DEOXYGUANOSIN 8 YL N ACETYLBENZIDINE FORMATION BY PEROXIDATIVE METABOLISM

N '-（3'-单磷酸脱氧鸟苷-8-基）-N-乙酰联苯胺（dGp-ABZ） 被认为在引发 联苯胺诱发的人类膀胱癌。 本报告评估了 这种加合物的可能形成的过氧化物活化的 N-乙酰联苯胺（ABZ）。 加合物形成通过以下测量： 32 P-标记后。 公羊精囊微粒体被用作 前列腺素H合成酶（PHS）的来源。 过氧化物酶活性 PHS进行了比较，辣根过氧化物酶。 两种过氧化物酶 将ABZ转化为dGp-ABZ，无论是DNA还是2 '-脱氧鸟苷 存在3 '-单磷酸（dGp）。 在32 P标记后， 从PEI-纤维素中提取酶和合成加合物 板，并显示具有相同的HPLC洗脱曲线， 二磷酸盐加合物（32 P-dpGp-ABZ）。 处理酶和 合成二磷酸与核酸酶P1加合物产生产物， 同时从HPLC（32 P-dpG-ABZ）洗脱。 额外 实验证明，PHS衍生的5’-单磷酸酯 （dpG-ABZ）和3 '-单磷酸（dGp-ABZ）加合物也相同 对应的合成标准品。 与可比数量的 在总ABZ代谢中，PHS产生的dGp-ABZ比 辣根过氧化物酶（1943 ± 339 vs 49 ± 7.8 fmol/mg dGp）。 加成物 形成依赖于过氧化物酶的存在， 特定的底物，即，花生四烯酸或H2 O2。 加合物形成 吲哚美辛（0.1 mM）、抗坏血酸（1 mM） 和谷胱甘肽（10 mM），但不是由DMPO（100 mM），自由基 清道夫 辣根过氧化物酶加合物的形成也受到抑制 抗坏血酸和谷胱甘肽。 此外，DMPO引起更大的 96%的抑制率。 结果表明， ABZ以形成dGp-ABZ。 本文还探讨了PHS和DGP-ABZ的形成机制。 辣根过氧化物酶可能不同。

N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is thought to play an important role in initiation of benzidine-induced bladder cancer in humans. This report assesses the possible formation of this adduct by peroxidatic activation of N-acetylbenzidine (ABZ). Adduct formation was measured by 32P-postlabeling. Ram seminal vesicle microsomes were used as a source of prostaglandin H synthase (PHS). The peroxidatic activity of PHS was compared to that for horseradish peroxidase. Both peroxidases converted ABZ to dGp-ABZ whether DNA or 2'-deoxyguanosine 3'-monophosphate (dGp) was present. Following 32P-postlabeling, the enzymatic and synthetic adduct were extracted from PEI-cellulose plates and shown to have the same HPLC elution profiles for the bisphosphate adduct (32P-dpGp-ABZ). Treatment of the enzymatic and synthetic bisphosphate adduct with nuclease P1 yielded a product which eluted at the same time from the HPLC (32P-dpG-ABZ). Additional experiment s demonstrated that the PHS-derived 5'-monophosphate (dpG-ABZ ) and 3'-monophosphate (dGp-ABZ) adducts were also identical to their corresponding synthetic standard. With comparable amounts of total ABZ metabolism, PHS produced about 40-fold more dGp-ABZ than horseradish peroxidase (1943 + 339 vs 49 + 7.8 fmol/mg dGp). Adduct formation was dependent upon the presence of peroxidase and the specific substrate, i.e., arachidonic acid or H2O2. Adduct formation by PHS was inhibited by indomethacin (0.1 mM), ascorbic acid (1 mM) and glutathione (10 mM), but not by DMPO (100 mM), a radical scavenger. Horseradish peroxidase adduct formation was also inhibited by ascorbic acid and glutathione. In addition, DMPO elicited greater than a 96% inhibition. Results demonstrate peroxidatic metabolism of ABZ to form dGp-ABZ. The mechanism of dGp-ABZ formation by PHS and horseradish peroxidase may be different.