1 μM of each probe, using the following program: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min (ABI 7900; Applied Biosystems). Quality was assured by including controls for each genotype, as well as negative controls, in each run, and repeating the genotyping on 5% of the samples. There was a perfect agreement between the original and repeat genotype runs for all three SNPs analyzed. Deviations from Hardy–Weinberg equilibrium were tested using the Chi square (χ2) test. In order to fulfil the criteria for parametric analysis, B-Cd, U-Cd and the biomarkers for renal dysfunction: UNAG, UB2M, www.selleckchem.com/products/Etopophos.html and UALB
were natural log-transformed. Each polymorphism was modeled as a categorical variable (zero, one, or two variant alleles). When the frequency of a variant homozygote genotype was low, this group was pooled with the heterozygotes.
To explore the influence of MT polymorphisms on B-Cd and U-Cd at different BAY 73-4506 datasheet levels of exposure, subjects were analyzed according to level of Cd pollution (high, moderate, and low). Analysis of variance (ANOVA) was employed to analyze, within the different exposure groups, the effects of genotype of each polymorphism on ln(B-Cd) or ln(U-Cd), as the dependent variables. Adjustments were made for other potentially influential variables (age, sex, and smoking). To account for multiplicative effect modification, a multivariate model was used with an interaction term between exposure group and genotype for ln(B-Cd) and ln(U-Cd) (both variables entered as categorical variables). To analyze the influence of MT polymorphisms on the association
between Cd exposure and excretion of low molecular weight proteins, ANOVA was employed to analyze, within the different exposure groups, the effects of genotype of each polymorphism on ln(UNAG), ID-8 ln(UB2M) or ln(UALB) as the dependent variables. Adjustments were made for other potentially influential variables (age, sex, and smoking). We then analyzed multiplicative effect modification in a multivariate model with an interaction term between ln(B-Cd/U-Cd) and genotype with ln(UNAG), or ln(UB2M) as dependent variables. For those analyses that demonstrated a significant interaction between genotype and ln(B-Cd) or ln(U-Cd), the analysis was stratified for genotype to obtain effect measures for different genotypes. Further, the influence of MT SNPs on the risk of having affected kidney function (measured as having UB2M or UNAG concentrations above 95th percentile of UB2M or UNAG concentrations of individuals from the control area (< 80 years)) was analyzed for individuals living in the medium and highly polluted areas. The strength of the associations between genotypes and risk of affected kidney function was estimated as odds ratios with 95% confidence intervals (CIs) by unconditional logistic regression. All statistical analyses were performed using SPSS software (version 15; SPSS, Chicago, IL, USA). Statistical significance refers to p < 0.05.