The purpose of this investigation was to assess the effect of chlormethiazole treatment on liver damage in the experimental rat intragastric ethanol-feeding model of alcoholic liver disease. Chlormethiazole has been used in the treatment of alcoholic withdrawal and has been shown to inhibit cytochrome P4502E1. Since treatment of experimental alcoholic liver disease with CYP2E1 inhibitors had an ameliorating effect on liver injury in the rat, chlormethiazole was used to see if it had a similar effect. Rats fed ethanol for 2 months had significantly less liver injury when chlormethiazole was added to the diet, fed intragastrically. The CYP2E1 apoprotein levels, which were increased by ethanol feeding, were also increased when chlormethiazole was fed with ethanol. Chlormethiazole inhibited the increase in the ethanol-induced CYP2E1 activity in vivo, as measured by chlorzoxazone 6-hydroxylation, but did not affect the level of CYP2E1 apoprotein. Likewise, the reduction in proteasome proteolytic enzyme activity produced by ethanol feeding was blunted in chlormethiazole-fed rats. These results support the conclusion that chlormethiazole treatment partially protects the liver from injury by inhibiting CYP2E1 activity in vivo.

This study was done to determine if a relationship exists between CYP2E1 induction by ethanol, lipid peroxidation, and liver pathology in experimental alcohol-induced liver disease in the rat. Rats were fed ethanol with or without diallyl sulfide (DAS) or phenethyl isothiocyanate (PIC) intragastrically for 1 month. CYP2E1 induction by ethanol was correlated with lipid peroxidation, liver microsomal CYP2E1 hydroxylation of paranitrophenol, and the liver pathology score using the data from the PIC-fed rats. Some of the data from the ethanol and DAS-fed rats were not included here because they have been reported elsewhere. Microsomal CYP2E1 protein levels induction by ethanol was decreased by PIC ingestion. Similarly, PIC reduced the increase microsomal reduced form of nicotinamide-adenine dinucleotide (NADPH)-dependent lipid peroxidation and p-nitrophenol hydroxylase (PNPH) activity, induced by ethanol feeding. The lipid peroxidation was reduced to below control levels; however, the pathology score was partially but not significantly reduced by isothiocyanate feeding. CYP2E1 messenger RNA (mRNA) was decreased by both inhibitors of CYP2E1. Immunohistochemical staining of liver for CYP2E1 protein showed that the lobular distribution of the isozyme changed from the centrilobular to a diffuse pattern, with an increase in the periportal region when the CYP2E1 inhibitors were fed with ethanol, and that this change correlated with the change in the distribution of fat in the lobule. The data support the idea that there is a link between CYP2E1 induction by ethanol and the early phase of ethanol-induced liver injury in this rat model. This link may involve lipid peroxidation, but other factors related to CYP2E1 induction must also be involved.

Rats were fed ethanol in combination with fish oil or a corn diet in order to evaluate the effect of fish oil feeding on liver injury, microsomal ethanol oxidation, and NADPH-dependent lipid peroxidation. The rats were maintained on the dietary regimen for 72 days, and for comparison, pair-fed controls were studied. The liver pathology score progressively worsened in rats fed alcohol, both in combination with fish oil and corn oil, but the severity of inflammation and focal fibrosis was greater in the ethanol fish oil fed rats as compared with the ethanol corn oil group, whereas the fatty change was greater in the ethanol corn oil fed rats. The alcohol treatment caused a 2-fold increase of the liver microsomal P450 content, and about a similar increase in the rate of microsomal NADPH oxidation. The amount of ethanol-inducible CYP2E1 was about 10-fold higher in alcohol-fed rats as compared with pair-fed controls. The NADPH-dependent lipid peroxidation in liver microsomes was about 10-fold higher in microsomes from alcohol-treated rats fed corn oil as compared with controls, but only 2- to 3-fold higher in alcohol-fed rats receiving fish oil than in pair-fed controls. This was due to a higher rate of NADPH-dependent lipid peroxidation in the control rats receiving fish oil. There was a pronounced correlation between the amount of CYP2E1 and the microsomal NADPH peroxidation in variously treated rats, and between the 2E1 levels and the pathology score. The data suggest that fish oil diet, like corn oil, supports ethanol-induced liver injury which is related to CYP2E1 induction and the presence of polyunsaturated fatty acids in the diet (i.e., either n-6 or n-3).

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The aim was to characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates. Thirteen human CYP probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were used in activity measurements. The metabolism of the probe substrates was compared in liver microsomes from male and female NMRI, CBA, C57bl/6, 129/SvJ and CD1 strains. The expression of proteins identified on Western blots with commonly available antibodies selective for specific human and rat CYP enzymes were compared in the different mouse strains. Males had higher metabolism than corresponding females for phenacetin O-deethylation (human marker for CYP1A2 activity), and a high correlation was found between phenacetin activity and immunoreactivity in Western blots produced with rat CYP1A2 antibodies. Protein detected by antibodies cross-reacting with human CYP2B6 and rat CYP2B1/2 antibodies was female specific except for the 129/SvJ strain, where it was absent in both genders. Females generally had a higher metabolism of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation (human markers for CYP2D activity). Bufuralol 1'-hydroxylation correlated with a female-dominant mouse CYP, which was detected with antibodies against rat CYP2D4. p-Nitrophenol 2-hydroxylation correlated better than chlorzoxazone 6-hydroxylation with the protein detected with antibodies against rat CYP2E1, indicating that p-nitrophenol is a more specific substrate for mouse CYP2E1.

1. Reconstituted membrane vesicles containing purified preparations of cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase effectively destroyed 2-deoxy-D-ribose in an NADPH-dependent process. 2. The destruction was mediated by hydroxyl radicals formed in an iron-catalysed Haber-Weiss reaction between superoxide anions and hydrogen peroxide liberated from the haemoprotein. 3. Administration of ethanol or benzene to rabbits, compounds known to be oxygenated by the hydroxyl radical-dependent mechanism, resulted in induction of a species of cytochrome P-450 effective in the radical-dependent metabolism of both chemicals. 4. Benzene treatment of rabbits also resulted in an enhanced hydroxyl radical-dependent metabolism of ethanol and benzene in liver microsomes. 5. It is suggested that, for certain substrates, hydroxyl radical-mediated cytochrome P-450-dependent oxygenation reactions are of importance for the microsomal metabolism of these compounds. 6. It is speculated that radical-producing species of cytochrome P-450 may contribute to hydroxyl radical-mediated cell damage.

The ethanol inducible isoform of cytochrome P450, CYP2E1, may play a role in ethanol-induced liver injury. Therefore, the factors which govern CYP2E1 degradation and turnover were investigated. These factors include cAMP, ubiquitin, proteasomal enzymes and CYP2E1 mRNA. Rats fed ethanol or pair-fed isocaloric dextrose were pair-fed with rats fed ethanol or dextrose treated with cAMP for 2 months. The liver pathology, regenerative activity, fatty acid composition, NFkappaB activation, ubiquitin conjugates and proteasomal enzymes were measured as were the apoprotein levels of CYP2E1, CYP3A, CYP4A and mRNA levels for CYP2E1 and ubiquitin expression. The results showed, that the cAMP treatment ameliorated the increase liver fat storage and changes in the fatty acid composition in the livers of ethanol fed rats. Other histologic features of alcoholic liver disease were not changed. Western blot quantitation showed that the amount of ubiquitin and ubiquitin conjugates were markedly reduced by ethanol treatment. Similarly, ethanol decreased the level of ubiquitin mRNA. cAMP ameliorated the inhibition of the proteasomal enzyme proteolysis caused by ethanol feeding. The ethanol-induced increase in the CYP2E1 protein was partially inhibited by cAMP treatment. cAMP treatment decreased CYP2E1 mRNA levels in both ethanol-fed and pair fed control rats. Likewise NFkappaB activation was not increased by ethanol but cAMP reduced the level of NFkappaB activation. CAMP treatment also reduced CYP4A but not CYP3A. The results support the concept that cAMP treatment partially protects the liver from ethanol-induced fatty liver by reducing CYP2E1 induction through cAMP's effects on CYP2E1 synthesis.