Comparison of the effects of piperine administered intragastrically and intraperitoneally on the liver and liver mixed-function oxidases in rats

Piperine, a major pungent constituent of black and red peppers, was administered to rats intragastrically and intraperitoneally to study whether it alters the activities of hepatic mixed-function oxidases (MFO) and serum enzymes as specific markers of hepatotoxicity. An intragastric dose of 100 mg/kg of piperine to adult, male Sprague-Dawley rats caused an increase in hepatic microsomal cytochrome P-450 and cytochrome b5, NADPH-cytochrome c reductase, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h following treatment. On the other hand, a 10 mg/kg dose given i.p. exhibited no effect on the activities of the aforementioned parameters of the hepatic drug-metabolizing enzyme system. However, when the intragastric and intraperitoneal doses were increased to 800 mg/kg and 100 mg/kg, respectively, the black pepper alkaloid produced a significant decrease in the levels of cytochrome P-450, benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 24 h after treatment. None of the treatments significantly elevated the activities of serum sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and isocitrate dehydrogenase (ICD), suggesting that piperine is not a hepatotoxic agent.     

Lack of in vitro and in vitro effects of fenbendazole on phase I and phase II biotransformation enzymes in rats, mice and chickens.

Intraperitoneal administration of 10 mg fenbendazole/kg bw daily for 5 d caused no significant alterations in the activities of hepatic microsomal drug-metabolizing enzymes viz aminopyrine N-demethylase, aniline hydroxylase and cytosolic glutathione S-transferase in rats, mice and chickens. Similarly no significant difference in the amount of microsomal cytochrome P-450 and NADPH-cytochrome c reductase was found between control and treated animals. In vitro incubation of fenbendazole with rat, mouse and chicken microsomes suggests that the drug neither binds to microsomal protein cytochrome P-450 nor inhibits the activities of aminopyrine N-demethylase and aniline hydroxylase. Similarly in vitro addition of fenbendazole to cytosolic glutathione S-transferase from the above species did not alter the activity of this enzyme. The results indicate that fenbendazole does not alter the activity of hepatic microsomal monooxygenase system significantly in rats, mice and chickens at a dosage level of 10 mg/kg body weight. In vitro studies also indicate that fenbendazole does not interact with the hepatic microsomal monooxygenase system, indicating it is not a substrate for cytochrome P-450-dependent monooxygenase system.     

Differences in the effects of piperine and piperonyl butoxide on hepatic drug-metabolizing enzyme system in rats

An i.p. administration of rats with piperine (100 mg/kg) and piperonyl butoxide (400 mg/kg) produced a significant decrease in hepatic cytochrome P-450, and activities of benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 1 hr after the treatment. Twenty-four hr later, these parameters along with cytochrome b5 and NADPH-cytochrome c reductase remained depressed only in piperine-treated rats. In contrast, piperonyl butoxide caused a significant induction of these parameters with the exception of cytochrome b5 and aminopyrine N-demethylase, which were up by 36 and 33% over their respective controls but not significantly. These results point up that effect of piperine on hepatic mixed-function oxidases is monophasic while that of piperonyl butoxide is biphasic.     

Differences in the Effects of Piperine and Piperonyl Butoxide on Hepatic Drug-Metabolizing Enzyme System in Rats

ABSTRACT

An i.p. administration of rats with piperine (100 mg/kg) and piperonyl butoxide (400 mg/kg) produced a significant decrease in hepatic cyotchrome P-450, and activities of benzphetamine N-demethylase, aminopyrine N-demethylase and aniline hydroxylase 1 hr after the treatment. Twenty-four hr later, these parameters along with cytochrome b5 and NADPH-cytochrome c reductase remained depressed only in piperine-treated rats. In contrast, piperonyl butoxide caused a significant induction of these parameters with the exception of cytochrome b5 and aminopyrine N-demethylase, which were up by 36 and 33% over their respective controls but not significantly. These results point up that effect of piperine on hepatic mixed-function oxidases is mono-phasic while that of piperonyl butoxide is biphasic.     

Studies on metabolism and disposition of sizofiran (SPG), an anti-tumor polysaccharide. II. Effects on hepatic drug-metabolizing enzyme system in rats

The effect of a single or multiple administration of sizofiran (SPG), an anti-tumor polysaccharide, on a hepatic drug-metabolizing enzyme system was studied in rats. When SPG was given intravenously at a single dose of 0.5 or 10 mg/kg, no alteration was observed in activities of aminopyrine (AP) N-demethylase and aniline hydroxylase, and in cytochrome P-450 (P-450) content in the livers of rats 48 h after dosing. However, only AP demethylase activity decreased by 34% after the administration of 200 mg/kg. Similarly, no change in the hepatic enzyme activities and P-450 content was observed for up to 180 d after a single dose of 10 mg/kg. Subcutaneous treatment of animals with either 10 or 40 mg/kg dose for 3 and 6 months resulted in no alteration in the enzyme activities and P-450 content. These results may indicate that the therapeutically effective dose of SPG has no effect on a hepatic drug-metabolizing enzyme system in rats.     

Contrasting effects of ethylenethiourea on hepatic monooxygenases in rats and mice

The effects of ethylenethiourea (ETU) on the hepatic xenobiotic metabolizing system in rats and mice were investigated. Male rats and male mice were given oral doses of 50 and 75, or 50, 75, 100, 500, and 1,000 mg/kg for 3 days. The microsomal enzymes studied were aminopyrine N-demethylase, aniline hydroxylase, and cytochrome P-450. In rats, the activity of aminopyrine N-demethylase was reduced to values between 60 and 70% of controls 24 h after treatment. A decrease in aniline hydroxylase activity and cytochrome P-450 content was observed on the 3rd day after exposure. In mice, treatment with ETU resulted in an increase of cytochrome P-450 at all dose levels. The activity of aniline hydroxylase was significantly elevated in the groups receiving doses of 100 mg/kg and higher. Aminopyrine N-demethylase was unaffected by the treatment. The results suggest that there are qualitative differences between rats and mice after ETU exposure with respect to the response of the hepatic monooxygenases.     

Induction of hepatic CYP2B1/2 by a teratogenic compound cis-1-[-4-(p-menthane-8-yloxy)phenyl]piperadine (YM9429) in rats

A teratogenic compound cis-1-[-4-(p-menthane-8-yloxy)phenyl]piperadine (YM9429) selectively induces skeletal malformations characterized by cleft palate in rat fetuses. In the present study, we investigated the effect of YM9429 on hepatic cytochrome P-450s and their activities in rats. Oral administrations of YM9429 at a dose of 250, 500 or 750 mg/kg daily for 3 days induced cytochrome P-450 contents in a dose-dependent manner. Concomitant induction of enzyme activities of benzphetamine N-demethylase, erythromycin N-demethylase and, to a lesser extent, aminopyrine N-demethylase was observed. Immunoblot analysis revealed that YM9429 up-regulated hepatic levels of CYP2B1/2 and CYP3A1/2 proteins. A single dose of YM9429 at 250 mg/kg induced CYP2B1/2 protein levels significantly. These results suggest that YM9429 is a strong inducer of cytochrome P-450 with characteristics resembling those of phenobarbital.     

Association between progesterone binding and cytochrome P-450 content of hepatic microsomes in the rat treated with cobalt-haem

The effect of cobalt protoporphyrin IX (Co-haem) given to male rats in single subcutaneous doses (25-100 mumol/kg body wt.) was studied. Co-haem decreased cytochrome P-450 content and aminopyrine N-demethylase activity, but increased progesterone content and 3H-progesterone binding in a dose-related manner. The effect of a single dose of 50 mumol/kg body wt. was reversible; cytochrome P-450 and progesterone content, and progesterone binding, returned to the normal level 24-40 d after injection but aminopyrine N-demethylase activity was only partially restored. The converse actions of Co-haem on microsomal progesterone and cytochrome P-450 content showed high correlation.     

Effect of tetrachlorophthalic anhydride on hepatic microsomal metabolism in rats and mice

The capacity for induction of microsomal metabolic enzymes by tetrachlorophthalic anhydride (TCPA) was evaluated in male Sprague-Dawley rats and male CD-1 mice. The rats were orally dosed for 7 d with TCPA suspended in corn oil at 25, 100, 250, or 500 mg/kg. Following this treatment a dose-dependent reduction in the zoxazolamine paralysis time occurred over the dose range 100-500 mg/kg in the rat. No effect on the hexobarbital sleep time was observed at any test level. TCPA was found to produce statistically significant increases in hepatic aminopyrine N-demethylase, aniline hydroxylase, and cytochrome P-450 in the rat at 500 mg per kg. In addition statistically significant increases were seen in aniline hydroxylase and cytochrome P-450 at 25 mg/kg. Mice were orally dosed with TCPA for 7 d at 250, 500, or 1000 mg/kg. There was no effect in the zoxazolamine paralysis time or the hexobarbital sleep time in this species. Hepatic microsomal enzyme levels were not measured in the mouse. These results suggest that following oral dosage TCPA is a weak inducer of microsomal enzymes in the rat. A similar effect was not observed in the mouse for the parameters tested.     

Altered activity of hepatic mixed-function mono-oxygenase enzymes in streptozotocin-induced diabetic rats.

1. In streptozotocin-induced diabetic male rats, hepatic microsomal aminopyrine N-demethylase activity was depressed, whereas aniline hydroxylase activity and cytochrome P-450 content were increased over control values. 2. In diabetic female rats, hepatic microsomal aminopyrine N-demethylase activity, aniline hydroxylase activity, biphenyl 4-hydroxylase activity, and cytochrome P-450 content were increased over control values. 3. Insulin treatment of diabetic male and female rats antagonized all physical and biochemical abnormalities of the diabetic state; 4. Methyl analogues of streptozotocin did not produce a diabetic state when injected into female rats, and resulted in no changes in aminopyrine N-demethylase activity, aniline hydroxylase activity, or cytochrome P-450 content. 5. Insulin treatment of non-diabetic female rats resulted in slight decreases in aminopyrine N-demethylase and aniline hydroxylase activities, but no changes in cytochrome P-450 content. These observations suggest that insulin primarily influences drug metabolism of diabetic animals through correction of the insulin-deficient diabetic state.     

Alterations in drug metabolising enzymes and lipid peroxidation in different rat tissues by fluoride

Sodium fluoride at a dose level of 5.0 mg/kg enhanced aminopyrine N-demethylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in rat liver, kidney, lung, intestine and testis, whereas acetanilide hydroxylase activity remained unchanged in kidney and lung and was increased in liver, intestine and testis. Sodium fluoride at 20.0 mg/kg caused a decrease in aminopyrine N-demethylase, acetanilide hydroxylase and NADPH cytochrome c reductase activities and cytochrome P450 and cytochrome b5 levels in all tissues, except for an increase in NADPH cytochrome c reductase activity in the intestine and testis. Fluoride at both dose levels produced only marginal changes in glutathione-S-transferase activity except for a 4-fold increase in the testis at 5.0 mg/kg. Sodium fluoride at 5.0 mg/kg increased lipid peroxidation in all tissues studied. At 20.0 mg/kg there was a decrease in lipid peroxidation in liver, lung and testis and an increase in kidney and intestine.     

Induction of xenobiotic biotransformation by the insecticide chlordimeform, a metabolite 4-chloro-o-toluidine and a structurally related chemical o-toluidine

Chlordimeform, 4-chloro-o-toluidine and o-toluidine have all been found to have carcinogenic properties. Due to an empirical link between such properties and alteration of some biotransformation enzymes, the abilities of these three chemicals to affect cytochrome P-450 mediated biotransformation, epoxide hydrolase and glutathione S-transferase have been examined. Chlordimeform had no effect on the cytochrome P-450 content, aniline p-hydroxylase or glutathione S-transferase activities, but induced ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase and epoxide hydrolase activities and decreased aldrin epoxidase and aminopyrine N-demethylase activities. The metabolite 4-chloro-o-toluidine increased cytochrome P-450, ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, glutathione S-transferase and epoxide hydrolase activities. o-Toluidine induced cytochrome P-450, ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase, and aldrin epoxidase activities. Ethoxy-resorufin-O-deethylase activity was induced approximately eight times by chlordimeform and 18 times by 4-chloro-o-toluidine and o-toluidine. Induction was seen at 50 mg/kg with chlordimeform and at 10 mg/kg with the other treatments. Chlordimeform increased the 7 alpha and 16 alpha androstenedione hydroxylase pathways. 4-Chloro-o-toluidine increased the 7 alpha, 16 beta and 16 alpha hydroxylase pathways, while o-toluidine increased the 7 alpha, 6 beta, 16 beta and 16 alpha hydroxylase pathways. All three chemicals marginally decreased the testosterone pathways. SDS-PAGE of rat microsomes revealed an increase in a protein band of MW c54,000 for the chlordimeform and 4-chloro-o-toluidine treated groups. Taken together with the increase in ethoxyresorufin-O-deethylase activity these observations are consistent with the induction of hepatic isozyme P-450d. Thus each chemical has been shown to induce various pathways of biotransformation with increases in the P-450c and P-450d specific substrate ethoxyresorufin-O-deethylase being a consistent finding.     

Hepatotoxicity of pulegone in rats: its effects on microsomal enzymes, in vivo

Oral administration of pulegone (400 mg/kg) to rats once daily for five days caused significant decreases in the levels of liver microsomal cytochrome P-450 and heme. Cytochrome b5 and NAD(P)H-cytochrome c-reductase activities were not affected. Massive hepatotoxicity accompanied by an increase in serum glutamate pyruvate transaminase (SGPT) and a decrease in glucose-6-phosphatase were observed upon treatment with pulegone. A significant decrease in aminopyrine N-demethylase was also noticed after pulegone administration. Menthone or carvone (600 mg/kg), compounds related to pulegone, when administered orally did not cause any decrease in cytochrome P-450 levels. The hepatotoxic effects of pulegone were both dose and time dependent. Pretreatment of rats with phenobarbital (PB) or diethylmaleate (DEM) potentiated the hepatotoxicity caused by pulegone, whereas, pretreatment with 3-methylcholanthrene (3-MC) or piperonyl butoxide protected from it. It appears that a PB induced cytochrome P-450 catalysed reactive metabolite(s) may be responsible for the hepatotoxicity caused by pulegone.     

Effect of caffeine on the hepatic microsomal mixed function oxidase system during phenobarbital and benzo[a]pyrene treatment in rats

Simultaneous administration of caffeine (100 mg/kg, i.p., 3 days) and phenobarbital (80 mg/kg, i.p., 3 days) to adult male rats resulted in a significant decrease in hepatic cytochrome P-450 and acetanilide hydroxylase activity, compared to phenobarbital administration alone. While simultaneous administration of caffeine and benzo[a]pyrene (20 mg/kg, i.p., 2 days) increased acetanilide hydroxylase, compared to benzo[a]pyrene administration, no change was seen in the cytochrome P-450 concentration. In vitro addition of 2.5 mM caffeine to microsomal incubations from untreated, phenobarbital- and benzo[a]pyrene-treated rats inhibited aminopyrine N-demethylase activity. No significant difference was seen in the extent of aminopyrine N-demethylase inhibition due to the in vitro addition of caffeine to microsomes from untreated or phenobarbital-treated rats, whereas inhibition in microsomes from benzo[a]pyrene-treated rats was greater.     

Effect of sizofilan, an immunomodulator, on hepatic microsomal mixed-function oxidase activities in rats

Mixed-function oxidase activities of hepatic microsomal preparations from rats were examined after intraperitoneal administration of sizofilan (SPG), an immunomodulator. Repeated doses of SPG (3 mg/kg/12 hr, 4 times) depressed the hepatic cytochrome P-450 content and the activities of aminopyrine N-demethylase and aniline hydroxylase.     

Depression of hepatic microsomal enzyme systems by lentinan in mice

Studies were performed to determine the effects of an immunopotentiating agent, lentinan, on the hepatic drug-metabolizing enzymes in mice. Lentinan was injected twice a day for two days, and the enzyme activities were determined 12 hr after the last injection of lentinan. A lentinan dose of over 0.25 mg/kg was required to cause a significant decrease (20-40%) in the hepatic microsomal aminopyrine N-demethylase and aniline hydroxylase activities. The loss of drug-metabolizing activity by the treatment with lentinan agreed with the loss of cytochrome P-450 content in many cases. Strain and substrate differences concerning the effect of lentinan on the metabolism of drug were also observed. That is to say, the loss of cytochrome P-450 content by the treatment with lentinan was observed in the ddY, C57BL/6 and BDF1 strain mice, but was not observed in the DBA/2, C3H/He and C57BL/10 strain mice. The decrease in the activities of 7-ethoxycoumarin O-deethylase and biphenyl 2-hydroxylase by the treatment with lentinan was considerably less than that of aminopyrine N-demethylase and aniline hydroxylase in ddY mice.     

Role of 3,5-dichlorophenyl methyl sulfone, a metabolite of m-dichlorobenzene, in the induction of hepatic microsomal drug-metabolizing enzymes by m-dichlorobenzene in rats

The increases in the hepatic microsomal aminopyrine N-demethylase activity and in the content of cytochrome P-450 produced by m-dichlorobenzene (m-DCB) occurred after increases in the hepatic concentration of 3,5-dichlorophenyl methyl sulfone, a minor metabolite. The extent of increases in aminopyrine N-demethylase activity and in the content of cytochrome P-450 at 48 hr after po administration of 200 mg/kg (1.36 mmol/kg) of m-DCB was almost equal to that 72 hr after the ip administration of 25 mumol/kg of the sulfone (Kimura et al., 1983). m-DCB in liver was not detectable at that time, and the concentration of sulfone was 63 to 70% of that 48 to 72 hr after the ip administration of 50 mumol/kg of sulfone. Administration of m-DCB (200 mg/kg) produced a significant reduction in hexobarbital sleeping time, but this reduction was less than that produced by administration of the sulfone (50 mumol/kg). The protein band patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the microsomes from rats treated with the sulfone and m-DCB were similar to those of phenobarbital-treated rats but were different from those of 3-methylcholanthrene-treated rats. The sulfone showed type I interaction with the cytochrome P-450 (Ks, 0.17 mM). The sulfone was formed from the sulfide but reduction of the sulfone was not observed when it was incubated in a hepatic microsomal preparation. The pattern of induction by the sulfone and m-DCB was similar to that by phenobarbital and differed from that by 3-methylcholanthrene. From these results, 3,5-dichlorophenyl methyl sulfone is considered to be a major contributing factor of the inducing activity of m-DCB and to be a potent phenobarbital-like inducer.     

Comparative incidence of oral ochratoxicosis and aflatoxicosis on the activity of drug-metabolizing enzymes in rat liver

Mycotoxicosis has been produced in the rat by daily oral administrations of ochratoxin A (1.5 mg/kg/day) or aflatoxin B1 (1 mg/kg/day). Hepatic microsomal cytochrome P-450 and b5 contents and many phase I and II biotransformation systems have been measured in the course of ochratoxicosis (4 to 15 dosings) and aflatoxicosis (1 to 8 dosings). In case of ochratoxicosis, decreases in cytochrome P-450 level, aminopyrine demethylase and aniline hydroxylase activities were observed in rats receiving 15 administrations of the toxin. Aflatoxicosis induced more severe decreases in cytochrome P-450, aminopyrine demethylase and ethoxycoumarin deethylase following 8 daily gavages. In the two studies, there was no significant change in activities of liver phase II biotransformation enzymes.     

Effect of monensin on liver glutathione, glutathione S-transferase and monooxygenases in rats.

Monensin administered ip to male rats at a dosage of 2.5 mg/kg/d for 3 consecutive days did not change the liver levels of glutathione, but depressed significantly the amount of cytochrome P-450 and the activities of aniline hydroxylase and a cytosolic CDNB-specific glutathione S-transferase. There was a marked decrease in the aminopyrine N-demethylase activity and a significant increase in the pentobarbital sleeping time in rats treated with monensin. In contrast, no change in these parameters was found 2 h after a single ip dose (7.5 mg/kg) of monensin. The results suggest that monensin-induced inhibition of the liver cytosolic glutathione S-transferase and microsomal monooxygenases is non-specific.     

Sex and age-related differences in aminopyrine N-demethylase activity in DA- and Wistar-strain rat liver microsomes. Effect of ovariectomy

1. Liver microsomal mixed-function oxidase components were studied in Wistar and Dark Agouti (DA) rats (4-45 weeks) with regard to sex- and age-related differences. Total cytochrome P-450 ranged from 0.29 to 1 nmol/mg in Wistar rats and from 0.21 to 1.27 nmol/mg in DA rats, males had higher levels than females (P less than 0.0025). Cytochrome b5 ranged between 0.42-1.37 nmol/mg and 0.42-1.56 nmol/mg in Wistar and DA strains, respectively, and NADPH-reductase activity ranged between 14-43 and 11-46 nmol/min per mg (Wistar and DA respectively). 2. Significant age-related differences were found in DA rats with four- to six-fold increase in N-demethylase activity from young to adult rats. Sex-related differences were found in both Wistar- and DA-strain rats, with males having higher (about twice) metabolic activity than females. In contrast, no significant sex- or age-related differences in cytochrome b5 content, or NADPH-reductase activity, were found. 3. Ovariectomy of 10-13-week-old females did not affect N-demethylase activity, cytochrome P-450, cytochrome b5 or NADPH-reductase activity in Wistar or DA rats. 4. Cytochrome P-450 content did not correlate (r = 0.35) with aminopyrine N-demethylase activity. 5. Results indicate that sex- and age-related differences are due to changes in the isozymic composition of cytochrome P-450, and that these changes are not subject to oestrogen regulation.