N-glucuronidation of 4-aminobiphenyl and the risk of urinary bladder cancer: Gender differences

The current study is aimed at investigating the role of N-glucuronidation in the organotropic differences between males and females using the bladder carcinogen, 4-aminobiphenyl (4-ABP). In order to assess the levels of male and female N-glucuronidation of 4-ABP we have developed a new HPLC method and optimized the N-glucuronidation assay. The method's maximum accuracy and precision were -12 % and 9.8 %, respectively. It has comparable or better sensitivity to the old methods which use the radioactive [C14]-UDPGA. Optimal incubation time of the UGT reaction was in the range of 20--90 minutes and the optimal amount of microsomal proteins was 0.1--0.5 mg/reaction. Alamethicin was the best reagent to activate microsomes at a concentration of 50 mug alamethicin/mg protein.; In the in vivo study, we treated male and female mice with 4-ABP after modulating their 4-ABP N-glucuronidation using the plant steroid, hecogenin and then determined the distribution of 4-ABP adducts in liver and bladder. In animals treated with 4-ABP only, males had statistically significant higher levels of DNA adducts in the bladder (p-value 0.0004) while females had statistically significant higher levels in the liver (p-value < 0.0001). Hecogenin co-treatment increased the levels of DNA adducts in the liver in both males and females but this increase was statistically significant only in males (p-value 0.0024). There was a slight decrease in the levels of DNA adducts in the bladder of both males and females with hecogenin co-treatment, but this decrease was statistically insignificant. Using two-way ANOVA, we found that gender and hecogenin treatment both had a statistically significant effect on liver DNA adduct levels, whereas only gender had statistically significant effect on bladder adduct levels where males have about 2.2-fold higher DNA adducts than females. The current data suggests that N-glucuronidation of 4-ABP may actually have an impact on the distribution of DNA damage. The fact that there was only a slight decrease in bladder adduct levels compared to the significant increase in the liver indicates that besides N -glucuronidation, conjugation and metabolic activation by other enzymes may also contribute to the transport of the proximate metabolites of 4-ABP to the bladder.