二氢二醇脱氢酶对加标大鼠肝脏亚细胞组分中（±）-反式-7,8-二羟基-7,8-二氢苯并[a]芘代谢的贡献。

Contribution of dihydrodiol dehydrogenase to the metabolism of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in fortified rat liver subcellular fractions.

作者信息

Shou M, Harvey R G, Penning T M

机构信息

Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.

出版信息

Carcinogenesis. 1992 Sep;13(9):1575-82. doi: 10.1093/carcin/13.9.1575.

DOI:10.1093/carcin/13.9.1575

PMID:1394842

Abstract

Dihydrodiol dehydrogenase (DD; EC 1.3.1.20) purified to homogeneity from rat liver cytosol will catalyze the NAD(P)(+)-dependent oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-diol) to yield benzo[a]pyrene-7,8-dione (BPQ). To verify that BPQ is a metabolite of B[a]P-diol in rat liver, an S100 fraction was supplemented with NAD+ and NADP+, and the formation of BPQ was followed by reverse-phase HPLC. The identity of BPQ was established by co-chromatography with an authentic standard (under different solvent conditions) and by RP-HPLC using a diode-array detector which established that the metabolite shared spectral identity with BPQ. The formation of BPQ in the S100 fraction was blocked by either a competitive inhibitor (indomethacin) or a suicide substrate [1-(4-nitrophenyl)-propen-1-ol] for DD, indicating that BPQ was being formed by this enzyme. To assess the contribution of DD to the metabolism of [3H]B[a]P-diol, subcellular fractions obtained from uninduced rat liver were fortified with co-factors to optimize the activity of enzymes that would compete for this proximate carcinogen. Under these conditions, S100 fractions fortified with NAD+ and NADP+ metabolized 25% of the B[a]P-diol, producing 731 +/- 154 pmol of BPQ. In contrast, rat liver microsomes fortified with an NADPH generating system metabolize 75% of the B[a]P-diol producing 2614 +/- 379 pmoles of benzo[a]pyrene-tetrahydrotetrols. Rat liver homogenates (S10) fortified with either uridine diphosphoglucuronic acid or phosphoadenosine phosphosulfate produced 180 +/- 56 and 95 +/- 31 pmoles of conjugates respectively, which were recovered as B[a]P-diol after treatment of the aqueous phase with either beta-glucuronidase or aryl sulfatase. Of the metabolites analyzed BPQ was formed in the second largest amount. These studies show that in uninduced rat liver DD may play a significant role in the metabolism of B[a]P-diol. The metabolic fate of BPQ remains to be determined.

摘要

从大鼠肝细胞溶胶中纯化至同质的二氢二醇脱氢酶（DD；EC 1.3.1.20）可催化（±）-反式-7,8-二羟基-7,8-二氢苯并[a]芘（B[a]P-二醇）的NAD(P)(+)依赖性氧化反应，生成苯并[a]芘-7,8-二酮（BPQ）。为了验证BPQ是大鼠肝脏中B[a]P-二醇的代谢产物，向S100组分中添加NAD⁺和NADP⁺，并通过反相高效液相色谱法跟踪BPQ的形成。通过与标准品共色谱（在不同溶剂条件下）以及使用二极管阵列检测器的反相高效液相色谱法确定BPQ的身份，该检测器确定代谢产物与BPQ具有相同的光谱特征。S100组分中BPQ的形成被DD的竞争性抑制剂（吲哚美辛）或自杀底物[1-(4-硝基苯基)-丙烯-1-醇]阻断，表明BPQ是由该酶形成的。为了评估DD对[³H]B[a]P-二醇代谢的贡献，用辅助因子强化从未诱导的大鼠肝脏获得的亚细胞组分，以优化竞争这种近端致癌物的酶的活性。在这些条件下，用NAD⁺和NADP⁺强化的S100组分代谢了25%的B[a]P-二醇，产生731±154皮摩尔的BPQ。相比之下，用NADPH生成系统强化的大鼠肝脏微粒体代谢了75%的B[a]P-二醇，产生2614±379皮摩尔的苯并[a]芘-四氢四醇。分别用尿苷二磷酸葡糖醛酸或磷酸腺苷磷酸硫酸强化的大鼠肝脏匀浆（S10）分别产生180±56和95±31皮摩尔的缀合物，在用β-葡糖醛酸酶或芳基硫酸酯酶处理水相后，这些缀合物以B[a]P-二醇的形式回收。在所分析的代谢产物中，BPQ的生成量位居第二。这些研究表明，在未诱导的大鼠肝脏中，DD可能在B[a]P-二醇的代谢中起重要作用。BPQ的代谢命运尚待确定。