人、兔和啮齿动物肝脏微粒体激活苯并[a]芘后体外形成的苯并[a]芘-DNA加合物的比较32P后标记分析。

Comparative 32P-postlabeling analysis of benzo[a]pyrene--DNA adducts formed in vitro upon activation of benzo[a]pyrene by human, rabbit and rodent liver microsomes.

作者信息

Roggeband R, Wolterbeek A P, Rutten A A, Baan R A

机构信息

TNO Medical Biological Laboratory, Department of Genetic Toxicology, Rijswijk The Netherlands.

出版信息

Carcinogenesis. 1993 Sep;14(9):1945-50. doi: 10.1093/carcin/14.9.1945.

DOI:10.1093/carcin/14.9.1945

PMID:8403223

Abstract

An interspecies comparison was made of the DNA-adducts formed in vitro upon incubation of rat liver DNA (RL-DNA) with benzo[a]pyrene (BP) in the presence of liver microsomes. Incubations were carried out with RL-DNA, BP (100 microM) and liver microsomes from hamsters, mice, rabbits, rats, 3-methylcholanthrene (3MC) pretreated rats and from humans. To analyse the adduct profiles, the 32P-postlabeling technique with the nuclease P1-enhancement procedure was used. The total amount of adduct formed varied greatly with the species; also the number of adduct spots detected was different, ranging from 1 to 5. In all incubations the BP-N2-deoxyguanosine adduct was formed. Relative to the total adduct level, the level of this adduct varied from 26% with rat, 54% with hamster, 56% with 3MC-pretreated rat, 58% with mouse and 75% with rabbit, to 100% with human liver microsomes. In human liver microsomes both the total amount of cytochrome P-450 per mg microsomal protein and the ethoxyresorufin O-deethylation (EROD) activity were low compared to that in animal liver microsomes. In microsomes from 3MC-pretreated rats the EROD activity was strongly induced. There was no correlation between EROD activity in non-induced microsomes and total adduct level. To compare BP-DNA adduct formation in human white blood cells (WBC) with that in RL-DNA, WBC were incubated with BP and 3MC-pretreated rat microsomes. The adduct profile in WBC-DNA differed from that observed after incubation of RL-DNA: the BP-N2-deoxyguanosine adduct in WBC-DNA accounted for 97% of the total adduct level. It is concluded that the 32P-postlabeling method is a suitable technique to investigate both qualitative and quantitative differences in BP-DNA adduct formation between species. Furthermore, the incubation of microsomes from the liver (or other sources) with a genotoxic agent and isolated DNA or cells can be a useful approach to study the formation and stability of reactive intermediates that are able to bind to DNA, also with respect to differences between species or tissue.

摘要

在肝微粒体存在的情况下，将大鼠肝脏DNA（RL-DNA）与苯并[a]芘（BP）进行体外孵育，对形成的DNA加合物进行了种间比较。使用来自仓鼠、小鼠、兔子、大鼠、经3-甲基胆蒽（3MC）预处理的大鼠以及人类的RL-DNA、BP（100微摩尔）和肝微粒体进行孵育。为了分析加合物图谱，采用了核酸酶P1增强程序的32P后标记技术。形成的加合物总量因物种而异；检测到的加合物斑点数量也不同，范围从1到5。在所有孵育中均形成了BP-N2-脱氧鸟苷加合物。相对于总加合物水平，该加合物的水平在大鼠中为26%，仓鼠中为54%，3MC预处理大鼠中为56%，小鼠中为58%，兔子中为75%，人类肝微粒体中为100%。与动物肝微粒体相比，人类肝微粒体中每毫克微粒体蛋白的细胞色素P-450总量和乙氧基试卤灵O-脱乙基酶（EROD）活性均较低。在3MC预处理大鼠的微粒体中，EROD活性被强烈诱导。未诱导微粒体中的EROD活性与总加合物水平之间没有相关性。为了比较人类白细胞（WBC）中BP-DNA加合物的形成与RL-DNA中的情况，将WBC与BP和3MC预处理的大鼠微粒体进行孵育。WBC-DNA中的加合物图谱与RL-DNA孵育后观察到的不同：WBC-DNA中的BP-N2-脱氧鸟苷加合物占总加合物水平的97%。结论是，32P后标记方法是研究物种间BP-DNA加合物形成的定性和定量差异的合适技术。此外，将来自肝脏（或其他来源）的微粒体与遗传毒性剂以及分离的DNA或细胞进行孵育，对于研究能够与DNA结合的反应性中间体的形成和稳定性，以及物种或组织之间的差异而言，可能是一种有用的方法。