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Biomedical subjects

Yedy Israel

Publications and source records attributed to Yedy Israel.

16 recordsLinked to original sources
PubMed2006

The UChA and UChB rat lines: metabolic and genetic differences influencing ethanol intake.

Ethanol non-drinker (UChA) and drinker (UChB) rat lines derived from an original Wistar colony have been selectively bred at the University of Chile for over 70 generations. Two main differences between these lines are clear. (1) Drinker rats display a markedly faster acute tolerance than non-drinker rats. In F2 UChA x UChB rats (in which all genes are 'shuffled'), a high acute tolerance of the offspring predicts higher drinking than a low acute tolerance. It is further shown that high-drinker animals 'learn' to drink, starting from consumption levels that are one half of the maximum consumptions reached after 1 month of unrestricted access to 10% ethanol and water. It is likely that acquired tolerance is at the basis of the increases in ethanol consumption over time. (2) Non-drinker rats carry a previously unreported allele of aldehyde dehydrogenase-2 (Aldh2) that encodes an enzyme with a low affinity for Nicotinamide-adenine-dinuclectide (NAD+) (Aldh2(2)), while drinker rats present two Aldh2 alleles (Aldh2(1) and Aldh2(3)) with four- to fivefold higher affinities for NAD+. Further, the ALDH2 encoded by Aldh2(1) also shows a 33% higher Vmax than those encoded by Aldh2(2) and Aldh2(3). Maximal voluntary ethanol intakes are the following: UChA Aldh2(2)/Aldh2(2) = 0.3-0.6 g/kg/day; UChB Aldh2(3)/Aldh2(3) = 4.5-5.0 g/kg/day; UChB Aldh2(1)/Aldh2(1) = 7.0-7.5 g/kg/day. In F2 offspring of UChA x UChB, the Aldh2(2)/Aldh2(2) genotype predicts a 40-60% of the alcohol consumption. Studies also show that the low alcohol consumption phenotype of Aldh2(2)/Aldh2(2) animals depends on the existence of a maternally derived low-activity mitochondrial reduced form of nicotinamide-adenine-dinucleotide (NADH)-ubiquinone complex I. The latter does not influence ethanol consumption of animals exhibiting an ALDH2 with a higher affinity for NAD+. An illuminating finding is the existence of an 'acetaldehyde burst' in animals with a low capacity to oxidize acetaldehyde, being fivefold higher in UChA than in UChB animals. We propose that such a burst results from a great generation of acetaldehyde by alcohol dehydrogenase in pre-steady-state conditions that is not met by the high rate of acetaldehyde oxidation in mitochondria. The acetaldehyde burst is seen despite the lack of differences between UChA and UChB rats in acetaldehyde levels or rates of alcohol metabolism in steady state. Inferences are drawn as to how these studies might explain the protection against alcoholism seen in humans that carry the high-activity alcohol dehydrogenase but metabolize ethanol at about normal rates.

Alcohol Drinking↗
PubMed2006

Hereditary hemochromatosis: an opportunity for gene therapy.

Levels of body iron should be tightly controlled to prevent the formation of oxygen radicals, lipoperoxidation, genotoxicity, and the production of cytotoxic cytokines, which result in damage to a number of organs. Enterocytes in the intestinal villae are involved in the apical uptake of iron from the intestinal lumen: iron is further exported from the cells into the circulation. The apical divalent metal transporter-1 (DMT1) transports ferrous iron from the lumen into the cells, while the basolateral transporter ferroportin extrudes iron from the enterocytes into the circulation. Patients with hereditary hemochromatosis display an accelerated transepithelial uptake of iron, which leads to body iron accumulation that results in cirrhosis, hepatocellular carcinoma, pancreatitis, and cardiomyopathy. Hereditary hemochromatosis, a recessive genetic condition, is the most prevalent genetic disease in Caucasians, with a prevalence of one in 300 subjects. The majority of patients with hereditary hemochromatosis display mutations in the gene coding for HFE, a protein that normally acts as an inhibitor of transepithelial iron transport. We discuss the different control points in the homeostasis of iron and the different mutations that exist in patients with hereditary hemochromatosis. These control sites may be influenced by gene therapeutic approaches; one general therapy for hemochromatosis of different etiologies is the inhibition of DMT1 synthesis by antisense-generating genes, which has been shown to markedly inhibit apical iron uptake by intestinal epithelial cells. We further discuss the most promising strategies to develop gene vectors and deliver them into enterocytes.

Adenoviridae↗
PubMed2005

Antisense gene delivered by an adenoassociated viral vector inhibits iron uptake in human intestinal cells: potential application in hemochromatosis.

Hereditary hemochromatosis (HH) is a condition in which intestinal iron absorption is greatly elevated. Present treatment is weekly phlebotomy, affecting quality of life and leading to recurrent infections. The iron transporter divalent metal transporter-1 (DMT-1) of enterocytes is responsible for iron uptake from the intestinal lumen; iron is further extruded into the blood by the basolateral transporter ferroportin-1. A therapeutic approach for HH could start with a long-term reduction of iron transport by reduction of DMT-1 levels. We designed an AAV vector coding for a short antisense RNA (AAV-DMT-1-AS) against DMT-1, which reduced iron uptake by 50-60% in human intestinal cells (Caco-2). At low infection levels, DMT-1 mRNA virtually disappeared, suggesting RNAi-like and/or RNase H antisense effects. DMT-1 mRNA levels returned to normal at higher infection levels, indicating that an additional mechanism of mRNA occupation, able to block DMT-1 translation and to avoid feedback regulation by iron responsive elements (IRE), also exists. Cell morphology was normal in all cases and no increases in the interferon-related responses, measured by (a) 2'-5' A oligo synthetase (b) IFITM1 and (c) ISGF3gamma mRNA levels, were observed. Studies presented herein indicate that enterocyte targeting with a gene coding for a short antisense against iron transport blocks enterocyte iron uptake, which may have therapeutic value.

Caco-2 Cells↗
PubMed2005

Complex I regulates mutant mitochondrial aldehyde dehydrogenase activity and voluntary ethanol consumption in rats.

Animals selectively bred for a desirable trait retain wanted genes but exclude genes that may counteract the expression of the former. The possible interactions between selected and excluded genes cannot be readily studied in transgenic or knockout animals but may be addressed by crossing animals bred for opposite traits and studying the F2 offspring. Ninety-seven percent of Wistar-derived rats selectively bred for their voluntary low-alcohol consumption display a mutated nuclear allele of aldehyde dehydrogenase Aldh22 that encodes an enzyme with a low affinity for NAD+, whereas rats bred for high-alcohol consumption do not present the Aldh22 allele. This enzyme is inserted into mitochondria, where NADH-ubiquinone oxidoreductase (complex I) regenerates NAD+. The possible influence of complex I on ALDH2 activity and voluntary ethanol intake was investigated. Homozygous Aldh22/Aldh22 rats derived from a line of high-drinker F0 females (and low-drinker F0 males) showed a markedly higher ethanol consumption (3.9=/-0.5 g x kg(-1) x day(-1)) than homozygous animals derived from a line of low-drinker F0 females (and high-drinker F0 males) (1.8+/-0.4 g x kg(-1) x day(-1)). Mitochondria of F2 rats derived from high alcohol-consuming females were more active in oxidizing substrates that generate NADH for complex I than were mitochondria derived from low alcohol-consuming females, leading in the former to higher rates of acetaldehyde metabolism and to a reduced aversion to ethanol. This is the first demonstration that maternally derived genes can either allow or counteract the phenotypic expression of a mutated gene in the context of alcohol abuse or alcoholism

Acetaldehyde↗
PubMed2005

Aldehyde dehydrogenase (ALDH2) activity in hepatoma cells is reduced by an adenoviral vector coding for an ALDH2 antisense mRNA.

BACKGROUND: Individuals carrying the Glu487Lys coding mutation in the gene for mitochondrial aldehyde dehydrogenase (ALDH2) have a diminished capacity to metabolize acetaldehyde. This deficiency leads to increases in blood acetaldehyde levels when they consume ethanol, which results in an aversion to alcohol and in marked protection against alcoholism. In the present studies, we aimed to mimic the high-acetaldehyde low-ALDH2 activity phenotype in a rat hepatoma cell line by inhibiting Aldh2 gene expression by an Aldh2 antisense-coding gene carried by an adenoviral vector. METHODS: We designed and produced elevated titers of adenoviral vectors (10 virions/ml) carrying Aldh2 cDNA cloned in the reverse orientation preceded by a CMV promoter and followed by a poly-A termination signal. Rat hepatoma cells were infected with these vectors. RESULTS: Studies showed that 1) the antisense gene is actively transcribed in the cells and high levels of antisense mRNA are attained, 2) the antisense gene reduced ALDH2 activity by 65%, and 3) when incubated with 10 mM ethanol, acetaldehyde accumulation by cells increased 8-fold to levels (80-90 microM) known to be aversive to animals and humans. CONCLUSIONS: Data presented show that antialcohol drugs that inhibit Aldh2 gene expression can be generated endogenously in liver cells infected by an adenoviral vector carrying an antisense-coding gene, thus mimicking the high-acetaldehyde phenotype that exists in humans carrying the Glu487Lys mutation who are protected against alcoholism.

Acetaldehyde↗
PubMed2005

Polymorphisms in the mitochondrial aldehyde dehydrogenase gene (Aldh2) determine peak blood acetaldehyde levels and voluntary ethanol consumption in rats.

Dependence on alcohol, a most widely used drug, has a heritability of 50-60%. Wistar-derived rats selectively bred as low-alcohol consumers for many generations present an allele (Aldh2(2)) of mitochondrial aldehyde dehydrogenase that does not exist in high-alcohol consumers, which mostly carry the Aldh2(1) allele. The enzyme coded by Aldh2(2) has a four- to five-fold lower affinity for NAD than that coded by Aldh2(1). The present study was designed to determine whether these polymorphisms account for differences in voluntary ethanol intake and to investigate the biological mechanisms involved. Low-drinker F0 Aldh2(2)/Aldh2(2) rats were crossed with high-drinker F0 Aldh2(1)/Aldh2(1) rats to obtain an F1 generation, which was intercrossed to obtain an F2 generation that segregates the Aldh2 alleles from other genes that may have been coselected in the breeding for each phenotype. Data show that, with a mixed genetic background, F2 Aldh2(1)/Aldh2(1) rats voluntarily consume 65% more alcohol (P<0.01) than F2 Aldh2(2)/Aldh2(2) rats. A major phenotypic difference was a five-fold higher (P<0.0025) peak blood acetaldehyde level following ethanol administration in the lower drinker F2 Aldh2(2)/Aldh2(2) compared to the higher drinker F2 Aldh2(1)/Aldh2(1) animals, despite the existence of identical steady-state levels of blood acetaldehyde in animals of both genotypes. Polymorphisms in Aldh2 play an important role in: (i) determining peak blood acetaldehyde levels and (ii) modulating voluntary ethanol consumption. We postulate that the markedly higher peak of blood acetaldehyde generated in Aldh2(2)/Aldh2(2)(2) animals is aversive, leading to a reduced alcohol intake in Aldh2(2)/Aldh2(2) versus that in Aldh2(1)/Aldh2(1) animals.

Acetaldehyde↗
PubMed2004

Inhibition of tumor necrosis factor alpha secretion and prevention of liver injury in ethanol-fed rats by antisense oligonucleotides.

Elevated serum tumor necrosis factor alpha (TNF-alpha) levels predict mortality in patients with alcoholic liver disease. Administration of anti-TNF-alpha antibodies, obliteration of Kupffer cells or gut sterilization protect against ethanol-induced hepatocellular injury in animal models. In this study, we evaluated the in vivo efficacy of an antisense phosphorothioate oligodeoxynucleotide (S-ODN) targeted against TNF-alpha mRNA (TJU-2755). Naive rats that were administered TJU-2755 (10 mg/(kg body weight (BW)/day) for 2 days) in the free form were challenged with LPS to induce TNF-alpha secretion. Antisense TJU-2755 treatment reduced serum TNF-alpha levels by 62%. A comparison of the efficacies of mismatched and random S-ODNs with that of TJU-2755 showed that some non-specific inhibition might accompany the sequence-specific effects of TJU-2755. To optimize the targeting of the S-ODN, TJU-2755 was encapsulated in pH-sensitive liposomes for in vivo delivery to macrophages. The efficacy of liposome-encapsulated TJU-2755 was assessed in ethanol-fed animals that were administered LPS to induce liver injury. Liposomal delivery of TJU-2755 allowed a much lower dose (1.9 mg/kg BW/day, for 2 days) of the S-ODN to reduce LPS-induced serum TNF-alpha (by 54%) and liver injury (by 60%) in ethanol-fed rats. These data indicate that liposome-encapsulated S-ODNs targeted against TNF-alpha have therapeutic potential in the treatment of alcoholic liver disease.

Animals↗
PubMed2004

Ethanol increases tumor necrosis factor-alpha receptor-1 (TNF-R1) levels in hepatic, intestinal, and cardiac cells.

Chronic ethanol consumption leads to cell injury in virtually every tissue. Tumor necrosis factor-alpha (TNF-alpha) constitutes a major factor in the development of alcohol-induced liver injury. In alcohol-dependent subjects, elevated levels of plasma TNF-alpha are strongly predictive of mortality. Binding of TNF-alpha to TNF-alpha receptor-1 (TNF-R1) activates death domain pathways, leading to necrosis and apoptosis in most tissues, and it also increases the expression of intercellular adhesion molecules (i.e., ICAM-1), which promote inflammation. We determined whether ethanol exposure leads to increases in cellular TNF-R1. We incubated HepG2 human hepatoma cells and H4-II-E-C3 rat hepatoma cells with 25, 50, and 100 mM ethanol for various intervals of time up to 48 h. Human colonic adenocarcinoma cells (Caco-2 cells) and neonatal rat primary cardiomyocytes were also incubated with different concentrations of ethanol. Levels of TNF-R1 were measured either by a sandwich enzyme-linked immunosorbent assay (ELISA) method or by determining the extracellular transmembrane domain of TNF-R1 by an intact-cell ELISA method. Ethanol exposure for 48 h increased TNF-R1 levels in human hepatoma cells in a dose-dependent manner. Levels increased significantly by 164% at 50 mM and by 240% at 100 mM ethanol. Effects were time dependent and did not reach a plateau at 48 h. Similar increases in TNF-R1 were also observed in rat hepatoma cells (90% at 50 mM and 230% at 100 mM ethanol). Under similar conditions, Caco-2 cells showed a significant 80% increase in TNF-R1 levels at 200 mM ethanol, a concentration found in intestine. Neonatal rat primary cardiomyocytes showed TNF-R1 increases of 36% at 50 mM and 44% at 100 mM ethanol. These results indicate that exposure of different cell types to pharmacologic concentrations of ethanol increases TNF-R1 levels and may augment TNF-alpha-mediated cell injury in different tissues.

Animals↗
PubMed2004

Effects of acute gamma-hexachlorocyclohexane intoxication in relation to the redox regulation of nuclear factor-kappaB, cytokine gene expression, and liver injury in the rat.

gamma-Hexachlorocyclohexane-induced hepatotoxicity is associated with oxidative stress. We tested the hypothesis that gamma-hexachlorocyclohexane triggers the redox activation of nuclear factor-kappaB (NF-kappaB), leading to proinflammatory cytokine expression. Liver NF-kappaB activation (electrophoretic mobility shift assay), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha (IL-1alpha) mRNA expression (reverse transcription-polymerase chain reaction), and their serum levels (enzyme-linked immunosorbent assay) were measured at different times after gamma-hexachlorocyclohexane treatment (50 mg/kg). The relationship between these and hepatic O(2) uptake, glutathione and protein carbonyl levels, and sinusoidal lactate dehydrogenase (LDH) efflux in liver perfusion studies was determined. gamma-Hexachlorocyclohexane increased liver NF-kappaB DNA binding at 14-22 h after treatment, concomitantly with significant glutathione depletion and an increase in the rate of O(2) consumption, the content of protein carbonyls, and the sinusoidal LDH efflux. In these conditions, the expression of TNF-alpha and IL-1alpha is enhanced, with maximal increases in their respective mRNA content and serum levels of the cytokines being elicited at 18 h after gamma-hexachlorocyclohexane treatment. All these changes are suppressed by the administration of alpha-tocopherol (100 mg/kg) or the Kupffer cell inactivator gadolinium chloride (10 mg/kg) prior to gamma-hexachlorocyclohexane. gamma-Hexachlorocyclohexane-induced TNF-alpha levels in serum are suppressed by pretreatment with an antisense oligonucleotide (ASO TJU-2755; daily doses of 10 mg/kg for 2 days) targeting the primary transcript for the cytokine, whereas those of IL-1alpha are not modified. It is concluded that gamma-hexachlorocyclohexane-induced liver oxidative stress triggers the DNA binding activity of NF-kappaB, with the consequent increase in the expression of NF-kappaB-dependent genes for TNF-alpha and for IL-1alpha, factors that may mediate the hepatotoxicity of the insecticide.

Animals↗
PubMed2003

Binding of acetaldehyde to a glutathione metabolite: mass spectrometric characterization of an acetaldehyde-cysteinylglycine conjugate.

BACKGROUND: Ethanol administration decreases hepatic glutathione levels and increases urinary sulfhydryl excretion. Ethanol-induced liver injury is blunted by the administration of glutathione precursors. Acetaldehyde generated in the metabolism of ethanol binds to a number of amino acid residues in proteins and peptides, but it does not react readily with glutathione. Due to the possible role of acetaldehyde in cysteine and glutathione homeostasis, we investigated the reaction of acetaldehyde to cysteinylglycine, the dipeptide generated in vivo in the hydrolysis of glutathione by gamma-glutamyltransferase. METHODS: A conjugate between acetaldehyde and cysteinylglycine was generated under physiologically relevant conditions, both in vitro and in vivo. It was separated by a new reverse-phase high-performance liquid chromatography method and identified by electrospray ionization/ion trap tandem mass spectrometric analysis. RESULTS: The conjugate with a stoichiometry of 1:1 between cysteinylglycine and acetaldehyde is most rapidly generated in vitro and was identified by mass spectroscopy as 2-methyl-thiazolidine-4-carbonyl-glycine. This thiazolidine derivative is stable in vitro and in biological fluids of rats. The conjugate was present in high concentrations in the bile of rats pretreated with ethanol and an inhibitor of aldehyde dehydrogenase. CONCLUSIONS: The sequestering of cysteinylglycine by acetaldehyde occurs rapidly under physiologic conditions. Long-lived sulfur-containing biomolecules that incorporate acetaldehyde might affect cysteine and glutathione homeostasis and may also play a protective role by reducing circulating acetaldehyde levels. The acetaldehyde conjugate or its metabolic products could potentially serve as markers of ethanol consumption.

Acetaldehyde↗
PubMed2002

Targeting Kupffer cells with antisense oligonucleotides.

During proinflammatory reactions such as endotoxemia, ischemia-reperfusion and immune reactions, excessive amounts of cytokines and prostanoids are released resulting in liver injury. In the liver, Kupffer cells are the primary source of cytokines and prostanoids. Obliteration of Kupffer cells prevents experimentally-induced liver damage, suggesting a major role for Kupffer in the pathogenesis of liver diseases. Pretreatment of experimental animals with antibodies directed against cytokines such as tumor necrosis alpha (TNF-alpha) prevented experimentally-induced liver damage. In recent years, antisense oligonucleotides (ASOs) directed against specific mRNAs have been tested as an alternative therapy to control the excessive production of inflammatory peptides. Although ASOs have great potential against gene expression, their design, in vivo delivery and stability, have posed significant challenges. Computer-aided configurational analysis and identification of viable motifs (GGGA) on the pre-mRNA transcripts have, in part, alleviated the problems in designing effective ASOs. However, the major challenge involves the in vivo delivery of an ASO to the target tissue. Additionally, it is critical that once taken up by the cells, the ASO is able to survive the lysosomal barrier and enter the cytoplasm. Anionic liposomes and lactosylated low-density lipoproteins (LDL) have been successively used as adjuvants for delivery of ASOs to Kupffer cells. In particular, pH-sensitive liposomes have shown great promise as delivering vehicles to target Kupffer cells. In summary, although ASOs are emerging as a new class of drugs against Kupffer cell-derived pro-inflammatory molecules, in vivo delivery still remains a challenge. pH-sensitive liposomes and LDL-based delivery systems show significant promise for specifically targeting Kupffer cells.

Animals↗
PubMed2002

The Research Society on Alcoholism.

Until the 1960s, the small amount of federal funding available in the alcohol field in the USA was provided by a division of the Alcohol, Drug and Mental Health Administration. In the late 1960s, the National Council on Alcoholism (NCA) joined the American Medical Society on Alcoholism (AMSA, which accepted only physicians). A group of physicians at NCA-AMSA felt that progress in our field needed the concerted effort of both physicians and nonphysician scientists, creating the Research Society on Alcoholism (RSA) in 1977. The National Institute on Alcohol Abuse and Alcoholism (NIAAA) had been created a few years earlier. From 250 members in 1978, the RSA has grown to 1500 in 2001, with a ratio of about three PhDs (with specialities that range from gene therapy to social policy) to each MD. The society holds yearly meetings-visitors can find more information on the web page (http://www.RSoA.org). The society publishes the journal Alcoholism: Clinical and Experimental Research. RSA committees are active in presenting the most recent findings to the public and to elected representatives, and in making recommendations on areas of research that need funding.

Alcoholism↗
PubMed2002

Increases in tumor necrosis factor-alpha in response to thyroid hormone-induced liver oxidative stress in the rat.

Thyroid hormone-induced calorigenesis contributes to liver oxidative stress and promotes an increased respiratory burst activity in Kupffer cells, which could conceivably increase the expression of redox-sensitive genes, including those coding for cytokines. Our aim was to test the hypothesis that L-3,3',5-triiodothyronine (T3)-induced liver oxidative stress would markedly increase the production of TNF-alpha by Kupffer cells and its release into the circulation. Sprague-Dawley rats receive a single dose of 0.1 mg T3/kg or vehicle (controls) and determinations of liver O2 consumption, serum TNF-alpha, rectal temperature, and serum T3 levels, were carried out at different times after treatment. Hepatic content of total reduced glutathione (GSH) and biliary glutathione disulfide (GSSG) efflux were measured as indices of oxidative stress. In some studies, prior to T3 injection animals were administered either (i) the Kupffer cell inactivator gadolinium chloride (GdCl3), (ii) the antioxidants alpha-tocopherol and N-acetyl-L-cysteine (NAC), or (iii) an antisense oligonucleotide against TNF-alpha (ASO TJU-2755). T3 elicited an 80-fold increase in the serum levels of TNF-alpha at 22h after treatment, which coincided with the onset of thyroid calorigenesis. Pretreatment with GdCl3, alpha-tocopherol, NAC, and ASO TJU-2755 virtually abolished this effect and markedly reduced T3-induced liver GSH depletion and the increases in biliary GSSG efflux. It is concluded that the hyperthyroid state in the rat increases the circulating levels of TNF-alpha by actions exerted at the Kupffer cell level and these are related to the oxidative stress status established in the liver by thyroid calorigenesis.

Acetylcysteine↗
PubMed2002

Proteomics in alcohol research.

The proteome is the complete set of proteins in an organism. It is considerably larger and more complex than the genome--the collection of genes that encodes these proteins. Proteomics deals with the qualitative and quantitative study of the proteome under physiological and pathological conditions (e.g., after exposure to alcohol, which causes major changes in numerous proteins of different cell types). To map large proteomes such as the human proteome, proteins from discrete tissues, cells, cell components, or biological fluids are first separated by high-resolution two-dimensional electrophoresis and multidimensional liquid chromatography. Then, individual proteins are identified by mass spectrometry. The huge amount of data acquired using these techniques is analyzed and assembled by fast computers and bioinformatics tools. Using these methods, as well as other technological advances, alcohol researchers can gain a better understanding of how alcohol globally influences protein structure and function, protein-protein interactions, and protein networks. This knowledge ultimately will assist in the early diagnosis and prognosis of alcoholism and the discovery of new drug targets and medications for treatment.

Alcoholism↗
PubMedDate not supplied

Use of an "acetaldehyde clamp" in the determination of low-KM aldehyde dehydrogenase activity in H4-II-E-C3 rat hepatoma cells.

The high-affinity (K(M)<1 microM) mitochondrial class 2 aldehyde dehydrogenase (ALDH2) metabolizes most of the acetaldehyde generated in the hepatic oxidation of ethanol. H4-II-E-C3 rat hepatoma cells have been found to express ALDH2. We report a method to assess ALDH2 activity in intact hepatoma cells that does not require mitochondrial isolation. To determine only the high-affinity ALDH2 activity it is necessary to keep constant low concentrations of acetaldehyde in the cells to minimize its metabolism by high-K(M) aldehyde dehydrogenases. To maintain both low and constant concentrations of acetaldehyde we used an "acetaldehyde clamp," which keeps acetaldehyde at a concentration of 4.2+/-0.4 microM. The clamp is attained by addition of excess yeast alcohol dehydrogenase, 14C-ethanol, and oxidized form of nicotinamide adenine dinucleotide (NAD(+)) to the hepatoma cell culture medium. The concentration of 14C-acetaldehyde attained follows the equilibrium constant of the alcohol dehydrogenase reaction. Thus, 14C-acetate is generated virtually by the low-K(M) aldehyde dehydrogenase activity. 14C-acetate is separated from the culture medium by an anionic resin and its radioactivity is determined. We showed that (1) acetate production is linear for 120 min, (2) addition of 160 microM cyanamide to the culture medium leads to a 75%-80% reduction of acetate generated, and (3) ALDH2 activity is dependent on cell-to-cell contact and increases after cells reach confluence. The clamp system allows the determination of ALDH2 activity in less than one million H4-II-E-C3 rat hepatoma cells. The specificity and sensitivity of the "acetaldehyde clamp" assay should be of value in evaluation of the effects of new agents that modify Aldh2 gene expression, as well as in the study of ALDH2 regulation in intact cells.

Acetaldehyde↗