-1) will be administered to hypophysectomized rats and hepatic 3alpha-HSD/DD mRNA levels will be measured by Northern analysis. mRNA levels will be correlated with changes in enzyme activity and enzyme amount measured by immunotitration. It will be determined whether IGF-1 is a common mediator for E2 and Gh effects. Nuclear-run off assays using (p3alpha-HSD/DD) plasmids as probes for RNA transcripts will be performed to show that hormonal treatment is associated with increased transcription of the 3alpha-HSD/DD gene. Promotor and E2, GH or IGF-1 responsive elements that may control transcription of the 3alpha-HSD/DD gene will be identified as follows: The structure of the leader sequence of 3alpha-HSD/DD mRNA will be determined by primer extension analysis and polymerase chain reaction amplification of the leader for dideoxysequencing. A rat liver genomic library (partial Sau 3 digest in EMBL3) will be screened with short 5' fragments (0.1 - 0.5 Kb) of the near full length [32P]-cDNA probe to isolate genomic clones that contain 5' flanking regions of the 3alpha- HSD/DD gene. Restriction mapping and Southern analysis of the positive clones, using the shortened probes, will identify genomic fragments (0.5 - 2.0 Kb corresponding to the 5' end for subcloning and sequencing. Sequences will be aligned to the leader sequence and compared to known consensus sites for promotors and responsive elements. Functional promotors of responsive elements that control 3alpha-HSD/DD gene expression will be identified by ligating DNA sequences into chloramphenicol acetyl-transferase (CAT) reporter gene constructs. Transient transfections will be performed in Hep G2 cells using pCAT-basic (no promotor and no enhancer), pCAT-basic (plus inserted promotor), pCAT- promotor (plus inserted enhancer), pCAT-control (SV40 promotor and enhancer). The level of CAT expression in the absence or presence of E2, GH, or IGF-1 will give a direct measure of either promotor or enhancer activity, respectively. Identification of hormones and/or their responsive elements that control transcription of the 3alpha-HSD/DD gene may provide clues to novel mechanisms that may regulate PAH carcinogenicity.
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