Background: Accurate risk assessment for carcinogens requires understanding how genetic variation modulates exposure. Following discovery of new polymorphic genes in environmental response pathways, it is necessary to characterize their impact on function (genotype/phenotype relationship).
Aims : 1) Characterize functional role of promoter and coding region polymorphisms of metabolism genes using standard biochemical, molecular and cellular bioassay techniques (e.g. GSTs, PGEs, COX).2) Develop model approaches for characterizing functional effects of polymorphisms in transcription factors, transcription factor binding sites, and other regulatory sequence elements using expression profiling.Accomplishments: 1. Cyclooxygenases (COX)-1 and -2 are the key enzymes in the conversion of arachidonic acid to prostaglandins. COX-2 appears to play an emerging role in inflammation and carcinogenesis. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of numerous diseases and reduce the risk of developing colorectal cancer. Polymorphisms in the COX-2 gene could alter enzyme expression, function, and/or the response to NSAIDs. Therefore, they could modify individual risks for developing cancer and other diseases or the occurrence of side effects or sensitivity toward selective or nonselective COX inhibitors. We sequenced the COX-2 gene of 72 individuals and identified rare polymorphisms in the promoter and the coding region. A COX-2 molecular model was used to locate the coding region polymorphisms relative to functional sites in the protein, and the COX-2 V511A polymorphism was very near to the active site. This variant protein was expressed, and function was evaluated, but no difference was detected in metabolism of the COX-2 substrates, arachidonic acid, linoleic acid, and 2- arachidonyl glycerol, compared with the wild type. The K(m) values for arachidonic acid showed no differences between the COX-2 wild type and V511A mutant. Inhibition with selective or nonselective COX inhibitors was essentially the same for the two enzymes. The absence of functionally important polymorphisms in the COX-2 gene may suggest that there has been selective pressure against those single nucleotide polymorphisms because of the critical role of this enzyme in maintenance of homeostasis.2. Cytochrome P450 1B1 (CYP1B1) is a central enzyme in the activation of the ubiquitous environmental carcinogen benzo[a]pyrene (B[a]P). We investigated six rare mutations associated with congenital glaucoma and four common polymorphisms for their effect of B[a]P metabolism. Five single amino acid substitutions associated with disease (Gly61Glu, Gly365Trp, Asp374Asn, Pro437Leu, and Arg469Tryp) dramatically decreased (between 3 and 12% of wild-type) the capacity of CYP1B1 to convert B[a]P-7,8-diol to B[a]P-9,10-epoxide. A 10 base-pair deletion resulting in a truncation mutation produced no detectible protein or activity. In contrast, proteins containing all possible combinations of four polymorphisms in CYP1B1 (Arg48Gly, Ala199Ser, Val432Leu, Asn453Ser) had modest effects on metabolism of B[a]P-7,8-diol. Michaelis-Menten analysis suggested that the two alleles containing Arg48, Ala119, either Val432 or Leu432, and Ser453 (RAVS and RALS) have a two-fold lower KM values compared to wild-type, 1.3+0.4 versus 2.8+0.8 ?M (pAims: 1) Characterize functional role of promoter and coding region polymorphisms of metabolism genes using standard biochemical, molecular and cellular bioassay techniques (e.g. GSTs, PGEs, COX).2) Develop model approaches for characterizing functional effects of polymorphisms in transcription factors, transcription factor binding sites, and other regulatory sequence elements using expression profiling.
