Hepatic levels of cytosolic, microsomal and 'mitochondrial' epoxide hydrolases and other drug-metabolizing enzymes after treatment of mice with various xenobiotics and endogenous compounds

This study was performed in order to study the response of epoxide hydrolases in different subcellular compartments of mouse liver to treatment with various compounds. Male C57BL/6 mice were treated with 31 different compounds--including traditional inducers of xenobiotic-metabolizing systems, liver carcinogens, stilbene derivatives, endogenous compounds and various other drugs and xenobiotics. The effects on liver somatic index; protein contents in 'mitochondria', microsomes and cytosol prepared from the liver; epoxide hydrolase activity towards trans- or cis-stilbene oxide in these three fractions; microsomal cytochrome P-450 content; cytosolic and 'mitochondrial' glutathione transferase activity and cytosolic DT-diaphorase activity were then determined. Cytosolic epoxide hydrolase activity was induced by chlorinated paraffins, di(2-ethylhexyl)phthalate and clofibrate and depressed by alpha-naphthylisothiocyanate, 3-methylcholanthrene, benzil and quercitin. Radial immunodiffusion revealed similar changes in the amount of enzyme protein present, except for two cases, where the increase in amount was larger; and the enzyme seems to be inhibited by benzil. Microsomal epoxide hydrolase activity was induced by these same compounds and several others as well, including dibenzoylmethane, butylated hydroxyanisole and polychlorinated biphenyls. 'Mitochondrial' epoxide hydrolase activity towards trans-stilbene oxide was not affected by those compounds which induced the cytosolic enzyme, but increased about two-fold after treatment with 2-acetylaminofluorene, DL-ethionine, aflatoxin B1 and phenobarbital. There does not seem to be any co-regulation of different forms of epoxide hydrolase in mouse liver. In general small effects were observed on liver weight and protein contents in the different subcellular fractions. Polychlorinated biphenyls were the most potent of the 8 compounds which induced cytochrome P-450, while butylated hydroxyanisole induced cytosolic glutathione transferase activity to the highest extent. 'Mitochondrial' glutathione transferase activity was most induced by certain of the stilbene derivatives. The most potent inducers of DT-diaphorase activity were 3-methylcholanthrene, polychlorinated biphenyls and dinitrotoluene.     

Differential time course of induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by Aroclor 1254

The time course of induction of rat liver microsomal cytochromes P-450a, P-450b + P-450e, P-450c, and P-450d and epoxide hydrolase has been determined in immature male rats administered a single large dose [1500 mumol (500 mg)/kg body wt] of the polychlorinated biphenyl mixture Aroclor 1254. Differential regulation of these xenobiotic-metabolizing enzymes was indicated by their characteristic patterns of induction. The rate of induction of cytochrome P-450a and epoxide hydrolase was relatively slow, and steady-state levels of these enzymes were maintained from approximately Days 9 to 15 after Aroclor 1254 treatment. In contrast, cytochrome P-450c was maximally induced 2 days after Aroclor 1254 treatment and remained at a constant level through Day 15. Steady-state levels of cytochrome P-450d, beginning 1 week after Aroclor 1254 treatment, were preceded by a fairly rapid rate of induction and possibly by a small decline from maximal levels observed around Days 4 to 5. Like those of the other cytochrome P-450 isozymes and epoxide hydrolase, the levels of cytochromes P-450b + P-450e were constant from Day 9 to 15 after Aroclor 1254 treatment. However, an unexpected but reproducible decline (approximately 25%) in total cytochrome P-450 content observed between Days 4 and 9 after Aroclor 1254 treatment principally reflected a dramatic and totally unanticipated decrease (approximately 45%) in the level of cytochromes P-450b + P-450e. This transient decline in the level of cytochromes P-450b + P-450e was not due to an unusual effect of a mixture of polychlorinated biphenyls, since identical results were obtained with two individual congeners, namely 2,3,4,5,4'-penta- and 2,3,4,5,3',4'-hexachlorobiphenyl, that induced the same isozymes as Aroclor 1254. In contrast, when rats were treated with 2,4,5,2',4',5'-hexachlorobiphenyl, which induces cytochromes P-450a and P-450b + P-450e and epoxide hydrolase but not cytochromes P-450c or P-450d, maximal levels of cytochromes P-450b + P-450e were attained on Day 4 and no decrease was observed over the next 11 days. These results suggest that there may be an interaction in the regulation of induction of certain individual cytochrome P-450 isozymes.     

Loss of cytochrome P-450 from hepatic nuclear membranes of rats fed 2-acetylaminofluorene

The cytochrome P-450 content of nuclear membranes isolated from the livers of male Sprague-Dawley rats fed a semipurified diet containing 0.05% w/w 2-acetylaminofluorene (AAF) for 3 weeks, was only about 20% of the values in control rats fed the same diet devoid of AAF. This effect was apparent after only 1 week of AAF treatment and persisted in nuclear membranes from isolated hyperplastic nodules (HPN) generated by 4 cycles of interrupted AAF-feeding. The microsomal cytochrome P-450 content, on the other hand, remained at control levels after 1 week of AAF treatment, and it was only slightly decreased after 3 weeks. In contrast, microsomes from HPN generated by prolonged AAF treatment had markedly decreased amounts of cytochrome P-450. The AAF treatment also caused changes in cholesterol epoxide hydrolase activity, which paralleled those observed for cytochrome P-450 content. Nuclear membranes from livers of rats fed AAF for 3 weeks, and from isolated HPN, had only 30-50% of the cholesterol epoxide hydrolase activity present in controls, whereas the microsomal enzyme activity remained at control levels after 3 weeks of AAF feeding but was 50% depressed in microsomes from HPN. The selective loss of cytochrome P-450 and of cholesterol epoxide hydrolase in hepatic nuclear membrane, but not in microsomes, of rats fed AAF for 3 weeks suggests independent control for these enzymes in these two membrane fractions. Cytochrome P-450 plays a role both in the activation of AAF (N-hydroxylation) as well as in its detoxification (ring hydroxylation) whereas cholesterol epoxide hydrolase initiates the detoxification of cholesterol epoxide. Therefore, our findings suggest the hypothesis that AAF treatment causes an early loss, at the surface of the nucleus, of the last line of defense for detoxification of transforming or promoting metabolites generated by microsomal activation of natural substances such as cholesterol and of xenobiotics such as AAF.     

Regulation of cytochrome P-450. Immunochemical quantitation of eight isozymes in liver microsomes of rats treated with polybrominated biphenyl congeners

Specific immunochemical techniques were used to quantitate the levels of eight isozymes of cytochrome P-450 (P-450) and epoxide hydrolase in liver microsomes of untreated rats and rats treated with phenobarbital, 3-methylcholanthrene, a mixture of these two compounds, nine individual polybrominated biphenyl (PBB) congeners, and a commercial mixture of PBBs. Levels of two 3-methylcholanthrene-inducible P-450s (designated P-450 beta NF-B and P-450 beta NF/ISF-G) varied over two orders of magnitude and were highly correlated. The levels of four phenobarbital-inducible P-450s (designated P-450PB-B, P-450PB-C, P-450PB-D, and P-450PB/PCN-E) were all correlated to each other. The level of one form, P-450UT-A, which was present at substantial levels in untreated rats, was inversely correlated to the levels of P-450 beta NF-B and P-450 beta NF/ISF-G. Among the PBB congeners which were examined, the presence of bromine at carbons o to the biphenyl bridge favored the induction of P-450PB-B, P-450PB-C, P-450PG-D, and P-450PB/PCN-E but did not necessarily eliminate the ability to induce P-450 beta NF-B and P-450 beta NF-ISF-G. PBB congeners with 2,2'-dibromo substitution induced P-450 beta NF-B and P-450 beta NF/ISF-G if one of the biphenyl rings contained bromines at positions 2,3, and 4. The induction of P-450UT-F was found to occur to a small extent with three of the compounds and is not readily explained in terms of structure-activity relationships. Although correlations were found among levels of some of the forms of P-450, several important exceptions were noted in relative levels of the individual enzymes. While the correlative data are useful in predicting induction patterns, all eight forms of P-450 appear to be independently regulated to some extent.     

Levels of cytochrome P-450, steroidogenesis and microsomal and cytosolic epoxide hydrolases in normal human adrenal tissue and corresponding tumors.

The levels of microsomal cytochrome P-450, steroidogenesis and microsomal and cytosolic epoxide hydrolase activities in normal human adrenal tissue (obtained from adult kidney transplant donors and autopsy material) and corresponding hyperplasia, adenomas and carcinomas (surgical biopsies) were determined. The increased steroid production demonstrated by most of the pathological tissue samples examined here was associated with either an unchanged or dramatically decreased specific microsomal content of cytochrome P-450. Furthermore, specific microsomal epoxide hydrolase activity was also found to be reduced in adrenocortical carcinomas, while the corresponding cytosolic activity was also decreased in at least two of these carcinomas. It is of interest to note in this connection that the level of microsomal epoxide hydrolase in slightly atropic adrenal cortex surrounding adrenocortical carcinomas was also found to be reduced. This would indicate that despite its appearance, this surrounding tissue is not normal in all respects. Thus, adrenocortical carcinomas fit into the common pattern in that their specific contents of microsomal cytochrome P-450 are dramatically decreased, but the simultaneous decrease in their microsomal epoxide hydrolase activity is more unusual.     

Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.     

Effects of ethanol and clofibrate on expression of cytochrome P-450 enzymes and epoxide hydrolase in cultures and cocultures of rat hepatocytes

Cultured and cocultured rat hepatocytes were used to study the effects of ethanol and clofibrate on cytochrome P-450 (P-450) enzymes and epoxide hydrolase. We showed that in the presence of ethanol, clofibrate or both compounds, rat hepatocytes were able to express, after 3 days of pure culture, quantitatively and qualitatively reasonable levels of most cytochrome P-450 enzymes and epoxide hydrolase, compared to freshly isolated hepatocytes. However, ethanol induced the P-450IA subfamily, and clofibrate the P-450-IVA subfamily. In cocultures, after 6 days, most P-450 enzymes were still expressed while P-450IIC11 was completely lost. Ethanol and clofibrate had the same effect as in pure culture. These results show, by modifying culture medium conditions and cell-cell interactions, that it is possible to maintain reasonable xenobiotic-metabolizing-enzyme expression; however, these conditions have to be improved in order to preserve better P-450 expression. The mechanism of these effects and the inducibility of these systems remain to be elucidated by a study at molecular level.     

Metabolism of 2-acetylaminofluorene by clara cells,type II cells and alveolar macrophages isolated from rabbit lung,and use of a new chamber incubation mutagenicity test system

Clara cells, alveolar type II cells and pulmonary alveolar macrophages (PAM) were isolated in high yield from rabbit lung. The purity of the cell fractions was 80–90%, 98% and above 99%, respectively. Cytochrome P-450 total content was determined in microsomes from freshly prepared cells. The Clara cells contained significantly more cytochrome P-450 than was found in whole lung microsomes. Furthermore, the cytochrome content of the Clara cells was 2 -fold higher than in the type II cells and 4 -fold higher than in the macrophages. 2-aminofluorene (AF) was the major metabolite in all preparations when intact cells were incubated with 2-acetylaminofuorene (AAF). The PAMs produced AF in the highest rates, while the Clara cells showed the largest rates of cytochrome P-450-dependent, ring hydroxylation of AAF. Mutagenic activation of AAF by isolated lung cells was assayed with a chamber-incubation method. The Clara cells were far more active than the type II cells in this respect, while the macrophages were inactive.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - DMSO dimethyl sulfoxide - NBT nitro blue tetrazolium - 7-OH-AAF 7-hydroxy-AAF - 9-OH-AAF 9-hydroxy-AAF     

Differential stereoselectivity of cytochromes P-450b and P-450c in the formation of naphthalene and anthracene 1,2-oxides. The role of epoxide hydrolase in determining the enantiomer composition of the 1,2-dihydrodiols formed

As is the case for cytochrome P-450c, arene 1,2-oxides have been identified as initial metabolites when naphthalene and anthracene are oxidized by cytochrome P-450b in a highly purified, reconstituted system. Overall rates of metabolism by cytochrome P-450b are greater than 3-fold and greater than 50-fold lower than the respective rates of metabolism by cytochrome P-450c. For both hydrocarbons, the (-)-(1S,2R)-oxide predominates (74%) with cytochrome P-450b as the terminal oxidant, based on trapping the labile arene oxides as N-acetyl-L-cysteine S-conjugates of known absolute configuration. This result is in marked contrast to data obtained with cytochrome P-450c where the (+)-(1R,2S)-oxides predominate (73-greater than 95%). In the absence of added epoxide hydrolase, the metabolically formed arene oxides rapidly isomerize to phenols. Addition of increasing amounts of epoxide hydrolase to the incubation medium results in the formation of trans-1,2-dihydrodiols at the expense of phenols from the common arene oxide intermediates. Evaluation of the kinetic parameters (Km and kcat) for the hydration of the (+)- and (-)-enantiomers of both arene oxides by epoxide hydrolase has indicated that the (+)-(1R,2S)-enantiomers exhibit lower values of Km (approximately 1 microM) whereas the values of kcat are similar for both enantiomers of a given arene oxide. These parameters have allowed construction of a mathematical model which predicts the enantiomer composition of the dihydrodiols formed from naphthalene in reconstituted systems containing specific epoxide hydrolase concentrations. The data reported argue against a selective functional coupling mechanism between cytochrome P-450c and epoxide hydrolase in the metabolism of naphthalene and anthracene to the 1,2-dihydrodiols.     

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes

A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.     

Identification of the forms of cytochrome P-450 induced in rat liver by 2-acetylaminofluorene using immunoblotting and partial purification

The amounts of 5 different forms of cytochrome P-450 in liver microsomes from rats treated with 2-acetylaminofluorene were determined and compared with the corresponding patterns in microsomes from control, 3-methylcholanthrene- and phenobarbital-treated animals. 2-Acetylaminofluorene was found to increase the amount of cytochromes P-450b + e 10-fold and of cytochrome P-450d 3-fold, while there was a 54% increase in the level of cytochrome P-450 PB/PCN-E. Cytochrome P-450c was increased from a level too low to detect (less than 0.001 pmol/mg protein) to 0.019 pmol/mg protein. These findings were also confirmed by partial purification of cytochromes P-450b + e and c after 2-acetylaminofluorene treatment.     

Induction of cytosolic and microsomal epoxide hydrolases in mouse liver by peroxisome proliferators, with special emphasis on structural analogues of 2-ethylhexanoic acid

Using dietary administration, mice were exposed to eight substances known to cause peroxisome proliferation (i.e. clofibrate clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, ICI-55.897, S-8527 and Wy-14.643) or the related substance p-chlorophenoxyacetic acid (group A). Other animals received di(2-ethylhexyl)phthalate, mono(2-ethylhexyl)phthalate, 2-ethylhexanoic acid, or one of 12 other metabolically and/or structurally related compounds (group B). The effects of these treatments on liver cytosolic and microsomal epoxide hydrolases, microsomal cytochrome P-450, cytosolic glutathione transferase activity, the liver-somatic index and the protein contents of the microsomal and cytosolic fractions prepared from liver were subsequently monitored. In general, peroxisome proliferation was accompanied by increases in cytosolic epoxide hydrolase activity. Many peroxisome proliferators also caused increases in microsomal epoxide hydrolase activity, although the correlation was poorer in this case. Immunochemical quantitation by radial immunodiffusion demonstrated that the increases observed in both of these enzyme activities reflected equivalent increases in enzyme protein, i.e. that induction truly occurred. Induction of total microsomal cytochrome P-450 was obtained after dietary exposure to clofibrate, clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, Wy-14.643, di(2-ethylhexyl)phthalate and di(2-ethylhexyl)phosphate. The most pronounced effects on cytosolic glutathione transferase activity were the decreases obtained after treatment with clofibrate, clofibric acid and Wy-14.643. Our results, together with those reported by others, suggest that the processes of peroxisome proliferation and induction of cytosolic epoxide hydrolase are intimately related. One possible explanation for this is presented.     

Immunoquantification of epoxide hydrolase and cytochrome P-450 isozymes in fetal and adult human liver microsomes

Epoxide hydrolase and three cytochrome P-450 isozymes were immunochemically determined in microsomes from adult and fetal human liver and tentatively correlated with some enzyme activities. The P-450 isozymes 5, 8 and 9 present in adult liver could not be positively correlated with the total cytochrome P-450 concentration spectrophotometrically determined. In fetal liver microsomes, isozyme 8 could not be detected by either electrophoretic or immunochemical procedures. Isozyme 5 was the major isozyme present in the fetal liver and its concentration increased in close relation with the total P-450 level. As shown previously, arylhydrocarbon hydroxylase activity was related to the concentration of isozyme 8 in adult liver. In fetal preparations, the absence of isozyme 8 was associated with a very low arylhydrocarbon hydroxylase activity. Aldrin epoxidase and benzphetamine-N-demethylase activities were correlated with isozyme 5 concentration, but with different slopes for adult and fetal microsomes: adult preparations catalyzed these two reactions more efficiently. Conversely, the dehydroepiandrosterone 16 beta-hydroxylase, also associated with isozyme 5 concentration, was more active in fetal than in adult microsomes. Moreover, if acetanilide hydroxylase increased with isozyme 5 concentration in adult samples, no correlation occurred between activity and P-450 isozyme level in fetal microsomes. Hydroxylations of lauric acid in positions 11 and 12 and of dehydroepiandrosterone in position 16 alpha increased with total P-450 concentration but not with isozyme concentrations whatever the age considered. Lastly, epoxide hydrolase activity towards benzopyrene 4,5-oxide was closely associated with its immunochemically determined level. These results clearly suggest that multiple mechanisms are involved in the regulation of different drug-metabolizing enzymes in the human fetus.     

Species differences in cytochromes P-450 and epoxide hydrolase: comparisons of xenobiotic-induced hepatic microsomal polypeptides in hamsters and rats

Cytochromes P-450 and epoxide hydrolase in hamsters were studied by using two-dimensional gel electrophoresis of hepatic microsomes from untreated animals and those treated with phenobarbital, 3-methylcholanthrene, beta-naphthoflavone, trans-stilbene oxide, and pregnenolone-16 alpha-carbonitrile. Coelectrophoresis with corresponding microsomes from rats and in situ peptide mapping were used to identify resolved microsomal polypeptides as cytochromes P-450 or epoxide hydrolase. Two forms of hepatic microsomal epoxide hydrolase were shown to exist in hamsters; these evidenced extensive structural homology with the corresponding enzyme in rats and were induced by the same xenobiotics. At least eight inducible polypeptides in microsomes from hamsters were tentatively identified as cytochromes P-450. Two of these were electrophoretically identical and structurally related with previously characterized forms of the enzyme in rats. Homologues of several major cytochromes P-450 induced by pregnenolone-16 alpha-carbonitrile and/or phenobarbital in the rat were apparently not present in the hamster. In most cases, putative forms of inducible cytochrome P-450 in the hamster existed at significant levels in microsomes from untreated animals whereas in rats the levels of most inducible forms of the enzyme were low in control microsomes, being more strictly dependent on xenobiotic pretreatment. In contrast with epoxide hydrolase, the molecular complexity of hepatic cytochrome P-450 seems to be comparable for rats and hamsters, but the structure and control of these hemoproteins appear to have markedly diverged.     

Mixtures of polycyclic aromatic compounds inhibit mutagenesis in the Salmonella/microsome assay by inhibition of metabolic activation

We observed that complex mixtures of aromatic compounds isolated from a coal-derived oil suppressed the mutagenic activity of the indirect mutagens benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 2-aminofluorene, and 2-acetylaminofluorene as measured in the Salmonella/microsome mutagenicity assay, using strain TA98 and metabolic activation with Aroclor-induced rat-liver S9 or microsomes. The mixture also inhibited S9-dependent benzo[a]pyrene metabolism and covalent binding to DNA in a cell-free system. The mixture did not suppress the activity of either the direct acting mutagens 2-nitrofluorene and benzo[a]pyrene diol-epoxide, or of the indirect mutagen N-hydroxy-2-acetylaminofluorene which requires a microsomal deacetylase for metabolic activation. Spectrophotometric measurements showed that components of the mixture bound to microsomal cytochrome P-450. The mixture did not inhibit microsomal NADPH-cytochrome c (P-450) reductase. These observations show that the mixtures inhibited metabolic activation by the microsomal monooxygenase system, probably by binding of unidentified components to cytochrome P-450. The resulting inhibition of mutagenesis may have implications for risk estimates for the mixtures we examined as well as for other types of complex mixtures for which similar inhibitory effects have been observed.     

Immunocytochemical evidence for the maintenance of cytochrome P-450 isozymes, NADPH cytochrome C reductase, and epoxide hydrolase in pure and mixed primary cultures of adult human hepatocytes

Specific polyclonal antibodies were used to investigate the distribution of two cytochrome P-450 isozymes (5 and 8), NADPH cytochrome c reductase, and epoxide hydrolase in adult human hepatocytes cultured alone or co-cultured with rat liver epithelial cells. The enzymes were localized by the indirect immunoperoxidase technique following fixation with a paraformaldehyde-glutaraldehyde mixture and membrane permeabilization with saponin. The pattern of distribution of the four enzymes after 24 hr in culture was similar to that found in vivo. Virtually all the hepatocytes exhibited nearly homogeneous positive staining for cytochrome P-450-8, whereas only 60-80% were positive for cytochrome P-450-5. Nearly homogeneous staining was also observed in all hepatocytes for NADPH cytochrome c reductase and epoxide hydrolase. During the first 12 days in pure culture, the intensity of staining, as well as the number of positively stained cells, decreased slightly except for epoxide hydrolase, which did not show any obvious change. In contrast, even after 15 days in co-culture the extent of staining for all the enzymes decreased less than in pure culture. These results indicate that adult human hepatocytes continue to express specific drug-metabolizing enzymes for several days in culture and provide further evidence that those cells are more stable than rodent hepatocytes in primary culture.     

Pulmonary microsomal cytochrome P-450 from 3-methylcholanthrene-treated hamsters: purification, characterization, and metabolism of benzo(a)pyrene

A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 165-fold from lung microsomes of 3-methylcholanthrene (MC)-treated hamsters. The purified preparation contained 14.2 nmol of cytochrome P-450 (P-450) per mg protein and was essentially free from NADPH-cytochrome P-450 (cytochrome c)-reductase (NADPH-reductase) and epoxide hydrolase. Pulmonary P-450MC exhibits an absorption maximum at 446.5 nm in the difference spectrum of reduced hemoprotein-CO complex, and a low-spin state of ferric iron in the heme. By sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 56,000. In a reconstituted system, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene (BP) hydroxylation, but showed low activities for 7-ethoxycoumarin O-deethylation and benzphetamine N-demethylation. In Ouchterlony double diffusion analysis, hamster pulmonary P-450MC reacted to the antibody prepared against rat hepatic P-450MC to form a faint precipitation line with a spur, indicating that the two P-450MCs have a common antigenic site but are not immunologically identical. When incubated with [14C]BP in a reconstituted system containing NADPH-reductase and epoxide hydrolase, hamster pulmonary P-450MC formed much higher amounts of BP diols, especially 7,8-diol, than were formed by rat pulmonary P-450MC.     

Differential inhibition of indirect immunofluorescence in rat liver sections incubated with antibodies against microsomal cytochromes P-450a, b, and c and epoxide hydrolase

Rat liver sections were incubated with antibodies (100-1000 micrograms IgG/ml) against microsomal cytochromes P-450a, P-450b, and P-450c, and epoxide hydrolase. Inhibition of indirect immunofluorescence, which progressed with higher concentrations of primary antibody, corresponded with antigen-enriched tissue in frozen liver sections from male and female rats. It was found in liver sections from phenobarbital-treated rats incubated with anti-P-450b and anti-epoxide hydrolase and from 3-methylcholanthrene-treated rats incubated with anti-P-450c. No inhibition was found in sections from untreated rats or rats receiving treatments that did not induce the specific antigen. No inhibition was found in sections incubated with anti-P-450a. Inhibition of immunofluorescence was abolished in frozen sections subjected to dehydration-rehydration protocols known to extract antigens, and was prevented by certain solvents and detergent-wash. Inhibition of immunofluorescence provides a unique method for confirming the antigen-rich regions of the liver lobules specific for microsomal expoxide hydrolase and the cytochrome P-450s.     

Multiplicity of induction patterns of rat liver microsomal mono-oxygenases and other polypeptides produced by administration of various xenobiotics.

Induction of hepatic microsomal mono-oxygenase species after administration of various xenobiotics is a well-documented phenomenon. To examine the number and specific species of rat liver microsomal membrane polypeptides involved in such responses, we have used sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to analyse microsomal fractions from animals treated with a number of important xenobiotics. The following are the principal points to have emerged from this study. 1. A minimum of twelve electrophoretically distinct patterns of induction of haemopolypeptides and other polypeptides could be distinguished after administration, either singly or in certain combinations, of phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls, 2-acetylaminofluorene, safrole (or isosafrole), pregnenolone-16 alpha-carbonitrile and ethanol. The patterns consisted of various permutations of the amounts of eight polypeptides of 47000-56000 mol.wt., of which at least three were haemopolypeptides. The possible identities of these polypeptides, which included species of cytochrome P-450, cytochrome P-448 and epoxide hydratase, are discussed. 2. Agents (3-methylcholanthrene, benzo[a]-pyrene, polychlorinated biphenyls, 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone) that result in the induction of cytochrome P-448 caused a marked increase in two polypeptides of 54000 and 56000 mol.wt., whereas safrole and isosafrole induced only the former polypeptide. 3. Administration of 2-acetylaminofluorene resulted in the induction of two polypeptides; evidence is presented that suggests that one of these is a species of epoxide hydratase [cf. Levin, Lu, Thomas, Ryan, Kizer & Griffin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3240-3243] ANd that the other may be a novel haemopolypeptide. 4. The overall results emphasize the complexity of the responses exhibited by rat liver microsomal fractions to the administration of xenobiotics.