| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Eight forms of cytochrome P450 1B1 (CYP1B1) allelic variants, namely Arg48Ala119Leu432Asn453, Arg48Ala119Val432Asn453, Gly48Ala119Leu432Asn453, Gly48Ala119Val432Asn453, Arg48Ser119Leu432Asn453, Arg48Ser119Val432Asn453, Gly48Ser119Leu432Asn453, and Gly48Ser119Val432Asn453, were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 7-ethoxyresorufin, 7-ethoxycoumarin, bufuralol, benzo[a]pyrene, and 17b-estradiol were determined. All the CYP1B1 variants expressed in bacterial membranes showed Fe2+・CO vs Fe2+ difference spectra with wavelength maxima at 446 nm and reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. Kinetic analysis showed that the Vmax/Km ratio of these eight CYP1B1 variants was the highest for 7-ethoxyresorufin O-deethylation (9-16 min-1μM-1 CYP1B1), followed by 7-ethoxycoumarin O-deethylation (0.2-0.7), 17b-estradiol 4-hydroxylation (0.3-0.6), 17b-estradiol 2-hydroxylation
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(0.1-0.5), benzo[a]pyrene metabolism (to the 7, 8-dihydoxy-7, 8-dihydrodiol ; 0.1-0.4, in the presence of epoxide hydrolase), and (±) bufuralol 1'- and 6-hydroxylations (0.001-0.01). Interestingly, the Vmax/Kmratio for 7-ethoxyresorufin O-deethylation by the eight CYP1B1 variants was rather constant, yet both Vmax and Km values were different among the eight forms. In contrast, Vmax values for 7-ethoxycoumarin O-deethylation were not very different in these CYP1B1 variants but Km values were, leading to large variations in Vmax/Km ratio. These results suggest that alterations in CYP1B1 structures have different effects on catalytic capacities depending on the substrates examined. The Vmax/Km ratio for 17b-estradiol 4-hydroxylation was found to be the highest with the Gly48Ser119Val432Asn453 variant. The ratio of Vmax/Km of 4-hydroxylation vs 2-hydroxylation of 17b-estradiol was higher in the Val432 compared to the Leu432 CYP1B1 variants. In contrast, Leu432-forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene to the 7, 8-dihydoxy-7, 8-dihydrodiol metabolite than did the Val432-froms. These results collectively indicate that polymorphic human CYP1B1 variants show altered catalytic specificities towards the substrates used and suggest that these polymorphisms may influence susceptibilities of individuals towards endogenous and exogenous carcinogens such as 17b-estradiol and benzo[a]pyrene. Polymorphisms of the genes whose products known to contribute to lung cancer development in cigarette smokers were analysed in lung cancer and control sample groups. These sample groups included thirty-six lung cancer patients who underwent thoracic surgery at the Princess Alexandra hospital in Brisbane, Queensland in 1996-1998 and thirty-five non-cancer cases drawn from the sample of Queensland residents who had died of accidental causes in 1997-1998. The two groups were not different in terms of frequencies of allelic variants of the AhR, GSTT1, GSTM1 and CYP1A1 genes. However, a statistically significant higher frequency of CYP1B1 m1 mutant allele was found in the lung cancer sample group (17%) compared to the control sample group (3%). This may suggest increased lung cancer risk in people who have homozygous m1 mutant allele of the CYP1B1 gene. Less
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