cLINICAL PHARMACOLOGY & THERAPEUTICS vOLUME 73, NUMBER2 American Society for Clinical Pharmacology and Therapeutics P59

PDI-B-6 GENETIC VARIATION AND HAPLOTYPE STRUCTURE OF

HUMAN MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 2 (MRP2). T.D. Nguven, C. C. Huang, M. Kawamoto, E. J. Carlson, I. Herskowitz, PhD, K. M. Giacomini, PhD, D. L. Kroetz, PhD, University of California, San Francisco, San Francisco, CA.

MRP2 is highly expressed on epithelial cells of the bile canaliculus and mediates the biliary elimination of anionic compounds. We hypothesized that genetic variation in MRP2 might contribute to interindividual variability in the elimination of MRP2 substrates. An ethnically diverse pool of 247 human DNA samples was screened for genetic variation in MRP2 using denaturing HPLC and direct se- quencing. A total of 55 variant sites were identified in MRP2, including 23 intronic SNPs, 15 synonymous SNPs, and t7 non- synonymous SNPs that resulted in an amino acid change. Haplotypes were statistically inferred for 476 chromosomes using PHASE and the 49 valiant sites in this population were organized into 71 distinct haplotypes. There were two major haplotypes, the reference sequence and a haplotype that contained a synonymous C3972T variant. The frequencies of these two haplotypes were 26.5% and 30.5% in Cau- casians (n 100) and 12.5% and 10.0% in African-Americans (n 93). There were 27 haplotypes found in more than 3 chromosomes ~nd these accounted for - 8 5 % of our population. There was also significant population substructure in the MRP2 haplotypes (FsT = 0.0676). Significant linkage disequilibrium was found across the entire MRP2 gene mad recombination was detected largely at the 3'-end of MRP2. The cellular and clinical phenotypes associated with these MRP2 haplotypes are currently being investigated. Supported by NIGMS 61390.

PDI-B-7 FUNCTIONAL CHARACTERIZATION OF MRP2 ALLELIC

VARIANTS USING OATP-C/MRP2 DOUBLE-TRANSFECTED HELA CELLS. R.H. Ho, MD, B. F. Leake, BSc, R. B. Kim, MD, Vanderbilt University, Nashville, TN.

A number of single nucleotide polymorphisms (SNPs) have been identified in multidrug resistance protein 2 (MRP2), an ATP-binding cassette (ABC) transporter involved in the efflux transport of numer- ous endobiotics and xenobiotics. Functional characterization of MRP2 allelic variants has been hampered by difficulties associated with the creation of cell lines expressing high levels of MRP2. We utilized site-directed mutagenesis to create multiple previously pub- lished MRP2 allelic variants, including those whose functional con- sequences ale unknown, as well as those associated with loss of function. We then co-expressed the drug uptake transporter, organic anion transporting polypeptide-C (OATP-C), with variant MRP2 transporters to enhance the intracellular concentration of the shared substrate, estradiol-17~-glucuronide. As expected, the Dubin- Johnson syndrome (DJS) mutations, MI and M2, resulted in a sig- nificant loss of MRP2 activity. In addition, we are now able to show M4 is associated with loss of transport activity while M3, M5, and M6 behave similarly to wild-type MRP2.

MRP2 Variant Base pair change Amino acid change

M1 * A4145G Q1382R M2* A3517T I l 173F M3 C2366T $789F M4 G4348A A1450T M5 G1249A V417I M6 G842A $281N

*DJS mutation

PDI-B-8 FUNCTIONAL CHARACTERIZATION OF GENETIC VARI-

ANTS OF THE NUCLEOSIDE TRANSPORTER, CNT1. J . H . Gray, R. P. Owen, T. J. Urban, PharmD, K. M. Giacomini, PhD, University of California, San Francisco, San Francisco, CA.

Purpose: The concentrative nucleoside transporter, CNT1, plays a role in tissue-specific uptake of nucleosides and nucleoside analogs, including the anti-cancer drug, gemcitabine. The goal of this study was to test the hypothesis that genetic variation in CNTI contributes to interindividual differences in cellular uptake of gemcitabine and other nucleoside analogs. Methods: Denaturing HPLC and sequenc- ing were used to analyze all 17 exons of CNTI in 247 DNA samples from ethnically diverse populations. All variants were constructed by site-directed mutagenesis and characterized in Xenopus laevis oo- cytes. Results: Four common non-synonymous variants (V1-V4) (allele frequencies ->20%) and three rare non-synonymous variants (V5-V7) (allele frequencies <1%) were identified. All four common variants were functional; however, V1 exhibited reduced function and V2 exhibited a reduced potency of interaction with gemcitabine (ICso of gemcitabine in inhibiting 3H-thymidine uptake in oocytes expressing V2 was 48 ~M and was 17 p~M for the reference CNT1). Two of the three rare variants (V6 and V7) were non-functional. Conclusions: These studies demonstrate that coding region variants of CNT1 may exhibit different functional characteristics and suggest that genetic variation in CNT1 may contribute to variation in re- sponse to gemcitabine.

PDI-C-1 THE INFLUENCE OF SMOKING, ALCOHOLISM AND GE-

NETICS ON CYP2B6 IN HUMAN BRAIN. S. Miksgs P ~ h D C. Lerman, PhD, P. Shields, MD, D. Mash, PhD, R. Tyndale, PhD, University of Toronto, Abramson Cancer Center at the University of Pennsylvania, Lombardi Cancer Center, Georgetown University, University of Miami, Toronto, Canada.

The enzyme cytocbrome P450 2B6 metabolizes drugs such as nicotine and bupropion, and many toxins and carcinogens. In hu- mans, genetic variation in CYP2B6 was associated with altered smok- ing cessation rates. We have previously shown in a small study (N=9) that CYP2B6 enzyme levels are higher in some brain regions of smokers. The aim of this study was to compare CYP2B6 expres- sion patterns in brains of smokers and non-smokers and alcoholics and non-alcoholics using immunocytocbemistry and immunoblotting of 26 human brain samples, and to determine the effect of CYP2B6 genotype on expression levels. Levels were significantly higher in 4 brain regions (hippocampus, caudate nucleus, putalnen and cerebel- lum) in smokers and alcoholics, particularly in cerebellar Purkinje cells and hippocampal pyramidal neurons, cells known to be dam- aged in alcoholics. In cerebellum the higher enzyme levels were associated with smoking alone. This influence of smoking and alco- holism on enzyme expression was modulated by the genetic variant C1459T (R487C), which has been associated with reduced hepatic enzyme levels, stability and activity. Smokers and alcoholics with the CC genotype had higher CYP2B6 brain levels than those with a T allele. Higher brain CYP2B6, resulting from an interaction of envi- ronmental and genetic influences, may cause altered sensitivity to centrally acting drugs, increased susceptibility to neurotoxins and carcinogenic xenobiotics and contribute to central tolerance to nico- tine.