Microsomal Drug-metabolizing Enzyme Activity Research Articles

Mitochondrial Lipid Peroxidation and Microsomal Drug-metabolizing Enzyme Activity of Rat Hepatotoxicity under Heavy Metals from Slag Waste Exposure

Heavy metals from slag waste (HMSWs) have attracted much attention because of their serious toxicity to the environment and human organs, especially hepatotoxicity. The aim of this study was to explore the effects of different HMSWs exposure on mitochondrial lipid peroxidation, microsomal drug metabolizing enzyme activities as well as their relationship in the rat liver injury. Based on toxicogenomic analysis, heavy metals including iron, copper, cobalt, nickel and manganese, might interfere with pathophysiological processes such as oxidative stress, cell death, and energy metabolism regulation in vivo, and participate in the regulation of HIF-1 signaling pathway, peroxisomes, drug metabolism-cytochrome P450, ferroptosis, and other signaling pathways. HMSWs exposure caused weight loss, and significantly increased lactate dehydrogenase (LDH), malondialdehyde (MDA), alanine transaminase (ALT), and aspartate transaminase (AST) in different groups of rat liver, suggesting the presence of mitochondrial lipid peroxidation damage. In addition, the ratios of AST/ALT and ALT/LDH were down-regulated, especially the ALT/LDH ratios were less than 1, indicating that hepatic ischemic injury occurred in the process of liver injury. The superoxide dismutase (SOD) and mitochondrial membrane potential (MMP) activities in rats also showed significant decreases, indicating the occurrence of hepatic oxidative/antioxidant dysfunction imbalance. Further decision tree analysis of live biochemical abnormalities suggested that AST > 58.78 U/gprot and MDA > 173.2 nmol/mgprot could be used for hepatotoxicity warning. Liver microsomal cytochrome P4501A2 (CYP1A2) and 3A1 (CYP3A1) enzymes were also involved in the hepatotoxic process of heavy metals. These results suggest that lipid peroxidation damage and metabolic damage in liver mitochondria and peroxisomes, may be one of the key events in heavy metal-induced liver injury.

Protection by Osbeckia aspera against carbon tetrachloride-mediated alterations in microsomal drug metabolizing enzyme activity.

Previous investigations have confirmed the protective effect of Osbeckia aspera leaf extract on carbon tetrachloride-mediated liver injury in rat models. It is well known that the earliest alterations in liver cell structure and function following carbon tetrachloride poisoning involve the endoplasmic reticulum and its drug metabolizing enzymes. Therefore, we investigated whether an aqueous leaf extract of O. aspera could offer protection against carbon tetrachloride-induced changes in the microsomal drug metabolizing enzymes aniline hydroxylase and p-aminopyrine N-demethylase. This enzyme activity was compared with phenobarbital-induced righting reflex and lipid peroxidation. Treatment of rats with the aqueous leaf extract of O. aspera (before or after the administration of carbon tetrachloride) resulted in a marked decrease in carbon tetrachloride-mediated alterations in aniline hydroxylase and p-aminopyrine N-demethylase activity, phenobarbital-induced loss of righting reflex and malondialdehyde formation due to lipid peroxidation. The Km value of these enzymes in control and Osbeckia-treated rats were the same. These results show that the plant extract can markedly decrease the carbon tetrachloride-mediated reduction in aniline hydroxylase and p-aminopyrine N-demethylase activity and inhibit peroxidative damage to the cell membrane. Phenobarbital-induced sleeping time in rats and kinetic enzyme studies suggested that the effects of the plant extract was neither due to an induction of the drug-metabolizing enzymes under investigation, nor due to an alteration in the Km values of these enzymes.

Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo

Indirubin and indigo, which are thought to be natural ligands for aryl hydrocarbon receptor (AhR), showed marked AhR ligand activities in a reporter gene assay using recombinant yeast. Their activities were comparable with or more potent than that of 2,3,7,8-tetrachlorodibenzo- p-dioxin. When indirubin and indigo were administered to mice, ethoxyresorufin- O-dealkylase and methoxyresorufin- O-dealkylase activities in the liver were increased, but subsequently decreased within 2 days. Indirubin was more potent than indigo. Levels of cytochrome P450 1A1/2 proteins and mRNAs in the liver of mice dosed with indirubin were also enhanced. These enhancing effects of indirubin and indigo were not observed in AhR knock-out mice. Ethoxyresorufin- O-dealkylase and methoxyresorufin- O-dealkylase activities in rat hepatocytes and HepG2 cells were enhanced by the addition of indirubin or indigo, but less potently than by 2,3,7,8-tetrachlorodibenzo- p-dioxin. Indigocarmine, a sulfate derivative of indigo, which is used as food additive, did not show these inducing effects on drug-metabolizing enzymes. Our results suggest that indirubin and indigo act as inducers for cytochrome P450 1A1/2 mediated by AhR in mammals in vivo.

Inhibition of Drug-Metabolizing Enzymes in Mouse Liver by a Water Soluble Fullerene C60

The microsomal cytochrome P450-dependent monooxygenase system is responsible for the metabolism a variety of xenobiotics including drugs and carcinogens. Fullerenol-1 is a water soluble polyhydroxylated C60 derivative synthesized by a sequence of reactions involving the electrophilic attack of nitronium tetrafluoroborate on fullerenes in the presence of organocarboxylic acids. The present study has evaluated the acute toxicity of fullerenol-1 and determined the effects of the C60 compound on the P450-dependent drug-metabolzing activities. Pretreatments with 0.5 and 1.0g/kg fullerenol-1 decreased P450 and cytochrome b5 contents, and NADPH-cytochrome P450 reductase, as well as drug metabolizing activities toward test substrates benzo[a]pyrene, 7-ethoxycoumarin, aniline, and erythromycin in liver microsomes. Pretreatments with 0.01 and 0.1 g/kg fullerenol-1 had no effect on these monooxygenases. Immunoblotting anlysis of the microsomal proteins revealed that pretreatments with fullerenol-1 did not alter the protein levels of P450s 1A, 2E and 3A. Additions of fullerenol-1 to mouse liver microsomes suppressed monooxygenases activities toward benzo[a]pyrene, 7-ethoxycoumarin, aniline, and erythromycin with IC50 values of 42, 94, 102 and 349 μM, respectively. These results demonstrate that fullerenol-1 can decrease the activities of microsomal drug-metabolizing enzymes in vivo and in vitro. Proper adjustment of the functionalized fullerene compound may facilitate the biomedical application of the new material.

Exposure of hemodialysis patients to di-2-ethylhexyl phthalate

The migration of di-2-ethylhexyl phthalate (DEHP) from dialyzers was studied in 21 patients with chronic renal failure undergoing maintenance hemodialysis. The circulating concentrations of DEHP were measured by high performance liquid chromatography in blood of patients obtained from the inlet and the outlet of the dialyzer during a 4-h dialysis session. During treatment of renal failure using plasticized tubing, the plasma level of DEHP increased. On average, an estimated 75.2 mg of DEHP was extracted from the dialyzer during a single dialysis session, with a range of 44.3–197.1 mg. On the other hand, the total amount of DEHP retained by the patient during the dialysis session was evaluated by the difference between the AUC out and the AUC in and ranged from 3.6 to 59.6 mg. The rate of extraction of DEHP from the dialyzer was correlated ( r=0.705, P<0.05) with serum lipid content (cholesterol and triglyceride). So, we confirmed that patients on hemodialysis are always regularly exposed to considerable amounts of DEHP. However, several metabolic effects have been reported in various animal species following treatment with DEHP, such as changes in lipid metabolism and in hepatic microsomal drug-metabolizing enzyme activities. DEHP is now a well-known hepatic peroxisomal proliferator in rodents and an inducer of many peroxisomal and non-peroxisomal enzymes. So, lipid metabolism modifications and hepatic changes observed in hemodialysis patients could be explained from chronic exposition to DEHP. In the coming years, it seems necessary to reconsider the use of DEHP as a plasticizer in medical devices. Highly unacceptable amounts of DEHP leached during the dialysis session could be easily avoided by careful selection of hemodialysis tubing.

Induction of radical scavenging ability and protection against radiation-induced damage to microsomal membranes following low-dose irradiation

Purpose: To investigate changes in rat liver cytosolic radical scavenging ability and in microsomal membrane function following low doses of radiation. Materials and methods: Wistar rats were irradiated with 1-50cGy of X-rays and liver cytosolic radical scavenging ability was determined using DPPH, a stable free radical. Liver microsomal drug metabolizing activity was determined using hexobarbital as a substrate. Cytosolic antioxidants and microsomal enzymes were spectrophotometrically determined. Results: Irradiation of rats at around 5-10cGy induced liver cytosolic radical scavenging ability and a considerable increase in this ability at 5cGy was observed for 3 days after irradiation. Glutathione reductase was suggested as a candidate of cytosolic antioxidants. Radiation-induced damage to liver microsomal drug metabolizing enzyme activity was suppressed by preirradiation with 5cGy, mainly by protecting cytochrome P-450. Conclusions: Low doses of radiation increased cytosolic radical scavenging ability and resulted in protection of microsomal membrane function which is easily damaged by radiation-induced free radicals.

Evaluation of the Effects of Risedronate on Hepatic Microsomal Drug Metabolizing Enzyme Activities Following Administration to Rats for 14 Days: Lack of an Induction Response

ABSTRACTRisedronate ([1-hydroxy-2-(3-pyridinyl)-ethylidene[bis]phosphonic acid] monosodium salt) was evaluated for induction of hepatic microsomal drug metabolizing enzymes in male and female Sprague Dawley rats (N-4/sex/dose group). Main study animals received water (vehicle control), risedronate (0.1, 0.8, 4, or 16 mg/kg/day) or phenobarbital (80 mg/kg/day, positive control) by daily oral gavage for 14 consecutive days. Recovery study animals received water, risedronate (16 mg/kg/day) or phenobarbital (80 mg/kg/day) by daily oral gavage for 14 consecutive days and then were maintained drug-free for 14 days to evaluate the reversibility of any observed effects. At the conclusion of each study the animals were sacrificed, the liver removed, weighed and the microsomal subcellular fraction prepared. the hepatic microsomal fraction was then evaluated for protein content, cytochrome P450, and the activities of aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase. Risedronate was well tolerated during the dosing phase of the study as evidenced by clinical observations, body weight gain and food consumption which were not significantly different from the vehicle controls. Risedronate did not significantly increase (P>0.05) liver weight, liver/body weight ratio, protein content, P450, aniline hydroxylase, aminopyrine N-demethylase, ethoxycoumarin O-deethylase or p-nitrophenol UDP-glucuronosyltransferase in rats of either sex when compared to vehicle controls. As expected, the hepatic microsomal enzyme inducer phenobarbital significantly increased (P>0.05) liver weight, liver/body weight ratio, protein content (males only), P450, aniline hydroxylase (males only), aminopyrine N-demethylase (males only), ethoxycoumarin O-deethylase and p-nitrophenol UDP-glucuronosyltransferase in rats relative to vehicle controls. Following the 14 day drug-free recovery period the induction parameters increased by phenobarbital reversed to vehicle control levels. the results obtained in this well controlled study indicate that risedronate is not an inducer of hepatic microsomal drug metabolizing enzymes in the rat.

Protective Effect of Rifampicin against Acute Liver Injury Induced by Carbon Tetrachloride in Mice

Rifampicin conferred significant protection against carbon tetrachloride (CCl4)-induced liver injury. Serum alanine transaminase (ALT) and aspartate transaminase (AST) activities were not markedly altered and only hepatocellular fatty degeneration was found in mice pretreated with rifampicin (200 mg/kg), whereas severe centrilobular necrosis was observed and serum ALT and AST activities were as high as 281 and 271 I.U./l, respectively, in the control group following administration of CCl4, (400 μl/kg). The contents and activities of microsomal drug-metabolizing enzymes in rifampicin-pretreated animals were also much higher than those of the controls. CCl4-mediated malondialdehyde (MDA) formation was increased in rifampicin-treated liver microsomes, demonstrating that rifampicin was capable of increasing the NADPH-dependent metabolism of CCl4 catalyzed by P-450 2E1 to produce free radicals. However, MDA formation was obviously depressed by rifampicin at varying concentrations from 2 to 32 × 10-6 M in an in vitro cytochrome P-450 (P-450) enzyme system. On the other hand, NADPH oxidation in the metabolism of CCl4, and aniline hydroxylation were not suppressed in the presence of rifampicin in this systems, suggesting that rifampicin did not influence the biotransformation of CCl4, by P-450 2E1 in vitro. Therefore, the protective effect of rifampicin against CCl4, hepatotoxicity appeared to result from the direct inhibition of lipid peroxidation generated by CCl4,-derived free radicals.

Activities of Liver Microsomal Drug-Metabolizing Enzymes by Oral Administration of Thiabendazole(TBZ) to Male and Female Mice.

Enzyme activities of liver microsomal cytochromes P-450 induced by thiabendazole (TBZ) which has been used as a food additive were investigated in 5 and 11 week-old mice (male and female). TBZ was suspended in olive oil and mice were given orally at 350, 700 or 1000 mg/kg/d for 3 d. Cytochromes P-450 were purified from mice liver microsomes by EAH Sepharose 4B and DEAE Sephadex A-50 chromatography. The catalytic activities of mice liver microsomes and purified cytochromes P-450 fractions on TBZ, aniline, aminopyrine and biphenyl were examined. TBZ hydroxylase, aniline hydroxylase and biphenyl 4-hydroxylase of liver microsomes were induced by TBZ in mice, but aminopyrine N-demethylase was not induced. TBZ hydroxylase activity was higher in female than male in 11 week-old mice. In a reconstituted system of cytochromes P-450, the fractions from TBZ-treated mice showed higher activities of TBZ and aniline hydroxylases than the corresponding fractions from untreated mice.

Enhanced teratogenic activity of di- n-butyltin diacetate by carbon tetrachloride pretreatment in rats

Di- n-butyltin diacetate (DBTA) has been shown previously to cause malformations such as cleft mandible, ankyloglossia and fused ribs in rat foetuses after oral treatment of dams on day 8 of gestation. In this study, effects of pretreatment with carbon tetrachloride (CCl 4) on the teratogenic activity of DBTA were examined in Wistar rats. Pregnant rats were pretreated sc with CCl 4 (0.5 ml/kg) on days 6 and 7 of gestation and were treated orally with DBTA on day 8 of gestation at doses of 0, 4.5, 10 or 22 mg/kg. Caesarean sections were performed on day 20 of gestation, and foetuses were examined for external and skeletal anomalies. Pretreatment with CCl 4 enhanced the incidence of external and skeletal malformations caused by DBTA. Concentrations of di- n-butyltin in embryos, maternal liver and maternal blood on day 9 of gestation were increased by CCl 4 pretreatment, which inhibited the activity of maternal hepatic microsomal drug-metabolizing enzymes. These results suggest that there is little probability that metabolites of DBTA make a critical contribution to the teratogenicity of DBTA.

Effects of Methylsulfonyl Metabolites of PCBs Accumulated in Tissues of Mammals on Activities of Hepatic Microsomal Drug-Metabolizing Enzymes in Rats (Proceedings of the 19th Symposium on Toxicology and Environmental Health)

Effects of Methylsulfonyl Metabolites of PCBs Accumulated in Tissues of Mammals on Activities of Hepatic Microsomal Drug-Metabolizing Enzymes in Rats (Proceedings of the 19th Symposium on Toxicology and Environmental Health)

Suppression of liver microsomal drug-metabolizing enzyme activities in adult female rats pretreated with cannabidiol.

The suppression by cannabidiol (CBD) of the liver microsomal drug-metabolizing enzyme activities in female rats was demonstrated and its mechanism was examined. Pretreatment of rats with CBD (10 mg/kg, i.p.) caused temporary decreases in contents of cytochrome P450 (P450) and b5 and NADPH-cytochrome c (P450) reductase activity compared with values from the vehicle control group. p-Nitroanisole O-demethylase, aniline hydroxylase, d-benzphetamine N-demethylase and delta 9-tetrahydrocannabinol 11-hydroxylase were also decreased by the CBD pretreatment. The latter two activities took a longer time to return to control levels than the former two. However, the CBD pretreatment, which reduced the protein level of P450 UT-2 (CYP2C11) in adult male rats, did not decrease the protein level of P450 F-1 (CYP2C6) or F-2 (CYP2C12) in liver microsomes from female rats. These results suggest that the mechanisms by which CBD suppresses liver microsomal drug-metabolizing enzyme activities are different in male and female rats.

Impairment of debrisoquine 4-hydroxylase and related monooxygenase activities in the rat following treatment with propranolol

The effect of repetitive oral administration of propranolol on hepatic microsomal drug metabolizing enzyme activities in the rat was investigated. Propranolol ring (4-, 5- and 7-)hydroxylase activities were markedly decreased, but, interestingly, N-desisopropylase activity was increased after propranolol administration. A marked decrease in enzyme activity after propranolol pretreatment was also observed with debrisoquine 4-hydroxylation. In addition, a similar decrease was observed with imipramine 2-hydroxylation which co-segregates with debrisoquine/sparteine type polymorphic drug oxidation, but not with imipramine N-demethylation. These results suggest the selective impairment of debrisoquine 4-hydroxylase by propranolol pretreatment.

肝障害ラットの血清ＧＰＴ値および肝薬物代謝酵素に及ぼすクロルプロマジンの影響

Effects of chlorpromazine (CPZ) on serum GPT (SGPT) and hepatic microsomal drug-metabolizing enzyme activities in the rat with liver injured by CCl4 were studied.Rats were intraperitoneally injected with CCl4 at a dose of 0.2ml/kg every 3 days, and with CPZ at a dose of 5 mg/kg once a day for 48 days. SGPT activity was gradually elevated from the beginning of CCl4-administration and reached to the maximum after 30 days.The elevation of SGPT value induced by CCl4-administration was inhibited by the administration of CPZ.In addition, decreases of cytochrome P-450 and cytochrome b5 by CCl4-administration were observed.However, CPZ restored the decrease of cytochrome P-450.These results indicate that CPZ protects liver cell damage induced by CCl4, resulting in that the effect of CPZ on cytochrome P-450 may be important for protecting from drug-induced hepatic injury.

Effects of lignans on hepatic drug-metabolizing enzymes

The effects of lignans, related to macelignan, on hepatic microsomal drug-metabolizing enzyme (DME) activity were evaluated to elucidate the structure-activity relationship in mice and rats. The compounds carrying the methylenedioxyphenyl nucleus were found to be the most potent among compounds tested; which not only produced a marked inhibition of DME with a single dose but a significant induction with repeated treatments. Lack of the methylenedioxy group caused marked decrease in the activity, implying that a methylenedioxy group is essential and of major importance eliciting DME modifying activity.

Effect of Melothria maderaspatana on carbon tetrachloride-induced changes in rat hepatic microsomal drug-metabolizing enzyme activity

Treatment with an aqueous extract of the aerial parts of Melothria maderaspatana, before or after CCl 4 administration in rats markedly decreased CCl 4-mediated reductions in aniline hydroxylase and p-aminopyrine N-demethylase activities. Phenobarbital-induced sleeping time in rats and kinetic enzyme studies showed that the effect of the plant was neither due to an induction of the drug metabolizing enzymes nor due to an alteration in the K m values of the enzymes.

Impairment of human antipyrine metabolism by piroxicam

The pharmacokinetics of a single oral dose of antipyrine was determined in healthy young volunteers (18-28 years), both 3 days before piroxicam, ketoprofen, or naproxen administration and on the following day of their discontinuation. In all subjects treated with piroxicam (10, 20, and 40 mg daily) for 5 consecutive days, the rate of salivary antipyrine elimination slowed. Antipyrine half-life was prolonged and metabolic clearance was reduced significantly (p less than 0.01) proportional to the dose administered. After piroxicam was discontinued, both pharmacokinetic parameters of antipyrine returned toward normal. No significant modification in antipyrine half-life or metabolic clearance rate was demonstrated after pretreatment with ketoprofen (50, 100, and 200 mg daily) or naproxen (250 and 500 mg daily). The impairment on antipyrine disposition produced by piroxicam has been interpreted as a consequence of a reduction in the activity of hepatic microsomal drug-metabolizing enzymes, particularly the cytochrome P-450 system. These results suggest the possibility of drug accumulation and toxicity when certain other therapeutic agents are administered simultaneously with piroxicam. For the same reason, it is recommended to bear in mind the potential danger of long-term piroxicam therapy on the oxidative degradation of steroid hormones and other endogenous compounds that are metabolized by the mixed-function oxidase system.

Effects of psoralen and angelicin on hepatic drug-metabolizing enzyme activities

The effects of psoralen and angelicin on hepatic microsomal drug-metabolizing enzyme (DME) activities were investigated to elucidate the mode of the interaction of furanocoumarins with DME system. A single administration (30mg/kg, i.p.) of both coumarins to mice caused a significant prolongation of hexobarbital-induced hypnosis as well as an increase in strychnine toxicity. The inhibitory potencies of both coumarins as measured by rat hepatic microsomal aminopyrine N-demethylase and hexobarbital hydroxylase activitiesin vitro were considerably weaker than those of other furanocoumarins which possess a side chain moiety. Both coumarins were found to have significant inducing effects on DME system, with repeated treatments of them. The activities of an angular coumarin were stronger than those of a linear coumarin.

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

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

Studies on the fate of thiabendazole(TBZ) in mice. II. Distribution of TBZ, 5-hydroxythiabendazole including its conjugates and the changes of liver microsomal drug-metabolizing enzyme activities in pregnant mice fed TBZ orally.

Thiabendazole (TBZ) suspended in olive oil or 3% gum arabic was given orally to pregnant ICR mice on d 15 or during d 6-15 of gestation at a dose of 1300 mg/kg/d, and TBZ including its metabolites, 5-hydroxythiabendazoles (5-OH TBZ and its conjugates) and the activities of the liver microsomal drug metabolizing enzymes were analyzed. The concentration of 5-OH TBZ was higher than that of its conjugates (glucuronide and sulfate) in the liver and placenta, whereas the levels of the conjugates were greater than that of the free form in the kidney 1 or 25 h after a single or repetitive administration of TBZ. Little sulfate of 5-OH TBZ was detected in the fetus. Aniline hydroxylase activity increased significantly after repetitive and a single administration (25 h after) of TBZ. The amount of cytochrome P-450 also increased significantly after repetitive administration of TBZ. Two vehicles (olive oil and 3% gum arabic) showed no significant difference in the data obtained above.