DuPont Haskell Global Centers for Health & Environmental Sciences, Newark, Delaware, USA.
Environ Sci Technol. 2010 Apr 15;44(8):3052-8. doi: 10.1021/es903909g.
Determination of biotransformation rates of xenobiotics in freshly isolated trout hepatocytes has been demonstrated to significantly improve the performance of bioaccumulation assessment models. In order to promote this in vitro approach, trout hepatocytes need to be cryopreserved to facilitate their availability while ensuring their metabolic competency. In the present study, we obtained basal level metabolic enzyme activities for cytochrome P450 (CYP) 1A, CYP3A, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase from trout hepatocytes cryopreserved for various periods of time up to three months and compared their values with those obtained from freshly isolated hepatocytes. Similarly, we compared intrinsic clearance (CL(int)) values determined in cryopreserved trout hepatocytes to those determined in freshly isolated hepatocytes for reference compounds molinate, michler's ketone, 4-nonylphenol, 2,4-ditert-butylphenol, benzo(a)pyrene, and pyrene. Our results show that cryopreserved trout hepatocytes maintained greater than 75% of their basal level enzyme activities and greater than 72% of xenobiotic biotransformation capabilities, regardless of the length of cryostorage. As a result, bioconcentration factors of the reference compounds were adequately predicted based on the CL(int) values. We simulated the condition for shipping cryopreserved trout hepatocytes and demonstrated that 24 h dry ice storage did not negatively affect the rates of xenobiotic biotransformation. We conclude that cryopreserved trout hepatocytes are suitable for biotransformation rate determination of xenobiotics in vitro, and therefore, are an acceptable alternative to freshly isolated trout hepatocytes in the application in bioaccumulation assessment.
已证实，测定新鲜分离的鳟鱼肝细胞中外源化学物质的生物转化速率，可显著提高生物累积评估模型的性能。为了促进这种体外方法，需要对鳟鱼肝细胞进行冷冻保存，以方便其使用，同时确保其代谢能力。在本研究中，我们测定了在长达三个月的不同冷冻保存时间内的鳟鱼肝细胞的基础代谢酶活性，包括细胞色素 P450（CYP）1A、CYP3A、谷胱甘肽-S-转移酶和尿苷 5'-二磷酸葡萄糖醛酸转移酶，并将其与新鲜分离的肝细胞的酶活性进行比较。同样，我们将冷冻保存的鳟鱼肝细胞测定的内在清除率（CL(int)）值与新鲜分离的肝细胞的 CL(int)值进行比较，以评估化合物灭草隆、米氏酮、壬基酚、2,4-二叔丁基苯酚、苯并(a)芘和芘。结果表明，无论冷冻保存时间长短，冷冻保存的鳟鱼肝细胞都能保持 75%以上的基础酶活性和 72%以上的外源化学物质生物转化能力。因此，参考化合物的生物浓缩因子可以根据 CL(int)值得到充分预测。我们模拟了运输冷冻保存的鳟鱼肝细胞的条件，并证明 24 小时干冰储存不会对外源化学物质生物转化速率产生负面影响。我们得出结论，冷冻保存的鳟鱼肝细胞适合用于体外测定外源化学物质的生物转化速率，因此，在生物累积评估的应用中，是新鲜分离的鳟鱼肝细胞的一种可接受的替代物。
