Hu Z, Wells P G
Department of Pharmacology, University of Toronto, Canada.
Toxicol Appl Pharmacol. 1994 Aug;127(2):306-13. doi: 10.1006/taap.1994.1166.
UDP-glucuronosyltransferases (UGTs) play a major role in the elimination of nucleophilic metabolites of xenobiotics, such the phenols and quinols of polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BP). In this way, UGTs may prevent the further oxidation of such metabolites to toxic reactive intermediates, which may react with lipids, DNA, RNA, and protein, thereby initiating such toxicities as cellular necrosis, birth defects, and cancer. We have shown previously in vivo and in hepatic microsomes that rats with a hereditary deficiency in bilirubin UGT (Gunn and RHA strains) have decreased glucuronidation of BP metabolites and enhanced BP covalent binding to hepatic DNA and microsomal protein, and enhanced BP embryotoxicity. We further hypothesized that a similar deficiency in BP glucuronidation, with increased BP bioactivation and covalent binding, might be observed in peripheral blood lymphocytes from UGT-deficient rats, which, if true, would suggest the utility of an analogous human lymphocyte model for evaluating the toxicological relevance of human UGT deficiencies. Such an in vitro approach is essential for human studies of environmental chemicals and drugs with a high toxicologic potential. In the current study, [7,10(-14)C]BP was preincubated with NADPH and hepatic microsomes from Wistar rats induced with beta-naphthoflavone. The supernatant from this preincubation, which contained BP reactive intermediates and hydroxylated BP metabolites, was further incubated with uridine diphosphate glucuronic acid and peripheral lymphocytes from heterozygous (j/+) and homozygous (j/j) RHA rats, which are genetically deficient in bilirubin UGT, and from congenic, homozygous (+/+) UGT-normal controls. In vivo, bilirubin glucuronidation was reduced in UGT deficiency, with progressively higher plasma concentrations of unconjugated bilirubin in j/+ and j/j UGT-deficient rats compared to +/+ UGT-normal controls (p < 0.05). In in vitro studies, glucuronide conjugates of BP were measured by high-performance liquid chromatography with a radioisotope detector, and the covalent binding of BP to microsomal protein was measured by liquid scintillation spectrometry. BP glucuronidation and covalent binding, respectively, were decreased and increased to a progressively greater degree in both hepatic microsomes and lymphocytes from j/+ and j/j UGT-deficient rats compared to +/+ UGT-normal controls (p < 0.05). Reduced BP glucuronidation in lymphocytes from UGT-deficient RHA rats correlated with elevated BP covalent binding (R2 = 0.85, p < 0.005). Both BP glucuronidation and BP covalent binding in lymphocytes significantly correlated with those reactions using hepatic microsomes from the same animals. These results in UGT-deficient lymphocytes also accurately reflected the in vivo biotransformation and covalent binding of BP in UGT-deficient rats.(ABSTRACT TRUNCATED AT 400 WORDS)
尿苷二磷酸葡萄糖醛酸基转移酶（UGTs）在清除外源性物质的亲核代谢产物中起主要作用，这些外源性物质如多环芳烃的酚类和醌类，包括苯并[a]芘（BP）。通过这种方式，UGTs可防止此类代谢产物进一步氧化为有毒的反应性中间体，这些中间体可能与脂质、DNA、RNA和蛋白质发生反应，从而引发细胞坏死、出生缺陷和癌症等毒性反应。我们之前在体内和肝微粒体中已表明，胆红素UGT存在遗传性缺陷的大鼠（Gunn和RHA品系），其BP代谢产物的葡萄糖醛酸化作用减弱，BP与肝DNA和微粒体蛋白的共价结合增强，且BP胚胎毒性增强。我们进一步推测，在UGT缺陷大鼠的外周血淋巴细胞中可能观察到类似的BP葡萄糖醛酸化缺陷，同时BP生物活化和共价结合增加，如果情况属实，这将表明类似的人类淋巴细胞模型可用于评估人类UGT缺陷的毒理学相关性。这种体外方法对于对具有高毒理学潜力的环境化学物质和药物进行人体研究至关重要。在当前研究中，[7,10(-14)C]BP与NADPH以及用β-萘黄酮诱导的Wistar大鼠的肝微粒体进行预孵育。该预孵育的上清液含有BP反应性中间体和羟基化BP代谢产物，其进一步与尿苷二磷酸葡萄糖醛酸以及来自杂合子（j/+）和纯合子（j/j）RHA大鼠的外周淋巴细胞一起孵育，这些大鼠在胆红素UGT方面存在基因缺陷，同时还与同基因、纯合子（+/+）UGT正常对照一起孵育。在体内，UGT缺陷时胆红素葡萄糖醛酸化作用降低，与+/+ UGT正常对照相比，j/+和j/j UGT缺陷大鼠中未结合胆红素的血浆浓度逐渐升高（p < 0.05）。在体外研究中，通过带有放射性同位素检测器的高效液相色谱法测量BP的葡萄糖醛酸结合物，通过液体闪烁光谱法测量BP与微粒体蛋白的共价结合。与+/+ UGT正常对照相比，j/+和j/j UGT缺陷大鼠的肝微粒体和淋巴细胞中，BP葡萄糖醛酸化作用分别降低且共价结合作用逐渐增强（p < 0.05）。UGT缺陷的RHA大鼠淋巴细胞中BP葡萄糖醛酸化作用降低与BP共价结合增加相关（R2 = 0.85，p < 0.005）。淋巴细胞中的BP葡萄糖醛酸化作用和BP共价结合作用均与使用同一只动物的肝微粒体进行的反应显著相关。UGT缺陷淋巴细胞中的这些结果也准确反映了UGT缺陷大鼠体内BP的生物转化和共价结合情况。（摘要截选至400字）
