Paolini M, Sapigni E, Hrelia P, Scotti M, Morotti M, Cantelli-Forti G
Dipartimento di Farmacologia, Universita' degli Studi, Bologna, Italy.
Carcinogenesis. 1991 May;12(5):759-66. doi: 10.1093/carcin/12.5.759.
The use of Aroclor 1254 to induce S9 liver fractions is a standard method for conducting short-term genotoxicity assays. An alternative induction procedure, using beta-naphthoflavone (beta-NF), as a safe (non-carcinogenic) substitute for polychlorinated biphenyls, combined with sodium phenobarbital (PB), was found to be equally effective. The aim of this work is to realize a novel schedule of induction for the preparation of metabolizing systems containing a wider spectrum of induced cytochrome P450s. Five inducers of different 'classes' such as PB (class IIB P450s), beta-NF (IA), isosafrol (IA2), ethanol (IIE1) and pregnenolone 16 alpha-carbonitrile (IIIA) were injected daily both separately (to achieve maximal monooxygenase induction) in male and female mice. Induction was monitored using specific P450-linked activities. In the optimal schedule for complete induction, the various monooxygenases were greater (2- to 4-fold) than those achieved by the classical schedule. More than a 14-fold increase of total P450 and 3.3-fold increase of NADPH-cytochrome (P450) c-reductase activity, over those uninduced, account for the above increase. For example, there was a marked increase in the deethylation of ethoxyresorufin (37-fold) compared to the uninduced mice that was considerably higher than classical induction (8-fold over uninduced). On the contrary, phase II reactions i.e. epoxide hydrolase, glutathione S-transferase, glutathione S-epoxide transferase and UDP-glucuronosyl transferase, examined to compare the phase I/phase II ratios in the traditional and proposed procedures, were increased to a lesser extent (2-fold over uninduced). No significant sex differences were seen. Five precarcinogens specifically metabolized by each of the induced P450s elicited a higher mutagenicity response in the presence of superinduced fractions with respect to the classical one, when tested on Salmonella typhimurium (cyclophosphamide, benzo[alpha]pyrene, 2-naphthylamine and dimethylnitrosamine) or Saccharomyces cerevisiae D7 strain (diethylstilbestrol). These novel metabolizing biosystems, with an enhanced spectrum of induced P450s and oxidative/post-oxidative reaction rates, are recommended for detecting unknown xenobiotics in genotoxicity studies.
使用多氯联苯混合物1254诱导S9肝匀浆是进行短期遗传毒性试验的标准方法。人们发现，一种替代诱导程序，即使用β-萘黄酮（β-NF）作为多氯联苯的安全（非致癌）替代品，并与苯巴比妥（PB）联合使用，同样有效。这项工作的目的是实现一种新的诱导方案，以制备含有更广泛诱导细胞色素P450的代谢系统。分别给雄性和雌性小鼠每日注射五种不同“类别”的诱导剂，如PB（IIB类细胞色素P450）、β-NF（IA类）、异黄樟素（IA2类）、乙醇（IIE1类）和孕烯醇酮16α-腈（IIIA类）（以实现最大程度的单加氧酶诱导）。使用与细胞色素P450相关的特定活性来监测诱导情况。在完全诱导的最佳方案中，各种单加氧酶的活性比传统方案更高（2至4倍）。与未诱导的情况相比，总细胞色素P450增加了14倍以上，NADPH-细胞色素（P450）c还原酶活性增加了3.3倍，这就解释了上述增加情况。例如，与未诱导的小鼠相比，乙氧芴香豆素的脱乙基作用显著增加（37倍），这比传统诱导（比未诱导的高8倍）要高得多。相反，为了比较传统方法和建议方法中的I相/II相比例而检测的II相反应，即环氧化物水解酶、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，增加的程度较小（比未诱导的高2倍）。未观察到明显的性别差异。当在鼠伤寒沙门氏菌（环磷酰胺、苯并[a]芘、2-萘胺和二甲基亚硝胺）或酿酒酵母D7菌株（己烯雌酚）上进行测试时，每种诱导的细胞色素P450特异性代谢的五种前致癌物在超诱导组分存在下比传统组分引发更高的诱变反应。这些具有增强的诱导细胞色素P450谱和氧化/氧化后反应速率的新型代谢生物系统，推荐用于遗传毒性研究中检测未知的外源化学物。
