Lindblad W J, Jackim E
Mutat Res. 1982 Sep;96(1):109-18. doi: 10.1016/0027-5107(82)90021-5.
A significant difference in mutation frequency has been observed in CHO cells exposed to benzo[a]pyrene with alternative activation systems. Each system employed rat-liver S9 homogenate with one using isocitrate dehydrogenase to provide reduced NADP, while the other method uses glucose-6-phosphate dehydrogenase. Total aryl hydrocarbon hydroxylase (AHH) activity was greater for the isocitrate dependent system, however, this yielded a lower level of HGPRT mutants. It was ascertained that this reduced mutation frequency may result from sequestering of B[a]P substrate by crystals in the medium, possibly calcium phosphate, which decreased the effective substrate concentration. This sequestration enhances B[a]P internalization, which would explain the dichotomy between the AHH values and the mutation frequency data. The production of specific B[a]P metabolites was also examined by reverse phase HPLC quantitation of extracts of solutions in which the two activation systems were used. The levels of 7,8 dihydroxybenzo[a]pyrene produced by the glucose-6-phosphate protocol were consistently greater than with isocitrate. This may also be a contributing mechanism for elevating the mutation frequency with this procedure. These results demonstrate several interactions between test compound, cells, and metabolizing system which must be considered with in vitro activation systems.
在使用不同活化系统将苯并[a]芘作用于中国仓鼠卵巢细胞（CHO）时，观察到了突变频率的显著差异。每个系统都采用大鼠肝脏S9匀浆，其中一个系统使用异柠檬酸脱氢酶来提供还原型辅酶Ⅱ（NADP），而另一种方法使用葡萄糖-6-磷酸脱氢酶。依赖异柠檬酸的系统中，总芳烃羟化酶（AHH）活性更高，然而，该系统产生的次黄嘌呤-鸟嘌呤磷酸核糖转移酶（HGPRT）突变体水平较低。已确定这种降低的突变频率可能是由于培养基中的晶体（可能是磷酸钙）对苯并[a]芘底物的隔离，这降低了有效底物浓度。这种隔离增强了苯并[a]芘的内化，这可以解释AHH值与突变频率数据之间的二分法。还通过反相高效液相色谱法定量分析了使用两种活化系统的溶液提取物，以此来检测特定苯并[a]芘代谢物的产生。葡萄糖-6-磷酸方案产生的7,8-二羟基苯并[a]芘水平始终高于异柠檬酸方案。这也可能是该程序提高突变频率的一个促成机制。这些结果表明，在体外活化系统中，必须考虑受试化合物、细胞和代谢系统之间的几种相互作用。
