Extrahepatic cytochrome P450s - Relation to cancer susceptibility

Abstract: Cytochrome P450 (CYP) enzymes in extrahepatic tissues may play important roles in regulating the capacity of individual cells to metabolize environmental carcinogens and hormones. In this thesis, we characterized the cytochrome P450 profile of the human normal breast and investigated whether polymorphisms in the genes encoding the estrogen metabolizing enzymes CYP1131 and Catechol-O-methyltransferase (COMT) were associated with breast or endometrial cancer risk. We also identified, cloned and characterized the novel extrahepatic cytochrome P450 2S1 (CYP2S1) enzyme with a possible role in metabolism of xenobiotics. Detected levels of CYP enzymes in extrahepatic tissues are generally lower than in the liver and thus difficult to measure. By partial purification of CYPs from breast reduction samples, we spectrally quantified the level in the human normal breast to be approximately 1000-fold lower than in the human liver. We characterized the CYP expression pattern by RTPCR and Western blot in 15 samples and found enzymes involved in the metabolism of environmental carcinogens, steroid hormones and drugs. A large interindividual variation in the expression of CYP1A1, 2A6, 2B6, 2D6 and 3A were shown whereas CYP1B1, 2C, 2E 1, 4A and 19 was present in most samples. Molecular epidemiological studies on the association between polymorphic variants of enzymes involved in estrogen biosynthesis and metabolism and hormonal cancer risk are published with rapidly increasing frequency. However, reported data are generally inconsistent, partly due to small sample sizes. We investigated the association between CYP1B1 and COMT genotypes, respectively, and breast and endometrial cancer (only CYP1B1) in a large population-based case-control study on Swedish postmenopausal women. We genotyped approximately 1 500 breast cancer cases, 690 endometrial cancer cases and 1 500 controls and calculated odds ratios and 95 percent confidence intervals from conditional logistic regression models. We found no overall association between CYP1B1 or COMT genotype and breast cancer risk. Stratified analyses indicated that the CYP1B1'3/'3 genotype may be of importance in conjunction with menopausal hormone use and that the low activity allele of COMT appeared associated with an increased risk for lobular breast cancer. CYP1B1 genotype had no effect on endometrial cancer risk, neither in overall nor in subgroup analyses. The CYP2S1 gene was identified through homology searches with known CYP sequences against the EST database and the high throughput genomic sequences database. The gene was found to be located together with the genes of subfamily CYP2A, CYP2B and CYP2F on chromosome 19. The predicted 504 amino acid sequence displayed 38-49 percent identity with other CYP2 family members and contained the typical structural CYP characteristics; the conserved cystein, the proline rich region and the N-terminal hydrophobic stretch. CYP2S1 mRNA was shown to be highly expressed in lung, trachea, and stomach. The transcript was detected in the liver but at lower levels. A rabbit peptide antiserum against the C-terminus of CYP2S1 recognized a single band in human lung with an apparent molecular weight of 50 kD. Subcellular localization and immunocytochemistry revealed CYP2S1 to be a microsomal protein. High expression in respiratory and gastrointestinal tract indicates a role for this enzyme in extrahepatic: xenobiotic metabolism. Unpublished observations from our lab show that small aromatic hydrocarbons appear to be substrates for CYP2S1. In summary, our work have contributed to the understanding of the presence of CYPs in extrahepatic tissues, particularly breast and lung, and evaluated a possible role for genetic polymorphisms in estrogen metabolism genes in relation to hormonal cancer susceptibility.