ANTIOXIDANTS AS CYTOCHROME P-450 STABILIZERS IN HEPATOCYTES K. N. Novikov, Ao M. Dudchenko, A. T. Ugolev, Z. I. Kuznetsova, L. D. Luk'yanova, and V. E. Kagan UDC 612.351.1.015.1.577.152.112]: 612.262].014.46:615.272.4.014. 425 KEY WORDS: hepatocytes; cytochrome P-450; lipid peroxidation; antioxidants. The technique of hemoperfusion has recently begun to be introduced into clinical prac- tice, and for detoxication it is customary to use hepatocytes, which contain a highly effec- tive system of mixed-function mono-oxygenases, whose catalytically active component is cyto- chrome Pm450 (terminal oxygenase) [i, 2, 7]. This raises the urgent task of obtaining native hepatocytes, developing methods of their long-term culture and maintenance of high mixed-func- tion oxygenase activity (in other words, a high level of cytochrome P-450). Lipid peroxidation (LPO) is known to play an essential role in the mechanism of injury to cytochrome P-450 [4, 5]. This suggests that antioxidants, inhibitors of free-radical oxi- dation, may behave as stabilizers of cytochrome P-450 in hepatocyte cultures. This paper describes the study of the action of two antioxidants -- water-soluble 2-ethyl- 6-methyl-3-hydroxypyridine (OP-6) and fat-soluble 4-methyl-2,6-di-tert-butylphenol (ionol) -- on cytochrome P-450 in rat hepatocytes. EXPERIMENTAL METHOD Intact noninbred albino rats weighing 150-180 g were used. Hepatocytes were isolated by the method in [i0] with some modifications. To isolate the cells Tyrode's solution of the following composition was used: 125 mM NaCI, 3 mM KCI, 1 mM MgCI2, 1.2 mM NaH2PO4, 2 mM CaCI2, 40 mM Tris, pH 7.4 (37~ After isolation the liver was perfused with this solution but without Caq-+ and the perfusate was aerated with carbogen (95% 02 + 5% CO=). For recirculat- ing perfusion a solution containing Ca++ and 0.3% collagenase (from Sigma, USA) was used. After dispersion of the liver tissue a suspension of hepatocytes was obtained by triple cen- trifugation (100g, 1-2 min) in Tyrode's solution containing 1.5% purified albumin. The hepato- cyte suspension was incubated in the same solution at 37~ with shaking once per second, The number of cells was counted in a Goryaev's chamber with trypan blue to estimate the num- ber of viable hepatocytes. The cytochrome P-450 concentration was determined by the method in [9]. LPO was induced in the hepatocyte suspension by a system of Fe++-ADP (5 • 10 -5 M) + NADPH (I0-~ M). Accumulation of malonic dialdehyde (MDA), a secondary product of LPO, was de- termined by the reaction with 2-thiobarbituric acid (TBA) [6]. Ionol was added to the hepato- cyte suspension in ethyl alcohol so that the concentration of alcohol did not exceed 1%, and OP-6 was dissolved in Tyrode's medium. The final concentration of the antioxidants was I0 -~ M. The protein concentration was determined by the biuret reaction, using bovine serum albu- min as the standard. The results were subjected to statistical analysis by Student's t test and by nonparametric methods. EXPERIMENTAL RESULTS The hepatocyte suspension contained 1 • 106-2 • 106 cells/ml (10-20 mg protein/ml), of which 60-90% were viable. The cytochrome P-450 concentration in the native hepatocytes was 0.053 • 0.006 nmole/mg protein, in agreement with data in the literature [8]. During incuba- tion of the hepatocyte suspension spontaneous destruction of cytochrome P-450 took place in the cells (Fig. la); the concentration fell by 50% during 3 h of incubation. Destruction of Laboratory of Physical Chemistry of Biomembranes, Faculty of Biology, M. V. Lomonosov Moscow University. Department of Functional Bioenergetics, Research Institute for Biological Testing of Chemical Compounds, Kupavna, Moscow Region. (Presented by Academician of the Acad- emy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Eksperimental' noi Biologii i Meditsiny, Vol. 96, No. ii, pp. 50-52, November, 1983. Original article sub- mitted December 28, 1982. 0007-4888/83/9611- 1551507.50 9 1984 Plenum Publishing Corporation 1551