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, 44 (2), 157-69

The Role of Ethanol Metabolism in Development of Alcoholic Steatohepatitis in the Rat

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The Role of Ethanol Metabolism in Development of Alcoholic Steatohepatitis in the Rat

Martin J Ronis et al. Alcohol.

Abstract

The importance of ethanol metabolism in the development of alcoholic liver disease remains controversial. The present study examined the effects of selective inhibition of the cytochrome P450 enzyme CYP2E1 compared with the inhibition of overall ethanol metabolism on the development of alcoholic steatohepatitis. Adult male Sprague-Dawley rats were fed via total enteral nutrition for 45 days with or without 10-12g/kg/d ethanol. Some groups were given 200mg/kg/d of the CYP2E1 inhibitor diallyl sulfide (DAS). Other groups were treated with 164mg/kg/d of the alcohol dehydrogenase (ADH) inhibitor 4-methylpyrazole (4-MP) and dosed at 2-3g/kg/d ethanol to maintain similar average urine ethanol concentrations. Liver pathology scores and levels of apoptosis were elevated by ethanol (P<.05) but did not differ significantly on cotreatment with DAS or 4-MP. However, liver triglycerides were lower when ethanol-fed rats were treated with DAS or 4-MP (P<.05). Serum alanine aminotransferase values were significantly lower in ethanol-fed 4-MP-treated rats indicating reduced necrosis. Hepatic oxidative stress and the endoplasmic reticulum (ER) stress marker tribbles-related protein 3 were increased after ethanol (P<.05); further increased by DAS but partly attenuated by 4-MP. Both DAS and 4-MP reversed ethanol increases in the cytokine, tumor necrosis factor-alpha (TNF-alpha), and the chemokine CXCL-2 (P<.05). However, neither inhibitors prevented ethanol suppression of interleukins IL-4 or IL-12. Moreover, neither inhibitors prevented ethanol increases in tumor growth factor-beta mRNA. Ethanol and DAS additively induced hepatic hyperplasia (P<.05). These data suggest that a significant proportion of hepatic injury after ethanol exposure is independent of alcohol metabolism. Ethanol metabolism by CYP2E1 may be linked in part to triglyceride accumulation, to induction of TNF-alpha, and to chemokine production. Ethanol metabolism by ADH may be linked in part to oxidative and ER stress and necrotic injury.

Figures

Figure 1
Figure 1
Representative urine ethanol concentration (UEC) profiles during TEN infusion of EtOH-containing diets with or without co-treatment with DAS or 4MP.
Figure 2
Figure 2
CYP2E1 activity and expression in liver microsomes of rats fed via TEN diets with or without EtOH and DAS or 4MP co-treatment. A. CYP2E1-dependent p-nitrophenol hydroxylase; B. CYP2E1-dependent carbon-tetrachloride-dependent lipid peroxidation – TBARS/mg microsomal protein/min; C. Representative Western immunoblots of CYP2E1 apoprotein, each lane represents a different animal; D. Immunoquantitation of CYP2E1 Western blots. Data represent mean ± SEM for N = 15 control; N = 18 EtOH; N = 7 DAS; N = 9 EtOH + DAS; N = 8 4MP and N = 15 EtOH + 4MP rats/group. * EtOH effect within group; ** DAS effect within group and # 4MP effect within group P<0.05 by two way ANOVA followed by Neuman-Keuls post-hoc multiple pair-wise comparisons comparing EtOH and DAS groups or EtOH and 4MP groups.
Figure 3
Figure 3
Representative H&E stained liver sections × 10 magnification. A. Control; B. EtOH; C. DAS; D. EtOH + DAS; E. 4MP; F. EtOH + 4MP.
Figure 4
Figure 4
CYP4A1 activity and expression in liver microsomes of rats fed via TEN diets with or without EtOH and DAS or 4MP co-treatment. A. Representative Western blots; B. immunoquantitation of CYP4A1 apoprotein in liver microsomes; and C. microsomal lauric acid ω-hydroxylase activity, comparing control (TEN) rats with EtOH-treated rats (EtOH) with or without DAS or 4MP co-treatment. Data represent mean ± SEM for N = 15 control; N = 18 EtOH; N = 7 DAS; N = 9 EtOH + DAS; N = 8 4MP and N = 15 EtOH + 4MP rats/group. * EtOH effect within group; ** DAS effect within group and # 4MP effect within group P<0.05 by two way ANOVA followed by Neuman-Keuls post-hoc multiple pair-wise comparisons comparing EtOH and DAS groups or EtOH and 4MP groups.
Figure 5
Figure 5
Real-time RT-PCR analysis of the effects of EtOH with or without DAS or 4MP co-treatment on relative expression of cytokine and chemokine mRNAs. A. TNFα; B. IL-4; C. IL-12 and D. CXCL2. Data represent mean ± SEM for N = 15 control; N = 18 EtOH; N = 7 DAS; N = 9 EtOH + DAS; N = 8 4MP and N = 15 EtOH + 4MP rats/group. * EtOH effect within group; ** DAS effect within group and # 4MP effect within group P<0.05 by two way ANOVA followed by Neuman-Keuls post-hoc multiple pair-wise comparisons comparing EtOH and DAS groups or EtOH and 4MP groups.
Figure 6
Figure 6
Real-time RT-PCR analysis of the effects of EtOH with or without DAS or 4MP co-treatment on relative expression of TGFβ mRNA and mRNAs for stellate cell activation markers smooth muscle actin (SMA) and PDGF receptor β (PDGFRβ). Data represent mean + SEM for N = 15 control; N = 18 EtOH; N = 7 DAS; N = 9 EtOH + DAS; N = 8 4MP and N = 15 EtOH + 4MP rats/group. * EtOH effect within group; ** DAS effect within group and # 4MP effect within group P<0.05 by two way ANOVA followed by Neuman-Keuls post-hoc multiple pair-wise comparisons comparing EtOH and DAS groups or EtOH and 4MP groups.
Figure 7
Figure 7
PCNA expression in liver homogenates of rats fed via TEN diets with or without EtOH and DAS or 4MP co-treatment. Data represent mean ± SEM for PCNA protein expression normalized against GAPDH, N = 4 rats/group. * EtOH effect within group; ** DAS effect within group and # 4MP effect within group P<0.05 by two way ANOVA followed by Neuman-Keuls post-hoc multiple pair-wise comparisons comparing EtOH and DAS groups or EtOH and 4MP groups.

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