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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES090604-07
Application #
6838618
Study Section
(LCBR)
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2003
Total Cost
Indirect Cost
Name
U.S. National Inst of Environ Hlth Scis
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Liu, Xuemei; Campbell, Michelle R; Pittman, Gary S et al. (2005) Expression-based discovery of variation in the human glutathione S-transferase M3 promoter and functional analysis in a glioma cell line using allele-specific chromatin immunoprecipitation. Cancer Res 65:99-104
Wang, Xuting; Tomso, Daniel J; Liu, Xuemei et al. (2005) Single nucleotide polymorphism in transcriptional regulatory regions and expression of environmentally responsive genes. Toxicol Appl Pharmacol 207:84-90
Lee, Su-Jun; Bell, Douglas A; Coulter, Sherry J et al. (2005) Recombinant CYP3A4*17 is defective in metabolizing the hypertensive drug nifedipine, and the CYP3A4*17 allele may occur on the same chromosome as CYP3A5*3, representing a new putative defective CYP3A haplotype. J Pharmacol Exp Ther 313:302-9
Grant, Delores J; Hall, Ingrid J; Eastmond, David A et al. (2004) Bilirubin UDP-glucuronosyltransferase 1A1 (UGT1A1) gene promoter polymorphisms and HPRT, glycophorin A, and micronuclei mutant frequencies in human blood. Mutat Res 560:1-10
Mammen, Jennifer S; Pittman, Gary S; Li, Ying et al. (2003) Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (-)benzo[a]pyrene-7R-trans-7,8-dihydrodiol. Carcinogenesis 24:1247-55
Fritsche, E; Baek, S J; King, L M et al. (2001) Functional characterization of cyclooxygenase-2 polymorphisms. J Pharmacol Exp Ther 299:468-76
Grant, D J; Bell, D A (2000) Bilirubin UDP-glucuronosyltransferase 1A1 gene polymorphisms: susceptibility to oxidative damage and cancer? Mol Carcinog 29:198-204