Abnormal Microsomal Cytochromes and Electron Transport in Morris Hepatomas

Abstract

SUMMARY Difference spectral assays of cytochromes P-450, P-420, and bs in Morris hepatoma microsomes are complicated by the presence of methemoglobin and hemoglobin. Alternate assays of cytochromes P-450 and P-420 have been developed. The specific contents of cytochrome P-450 present in micro- somes obtained from tumor lines 5123C, 7777, and R3B are: 0.31, 0.014, and 0.10 nmoles per mg of protein, respectively; the specific contents of cytochrome P-420 are: 0.038,0.14, and 0.042 nmoles per mg of protein, respectively. Cytochrome bs is present in all samples of tumor microsomes; the specific content is somewhat higher in microsomes from tumors that contain greater amounts of cytochrome P-450. Although rat hepatic cytochrome P-450 exists in at least three spectrally identifiable forms, only two forms are found in microsomes from the slower growing tumor line, 5123C, in which cytochrome P-450 is relatively abundant. In the rapidly growing tumor line, 7777, there is only one form of cytochrome P-450. Only one spectrally discernible form of the very abundant cytochrome P-420 is present in tumor line 7777, and this cytochrome P-420 is no longer converted to cytochrome P-450 simply by the addition of glycerol. The amounts of flavoproteins of the NADH- and NADPH- oxidative pathways of microsomes in the tumor microsomes are somewhat lower than those of liver. However, NADH- and NADPH-dependent reductions of cytochrome c and ferri- cyanide are relatively rapid, and the properties of flavoproteins of tumor are similar to hepatic microsomes. The rate of NADPH-dependent oxygen uptake by micro- somes from tumor line 7777 is too slow to measure; aerobic oxidation of NADPH is unmeasurable. Demethylation of aminopyrine and benzphetamine occurs at only very slow rates. Furthermore, phenobarbital and methylcholanthrene induce mixed function oxidase activity in liver of the host rats but not in microsomes prepared from tumor line 7777. There is a striking appearance of cytochrome P-448 (Pi-450) in * This work was supported in part by funds from Research Grant AM-10767 from the National Institute of Arthritis, Metab- olism, and Digestive Diseases, Grant CA-10729 from the National Cancer Institute, United States Public Health Service, and in part by funds made available through the State University of New York. $ On leave from the Department of Biochemistry, University of Osaka Medical School, Osaka, Japan. tumor microsomes obtained from methylcholanthrene-treated rats. However, cytochrome P-448-dependent arylhydro- carbon hydroxylase activity is undetectable, even in these induced preparations. Since the cytochromes and flavoproteins are present in tumor microsomes, inactivity of mixed function oxidases may be ascribed either to absence of a hitherto unknown compo- nent or to undetected alterations in one of the known com- ponents. Since the cytochromes apparently may not function as components of mixed function oxidases in this tissue, alternate functions of these microsomal hemoproteins must be considered. Liver microsomes contain mixed function oxidases that cata- lyze the enzymic attack of drugs, carcinogenic hydrocarbons, insecticides, and several other classes of xenobiotics that have the common property of being relatively hydrophobic (1, 2). The oxidases are relatively complex multienzymic systems, being composed of cytochrome and flavoprotein; in addition, they may require phospholipids, which are selectively attacked by phos- pholipases (3). The complex systems may be chemically induced in wivo (1,4, 5). Induction experiments have yielded considerable informa- tion about the nature of microsomal oxidases; however, the in- duction process is complex, and results may be beyond interpre- tation. Alternatively, hepatic microsomal mixed function oxi- dases have been studied by genetic selection (6), resolution and reconstitution of enzymic components (7), electron spin reso- nance (8) and visible spectral studies of components (9), studying oxidation-reduction potentials (lo), investigating changes upon combination with substrate (11, 12), and immunological methods (13). A different approach has been the investigation of several uninduced oxidases from sources ot.her than liver, e.g. adrenal gland mitochondria (14), kidney (15), bacteria (16), yeast (17), plants (18). Since the Morris hepatoma is derived from liver hepatocytes, comparisons to microsomal enzymes of liver are particularly attractive. The terminal oxidase in these multienzymic systems is cyto- chrome P-450. Several lines evidence suggest that cytochrome 1980