Species differences in beta-oxidative metabolism of a thromboxane A2-receptor antagonist [(+)-S-145] in rat, dog and monkey.

1. The formation of beta-oxidized metabolites from (+)-S-145 [(+)-(Z)-7-[(1R, 2S, 3S, 4S)-3-(benzenesulphonamide)bicyclo-[2.2.]-hept-2-yl]-5-heptenoic acid] by liver homogenates were compared between rat, dog and monkey. Species differences were found in hepatic beta-oxidation capacities. The results agree with the qualitative and quantitative differences in beta-oxidized metabolite proportions among these species observed in vivo. 2. The activities of microsomal (+)-S-145-CoA synthesis, the initial step of the beta-oxidation, were determined. Species differences in their intrinsic clearances primarily agreed with those of the beta-oxidized metabolite formation. 3. (+)-S-145-CoA oxidation activities towards (+)-S-145-CoA by liver homogenates were much higher than the beta-oxidized metabolite formation in all species, indicating that formed (+)-S-145-CoA was immediately beta-oxidized in peroxisomes. The species differences were inconsistent with those of beta-oxidized metabolite formation in vitro. 4. Therefore, quantitative differences of hepatic (+)-S-145 beta-oxidation capacity in rat, dog and monkey were considered to be mainly due to the species difference in (+)-S-145-CoA formation.

Identification of rat and human cytochrome P450 forms involved in the metabolism of the thromboxane A2 receptor antagonist (+)-S-145.

(+)-S-145 [5-(+)-(Z)-7-[(1R, 2S, 3S, 4S)-3-phenylsulfonylaminobicyclo[2.2.1]hept-2-yl]-heptenoic acid] and its beta-oxidized metabolites [two [bisnor or dihydro (DH)-bisnor] or four (tetranor) carbon-shortened products at the carboxyl side chain] are hydroxylated at the C-5 or C-6 position of the bicyclo ring by microsomal monooxygenases. We investigated the oxidative metabolism of (+)-S-145 and its beta-oxidized metabolites with liver microsomes from rats and humans to identify which cytochrome P450 (P450) forms are involved in these reactions. In rats, phenobarbital or dexamethasone treatment significantly increased 5- and 6-hydroxylation activities toward (+)-S-145 and its beta-oxidized metabolites, suggesting the involvement of P4503A forms. Immunoinhibition studies suggested that P4503A2 was mainly responsible for the 5-hydroxylation of (+)-S-145, bisnor, and DH-bisnor and the 6-hydroxylation of bisnor and tetranor. Furthermore, P4502C6, a phenobarbital-inducible 2C form in the rat, was involved in the 6-hydroxylation of (+)-S-145, bisnor, and DH-bisnor. P4502C11, the major constitutive form (male rats), was partly involved in the 5-hydroxylation of DH-bisnor and the 6-hydroxylation of bisnor and DH-bisnor. Reconstitution studies with purified human enzymes and immunoinhibition studies suggest that P4503A4 is primarily involved in the 5-hydroxylation of (+)-S-145 and bisnor and the 6-hydroxylation of tetranor; P4502C9/10 mainly catalyzed the 5-hydroxylation of tetranor and the 6-hydroxylation of (+)-S-145. Results of the present study indicated that the same subfamily P450 forms are responsible for the oxidative metabolism of (+)-S-145 in rats and humans. P4503A enzymes were shown to be involved in the formation of 6-hydroxy tetranor, the main metabolite of S-1452 in vivo.

Sex differences in the metabolism of (+)-S-145, a novel thromboxane A2 receptor antagonist in rat.

1. After the oral administration of 5 mg/kg S-1452 to rat, the plasma levels of (+)-S-145 were similar between the male and female, but there were sex differences in the profiles of its beta-oxidized and hydroxylated metabolites in plasma. 2. beta-Oxidation of (+)-S-145 determined in vitro was slightly higher in the female than in the male, and agreed with the plasma levels of the beta-oxidized metabolites. 3. 5-Hydroxylation activities of (+)-S-145 and beta-oxidized metabolites by rat liver microsomes were significantly higher in the male than in the female, but marked sex differences were not observed in 6-hydroxylation activities. These results revealed that differences in monooxygenase activities directly account for the sex differences in the plasma level of 5-hydroxylated metabolites, and that the peroxisomal beta-oxidation enzyme system also affected the plasma level of 6-hydroxylated metabolites. 4. Biliary excretion was higher in the male than in the female, and quantitative identification of metabolites in bile indicated that this was based on the prominent excretion of taurine conjugates in the male rat. This conclusion was supported by the fact that taurine conjugation activity was higher in male liver homogenates than in the female.

In vitro studies to elucidate the metabolic pathway of (+)-S-145, a thromboxane A2 receptor antagonist, in rats. Evidence for two independent pathways in peroxisomal beta-oxidation.

The metabolism of (+)-S-145, a thromboxane A2 receptor antagonist, was investigated in vitro using isolated hepatocytes, liver homogenates, and subcellular fractions prepared from rats. The cofactor requirement and subcellular distribution of beta-oxidation and hydroxylation suggested that the chain shortening of the carboxyl side chain of (+)-S-145 was catalyzed by beta-oxidation enzyme systems in peroxisomes and hydroxylation at the C-5 and C-6 positions of the bicyclo ring was catalyzed by monooxygenases in microsomes, respectively. In the initial stage of metabolism of (+)-S-145, the potential of activation to its coenzyme A (CoA) thio ester was prominent, compared with that of the hydroxylation. The resulting (+)-S-145-CoA was beta-oxidized. There seems to be two metabolic pathways in the metabolism of (+)-S-145-CoA. One is the biotransformation of (+)-S-145-CoA to bisnor-(+)-S-145 and tetranor-(+)-S-145 in the beta-oxidation cycle, and the other is the reduction of (+)-S-145-CoA to dihydro-(+)-S-145-CoA by NADPH dependent delta 5-reductase followed by beta-oxidation to dihydrobisnor-(+)-S-145, which was scarcely beta-oxidized to tetranor-(+)-S-145. Finally, these beta-oxidized metabolites are hydroxylated by monooxygenases in microsomes at the 5- or 6-position of their bicyclo ring, whereas beta-oxidation activity of hydroxylated metabolites of (+)-S-145 was not observed in the light mitochondrial fraction nor in isolated hepatocytes.

Pathological studies on chlamydiosis in parakeets (Psittacula krameri manillensis).

Histological, immunohistochemical and ultrastructural findings are described in 20 parakeets (Psittacula krameri ntanillensis) affected with chlamydiosis. The main histological lesions consisted of focal necrosis in the liver and adrenal gland, lymphocytic depletion with fibrin exudation in the splenic sinuses and follicles and fibrinopurulent airsacculitis. In these lesions basophilic chlamydial inclusion bodies were found. Macrophages and plasma cells increased mainly in the liver and spleen. Immunohistochemically. more chlamydial inclusion bodies were observed in cells of various organs and tissues including epithelial cells, capillary endothelium and proliferated macrophages. With an electron microscope, the chlamydial inclusion bodies were shown to consist of chlamydial organisms in developmental stages. Concurrent lesions of pulmonary herpesvirus infection appeared frequently in the present cases and seemed to have a close relationship with the chlamydiosis onset.

Species and sex differences in the inhibitory action of the corticosteroid alclometasone dipropionate on the hepatic drug-metabolizing system.

The effect of successive administration of the corticosteroid alclometasone dipropionate (ACM) on the hepatic drug-metabolizing system was examined using male and female rats. Although some pharmacological changes such as increases in plasma enzyme activity, lipid level and protein concentration appeared similarly in ACM-treated male and female rats, the activities of 7-alkoxycoumarin O-dealkylase, especially the O-depropylation activity, decreased dose-dependently by ACM administration only in male rats. ACM did not affect the hepatic drug-metabolizing activity in female rats and mice of both sexes. Also, ACM did not inhibit androgen-independent aniline hydroxylase activity even in male rats. The time course of changes of the drug-metabolizing system in male rats showed a rapid decrease in cytochrome P-450 content and O-depropylation activity following successive treatments with ACM, but there was a slow onset in the decreases of the O-demethylation and O-deethylation activities of 7-alkoxycoumarin. When ACM was withdrawn, the O-demethylation and O-deethylation activities rapidly returned to their control levels, while recovery of the O-depropylation activity was slow. These results suggested that ACM inhibits the hepatic drug-metabolizing enzyme activity associated with a specific form(s) of androgen-dependent cytochrome P-450 in male rats.

Depression of liver microsomal vitamin K epoxide reductase activity associated with antibiotic-induced coagulopathy.

Hypoprothrombinemic changes in blood coagulation parameters, such as prolongation of prothrombin time, increase in the level of plasma protein induced by vitamin K absence, and decrease in plasma prothrombin level, were detected in rats fed a vitamin K-deficient diet. These changes were enhanced by the administration of beta-lactam antibiotics containing N-methyltetrazolethiol, thiadiazolethiol or methyl-thiadiazolethiol. Microsomal vitamin K epoxide reductase activity was suppressed with the maximum effect at 1-2 days after the treatment and with recovery, thereafter, gradually to the normal level after 5-7 days. Hypoprothrombinemic alterations in blood coagulation parameters following a single administration of antibiotic to vitamin K-deficient rats were somewhat delayed compared with the change in the epoxide reductase activity, but the effects of the antibiotic on both blood coagulation parameters and the enzyme activity disappeared completely 7 days after the antibiotic treatment. Antibiotic-induced depression of the epoxide reductase activity was observed even in the vitamin K sufficient rats, although the hypoprothrombinemic changes in the blood coagulation parameters did not develop. Vitamin K administration could normalize the blood coagulation parameters in the hypoprothrombinemic rats caused by treatment with the antibiotics but without recovery of the decreased epoxide reductase activity. These results suggest that some antibiotics inhibit liver microsomal vitamin K epoxide reductase, which causes hypoprothrombinemia to develop under vitamin K-deficient conditions.

Inclusion bodies containing adenovirus-like particles in the kidneys of psittacine birds.

Two types of intranuclear inclusion bodies were found in the epithelial cells of renal tubules or rami ureterici of 12 psittacine birds. One type was large and stained moderately with haematoxylin, poorly with eosin and contained a few granules which stained with haematoxylin. The other was medium in size, circular, almost filling the entire nucleus or irregularly shaped with a clear halo, and stained homogeneously with eosin. Ultrastructurally, the large inclusions consisted of virus particles and fine filamentous materials. The particles exhibited an oval or hexagonal configuration, ranging from 55 to 88 nm in diameter, morphologically characteristic of adenovirus particles. The medium-sized inclusions were composed of aggregates of fine granules. The adenovirus infections were regarded as opportunistic infections.

[Effect of the nonsteroidal anti-inflammatory drug 480156-S on hepatic drug-metabolizing activity and pharmacological action of diazepam and pentobarbital in rats].

Effect of the nonsteroidal anti-inflammatory drug 480156-S on liver drug-metabolizing activity was studied in rats, and its effect was compared with that of cimetidine. Cytochrome P-450-dependent 7-alkoxycoumarin O-dealkylase activity was not affected by a single administration of 480156-S, but the activity, especially the O-demethylase but not the O-depropylase, was suppressed dose-dependently by multiple administrations. Pretreatment of rats with phenobarbital caused a diminution of the inhibitory action of 480156-S. Treatment of rats with cimetidine resulted in a marked decrease in the activity, although it recovered 24 hr later. After the pretreatment of animals with 480156-S or reference drugs, the pharmacological action of diazepam was determined using muscle relaxation and inhibitions of electroshock-induced convulsion and pentetrazole-induced clonic convulsion as the indicators. Prolonged pharmacological activity of diazepam was observed when liver drug-metabolizing activity was lowered by the pretreatment. On the other hand, pentobarbital-induced anesthesia was prolonged by the pretreatment of rats with cimetidine, but the anesthesia was not modified by the administration of 480156-S. These results suggest the inhibitory action of 480156-S on a specific form(s) of P-450 isozyme.

In vitro effect of beta-lactam antibiotics and N-methyltetrazolethiol on microsomal vitamin K epoxide reductase in rats.

Liver microsomal vitamin K epoxide reductase activity was determined by measuring the formation of menaquinone-4 from the substrate menaquinone-4 2,3-epoxide. The enzyme was active when dithiothreitol (DTT) was used as a reducing agent, and the activity increased gradually with increasing concentrations of DTT. Glutathione and cysteine also functioned as reductants, but these physiological reductants showed less than 15% of the activity detected with 0.5 mM DTT. Addition of various beta-lactam antibiotics to the assay mixture for vitamin K epoxide reductase caused a slight inhibition of the activity. N-Methyltetrazolethiol (NMTT) and other heterocyclic thiol compounds also inhibited the enzyme activity in vitro depending on their concentrations. Most of these antibiotics and heterocyclic thiol compounds inhibited the enzyme activity only 10-25% in the in vitro assay system even when higher concentrations were added (5-10 mM). Among the compounds tested, methyl-thiadiazolethiol was the only compound that caused 50% inhibition of the enzyme activity. NMTT-induced inhibition was diminished gradually by increasing DTT concentrations. Kinetic analysis of the inhibitory action of heterocyclic thiol compounds showed competitive inhibition against the reductant DTT and non-competitive inhibition against the substrate. On the other hand, warfarin, a typical anticoagulant, showed different patterns in the inhibitory action: non-competitive inhibition against DTT and mixed-type inhibition against the substrate.

In vivo effects of beta-lactam antibiotics and heterocyclic thiol compounds on vitamin K-dependent carboxylation activity and blood coagulation factors in vitamin K-deficient rats.

The in vivo effects of heterocyclic thiol compounds, corresponding to the 3'-position substituents of several beta-lactam antibiotics, on blood coagulation factors and on liver microsomal gamma-glutamylcarboxylation (gamma-carboxylation) activity were evaluated in rats maintained on a vitamin K-deficient diet. These rats, when compared to normal control animals, exhibited hypoprothrombinemic changes: prolongation of both prothrombin time and activated partial thromboplastin time, decreases in factor VII and plasma prothrombin, and increases in PIVKA II (descarboxyprothrombin) both in plasma and liver. They also displayed a marked increase in liver microsomal gamma-carboxylation activity. These blood coagulation variables could be altered markedly by administering various heterocyclic thiol compounds to the vitamin K-deficient rats, although these compounds did not inhibit gamma-carboxylation activity in an assay system using phylloquinone. A similar pattern of alteration was observed when some beta-lactam antibiotics were administered. Increased microsomal gamma-carboxylation activity in antibiotic-treated vitamin K-deficient rats was normalized by the administration of vitamin K, concomitant with the recovery of blood coagulation variables to the normal range. The results indicate that antibiotic-induced hypoprothrombinemia in vivo is not caused by inhibition of enzymes of the gamma-carboxylation system, such as vitamin K reductase and gamma-glutamylcarboxylase, but is related to the endogenous vitamin K level.

In vitro effects of various heterocyclic thiol compounds and beta-lactam antibiotics on vitamin K-dependent gamma-glutamylcarboxylation activity in liver microsomes.

The in vitro effect of N-methyltetrazolethiol (NMTT), one of the common substituents at the 3'-position of the cephem in various beta-lactam antibiotics, on liver microsomal gamma-glutamylcarboxylation (gamma-carboxylation) activity was examined using solubilized rat liver enzyme. The enzyme activity was inhibited by coexisting with NMTT and NADH, and this inhibitory activity could be suppressed by the addition of a sulfhydryl compound such as dithiothreitol (DTT), glutathione or cysteine. Various five-membered heterocyclic thiol compounds exhibited concentration-dependent inhibition of microsomal gamma-carboxylation activity. These inhibitory actions diminished markedly in the presence of 1 mM DTT. In vitro gamma-carboxylation activity also decreases upon addition of various beta-lactam antibiotics at 1 or 10 mM, depending upon the concentration of the drug. Among the heterocyclic thiol compounds, there is a correlation between their inhibitory activities and hydrophobicities. Thus, the in vitro inhibitory activity of heterocyclic thiol compounds and beta-lactam antibiotics on microsomal gamma-carboxylation activity is not correlated with their molecular structures, but rather depends on their hydrophobicities and with the concentrations in the reaction mixture.

Effect of change of hepatic drug-metabolizing activity on plasma concentrations of major metabolites of the new sleep inducer 450191-S, a 1H-1,2,4-triazolyl benzophenone derivative.

Plasma concentrations of the major metabolites of 450191-S, a new sleep inducer which is a 1H-1,2,4-triazolyl benzophenone derivative, were determined in rats. Under the HPLC conditions employed, several major metabolites were detected in plasma, and thus the plasma concentration-time profiles for these metabolites were checked in rats in various states. When the animals were pretreated with high doses of 450191-S (200 or 600 mg/kg for 5 or 3 days, respectively) to induce hepatic drug-metabolizing enzymes, plasma concentrations of the metabolites after oral administration of a dose of 200 mg/kg of 450191-S decreased markedly depending on the induced enzyme activity. Pretreatment of rats with phenobarbital also caused decreased plasma levels of metabolites, which were almost the same as those in 450191-S-pretreatment. On the other hand, administration of beta-naphthoflavone to rats led to higher plasma levels of metabolites, and slower elimination compared with those in the control and 450191-S- or phenobarbital-pretreated rats. These results indicate that plasma levels of metabolites are regulated by the drug-metabolizing enzymes in the liver. It also suggests the participation of some specific forms of cytochrome P-450 in the biotransformation of 450191-S and its metabolites.

Effect of N-methyltetrazolethiol on liver microsomal gamma-glutamylcarboxylation: modification of the in vitro action of N-methyltetrazolethiol.

Vitamin K-dependent gamma-glutamylcarboxylation activity in rat liver microsomes was monitored using the incorporation of 14CO2 into exogenous pentapeptide and endogenous protein substrates as indicators. Detergent solubilization of the microsomal enzymes was required for the activity to develop, but higher concentrations of detergent inhibited the enzymatic reaction. Pyridoxal-5'-phosphate (PAL-P) and dithiothreitol (DTT) stimulated the enzyme activity. The enzyme activity was observed when the hydroquinone form of vitamin K or the quinone form plus NADH was employed as the cosubstrate, but little activity was detected in the reaction system containing vitamin K-epoxide plus NADH plus DTT. N-Methyltetrazolethiol (NMTT), the substituent at the 3'-position of several beta-lactam antibiotics, inhibited the enzyme activity in vitro only in the reaction system containing NADH. Addition of DTT diminished the in vitro action of NMTT, while PAL-P and detergent did not affect it. The results indicate that the in vitro inhibitory action of NMTT is observable under some specific and restricted assay conditions. This paper also discusses the differences between the in vitro action and the in vivo effect of NMTT.

Autoinduction of 450191-S, a new sleep inducer of 1H-1,2,4-triazolyl benzophenone derivative, in dogs.

Alterations of plasma metabolite profiles were studied following single or multiple oral administrations of 5-[(2-aminoacetamido) methyl]-1-[p-chloro-2-(o-chlorobenzoyl)phenyl]-N, N-dimethyl-1H-1, 2,4-triazole-3-carboxamide hydrochloride dihydrate (450191-S) in Beagle dogs. In plasma, unchanged 450191-S was not detected, but active metabolite, 8-chloro-6-(2-chlorophenyl)-2-(N, N-dimethylcarbamoyl)-4H-1,2, 4-triazolo [1, 5-a] [1, 4] benzodiazepine (M-1) appeared first, followed by four active metabolites that were hydroxylated or demethylated in the N, N-dimethylcarbamoyl side chain of M-1. At single doses of 5 to 50 mg/kg, the areas under the plasma concentration-time curves (AUCs) of the metabolites were linearly increased, showing that there was no saturable process in the steps of absorption, distribution, metabolism and excretion. After multiple administrations (50 mg/kg/d for 15 d), the same metabolites appeared in the plasma but the patterns of the plasma metabolite profiles were considerably different from those after single administrations. The peak plasma levels of M-1 and its hydroxylated metabolites in the carbamoyl side chain were attained more rapidly in the multiple administrations, demonstrating higher peak values compared to those in the single administrations, and the eliminations of these metabolites from plasma were also rapid. However, no difference in the values of the AUCs were observed between single and multiple administrations. With the other active metabolites, the peak plasma levels after multiple administrations were considerably lowered by rapid elimination, resulting in a marked increase in AUCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of rat liver microsomal drug-metabolizing enzymes by a new sleep inducer 450191-S and plasma levels of 450191-S-metabolites.

Liver microsomal 7-alkoxycoumarin O-dealkylase activities in rats were stimulated by the administration of large doses of 5-[(2-aminoacetamide)methyl]-1-[4-chloro-2-(o-chlorobenzoyl)phenyl ]-N, N-dimethyl-1 H-s-triazole-3-carboxamide hydrochloride dihydrate (450191-S), a new sleep inducer which is a 1H-1,2,4-triazolyl benzophenone derivative. To obtain the correlation between the stimulation or induction of hepatic enzymes and the plasma level of the metabolites of 450191-S, various amounts of 450191-S were administered orally to rats and the metabolites in plasma were determined by high performance liquid chromatography. Plasma concentration-time profiles for metabolites in rats showed the appearance of metabolites in plasma followed by their rapid disappearance from blood when the animals received non-inducing amounts of 450191-S. On the other hand, the profiles of metabolites in rats administered higher amounts of the drug showed very high plasma concentrations of metabolites, especially 8-chloro-6-(2-chlorophenyl)-N-methyl-4H-1,2,4-triazolo [1,5-a] [1,4]benzodiazepine-2-carboxamide (M-2) and 8-chloro-6-(2- chlorophenyl)-N-hydroxymethyl-4H-1,2,4-triazolo [1,5-a] [1,4]benzodiazepine-2-carboxamide (M-A), which were maintained for a long time with slow elimination. These results led to the conclusion that the induction of hepatic drug-metabolizing enzymes is closely correlated with the high plasma concentrations of metabolites and their prolonged existence in plasma.

[Effect of a new sleep inducer, 1H-1,2,4-triazolyl benzophenone derivative 450191-S on rat liver drug-metabolizing enzyme system].

The effect of a new sleep inducer, 450191-S, on the hepatic drug-metabolizing enzyme system was examined using rats and compared with those of nitrazepam and phenobarbital. Cytochrome P-450-dependent 7-alkoxycoumarin O-dealkylation activity determined using liver homogenate and isolated microsomes increased after successive oral administrations of 450191-S, and induction of the enzyme system was observed by administration of 150 approximately 200 mg/kg of the drug for at least 3 approximately 5 days. Normal activity was recovered with withdrawal of the drug for 3 approximately 5 days after induction of the hepatic enzyme system. Administration of higher amounts of 450191-S (200 approximately 600 mg/kg/day) for 3 days caused remarkable increases in the O-dealkylase and UDPGA-glucuronyltransferase activities and cytochrome P-450 and b5 contents. Similar changes in the hepatic enzyme system were observed with administration of nitrazepam (200 approximately 600 mg/kg for 3 days, p.o.) or phenobarbital (10 approximately 40 mg/kg for 3 days, i.p.). We concluded that the inducing activity of 450191-S is almost the same as that of nitrazepam, but weaker than that of phenobarbital. When the hepatic enzyme system was induced by the administration of either 450191-S or phenobarbital, the pentobarbital-induced sleeping time was shortened with increasing doses of the drugs. On the other hand, sleeping time was prolonged by the administration of another type of inducer, beta-naphthoflavone. The results suggest that the inductive pattern of 450191-S is similar to that of phenobarbital.

Hepatic 7-alkoxycoumarin O-dealkylase in mice: induction by beta-naphthoflavone of hepatic enzyme activity.

Hepatic 7-alkoxycoumarin O-dealkylation activities in control and beta-naphthoflavone-pretreated mice were determined. The O-demethylation and O-deethylation activities of 7-alkoxycoumarin in control mice were almost the same values, while the O-depropylation activity was lower than those of the other reactions. The O-dealkylase activity varied markedly among the 18 strains of mice surveyed, and strain-dependent differences in the cytochrome P-450 content were also detected among the strains. beta-Naphthoflavone induced O-dealkylation activity, especially O-deethylation and O-depropylation activities, only in ddY, DS and its substrains (A2-3, A3-1, C1-2 and Nh/+). C3H/He and C57BL/6J strains, but not in DBA/2, BALB/c and KYF/2 strains. The former strains of mice are thus classified as "responsive strains" to beta-naphthoflavone and the latter as "non-responsive strains". The O-dealkylase activity in other strains of mice were not clear in responsiveness to beta-naphthoflavone. The hepatic cytochrome P-450 content in responsive strains also increased upon pretreatment of the animals with beta-haphthoflavone. The results indicate marked strain differences in basal and beta-naphthoflavone-induced activity of hepatic 7-alkoxycoumarin O-dealkylase in mice.

Carbon tetrachloride-induced hepatotoxicity in rats: evidence for different susceptibilities of rat liver lobes.

The hepatotoxic effect of carbon tetrachloride (CCl4), reflected by augmented blood aspartate aminotransferase and alanine aminotransferase activities and the extent of histological liver damage, was observed following oral administration of CCl4 to rats. A marked increase of blood transaminase activities and severe degeneration of hepatocytes in the centrilobular region were detected 1-2 days after the administration, while the cytochrome P-450 content and the drug metabolizing activity in livers were depressed immediately after the administration. Based on these results, the effect of CCl4 on hepatic cytochrome P-450 and the histological pattern of liver cells was observed using tissue samples obtained from various liver lobes of rats given CCl4 24 hr previously. Dose-dependent inactivation of cytochrome P-450 by the administration of CCl4 was observed throughout the liver, with the most extensive decrease in the cytochrome content in the median lobe. The extent of liver damage (hydropic swelling degeneration and central necrosis in lobule) was also greater in the median and right liver lobes than in the left lobe. When a small amount of CCl4 was administered, degeneration of liver cells was detected only in the median and right lobes with only slight degeneration in the left lobe. These results indicate different susceptibilities of rat liver lobes to CCl4.

Biotransformation of coumarin derivatives. (2). Oxidative metabolism of 7-alkoxycoumarin by microsomal enzymes and a simple assay procedure for 7-alkoxycoumarin O-dealkylase.

The in vitro biotransformation of 7-alkoxycoumarin by rat liver microsomes was studied to develop a simple and accurate assay procedure for 7-alkoxycoumarin O-dealkylase. 7-Alkoxycoumarin was converted to the O-dealkylated metabolite, 7-hydroxycoumarin, by aerobic incubation of the parent compound with microsomes and NADPH, but the decreased amount of 7-alkoxycoumarin in the reaction mixture was several times higher than that of the 7-hydroxycoumarin produced during the incubation. The thin-layer chromatogram of the ether extractable metabolites in the reaction mixture showed the existence of several fluorescent metabolites including 7-hydroxycoumarin. Fluorescent properties of the parent compound, 7-alkoxycoumarin, and most of the metabolites differed from that of 7-hydroxycoumarin, but the reaction cofactor, NADPH, showed similar properties. Treatment of the reaction mixture with perchloric acid resulted in conversion of NADPH to the non-fluorescent form without any effect upon the fluorescent properties of 7-hydroxycoumarin and its related compounds. Based on these properties, an improved and simple in vitro fluorometric assay of the O-dealkylation of 7-alkoxycoumarin was developed. The method is applicable to routine determination of O-dealkylase activity in both isolated microsomes and whole homogenate. Species differences in the substrate specificity of the O-dealkylation reaction and in the responsiveness of animals to the inducer were observed even with use of the liver homogenate obtained from untreated and phenobarbital- or beta-naphthoflavone-pretreated animals, similar to what was observed with the microsomal system.