Microsomal Ethanol Oxidizing System Activity Research Articles

Biochemical Aspects of the Hepatic Microsomal Ethanol-oxidizing System (MEOS): Resolved Initial Controversies and Updated Molecular Views

The hepatic microsomal ethanol-oxidizing system (MEOS) was initially confronted with much uncertainty, skepticism, scientific antagonism, and heavy discussions. Viewed as scientific challenges, this stimulated further research, and led to its successful separation from both, alcohol dehydrogenase and catalase, and its reconstitution that allowed defining the individual components of MEOS: cytochrome P450 (CYP), reductase, and phospholipids. Subsequently, it was challenging to elucidate the molecular basis of the microsomal ethanol oxidation. Unlike a usual dehydrogenation or simple oxidation process, ethanol oxidation via MEOS proceeds via reactive intermediates, commonly known as reactive oxygen species (ROS) and generated by various microsomal CYP isoenzymes including CYP 2E1, all of which are established components of MEOS. Due to its radical scavenging properties, ethanol combines with a small fraction of hydroxyl radicals and undergoes oxidation while the remaining radicals attack phospholipids of liver cell membranes. Chronic alcohol use enhances MEOS activity by upregulating CYP 2E1 combined with ROS generation, and thereby increases the metabolism of ethanol to acetaldehyde, its first metabolite with a high hepatotoxic potential. Considering the involvement of various CYP isoenzymes as constituents, MEOS is now best defined as a multi-CYP isoenzyme system, participating in ethanol metabolism and responsible for the molecular-based alcoholic liver disease.

눈꽃동충하초 물추출물이 흰쥐의 알코올대사에 미치는 영향

본 연구에서는 눈꽃동충하초 물추출물이 알코올 대사에 미치는 효과를 동물실험을 통하여 살펴보았다. 알코올과 그대사산물인 아세트알데히드의 혈중 농도를 측정한 결과, 눈꽃동충하초 물추출물의 경구투여는 흰쥐의 혈중 알코올 농도에는 영향을 미치지 않았으나, 3, 6, 9시간째의 혈중 아세트알데히드 농도를 유의적으로 감소시켰다. 특히 100 ㎎/㎏ 농도의 눈꽃동충하초 물추출물 투여 시 혈중 아세트알데히드의 농도-시간 곡선하 면적에서 유의적인 감소를 보였다. 반면에 알코올 투여 후 9시간째 측정한 간의 알코올 탈수소효소, 알데히드 탈수소효소, MEOS의 활성은 눈꽃동충하초물추출물 투여에 따른 변화를 보이지 않았다. 종합적으로 눈꽃동충하초 물추출물은 혈중 알코올 농도에는 영향을 미치지 않으나 혈중 아세트알데히드 농도를 감소시키는 것으로 보인다. 눈꽃동충하초 물추출물이 혈중 아세트알데히드 수준을 낮추는 작용기전과 관련하여, 경시적인 알코올대사 효소활성의 변화와 간 손상 관련 지표 조사 등에 관한 추가적인 연구가 필요할 것으로 사료된다.

Dilinoleoylphosphatidylcholine Decreases Ethanol-Induced Cytochrome P4502E1

Cytochrome P4502E1 (CYP2E1) induction by ethanol contributes to alcoholic liver disease and we found that a mixture of polyunsaturated phosphatidylcholines (PPC), which protects against alcohol-induced liver injury, also decreases CYP2E1. Since dilinoleoylphosphatidylcholine (DLPC) is the major component of PPC, we assessed here whether it is responsible for the protection of PPC by feeding rats for 8 weeks our liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either DLPC (1.5 g/1000 cal), PPC (3 g/1000 cal), or linoleate. CYP2E1 was assessed by Western blots and by two of its enzyme activities: the microsomal ethanol-oxidizing system (MEOS) and p-nitrophenolhydroxylase (PNP). With ethanol, CYP2E1 increased 10-fold, with corresponding rises in PNP and MEOS activities. Compared to linoleate, DLPC significantly decreased cytochrome b5, total cytochromes P450, CYP2E1 content and its corresponding activities. DLPC decreases ethanol-induced CYP2E1 and should be considered for the prevention of alcoholic liver disease.

Polyenylphosphatidylcholine Opposes the Increase of Cytochrome P‐4502E1 by Ethanol and Corrects Its Iron‐Induced Decrease

Dietary iron overload damages membrane phospholipids and decreases microsomal cytochromes P-450. We wondered whether this might also pertain to cytochrome P-4502E1 (2E1) and whether polyenylphosphatidylcholine (PPC), a 94-96% pure mixture of linoleate-rich polyunsaturated phosphatidylcholines that protects against alcohol-induced liver injury, also affects 2E1, either in the presence or absence of iron. Accordingly, rats were fed for 8 weeks our standard liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either PPC (3 g/1000 Cal) or equivalent amounts of linoleate (as safflower oil). 2E1 was assessed by Western blots and by two of its characteristic enzyme activities: the microsomal ethanol oxidizing system (MEOS), evaluated by the conversion of ethanol to acetaldehyde (determined by head space GC), and p-nitrophenolhydroxylase (PNP) activity, measured by HPLC with UV detection of 4-nitrocatechol. With ethanol (36% of energy) replacing carbohydrates, 2E1 content increased 10-fold, with a corresponding increase in PNP and MEOS activities, but when carbonyl iron (5 g/1000 Cal) was added, the induction was significantly reduced. This iron-induced decrease was corrected by PPC. PPC is rich in linoleate, but when the latter was given as triglycerides (safflower oil), there was no effect, whereas hepatic nonheme iron content was the same in both these groups. It also was found that in the absence of iron, the ethanol-mediated induction of 2E1 and its corresponding enzyme activities were significantly less with PPC (< 0.001) than with safflower oil. In addition, in alcohol-fed animals, PPC decreased the oxidative stress (as determined by F2-isoprostanes), which reflects yet another hepatoprotective effect of PPC.

INHIBITION OF BACTERIOCOLONIC PATHWAY FOR ETHANOL OXIDATION BY CIPROFLOXACIN IN RATS

Many colonic bacteria possess marked alcohol dehydrogenase (ADH) activity and are capable of oxidizing ethanol to acetaldehyde both in vitro and in vivo. We have recently shown that part of ingested ethanol is metabolized to acetaldehyde in the colon during normal alcohol metabolism. To assess the contribution of this bacteriocolonic pathway for ethanol oxidation to total ethanol metabolism, the elimination rate of ethanol, faecal aerobic flora and faecal ADH activity were determined in rats before and after the treatment with ciprofloxacin (200mg/kg/day) for four days. Ciproflaxacin treatment decreased ethanol elimination rate from 310±9 to 282±13 mg/kg/h (mean±SE; p<0.02), markedly reduced faecal aerobic flora, and also lowered faecal ADH activity from 63±17 to 17±7 nmol/min/mg faeces (p<0.05). Neither hepatic ADH nor microsomal ethanol oxidizing system activities were affected by the ciprofloxacin treatment. On the contrary, an acute intraperitoneal dose of ciprofloxacin had no effect on the rate of ethanol elimination. These results support the significant role of the bacteriocolonic pathway in total ethanol elimination, and open a new, microbiological, perspective for studies on ethanol metabolism and pathogenesis of alcohol related organ damages.

Microsomal ethanol oxidizing system activity by human hepatic cytochrome P450S

Microsomal ethanol oxidizing system activity by human hepatic cytochrome P450S

Contribution of cytochrome P450s to MEOS (microsomal ethanol-oxidizing system): a specific and sensitive assay of MEOS activity by HPLC with fluorescence labeling.

The contribution of cytochrome P450s to MEOS (microsomal ethanol-oxidizing system) in rat hepatic microsomes was studied with a modified assay method for MEOS activity. Acetaldehyde produced by MEOS was converted into the fluorescent derivative with cyclohexane-1,3-dione and analyzed by HPLC with a fluorescence detector. Acetaldehyde production by hepatic microsomes of rats treated with ethanol was higher by 75% than that by control rats. Ethanol oxidation activity of eight forms of P450 was investigated in a reconstituted system. CYP1A2 and CYP2E1 had high rates of acetaldehyde formation. The Km value of CYP2E1 for ethanol oxidation was 9.48 mM, similar to that of hepatic microsomes from rats treated with ethanol. The Km value of CYP1A2 was higher than that of CYP2E1, indicating the CYP1A2 with lower affinity for ethanol compared with CYP2E1. From the results of an inhibition study with antibodies, CYP2E1 was found to be a main contributor to MEOS induced by ethanol in rats, but CYP1A2 was considered to play an important role for MEOS when CYP2E1 was not induced. These results present the possibility that CYP1A2 and CYP2E1 contribute to MEOS activity.

Immunohistochemical Demonstration of Ethanol-Inducible

The microsomal ethanol-oxidizing system (MEOS) is a P450-dependent pathway for ethanol oxidation in hepatic microsomes. The bulk of MEOS activity is catalyzed by P450 2E1 (an ethanol-inducible isozyme of P450) in animal livers treated with ethanol. Rat brains also metabolize certain drugs, and it is theorized that a mechanism for drug metabolism exists within the brain which acts on P450. We compared immunohistochemical localization of P450 2E1 between ethanol-treated rats and pair-fed control rats. In control rats, immunoreactive P450 2E1 was detected in minute amounts in both basal ganglia and cerebellar cortices. After ethanol treatment, the content of P450 2E1 increased in the basal ganglia, and the enzyme was also induced in the cerebellar cortices, substantia nigra and hippocampus. We speculate that the rat brain metabolizes ethanol by P450 2E1.

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.

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 less than 0.01). In TF-ALC rats, MEOS activity was increased to 2.6 times compared to that of TF-CTL (P less than 0.01). ApoCYP2E1 in CF-ALC and TF-ALC were 818 +/- 63 and 433 +/- 1- pmol/mg protein, respectively, and these values were significantly higher when compared with those of the pair-fed controls (P less than 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 apoCYP2B1 (2x) and PH (6-8x) were the same for CF and TF (2x); but not for apoCYP2E1 (21 and 8x, respectively) and MEOS activity (8 and 2.6x, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Respective roles of the microsomal ethanol oxidizing system and catalase in ethanol metabolism by deermice lacking alcohol dehydrogenase

To evaluate the roles of MEOS (microsomal ethanol oxidizing system) and catalase in non-alcohol dehydrogenase (ADH) ethanol metabolism, MEOS and catalase activities in vitro and ethanol oxidation rates in hepatocytes from ADH-negative deermice were measured after treatment with catalase inhibitors and/or a stimulator of H 2O 2 generation. Inhibition of ethanol peroxidation by 3-amino-1,2,4-triazole (aminotriazole) was found to be greater than 85% up to 3 h and 80% at 6 h in liver homogenates. Urate (1 m m) which stimulates H 2O 2 production in living systems, increased ethanol oxidation fourfold in control but not in cells from aminotriazole-treated animals, documenting effective inhibition of catalase-mediated ethanol peroxidation by aminotriazole. While aminotriazole slightly depressed (15%) basal ethanol oxidation in hepatocytes, in vitro experiments showed a similar decrease in MEOS activity after aminotriazole pretreatment. Azide (0.1 m m), a potent inhibitor of catalase in vitro, also did not affect ethanol oxidation in control cells. By contrast, 1-butanol, a competitive inhibitor of MEOS, but neither a substrate nor an inhibitor of catalase, decreased ethanol oxidation rates in a dose-dependent manner. These results show that, in deermice lacking ADH, catalase plays little if any role in hepatic ethanol oxidation, and that MEOS mediates non-ADH metabolism.

The deuterium isotope effect on ethanol metabolism in perfused rat liver: effect of reversed perfusion on ethanol and oxygen uptake.

Livers from rats fasted for 24 hr were subjected to nonrecirculating perfusion with Krebs-Ringer bicarbonate solution containing 10 mM ethanol. The deuterium isotope effect was measured using (1-R)-[1-14C,1-2H]ethanol. A value of 2.57 +/- 0.09 (SD) was obtained independent of the direction of perfusion. Oxygen uptake and ethanol metabolism in contrast were significantly increased when reverse perfusion (i.e., from vena cava to vena portae) was used. The magnitude of the isotope effect indicates that contribution from microsomal ethanol-oxidizing system if this is the only supplementary system is 9.8% under the experimental conditions. At high ethanol concentrations, the contribution would approach 18%. Equal activities of microsomal ethanol-oxidizing system and catalase under the experimental conditions would mean that both contribute 7.3% of the total ethanol metabolism. At high ethanol concentrations (80 mM), however, catalase will be 6.8% and microsomal ethanol-oxidizing system is calculated to 13.3%. Preliminary experiments with rats pretreated with phenobarbital showed no change in the isotope effect or in the rate of ethanol metabolism, but a 40-50% increase in oxygen consumption. The acetaldehyde concentration in the effluent medium was below 1 microM.

Microsomal ethanol oxidizing system (MEOS): Interaction with ethanol, drugs and carcinogens

Several studies in our unit showed that in men, baboons, rats and deermice, blood ethanol clearance is significantly accelerated at ethanol concentrations higher than the levels needed to effectively saturate the low K m forms of ADH present in animals, thereby incriminating a high K m non-ADH system such as microsomal ethanol oxidizing system (MEOS). Furthermore, kinetics of blood ethanol clearance were consistent with the K m of MEOS. After chronic ethanol consumption, there was an increase in rates of ethanol elimination and in the activity of MEOS. There was an associated rise in microsomal cytochrome P-450, including a form (different from that induced by phenobarbital and methylcholanthrene) which had a high affinity for ethanol in a reconstituted system. The role of a non-ADH pathway of ethanol metabolism and its increase after chronic ethanol consumption was most conclusively shown in ADH-negative deermice. Microsomal induction was also associated with enhanced metabolism of other drugs, resulting in metabolic drug tolerance. In addition, there was increased activation of known hepatotoxic agents (such as CCl 4 and acetaminophen) which may explain the enhanced susceptibility of alcoholics to the toxicity of solvents and commonly used drugs. There was enhanced activation of procarcinogens, sometimes at concentrations much lower than those required for other microsomal inducers. Moreover, catabolism of retinoic acid was accelerated possibly contributing to hepatic vitamin A depletion. In conclusion, after chronic ethanol consumption, enhanced MEOS activity and concomitant cytochrome P-450 changes may contribute to accelerated ethanol and drug metabolism and associated activation of hepatotoxic agents and carcinogens.

Effect of thyrotoxicosis on ethanol metabolism and on hepatic ethanol oxidation enzymes.

The influence and mechanism of action of T4 treatment on ethanol metabolism was investigated by the determination of ethanol elimination in intact rats and by the estimation of alcohol dehydrogenase and the microsomal ethanol oxidizing system activity in isolated rat liver. The rate of ethanol degradation was significantly decreased to 65% of control values in T4-treated rat liver. Similarly blood ethanol disappearance rate was significantly inhibited in thyrotoxic rats as compared to euthyroid animals. Hepatic alcohol dehydrogenase activity was reduced to 39% of control values as a result of T4 treatment; however, the microsomal ethanol oxidizing system activity was not affected. These data suggest that reduction in blood ethanol disappearance rate is associated with inhibition of hepatic alcohol dehydrogenase activity in thyrotoxicosis.

Induction of hepatic microsomal ethanol-oxidizing system activity following chronic ethanol consumption: Respective roles of ethanol and carbohydrates

Induction of hepatic microsomal ethanol-oxidizing system activity following chronic ethanol consumption: Respective roles of ethanol and carbohydrates

Effect of thyroid hormones on the activities of hepatic alcohol metabolizing enzymes

Treatment with thyroxine or triiodothyronine for 7 days in order to simulate a hyperthyroid state results in an enhanced activity of the microsomal ethanol oxidizing system. Conversely, a decrease of hepatic alcohol dehydrogenase activity was observed under these experimental conditions, whereas hepatic catalase activity remained unchanged. These findings suggest that if chronic ethanol consumption simulates a “hyperthyroid hepatic state”, increased rates of ethanol metabolism observed following prolonged alcohol intake might therefore be attributed at least in part to an induction of microsomal ethanol oxidizing system activity in the liver.

Metabolism of alcohol at high concentrations: role and biochemical nature of the hepatic microsomal ethanol oxidizing system.

At intermediate and higher alcohol concentrations, ethanol metabolism proceeds via alcohol dehydrogenase (ADH) and the microsomal ethanol oxidizing system (MEOS), whereas catalase plays no significant role. Following prolonged ethanol consumption, an enhancement of both MEOS activity as well as the rates of ethanol metabolism occurs; the latter persisted despite inhibition of ADH by pyrazole and catalase by sodium axide, suggesting the involvement of MEOS in the adaptive increase. MEOS exhibits characteristics similar to those of other microsomal drug metabolizing enzymes and can be differentiated and isolated from both ADH and catalase activities. Reconstitution of MEOS activity was achieved with partially purified cytochrome P-450 and NADPH-cytochrome c reductase in the presence of synthetic phospholipid.

EFFECT OF CARBON TETRACHLORIDE ON ETHANOL METABOLISM

Treatment of animals with a potent hepatotoxin carbon tetrachloride (CCl4) was found to decrease drastically the enzymatic activity of the hepatic microsomal ethanol oxidizing system (MEOS). CCl4 treatment, however, appeared to have no effect on ethanol metabolism in vivo, as estimated from the elimination rate of blood ethanol in rabbits and rats, or from the disappearance rate of ethanol from the whole body in mice. In contrast to the reduced MEOS activity, the cytoplasmic alcohol dehydrogenase (ADH) activity was not influenced by CCl4 treatment. In confirmation of other reports, it is concluded that the elimination rate of ethanol from the blood is not an adequate index for the liver damage and the hepatic microsomes do not play a significant role in ethanol metabolism in vivo.

Hepatic microsomal ethanol-oxidizing system: Solubilization, isolation, and characterization

The solubilization and subsequent separation of the hepatic microsomal ethanol-oxidizing system from alcohol dehydrogenase and catalase activities by DEAE-cellulose column chromatography is described. Absence of alcohol dehydrogenase in the column eluates exhibiting microsomal ethanol-oxidizing system activity was demonstrated by the failure of NAD + to promote ethanol oxidation at pH 9.6. Differentiation of the microsomal ethanol-oxidizing system from alcohol dehydrogenase was further shown by the apparent K m for ethanol (7.2 m m, insensitivity of the microsomal ethanol-oxidizing system to the alcohol dehydrogenase inhibitor pyrazole (0.1 m m) and by the failure of added alcohol dehydrogenase to increase the ethanol oxidation. Absence of catalatic activity in these fractions was demonstrated by spectrophotometric and polarographic assay. Differentiation of the microsomal ethanol-oxidizing system from the peroxidatic activity of catalase was shown by the apparent K m for oxygen (8.3 μ m), insensitivity of the microsomal ethanol-oxidizing system to the catalase inhibitors azide and cyanide, and by the lack of a H 2O 2-generating system (glucose-glucose oxidase) to sustain ethanol oxidation in the eluates. The oxidation of ethanol to acetaldehyde by the alcohol dehydrogenase- and catalase-free fractions required NADPH and oxygen and was inhibited by CO. The column eluates showing microsomal ethanol-oxidizing system activity contained cytochrome P-450, NADPH-cytochrome c reductase, and phospholipids and also metabolized aminopyrine, benzphetamine, and aniline.