Altered regulation of cytochrome P-450 enzymes in choline-deficient cirrhotic male rat liver: impaired regulation and activity of the male-specific androst-4-ene-3,17-dione 16 alpha-hydroxylase, cytochrome P-450UT-A, in hepatic cirrhosis

Total microsomal cytochrome P-450 levels were decreased, to about 50% of control, in liver of male rats made cirrhotic by the prolonged intake of a choline-deficient diet. We have suggested previously that this decrease in cytochrome P-450 levels is not a generalized one, but is selective for certain forms of the enzyme. In the present study, levels of six cytochrome P-450 forms including the sex-specific cytochrome P-450 forms, P-450UT-A, P-450PCN-E, and P-450UT-l, were quantitated immunochemically in hepatic microsomes prepared from control and cirrhotic male rats and were related to changes in the regioselectivity of cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation in these fractions. The principal finding of this study was that the male-specific androst-4-ene-3,17-dione 16 alpha-hydroxylase was decreased in cirrhotic microsomes to about 20% of control. The content of P-450UT-A decreased concurrently from about 0.40 to less than 0.01 nmol/mg of microsomal protein. Other pathways of androst-4-ene-3,17-dione hydroxylation were also affected, but to different extents than the 16 alpha-hydroxylase. 6 beta-Hydroxylation decreased in cirrhotic microsomes to about 45% of control, despite a marked decrease in P-450PCN-E from 0.27 to less than 0.002 nmol/mg of microsomal protein. The rate of androst-4-ene-3,17-dione 7 alpha-hydroxylation underwent a less pronounced reduction in cirrhosis to about two-thirds of control microsomal activity, and levels of the cytochrome P-450 associated with this activity, P-450UT-F, were decreased in proportion with the decrease in total microsomal cytochrome P-450. 16 beta-Hydroxylase activity was unaffected by the cirrhotogenic process. From spectral binding studies it was apparent that androst-4-ene-3,17-dione elicited a high affinity type I interaction in control microsomal fractions (Ks = 4.5 microM), whereas no interaction was apparent in cirrhotic liver microsomes. Levels of three other forms of cytochrome P-450--P-450PB-C (a constitutive form inducible by phenobarbital), P-450ISF-G (a major isosafrole-inducible form), and P-450UT-I (the major female sexually-differentiated isozyme)--were apparently unaltered in cirrhosis. These findings are consistent with the assertion that specific forms of cytochrome P-450 are subject to altered regulation in hepatic cirrhosis.     

Effects of long-term choline deficiency on hepatic microsomal cytochrome P-450-mediated steroid and xenobiotic hydroxylases in the female rat

Total cytochrome P-450 levels decreased to about 80% of control in hepatic microsomes from female rats maintained for 30 weeks on a choline-deficient diet. Livers from these rats were fibrotic and had extensive fatty infiltration but, unlike livers of male rats on the same regimen, were not cirrhotic. Steroid hydroxylase activities were assessed in microsomes of female rats that received the choline-deficient diet and it was noted that the activity of the cytochrome P-450 UT-F-mediated steroid 7 alpha-hydroxylase was decreased to about 50% of the activity present in choline-supplemented control rat microsomes. Similar decreases were observed for microsomal androstenedione 6 beta-hydroxylase and aniline 4-hydroxylase activities. In female rat hepatic microsomes these two activities are probably mediated by the isozyme cytochrome P-450 ISF-G. In contrast to these findings, the activities of four other xenobiotic metabolising enzymes, as well as rates of microsomal steroid 16 alpha- and 16 beta-hydroxylation, were unchanged from control. Thus, in hepatic microsomes from choline-deficient female rats, it appears likely that levels of the non-sexually differentiated cytochromes P-450 UT-F and ISF-G are decreased. Unlike the situation in male rats, long term choline deficiency does not appear to influence levels of sexually-differentiated P-450 enzymes in the female rat.     

Regulation of renal cytochrome P-450. Effects of two-thirds hepatectomy, cholestasis, biliary cirrhosis and post-necrotic cirrhosis on hepatic and renal microsomal enzymes

The possibility of a relationship between hepatic and renal cytochrome P-450 contents was assessed in rats with liver disease. In rats killed 3 days after two-thirds hepatectomy (a model for hepatocellular insufficiency), the total microsomal cytochrome P-450 content of the whole liver was decreased by 60% as compared to that in control rats; renal cytochrome P-450 was increased by 30% while the 7-ethoxycoumarin deethylase activity of kidney microsomes was increased by 80%. In rats killed 7 days after bile duct ligation (a model for cholestasis) or 35 days after bile duct ligation (a model for biliary cirrhosis), hepatic cytochrome P-450 was decreased by 60% and 45%, respectively, while renal cytochrome P-450 content was increased by 50% and 150%, respectively. In contrast, in rats killed 15 days after the last dose of carbon tetrachloride, 1.3 ml/kg twice weekly for 3 months (a model for post-necrotic cirrhosis), both hepatic and renal cytochrome P-450 contents remained unchanged. Phenobarbital (80 mg/kg daily for 3 days) was a poor inducer of renal cytochrome P-450 in sham-operated rats but became a potent inducer of renal cytochrome P-450 in rats with two-thirds hepatectomy. We conclude that renal cytochrome P-450 is increased in three models in which hepatic cytochrome P-450 contents are decreased (two-thirds hepatectomy, cholestasis and biliary cirrhosis), but remains unchanged in a model of severe liver pathology, in which hepatic cytochrome P-450 content is not modified (late, post-necrotic cirrhosis). The hypothetical role of endogenous inducer(s) is discussed.     

Induction of rat liver microsomal and nuclear cytochrome P-450 by dietary 2-acetylaminofluorene and butylated hydroxytoluene

The influence of dietary 2-acetylaminofluorene (AAF) on the cytochrome P-450 content of rat liver microsomal and nuclear fractions was immunochemically probed with monoclonal and polyclonal antibodies to cytochromes P-450c and P-450d. Cytochrome P-450d but not P-450c was immunodetected in microsomes, nuclear envelopes, and nuclei from untreated rats. The levels of both cytochromes P-450c and P-450d were elevated after a diet of either 0.1% AAF for 1 week or 0.05% AAF for 3 weeks. However, the level of cytochrome P-450c relative to P-450d was lower after the more prolonged AAF feeding. Supplementation of AAF-containing diets with 0.3% butylated hydroxytoluene (BHT), which affords protection against AAF hepatocarcinogenesis in high-fat fed rats, protected and/or induced total (spectral) nuclear envelope cytochrome P-450 content. Immunochemical studies of liver fractions showed that BHT enhanced the AAF-dependent induction of cytochrome P-450c, but not of P-450d. This was a concerted effect of AAF + BHT since dietary BHT by itself did not affect the levels of cytochrome P-450c or P-450d as compared to control rats. Since 1- to 3-week dietary AAF had little effect on total (spectral analyses) microsomal cytochrome P-450 but markedly reduced total P-450 in nuclear envelopes, the coordinated induction of specific cytochrome P-450s in the different fractions suggests selective induction and depression of different forms of cytochrome P-450 and provides additional evidence for independent regulation of the drug-metabolizing system in nuclear envelope and microsomes. In addition, these results suggest that regulation of cytochrome P-450 may play a crucial role in the nutritional modulation of AAF hepatocarcinogenesis.     

Cytochrome P-450 and oxidative metabolism in molluscs

1. Cytochrome P-450 and the associated components and oxidative activities of a mixed-function oxidase (MFO) system are localized primarily in the microsomes of the digestive gland of molluscs. 2. Cytochrome P-450 and putative cytochrome P-450-catalysed oxidative activities, measured in vitro and/or in vivo, have variously been detected in 23 species of mollusc. 3. Cytochrome P-450 and other MFO components and activities may be increased by exposure to xenobiotics, but the results are variable and no correlation is obvious between changes in cytochrome P-450 content and measured MFO activities (benzo[a]pyrene hydroxylase (BPH) and 7-ethoxycoumarin O-deethylase (ECOD)). 4. Type II binding compounds (clotrimazole, miconazole, ketoconazole, metyrapone and pyridine) give type II difference spectra with mussel digestive gland microsomal P-450, whereas type I binding compounds (testosterone, 7-ethoxycoumarin, alpha-naphthoflavone, SKF525-A) give apparent reverse type I difference spectra. 5. The existence of multiple or particular forms (P450 IVA or LAw) of cytochrome P-450 is indicated from enzyme kinetics and inhibition studies, seasonality, purification studies and cDNA probes. 6. Microsomal MFO activities are observed even in the absence of added or generated NADPH, and the NADPH-independent BPH, ECOD and N,N-dimethylaniline N-demethylase activities are inhibited by reducing agents, including NADPH. 7. The major metabolites of microsomal benzo[a]pyrene metabolism are quinones. 8. One-electron oxidation is considered to be one possible mechanism of molluscan cytochrome P-450 catalytic action.     

Spectral properties of substrate-cytochrome P-450 interaction and catalytic activity of xenobiotic metabolizing enzymes in isolated rainbow trout liver cells

A method for the isolation of intact viable rainbow trout liver cells in high numbers is described. The technique involves perfusion of collagenase through the liver. A major part of the cytochrome P-450 in isolated liver cells was present in the oxidized non-substrate bound form. It was observed that 7-ethoxycoumarin was rapidly taken up by the liver cells and bound to cellular cytochrome P-450. The substrate binding spectrum for isolated trout liver cells was slightly modified compared with that obtained with trout liver microsomes. The microsomal affinity of 7-ethoxycoumarin, calculated as the apparent spectral dissociation constant (ks), was elevated 11-fold after fish were treated with beta-naphthoflavone, indicating a qualitative alteration in the nature of the constitutive cytochrome P-450. The metabolism of 7-ethoxycoumarin in isolated liver cells was found to be of a comparable rate to that obtained in liver microsomes. Pretreatment of fish with Clophen A50 or beta-naphthoflavone significantly increased the content of cytochrome P-450 and elevated the rate of 7-ethoxycoumarin deethylation in isolated liver cells. Furthermore, the rate of conjugation of 7-hydroxycoumarin was significantly elevated in liver cells isolated from beta-naphthoflavone treated fish when compared with the control rate. In isolated liver cells, 90% of the 7-hydroxycoumarin formed from deethylation of 7-ethoxycoumarin was further metabolized to conjugated products. However, in beta-naphthoflavone of Clophen A50 treated fish the fraction of conjugated metabolites was markedly decreased, indicating a changed balance between cytochrome P-450 dependent reactions and conjugation reactions in the cell.     

Responses of cytochrome P-450 isozymes of rat lung to in vivo exposure to ozone

The effects of 2 weeks exposures of rats to 0.2 ppm O3 on pulmonary cytochrome P-450 isozymes were investigated. Two main types of cytochrome P-450 isozymes (cytochromes P-450FI and P-450FII) were separated from lung microsomes of control rats on an anion exchange cellulose column. Antibody raised against cytochrome P-450b, which is the main isozyme of liver obtained from phenobarbital-treated rats, cross-reacted with pulmonary cytochrome P-450FII, but not with cytochrome P-450FI. Ozone exposures caused increases in the content of both cytochromes P-450FI and P-450FII to the same extent two times greater than those of control rats without changes in immunological properties of these isozymes. No pulmonary cytochrome P-450 isozyme other than cytochromes P-450FI and P-450FII was observed in eluate through anion exchange column chromatography. These results indicate that increases in the content of pulmonary cytochrome P-450 of rats exposed to O3 can be ascribed to increase in constitutive types of the isozymes, but not to induction of other types of isozyme.     

Different effects of cyanide on the activities of 7-ethoxycoumarin O-deethylation catalyzed by two forms of cytochrome P-450 purified from 3-methylcholanthrene-treated rats

The effect of cyanide on 7-ethoxycoumarin O-deethylation by two cytochrome P-450 isozymes obtained from 3-methylcholanthrene treated rat liver microsomes was investigated. 7-Ethoxycoumarin O-deethylation was stimulated by the addition of cyanide to a reconstituted monooxygenase system consisting of NADPH, dilauroyl 3-L-phosphatidylcholine, NADPH-cytochrome P-450 reductase and MC P-448(2) (low spin form of cytochrome). In contrast, a weak inhibitory effect of cyanide on 7-ethoxycoumarin O-deethylation was observed when MC P-448(1) (high spin form of cytochrome) was used in the reconstituted system. Cyanide did not influence the apparent Km for 7-ethoxycoumarin when either form of cytochrome P-450 was used in the reconstituted system and did not stimulate the cumene hydroperoxide dependent O-deethylation by MC P-448(2). The stimulatory effect of cyanide on O-deethylation by MC P-448(2) was decreased with increasing the concentration of the reductase added to the reconstituted system. On the other hand, the effect of cyanide on O-deethylation by MC P-448(1) was virtually independent on the amount of the reductase added.     

Metabolism of diamantane by rat liver microsomal cytochromes P-450

1. Diamantane binds to liver microsomes from phenobarbital-treated rats with an apparent Ks value of 5.2 x 10(-7) mol/l. This value being lower than that obtained for perhydrophenanthrene indicates that diamantane is very strongly bound to microsomal cytochrome P-450. 2. Metabolic studies show that liver microsomes from phenobarbital-treated rats readily metabolize diamantane to mono-, di- and possibly tri-hydroxy derivatives, whereas liver microsomes from beta-naphthoflavone-induced rats do not bind this hydrocarbon or metabolize it. 3. Reconstituted cytochromes P-450 b and e were more efficient in the hydroxylation of diamantane than liver microsomes; metabolites formed by the reconstituted system do not include all the products formed by microsomes, which indicates the involvement of forms of cytochrome P-450 other than the isozymes b and e.     

Effects of primaquine on hepatic microsomal haemoproteins and drug oxidation

Administration of the antimalarial agent primaquine to male rats (50 mg/kg i.p. daily for 4 days) resulted in a 30% decrease in hepatic microsomal cytochrome P-450 (P-450) content; levels of other microsomal haemoproteins were unaltered. Kinetic analysis of 2 mixed-function oxidase activities (aminopyrine N-demethylase and aniline p-hydroxylase) in primaquine-pretreated rat liver microsomes revealed a significant and similar decrease in the maximal reaction velocities of these enzymes (Vmax), but the apparent Michaelis constants (Km) were not changed. The activity of mitochondrial delta-aminolaevulinic acid synthetase (the rate-limiting step in haem biosynthesis) was normal in primaquine-pretreated rat liver but haem oxygenase activity (the rate-limiting step in haem degradation) was elevated approximately 2-fold. Haem availability for haemoprotein assembly (determined as the haem-saturation ratio of the cytosolic haemoprotein tryptophan pyrrolase) was also normal although the absolute activity of tryptophan pyrrolase was decreased after primaquine pretreatment. In order to facilitate an analysis of the P-450 isozyme profile in control and primaquine-treated rat liver, total microsomal P-450 was isolated by hydrophobic affinity chromatography on n-octylamino-Sepharose 4B. Densitometry of stained polyacrylamide gels following electrophoresis of these partially-purified P-450 fractions indicated that primaquine exposure did not selectively decrease any of the 3 protein bands in the P-450 molecular weight region (48-56 kD). These observations, when considered together, suggest that primaquine may affect P-450 and mixed-function oxidase activity by inhibition of protein synthesis. The characteristic rapid turnover rates of P-450 isozymes may predispose these haemoproteins to the toxic effects of primaquine whereas those haemoproteins that turn over less rapidly, such as cytochrome b5, appear to be less susceptible. Microsomal haem oxygenase activity may be elevated after primaquine administration since lowered haemoprotein requirements for haem could result in excess haem levels within the hepatocyte.     

Role of isozymes of cytochrome P-450 in the metabolism of N,N-dimethyl-4-aminoazobenzene in the rat

The metabolism of N,N-dimethyl-4-aminoazobenzene (DAB) was investigated in vitro by use of hepatic 10,000g supernatant fraction, microsomes, and purified cytochromes P-450 prepared from rats. Position-selective metabolism was studied in response to induction by 3-methylcholanthrene (MC), phenobarbital (PB), beta-naphthoflavone (BNF), and pregnenolone-16 alpha-carbonitrile (PCN) as well as inhibition by SKF 525-A, metyrapone, alpha-naphthoflavone, and piperonyl butoxide. The principal phase I pathways are demethylation of the tertiary (DAB) and secondary (MAB) amines and ring hydroxylation. When metabolism was measured with 10,000g supernatant fractions, each pathway responded differently and often independently to the inducers and inhibitors, suggesting that they are catalyzed preferentially by different isozymes of cytochrome P-450. Microsomes from PB-treated animals demethylated and hydroxylated DAB at the same rate as did control microsomes, based on cytochrome P-450 content, whereas microsomes from BNF- or MC-treated animals demethylated more rapidly and hydroxylated more slowly. Microsomes from PB-treated animals demethylated the secondary amine, MAB, more rapidly than the tertiary amine, DAB. Purified cytochrome P-448 from MC-treated animals catalyzed DAB demethylation very readily but hydroxylation very poorly. The turnover number was 10 times that seen in microsomes from MC-treated animals. Only one of the four cytochrome P-450 fractions isolated from PB-treated animals had significant activity with DAB and the turnover number of one of these (fraction B) was approximately that seen in microsomes. This study supports the concept of selectivity of various isozymes of cytochrome P-450 for the different steps in phase I metabolism of DAB. Furthermore, it is apparent that the association of certain inhibitors with specific isozymes of cytochrome P-450 is a generalization that requires qualification in terms of the substrates(s) involved.     

Cimetidine interaction with liver microsomes in vitro and in vivo. Involvement of an activated complex with cytochrome P-450

The O-deethylation of 7-ethoxycoumarin was inhibited in a mixed type manner by cimetidine in vitro and in microsomes isolated from rats treated with cimetidine in vivo. It was found that the inhibition was even greater if cimetidine was preincubated with the microsomal suspension in the presence of an NADPH-generating system prior to the addition of substrate. In vitro the decrease in activity was accompanied by a decrease in cytochrome P-450 content. This decrease was unaffected by the addition of EDTA to the microsomal suspensions, eliminating the possibility that free radical production was responsible for the decrease in cytochrome P-450. The decrease in activity and cytochrome P-450 content following preincubation of microsomal suspensions with cimetidine could be attenuated if potassium ferricyanide was added to the suspensions. The deethylation activity and cytochrome P-450 content of liver microsomes prepared from cimetidine-treated rats was decreased compared to control animals. The activity and cytochrome P-450 content of microsomes from cimetidine-treated rats could also be restored if microsomes were washed with potassium ferricyanide prior to incubation with substrate. It is proposed that an intermediate complex of cimetidine and cytochrome P-450 could be involved in the inhibition of microsomal metabolism by cimetidine.     

Cytochrome P-450 and oxidative metabolism in molluscs

1. Cytochrome P-450 and the associated components and oxidative activities of a mixed-function oxidase (MFO) system are localized primarily in the microsomes of the digestive gland of molluscs.

2. Cytochrome P-450 and putative cytochrome P-450-catalysed oxidative activities, measured in vitro and/or in vivo, have variously been detected in 23 species of mollusc.

3. Cytochrome P-450 and other MFO components and activities may be increased by exposure to xenobiotics, but the results are variable and no correlation is obvious between changes in cytochrome P-450 content and measured MFO activities (benzo[a]pyrene hydroxylase (BPH) and 7-ethoxycoumarin O-deethylase (ECOD)).

4. Type II binding compounds (clotrimazole, miconazole, ketoconazole, metyrapone and pyridine) give type II difference spectra with mussel digestive gland microsomal P-450, whereas type I binding compounds (testosterone, 7-ethoxycoumarin, α-naphthoflavone, SKF525-A) give apparent reverse type I difference spectra.

5. The existence of multiple or particular forms (P450 IVA or LAw) of cytochrome P-450 is indicated from enzyme kinetics and inhibition studies, seasonality, purification studies and cDNA probes.

6. Microsomal MFO activities are observed even in the absence of added or generated NADPH, and the NADPH-independent BPH, ECOD and N,N-dimethylaniline N-demethylase activities are inhibited by reducing agents, including NADPH.

7. The major metabolites of microsomal benzo[a]pyrene metabolism are quinones.

8. One-electron oxidation is considered to be one possible mechanism of molluscan cytochrome P-450 catalytic action.     

A novel form of cytochrome P-450 in beagle dogs. P-450-D3 is a low spin form of cytochrome P-450 but with catalytic and structural properties similar to P-450d

A form of cytochrome P-450, P-450-D3, cross reactive with antibodies to rat P-450d was purified from liver microsomes of polychlorinated biphenyl (PCB)-treated female Beagle dogs to an electrophoretic homogeneity. Judging from the result of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of P-450-D3 was estimated to be 54,000. The oxidized form of P-450-D3 showed a peak at 416 nm indicating that the cytochrome is mostly in a low spin state. The carbon monoxide bound reduced form of P-450-D3 showed a peak at 448 nm. In a reconstituted system, P-450-D3 catalyzed drug oxidations including benzphetamine and aminopyrine N-demethylations, 7-ethoxycoumarin and p-propoxyaniline O-dealkylations, and aniline and benzo(a)pyrene hydroxylations. The rate of aniline hydroxylation catalyzed by P-450-D3 was similar to that catalyzed by P-450c which is a low spin form of cytochrome P-450 purified from liver microsomes of PCB-treated rats, whereas the catalytic activities of P-450-D3 for 7-ethoxycoumarin O-deethylation and benzo(a)pyrene hydroxylation were considerably lower than those of P-450c. The amino terminal portion of P-450-D3 was found to be highly similar to those of P-450d, human P3-450 and P3-450 when four amino acid deletions were tentatively inserted between fifth and sixth amino acids from the N-terminal, but not that of P-450c which is a low spin form of cytochrome P-448 purified from rat liver microsomes. These results indicate that Beagle dogs possess a low spin form of cytochrome P-450 with spectral properties similar to P-450c but with catalytic and structural properties similar to P-450d.     

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.     

Inhibition by SKF 525-A of 7-ethoxycoumarin O-deethylation in microsomal and the reconstituted monooxygenase systems from PCB-treated rat livers

Addition of diethylaminoethyl 2,2-diphenylvalerate-HCl (SKF 525-A) to the incubation mixture containing liver microsomes or purified cytochrome P-450 (PCB P-450) from PCB (KC-500)-treated rats resulted in non-competitive inhibition of 7-ethoxycoumarin O-deethylation activity whereas the addition to the incubation mixture containing purified cytochrome P-448 (PCB P-448) showed a competitive inhibition. Fortification of PCB-induced microsomes with purified NADPH-cytochrome P-450 reductase enhanced the O-deethylation activity. With the reductase-fortified microsomes, SKF 525-A inhibited the O-deethylation in a competitive manner. Based on these results, we confirmed that SKF 525-A inhibits non-competitively and competitively, depending on the species of cytochrome P-450. Our results also support the view that in microsomes from PCB-treated rats, PCB P-450 rather than PCB P-448 is mainly involved in the O-deethylation reaction, presumably due to the presence of a limited amount of NADPH-cytochrome P-450 reductase in microsomes.     

Depression of mouse liver microsomal mixed function oxidase enzymes by adriamycin

Levels of cytochrome P-450 and cytochrome b5, and activities of NADPH cytochrome c reductase and 7-ethoxycoumarin O-deethylase, were found to be significantly decreased in hepatic microsomes prepared from mice killed 24 h after administration of a single intraperitoneal (i.p.) dose of adriamycin (ADR, 5 mg/kg). In contrast, both ascorbate-induced lipid peroxidation and conjugated dienes were increased in the same preparations. In vitro addition of ADR (5 micrograms/ml) to hepatic microsomal preparations (1 mg/ml protein) from the control mice also led to a substantial decrease in the mixed function oxidase (MFO) enzymes. A characteristic spectral change with an absorption peak at 408 nm and trough at 422 nm was associated with this in vitro interaction. It is suggested that the loss of cytochrome P-450 and related MFO enzymes due to ADR treatment is related to the generation of free radicals and subsequent lipid peroxidation in the liver.     

Effects of acetone and methyl n-butyl ketone on hepatic mixed-function oxidase

The administration of ketones potentiates CCl4 hepatotoxicity; however, the potencies of the ketones differ. The aim of the present study was to assess potential differences between acetone and methyl n-butyl ketone (MnBK) on cytochrome P-450. The effects of single and repetitive doses of acetone and MnBK were determined in male rats by estimating the rate of metabolite formation of three substrates and the hepatic content of cytochrome P-450. A single treatment with acetone (13.5 mmol/kg or greater) enhanced the oxidation of aniline and 7-ethoxycoumarin, whereas repetitive treatments also increased aminopyrine demethylation and cytochrome P-450 content. Single and repetitive treatments of MnBK (15 mmol/kg) augmented the oxidation of all three substrates and increased cytochrome P-450 content. The effects of the ketones on cytochrome P-450 isozymes were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetone and MnBK increased the 52.1 and 54.1 kD forms and, in addition, MnBK tended to increase the 50.6 kD species. The data indicate that ketones differ in the type of isozymes induced and in the degree of induction. The higher potency of MnBK, compared to acetone, is probably associated with the fact that MnBK affects a greater number of isozymes than acetone.     

Interspecies features of hepatic cytochromes P450 IA1 and P450 IA2 in rodents

1. Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) both inhibit the O-deethylation of 7-ethoxy-resorufin (ER) in liver microsomes of benzo(a)pyrene-induced (BP) mice but do not inhibit the O-deethylase activity in liver microsomes of BP-induced rats. 2. Anti-P3-450 and anti-P-450d-c inhibit BP hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this rection at all in BP-induced rat liver microsomes. 3. Isolated cytochrome P3-450 in a reconstituted monooxygenase system metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, does not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min per nmol cytochrome. 4. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes corresponding to cytochrome P1-450, 55.0 kDa, and cytochrome P3-450, 54.5 kDa. There appeared a single band (cytochrome P3-450) in interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c.     

The hepatic microsomal mixed-function oxygenase (MFO) system of Alligator mississippiensis: induction by 3-methylcholanthrene (MC)

1. Pretreatment of alligators i.p. with 3-methylcholanthrene (MC) resulted in a 1.6-fold increase (P<0.001) in cytochrome P-450 specific content and a bathochromic shift in the absorption maximum of reduced, CO-liganded microsomes (448?nm).

2. Control and MC microsomal cytochrome P-450 binding spectra with a number of type I and type II ligands were similar.

3. MC treatment of alligators resulted in a 12-fold increase in benzo[a]pyrene hydroxylase activity, which was inhibited 82% by 0.1 mM α-naphthoflavone. The turnover number (units/nmol P-450) of aminopyrine N-demethylase and 7-ethoxycoumarin O-deethylase were unaffected by MC treatment.

4. The O-dealkylation (OD) of a series of alkoxyresorufins (ethoxyresorufin (ER), methoxyresorufin (MR), benzyloxyresorufin (BR), and pentoxyresorufin (PR)) was investigated. MC treatment resulted in a significant (P<0.001) increase in turnover number of EROD, MROD, and BROD over control values. The turnover number of PROD was unaltered by MC treatment.

5. Western blots showed that control alligator microsomes contain a protein band of lower mol. wt. than either rat cytochrome P-450c (P450 IA1) or P-450d (P450 IA2), which was recognized by antibodies to both P-450c and P-450d but preferentially by that against P-450c. This protein band was induced 3-4-fold by MC. MC treatment induced a second protein band in alligator microsomes of the same mol. wt. as rat P-450d, recognized preferentially by antibodies to rat cytochrome P-450d.

6. These results illustrate that the alligator mixed-function oxidase (MFO) system responds to MC in a similar manner as described in mammals, i.e. induction in P-450 content, increases in specific MFO activities, and the apparent expression of different P-450 isoenzymes.