Hatcher J F, Swaminathan S
University of Wisconsin Comprehensive Cancer Center, Madison 53792, USA.
Carcinogenesis. 1995 Sep;16(9):2149-57. doi: 10.1093/carcin/16.9.2149.
32P-Postlabeling analysis of the bisphosphate derivatives was conducted to characterize the DNA adducts generated from the peroxidase-mediated activation of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP). Autoradiography of the D1 chromatogram of the postlabeled DNA hydrolysate revealed a major adduct (adduct 1) that migrated at Rf 0.15. An adduct with similar chromatographic characteristics was also obtained by postlabeling the products generated by chemical interaction of: (i) 2',6'-dichlorobenzoyloxy-4-acetylaminobiphenyl with the 3'-monophosphate of deoxyguanosine, and (ii) N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP) with calf thymus DNA. The adduct derived from chemical reaction exhibited the same mobilities on two-dimensional TLC as that obtained from the peroxidase-mediated DNA binding of N-OH-AABP. Moreover, on HPLC analyses, these bisphosphate derivatives exhibited identical retention times, suggesting that structurally they might be the same. Furthermore, adduct 1 was insensitive to digestion with nuclease P1. In addition to adduct 1, another minor adduct (adduct 2) was also detected in the peroxidase-mediated DNA binding of N-OH-AABP. The adduct 2 in D1 exhibited an Rf of 0.66. Adduct 2 was also observed in the DNA sample chemically interacted with N-OAc-AABP. Both these adducts retained the acetyl moiety, which was confirmed by the presence of radioactivity in the hydrolysate of DNA derived by interaction with N-OAc-[14C-acetyl]AABP (labeled at the N-acetyl group). Based on proton NMR and MS analyses of the 5'-phospho analogs of adducts 1 and 2, the structures of these have been identified as 3-(deoxyguanosine-N2-yl)-4-acetylaminobiphenyl (dG-N2-AABP) and N-(deoxyguanosine-8-yl)-4-acetylaminobiphenyl (dG-C8-AABP). Analyses of the DNA samples obtained from human uroepithelial cells following exposure to N-OH-AABP revealed primarily the non-acetylated derivative N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-ABP) with trace amounts of dG-N2-AABP. These results suggest that in the target cells for 4-aminobiphenyl carcinogenesis, the prevalence of the peroxidase mediated activation reaction of N-OH-AABP is relatively minor compared to the acetyltransferase pathway.
对双磷酸盐衍生物进行了³²P后标记分析，以表征由过氧化物酶介导的N-羟基-4-乙酰氨基联苯（N-OH-AABP）活化产生的DNA加合物。后标记DNA水解产物的D1色谱图的放射自显影显示出一种主要加合物（加合物1），其比移值（Rf）为0.15。通过对以下化学相互作用产生的产物进行后标记，也获得了具有相似色谱特征的加合物：（i）2',6'-二氯苯甲酰氧基-4-乙酰氨基联苯与脱氧鸟苷的3'-单磷酸，以及（ii）N-乙酰氧基-4-乙酰氨基联苯（N-OAc-AABP）与小牛胸腺DNA。化学反应产生的加合物在二维薄层层析上的迁移率与过氧化物酶介导的N-OH-AABP与DNA结合所获得的加合物相同。此外，在高效液相色谱分析中，这些双磷酸盐衍生物表现出相同的保留时间，表明它们在结构上可能相同。此外，加合物1对核酸酶P1的消化不敏感。除了加合物1，在过氧化物酶介导的N-OH-AABP与DNA结合中还检测到另一种次要加合物（加合物2）。D1中的加合物2的Rf为0.66。在与N-OAc-AABP发生化学相互作用的DNA样品中也观察到了加合物2。这两种加合物都保留了乙酰基部分，这通过与N-OAc-[¹⁴C-乙酰基]AABP（在N-乙酰基处标记）相互作用衍生的DNA水解产物中存在放射性得到证实。基于对加合物1和2的5'-磷酸类似物的质子核磁共振和质谱分析，已将它们的结构鉴定为3-（脱氧鸟苷-N²-基）-4-乙酰氨基联苯（dG-N²-AABP）和N-（脱氧鸟苷-8-基）-4-乙酰氨基联苯（dG-C⁸-AABP）。对人尿上皮细胞暴露于N-OH-AABP后获得的DNA样品进行分析，主要发现了非乙酰化衍生物N-（脱氧鸟苷-8-基）-4-氨基联苯（dG-C⁸-ABP），以及痕量的dG-N²-AABP。这些结果表明，在4-氨基联苯致癌作用的靶细胞中，与乙酰转移酶途径相比，N-OH-AABP的过氧化物酶介导的活化反应发生率相对较低。
