Acetaldehyde, a highly reactive intermediate of alcohol metabolism, has been shown to form adducts with liver proteins in rats fed alcohol for long periods. In this report, the zonal distribution of liver protein-acetaldehyde adducts that formed in vivo was studied by means of histoimmunostaining. Rats were pair-fed alcohol-containing and alcohol-free AIN'76 liquid diets for 2 or 11 wk before they were killed and subjected to whole body perfusion with paraformaldehyde. Each liver was cut into 60-microns-thick slices. Slices were first treated with 10% hydrogen peroxide to eliminate endogenous peroxidase activity. They were then incubated sequentially with rabbit antihemocyanin-acetaldehyde adduct, goat antirabbit serum IgG and rabbit peroxidase-antiperoxidase complex. The liver slices were stained with diaminobenzidine and counterstained with methylgreen. In the livers of rats fed alcohol for 2 wk, peroxidase activity was evident in the perivenous zone but not the periportal zone. No staining was obtained when the primary antibody had been preabsorbed with immobilized hemocyanin-acetaldehyde adduct or if the liver slices were incubated with the unimmunized rabbit IgG. Slight staining of the perivenous zone was seen in the livers of control rats, presumably because of minimal protein-acetaldehyde adduct formation emanating from endogenous acetaldehyde. When rats were fed alcohol for longer periods (e.g., 11 wk), protein-acetaldehyde adducts were still seen predominantly in the perivenous zone, but the distribution pattern was more diffuse than that observed in the livers of rats fed alcohol for only 2 wk. More liver cells produced protein-acetaldehyde adducts when rats were fed the alcohol-containing diet supplemented with cyanamide.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously reported that a 37-kD liver protein formed an adduct with acetaldehyde in vivo when rats were fed alcohol chronically. To understand the mechanism of the formation of this protein-acetaldehyde adduct, rat hepatocytes in primary culture were treated with ethanol in vitro for several days. When cultured in hormone-enriched and trace metal-enriched Waymouth's medium, alcohol dehydrogenase activities in hepatocytes decreased only about 30% during 6 days of culture. At the end of the specified time, protein extracts of hepatocytes were immunotransblotted with rabbit immunoglobulin G that recognized acetaldehyde adduct as an epitope. The 37-kD protein-acetaldehyde adduct band could be detected within 3 days in cells that had been treated with alcohol at a steady-state concentration as low as 5 mmol/L. Although the maximal intensity was obtained at approximately 10 to 40 mmol/L ethanol, addition of cyanamide (an inhibitor of aldehyde dehydrogenase) further increased the intensity of this protein-acetaldehyde adduct band by more than twofold. A good correlation existed between acetaldehyde concentration in the medium and the intensity of the 37-kD protein-acetaldehyde adduct band. Formation of the 37-kD liver protein-acetaldehyde adduct is thus dependent on acetaldehyde, and the 37-kD protein is apparently unusually susceptible to chemical modification by acetaldehyde.

We recently identified ⌬ 4 -3-ketosteroid-5␤-reductase as the 37 kd liver protein which is highly susceptible to acetaldehyde modification in rats continuously fed alcohol. The 5␤-reductase is a key enzyme involved in bile acid synthesis. We report here that the ability to degrade 7␣-hydroxy-4-cholesten-3-one (HCO) was lower in the liver cytosol of alcohol-fed rats than in control animals, suggesting an inhibition of the 5␤-reductase enzyme activity by acetaldehyde modification. We also showed that HCO exhibited a time-and concentration-dependent cytotoxicity to HepG2 cells. HCO cytotoxicity was noticeable at a concentration of 2.5 g/mL. When 10 g/mL of HCO was added to confluent cell monolayers, 57% and 37% of cells remained viable after 24 and 48 hours of treatment. The decrease in cell viability was accompanied by an increased lactic dehydrogenase activity in the culture medium. DNA extracted from HCO-treated cells showed no evidence of DNA fragmentation when analyzed by agarose gel electrophoresis. Staining with propidium iodide showed no nuclear condensation in cells. Thus, cell death by HCO treatment was caused by necrosis and not by apoptosis. Various agents, including, serum proteins, hormones, bile acids, antioxidants, Ca ϩϩ -chelators, Fe ϩϩ -chelator, CYP450 inhibitor, adenylate cyclase inhibitor, protease inhibitors, and nitric oxide synthase inhibitor, did not protect against HCO cytotoxicity. We speculate that HCO concentrations may be elevated around the pericentral area in the liver after chronic alcohol ingestion, causing local cell necrosis. The release of cellular contents and protein-acetaldehyde adducts (PAAs) may activate nonparenchymal cells and provoke autoimmune reaction. Thus, the formation of the 37 kd-PAA may play an important role in the initiation of alcoholic liver injury. (HEPATOLOGY 1998;27:100-107.)Alcohol is metabolized in the liver mainly by alcohol dehydrogenase (ADH). 1 With prolonged alcohol consumption, the metabolic pathway through cytochrome P450IIE1 becomes important. 2 Both ADH and CYPIIE1 generate acetaldehyde from ethanol.

After alcohol ingestion, ethanol is oxidized to acetalAcetaldehyde, the first product of alcohol metabolism, is highly reactive. Several proteins have been shown to dehyde in the liver mainly by the action of alcohol dehybe covalently modified by acetaldehyde in vivo. We have drogenase 1 and, on prolonged alcohol consumption, by previously reported the detection of a cytosolic 37-kd microsomal cytochrome P450IIE1. 2 Little acetaldehyde protein-acetaldehyde adduct (-AA) in the liver of alcohol-accumulates during alcohol oxidation because this infed rats. The liver extract from an alcohol-fed rat was termediate is rapidly converted to acetate by aldehyde subjected to 2-dimensional (2D) sodium dodecylsulfate dehydrogenase. Nevertheless, acetaldehyde is highly (SDS)-polyacrylamide gel electrophoresis (PAGE), trans-reactive. For many years, investigators have shown ferred to polyvinylidene difluoride (PVDF) membrane, that acetaldehyde can covalently modify proteins in and the 37-kd protein-AA spot was digested with trypsin vitro to form protein-acetaldehyde adducts (-AAs). 3 The and sequenced for amino acids. Degenerate oligonucleoformation of protein-AAs in vivo has been demontides corresponding to a peptide sequence of the proteinstrated more recently. 4-9 Our laboratory was the first AA were used as the probe to screen a lgt11 rat liver to report the detection of a liver protein-AA (apparent complementary DNA (cDNA) library. A clone that extended to a potential ATG start codon was identified. MW, 37,000) by Western immunoblot in rats fed alcohol The open reading frame was 978 nucleotides long, encod-chronically. 4 The 37-kd liver protein-AA can be deing 326 amino acid residues. The sequence matched that tected in rats within 1 week of alcohol feeding (either of rat liver D 4 -3-ketosteroid 5b-reductase. The cloned the high-fat Lieber-DeCarli or the low-fat AIN'76 liquid cDNA was expressed in Escherichia coli using pGEX-KG diet containing alcohol [Bio-Serv, Inc., Frenchtown, as the vector. The expressed protein was found to be of NJ]). 10 The bond between the 37-kd protein and acetalcorrect molecular weight. It reacted with an antibody dehyde was very stable, and its stability was not enthat recognized the unmodified liver 37-kd protein by hanced by incubating with cyanoborohydride as a reWestern blotting. Peptide profiles of tryptic-digested re-ducing agent. 4 Adding pyrazole (an inhibitor of alcohol combinant protein and the purified rat liver 37-kd pro-dehydrogenase) 11,12 to the alcohol-containing diet abro- dehydrogenase) 14 raised plasma acetaldehyde concentrations more than sixfold and greatly increased the intensity of the 37-kd protein-AA band on immunoblots. 13 These experiments therefore provide strong Abbreviations: AA, acetaldehyde adduct; KLH, keyhole limpet hemocyanin; evidence that the 37-kd band detected by immunoblot-SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; 2D, ting is truly a protein adduct of acetaldehyde. On subtwo-dimensional; Ig, immunoglobulin; PVDF, polyvinylid...

Acetaldehyde, a highly reactive intermediate of ethanol metabolism, has been shown to form adducts with liver proteins (e.g., a cytosolic 37 kDa protein and the microsomal cytP450IIE1) in rats fed alcohol chronically. In this study, flow cytometry was utilized to test for the presence of protein-acetaldehyde adducts (-AAs) on the surface of hepatocytes and immunotransblot was used to detect for the 37 kDa protein-AA in cytosol as was previously described. For flow cytometric analysis, rabbit anti-hemocyanin-AA IgG and fluorescein isothiocyanate-conjugated goat anti-rabbit serum IgG were used as the primary and secondary antibodies to label surface protein-AAs on hepatocytes at 0 degrees to 4 degrees C. After labeling and washing, hepatocytes were fixed with paraformaldehyde-cacodylate and analyzed with a flow cytometer. In an experiment wherein hepatocytes isolated from rats pair-fed liquid diets with and without ethanol were treated by adding both the primary and secondary IgGs, some hepatocytes from both alcohol-fed and control rats exhibited positive fluorescence but no significant difference in fluorescence intensity was noted. In another experiment, hepatocytes were isolated from rats pair-fed cyanamide (a selective aldehyde dehydrogenase inhibitor) with and without ethanol. The number of hepatocytes showing positive fluorescence in the presence of both primary and secondary IgGs was significantly higher in rats fed cyanamide plus ethanol than in rats fed cyanamide only. Of note, the 37 kDa protein-AA could be detected by immunotransblot in liver cytosol of alcohol-fed rats but not in the controls of both experiments with and without cyanamide supplementation.(ABSTRACT TRUNCATED AT 250 WORDS)