Paolini M, Barone E, Corsi C, Paganin C, Revoltella R P
Istituto di Mutagenesi e Differenziamento, C.N.R., Pisa, Italy.
Cancer Res. 1991 Jan 1;51(1):301-9.
Four novel nontransformed epithelial cell lines, isolated from fetal or adult mouse liver, were tested: (a) to determine the profile of xenobiotic metabolizing enzymes; (b) to evaluate the inducibility of the polysubstrate (cytochrome P-450-dependent) monooxygenase system by various classes of inducers; and (c) to assess the capacity of the cells to metabolize structurally different procarcinogens. With regard to the phase I pathway, the cells expressed various P-450 (class IA, IA2, IIB, IIE1, IIIA) and flavin adenine dinucleotide-containing monooxygenase-dependent bio-transformation enzyme activities at levels (in lines C2.8 and C6) comparable with those present in murine adult liver preparations. The expression of various P-450s was demonstrated also by immunoprecipitation assays using rabbit polyclonal antibodies. For the phase II pathway, cells expressed substantial levels of glutathione S-transferase, glutathione S-epoxide transferase, and UDP-glucuronosyltransferase. Low expression of epoxide hydrolase was observed. Induction of P-450 function by sodium phenobarbital, beta-naphthoflavone, isosafrole, ethanol, and pregnenolone 16 alpha-carbonitrile, monitored using specific P-450-linked activities, was considerably elevated (over 5-fold in class IIB with the C2.8 and C6 cell lines). The most competent C2.8 and C6 cell lines were able to activate benzo(a)pyrene, cyclophosphamide, dimethylnitrosamine, diethylstilbestrol, and 2-naphthylamine as shown by the significantly increased frequencies of mitotic gene conversion, mitotic crossing-over, and point [reverse] mutation in the diploid D7 strain of Saccharomyces cerevisiae after 4 [cyclophosphamide], 24 [benzo(a)pyrene,2-naphthylamine, dimethylnitrosamine] or 48 [diethylstilbestrol], h of exposure in the presence of 3 x 10(6) cells/flask. The degree of conservation and the inducibility of representative oxidative and postoxidative reactions in the novel epithelial cell lines C2.8 and C6, together with their ability to activate a wide spectrum of procarcinogens, offers a means to study the potential of chemicals for inducing DNA damage in short-term genotoxicity testing. In addition the cells may be suitable for analyzing the metabolic disposition of compounds and the multistage process of carcinogenesis.
(a) 确定外源性代谢酶的概况；(b) 评估各类诱导剂对多底物（细胞色素P-450依赖型）单加氧酶系统的诱导能力；(c) 评估这些细胞代谢结构不同的前致癌物的能力。关于I相途径，这些细胞表达了多种P-450（IA类、IA2、IIB、IIE1、IIIA）以及含黄素腺嘌呤二核苷酸的单加氧酶依赖性生物转化酶活性，其水平（在C2.8和C6细胞系中）与成年小鼠肝脏制剂中的相当。使用兔多克隆抗体的免疫沉淀试验也证实了多种P-450的表达。对于II相途径，细胞表达了大量的谷胱甘肽S-转移酶、谷胱甘肽S-环氧化物转移酶和UDP-葡萄糖醛酸基转移酶。观察到环氧水解酶的表达较低。使用特定的P-450相关活性监测苯巴比妥钠、β-萘黄酮、异黄樟素、乙醇和孕烯醇酮16α-腈对P-450功能的诱导作用，其显著升高（在C2.8和C6细胞系中，IIB类超过5倍）。最具活性的C2.8和C6细胞系能够激活苯并(a)芘、环磷酰胺、二甲基亚硝胺、己烯雌酚和2-萘胺，这表现为在3×10(6)个细胞/培养瓶存在的情况下，暴露4小时（环磷酰胺）、24小时（苯并(a)芘、2-萘胺、二甲基亚硝胺）或48小时（己烯雌酚）后，酿酒酵母二倍体D7菌株中有丝分裂基因转换、有丝分裂交换和点[反向]突变的频率显著增加。新的上皮细胞系C2.8和C6中代表性氧化反应和氧化后反应的保守程度及诱导能力，以及它们激活多种前致癌物的能力，为在短期遗传毒性测试中研究化学物质诱导DNA损伤的潜力提供了一种手段。此外，这些细胞可能适用于分析化合物的代谢处置和致癌作用的多阶段过程。
