Molecular Mechanisms of Acetaldehyde-Mediated Carcinogenesis in Squamous Epithelium

Acetaldehyde is a highly reactive compound that causes various forms of damage to DNA, including DNA adducts, single- and/or double-strand breaks (DSBs), point mutations, sister chromatid exchanges (SCEs), and DNA–DNA cross-links. Among these, DNA adducts such as N2-ethylidene-2′-deoxyguanosine, N2-ethyl-2′-deoxyguanosine, N2-propano-2′-deoxyguanosine, and N2-etheno-2′-deoxyguanosine are central to acetaldehyde-mediated DNA damage because they are associated with the induction of DNA mutations, DNA–DNA cross-links, DSBs, and SCEs. Acetaldehyde is produced endogenously by alcohol metabolism and is catalyzed by aldehyde dehydrogenase 2 (ALDH2). Alcohol consumption increases blood and salivary acetaldehyde levels, especially in individuals with ALDH2 polymorphisms, which are highly associated with the risk of squamous cell carcinomas in the upper aerodigestive tract. Based on extensive epidemiological evidence, the International Agency for Research on Cancer defined acetaldehyde associated with the consumption of alcoholic beverages as a “group 1 carcinogen” (definite carcinogen) for the esophagus and/or head and neck. In this article, we review recent advances from studies of acetaldehyde-mediated carcinogenesis in the squamous epithelium, focusing especially on acetaldehyde-mediated DNA adducts. We also give attention to research on acetaldehyde-mediated DNA repair pathways such as the Fanconi anemia pathway and refer to our studies on the prevention of acetaldehyde-mediated DNA damage.


Acetaldehyde, Acetaldehyde Metabolism, and Risk of Cancers
Acetaldehyde, a low molecular weight organic aldehyde with the formula CH 3 CHO, is a highly reactive compound that causes DNA damage [1,2]. It is found in food and drinks such as yogurt, ripe fruits, cheese, coffee, and alcoholic beverages [3,4], and in tobacco smoke [5]. In addition, acetaldehyde can be produced by microorganisms such as yeasts and bacteria in the human oral cavity [6][7][8]. Thus, acetaldehyde can be ingested orally in a variety of ways. In particular, alcoholic beverages such as Calvados and other spirits contain high quantities of "free" acetaldehyde (e.g., Calvados: 1781 ± 861 µM), and frequent consumption of these beverages is associated with an increased risk of esophageal squamous cell carcinoma (ESCC) [4,9], although "free" acetaldehyde present in alcoholic beverages appears to cause only a short time (1-2 min) direct exposure to the organs [10].
More importantly, acetaldehyde is also generated endogenously by alcohol metabolism. Ingested alcohol is absorbed from the upper gastrointestinal tract and transported to the liver, where it is mainly metabolized into acetaldehyde by alcohol dehydrogenase 1B (ADH1B), and then detoxified to acetic acid by aldehyde dehydrogenase 2 (ALDH2) (Figure 1) [11,12]. Genetic polymorphisms in ADH1B and/or ALDH2 can result in different enzymatic activities that have a major impact on the risk of ESCC as well as head and neck squamous cell carcinoma (HNSCC) [13][14][15][16].
Thus, extensive epidemiological evidence suggests that acetaldehyde is deeply involved in the carcinogenesis of the squamous epithelium of the esophagus, and head and neck. In addition, the International Agency for Research on Cancer has defined acetaldehyde associated with the consumption of alcoholic beverages as a "group 1 carcinogen" (definite carcinogen) for the esophagus and/or head and neck [28]. More importantly, acetaldehyde is also generated endogenously by alcohol metabolism. Ingested alcohol is absorbed from the upper gastrointestinal tract and transported to the liver, where it is mainly metabolized into acetaldehyde by alcohol dehydrogenase 1B (ADH1B), and then detoxified to acetic acid by aldehyde dehydrogenase 2 (ALDH2) (Figure 1) [11,12]. Genetic polymorphisms in ADH1B and/or ALDH2 can result in different enzymatic activities that have a major impact on the risk of ESCC as well as head and neck squamous cell carcinoma (HNSCC) [13][14][15][16].
Thus, extensive epidemiological evidence suggests that acetaldehyde is deeply involved in the carcinogenesis of the squamous epithelium of the esophagus, and head and neck. In addition, the International Agency for Research on Cancer has defined acetaldehyde associated with the consumption of alcoholic beverages as a "group 1 carcinogen" (definite carcinogen) for the esophagus and/or head and neck [28]. Ethanol and acetaldehyde metabolism after alcohol ingestion. Ethanol is metabolized to acetaldehyde by alcohol dehydrogenase 1B (ADH1B), and then acetaldehyde is degraded to acetic acid by aldehyde dehydrogenase 2 (ALDH2).

Field Cancerization in the Esophagus, and Head and Neck
In some patients, ESCC occurs synchronously and/or metachronously in conjunction with HNSCC ( Figure 2A) [12,29]. In such patients, widespread epithelial oncogenic alterations are frequently observed in the esophagus and can be visible as multiple Lugol-voiding lesions (LVLs) by Lugol chromoendoscopy ( Figure 2B) [30,31]. Thus, multiple occurrences of neoplastic changes in the upper aerodigestive tract have been explained by the phenomenon of "field cancerization" [32]. We reported previously that the ALDH2*2 allele is the strongest contributing factor (OR: 17.6) for the development of multiple LVLs [29]. Our recent prospective cohort study also revealed that the severity of LVLs is associated with the amount of average alcohol consumption, and individuals with multiple LVLs in their esophagus are especially at high risk for metachronous multiple ESCC and HNSCC [33]. Thus, alcohol consumption in individuals with the ALDH2*2 allele is proven to be associated with the development of field cancerization in the esophagus, and head and neck. Ethanol and acetaldehyde metabolism after alcohol ingestion. Ethanol is metabolized to acetaldehyde by alcohol dehydrogenase 1B (ADH1B), and then acetaldehyde is degraded to acetic acid by aldehyde dehydrogenase 2 (ALDH2).

Field Cancerization in the Esophagus, and Head and Neck
In some patients, ESCC occurs synchronously and/or metachronously in conjunction with HNSCC ( Figure 2A) [12,29]. In such patients, widespread epithelial oncogenic alterations are frequently observed in the esophagus and can be visible as multiple Lugol-voiding lesions (LVLs) by Lugol chromoendoscopy ( Figure 2B) [30,31]. Thus, multiple occurrences of neoplastic changes in the upper aerodigestive tract have been explained by the phenomenon of "field cancerization" [32]. We reported previously that the ALDH2*2 allele is the strongest contributing factor (OR: 17.6) for the development of multiple LVLs [29]. Our recent prospective cohort study also revealed that the severity of LVLs is associated with the amount of average alcohol consumption, and individuals with multiple LVLs in their esophagus are especially at high risk for metachronous multiple ESCC and HNSCC [33]. Thus, alcohol consumption in individuals with the ALDH2*2 allele is proven to be associated with the development of field cancerization in the esophagus, and head and neck.

Blood and Salivary Acetaldehyde Level after Alcohol Intake
Alcohol consumption increases acetaldehyde concentrations in the blood, saliva, and breath [29,34,35]. In particular, acetaldehyde concentration reaches a very high level in saliva compared with blood [6]. When ALDH2*1/*1 or ALDH2*1/*2 carriers drink 0.6 g ethanol/kg body weight, salivary acetaldehyde concentrations immediately reach 24 to 53 μM in ALDH2*1/*1 carriers and 37 to 76 μM in ALDH2*1/*2 carriers, respectively [36]. The reason for the high acetaldehyde concentrations in saliva is considered to be associated with the formation of acetaldehyde from ethanol via microbial [6] and/or mucosal ADH [37]. Moreover, secretion from salivary glands also influences acetaldehyde concentration in saliva. Indeed, alcohol drinking (0.5 g ethanol/kg body weight) increases acetaldehyde concentrations in parotid duct saliva on ALDH2*1/*2 carriers, while it does not affect those on ALDH2*1/*1 carriers [38]. Furthermore, breath acetaldehyde is also thought to dissolve into saliva. The acetaldehyde concentrations in the oral cavity thus produced are equivalent to the concentration that can induce DNA damage in vitro [6,38]. Therefore, alcohol consumption in ALDH2*1/*2 carriers could promote the direct contact of high acetaldehyde-containing saliva to the surface of the oropharynx, hypopharynx, and esophagus and has the potential to induce DNA damage in the squamous epithelium. Taken together, sustained high acetaldehyde-containing saliva is considered to play an important role in the carcinogenesis of upper digestive tract cancers and it could be involved in "field cancerization."

Blood and Salivary Acetaldehyde Level after Alcohol Intake
Alcohol consumption increases acetaldehyde concentrations in the blood, saliva, and breath [29,34,35]. In particular, acetaldehyde concentration reaches a very high level in saliva compared with blood [6]. When ALDH2*1/*1 or ALDH2*1/*2 carriers drink 0.6 g ethanol/kg body weight, salivary acetaldehyde concentrations immediately reach 24 to 53 µM in ALDH2*1/*1 carriers and 37 to 76 µM in ALDH2*1/*2 carriers, respectively [36]. The reason for the high acetaldehyde concentrations in saliva is considered to be associated with the formation of acetaldehyde from ethanol via microbial [6] and/or mucosal ADH [37]. Moreover, secretion from salivary glands also influences acetaldehyde concentration in saliva. Indeed, alcohol drinking (0.5 g ethanol/kg body weight) increases acetaldehyde concentrations in parotid duct saliva on ALDH2*1/*2 carriers, while it does not affect those on ALDH2*1/*1 carriers [38]. Furthermore, breath acetaldehyde is also thought to dissolve into saliva. The acetaldehyde concentrations in the oral cavity thus produced are equivalent to the concentration that can induce DNA damage in vitro [6,38]. Therefore, alcohol consumption in ALDH2*1/*2 carriers could promote the direct contact of high acetaldehyde-containing saliva to the surface of the oropharynx, hypopharynx, and esophagus and has the potential to induce DNA damage in the squamous epithelium. Taken together, sustained high acetaldehyde-containing saliva is considered to play an important role in the carcinogenesis of upper digestive tract cancers and it could be involved in "field cancerization."
CrPdG is generated by the reaction of two molecules of acetaldehyde with DNA [53] and exists in a ring-opened or ring-closed form [54,55]. Here, two molecules of acetaldehyde are converted into crotonaldehyde and then react with DNA to form CrPdG [56]. The levels of CrPdG are also related to the amount of acetaldehyde produced [57].
CrPdG is generated by the reaction of two molecules of acetaldehyde with DNA [53] and exists in a ring-opened or ring-closed form [54,55]. Here, two molecules of acetaldehyde are converted into crotonaldehyde and then react with DNA to form CrPdG [56]. The levels of CrPdG are also related to the amount of acetaldehyde produced [57].

DNA Adducts Induce Severe DNA Damage
N 2 -Et-dG blocks DNA synthesis and induces DNA mutations [60][61][62][63]. Moreover, N 2 -Et-dG inhibits translesion DNA synthesis (TLS), which leads to a majority of frameshift deletions and a minority of G:C > T:A transversions in human cells [62]. N 2 -Et-dG can rotate around the exocyclic nitrogen and the alpha carbon of acetaldehyde because it has a single bond, whereas N 2 -ethylidene-dG has a double bond, which makes it more hydrophobic than N 2 -Et-dG. These differences may result in significantly different mutagenic potential between N 2 -Et-dG and N 2 -ethylidene-dG [2].
CrPdG induces DNA interstrand [64] and intrastrand cross-links [65]. The ring-opened form of CrPdG can react with dG on the opposite strand of the DNA to form DNA interstrand cross-links [66]. A similar mechanism has been suggested for the formation of DNA intrastrand cross-links [2]. Whereas the ring-closed form of CrPdG would prevent Watson-Crick base pairing with cytosine in the anti conformation, Hoogsteen base pairing with cytosine would be possible in the syn conformation [55]. CrPdG-mediated disruption of the DNA replication process is thought to cause DNA damage [55,[67][68][69].
NεG inhibits a replicative polymerase δ in complex with proliferating cell nuclear antigen (PCNA) while translesion polymerases η, ι, and κ can bypass the lesion with varying mutagenic consequences [70][71][72]. In cells, replication of a plasmid containing a site-specific NεG induces base-pair mutations at the NεG site as well as deletions, rearrangements, double mutants, and base-pair substitutions near the NεG site [73]. These mutations near the NεG site could be triggered by error-prone processing of DNA double-strand breaks (DSBs) resulting from a replication fork collapse caused by NεG [2]. Certainly, acetaldehyde blocks DNA replication and increases the level of phosphorylated histone H2AX (γ-H2AX), a DSB marker, in cells [74].
Acetaldehyde exposure of human cells increases rates of sister chromatid exchange (SCE) [75]. SCE is thought to result from replication-blocking DNA lesions [76]. Although CrPdGs, NεG, and interstrand cross-links are shown to inhibit replication, the adducts or cross-links that relate to the formation of SCEs have not been elucidated.

Carcinogenic Effects of Acetaldehyde
To elaborate on details mentioned previously in part, acetaldehyde causes DNA adducts [39][40][41][42], DNA single-strand breaks, DSBs [77], point mutations [69], SCEs [78][79][80], DNA-DNA cross-links [81], micronuclei [82], and gross chromosomal aberrations [65,80]. Accumulations of these genetic abnormalities are considered to proceed cancer development. Exposure of acetaldehyde directly induces mutations, most frequently G:C > A:T transitions in the TP53 gene [83]. This transition pattern is consistent with that found in a study of the HPRT reporter gene [69]. In addition, G:C > T:A transversions are the most frequent miscoding events induced by CrPdG, followed by G:C > C:G and G:C > A:T mutations [67][68][69]. This spectrum of mutations corresponds with the gene variation pattern observed in ESCC [84,85] and HNSCC [86]. Furthermore, inhalation of acetaldehyde causes nasal and respiratory squamous cell carcinoma in rats and hamsters [87,88]. These results indicate that acetaldehyde has direct carcinogenic effects in animals.

Repair Pathways of Acetaldehyde-Mediated DNA Damage
Recent research has revealed that cells coordinate multiple processes, such as the Fanconi anemia (FA) pathway, nucleotide excision repair (NER), homologous recombination (HR), TLS, base excision repair (BER), fork protection complex, and ATR-dependent cell cycle checkpoint activation, to prevent and repair acetaldehyde-mediated DNA damage [89].
The specific repair processes for N 2 -ethylidene-dG and N 2 -Et-dG remain unknown. The efforts to identify the repair mechanism for N 2 -Et-dG are reported to be unsuccessful [2,90].
The most plausible repair pathway of CrPdG is NER [91]. CrPdG generates interstrand cross-links [64], which can be repaired by the FA pathway [2]. This pathway is composed of at least 19 genes (FANCA, B, C, D1, D2, E-G, I, J, L-T) and the deficiency of these genes can cause FA [92]. FANCA, B, C, E-G, L, and M form a core complex at the site of interstrand cross-links and then promote ubiquitination of the FANCD2-FANCI complex. This ubiquitination leads to the activation of downstream effector proteins, FANCD1, O, P, and Q. They promote the nucleolytic processing of interstrand cross-links, followed by DNA repair via HR [93][94][95][96][97]. Indeed, the FA-BRCA network is activated when cells are treated with ethanol or aldehyde [98,99]. Cells derived from an FA patient are hypersensitive to acetaldehyde exposure [99,100]. Cells deficient for FANCG, FANCQ, or HR protein Rad51D also show many chromosomal aberrations in response to acetaldehyde, while cells deficient for BER and nonhomologous end-joining show subtle increases in chromosome aberration [101,102]. In vivo, when mice with disrupted Aldh2 locus (Aldh2 +/− or Aldh2 −/− ) and Fancd2 heterozygosity (Fancd2 +/− ) are crossed and then challenged with ethanol exposure, the numbers of double-knockout offspring (Aldh2 −/− , Fancd2 −/− ) are significantly reduced [103]. Treatment with ethanol in adult double-knockout mice (Aldh2 −/− , Fancd2 −/− ) results in dramatic reductions of bone marrow cells. Moreover, these mice develop leukemia, even without ethanol administration [103]. These results indicate that Fancd2 plays an important role in the protection from acetaldehyde-induced genotoxicity.

Prevention of Acetaldehyde-Mediated DNA Damage
Acetaldehyde-mediated DNA damage is influenced by ALDH2 expression level [52]. ALDH2 is known to express in various tissues including the liver, kidney, muscle, and heart [104]. Recently, we found that alcohol consumption in mice promoted ALDH2 protein production in esophageal epithelium [52]. In vitro experiments revealed that ALDH2 is induced by acetaldehyde exposure in esophageal keratinocytes. ALDH2 knockdown resulted in an increase of susceptibility to acetaldehyde. Conversely, ALDH2 overexpression prevented acetaldehyde-mediated DNA damage in esophageal keratinocytes, although overexpression of mutant ALDH2 (ALDH2*2) offered no protection. Thus, enhancement of ALDH2 expression level may prevent acetaldehyde-mediated DNA damage.

Conclusions
Previous studies have provided substantial evidence that acetaldehyde induces various forms of DNA damage leading to cancer development ( Figure 4). DNA adduct formation might be the key to acetaldehyde-mediated DNA damage; however, the role of DNA adducts in carcinogenesis has not been completely elucidated. Further studies are necessary to reveal the complete mechanisms of acetaldehyde-mediated cancer development. promote ubiquitination of the FANCD2-FANCI complex. This ubiquitination leads to the activation of downstream effector proteins, FANCD1, O, P, and Q. They promote the nucleolytic processing of interstrand cross-links, followed by DNA repair via HR [93][94][95][96][97]. Indeed, the FA-BRCA network is activated when cells are treated with ethanol or aldehyde [98,99]. Cells derived from an FA patient are hypersensitive to acetaldehyde exposure [99,100]. Cells deficient for FANCG, FANCQ, or HR protein Rad51D also show many chromosomal aberrations in response to acetaldehyde, while cells deficient for BER and nonhomologous end-joining show subtle increases in chromosome aberration [101,102]. In vivo, when mice with disrupted Aldh2 locus (Aldh2 +/− or Aldh2 −/− ) and Fancd2 heterozygosity (Fancd2 +/− ) are crossed and then challenged with ethanol exposure, the numbers of double-knockout offspring (Aldh2 −/− , Fancd2 −/− ) are significantly reduced [103]. Treatment with ethanol in adult double-knockout mice (Aldh2 −/− , Fancd2 −/− ) results in dramatic reductions of bone marrow cells. Moreover, these mice develop leukemia, even without ethanol administration [103].
These results indicate that Fancd2 plays an important role in the protection from acetaldehydeinduced genotoxicity. Acetaldehyde-mediated DSB is repaired by HR [74]. Acetaldehyde accumulates γ-H2AX, which colocalizes with foci of the HR protein Rad51 in cells [74]. Moreover, recombination-defective cells are hypersensitive to acetaldehyde [74].

Prevention of Acetaldehyde-Mediated DNA Damage
Acetaldehyde-mediated DNA damage is influenced by ALDH2 expression level [52]. ALDH2 is known to express in various tissues including the liver, kidney, muscle, and heart [104]. Recently, we found that alcohol consumption in mice promoted ALDH2 protein production in esophageal epithelium [52]. In vitro experiments revealed that ALDH2 is induced by acetaldehyde exposure in esophageal keratinocytes. ALDH2 knockdown resulted in an increase of susceptibility to acetaldehyde. Conversely, ALDH2 overexpression prevented acetaldehyde-mediated DNA damage in esophageal keratinocytes, although overexpression of mutant ALDH2 (ALDH2*2) offered no protection. Thus, enhancement of ALDH2 expression level may prevent acetaldehyde-mediated DNA damage.

Conclusions
Previous studies have provided substantial evidence that acetaldehyde induces various forms of DNA damage leading to cancer development ( Figure 4). DNA adduct formation might be the key to acetaldehyde-mediated DNA damage; however, the role of DNA adducts in carcinogenesis has not been completely elucidated. Further studies are necessary to reveal the complete mechanisms of acetaldehyde-mediated cancer development.

Conflicts of Interest:
The authors declare that they have no competing interests.