Glutathione S-transferase (GST), a member of the phase II group of xenobiotic metabolizing enzymes, has been intensively studied at the levels of phenotype and genotype. The GST 1 (GSTM1) and GST 1 (GSTT1) genes have a null-allele variant in which the entire gene is absent. The null genotype for both enzymes has been associated with many different types of tumors. The aim of this study was to determine the possible differences in increased oxidative stress susceptibility to smoking within the GSTM1 and GSTT1 genotypes and the impact of high tea drinking on this. We designed a Phase II randomized, controlled, three-arm tea intervention trial to study the effect of high consumption (4 cups/day) of decaffeinated green or black tea, or water on oxidative DNA damage, as measured by urinary 8-hydroxydeoxyguanosine (8-OHdG), among heavy smokers over a 4-month period and to evaluate the roles of GSTM1 and GSTT1 genotypes as effect modifiers. A total of 133 heavy smokers (100 females and 33 males) completed the intervention. GSTM1 and GSTT1 genotype statuses were determined with a PCR-based approach. Multiple linear regression models were used to estimate the main effects and interaction effect of green and black tea consumption on creatinine-adjusted urinary 8-OHdG, with or without adjustment for potential confounders. Finally, we studied whether the effect of treatment varied by GSTM1 and GSTT1 status of the individual. Although there were no differences in urinary 8-OHdG between the groups at baseline, the between-group 8-OHdG levels at month 4 were statistically significant for GSTM1-positive smokers (P ؍ 0.05) and GSTT1-positive smokers (P ؍ 0.02). GSTM1-positive and GSTT1-positive smokers consuming green tea showed a decrease in urinary 8-OHdG levels after 4 months. Assessment of urinary 8-OHdG after adjustment for baseline measurements and other potential confounders revealed significant effect for green tea consumption (P ؍ 0.001). The change from baseline was significant in both GSTM1-positive (t ؍ ؊2.99; P ؍ 0.006) and GSTT1-positive (P ؍ 0.004) green tea groups, but not in the GSTM1-negative (P ؍ 0.07) or GSTT1-negative (P ؍ 0.909) green tea groups. Decaffeinated black tea consumption had no effect on urinary 8-OHdG levels among heavy smokers. Our data show that consumption of 4 cups of tea/day is a feasible and safe approach and is associated with a significant decrease in urinary 8-OHdG among green tea consumers after 4 months of consumption. This finding also suggests that green tea intervention may be effective in the subgroup of smokers who are GSTM1 and/or GSTT1 positive.
used in combination with any other M13-tailed forward primer.We then tested whether the method could be extended to analysis on an automatic capillary sequencer. Three M13 primers end-labeled with WellRED fluorescent dyes D2, D3, and D4 (Beckman Coulter, Fullerton, CA, USA) were ordered from Research Genetics (Huntsville, AL, USA). These primers were used for the amplification of labeled alleles using the same protocol described previously for 32 P-labeled primers. After amplification, the PCR products were pooled (i.e., up to nine different products, three size ranges, and three dyes). For desalting, wells of a MultiScreen -FB filter plate (Millipore, Bedford, MA, USA) were pre-wetted with binding buffer [7 M guanidine-HCl and 200 mM 2-(N-morpholino) ethanesulfonic acid, pH 5.6], the pooled PCR products were then mixed in a 1:1 ratio with binding buffer and loaded onto individual wells by centrifugation at 1000× g for 5 min. The wells were then washed twice with 80% ethanol, and the products were eluted with 50 µL TE buffer (0.1 mM EDTA and 10 mM Tris-HCl, pH 8.0). Fifteen microliters of eluate were mixed with 25 µL deionized formamide and 0.5 µL size standards (Beckman Coulter) labeled with WellRED fluorescent dye D, then loaded and analyzed on a CEQ 2000XL capillary DNA analysis system (Beckman Coulter). Figure 2 shows an analysis of a run containing six multiplexed PCR products. Genomic DNA from strain A amplified by three markers along with M13 primer end-labeled with WellRED fluorescent dye D2 is compared with genomic DNA from strain B amplified with the same markers along with M13 primer end-labeled with fluorescent WellRED dye D3. As observed with 32 P-labeled markers, PCR products migrate as clean-laddered fluorescence peaks without interfering artifactual bands and, thus, allow clear identification of the origin of the alleles. We have obtained similar results when multiplexing was initiated at the level of PCR, by amplifying genomic DNA with several primer combinations simultaneously. It is reasonable to anticipate that this method could be used with dyes designed for other sequencing platforms. For instance, M13 primers labeled with
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