Paolini M, Mesirca R, Pozzetti L, Silingardi P, Grilli S, Cantelli-Forti G
Dipartimento di Farmacologica, Università degli Studi di Bologna, Italy.
Carcinogenesis. 1992 Aug;13(8):1403-8. doi: 10.1093/carcin/13.8.1403.
The aim of this work was to optimize the ionic strength (tau) in the liver microsomal assay (LMA) in performing short-term genotoxicity tests. tau optimization would increase the sensitivity (i.e. decrease false negatives) and at the same time increase the specificity (decrease false positives). Such optimization depends upon the relative activities and stabilities of the liver polysubstrate cytochrome P450- and FAD-containing monooxygenase-dependent metabolizing enzymes present in the incubation mixtures. With regard to phase-I pathway, the expression of various P450-like activities (IA1, IA2, IIB1, IIE1, IIIA P450 classes) and thiobenzamide s-oxidase (as FAD-MFO marker), were examined in terms of their exact incubation conditions for the LMA during a period of preincubation (1 h) over the tau range 0.06-1.40. As a comparison with the phase-II pathway, the behaviour of glutathione S-transferases (total and pi class), glutathione S-epoxide transferase, epoxide hydrolase and UDP-glucuronosyl transferase were studied. Lipid peroxidation (LP) was also determined. Experiments were performed on S9 fractions derived from sodium phenobarbital, beta-naphthoflavone, isosafrol, ethanol and pregnenolone 16-alpha carbonitrile super-induced mouse liver. The maximal value of the mean specific activity (Asp), up to a 46% increase, was found at tau = 0.864 for oxidative reactions considered. On the contrary, a slight modulation of Asp for post-oxidative reactions was seen. LP was not changed appreciably by varying tau. In vitro DNA binding of the well-known premutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at tau = 0.864 (2.25-fold) compared to the usual tau (0.06) used. Additional confirmation of these results stems from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. Indeed, a significant enhancement of mitotic gene conversion (up to 1.8-fold), mitotic crossing-over (2.6-fold) and reverse point mutation (2.6-fold) frequencies was achieved at tau = 0.86 compared to tau = 0.06 (traditional). These data show that tau = 0.86 can provide more convenient conditions for in vitro bioactivation (as exemplified by an increased Asp phase-I/Asp phase-II ratio), as well as DNA binding and genotoxic response.
这项工作的目的是在进行短期遗传毒性测试时优化肝脏微粒体试验（LMA）中的离子强度（tau）。tau优化将提高灵敏度（即减少假阴性），同时提高特异性（减少假阳性）。这种优化取决于孵育混合物中存在的肝脏多底物细胞色素P450和含FAD的单加氧酶依赖性代谢酶的相对活性和稳定性。关于I相途径，在tau范围为0.06 - 1.40的预孵育期（1小时）内，根据LMA的确切孵育条件，检测了各种P450样活性（IA1、IA2、IIB1、IIE1、IIIA P450类别）和硫代苯甲酰胺s -氧化酶（作为FAD - MFO标志物）的表达。作为与II相途径的比较，研究了谷胱甘肽S -转移酶（总类和pi类）、谷胱甘肽S -环氧化物转移酶、环氧化物水解酶和UDP -葡糖醛酸基转移酶的行为。还测定了脂质过氧化（LP）。实验是在源自苯巴比妥钠、β -萘黄酮、异黄樟素、乙醇和孕烯醇酮16 -α腈超诱导小鼠肝脏的S9组分上进行的。对于所考虑的氧化反应，在tau = 0.864时发现平均比活性（Asp）的最大值，增加高达46%。相反，对于氧化后反应，Asp有轻微调节。改变tau对LP没有明显影响。由小鼠肝微粒体酶介导的著名前诱变剂[14C]二甲基亚硝胺（[14C]DMNA）的体外DNA结合显示，与通常使用的tau（0.06）相比，在tau = 0.864时比活性显著增加（2.25倍）。这些结果的进一步证实来自于使用DMNA对酿酒酵母二倍体D7菌株进行诱变实验作为生物测试系统。实际上，与tau = 0.06（传统值）相比，在tau = 0.86时有丝分裂基因转换（高达1.8倍）、有丝分裂交换（2.6倍）和反向点突变（2.6倍）频率显著提高。这些数据表明，tau = 0.86可以为体外生物活化（如通过增加的Asp I相/Asp II相比值举例说明）以及DNA结合和遗传毒性反应提供更便利的条件。
