Stone E M, Williams J A, Grover P L, Gusterson B A, Phillips D H
Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, UK.
Carcinogenesis. 1998 May;19(5):873-9. doi: 10.1093/carcin/19.5.873.
The heterocyclic amines, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are pyrolysis products formed when meat is cooked and are rodent mammary carcinogens. They are thought to be metabolically activated by N-hydroxylation, catalysed by cytochrome P450 (CYP), followed by O-acetylation catalysed by N-acetyltransferases. Primary cultures of human mammary epithelial cells (HMECs) prepared from up to 26 individuals for each compound, were treated with IQ, MeIQ, or PhIP (500 microM) or with N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) or N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ) (20 microM) and the levels of adduct formation in their DNA analysed by 32P-post-labelling. In order to investigate whether pharmacogenetic polymorphisms influence DNA adduct formation, the NAT2 genotype of each individual was determined by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method that distinguishes between the wild-type and four variant alleles. Presence of two variant alleles designates a slow NAT2 acetylator, whereas individuals with one or two wild-type alleles are designated fast NAT2 acetylators. Interindividual variations in total DNA adduct levels ranged for IQ from 0.64-63.1 DNA adducts per 10(8) nucleotides (mean 7.80), for MeIQ from 1.99-17.8 (mean 6.63), for PhIP from 0.13-4.0 (mean 0.96), for N-OH-PhIP from 6.32-497 (mean 176) and for N-OH-IQ from 0.92-30.6 (mean 9.24). The higher adduct levels observed in cells treated with the N-OH metabolites suggests that N-hydroxylation is the rate-limiting step in HMECs and this may be due to low CYP levels. In contrast, the Phase II reaction catalysed by N-acetyltransferases is probably the major step in the metabolic activation of heterocyclic amines that occurs in the breast. Higher mean levels of heterocyclic amine-DNA adduct formation were detected in the cells of NAT2 fast acetylators compared with slow acetylators, with mean adduct levels per 10(8) nucleotides following IQ treatment, of 12.74 and 3.57 respectively, following PhIP treatment, of 1.20 and 0.74, respectively, following MeIQ treatment, of 7.90 and 5.08, respectively and following N-OH-PhIP-treatment, of 243.1 and 130.0, respectively. However, due to the large variations in adduct levels, these differences in mean values were not statistically significant with the limited number of individuals studied. This appears to be the first pilot study to demonstrate interindividual variations in the metabolic activation of heterocyclic amines and their metabolic intermediates in primary cultures of HMECs in vitro.
杂环胺，2-氨基-3-甲基咪唑[4,5-f]喹啉（IQ）、2-氨基-3,4-二甲基咪唑[4,5-f]喹啉（MeIQ）和2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶（PhIP）是肉类烹饪时形成的热解产物，也是啮齿动物乳腺致癌物。它们被认为通过细胞色素P450（CYP）催化的N-羟基化作用进行代谢活化，随后由N-乙酰转移酶催化O-乙酰化。针对每种化合物，从多达26名个体中制备人乳腺上皮细胞（HMEC）原代培养物，用IQ、MeIQ或PhIP（500微摩尔）或用N-羟基-2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶（N-OH-PhIP）或N-羟基-2-氨基-3-甲基咪唑[4,5-f]喹啉（N-OH-IQ）（20微摩尔）处理，并用32P后标记法分析其DNA中加合物形成水平。为了研究药物遗传多态性是否影响DNA加合物形成，通过聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法确定每个个体的NAT2基因型，该方法可区分野生型和四个变异等位基因。存在两个变异等位基因表明是NAT2慢乙酰化者，而具有一个或两个野生型等位基因的个体被指定为NAT2快乙酰化者。每10^8个核苷酸中总DNA加合物水平的个体间差异范围为：IQ为0.64 - 63.1个DNA加合物（平均7.80），MeIQ为1.99 - 17.8（平均6.63），PhIP为0.13 - 4.0（平均0.96），N-OH-PhIP为6.32 - 497（平均176），N-OH-IQ为0.92 - 30.6（平均9.24）。在用N-羟基代谢物处理的细胞中观察到的较高加合物水平表明，N-羟基化是HMEC中的限速步骤，这可能是由于CYP水平较低。相比之下，N-乙酰转移酶催化的II相反应可能是乳腺中发生的杂环胺代谢活化的主要步骤。与慢乙酰化者相比，在NAT2快乙酰化者的细胞中检测到更高的杂环胺-DNA加合物形成平均水平，IQ处理后每10^8个核苷酸的平均加合物水平分别为12.74和3.57，PhIP处理后分别为1.20和0.74，MeIQ处理后分别为7.90和5.08，N-OH-PhIP处理后分别为243.1和130.0。然而，由于加合物水平差异很大，在所研究个体数量有限的情况下，这些平均值差异无统计学意义。这似乎是第一项在体外HMEC原代培养物中证明杂环胺及其代谢中间体代谢活化存在个体间差异的初步研究。
