Molecular Mechanisms of Formaldehyde Toxicity and Tolerance in Budding Yeast Saccharomyces cerevisiae
2026 Volume 14 Issue 4 Pages 18-29
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2026 Volume 14 Issue 4 Pages 18-29
Formaldehyde is a highly reactive one‑carbon aldehyde that induces cytotoxicity through DNA–protein crosslinks, RNA adduct formation, and disruption of cellular redox homeostasis. Because all living cells must counteract these lesions to survive, elucidating formaldehyde detoxification and tolerance mechanisms is essential for understanding formaldehyde stress biology. The budding yeast Saccharomyces cerevisiae provides a genetically tractable model for dissecting formaldehyde‑induced molecular damage and the pathways that mitigate it. This review summarizes key mechanisms of formaldehyde tolerance in budding yeast, including glutathione‑dependent formaldehyde detoxification, aldehyde and alcohol metabolic networks, DNA and RNA repair pathways, and other protective mechanisms. As many of these mechanisms are evolutionarily conserved, insights gained from budding yeast offer a valuable framework for understanding formaldehyde toxicity across eukaryotes and may contribute to the development of strategies to mitigate formaldehyde‑related cellular damage.
Formaldehyde is the simplest aldehyde, characterized by a formyl group within its molecular structure. It is also known as methanal or methylene oxide, and its aqueous solution is commonly referred to as formalin. Formaldehyde is a colorless compound with a pungent odor and is highly volatile and flammable.
In the context of organic synthesis, the carbonyl carbon of formaldehyde is highly susceptible to nucleophilic attack. As a C1-symmetric electrophile, formaldehyde participates in a wide range of reactions, including Schiff base formation with primary amines, aldol reactions [1], Wittig reactions, Cannizzaro reactions [2], Biginelli reactions [3], Ugi reactions, Passerini reactions [4], and Prins reactions [5, 6]. Beyond its chemical reactivity, formaldehyde serves as an essential industrial precursor for resin materials. It is a key raw material for producing polymers and resins such as phenolic, melamine, and urea resins [7, 8] and is also widely used in the manufacture of adhesives [8, 9].
Because formaldehyde is highly reactive toward nucleophiles, it readily forms covalent adducts with proteins and nucleic acids, leading to pronounced cytotoxicity, genotoxicity, and reproductive toxicity [10]. For this reason, the International Agency for Research on Cancer (IARC) has classified formaldehyde as a Group 1 carcinogen, indicating sufficient evidence of carcinogenicity in humans [11]. Human exposure to formaldehyde occurs primarily through inhalation but may also result from oral ingestion, dermal contact, and mucosal absorption. Acute exposure to high concentrations of formaldehyde is known to induce pulmonary edema [12, 13], renal failure [13, 14, 15], and inflammatory responses [12]. Chronic exposure has been significantly associated with an increased risk of cancer, including myeloid leukemia [16]. Owing to its pronounced toxicity, strict regulatory control and prevention of occupational formaldehyde exposure have been implemented worldwide, including in the United States [17, 18].
Major sources of environmental formaldehyde exposure include volatilization from construction materials and formaldehyde-containing furniture or household products, as well as combustion-related emissions such as tobacco smoke, automobile exhaust, and incomplete combustion of fossil fuels [19, 20]. Industrial facilities, research laboratories, and waste treatment plants may also release formaldehyde into the environment [21, 22, 23]. In addition to exogenous formaldehyde derived from environmental exposure, endogenous formaldehyde is continuously generated in living organisms at low levels through intracellular metabolic reactions. Endogenous formaldehyde arises during nutritional metabolism, including folate degradation, as well as during demethylation reactions of nucleic acids and proteins [24, 25, 26, 27]. Although endogenous formaldehyde has traditionally been considered less harmful than exogenous formaldehyde, accumulating evidence indicates that it can exert genotoxic and carcinogenic effects in embryonic stem cells and hematopoietic stem cells [28, 29]. Therefore, efficient detoxification of formaldehyde is essential for maintaining cellular function and ensuring proper cell differentiation.
A comprehensive understanding of formaldehyde-induced cytotoxicity and cellular stress response mechanisms is indispensable for elucidating formaldehyde detoxification pathways and for developing strategies to confer formaldehyde tolerance. Studies of eukaryotic stress responses have been extensively conducted using budding yeast and fission yeast, yielding a wealth of foundational knowledge. The budding yeast Saccharomyces cerevisiae is a fully sequenced model organism equipped with well-established databases and genetic manipulation tools [30, 31, 32]. Gene knockout and heterologous gene expression are technically straightforward [33, 34], and unlike mammalian systems, yeast experiments are free from ethical constraints and benefit from rapid growth rates. These advantages have made yeast a widely used model for investigating stress responses. Numerous studies have also employed yeast to explore formaldehyde stress responses, contributing significantly to the elucidation of formaldehyde toxicity mechanisms and resistance acquisition.
In this review, we focus on the cytotoxic effects of formaldehyde and recent advances in understanding formaldehyde stress responses, particularly in yeast cells, and discuss the molecular mechanisms by which yeast cells evade formaldehyde-induced toxicity.
The toxicity of formaldehyde has long been recognized through animal administration and exposure studies [22, 35]. In rats, the reported LD50 values are 800 mg/kg for oral administration and 0.578 mg/L (4 h) for inhalation exposure. For dermal exposure, available data are primarily obtained from rabbits, with a reported LD50 of 270 mg/kg. The principal mechanisms underlying formaldehyde-induced cytotoxicity are currently recognized as: (i) the formation of adducts with intracellular biomolecules, and (ii) disruption of intracellular redox homeostasis due to reduced glutathione (GSH) depletion (Figure 1) [36, 37, 38].
A particularly noteworthy feature of formaldehyde toxicity is that its fundamental mechanisms are conserved from yeast to mammalian cells (Table 1). This conservation suggests that mechanistic insights obtained from model organisms such as yeast can be effectively translated to higher eukaryotes, thereby facilitating the elucidation of formaldehyde toxicity pathways and the development of strategies to enhance formaldehyde tolerance.
Formaldehyde possesses strong electrophilicity owing to the low electron density of its carbonyl carbon. This high reactivity enables formaldehyde to form Schiff base–like adducts with nucleophilic biomolecules such as nucleic acids, amino acids, and sugars. Once internalized, formaldehyde forms crosslinked structures with nucleic acids and proteins, thereby exerting pronounced genotoxic and cytotoxic effects. Formaldehyde initially reacts with nucleophilic sites, including amine and amide groups within nucleic acids or proteins, to generate methylol adducts [39, 40]. These methylol intermediates subsequently undergo further reactions with additional nucleophilic sites, resulting in the formation of DNA interstrand crosslinks (ICLs), DNA–protein crosslinks (DPCs), RNA–protein crosslinks (RPCs), and related adducts [39]. Formaldehyde-induced ICLs and DPCs impede replication fork progression, promote DNA double-strand breaks, and inhibit transcription. Similarly, RPCs have been shown to stall ribosomal progression, thereby suppressing translation [41].
| Formaldehyde metabolic enzymes | S. cerevisiae | human | mouse | rat |
|---|---|---|---|---|
| GSH-dependent formaldehyde dehydrogenase | SFA1 | ADH5 | Adh5 | Adh5 |
| Mitochondrial aldehyde dehydrogenase | ALD4/ALD5 | ALDH2 | Aldh2 | Aldh2 |
| Esterase/S-Formylglutathione hydrolase | YJL068C | ESD | Esd | Esd |
| Formate dehydrogenase | FDH1/FDH2 | ALDH1L1/ALDH1L2 | Aldh1l1/Aldh1l2 | Aldh1l1/Aldh1l2 |
In addition to direct macromolecular damage, formaldehyde toxicity is closely associated with redox imbalance arising during formaldehyde metabolism. GSH functions as a major radical scavenger, and the primary formaldehyde detoxification pathway in budding yeast and mammalian cells is GSH dependent. Intracellular free formaldehyde reacts non-enzymatically with GSH to form S-hydroxymethylglutathione (HMGSH). This conjugation consumes intracellular GSH, leading to alterations in the GSH/GSSG ratio [38]. Depletion of GSH promotes the accumulation of reactive oxygen species (ROS), thereby exposing cells to oxidative stress. Indeed, knockout of the formaldehyde-detoxifying enzyme gene ADH5 in human colorectal carcinoma HCT116 cells results in ROS accumulation, demonstrating that formaldehyde exposure induces oxidative stress [38]. Moreover, supplementation with GSH precursors such as N-acetyl L-cysteine (NAC) or GSH monomethyl ester (GSH MEE) has been shown to attenuate formaldehyde-induced toxicity [38].
Beyond adduct formation and oxidative stress, the generation of formic acid, an oxidized metabolite of formaldehyde, may also contribute to formaldehyde cytotoxicity. Formic acid is produced as an intermediate during formaldehyde detoxification [43] and exerts toxicity by inhibiting respiratory cytochromes [43], inducing intracellular acidification [44, 45], and consequently reducing the proton motive force [46]. Transcriptomic analyses in S. cerevisiae exposed to formic acid have revealed induction of oxidative stress responses, inhibition of protein synthesis, activation of autophagy, and alterations in cellular energy metabolism [47]. Because formic acid is an obligatory intermediate in the complete oxidation of formaldehyde, detoxification-derived formic acid may contribute to overall formaldehyde toxicity. However, no studies have directly examined secondary formic acid accumulation following formaldehyde exposure, and thus the precise contribution of formic acid to formaldehyde stress remains to be clarified.
As a genetically tractable eukaryotic model, budding yeast such as S. cerevisiae has provided a rich foundation for dissecting cellular stress responses. Extensive genetic and molecular studies have established it as one of the most informative systems for analyzing formaldehyde tolerance. Here, we highlight key molecular mechanisms identified through yeast research that contribute to formaldehyde resistance.
3.1 Function of glutathione-dependent formaldehyde dehydrogenase in formaldehyde tolerance of budding yeastOxidative detoxification represents the most critical mechanism by which cells evade formaldehyde toxicity (Figure 2). Formaldehyde detoxification involves its conversion to formic acid, followed by complete oxidation to carbon dioxide. In S. cerevisiae, this sequential process is catalyzed by glutathione (GSH) dependent formaldehyde dehydrogenase encoded by SFA1, and S-formylglutathione hydrolase encoded by YJL068C [44]. Sfa1p utilizes S-hydroxymethylglutathione (HMGSH)—formed non enzymatically from formaldehyde and GSH—as its substrate. This enzyme catalyzes the oxidation of HMGSH to S-formylglutathione (GSCHO). GSCHO is subsequently hydrolyzed by S-formylglutathione hydrolase (YJL068C), an esterase specific for S-formylglutathione, producing formic acid and regenerating GSH [48]. The recycled GSH is reutilized within the cell. Formic acid is then fully oxidized to CO2 by NAD⁺ dependent formate dehydrogenase (FDH) encoded by FDH1, thereby completing the detoxification process before CO2 is released.
This formaldehyde detoxification pathway is highly conserved from fungi to human cells. Human ADH5 and ESD are homologs of yeast SFA1 and YJL068C, respectively [49, 50, 51, 52]. The importance of these enzymes in
formaldehyde tolerance has been demonstrated through gene deletion and overexpression studies. The sfa1Δ strain remains viable under non stress conditions but exhibits markedly reduced formaldehyde tolerance [42, 48]. Conversely, overexpression of SFA1 from a multicopy plasmid enhances formaldehyde resistance [42], indicating that SFA1 constitutes a central determinant of formaldehyde tolerance in yeast.
In addition, SFA1 overexpression has been reported to improve ethanol production when fermentable sugars or lignocellulosic hydrolysates are used as carbon sources [53, 54], suggesting that the bifunctional properties of formaldehyde dehydrogenase—including alcohol dehydrogenase-related activity—may contribute to this phenotype. SFA1 has also been implicated in the metabolism of the glycolytic intermediate dihydroxyacetone (DHA) [55], indicating that SFA1 not only catalyzes formaldehyde oxidation but also interacts with central carbon metabolism through its broad substrate specificity toward aldehydes. To our knowledge, however, there are currently no reports demonstrating that SFA1 or its homologs are directly involved in central carbon metabolism in organisms other than S. cerevisiae.
Like the sfa1Δ strain, the yjl068cΔ strain is viable but exhibits pronounced hypersensitivity to formaldehyde [48]. Moreover, enhanced expression of formate dehydrogenase likewise strengthens formaldehyde tolerance in yeast. Sasnauskas et al. identified CmFDH1, which participates in formaldehyde tolerance in Candida maltosa, by shotgun cloning using the formaldehyde tolerance indicator of the S. cerevisiae strain DBY746 (MATα, his3-Δ1, leu2-3,112, ura3-52, trp1-289) [56]. CmFDH1 is an ortholog of S. cerevisiae ADH1, and its heterologous expression in S. cerevisiae without replacing the endogenous CmFDH1 was shown to enhance formaldehyde tolerance [56]. This finding underscores that formate toxicity during formaldehyde detoxification is not merely secondary; rather, efficient formate oxidation is essential for mitigating overall formaldehyde toxicity.
The yeast formaldehyde metabolism genes have homologs in many other organisms, and the GSH dependent formaldehyde detoxification pathway is broadly conserved across eukaryotes, including mammals. Thus, yeast research provides a useful framework for understanding formaldehyde tolerance mechanisms in diverse organisms. However, some formaldehyde detoxification pathways found in mammals, such as mitochondrial aldehyde dehydrogenase 2 (ALDH2), are absent or not functionally characterized in yeast. In mammalian cells, ALDH2 directly converts formaldehyde to formic acid using NADP⁺/NAD⁺ [57]. Mice deficient in both ADH5 and ALDH2 (Aldh2⁻/⁻ Adh5⁻/⁻) exhibit hypersensitivity to endogenous formaldehyde, demonstrating that mitochondrial aldehyde dehydrogenases also contribute to formaldehyde detoxification in animals [58]. Although S. cerevisiae encodes an ALDH2 homolog ALD4 and ALD5, encoding genes for mitochondrial aldehyde dehydrogenase, their involvement in formaldehyde detoxification has not yet been established. Therefore, elucidating formaldehyde tolerance mechanisms require comparative analysis of formaldehyde detoxification genes and pathways across yeast and the target organism.
3.2 Alternative mechanisms of formaldehyde tolerance in budding yeastIn addition to formaldehyde metabolizing enzymes, genes involved in the central ethanol production pathway of yeast also contribute to formaldehyde tolerance. ADH1, which encodes an alcohol dehydrogenase that catalyzes the reduction of acetaldehyde to ethanol, has been shown to influence formaldehyde resistance (Figure 2). Overexpression of ADH1 decreases intracellular formaldehyde levels and enhances formaldehyde tolerance, whereas the adh1Δ strain displays increased formaldehyde sensitivity relative to the wild type [59]. Interestingly, 14C labeled formaldehyde tracing experiments revealed that ADH1 overexpressing strains exhibit reduced intracellular formaldehyde levels and lower CO2 production compared with SFA1 overexpressing strains [59]. These observations suggest that ADH1 may detoxify formaldehyde by reducing it to methanol—a less toxic metabolite—rather than promoting its complete oxidation.
Furthermore, overexpression of ALD6, which encodes a cytoplasmic aldehyde dehydrogenase that catalyzes the oxidation of acetaldehyde to acetate, has also been reported to enhance formaldehyde tolerance [60]. Unlike Sfa1p, however, Ald6p does not directly oxidize formaldehyde, although it mainly catalyzes acetaldehyde oxidation [62]. Instead, its protective effect is thought to arise indirectly through modulation of metabolic fluxes and maintenance of intracellular redox balance via NADP+/NADPH homeostasis. Such conditions may arise during fermentative growth or stress conditions, where multiple aldehydes, including acetaldehyde, can accumulate as metabolic intermediates. Moreover, an amino acid–mediated formaldehyde trapping system has been described in mammals [61], suggesting that additional, as-yet-unidentified formaldehyde tolerance mechanisms may also exist in yeast.
3.3 Genome wide screening and transcriptomic insights into formaldehyde tolerance in budding yeastAs described above, enhanced formaldehyde tolerance in yeast has been achieved through overexpression of SFA1, which catalyzes formaldehyde scavenging [42], and through elevated expression of FDH1, which promotes complete oxidation of formic acid [56]. In recent years, DNA microarray analyses and genome wide screening approaches in budding yeast have further expanded the repertoire of genes implicated in formaldehyde tolerance [62, 63, 64], suggesting that, beyond formaldehyde metabolism itself, additional cellular processes are critical for survival under formaldehyde stress (Figure 2).
Yasokawa et al. performed a DNA microarray analysis to compare the toxic effects of methanol and formaldehyde in S. cerevisiae [63]. Among approximately 6,000 open reading frames (ORFs), formaldehyde treatment resulted in induction (≥2 fold) of 622 genes and repression (≤0.5 fold) of 610 genes relative to untreated controls. Many of the upregulated genes were annotated to gene ontology categories associated with oxidative stress responses, radical scavenging, protein processing, and DNA repair. Notably, genes directly involved in formaldehyde detoxification, such as SFA1 and YJL068C, were strongly induced, reinforcing the conclusion that formaldehyde detoxification is essential for yeast survival under formaldehyde stress (Figure 2).
In addition, members of the AAD gene family (AAD16, AAD6, AAD14, and AAD3) were upregulated. The AAD genes encode putative aryl alcohol dehydrogenases and have been implicated in intracellular aldehyde metabolism; they are also known to be induced under oxidative stress [65, 66]. Thus, rather than participating directly in formaldehyde removal, AAD proteins are more likely to mitigate formaldehyde induced oxidative stress. Altered expression was also observed among alcohol dehydrogenase (ADH) genes: ADH6 was induced whereas ADH1 was repressed. Although ADH6 belongs to the cinnamyl alcohol dehydrogenase family of medium chain alcohol dehydrogenases [67], its ability to reduce short chain aldehydes such as formaldehyde remains unclear. It is interesting that, although ADH1 has previously been implicated in formaldehyde detoxification and tolerance [59], its transcriptional level is repressed during formaldehyde stress. The mechanism underlying this repression is still unknown.
Furthermore, methionine biosynthesis genes (MET17, MET3, MET16, MET10, and MET6) and methionine permease genes (MUP1 and MUP3) were strongly induced upon formaldehyde exposure. Given that methionine biosynthesis is closely linked to GSH production [68, 69, 70], the upregulation of these genes implies reinforcement of intracellular GSH synthesis to counteract formaldehyde‑induced redox imbalance (Figure 2). Collectively, these findings indicate that, in addition to direct formaldehyde detoxification, genes involved in oxidative stress management, DNA repair, and maintenance of cellular homeostasis play major roles in determining survival under formaldehyde stress.
De Graaf et al. conducted a genome wide screen using approximately 5,000 deletion mutants of S. cerevisiae to identify genes required for formaldehyde tolerance under chronic (1.5 mM) and acute (60 mM) exposure [62]. Their analysis revealed that genes involved in homologous recombination (HR) repair and nucleotide excision repair (NER) are critical for formaldehyde tolerance, as deletion of these genes caused pronounced formaldehyde sensitivity (Figure 2). Moreover, acute formaldehyde exposure led to accumulation of DPCs across the genome, and these lesions were repaired in a dose dependent manner following formaldehyde removal, primarily via HR and NER pathways (Figure 2).
Similarly, North et al. performed a genome wide screen using a pooled collection of 4,607 null mutants exposed to formaldehyde concentrations ranging from 0.15 to 0.6 mM for 5 to 15 generations [64]. Differential strain sensitivity analysis, based on TAG4 array hybridization, revealed that genes with significant fitness defects under formaldehyde stress were enriched for functions related to DNA double strand break repair, stress response, and chromosome organization [64, 71]. Subsequent validation experiments demonstrated that mutants defective in RNA degradation (SKI complex), HR repair, single strand annealing, and post replication repair exhibited increased formaldehyde sensitivity [64].
Importantly, the identification of RNA degradation pathways as determinants of formaldehyde tolerance highlights the physiological relevance of RPCs and RNA adducts formed under formaldehyde stress. Efficient removal or turnover of damaged RNA appears essential for maintaining translational capacity and cell viability. In contrast to the findings of De Graaf et al. [62], NER mutants were not prominently identified in North’s screen, likely reflecting differences in formaldehyde concentration and exposure duration [64]. It has been proposed that repair pathway utilization depends on formaldehyde dose, with HR playing a predominant role under low, chronic exposure, whereas NER becomes more critical under high, acute exposure.
Interestingly, North’s analysis also suggested that, in addition to formaldehyde detoxification and DNA repair genes, pathways related to osmotic regulation and protein trafficking contribute to survival under formaldehyde stress. Deletion of key components of the high osmolarity glycerol (HOG) pathway, including SSK1, SSK2, and HOG1, resulted in reduced formaldehyde tolerance [64]. However, the mechanistic basis of HOG pathway involvement in formaldehyde resistance remains unclear and warrants further investigation.
Collectively, transcriptomic analyses and genome wide functional screens have expanded our understanding of formaldehyde tolerance in yeast beyond classical detoxification pathways. These studies underscore the importance of RNA degradation, DNA repair, and oxidative stress responses in determining survival under formaldehyde stress. Specifically, transcriptional induction data and mutant phenotypes demonstrate that HR and NER pathways are essential for maintaining DNA integrity following DPC formation, whereas RNA degradation pathways alleviate translation inhibition caused by RPCs. The importance of DNA repair pathways under formaldehyde stress has also been demonstrated in mammalian cells [26, 27, 72, 73], suggesting that insights obtained from yeast may have significant implications for formaldehyde tolerance research across species. Moreover, large scale screening approaches have implicated additional factors, including protein transporters, AAD family members, ADH genes, and the MAP kinase Hog1, in formaldehyde tolerance. Elucidating the roles of these genes may reveal previously unrecognized mechanisms of formaldehyde resistance and further refine our understanding of cellular adaptation to formaldehyde stress.
The currently understood mechanisms of formaldehyde response in yeast cells exhibit a high degree of conservation with those characterized in other organisms. In particular, key components of formaldehyde metabolism, such as SFA1 and its human homolog ADH5, and YJL068C and its human homolog ESD, perform analogous functions in mammalian cells. This evolutionary conservation suggests that mechanistic insights obtained from yeast models are broadly applicable across species.
Consequently, continued investigation of formaldehyde tolerance mechanisms in budding yeast is likely to facilitate the identification of novel formaldehyde response pathways and reveal molecular targets relevant to the treatment and prevention of formaldehyde associated diseases. Insights from yeast research may therefore have wide ranging implications for understanding formaldehyde toxicity across species and for developing strategies to mitigate its harmful effects.
Yuichiro Ikawagawa: Conceptualization, Literature review, Writing–original draft. Tomoyuki Nakagawa: Conceptualization, Writing–review & editing, Supervision. All authors have read and approved the final manuscript.
This research was funded in part by the Make New Standards Program for Next Generation Researchers, Gifu University, to YI, and by the 2022 Grant for Environmental Research Projects from the Sumitomo Foundation to TN.
The authors declare no conflict of interest.
