Immunohistochemical Determination of Hepatic Cytochrome P-4502E1 in Formalin-Fixed, Paraffin-Embedded Sections

Abstract

Cytochrome P-4502E1 (2E1) is inducible by chronic ethanol consumption that results in enhanced activation of anesthetics and commonly used drugs (such as acetaminophen) to hepatotoxins. Therefore, assessment of hepatic 2E1 is needed in prescribing these drugs for the management of alcoholic patients. Currently, measurement of 2E1 requires either immunohistochemistry on frozen sections or Western blot (WB) analysis of homogenized tissue in excess of that needed for pathology. To obtain a more widely applicable method, we developed a procedure to detect 2E1 by immunohistochemistry in formalin-fixed, paraffin-embedded liver biopsies obtained routinely for diagnosis. Data were collected from rats fed ethanol-containing or control liquid diets for 3 weeks. Immunostaining was performed using anti-human rabbit 2E1 antibody as the primary antibody, and the immunoreaction was detected by the avidin-biotin immunoperoxidase method after treating sections with target unmasking fluid, an antigen retrieval buffer that enhanced the staining of 2E1. In control rats, 2E1 staining was weak and perivenular. After ethanol feeding, it showed a lobular gradient, strongest perivenular and weakest periportal, similar to that seen in frozen sections. The staining intensity was scored as: 0 (no staining) to 3 (strong staining). The zonal staining was scored as follows: 1 = perivenular zonal staining, 2 = midzonal, and 3 = panlobular. With the product of the two scores, a significant difference was found between alcohol-fed and control rats (5.1 +/- 0.3 vs. 0.8 +/- 0.2, p < 0.001). 2E1 assessments by WB were also significantly different for these rat pairs (68.5 +/- 2.1 vs. 7.9 +/- 0.8 arbitrary units/mg protein, p < 0.001), with a parallel increase of immunostaining scores and WB measurement of 2E1 content. This immunohistochemical method was then validated in 14 paraffin-embedded percutaneous human liver biopsy samples. In livers of nonalcoholics, 2E1 staining was seen in the perivenular zone only, whereas in samples of alcoholics, the staining was perivenular to midzonal and sometimes periportal. A significant correlation between the zonal staining scores (rs = 0.67, p < 0.005) or intensity x zonal staining scores (rs = 0.79, p < 0.001) and WB analysis was found. The immunohistochemical assessments of 2E1 expression in formalin-fixed, paraffin-embadded sections from livers of alcoholics was found to correlate with WB analysis, and lobular distribution was consistent with that seen in frozen sections. The proposed method should therefore be useful for the assessment of 2E1 content in paraffin-embedded liver samples, thereby aiding in the management of heavy drinkers.

Increased dietary fat contributes to dysregulation of the LKB1/AMPK pathway and increased damage in a mouse model of early-stage ethanol-mediated steatosis

“…This is in agreement with previous studies that indicated that decreasing dietary fat along with increased carbohydrates resulted in reduced ALT, reduced triglyceride accumulation and reduced CYP2E1 induction [33]. During ALD, CYP2E1 induction occurs primarily in the perivenous region [31; 34]. In this study, tissue examination of the MF+E and HF+E mice revealed less intense staining of CYP2E1 in the MF+E animals when compared to HF+E animals.…”

“…In ALD, CYP2E1 is involved in the detoxification of ethanol [18; 31]. Previous reports have suggested an effect of dietary fat and CYP2E1 expression in rats [30; 32].…”

Early Alcoholic Liver Injury: Formation of Protein Adducts with Acetaldehyde and Lipid Peroxidation Products, and Expression of CYP2E1 AND CYP3A

“…''~'~ It is notable that the first typical histological lesions of alcohol-induced liver injury in man also develop in the centrilobular regi0n.2~ Cytochrome P-450 family of proteins are known to be of critical importance in the oxidative, peroxidative, and reductive metabolism of numerous xenobi~tics.~' Chronic alcohol consumption is known to lead to an increase in the content of ethanol-inducible G'HTE1 in the liver and to enhance its catalytic activity in the microsomal fraction. [16][17][18]31 Induction of CYP2E1 in alcohol-fed animals has been shown to increase the sensitivity of hepatic microsomes to lipid peroxidation. 16 On the other hand, inhibition of G"2E1 activity may lead to clinical improvement in the early phase of alcoholic liver injury."…”