Degawa M, Stern S J, Martin M V, Guengerich F P, Fu P P, Ilett K F, Kaderlik R K, Kadlubar F F
Office of Research (HFT-100), National Center for Toxicological Research, Jefferson, Arkansas 72079.
Cancer Res. 1994 Sep 15;54(18):4915-9.
Putative carcinogen-DNA adducts in human larynx tissues (n = 25) from smoker and non/ex-smoker patients were examined by 32P-postlabeling and compared with the metabolic activation capacity of larynx microsomes and cytosols from the same tissues. Hydrophobic DNA adducts were evident only in smokers, and chromatographic profiles of the adducts were similar using either the butanol extraction or nuclease P1 enhancement method, which suggested that the adducts may be derived from polycyclic aromatic hydrocarbons but not aromatic amines. Immunoblots of larynx microsomes using anti-cytochrome P450 1A1/1A2, 2C, 3A4, 2E1, and 2A6 antibodies showed intensities ranging from 1-10% of that typically observed with human liver microsomes. Enzymatic assays of larynx microsomes showed appreciable activity for benzo(a)pyrene hydroxylation (P450 1A1 and 2C) but not for 4-aminobiphenyl N-oxidation (P450 1A2), which indicated that the observed immunoreactivity was for P450 1A1; this represents the highest level of this P450 yet detected in human extrahepatic tissues. Accordingly, total DNA adduct levels in the larynx correlated strongly with levels of P450 2C, 1A1, and 3A4 but not with P450 2E1 or 2A6. Larynx cytosols also showed appreciable aromatic amine N-acetyl-transferase activity for p-aminobenzoic acid (NAT-1) but not for sulfamethazine (NAT-2); however, NAT-1 activity was not correlated with total DNA adducts, which is again consistent with the lack of aromatic amine-DNA adducts detected by 32P-postlabeling. Thus, these results suggest that the DNA adducts detected in human larynx are largely derived from metabolic activation of polycyclic aromatic hydrocarbons in cigarette smoke by P450 2C, 3A4, and/or 1A1.
采用³²P后标记法检测了25例吸烟者及非吸烟者/已戒烟者的人喉组织中的推定致癌物 - DNA加合物，并将其与来自相同组织的喉微粒体和胞质溶胶的代谢活化能力进行了比较。疏水性DNA加合物仅在吸烟者中明显，并且使用丁醇萃取法或核酸酶P1增强法得到的加合物色谱图相似，这表明这些加合物可能来源于多环芳烃而非芳香胺。使用抗细胞色素P450 1A1/1A2、2C、3A4、2E1和2A6抗体对喉微粒体进行免疫印迹分析，结果显示其强度为人肝微粒体中典型观察值的1 - 10%。对喉微粒体的酶活性测定表明，其对苯并(a)芘羟基化（P450 1A1和2C）有明显活性，但对4 - 氨基联苯N - 氧化（P450 1A2）无活性，这表明观察到的免疫反应性是针对P450 1A1的；这是在人肝外组织中检测到的该P450的最高水平。因此，喉组织中的总DNA加合物水平与P450 2C、1A1和3A4的水平密切相关，但与P450 2E1或2A6无关。喉胞质溶胶对对氨基苯甲酸（NAT - 1）也显示出明显的芳香胺N - 乙酰转移酶活性，但对磺胺二甲嘧啶（NAT - 2）无活性；然而，NAT - 1活性与总DNA加合物无关，这再次与³²P后标记法未检测到芳香胺 - DNA加合物一致。因此，这些结果表明，在人喉中检测到的DNA加合物主要来源于香烟烟雾中的多环芳烃经P450 2C、3A4和/或1A1的代谢活化。
