Bevan D R, Sadler V M
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308.
Carcinogenesis. 1992 Mar;13(3):403-7. doi: 10.1093/carcin/13.3.403.
[3H]Benzo[a]pyrene (B[a]P) was administered to male Sprague-Dawley rats via intratracheal instillation, and bile was collected over a period of 6 h. Conjugated metabolites of B[a]P in bile were separated by paper chromatography or reversed-phase ion-pair HPLC and quantified by liquid scintillation spectrometry. In paper chromatographic analysis, a class of conjugates more polar than thioether conjugates was recognized. These conjugates were identified as quinol diglucuronides by hydrolyzing with beta-glucuronidase and analyzing products of the hydrolysis with HPLC, and by migration on paper relative to a standard of 3,6-quinol diglucuronide. From this analysis, relative amounts of conjugated metabolites of B[a]P in bile were 37.3% quinol diglucuronides, 19.9% thioether conjugates, 33.3% monoglucuronide and sulfate conjugates, and 9.4% unconjugated metabolites. Analysis by reversed-phase ion-pair HPLC provided improved resolution among the conjugates in bile. In particular, the 3,6-quinol diglucuronide was resolved from the 1,6- and 6,12-quinol diglucuronides, with identification of peaks being based on sensitivity to hydrolysis with beta-glucuronidase and elution of standards of these diglucuronides. The elution position of thioether conjugates was identified by their insensitivity to hydrolysis with beta-glucuronidase and arylsulfatase and by synthesis of thioether conjugates in V79 (XEM-2) cells, which express cytochrome P450IA1 and have relatively high levels of glutathione S-transferases but low levels of UDP-glucuronyltransferases and sulfotransferases. From the reversed-phase ion-pair HPLC analysis, relative amounts of conjugates in bile were 10.4% 1,6- and 6,12-quinol diglucuronides, 20.8% 3,6-quinol diglucuronide, 30.4% thioether conjugates, 17.8% monoglucuronides, 6.2% sulfate conjugates, and 14.4% unconjugated metabolites. These studies provide the first report of the biosynthesis of quinol diglucuronide conjugates of B[a]P in vivo and demonstrate that they are excreted into bile in significant quantities.
通过气管内滴注法给雄性斯普拉格 - 道利大鼠施用[3H]苯并[a]芘（B[a]P），并在6小时内收集胆汁。通过纸色谱法或反相离子对高效液相色谱法分离胆汁中B[a]P的结合代谢物，并通过液体闪烁光谱法进行定量。在纸色谱分析中，识别出一类比硫醚结合物极性更强的结合物。通过用β-葡萄糖醛酸酶水解并用高效液相色谱法分析水解产物，以及相对于3,6 - 喹啉二葡萄糖醛酸苷标准品在纸上的迁移情况，将这些结合物鉴定为喹啉二葡萄糖醛酸苷。通过该分析，胆汁中B[a]P结合代谢物的相对含量为：喹啉二葡萄糖醛酸苷37.3%、硫醚结合物19.9%、单葡萄糖醛酸苷和硫酸盐结合物33.3%以及未结合代谢物9.4%。反相离子对高效液相色谱分析提高了胆汁中结合物之间的分离度。特别是，3,6 - 喹啉二葡萄糖醛酸苷与1,6 - 和6,12 - 喹啉二葡萄糖醛酸苷得以分离，峰的鉴定基于对β-葡萄糖醛酸酶水解的敏感性以及这些二葡萄糖醛酸苷标准品的洗脱情况。硫醚结合物的洗脱位置通过其对β-葡萄糖醛酸酶和芳基硫酸酯酶水解不敏感以及在表达细胞色素P450IA1且谷胱甘肽S - 转移酶水平相对较高但尿苷二磷酸葡萄糖醛酸转移酶和磺基转移酶水平较低的V79（XEM - 2）细胞中硫醚结合物的合成来确定。根据反相离子对高效液相色谱分析，胆汁中结合物的相对含量为：1,6 - 和6,12 - 喹啉二葡萄糖醛酸苷10.4%、3,6 - 喹啉二葡萄糖醛酸苷20.8%、硫醚结合物30.4%、单葡萄糖醛酸苷17.8%、硫酸盐结合物6.2%以及未结合代谢物14.4%。这些研究首次报道了B[a]P的喹啉二葡萄糖醛酸苷结合物在体内的生物合成，并证明它们大量排泄到胆汁中。
