Enhancement of acetaldehyde-protein adduct formation by L-ascorbate

The effect of L-ascorbate on the binding of [14C]acetaldehyde to bovine serum albumin was examined. In the absence of ascorbate, acetaldehyde reacted with albumin to form both unstable (Schiff bases) and stable adducts. Ascorbate (5 mM) caused a time-dependent increase in the formation of total acetaldehyde-albumin adducts, which were comprised mainly of stable adducts. Significant enhancement of adduct formation by ascorbate was observed at acetaldehyde concentrations as low as 5 microM. An ascorbate concentration as low as 0.5 mM was still effective in stimulating stable adduct formation. The electron acceptor, 2,6 dichlorophenolindophenol, prevented the ascorbate-induced increase in albumin-adduct formation. Ascorbate also caused enhanced acetaldehyde adduct formation with other purified proteins, including cytochrome c and histones, as well as the polyamino acid, poly-L-lysine. These results indicate that ascorbate, acting as a reducing agent, can convert unstable acetaldehyde adducts to stable adducts, and can thereby increase and stabilize the binding of acetaldehyde to proteins.     

Differential modification of hemoglobin chains by acetaldehyde

Acetaldehyde-hemoglobin adducts have been suggested as potential markers for alcohol consumption. These adducts were formed in vitro with [14C]acetaldehyde and separated into hemoglobin subunits by cation-exchange chromatography to examine the relative modification of the alpha- and beta-chains. The effect of varying concentrations of acetaldehyde on the relative amounts of polypeptide adducts and on the specific radioactivities of undissociated hemoglobin (Hb) following reaction with hydroxymercurybenzoate (HMB) was also studied. There were linear relationships (P less than 0.05) between increasing levels of [14C]acetaldehyde (0.0, 0.1, 0.2, 0.5 mM) and the radioactivities of the alpha- and one of the two beta-chain adducts (22, 25, 53 dpm/mg Hb and 151, 272, 626 dpm/mg Hb, respectively). Increases in radioactivities of a minor unidentified hemoglobin adduct fraction were also observed. The ratios of specific radioactivities of beta-to alpha-chain (8.8 +/- 1.2 SEM) did not vary with the concentrations of acetaldehyde. Although the amounts of undissociated hemoglobin following reaction with HMB did not increase with increasing concentrations of acetaldehyde, the significant increase of specific radioactivities of this fraction (152, 1967, and 6562 dpm/mg Hb for 0.1, 0.2, and 0.5 mM acetaldehyde, respectively) suggested possible crosslinks within the tetramer or dimer. The amino acid analysis of alpha- and beta-subunit adducts formed with 0.1 and 0.5 mM acetaldehyde showed that unreacted cysteine residues were more often detected at the higher acetaldehyde concentration consistent with the formation of cysteine adducts labile to acid hydrolysis or the shielding of cysteine residues in acetaldehyde-modified Hb against the subunit separation by HMB treatment. Thus acetaldehyde reacts differentially with the alpha- and beta-hemoglobin subunits and with the undissociated hemoglobin molecule.     

Reaction of acetaldehyde with hemoglobin

Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.     

Acetaldehyde adducts with proteins: Binding of [14C]acetaldehyde to serum albumin

Acetaldehyde, the immediate oxidation product of ethanol metabolism, was assessed for its ability to bind covalently to a purified protein in solution. Bovine serum albumin (BSA)² was used as the model protein incubated in the presence of 0.2 mm [¹⁴C]acetaldehyde at pH 7.4 and at 37 °C. Acetaldehyde formed both stable and unstable adducts with serum albumin. Unstable adducts were identified following stabilization with the reducing agent sodium borohydride. We examined both types of binding using trichloroacetic acid precipitation, gel filtration, and dialysis as means to separate bound from free acetaldehyde. All three methods of analysis gave comparable results except that the number of stable acetaldehyde adducts with albumin were significantly lower following separation by dialysis. The effects of l-cysteine, l-lysine, and reduced glutathione were assessed for their abilities as competitive reagents to decrease binding of [¹⁴C]acetaldehyde to BSA. Addition of cysteine caused a rather dramatic concentration-dependent reduction in [¹⁴C]acetaldehyde binding to BSA when compared to that caused by lysine which displayed a relatively mild effect on covalent binding. The presence of glutathione caused a concentration-dependent decrease in protein-bound radioactivity that was stronger than that by lysine but not as effective as cysteine. The ability of each reagent to reverse the formation of preformed acetaldehyde adducts with BSA was also examined. Only l-cysteine effectively decreased the number of unstable acetaldehyde adducts with BSA while stable binding of acetaldehyde remained essentially unaffected by any of the three reagents. These results indicate that acetaldehyde can covalently bind to protein and form unstable as well as stable adducts.     

Covalent binding of acetaldehyde to tubulin: evidence for preferential binding to the alpha-chain

The covalent binding of [14C]acetaldehyde to purified beef brain tubulin was characterized. As we have found for several other proteins, tubulin bound acetaldehyde to form both stable and unstable adducts. Unstable adducts (Schiff bases) were stabilized, and rendered detectable, by treating incubated reaction mixtures with the reducing agent sodium borohydride. In short-term incubations, the majority of the adducts formed were unstable, but the percentage of total adducts that were stable gradually increased with time. Stable adduct formation was greatly increased by the inclusion of sodium cyanoborohydride in reaction mixtures (reductive ethylation). When reaction mixtures were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the alpha- and beta-chains of the heterodimeric tubulin molecule, the alpha-chain of free tubulin, but not intact microtubules, was the preferential site of stable adduct formation under both reductive and nonreductive conditions. Denaturation studies showed that the native tubulin conformation was necessary for the alpha-chain to show enhanced reactivity toward acetaldehyde. Competition binding studies showed that alpha-tubulin could effectively compete with beta-tubulin and bovine serum albumin for a limited amount of acetaldehyde. Unstable acetaldehyde adducts with free tubulin or microtubules did not exhibit alpha-chain selectivity. Analysis of reaction mixtures indicates that lysine residues are the major group of the protein participating in adduct formation. These data indicate that the alpha-chain of free tubulin is the preferential site of stable acetaldehyde-tubulin adduct formation. Further, these data raise the possibility that alpha-tubulin may be a selective target for acetaldehyde adduct formation in cellular systems.     

Acetaldehyde binding increases the catabolism of rat serum low-density lipoproteins

Acetaldehyde was found to form adducts with rat serum lipoproteins. The binding of [14C]acetaldehyde to lipoproteins was studied at low concentrations which are known to exist during ethanol oxidation. The amount of lipoprotein adducts was a linear function of acetaldehyde concentration up to 250 microM. Incubation of rat plasma low-density lipoproteins (LDL) with 200 microM acetaldehyde increased the disappearance rate of the 3H-label from the cholesterol ester moiety of LDL injected into normal rats. The data show that even low concentrations of acetaldehyde are capable of affecting LDL metabolism. These findings may provide an explanation for the low concentrations of serum LDL in alcoholics.     

Increased covalent binding of acetaldehyde to calmodulin in the presence of calcium

The regulatory protein, calmodulin, undergoes major conformational changes in response to changes in intracellular calcium concentration. Furthermore, calmodulin has been reported to have lysine residues which markedly increase their reactivity toward electrophilic substances in the calcium-loaded state. We found that calmodulin formed two to three times more stable adducts with acetaldehyde in the calcium-loaded state as compared to the calcium-free state. Competition-binding studies showed that calmodulin could preferentially compete with albumin for acetaldehyde in the presence, but not in the absence, of calcium. When calmodulin was in the calcium-loaded state, trifluoperazine, an inhibitor of calmodulin activity, significantly decreased the stable binding of acetaldehyde to the protein, whereas in the calcium-free state, minimal effects on binding were observed. Since calmodulin is involved in regulation of multiple important processes in the cell, it is possible that acetaldehyde-calmodulin adducts could contribute to liver injury by perturbation of calcium-dependent homeostatic mechanisms within the hepatocyte.     

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n = 24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n = 16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.     

Reactions of malonaldehyde and acetaldehyde with calf thymus DNA: formation of conjugate adducts

Our previous work has shown that treatment of nucleosides with malonaldehyde simultaneously with acetaldehyde affords stable conjugate adducts. In the present study we demonstrate that conjugate adducts are also formed in calf thymus DNA when incubated with the aldehydes. The adducts were identified in the DNA hydrolysates by their positive ion electrospray MS/MS spectra, by coelution with the 2'-deoxynucleoside standards, and, in the case of adducts exhibiting fluorescent properties, also by LC using a fluorescence detector. In the hydrolysates of double-stranded DNA (ds DNA), two deoxyguanosine and two deoxyadenosine conjugate adducts were detected and in single-stranded DNA (ss DNA) also, the deoxycytidine conjugate adduct was observed. The guanine base was the major target for the malonaldehyde-acetaldehyde conjugates and 2'-deoxyguanosine adducts were produced in ds DNA at levels of 100-500 adducts/10(5) nucleotides (0.7-3 nmol/mg DNA).     

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n?=?24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n?=?16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.     

The effect of acetaldehyde concentrations on the relative rates of formation of acetaldehyde-modified hemoglobins

The effect of various concentrations of acetaldehyde (0, 0.05, 0.1, 0.25, 0.5, 1.0, and 5.0 mM) on the relative rates of formation of hemoglobin acetaldehyde adducts detected in fractions eluted from cation exchange high-pressure liquid chromatography (HPLC) was investigated. When the hemoglobin and acetaldehyde mixtures were incubated at 37 degrees C for various time intervals up to 24 hr, increased amounts of HbA1c could be observed after 2 hr incubation with 1 mM or greater concentrations of acetaldehyde, or after 4 hr incubation with at least 0.5 mM acetaldehyde. An increase in the HbA1a + b fraction was not observed with 4 hr incubation time until the acetaldehyde level reached 1 mM. The HPLC method detected no difference in minor hemoglobins from alcoholic and normal subjects. Incubation of red blood cells at 37 degrees C for 1 hr with six consecutive pulses of 0.05 mM [14C]acetaldehyde showed no differences in the amounts of minor hemoglobins determined chromatographically at various pulse intervals. However, the measure of the 14C-label incorporation into hemoglobin showed that adducts eluting in the HbA1a+b fraction were formed at a faster rate than those eluting in the HbA1c or HbA0 fraction, respectively. The specific activities of the HbA1a+b fractions at 2, 4, and 6 pulses were 34, 128, and 949 cpm/mg hemoglobin; those of the HbA1c fraction were 15, 58, and 174 cpm/mg hemoglobin. This evidence of modification of hemoglobin by physiological levels of acetaldehyde from 14C-label incorporation suggests that an assay more sensitive than chromatographic separation of adducts might be clinically useful in detecting alcoholism or monitoring alcohol detoxification programs.     

Various enzymatic and nonenzymatic pathways of acetaldehyde transformation

In blood acetaldehyde is bound to hemoglobin and serum albumin and interacts with low-molecular-weight components--cysteine, glutathione. The association constants for human serum albumin and human hemoglobin are about 30 M-1 and 410 M-1, respectively. The SH-groups of hemoglobin play an essential role in the interaction of protein with acetaldehyde. The reversible complexes of acetaldehyde with proteins can transform into stable adducts after long incubation with albumin, some other blood proteins but not with immunoglobulins. Acetaldehyde participates as a substrate in the reaction of acetoin formation in homogenates of different tissues. The rate of acetoin formation is about 15 +/- 3 mmol/min per 1 gram of the tissue and essentially increases in the presence of pyruvate.     

Aldehyde-modified proteins as mediators of early inflammation in atherosclerotic disease

Inflammation is widely accepted to play a major role in atherosclerosis and other cardiovascular diseases. However, the exact mechanism(s) by which inflammation exerts its pathogenic effect remains poorly understood. A number of oxidatively modified proteins have been associated with cardiovascular disease. Recently, attention has been given to the oxidative compound of malondialdehyde and acetaldehyde, two reactive aldehydes known to covalently bind and adduct macromolecules. These products have been shown to form stable malondialdehyde–acetaldehyde (MAA) adducts that are reactive and induce immune responses. These adducts have been found in inflamed and diseased cardiovascular tissue of patients. Antibodies to these adducted proteins are measurable in the serum of diseased patients. The isotypes involved in the immune response to MAA (i.e., IgM, IgG, and IgA) are predictive of atherosclerotic disease progression and cardiovascular events such as an acute myocardial infarction or coronary artery bypass grafting. Therefore, it is the purpose of this article to review the past and current knowledge of aldehyde-modified proteins and their role in cardiovascular disease.     

Conformations and selective photodissociation of heterogeneous benzo(a)pyrene diol epoxide enantiomer-DNA adducts

The covalent binding of the tumorigenic (+) enantiomer and the nontumorigenic (-) enantiomer of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,19-tetrahydrobenzo(a)pyrene (BPDE) to double-stranded native DNA gives rise to heterogeneous adducts, especially in the case of (-)-BPDE. The covalent (+)-BPDE-DNA adducts are predominantly of the external site II type, while the (-)-BPDE-DNA adducts are predominantly of the quasi-intercalative, site I type (65%), with 35% of site II adducts. The site I adducts can be selectively photodissociated with near-ultraviolet light (quantum yields in the range 0.0003-0.005); the external site II adducts (photodissociation quantum yield 3 X 10(-5) are 10-100-times more stable. The photolability of covalent (-)-BPDE-DNA adducts accounts for the discrepancies in the linear dichroism properties of these complexes reported previously. Fluorescence quenching data, previously utilized to assess the degree of solvent exposure of the pyrenyl residues in covalent adducts, were in some cases significantly influenced by the presence of highly fluorescent tetraol dissociation products. After correcting for this effect, it is shown that the fluorescence of the external site II (+)-BPDE-DNA adducts is sensitive to acrylamide, while the fluorescence of the dominant site I (-)-BPDE-DNA adducts is not affected by this fluorescence quencher, as expected for adducts with considerable carcinogen-base stacking interactions.     

Distribution of ethanol-induced protein adducts in vivo: relationship to tissue injury

Generation of oxygen free radicals and reactive aldehydes as a result of excessive ethanol consumption has been well established. Recent studies in human alcoholics and in experimental animal models have indicated that acetaldehyde, the first metabolite of ethanol, and the aldehydic products of lipid peroxidation can bind to proteins in tissues forming stable adducts. The demonstration of such adducts in zone 3 hepatocytes in alcoholics with an early phase of histological liver damage indicates that adduct formation may have an important role in the sequence of events leading to alcoholic liver disease. There may be interference with cellular functions, stimulation of fibrogenesis, and immunological responses. Autoantibodies towards distinct types of adducts have been shown to be associated with the severity of liver disease in alcoholic patients. High fat diet and/or iron supplementation combined with ethanol may increase the amount of aldehyde-derived epitopes and promote fibrogenesis in the liver. Recently, ethanol-derived protein modifications have also been found from other tissues exposed to ethanol and acetaldehyde, including rat brain after lifelong ethanol administration, pancreas, and rat muscle. Elevated adduct levels also occur in erythrocytes of alcoholics, which may be related to ethanol-induced morphological aberrations in hematopoiesis.     

Non-oxidative mechanisms are responsible for the induction of mutagenesis by reduction of Cr(VI) with cysteine: role of ternary DNA adducts in Cr(III)-dependent mutagenesis

Intracellular reduction of carcinogenic Cr(VI) generates Cr-DNA adducts formed through the coordination of Cr(III) to DNA phosphates (phosphotriester-type adduct). Here, we examined the role of Cr(III)-DNA adducts in mutagenesis induced by metabolism of Cr(VI) with cysteine. Reduction of Cr(VI) caused a strong oxidation of 2', 7'-dichlorofluoroscin (DCFH) and extensive Cr-DNA binding but no DNA breakage. Cr-DNA adducts induced unwinding of supercoiled plasmids and structural distortions in the DNA helix as detected by decreased ethidium bromide binding. Propagation of Cr-treated pSP189 plasmids in human fibroblasts led to a dose-dependent formation of the supF mutants and inhibition of replication. Blocking of Cr(III)-DNA binding by occupation of DNA phosphates with Mg(2+) or by sequestration of Cr(III) by inorganic phosphate or EDTA eliminated mutagenic responses and restored a normal yield of replicated plasmids. Dissociation of Cr(III) from DNA by a phosphate-based reversal procedure returned mutation frequency to background levels. The mutagenic responses at the different phases of the reduction reaction were unrelated to the amount of reduced Cr(VI) but reflected the number and the spectrum of Cr(III)-DNA adducts that were formed. Ternary cysteine-Cr(III)-DNA adducts were approximately 4-5 times more mutagenic than binary Cr(III)-DNA adducts. Although intermediate reaction products (CrV/IV, thiyl radicals) were capable of oxidizing DCFH, they were insufficiently reactive to damage DNA. Single-base substitutions at G/C pairs were the predominant type of Cr-induced mutations. The majority of mutations occurred at the sites where G had adjacent purine in the 3' or 5' position. Overall, our results present the first evidence that Cr(III)-DNA adducts play the dominant role in the mutagenicity caused by the metabolism of Cr(VI) by a biological reducing agent.     

Oxygen radicals photo-induced by ferric nitrilotriacetate complex

This study examined the photo-induced generation of reactive oxygen species (ROS) by the carcinogenic iron(III)-NTA complex. Iron(III)-NTA complex (1:1) has three conformations (type (a) in acidic conditions of pH 1-6, type (n) in neutral conditions of pH 3-9, and type (b) in basic conditions of pH 7-10) with two pK(a) values (pK(a1) approximately 4, pK(a2) approximately 8). The iron(III)-NTA complex was reduced to iron(II) under cool-white fluorescent light without the presence of any reducing agent, and the reduction rates of the three conformations of iron(III)-NTA were in the order type (a)>type (n)>type (b) as reported previously (Akai K. et al., Free Radic. Res. 38, 951-962, 2004). ROS generation was investigated by electron paramagnetic resonance (EPR) spectroscopy with a spin-trapping technique. Apparent EPR signals attributed to PBN/*(13)CH(3) and PBN/*OCH(3) spin adducts were observed after incubation of the iron(III)-NTA complex was mixed with alpha-phenyl-tert-butylnitrone (PBN) and (13)C-DMSO in an aerobic condition. The addition of catalase effectively attenuated the PBN adducts, but superoxide dismutase enhanced them. Taken together, these results indicate that the iron(III)-NTA complex is spontaneously reduced to the iron(II)-NTA complex by light under acidic to neutral pH, and in turn transfers an electron to molecular oxygen to form ROS.     

Effect of Kupffer cell inactivation on ethanol-induced protein adducts in the liver

Tissue deposition of protein adducts derived from ethanol metabolism and lipid peroxidation, has been suggested to play a role in the initiation of alcoholic liver disease. The mechanisms modulating adduct formation have, however, remained unclear. We used immunohistochemical methods to examine acetaldehyde (AA) and malondialdehyde (MDA) adducts and cytochrome P4502E1 and P4503A2 expression in rats after administration of (i) an ethanol-diet (n = 6), (ii) ethanol-diet plus gadolinium chloride (GdCl(3)), a selective Kupffer cell toxicant (n = 7), or (iii) control diet (n = 6). A 4 week ethanol treatment resulted in liver steatosis, necrosis, and inflammation and deposition of protein adducts with both AA and MDA, which colocalized with areas of fatty change. The intensities (mean +/- SD) of the immunohistochemical reactions for both AA (2.50 +/- 1.23) and MDA (3.00 +/- 1.10) adducts were significantly higher in the ethanol-fed animals than in the controls (0.083 +/- 0.20) (0.16 +/- 0.25) (p <.001). GdCl(3) prevented adduct accumulation, the mean immunohistochemistry scores being 0.86 +/- 1.07 for AA and 1.64 +/- 0.63 for MDA, the former showing a more striking reduction (p <.01). The hepatic cytochrome enzymes were not different in the ethanol-fed groups with or without GdCl(3). The data indicates that Kupffer cells are involved in the generation of protein adducts with both acetaldehyde and ethanol-induced lipid peroxidation products in alcoholic liver disease.     

Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast

Acetaldehyde, a primary metabolite of alcohol, forms DNA adducts and disrupts the DNA replication process, causing genomic instability, a hallmark of cancer. Indeed, chronic alcohol consumption accounts for approximately 3.6% of all cancers worldwide. However, how the adducts are prevented and repaired after acetaldehyde exposure is not well understood. In this report, we used the fission yeast Schizosaccharomyces pombe as a model organism to comprehensively understand the genetic controls of DNA damage avoidance in response to acetaldehyde. We demonstrate that Atd1 functions as a major acetaldehyde detoxification enzyme that prevents accumulation of Rad52-DNA repair foci, while Atd2 and Atd3 have minor roles in acetaldehyde detoxification. We found that acetaldehyde causes DNA damage at the replication fork and activates the cell cycle checkpoint to coordinate cell cycle arrest with DNA repair. Our investigation suggests that acetaldehyde-mediated DNA adducts include interstrand-crosslinks and DNA-protein crosslinks. We also demonstrate that acetaldehyde activates multiple DNA repair pathways. Nucleotide excision repair and homologous recombination, which are both epistatically linked to the Fanconi anemia pathway, have major roles in acetaldehyde tolerance, while base excision repair and translesion synthesis also contribute to the prevention of acetaldehyde-dependent genomic instability. We also show the involvement of Wss1-related metalloproteases, Wss1 and Wss2, in acetaldehyde tolerance. These results indicate that acetaldehyde causes cellular stresses that require cells to coordinate multiple cellular processes in order to prevent genomic instability. Considering that acetaldehyde is a human carcinogen, our genetic studies serve as a guiding investigation into the mechanisms of acetaldehyde-dependent genomic instability and carcinogenesis.