Effects of chronic ethanol exposure on acetaldehyde and free radical production by astrocytes in culture.

In a previous study, the production of acetaldehyde and free radicals derived from ethanol was characterized in astrocytes in primary culture. In the present study, the effects of chronic exposure on the production of both compounds as well as on the main antioxidant system were compared with those of an acute exposure. This was done to better understand the different ways the brain reacts to these modes of exposure. Under these conditions, both a time-dependent increase in the accumulation of acetaldehyde and a decreased formation of the alpha-hydroxyethyl radical were shown. This was associated with increased activities of catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX) and with decreased glutathione (GSH) content. These effects, which counteract reactive oxygen species (ROS) formation by stimulating the main enzymes of the antioxidant system, were also associated with the reduced amount of radicals derived from ethanol. This could be a beneficial effect, but this was counter-balanced by the increased rate of acetaldehyde accumulation, whose high toxicity is well known. All these effects underline the crucial role played by catalase which, on one hand converts hydrogen peroxide to water and, on the other hand, ethanol to acetaldehyde.     

Electron spin resonance study of free radicals produced from ethanol and acetaldehyde after exposure to a Fenton system or to brain and liver microsomes.

Free radical formation from ethanol and acetaldehyde was studied in the presence of a spin-trap and a NADPH generating system with a chemical model, Fenton's reagent, or by enzymatic oxidation of these solvents by rat liver and brain microsomes. The free radicals were detected by electron spin resonance spectroscopy (E.S.R.), using the spin-trapping agent, alpha-(4-pyridyl l-oxide)-N-tertbutyl-nitrone (POBN). Under such conditions, the hydroxyethyl radical derived from ethanol was obtained after both incubation in liver and brain microsomes as well as after exposure to the Fenton system. Enzymatic inhibition and activation showed that the mixed function oxidase system plays an important role in the generation of such a radical, even in the brain. Under all the experimental conditions acetaldehyde could also generate a free radical deriving directly from the parent molecule and modified by enzymatic activation or inhibition. A second, longer lasting radical was also observed in the presence of acetaldehyde. On the basis of a comparative study to a known process causing lipoperoxidation, its lipidic origin was suggested.     

Acute exposure of cultured neurones to ethanol results in reversible DNA single-strand breaks; whereas chronic exposure causes loss of cell viability

AIMS: Ethanol can create progressive neuropathological and functional alterations of neurones. However, the influence of exposure duration is still debated. It is difficult to specify the level of alcohol consumption leading to alcohol-induced brain damage. Moreover, the mechanism of toxicity is assumed to combine direct and metabolically induced effects, although numerous uncertainties remain. Finally, the genotoxic power of ethanol has not fully been investigated in the brain. In the experiment reported herein, primary cultures of neurones were exposed either chronically or acutely to doses of ethanol within the range of blood alcohol levels in intoxicated humans. The impact on the integrity of neurones was assessed by cytotoxicity tests and DNA alterations by single-cell gel electrophoresis (Comet assay) and flow cytometry. Chronic ethanol exposure, even at a low dose, was more harmful to neurones than acute exposure. Both significant reductions in cell viability and DNA alterations were observed in this condition. On the other hand, DNA repair capacities seemed to be preserved as long as the viability measured by specific tests was not affected. Instead, neurones entered a death cell process compatible with apoptosis.     

Supraphysiological acetaldehyde levels suppress growth in chicken embryos.

Although exposure to ethanol is known to cause growth inhibition in a developing embryo, the contributing effect of acetaldehyde on growth is not as well documented. In this study, we measured acetaldehyde-induced growth suppression in three different chicken strains: Peterson x Hubbard, HY x Hubbard, and W36 Ginther White Leghorn. The chicken embryo provides a useful model for studying fetal alcohol syndrome (FAS) and has been used extensively in our laboratory. The current study was undertaken to determine whether the chicken embryo could serve as a model for studying the effects of acetaldehyde on growth. Acetaldehyde caused a significant reduction in embryonic weights only at the higher acetaldehyde concentrations. Torso-to-head ratios were unchanged at every acetaldehyde dose for all strains, supporting the suggestion that acetaldehyde-induced growth suppression was generalized in all tissues, rather than being exhibited as a selective decrease of neuronal tissue. All strains experienced a significant decrease in viability only at higher acetaldehyde concentrations, but differences in viability were evident among the strains. These results support findings obtained from previous work done on ethanol-induced differences among chicken strains by supporting the suggestion that the strain of chicken is important when studying the effects of teratogens on growth and viability. More importantly, the supraphysiological concentrations of acetaldehyde necessary to induce growth suppression seem to indicate that the chicken embryo may not be a viable model of FAS for studying the direct effects of acetaldehyde on embryonic growth.     

Hypothalamic and serum catecholamines in ethanol and acetaldehyde treated guinea-pigs. Relation to moderate short-term cold exposure

Adult guinea-pigs were treated with ethanol (2.5 g/kg, IP) or acetaldehyde (100 mg/kg, IP) and exposed to moderate cold (+4 degrees C) for 50 minutes. Controls were given 0.9% NaCl solution. The hypothalamic catecholamines norepinephrine (NE) and dopamine (DA) and also norepinephrine and epinephrine (E) in the serum were analyzed by high-performance liquid chromatography with an electrochemical detector. Blood glucose, free fatty acids and glycogen in the liver and skeletal muscle were also measured. Acetaldehyde caused a similar drop in colon temperature as did ethanol, but neither could prevent cold-induced vasoconstriction in the ear lobe. Ethanol significantly reduced the concentration of NE in the hypothalamus compared to the controls. Acetaldehyde had a tendency to lower hypothalamic NE. There was no significant difference between drug-treated groups in NE concentration. Neither ethanol nor acetaldehyde had any effect on hypothalamic DA. In the ethanol group serum E and glucose were significantly elevated compared to the acetaldehyde group. Serum glucose was also higher compared to the controls, and the difference in serum E concentration near the level of significance. No significant differences were found between the groups in serum NE, FFA or skeletal muscle and liver glycogen concentration. The results point to a possible central effect of ethanol during a short-term moderate cold exposure. The effects of acetaldehyde on neuronal tissue remain speculative, but a possible effect on noradrenergic neurons cannot be ruled out. Although the hypothermic effect of acetaldehyde corresponded that of ethanol, further experiments are required to elucidate the role of acetaldehyde in ethanol-induced hypothermia.     

Urinary 8-hydoxydeoxyguanosine (8-OHdG) and plasma malondialdehyde (MDA) levels in Aldh2 knock-out mice under acetaldehyde exposure

To clarify the carcinogenicity of acetaldehyde when associated with ALDH (aldehyde dehydrogenase) 2 polymorphism, Aldh2 knock-out (Aldh2-/-) mice and their wild type (Aldh2+/+) mice were exposed to two different concentrations of acetaldehyde (125 ppm and 500 ppm) for two weeks. Aldh2-/- mice, which have the same genetic background as C57BL/6J (wild mice) except for the Aldh2 gene, were used as models of humans who lack ALDH2 activity. Urinary 8-hydroxydeoxyguanosine (8-OHdG) and plasma malondialdehyde (MDA) levels were measured as indicators of oxidative DNA damage and lipid peroxidation, respectively. At 125 ppm acetaldehyde exposure for 12 d, urinary 8-OHdG levels in Aldh2+/+ mice did not increase. However, urinary 8-OHdG levels in Aldh2-/- mice were slightly increased by the end of the exposure. On the other hand, plasma MDA levels did not increase in either Aldh2-/- orAldh2+/+ mice. At 500 ppm, urinary 8-OHdG levels in both Aldh2-/- and Aldh2+/+ mice significantly increased after 6 and 12 d, but there was no genetic difference. On the other hand, plasma MDA levels in Aldh2+/+ and Aldh2-/- mice did not increase at either 125 ppm or 500 ppm after two weeks of exposure. In conclusion, it is suspected that DNA was damaged by acetaldehyde inhalation, and that susceptibility to acetaldehyde varies according to Aldh2 genotype.     

Dietarily-induced changes in voluntary ethanol consumption and ethanol metabolism in the rat

1. The voluntary ethanol consumption, ethanol elimination rate and blood acetaldehyde level after intraperiotoneal injection of ethanol were studied in rats receiving diets with highly imbalanced proportions of dietary protein, carbohydrate and fat. 2. The rats, which received the low-protein diet containing 0.05 of the total energy as protein, 0.80 as carbohydrate and o.15 as fat, drank only approximately half as much ethanol as did the control group, which received 0.30 of its total food energy from protein, 0.55 from carbohydrate and 0.15 from fat. Ethanol elimination rate in the low-protein group was decreased and the blood acetaldehyde level after ethanol injection was markedly increased. 3. On the high-fat diet, which contained 0.30 of the total energy from protein, 0.05 from carbohydrate and 0.65 from fat, the rats drank significantly more ethanol than did the rats on the control diet; their ethanol elimination rate was decreased but their blood acetaldehyde level was not affected. 4. In conclusion, the strong decrease in voluntary ethanol drinking by the low-protein group may have been caused by the increased acetaldehyde accumulation in the blood, but a particularly low blood acetaldehyde level was not one of the factors inducing excessive ethanol drinking in the high-fat group.     

Microbes and mucosa in the regulation of intracolonic acetaldehyde concentration during ethanol challenge

— Aims: The bacteriocolonic pathway for ethanol oxidationleads to high intracolonic levels of carcinogenic acetaldehyde.The respective roles of colonic mucosal cells and gut florain the regulation of intracolonic acetaldehyde concentrationare not known. Disulfiram inhibits hepatic acetaldehyde oxidationand may have an effect on colonic mucosal cells. On the otherhand, metronidazole treatment leads to overgrowth of acetaldehyde-producingaerobic flora in the large intestine. The aim of this studywas to characterize by means of disulfiram and metronidazolethe contribution of colonic mucosal cells and intracolonic microbesto the regulation of intracolonic acetaldehyde concentrationduring ethanol oxidation in rats. Methods: Forty male Wistarrats were used. Three groups of 10 rats each received metronidazole,disulfiram, or both for 5 days, and a fourth group of 10 ratsserved as controls and did not receive any premedication. Faecalsamples were taken for the ALDH (aldehyde dehydrogenase) determinationbefore the injection of ethanol, after which all rats receivedethanol (1.5 g/kg) 2 h prior to taking samples from blood, liver,colonic mucosa and colonic contents. Results: Disulfiram decreasedsignificantly hepatic and colonic mucosal ALDH activities, andresulted in increased blood and intracolonic acetaldehyde levels.In disulfiram-treated rats, mean intracolonic acetaldehyde levelwas 8-fold higher than that in the blood. Metronidazole inhibitedonly colonic mucosal high K_M ALDH and increased intracolonic,but not blood, acetaldehyde levels. Faecal ALDH activity wasnot detectable in any of the groups. Conclusions: This studydemonstrates that during ethanol challenge, intracolonic acetaldehydelevel is regulated not only by intracolonic microbes, but alsoby colonic mucosal cells.     

Acetaldehyde production and metabolism by human indigenous and probiotic Lactobacillus and Bifidobacterium strains

Many human gastrointestinal facultative anaerobic and aerobic bacteria possess alcohol dehydrogenase (ADH) activity and are therefore capable of oxidizing ethanol to acetaldehyde. We examined whether human gastrointestinal lactobacilli (three strains), bifidobacteria (five strains) and probiotic Lactobacillus GG ATCC 53103 are also able to metabolize ethanol and acetaldehyde in vitro. Acetaldehyde production by bacterial suspensions was determined by gas chromatography after a 1-h incubation with 22 mM ethanol. To determine the acetaldehyde consumption, the suspensions were incubated with 50 microM or 500 microM acetaldehyde as well as with 500 microM acetaldehyde and 22 mM ethanol, i.e. under conditions resembling those in the human colon after alcohol intake. The influence of growth media and bacterial concentration on the ability of lactobacilli to metabolize acetaldehyde and to produce acetate from acetaldehyde were determined. ADH and aldehyde dehydrogenase (ALDH) activities were determined spectrophotometrically. Neither measurable ADH nor ALDH activities were found in aerobically grown Lactobacillus GG ATCC 53103 and Lactobacillus acidophilus ATCC 4356 strains. All the lactobacilli and bifidobacteria strains revealed a very limited capacity to oxidize ethanol to acetaldehyde in vitro. Lactobacillus GG ATCC 53103 had the highest acetaldehyde-metabolizing capacity, which increased significantly with increasing bacterial concentrations. This was associated with a marked production of acetate from acetaldehyde. The type of the growth media had no effect on acetaldehyde consumption. Addition of ethanol to the incubation media diminished the acetaldehyde-metabolizing capacity of all strains. However, in the presence of ethanol, Lactobacillus GG ATCC 53103 still demonstrated the highest capacity for acetaldehyde metabolism of all strains. These data suggest a beneficial impact of Lactobacillus GG ATCC 53103 on high gastrointestinal acetaldehyde levels following alcohol intake. The possible clinical implications of this finding remain to be established in in vitro studies.     

Effects of ethanol on an acetaldehyde drug discrimination with a conditioned taste aversion procedure.

The current study was designed to investigate whether ethanol shares stimulus properties with acetaldehyde with the use of a discriminative taste aversion procedure. Animals were trained to discriminate a dose of acetaldehyde (0.2 or 0.3 g/kg, i.p.) from saline in eight consecutive cycles consisting of a pairing day (PD) and three nonpairing days (NPDs). On PDs, all animals were injected with a particular dose of acetaldehyde 30 min before a 20-min limited access to a saccharin solution [0.1% (wt./vol.)] and then injected immediately with either LiCl (0.15 M, 1.8 mEq) or saline. On the three following NPDs, animals were injected with saline and 30 min later were presented with the same saccharin solution for 20 min. All animals in each acetaldehyde training dose group acquired discriminative stimulus control before generalization tests were conducted. Results of generalization tests (ethanol doses of 0.8, 1.2, 1.6, and 2.0 g/kg) revealed that ethanol substituted for acetaldehyde at doses of 1.2 and 1.6 g/kg for both training doses of acetaldehyde. The findings for this study indicate that acetaldehyde and ethanol may share some stimulus properties.     

酒精对鼠胚胎神经胶质细胞c-fos基因表达的影响

目的 探讨酒精致脑发育异常机制。方法 从孕19d鼠胚胎脑组织分离培养星形、少突胶质细胞体外分别施不同剂量酒精及其代谢产物乙醛,应用免疫细胞化学技术研究二作用不同时间对星形、少突胶质细胞原癌基因c-fos表达影响。结果 酒精对星形、少突胶质细胞c-fos表达与时间和剂量有关。各剂量酒精、乙醛作用lh既可影响两种细胞c-fos基因表达,2h表达至峰值,72h恢复正常呈现特异时相性。结论酒精、乙醛均可致星形、少突胶质细胞c-fos表达增强。c-fos异常表达很可能在酒精所致脑发育异常担当重要作用。     

Metabolism of ethyl alcohol in the human body

More than 90% of ingested ethanol is metabolized in the body to acetaldehyde and acetate. Ethanol is metabolized in the liver via three distinct enzymatic pathways: alcohol dehydrogenase (ADH), the microsomal ethanol oxidizing system (MEOS) and catalase. It is generally accepted that alcohol dehydrogenase is the predominant pathway for hepatic ethanol oxidation. Acetaldehyde is metabolized to acetate by a group of dehydrogenase enzymes called aldehyde dehydrogenase.     

Breath acetaldehyde following ethanol consumption.

Five pairs of volunteers were studied to determine the effect of drinking ethanol on breath acetaldehyde levels. On a given study day, samples of breath were obtained for measurement of acetaldehyde and ethanol from both participants at t = -1 h, t = -0.5 h, and at t = 0 to obtain baseline values. The drinkers were then given ethanol (0.3 g/kg body weight), and the controls given an equal volume of tap water. Breath samples were then taken at 0.5, 1, 1.5, 2 h, and hourly until t = 6 h. The last sample taken was at t = 23.5 h. Acetaldehyde levels in breath were quantified with a fluorigenic high-performance liquid chromatographic assay. Blood ethanol was approximated using a breath analyzer. Acetaldehyde in breath rose 50-fold at the 0.5-h, time point and returned to levels not significantly different from baseline values by 3-4 h. The mean peak blood ethanol values reached 0.055%. The t 1/2 elimination for ethanol was 1.6 h, and that for acetaldehyde was 2.25 h. Elimination of both acetaldehyde and ethanol in breath were initially 0 order. A significant correlation (r = 0.74) was found between baseline breath acetaldehyde levels and peak acetaldehyde levels. We conclude that acetaldehyde resulting from ethanol intake rapidly partitions into breath. The correlation of baseline breath acetaldehyde values with peak values found after an ethanol challenge indicate that measurement of breath acetaldehyde may be useful in the identification of individual differences in ethanol metabolism.     

The effects of aminotriazole and acetaldehyde on an ethanol drug discrimination with a conditioned taste aversion procedure.

The present study was designed to investigate whether acetaldehyde shares stimulus properties with ethanol using the conditioned taste aversion (CTA) baseline of drug discrimination learning. Animals were trained to discriminate ethanol (0.8 g/kg, i.p.) from saline using 11 consecutive cycles consisting of a pairing day and three nonpairing days. On pairing days, all animals were injected with ethanol 30 min prior to a 20-min limited access to a saccharin solution (0.1% w/v) and then immediately injected with either LiCl (0.15 M, 1.8 meq) or distilled water. On the three following nonpairing days, animals were injected with saline and 30 min later presented with the same saccharin solution for 20 min. No injections followed on these nonpairing days. Results showed that animals acquired discriminative stimulus control for ethanol after seven pairings. Pretreatment with the catalase inhibitor did not alter the discriminative control for ethanol. Generalization tests revealed that acetaldehyde substituted for ethanol at a dose of 0.3 g/kg. The results of the present study suggest that catalase inhibition did not reverse or alter the discriminative stimulus effects of ethanol. However, generalization tests showed that acetaldehyde (0.3 g/kg) will substitute for ethanol suggesting that these two drugs share some similar properties.     

Ethanol and acetaldehyde action on central dopamine systems: mechanisms, modulation, and relationship to stress

There has been a great deal of activity in recent years in the study of the direct effects of ethanol on the dopamine reward system originating in the ventral tegmental area (VTA). In addition, recent evidence suggests that acetaldehyde formed from ethanol in the brain or periphery may be a crucial factor in the central effects of ethanol. This critical review examines the actions of ethanol and acetaldehyde on neurons of the VTA and the possible interactions with stress, with a focus on electrophysiological studies in vivo and in vitro. Ethanol has specific effects on dopamine neurons and there is recent evidence that some of the in vivo and in vitro effects of ethanol are mediated by acetaldehyde. Stress has some analogous actions on neuronal activity in the VTA, and the interactions between the effects of stress and alcohol on VTA neurons may be a factor in ethanol-seeking behavior. Taken together, the evidence suggests that stress may contribute to the activating effects of ethanol on dopamine VTA neurons, that at least some actions of ethanol on dopamine VTA neurons are mediated by acetaldehyde, and that the interaction between stress and alcohol could play a role in susceptibility to alcoholism. The link between acetaldehyde and ethanol actions on brain reward pathways may provide a new avenue for the development of agents to reduce alcohol craving.     

Alcohol and liver cancer.

Hepatocellular carcinoma is the eighth most frequent cancer in the world, accounting for approximately 500,000 deaths per year. Unlike many malignancies, hepatocellular carcinoma occurs predominantly within the context of known risk factors, with hepatic cirrhosis being the most common precursor to the development of hepatocellular carcinoma. After ethanol ingestion, the liver represents the major site of metabolism. Ethanol metabolism by alcohol dehydrogenase leads to the generation of acetaldehyde and free radicals that bind rapidly to numerous cellular targets, including components of cell signaling pathways and DNA. In addition to direct DNA damage, acetaldehyde depletes glutathione, an antioxidant involved in detoxification. Chronic ethanol abuse leads to induction of hepatocyte microsomal cytochrome P450 2E1, an enzyme that metabolizes ethanol to acetaldehyde and, in doing so, causes further free radical production and aberrant cell function. Cytochrome P450 2E1-dependent ethanol metabolism is also associated with activation of procarcinogens, changes in cell cycle, nutritional deficiencies, and altered immune system responses. The identification of oxidative stress in mediating many deleterious effects of ethanol in the liver has led to renewed interest in the use of dietary antioxidants as therapeutic agents. Included in this group are S-adenosyl-L-methionine and plant-derived flavanoids.     

Evidence of acetaldehyde-protein adduct formation in rat brain after lifelong consumption of ethanol

Acetaldehyde, the first metabolite of ethanol, has been shown to be capable of binding covalently to liver proteins in vivo, which may be responsible for a variety of toxic effects of ethanol. Acetaldehyde-protein adducts have previously been detected in the liver of patients and experimental animals with alcoholic liver disease. Although a role for acetaldehyde as a possible mediator of ethanol-induced neurotoxicity has also been previously suggested, the formation of protein-acetaldehyde adducts in brain has not been examined. This study was designed to examine the occurrence of acetaldehyde-protein adducts in rat brain after lifelong ethanol exposure. A total of 27 male rats from the alcohol-preferring (AA) and alcohol-avoiding (ANA) lines were used. Four ANA rats and five AA rats were fed 10-12% (v/v) ethanol for 21 months. Both young (n = 10) and old (n = 8) rats receiving water were used as controls. Samples from frontal cortex, cerebellum and liver were processed for immunohistochemical detection of acetaldehyde adducts. In four (two ANA, two AA rats) of the nine ethanol-exposed rats, weak or moderate positive reactions for acetaldehyde adducts could be detected both in the frontal cortex and cerebellum, whereas no such immunostaining was found in the remaining five ethanol-treated rats or in the control rats. The positive reaction was localized to the white matter and some large neurons in layers 4 and 5 of the frontal cortex, and to the molecular layer of the cerebellum. Interestingly, the strongest positive reactions were found among the ANA rats, which are known to display high acetaldehyde levels during ethanol oxidation. We suggest that acetaldehyde may be involved in ethanol-induced neurotoxicity in vivo through formation of adducts with brain proteins and macromolecules.     

Possible role of acetyl-CoA in the inhibition of CoA biosynthesis by ethanol in rats

Ethanol, both administered to rats in vivo and added to cultured hepatocyte incubations, inhibits the conversion of [14C]pantothenate to coenzyme A (CoA). Data suggesting that the inhibition by ethanol involves its oxidation to acetate were obtained with rat hepatocytes maintained in primary culture. Ethanol, acetaldehyde and acetate were approximately equally effective inhibitors of [14C]pantothenate conversion to CoA (46-71%) and had no effect on uptake of [14C]pantothenate by hepatocytes. In the presence of saturating levels of acetate, acetaldehyde had no additional inhibitory effect. Cyanamide and diethyldithiocarbamate decreased the inhibition by acetaldehyde at the same concentration (10 microM), which saturated their ability to inhibit acetaldehyde oxidation. Studies with an isolated pantothenate kinase preparation showed that, of the ethanol metabolites, only acetyl-CoA was an effective inhibitor. Acetate and butyrate, which were both inhibitors of [14C]pantothenate conversion to CoA, increased the acetyl-CoA and decreased the free, unacylated CoA (CoASH) content of the cultured hepatocytes. The data were consistent with a mechanism for the inhibitory effect of ethanol that involves inhibition of pantothenate kinase by acetyl-CoA, but did not exclude a possible role of additional regulatory factors.     

A COMPARATIVE STUDY OF ETHANOL ABSORPTION IN THE CANINE JEJUNUM AFTER PRETREATMENT WITH CYANAMIDE OR PYRAZOLE

This report describes the retardation of ethanol absorptionfrom the intestinal tract and reduction of portal blood flowby high acetaldehyde concentrations in dogs using a jejunalsegment with the vascular supply intact. The cyanamide-pretreatmentgroup (CY), in which an extremely high acetaldehyde concentrationdeveloped, in comparison with the control and pyrazole-pretreated(PY) groups, showed a gradual increase of portal blood ethanol,a 25% reduction in the amount of absorbed ethanol, and an 85%smaller absorption rate constant value (K_a). These facts indicatethat the presence of a high acetaldehyde concentration in theblood results in a reduction of ethanol absorption and retardationof ethanol reaching the systemic circulation. The rapid reductionof portal blood flow and the lower ethanol level in the portalvein observed in the CY group, in comparison with the othertwo groups, also indicate that the reduction of ethanol permeabilitythrough the absorption site to the blood is an important retardingfactor induced by acetaldehyde.     

Protein adduct species in muscle and liver of rats following acute ethanol administration

AIMS: Previous immunohistochemical studies have shown that the post-translational formation of aldehyde-protein adducts may be an important process in the aetiology of alcohol-induced muscle disease. However, other studies have shown that in a variety of tissues, alcohol induces the formation of various other adduct species, including hybrid acetaldehyde-malondialdehyde-protein adducts and adducts with free radicals themselves, e.g. hydroxyethyl radical (HER)-protein adducts. Furthermore, acetaldehyde-protein adducts may be formed in reducing or non-reducing environments resulting in distinct molecular entities, each with unique features of stability and immunogenicity. Some in vitro studies have also suggested that unreduced adducts may be converted to reduced adducts in situ. Our objective was to test the hypothesis that in muscle a variety of different adduct species are formed after acute alcohol exposure and that unreduced adducts predominate. METHODS: Rabbit polyclonal antibodies were raised against unreduced and reduced aldehydes and the HER-protein adducts. These were used to assay different adduct species in soleus (type I fibre-predominant) and plantaris (type II fibre-predominant) muscles and liver in four groups of rats administered acutely with either [A] saline (control); [B] cyanamide (an aldehyde dehydrogenase inhibitor); [C] ethanol; [D] cyanamide+ethanol. RESULTS: Amounts of unreduced acetaldehyde and malondialdehyde adducts were increased in both muscles of alcohol-dosed rats. However there was no increase in the amounts of reduced acetaldehyde adducts, as detected by both the rabbit polyclonal antibody and the RT1.1 mouse monoclonal antibody. Furthermore, there was no detectable increase in malondialdehyde-acetaldehyde and HER-protein adducts. Similar results were obtained in the liver. CONCLUSIONS: Adducts formed in skeletal muscle and liver of rats exposed acutely to ethanol are mainly unreduced acetaldehyde and malondialdehyde species.