Localization of Protein‐Acetaldehyde Adducts on Cell Surface of Hepatocytes by Flow Cytometry

Abstract

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)

Acute effect of EtOH on Mg2+homeostasis in liver cells: evidence for the activation of an Na+/Mg2+exchanger

“…The formation of acetaldehyde-protein adducts in the cytosol of liver cells and their subsequent migration to the plasma membrane has been observed (31). If acetaldehyde has a role in modulating the activity of the Na ϩ /Mg 2ϩ exchanger, the extrusion of Mg 2ϩ from EtOHtreated livers should be potentiated when CyN is present to inhibit the metabolic conversion of (acet)aldehyde to acetic acid.…”

Evaluation of the Effect of Ethanol’s Toxic Metabolite Acetaldehyde on the Gastrointestinal Oligopeptide Transporter, PEPT1: In Vitro and in Vivo Studies

“…bestatin) (Bhardwaj et al., 2005; Covitz et al., 1996; Irvine et al., 1999; Sun et al., 2002; Tuma et al., 2001). Thus, PEPT1 is a key transporter protein involved in mediating the oral bioavailability of various therapeutic drug classes and has been implicated in drug‐drug interactions (Bailey et al., 2000; Lin et al., 1992). Therefore, PEPT1 presents an excellent model protein for the study of the effects of AcH on transporter protein function in vitro as well as in vivo .…”

Cellular and subcellular localization of acetaldehyde-protein adducts in liver biopsies from alcoholic patients.

“…However, our study, in accordance with other ones (Niemela et al, 1990a), do not confirm this hypothesis, since we found the hepatocyte plasma membrane consistently unstained. It is of note that the experimental conditions used by Lin et al (1992) were very different from those observed in human alcoholics, which could explain discordant results. Our data suggest that Ito and myofibroblast cells, by expressing APAs on their membranes, could trigger the immune reaction.…”

“…It has been suggested that an immune-mediated reaction may be triggered to acetaldehyde-modified proteins bound to hepatocyte membrane, The presence of APAs on the hepatocyte surface has been reported by Lin et al (1992) by using flow cytometry in an experimental model. However, our study, in accordance with other ones (Niemela et al, 1990a), do not confirm this hypothesis, since we found the hepatocyte plasma membrane consistently unstained.…”