Kohn J, Durham H D
Montreal Neurological Institute, McGill University, Quebec, Canada.
Neurotoxicology. 1993 Winter;14(4):381-6.
Methods to evaluate the neurotoxicity of chemicals in vitro must take into account that many xenobiotics exert their toxic effects through metabolites. S9 fraction of liver homogenate has been used in cultures of bacterial and mammalian cells as a system for metabolic activation. The suitability of the S9 activation system for long-term neurotoxicity studies in vitro was investigated in dissociated cultures of murine spinal cord-dorsal root ganglia. Exposure to amounts of S9 greater than 0.07 mg S9 protein/ml of culture medium for 4 days or longer was cytotoxic to all types of neurons in the cultures. Non-neuronal cells tolerated higher exposures, but contained numerous cytoplasmic inclusions when 0.35 mg S9 protein was included in the medium. It was demonstrated that cytotoxicity was caused by the particulate, microsomal fraction of S9. It is concluded that direct incorporation of S9 fraction in the growth medium (0.07 mg S9 protein/ml or greater) is not a suitable method of generating metabolites in dissociated cultures of central nervous system when several days are required to elicit a biological response. Cytotoxicity can be prevented by using tissue culture inserts to separate cultured cells from S9 particulate fraction by a microporous membrane.
体外评估化学物质神经毒性的方法必须考虑到许多外源性物质是通过代谢产物发挥其毒性作用的。肝脏匀浆的S9组分已被用于细菌和哺乳动物细胞培养中作为代谢活化系统。在小鼠脊髓-背根神经节的解离培养物中研究了S9活化系统用于体外长期神经毒性研究的适用性。在培养基中暴露于大于0.07 mg S9蛋白/ml的S9量4天或更长时间对培养物中的所有类型神经元具有细胞毒性。非神经元细胞能耐受更高的暴露量，但当培养基中含有0.35 mg S9蛋白时，细胞内含有大量细胞质内含物。已证明细胞毒性是由S9的微粒体部分引起的。得出的结论是，当需要数天来引发生物学反应时，在中枢神经系统解离培养物中直接将S9组分掺入生长培养基（0.07 mg S9蛋白/ml或更高）不是产生代谢产物的合适方法。通过使用组织培养插入物通过微孔膜将培养细胞与S9微粒体部分分开，可以防止细胞毒性。
