Flowers-Geary L, Harvey R G, Penning T M
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Carcinogenesis. 1995 Nov;16(11):2707-15. doi: 10.1093/carcin/16.11.2707.
Dihydrodiol dehydrogenase (DD) has been shown to catalyze the oxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo [a]pyrene (BP-diol) to yield benzo[a]pyrene-7,8-dione (BPQ) in uninduced fortified rat liver S100 fractions but the formation of BPQ has not been observed in whole cells. In these studies [3H]BP-diol was incubated with isolated hepatocytes from uninduced rats for 0-20 min at 37 degrees C. Organic-extractable radioactivity in the cell media accounted for 20% of the total [3H]BP-diol added. Reverse phase (RP)-HPLC analysis of this fraction revealed the formation of an unknown metabolite that co-chromatographed with an authentic synthetic standard of BPQ. The identity of the unknown metabolite was further established by: (i) co-chromatography with synthetic BPQ under both RP- and normal phase-HPLC conditions using diode array detection, which indicated that metabolite shared UV/vis spectral identity with standard BPQ; and by (ii) electron impact mass spectrometry of the unknown metabolite which gave the same parent and fragment ions as the synthetic standard. The formation of BPQ by isolated hepatocytes was found to be 0.50 nmol/3 x 10(6) cells/10 min, and represented 7% of the total organic-soluble metabolites in the extracellular media. Its formation was abolished by the addition of indomethacin, a competitive inhibitor of DD, indicating that this enzyme was responsible for BPQ formation. Other organic-soluble metabolites formed corresponded to BP-tetraols (hydrolysis products of the anti- and syn-diol epoxides). Examination of the aqueous phase of the extracellular media indicated that a large portion of BP-diol was converted to glucuronide and sulfate conjugates. Under the conditions employed BP-tetraols and BPQ were formed to an equal extent implying that in hepatocytes isolated from uninduced rats, DD and CYP1A1 contributed equally to the metabolism of BP-diol.
二氢二醇脱氢酶（DD）已被证明可催化（±）-反式-7,8-二羟基-7,8-二氢苯并[a]芘（BP-二醇）氧化，在未诱导的强化大鼠肝脏S100组分中生成苯并[a]芘-7,8-二酮（BPQ），但在全细胞中未观察到BPQ的形成。在这些研究中，[3H]BP-二醇与未诱导大鼠分离的肝细胞在37℃下孵育0 - 20分钟。细胞培养基中有机可提取放射性占添加的总[3H]BP-二醇的20%。对该部分进行反相（RP）-HPLC分析，发现形成了一种未知代谢物，其与BPQ的真实合成标准品共色谱。通过以下方法进一步确定了未知代谢物的身份：（i）在使用二极管阵列检测的RP-和正相-HPLC条件下，与合成BPQ共色谱，这表明代谢物与标准BPQ具有相同的紫外/可见光谱特征；以及（ii）对未知代谢物进行电子轰击质谱分析，其产生的母离子和碎片离子与合成标准品相同。发现分离的肝细胞形成BPQ的量为0.50 nmol/3×10(6)个细胞/10分钟，占细胞外培养基中总有机可溶性代谢物的7%。加入吲哚美辛（DD的竞争性抑制剂）后，其形成被消除，表明该酶负责BPQ的形成。形成的其他有机可溶性代谢物对应于BP-四醇（反式和顺式二醇环氧化物的水解产物）。对细胞外培养基水相的检查表明，大部分BP-二醇转化为葡萄糖醛酸和硫酸盐结合物。在所采用的条件下，BP-四醇和BPQ的形成程度相同，这意味着在从未诱导大鼠分离的肝细胞中，DD和CYP1A1对BP-二醇的代谢贡献相等。
