On the aromatic hydroxylation of amphetamine in rat liver microsomes and perfused liver preparations: effects of long-term administration.

The liver microsomal p-hydroxylation of amphetamine to parahydroxyamphetamine (pOHA) was dependent on NADP and inhibited by carbon monoxide indicating the involvement of cytochrome P-450, SKF 525-A, fenfluramine and desmethylimipramine were the most effective inhibitors of this pathway of amphetamine metabolism. Repeated administraion of phenobarbital resulted in reduced p-hydroxylation of amphetamine in vitro. Chronic administration of amphetamine reduced the microsomal p-hydroxylation of amphetamine without apparent changes in the cytochrome P-450 levels or in the activity of NADPH-cytochrome c reductase. The aromatic hydroxylation of aniline and the demethylation of ethylmorphine was not affected by this treatment. However, the 455 nm complex formed during the microsomal metabolism of N-hydroxy-amphetamine was increased by the long-term administration of amphetamine. These results indicate some pecularities of the in vitro hydroxylation of amphetamine by rat liver microsomes. Amphetamine disappeared from the perfusate of the perfused liver at the same rate in rats given a single dose of amphetamine and in rats given amphetamine orally for four weeks. The excretion of pOHA and its conjugate increased at 60 and 90 min. and 30, 60 and 90 min. respectively in the perfusate of the same experiment as compared to the controls. The total excretion of radioactive amphetamine metabolites at the end of the perfusion was increased in the perfusate and reduced in the bile compared to the control experiment.     

Effect of spin traps in isolated rat hepatocytes and liver microsomes

Spin traps are increasingly employed in the detection of free radicals in biological systems, including liver microsomes and isolated hepatocytes. Two spin traps phenyl-t-butyl nitrone (PBN) and 4-pyridyl-l-oxide-t-butyl nitrone (4-POBN) have been tested for their effects on hepatocyte viability and mixed-function oxidase activity. High concentration of PBN but not of 4-POBN proved to moderately affect liver cell integrity, without interfering with intracellular ATP or cytochrome P-450 content. PBN also decreased hepatocyte GSH content, probably as the result of its metabolism to benzaldehyde. The two spin traps were found to inhibit aminopyrine demethylase and ethoxycoumarin deethylase activity in hepatocytes and microsomes. At low concentrations (1-5 mM) PBN enhanced aniline hydroxylase while high concentrations of the spin trap inhibited this activity. The inhibition of the monooxygenase system was not caused by damage of microsomal enzymes, but rather by competition with other substrates for the binding to the haemoprotein. The effects of spin traps on mixed function oxidase systems should be taken into account when evaluating the results of spin trapping experiments.     

The p-hydroxylation of amphetamine and phentermine by rat liver microsomes.

1. The products of p-hydroxylation of amphetamine and phentermine by two different preparations of rat liver microsomes were identified and quantitatively determined. At low concentrations (muM) significant proportions of the substrates were metabolized to the p-hydroxy derivatives by an NADPH-dependent system. The enzyme system was inhibited by higher substrate concentrations (mM) and was not induced by either phenobarbital or 3-methylcholanthrene. 2. The properties of this in vitro system are consistent with reports on in vivo studies of this reaction.     

Study of hepatotoxicity in isolated perfused liver versus cultures of rat hepatocytes

Isolated perfused liver and cultures of rat hepatocytes were assessed for the quantitative evaluation of hepatotoxicity. Release of de novo biosynthesized plasma proteins and acid hydrolases into perfusion or culture media was taken as an indication of the integrity of hepatocytes in both systems. The activities of six acid hydrolases, alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-N-acetylgalactosaminidase, beta-D-N-acetylglucosaminidase, and cathepsin D, were assayed in collagenase-segregated hepatocytes and in monolayer cultures of rat liver cells obtained via collagenase perfusion of rat liver. In situ, liver perfusion with collagenase led to a loss of 45 +/- 5% of the total acid hydrolase activity in the mitochondrial-lysosomal pellet of the liver cells with concomitant increase of these enzymes in the cytosol. In monolayer cultures over a period of 30 h, increased activity of cathepsin D, beta-D-galactosidase, and beta-D-N-acetylglucosaminidase in the mitochondrial-lysosomal pellet and the cytosol fraction was evident with concurrent biosynthesis of plasma proteins. The use of radioactive tracing techniques with the isolated perfused liver revealed that the rate of catabolism of intracellular protein was approximately 5 times that of plasma protein synthesis. Both methods described here are suitable for the study of the effects of toxins on the function of hepatocytes.     

Disposition of ethimizol, a xanthine-related nootropic drug, in perfused rat liver and isolated hepatocytes

Ethimizol, 4,5-di(methylcarbamoyl)-1-ethyl-imidazole, was metabolized into at least six metabolites in an isolated perfused rat liver preparation. Based on TLC and mass spectrometry, 4-carbamoyl-5-methylcarbamoyl-1-ethyl-imidazole and 4,5-di(methylcarbamoyl)-imidazole were identified as the primary metabolites of ethimizol. These undergo further biotransformation: both can form 4(5)-carbamoyl-5(4)-methylcarbamoyl-imidazole, and, moreover, the former can be hydroxylated. Besides the identified metabolites, two polar ones of unknown structure were detected. Dose-dependent elimination of ethimizol was observed when the drug was added to the liver perfusion recirculating medium in initial reservoir concentrations of 39, 48, 85, 165, and 240 microM. The observed nonlinearity appeared to be a result of a competitive product inhibition. Similar to the parent drug, ethimizol primary metabolites were formed and eliminated in a dose-dependent manner. The uptake of ethimizol by isolated hepatocytes was extremely rapid, independent of drug concentration, over the range 0 to 250 microM, unaffected by inhibitors and independent of temperature.     

Metabolic activation of pioglitazone identified from rat and human liver microsomes and freshly isolated hepatocytes.

Pioglitazone is in the class of compounds known as the thiazolidinediones and is used to treat type 2 diabetes mellitus. The first in its class compound, troglitazone, was withdrawn from the U.S. market in 2000 due to a high incidence of hepatotoxicity and drug-induced liver failure. Reactive ring-opened products of troglitazone have been identified and evidence suggests that these reactive intermediates might be a potential cause of hepatotoxicity. The present work shows that pioglitazone has a reactive ring-opened product which was trapped by glutathione and positively identified by high performance liquid chromatography with tandem mass spectrometry accurate mass measurements. The novel thiazolidinedione ring-opened products of pioglitazone were identified in rat and human liver microsomes and in freshly isolated rat but not human hepatocytes.     

Metabolite kinetics of ondansetron in rat. Comparison of hepatic microsomes,isolated hepatocytes and liver slices,with in vivo disposition

1. The kinetics of hydroxylation and N-demethylation of ondansetron have been determined in freshly isolated hepatocytes, hepatic microsomes and precision-cut liver slices from the male Sprague-Dawley rat. In vivo studies have also been carried out to characterize the pharmacokinetics of ondansetron and in vitro data have been assessed for their value as predictors of hepatic clearance.

2. In the three in vitro systems, the formation of hydroxylated and demethylated metabolites were characterized as a function of substrate concentration by a high-affinity, low-capacity site and a low-affinity, high-capacity site which was not saturated over the concentration range studied (2.5–500 μM). Slices gave consistently higher K_m's (20 and 30 μM for hydroxylation and demethylation respectively) than hepatocytes (3 and 13 μM respectively) and microsomes (2 and 5 μM respectively). The rank order of V_max and CL_int was the same for each system; hydroxylation rates exceeding demethylation rates. Although two hydroxylations (7- and 8-hydroxy metabolites) occurred exclusively in microsomes, these are believed to originate from a common precursor.

3. The high CL_int of ondansetron (150 ml/min/SRW, where SRW is a standard rat weight of 250 g) is well predicted by scaling either microsomal clearance for microsomal protein recovery or hepatocyte clearance for hepatocellularity (212 and 135 ml/min/SRW respectively). In contrast, the use of liver slice data scaled to a whole liver substantially underestimates CL_int (9 ml/min/SRW).     

Hydroxylation of amphetamine to parahydroxyamphetamine by rat liver microsomes

Rat liver microsomes were shown to hydroxylate d- and l-amphetamine at the para position of the benzene ring. The identity of the product, p-hydroxyamphetamine, was established by the use of three different methods, paper chromatography, radio gas-chromatography and mass-spectral analysis. The hydroxylation reaction was found to be linear with time to 15 min and protein concentration to 2 mg/ml. The reaction followed Michaelis-Menten kinetics only at low substrate concentrations. When the concentration of d-amphetamine exceeded 0·5 M the plot indicated substrate inhibition. According to the Linewaaver-Burk plot the following apparent kinetic data were found: d-amphetamine, K_m = 2·$ × 10^?5 M, V_max = 3·7 moles × 10 min; l-amphetamine. K_m = 1·1 × 10⁶ M, V_max = 2·8 nmoles × mg proteins × 10 min^?1.     

Differential inhibition of biphenyl hydroxylation in perfused rat liver

Summary A differential inhibition of biphenyl hydroxylation by -naphthoflavone and metyrapone was observed in isolated pefused rat liver. -Naphthoflavone inhibited 2- and 4-hydroxylation in livers from -naphthoflavone-pretreated animals but had no effect on both reactions in livers from phenobarbital-pretreated animals. Metyrapone inhibited 2- and 4-hydroxylation in phenobarbital-stimulated livers, but only insignificant inhibition of 2-hydroxylation and a slight enhancement of 4-hydroxylation by metyrapone was observed in -naphthoflavone-stimulated livers.Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl preferentially formed sulphates in livers from untreated animals but after induction glucuronidation was as effective as sulphation or even exceeded sulphation. Only glucuronic acid conjugates of 2-hydroxybiphenyl were detected.     

Metabolism of a benzothiazine compound (SQ 11,579) by the intact rat, isolated perfused rat liver, and rat-liver microsomes.

1. The metabolic dispositions of a benzothiazine compound (SQ 11,579) by the intact rat, isolated perfused rat liver, and rat-liver microsomes have been investigated, and the results compared. 2. The drug was well absorbed after oral administration to rats and was widely distributed in all tissues, which, with the exception of brain, had higher concentrations of the drug, its metabolites, or both, than did plasma. 3. Metabolism by rat-liver microsomes included N-oxidation, N-demethylation, S-oxidation and aryl hydroxylation. Metabolites hydroxylated in the aromatic ring were excreted only in bile, both by the isolated perfused rat livers and by anaesthetized bile-duct-cannulated rats. 4. Liver perfusion of the benzothiazine or its monodesmethyl analogue (V) resulted in temporary cessation of the flow of perfusate through the organ. The benzothiazine sulphoxide (IV) had only a slight effect on the flow of liver perfusate, but IV followed by I caused the flow of perfusate to cease.     

Histamine inhibition of mixed function oxidase activity in rat and human liver microsomes and in the isolated perfused rat liver

The imidazole ring is a common structural feature of some xenobiotics that inhibit cytochrome P-450-catalysed reactions. Histamine is a 4-substituted imidazole and a preliminary study has shown it to be an inhibitor of rat liver microsomal drug oxidation. This work has now been extended. Histamine appears to be a competitive inhibitor of the alpha-hydroxylation (HM) (Ki = 164 microM; IC50 at 20 microM = 308 microM) and O-demethylation (ODM) (Ki = 243 microns; IC50 at 20 microM = 400 microM) of metoprolol in rat liver microsomes. Of the metabolites of histamine only N-acetylhistamine showed comparable inhibitory potency to that of the parent compound. Histamine impaired the disappearance of lignocaine when incubated with rat liver microsomes. This was accompanied by a corresponding inhibition of 3-hydroxy-lignocaine appearance. Histamine produced a type II spectral interaction with rat liver microsomes (lambda max = 432 nm, lambda min = 408 nm; Ks = 0.11 mM). When histamine was incubated alone with rat liver microsomes no loss of substrate was observed. The oxidation of metoprolol by human liver microsomes was impaired by histamine (IC50 values for ODM appearance at 25 microM: liver HL1 greater than 10, HL3 = 3.8 and HL4 = 3.7 mM). In comparison, cimetidine had an IC50 value of 1.5 mM using microsomes from liver HL3. Addition of histamine impaired the elimination of metoprolol by the isolated perfused rat liver in a dose-dependent manner (P less than 0.001, one-way analysis of variance). These data demonstrate that histamine can enter hepatocytes, interact with cytochrome P-450 and inhibit some drug oxidation reactions. The physiological relevance of inhibition of drug metabolism by histamine remains to be determined.     

Hepatic drug transport in the rat: A comparison between isolated hepatocytes,the isolated perfused liver and the liver in vivo

The hepatic transport of three different drugs, the organic anion dibromosulphophthalein, the organic cation d-tubocurarine and the uncharged compound ouabain was studied in vivo in the isolated perfused rat liver and isolated hepatocytes. The respective clearances by uptake were determined for the various substrates and corrected for differences in hepatic blood flow and extracellular protein binding in the three liver preparations. The corrected uptake values in the intact organ, in vivo and in the isolated perfused liver were highly comparable; for dibromosulphophthalein a clearance of 2.1ml/minper 10⁶ hepatocytes was found in vivo, whereas in perfusion a value of 2.4 ml/min per 10⁶ cells was calculated. For d-tubocurarine, the values were 34 × 10^?4 and 55 × 10^?4ml/min per 10⁶ cells obtained in vivo and in the isolated perfused organ, respectively. With ouabain as the substrate, the in vivo clearance amounted to 5.1 × 10^?2, whereas in the isolated perfused liver a value of 4.8 × 10^?2ml/min per 10⁶ cells was calculated. The clearance by uptake obtained for dibromosulphophthalein and ouabain in the isolated hepatocytes appeared to be a factor of 2–3 lower than in the intact organ. In the case of d-tubocurarine however the clearance was identical to that in vivo and the isolated perfused liver.The rate of secretion from isolated hepatocytes was, for dibromosulphophthalein identical to, and for d-tubocurarine and ouabain lower than that in the intact organ, especially as compared with the in vivo preparation.It is concluded that transport function is well preserved in the isolated perfused liver and isolated hepatocytes. For certain substrates freshly isolated hepatocytes may exhibit a somewhat lower uptake and/or secretion rate, in spite of a good cell quality as judged by generally accepted criteria for cell viability. Whether this is due to changes in membrane composition (not detected by our viability tests) or a selection of a subpopulation of hepatocytes, is discussed.     

Aromatic hydroxylation of methylenedioxybenzene (MDB) and methylenedioxymethamphetamine (MDMA) by rabbit liver microsomes.

1. Metabolites formed during incubation of methylenedioxybenzene (MDB) and methylenedioxymethamphetamine (MDMA) with rabbit liver microsomes were examined by h.p.l.c.-electrochemical detection and g.l.c.-mass spectrometry. 2. The trifluoroacetyl derivative of metabolite M-1, obtained from MDB, had a molecular ion at m/z 234 and was identified as 3,4-methylenedioxy-6-hydroxybenzene (sesamol) by comparison with authentic material. 3. The trifluoroacetyl derivative of metabolite M-2, obtained from MDMA, exhibited a molecular ion at m/z 401. Experiments with the deuterium substituted variants of MDMA indicated that the product was hydroxylated on the aromatic ring. 4. The formation of these hydroxylated metabolites required NADPH and was inhibited by carbon monoxide, indicating the possible participation of cytochrome P-450. Phenobarbital (PB) induction caused a marked enhancement of MDP hydroxylase activity whereas MDMA hydroxylation was not affected. 5. The aromatic hydroxylation of MDB and MDMA was also observed in a reconstituted system with cytochrome P-450 isozyme IIB4.     

Effect of oxygen concentration on the reaction of halothane with cytochrome P450 in liver microsomes and isolated perfused rat liver

The influence of oxygen on the complex formation of reduced cytochrome P450 with halothane has been investigated with liver microsomes and perfused livers from phenobarbital-pretreated rats. The reductive formation of the trifluoro carbene complex from halothane in liver microsomes was inhibited at high oxygen concentrations but started to appear below 50 μM oxygen and was maximal under anaerobic conditions. Metyrapone was an efficient inhibitor of the carbene complex formation. Organ spectrophotometry of isolated perfused livers established that the complex appeared already under slightly hypoxic conditions and that metyrapone addition to the perfusion medium abolished its formation. The results indicate the possibility of a reductive in vivo-metabolism of halothane to reactive intermediates when the oxygen concentration of the cell becomes lower than about 50 μM.     

Phalloidin uptake by the liver of cholestatic rats in vivo,in isolated perfused liver and isolated hepatocytes

Summary Contractions of the guinea-pig ileum elicited by GABA were reversibly antagonised by various concentrations of picrotoxinin and bicuculline. This antagonism was specific for GABA-induced responses, responses to ACh, BK, and HA being unaffected. The chloride ion channel blockers, furosemide, 2×10^–4 mol/l, and piretanide, 5.5×10^–5 mol/l, substantially reduced GABA-induced contractions of the ileum but not those elicited by ACh or electrical stimulation of the cholinergic nerves. The action of GABA in stimulating the intrinsic cholinergic nerves appears to be Cl^– dependant.     

The effects of primaquine stereoisomers and metabolites on drug metabolism in the isolated perfused rat liver and in vitro rat liver microsomes

The effect of the antimalarial drug primaquine, its stereoisomers and its proposed metabolites, on the metabolism of substrates for mixed function oxidase, has been studied in isolated perfused rat livers (IPRL) and/or in vitro microsomal suspension. Following acute administration to an IPRL preparation, racemic primaquine produced a dose related reduction in the hepatic clearance of antipyrine which at the highest dose of primaquine (5.0 mg) represented a decrease to 46% of control values. Antipyrine clearance was reduced to a comparable extent by the (+) and (-) isomers and the racemic mixture (each at a dose of 2.5 mg) with mean reductions of 45, 49 and 47%, respectively. These changes in clearance were reflected by significant increases in half-life relative to control. The apparent volume of distribution of antipyrine was unchanged in all experiments. Racemic primaquine and its (+) and (-) isomers were equipotent in inhibiting aminopyrine N-demethylase activity, producing reductions of 56, 59 and 55%, respectively, relative to control values. These three compounds also produced corresponding reductions of 73, 58 and 73% in ethoxyresorufin O-deethylase activity. The N-acetyl and 5-hydroxy derivative of primaquine produced inhibitory effects comparable to that seen for the parent drug. In contrast the carboxylic acid metabolite of primaquine, 6-desmethylprimaquine and 5-hydroxy-6-desmethyl primaquine did not influence aminopyrine N-demethylase activity. These results indicate that the propensity to inhibit drug metabolism by these primaquine related substances, is influenced by functional group substitution rather than the optical activity of the parent drug.