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. CYPs, GSTs, PGEs). 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. This project focuses on the functional characterization of variation in environmental response genes. These projects tend to be labor intensive and time consuming. We have recently developed methods which will improve productivity in this area (see future plans). Accomplishments: 1. 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 uM.
