The formation and stability of imidazolidinone adducts from acetaldehyde and model peptides. A kinetic study with implications for protein modification in alcohol abuse.

The kinetics of the reaction of acetaldehyde (AcH) with the alpha-amino group of several di- and tripeptides to form 2-methylimidazolidin-4-one adducts were determined at pH 7, 4, 37 degrees C, using reverse phase HPLC to separate peptides from adducts. The imidazolidin-4-one structure of the adducts was confirmed by 13C NMR spectroscopy. The reaction of val-gly-gly with AcH was shown to follow second-order kinetics over a wide range of concentrations of both reactants, with k2 = 0.734 +/- 0.032 M(-1) min(-1). Under conditions similar to those in the liver of an alcoholic during chronic ethanol oxidation ([Ach]o = 50-910 microm; [free peptide alpha-amino groups]o = 1.5 mM), the reaction proceeded until effectively all of the AcH had been consumed. The side chain of the N-terminal amino acid was shown not to have a marked effect on the rate of imidazolidinone formation. The decomposition of the imidazolidinone adduct of val-gly-gly and AcH was observed at 60-100 degrees C. Extrapolation of an Arrhenius plot to 37 degrees C provided an estimate of K(obs) of 0.002 h-1 (t1/2 approximately 14 days). Based on these kinetic studies, it is concluded that imidazolidinone adducts of AcH with proteins may be present in the liver and, possibly, in the blood of alcoholics.

FOS and JUN as markers for ethanol-sensitive pathways in the rat brain.

The expression of proteins coded by the immediate early genes of the fos family and c-jun was used to study the effect of acute ethanol administration on convulsant-induced neuronal activity in rat brain. Immunoreactivity for both types of protein was induced by either SC injection of pentylenetetrazole or by IP injection of N-methyl-D-aspartic acid. Both agents elicited distinct patterns of behaviour and a high level of FOS-immunoreactivity in the cerebral cortex and hippocampus. Acute IP doses of ethanol (1.0-3.0 g/kg) significantly reduced the behaviours and FOS-immunoreactivity induced in the cerebral cortex by both pentylenetetrazole and N-methyl-D-aspartic acid. Pentylenetetrazole-induced FOS-immunoreactivity in the hippocampus was also inhibited by ethanol. In contrast, N-methyl-D-aspartic acid-induced FOS-immunoreactivity in the hippocampus was not inhibited by any dose of ethanol. c-JUN immunoreactivity showed a distinct pattern of induction in the hippocampus after injection of N-methyl-D-aspartic acid. Ethanol (3.0 g/kg) inhibited N-methyl-D-aspartic acid-induced c-JUN-immunoreactivity in the hippocampus and cerebral cortex. The differences in inhibition of immunoreactivity suggest that the sensitivity of the NMDA- and GABAA-related neuronal pathways to ethanol varies among different anatomical structures.

Antibodies made against a formaldehyde-protein adduct cross react with an acetaldehyde-protein adduct. Implications for the origin of antibodies in human serum which recognize acetaldehyde-protein adducts.

Acetaldehyde, the major metabolite of ethanol, reacts with lysine and other free amino groups on proteins to form acetaldehyde-protein adducts. The presence of antibodies which recognize such acetaldehyde-protein adducts in sera from alcoholics has been attributed to an immune response to such adducts. Complicating this conclusion is the finding that sera from non-alcoholic control subjects also contain antibodies which recognize acetaldehyde-protein adducts. In the current research we sought to determine whether antibodies which recognize epitopes formed by the reaction of a protein with acetaldehyde can be formed in response to a protein modified with a structurally related protein adduct. We modified lysine residues on apolipoprotein (apo) B-100 with acetaldehyde and formaldehyde under reducing conditions, to form epsilon-N-methyl- and epsilon-N-ethyl-lysine residues, and with acetic anhydride to form epsilon-N-acetyl-lysine residues, and made antibodies against these modified proteins in guinea-pigs. In ELISA assays antibodies made against methylated apoB-100 (Me-apoB) cross-reacted effectively with ethylated apoB-100 (Et-apoB), while antibodies made against acetic anhydride-modified apoB-100 did not cross-react. We conclude that methyl-lysine shares one or more immunoreactive epitopes with ethyl-lysine, and that antibodies which recognize acetaldehyde-modified proteins can be formed in response to formaldehyde-modified proteins. We demonstrate that sera from both alcoholics and non-drinkers contain antibodies which recognize Me-apoB and Et-apoB and that the titres of these antibodies are comparable. These data raise the possibility that some human serum antibodies which recognize acetaldehyde-modified protein epitopes may have been made against formaldehyde-modified protein epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-acetaldehyde adduct antibodies generated by ethanol-fed rats react with reduced and unreduced acetaldehyde-modified proteins.

We have previously shown that rats fed ethanol for prolonged periods generate antibodies reactive with proteins modified by acetaldehyde in vitro. In this report we demonstrate that these antibodies react with two groups of adducts: those formed when acetaldehyde reacts with proteins at 37 degrees C for 24 hr ('unreduced' adducts) and those formed by a 1 hr incubation followed by the addition of sodium cyanoborohydride (a reducing agent specific for Schiff bases) to the reaction mixture ('reduced' adducts). These data suggest that adducts from both of these groups are formed in vivo as a result of ethanol ingestion by rats.

Ethanol and protein kinase C in rat brain.

The effect of chronic ethanol consumption on the catalytic activity of protein kinase C isolated from rat brain was studied in two different ways. Enzyme activity was first measured by phosphorylation of Histone IIIS in vitro. There was no change in the activity of the cytosolic enzyme. Membrane-associated enzyme activity was reduced in the ethanol-treated animal. This difference was not evident if the enzyme was stimulated by arachidonate. The reduction in enzyme activity was confirmed by analysis of the phosphorylation of endogenous substrates in intact synaptosomes. When the binding of the ligand [3H]phorbol dibutyrate was measured by quantitative autoradiography, increased binding to membrane-associated protein kinase C was observed in the CA1 region of the hippocampus but not in other brain regions. These results indicate that ethanol treatment results in a general reduction in membrane-associated protein kinase C activity as measured in vitro but the effect may not be consistent in all brain regions. The differential effect in the CA1 region of the hippocampus may be a reflection of a disruption in the normal regulation of protein kinase C activity in this area and may indicate that this region is a sensitive target for the action of ethanol.

Increased sensitivity of the hippocampus in ethanol-dependent rats to toxic effect of N-methyl-D-aspartic acid in vivo.

Chronic administration of ethanol in animals leads to CNS tolerance and physical dependence. Subsequent withdrawal of ethanol causes hyperexcitability which is thought to be related to increased sensitivity of N-methyl-D-aspartic acid (NMDA) receptors. The purpose of this study was to investigate sensitivity to NMDA in ethanol-treated animals by detecting damage after intrahippocampal injection of NMDA. Choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) specific activity was used as markers of cholinergic and gamma-aminobutyric acid neurons, respectively. Ethanol-dependent animals were more liable to die following intrahippocampal injection of either 120 or 240 nmol of NMDA. There was a significantly greater decrease in hippocampal GAD but not ChAT specific activity in the surviving animals. These data support the hypothesis that ethanol dependence is associated with increased sensitivity to NMDA which may be responsible for excitotoxic brain damage and death.

Ethanol inhibits NMDA-receptor mediated regulation of immediate early gene expression.

The expression of c-fos mRNA in rat brain was induced by intraperitoneal (ip) administration of N-methyl-D-aspartate (NMDA) and kainic acid, agonists of different classes of glutamate receptors and by caffeine, an antagonist of adenosine receptors. The actions of NMDA but not kainic acid or caffeine were blocked by ethanol. Chronic exposure to ethanol vapour did not increase c-fos expression. However, ethanol-withdrawn rats showed a marked increase in c-fos expression. In situ hybridisation and immunohistochemistry indicated the expression was widely distributed in the brain but particularly in the cortex, hippocampus and the cerebellum. There was no marked change in the sensitivity of the NMDA receptor to inhibition by ethanol after chronic ethanol treatment. The withdrawal-induced expression of c-fos mRNA could be inhibited by prior administration of MK801. These results emphasise the NMDA receptor as an important site of ethanol action and the potential use of immediate early gene expression in neuropharmacology in vivo.

Chronic ethanol administration sensitizes hippocampal neurons to neurotoxicity of N-methyl-D-aspartic acid.

The hyperexcitability observed following ethanol withdrawal in dependent animals is thought to be related to increased sensitivity of glutamate receptors. This may predispose to 'excitotoxic' neural damage. The aim of this study was to test this hypothesis by intrahippocampal injection of N-methyl-D-aspartic acid (NMDA) in ethanol-dependent rats and to quantify brain damage using enzymic neuronal markers, viz. choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD). Specific activity of GAD but not ChAT was found to be significantly decreased in hippocampi of ethanol-dependent animals following injection of NMDA, suggesting that chronic ethanol administration sensitizes GABAergic neurons to the toxic effects of excitatory amino acid transmitters.

Involvement of gamma-aminobutyric acid and N-methyl-D-aspartate receptors in the inhibitory effect of ethanol on pentylenetetrazole-induced c-fos expression in rat brain.

The expression of c-fos mRNA in rat brain was induced by intraperitoneal administration of pentylenetetrazole (PTZ) and picrotoxin, which act on the picrotoxin binding site of the gamma-aminobutyric acid-benzodiazepine (GABA-BZ) receptor complex, by N-methyl-D-aspartate (NMDA) and kainic acid, agonists of different classes of glutamate receptors and by caffeine, an antagonist of adenosine receptors. The actions of PTZ and picrotoxin but not that of NMDA were blocked by ethanol and the NMDA-receptor antagonist, MK-801. Ro 15-4513 partially reversed the inhibitory effect of ethanol on PTZ-induced c-fos mRNA synthesis. Acute ethanol administration blocked the actions of PTZ and NMDA without affecting the response to kainic acid or caffeine. Taken together, these data suggest that ethanol blocks c-fos gene activation by noncompetitive antagonists of the GABA-BZ receptor via actions on both the NMDA and GABA receptors.

Effects of ethanol and its fluorinated analogues on the calcium ATPase of sarcoplasmic reticulum.

The effects of ethanol, monofluoro- and trifluoroethanol on the hydrolysis of ATP and coupled Ca2+ translocation by sarcoplasmic reticulum Ca(2+)-ATPase (EC 3.6.1.38) were measured. All three alcohols had parallel effects on the enzyme activities at concentrations up to 200 mM, suggesting a similar mechanism of action. 19F nuclear magnetic resonance spectroscopy was used to investigate their site of action in the purified enzyme.

Antibodies against acetaldehyde-modified epitopes: an elevated IgA response in alcoholics.

Several recent reports have shown that antibodies reactive with acetaldehyde (AcH)-modified epitopes are present in alcoholics. However, similar antibodies have also been found in patients with non-alcoholic liver disease and control subjects. In each of these studies total immunoglobulin binding to the AcH-modified proteins was measured, with no attempt being made to identify the classes of immunoglobulin involved. In the present study we employed an enzyme-linked immunosorbent assay (ELISA) to assess the classes of immunoglobulin involved in this response, using plasma samples from 97 alcoholics with varying degrees of liver disease, 35 patients with non-alcoholic liver disease and 33 control subjects. All three groups exhibited a large IgM response and a negligible IgG response. However, the alcoholics exhibited a significantly higher IgA response than either of the other groups. This suggests that the measurement of the IgA response to AcH-modified epitopes may be a specific marker of ethanol abuse.

Detection of stable acetaldehyde-modified proteins in the livers of ethanol-fed rats.

Liver cytosolic fractions, obtained from rats pair-fed diets supplemented with either ethanol or an isocaloric amount of sucrose for periods from 3 weeks to 27 months, were tested for the presence of acetaldehyde-modified proteins by immunoblotting, using a partially purified antiserum raised in rabbits against proteins modified by acetaldehyde in vitro. The antiserum reacted with a large number of proteins in cytosolic fractions from ethanol-fed rats but did not react with any proteins in the same fraction from control animals. The duration of the ethanol-containing diet did not appear to influence the number of proteins modified or the total amount of modification within the time range tested. These results indicate that many proteins in rat liver cytosol are targets for modification by acetaldehyde in vivo.

Alcohol and the brain: recent advances in biomedical research.

Considerable progress has been made in our understanding of the cellular mechanisms of action of alcohol on the brain, particularly in relation to tolerance and dependence. Tolerance is associated with changes in the composition of the gamma-aminobutyric acid-benzodiazepine receptor, making it less sensitive to alcohol. An increased density of excitatory glutamate receptors may underlie certain withdrawal manifestations. Other neuroadaptive mechanisms include an increase in presynaptic calcium influx on depolarization and effects on adenylate cyclase. Alcohol also influences the expression of a proto-oncogene which plays an important role in neural development. Further advances in neurobiology offer the prospect of elucidating the mechanisms of tolerance and brain damage.

Acute administration of ethanol suppresses pentylenetetrazole-induced c-fos expression in rat brain.

The effect of acute ethanol administration on pentylenetetrazole-induced c-fos expression in rat brain was studied. Pentylenetetrazole induced the rapid and transient expression of c-fos mRNA in rat brain. Maximal induction at a dose of 30 mg/kg was detected within 30 min and persisted for 60 min. Thereafter c-fos gene expression decreased to control levels by 180 min. No increase in c-fos mRNA was evident at doses of pentylenetetrazole < or = 20 mg/kg, whereas maximal elevation was seen at 30 or 40 mg/kg. This action was inhibited by acute ethanol treatment (blood alcohol level > or = 100 mg/dl). Acute ethanol treatment alone had no effect on c-fos gene expression.

Acetaldehyde-modified proteins and associated antibodies in ethanol-fed rats.

Rats fed ethanol for periods of 3-27 months were found to produce an immune response to acetaldehyde-modified proteins, whereas treatment for a shorter period (3 weeks) did not lead to such a response. The antibodies from the ethanol-fed animals were shown to be reactive with a series of proteins modified by acetaldehyde in vitro. This suggests that reactivity towards the modified proteins is independent of the carrier protein, with antibodies being raised which interact solely with the acetaldehyde-containing adduct and not the protein itself. We have also been able to demonstrate the presence of modified proteins in the liver cytosol of these ethanol-fed rats. However, unlike previous reports which suggested that only one protein was modified, we found that many cytosolic proteins were modified. This suggests that the modification of proteins by acetaldehyde is less specific than was previously thought.

Alcohol abusers exhibit a higher IgA response to acetaldehyde-modified proteins.

In the present study we have used an enzyme-linked immunosorbent assay to measure the immunoreactivity towards acetaldehyde-modified proteins in plasma from alcoholics, patients with non-alcoholic liver disease and control social drinkers. All three groups showed a response to modified proteins when total immunoglobulin binding was measured. However, when the assay was modified such that class-specific immunoglobulin binding was measured, it was found that the alcoholic response had a significantly greater immunoglobulin A component than patients with non-alcoholic liver disease or controls who were social drinkers. Thus measurement of IgA binding to acetaldehyde-modified proteins may be a marker for alcohol abuse.

Interaction of chronic ethanol consumption and aging on brain muscarinic cholinergic receptors.

It has been proposed that ethanol and aging may interact synergistically to impair brain function through effects on central muscarinic receptors. Previous studies have investigated the effect of either chronic ethanol treatment or aging, but not both factors simultaneously, on brain muscarinic receptors. We have studied brain muscarinic receptors in animals treated with ethanol for up to 25 months. Ethanol consumption for 3 and 9 months resulted in increased density of quinuclidinyl benzilate (QNB) binding sites in cortex, coinciding with an increase in high-affinity pirenzepine binding sites and low-affinity carbachol binding sites. Upregulation of QNB binding sites in striatum and hippocampus became obvious after further ethanol treatment (15 and 21 months, respectively). Affinity of QNB binding sites and carbachol binding sites was not altered by ethanol treatment. However, there was an ethanol-related decrease in affinity of low-affinity pirenzepine binding sites in cerebral cortex. Density of QNB binding sites and low-affinity pirenzepine binding sites decreased with age in three brain areas investigated. There were age-related changes in receptor affinity in hippocampus and striatum, but not in cortex. Ethanol-related upregulation of muscarinic receptors was superimposed on age-related loss of receptors. We conclude that acceleration of the aging process associated with ethanol abuse is unlikely to be explained on the basis of alterations in receptor density or affinity.

Antibodies against acetaldehyde-modified epitopes: presence in alcoholic, non-alcoholic liver disease and control subjects.

Several studies have recently shown the presence of antibodies against acetaldehyde-modifications in the sera of alcoholic patients. To assess the specificity of this immune response, plasma immunoreactivity with proteins modified in vitro by acetaldehyde was measured using an enzyme-linked immunosorbent assay in 97 alcoholic patients with varying degrees of alcoholic liver disease, in 35 patients with non-alcoholic liver diseases and in 33 control subjects who were social drinkers. All three groups showed some response against acetaldehyde-modified epitopes. The highest plasma reactivities were found in alcoholics, especially those with steatosis and alcoholic hepatitis. Plasma from patients with non-alcoholic liver disease and control subjects also reacted with the acetaldehyde conjugates, but to a lesser extent than plasma from alcoholics. The reliability of these antibodies as markers of either alcohol abuse or alcoholic liver disease therefore appears to be low. However, further studies using more precisely modified proteins or elucidation of the classes of immunoglobulin involved in the immune response against the modified proteins may give clearer differences between the three groups.

Chronic ethanol administration alters binding of [(35)S]t-butylbicyclophosphorothionate to the GABA-benzodiazepine receptor complex in rat brain.

Chronic treatment of male Wistar rats with ethanol (15% v/v) in drinking fluid for a period of 3 months affected the binding of the chloride channel antagonist, [(35)S]TBPS, to well-washed synaptic membranes and slide-mounted brain slices, the affinity for [(35)S]TBPS in brains of ethanol-treated animals was significantly decreased in comparison to controls while receptor density was increased (P < 0.001). However, other well described treatments, viz. an ethanol-containing liquid diet and chronic inhalation of ethanol failed to demonstrate changes in the binding of [(35)S]TBPS in brain preparations. Our findings suggest that long-term administration of ethanol can induce alterations in the characteristics of the ionophore component of the GABA-benzodiazepine receptor complex. This may have relevance to ethanol-induced neuronal damage.