Berberine protects liver from ethanol-induced oxidative stress and steatosis in mice

Alcohol consumption is customary in many cultures and it is a common human behavior worldwide. Binge ethanol and chronic alcohol consumption, two usual drinking patterns of human beings, produce a state of oxidative stress in liver and disturb the liver function. However, a safe and effective therapy for alcoholic liver disease in humans is still elusive. This study identified the natural product berberine as a potential agent for treating or preventing ethanol-induced liver injury. We demonstrated that berberine attenuated oxidative stress resulted from binge drinking in liver by reducing hepatic lipid peroxidation, glutathione exhaust and mitochondrial oxidative damage. Furthermore, berberine also prevented the oxidative stress and macrosteatosis in response to chronic ethanol exposure in mice. Either the total cytochrome P450 2E1 or the mitochondria-located cytochrome P450 2E1, which is implicated in ethanol-mediated oxidative stress, was suppressed by berberine. On the other hand, berberine significantly blunted the lipid accumulation in liver due to chronic alcohol consumption, at least partially, through restoring peroxisome proliferator-activated receptor α/peroxisome proliferator-activated receptor-gamma Co-activator-1α and hepatocyte nuclear factor 4α/microsomal triglyceride transfer protein pathways. These findings suggested that berberine could serve as a potential agent for preventing or treating human alcoholic liver disease.     

Inhibition of CYP2E1 catalytic activity in vitro by S-adenosyl-L-methionine

The objective of this work was to evaluate the possible in vitro interactions of S-adenosyl-l-methionine (SAM) and its metabolites S-(5'-Adenosyl)-l-homocysteine (SAH), 5'-Deoxy-5'-(methylthio)adenosine (MTA) and methionine with cytochrome P450 enzymes, in particular CYP2E1. SAM (but not SAH, MTA or methionine) produced a type II binding spectrum with liver microsomal cytochrome P450 from rats treated with acetone or isoniazid to induce CYP2E1. Binding was less effective for control microsomes. SAM did not alter the carbon monoxide binding spectrum of P450, nor denature P450 to P420, nor inhibit the activity of NADPH-P450 reductase. However, SAM inhibited the catalytic activity of CYP2E1 with typical substrates such as p-nitrophenol, ethanol, and dimethylnitrosamine, with an IC(50) around 1.5-5mM. SAM was a non-competitive inhibitor of CYP2E1 catalytic activity and its inhibitory actions could not be mimicked by methionine, SAH or MTA. However, SAM did not inhibit the oxidation of ethanol to alpha-hydroxyethyl radical, an assay for hydroxyl radical generation. In microsomes engineered to express individual human P450s, SAM produced a type II binding spectrum with CYP2E1-, but not with CYP3A4-expressing microsomes, and SAM was a weaker inhibitor against the metabolism of a specific CYP3A4 substrate than a specific CYP2E1 substrate. SAM also inhibited CYP2E1 catalytic activity in intact HepG2 cells engineered to express CYP2E1. These results suggest that SAM interacts with cytochrome P450s, especially CYP2E1, and inhibits the catalytic activity of CYP2E1 in a reversible and non competitive manner. However, SAM is a weak inhibitor of CYP2E1. Since the K(i) for SAM inhibition of CYP2E1 activity is relatively high, inhibition of CYP2E1 activity is not likely to play a major role in the ability of SAM to protect against the hepatotoxicity produced by toxins requiring metabolic activation by CYP2E1 such as acetaminophen, ethanol, carbon tetrachloride, thioacetamide and carcinogens.     

The discovery of the microsomal ethanol oxidizing system and its physiologic and pathologic role

Oxidation of ethanol via alcohol dehydrogenase (ADH) explains various metabolic effects of ethanol but does not account for the tolerance. This fact, as well as the discovery of the proliferation of the smooth endoplasmic reticulum (SER) after chronic alcohol consumption, suggested the existence of an additional pathway which was then described by Lieber and DeCarli, namely the microsomal ethanol oxidizing system (MEOS), involving cytochrome P450. The existence of this system was initially challenged but the effect of ethanol on liver microsomes was confirmed by Remmer and his group. After chronic ethanol consumption, the activity of the MEOS increases, with an associated rise in cytochrome P450, especially CYP2E1, most conclusively shown in alcohol dehydrogenase negative deer mice. There is also cross-induction of the metabolism of other drugs, resulting in drug tolerance. Furthermore, the conversion of hepatotoxic agents to toxic metabolites increases, which explains the enhanced susceptibility of alcoholics to the adverse effects of various xenobiotics, including industrial solvents. CYP2E1 also activates some commonly used drugs (such as acetaminophen) to their toxic metabolites, and promotes carcinogenesis. In addition, catabolism of retinol is accelerated resulting in its depletion. Contrasting with the stimulating effects of chronic consumption, acute ethanol intake inhibits the metabolism of other drugs. Moreover, metabolism by CYP2E1 results in a significant release of free radicals which, in turn, diminishes reduced glutathione (GSH) and other defense systems against oxidative stress which plays a major pathogenic role in alcoholic liver disease. CYP1A2 and CYP3A4, two other perivenular P450s, also sustain the metabolism of ethanol, thereby contributing to MEOS activity and possibly liver injury. CYP2E1 has also a physiologic role which comprises gluconeogenesis from ketones, oxidation of fatty acids, and detoxification of xenobiotics other than ethanol. Excess of these physiological substrates (such as seen in obesity and diabetes) also leads to CYP2E1 induction and nonalcoholic fatty liver disease (NAFLD), which includes nonalcoholic fatty liver and nonalcoholic steatohepatitis (NASH), with pathological lesions similar to those observed in alcoholic steatohepatitis. Increases of CYP2E1 and its mRNA prevail in the perivenular zone, the area of maximal liver damage. CYP2E1 up-regulation was also demonstrated in obese patients as well as in rat models of obesity and NASH. Furthermore, NASH is increasingly recognized as a precursor to more severe liver disease, sometimes evolving into "cryptogenic" cirrhosis. The prevalence of NAFLD averages 20% and that of NASH 2% to 3% in the general population, making these conditions the most common liver diseases in the United States. Considering the pathogenic role that up-regulation of CYP2E1 also plays in alcoholic liver disease (vide supra), it is apparent that a major therapeutic challenge is now to find a way to control this toxic process. CYP2E1 inhibitors oppose alcohol-induced liver damage, but heretofore available compounds are too toxic for clinical use. Recently, however, polyenylphosphatidylcholine (PPC), an innocuous mixture of polyunsaturated phosphatidylcholines extracted from soybeans (and its active component dilinoleoylphosphatidylcholine), were discovered to decrease CYP2E1 activity. PPC also opposes hepatic oxidative stress and fibrosis. It is now being tested clinically.     

混合探针底物法测定五味子成分对大鼠肝脏细胞色素P450同工酶的影响

研究大鼠多次口服五味子成分和体外对肝脏细胞色素P450酶(CYP450)6种同工酶的影响。采用超速离心法制备正常及多次口服五味子醇/水提物的大鼠肝微粒体并与探针药进行体外温孵,应用液相色谱?串联质谱分析方法测定CYP450的6种同工酶特异性探针底物非那西丁(CYP1A2)、右美沙芬(CYP2D2)、双氯芬酸钠(CYP2C6)、美芬妥英(CYP2C11)、氯唑沙宗(CYP2E1)、咪达唑仑(CYP3A1/2)在大鼠肝微粒体的代谢产物生成(对乙酰氨基酚、右啡烷、4-羟基双氯芬酸钠、4-羟基美芬妥英、6-羟基氯唑沙宗、1-羟基咪达唑仑)以反映各CYP450同工酶活性。五味子醇提物(28～120μg.mL-1)体外对大鼠肝微粒体CYP450的6个同工酶均有不同程度的抑制作用。大鼠多次口服五味子醇提物(1.5 g.kg-1,qd×7d)对肝脏CYP3A1/2和CYP2E1有显著诱导作用,对CYP2D2和CYP2C11有明显抑制作用,而对CYP2C6和CYP1A2无明显影响。五味子水提物(100～500μg.mL-1)体外对大鼠肝脏CYP450同工酶亦有抑制作用;体内多次给药(1.5 g.kg-1,qd×7d)对肝脏CY...     

淫羊藿苷对不同月龄大鼠肝微粒体细胞色素P450的影响

目的 揭示淫羊藿苷(Ica)对大鼠肝微粒体细胞色素P450的含量及部分亚型的影响,并比较月龄的差异.方法 ig给予6月龄和18月龄的♂SD大鼠Ica( 60 mg· kg -1),4周后取肝脏,用钙沉淀法提取肝微粒体,BCA法测定微粒体蛋白浓度;用一氧化碳还原差示光谱法测定CYP450的含量;用ELISA法测定CYP1 A1、CYPb5的含量;用比色法测定苯胺羟化酶(反映CYP2E1活性)和红霉素-N-脱甲基酶(反映CYP3A活性)的活性;用real - time RT - PCR检测CYP1 A1、CYP2A3、CYP2E1、CYP3A1、CYP3A2和CYP4B1 mRNA的表达.结果 60 mg· kg-1 Ica明显增加了CYP450的总酶和CYP1 A1的含量、CYP3A的活性及CYP1 A1、CYP3A1、CYP3A2 mRNA的表达,降低了CYP2E1的活性及其mRNA的表达;但Ica对上述各指标的诱导或抑制作用在大鼠月龄方面差异不明显;Ica对CYPb5的含量及CYP2A3、CYP4B1 mRNA的表达未见明显影响.结论 Ica对大鼠肝微粒体CYP450总酶、CYPI A1和CYP3A具有诱导作用,对CYP2E1具有抑制作用,该作用未见明显月龄差异.     

慢性间断性低氧对大鼠肝脏细胞色素P450的影响

目的观察慢性间断性低氧对大鼠肝脏P450同工酶的影响。方法♂SD大鼠随机分为对照组和实验组,实验组分别低氧3、7、14、28d。采用酶法测定血清丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST)活性,分光光度法测定大鼠肝微粒体红霉素N-脱甲基酶(ERD)、苯胺羟化酶(ANH)活性,半定量逆转录聚合酶链式反应(RT-PCR)检测大鼠肝脏细胞色素P4503A2、2E1的mRNA表达水平。结果慢性间断性低氧对血清ALT和AST活性无明显影响;低氧7d后,大鼠肝脏ERD和ANH活性明显升高,28d时诱导率分别为155·5%和42·2%;同时CYP3A2和CYP2E1mR-NA的表达水平,也分别增加了220·5%和102·8%。结论慢性间断性低氧能明显增加大鼠肝脏ERD(CYP3A2)和ANH(CYP2E1)活性,其机制可能与其在转录水平上提高肝脏CYP3A2和CYP2E1的基因表达水平有关。     

CYP2E1-dependent oxidative stress and toxicity: role in ethanol-induced liver injury

Ethanol-induced oxidative stress plays a major role in the mechanisms by which ethanol causes liver injury. Many pathways contribute to how ethanol induces a state of oxidative stress. One central pathway appears to be the induction, by ethanol, of the CYP2E1 form of cytochrome P450 enzymes. CYP2E1 is of interest because it metabolises and activates many toxicological substrates, including ethanol, to more reactive products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions. CYP2E1 is an effective generator of reactive oxygen species. This review summarises some of the biochemical and toxicological properties of CYP2E1, and briefly describes the use of HepG2 cell lines in assessing the actions of CYP2E1. Future directions, which may help to better understand the actions of CYP2E1 and its role in alcoholic liver injury, are suggested.     

Altered expression of cytochrome P450 and possible correlation with preneoplastic changes in early stage of rat hepatocarcinogenesis

Aim: Correlation of cytochrome P450 (CYPs) with preneoplastic changes in the early stage of hepatocarcinogenesis is still unclear. To detect the expression of carcinogen-metabolizing related microsomal P450 enzymes, namely the CYP1A1,CYP1A2, CYP2B 1/2, CYP2E1, and CYP3A, we performed the medium-term bioassay of Ito‘s model in Sprague-Dawley rats. Methods: The amount and activity of CYP were assessed by biochemical and immunohistochemical methods in week 8.The correlation between CYP expression and microsomal oxidative stress was investigated by comparing the generation of microsomal lipid peroxidation in the presence or absence of specific CYP inhibitor. Results: In the DEN-2-AAF and 2-AAF alone groups, the expression of CYP1A1 and CYP2E1 were up-regulated and the expression of CYP2B 1/2 and CYP1 A2 were quite the contrary. Strong staining of CYP2E1 and CYP2BI/2 was found around the centrolobular vein and weak staining in the altered hepatic foci revealed by immunohistochemical procedure.There was no significant change in the activity of CYP3A among the 4 groups.Altered hepatic tissue bore more microsomal NADPH (nicotinamide adenine dimucleotide phosphate,reduced form)-dependent lipid peroxidation than normal tissue. And the difference among the 4 groups disappeared when CYP2E1 was inhibited. More microsomal lipid peroxidation was generated when incubated with CYP1A inhibitor α-naphthoflavone. Conclusion: CYP altered their expression levels and these alterations can play important roles in the alteration of cell redox status of preneoplastic tissue in the early stage of hepatocarcinogenesis.     

Altered activity of cytochrome P450 in alcoholic fatty liver exposed to ischemia/reperfusion

Ischemia/reperfusion (I/R) injury is a major cause of morbidity and mortality in liver surgery and transplantation, and fatty livers are susceptible to greater I/R injury and a higher incidence of primary graft nonfunction after transplantation. Because alcohol intake and obesity are major causes of fatty liver, this study was initiated to investigate the effect of chronic ethanol consumption on hepatic microsomal cytochrome P450 (CYP) activity after I/R. Rats were fed an alcohol liquid diet or a control isocaloric diet for 4 weeks, and then subjected to 60 min of hepatic ischemia and 5 h of reperfusion. It was found that, chronic ethanol consumption significantly increased liver weight, serum triglyceride (TG), liver TG, and serum aminotransferase activities. Moreover, alcoholic fatty livers exposed to I/R showed significantly higher levels of aminotransferase activities than the controls. No significant differences in microsomal CYP content or CYP1A1 activity were found between I/R treated animals fed a control diet (the CD + I/R group) and I/R treated animals fed an ethanol containing diet (the ED + I/R group). Moreover, whereas CYP1A2 activity was decreased in the ED + I/R group versus the CD + I/R group, CYP2E1 activity was elevated. Additionally, chronic alcohol consumption up-regulated TNF-alpha and IL-6 mRNA levels immediately after I/R. In conclusion, chronic ethanol consumption was found to potentiate hepatocellular damage as indicated by abnormalities in microsomal drug metabolizing function during I/R.     

Effects of Dietary Fat Composition on Activities of the Microsomal Ethanol Oxidizing System and Ethanol-Inducible Cytochrome P450 (CYP2E1) in the Liver of Rats Chronically Fed Ethanol

Abstract: We studied the effects of dietary fat composition on the activities of the microsomal ethanol oxidizing system (MEOS), paranitrophenol hydroxylase (PH) activity and ethanol-inducible cytochrome P450 isozymes (CYP2E1 and CYP2B1) in the liver of rats to determine the role of this ethanol metabolizing pathway in the pathogenesis of alcoholic liver disease (ALD). Wistar male rats were pair-fed a liquid diet, containing either tallow (TF) or corn oil (CF) as the fat component, and ethanol or an isocaloric amount of dextrose, through an implanted intragastric cannula. Liver pathology of rats fed ethanol (CF-ALC) and CF diet showed severe fatty change whereas the rats fed TF-alcohol and the TF and CF controls did not. MEOS activity of the CF-ALC group was 8 times of that in the CF-CTL group (P < 0.01). In TF-ALC rats, MEOS activity was increased to 2.6 times compared to that of TF-CTL (P< 0.01). ApoCYP2E1 in CF-ALC and TF-ALC were 818 ± 63 and 433 ± 17 pmol/mg protein, respectively, and these values were significantly higher when compared with those of the pair-fed controls (P< 0.005). In contrast, apoCYP2B1 was increased to an equal degree in both CF-ALC and TF-ALC. When PH-activity was measured, the level of activity on TF-ALC rats did not differ from that of CF-ALC rats. Thus, ethanol-induction of apoCYP2Bl (2 x) and PH (6–8 x) were the same for CF and TF (2 x); but not for apoCYP2E1 (21 and 8 x, respectively) and MEOS activity (8 and 2.6 x, respectively). These results indicate that the dietary effect on the expression of CYP2E1 correlates with the induction of centrilobular liver damage seen in the corn oil fed rats. The centrilobular distribution of this isozyme also correlates with the site of liver cell injury further suggesting a pathogenic link to alcohol-induced cell injury.     

An improved in vitro method for screening toxin and medicine targeting CYP2E1

The cytochrome P450 enzyme 2E1 (CYP2E1) presents in both microsome and mitochondrion, which influences the metabolism of many xenobiotics. The mice active liver homogenate was prepared for the medicinal incubation and mitochondrion was extracted for chemical screening targeting CYP2E1 enzyme. Representative CYP2E1 inducers (ethanol and pyrazole) and inhibitors (diallyldisulfide and kaempferol) were applied to evaluate the effectiveness of homogenate-mitochondrial system. In parallel, the in-vitro microsomal method targeting CYP2E1 was also operated for comparison. The results showed that in homogenate-mitochondrial method, the protein level and activity of CYP2E1 were increased by ethanol and pyrazole; reduced by diallyldisulfide and kaempferol, and this homogenate-mitochondrial method is convenient with good repeatability and reproducibility in screening chemicals targeting CYP2E1, especially for the inducers. Thus, the homogenate-mitochondrial method might be effective in screening both CYP2E1 inhibitor and inducer.     

One injection of estradiol valerate induces dramatic changes in rats' intake of alcoholic beverages

A series of experiments investigated the effects of a single injection of estradiol valerate (EV) on female rats' consumption of alcoholic beverages. EV provides sustained release of estradiol. Just after an injection of EV, rats' intake of a palatable alcoholic beverage, which had been taken regularly before, is reduced dramatically. Subsequently, rats' intake of alcoholic beverage returns to baseline levels. With continued opportunity to drink, rats take more ethanol than controls. When EV was given 15 and 31 days before the first opportunity to drink an alcoholic beverage, female rats markedly enhanced their intake of ethanol. Once enhanced intakes emerged, they were observed with different kinds of alcoholic beverages and endured for months.     

Novel metabolic pathway of estrone and 17beta-estradiol catalyzed by cytochrome P-450.

We have already reported that the quinol formation from some para-alkylphenols, which is a novel metabolic pathway catalyzed by cytochrome P-450, occurs in a rat liver microsomal system (). In the present study, we investigated whether estrone and 17beta-estadiol, each of which contains a p-alkylphenol moiety, are also oxidized into the corresponding quinols by cytochrome P-450. Six recombinant human cytochrome P-450 enzymes, CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2E1, and CYP3A4, were tested. The results show that estrone and 17beta-estadiol were converted into the corresponding quinols by CYP1A1, CYP2B6, and CYP2E1.     

Effects of coffee and its chemopreventive components kahweol and cafestol on cytochrome P450 and sulfotransferase in rat liver.

Coffee drinking appears to reduce cancer risk in liver and colon. Such chemoprevention may be caused by the diterpenes kahweol and cafestol (K/C) contained in unfiltered beverage. In animals, K/C treatment inhibited the mutagenicity/tumorigenicity of several carcinogens, likely explicable by beneficial modifications of xenobiotic metabolism, particularly by stimulation of carcinogen-detoxifying phase II mechanisms. In the present study, we investigated the influence of K/C on potentially carcinogen-activating hepatic cytochrome P450 (CYP450) and sulfotransferase (SULT). Male F344 rats received 0.2% K/C (1:1) in the diet for 10 days or unfiltered and/or filtered coffee as drinking fluid. Consequently, K/C decreased the metabolism of four resorufin derivatives representing CYP1A1, CYP1A2, CYP2B1, and CYP2B2 activities by approximately 50%. For CYP1A2, inhibition was confirmed at the mRNA level, accompanied by decreased CYP3A9. In contrast to K/C, coffee increased the metabolism of the resorufin derivatives up to 7-fold which was only marginally influenced by filtering. CYP2E1 activity and mRNA remained unchanged by K/C and coffee. K/C but not coffee decreased SULT by approximately 25%. In summary, K/C inhibited CYP450s by tendency but not universally. Inhibition of CYP450 and SULT may contribute to chemoprevention with K/C but involvement in the protection of coffee drinkers is unlikely. The data confirm that the effects of complex mixtures may deviate from those of their putatively active components.     

Metabolic oxidation and toxification of N-methylformamide catalyzed by the cytochrome P450 isoenzyme CYP2E1.

Alkylformamides, for example N-methylformamide, are hepatotoxic in rodents and humans. The mechanism by which N-methylformamide exerts its hepatotoxicity involves metabolic oxidation at the formyl moiety to yield a short-lived intermediate, perhaps methyl isocyanate, which reacts with glutathione to afford S-(N-methylcarbamoyl)glutathione. The hypothesis that the cytochrome P450 isozyme CYP2E1 catalyzes the metabolic toxification of N-methylformamide was tested. Hepatocytes obtained from mice that had received acetone, an inducer of CYP2E1, were incubated for up to 4 hr with N-methylformamide (5 and 10 mM). Whereas N-methylformamide caused cytotoxicity in these cells, as measured by release from the cells of lactate dehydrogenase, it was barely toxic, under these conditions, to cells from untreated mice. Coincubation of N-methylformamide with dimethylsulfoxide (10 mM), a CYP2E1 inhibitor, for 4 or 6 hr abolished the hepatocytotoxicity of N-methylformamide. Metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was measured in incubates with liver microsomes from rats, mice, or humans in the presence of glutathione. Pretreatment of rodents with acetone or ethanol induced the rate of metabolism of N-methylformamide and of p-nitrophenol, a known CYP2E1 substrate, but it did not increase aminopyrine N-demethylation. Metabolism of N-methylformamide and p-nitrophenol was elevated in microsomes from animals that had received acetone (1%) in their drinking water for 1 week to 230% and 200%, respectively, of control values in mouse microsomes and to 310% and 240%, respectively, of control values in rat microsomes. Pretreatment of animals with 4-methylpyrazole (200 mg/kg intraperitoneally, once daily for 3 days) increased metabolism of N-methylformamide to 410% of control values in rat liver microsomes but was without effect on murine microsomal metabolism of N-methylformamide. The metabolism of this compound was strongly inhibited by the CYP2E1 substrates or inhibitors dimethylsulfoxide (1-100 mM), p-nitrophenol (100 microM), and diethyldithiocarbamate (100 microM), which did not affect aminopyrine N-demethylation. A polyclonal antibody against rat CYP2E1 (10 mg of IgG/nmol of cytochrome P450) inhibited N-methylformamide metabolism in liver microsomes from rats and from a human by 75% and 80%, respectively. The rate of metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was determined in liver microsomes from six humans and correlated with extent of metabolic hydroxylation of chlorzoxazone, a CYP2E1 probe, and with amount of immunodetectable enzyme using an anti-rat CYP2E1 antibody (r = 0.81 and 0.80, respectively). The results suggest that CYP2E1 is the predominant, if not sole, cytochrome P450 isozyme responsible for the metabolic toxification of hepatotoxic N-alkylformamides.     

罗格列酮对大鼠肝细胞色素P-4502E1活性的影响

目的 :探讨罗格列酮对细胞色素P 4 5 02E1活性的影响。方法 :在正常大鼠肝细胞微粒体中加入浓度为 0 ,0 .10 ,0 .2 5 ,0 .5 0 ,1.0 0mmol·L- 1罗格列酮 ,以N 二甲基亚硝胺为底物 ,测定细胞色素P 4 5 0 2E1活性。另外用 0 ,0 .2 ,2 ,2 0mg·kg- 1剂量的罗格列酮分别对用乙醇诱导和未用乙醇诱导的大鼠灌胃 ,然后取大鼠肝细胞微粒体 ,以N 二甲基亚硝胺为底物 ,测定细胞色素P 4 5 0 2E1活性。结果 :在体外试验中 ,加入不同浓度罗格列酮的大鼠肝微粒体中细胞色素P 4 5 0 2E1活性差异无显著意义。在体内试验中 ,给予不同剂量罗格列酮的大鼠的肝细胞色素P 4 5 0 2E1活性差异也无显著意义 ;结论 :罗格列酮对大鼠的肝细胞色素P 4 5 0 2E1活性无明显影响     

Effects of normothermic hepatic ischemia-reperfusion injury on the in vivo, isolated perfused liver, and microsomal disposition of chlorzoxazone, a cytochrome P450 2E1 probe, in rats

In vitro studies have shown that the activities of cytochrome P450 (P450) enzymes may be altered after hepatic ischemia-reperfusion (IR) injury. Here, we investigated the effects of 1 h of partial ischemia, followed by 3 (IR3) or 24 (IR24) h of in vivo reperfusion, on the in vivo, isolated perfused rat liver (IPRL), and microsomal disposition of chlorzoxazone (CZX) and its cytochrome P450 2E1 (CYP2E1)-mediated metabolite, 6-hydroxychlorzoxazone (HCZX), in rats. Although IR3 caused a 30% reduction in the in vivo clearance of CZX, the area under the plasma concentration-time curve of HCZX was not affected. IPRL experiments showed that IR3, in addition to a 30% reduction in the clearance of CZX, causes a 70% decrease in the biliary clearance of HCZX. Microsomal data revealed a 50% decline in the intrinsic clearance of HCZX formation due to an IR3-induced significant decline in maximum velocity. Although IR3 did not affect the microsomal CYP2E1 protein, it caused approximately 30% reduction in the cytochrome P450 reductase activity. IR24 did not have any effect on the disposition of CZX or HCZX. In conclusion, metabolism of xenobiotics and endogenous compounds that are substrates for CYP2E1, and possibly other P450 isoenzymes, may be reduced shortly after surgical procedures that require transient interruption of the hepatic blood flow.     

CYP2E1--biochemical and toxicological aspects and role in alcohol-induced liver injury

Ethanol-induced oxidative stress appears to play a major role in mechanisms by which ethanol causes liver injury. 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. CYP2E1 is of interest because of its ability to metabolize and activate many toxicological substrates, including ethanol, to more reactive toxic products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions, and after acute and chronic alcohol treatment. CYP2E1 is also an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide, and in the presence of iron catalysts, it produces powerful oxidants such as the hydroxyl radical. This review article summarizes some of the biochemical and toxicological properties of CYP2E1, and briefly describes the use of HepG2 cell lines developed to constitutively express the human CYP2E1 in assessing the actions of CYP2E1. Regulation of CYP2E1 is quite complex and will be briefly reviewed. Future directions in research, which may help clarify the actions of CYP2E1 and its role in alcoholic liver injury, are suggested.     

Induction and inhibition of cytochrome P450 and drug-metabolizing enzymes by climbazole

To determine the effect of climbazole on hepatic microsomal cytochrome P450 (P450) and drug-metabolizing enzymes, four different P450 isoforms (CYP2B1, 3A2, 2E1, and 2C12) were examined in female Long-Evans rats. Treatment of rats with climbazole resulted in the induction of P450 content. Climbazole both induced and inhibited aminopyrine N-demethylase activity, but not erythromycin N-demethylase activity. Uridine 5'-phosphate (UDP)-glucuronosyl transferase and glutathione S-transferase activities were also increased with climbazole treatment. Immunoblot analyses revealed that climbazole induces CYP2B1 and CYP3A2 at the lower dose examined, but it failed to increase CYP2B1 at the higher dose. Northern blot analysis revealed that climbazole markedly increases P450 2B1 mRNA. These results indicate that climbazole induces and inhibits P450-dependent drug-metabolizing enzymes in vivo and may have the dose-differential effect on CYP2B1 in rat liver.     

Combined effect of ethanol and carbon disulphide on cytochrome P-450 mono-oxygenase, lipid peroxidation and ultrastructure of the liver in chronically exposed rats

A 5-month treatment of rats with ethanol (10% solution in drinking water) stimulated aniline p-hydroxylase and the microsomal ethanol oxidizing system (MEOS) by 140 and 70%, respectively, cytochrome P-450 by 22% and accompanied by lipid peroxidation by 40% in microsomes. It also caused smooth endoplasmic reticulum (SER) proliferation and rough endoplasmic reticulum (RER) degranulation in hepatocytes. Repeated inhalatory exposure of rats to 1.5 g/m3 of CS2, 5 h daily, 5 days a week for 5 months decreased aniline p-hydroxylase and MEOS by 70 and 55% respectively, doubled hexobarbital sleeping time and depressed cytochrome P-450 by 30% and its conversion to cytochrome P-420; these effects were accompanied by the appearance of cytochrome P-420, the intensification of lipid peroxidation in microsomes and some degranulation of RER in hepatocytes. Combined exposure of rats to ethanol and CS2 resulted in a significant potentiation of the inhibitory effects of CS2 on cytochrome P-450 mono-oxygenase and MEOS but with enhancement of CS2 effects on the liver microsomal mono-oxygenase, but CS2 decreased the effect of ethanol on SER proliferation. The interaction both on the biochemical and the morphological level can be explained with the ethanol-stimulated biotransformation of CS2 to reactive electrophilic derivative(s), the subsequent destruction of cytochrome P-450 to cytochrome P-420 and the intensification of lipid peroxidation.