Hepatic Microsomal Cytochrome Research Articles

Mode of action analysis for fluxapyroxad-induced rat liver tumour formation: evidence for activation of the constitutive androstane receptor and assessment of human relevance

The fungicide fluxapyroxad (BAS 700 F) has been shown to significantly increase the incidence of liver tumours in male Wistar rats at dietary levels of 1500 and 3000 ppm and in female rats at a dietary level of 3000 ppm via a non-genotoxic mechanism. In order to elucidate the mode of action (MOA) for fluxapyroxad-induced rat liver tumour formation a series of in vivo and in vitro investigative studies were undertaken. The treatment of male and female Wistar rats with diets containing 0 (control), 50, 250, 1500 and 3000 ppm fluxapyroxad for 1, 3, 7 and 14 days resulted in a dose-dependent increases in relative weight at 1500 and 3000 ppm from day 3 onwards in both sexes, with an increase in relative liver weight being also observed in male rats given 250 ppm fluxapyroxad for 14 days. Examination of liver sections revealed a centrilobular hepatocyte hypertrophy in some fluxapyroxad treated male and female rats. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 1500 and 3000 ppm fluxapyroxad for 3 and 7 days and in female rats given 50–3000 ppm fluxapyroxad for 7 days and 250–3000 ppm fluxapyroxad for 3 and 14 days; the maximal increases in RDS in both sexes being observed after 7 days treatment. The treatment of male and female Wistar rats with 250–3000 ppm fluxapyroxad for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content and CYP2B subfamily-dependent enzyme activities. Male Wistar rat hepatocytes were treated with control medium and medium containing 1–100 μM fluxapyroxad or 500 μM sodium phenobarbital (NaPB) for 4 days. Treatment with fluxapyroxad and NaPB increased CYP2B and CYP3A enzyme activities and mRNA levels but had little effect on markers of CYP1A and CYP4A subfamily enzymes and of the peroxisomal fatty acid β-oxidation cycle. Hepatocyte RDS was significantly increased by treatment with fluxapyroxad, NaPB and 25 ng/ml epidermal growth factor (EGF). The treatment of hepatocytes from two male human donors with 1–100 μM fluxapyroxad or 500 μM NaPB for 4 days resulted in some increases in CYP2B and CYP3A enzyme activities and CYP mRNA levels but had no effect on hepatocyte RDS, whereas treatment with EGF resulted in significant increase in RDS in both human hepatocyte preparations. Hepatocytes from male Sprague-Dawley wild type (WT) and constitutive androstane receptor (CAR) knockout (CAR KO) rats were treated with control medium and medium containing 1–16 μM fluxapyroxad or 500 μM NaPB for 4 days. While both fluxapyroxad and NaPB increased CYP2B enzyme activities and mRNA levels in WT hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both fluxapyroxad and NaPB only increased RDS in WT and not in CAR KO rat hepatocytes, whereas treatment with EGF increased RDS in both WT and CAR KO rat hepatocytes. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that fluxapyroxad is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for fluxapyroxad-induced rat liver tumour formation has been established. Based on the lack of effect of fluxapyroxad on RDS in human hepatocytes, it is considered that the MOA for fluxapyroxad-induced liver tumour formation is qualitatively not plausible for humans.

Pharmacokinetic and pharmacodynamic drug-drug interaction of Nomilin with atorvastatin in hyperlipidemic mice

Atorvastatin (Atv) is widely used to lower cholesterol levels and treat hyperlipidemia in clinical application. Nomilin (Nom) is a kind of limonoids, which is found and isolated from the citrus herbs of Rutaceae family, which are widely used as patent medicines, functional foods, and nutritional supplements in many countries. In previous studies, Nom has the effect of anti-obesity and curing other metabolic diseases. Nevertheless, in recent years, the drug-drug interaction (DDI) caused by the administration of drugs with synergistic effects have raised worldwide concerns. To investigate the DDI of Nom and Atv in vivo, the pharmacokinetic studies were performed with using C57BL/6 mice. The plasma concentrations of Nom and Atv were measured after oral administration of different drug combinations by a simple and sensitive UHPLC-MS/MS method. The experimental mice were randomly divided into five groups, including control group, model group, administered Nom individually group, administered Atv individually group and co-administered of Nom and Atv group. The lipid levels including total cholesterol (TC), triglycerides (TG), high density lipoproteins-cholesterol (HDL-C), low density lipoproteins-cholesterol (LDL-C) were measured for pharmacodynamic study. The hepatic microsomal Cytochrome P450 (CYP1A2, CYP2E1 and CYP3A11) activities were probed using cocktail assay. The gene and protein expressions of CYP3A11 were detected via qPCR and Western blot method. The results shown that the area under the plasma concentration-time curve (AUC) of Atv in administered Atv individually group was 69.30 ± 15.45 ng/mL × h, while that of combined Nom with Atv group was 42.37 ± 10.15 ng/mL × h (p＜0.05). The degree of reduction in lipid levels of mice treated with co-administration of Atv and Nom was less than that of mice treated with Atv alone. In addition, Nom could cause an increased hepatic microsomal CYP3A11 activity significantly, and induce the gene levels and protein expressions of CYP3A11 elevated in mice livers. In conclusion, Nom could up-regulate CYP3A11 activity, thereby impacting on the pharmacokinetic profile and pharmacodynamic effect of Atv. The findings provide more insight for the use risk of these two drugs to treat hyperlipidemia diseases.

Treatment of Drug-Induced Liver Injury.

Current pharmacotherapy options of drug-induced liver injury (DILI) remain under discussion and are now evaluated in this analysis. Needless to say, the use of the offending drug must be stopped as soon as DILI is suspected. Normal dosed drugs may cause idiosyncratic DILI, and drugs taken in overdose commonly lead to intrinsic DILI. Empirically used but not substantiated regarding efficiency by randomized controlled trials (RCTs) is the intravenous antidote treatment with N-acetylcysteine (NAC) in patients with intrinsic DILI by N-acetyl-p-aminophenol (APAP) overdose. Good data recommending pharmacotherapy in idiosyncratic DILI caused by hundreds of different drugs are lacking. Indeed, a recent analysis revealed that just eight RCTs have been published, and in only two out of eight trials were DILI cases evaluated for causality by the worldwide used Roussel Uclaf Causality Assessment Method (RUCAM), representing overall a significant methodology flaw, as results of DILI RCTs lacking RUCAM are misleading since many DILI cases are known to be attributable erroneously to nondrug alternative causes. In line with these major shortcomings and mostly based on anecdotal reports, glucocorticoids (GCs) and other immuno-suppressants may be given empirically in carefully selected patients with idiosyncratic DILI exhibiting autoimmune features or caused by immune checkpoint inhibitors (ICIs), while some patients with cholestatic DILI may benefit from ursodeoxycholic acid use; in other patients with drug-induced hepatic sinusoidal obstruction syndrome (HSOS) and coagulopathy risks, the indication for anticoagulants should be considered. In view of many other mechanistic factors such as the hepatic microsomal cytochrome P450 with a generation of reactive oxygen species (ROS), ferroptosis with toxicity of intracellular iron, and modification of the gut microbiome, additional therapy options may be available in the future. In summation, stopping the offending drug is still the first line of therapy for most instances of acute DILI, while various therapies are applied empirically and not based on good data from RCTs awaiting further trials using the updated RUCAM that asks for strict exclusion and inclusion details like liver injury criteria and provides valid causality rankings of probable and highly probable grades.

Heavy Metals, Halogenated Hydrocarbons, Phthalates, Glyphosate, Cordycepin, Alcohol, Drugs, and Herbs, Assessed for Liver Injury and Mechanistic Steps.

Aluminum, arsenic, cadmium, chromium, cobalt, copper, iron, lead, mercury, nickel, thallium, titanium, zinc, carbon tetrachloride, phthalates, glyphosate, alcohol, drugs, and herbs are under discussion having the potential to injure the human liver, but allocation of the injury to the hepatotoxicant as exact cause is difficult for physicians and requires basic clinical knowledge of toxicology details. Liver injury occurs at a variable extent depending on the dose, mostly reproducible in animal models that allow studies on molecular steps leading to the hepatocellular injury. These exogenous hepatotoxins may cause an overproduction of reactive oxidative species (ROS), which are generated during microsomal or mitochondrial oxidative stress from incomplete oxygen split and trigger the injury if protective antioxidant capacities are reduced. Primary subcelluar target organelles involved are liver mitochondria through lipid peroxidation of membrane structures and the action of free radicals such as singlet radical 1O2, superoxide radical HO•2, hydrogen peroxide H2O2, hydroxyl radical HO•, alkoxyl radical RO•, and peroxyl radical ROO•. They attempt covalent binding to macromolecular structural proteins. As opposed to inorganic chemicals, liver injury due to chemicals with an organic structure proceedes via the hepatic microsomal cytochrome P450 with its different isoforms. In sum, many exogenous chemicals may have the potential of liver injury triggerd by overproduced ROS leading primarily to impairment of mitochondial functions in the course of structural mitochondial membrane dearrangement. As clinical data were often incomplete, future clinical prototols should focus on meeting liver injury criteria, exclusion of alternative causes, a robust causality evaluation management, and obtaining liver histology if clinically indicated and of benefit for the patient.

Amelioration of cyclophosphamide toxicity via modulation of metabolizing enzymes by avocado (Persea americana) extract.

Cyclophosphamide (CPA) is highly effective in treating several human tumours and autoimmune disorders; but, it triggers deleterious side effects. Avocado, Persea americana (Mill.), is a widely consumed fruit with pronounced nutritional and medicinal value. Though many studies examined the protective mechanisms of natural products against CPA toxicity, almost none investigated the modulation of CPA metabolism as a potential underlying mechanism for protection. Here, we investigated the modulating effect of avocado extract (AE) on certain CPA metabolizing enzymes and its correlation with the extent of CPA-induced pulmonary toxicity and urotoxicity. Rats received oral AE (0.9 g/kg body weight/day) 7 days before a single CPA injection (150 mg/kg body weight) and continued AE intake for 2, 7 or 28 days to study three phases of CPA-induced urotoxicity and pulmonary toxicity. CPA acutely elevated then reduced hepatic microsomal cytochrome P450 2B6 (CYP2B6) content and significantly suppressed bladder and lung glutathione-S-transferase activity. Furthermore, CPA elevated lung myeloperoxidase activity, DNA content and hydroxyproline level and bladder blood content. AE ameliorated CPA-induced derangements through suppression of CYP2B6 and myeloperoxidase and augmentation of glutathione-S-transferase activity in CPA-treated rats. AE modulation of CPA metabolizing enzymes and potential anti-inflammatory effect may mitigate CPA-induced toxicity.

Extraction of Ethyl-p-methoxycinnamate from Kaempferia galanga L.

In this study, ethyl-p-methoxycinnamate (EPMC) was extracted from Kaempferia galanga L. by different methods. The study results show that vacuum drying process led to a significant decrease in EPMC content in the herb (about 7%); EPMC obtained by water distillation not only had a high purity and yield but could also be applied for both fresh and dried herb; whereas, Soxhlet extraction with n-hexane solvent brought the highest extraction yield (1.99% on dried herb weight) despite lower purity when compared to that by water distillation method.
 Keywords
 Ethyl-p-methoxycinnamate, EPMC, Kaempferia galanga L., extraction.
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Piperonyl butoxide: Mode of action analysis for mouse liver tumour formation and human relevance

In a 79 week bioassay the pesticide synergist piperonyl butoxide (PBO) was shown to significantly increase the incidence of hepatocellular adenoma (but not hepatocellular carcinoma) in male CD-1 mice at dietary levels of 100 and 300 mg/kg/day PBO and in female mice at a dietary level of 300 mg/kg/day. As PBO is not a genotoxic agent, a series of investigative studies were undertaken to elucidate the mode of action (MOA) for PBO-induced mouse liver tumour formation. Male CD-1 mice were fed diets to provide intakes of 0 (control), 30, 100 and 300 mg/kg/day PBO and for purposes of comparison 500 ppm sodium phenobarbital (NaPB), a known constitutive androstane receptor (CAR) activator, for 7 and 14 days. Treatment with 100 and 300 mg/kg/day PBO and 500 ppm NaPB increased relative liver weight which was associated with hepatocyte hypertrophy, with hepatocyte replicative DNA synthesis (RDS) being increased after 7 days treatment. The treatment of CD-1 mice with 30−300 mg/kg/day PBO for 14 days resulted in significant dose-dependent increases in hepatic microsomal cytochrome P450 (CYP) content and 7-pentoxyresorufin O-depentylase (PROD) activity and in hepatic Cyp2b10 mRNA levels. In contrast, PBO produced a biphasic effect on markers of activation of the peroxisome proliferator-activated receptor alpha (PPARα), with small increases in microsomal lauric acid 12-hydroxylase activity and hepatic Cyp4a10 mRNA levels being observed in mice given 100 mg/kg/day with PBO, with either no increase or a significant inhibition being observed in mice given 300 mg/kg/day PBO. The hepatic effects of PBO in male CD-1 mice were generally similar to those produced by NaPB and were reversible after the cessation of treatment for 28 days. Studies were also performed in male C57BL/6J (wild type) mice and in hepatic CAR and pregnane X receptor (PXR) knockout mice (CAR KO/PXR KO mice), where in the CAR KO/PXR KO mice PBO had little effect on markers of CAR activation, but produced some increases in markers of PPARα activation. The treatment of male CD-1 mouse hepatocytes for 4 days with 5−50 μM PBO, 10−1000 μM NaPB and 25 ng/mL epidermal growth factor (EGF) resulted in significant increases in hepatocyte RDS. While treatment of hepatocytes from one male and one female human donor with 5−500 μM PBO and 10−1000 μM NaPB for 4 days had no effect on hepatocyte RDS, treatment with EGF resulted in significant increases in RDS in both human hepatocyte preparations. In summary, PBO is predominantly a hepatic CAR activator at carcinogenic dose levels in CD-1 mice, with activation of hepatic CAR resulting in a suppression of the effect of PBO on hepatic PPARα. A robust MOA for PBO-induced mouse liver tumour formation has been established, this MOA being similar to that previously identified for NaPB and some other rodent liver CAR activators. Based on the lack of effect of PBO on RDS in human hepatocytes, it is considered that the MOA for PBO-induced mouse liver tumour formation is qualitatively not plausible for humans.

Metazachlor: Mode of action analysis for rat liver tumour formation and human relevance

In a 2-year study the herbicide metazachlor (BAS 479H) was shown to significantly increase the incidence of liver tumours in female Wistar rats at a dietary level of 8000 ppm. As metazachlor is not a genotoxic agent, a series of in vivo and in vitro investigative studies were undertaken to elucidate the mode of action (MOA) for metazachlor-induced female rat liver tumour formation. Male and female Wistar rats were given diets containing 0 (control), 200 and 8000 ppm metazachlor for 3, 7, 14 and 28 days. The treatment of male rats with 200 and 8000 ppm metazachlor and female rats with 8000 ppm metazachlor resulted in significant increases in relative liver weight, which was associated with a centrilobular hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis (RDS) was significantly increased in male rats given 8000 ppm metazachlor for 3 and 7 days and in female rats given 200 ppm metazachlor for 7–28 days and 8000 ppm metazachlor for 3–28 days. Significant increases in relative liver weight, centrilobular hepatocyte hypertrophy and hepatocyte RDS were also observed in male and female Wistar rats given and 500 ppm sodium phenobarbital (NaPB) for 3–28 days. The treatment of female Wistar rats with either 8000 ppm metazachlor for 7 days or with 500 ppm NaPB for 3 and 7 days resulted in the nuclear translocation of the hepatic constitutive androstane receptor (CAR). Treatment of male and female Wistar rats with 8000 ppm metazachlor for 14 days resulted in significant increases in hepatic microsomal total cytochrome P450 (CYP) content, CYP2B subfamily-dependent enzyme activities and mRNA levels, together with some increases in CYP3A enzyme activity and mRNA levels. The treatment of male Wistar rat hepatocytes with metazachlor (concentration range 0.5–50 μM) and NaPB (500 μM) for 4 days resulted in increased CYP2B enzyme activities and mRNA levels; with metazachlor and NaPB also producing significant increases in hepatocyte RDS levels. Studies were also performed with hepatocytes from male Sprague-Dawley wild type (WT) rats and CAR knockout (CAR KO) rats. While both treatment with metazachlor and NaPB for 4 days increased CYP2B enzyme activities and mRNA levels in WT rat hepatocytes, only minor effects were observed in CAR KO rat hepatocytes. Treatment with both metazachlor and NaPB only increased RDS in WT but not in CAR KO rat hepatocytes. The treatment of hepatocytes from two male human donors with 0.5–25 μM metazachlor or 500 μM NaPB for 4 days resulted in increases in CYP2B6 and CYP3A4 mRNA levels but had no effect on hepatocyte RDS. EGF as concurrently used positive control demonstrated the expected RDS response in all rat and human hepatocyte cultures. In conclusion, a series of in vivo and in vitro investigative studies have demonstrated that metazachlor is a CAR activator in rat liver, with similar properties to the prototypical CAR activator phenobarbital. A robust MOA for metazachlor-induced female rat liver tumour formation has been established. Based on the lack of effect of metazachlor on RDS in human hepatocytes, it is considered that the MOA for metazachlor-induced rat liver tumour formation is qualitatively not plausible for humans.

Impact of Ethnicity-Specific Hepatic Microsomal Scaling Factor, Liver Weight, and Cytochrome P450 (CYP) 1A2 Content on Physiologically Based Prediction of CYP1A2-Mediated Pharmacokinetics in Young and Elderly Chinese Adults.

The vast majority of physiological and biological data required for physiologically based predictions are primarily available in Caucasians rather than other ethnic populations, which leads to a lack of confidence in the application of physiologically based pharmacokinetic (PBPK) modeling for ethnicity-specific prediction of pharmacokinetics in the Chinese population. In this study we recalibrate the system parameters of Chinese-specific PBPK modeling and explore for the first time the relative importance of ethnicity-specific microsomal protein per gram of liver (MPPGL), liver weight, and cytochrome P450 (CYP) 1A2 abundance to the projection of drug disposition mediated by CYP1A2 in young and elderly Chinese adults. Chinese MPPGL levels and associated variability were parameterized and incorporated for the first time into ethnicity-specific PBPK models for the Chinese adults. Parameterization of Chinese liver weights was also recalibrated on the basis of autopsy data from Chinese individuals (n = 4081) across the entire adult age range. Uncertainty surrounding the Chinese-specific CYP1A2 content has also been explored and clarified by conducting ethnicity-related PBPK simulations under different scenarios. Various ethnicity-related or 'what-if' scenarios for PBPK modeling were implemented to assess the predictive performance and explore the relative importance of ethnicity-specific MPPGL and liver weight to the projection of drug disposition mediated by CYP1A2 in terms of two typical CYP1A2 substrates, caffeine and theophylline, in young and elderly Chinese adults by comparing the predicted concentration-time data and associated pharmacokinetic parameter estimates with observations. Compared with 0.85, the liver scalar of 0.9 generally produced more accurate liver weight levels in virtual Chinese peers. Additionally, simulated MPPGL levels on the basis of Caucasian data were not able to reflect the age-independent pattern observed in Chinese adults, dissimilar to that on the basis of Chinese-specific adult MPPGL data. The modeling Scenarios A and B provided similar predictions for theophylline pharmacokinetics in young Chinese adults across different age groups, while Scenario B provided the most accurate prediction for theophylline pharmacokinetics in elderly Chinese adults. However, the use of a stratified value of CYP1A2 content derived from a Han Chinese cohort with a small sample size instead of the pooled value of all Chinese cohorts involved regardless of Chinese sub-ethnicity resulted in inadequate prediction of CYP1A2-mediated pharmacokinetics in terms of caffeine and theophylline in either young or elderly Chinese subjects. Additionally, the impact of ethnic-specific MPPGL on predictive accuracy of theophylline pharmacokinetics in elderly Chinese subjects is more evident than that of liver weight. We provided quantitative information pertaining to Chinese-specific levels of liver weight and MPPGL, and recalibrated these system parameters for PBPK modeling for young and elderly Chinese subjects. Uncertainty surrounding the Chinese-specific CYP1A2 content has also been clarified. PBPK modeling based on the recalibrated system parameters can accurately simulate CYP1A2-mediated pharmacokinetics in both young and elderly Chinese adults, particularly in elderly individuals.

Pharmacokinetic implications of albendazole biotransformation in goats following interaction with curcuma longa

To test the hypothesis that Curcuma longa could act as hepatic microsomal cytochrome P450 inhibitor resulting in increased bioavailability of albendazole, a study was undertaken to understand the pharmacokinetics and efficacy of albendazole in goats with and without coadministration of albendazole with hydroalcoholic extract of Curcuma longa at 7.5 and 500.0 mg per kg body weight, respectively. The comparative pharmacokinetic values (Mean±S.E.) of albendazole sulphoxide in goats using albendazole and albendazole combination revealed significantly (P<0.05) reduced values of concentration maximum, area under concentration-time curve and total area under the first movement of plasma drug concentration-time curve (P<0.001) in goats receiving albendazole and Curcuma long a. Significantly lower values of under concentration-time curve in goats receiving albendazole in combination with Curcuma longa had resulted in relative bioavailability of 0.57. Estimated drug-parasite contact of albendazole sulphoxide in goats revealed significantly decreased values in goats receiving ABZ combination. Nematode parasites of goats dominated by Haemonchus contortus also exhibited resistant against benzimidazole following administration of albendazole alone and in combination with Curcuma longa. Simultaneous administration of hydroalcoholic extract of Curcuma longa, contrary to the hypothesis, resulted in reduced bioavailability of albendazole.

Effects of Dendrobium huoshanense on expressions and activities of hepatic microsomal cytochrome P450s in mice

This study was carried out to investigate the effect of oral administration of Dendrobium huoshanense on the expressions and activities of hepatic microsomal cytochrome P450s in mice, and to provide a reference for the evaluation of drug-drug interactions between D. huoshanense and clinical drugs. The C57BL/6 mice were randomly divided into blank control group, D. huoshanense low dose group (crude drug 1.25 g·kg⁻¹), D. huoshanense high dose group (crude drug 7.5 g·kg⁻¹), and phenobarbital positive control group (0.08 g·kg⁻¹). Each group was intragastrically administered with drugs for 2 weeks. The mice were sacrificed and their liver microsomes were prepared. The expressions of major subtypes of P450 enzyme were determined by Western blot and the probe drugs were used to detect the enzyme activities of P450 subtypes with protein expression changes. Western blot analysis showed that the protein expressions of CYP1A1, CYP1A2 and CYP2B in liver tissues were up-regulated in D. huoshanense-treated group. In vitro enzyme activity tests showed that there were no significant difference in metabolism of 7-ethoxyresorufin (a probe drug for CYP1A1) and bupropion (a probe drug for CYP2B) between D. huoshanense group and control group. The metabolism of phenacetin (a probe drug for CYP1A2) showed a statistical difference in rate Vmax, and it was significantly increased by approximately 20% in D. huoshanense group as compared with the blank control group, and the clearance CLint in treated group was also increased by about 32%. Therefore, oral administration of D. huoshanense had no effects on the activities of most hepatic P450 enzymes in mice, with no drug-drug interaction related to the P450 enzyme system in most clinical drugs theoretically. However, oral administration of D. huoshanense may accelerate the metabolism of CYP1A2-catalyzed drugs, which needs to be considered in clinical practice.

Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats

This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16β- (CYP2B1), and 6β-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1β, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P<0.05; fold change>1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury.

Impact of hepatic P450-mediated biotransformation on the disposition and respiratory tract toxicity of inhaled naphthalene

We determined whether a decrease in hepatic microsomal cytochrome P450 activity would impact lung toxicity induced by inhalation exposure to naphthalene (NA), a ubiquitous environmental pollutant. The liver-Cpr-null (LCN) mouse showed decreases in microsomal metabolism of NA in liver, but not lung, compared to wild-type (WT) mouse. Plasma levels of NA and NA-glutathione conjugates (NA-GSH) were both higher in LCN than in WT mice after a 4-h nose-only NA inhalation exposure at 10ppm. Levels of NA were also higher in lung and liver of LCN, compared to WT, mice, following exposure to NA at 5 or 10ppm. Despite the large increase in circulating and lung tissue NA levels, the level of NA-GSH, a biomarker of NA bioactivation, was either not different, or only slightly higher, in lung and liver tissues of LCN mice, relative to that in WT mice. Furthermore, the extent of NA-induced acute airway injury, judging from high-resolution lung histopathology and morphometry at 20h following NA exposure, was not higher, but lower, in LCN than in WT mice. These results, while confirming the ability of extrahepatic organ to bioactivate inhaled NA and mediate NA's lung toxicity, suggest that liver P450-generated NA metabolites also have a significant, although relatively small, contribution to airway toxicity of inhaled NA. This hepatic contribution to the airway toxicity of inhaled NA may be an important risk factor for individuals with diminished bioactivation activity in the lung.

The role of microsomal oxidation in the regulation of monoamine oxidase activity in the brain and liver of rats

It has been shown in our previous study that monoamine oxidase (MAO) activity in different brain regions are correlated with a microsomal oxidation phenotype. The data obtained in this study, using the microsomal oxidation inhibitor SKF525, and using animals with different duration of hexobarbital sleep, has shown that increased intensity of microsomal oxidation might be associated with increased MAO activity. Since the rats with short hexobarbital sleep time had higher content of hepatic microsomal cytochrome P450 than did rats with long hexobarbital sleep time. In addition, the rats with higher hepatic content of CYP450 had higher activities of MAO-A and MAO-B. Moreover, the microsomal oxidation inhibitor SKF-525 reduced brain and liver activities of MAOA and MAO-B. Consequently, MAO activities in a brain and a liver depend on the microsomal oxidation process.

The immature rat as a potential model for chemical risks to children: Ontogeny of selected hepatic P450s

Concern about potential susceptibilities of infants and children to chemicals has led to the consideration of immature rodents as potential test surrogates. Maturation of some hepatic microsomal cytochrome P450s (CYPs), that participate in metabolic activation of organic solvents and polycyclic aromatic hydrocarbons (PAHs), may differ significantly between humans and rodents. The present investigation was undertaken to delineate the ontogeny of selected hepatic CYPs in male and female Sprague-Dawley (S-D) rats, and to contrast them with developmental profiles in humans. Microsomes were prepared from the liver of sexed and unsexed postnatal day (PND) 1–90 rats, and total CYP450 levels, as well as CYP1A1/2, CYP2E1 and CYP2B1/2 activities and protein, were quantified. CYP1A1/2 and CYP2E1 activity and expression rose rapidly after birth, peaked from PND 21–40/50, and declined substantially to adult values by PND 90. The same ontogenic profiles were manifested when the enzyme activities were expressed per entire liver or liver normalized to body weight. CYP1A1/2 and CYP2E1 activity and protein expression were well correlated. CYP2B1/2 activity peaked abruptly on PND 21 and declined irregularly to adult values. These patterns are in contrast to human CYP1A2 and CYP2E1, which are reported to progressively increase in liver during the first few months to years of life. The three CYP protein developmental profiles were largely gender independent in rats. The immature rat does not appear to be a suitable model for assessing risks posed to infants and children by chemicals metabolically activated by CYP2E1, based on the findings of greater carbon tetrachloride hepatotoxicity in preweanlings and weanlings than in adult animals. Additional studies are required to determine whether immature S-D rats may be used as an animal model for substrates of other CYPs, as total CYP450 levels in the liver progressively rose during maturation, similarly to humans.

Evaluating toxic endpoints of exposure to a commercial PCB mixture: an &lt;em&gt;in vivo&lt;/em&gt; laboratory study

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that produce a wide range of toxic effects. To determine sensitive endpoints in various organ systems, the effects of Aroclor 1260 on immune, endocrine, and hepatic systems were evaluated in a dose-response study. Nine-week old male rats were treated with Aroclor 1260 by oral gavage at dosages ranging from 0.025 to 156 mg/kg/day for 10 consecutive days and killed two days after the last treatment. Eight days prior to sacrifice, rats were injected i.v. with sheep red blood cells (SRBC) for determination of humoral immunity. No observable adverse effect level (NOAEL) and lowest observable adverse effect level (LOAEL) were determined for liver, thymus and genital organ weights, body weight, serum luteinizing hormone (LH), testosterone, thyroxine and thyroid-stimulating hormone (TSH) concentrations, hepatic microsomal testosterone hydroxylase activities, and hepatic microsomal cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1 and CYP2B2 protein levels. Treatment with Aroclor 1260, at all dosages, had no effect on testis, seminal vesicle or ventral prostate weights, on thymus weight or on serum LH or testosterone levels. Among the endpoints altered by Aroclor 1260, the most sensitive, with a LOAEL of 1.25 mg/kg/day, were increased testosterone 16β-hydroxylase activity and androstenedione formation. The LOAEL for increased liver weight, testosterone 16α-hydroxylase activity and CYP2B1 protein content was 3.13 mg/kg/day, while the LOAEL for decreased serum thyroxine levels and anti-SRBC IgM titer was 6.25 mg/kg/day. Less sensitive responses, as reflected by larger LOAEL values, included CYP1A enzyme induction and decreased body weight. In summary, comparison of NOAEL and LOAEL values indicated that hepatic CYP2B-mediated activities were a more sensitive response to Aroclor 1260 exposure in male rats than immune or endocrine endpoints.

Regioselective Versatility of Monooxygenase Reactions Catalyzed by CYP2B6 and CYP3A4: Examples with Single Substrates.

Hepatic microsomal cytochrome P450 (CYP) enzymes have broad and overlapping substrate specificity and catalyze a variety of monooxygenase reactions, including aliphatic and aromatic hydroxylations, N-hydroxylations, oxygenations of heteroatoms (N, S, P and I), alkene and arene epoxidations, dehalogenations, dehydrogenations and N-, O- and S-dealkylations. Individual CYP enzymes typically catalyze the oxidative metabolism of a common substrate in a regioselective and stereoselective manner. In addition, different CYP enzymes often utilize different monooxygenase reactions when oxidizing a common substrate. This review examines various oxidative reactions catalyzed by a CYP enzyme acting on a single substrate. In the first example, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a halogenated aromatic environmental contaminant, was oxidatively biotransformed by human CYP2B6. Nine different metabolites of BDE-47 were produced by CYP2B6 via monooxygenase reactions that included aromatic hydroxylation, with and without an NIH-shift, dealkylation and debromination. In the second example, lithocholic acid (3α-hydroxy-5β-cholan-24-oic acid), an endogenous bile acid, served as a substrate for human CYP3A4 and yielded five different metabolites via aliphatic hydroxylation and dehydrogenation reactions.

Xenobiotic metabolism modulation after long-term temperature acclimation in juveniles of Solea senegalensis

The Senegalese sole, Solea senegalensis, originates from subtropical waters and displays great adaptability to environmental factors such as temperature. A comprehensive study on the effect of long-term temperature acclimation on xenobiotic metabolism, along with the assessment of other parameters related to physiological status, was designed to characterize the response of this species to temperature fluctuations within a realistic range. S. senegalensis juveniles were acclimated for a period of 60 days to two different ambient temperatures, 15 and 20 °C. Several hepatic, gill, muscular and plasmatic parameters were measured over time at the two temperatures. The lower temperature triggered, over time, the synthesis of hepatic microsomal cytochrome P450-related enzymes (e.g. 7-ethoxyresorufin O-deethylase (EROD), carboxylesterases, and the conjugating enzyme uridine diphosphate glucuronosyltransferase and, more significantly, EROD activity in gills. The antioxidant enzyme activities: catalase and glutathione reductase in liver were positively correlated to temperature. Plasmatic parameters (glucose, lactate, triglycerides and osmolality) were consistent with a good physiological status of the experimental fish. The expression of heat shock proteins in muscle did not significantly change in the two temperature groups. The results evidenced that the subtropical species S. senegalensis also uses the temperature compensation strategy to different degrees for most biotransformation enzymes; this response was more intense and faster in gills than in liver. This compensatory strategy did not apply to antioxidant enzymes and GST. The present findings highlight the need to consider the thermal history of the fish when using S. senegalensis as a sentinel in a biomarker-based pollution monitoring study. The fish plasticity on its strategy of physiological adaptation to temperature changes could contribute to explain the success in the geographical expansion of this species.

Role of Kupffer cells in ischemic injury in alcoholic fatty liver

BackgroundThis study was designed to evaluate the role of Kupffer cells (KCs) in hepatic drug metabolizing dysfunction after hepatic ischemia–reperfusion (IR) in alcoholic fatty liver. Materials and methodsRats were fed the Lieber-DeCarli diet for 5 wk to develop alcoholic fatty liver, then were subjected to 90 min of hepatic ischemia and 5 h of reperfusion. For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl3) 48 and 24 h before the IR procedure. ResultsAfter the IR procedure, ethanol diet (ED)-fed rats had higher serum aminotransferase activity compared with the control diet-fed rats. These changes were attenuated by GdCl3. The ED-fed rats exhibited increased hepatic microsomal total cytochrome P450 (CYP) content and nicotinamide adenine dinucleotide phosphate-CYP reductase and CYP1A1, 1A2, 2B1, and 2E1 isozyme activity. After hepatic IR, these increases were reduced to lower levels than observed in the sham group, except CYP2E1 activity. Increases in CYP2E1 activity and its expression were augmented after hepatic IR in ED-fed animals, but were attenuated by GdCl3. Finally, toll-like receptor 4 and myeloid differentiation primary response gene 88 protein expression, nuclear translocation of nuclear factor-κB and activator protein 1, and levels of proinflammatory mediators were further increased in ED-fed animals compared with control diet-fed animals after IR. These increases were attenuated by GdCl3. ConclusionsWe suggest that KCs contribute to hepatic drug metabolizing dysfunction during hepatic IR in alcoholic fatty liver via the toll-like receptors 4-mediated inflammatory response.

Pharmacokinetic changes of drugs in a rat model of liver cirrhosis induced by dimethylnitrosamine, alone and in combination with diabetes mellitus induced by streptozotocin.

Rats with liver cirrhosis induced by N-dimethylnitrosamine (LC) and rats with LC with diabetes mellitus induced by streptozotocin (LCD) have been developed as animal models for human liver cirrhosis and liver cirrhosis with diabetes mellitus, respectively. Changes in the pharmacokinetics of drugs (mainly non-renal clearance, CLNR) in LC and LCD rats reported in the literature compared with respective control rats were reviewed. This review mainly explains the changes in the CLNRs of drugs (which are mainly metabolized via hepatic microsomal cytochrome P450s, CYPs) in LC and LCD rats, in terms of the changes in in vitro hepatic intrinsic clearance (CLint; mainly due to the changes in CYPs in the disease state), free (unbound) fraction of a drug in the plasma (fp) and hepatic blood flow rate (QH) depending on the hepatic excretion ratio of the drug. Generally, changes in the CLNRs of drugs in LC and LCD rats could be well explained by the above-mentioned three factors. The mechanism of urinary excretion of drugs (such as glomerular filtration or renal active secretion or reabsorption) in LC and LCD rats is also discussed. The pharmacokinetics of the drugs reported in the LC and LCD rats were scarce in humans. Thus, the present rat data should be extrapolated carefully to humans.