Cytochrome P450-dependent Mixed Function Research Articles

Cytochrome P450-dependent mixed-function oxidase and glutathione S-transferase activities in spontaneous obesity-diabetes

The effect of non-insulin-dependent diabetes on the hepatic microsomal cytochrome P450-dependent mixed-function oxidase system and on cytosolic glutathione S-transferase activity was determined using the spontaneously obese-diabetic (ob/ob) mouse model. The activities of the xenobiotic-metabolizing cytochrome P450 proteins were monitored by the use of chemical probes. Non-insulin-dependent diabetes did not influence the hepatic metabolism of substrates associated with the P450 I, IIB, IIE, III and IV families of cytochromes. In contrast, cytosolic glutathione S-transferase activity was markedly reduced and glutathione levels were significantly lowered. These findings raise the possibility that patients suffering from this disease may be more susceptible to chemicals that rely on glutathione conjugation for their deactivation.

An Overview of the Mechanisms of Action of Herbicide Safeners

Abstract Herbicide safeners are chemicals used for manipulating the tolerance of large-seeded grass crops to selected soil-applied herbicides. The physiological interactions of herbicides and their respective safeners are characterized by the following facts: a) safeners are most effective when applied prior to or simultaneously with the herbicides whose injury they prevent; b) safeners exhibit a high degree of botanical and chemical specificity protecting only certain grasses against injury caused from specific classes of herbicides; and c) protected grass crops are moderately tolerant to the antagonized herbicides. At the biochemical level, safeners may act either as “bioregulators” regulating the amount of a given herbicide that reaches its target site in an active form or as “antagonists” of herbicidal effects at a similar site of action. A safener-induced enhancement of herbicide detoxication in protected plants is currently viewed as the most apparent mechanism for the action of the currently available safeners. Safeners enhance the conjugation of carbamothioate and chloroacetanilide herbicides with glutathione either by elevating the levels of reduced glutathione (GSH) or by inducing the activity of specific glutathione S̱-transferases (GSTs). A safener-induced enhancement of the activity of other degradative enzymes such as the cytochrome P450-dependent mixed function oxidases or UDP-glucosyl transferases seems to be important for the protective action of safeners against injury from aryloxyphenoxypropionate, imidazolinone, and sulfonylurea herbicides. Metabolic processes related to acetyl-CoA metabolism have been implicated as likely target sites for a competitive antagonism between safeners and chloroacetanilide or carbamothioate herbicides. At the molecular level, the “gene activation” and “gene amplification” theories offer a likely explanation for the action of safeners.

The effects of dimethylsulphoxide and 5-aminolaevulinic acid on the activities of cytochrome P450-dependent mixed function oxidase and UDP-glucuronosyl transferase activities in human Hep G2 hepatoma cells

The effects of dimethylsulphoxide and 5-aminolaevulinic acid on the activities of cytochrome P450-dependent mixed function oxidase and UDP-glucuronosyl transferase activities in human Hep G2 hepatoma cells

The urotoxic effects of N,N′-dimethylaminopropionitrile : 2. In vivo and in vitro metabolism

The urotoxicity and metabolism of N,N′-dimethylaminopropionitrile (DMAPN) were investigated in male Sprague-Dawley rats. Animals treated with 525 mg DMAPN/kg or equimolar doses of commercially available potential DMAPN metabolites showed varying levels of urinary retention. About 44% of the administered dose of DMAPN was excreted unchanged in 5 days. β-Aminopropionitrile and cyanoacetic acid were identified as urinary metabolites. The urinary excretion of cyanoacetic acid was nonlinearly proportional to the volume of urine retained in the bladders. In vitro, the metabolism of DMAPN to cyanide, formaldehyde, and cyanoacetic acid was localized mostly in the microsomal fraction of liver, kidney, and urinary bladders. This reaction required NADPH and oxygen for maximal activity. Metabolism of DMAPN was increased in hepatic microsomes obtained from phenobarbital-treated rats (220% of control) and decreased following CoCl 2 treatments (73% of controls). Addition of SKF 525-A to the incubation mixtures inhibited the metabolism of DMAPN to formaldehyde (47–64% of controls). Addition of sulfhydryl compounds (glutathione and cysteine) to the incubation mixtures did not affect the rate of these reactions. These findings indicate that DMAPN is primarily metabolized via a cytochrome P450-dependent mixed-function oxidase system and that the urotoxic effects of DMAPN may be related to this metabolism.

Rat adult hepatocytes in primary pure and mixed monolayer culture: Comparison of the maintenance of mixed function oxidase and conjugation pathways of drug metabolism

The stabilities of several drug oxidation and conjugation pathways in adult rat hepatocytes were investigated in two systems: a primary pure culture lasting 3 days and a primary mixed culture (hepatocytes co-cultured with epithelial cells) lasting 10 days. The cytochrome P450 content in hepatocytes drastically declined within 48 hr in both culture systems. Cytochrome P450-dependent mixed function oxidase was measured by the O-dealkylation of ethoxyresorufin (EROD) and of pentoxyresorufin (PROD). UPD-glucuronosyl transferase (UDP-GT) activity was measured using 1-naphthol and morphine as substrates. In both culture systems, the activities of enzymes belonging to the 3-methylcholanthrene-inducible family, namely EROD and 1-naphthol UDP-GT, were much better maintained than those of PROD and morphine UDP-GT, which belong to the phenobarbitone-inducible family: in pure cultures, EROD and 1-naphthol UDP-GT activities declined to 60% of initial values within 3 days; in mixed cultures, EROD activity was stable throughout the 10 day culture period, whereas that of 1-naphthol UDP-GT was stable until day 4 but had declined to 70% of the initial value by day 8. In contrast, PROD and morphine UDP-GT activities declined to approx. 30% of the initial values within 2 days in both culture systems, and had dropped to approx. 10% of the initial value within 8 days in mixed culture. Reduced glutathione (GSH) levels fluctuated, but remained high throughout culture. GSH conjugation declined to 40% of initial values within 3 days in pure culture, whereas it remained relatively constant in mixed culture. Comparison of these two culture systems therefore showed that although the inclusion of epithelial cells did prolong hepatocyte viability, there was a change in relative enzyme activities in both systems, suggesting a shift towards a more dedifferentiated drug metabolism pattern.

Identification of the cytochrome P450 IIIA family as the enzymes involved in the N-demethylation of tamoxifen in human liver microsomes

The antiestrogen tamoxifen (Tam or Nolvadex, ICI)—Z-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1,2-diphenyl-1-butene is widely used in treatment of hormone-dependent breast cancer. The drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-desmethyl metabolite (DMT). This study has been carried out to determine the P450 enzyme involved in the N-oxidative demethylation of Tam in microsomal samples from 25 human livers (23 adults, two children). This metabolic step was inhibited by carbon monoxide up to 75%. Tam was demethylated into DMT with an apparent K m of 98 ± 10 μM; rates varied between 37 and 446 pmol/min/mg microsomal protein. These metabolic rates were strongly correlated with 6β-hydroxylation of testosterone ( r = 0.83) and erythromycin N-demethylase ( r = 0.75), both activities known to be associated with P450 IIIA enzyme. To further assess whether or not the Tam demethylation pathway is catalysed by the same P450, the inhibitory effect of TST on this reaction was determined. The competitive inhibition had an apparent K i of 100 ± 10 μM. Drugs such as erythromycin, cyclosporin, nifedipine and diltiazem were shown to inhibit in vitro the metabolism of tamoxifen. Furthermore the P450 IIIA content of liver microsomal samples, measured by Western blot technique using a monoclonal P450NF (nifedipine) antibody, was strongly correlated with DMT formation ( r = 0.87). Tam N-demethylase activity was inhibited by more than 65% with polyclonal anti-human anti-P450NF. All these in vitro observations establish that a P450 enzyme of the IIIA sub-family is involved in the oxidative demethylation of tamoxifen in human liver.

Biochemical toxicology of argemone oil. I. effect on hepatic cytochrome P‐450 and xenobiotic metabolizing enzymes

The in vivo effect of argemone oil on hepatic xenobiotic metabolizing enzymes was investigated in albino rats following either a single (10 ml kg-1 body wt.) or multiple intraparenteral doses (5 ml kg-1 body wt.) for three days. Animals sacrificed 72 h after a single intraparenteral dose of argemone oil exhibited a significant loss of hepatic cytochrome P-450 (35%) and cytochrome b5 (34%) contents and inhibition of aminopyrine-N-demethylase (APD), aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin-O-deethylase (ECD) activities (21-39%). Three successive 24-hourly intraparenteral injections of argemone oil followed by sacrificing the animals after 24 h of the last injection, showed a greater degree of inhibition of the content of cytochrome P-450 (58%) and its dependent mixed-function oxidases (35-63%). Also, multiple treatment of argemone oil caused a depletion of endogenous hepatic glutathione (GSH) content (72%) with a concomitant increase in lipid peroxidation (177%) and decrease in glutathione-S-transferase (GST) activity (30%). A significant decrease in relative liver weight (39%) was observed in animals treated with multiple treatment of argemone oil. These results suggest that argemone oil can alter both membrane and cytosolic defences and destabilizes the hepatic cytochrome P-450 dependent mixed-function oxidase system, so that it tips in the direction of autooxidative peroxidation of lipids.

Effects of the immunosuppressant FK-506 and its analog FK-520 on hepatic and renal cytochrome P450 mixed-function oxidase

The novel immunosuppressant FK-506 and its analog FK-520 were found to inhibit the hepatic microsomal mixed-function oxidase system in male Sprague-Dawley rats. At 5 and 10mg/kg/ day, s.c., for 6 days they caused 30–80% decreases in cytochrome P450 levels, NADPH-cytochrome P450 reductase, and benzphetamine N-demethylase activities. The metabolism of FK-506 itself was inhibited by 50%. FK-506 and FK-520 had a minimal effect on the renal cytochrome P450 levels unlike cyclosporin A which produced a 67% increase after six daily 25 mg/kg doses. A single dose of FK-506 (25 mg/kg, s.c.) had a minimal effect on the hepatic or renal metabolizing enzyme system. In vitro, addition of FK-506 and FK-520 to human and control rat liver microsomes resulted in a concentration-dependent inhibition of benzphetamine N-demethylation (10–20% at 50 μM, 60–75% at 250 μM). We suggest that in view of its potential to inhibit hepatic cytochrome P450-dependent mixed-function oxidase, resulting in the inhibition of its own metabolism, FK-506 should be administered with caution to transplant patients.

Characteristics of the 25-hydroxyvitamin D 3- and 1,25-dihydroxyvitamin D 3-24-hydroxylase(s) from HL-60 cells

1,25-Dihydroxyvitamin D 3 induces the human promyelocyte leukemia cell line, HL-60, to differentiate into macrophages/monocytes via a steroid-receptor mechanism. This system is a relevant one for an investigation of the molecular mechanism of 1,25-dihydroxyvitamin D 3. We have now examined the effect of 1,25-dihydroxyvitamin D 3 on the induction of 1,25-dihydroxyvitamin D 3- and 25-hydroxyvitamin D 3-24-hydroxylase activities in HL-60 cells. The hydroxylase activities were measured by a periodate-based assay, which was validated by comparison with well-established HPLC analysis. HPLC analysis also suggested that 1,25-dihydroxyvitamin D 3 induces a 23-hydroxylase in addition to the 24-hydroxylase. 1,25-Dihydroxyvitamin D 3- and 25-hydroxyvitamin D 3-24-hydroxylase activities were stimulated as early as 4 h after the addition of 10 −7 m 1,25-dihydroxyvitamin D 3 and became maximal by 24 h. 1,25-Dihydroxyvitamin D 3 stimulated both activities in a dose-dependent manner up to 10 −6 m. The K m of 24-hydroxylase for 1,25-dihydroxyvitamin D 3 and 25-hydroxyvitamin D 3 were 2 × 10 −8 m and 5.2 × 10 −7 m, respectively. Cycloheximide (5 μ m) inhibited 1,25-dihydroxyvitamin D 3-mediated stimulation of 24-hydroxylase activity. Other differentiation inducers, such as retinoic acid and phorbol ester, did not induce either activity. 1,25-Dihydroxyvitamin D 3-24-hydroxylase in HL-60 mitochondria was solubilized with 0.6% cholate and reconstituted with NADPH, beef adrenal ferredoxin, and beef adrenal ferredoxin reductase, each component being essential for 24-hydroxylase activity. These results strongly suggest that the 24-hydroxylase in HL-60 cells is a three-component cytochrome P450-dependent mixed-function oxidase.

The Effect of Tetrahydrofuran on Biological Systems: Does a Hepatotoxic Potential Exist?

Tetrahydrofuran (THF) is a widely used solvent in industry and research. THF is a weak toxin, with approximate acute LD50s in the range of 2 to 3 g/kg, 8 to 20 mg/L, and 800 mg/kg following oral, inhalation, and i.v. routes, respectively. The two primary signs of intoxication are narcosis and hepatocellular dysfunction, both occurring at doses of approximately one-half of the lethal dose. Little is known concerning the pharmacokinetics of THF. No evidence exists for genotoxicity due to THF. Ongoing carcinogenicity bioassay tests sponsored by the National Toxicology Program have not yet been completed. Two significant interactions of THF with cellular components have been studied; first, THF inhibits a number of enzymatic reactions at concentrations ranging from 10 to 100 mM. Most notably, THF is an inhibitor of a number of cytochrome P450 (P450) dependent mixed function oxidase activities, with a particular affinity for the alcohol-induced isozyme (P450IIE1). Second, THF has been noted to enhance the toxic action of a number of compounds (i.e. solvent effect), in particular stimulating the more rapid absorption of reactive metabolites. A third factor to be considered is THF's ability to form peroxides upon standing. Little is known concerning the toxicity of THF peroxides. Therefore, while there is little evidence to suggest that THF would be a direct (Type I) hepatotoxin at relatively low doses, its ability to inhibit drug-metabolizing reactions (perhaps more so in alcohol users), and enhance the absorbance of reactive metabolites, are relatively unexplored avenues for THF to contribute to a hepatotoxic response. As a great deal of the human hepatotoxicity (Type II) is "idiosyncratic" and cannot be readily reproduced in experimental animals, this potential route of hepatotoxicity should be considered in future evaluations of human exposure to THF.

In vivo and in vitro effects of helenalin on mouse hepatic microsomal cytochrome P450

Helenalin, a natural plant product with significant antitumor activities, decreased male BDF1 mouse hepatic microsomal cytochrome P450 contents in vivo and in vitro. A single i.p. dose of 25 mg helenalin/kg body weight significantly (P < 0.05) decreased microsomal cytochrome P450 contents and inhibited cytochrome P450-dependent mixed-function oxidase activities within 1–2 hr post-exposure. Helenalin (1.0 mM) decreased microsomal cytochrome P450 contents in vitro by 11% in the absence of NADPH and by 32% in the presence of NADPH. These in vitro and in vivo decreases in cytochrome P450 were accompanied by comparable decreases in total microsomal heme contents. Helenalin (1.0 mM) increased mouse hepatic microsomal oxygen consumption and NADPH utilization by 3.2 and 5.4 nmol/min/mg protein respectively. Helenalin (1.0 mM) significantly (P < 0.05) increased microsomal lipid peroxidation in vitro, and this helenalin-induced increase in lipid peroxidation was inhibited completely by the addition of 0.05 mM EDTA. However, microsomal cytochrome P450 contents were equally affected by helenalin in the presence or absence of EDTA, suggesting that lipid peroxidation did not contribute to the helenalin-induced decrease in cytochrome P450. The addition of 0.05 mM hemin to microsomes treated in vitro with 1.0 mM helenalin resulted in a 58% recovery of cytochrome P450 contents. This ability of hemin to reconstitute cytochrome P450 in helenalin-treated microsomes suggests that helenalin produced a selective loss of heme from the cytochrome P450 holoprotein, and that the resulting cytochrome P450 apoprotein remained intact after helenalin treatment. The increased loss of microsomal cytochrome P450 produced by helenalin in the presence of NADPH suggests that a helenalin metabolite may be responsible for heme loss and the in vitro destruction of cytochrome P450.

Characterization of microsomal oxidative activities in a wild-type and in a DDT resistant strain of Drosophila melanogaster

Resistance of a laboratory selected DDT strain of Drosophila melanogaster (RalDDT R) has been found to be monofactorial and correlated to an increased level of activity of the cytochrome P450-dependent mixed function oxidase (MFO). Both strains metabolize DDT and deltamethrin via MFO activity. However, the resistant strain does it more rapidly. The amount of DDT metabolites, including kelthane, bis-4-chlorophenyl acid, bis-4-chlorophenyl-ethanol, and 1,1-bis ( p-chlorophenyl)2,2-dichloroethane, is approximately 9-fold greater with RalDDT R microsomes than with the wild-type strain Raleigh (Ral). Production of deltamethrin metabolites is 2.7-fold higher within the resistant strain. As compared to insecticides, lauric acid and the two steroids used as substrates in this study present many more sites for MFO metabolic action. Lauric acid is hydroxylated on positions 11 and 12 by both strains, but the amount of metabolites formed is 10-fold higher with RalDDT R microsomes. The 2,22-dideoxyecdysone is converted to two polar metabolites when incubated with RalDDT R microsomal preparations. These unidentified metabolites are neither 2-deoxyecdysone nor ecdysone. Also reported for the first time is the metabolization of testosterone by insect microsomes, which gives 13 oxiderivatives formed at different rates, depending on the strains.

Ring-methyl hydroxylation of chlortoluron by an inducible cytochrome P450-dependent enzyme from maize

An enzyme catalysing ring-methyl hydroxylation of the substituted phenylurea herbicide chlortoluron was detected in isolated microsomes from germinating maize. Chlortoluron hydroxylation was absolutely dependent on NADPH and molecular oxygen. The enzyme was inhibited by carbon monoxide in the presence of oxygen and this inhibition could be reversed by light. Several known inhibitors of cytochrome P450 enzymes inhibited chlortoluron hydroxylation to varying degrees. It is concluded that the newly detected enzyme is a cytochrome P450-dependent mixed function oxidase. Enzyme activity was stimulated 15-fold by treatment of maize seeds with the herbicide antidote CGA 154281 [4-(dichloroacetyl)-3,4-dihydro-3-methyl-2H-1,4-benzoxazine].

Monoterpene biosynthesis: Specificity of the hydroxylations of (−)-limonene by enzyme preparations from peppermint ( Mentha piperita), spearmint ( Mentha spicata), and perilla ( Perilla frutescens) leaves

Microsomal preparations from the epidermal oil glands of Mentha piperita, Mentha spicata, and Perilla frutescens leaves catalyze the NADPH- and O 2-dependent allylic hydroxylation of the monoterpene olefin (−)-limonene at C-3, C-6, and C-7, respectively, to produce the corresponding alcohols, (−)- trans-isopiperitenol, (−)- trans-carveol, and (−)-perillyl alcohol. These transformations are the key steps in the biosynthesis of oxygenated monoterpenes in the respective species, and the responsible enzyme systems meet most of the established criteria for cytochrome P450-dependent mixed function oxygenases. The reactions catalyzed are completely regiospecific and, while exhibiting only a modest degree of enantioselectivity, are highly specific for limonene as substrate. Of numerous monoterpene olefins tested, including several positional isomers of limonene, only the 8,9-dihydro analog served as an alternate substrate for ring (C-3 and C-6) hydroxylation, but not side chain (C-7) hydroxylation. In addition to the regiospecificity of the allylic hydroxylation, these enzymes are also readily distinguishable based on differential inhibition by substituted imidazoles.

Mechanism of the in vitro antimutagenic action of retinol

The antimutagenic action of retinoids against three amino-imidazoazaarene pre-carcinogens, i.e. 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), 2-amino-3,4-dimethylimidazo(4,5-f)quinoline (MeIQ) and 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx), was investigated using the Ames test and hepatic activation systems derived from rats pretreated with Aroclor 1254. Both retinol and retinal, when incorporated into the S9 activation system, gave rise to a concentration-dependent decrease in the mutagenicity of all three mutagens, retinol being generally the more effective. Retinol suppressed the mutagenic activity of IQ even when isolated microsomes were used as activation systems. Moreover, retinol gave rise to a concentration-dependent inhibition of the microsomal dealkylations of pentoxy- and benzyloxy- and, especially, ethoxy-resorufin, but had no effect on the NADPH-dependent reduction of cytochrome c. Exposure of the bacteria to retinol with subsequent removal of the vitamin did not influence the mutagenicity of IQ. It is concluded that retinoids suppress the mutagenicity of aminoimidazoazaarenes and this is achieved through inhibition of their cytochrome P450-dependent metabolic activation. Retinol is a non-selective in vitro inhibitor of the hepatic cytochrome P450-dependent mixed function oxidase system as predicted by a computer graphic analysis of its molecular shape.

The role of reductive and oxidative metabolism in the toxicity of mitoxantrone, adriamycin and menadione in human liver derived Hep G2 hepatoma cells

The cytotoxic properties of quinones, such as menadione, are mediated through one electron reduction to yield semi-quinone radicals which can subsequently enter redox cycles with molecular oxygen leading to the formation of reactive oxygen radicals. In this study the role of reduction and oxidation in the toxicity of mitoxantrone was studied and its toxicity compared with that of adriamycin and menadione. The acute toxicity of mitoxantrone was not mediated through one-electron reduction, since inhibition of the enzymes glutathione reductase and catalase, responsible for protecting the cells against oxidative damage, did not affect its toxicity. Adriamycin was the most potent inhibitor of protein and RNA synthesis of the three quinones. Menadione, at concentrations up to 25 microM, did not inhibit either protein or RNA synthesis unless dicoumarol, an inhibitor of DT-diaphorase, was also present. The two-electron reduction of menadione by DT-diaphorase is therefore a protective mechanism in the cell. This enzyme also protected against the toxicity of high concentrations (100 microM) of mitoxantrone. The inhibitory effect of mitoxantrone, but not of menadione or adriamycin, on cell growth was prevented by inhibiting the activity of cytochrome P450-dependent mixed function oxidase (MFO) system using metyrapone. This suggests that mitoxantrone is oxidised to a toxic intermediate by the MFO system.

Suppression of the hepatic microsomal cytochrome P-450 dependent mixed function oxidase. Activities in golden hamster during Leishmania donovani infection

Experimental infection of golden hamsters with Leishmania donovani caused significant alterations in the hepatic microsomal mixed function oxidase system. Gross examination of liver indicated hepatomegaly. Microsomal protein contents were only marginally elevated. Cytochrome P-450 as well as haem contents were significantly decreased and it directly correlated with the degree of infection. Cytochrome b 5 exhibited elevation at lower degrees of infection which came down to control levels at the peak infection. Concomitant suppression was also noticed in cytochrome P-450 dependent monooxygenase activities, viz. aniline hydroxylase, benzo[ a]pyrene hydroxylase and aminopyrine N-demethylase. No significant change was observed in NADH-cytochrome b 5 reductase and NADPH-cytochrome c reductase. The results indicate suppression of hepatic microsomal MFO activities during visceral leishmaniasis.

Effect of a Prudhoe Bay crude oil on hepatic and renal peroxisomal beta-oxidation and mixed-function oxidase activities in rats.

The effect of oral administration of a Prudhoe Bay crude oil (PBCO) to male rats (PBCO, 2.6 g/kg body weight, daily) for 5-12 days on hepatic and renal microsomal monooxygenase activities and peroxisomal beta-oxidation has been investigated. PBCO administration leads to liver enlargement. This is associated with induction of microsomal cytochrome P-450 levels (1.6- to 2.0-fold) and dependent mixed-function oxidase activities (7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-depentylase, representing cytochrome P-450I and cytochrome P-450IIB isoenzymes respectively, 9- to 15-fold; omega-oxidation of lauric acid representing the cytochrome P-450IVA1 isoenzyme, 1.4- to 1.5-fold) along with peroxisomal beta-oxidation (palmitoyl CoA oxidation, 2- to 5-fold). It was observed that rats exposed to PBCO showed an increase in renal microsomal cytochrome P-450 content (1.6- to 2.3-fold), cytochrome P-450I activity (5- to 8-fold) and omega-oxidation activity (1.3- to 1.4-fold). However, renal peroxisomal beta-oxidation was unaltered. Serum total triglycerides were lowered by 41-46% after PBCO exposure. These results suggest that induction of peroxisomal beta-oxidation and possibly mono-oxygenases may be related to the carcinogenic/tumorigenic potential of crude oil.

Effects of bile duct obstruction and decompression on hepatic microsomal mixed function oxidase system in rats

The effects of biliary obstruction and drainage on the hepatic microsomal mixed function oxidase system were studied in rats. Bile duct obstruction produced a significant reduction in the hepatic cytochrome P-450 dependent mixed function oxidase system. After release of the bile duct obstruction, the reduction in microsomal enzymes was practically reversible; however, the process of recovery was slow and differed with the microsomal enzymes in question. Increases in cytochrome b 5 content and NADH-cytochrome b 5 reductase activity were slower than increases in cytochrome P-450 content and NADPH-cytochrome c reductase activity. Aniline hydroxylase activity increased more rapidly and corresponded to cytochrome P-450 contents more so than did the aminopyrine demethylase activity. After the release of bile duct obstruction, however, the bile acids which had accumulated in the liver during cholestasis were reduced rapidly, to a normal range. These results suggest that there is a discrepancy between reductions in hepatic bile acids and those in the hepatic microsomal mixed function oxidase system after biliary decompression.

Biophysical and catalytic properties of the phenobarbital p-hydroxylase--a cytochrome P-450 dependent mixed function oxidase.

1. A sensitive method to detect and quantify the products of phenobarbital (PB) hydroxylation by model chemical systems and by biological systems has been developed. 2. Chemical model systems hydroxylate PB in the p-position of the phenyl ring and form one or two additional oxidation products, while in vitro and in vivo (bile fistula rats) biological systems hydroxylate PB only in the p-position. 3. Phenobarbital hydroxylation rates in vitro are of the order of 0.007 nmol/nmol cytochrome P-450 per min. These values are decreased by pretreatment of the rats with inducing doses of phenobarbital. 4. Enzymes catalysing the p-hydroxylation reaction of phenobarbital are localized in the microsomes and have the biophysical and chemical properties that are usually associated with cytochrome P-450-dependent mixed function oxidases.