DNA损伤剂与ADPRT介导的细胞NAD含量下降

本文观察了一系列已知致癌物及其非致癌性类同物对经β—萘黄酮诱导过的或未经诱导的人FL细胞中的作用,以验证有ADPRT介导的,即可被ADPRT抑制剂防止的细胞NAD含量下降作为判断终点来检测化学物质诱发的DNA损伤的可能性。实验表明在MFO诱发过的FL细胞中AFB_1(1～100μmol/L),B(a)P(10～1000μmol/L),芘(1～1000μmol/L),DMA(10～100μmol/L),2—AAF(100～1000μmol/L)及 EC(10～1000μmol/L)可引起剂量依存性ADPRT介导的细胞NAD含量下降,而蒽(0.1～1000μmol/L),4—AAF(0.1～1000μmol/L),IsoPC(1～100μmol/L),黄樟素0.1～100μmol/L)及 Cy(1～100μmol/L)则否。直接作用致癌物ProLac(1～100μmol/L)及其非致癌性类同物BuLac(1～1000μmol/L)在MFO未经诱导的FL细胞中皆不能诱发ADPRT介导的细胞NAD含量降低。与非程序性DNA合成试验结果相对比,上述结果表明这一以ADPRT介导的细胞NAD含量下降作为判断终点的新检测法是一个检测化学诱变剂/致癌物特异而价廉的方法,其特异性与灵敏度与UDS试验相仿。

DNA damaging agents and the ADPRT mediated decrease of cellular NAD content

It was reported previously that the DNA-damaging agents,N-methyl-N'-nitro-N-nitrosoguanidine,methyl methanesulphonate and 4-nitroquinoline-N-oxide could stimulate the ADP-ribosyl transferase(ADPRT)activity and lower the cellular nicotinamide adenine dinucleotide(NAD)content in a dose-dependent way.It was also demonstrated previously in this laboratory that the observed lowering of NAD,the only substrate of ADPRT could be partially or completely prevented by ADPRT inhibitors,3-aminobenzamide or nicotinamide,which showed no influence on the metabolic blocking agent-induced NAD lowering of cells.In this paper,the possibility of using ADPRT mediated(ADPRT inhibitor preventied)decrease of cellular NAD content as an endpoint for detecting the chemical-induced DNA damage was validated by observing the action of a variety of known carcinogens and their non-carcinogenic analogues in β-naphthoflavon,a mixed function oxygenase inducer,induced or uninduced human FL cells.It was found that in mixed-functional oxidase(MFO)-induced FL cells,24 h exposure of aflatoxin B1(AFB1)1-100,benzo(a)pyrene(B(a)P)10-1000,pyrene 1-1000,10-dimethylanthracene10-1000,2-acetylaminofluorene(2-AAF)100-1000 and ethylcarbamate(EC)10-1000μmol/L could induce the ADPRT mediated reduction of the cellular NAD content in a dose-dependent way,while anthracene 0.1-1000,4-acetylaminofluorene(4-AAF)0.1-1000,isopropyl-N-(3-chlorophenyl)-carbamate(IsoPC)1-100,safrol 0.1-100 and cyclophosphamide(Cy)1-100μmol/L could not.Both β-propiolactone(1-100μmol/L)and γ-butyrolactone(1-1000μmol/L)could not induce an ADPRT-mediated decrease of cellular NAD content in MFO uninduced FL cells.These results as compared with those obtained from unscheduled DNA synthesis(UDS)assay indicate that this new assay,taking the ADPRT-mediated decrease of the cellular NAD content as an endpoint,is a specific and inexpensive method for detecting DNA damage induced by chemical mutagens/carcinogens with a specificity and sensitivity at nearly the same grade as the validated UDS assay.