Biomedical subjects
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Streptozotocin-diabetes in rats leads to a decrease of cytochrome P-450 UT-A (the major form in control rats) and an increase of cytochrome P-450 PB-B (the major one induced by phenobarbital treatment) in liver microsomes. The increased benzphetamine-N-demethylase activity can be related to the induction of cytochrome P-450 PB-B.
Macrolide antibiotics like Erythromycin and Tri-acetyl oleandomycin (TAO) are metabolized to nitrosoderivatives which cause inactivation of Cytochrome P-450 by forming stable complex with the Iron of the hemoporphyrin. Several derivatives of erythromycin having lost their cladinose moiety are stronger inducer of liver cytochrome P-450 itself. The major form of cytochrome P-450 induced by all these macrolides in rat liver electrophoretically and immunologically indistinguishable from the major form induced by pregnenolone 16 alpha carbonitrile (PCN). This form is particularly able to metabolize macrolide and to lead to the corresponding 456 nm absorbing cytochrome P-450 complexes in vivo and in vitro.
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.
The overall cytochrome P-450 content and the immunochemically determined concentrations of constitutive isoenzymes UT-A and UT-I and isoenzymes induced by phenobarbital (PB-B), pregnenolone 16 alpha-carbonitrile (PCN-E), beta-naphthoflavone (BNF-B) and isosafrole (ISF-G) were investigated in liver microsomes from developing rats and tentatively correlated with mono-oxygenase activities. In fetuses of untreated rat, although the cytochrome P-450 was easily quantified spectrally, none of the isoenzymes tested could be immunochemically detected. After birth, each isoenzyme develops in untreated animals with its own pattern of evolution. Mono-oxygenase activities exhibited different developmental pictures. Benzphetamine-N-demethylase and lauric acid 11-hydroxylase activities progressively increased up to adult values, aniline hydroxylase activity was maximal in 15-day-old animals and benzopyrene hydroxylase and lauric acid 12-hydroxylation were increased after birth until 15 days of age in both males and females and underwent a second increase in males. Pretreatment with phenobarbital resulted in the precocious onset of PB-B in fetal and neonatal rat liver accompanied by an increase in classically associated monooxygenase activities. The PCN-E concentration was enhanced by phenobarbital pretreatment only at 15 days and in older animals. BNF-B and benzo(a)pyrene hydroxylase activity were significantly increased by 3-methylcholanthrene whatever the age considered, whereas this inductive effect upon ISF-G concentration became effective only after 2 weeks of age. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, UT-A was faint in early neonates and intensified at 15 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Monoclonal hybridomas which produce antibodies against human liver microsomal cytochrome P-450 were developed. Three similar hybridomas produced antibodies which recognized an epitope specific to a family of human P-450 isozymes (P-450(5)). This epitope was also present on cytochrome P-450 PCN-E (pregnenolone-16 alpha-carbonitrile induced) from rat liver microsomes, but this isozyme differed from the human P-450(5) by its molecular weight. These antibodies enabled us to quantify cytochrome P-450(5) in human liver microsomes and to demonstrate an important quantitative polymorphism in the human liver monooxygenase system.
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.
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Using a classical methodology of purification consisting of three chromatographic steps (Octyl-Sepharose, DEAE-cellulose, CM-cellulose) we have purified a new cytochrome P-450 from human liver microsomes. It was called cytochrome P-450(9). It has been proven to be different from all precedingly purified human liver microsomal cytochrome P-450 isozymes by its immunological and electrophoretical properties. It does not cross-react with any rat liver cytochrome P-450 and anti-cytochrome P-450(9) does not recognize rat liver microsomes; thus this cytochrome P-450(9) is specific to humans. This cytochrome P-450 isozyme exists in low amounts in human liver microsomes and exhibits an important quantitative polymorphism. In reconstituted system, cytochrome P-450(9) is able to hydroxylate all substrates tested but is not specific of any; its exact role in xenobiotic metabolism in man remains to be elucidated.
Erythromycin derivatives having lost the cladinose moiety, erythralosamine and its mono- and diacetate, are strong inducers of liver cytochrome P-450, better than troleandomycin, in rats. The major cytochrome P-450 form induced by all these macrolides is electrophoretically and immunologically indistinguishable from the major form induced in rats by pregnenolone carbonitrile. This form is particularly able to metabolize the macrolides and to lead to the corresponding 456 nm absorbing cytochrome P-450 metabolite complexes in vivo and in vitro.
The continuous infusion of a low dose of glucagon (35 micrograms/kg/d, for 5 d) constitutes, in view of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase activities, a reliable experimental model of hyperglucagonemia. By conjunction of monooxygenase assays and immunoquantitation of specific isozymes of cytochrome P-450, the actual inducing ability of glucagon has been shown and it might explain some of the modifications of the drug metabolizing system in diabetic mice. The isozymic pattern of cytochrome P-450 of liver microsomes from diabetic mice appears very different from that produced by classical inducers.
The mutagenicity of several test compounds was verified by the Salmonella/microsome mutagenicity test (Ames test), using both human liver and rat liver (untreated or pretreated with Aroclor 1254) S9 under identical experimental conditions. Aflatoxin B1, 3-methylcholanthrene, and cigarette-smoke condensate were less mutagenic in the presence of human-liver S9 than in the presence of rat-liver S9 (particularly after treatment with Aroclor 1254). The opposite was observed with 2-aminonanthracene and to a lesser degree with 2-aminofluorene; correlation studies indicate that the two compounds were activated by the same or by very similar enzymes, probably cytochrome P-450s. These results clearly indicate that human-liver S9, as an activating system, behaves differently than rat-liver S9; therefore, it may constitute a useful, additional tool for the study of mutagenicity and probably, carcinogenicity in man.
Twenty-eight samples of human liver have been characterised for cytochrome P-450 content, aldrin epoxidase, debrisoquine 4-hydroxylase and bufuralol 1'-hydroxylase activities. Evidence is presented here and elsewhere that bufuralol 1'-hydroxylase and debrisoquine 4-hydroxylase are activities catalysed by the same form of cytochrome P-450 in man, and that this form is different from that catalysing the epoxidation of aldrin. Attempts to phenotype liver samples in vitro, in the absence of any metabolic data in vivo for debrisoquine 4-hydroxylation status, met with limited success. A combination of enzyme assays will most probably be required in any such phenotyping of human liver samples.
An enzyme linked immunosorbent assay (ELISA) using monoclonal and polyclonal antibodies has been developed to quantify individual cytochrome P-450 isoenzymes in microsomal preparations, namely UT-A and PB-B. This very sensitive method can be used for the rapid processing of large quantities of determinations and requires only limited amounts of antibodies.
Monoclonal antibodies against cytochrome P-450 were prepared from phenobarbital-induced rat liver microsomes. The immunoglobulin classes and subclasses, as well as the binding capacity to cytochrome P-450, of the different antibodies were characterized. Their specificity was verified by various techniques and seemed to correspond to a single form of cytochrome P-450, the major phenobarbital-inducible form. However, the antibodies were unable to inhibit completely the monooxygenase activities investigated. These antibodies may constitute very specific and powerful analytical tools for characterizing and quantifying cytochrome P-450 isoenzymes.
Six cytochrome P-450 (P-450) isozymes were purified to electrophoretic homogeneity from the livers of four human organ donors, with three of these isozymes purified from a single individual. Differences were noted between all six P-450s for some or all of the parameters determined by the techniques of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, spectral analysis of ferrous-carbon monoxide complexes, double-diffusion immunoprecipitin analysis or crossed immunoelectrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis/peroxidase-coupled staining) with rabbit antisera raised to five of the P-450s, or catalytic activity toward d-benzphetamine, benzo[a]pyrene, acetanilide, debrisoquine, (R)- and (S)-warfarin, and 1-naphthylamine. While NADPH-fortified human liver microsomal preparations showed catalytic activity toward trichloroethylene, 7-ethoxycoumarin, 2-naphthylamine, and 2-aminofluorene in addition to the other substrates mentioned, none of the P-450s which we purified from these microsomes catalyzed the oxidation of these compounds in reconstituted enzyme systems containing purified rat liver NADPH-P-450 reductase. Antibodies raised against one of the purified P-450s inhibited d-benzphetamine N-demethylase activity in microsomal incubations but did not inhibit the metabolism of 7-ethoxycoumarin, acetanilide, benzo[a]pyrene, or debrisoquine. The data provide a strong biochemical basis for the view that distinct isozymes of P-450 exist in humans and that these isozymes differ in catalytic activity toward drugs and carcinogens.
This preliminary study reports the functional capacities of freshly isolated human hepatocytes in regard to their energetic metabolism and monooxygenase activities. Incubated for 30 or 60 min, isolated cells maintain their membrane integrity, ATP and reduced glutathione content and redox potential estimated by means of lactate to pyruvate and beta-hydroxypyruvate to acetoacetate ratios. Three monooxygenase activities, supported by different isoenzymes of cytochrome P-450 are determined by the accumulation of unconjugated metabolites: their relative magnitudes are similar to those observed in microsomes, indicating a good preservation of hydroxylase activities during cell isolation and incubation. Although incubations did not exceed 60 min, one can conclude that human hepatocytes maintain their viability and metabolic capacities after isolation and might be considered in transplantation process for the treatment of acute hepatic failure. Isolated human hepatocytes might be also used as a tool for studying biochemical and toxicological effects of a drug.
Drug-metabolizing enzyme activities were determined in liver microsomes from six kidney-transplant donors, one tricyclic antidepressant-treated and five untreated donors. The tricyclic antidepressant treatment modifies neither the overall cytochrome P-450 content of the liver, nor enzymatic activities of 4-nitroanisole demethylase, aniline hydroxylase, epoxide hydrolase and glutathione S-transferase. Only benzphetamine and ketotifen demethylation and conjugation of bilirubin with UDP-glucuronic acid are markedly augmented (more than two-fold). Separation of the different cytochrome P-450 fractions on a DEAE cellulose column indicates a modification of the elution pattern: the fraction increased by tricyclic antidepressants is responsible for the enhanced monooxygenase activity towards benzopyrene and benzphetamine.
A pharmacokinetic study of sodium valproate (DPA) was undertaken in children treated with this drug after a febrile convulsion. In infants, after a single oral dose of 20 mg/kg body weight in the first morning meal, plasma kinetic data were as follows: T max 2.4 hr +/- 1.1; total DPA maximum plasma concentration: 94 micrograms/ml +/- 28; T 1/2: 12.4 hr +/- 4.9; the distribution volume was 0.25 l/kg +2- 0.07 and plasma clearance was 0.014 l/kg/hr +2- 0.004. Control of prolonged treatment showed that the measured plasma concentrations were not correlated with the daily dosage. Furthermore, when febrile convulsions are concerned, one should carefully weight the recently reported hepatic toxicity of DPA, against the usually good prognosis of febrile convulsions.