Phenobarbital-pretreated Rats Research Articles

Pretreatment with the inducers rifampicin and phenobarbital alters ivermectin gastrointestinal disposition

The goal of the study was to evaluate the effects of rifampicin (RFP) and phenobarbital (PBT) on the plasma and gastrointestinal disposition kinetics of ivermectin (IVM) subcutaneously administered to Wistar rats. Fifty seven rats were used. Animals in Group I were the noninduced (control) group. Those in Groups II and III received a treatment with RFP (160 mg/day) and PBT (35 mg/day), respectively, both given orally during eight consecutive days as induction regimen. The IVM pharmacokinetic study was started 24 h after the RFP and PBT last administration. Animals received IVM (200 microg/kg) by subcutaneous injection. Rats were sacrificed between 6 h and 3 days after IVM administration. Blood and samples of liver tissue, intestinal wall and luminal content of jejunum were collected from each animal. IVM concentrations were measured by high performance liquid chromatography. IVM disposition kinetics in plasma and tissues was significantly modified by the PBT treatment, but not by RFP. Despite the enhanced CYP3A activity observed after the pretreatment with RPF and PBT, there were no marked changes on the percentages of IVM metabolites recovered from the bloodstream in induced and noninduced animals. An enhanced P-glycoprotein-mediated intestinal transport activity in pretreated animals (particularly in PBT pretreated rats) may explain the drastic changes observed on IVM disposition.

A new metabolite of nodakenetin by rat liver microsomes and its quantification by RP‐HPLC method

The biotransformation of nodakenetin (NANI) by rat liver microsomes in vitro was investigated. Two major polar metabolites were produced by liver microsomes from phenobarbital-pretreated rats and detected by reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. The chemical structures of two metabolites were firmly identified as 3'(R)-hydroxy-nodakenetin-3'-ol and 3'(S)-hydroxy-nodakenetin-3'-ol, respectively, on the basis of their (1)H-NMR, MS and optical rotation analysis. The latter was a new compound. A sensitive, selective and simple RP-HPLC method has been developed for the simultaneous determination of NANI and its two major metabolites in rat liver microsomes. Chromatographic conditions comprise a C(18) column, a mobile phase with MeOH-H(2)O (40 : 60, v/v), a total run time of 40 min, and ultraviolet absorbance detection at 330 nm. In the rat heat-inactivated liver microsomal supernatant, the lower limits of detection and quantification of metabolite I, metabolite II and NANI were 5.0, 2.0, 10.0 ng/mL and 20.0, 5.0, 50.0 ng/mL, respectively, and their calibration curves were linear over the concentration range 50-400, 20-120 and 150-24000 ng/mL, respectively. The results provided a firm basis for further evaluating the pharmacokinetics and clinical efficacy of NANI.

METABOLISM OF TERRITREM A IN LIVER MICROSOMES FROM MALE WISTAR RATS: 3. CYTOCHROME P-450 ISOFORMS CATALYZING TRA METABOLISM

The cytochrome P-450 isoforms involved in territrem A (TRA) metabolism in liver microsomes of male Wistar rats have been characterized. Pretreatment with phenobarbital (PB) or dexamethasone (DEX) resulted in a similar significant increase in TRA metabolic activity. Although PB treatment resulted in a significant elevation in CYP2B, CYP2C11, and CYP3A levels, only CYP3A levels were significantly increased by DEX treatment. Cimetidine markedly reduced the formation of the TRA metabolites 4 g -hydroxymethyl-4 g -demethylterritrem A (MA 1 ), 4 g -oxo-4 g -demethylterritrem A (MAX) and 2-dihydro-4 g -demethylterritrem A (MA 2 ) in liver microsomes from 2-wk-old rats (mainly containing CYP3A2) and 7-wk-old rats (containing CYP2B, CYP2C11, and CYP3A2). SKF 525A, which inhibits CYP2B, CYP2C11, and CYP3A2, and orphenadrine, which inhibits CYP2B, also decreased MA 2 formation in liver microsomes from 7-wk-old phenobarbital-pretreated rats. The formation of MA 1 and MAX was not affected. Furthermore, an immunoinhibition study demonstrated that anti-CYP3A2 antibody reduced MA 1 , MAX, and MA 2 formation to nondetectable levels in liver microsomes from 2- and 7-wk-old rats, whereas anti-CYP2C11 or anti-CYP2B antibody, respectively, had no marked effect on MA 1 , MAX, and MA 2 formation in liver microsomes from 7-wk-old untreated or PB-treated rats. These results suggest that the CYP3A isoform is mainly responsible for MA 1 , MAX, and MA 2 formation in liver microsomes in male Wistar rats.

Formation of cyclopropanone during cytochrome P450-catalyzed N-dealkylation of a cyclopropylamine.

The role of single electron transfer (SET) in P450-catalyzed N-dealkylation reactions has been studied using the probe substrates N-cyclopropyl-N-methylaniline (2a) and N-(1'-methylcyclopropyl)-N-methylaniline (2b). In earlier work, we showed that SET oxidation of 2a by horseadish peroxidase leads exclusively to products arising via fragmentation of the cyclopropane ring [Shaffer, C. L.; Morton, M. D.; Hanzlik, R. P. J. Am. Chem. Soc. 2001, 123, 8502-8508]. In the present study, we found that liver microsomes from phenobarbital pretreated rats (which contain CYP2B1 as the predominant isozyme) oxidize [1'-(13)C, 1'-(14)C]-2a efficiently (80% consumption in 90 min). Disappearance of 2a follows first-order kinetics throughout, indicating a lack of P450 inactivation by 2a. HPLC examination of incubation mixtures revealed three UV-absorbing metabolites: N-methylaniline (4), N-cyclopropylaniline (6a), and a metabolite (M1) tentatively identified as p-hydroxy-2a, in a 2:5:2 mole ratio, respectively. 2,4-Dinitrophenylhydrazine trapping indicated formation of formaldehyde equimolar with 6a; 3-hydroxypropionaldehyde and acrolein were not detected. Examination of incubations of 2a by (13)C NMR revealed four (13)C-enriched signals, three of which were identified by comparison to authentic standards as N-cyclopropylaniline (6a, 33.6 ppm), cyclopropanone hydrate (11, 79.2 ppm), and propionic acid (12, 179.9 ppm); the fourth signal (42.2 ppm) was tentatively determined to be p-hydroxy-2a. Incubation of 2a with purified reconstituted CYP2B1 also afforded 4, 6a, and M1 in a 2:5:2 mole ratio (by HPLC), indicating that all metabolites are formed at a single active site. Incubation of 2b with PB microsomes resulted in p-hydroxylation and N-demethylation only; no loss or ring-opening of the cyclopropyl group occurred. These results effectively rule out the participation of a SET mechanism in the P450-catalyzed N-dealkylation of cyclopropylamines 2a and 2b, and argue strongly for the N-dealkylation of 2a via a carbinolamine intermediate formed by a conventional C-hydroxylation mechanism.

Hydroxylation of fatty acids by microsomal and reconstituted cytochrome P450 2B1

Understanding the mechanisms by which cytochrome(s) P450 (CYP) discriminate good from poor substrates, and orient them for highly regio- and stereoselective oxidation, has considerable intrinsic and practical importance. Here we present results of a study of fatty acid hydroxylation by CYP2B1 in a reconstituted system and in microsomes from phenobarbital-pretreated rats. The results indicate that 2B1 prefers decanoic acid as the optimum fatty acid substrate (among C 8–C 16) and that it hydroxylates all positions five or more methylene groups distant from the carboxylate carbon. That hydroxylation does not occur at carbon atoms closer to the carboxyl group than the C 6 position suggests that these carbons may not reach the ferryl oxygen because the carboxyl group is anchored to a specific site at a fixed distance from the heme iron.

The role of enzyme induction and inhibition on cypermethrin hepatotoxicity

Cypermethrin at different concentrations (100, 200, 400 and 800 ng ml−1) was incubated with a primary culture of rat hepatocytes. Cypermethrin was cytotoxic to rat hepatocytes at concentrations of 200 ng ml−1or greater. Toxicity was measured by a decrease in cell viability and leakage of ALT and AST enzymes into the culture medium. The role of cytochrome P450 in the hepatotoxicity of cypermethrin insecticide was investigated in fresh hepatocytes isolated either from phenobarbital pretreated rats or control rats and coincubated with SKF525A. Pretreatment with phenobarbital strongly protected the hepatocytes against the cypermethrin induced loss of cell viability percentage and increased enzyme leakage percentage. Coincubation of the hepatocytes with SKF525A, a well-known cytochrome P450 inhibitor, substantially potentiated the effect of cypermethrin on cell viability and enzyme leakage. These results suggest that the cytocidal hepatotoxicity of cypermethrin in primary hepatocyte culture depends on its parent compound and phenobarbital, as a cytochrome P450 inducer, could be of therapeutic value.

Differential effects of isoflurane and halothane on the induction of heat shock proteins

Isoflurane is considered to be a less hepatotoxic volatile anesthetic than halothane since it not only undergoes quantitatively much less metabolism to form toxic reactive intermediates, but also preserves better hepatic blood flow. However, the biochemical basis for the reduced hepatotoxicity has not been elucidated. In this study, we examined the induction of two heat shock proteins, heat shock protein 70 (HSP70) and heme oxygenase-1 (HO-1), in the livers of rats pretreated with or without phenobarbital, followed by exposure to isoflurane or halothane under hypoxic conditions. In the phenobarbital-pretreated rats, the maximal induction of HSP70 was observed by halothane-hypoxia treatment, followed by a half-maximal induction by isoflurane-hypoxia treatment, and less than 30% induction by hypoxia treatment alone. Serum alanine aminotransferase (ALT) activity, an indicator of hepatic dysfunction, which correlated well with the extent of centrilobular necrosis, showed similar changes with increases in HSP70 mRNA. In contrast, HO-1 mRNA was induced only by treatment with halothane-hypoxia. In addition, changes in the expression of HSP70 and HO-1 mRNAs were correlated with their protein expression in the liver. In non-pretreated rats, neither isoflurane-hypoxia exposure nor halothane-hypoxia exposure caused apparent hepatic injury. There was also no induction of HSP70 or HO-1 mRNA by these treatments in non-pretreated animals. These findings demonstrate that there is a significant difference in hepatic injury, and in the induction of HO-1 and HSP70 between halothane-hypoxia and isoflurane-hypoxia treatments. Isoflurane is known to be safer than halothane, which may, in part, be accounted for by the generation of less oxidative stress in the presence of isoflurane, as assessed by reduced induction of heat shock proteins compared with halothane treatment.

Understanding enantioselective processes: a laboratory rat model for alpha-hexachlorocyclohexane accumulation.

Since cyclodextrin gas chromatography columns became popular for chiral separations, many researchers have noticed high enantiomeric ratios [ER: (+)-enantiomer/(-)-enantiomer] for alpha-HCH in the brains of wildlife. This investigation used the laboratory rat as a model for these phenomena. Rats were either pretreated with phenobarbital (PB) or left untreated and then dosed with alpha-HCH. Animals were sacrificed after 1 or 24 h. The ER averaged 0.95 +/- 0.01 in blood, 1.29 +/- 0.02 in fat, and 0.77 +/- 0.004 in liver. ERs in brain ranged from 2.8 +/- 0.5 to 13.5 +/- 0.4. Both the tissue concentration distribution and the ERs agree well with those previously reported in wildlife. To determine whether high brain ERs were due to enantioselective metabolism or transport through the blood-brain barrier, alpha-HCH exposed brain and liver tissue slices were compared. Concentrations in the brain slices did not decrease with PB pretreatment but did decrease in the liver slices. Enantiomeric ratios in the brain slices averaged 1.11 +/- 0.02 and were 0.76 +/- 0.03 in liver slices for the PB pretreated rats. These data indicate that the enantioselective metabolism of alpha-HCH by the brain is not the mechanism responsible for high ERs in this tissue.

Effect of N-acetylcysteine and deferoxamine on endogenous antioxidant defense system gene expression in a rat hepatocyte model of cocaine cytotoxicity

In the present study we investigated on cultures of hepatocytes from phenobarbital-pretreated rats, the effect of the antioxidants, 0.5 mM N-acetylcysteine (NAC) or 1.5 mM deferoxamine (DFO), previously incubated for 24 h and coincubated with cocaine (0–1000 μM) for another 24 h. Cocaine cytotoxicity was monitored by either the lysis of the cell membranes or apoptosis. Lysis of the cell membranes was evidenced by lactate dehydrogenase leakage, apoptosis was observed by detecting a hypodiploid peak (<2C) in DNA histograms obtained by flow cytometry, peroxide production was quantified with 2′,7′-dichlorodihydrofluorescein diacetate and gene expression of the antioxidant enzymes: Mn- and Cu,Zn-superoxide dismutases, catalase and glutathione peroxidase were measured by Northern blot analysis. NAC and DFO significantly decreased the extent of lysis of cell membranes and apoptosis, and the antiapoptotic effect was parallel to peroxide generation. By the effect of NAC and DFO, significant increases were detected in the levels of mRNA of catalase, manganese superoxide dismutase and glutathione peroxidase. From these results we conclude that NAC or DFO, when incubated in the presence of cocaine, exerted a protective effect against cocaine toxicity at the level of both lysis of the membranes and apoptosis. This protective effect, in the case of NAC, was directed towards an increase in antioxidant enzyme expression, and in the case of DFO against reactive oxygen species generation.

Prevention of halothane-induced hepatotoxicity by hemin pretreatment: Protective role of heme oxygenase-1 induction

Reductive metabolism of halothane in phenobarbital-pretreated rats is known to increase free radical formation that results in hepatotoxicity. It also is associated with a marked induction of microsomal heme oxygenase-1 (HO-1), suggesting that there is an alteration in heme metabolism. In this study, we examined heme metabolism in rats pretreated with phenobarbital, followed by exposure to halothane–hypoxia. In this model, there was a significant decrease in microsomal cytochrome P450 content in the liver, followed by a rapid increase in free heme concentration and a decrease in the level of mRNA for the nonspecific δ-aminolevulinate synthase. A transient but dramatic induction of HO-1 mRNA and a prolonged induction of heat shock protein 70 mRNA also occurred. The HO-1 protein was detected principally in the hepatocytes around the central vein. Serum alanine transaminase (ALT) activity, an indicator of hepatic dysfunction, increased continuously throughout the experiment. Hemin pretreatment induced hepatic HO-1 with abrogation of the halothane-induced hepatotoxicity in this model, as judged by ALT activity and normal histology. Our findings in this study thus indicate that halothane-induced hepatotoxicity is due not only to its reductive metabolite formation, but also to an increase in hepatic free heme concentration, which is a potent prooxidant; HO-1 induction is an important protective response against such changes. This is also the first study to demonstrate that hemin pretreatment, which induces HO-1 prior to exposure to halothane, effectively prevents halothane-induced hepatotoxicity.

Sex-related effect of hemin on cytochrome P450 and drug-metabolizing enzymes in rat liver.

We examined the effect of hemin on rat hepatic microsomal cytochrome P450 (P450) and its molecular species (P450 2E1, 3A2, 2C11 and 2C12) under the induction of heme oxygenase activity in male and female rats. Hemin produced an inverse relationship between the induction of heme oxygenase activity and the decrease of P450 content in a dose-dependent manner. A time course study revealed that hemin produced a significant decrease in total P450 content in male rats to about 37% that of the controls at 24 hr after its administration. Western and Northern blot analyses revealed that the increase in both heme oxygenase-1 (HO-1) protein and HO-1 mRNA reached a maximum at 24 hr and returned to control levels within 120 hr in both sexes. With respect to P450-dependent monooxygenase activities, we found that there was a significant decrease of aniline p-hydroxylase activity to about 30% of the control animals, but not in erythromycin N-demethylase activity at various time intervals. Immunoblot analysis revealed that P450 2E1 in male rat liver was dramatically decreased at 24 hr to about 20% of the controls, but not P450 3A2. Parallel to the decrease of androstenedione 16 alpha-hydroxylase activity, there was a marked decrease of P450 2C11 or 2C12 in male or female rats, respectively, at 72 hr after the treatment; however, hemin did not decrease androstenedione 16 alpha-hydroxylase activity in phenobarbital-pretreated rat liver. These findings suggest that hemin predominantly affects constitutively expressed isozymes rather than inducible P450 species in rat liver.

Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment in rats. Inducibility of FAD monooxygenase system and age effect

The ability of phenobarbital to induce the expression and activity of microsomal drug monooxygenases in the liver presents one of the most important issues in the field of chemical interactions and in the toxicity of xenobiotics. The model of rat liver injury induced by a single dose of thioacetamide (500 mg/kg intraperitoneally) was used to study the effect of phenobarbital (80 mg/kg/day intraperitoneally) for 5 days prior to thioacetamide. Serum parameters of liver injury such as aspartate aminotransferase activity, gamma-glutamyl transferase activity and the total bilirubin levels, as well as the activities of hepatic FAD and cytochrome P450 microsomal monooxygenases, were assayed in 2- and 12-month-old rats. Samples of blood and liver were obtained from controls (injected at 0 h with 0.5 ml of 0.9% NaCl) and at 12, 24, 48, 72 and 96 h of thioacetamide intoxication either to non-treated or phenobarbital pretreated rats. Potentiation of thioacetamide hepatotoxicity by phenobarbital pretreatment was demonstrated at morphological level, and by significant increases in the activities of serum aspartate aminotransferase and gamma-glutamyl transferase, and in the levels of total bilirubin. The extent of potentiation of thioacetamide-induced liver injury by phenobarbital pretreatment was similar in both age groups. Microsomal FAD monooxygenase activity, the enzyme responsible for thioacetamide biotransformation, was significantly enhanced (twofold) by phenobarbital pretreatment, and also underwent a further increase following thioacetamide, preceding the peak of necrosis. Cytochrome P-450 monooxygenases were induced by phenobarbital pretreatment more than sixfold, and sharply decreased when phenobarbital was withdrawn and thioacetamide administered, showing at 48 h intoxication values close to basal. Phenobarbital pretreatment potentiated thioacetamide necrogenicity, and this potentiation was parallel to the induction of the microsomal FAD monooxygenase system, both by phenobarbital and by thioacetamide itself. The extent of thioacetamide-induced liver injury was significantly higher in 12-month-old rats, but the effect of phenobarbital pretreatment was similar in both age groups.

Cytochrome P450 destruction by quinones:: Comparison of effects in rat and human liver microsomes

Exposure to benzene was recently reported to lower the cytochrome P450 (CYP) content in phenobarbital-pretreated rats in vivo (Gut et al., Environ. Health Perspect. 104 (1996) 1211–1218). This study followed the ability of quinonic benzene metabolites (catechol, hydroquinone, and benzoquinone) to destroy CYP in liver microsomes from rats pretreated with various inducers and in human liver microsomes. Sensitivity of CYP isoforms to destruction was revealed and the interspecies differences assessed. The spectrophotometric evaluations of the total CYP content, assay of CYP marker activities, and electrophoresis with immunoblotting after incubation of microsomes with quinones revealed that: (1) rat liver CYP activities markedly differed in sensitivity to quinone-mediated destruction in vitro, CYP 1A and 3A being the most sensitive isoforms; (2) differences in OH radicals formation and lipid peroxidation among microsomes from rats pretreated with various CYP inducers were also observed; (3) semiquinone radical formation, OH radical production, and induction of lipid peroxidation did not contribute significantly to CYP destruction by quinones; (4) the main mechanism of CYP destruction is covalent binding of the oxidized quinone form to protein and heme moieties of CYP; (5) quinones, mainly benzoquinone, destroy human CYP isoforms to a much greater extent than rat enzymes and thus humans may be much more susceptible to the deleterious effect of benzene metabolism. In conclusion, it is suggested that CYP destruction may be another consequence of benzene exposure and should be taken into consideration when evaluations of possible health risks are performed.

Bioactivation of benzo(a)pyrene and formation of DNA adducts in long-term cultures of isolated hepatocytes from spot ( Leiostomus xanthurus)

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (−)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (−)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (−)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (−)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ∼30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ∼5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.

Effect of phenobarbital, 3-methylcholanthrene, and chloramphenicol pretreatment on the pharmacokinetics and pharmacodynamics of azosemide in rats.

The effects of pretreatment with the enzyme inducers phenobarbital (PB) and 3-methylcholanthrene (3-MC) and the enzyme inhibitor chloramphenicol (CM) on the pharmacokinetic and pharmacodynamic parameters of azosemide were examined after intravenous (i.v.) administration of azosemide, 10 mg kg-1, to rats. The nonrenal clearance (1.63 versus 3.30 mL min-1 kg-1) of azosemide increased significantly in 3-MC pretreated rats. This suggested that the nonrenal metabolism of azosemide increased by pretreatment with 3-MC. This relationship was supported by the significant decrease in 24 h urinary excretion of unchanged azosemide in 3-MC pretreated rats (54.1 versus 41.1% of i.v. dose). This relationship was also supported at least in part by the results of a liver homogenate study; the amount of azosemide remaining per gram of liver decreased significantly (48.2 versus 43.0 micrograms) and the amount of M1 formed increased significantly (4.88 versus 6.66 micrograms when expressed in terms of azosemide) in 3-MC pretreated rats after 30 min incubation of 50 micrograms azosemide in 9000 g supernatant fractions of liver homogenates. The content of hepatic cytochrome P-450 (0.751 versus 1.57 nmol/mg protein) and the weight of liver (3.53 versus 4.20% of body weight) increased significantly in 3-MC pretreated rats, suggesting that the metabolizing enzyme(s) for azosemide seemed to be induced by pretreatment with 3-MC. The 8 h urine output (29.2 versus 18.1 mL) and 8 h urinary excretion of sodium (4.02 versus 2.39 mmol) and chloride (4.01 versus 2.73 mmol) per 100 g body weight decreased significantly in 3-MC pretreated rats. However, the diuretic, natriuretic, kaluretic, and chloruretic efficiencies were not significantly different between the control and 3-MC pretreated rats. The pharmacokinetic and pharmacodynamic parameters of azosemide were not significantly different between the control and PB pretreated rats, and similar results were also obtained from the control and CM pretreated rats. The above data indicate that the metabolizing enzyme(s) for azosemide seem(s) to be neither induced by PB pretreatment nor inhibited by CM pretreatment. However, the content of hepatic cytochrome P-450 and the weight of liver increased significantly in PB pretreated rats, while the values were not significantly different between the control and CM pretreated rats.

EFFECT OF PHENOBARBITAL, 3-METHYLCHOLANTHRENE, AND CHLORAMPHENICOL PRETREATMENT ON THE PHARMACOKINETICS AND PHARMACODYNAMICS OF AZOSEMIDE IN RATS

The effects of pretreatment with the enzyme inducers phenobarbital (PB) and 3-methylcholanthrene (3-MC) and the enzyme inhibitor chloramphenicol (CM) on the pharmacokinetic and pharmacodynamic parameters of azosemide were examined after intravenous (IV) administration of azosemide, 10 mg kg−1, to rats. The nonrenal clearance (1·63 versus 3·30 mL min−1 kg−1) of azosemide increased significantly in 3-MC pretreated rats. This suggested that the nonrenal metabolism of azosemide increased by pretreatment with 3-MC. This relationship was supported by the significant decrease in 24 h urinary excretion of unchanged azosemide in 3-MC pretreated rats (54·1 versus 41·1% of IV dose). This relationship was also supported at least in part by the results of a liver homogenate study; the amount of azosemide remaining per gram of liver decreased significantly (48·2 versus 43·0 μg) and the amount of M1 formed increased significantly (4.88 versus 6.66 μg when expressed in terms of azosemide) in 3-MC pretreated rats after 30 min incubation of 50 μg azosemide in 9000 g supernatant fractions of liver homogenates. The content of hepatic cytochrome P-450 (0·751 versus 1·57 nmol/mg protein) and the weight of liver (3.53 versus 4·20% of body weight) increased significantly in 3-MC pretreated rats, suggesting that the metabolizing enzyme(s) for azosemide seemed to be induced by pretreatment with 3-MC. The 8 h urine output (29·2 versus 18·1 mL) and 8 h urinary excretion of sodium (4·02 versus 2·39 mmol) and chloride (4·01 versus 2·73 mmol) per 100 g body weight decreased significantly in 3-MC pretreated rats. However, the diuretic, natriuretic, kaluretic, and chloruretic efficiencies were not significantly different between the control and 3-MC pretreated rats. The pharmacokinetic and pharmacodynamic parameters of azosemide were not significantly different between the control and PB pretreated rats, and similar results were also obtained from the control and CM pretreated rats. The above data indicate that the metabolizing enzyme(s) for azosemide seem(s) to be neither induced by PB pretreatment nor inhibited by CM pretreatment. However, the content of hepatic cytochrome P-450 and the weight of liver increased significantly in PB pretreated rats, while the values were not significantly different between the control and CM pretreated rats. © 1997 John Wiley & Sons, Ltd.

Effect of sulfamethazine on phenobarbital and benzo[a]pyrene induced hepatic microsomal mixed function oxidase system in rats

Administration of sulfamethazine (300 mg/kg, i.p., single dose) to phenobarbital (80 mg/kg, i.p., 3 days) pretreated rats showed significant decrease in microsomal protein, electron transport components and drug metabolizing enzyme activities, compared with phenobarbital administration alone. Induction of mixed function oxidase enzymes due to phenobarbital was not affected by the pretreatment of sulfamethazine. Sulfamethazine administration to benzo[a]-pyrene (20 mg/kg, i.p., 2 days in oil) pretreated rats showed no significant change, but there was a slight decrease in cytochrome P450 and aminopyrine N-demethylase activity, compared with benzo[a]pyrene administration alone. A significant inhibition was observed in aminopyrine N-demethylase activity due to in vitro addition of sulfamethazine (3.5 mM) to microsomal incubations from untreated, sulfamethazine, phenobarbital and benzo[a]pyrene-treated rats. The results indicate that the phenobarbital induced cytochrome P450 is more susceptible to sulfamethazine than benzo[a]pyrene induced cytochrome P450.

Glutathione depletion in human erythrocytes and rat liver: a study on the interplay between bioactivation and inactivation functions of liver and blood

The interplay between bioactivation and inactivation functions of human erythrocytes and rat liver was studied. Glutathione depletion was used as a measure of the amount of reduced glutathione (GSH)-reactive compound. Iodoacetamide (IAcA), N-ethylmaleimide (NEM) and diethyl maleate (DEM), which are electrophiles that need no metabolic activation, were able to deplete GSH in incubations with either aqueous GSH solution or erythrocytes. These results indicate that these compounds can pass the erythrocyte membrane. Cyclophosphamide (CP), 3-hydroxyacetanilide (3-HAA) and 2-methylfurane (2-MF) needed metabolic activation by rat liver microsomes to deplete glutathione in incubations with aqueous GSH solution or erythrocytes. By measuring the sum of both reduced and oxidized glutathione [ = total glutathione (GT)] it became clear that GSH-reactive metabolites are generated out of CP, 3-HAA and 2-MF by the action of microsomes and that these metabolites can pass through the erythrocyte membrane. As GT depletion was higher when microsomes of phenobarbital-pretreated rats were used, the metabolites were (are expected to be) generated by phenobarbital-inducible enzymes. GT was also depleted in incubations with haemolysate and 3-HAA or 2-MF but not in incubations with aqueous GSH solution, which indicates that erythrocyte cytosol can metabolize 3-HAA and 2-MF into GSH-reactive compounds. The pesticides monuron and monulinuron did not affect GT concentrations when aqueous GSH solution, haemolysate or erythrocytes with or without microsomal activating system were tested. When hepatocytes were incubated with 3-HAA or CP (2 m m), about 2 m m of internal GT concentration was depleted. The hepatocytes excreted GSH-reactive metabolites generated from 3-HAA and CP (about 20% of the metabolites formed for 3-HAA). Erythrocyte GT was not depleted in co-incubations of hepatocytes and erythrocytes with 3-HAA. This can be explained by the amounts of GSH-reactive metabolites excreted by the hepatocytes, which would require very effective uptake by the erythrocytes in order to be detectable.

Demonstration of halothane-induced hepatic lipid peroxidation in rats by quantification of F2-isoprostanes.

Halothane can be reductively metabolized to free radical intermediates that may initiate lipid peroxidation. Hypoxia and phenobarbital pretreatment in Sprague-Dawley rats increases reductive metabolism of halothane. F(2)-isoprostanes, a novel measure of lipid peroxidation in vivo, were used to quantify halothane-induced lipid peroxidation in rats. Rats were exposed to 1% halothane or 14% O(2) for 2 h. Pretreatments included phenobarbital, isoniazid, or vehicle. Rats also were exposed to halothane, enflurane, and desflurane at 21% O(2). Lipid peroxidation was assessed by mass spectrometric quantification of F(2)-isoprostanes. Exposure of phenobarbital-pretreated rats to 1% halothane at 21% O(2) for 2 h caused liver and plasma F(2)-isoprostane concentrations to increase fivefold compared to nonhalothane control rats. This halothane-induced increase was enhanced by 14% O(2), but hypoxia alone had no significant effect. Alanine aminotransferase activity at 24 h was significantly increased only in the 1% halothane/14% O(2) group. The effect of cytochrome P450 enzyme induction on halothane-induced F(2)-isoprostane production and liver injury was determined by comparing the effects of isoniazid and phenobarbital pretreatment with no pretreatment under hypoxic conditions. Halothane caused 4- and 11-fold increases in plasma and liver F(2)-isoprostanes, respectively, in non-pretreated rats, whereas isoniazid pretreatment had no effect. Phenobarbital pretreatment potentiated halothane-induced lipid peroxidation with 9- and 20-fold increases in plasma and liver F(2)-isoprostanes, respectively. Alanine aminotransferase activity was increased only in this group. At ambient oxygen concentrations, halothane but not enflurane or desflurane, caused F(2)-isoprostanes to increase. Specific halothane-induced lipid peroxidation was demonstrated in Sprague-Dawley rats using quantification of F(2)-isoprostanes and was increased by hypoxia and phenobarbital pretreatment, but not isoniazid pretreatment.

Enhanced arsenite-induced hepatic morphological and biochemical changes in phenobarbital-pretreated rats.

Changes in liver morphology and biochemistry have been assessed 16 hr after a sc injection of sodium arsenite [As(III), 75 mumol/kg] to control and phenobarbital (PB)-pretreated (80 mg/kg, ip daily for 3 days) adult male Wistar rats. As(III) administration to PB-pretreated rats [PB + As(III)] caused hydropic degeneration, total loss of glycogen, necrosis in some centrilobular zones, and an increase in lipid vacuoles around the periportal area. Electron microscopy showed an increased number of vacuoles and autophagosomes containing organelle-like material. There was a 30% decrease in total hepatic cytochrome P-450 (CYP450). O-dealkylation of ethoxy- and pentoxyresorufin and N-demethylation of benzphetamine decreased to 42, 32, and 30% of control values, respectively. 5-Aminolevulinic acid dehydrase decreased 25% from controls, and metal chelatase activities decreased to 25% of the PB-treated group. Injection of As(III) alone resulted in a mild increase in lipid-containing vacuoles around the periportal zone, a moderate loss of glycogen in midzonal areas, and, by electron microscopy, a dilatation of the bile canaliculi and an increase of the number of myelin-like structures. CYP450 content and the O-dealkylation of ethoxy- and pentoxyresorufin and N-demethylation of benzphetamine all decreased between 30 and 50%. These results demonstrate the greater susceptibility of the liver to injury following PB with compounds not requiring metabolic activation. The metabolic basis of these changes are unclear but may result from an increased demand for metabolic energy due to PB induction and decreased adenosine triphosphate synthesis caused by arsenic.