Differential stabilization of cytochrome P-450 isoenzymes in primary cultures of adult rat liver parenchymal cells

Summary Cytochrome P-450 dependent hydroxylation of testosterone was measured in 7-day-old cultures of primary rat liver parenchymal cells. Determinations were carried out in monocultures of parenchymal cells and co-cultures of parenchymal cells with rat liver nonparenchymal epithelial cells, or mouse embryo fibroblasts. In the monoculture system, testosterone metabolism was drastically reduced and hardly measurable after 7 days in culture. In the co-culture systems, individual P-450 isoenzymes were stabilized on different levels. P-450sp and presumablyc were well preserved, P-450a was reduced but clearly measurable, P-450h was totally lost whereas P-450sb ande were not measurable after 7 days (the activities of these isoenzymes however were already low in freshly isolated parenchymal cells). The results were independent of the cell line used for co-cultivation and of the method of parenchymal cell isolation, that is whether collagenase or EDTA was used as the agent for dissociating the cells from the liver. The results showed that the co-cultivation of liver parenchymal cells with other nonparenchymal cells significantly improved the differentiated status of the former. In this cell culture system however, not every parameter was equally well stabilized.     

Different requirement for cytochrome b5 in NADPH-supported O-deethylation of p-nitrophenetole catalyzed by two types of microsomal cytochrome P-450

The role of cytochrome b₅ in the NADPH-supported O-deethylation of p-nitrophenetole catalyzed by cytochrome P-450 was studied with reconstituted systems using two types of cytochrome P-450 (P-450_PB and P-450_MC) purified from rat liver microsomes. The O-deethylation by P-450_PB absolutely required the presence of cytochrome b₅, whereas the same reaction catalyzed by P-450_MC did not require cytochrome b₅. These effects of cytochrome b₅ on the activities of reconstituted systems were confirmed by the use of antibodies to cytochrome b₅. On the other hand, the oxidations of ethylmorphine and aniline by these two types of cytochrome P-450 did not show significant dependence on cytochrome b₅. These observations suggest that the requirement for cytochrome b₅ in NADPH-supported drug oxidations depends not only on the species of cytochrome P-450 catalyzing the reactions, but also on the substrates oxidized.     

Role of cytochrome b5 in the cytochrome P-450-mediated C21-steroid 17,20-lyase reaction

The cytochrome P-450 (P-450_sccII) and its reductase, NADPH-cytochrome reductase [EC 1.6.2.4], associated with conversion of progesterone to 4-androstene-3,17-dione, were extensively purified from pig testis microsomes. Higher lyase activity (turnover number of 15 mol of the product formed/min/mol of P-450) could be restored by mixing the P-450_sccII, its reductase, pig liver cytochrome b₅ and cytochrome b₅-reductase [EC 1.6.2.2], and phospholipid in the presence of NADPH, NADH, and O₂. Omission of either cytochrome b₅ or NADH resulted in a significant loss of the lyase activity indicating actual participation of cytochrome b₅ in this P-450-mediated steroidogenic system in the testis.     

Benzo(a)pyrene metabolism by purified forms of rabbit liver microsomal cytochrome P-450, cytochrome b5 and epoxide hydrase in reconstituted phospholipid vesicles

The roles of rabbit liver cytochrome b₅, epoxide hydrase and various forms of cytochrome P-450 in the NADPH-dependent metabolism of benzo(a)pyrene were examined. After incorporation of the purified enzymes into phospholipid vesicles, using the cholate gel filtration technique, the various types of cytochrome P-450 did exhibit different stereospecificities in the oxygenation of the substrate. Cytochrome P-450_LM2 was found to efficiently convert benzo(a)pyrene in the presence of epoxide hydrase to 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene whereas cytochrome P-450_LM4 primarily participated in the formation of 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene. By contrast, benzo(a)pyrene was not metabolized by cytochrome P-450_LM3. Cytochrome b₅ enhanced cytochrome P-450_LM2-catalyzed oxygenations 5-fold, whereas cytochrome P-450_LM4-dependent oxygenations proceeded at a 3 times higher rate when cytochrome b₅ was present in the membrane.     

Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo

Degradation of cytochrome P-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome P-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome P-450 in vivo. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin in vitro, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome P-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome P-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal (14)C-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome P-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome P-450 degradation in vivo. After administration of bromobenzene, the degradation of the haem moiety of cytochrome P-450 was accelerated in vivo in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome P-450. However, the drug failed to affect degradation of haem not associated with cytochrome P-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome P-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state in vivo, as stimulated by bromobenzene in vivo, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring.     

Comparison of highly-purified microsomal cytochromes P-450 and NADPH-cytochrome P-450 reductases by peptide mapping

Considerable differences were observed among several cytochrome P-450 species upon mapping of proteolytic digests, although certain preparations derived from rabbits were not distinguished by this technique. Several NADPH-cytochrome P-450 reductases gave peptide maps rather similar to each other. The results support and extend other evidence that microsomal cytochromes P-450 are not necessarily closely-related polypeptides.     

Selective induction of cytochrome b5 and NADH cytochrome b5 reductase by propylthiouracil

Both the cytochrome b₅ level and NADH cytochrome b₅ reductase activity in rat liver microsomes were increased 2-fold by repeated i.p. administration of 1.5 mmol/kg propylthiouracil (PTU) for 2 weeks, but neither the cytochrome P-450 level nor NADPH cytochrome P-450 reductase activity were affected by the treatment. Liver microsomes from PTU-treated rats showed a significant decrease in aminopyrine N-demethylation, but not in benzphetamine N-demethylation, aniline hydroxylation or 7-ethoxycoumarin O-deethylation. A single administration of the compound had no effect on any components of the system. $\text{In vitro}$, drug hydroxylation activities were not affected by PTU up to 1.0 mM. From the above evidence, repeated administration of PTU selectively induced cytochrome b₅ and NADH cytochrome b₅ reductase in rat liver microsomes.     

Primary and secondary progesterone hydroxylation by the rabbit liver microsomal cytochromes P-450 system

Progesterone metabolism by the rabbit liver microsomal cytochromes P-450 system has been investigated with reverse phase (C18) high performance liquid chromatography. Time-course studies indicated that 6 beta-OHDOC accumulated as a secondary metabolite produced by the sequential 6 beta- and 21-hydroxylation of progesterone. When 21-hydroxylation occurred first, DOC accumulated and 6-hydroxylation was reduced. 16-OHP was resistant to secondary metabolism at both the 6- and 21-positions. The variable rates of 21-hydroxylase activity between different rabbits was further influenced by the nature of C-6-function in the order of 6-oxo greater than 6 beta-OH greater than 6 beta-OH greater than 6 alpha-OH greater than 6-H greater than 6 beta-acetoxy. These results indicate the potential interaction of the microsomal cytochromes P-450 and/or products in the sequential hydroxylation of a single steroid substrate at multiple sites.     

Age-related changes in the iron spin state of testosterone-binding rat liver microsomal cytochromes P-450

The aging process is accompanied by decreased drug metabolism as well as lower levels of sex hormones such as testosterone. We examined the age-dependence of liver microsomal cytochromes P-450 from young (3 months) and old (24 months) male rats by absorption and ESR spectroscopy. Spectral perturbations by testosterone were used to identify testosterone-specific P-450 forms. Absorption difference spectra indicated that testosterone induced a greater conversion of P-450 to the high spin form in young rats than in old rats. ESR signals corresponding to total low spin P-450 were of higher intensity in the young rats and were increased by testosterone. Testosterone also interconverted one low spin P-450 species to another. These results demonstrate age-related differences in the types and amounts of testosterone-specific P-450's in rats.     

Studies on the molecular organization of cytochromes P-450 and b5 in the microsomal membrane.

1. The relative orientations of the heme groups of cytochromes P-450 and b5 in the microsomal membrane have been studied by the technique of electron paramagnetic resonance. The results show that the heme plane of cytochrome P-450 lies in the same plane as the membrane surface, whereas the cytochrome b5 heme plane has a random orientation. 2. No significant broadening or change in relaxation properties of the gz component of low spin cytochrome P-450 occurred when cytochrome b5 was reduced by redox poising. It is concluded that there is little or no paramagnetic coupling between the heme groups of the two species. 3. The results favor a model in which no tight complex between cytochromes P-450 and b5 is present, the species being independent and interacting only by random molecular collisions or via other intermediate species.     

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.     

Immunohistochemical localizations of cytochromes P-450 in rat liver

Antibodies produced against two forms of cytochrome P-450, PB-B and MC-B, which were purified to apparent homogeneity from hepatic microsomes of rats pretreated with phenobarbital and 3-methylcholanthrene, respectively, have been employed to localize these hemoproteins immunohistochemically at the light microscopic level in the livers of untreated rats. Using these antibodies in an unlabeled antibody peroxidase-antiperoxidase technique, immunohistochemical staining for the cytochromes P-450 was detected in parenchymal cells throughout the liver lobule. The patterns of immunohistochemical staining intensity observed with the two antibodies, however, were quite different. Exposure of liver sections to the antibody to cytochrome P-450 PB-B resulted in intense immunostaining within the centrilobular regions but produced staining of considerably weaker intensity in the peripheral regions of the lobule. In contrast to these observations, the antibody to cytochrome P-450 MC-B yielded a more uniform pattern of immunohistochemical staining, with the intensity of staining being only slightly greater in the centrilobular regions. The results of this immunohistochemical study thus demonstrate that different patterns of distribution exist for different forms of cytochrome P-450 within the liver lobule and that the greatest concentration of cytochrome P-450 occurs within the centrilobular regions of the liver.     

Studies on the maintenance of cytochromes P-450 and b5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes

The cytochrome P-450 content of rat hepatocytes declined rapidly over 72 h in culture, due primarily to denaturation to cytochrome P-420. Six different media were investigated for their ability to conserve cytochrome P-450 during culture, and the most successful was a modified Earle's medium. After 72 h culture in this medium, cytochromes P-450 and b5, NADH-cytochrome b5- and NADPH-cytochrome c-reductases were maintained at 40, 100, 35 and 52% of fresh cell values, respectively. Cytochrome P-450 showed differential functional stability during culture with ethoxyresorufin O-deethylation being more stable than either pentoxyphenoxazone O-depentylation or biphenyl 4-hydroxylation. Monooxygenase than did cytochrome P-450 content. This discrepancy was not explained by loss of flavin nucleotides, FMN or FAD.     

Effects of emulsified perfluorochemicals on liver cytochromes P-450 in rats

1. The effects of intravenous (i.v.) injection of various perfluorochemical (PFC) emulsions or different fractions of the non-ionic poloxamer surfactant, Pluronic F-68, have been studied separately in male and female rats. 2. Injection of 10 ml/kg body wt of either Fluosol-DA 20% (F-DA) or a novel perfluorodecalin emulsion containing a C-16 oil additive in male rats increased liver weight up to 7 days later; no corresponding effect occurred in response to injection of Oxypherol (FC-43). 3. Liver weight was also increased in female rats at 72 hr after injection of the novel emulsion but this was less pronounced than in males; liver weight in female rats was unchanged in response to injection of either F-DA or FC-43. 4. Mean liver microsomal cytochromes P-450 concentrations in male rats were increased 2-3 fold at 72 hr after injection of either F-DA or the novel emulsion with a less pronounced increase also seen at 7 days in animals receiving the novel emulsion. No significant alterations in cytochrome concentration occurred in response to injection of FC-43 or either commercial grade or purified pluronic solution. 5. Liver cytochromes P-450 concentrations in female rats were unaffected by any of the experimental treatments. 6. These results show that injection of a single low dose of emulsified PFCs into male rats can increase hepatic microsomal cytochromes P-450 concentration but the response is highly variable, depending on composition of emulsion injected.