Pyrazole treatment of rats potentiates CCL4-but not CHCL3-hepatotoxicity

Rats were treated with pyrazole to increase the liver content of the "alcohol-inducible" form of cytochrome P-450. This treatment increased the sensitivity of these animals to CCl4-hepatotoxicity assessed by increases in SGPT and SGOT levels and decreases in microsomal cytochrome P-450 and aniline p-hydroxylase activity. However, the hepatotoxicity of CHCl3 was not increased by pyrazole-treatment. These data are consistent with the hypothesis that the "alcohol-inducible" form of cytochrome P-450 is capable of CCl4- but not CHCl3-activation.     

Vitamin A and lipid peroxidation: effect of retinol deficiency

The effect of alimentary vitamin A deficiency on some parameters of lipid peroxidation (LPO) in young rats was studied. It was found that under vitamin A deficiency the content of diene conjugates in liver homogenates and microsomes diminishes, whereas that of malonic aldehyde in small intestinal mucosa, liver and testis homogenates is unaffected. However, the malonic aldehyde production in liver homogenates and microsomes decreases after 60 min incubation at 37 degrees C without addition of prooxidants. At the same time, enzymatic NADPH-dependent and nonenzymatic ascorbate-dependent LPO in liver microsomes of vitamin A-deficient rats does not change significantly. The decrease of LPO intensity in vitamin A-deficient animals may be due to the reduced content in liver microsomes of the main LPO substrates, i.e., arachidonic and linoleic acids, as well as to the decrease of cytochrome P-450 level in rat liver.     

Effect of vitamin A deficiency on hepatic and intestinal drug metabolizing enzymes in rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks caused significant reduction in the hepatic cytochrome P-450, cytochrome b5, aminopyrine N-demethylase and arylhydrocarbon hydroxylase activities. Contrary to this, the levels of these Phase I enzymes were found to be significantly elevated in all the 3 portions (proximal, middle and distal) of the intestine in deficient animals as compared to corresponding pair-fed controls. Of the Phase II enzymes studied, UDP-glucuronyltransferase showed a significant decrease whereas glutathione S-transferase showed a significant increase in vitamin A-deficient rat liver and small intestine. The study suggests that vitamin A deficiency causes an imbalance between the Phase I and phase II drug metabolizing enzyme systems which may decrease the capacity of the organism to withstand the neoplastic effects of chemical carcinogens in vitamin A deficiency.     

Liver cytochrome P-450 system in rats with a varying susceptibility to the development of experimental alcoholism

The cytochrome P-450-dependent monooxygenase system of the liver was studied in laboratory noninbred male rats selected according to the intensity of their initial alcohol motivation and the dynamics of these parameters was followed up during 10-day alcoholisation. It was shown that in the animals inclined to the development of alcoholism the activity of the monooxygenase system (cytochrome P-450, B5; enzymes: aminopyrine N-demethylase, aniline p-hydroxylase, NADPH-cytochrome c-reductase) is higher than in the animals noninclined to the development of this disease. 10-day alcohol consumption in the free-choice situation between water and 15% ethanol solution did not change the parameters investigated. The only exception was NADPH-cytochrome c-reductase: its activity grew in both the groups of the animals by 40-75%.     

Effect of DDT on hepatic mixed-function oxidase activity in normal and vitamin A-deficient rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks resulted in significant decrease in the body weight and marked reduction in the hepatic vitamin A content. The levels of hepatic phase I microsomal enzymes cytochrome P-450, cytochrome b5, aminopyrine N-demethylase and arylhydrocarbon hydroxylase were found to be substantially reduced by vitamin A-deficiency. Also, the activity of phase II microsomal UDP - glucuronyl transferase enzyme was significantly decreased in deficient animals. Following repeated oral administration of DDT (15 mg/kg/body wt/day) for 21 days, the phase I microsomal enzymes were induced to a greater extent in controls as compared to deficient animals. UDP - glucuronyltransferase remained insensitive to DDT induction. The results imply that the capacity for induction of the hepatic mixed-function oxidase enzyme system is impaired in deficient animals concurrently exposed to DDT.     

A model for the regulation of δ-aminolaevulinate synthetase induction in rat liver

A reciprocal relationship exists between the cytochrome P-450 content and delta-aminolaevulinate synthetase activity in adult rats. In young rats the basal delta-aminolaevulinate synthetase activity is higher and the cytochrome P-450 content is lower compared with the adult rat liver. Administration of allylisopropylacetamide neither induces the enzyme nor causes degradation of cytochrome P-450 in the young rat liver, unlike adult rat liver. Allylisopropylacetamide fails to induce delta-aminolaevulinate synthetase in adrenalectomized-ovariectomized animals or intact animals pretreated with successive doses of the drug, in the absence of cortisol. The cortisol-mediated induction of the enzyme is sensitive to actinomycin D. Allylisopropylacetamide administration degrades microsomal haem but not nuclear haem. Haem does not counteract the decrease in cytochrome P-450 content caused by allylisopropylacetamide administration, but there is evidence for the formation of drug-resistant protein-bound haem in liver microsomal material under these conditions. Phenobarbital induces delta-aminolaevulinate synthetase under conditions when there is no breakdown of cytochrome P-450. On the basis of these results and those already published, a model is proposed for the regulation of delta-aminolaevulinate synthetase induction in rat liver.     

Ring-andN-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system.

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.     

Effect of vitamin A deficiency on the pulmonary and hepatic drug-metabolizing enzymes in rat

The effect of vitamin A deficiency on the drug-metabolizing enzyme system of the lung and liver was analyzed in rats fed diets with or without vitamin A for 5-6 weeks. The hepatic level of vitamin A was significantly reduced in vitamin A deficient animals. The hepatic cytochrome P-450 and b5 contents and activity of benzo(a)pyrene hydroxylase was significantly reduced in deficient animals. Contrary to this, pulmonary cytochrome P-450 and b5 contents were above the control values. No alteration in pulmonary benzo(a)pyrene hydroxylase was noted. The uridine diphosphate-glucuronosyltransferase activity of digitonin-treated microsomal membranes was below the control values both in lung and liver. However, the native uridine diphosphate-glucuronosyltransferase activity remained unchanged in the liver and was below control values in the lung.     

Differences in the metabolism of vitamin D2 and vitamin D3 by subcellular fractions from rat liver

The 25-hydroxylation of vitamin D2 and vitamin D3 was studied in the mitochondrial fraction from rat liver and in a reconstituted system containing cytochrome P-450 from rat liver microsomes. The mitochondrial fraction catalyzed the 25-hydroxylation of vitamin D3 at least two times more effectively than the 25-hydroxylation of vitamin D2. Microsomal cytochrome P-450 catalyzed an efficient 25-hydroxylation of vitamin D3, but no 25-hydroxylation of vitamin D2 could be detected. The present results show a difference in the 25-hydroxylation of vitamin D2 and vitamin D3 in rat liver in vitro.     

25-hydroxylation of C27-steroids and vitamin D3 by a constitutive cytochrome P-450 from rat liver microsomes

A constitutive cytochrome P-450 catalyzing 25-hydroxylation of C27-steroids and vitamin D3 was purified from rat liver microsomes. The enzyme fraction contained 16 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum molecular weight of 51,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified cytochrome P-450 catalyzed 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, and 1 alpha-hydroxyvitamin D3 up to 50 times more efficiently, and 25-hydroxylation of vitamin D3 about 150 times more efficiently than the microsomes. The cytochrome P-450 showed no detectable 25-hydroxylase activity towards vitamin D2 and was inactive in cholesterol 7 alpha-hydroxylation as well as in 12 alpha- and 26-hydroxylations of C27-steroids. It catalyzed hydroxylations of testosterone and demethylation of ethylmorphine at the same rates as, or lower rates than, microsomes. The 25-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and vitamin D3 with the purified cytochrome P-450 was not stimulated by addition of phospholipid or cytochrome b5 to the reconstituted system. Emulgen inhibited 25-hydroxylase activity towards both substrates. The possibility that 25-hydroxylation of C27-steroids and vitamin D3 is catalyzed by the same species of cytochrome P-450 is discussed.     

25-Hydroxylation of vitamin D3 by a cytochrome P-450 from rabbit liver mitochondria.

A cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 9 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 52,000 upon SDS/polyacrylamide-gel electrophoresis. The preparation showed a single protein spot with an apparent isoelectric point of 7.8 and an Mr of approx. 52,000 upon two-dimensional isoelectric-focusing-polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 5000 times more efficiently than did the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 catalysed, in addition to 25-hydroxylation of vitamin D3, the 25-hydroxylation of 1 alpha-hydroxyvitamin D3 and the 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol. The enzyme did not catalyse side-chain cleavage of cholesterol, 11 beta-hydroxylation of deoxycorticosterone, 1 alpha-hydroxylation of 25-hydroxyvitamin D3, hydroxylations of lauric acid and testosterone or demethylation of benzphetamine. The results raise the possibility that the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of C27 steroids are catalysed by the same species of cytochrome P-450 in liver mitochondria. The possible role of the liver mitochondrial cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

25-Hydroxylation of vitamin D3 by a reconstituted system from rat liver microsomes.

Using isotope dilution—mass fragmentography as assay technique, it was shown that highly purified preparations of cytochrome P-450 from rat liver microsomes catalyzed 25-hydroxylation of vitamin D₃ when combined with NADPH-cytochrome P-450 reductase and a phospholipid. The rate of conversion was approximately linear with the amount of cytochrome P-450, and was considerably higher than the rate of conversion obtained with crude liver microsomes. The possibility is discussed that the microsomal fraction contains inhibitors of 25-hydroxylase activity, which may be of regulatory importance in vitamin D₃ metabolism.     

Role of splenin and its active factor in regulating liver monooxygenase system in experimental immunodeficiency and hepatosohepatitis

An influence of splenin and its non-peptide factor of splenin (NFS) on the state of cytochrome P-450 dependent monooxygenase system (MOS) of liver microsomes in healthy animals under immunodeficiency (splenectomy, administration of gamma-aminobutyric acid (GABA) and toxic hepatosohepatitis was studied. The stimulating action of splenin and NFS on cytochrome P-450 content and MOS activity of liver microsomes in healthy animals has been established. The indices studied markedly decreased after splenectomy. The splenin or NFS administrations promote the recovery of these indices up to starting level in asplenic animals. A decrease in thymic mass dependent in GABA administration is prevented by NFS pretreatment of animals; there is no any effect of mediator acid on cytochrome P-450 content and MOS activity was noted. The preliminary administration NFS potentiates hepatotoxic effect of carbon tetrachloride and increases its inhibitory effect on P-450 dependent MOS of liver microsomes. Under the NFS action the effect in activity of the last is caused by the factor influence on the reparative processes in the liver.     

Liver cytochrome P-450 induction in rats by drug preparations and the body vitamin A allowance

The content of P-450 cytochrome and vitamin A was determined in the liver of mature male rats who received for a month daily parenteral administrations of phenobarbital solutions (Pb; 40 mg/kg), rheopyrine (a mixture of equal aliquots of amidopyrine and butadione; 200 mg/kg), amidopyrine (100 mg/kg) or water (control). The animals were kept on a semisynthetic diet, receiving once, every week 400 IU of retinol-palmitate per rat. Pb administration markedly (more than threefold) increased P-450 cytochrome content in the liver. Rheopyrine and amidopyrine also elevated its level, but to a lesser extent than Pb. Pb and rheopyrine also depressed vitamin A levels in the liver and caused either a trend towards its decrease (Pb) or a significant decrease (rheopyrine) of its overall content in the liver. The effect of amidopyrine on the concentration and storage of retinol in the liver was less pronounced. The data obtained suggest that the drugs inducing P-450 cytochrome are capable of disturbing vitamin A content in the body.     

Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells.

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.     

Effects of dietary benzo(a) pyrene on intestinal phase I and phase II drug metabolizing systems in normal and vitamin A-deficient rats

Feeding of vitamin A-deficient diet to male weanling rats for 10 weeks caused significant increase in the activities of Phase I enzyme system, i.e., cytochrome P-450, cytochrome b5 and arylhydrocarbon hydroxylase in the proximal, middle and distal segments of the intestine. Of the Phase II enzymes studied, UDP-glucuronyltransferase showed significant decrease whereas glutathione S-transferase showed significant increase. Treatment with benzo(a)pyrene caused greater induction in the levels of Phase I enzymes in deficient animals as compared to controls. In contrast to this, benzo(a)pyrene treatment induced the level of UDP-glucuronyltransferase in control rats more than in deficient rats. Intestinal NADPH cytochrome C-reductase and glutathione S-transferase remained insensitive to benzo(a)pyrene induction.     

Effect of vitamin A deficiency on pulmonary and hepatic in vitro metabolism of benzo(a)pyrene in rat

The metabolism of (3H)-benzo(a)pyrene and the activities of enzymes involved in its metabolism were studied in rat lung and liver in vitamin A deficiency. Deficiency of vitamin A resulted a significant decrease in the overall metabolism of benzo(a)pyrene in the liver in vitro, whereas no significant difference was evident in the lung. The ethyl acetate-soluble metabolites of benzo(a)pyrene formed by lung and liver preparations were unaltered qualitatively by vitamin A deficiency. However, quantitative analysis revealed that vitamin A deficiency decreased the yield of dihydrodiols, quinones and phenols in liver, and dihydrodiols in lung. The hepatic cytochrome P-450 content, arylhydrocarbon hydroxylase and uridine diphosphate-glucuronosyl transferase activities were reduced, whereas glutathione S-transferase activity was increased in the vitamin A deficient animals. Contrary to this, pulmonary cytochrome P-450 content was above the control values (p less than 0.01) and no alteration in pulmonary arylhydrocarbon hydroxylase activity was observed in vitamin A deficient rats. Uridine diphosphate-glucuronosyltransferase and glutathione S-transferase activities were impaired in lung by inducing vitamin A deficiency. However, no significant difference was evident in the overall metabolism of benzo(a)pyrene by lung supernatants from the two groups.     

Reconstituted hamster liver microsomal enzyme system for N-hydroxylation of the carcinogen 2-acetylaminofluorene

A procedure is described for the isolation of cytochrome P-450 fraction from hamster liver microsomes. It involves removal of NADPH-cytochrome c reductase activity by treatment with bacterial protease before solubilization with Triton X-100 and precipitation with ammonium sulfate. Reconstitution studies indicate that 2-acetylaminofluorene $\text{N}$-and ring-hydroxylation require both cytochrome P-450 fraction and the reductase fraction. $\text{N}$-hydroxylation activity of cytochrome P-450 fraction from 3-methylcholanthrene pretreated hamsters is different and severalfold greater than that of cytochrome P-450 fraction from controls. These results demonstrate for the first time an activation of a chemical carcinogen by a reconstituted cytochrome P-450 enzyme system.     

Purification of a cytochrome P-450 from pig kidney microsomes catalysing the 25-hydroxylation of vitamin D3.

Cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 was purified from pig kidney microsomes. The enzyme fraction contained 7 nmol of cytochrome P-450/mg of protein and showed only one protein band with an apparent Mr of 50,500 upon SDS/polyacrylamide-gel electrophoresis. The purified cytochrome P-450 catalysed 25-hydroxylation of vitamin D3 up to 1,000 times more efficiently, and 25-hydroxylation of 1 alpha-hydroxyvitamin D3 up to 4000 times more efficiently, than the microsomes. The cytochrome P-450 required microsomal NADPH-cytochrome P-450 reductase for catalytic activity. Mitochondrial ferredoxin and ferredoxin reductase could not replace microsomal NADPH-cytochrome P-450 reductase. The enzyme preparation showed no detectable 25-hydroxylase activity towards vitamin D2 or 1 alpha-hydroxylase activity towards 25-hydroxyvitamin D3. CO inhibited the 25-hydroxylation by more than 85%. Mannitol, hydroquinone, catalase and superoxide dismutase did not affect the 25-hydroxylation. The possible role of the kidney microsomal cytochrome P-450 in the metabolism of vitamin D3 is discussed.     

Purification of hepatic microsomal cytochromes P-450 from beta-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

Four isozymes of cytochrome P-450 were purified to varying degrees of homogeneity from liver microsomes of cod, a marine teleost fish. The cod were treated with beta-naphthoflavone by intraperitoneal injection, and liver microsomes were prepared by calcium aggregation. After solubilization of cytochromes P-450 with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate, chromatography on Phenyl-Sepharose CL-4B, and subsequently on DEAE-Sepharose, resulted in two cytochrome P-450 fractions. These were further resolved on hydroxyapatite into a total of four fractions containing different isozymes of cytochromes P-450. One fraction, designated cod cytochrome P-450c, was electrophoretically homogeneous, was recovered in the highest yield and constituted the major form of the isozymes. The relative molecular mass of this form (58 000) corresponds well with a protein band appearing in cod liver microsomes after treatment with beta-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56000) showed activity towards 7-ethoxyresorufin in a reconstituted system containing rat liver NADPH-cytochrome P-450 reductase and phospholipid. Differences between these two forms were observed in the rate and optimal pH for conversion of this substrate, and in optical properties. Rabbit antiserum to cod cytochrome P-450c did not show any cross-reactions with cod cytochrome P-450a (Mr 55000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identity with cod cytochrome P-450b (Mr 54000), possibly as a result of contaminating cytochrome P-450c in this fraction.