Microsomal Cytochrome P-450 Enzyme System Research Articles

Suppressive effect of accumulated aluminum trichloride on the hepatic microsomal cytochrome P450 enzyme system in rats

Aluminum (Al) is a low toxicological metal and can accumulate in the liver. The hepatic microsomal cytochrome P450 enzyme system (CYPS) plays important role in the transformation of the toxic materials. It is not clear if the CYPS is affected by Al exposure. Thus, the aim of this study is to investigate the effects of aluminum trichloride (AlCl3) on CYPS in rats. Forty male Wistar rats (5weeks old) weighing 110–120g were randomly allocated and orally exposed to 0, 64.18, 128.36 and 256.72mg/kg body weight (BW) AlCl3 in drinking water for 120days. The body weight (BW) of rats, hepatosomatic index (HSI), hepatic Al content, the concentrations of cytochrome P450 (CYP450), cytochrome B5 (B5), microsomal protein and the activities of NADPH-cytochrome c reductase (CR), aminopyrin N-demethylase (AND), erythromycin N-demethylase (ERND) and aniline-4-hydeoxylase (AH) were assessed at the end of the experiment. The results showed that the increase in Al concentration decreased BW, HIS, concentrations of CYP450, B5, microsomal protein and the activity of CR, AND, ERND and AH in hepatic microsomes. The results revealed that exposure to AlCl3 inhibited the microsomal CYP450 dependent enzyme system of liver. Our findings suggest that long term daily exposure of AlCl3 exerts the suppressive effects and thus may cause dysfunction of hepatic CYP450 dependent enzyme system of rat.

The effect of immunosuppressive agents (FK-506, rapamycin) on renal P450 systems in rat models.

It is well known that cyclosporin, rapamycin and FK-506 (tacrolimus) are metabolized by the liver microsomal cytochrome P450 enzyme system. Although there have been reports of interaction between these drugs and the renal P450 enzyme system, differences among these immunosuppressants has not been comprehensively demonstrated. We have studied the individual capacities of these immunosuppressants to induce renal microsomal P450 enzymes similar to CYP2B4 and CYP4A2 by examining renal function in treated rats, and have correlated the results by means of biochemical, immunological and immunohistochemical assays of renal P450 enzymes. Cyclosporin caused impairment of renal function with an increase in renal-specific P450 content, but FK-506 and rapamycin did not. Laurate omega- and (omega-1)-hydroxylase activity increased in rats treated with rapamycin but decreased in those treated with FK-506. Prostaglandin A1 (PGA1) omega-hydroxylase activity increased in rats treated with FK-506 but was reduced by treatment with cyclosporin. Aminopyrine N-demethylase activity increased in rats treated with cyclosporin or FK-506, but not in those treated with rapamycin. Western-blot analysis revealed significant induction of P450, (similar to CYP2B4 of the rabbit P450 isozyme) in kidneys from rats treated with cyclosporin but not in those from rats receiving FK-506 or rapamycin. Histochemical studies clearly demonstrated a form of P450 such as CYP4A2 in the proximal tubules of rats treated with cyclosporin, but not in those of rats treated with FK-506 or rapamycin. These results show that although cyclosporin has a strong effect on renal P450 systems and induces such a system in kidney cortex (microsomal P450), FK-506 and rapamycin have no substantial effect on the induction of renal P450. These findings might clarify the nephrotoxicity induced by these immunosuppressive drugs.

Optimization of the benzoate-inducible benzoate p-hydroxylase cytochrome P450 enzyme system in Aspergillus niger.

Introduction in the fungus Aspergillus niger of multiple copies of the A. niger bphA gene, encoding the cytochrome P450 enzyme benzoate p-hydroxylase, did not result in increased activities of this enzyme [Gorcom RFM van, et al. Mol Gen Genet (1990) 223: 192-197] probably because of low expression levels of the gene encoding the second component of the microsomal cytochrome P450 enzyme system, cytochrome P450 reductase. For improvement of this and other cytochrome-P450-dependent reactions, A. niger strains were constructed in which the copy number of the A. niger cprA gene (encoding cytochrome-P450 reductase) or the copy numbers of both cprA and the cytochrome-P450-encoding gene were increased. Expression of both genes was controlled by their own transcription control regions. Benzoate p-hydroxylase activity of different transformants was determined in microsomal fractions using a newly developed indirect in vitro assay. In transformants containing multiple copies of both genes, benzoate p-hydroxylase activity was significantly higher than in the wild-type strain or in transformants in which the copy number of only one of the genes was increased. These results clearly indicate the importance of co-expression of cytochrome-P450 reductase for achieving maximal cytochrome P450 activities in cytochrome-P450-overproducing filamentous fungi.

Metabolism of formosanin-C by cytochrome P-450

Fonnosanin c is a steroidal glycoside isolated from Paris fonnosana Hayata. It has been known from the previous studies, the growth of solid MH134 mice hepatoma was retarded by 1-2.5 mg/kg Fonnosanin c, and also significant regression in tumor SK-OV-3 (ovary) and HT-29 (colon) was induced by PF-3. The biotransfonnation of fonnosanin c by hepatic cytochrome P-450 has been investigated. The hepatic microsomal cytochrome P450 enzyme system is capable of binding and metabolising formosanin c which is shown to bind the type II site (oxygen binding site) of the enzyme at the concentrations of 150 μM. The apparent binding constants (Ks) was 78μM. The observed effect of induction of phenobarbital on A max and V max suggests that several of the multiple forms of cytochrome P450 bind formosanin c. The incubation of Formosanin c with hepatic microsomes of rats in the presense of an NADPH-generating system led to decrease of cytochrome P-450 measured by its carbon monoxide difference spectra, and the decrease was enhanced by phenobarbital treatment.

Characterization of cytochrome P-450-dependent activities in hagfish, dogfish, perch and spectacle caiman

1. 1. Owing to interest in the original function and evolution of cytochrome P-450 this enzyme system was studied in four species each at different levels on the phylogenetic tree, representing Agnatha, Schlachi, Teleostei, and Reptilia. 2. 2. All species contained considerable amounts of hepatic microsomal cytochrome P-450. The content in dogfish, perch and spectacle caiman was 0.2–0.3 while in the dogfish it was 0.6 nmol per mg protein. 3. 3. Hepatic microsomal cytochrome P-450 dependent ethylmorphine demethylase, benzo(a)pyrene hydroxylase, 7-ethoxycoumarin-O-deethylase and 7-ethoxyresorufin-O-deethylase activities have been measured and the assays were carefully characterized with respect to pH-, protein content-, incubation time- and temperature-activity relationships. 4. 4. The study revealed species-dependent differences in the liver microsomal cytochrome P-450 enzyme system, which could be due to differences in cytochrome P-450 composition or in the microenvironment around the enzyme.

Prizidilol: Metabolism by cytochrome P-450 and acetyltransferase

The hepatic microsomal cytochrome P-450 enzyme system bound and metabolized the experimental drug prizidilol. Prizidilol bound to two distinct sites on cytochrome P-450. At low concentrations (less than ca 20 μM), prizidilol bound to the substrate binding site of the enzyme and produced a Type I difference spectrum. At higher concentrations (25–190 μM), prizidilol bound to the oxygen binding site of the enzyme and produced a type II difference spectrum. Prizidilol stimulated hepatic microsomal CO-inhibitable NADPH oxidation. Prizidilol metabolism by hepatic microsomes assessed by prizidilol disappearance was inhibited by CO:O 2 (80:20; v/v), SKF 525-A and metyrapone. Prizidilol disappearance was monitored using a newly developed TLC assay for prizidilol following derivatization with quinolin-3-al. The apparent binding constants ( K s), maximum extents of binding ( ΔA max), Michaelis constants ( K m) and maximum velocities ( V max) for the interaction of prizidilol with hepatic microsomal cytochrome P-450 were assessed in rats pretreated or not with the inducing agents phenobarbital, β-naphthoflavone and pregnenolone-16α-carbonitrile. For the differently pretreated rats the apparent K s values for the type I site and the type II site and the apparent K m were ca 3 μM, 150 μM and 2 μM, respectively. Apparent V max values varied from 20 to 70 pmol per min per mg microsomal protein. The observed effects of induction on the apparent equilibrium constants and maximum extents of binding and metabolism of prizidilol indicate that the forms of cytochrome P-450 induced by phenobarbital, pregnenolone-16α-carbonitrile or β-naphthoflavone do not play a major role in the metabolism of prizidilol. Prizidilol was also metabolized by hepatic cytosolic N-acetyltransferase. The apparent K m values for prizidilol and acetyl CoA were 0.8 and 22 μM. Apparent V max values were 50 and ca 2 pmol per min per mg protein for partially purified transferase and cytosol, respectively. It is concluded that the rates of oxidation and acetylation of this drug would be expected to be relatively low, being limited by low apparent V max values for both oxidation and acetylation.

Inhalation of butanols: changes in the cytochrome P-450 enzyme system.

After inhalation of different butanol isomers for 3 days (2000 ppm) and 5 days (500 ppm), liver and kidney parameters of the microsomal cytochrome P-450 enzyme system were increased. sec-Butanol caused the highest increase in cytochrome P-450 concentration with a 47% rise in the kidneys (500 ppm for 5 days) and 33% in the liver (2000 ppm for 3 days). A concomitant increase of the in vitro n-hexane metabolism in liver microsomes was observed with a 77% increased formation of the preneurotoxic metabolite 2-hexanol compared with control. iso-Butanol did not alter total cytochrome P-450 concentration but caused a significant 30% decrease in the formation of 2-hexanol. Inhalation of all butanols slightly decreased the enzyme levels in the lung. Changes in microsomal enzymes did not correlate with measured serum concentrations of the different butanols showing different inducing capacities among the butanol isomers themselves or the participation of metabolites in the inducing process. As a conclusion sec-butanol, probably through its metabolite methyl-ethyl-ketone, is the most potent inducer of microsomal cytochrome P-450 in liver and kidney while iso-butanol does not alter total cytochrome P-450.

The role of metabolic activation of analgesics and non-steroidal anti-inflammatory drugs in the development of renal papillary necrosis and upper urothelial carcinoma

There has been no cogent hypothesis to explain the molecular basis of analgesic and non-steroidal anti-inflammatory drug (NSAID) associated renal papillary necrosis (RPN) and upper urothelial carcinoma (UUC). The microsomal cytochrome P-450 enzyme system may generate reactive intermediates which promote pathophysiological effects in the lung, liver and renal cortex, but the absence of P-450 activity in the medulla suggests that it is unlikely that similar events lead to RPN and UUC. Other enzymes (eg. peroxidases) convert substituted aromatics into benzoquinone imines (an intermediate that has previously been defined in P-450-mediated toxicity). The medulla is rich in fatty acid peroxidases involved in the metabolism of arachidonic acid. NSAID and analgesics interact with key enzymes in this pathway, which could lead to the cooxygenation of exogenous and endogenous compounds via the peroxidase, lipoxygenase, or prostaglandin hydroperoxidase enzymes. The generation of reactive molecules in the medulla could explain both RPN and UUC via the alkylation of macromolecules. The formation of free radicals would give rise to extensive lipid peroxidation, (there are large quantities of free polyunsaturated fatty acids in the medullary interstitial cells), an event of major potential importance to local cell destruction and genotoxic effects. At present this proposed mechanism of cooxygenation offers the most attractive working hypothesis to explain the molecular pathogenesis of both RPN and UUC.

The mechanistic plurality of cytochrome P-450 and its biological ramifications.

The mechanistic plurality of the microsomal cytochrome P-450 enzyme system is illustrated by studies of the oxidative metabolism of benzo[a]pyrene, 3-hydroxybenzo[a]pyrene and arachidonic acid. Rat liver microsomal metabolism of benzo[a]pyrene or 3-hydroxy-benzo[a]pyrene, supported by cumene hydroperoxide, generates benzo[a]pyrene quinones via molecular oxygen-dependent and -independent pathways. Arachidonic acid is metabolized by rat liver microsomal fractions to a variety of oxygenated products, including cis-trans diene conjugated monohydroxy-acids, epoxy-acids as well as omega- and omega-1-oxidation products. The chemistry of the different reaction products is discussed in terms of the possible mechanisms responsible for their formation and the role of the haemoprotein during catalysis. An integrated view for the reaction cycle of cytochrome P-450 is presented.

Ring- and N-hydroxylation of 2-acetylaminofluorene by reconstituted mouse liver microsomal cytochrome P-450 enzyme system

The N- and ring-hydroxylation of 2-acetylaminofluorene (AAF) are examined with a reconstituted cytochrome P-450 enzyme system from liver microsomal fractions from both control and 3-methylcholanthrene (MC)-pretreated mice. Partial purification of cytochrome P-450 fraction is achieved by bacterial protease treatment of microsomes followed by Triton X-100 solubilization and ammonium sulfate precipitation. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions are required for optimum oxidative activity. Hydroxylation activity is determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from MC-pretreated mice is several fold more active than that from controls.

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B 1 and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.

Phospholipid requirement for dimethylnitrosamine demethylation by hamster hepatic microsomal cytochrome P-450 enzyme system.

Extraction with butan-1-ol of freeze-dried microsomal fractions from livers of 3-methyl-cholarthrene-pre-treated hamsters removed about 90% of the total lipid content, but the lipid remaining proved sufficient for the cytochrome P-450 enzyme system to retain about 15-40% of its original catalytic activity for dimethylnitrosamine demethylation. Addition of butan-1-ol-extracted total phospholipid or phosphatidylcholine could not restore any activity, whereas the addition of the synthetic phospholipid dilauroyl phosphatidylcholine was able to restore almost complete activity. Synthetic dipalmitoyl or distearoyl phosphatidylcholine was ineffective in restoring the activity in this reconstituted system.