No abstract

Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert cytotoxicity through DNA and DNA-protein crosslinking, likely contributing to the onset of the human DNA repair condition Fanconi Anaemia. Mutations in the genes coding for FA detoxifying enzymes underlie a human inherited bone marrow failure syndrome (IBMFS), even in the presence of functional DNA repair, raising the question of whether FA causes relevant cellular damage beyond genotoxicity. Here, we report that FA triggers cellular redox imbalance in human cells and in Caenorhabditis elegans. Mechanistically, FA reacts with the redox-active thiol group of glutathione (GSH), altering the GSH:GSSG ratio and causing oxidative stress. FA cytotoxicity is prevented by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which metabolizes FA-GSH products, lastly yielding reduced GSH. Furthermore, we show that GSH synthesis protects human cells from FA, indicating an active role of GSH in preventing FA toxicity. These findings might be relevant for patients carrying mutations in FA-detoxification systems and could suggest therapeutic benefits from thiol-rich antioxidants like N-acetyl-L-cysteine.

Writing in Nature communications, Zhu and collaborators reported the development of a genetically encoded sensor for the detection of formaldehyde in cells and tissues. This tool has great potential to transform formaldehyde research; illuminating a cellular metabolite that has remained elusive in live structures. Formaldehyde, a ubiquitous and reactive metabolite Cells obtain energy and molecules required for sustaining life from multiple interconnected biochemical reactions known as metabolic pathways. These reactions can also generate additional reactive metabolites that might damage key biomolecules such as DNA and proteins. One toxic metabolite is formaldehyde (FA), the simplest and one of the most reactive aldehydes. FA is generated endogenously from vital pathways such as protein and nucleic acid demethylation, the one-carbon cycle and from the oxidative degradation of the one-carbon carrier tetrahydrofolate (THF) and some of its derivatives, among others 1. Dietary supplements like the sweetener aspartame or fruit juices rich in methyl-pectins can be metabolized to methanol, which is further oxidized to FA in the liver 2. Furthermore, pollution, cigarette smoke and certain chemicals are common environmental sources of FA, likely contributing to the~50 micromolar FA concentrations reported in human blood (Fig. 1a) 2,3. FA might be implicated in cancer development in patients carrying mutations in the tumour suppressor BRCA2, and in the onset of some human conditions such as Fanconi Anaemia. This rare genetic disorder originates from mutations in genes coding for factors that repair DNAinterstrand crosslinks, one of the lesions inflicted by FA on the DNA (Fig. 1a) 4,5. The World Health Organization (WHO) recognizes FA as an environmental human carcinogen, described to increase the risk of developing nasopharyngeal cancer 6. Consequently, FA levels inside cells need to be strictly controlled by specific mechanisms that metabolize it into less reactive molecules, such as formate. Cytosolic FA metabolism initiates with the cellular antioxidant glutathione (GSH), which spontaneously reacts with FA producing S-hydroxymethyl-GSH (HSMGSH) 7. This compound is oxidized by the enzyme Alcohol dehydrogenase 5 (ADH5), thus keeping a lowstill unknownintracellular concentration of free FA, and limiting redox balance disruption by recycling GSH 7,8. Chasing in the gloom A few methods including colorimetric assays, chromatography and mass spectrometry have been developed to measure FA in biological samples. However, these techniques often require sample destruction and time-consuming sample preparation. Hence, the in vivo study of FA has remained limited due to the lack of tools that are able to detect its real-time concentration and location in a non-invasive manner. Recently, some small fluorescent chemical molecules have been developed to measure FA both in vitro and in living cells. For example, the Aza-Cope-based probes are highly sensitive and specific to FA; however, their reaction kinetics are slow and

25Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert 26 cytotoxicity through DNA and DNA-protein crosslinking. We show here that FA can cause cellular damage 27 beyond genotoxicity by triggering oxidative stress, which is prevented by the enzyme alcohol dehydrogenase 28 5 (ADH5/GSNOR). Mechanistically, we determine that endogenous FA reacts with the redox-active thiol group 29 of glutathione (GSH) forming S-hydroxymethyl-GSH, which is metabolized by ADH5 yielding reduced GSH thus 30 preventing redox disruption. We identify the ADH5-ortholog gene in Caenorhabditis elegans and show that 31 oxidative stress also underlies FA toxicity in nematodes. Moreover, we show that endogenous GSH can protect 32 cells lacking the Fanconi Anemia DNA repair pathway from FA, which might have broad implications for 33 Fanconi Anemia patients and for healthy BRCA2-mutation carriers. We thus establish a highly conserved 34 mechanism through which endogenous FA disrupts the GSH-regulated cellular redox homeostasis that is 35 critical during development and aging. 36 37 hydroxymethylglutathione, Cancer, Genotoxicity. 38 39 BMF, liver and kidney dysfunction and early cancer onset 9 , indicating that endogenous FA can drive cancer 50 initiation and Fanconi Anemia phenotypes. 51Genotoxicity has been widely indicated as the main consequence of FA reactivity in cells 4 . However, 52 the strong reactivity of the FA carbonyl group might also affect other molecules than DNA. In vitro, the 53 spontaneous electrophilic attack of the FA carbonyl group to the thiol-group of GSH leads to the formation of 54 the covalent product S-hydroxymethyl-GSH (HSMGSH) 12 . This reaction might be strongly favored inside cells, 55where GSH levels are in the millimolar range 13 . Accordingly, ADH5 metabolizes HSMGSH yielding formate, 56 which is directed to the one-carbon cycle for nucleotide synthesis 3 (Fig. 1A). 57Considering the electrophilicity of FA and the abundance of GSH we hypothesize that the reaction 58 between FA and GSH might affect the GSH pool having detrimental biological consequences. Indeed, 59 alterations in GSH homeostasis have been reported in multiple pathologies such as hemolytic anemia, 60 diabetes, liver diseases, cystic fibrosis, neurodegeneration and cancer [14][15][16][17] . GSH not only neutralizes reactive 61 oxygen species (ROS), but can also promote chemoresistance by forming GSH-xenobiotic conjugates that are 62 pumped out of the cell via multiple resistance-associated protein transporters (MRP) 18 . To replenish 63 intracellular GSH, cells synthesize GSH in a two-step metabolic pathway centered on the rate-limiting enzyme 64 glutamate cysteine ligase (GCL), which is composed of a catalytic (GCLC) and a regulatory (GCLM) subunit, and 65 the GSH synthetase (GS) ( Fig. 1a) 18 . Cells might thus also need to maintain the balance between GSH and the 66 oxidized GSH disulfide form (GSSG) -GSH:GSSG-to limit free FA and to prevent redox disruption. 67We report here that FA toxicity is inf...