Microsomal Protein Content Research Articles

The effects of prochloraz on some microsomal parameters in rat liver.

The antifungal agent prochloraz [l-n-propyl-N(2,4,6-trichlorophenoxy ethylcarbamoyl)-imidazole] induces cytochrome P-450 in the liver of vertebrates and interacts with lipid metabolism (Riviere et al. 1984; Laignelet et al. 1986). A study was undertaken to detect changes in lipid composition and fluidity of microsomal membranes. Sprague-Dawley rats (180-190g) were injected i.p. with either 600 Ixmol/kg prochloraz (95% pure; Schering Co.) in corn oil or with corn oil only, 3 days before sacrifice. In the liver homogenates vitamin A was measured (Olson 1979). The following assays were carried out with the microsomal suspensions: benzo(a)pyrene monooxygenase activity (BaPMO) (Depierre et al. 1975), epoxide hydrolase activity (EH) (Oesch et al. 1971), microsomal protein content (Lowry et al. 1951) and cytochrome P-450 content (Omura and Sato 1964). Microsomal lipids were extracted (Bligh and Dyer 1959) and quantitatively determined (Canal et al. 1973); cholesterol content was also measured (Boehringer Mannheim standard kit). Individual phospholipids were separated by two-dimensional TLC (Wolff et al. 1984) and total lipid phosphorus as well as individual phospholipids were thus determined (Ames 1966). Fatty acids were derivatized to their methyl esters (Morrison and Smith 1964) and analysed by GLC. Microsomal fluidity was studied at 37~ by fluorescence depolarization measurement of diphenyl hexatriene (DPH) according to Schinitzki and Barenholdz (1978). The results are summarized in Table 1 and all the differences indicated below are significant (at p < 0.01 level; t test calculation). Prochloraz significantly increased liver weight (+ 27%) and decreased the vitamin A liver burden (-17%). In treated animals the microsomal protein content of whole liver was significantly increased (nearly 40%). In microsomes of treated animals cytochrome P-450 content and BaPMO activity were significantly increased (+21% and + 121%, respectively), although EH activity was not altered. Prochloraz did not change the total lipid and phospholipid contents in microsomal membranes. However, the cholesterol level in these membranes and thus the phospholipid/cholesterol molar ratio were decreased ( -22% and -20%, respectively). Consequently, the microviscosity, evaluated in terms of fluorescence with the probe DPH, was significantly decreased ( 12%). It was reported that prochloraz inhibited lanosterol 14-demethylation (a cytochrome P-450 dependent reaction) in fungi (Schwinn 1983) and increased lanosterol liver content in Japanese quails (Riviere et al. 1984). More-

Determination of microsomal lauric acid hydroxylase activity by HPLC with flow-through radiochemical quantitation

An assay for the microsomal hydroxylation of lauric acid (LA), based on HPLC with flow-through radiochemical detection, has been developed. Conditions were optimized for resolution and quantitation of three microsomal metabolites of LA, one of which has not been reported previously as a metabolite of LA in mammalian microsomal incubations. These products, 12-(ω)-hydroxy-LA, 11-(ω-1)-hydroxy-LA, and a novel metabolite, 10-(ω-2)-hydroxy-LA, were isolated by HPLC and identified by gas chromatography/mass spectrometry. In the presence of NADPH, the formation of all three metabolites was linear with time and microsomal protein concentration. Hydrogen peroxide also supported the microsomal metabolism of LA, although the ratio of metabolites was substantially different than that produced by NADPH-supported microsomes. Several biochemical probes (metyrapone, α-naphthoflavone, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, and 10-undecynoic acid) were used to dissociate the three LA hydroxylase activities. These experiments suggest that the site-specific hydroxylation [ω-, (ω-1)-, (ω-2)-] of LA may be catalyzed by different isozymes of cytochrome P-450.

The effect of hypoxia on hepatic cytochromes and heme turnover in rats in vivo.

We evaluated the effect of hypoxia (7% v/v) on hepatic heme turnover in vivo and microsomal heme protein content in male Sprague-Dawley rats. Hepatic heme protein turnover, measured as 14CO-production during continuous infusion of 5-14C-aminolevulinic acid, a precursor of nonerythrogenic heme, was decreased 60% during hypoxia and returned to control levels promptly after reoxygenation. Hepatic cytochrome P-450 content was decreased in hypoxic and 24-h reoxygenated animals. We conclude that normobaric hypoxia decreases hepatic cytochrome P-450 which could contribute to decreased drug metabolism in hypoxia. This decrease is probably due to heme oxygenase-independent breakdown of hepatic heme.

Disposition of d-fenfluramine in lean and obese rats

The anorectic drug D-fenfluramine (D-F) was administered as single i.v. doses of 1.25 and 6.25 mg/kg to lean female Sprague-Dawley and lean and obese female Zucker rats. Blood samples were collected serially and analysed by electron capture gas-liquid chromatography for D-F and its main metabolite, D-norfenfluramine (D-NF). At the lowest dose the disappearance of D-F followed an apparent first-order process with mean elimination half-life (T1/2) of approximately 2 h in female Sprague-Dawley and 4 h in lean and obese Zucker rats. Mean absolute steady-state volume of distribution (Vss) was the same in the lean female of both strains but total clearance (Cl) was significantly lower in the Zucker rats. Therefore elimination T1/2 of D-F was longer in female Zucker than Sprague-Dawley animals. Obese rats presented lower relative Cl and Vss but no change in absolute Cl and Vss and elimination T1/2 of the drug. Intra- and inter-strain differences were observed in hepatic microsomal protein and P-450 content. As in the case of D-F the elimination T1/2 of D-NF was also longer in Zucker than Sprague-Dawley rats. No differences were observed between lean and obese rats but in all cases the elimination T1/2 of the metabolite was much longer than that of its parent drug. After larger doses (6.25 mg/kg) the kinetics of the drug were not linear. The apparent Cl declined changing the metabolite-to-parent drug ratios in all types of rats, but more evidently in Zucker than Sprague-Dawley rats and in obese than lean animals. Inter- and intra-strain differences in D-F and D-NF kinetics should be considered in neurochemical studies of the drug and extrapolation of data across animal species requires consideration of dose dependence in the rat.

Effects of chronic acetone administration on ethanol-inducible monooxygenase activities in the rat

Liver microsomal monooxygenase activities known to be ethanol-inducible were determined in female Sprague-Dawley rats after 2-week treatment with 1% v/v) acetone. Daily acetone intake was in the order of 1.2 g/kg. The final body weight, liver weight and microsomal protein content of acetone-treated rats were identical to those of untreated controls. Microsomal NADPH-cytochrome c reductase activity was also unaffected, while cytochrome P-450 content was only increased 12–18%. Ethanol-inducible p-nitrophenol hydroxylation, aniline hydroxylation and 7-ethoxycoumarin Odeethylation activities were enhanced 5.3-, 4.4- and 2.6-fold, respectively, by chronic acetone treatment. The sex-dependent inducing effect of ethanol on benzphetamine N-demethylation activity in female rats was not observed however, after acetone. Addition of acetone in vitro had a stimulatory effect on aniline hydroxylation by microsomes from control and acetone-induced rats. Acetone, however, was found to be a competitive inhibitor of p-nitrophenol hydroxylation activity (apparent K i = 1.8 mM), an observation suggesting that p-nitrophenol is a more selective substrate than aniline for rat liver ethanol- and acetoneinducible cytochrome P-450j. Interruption of the chronic acetone treatment for 24 hr resulted in the almost complete disappearance of its inducing affects, this treatment apparently reproducing only the rapidly reversible preferential inducing effects of chronic ethanol administration. This experimental model of induction by acetone in the rat, when compared to chronic ethanol administration, would thus permit a more selective look at the consequences of these common inducing effects in particular, with respect to drug metabolism and toxicity in vivo, and this, in the absence of the hepatotoxic effects of ethanol itself.

Effect of cadmium on pulmonary and renal microsomal drug metabolizing enzymes of the guinea-pig

1. 1. The effect of acute cadmium (Cd) treatment on pulmonary and renal microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase enzyme activities of adult male guinea-pigs were assessed 72 hr following a single dose of Cd ion (2mg Cd 2+/kg i.p.). Tissue and microsomal Cd levels were also determined. 2. 2. There were no significant differences between either lung or kidney tissue weights, microsomal protein contents or enzyme activities of Cd treated and control animals. 3. 3. The tissues and microsomes of Cd-treated animals were found to have significantly higher levels of Cd than those of control animals. In Cd treated animals, tissue and microsomal Cd levels of kidney were found to be higher than that of lung. 4. 4. In vitro addition of cadmium chloride (CdCl 2) to incubation mixtures produced concentration related inhibitions of microsomal drug metabolizing enzymes in each tissue. However, in vitro effect of CdCl 2 was found to be stronger on drug metabolizing enzymes of kidney than those of lung. In addition, while the strength of Cd effect was more pronounced on the activity of ethylmorphine N-demethylase than that of aniline 4-hydroxylase in the lung, the opposite was observed in the kidney.

Properties of microsomal acyl-CoA: lysophosphatidylcholine acyltransferase from liver of thermally-acclimated rainbow trout,Salmo gairdneri

Acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT) (EC 2.3.1.23) activity was assayed in liver microsomes from rainbow trout,Salmo gairdneri, acclimated to 5°C and 20°C to assess its contribution to the temperature-induced restructuring of phospholipid acyl chain composition. The synthesis of phosphatidylcholine (PC) (from lyso-PC) was threefold the synthesis of phosphatidylethanolamine (PE) (from lyso-PE) under similar assay conditions. LPCAT activity (i) displayed an absolute requirement for lysophosphatidylcholine (LPC) and was enhanced by the presence of ATP, MgCl2 and CoA (which reduced the impact of endogenous acyl-CoA hydrolase activity by regenerating the acyl-CoA substrate) in the assay medium; (ii) remained linear with time up to 30 min; and (iii) increased linearly with microsomal protein concentration up to 0.2 mg/ml for the 20°C assay and 0.4 mg/ml for the 5°C assay. There was no difference in Km or Vmax values due to the acclimation history of the fish, but there were obvious differences due to assay temperature. The apparent Km values for LPC were 58.54±7.24 μM and 12.26±2.14 μM when assayed at 5°C and 20°C respectively; values for oleoyl-CoA were 9.11±0.78 μM and 1.23±0.25 μM under the same assay conditions. Activity was 1.99±0.31 nmol min−1 mg protein−1 when assayed at 5°C, and 3.8±0.45 nmol min−1 mg protein−1 when assayed at 20°C. These findings indicate that adjustments in the activity of LPCAT play no significant role in the temperature-induced restructuring of PC molecular species composition. However, the marked temperature dependence of the Km values for LPC and oleoyl CoA suggest that patterns of fatty acid incorporation (i.e. substrate preference) may vary with assay temperature, and in this way LPCAT could contribute to the restructuring response.

Age dependent expression of cytochrome P-450b and metabolism of the potent carcinogen 2-nitrofluorene in the rat lung.

Northern and Western blot analyses, and analyses of microsomal metabolism of the carcinogen 2-nitrofluorene (NF) were conducted with the aim of studying age dependent cytochrome P-450b levels in the rat lung. The level of P-450b homologous mRNA and corresponding protein is very low in lungs from fetal and newborn rats. The levels then increase between 3 and 4 weeks of age, and reach adult levels at 6-8 weeks. No sex differences were detected with regard to lung P-450b mRNA levels or catalytical activities. Lung microsomal metabolism of NF increased in parallel with the accumulation of P-450b homologous mRNA and microsomal cytochrome P-450b protein concentration. Formation of the major metabolite, and potent mutagen, 9-hydroxy-2-nitrofluorene (9-OHNF) was significantly inhibited by addition of polyclonal anti-P-450b-IgG, and by addition of the inhibitor proadifen to incubations with lung microsomal protein. It is postulated that the observed, profound age-related differences in level and activity of lung cytochrome P-450b are likely to affect both availability and the ratio of metabolic detoxification and activation of chemical carcinogens deposited in the lung.

Cadmium sensitivity differences between liver microsomal drug metabolizing enzyme systems of guinea-pig and rat

1. 1. Male guinea-pigs (400–500 g) and rats (225–275 g) were given a single dose of cadmium chloride (CdCl 2) (2 mg Cd 2+/kg i.p.) and 72 hr later the liver microsomal drag metabolizing enzyme activities and Cd levels of tissues and microsomes were determined. 2. 2. No significant differences were noted between Cd treated and control animal tissue weights of microsomal protein contents in either guinea-pigs or rats. 3. 3. Cd treatment exhibited significant inhibition of the activities of aniline 4-hydroxylase and ethyl-morphine N-demethylase and on the levels of cytochrome P-450 and cytochrome b 5 of liver of both species but the degree of inhibition were not the same in the species; they were 23, 34, 16 and 10% in guinea-pigs and 58, 57, 25 and 13% in rats, respectively. 4. 4. No activity changes were observed in liver NADPH-cytochrome c reductase of the species by Cd treatment. 5. 5. The duration of hexobarbital sleeping time was significantly prolonged in both species. However, the prolongation was 1.6 fold in guinea-pigs but 3.4 fold in rats. 6. 6. No significant differences were found between either tissue or microsomal Cd levels of guinea-pigs and rats.

Effect of diisopropyl 1,3-dithiolan-2-ylidenemalonate(NKK-100) on the components of hepatic microsomal mixed function oxidase in male broiler chicks.

Effect of oral administration of diisopropyl 1, 3-dithiolan-2-ylidenemalonate (NKK-100) on the components of mixed function oxidase system in hepatic microsomes was investigated in male broiler chicks. Feeding NKK-100 increased relative liver weight and microsomal protein content of the liver. Contents of cytochrome P 450 and b 5 per mg microsomal protein and g liver increased with NKK-100 levels. The activities of NADPH-cytochrome c reductase and NADH-cytochrome b 5 reductase per g liver were enhanced by feeding NKK-100, while those activities per mg microsomal protein remained unchanged. Pretreatment with clopidol (an anticoccidial agent) and colistin (an antibiotic) did not affect the components of MFO. These results suggests that NKK-100 has a property to activate the hepatic microsomal MFO and the effect of NKK-100 are more prominent in the increases in specific activities of cytochrome P 450 and b 5 rather than in those of NADPH-cytochrome c reductase or NADH-cytochrome b 5 reductase.

Metabolic activation of α-naphthoflavone by 2,3,7,8-tetrachlorodibenzodioxin-induced rat liver microsomes

Previous studies in our laboratory had demonstrated that addition of α-naphthoflavone (ANF) to lymphocytes from smokers or polychlorinated biphenyls (PCB)s-exposed individuals caused an increase in sister chromatid exchange (SCE) frequency whereas lymphocytes from controls were relatively unaffected. In order to investigate the mechanism responsible, metabolism of ANF by uninduced and 2,3,7,8-tetrachlorodibenzodioxin (TCDD)-induced microsomes was studied as a function of microsomal protein concentration and incubation time. Nonpolar metabolites were analyzed and the amount of conjugated (polar) and protein-bound metabolites determined. The initial ANF-metabolism rate was 10-fold higher in TCDD-induced microsomes (4.9 ± 0.6 nmol/min per mg TCDD-induced microsomal protein vs. 0.5 ± 0.2 nmol/min per mg uninduced microsomal protein) than in uninduced microsomes. Moreover, uninduced microsomes no longer metabolize ANF after 30–40 min while TCDD-induced microsomes metabolize ANF for longer than 2 h or until all the ANF is gone. In addition to the metabolites formed by uninduced microsomes [7,8-dihydro-7,8-dihydroxy-ANF (7,8-dihydrodiol); 5,6-dihydro-5,6-dihydroxy-ANF (5,6-dihydrodiol); 5,6-oxide-ANF and 6-hydroxy-ANF], TCDD-induced microsomes from unidentified metabolites. When TCDD-induced microsomes and 40 μM ANF were added to Chinese hamster ovary (CHO) cells, we found a correlation between the concentration of 5,6-oxide-ANF and clastogenicity to CHO cells. However, purified 5,6-oxide-ANF did not induce SCEs in CHO cells in the absence or presence of TCDD-induced microsomes. However, a minor metabolite (identified as the 9,10-dihydro-9,10-dihydroxy-ANF by acid dehydration) formed with TCDD-induced microsomes produces clastogenicity in CHO cells. These data indicate that a minor metabolite of ANF is a potent clastogen which suggests that this metabolite may be responsible for the ANF-mediated increases in SCE frequency in lymphocytes from smokers or PCB-exposed individuals.

Hepatic microsomal mixed-function oxidase activity in the japanese medaka (Oryzias latipes)

Growing interest in the use of the Japanese medaka (Oryzias latipes), a small aquarium fish, for carcinogenicity and toxicological studies necessitates an understanding of their xenobiotic metabolizing capabilities. Historically, hepatic mixed-function oxidase activity has been characterized using inducing agents such as beta-naphthoflavone (BNF). Hepatic cytochrome P450, 7-ethoxy-resorufin o-deethylase (EROD) activity, and NADPH-cytochrome c reductase activity were increased in male and female adult medaka following dietary exposure to BNF. In noninduced male and female adult medaka, EROD activity was not detectable. The EROD activity of the male medaka increased to 1.05 nmol/min/mg protein on day 4. Female EROD activity reached a maximum of 0.65 nmol/min/mg protein on day 2. NADPH-cytochrome c reductase activity in the males increased from a control level of 33.02 to 81.9 nmol/min/mg protein by day 4 of BNF exposure. NADPH-cytochrome c reductase activity in females reached a maximum rate of 63.4 nmol/min/mg protein within 48 h of exposure, but returned to noninduced levels (49.6 nmol/min/mg protein) by day 4. Cytochrome P 450 concentration, liver-to-body-weight ratios, and microsomal protein concentration increased in a time-dependent fashion for both sexes following BNF exposure.

Tolbutamide 4-hydroxylase activity of human liver microsomes: effect of inhibitors.

Eight samples of human liver have been characterised for microsomal protein content, cytochrome P-450 content, tolbutamide 4-hydroxylase and ethinyloestradiol 2-hydroxylase activities. Cytochrome P-450 content correlated significantly with ethinyloestradiol 2-hydroxylase activity but not with tolbutamide 4-hydroxylase activity. There was no significant correlation between ethinyloestradiol 2-hydroxylase and tolbutamide 4-hydroxylase activities. The maximum tolbutamide 4-hydroxylase activity was 0.45 nmol min-1 mg-1 microsomal protein, with a Km value of 74 microM. A number of compounds were tested for their ability to inhibit tolbutamide metabolism. All the compounds showing inhibition were either non-competitive or mixed non-competitive inhibitors of tolbutamide 4-hydroxylation. These studies suggest that tolbutamide is metabolised by an isozyme of cytochrome P-450 which appears to be distinct from those isozymes metabolising many other drugs.

The effects of dicofol on induction of hepatic microsomal metabolism in rats

In a comparative study, the induction effects of dicofol, technical Kelthane, and DDT on hepatic microsomal and cytosolic enzyme activities in rats were compared with those effects produced by phenobarbital (PhB) and β-naphthoflavone (BNF). Male rats (ca. 250 g) were injected (ip) for 4 consecutive days with 1.0 ml of vehicle containing either dicofol (1.5, 15.0, 29.5, or 59.0 m M, Kelthane (dicofol content equal to 29.5 or 59.0 m M), DDT (59.0 m M), or BNF (36.7 m M). Liver weights, microsomal protein, and cytochrome P-450 concentrations and microsomal and cytosolic enzyme specific activities were measured. Dicofol produced dose-related increases in all of the parameters measured except liver weight and cytosolic epoxide hydrolase activity. At a concentration of 59.0 m M, dicofol increased the concentrations of microsomal protein (1.7-fold) and cytochrome P-450 (2.9-fold), and the specific activities of cytochrome c reductase (1.6-fold), ethoxycoumarin O-deethylase (2.3-fold), aminopyrine N-demethylase (3.0-fold), microsomal epoxide hydrolase (2.6-fold), and glutathione S-transferase (2.9-fold). The induction potency of dicofol was equivalent to Kelthane, DDT, and PhB at equimolar (59.0 m M) concentrations of chemical.

Effects of in vivo administration of detergents on the hepatic microsomal cytochrome P-450 system in rat.

The influence of in vivo administration of detergents on the hepatic microsomal cytochrome P-450 system was studied in rat. Male Wistar rats were administered detergents, Emulgen 913 (50 mg or 100 mg kg-1 of body weight (B.W.], or sodium dodecylsulfate (SDS, 25 mg or 50 mg kg-1 of B.W.) intraperitoneally once a day for 3 days. Cytochrome P-450 content in liver microsomes was significantly decreased to 85% and 73% of control by the administration of 50 mg or 100 mg Emulgen, respectively, but the microsomal protein concentration was not changed by these administrations. The content of cytochrome P-450 was also reduced to 76% and 70% of control by the administration of 25 mg or 50 mg SDS/kg of B.W., respectively. The total hydroxylation activity (the sum of omega- and (omega-1)-hydroxylase activity) of laurate almost paralleled the decrease in cytochrome P-450 in detergent-treated rats. However, the omega/omega-1-hydroxylation ratio was not changed. These results suggest that the administration of these detergents lowered the level of cytochrome P-450 species catalyzing omega- and (omega-1)-hydroxylation of laurate to a similar extent. On the other hand, aminopyrine N- and p-nitroanisole O-demethylation activities in Emulgen 913-treated rats was decreased while those in SDS-treated rats did not change, though the content of cytochrome P-450 was decreased by both administrations. Thus, it was demonstrated that the livers of rats responded to exogenous detergents in different manners.

Loss of NADPH during assays of HMG-CoA reductase: implications and approaches to minimize errors.

In assays of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, preincubation of isolated washed microsomes with NADPH led to a time- and protein concentration-dependent loss of enzyme activity. This occurred despite the presence of an NADPH regenerating system. Addition of fresh NADP, glucose 6-phosphate and glucose 6-phosphate dehydrogenase restored activity. Of the individual components, only NADP was effective. Errors due to loss of NADPH are most pronounced in assays using high microsomal protein, low NADPH levels and preincubation with NADPH and when glutathione rather than dithiothreitol is present. To minimize the effects of NADPH depletion, it is recommended that (i) NADP and NADPH not be present during the preincubation period; (ii) incubation periods be relatively short; (iii) microsomal protein concentrations be less than 1 mg; and (iv) NADPH concentrations be 1 to 2 mM.

Development of a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay for measurement of human leukocyte microsomal HMG-CoA reductase protein concentration.

Methods were developed for determination of human mononuclear leukocyte HMG-CoA reductase protein concentration by a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay procedure. Leukocyte microsomal HMG-CoA reductase, first immobilized onto a nitrocellulose filter, is sequentially reacted with 1) monospecific, polyclonal rabbit anti-rat liver HMG-CoA reductase antiserum, which crossreacts with the human liver and leukocyte enzymes; 2) biotinylated donkey anti-rabbit immunoglobulin; 3) a streptavidin-horseradish peroxidase conjugate; and 4) 4-chloro-1-naphthol and H2O2 to visualize the quantity of horseradish peroxidase bound to the immunocomplex. Color development was proportional to the quantity of either purified liver or leukocyte microsomal HMG-CoA reductase applied to the nitrocellulose. Color development was not observed, however, when HMG-CoA reductase was omitted from the nitrocellulose, when one of the reactant species was omitted from the incubation reactions, or when anti-rat liver HMG-CoA reductase antiserum was pre-absorbed with either rat liver or human leukocyte HMG-CoA reductase. Immunoreactivity of microsomal HMG-CoA reductase was independent of the phosphorylation state of the enzyme, but was inversely related to the concentration of thiol-reducing agents present in the microsomal preparation up to 4 mM. Further increases in thiol-reductant failed to produce changes in immunoreactivity. Freshly isolated mononuclear leukocyte microsomal HMG-CoA reductase protein concentration in leukocytes from 31 healthy, normocholesterolemic subjects was a linear function of HMG-CoA reductase activity (R = 0.65; P less than 0.001). The catalytic efficiency of the freshly isolated mononuclear leukocyte enzyme was 313 +/- 34 pmol of mevalonate formed per min of incubation at 37 degrees C per mg immunoreactive protein. This methodology, in conjunction with that recently developed to measure human leukocyte HMG-CoA reductase activity (1984. J. Lipid Res. 25: 967-978), should prove useful in discriminating between HMG-CoA reductase regulatory mechanisms involving changes in enzyme protein concentration and those resulting from changes in enzyme catalytic efficiency.

Biphasic effect of methadone on hepatic drug metabolism.

When methadone HCl (30 mg/kg, po) was given acutely to mice, it was found to inhibit drug metabolism as evidenced by a prolongation of hexobarbital sleeping time and zoxazolamine paralysis time. Pharmacokinetic studies revealed that this acute dose of the narcotic analgesic could also prolong the plasma half-life of aminopyrine without any change in its volume of distribution. When added to the incubation mixture containing 10,000 g mouse liver supernatant fraction and a complete system for measuring aminopyrine N-demethylase or aniline hydroxylase, methadone showed a dose-dependent inhibition of the enzymes; the former enzyme was inhibited to a greater extent than the latter one. However, subacute treatment of mice with methadone HCl (30 mg/kg, po, twice daily for 3 days) resulted in increases in liver weight, microsomal protein, and cytochrome P-450 content in consonant with the increased activities of four hepatic drug-metabolizing enzymes: aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole, O-demethylase, and benzphetamine N-demethylase. Moreover, both hexobarbital sleeping time and zoxazolamine paralysis time were shortened. The plasma half-life of aminopyrine was decreased. These changes were prevented by simultaneous administration of puromycin diHCl (80 mg/kg, ip). Methadone thus seems to act in a manner very similar to that of propoxyphene or SKF-525A, acting as a potent inhibitor of hepatic drug metabolism when given acutely and as an inducer when given subacutely.

Effect of Dietary β-Carotene on Hepatic Drug-Metabolizing Enzymes in Mice

The effects of dietary supplements of β-carotene (20-500mg per kg diet) on hepatic microsomal drug-metabolizing enzyme activities were studied in mice. Supplementation for 14 days resulted in marked reductions in the concentrations of cytochrome P-450 and biphenyl 4-hydroxylase. The antipyrine N-demethylase and p-nitroanisol O-demethylase activities, however, were unchanged. Also apparently unchanged were the hepatic concentrations of microsomal protein, lipid peroxides, and superoxide dismutase. Supplemental β-carotene was weakly protective against the acute toxic effects of an injection of 1, 2-dimethylhydrazine (DMH), as indicated by a lowered mortality. This anti-carcinogenic action of β-carotene, including a protection against DMH-induced colon carcinogenesis, suggests an alteration by this carotenoid in the metabolism of carcinogens by the liver.

No evidence for an interaction of the bacterial immunomodulator trehalose dimycolate (TDM) with liver drug-metabolizing system in mice.

Trehalose dimycolate (TDM), an immunomodulatory glycolipid component of Mycobacteria, was tested from the point of view of possible effects on drug metabolism. TDM was given intraperitoneally as a single 0.1 mg dose to mice. Basic parameters of the liver mixed-function oxidase system were assayed 1 or 7 days later. No significant changes were found in contents of cytochromes P-450 and b5, as well as in specific activities of microsomal enzymes--aniline hydroxylase, ethylmorphine demethylase, and glucuronide transferase. Similarly, liver microsomal protein concentration and activities of serum aspartate and alanine transaminases remained unchanged. The susceptibility of microsomal membranes to lipid peroxidation was significantly decreased 7 days after TDM administration. TDM may thus be presumed not to influence range and magnitude of effects of concurrently administered drugs.