Hepatic Biotransformation System Research Articles

Bioaccumulation and the expression of hepatic cytochrome P450 genes in marine medaka (Oryzias melastigma) exposed to difenoconazole

This study was conducted to assess the effects of difenoconazole (DFZ), a triazole fungicide, on the hepatic biotransformation system and its bioaccumulation in marine medaka (Oryzias melastigma). Fish were exposed to DFZ (1, 10, 100, 1000ng/L) for 180days. The results showed that: (1) The mRNA levels of hepatic CYP1A1, CYP1B, CYP1C1, CYP27B and CYP3A40 were up-regulated, but those of CYP3A38 and CYP27A1 were down-regulated. (2) The activity of ethoxyresorufin-O-deethylase (EROD) and the content of reduced glutathione (GSH) in the liver were increased in the DFZ-treated groups, and glutathione S-transferase (GST) activity was increased in the 100 and 1000ng/L groups. (3) DFZ was accumulated in the muscle and the biological concentration factors in the 10, 100, and 1000ng/L groups were respectively 149, 81 and 25. These results suggested that long-term exposure to DFZ at low concentrations would result in a bioaccumulation of this compound and disturb the biotransformation system.

THE PROTECTIVE EFFECT OF COFFEE AGAINST PARACETAMOL-INDUCED HEPATIC INJURY IN RATS

ABSTRACT The effect of daily coffee ingestion on the hepatic detoxifying enzymes of rats challenged with high doses of paracetamol (PCM) was investigated. Two groups of male Wistar rats fed, respectively, chow diets supplemented or not with a coffee extract, were sacrificed at 75 days of age, 48 h after injection of saline or PCM (700 mg/kg body wt). The hepatocytes were isolated. Coffee decreased the lipid peroxidation (29%), increased glutathione (GSH) content (46%) and abolished the increased peroxidation caused by PCM (25%). PCM reduced the hepatic GSH content (62%), but in the animals fed the coffee supplemented diet, the reduction was less pronounced (32%). Coffee increased liver UDP-glucuronosyl-transferases UGT1 (75%) and UGT2 (38%), glutathione transferase (46%) and glutathione reductase (72%) activities and decreased glutathione peroxidase (31%). Therefore, it can be concluded that coffee ingestion modulates phase II of the hepatic biotransformation system improving the detoxification ability of the organism against toxic xenobiotics. PRACTICAL APPLICATIONS Coffee is a very popular beverage, which is consumed worldwide and consists of many substances that are suggested to be potential chemoprotectors in biological systems. Among the constituents, caffeine, phenolic polymers, chlorogenic acids, lipids, kahweol and cafestol, all of which have pharmacological properties that lead to adaptation of the organism to its environment, can be cited. Coffee, therefore, is able to protect the human being against diseases and xenobiotic challenges. The results presented in this study demonstrate the effect of coffee as a modulator of the hepatic detoxifying enzyme system. Therefore, this beverage can be classified as a functional food. In addition to the reported application of coffee for the protection against degenerative diseases, free radical damage, cancer and other metabolic alterations, it also acts by modulating the hepatic enzyme system, thereby improving the detoxification capacity of the liver.

Effects of 17α-ethynylestradiol on hormonal responses and xenobiotic biotransformation system of Atlantic salmon ( Salmo salar)

Pharmaceuticals are ubiquitous pollutants in the aquatic environment where their potential effects on non-target species like fish has only recently become subject of systematic investigations. In the present study, experiments were undertaken to examine the effects of a synthetic pharmaceutical endocrine disruptor, ethynylestradiol (EE2), given in water at 5 or 50 ng/L and sampled at days 0 (control), 3 and 7 after exposure, on hepatic phase I and II biotransformation and hormonal pathways of juvenile salmon using quantitative (real-time) polymerase chain reaction (qPCR), Vtg ELISA and 7-ethoxyresorufin O-deethylase (EROD) catalytic activity. Our data show that EE2 produced time- and concentration-specific modulation of estrogen receptor isoforms (ERα, ERβ) and androgen receptor-β (ARβ). EE2 produced a concentration-specific induction of vitellogenin (Vtg) and zona radiata protein ( Zr-protein) at day 3 after exposure. At day 7, Vtg and Zr-protein mRNA (and plasma Vtg protein) expression were significantly decreased in the group given 5 ng EE2/L, compared to dimethyl sulfoxide (DMSO) control group. In the xenobiotic biotransformation pathway, EE2 produced a significant increase of aryl hydrocarbon receptor-α (AhRα) at day 3 in the group given 5 ng EE2/L and AhRβ was decreased at the same concentration at day 7. While CYP3A was not significantly affected by EE2 exposure, the CYP1A1, AhR nuclear translocator (Arnt) and AhR repressor (AhRR) mRNA showed an apparent EE2 concentration and time-dependent decrease. The expression of uridine diphosphoglucuronosyl transferase (UGT) and glutathione S-transferase class pi-like (GST pi-like) mRNA were decreased after exposure to 50 ng EE2/L at both day 3 and 7 after exposure. The effect of EE2 on the CYP1A1 gene expressions paralleled effect on EROD and AhRR mRNA, suggesting a direct role of EE2 in controlling cellular detoxification machinery. Interestingly, the carrier vehicle, DMSO produced significant time-dependent induction of estrogenic (ERα, Vtg and Zr-protein) responses, compared with blank (i.e. without DMSO) controls at day 7 post-exposure. The effect of DMSO totally underscored the observed EE2 effect at day 7 after exposure. In general, these findings support previous reports on the endocrine effects of EE2, in addition to effects on hepatic biotransformation system. In view of the data presented here and our recent studies, the use of DMSO as carrier vehicle in endocrine toxicological experimental studies should be re-evaluated.

Modulatory influence of noscapine on the ethanol-altered hepatic biotransformation system enzymes, glutathione content and lipid peroxidation in vivo in rats

The modulatory potential of noscapine, an opium alkaloid was assessed on the ethanol-induced changes in hepatic drug metabolizing enzyme systems, glutathione content and microsomal lipid peroxidation. Noscapine was administered orally to male Wistar rats at a dose level of 200 mg/kg bw alone as well as in combination with 50% ethanol (v/v) for 5 days. Noscapine administration was associated with a approximately 91% decrease in hepatic microsomal cytochrome P-450 content. A decline of approximately 36% was observed in the NADPH-cytochrome c reductase activity on noscapine administration. The lowering of cytochrome P-450 levels on noscapine administration was accompanied by a concomitant increase in heme oxygenase activity as well as serum bilirubin levels. Our results indicate that the combination dosage of noscapine and ethanol antagonised the ethanol-induced elevation of cytochrome P-450 levels. Noscapine fed rats had decreased glutathione (GSH) content and enhanced lipid peroxidation compared to control rats as indexed by MDA method. Further, noscapine and ethanol coexposure produced a more pronounced elevation in lipid peroxidation and the glutathione levels also decreased significantly. We speculate on the basis of our results that the significant enhancement of lipid peroxidation on combination dosage of noscapine and ethanol is a consequence of depletion of glutathione to certain critical levels. The inhibition of glutathione-S-transferase (GST) as well as lowering of cytochrome P-450 suggests that the biotransformation of noscapine and ethanol is significantly altered following acute coexposures.

Inmunoferon, an immunomodulator of natural origin, does not affect the rat liver cytochrome P-450 and phase II conjugation enzymes.

Inmunoferon is a glycoconjugate of natural origin with immunomodulatory properties. It has recently been shown to regulate TNF-alpha expression induced by lipopolysaccharide (LPS) challenge through a hypothalamo-pituitary-adrenal (HPA)-dependent mechanism. Inmunoferon is orally administered to immunocompromised patients as an adjuvant during immune therapy such as vaccination or infectious diseases treatment. Due to its mainly adjuvant nature, it is necessary to determine if coadministration of Inmunoferon affects the activity of other drugs. In this study we analyzed the possible modification of the hepatic drug biotransformation system by using Inmunoferon in a rat model, which may result in changes in the biological activity of other drugs administered simultaneously. Inmunoferon-treated animals showed no differences to control littermates in antipyrine metabolism. No differences were found in either cytochrome P-450 and b5 levels or cytochrome P-450-dependent activities and phase II conjugation enzymes in lysates from Inmunoferon-treated rat hepatic cells. The same treatment reduced levels of serum TNF-alpha in LPS-challenged animals. In summary, Inmunoferon is unable to affect the hepatic bioconjugation system during administration and thus seems unlikely to interact with, or modify the effect of, coadministered drugs.

Severe and prolonged sedation in five neonates due to persistence of active diazepam metabolites.

Five neonates who suffered from an unexpected long period of respiratory failure, muscular hypotonia, and drowsiness were observed in a retrospective study. Prior to this general depression, unusually high doses of diazepam were administered to all patients via intravenous bolus injection. Serum levels of diazepam and its active metabolites were substantially elevated in the course of the disease. The persistence of the very long-acting N-desmethyldiazepam with considerable extension in neonates and even more exaggerated in premature infants is emphasized due to the reduced capacity of the hepatic biotransformation system. The intravenous application of diazepam imposes a risk of marked and prolonged general depression in neonates. Pronounced adverse effects are to be expected for prematures even after a single diazepam intravenous bolus if the dosage is not appropriate. Diazepam should not be used for short sedation and is not the drug of choice for anticonvulsant therapy in neonates.

Modulatory influence of arecoline on the phytic acid-altered hepatic biotransformation system enzymes, sulfhydryl content and lipid peroxidation in a murine system.

The potential of arecoline alkaloid, by direct or translactational exposure, to modify the chemopreventive efficacy of phytic acid, via modulation of hepatic biotransformation system enzymes and antioxidant defence mechanism, was assessed in a murine system. Phytic acid (500 or 1000 mg/kg b.w. per day) induced a statistically significant increase in the hepatic levels of glutathione S-transferase (GST) and sulfhydryl (-SH) in murine females and suckling neonates. The elevated levels of hepatic cytochrome b5 (Cyt. b5), cytochrome P-450 (Cyt. P-450) and the depleted level of malondialdehyde (MDA) were observed in the lactating mice. Arecoline (20 mg/kg b.w. per day) alone did not modulate the hepatic GST and -SH levels although it induced a statistically significant increase in the levels of Cyt. b5, Cyt. P-450 and MDA in the murine system. Phytic acid-modulated hepatic levels of phase II components were depressed whereas phase I enzymes and lipid peroxides were further elevated by arecoline-plus-phytic acid treatment. The implications of direct or translactational modulation in the competing potential pathways of biotransformation system enzymes in the process of chemical carcinogenesis are discussed.

Effect of arecoline on the curcumin-modulated hepatic biotransformation system enzymes in lactating mice and translactationally exposed F1 pups.

The present study assesses the potential of arecoline alkaloid to translactationally modify the chemopreventive efficacy of curcumin (diferuloyl methane) via neonatal modulation of hepatic biotransformation system enzymes. Curcumin (0.4 g/kg body wt/day) induced a significant increase in the hepatic levels of glutathione-S-transferase (GST), acid-soluble sulfhydryl (SH), cytochrome b5, and cytochrome P-450 in lactating dams and F1 pups at 14 or 21 days. Arecoline (20 mg/kg body wt/day) could not modulate the hepatic GST and SH levels, although significant induction was observed in the levels of cytochrome b5 and cytochrome P-450 in dams and suckling pups. Significant enhancement of hepatic GST, SH, cytochrome b5, and cytochrome P-450 levels was observed in groups treated with curcumin+arecoline. Curcumin-induced levels of GST and SH were depressed whereas cytochrome b5 and cytochrome P-450 were further elevated by curcumin+arecoline treatment. The elevated levels of Phase I enzymes were more significant with exposure to curcumin+arecoline than with arecoline exposure alone. Modulation in competing potential pathways of biotransformation system enzymes in lactating dams may affect the rate and extent of maternal detoxication and thus influence the passage of metabolites of administered xenobiotics to the suckling neonate.

Postnatal effect of arecoline on chlorophyllin-modulated hepatic biotransformation system enzymes in suckling neonate and lactating mice.

The present study evaluates the potential of arecoline alkaloid on chlorophyllin (CHL) modulated levels of hepatic biotransformation system enzymes in suckling neonate and lactating mice. CHL [50, 100, or 200 mg/kg body weight (b.w.)/day] induced significant increases in the hepatic levels of glutathione S-transferase (GST) and sulfhydryl (-SH) in lactating dams and suckling pups of 14 or 21 days. The depleted level of hepatic Cytochrome (Cyt.) P-450 was observed only in lactating dams given 200 mg/kg b.w. CHL. Arecoline (20 mg/kg b.w.) could depress the CHL-induced levels of hepatic GST and -SH, while Cyt. P-450 and Cyt. b5 levels remained unaltered by arecoline alone or arecoline plus CHL treatment. In lactating dams the modulated levels of hepatic biotransformation system enzymes potentially could affect the detoxication efficacy of administered chemicals besides influencing the rate and extent of passage of metabolites to suckling neonate.

Postnatal effect of arecoline on chlorophyllin‐modulated hepatic biotransformation system enzymes in suckling neonate and lactating mice

The present study evaluates the potential of arecoline alkaloid on chlorophyllin (CHL)-modulated levels of hepatic biotransformation system enzymes in suckling neonate and lactating mice. CHL [50, 100, or 200 mg/kg body weight (b.w.)/day] induced significant increases in the hepatic levels of glutathione S-transferase (GST) and sulfhydryl (-SH) in lactating dams and suckling pups of 14 or 21 days. The depleted level of hepatic Cytochrome (Cyt.) P-450 was observed only in lactating dams given 200 mg/kg b.w. CHL. Arecoline (20 mg/kg b.w.) could depress the CHL-induced levels of hepatic GST and -SH, while Cyt. P-450 and Cyt. b5 levels remained unaltered by arecoline alone or arecoline plus CHL treatment. In lactating dams the modulated levels of hepatic biotransformation system enzymes potentially could affect the detoxication efficacy of administered chemicals besides influencing the rate and extent of passage of metabolites to suckling neonate. © 1996 Wiley-Liss, Inc.

Postnatal modulation of hepatic biotransformation system enzymes via translactational exposure of F1 mouse pups to turmeric and curcumin.

The potential for the transfer of active principle(s) of turmeric (Curcuma longa L.) and curcumin (major pigment in turmeric) via translactational route and its modulatory influence on the hepatic biotransformation system enzymes in the lactating dams and their suckling offspring was assessed. Turmeric (4 g/kg b.w. per day) and curcumin (0.4 g/kg b.w. per day) induced significant (P < 0.01) increase in the hepatic levels of glutathione S-transferase (GST) and acid soluble sulfhydryl (-SH) after 14 or 21 days treatment in lactating dams and translactationally exposed F1 pups. However the lower dose of curcumin (0.2 g/kg b.w. per day) could modulate hepatic GST activity (P < 0.05) and -SH (P < 0.01) only after 21 days of treatment in dams and pups. Cytochrome b5 and cytochrome P450 levels were significantly elevated (P < 0.05) in the dams as well as their suckling pups of both 14 and 21 days age groups at the selected dose levels of turmeric (4 g/kg b.w.) and curcumin (0.4 g/kg b.w.). The induction in hepatic biotransformation system enzymes in lactating dams and F1 progeny suggests the passage of active constituents and/or metabolites of turmeric and curcumin via the translactational route.

Induction of the Hepatic Biotransformation System of Golden Ide ( Leuciscus idus (L.)) after Exposure in the River Elbe

The xenobiotic-metabolizing enzyme system of hatchery golden ide [ Leuciscus idus (L.)] were tested for applicability in biological monitoring in rivers. The golden ide, cyprinid fish used for toxicity testing, were caged for 1 to 3 weeks in flowthrough systems at various locations of the river Elbe. The activities of 7-ethoxycoumarin-O-deethylase (ECOD) and the cytochrome P450 (P450) concentrations in the livers were analyzed. Exposure in the river Elbe increased the ECOD activity and the P450 concentration. In a flowthrough basin located in the harbor of Hamburg the ECOD activity increased up to 3.5 times, and the P450 concentration increased slightly by a factor of 1.5. During exposure of the golden ide at two Elbe stations upstream and downstream the city of Hamburg, levels of P450 in the livers did not change significantly when compared with laboratory controls. There was, however, a significant increase of ECOD activity between stations Bunthaus (upstream) and Blankenese (downstream). The ECOD activity in the livers of golden ide caged downstream were significantly higher compared to those at the upstream station. The results presented in this study suggest that golden ide can be employed as experimentals for routine measurements in limnic habitats using biochemical assays.

Evaluation of the modulatory influence of black pepper ( Piper nigrum, L.) on the hepatic detoxication system

The present paper assesses the modifying potential of black pepper on the hepatic biotransformation system in mice. The modulatory effect was assessed on glutathione S-transferase (GST), cytochrome b 5 (cyt. b 5), cytochrome P-450 (cyt. P-450), acid-soluble sulfhydryl (-SH) content and malondialdehyde (MDA) level. Swiss albino mice of either sex (eight weeks old) were fed on a diet containing 0.5%, 1% and 2% black pepper (w/w) for 10 and 20 days. The findings revealed a significant and dose-dependent increase in GST and -SH content in the experimental groups except the one maintained on 0.5% black pepper diet for 10 days. Elevated levels of cyt. b 5 and cyt. P-450 were also statistically significant and dosedependent. The level of MDA was lowered in the group fed on 2% black pepper diet for 20 days. Being a potential inducer of detoxication system, the possible chemopreventive role of black pepper in chemical carcinogenesis is suggested.

Effects of glucosinolate breakdown products on the hepatic biotransformation system in male rats.

The effects of 5-vinyloxazolidine-2-thione (VOT), 1-cyano-3-butene (CYB) and various isothiocyanates on parameters of hepatic phase I and phase II biotransformation were investigated in male rats after oral treatment for 3 consecutive days. The compounds with the exception of CYB caused increases in liver weight and glutathione S-transferase activity. Cytochrome P-450 level and monooxygenase activities were decreased by the isothiocyanates and CYB. Administration of VOT resulted in contrasting response of monooxygenases: reduced aminopyrine N-demethylation and enhanced aniline hydroxylation. Possible mechanisms were discussed.

Drug metabolizing enzymes in rat hepatocytes co-cultured with cell lines

We have developed new co-cultures of continuous cell lines 3T3 (clone A31) and C3H/10T1/2 (clone 8) with hepatocytes as an alternative to co-cultures with noncontinuous epithelial cells. In this biological system we studied in detail the expression of the hepatic biotransformation system. After 7 d in culture, total cytochrome P-450 content and the monooxygenase activities aryl hydrocarbon hydroxylase and 7-ethoxycoumarin o-deethylase still maintained about 30% of their initial value, whereas in pure cultured hepatocytes these activities were undetectable. A significant response to induction by methylcholanthrene and phenobarbital of monooxygenase activities was observed in co-cultures for 7 d. NADPH-cytochrome c reductase activity remained unchanged for at least 7 d in co-cultured hepatocytes, whereas in pure cultures this activity was reduced to about 75% of the initial value after only 24 h. Finally, the activity of the conjugating enzymes UDP-Gt and GSH-t was maintained at nearly the initial levels during the complete period of study. The easy handling of continuous cell lines and the maintenance of the biotransformation system of hepatocytes in co-culture make this approach simpler and easier to standardize.

Drug metabolism components in liver microsomes from Hypostomus punctatus, a brazilian benthic fish (cascudo)

1. The major components of hepatic drug biotransformation system were identified in a Brazilian freshwater benthic fish. 2. Cytochrome P-450 difference spectra were obtained adding 0.02 mM phenazine ethosulphate and 2 mM ascorbate to microsomal suspensions. Basal levels of P-450 were high (0.9 nmol/mg of microsomal protein) and were not induced by 3-MC. 3. Microsomal NADPH-cytochrome C reductase activity was determined in presence of 1.3 × 10 −4M NADPH, 3.3 × 10 −5 M cytochrome C, 1.0 × 10 − M EDTA, 66 μg of microsomal protein per ml in a 0.3 M Tris-HCl buffer, pH8.6. Basal levels of NADPH-cytochrome C were 152.7nmoles/min/mg of microsomal protein.

Effect of portal arterialization on hepatic cytochrome P-450 in rats with portacaval shunt.

Fractional clearance of colloidal gold particles (k), liver weight and hepatic cytochrome P-450 were measured in rats with portacaval shunt and in rats with portacaval shunt plus arterialization of the hepatic stump of the portal vein. The effects of enzyme induction by phenobarbital was studied in both groups. Total arterialization of the liver provides a probably normal hepatic blood flow and seems to protect the liver from post-shunt atrophy. Nevertheless, in both arterialized and shunted rats, the cytochrome P-450 concentration was significantly lower than in controls. The same results were obtained after treatment by phenobarbital. These findings suggest that normal hepatic blood flow and oxygen supply are not the main determinant of a normal activity of the hepatic microsomal biotransformation system. Substances present in portal blood would probably be necessary in maintaining hepatic cytochrome P-450 level.