Steward A R, Dannan G A, Guzelian P S, Guengerich F P
Mol Pharmacol. 1985 Jan;27(1):125-32.
We prepared primary monolayer cultures of adult rat hepatocytes and measured the losses of cytochromes P-450 with the use of specific antibodies directed against purified forms of hepatic cytochrome P-450 which predominate in untreated rats (P-450UT-A, P-450UT-F) or in rats treated with phenobarbital (P-450PB-B/D, P-450PB-C, P-450PB/PCN-E) or with 3-methylcholanthrene (P-450 beta NF-B, P-450 beta NF/ISF-G). In hepatocytes prepared from an untreated rat and incubated in control medium, total cytochrome P-450, measured spectrally as CO-binding hemoprotein, declined 68% during the first 72 hr in culture. However, the sum of the immunoreactive cytochromes P-450 declined only 24%, indicating that loss of heme rather than of protein accounts for much of the well-known loss of cytochromes P-450 in hepatocyte cultures. In cultures prepared from untreated rats or from rats treated with phenobarbital or with 3-methylcholanthrene, individual forms of cytochrome P-450 declined at markedly differing rates. Incubation of cultures in three different media previously reported to maintain levels of total cytochrome P-450 failed to prevent the decline in total cytochrome P-450 during the first 24 to 72 hr in culture. However, in cultures incubated in medium containing metyrapone, the level of holocytochrome P-450 was maintained at the initial value during the first 72 hr, apparently by preventing the net loss of cytochrome P-450 heme and by increasing the concentrations of immunoreactive P-450PB/PCN-E and P-450 beta NF-B. Medium containing nicotinamide increased the proportion of P-450 beta NF-B relative to the other forms of cytochrome P-450, whereas cysteine-free medium increased P-450UT-F. We conclude that loss of cytochrome P-450 in cultured hepatocytes involves loss of its heme moiety coupled with changes in the concentrations of the individual forms. Recognition of these changes as influenced by specific components of the culture medium is important when using primary hepatocyte cultures for study of xenobiotic metabolism and toxicity in the liver.
我们制备了成年大鼠肝细胞的原代单层培养物，并使用针对纯化形式的肝细胞色素P - 450的特异性抗体来测量细胞色素P - 450的损失，这些细胞色素P - 450在未处理的大鼠（P - 450UT - A、P - 450UT - F）、用苯巴比妥处理的大鼠（P - 450PB - B/D、P - 450PB - C、P - 450PB/PCN - E）或用3 - 甲基胆蒽处理的大鼠（P - 450βNF - B、P - 450βNF/ISF - G）中占主导地位。在从未处理的大鼠制备并在对照培养基中孵育的肝细胞中，以与一氧化碳结合的血红蛋白光谱法测量的总细胞色素P - 450在培养的最初72小时内下降了68%。然而，免疫反应性细胞色素P - 450的总和仅下降了24%，这表明肝细胞培养物中细胞色素P - 450的大量损失是由于血红素的损失而非蛋白质的损失。在从未处理的大鼠或用苯巴比妥或3 - 甲基胆蒽处理的大鼠制备的培养物中，细胞色素P - 450的各个形式以明显不同的速率下降。在先前报道能维持总细胞色素P - 450水平的三种不同培养基中孵育培养物，并不能防止培养最初24至72小时内总细胞色素P - 450的下降。然而，在用甲吡酮培养基孵育的培养物中，全细胞色素P - 450的水平在最初72小时内维持在初始值，显然是通过防止细胞色素P - 450血红素的净损失以及增加免疫反应性P - 450PB/PCN - E和P - 450βNF - B的浓度来实现的。含有烟酰胺的培养基增加了P - 450βNF - B相对于其他形式细胞色素P - 450的比例，而不含半胱氨酸的培养基增加了P - 450UT - F。我们得出结论，培养的肝细胞中细胞色素P - 450的损失涉及血红素部分的损失以及各个形式浓度的变化。当使用原代肝细胞培养物研究肝脏中的外源化合物代谢和毒性时，认识到这些受培养基特定成分影响的变化非常重要。
