The dominant alcohol component of alcoholic beverages is usually ethyl alcohol. Additionally, beverages contain other alcohols (such as methanol or 1-propanol) and non-alcoholic congeners (for example, acetone, tannins, and resveratrol) present in different amounts in recorded and unrecorded alcoholic preparations with potential benefit or harm dirrctly or after metabolic transformation. The accompanying substances may exert a coadjuvant or independent role [9,10,11]. The heterogeneous compositions of alcoholic drinks add complexity to studying the effects only attributable to ethanol on human health.

Alcohol enters the oral cavity and descends through the esophagus and the gastrointestinal tract, contacting gut epithelia and microbiomes before reaching cells in all organs and tissues. It exerts an irritant effect on the mucosa, and the resident microorganisms absorb and metabolize the substance with consequences both in situ and systemically [12]. In oral human carcinoma samples, Marttila et al. found an increase in the expression of mutated tumor suppressor P53, associated with augmented acetaldehyde production by the microbiome [13]. Alcohol and tobacco alter the composition of the commensal and pathogenic microbiome resident in the oral cavity and gastrointestinal tract, contributing to cancer [14]. Additionally, ethanol and its metabolites and the imbalance in the microbiome populations may affect gut permeability, permitting the passage of bacterial metabolites and endotoxins to the general circulation and inducing inflammation both locally and in distant organs [15]. As a representative example, Fusobacterium nucleatum, a bacteria resident in the oral cavity, secretes immune modulators, virulence factors, and microRNAs linked to the initiation and progression of the oral cavity [16,17], esophagus [18], or colorectal [19,20] cancer. In biopsies from patients suffering from colorectal cancer, the expanded bacteria population significantly correlated with alcohol consumption [20]. F. nucleatum may also influence tumor growth and metastatic progression in tissues outside the gut, such as the breast tissues. In an in vivo experiment, mice inoculated with lectin Fap-2, expressing F. nucleatum, colonize mammary tumors and promote growth and metastasis by blocking the buildup of T cells. The observed effect disappears with antibiotics [21].

The relationship between heavy episodic alcohol drinking (HED) and cancer development depends on covariates (age, diet, smoking, genetic background, environmental conditions, nutrition state, exercise routine, microbiome, and cell´s microenvironment), and different mechanisms may confound the impact of a single factor [22,23,24,25,26,27]. The difficulties compound due to the apparent restrictions of experiments on humans. Separating the biochemical routes directly compromised by alcohol and its metabolites is necessary for effective prevention and therapy endeavors. Epidemiological and data-mining studies and animal experiments are also valuable for this purpose [27,28,29,30].

One link that associates chronic alcohol consumption with cancer development is the formation of metabolic products that may induce direct cell dysfunction or cell damage. The metabolic transformation of ethanol stresses many metabolic pathways, which, in turn, may lead to metabolic deficiencies with consequent harmful effects. Next, we review ethanol’s metabolic pathways, emphasizing the role of the metabolites in altering cell function, causing cancer.

The ingestion of ethanol activates metabolic pathways that move electrons across metabolites (oxidative metabolism) or incorporate ethyl alcohol into other chemical structures (nonoxidative metabolism). 

The oxidative metabolism of ethanol predominates over nonoxidative conversions. Ethanol oxidates to acetate in a two-step process, sequentially catalyzed by alcohol dehydrogenase (ADH, EC 1.1.1.1) and aldehyde dehydrogenase (ALDH, EC 1.2.1.3). The final product is acetate. The main enzyme responsible for converting ethanol into acetaldehyde is ADH. The reaction uses NAD⁺ as a coenzyme that reduces to NADH, whereas ethanol oxidates to acetaldehyde. ADH is widely present in nature, from bacteria and thermophilic archaea [48] to eucaryote [49]. In mammals, ADH is a family of cytosolic enzymes that shows broad substrate specificity and is present in many tissues [50]. The reaction catalyzed by mammalian ADH requires zinc to stabilize the enzyme’s active center’s essential amino acid residues. Once ingested, gastric ADH oxidates some alcohol and, depending on several factors (sex, enzyme variants, empty stomach, or accompanying medication), the proportion of oxidized alcohol may vary. From the stomach and the small intestine, alcohol is absorbed into the blood and distributed into different tissues. The oxidation of ethanol to produce acetaldehyde mainly occurs in hepatocytes. The reaction consumes the redox coenzyme NAD⁺; therefore, the NADH/NAD⁺ proportion increases.

The fraction of acetaldehyde obtained by other enzymes’ catalytic activity, namely catalase (EC 1.11.1.21) and CYP2E1 (cytochrome P450 2E1, EC 1.14.14.1), represents a small fraction of the total. Catalase is a peroxidase that works in the presence of hydrogen peroxide and ethanol to produce acetaldehyde and water. Although this heme-containing enzyme is present in many tissues, it does not represent the main route for alcohol elimination [51,52]; however, its role in brain tissue is relevant [47]. CYP2E1 belongs to the microsomal ethanol oxidizing system (MEOS) [53]. It also localizes in mitochondria [35]. Where oxygen is present, the enzyme oxidizes ethanol to acetaldehyde and reduces oxygen to water [53]. CYP2E1 has a heme pocket, the polarity of which plays an essential role in its mechanistic adaptation to oxidate alcohol when alcohol concentration increases [54]. The catalytic efficiency of CYP2E1 is very low, showing a higher value for Km compared to ADH. This enzyme also acts on other xenobiotic chemicals, such as anesthetics and halogenated hydrocarbons [55,56]. Additionally, CYP2E1 oxidizes acetaldehyde to acetate with a higher kcat/Km value than that observed in ethanol’s oxidation to acetaldehyde [57]. One consequence of the activation of CYP2E1 by ethanol is the accelerated retinol inactivation, eventually affecting cell proliferation [58].

Two molibdo-flavoproteins, xanthine oxidoreductase (EC 1.17.3.2) and aldehyde oxidase (EC 1.2.3.1) may also oxidize acetaldehyde and propagate reactive oxygen species [59].

Acetaldehyde rapidly converts into acetate, where active ALDH is available. This reaction also reduces NAD⁺ to NADH and increases the index NADH/NAD⁺. Human ALDHs are a family of enzymes with different preferred substrates and subcellular locations [60]. The isozyme ALDH2 is abundant in liver cells, localizes in mitochondria, and is responsible for acetaldehyde oxidation with a Km value in the lower micromolar range [34]. Its contribution to ethanol removal, ALDH2, also participates in other detoxifying pathways, including eliminating endogenous aldehydes and other metabolites [60,61]. The liver feeds the acetate to other tissues, where it converts to acetyl-CoA, which eventually fully oxidates to carbon dioxide through the citric acid cycle.

Acetaldehyde is not only a metabolite of alcohol oxidation but is also present in alcoholic beverages, viands, and tobacco smoke and is a chemical resulting from alcoholic fermentation [62]. Its electrophilic character facilitates reactions with different molecules to produce adducts or secondary products, such as salsolinol, when it condensates with dopamine [63]. Salsolinol may cause DNA damage through reactive oxygen species [64]. The formation of protein adducts by acetaldehyde reacting with amino groups (for example, ε-amino groups of lysine residues) and forming Schiff bases alters many proteins’ structures and functions, including the histones of the nucleosome core [65]. Acetaldehyde also interferes with retinoic acid metabolism and disrupts retinoic acid signaling with consequent alteration in gene expression and cell differentiation processes [66,67]. Acetaldehyde may form DNA adducts that impair proper DNA function if not eliminated or repaired [68,69,70]. In vitro studies with human lung fibroblasts indicated that acetaldehyde induces clastogenic effects with detected DNA breaks at telomeric regions, arresting cells at the G2/M phase and negatively affecting cell viability [71]. The increase in acetate units resulting from ethanol’s active oxidative metabolism, accompanied by food intake restriction, depleted glycogen reserves, and increased NADH concentrations, favors ketone bodies’ synthesis from acetate.

Acetaldehyde depletes glutathione (GSH) and, hence, intracellular redox eucrasia. Different mechanisms may participate: sequestering of GSH pools by non-enzymatic binding [72], reduction in GSH peroxidase (EC 1.11.1.9) activity [73], conjugation with GSH metabolites, such as cysteinyl glycine [74,75], or disruption of the transsulfuration pathway [76]. However, in a murine experimental model of forced GSH depletion obtained by disrupting a glutamate-cysteine ligase modifier (GCLM) gene, Chen et al. [77] showed that low levels of GSH protected liver tissue against the insult of ethanol by adapting the metabolic flux of several compounds to resist alcohol insult. Hence, the availability of GSH needs precise modulation to manage volatile metabolic environments.

Among the agents classified by the International Agency for Research of Cancer (IARC) monographs, ethanol, and acetaldehyde appear in group 1 (carcinogenic to humans) [78].

Several enzymes catalyze ethanol’s nonoxidative incorporation into different molecules, including glucuronic acid, sulfate, phosphatidylcholine, or fatty acids. These reactions quantitatively represent a minority compared to the oxidative pathways but may have pathological significance due to the modification of metabolism homeostasis [37]. Furthermore, the determination of these alcohol metabolites in different tissues may help ascertain alcohol consumption and unveil unreported alcohol drinking [86].

The transphosphatidylation reaction, prevailing over hydrolytic activity, uses phospholipids, preferentially phosphatidylcholine and ethanol, to produce phosphatidyl ethanol (Peth). The phospholipid forms after alcohol exposure [33,36] by the intervention of phospholipase D (PLD, EC 3.1.4.4) [36,39]. Consequently, Peth, an anionic phospholipid, interferes with phosphatidic acid (PA) synthesis, and its buildup in cell membranes induces changes that may affect membrane-bonded events (cell adhesion, cell signaling, cell trafficking, or cell proliferation) [87,88].

The enzymatic esterification of ethanol and fatty acids generates fatty acid ethyl esters (FAEEs) in different tissues after alcohol exposure [46,89]. Two catalysts participate in this synthesis: FAEE synthase (EC 3.1.1.67) conjugates ethanol with free fatty acids, and (-CoA ethanol-O-acyltransferase (AEAT, EC 2.3.1.84) esterifies acyl chains from acyl-CoA to ethanol [40,89,90]. Carboxyl ester lipase (CEL, EC 3.1.1.3), acting on triglycerides and ethanol, is another source of FAEE, following an ethanolysis (instead of hydrolysis) reaction mechanism [41,42]. In tissues where the oxidative capacity is low, the formation of nonoxidative products, including FAEE, can be relevant, as they may amass in mitochondrial membranes and alter their function [91]. The accumulation of FAEE may also change the activity of key enzyme activities. For example, although it may not appear as the only mechanism, it disrupts the work of adenosine monophosphate-activated protein kinase (AMPKα), a sensor and keeper of energy equilibrium [92]. FAEE leads to the deterioration of cell bioenergetics in human pancreatic acinar cells and causes cell damage since the activation of AMPKα with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) attenuated FAEE formation, improved mitochondrial bioenergetics, and alleviated inflammatory responses [93].

Broadly expressed UDP-glucuronosyltransferases (EC 2.4.1.17) catalyze the conjugation of ethanol with glucuronic acid by using 5’-uridine diphosphate (5’-UDP-glucuronic acid) as substrate donor to generate ethyl glucuronide (EtG) as an end product [94]. Sulfotransferases (STs, EC 2.8.2.2) activate the sulfonation of ethanol with 3’-phosphoadenosine-5’-phosphosulfate (PAPS) to produce ethyl sulfate [95]. Some reports established a putative biological role of EtG and other glucuronide conjugates in the activation of Toll-like receptor proteins [38] or the in vitro association of EtG with increased oxidant stress in red blood cells [96]. However, we lack in vivo evidence that both chemicals may have salient biological significance [37]. Because they are water-soluble excretion metabolites, they may serve as biomarkers of alcohol consumption and abstinence vigilance due to their detection after ethanol blood concentrations reach zero value [43,97,98].

Tunneling ethanol through oxidation pathways leads to a depletion of oxidated NAD (NAD⁺) at the expense of increasing its reduced form (NADH). Therefore, the ratio [free NAD^+]/[free NADH] decreases.

Hundreds of metabolic reactions (glycolysis, pyruvate fermentation, oxidation of pyruvate, citric acid cycle reactions, mitochondrial electron transport chain reactions, oxidation of amino acids, and fatty acids oxidations) use NAD⁺or NADH as redox coenzymes. Additionally, NAD⁺ is a substrate in other metabolic reactions, such as the deacetylation of histones, polymerization of nucleotides, post-translational modifications of proteins, synthesis of cADP-ribose, or synthesis of NADP⁺ [45,99,100]. Therefore, NAD⁺ –coupled enzymes influence the coenzyme’s bioavailability and energy metabolism [101]. An imbalance in the redox couple concentrations can have consequences that globally affect cell function. An acute outcome is that gluconeogenesis from lactate, serine, and alanine is inhibited [102], and carbohydrate metabolism is significantly compromised. Additionally, alcohol helps switch the metabolism of acetate units to fatty acids storage deposits, aided by the malic enzyme (EC 1.1.1.40) that oxidizes malate to pyruvate and increases reducing equivalents in the form of NADPH [103]. In addition, the altered homeostasis of nicotinamide adenine dinucleotide coenzymes impacts the utilization of nutrients by tumor cells [100].

Depletion of NAD⁺ affects ADP-ribosylation of different protein substrates and histones’ deacetylation by sirtuins (EC 3.5.1.98; EC 2.4.2.31), with consequent cellular stress. Sirtuins are sensors that link energy metabolism with transcriptional regulation and, depending on the context, may behave as mitochondrial tumor suppressors or tumor-promoting agents, as is the case for sirtuin Sirt 4 [104]. Deficits in sirtuin Sirt1 deacylase activity disrupt lipid metabolism and alter specific transcriptional factors, favoring alcohol liver damage progression [105].

The poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) enzymes also depend on NAD⁺ availability. In experimental models of alcohol-induced steatohepatitis, Mukhopadhyay et al. [106] reported that PARP inhibition restored Sirt1 activity and ameliorated the induced inflammatory and oxidative stress responses. The results associate PARP and Sirt1 actions through the use of NAD⁺ in their reactions.

The free AMP concentration also regulates the enzyme AMPK. This enzyme promotes ATP synthesis by increasing ATP catabolic production and inhibiting ATP anabolic needs. It also regulates several metabolic pathways that maintain a state of metabolic homeostasis and adequate cell function. One consequence of ethanol oxidation is the augmented [free NADH]/[free NAD⁺] proportion and the cell’s energy state. Consequently, free AMP concentrations decay and AMPK activation is blocked. Therefore, the metabolic activities, depending on AMPK’s proper functioning, are affected [44]. Given the essential roles of AMPK in anabolic metabolism, cell growth, proliferation, autophagy, and DNA repair, it plays a central role in carcinogenesis control [107].

NAD⁺ is a chemical beacon regulating many of the enzyme activities responsible for essential metabolic control and disease resilience [108]. Alcohol ingestion may affect cell functioning, as well as other mechanisms, by disabling the delicate balance of [free NAD⁺]/[free NADH] and the energy state of the cell [109].

The term C1 metabolism refers to the circuitry of metabolic pathways, essential for cell proliferation and endurance (nucleotide synthesis, Ser Gly and Met metabolism, methylation reactions, synthesis of polyamines, redox reactions, and transsulfuration pathway), which manage the provision of one-carbon modules (methyl groups) and connects with the control of the cellular redox state [76,112,113,114,115,116]. Folate (pteroil-L-glutamate) metabolism distributes one-carbon components through redox reactions. It also couples with metabolic routes where a carbon atom moves between different substrates through transfer reactions essential for cellular proliferation and epigenetic regulation [112,114,115,116]. Many reports analyze valuable information concerning the role of one-carbon metabolism in cell proliferation, differentiation, and growth [112,113,116,117,118,119,120,121].

Several studies report that the chronic ingestion of alcohol affects folate availability and metabolism, not restored after folate’s additional dietary supply [122]. In humans, many mechanisms possibly contribute, including reduced intestinal absorption [122,123,124], augmented renal excretion [125], and the oxidative disruption of the molecule through the effect of acetaldehyde or free radicals derived from their oxidation [126]. Sustained alcohol consumption impairs an intestinal γ-glutamate carboxypeptidase (GCP, EC 3.4.17.11), which hydrolyzes polyglutamate forms of folate to absorbable forms. It also reduces the action of folate binding proteins, which is also essential for absorption [124,127]. Additionally, ethanol may interfere with metabolic pathways, involving the movement of one-carbon units by affecting essential enzyme activities, such as methionine synthase (MS, EC 2.1.1.13). GSH also influences it since homocysteine is a source for the synthesis of GSH by the transsulfuration route [76,128]. Ethanol-treated micropigs during 14 weeks showed reduced liver methionine synthase activity, lowered S-adenosyl methionine (SAM) and GSH, and elevated plasma concentrations of malondialdehyde (MDA). These findings correlated with detecting acetaldehyde adducts, DNA damage, and liver injury [124]. Methionine deficiency affects the levels of other metabolite pools, including the methyl donor SAM, thus negatively impacting essential methylation reactions [129]. Additionally, 5-methyl-THF accumulates in a trapped pool that cannot convert into other folate forms, as methyleneTHF reductase (EC 1.5.1.20) catalyzes an irreversible reaction. This effect has consequences on different reactions responsible for DNA synthesis and DNA-defect repair [122,130,131]. However, the evidence obtained points to variable impact, depending on the organ exposed. For example, experimental chronic alcohol treatment in rats differentially affects one-carbon metabolism in liver and brain tissue [132].

The relationship of ethyl alcohol with carcinogenesis and tumor progression is complex and manifold. The influence of alcohol on folate metabolism plays a prominent and not yet well-understood role. The altered metabolism may induce malignant transformation, but cell proliferation in tumors requires complete folate metabolism to support the synthetic demands of the transformed cells [26,27,81,133,134,135,136]. Consequently, chemicals designed to target folate metabolism’s crucial steps are essential tools when treating or preventing cancer, whether ethanol or other known etiological factors play their part.

Oxidative stress refers to a state characterized by increased production of highly reactive species. Two reactive species classes [140] are, on the one hand, radicals or short half-life molecules with unpaired electrons in their atoms’ outer orbitals (examples include superoxide, oxygen radical, hydroxyl, alkoxy radical, peroxyl radical, and nitric oxide). On the other hand, non-radical molecules (for example, hydrogen peroxide, singlet oxygen, and nitrosyl cation) can easily give rise to radicals. The balance of the concentration of these molecules is paramount to serving cell function since reactive species’ production does not always associate with cell damage, and essential activities depend on the action of these molecules [141,142,143]. These compounds can be intracellularly generated or may have an exogenous origin [140]. Mitochondrial enzymatic activities carried out by the mitochondrial electron transfer chain, monoamine oxidases, and several dehydrogenases are a primary source of reactive oxygen species (ROS) [144,145,146]. Reactive oxygen and nitrogen species (RONS) derive from nitric oxide synthase and NADPH oxidase enzymatic reactions [147,148].

Antioxidant enzymes depict a paradoxical role and may benefit or derange redox homeostasis [157]. They encompass specialized enzymes, reductive compounds, or chelates. For example, the enzyme SOD converts superoxide anion (O2.-) into hydrogen peroxide and water. Catalase reduces two hydrogen peroxide molecules to water and molecular oxygen. Glutathione peroxidases reduce many substrates, including hydrogen peroxide, by the concomitant oxidation of reduced glutathione to oxidized glutathione (GSSG). Peroxiredoxins (EC 1.11.1.15) oxidize their N-terminal Cys residue to reduce hydrogen peroxide [157,158].

The dysregulation of antioxidant barriers may also account for the harmful effect of acetaldehyde. Researchers found a diminished serum antioxidant activity in rats treated with alcohol (an oral diet containing 15% v/v) for two months [159]. The direct exposure of endothelial cells of human origin to alcohol and acetaldehyde increases RONS production and augments SOD and catalase activities [160]. Many factors play a part in gastric and colon cancer; the generation of ROS caused by the suppression of enzymes with antioxidant capacity contributes significantly [161]. Chronic, but not acute, ethanol exposure induces an oxidative milieu that may facilitate the progression from chronic hepatitis to cirrhosis and hepatocellular carcinoma [162]. Many studies report contradictory results regarding how and to what extent alcohol influences the cells’ antioxidant barrier status [163,164,165,166,167]. Different results may reflect the different experimental approaches (mostly related to the amount and pattern of exposure and the experimental specimen under analysis).

Another aspect worth considering is that ethanol can promote cancer development in different tissues and contribute to cancer cell survival. In malignant colon cells, ethanol promotes cell survival. It favors a more malignant phenotype in an oxidative environment by activating nuclear factor erythroid 2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) recruitment to the nucleus that eventually provokes the activation of antioxidant enzymes and bolsters malignant cell survival [168].

Additionally, the failure of complete oxidation by ethanol metabolizing enzymes may generate acetaldehyde radicals (ethoxy, hydroxymethyl, or acetyl) with potential toxic effects [59,169].

Another origin of metabolic changes is genetic variants of enzymes that participate directly in ethanol elimination, exhibit an antioxidant task, or are key in different metabolic routes that maintain cell function. Gene variants relate to sensitivity to alcohol and its metabolites [170,171,172,173].

When analyzing ethanol’s influence as a risk or causal factor of certain types of cancer, one should consider that some gene polymorphisms linked to alcohol metabolism may contribute to maintaining harmful drinking behavior, which sustains and prolongs damage. The expression of some genes and variants may also be affected by alcohol or its metabolites. Other genes or variants not influenced by alcohol can contribute as primary risk elements independently or in an additive or synergistic manner combined with alcohol drinking. Therefore, the dissection of specific mechanisms accountable for direct or selected damage is challenging; however, establishing relationship patterns may clear these interactions and their relevance in delineating prevention and treatment strategies.

Gene variations of two ethanol metabolizing enzymes, ADH and ALDH, may facilitate acetaldehyde accumulation. Some polymorphisms of these two enzymes are associated with the enhanced or attenuated drinking behavior of imbibers. The distribution of these polymorphisms varies among ethnic and geographical groups, adding complexity to the unveiling of their impact on pathologies, including cancer [174,175,176,177,178,179,180]. Cancer cells of the alcohol-metabolizing organs of the gastrointestinal system show activated ADH and attenuated ALDH activities compared to healthy tissues. This observation relates to different isoenzymes acting in cancer cells [177]. Furthermore, evaluating enzyme polymorphisms as a risk for the development and progression of malignancies needs attention within the context of other factors (nutrition, smoking habit, sex, age, diabetes, and obesity) that may also contribute.

Human alcohol dehydrogenases (ADHs) can be arranged into five classes. Class I includes ADH1A, ADH1B, and ADH1C isoenzymes [181], which may form homodimers or heterodimers responsible for almost all ethanol oxidation in the liver [50,182]. Single nucleotide polymorphisms (SNPs) in coding and noncoding regions influence the function or the expression of the corresponding protein, affecting its overall performance [50,183]. ADBH1B variations ADH1B*2 (rs1229984, Arg48His), ADH1B*3 (rs2066702, Arg370Cys), and ADH1C (rs698, Ile350Val, and rs1693482, Arg272Gln) manifest higher activity compared to normally functioning alleles, thus accelerating ethanol’s oxidation to acetaldehyde [174,184]. However, this effect may disappear if a robust form of ALDH acts on increasing acetaldehyde [50].

Human ALDHs form a family of nineteen members. Only three of them, ALDH1A1, ALDH1B1, and ALDH2, are relevant for acetaldehyde oxidation to acetate [50,185]. Drinking patterns and alcohol metabolism are affected by the allele variation rs671 (Glu487Lys) of isoenzyme ALDH2, ALDH2*2, observed mainly in Asian populations. The amino acid change (acidic to basic) blocks the enzyme, and, consequently, acetaldehyde accumulates. The variation may protect the carrier from excessive alcohol ingestion to avoid the unpleasant effects caused by acetaldehyde [186,187].

The combination of more effective ADH variants and deficient ALDH activities favors the exposure of cells to acetaldehyde. The epidemiological analysis of specific human communities relates to these gene variants with a higher risk of gastrointestinal tract cancer [188,189,190,191,192]. Additionally, polymorphisms of ethanol-metabolizing enzymes and gastric colonization by bacteria (Helicobacter pylori and others) contribute to augmented acetaldehyde concentrations in the stomach and colon [161].

Cytochrome CYP2E11 belongs to the Cytochrome P450 subfamily of enzymes. The single nucleotide variation (SNV) of the gene CYP2E11, rs2031920, associates with colorectal cancer risk [193]. Polymorphisms rs2031920 and rs6413432 associate with prostaglandin G/H synthase 2 gene (PTGS2) polymorphisms for lung cancer risk in specific human groups [194]. Two variants, rs72559710 (Arg76His) and rs55897648 (Val389Ile) [195] may affect the function of the enzyme, although its relationship with cancer risk is not well-defined and needs more in-depth analysis [55].

Additional enzyme variants may associate with cancer risk. Compared to the most common genotype, the homozygous rs1801133 (Ala222Val) variant of the enzyme methylenetetrahydrofolate reductase (MTHFR) shows reduced activity. Many studies analyzed the link between excessive alcohol drinking and this variant of MTHFR with risk of head and neck, gastrointestinal tract, or lung cancer in different human populations [174,198,214,216,217,218,219,220,221,222]. SOD variants rs4998557 and rs4880 (Ala16Val), Helicobacter pylori infection history, and alcohol drinking correlate with gastric cancer risk [209]. The SOD rs4880 variant is associated with the epidermal growth factor receptor (EGFR) Leu858Arg mutation in patients with non-small-cell lung cancer and primary brain tumors [208].

Lipid metabolism is also affected by alcohol. In liver tissue, fat accumulates, leading to liver steatosis and fibrosis [223]. An enzyme associated with these processes is patatin-like phospholipase domain-containing 3 or adiponutrin (PNPLA3, EC 2.3.1.51), a protein that catalyzes coenzyme A (CoA)-dependent acylation of 1-acyl-sn-glycerol 3-phosphate to produce phosphatidic acid (PA). Additionally, it shows triacylglycerol lipase (EC 3.1.1.3) and CoA-independent acylglycerol transacylase (2.3.1.147) activities [224,225]. Genetic variation in PNPLA gene rs738409 is associated with steatosis and fibrosis in individuals with alcoholic liver disease [69,211,212]. The occurrence of single nucleotide variants rs738409 in the PNPLA gene and rs58542926 in transmembrane 6 superfamily member 2 gene (TM6SF2) associate with the risk of hepatocellular carcinoma, whereas variant rs4607179 of hydroxysteroid 17-beta dehydrogenase gene (HSD17B13) reduces the risk [213]. In a genome-wide association study, variants rs738409 of PNPLA gene and rs4607179 of HSD17B13 gene showed significant association with the appearance of alcohol-associated liver cirrhosis, and rs374702773 of Fas-associated factor family member 2 gene (FASF2) showed a protective association [206].

Additional gene variants of proteins (phosphatases, phospholipases, kinases, receptors, and DNA-repair enzymes) are statistically associated with developing different types of cancer in the context of alcohol drinking [178,210,226,227,228].

Polymorphism studies are relevant to ascertaining cancer risk in certain tissues and specific populations. The analysis of polymorphisms in selected alcoholic cohorts also provides essential information. Nevertheless, we need broader analysis and studies that link these relationships with experimental evidence at the molecular level. The task is not easy but is necessary. Alcohol may represent an independent risk factor that may act in an additive or synergistic manner with the polymorphic background of certain enzymes found in selected human communities. In this regard, genome-wide association studies (GWAS) and gene-environment-wide interaction studies (GWIS) [178] may serve, together with biochemical analyses, to offer a clearer picture.

Humans usually ingest alcohol per os, exerting a direct irritant impact on mucoses. Additionally, resident oral and gut microbiota populations destabilize and locally metabolize the substance to generate acetaldehyde. Local effects of ethanol include changes in the permeability of the intestinal wall and the pass of bacterial endotoxins and metabolites that may induce inflammation. Once ethanol reaches the general circulation, it arrives at cells in different organs, but the liver is the central station of arrival. Cells may metabolize ethanol through oxidative and nonoxidative pathways, producing noncommon molecules that alter the intermediary metabolism, break the proportion of essential coenzymes NAD⁺ and NADH, detour energy production mechanisms, alter substrate fluxes in one-carbon unit pathways, and generate free radicals. These transformations may enhance oncogenic transformation by damaging DNA. To the modifications mentioned above, gene variants affecting a diversity of enzymes and proteins add complexity to the effects of ethanol on human health. The elements contributing to the described framework of alcohol capacities may facilitate cell, tissue, and organ suffering, leading to disease. Poblational studies identify alcohol and its metabolites as necessary contributors to cancer development. This association receives support from basic research using in vivo and in vitro models analyzing the impact of ethanol on specific targets. Unfortunately, we cannot precisely define ethanol as a specific agent leading to carcinogenesis, but indirect evidence is overwhelming as we relate in this review. According to the defining hallmarks of cancer [8], alcohol metabolism may result directly in carcinogenesis or accompanied by other agents and mechanisms due to its capacity to induce inflammation, DNA damage with subsequent oncogenic activation, and protein/enzyme malfunction.

Direct or indirect alcohol metabolism products may react with proteins, lipids, and DNA, affecting their architecture and function with consequences on the activation of cell proliferation or inhibition of tumor-suppressor mechanisms [26,27,62,68,79,83,150,229,230,231]. One of these notable metabolites is acetaldehyde. Additionally, reactive oxygen and nitrogen species, lipid peroxidation products [68], and folate and estrogens’ altered metabolism play a significant role [26].

Acetaldehyde is an electrophile that reacts with nucleophiles to generate covalent adducts with lipids, proteins, and nucleic acids.

Although acetaldehyde rapidly oxidizes to acetate by ALDH, its concentration can reach significant levels in individuals carrying poorly active ALDH2 variants, facilitate the formation of adducts [65,232], and augment cancer risk [200]. Experimentally increased acetaldehyde concentrations obtained in ALDH knockout mice exposed to ethanol elicited an increase in acetaldehyde-DNA adducts [233]. Furthermore, the disruption of DNA repair mechanisms, such as the tumor suppressor OVCA2 [234] or tumor suppressor p53 [235], dramatically influences acetaldehyde tolerance. The aldehyde reacts with amino groups in proteins (ε-amino of lysines and α-amino of the amino acids at the N-terminus). Firstly, unstable, reversible Schiff base forms—later, stable adducts—may derive in intrachain and interchain crosslinked structures [83].

Acetaldehyde also forms DNA adducts by reacting with nucleophilic amino groups. Reaction with guanine’s purine ring produces N2-ethylidene-2´deoxyguanosine, which converts to the reduced, more stable adduct, N2-ethyldeoxyguanosine [65,70,151,236]. Additionally, acetaldehyde can generate intrastrand crosslinked structures between two adjacent guanines and significantly disturb the double helix’s topography [237]. Another DNA adduct, α-methyl-γ-hydroxy-1, N2-propane-2’-deoxyguanosine (detected as an open aldehyde or closed-ring form), is the product of the reaction of acetaldehyde with the guanine ring occurring in a basic environment (e.g., arginine and lysine basic groups on the neighbor histones) [68,238]. This guanosine adduct also appears after the nucleophilic attack of prop-2-enal (a lipid peroxidation product) [239].

High concentrations and chronic consumption of ethanol activate CYP2E11 with consequent local accumulations of ROS [55], causing the lipid peroxidation of biomembranes that yield reactive aldehydes—4-hydroxynonenal, prop-2-enal, 4-oxo-2-nonenal, and malondialdehyde—and their epoxides from oxidized lipids containing polyunsaturated fatty acids (PUFAs) [68,240,241]. These compounds non-enzymatically react with proteins through Michael additions and Schiff base formation and generate lipid and DNA adducts [241,242].

They also form adducts with membrane lipids, such as phosphatidylethanolamine or phosphatidylserine, to produce phospholipids exhibiting modified solubility that influences the membrane’s biophysical properties [81,244]. Additionally, acetaldehyde and malondialdehyde (a lipid peroxidation product) may generate malondialdehyde-acetaldehyde (MAA) protein hybrid adducts [83,245,246].

Many adducted proteins are covalently modified after reacting with aldehydes, and they have numerous activities [65]. Covalent modifications may alter function (often by inactivation), depending on the protein’s targeted amino acid(s). Moreover, these modifications block enzymes involved in ethanol metabolism. In this situation, they serve as a control mechanism to regulate ROS and acetaldehyde accumulation and modify drinking behavior [65,80,247]. Protein adducts may trigger an immune response directed against specifically modified epitopes (adduct epitopes) acting as neoantigens [245,248,249,250]. This phenomenon affects the immune responses that are essential for controlling cell damage and extracellular milieu homeostasis.

DNA modifications lead to secondary reactions carried out by exposed carbonyl or hydroxyl groups, which result in intrastrand and interstrand crosslinks and DNA-protein structures crosslinked that obstruct proper function. DNA lesions, if not repaired, propagate as mutations after DNA replication. Specific mutations in genes that code for tumor-suppressor proteins, transcription factors, or proteins responsible for cell cycle surveillance may lead to cell decline or uncontrolled cell proliferation, cell transformation, and malignancy [229,230,235]. Furthermore, the cell’s difficulties in repairing damage by canonical mechanisms amplify damage [68,243]. Analytical assessment of these adducts represents a valuable tool, combined with other biomarkers’ analyses, to better monitor AUD patients and predict reliable disease risk profiles [65].

As a consequence of DNA damage, mutations of oncogenic routes located at the so-called fragile sites accumulate and enhance their activity. Their mutated products compromise DNA repair and trigger the onset of cell transformation by different pathways. Specific oncogenic routes associate with several cancer types and define mutational signatures [251]. Specifically, among these signatures, tumor suppressor p53 (TP53), cullin 3 (CUL3), and nuclear receptor binding SET domain protein 1 (NSD) contribute to profiles related to alcohol consumption of cancer patients [251]. Examples of population studies observe the activation of oncogenic routes such as Kirsten rat sarcoma viral oncogene homolog gene (KRAS) [252], tumor suppressor genes adenomatous polyposis coli (APC) and TP53 [13,253], cyclin D1 (CCND-1), or matrix metalloprotease 2 (MMP2) [254], in different cancers types influenced, at least in part, by alcohol consumption [255]. In some types of cancer, however, the association is unclear [253].

Epigenetic mechanisms (dependent on DNA methylation, noncoding RNA signaling and chromatin covalent modifications) play a significant role in cancer development by silencing tumor suppressor genes or facilitating unstable genomes [256]. Observed modifications (described previously) induced by ethanol in one-carbon metabolism, antioxidant barriers, NADH concentration, noncoding RNAs regulatory function, and altered autophagy mechanisms may affect DNA regulation histone architecture and compromise gene expression [138,257,258,259,260,261,262,263,264]. Other intrinsic and extrinsic factors may determine these epigenetic shifts where alcohol may synergize with the injury process [265,266,267].