Microsomal System Research Articles

Discovery of benzo[g]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor

The Keap1-Nrf2 system is an attractive target for drug discovery regarding various unmet medical needs. Only covalent inhibitors for protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 have been approved or are under clinical trials, but such electrophilic compounds lack selectivity. Therefore, specific non-covalent Keap1-Nrf2 PPI inhibitors are expected to be safer Nrf2 activators. We found a novel class of non-covalent Keap1-Nrf2 PPI inhibitor that has a benzo[g]indole skeleton and an indole-3-hydroxamic acid moiety and that exhibits significant PPI inhibitory activity. Additionally, the benzo[g]indole-3-carbohydrazide derivatives were newly prepared. The benzo[g]indole derivatives showed a stronger Keap1-Nrf2 PPI inhibitory activity than Cpd16, a previously reported non-covalent PPI inhibitor. Moreover, most of the PPI inhibitors showed a high metabolic stability in a human microsome system with a low cytotoxicity against HepG2 cell lines, which suggests that novel benzo[g]indole-type Keap1-Nrf2 PPI inhibitors are expected to be biological tools or lead compounds for Nrf2 activators.

Traumatic brain injury is unlikely precipitating Leigh syndrome due to the GJB2 mutation c.35delG.

With interest we read the article by Ashrafi et al. about a 14-year-old female who is regarded to have developed Leigh syndrome (LS) after traumatic brain injury (TBI). We have the following comments and concerns:We do not agree with the notion that traumatic brain injury was the precipitating factor for LS. The patient had a history of hypoacusis, which is a typical clinical manifestation of a mitochondrial disorder (MID). Hypoacusis obviously had developed long before the TBI. Additionally, the patient was diagnosed with neuropathy of the peripheral nerves two months after TBI. It is rather unlikely that neuropathy was triggered by TBI and more likely it was already present before the trauma. Thus, the initial manifestations of LS in the presented patient were most likely hypoacusis followed by neuropathy and TBI only might have triggered the seizure but not the MID. Why was the patient put on phenytoin, which is well-known to be mitochondrion-toxic? Phenytoin may worsen epilepsy and MID in general and it is conceivable that in fact phenytoin was responsible for worsening of the phenotype and not the TBI. In a 16-year-old female with MELAS syndrome due to the mutation m.3243A>G, phenytoin caused intestinal pseudo-obstruction one month after intravenous phenytoin for status epilepticus. In a patient with Kearns-Sayre syndrome phenytoin decreased cerebrospinal fluid (CSF) folate levels. In rat hepatocytes, phenytoin increased reactive oxygen species (ROS) formation, decreased intracellular reduced glutathione, increased intracellular oxidised glutathione, and enhanced lipid peroxidation and mitochondrial damage. In a hepatic microsomal system, phenytoin decreased state-3 respiration, ATP synthesis, and the mitochondrial membrane potential. In this model, phenytoin increased state-4 respiration, impaired Ca++-uptake and release, and inhibited Ca++-induced swelling. It would be interesting to know how the GJB2 mutation was detected. Was whole exome or panel sequencing carried out? Did the authors choose a gene by gene sequencing approach? Did the parents undergo genetic investigations? Did each of them carry the mutation in the heterozygous state? Did either of the parents manifest clinically? We should be informed about the exact sequence of the TBI. Is it conceivable that the fall was already due to a seizure or syncope? Was the patient unconscious after the TBI? The authors mention that basal ganglia lesions were “negative” on DWI. However, basal ganglia appear hyperintense on DWI in figure 2.  What was the cause of respiratory insufficiency at the second admission? Was it attributable to the cerebral lesions, to myopathy affecting the respiratory muscles, to lactic acidosis, or to a pulmonary infection? Simultaneous application of a bunch of drugs is not advisable since it cannot be differentiated which of them is effective in case the administration is followed by a beneficial response. Overall, the report is not convincing with regard to TBI as the precipitating factor of LS. Most likely, worsening of LS was a random event or triggered by phenytoin. It is also conceivable that stress from TBI triggered the production of ROS, which were mitochondrion-toxic to such a degree that a previously subclinical condition became gradually symptomatic.

New Method For Early Diagnostics of Pathological States under the Action of Physiologically Active Substances with Genotoxic Properties upon Organism

The article is dedicated to the new method for early diagnostics of pathological conditions under the action of physiologically active substances with genotoxic properties. The method allows the evaluation of microsomal system of the liver under the action of xenobiotic alkylating type and the condition of microsomal oxidiation system in vivo. It is supposed to asses preliminary the norm of the reaction of organism (phenotype of bio-object) by the intensity of microsomal oxidiation during the study of the toxicological characteristics of physiologically active substances influencing the organism at low doses. The method includes the characteristics of hereditary intensity of epoxidiation (hydroxyliation) reactions of metabolism. Carbamazepine is used as a pharmacological probe. The article formulates the definition of the concept of norm, threshold for the harmful effect and prepatological state of organism

Strategic Drug Design to Avoid the Metabolic Activation of Hepatotoxic Drugs.

Adverse reactions are one of the most important issues in drug development, as well as in the therapeutic usage of drugs during the post-approval stage. Specifically, idiosyncratic adverse drug reactions (IDR) occur in only a small group of patients who are treated with certain drugs, and are unpredictable. It is widely accepted that drug-induced IDR is often associated with CYP-mediated bioactivation. Benzbromarone (BBR) is effective in the treatment of hyperuricemia, and has been used as an effective drug in Japan for a long time. However, BBR has been associated with hepatotoxicity, including fatal liver injury. We identified 2,6-dibromohydroquinone (DBH) and mono-debrominated catechol (CAT) as novel metabolites of BBR in human and rat liver microsomal systems, by comparison with chemically synthesized authentic compounds via ipso-substitution, which we previously discovered to be a unique metabolic reaction of substituted phenols by CYP. Furthermore, CAT, DBH and the oxidized form of DBH (DBBQ) were highly cytotoxic in human hepatocellular carcinoma cells, compared with BBR. We consider that the formation of these metabolites from BBR is linked to the mechanism involved in BBR-induced hepatotoxicity because catechols, hydroquinones, and their oxidized forms are known to be toxic.

Thermodynamics of interactions between mammalian cytochromes P450 and b5

Cytochromes P450 (CYPs) play an important role in the metabolism of xenobiotics and various endogenous substrates. Being a crucial component of the microsomal monooxygenase system, CYPs are involved in numerous protein-protein interactions. However, mechanisms underlying molecular interactions between components of the monooxygenase system still need better characterization. In this study thermodynamic parameters of paired interactions between mammalian CYPs and cytochromes b5 (CYB5) have been evaluated using a Surface Plasmon Resonance (SPR) based biosensor Biacore 3000. Analysis of 18 pairs of CYB5-CYP complexes formed by nine different isoforms of mammalian CYPs and two isoforms of human CYB5 has shown that thermodynamically these complexes can be subdivided into enthalpy-driven and entropy-driven groups. Formation of the enthalpy-driven complexes was observed in the case of microsomal CYPs allosterically regulated by CYB5 (CYB5A-CYP3A4, CYB5A-CYP3A5, CYB5A-CYP17A1). The entropy-driven complexes were formed when CYB5 had no effect on the CYP activity (CYB5A-CYP51A1, CYB5A-CYP1B1, CYB5B-CYP11A1). Results of this study suggest that such interactions determining protein clustering are indirectly linked to the monooxygenase functioning. Positive ΔH values typical for such interactions may be associated with displacement of the solvation shells of proteins upon clustering. CYB5-CYP complex formation accompanied by allosteric regulation of CYP activity by CYB5 is enthalpy-dependent.

Protective effect of carnosic acid against acrylamide-induced toxicity in RPE cells

Acrylamide is a substance that can be neurotoxic in humans and experimental animals. It is formed at different rates in starchy foods cooked at temperatures above 120 °C as a result of interaction between monosaccharides and the amino acid asparagine. Carnosic acid accounts for over 90% of the antioxidant properties of rosemary extract and is a powerful inhibitor of lipid peroxidation in microsomal and liposomal systems. Carnosic acid has been shown to protect against oxidative and inflammatory effects. In order to investigate the protective properties of carnosic acid against acrylamide-induced toxicity in human retinal pigment epithelium (RPE) cells, ARPE-19 cells were pre-treated with 10 μM carnosic acid for 24 h followed by treatment with acrylamide (0.7 or 1 mM) for 24 h. ARPE-19 cells pre-treated with 10 μM carnosic acid showed significantly increased cell viability and decreased cell death rate when compared to ARPE-19 cells treated with acrylamide alone. Activities of SOD and catalase and the level of GSH and expression of NRF2 and a number of anti-oxidant genes were significantly decreased in ARPE-19 cells, while there were significant increases in ROS and MDA; pre-treatment with carnosic acid significantly counteracted these changes. Our results suggest that carnosic acid protected RPE cells from acrylamide-induced toxicity.

Triptolide Induces hepatotoxicity via inhibition of CYP450s in Rat liver microsomes

BackgroundTriptolide (TP), an active constituent of Tripterygium wilfordii, possesses numerous pharmacological activities. However, its effects on cytochrome P450 enzymes (CYP450s) in rats remain unexplored.MethodsIn this study, the effects of triptolide on the six main CYP450 isoforms (1A2, 2C9, 2C19, 2D6, 2E1, and 3A) were investigated both in vivo and in vitro. We monitored the body weight, survival proportions, liver index, changes in pathology, and biochemical index upon TP administration, in vivo. Using a cocktail probe of CYP450 isoform-specific substrates and their metabolites, we then carried out in vitro enzymatic studies in liver microsomal incubation systems via ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Finally, we verified our results at the messenger ribonucleic acid (mRNA) and protein level through quantitative real-time polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemical detection.ResultsThe in vivo toxicity study confirmed that Sprague-Dawley (SD) rats exhibited dose-dependent hepatotoxicity after intragastric administration of TP [200, 400, and 600 μg/(kg.day)] for 28 days. In case of the CYP450 isoforms 3A, 2C9, 2C19, and 2E1, the in vitro metabolic study demonstrated a decrease in the substrate metabolic rate, metabolite production rate, and Vmax, with an increase in the Km value, compared with that observed in the control group. Additionally, a TP dose-dependent decrease in the mRNA levels was observed in the four major isoforms of CYP3A subfamily (3A1/3A23, 3A2, 3A9, and 3A62) and CYP2C9. A similar effect was also observed with respect to the protein levels of CYP2C19 and CYP2E1.ConclusionsThis study suggests that TP can cause hepatotoxicity by reducing the substrate affinity, activity, and expression at the transcriptional and protein levels of the CYP450 isoforms 3A, 2C9, 2C19, and 2E1. TP also has the potential to cause pharmacokinetic drug interactions when co-administered with drugs metabolized by these four isoforms. However, further clinical studies are needed to evaluate the significance of this interaction.

Role of Cytochrome P450 and Oxidative Stress in Alcohol-Induced Liver Injury

Alcohol-induced liver injury is a significant global health problem. The mechanisms by which alcohol causes cell injury are still not clear. Many of the hepatic toxic effects of ethanol have been linked to its metabolism in the liver and shown to correlate with cytochrome P450 2E1 (CYP2E1) levels, lipid peroxidation, and elevated formation of reactive oxygen species (ROS). CYP2E1 is induced under a variety of pathophysiological conditions, by drugs, and by alcohol. The goal of this review is to describe and characterize the liver cytochrome P450 microsomal mixed function oxidase system including the generation of ROS. The review then discusses biochemical and toxicological properties of CYP2E1 and its contribution to ethanol-elevated oxidant stress and liver pathology.

Cytochrome P450 Mediated Bioactivation of Saracatinib.

Saracatinib is a highly selective Src kinase inhibitor against all Src kinase family members and has demonstrated anticancer effects in preclinical models. Unfortunately, it has shown multiple adverse effects during its clinical trials, along with time-dependent inhibition of P450 enzymes. The major objective of this study was to identify reactive metabolites of saracatinib in vitro and in vivo. Four oxidative metabolites (M1-M4) were detected in rat and human liver microsomal incubation systems after exposure to saracatinib. An ortho-quinone-derived reactive metabolite existing as a GSH conjugate (M5) was found in microsomes fortified with GSH as a trapping agent. The formation of the metabolites detected was NADPH dependent. Metabolites M2-M4 were also observed in bile and urine of rats given saracatinib, and M5 was only detected in bile. Inhibition and recombinant P450 enzyme incubation studies demonstrated that P450 3A4 was the primary enzyme responsible for the metabolic activation of saracatinib. The metabolism study facilitates the understanding of correlation between saracatinib-induced hepatotoxicity and bioactivation.

Construction of Metabolism Prediction Models for CYP450 3A4, 2D6, and 2C9 Based on Microsomal Metabolic Reaction System.

During the past decades, there have been continuous attempts in the prediction of metabolism mediated by cytochrome P450s (CYP450s) 3A4, 2D6, and 2C9. However, it has indeed remained a huge challenge to accurately predict the metabolism of xenobiotics mediated by these enzymes. To address this issue, microsomal metabolic reaction system (MMRS)—a novel concept, which integrates information about site of metabolism (SOM) and enzyme—was introduced. By incorporating the use of multiple feature selection (FS) techniques (ChiSquared (CHI), InfoGain (IG), GainRatio (GR), Relief) and hybrid classification procedures (Kstar, Bayes (BN), K-nearest neighbours (IBK), C4.5 decision tree (J48), RandomForest (RF), Support vector machines (SVM), AdaBoostM1, Bagging), metabolism prediction models were established based on metabolism data released by Sheridan et al. Four major biotransformations, including aliphatic C-hydroxylation, aromatic C-hydroxylation, N-dealkylation and O-dealkylation, were involved. For validation, the overall accuracies of all four biotransformations exceeded 0.95. For receiver operating characteristic (ROC) analysis, each of these models gave a significant area under curve (AUC) value >0.98. In addition, an external test was performed based on dataset published previously. As a result, 87.7% of the potential SOMs were correctly identified by our four models. In summary, four MMRS-based models were established, which can be used to predict the metabolism mediated by CYP3A4, 2D6, and 2C9 with high accuracy.

In vitro effects of Genkwa Flos chloroform extract on activity of human liver microsomes UGTs and UGT1A1

To predict the mechanism of liver injury induced by Genkwa Flos, we investigated the effect of chloroform extract on UGTs and UGT1A1 activities of the liver microsomes in rat and human. In the present study, 4-nitrophenol(4-NP) and β-estradiol were elected as substrates to determine activities of UGTs and UGT1A1 by UV and HPLC. The results showed that there were 1.00% of apigenin, 6.40% of hydroxygenkwanin and 18.38% of genkwanin in chloroform extract; and total diterpene mass fraction was 31.40%. Compared with the control group, chloroform extract could significantly inhibit the activity of UGTs in rat liver microsomes(RLM) system, while the inhibitory effect was not obvious in human liver microsomes(HLM) system. UGT1A1 activity was inhibited by chloroform extract in rat liver microsomes and human liver microsomes (based on genkwanin, IC₅₀=8.76, 10.36 μmol•L⁻¹). The inhibition types were non-competitive inhibition(RLM) and uncompetitive inhibition(HLM). In conclusion, the results indicated that chloroform extract showed different inhibitory effects on UGTs and UGT1A1 activity, which may be one of the mechanisms of liver injury induced by Genkwa Flos.

Metabolism of Carfentanil, an Ultra-Potent Opioid, in Human Liver Microsomes and Human Hepatocytes by High-Resolution Mass Spectrometry.

Carfentanil is an ultra-potent synthetic opioid. No human carfentanil metabolism data are available. Reportedly, Russian police forces used carfentanil and remifentanil to resolve a hostage situation in Moscow in 2002. This alleged use prompted interest in the pharmacology and toxicology of carfentanil in humans. Our study was conducted to identify human carfentanil metabolites and to assess carfentanil's metabolic clearance, which could contribute to its acute toxicity in humans. We used Simulations Plus's ADMET Predictor™ and Molecular Discovery's MetaSite™ to predict possible metabolite formation. Both programs gave similar results that were generally good but did not capture all metabolites seen in vitro. We incubated carfentanil with human hepatocytes for up to 1h and analyzed samples on a Sciex 3200 QTRAP mass spectrometer to measure parent compound depletion and extrapolated that to represent intrinsic clearance. Pooled primary human hepatocytes were then incubated with carfentanil up to 6 h and analyzed for metabolite identification on a Sciex 5600+ TripleTOF (QTOF) high-resolution mass spectrometer. MS and MS/MS analyses elucidated the structures of the most abundant metabolites. Twelve metabolites were identified in total. N-Dealkylation and monohydroxylation of the piperidine ring were the dominant metabolic pathways. Two N-oxide metabolites and one glucuronide metabolite were observed. Surprisingly, ester hydrolysis was not a major metabolic pathway for carfentanil. While the human liver microsomal system demonstrated rapid clearance by CYP enzymes, the hepatocyte incubations showed much slower clearance, possibly providing some insight into the long duration of carfentanil's effects.

Liquid chromatography separation of the chiral prodrug eslicarbazepine acetate and its main metabolites in polar organic mode. Application to their analysis after in vitro metabolism

A LC method using a chiral stationary phase (CSP) with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector in polar organic mode (POM) was developed for the separation of the biopharmaceutic classification system (BCS) class II chiral prodrug eslicarbazepine acetate (ESL) and its main metabolites, namely eslicarbazepine, its optical antipode, (R)-licarbazepine, and the achiral oxcarbazepine (OXC). The percentage of methanol (MeOH) in the mobile phase containing acetonitrile (ACN) as the main solvent was found to significantly influence analyte retention and resolution. A reversal of elution order of OXC and (R)-licarbazepine was observed, depending on the MeOH percentage in the mobile phase. The optimized mobile phase consisted of ACN/MeOH/acetic acid/diethylamine (95/5/0.2/0.07; v/v/v/v). The potential of this chemo- and enantioselective LC method combined with solid-phase extraction (SPE) was then evaluated for in vitro metabolism studies using ESL as a model case. Only eslicarbazepine could be detected after incubation of ESL in human liver microsome systems.

The use of juvenile Solea solea as sentinel in the marine platform of the Ebre Delta: in vitro interaction of emerging contaminants with the liver detoxification system.

Juveniles of Solea solea were sampled during the spring season in three consecutive years at a marine site by the mouth of the Ebre river. The aim was to assess if the extractive works from the toxic load upstream the river could be reflected on the health status of the fish living at the immediate sea. The biomarkers selected for the in vivo field study are commonly used as indicators of chemical exposures. They include activities of energy metabolism: lactate dehydrogenase (LDH) and citrate synthase (CS); neurotoxicity: cholinesterases (ChE); xenobiotic metabolism: cytochrome P450 (CYP)-dependent: EROD and BFCOD, carboxylesterase (CbE), glutathione S-transferase (GST) and uridine diphosphate glucuronyltransferase (UDPGT); and oxidative stress parameters such as catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPX) as well as levels of lipid peroxidation (LPO). These biomarkers were mostly analysed in liver but also in gills and muscle depending on their particular tissue distribution and role. A complementary in vitro approach was also sought to see the capacity of common emerging contaminants (pharmaceuticals and personal care products; PPCPs) to interact with the liver microsomal detoxification system of the fish (EROD, BFCOD and CbE activities). The results indicated that in fish sampled in 2015 there was an enhancement in detoxification parameters (EROD, BFCOD and gill GR), muscular ChEs and gill CS, but a decrease in CbE activity and a marked oxidative stress situation (increased LPO and decreased CAT activity). Also, 4 out of the 10 PPCPs tested in vitro were able to interact with the CYP3A4 (BFCOD) enzymatic system while the lipid regulators simvastatin and fenofibrate inhibited CbE activity, as it occurs in higher vertebrates. The in vivo results support the use of a multibiomarker approach when assessing the disturbances due to chemical exposures, not only spatially but also over time, once the influence of other variables has been taken into consideration. The in vitro results highlight the importance of the CYP3A4 and CbE pathway in pharmaceutical metabolism, also in fish.

Effects of phillyrin and forsythoside A on rat cytochrome P450 activities in vivo and in vitro

1. Phillyrin and forsythoside A are two important active ingredients in Forsythia suspensa. However, the effects of phillyrin and forsythoside A on the activities of cytochrome P450 (CYP450) remain unclear.2. This study aimed to investigate the effects of phillyrin and forsythoside A on the activities of CYP1A2, CYP2C11, CYP2D1 and CYP3A1/2 by cocktail probe drugs in rats both in vivo and in vitro.3. Many pharmacokinetic parameters of caffeine and metoprolol in phillyrin pretreatment group, caffeine and tolbutamide in forsythoside A pretreatment group were affected significantly. In rat liver microsomal incubation system, the concentrations of acetaminophen and dextrophan in the phillyrin pretreatment group are higher than blank control group by 207.69% and 125.00%, however, the concentrations of 4-hydroxytolbutamide and 6β-hydroxytestosterone were not significantly altered. The concentrations of acetaminophen and 4-hydroxytolbutamide in the forsythoside A pretreatment group are higher than blank control group by 223.07% and 154.16%, whereas the concentrations of dextrophan and 6β-hydroxytestosterone were not significantly altered.4. These results indicated that Phillyrin had potential inductive effects on rat CYP1A2 and CYP2D1 activities, without affecting CYP2C11 and CYP3A1/2 activities. Moreover, forsythoside A had inductive effects on the activities of CYP1A2 and CYP2C11, without affecting CYP2D1 and CYP3A1/2 activities.

Flavokawain A inhibits Cytochrome P450 in in vitro metabolic and inhibitory investigations

Ethnopharmacological relevanceFlavokawain A, the major chalcone in kava extracts, was served as beverages for informal social occasions and traditional ceremonials in most South Pacific islands. It exhibited strong antiproliferative and apoptotic effects against human prostate and urinary bladder cancer cells. Aim of the studyThe current study was purposed to investigate the interaction between Flavokawain A and Cytochrome P450, including the inhibitory effects of Flavokawain A on predominant CYP450 isotypes and further clarified the inhibitory mechanism of FKA on CYP450 enzymes. Besides, study about identifying the key CYP450 isotypes responsible for the metabolism of FKA was also performed. Materials and methodsIn this study, probe-based assays with rat liver microsome system were used to characterize the inhibitory effects of FKA. Molecular docking study was performed to further explore the binding site of FKA on CYP450 isoforms. In addition, chemical inhibition experiments using specific inhibitors (a-naphthoflavone, quinidine, sulfamethoxazde, ketoconazole, omeprazole) were performed to clarify the individual CYP450 isoform that are responsible for the metabolism of FKA. ResultsFKA showed significant inhibition on CYP1A2, CYP2D1, CYP2C6 and CYP3A2 activities with IC50 values of 102.23, 20.39, 69.95, 60.22μmol/L, respectively. The inhibition model was competitive, mixed-inhibition, uncompetitive, and noncompetitive for CYP1A2, CYP2D1, CYP2C6 and CYP3A2 enzymes. Molecular docking study indicated the ligand-binding conformation of FKA in the active site of CYP450 isoforms. The chemical inhibition experiments showed that the metabolic clearance rate of Flavokawain A decreased to 19.84%, 50.38%, and 67.02% of the control in the presence of ketoconazole, sulfamethoxazde and a-naphthoflavone. ConclusionThe study showed that Flavokawain A has varying inhibitory effect on CYP450 enzymes and CYP3A2 was the principal CYP isoform contributing to the metabolism of Flavokawain A. Besides, CYP2C6 and CYP1A2 isoforms also play important roles in the metabolism of FKA. Our results provided a basis for better understanding the biotransformation of FKA and prediction of drug-drug interaction of FKA.

Predicting anxiety responses to halogenated glucocorticoid drugs using the hexobarbital sleep time test

Glucocorticoids (GCs) are used to treat numerous diseases, but their use in limited by adverse side effects. One such effect is occasional increased anxiety. Since the intensity of hepatic microsomal oxidation has been shown to alter responses to GC, we examined the possibility that rats with lower rates of hepatic GC metabolism would have increased anxiety. We hypothesized that the resulting, excessive GC would stimulate brain monoamine oxidase A (MAO-A), which would reduce brain serotonin, and thereby increase anxiety. Hepatic microsomal oxidative intensity was evaluated by the hexobarbital sleep time (HST) test. Results showed that rats with lower rates of hepatic GC metabolism had elevated brain MAO-A activity, reduced brain serotonin, and more anxiety than rats with higher rates of hepatic GC metabolism. We suggest that the HST test, as an integrative test of microsomal oxidation status, should be useful for predicting individual sensitivity to GC and to other drugs metabolized by the hepatic microsomal oxidation system.

Chronic ethanol treatment depletes glutathione regeneration capacity in hepatoma cell line HepG2

Chronic alcohol abuse is the leading cause of liver cirrhosis in western countries. Ethanol, even at lower concentrations, can cause pleiotropic effects at the cellular level such as the formation of reactive oxygen species (ROS) and DNA damage. Ethanol is oxidized to acetaldehyde by liver alcohol dehydrogenase and by a microsomal ethanol-oxidizing system which is dependent on CYP2E1. There are numerous reports on CYP2E1-mediated formation of ROS. These lower the levels of intracellular glutathione, an efficient antioxidant. We were interested to investigate ethanol effects on glutathione levels independent of CYP2E1. We chose human hepatoma cell line HepG2 as a model known to lack physiological CYP2E1 expression. We found that 40 mM ethanol, a dosage comparable to blood ethanol concentration after heavy alcohol drinking, reduced intracellular HepG2 glutathione levels only by 18%. Within 24 hours, this effect could be normalized by the glutathione regeneration system. When HepG2 cells were exposed to 40 mM ethanol for one week, the cells gradually lost their ability to regenerate intracellular glutathione stores. We conclude that chronic ethanol exposure has a substantial effect on the glutathione regeneration capacity in liver cells which might contribute to alcohol-induced liver disease.

Effects of aqueous extract of Cassiae Semen on activity hepatic microsomal CYP450 isozymes in rats

To study the effect of aqueous extract of Cassiae Semen on the activity, mRNA and protein expressions of cytochrome P450(CYP450) system in rat liver microsomes, microsomes of rat liver were prepared after the oral administration with aqueous extract of Cassiae Semen for 14 days. The enzyme activity was quantified by Cocktail method. Meanwhile, the mRNA and protein expressions of CYP1A2, CYP2B1, CYP2C11, CYP2D2, CYP2E1 and CYP3A1 in the livers were detected by RT-PCR and Western blot. The result of this experiment was that aqueous extract of Cassiae Semen obviously induced the enzyme activities of CYP1A2, CYP2B1, CYP2C11, CYP2D2, CYP2E1 and CYP3A1. Low dose of aqueous extract of Cassiae Semen significantly reduced the activity of CYP2D2, but the activity of CYP2D2 was significantly induced by middle dose and high dose of aqueous extract of Cassiae Semen. These subtypes were increased in a dose-dependent manner except for CYP3A1. The mRNA levels of CYP1A2, CYP2C11, CYP2D2 and CYP2E1 were also induced in rats treated with aqueous extract of Cassiae Semen, but with no significant effect in CYP2B1 and CYP3A1 mRNA expressions. The protein levels of CYP2C11 and CYP2E1 were also induced in rats treated with aqueous extract of Cassiae Semen, but with no significant difference. Since the enzyme activity, mRNA and protein expressions of CYP450, particularly CYP2C11and2E1subtypes, were induced or inhibited by aqueous extract of Cassiae Semen to varing degrees, suggesting the potential drug-drug interactions should be concerned.

Factors limiting the extent of absolute bioavailability of pradefovir in rat

1. Pradefovir was designed as an oral liver target prodrug of 9-(2-phosphonylmethoxyethyl) adenine (PMEA). Liver targeting arises through first pass hepatic metabolism by cytochrome P-450 3A4 (CYP3A4). For CYP3A4 primarily exists in intestines and liver, intestinal metabolism may impair its liver selectivity and oral bioavailability, and then impair its efficacy and safety. It was important to reveal details of the disposition of pradefovir in intestines and liver in a preclinical study.2. The absolute bioavailability of pradefovir was 4.75% based on the intravenous and oral AUC0–24 h in rats. Pradefovir was stable in intestinal segments and microsomes. The fractions of the dose absorbed from the GI tract were 20.3% and 15.3% from intravenous and oral administration of pradefovir in rats and portal vein-cannulated rat models, respectively. The liver extraction ratio was predicted to be 49.2% from liver microsomes system, based on the monitoring substrate loss rate. Rat intestines’ Ussing chamber experiment indicated that P-glycoprotein (P-gp) transporter and paracellular pathway may involve in intestinal transportation.3. Activation of pradefovir mainly occurs in the liver. Low intestinal absorption was the main reason of low bioavailability of pradefovir in rats. The result was suggestive for the disposition of pradefovir in human intestine and liver.