Induction Of CYP2E1 Research Articles

Modulation of hepatic and renal metabolism and toxicity of trichloroethylene and perchloroethylene by alterations in status of cytochrome P450 and glutathione

The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by the cytochrome P450 (P450) and glutathione (GSH) conjugation pathways in their acute renal and hepatic toxicity was studied in isolated cells and microsomes from rat kidney and liver after various treatments to modulate P450 activity/expression or GSH status. Inhibitors of P450 stimulated GSH conjugation of Tri and, to a lesser extent, Perc, in both kidney cells and hepatocytes. Perc was a more potent, acute cytotoxic agent in isolated kidney cells than Tri but Perc-induced toxicity was less responsive than Tri-induced toxicity to modulation of P450 status. These observations are consistent with P450-dependent bioactivation being more important for Tri than for Perc. Incubation of isolated rat hepatocytes with Tri produced no acute cytotoxicity in isolated hepatocytes while Perc produced comparable cytotoxicity as in kidney cells. Modulation of P450 status in hepatocytes produced larger changes in Tri- and Perc-induced cytotoxicity than in kidney cells, with non-selective P450 inhibitors increasing toxicity. Induction of CYP2E1 with pyridine also markedly increased sensitivity of hepatocytes to Tri but had little effect on Perc-induced cytotoxicity. Increases in cellular GSH concentrations increased Tri- and Perc-induced cytotoxicity in kidney cells but not in hepatocytes, consistent with the role of GSH conjugation in Tri- and Perc-induced nephrotoxicity. In contrast, depletion of cellular GSH concentrations moderately decreased Tri- and Perc-induced cytotoxicity in kidney cells but increased cytotoxicity in hepatocytes, again pointing to the importance of different bioactivation pathways and modes of action in kidney and liver.

Involvement of hepatic xenobiotic related genes in bromadiolone resistance in wild Norway rats, Rattus norvegicus (Berk.)

To examine the role of xenobiotic relevant genes in bromadiolone resistance in wild Norway rats ( Rattus norvegicus) we compared the constitutive liver gene expression and expression upon bromadiolone administration in bromadiolone resistant and anticoagulant susceptible female rats using a LNA microarray and quantitative PCR. Resistant rats showed significantly higher constitutive expression of the cytochrome P450 genes Cyp2c13 and Cyp3a2 and lower expression of Cyp2e1 and Gpox1 compared to the susceptible rats. The Cyp1a2, Cyp2c13, Cyp2e1, Cyp3a2 and Cyp3a3 genes were significantly higher expressed in resistant than susceptible rats upon bromadiolone exposure. To establish how bromadiolone affected xenobiotic gene expression in the two strains we compared bromadiolone expression profiles to saline profiles of both strains. Bromadiolone mediated significant up-regulation of Cyp2e1 and Cyp3a3 expression in the resistant rats whereas the rodenticide conferred down-regulation of Cyp2e1, Cyp3a3 and Gpox1 and induction of Cyp2c12 expression in susceptible rats. Cyp2c13 and Cyp3a2 expression were markedly suppressed in both strains upon treatment. This suggests that xenobiotic relevant enzymes play a role in bromadiolone resistance in the Norway rat. A high constitutive expression of Cyp2c13 and Cyp3a2 and induction of Cyp1a2, Cyp2e1 and Cyp3a3 expression during bromadiolone exposure may increase the resistance to bromadiolone presumably by facilitating increased detoxification and decreased liver injury.

COMMON PATHOGENIC MECHANISM IN DEVELOPMENT PROGRESSION OF LIVER INJURY CAUSED BY NON-ALCOHOLIC OR ALCOHOLIC STEATOHEPATITIS

This review showed the common pathogenic mechanism in the development of non-alcoholic or alcoholic steatohepatitis. In particular, we describe the role of innate immune system and oxidative stress caused by gut-derived endotoxin. Gut-derived endotoxin plays an important role in alcoholic liver injury. It was reported that acute ethanol administration reduced activation of Kupffer cells. It is therefore possible that alcohol-induced hepatocellular damage occurs as a result of bacterial or endotoxin translocation under a reduction of the reticuloendothelial system (RES) function in alcoholic liver disease (ALD). On the other hand, recently, attention has been directed toward the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut-derived endotoxin via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol-induced liver injury. It is generally accepted that activation of the innate immune system and increased release of proinflammatory cytokines and other mediators plays an important role in the development of ALD. It was shown that Kupffer cells activation by endotoxin via Toll-like receptor (TLR-4) is involved in alcohol-induced liver injury and that ethanol-induced oxidative stress is important in the regulation of transcription factor NF-kappaB activation and that cytokine production by Kupffer cells. TNF-alpha and free radicals are produced in early alcohol-induced liver injury. In support of this finding, the pathology caused by alcohol was blocked nearly completely in TNF-alpha receptor 1. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of the CYP2E1 form of cytochrome P450 enzymes by ethanol. Initial efforts to clarify the mechanisms that promote the progression from steatosis to steatohepatitis somewhat artificially divides disease mechanisms into "first and second" hit. The best candidates for these second hits were considered to be oxidative stress (CYP2E1 induction) and associated lipid peroxidation and cyokines, principally, TNF-alpha. Some of the most definitive data on the importance of the innate immune system or oxidative stress in the pathogenesis of liver disease come from studies of alcoholic and non-alcoholic steatohepatitis in animals.

β‐Catenin is essential for liver regeneration following acetaminophen‐induced acute liver failure.

Overdose of acetaminophen (APAP) is the leading cause of acute liver failure (ALF). Understanding the mechanisms of liver regeneration following APAP-induced liver injury are of high therapeutic relevance. To investigate the role of Wnt/β-catenin, an important pathway in liver biology, in liver regeneration after APAP-induced ALF, male CD-1 mice were treated with a non-lethal dose of APAP (500 mg/kg), which induced extensive liver injury and subsequent regeneration. Rapid induction in the total and active β-catenin was evident at 1–3 hr after APAP treatment, along with its nuclear translocation at 6 hr. Further studies revealed that both canonical (GSK-3β-dependent) and non-canonical (c-met-dependent) pathways were involved in β-catenin activation. Administration of a lethal dose of APAP (700 mg/kg) known to inhibit liver regeneration completely suppressed β-catenin activation. Further, β-catenin conditional knockout mice, which have low CYP2E1 expression, exhibited increased APAP-mediated injury and decreased cell proliferation following CYP2E1 induction. Furthermore, suppression of β-catenin activation was observed in human APAP-induced ALF liver samples along with suppression of liver regeneration. Taken together, these data indicate that β-catenin activation plays an important role in stimulation of liver regeneration following APAP overdose and might have therapeutic and prognostic implications in APAP ALF.

Effects of diabetes on rabbit kidney and lung CYP2E1 and CYP2B4 expression and drug metabolism and potentiation of carcinogenic activity of N-nitrosodimethylamine in kidney and lung

There are limited number of studies regarding the influence of diabetes on the regulation of cytochrome P450s and associated drug metabolizing enzyme activities especially in extrahepatic tissues such as kidney. However, there is almost no such study in lung. Alloxan-induced diabetes did not change CYP2B4 expression as measured with immunoblot analysis and associated enzyme, benzphetamine N-demethylase, activity in rabbit kidney and lung. Induction of cytochrome P4502E1 by diabetes was identified by immunochemical detection on Western blots in the lung and kidney microsomes of rabbits. In parallel to CYP2E1 induction, aniline 4-hydroxylase and p-nitrophenol hydroxylase activities were markedly increased in diabetic rabbit lung and kidney. CYP2B4 and CYP2E1 dependent drug metabolism did not show any tissue variation in diabetic rabbit. These findings are in contrast to those of rats, mice and hamster. The results of the present work, in combination with those of the previous work [Arinç, E., Arslan, Ş., Adali, O., 2005. Differential effects of diabetes on CYP2E1 and CYP2B4 proteins and associated drug metabolizing enzyme activities in rabbit liver. Arch. Toxicol. 79, 427–433], indicate the existence of species-dependent response of CYP-dependent drug metabolizing enzymes to diabetes. A procarcinogen and food contaminant, N-nitrosodimethylamine (NDMA), is converted to its carcinogenic form after it is activated with NDMA N-demethylase. In the current study, a statistically significant increase of liver, kidney and lung NDMA N-demethylase activity associated with CYP2E1 was shown in diabetic rabbit. Thus, it is expected that, the risk of nitrosamine induced carcinogenesis will be greater in liver, kidney and lung of the diabetic subjects.

Role of CYP2E1 and saturation kinetics in the bioactivation of thioacetamide: Effects of diet restriction and phenobarbital

Thioacetamide (TA) undergoes saturation toxicokinetics in ad libitum (AL) fed rats. Diet restriction (DR) protects rats from lethal dose of TA despite increased bioactivation-mediated liver injury via CYP2E1 induction. While a low dose (50 mg TA/kg) produces 6-fold higher initial injury, a 12-fold higher dose produces delayed and mere 2.5-fold higher injury. The primary objective was to determine if this less-than-expected increase in injury is due to saturation toxicokinetics. Rats on AL and DR for 21 days received either 50 or 600 mg TA/kg i.p. T(1/2) and AUCs for TA and TA-S-oxide were consistent with saturable kinetics. Covalent binding of (14)C-TA-derived-radiolabel to liver macromolecules after low dose was 2-fold higher in DR than AL rats. However, following lethal dose, no differences were found between AL and DR. This lack of dose-dependent response appears to be due to saturation of bioactivation at the higher dose. The second objective was to investigate the effect of phenobarbital pretreatment (PB) on TA-initiated injury following a sub-lethal dose (500 mg/kg). PB induced CYP2B1/2 approximately 350-fold, but did not increase covalent binding of (14)C-TA, TA-induced liver injury and mortality, suggesting that CYP2B1/2 has no major role in TA bioactivation. The third objective was to investigate the role of CYP2E1 using cyp2e1 knockout mice (KO). Injury was assessed over time (0-48 h) in wild type (WT) and KO mice after LD(100) dose (500 mg/kg) in WT. While WT mice exhibited robust injury which progressed to death, KO mice exhibited neither initiation nor progression of injury. These findings confirm that CYP2E1 is responsible for TA bioactivation.

Role of intracellular calcium and phospholipase A2 in arachidonic acid-induced toxicity in liver cells overexpressing CYP2E1

Liver cells (HepG2 and primary hepatocytes) overexpressing CYP2E1 and exposed to arachidonic acid (AA) were previously shown to lose viability together with enhanced lipid peroxidation. These events were blocked in cells pre-incubated with antioxidants (α-tocopherol, glutathione ethyl ester), or in HepG2 cells not expressing CYP2E1. The goal of the current study was to evaluate the role of calcium and calcium-activated hydrolases in these CYP2E1–AA interactions. CYP2E1-expressing HepG2 cells treated with AA showed an early increase in cytosolic calcium and partial depletion of ionomycin-sensitive calcium stores. These changes in calcium were blocked by α-tocopherol. AA activated phospholipase A2 (PLA2) in CYP2E1-expressing liver cells, and this was inhibited by PLA2 inhibitors or α-tocopherol. PLA2 inhibitors prevented the cell death caused by AA, without affecting CYP2E1 activity or lipid peroxidation. AA toxicity and PLA2 activation were inhibited in calcium-depleted cells, but not by removal of extracellular calcium alone. Removal of extracellular calcium inhibited the early increase in cytosolic calcium caused by AA. CYP2E1 overexpressing HepG2 cells exposed to AA showed a decrease in mitochondrial membrane potential, which was prevented by the PLA2 inhibitors. These results suggest that AA-induced toxicity to CYPE1-expressing cells: (i) is associated with release of Ca 2+ from intracellular stores that depends mainly on oxidative membrane damage; (ii) is associated with activation of PLA2 that depends on intracellular calcium and lipid peroxidation; (iii) does not depend on increased influx of extracellular calcium, and (iv) depends on the effect of converging events (lipid peroxidation, intracellular calcium, activation of PLA2) on mitochondria to induce bioenergetic failure and necrosis. These interactions may play a role in alcohol liver toxicity, which requires polyunsaturated fatty acids, and involves induction of CYP2E1.

Obesity-induced increase of CYP2E1 activity and its effect on disposition kinetics of chlorzoxazone in Zucker rats

This study was designed to investigate the induction of CYP2E1 in obese Zucker rats and its effect on the disposition kinetics of chlorzoxazone (CZX). CZX 20 mg/kg was administered to three groups of rats: normal Zucker rats fed a normal diet (ND), normal Zucker rats fed a high-fat diet (HF), and genetically obese Zucker rats fed a normal diet (OB). The values of the area under the plasma concentration–time curve from 0 to ∞ (AUC ∞) of CZX were in the order of ND > HF > OB rats. The AUC ∞ values of total 6-hydroxychlorzoxazone (6OHCZX-T), which is considered to be a CYP2E1 metabolic marker, were in the opposite order. The values of the AUC ∞ ratio (6OHCZX–T/CZX) in ND, HF and OB rats were approximately 0.2, 0.3 and 0.4, respectively. The CZX concentration in fat was much higher than the concentrations in plasma, liver and kidney in all groups. Induction of CYP2E1 protein was greater in both liver and fat of OB rats than in those of HF rats. Microsomal activity of CYP2E1 in liver and fat was also in the order of OB > HF > NM rats. These results suggest that CYP2E1 may be induced in liver and fat of obese patients, thereby potentially altering the disposition kinetics of not only CZX, but also other lipophilic drugs metabolized by CYP2E1.

Induction of CYP2B and CYP2E1 in precision-cut rat liver slices cultured in defined medium

Many drugs and endogenous substances undergo biotransformation by cytochrome P450s (CYPs), and some drugs are also capable of modulating the expression of various CYPs. Knowledge of the potential of a drug to modulate CYPs is useful to help predict potential drug interactions. This study utilized precision-cut rat liver slices in dynamic organ culture to assess the effects of various media on the viability of rat liver slices and the expression of CYP2B and CYP2E1 when the slices are exposed to phenobarbital and isoniazid, which are drugs capable of inducing these respective CYPs. Liver slices were maintained in serum supplemented Waymouths medium and two different serum-free media, Hepatozyme (Life Technologies) and a new defined medium, which is named BPM. While Hepatozyme is considered a suitable medium to support primary hepatocyte cultures, this product did not maintain viable liver slices, even for 24 h. The serum containing and new defined media maintained viable liver slices for up to 96 h in culture. Phenobarbital (0.5 mM) and isoniazid (0.1 or 0.6 mM) did not affect viability in this model. In the absence of phenobarbital or isoniazid, liver slices maintained for 96 h in the new BPM medium maintained the respective levels of CYP2B and 2E1 protein at 1.8 and 1.9-fold higher than in slices maintained in the serum-containing medium. Phenobarbital exposure (0.5 mM) for 96 h induced CYP2B protein 5.2-fold in the BPM medium and 2.5-fold in the serum-containing medium. Isoniazid exposure (0.1 and 0.5 mM) for 96 h induced CYP2E1 protein 1.9 and 2.1-fold (respectively) in the BPM medium and 2.1 and 2.0-fold in the serum-containing medium. The respective CYP enzymatic activities were also increased by these drugs in a similar manner. Thus, the new defined BPM medium provides suitable conditions for maintaining CYP2B and 2E1 in liver slices and supports the investigation of drug-induced modulation of these enzymes.

Effects of glucose supplementation on the pharmacokinetics of intravenous chlorzoxazone in rats with water deprivation for 72 h

It was reported that in rats with water deprivation for 72 h with food (dehydration rat model), the expression of CYP2E1 was 3-fold induced with an increase in mRNA level and glucose supplementation instead of food during 72-h water deprivation (dehydration rat model with glucose supplementation) inhibited the CYP2E1 induction in dehydration rat model. It was also reported that chlorzoxazone (CZX) is metabolized to 6-hydroxychlorzoxazone (OH–CZX) mainly via CYP2E1 in rats. Hence, the effects of glucose supplementation on the pharmacokinetics of CZX and OH–CZX were investigated after intravenous administration of CZX at a dose of 25 mg/kg to control male Sprague–Dawley rats and dehydration rat model and dehydration rat model with glucose supplementation. Based on the above mentioned results of CYP2E1, it could be expected that increased formation of OH–CZX in dehydration rat model could decrease in dehydration rat model with glucose supplementation. This was proven by the following results. In dehydration rat model with glucose supplementation, the AUC of OH–CZX was significantly smaller (1900 versus 1050 μg min/ml), AUC OH–CZX/AUC CZX ratio was considerably smaller (105 versus 34.3%), C max was significantly lower (20.6 versus 8.08 μg/ml), total amount excreted in 24-h urine as unchanged OH–CZX was significantly smaller (62.3 versus 42.7% of intravenous dose of CZX), and in vitro V max (2.18 versus 1.20 nmol/min/mg protein) and CL int (0.0285 versus 0.0171 ml/min/mg protein) were significantly slower than those in dehydration rat model.

Enhancement by pyrazole of lipopolysaccharide-induced liver injury in mice: Role of cytochrome P450 2E1 and 2A5

The mechanisms by which alcohol causes liver injury are still not certain. Either LPS or CYP2E1 are considered independent risk factors involved in alcoholic liver disease, but mutual relationships or interactions between them are unknown. In the present study, the possible synergistic action of CYP2E1 and LPS in liver injury was investigated by evaluating the effects of pyrazole (inducer of CYP2E1), Chlormethiazole (CMZ), an inhibitor of CYP2E1, and CYP2E1-knockout mice. Mice were injected with pyrazole (150 mg/kg, ip) daily for 2 days, followed by LPS injection (4 mg/kg, ip). CMZ (50mg/kg, ip) was administered 15 h before and 30 min after LPS treatment, respectively. LPS-induced liver injury was enhanced by pyrazole, as indicated by pathological changes and increases in ALT and AST, and positive TUNEL staining. LPS-induced oxidative stress was also enhanced by pyrazole as indicated by increases in 4-hydroxy-2-nonenal and 3-nitrotyrosine adduct formation. CMZ protected against the pyrazole enhanced LPS liver injury and oxidative stress. CYP2E1 but also CYP2A5 were increased by the pyrazole/LPS treatment. CMZ decreased the elevated CYP2E1 activity by 90%, but CYP2A5 activity was also lowered (30%-50%). CYP2E1-knockout mice exhibited only minor liver injury after treatment with pyrazole/LPS, but wild-type mice exhibited severe liver injury. While no CYP2E1 was present in the CYP2E1 knockout mice, CYP2A5 activity was also lower. In conclusion, induction of CYP2E1 plays an important role in the enhancement of LPS liver injury by pyrazole, but some contribution by CYP2A5 cannot be excluded.

Differences in sensitivity of cultured rat brain neuronal and glial cytochrome P450 2E1 to ethanol

The expression of the cytochrome P450s (CYPs) may vary in the different brain cells depending on their specialization and the presence of different endogenous factors. The present study was initiated to investigate the expression and catalytic activity of the constitutive and inducible forms of CYP2E1, the major ethanol inducible CYP, in cultured rat brain neuronal and glial cells. These cells exhibited relatively two-fold higher activity of N-nitrosodimethylamine demethylase (NDMA-d) when compared with the liver enzyme. Pretreatment with ethanol revealed a significant time and concentration dependent induction in NDMA-d activity in both cell types. Western blot, immunocytochemistry and RT-PCR also indicated significant induction of CYP2E1 in the cultured brain cells. Interestingly, the neuronal cells exhibited greater magnitude of induction than the glial cells. The relatively higher degree of induction in cultures of neurons has indicated enhanced sensitivity of neurons to the inductive effects of ethanol. This enhanced induction of CYP2E1 in neuronal cells has indicated that like regional specificity, cell specificity also exists in the induction of CYP2E1 and other CYPs.

Effects of enzyme inducers and inhibitors on the pharmacokinetics of intravenous ipriflavone in rats

In order to find out what types of the hepatic microsomal cytochrome P450 (CYP) isozymes are involved in the metabolism of ipriflavone, ipriflavone at a dose of 20 mg kg(-1) (or 15 mg kg(-1)) was infused in male Sprague-Dawley rats. In rats pretreated with SKF 525-A (a non-specific CYP isozyme inhibitor in rats), the total body clearance (CL) of ipriflavone was significantly slower (29.9% decrease) than that in control rats. This indicates that ipriflavone is metabolized via CYP isozymes in rats, hence various enzyme inducers and inhibitors were used in in-vitro or in-vivo studies in rats. In rats pretreated with 3-methylcholanthrene and phenobarbital (main inducers of CYP1A1/2 and 2B1/2 in rats, respectively), the CL values were significantly higher (153 and 67.2% increases, respectively). In rats pretreated with sulfaphenazole (a main inhibitor of CYP2C11 in rats), the CL was significantly slower (22.5% decrease) than that in control rats. On addition of furafylline (a main inhibitor of CYP1A2 in rats), the in-vitro intrinsic clearance for the disappearance of ipriflavone was significantly slower (50.8% decrease) than that without furafylline. However, the CL values were not significantly different in rats pretreated with orphenadrine and isoniazid (a main inducer of CYP2E1 in rats), and quinine and troleandomycin (main inhibitors of CYP2D1 and 3A23/2 in rats, respectively) compared to controls. These data suggest that ipriflavone could be metabolized mainly via CYP1A1/2, 2B1/2 and 2C11 in rats.

Overexpression of CYP2E1 in Mitochondria Sensitizes HepG2 Cells to the Toxicity Caused by Depletion of Glutathione

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.

INDUCTION AND RECOVERY TIME COURSE OF RAT BRAIN CYP2E1 AFTER NICOTINE TREATMENT

CYP2E1, the primary ethanol-metabolizing cytochrome P450, metabolizes endogenous substrates (e.g., arachidonic acid) and drugs (e.g., acetaminophen, chlorzoxazone) and bioactivates procarcinogens (e.g., tobacco-specific nitrosamines) and toxins (e.g., carbon tetrachloride). Nicotine from tobacco smoke may contribute to the enhanced hepatic CYP2E1 activity in smokers. We have previously shown that chronic nicotine treatment can increase CYP2E1 in rat liver and brain. In this study, induction of brain CYP2E1 was assessed after a single acute or a 7-day chronic treatment with saline or nicotine (1 mg/kg s.c.), with sacrifice performed at various times after the last injection. Chronic 7-day nicotine treatment showed the highest levels of CYP2E1 12 h after the last injection in frontal cortex (1.4-fold, p < 0.05) versus 8 h in hippocampus (1.8-fold, p < 0.01) and cerebellum (1.4-fold, p < 0.05), returning to basal levels by 24 h. In contrast, acute nicotine treatment did not induce CYP2E1 in frontal cortex and hippocampus but increased CYP2E1 in cerebellum 8 h after treatment (1.6-fold, p < 0.01). Brain CYP2E1 mRNA levels did not increase after chronic nicotine treatment, suggesting nontranscriptional regulation. Thus, humans exposed to nicotine may have altered CYP2E1-mediated metabolism of centrally acting drugs and toxins as well as altered toxicity because of oxidative stress caused by CYP2E1. Those affected may include current and passive smokers and people that may be treated with nicotine such as smokers and, potentially, patients with Alzheimer's, Parkinson's disease, or ulcerative colitis.

Dietary Steatotic Liver Attenuates Acetaminophen Hepatotoxicity in Mice

To determine whether hepatic steatosis is susceptible to acetaminophen (APAP) hepatotoxicity. Male C57Bl/6 mice were fed a "Western-style" diet (high fat and high carbohydrate) for 4 months to develop severe hepatic steatosis with mild increases in alanine aminotransferase (ALT) levels. These were compared to mice fed a standard chow diet. Treatment with APAP (300 mg/kg, orally) to mice fed a regular chow increased ALT levels (519-fold) and caused hepatic centrilobular injury at 6 h. APAP increased hepatic cytochrome-P (CYP)-2E1 mRNA levels (17-fold). In vivo microscopic studies showed that APAP caused a 30% decrease in sinusoidal perfusion and the infiltration of red blood cells into the space of Disse. Electron microscopy demonstrated that numerous gaps were formed in sinusoidal endothelial cells. Mice fed the "Western-style" diet were protected from APAP hepatotoxicity as evidenced by 89% decrease in ALT levels and less centrilobular injury, which was associated with 42% decrease in CYP2E1 mRNA levels. The APAP-induced liver microcirculatory dysfunction was minimized in mice fed the "Western-style" diet. These results suggest that hepatic steatosis elicited by the "Western-style" diet attenuated APAP-induced hepatotoxicity by inhibiting CYP2E1 induction and by minimizing sinusoidal endothelial cell injury, leading to protection of liver microcirculation.

Quantification of the expression and inducibility of 12 rat cytochrome P450 isoforms by quantitative RT–PCR

The administration of xenobiotics may significantly alter the expression of cytochromes P450 (CYPs), thereby leading to potentially toxic cellular, physiologic, and pharmacologic responses. Indeed, an important task in the development of new therapeutic entities is to evaluate efficiently and quantitatively their potential effects on the expression level of different CYPs. In this report, reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure basal and induced mRNA of a wide range of rat CYP isoforms. Rats (n=3 per treatment) were treated with five prototype inducers of CYP isoforms or with vehicle only. RT and PCR efficiencies were determined using appropriate RNA and DNA standards. Messenger RNA was quantified by PicoGreen standard curves and normalized to cyclophilin. Quantitative RT-PCR was used successfully to demonstrate that CYP isoforms were induced at the mRNA level following drug administration. Notably, phenobarbital resulted in significant induction of CYP2B1, CYP2B2, CYP2C6, CYP2C13, CYP2E1, CYP3A1, and CYP3A2. 3-Methylcholanthrene induced CYP1A1, CYP1A2, and CYP1B1. CYP2C11 expression was highly variable and suppressed by pyridine, whereas the expression of CYP2E1 was suppressed by dexamethasone. We demonstrated that quantitative RT-PCR can be used to evaluate efficiently the effect of compounds on the expression of a wide range of CYP isoforms. The technique is advantageous over others in that it is very sensitive, efficient and applicable to highly homologous CYP isoforms.

Alcohol Upregulation of CYP2A5: Role of Reactive Oxygen Species

Hepatic cytochrome P450 (CYP) 2E1 and CYP2A5 activate many important drugs and hepatotoxins. CYP2E1 is induced by alcohol, but whether CYP2A5 is upregulated by alcohol is not known. This article reviews recent studies on the induction of CYP2A5 by alcohol and the mechanism and role of reactive oxygen species (ROS) in this upregulation. Chronic feeding of ethanol to wild type mice increased CYP2A5 catalytic activity and protein and mRNA levels. This induction was blunted in CYP2E1 knockout mice and by a CYP2E1 inhibitor, but was restored in CYP2E1 knockin mice, suggesting a role for CYP2E1 in the induction of CYP2A5 by alcohol. Since CYP2E1 actively generates ROS, the possible role of ROS in the induction of CYP2A5 by alcohol was determined. ROS production was elevated by ethanol treatment. The antioxidants N-acetyl cysteine and vitamin C lowered the alcohol-induced elevation of ROS and blunted the alcohol-mediated induction of CYP2A5. These results suggest that ROS play a novel role in the crosstalk between CYP2E1 and CYP2A5. Alcohol treatment activated nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2), a transcription factor which up-regulates expression of CYP2A5. The antioxidants blocked the activation of Nrf2. The alcohol-induced elevation of CYP2A5, but not CYP2E1, was lower in Nrf2 knockout mice. We propose that increased generation of ROS from the alcohol-induced CYP2E1 activates Nrf2, which subsequently up-regulates the expression of CYP2A5. Thus, a novel consequence of the alcohol-mediated induction of CYP2E1 and increase in ROS is the activation of redox-sensitive transcription factors, such as Nrf2, and expression of CYP2A5. Further perspectives on this alcohol-CYP2E1-ROS-Nrf2-CYP2A5 pathway are presented.

Nrf2 is increased by CYP2E1 in rodent liver and HepG2 cells and protects against oxidative stress caused by CYP2E1†

Induction of CYP2E1 by ethanol is one pathway through which ethanol generates oxidative stress. Nrf2 is a transcription factor that regulates important antioxidant and phase II detoxification genes. Nrf2 induction by CYP2E1 and its importance in the adaptive response to increased oxidative stress caused by CYP2E1 was studied. Increases in Nrf2 protein and mRNA were observed in livers or hepatocytes of chronic alcohol-fed mice or rats and of pyrazole-treated rats or mice, conditions known to elevate CYP2E1. HepG2 cells expressing CYP2E1 (E47 cells) showed increased Nrf2 mRNA and protein expression compared with control HepG2 C34 cells. Nrf2 is activated in E47 cells as shown by an increase in nuclear Nrf2 levels and Nrf2-antioxidant-responsive element binding activity, and upregulation of Nrf2-regultated genes, glutamate cysteine ligase catalytic subunit (GCLC), and heme oxygenase 1 (HO-1). Increases in Nrf2 protein and mRNA are blocked by inhibitors of CYP2E1 activity and a reactive oxygen species (ROS) scavenger, N-acetylcysteine, which decrease ROS levels as well as Nrf2 mRNA induction. Upregulation of GCLC and HO-1 in E47 cells is dependent on Nrf2 and is prevented by siRNA-Nrf2. Blocking Nrf2 by siRNA-Nrf2 decreases glutathione and increases ROS and lipid peroxidation, resulting in decreased mitochondrial membrane potential and loss of cell viability of E47 cells but not C34 cells. These results suggest that Nrf2 is activated and that levels of protein and mRNA are increased when CYP2E1 is elevated. In conclusion, Nrf2 plays a key role in the adaptive response against increased oxidative stress caused by CYP2E1.

S-Adenosyl-l-methionine Attenuates Hepatotoxicity Induced by Agonistic Jo2 Fas Antibody following CYP2E1 Induction in Mice

S-Adenosyl-l-methionine (SAM) has been shown to be hepatoprotective against many toxic agents. Its possible effectiveness in protecting against CYP2E1-dependent toxicity is not known. We recently reported that treatment of mice with pyrazole to induce CYP2E1 increased hepatotoxicity produced by Fas agonistic Jo2 antibody. The current study was designed to investigate the effect of exogenous administration of SAM on the synergistic hepatotoxicity produced by Fas agonistic Jo2 antibody plus CYP2E1 following pyrazole pretreatment in C57BL/6 mice. Suboptimal administration of Jo2 Fas antibody combined with pyrazole pretreatment caused severe hepatotoxicity as determined by elevations in serum transaminase levels and histopathology. Exogenous administration of SAM (50 mg i.p./kg body weight every 12 h for 3 days) significantly decreased serum transaminases and ameliorated morphological changes of the liver. Addition of SAM elevated hepatic SAM and total reduced glutathione levels and inhibited CYP2E1 activity. SAM also lowered the elevated oxidative stress (lipid peroxidation, protein carbonyls, and superoxide production) and nitrosative stress (induction of inducible nitric-oxide synthase and 3-nitrotyrosine adducts) and increases in caspase-8 and -3 activation produced by the pyrazole plus Jo2 treatment. SAM did not prevent the increase in serum TNF-alpha levels or the decrease in catalase activity in this model. These results indicate that SAM can have an important hepatoprotective role as an effective reagent against Fas plus CYP2E1-induced hepatotoxicity by lowering oxidative and nitrosative stress.