Project title: Functional Significance of Polymorphic Variants of CYP1B1 and COMT in Estrogen Metabolism and Their Relevance to Endometrial Cancer
Completed in: 2004 | Faculty advisor: David L. Eaton
Polymorphic enzymes involved in oxidative estrogen metabolism may influence endometrial cancer risk. Cytochrome P450s oxidize estrogen to catechols (CEs) that may be further oxidized to DNA damaging semiquinones and quinones. Catechol O-methyltransferase (COMT), the glutathione S-transferases (GSTs), and NADPH-quinone reductase (NQO1) may attenuate the damage by CE methylation, quinone-GSH conjugation, and quinone reduction, respectively. Combinations of these variants may modify endometrial cancer risk. To investigate this, correlations between DNA damage and CYP1A1, CYP1B1, COMT, GSTmu, theta, and pi, and NQO1 genotypes were evaluated in human endometrial tissues of non-cancer origin. Transcript levels for all genes except NQO1, Western blots for CYP1B1 and COMT, and activity assays measuring E2 hydroxylation and CE methylation proved the genes were expressed and active in human endometrial tissues. A particular focus was placed on CYP1B1 and COMT, as CYP1B1 was thought to be very important in E2 activation and COMT catalyzes the first reaction detoxifying E2 oxidative metabolites.
To complement the above investigation, yeast trp reversion and Ames mutation assays were developed with hopes of detecting CYP1B1-mediated E2 metabolite-induced DNA damage and distinguishing between the relative protective abilities of the COMT variants. Neither system produced measurable CYP1B1-mediated DNA damage resulting from E2 oxidation. The relative protection conferred by the COMT variants could therefore not be evaluated.
No correlations were found between DNA damage and the enzymes variants involved in oxidative E2 metabolism. RTPCR revealed that CYP1A1 was not expressed, whereas CYP1B1 was expressed, but only in small amounts, in human endometrial tissue. CYP3A5 transcripts were definitely present. CE methylation and COMT protein levels did not vary by COMT genotype. However, E2 hydroxylating activity in the tissues was associated with the CYP3A5*1 genotype. Samples homozygous for CYP3A5 *3 had E2 hydroxylating activities below the assay limit of detection. Given that CYP3A5 is relatively highly expressed and its activity varied by *1 genotype, CYP3A5*1 status may influence the risk for developing endometrial cancer. Unfortunately, the scope of the project did not include CYP3A5 is the genotype/DNA damage phenotype assessment. The possible role of CYP3A5 in the etiology of endometrial cancer should be investigated further.