A novel role of an old friend: glutathi- one protects cells from formaldehyde tox- icity

Abstract

Formaldehyde (FA) is produced inside cells as a byproduct of es-sential biological processes such as epigenetic demethylations andthe one carbon cycle. We have previously shown that human col-orectal carcinoma cells lacking the FA-metabolizing enzyme alcoholdehydrogenase 5 (ADH5) cannot cope with blood concentrationsof FA, which can be reverted by thiol-containing antioxidants likeN-acetylcysteine (NAC) or glutathione monoethyl ester (GSH-MEE).The rescue of FA toxicity by thiol-rich antioxidants might indicate thatglutathione (GSH) participates in limiting the toxicity of FA. To ad-dress this hypothesis, we used CRISPR/Cas9 to inactivate the genecoding for the regulatory unit (GCLM) of the enzyme γ-glutamate-cysteine ligase (GCL), which conforms the GSH synthetic pathway,in HCT116 human colorectal carcinoma cells. The normalized viabil-ities against 150uM FA were WT= 72.5±7.4 μM; ∆ADH5= 26.0±5.1μM and ΔGCLM= 46.9±7.1 μM (p=0.002, Tukey comparison vs WT;n=5, mean ± SEM). Moreover, the normalized plating efficiencies(PE) in presence of 25 μM FA were WT= 90.1±6.3 %; ∆ADH5=7.9±2.7 % (P<0.0001) and ΔGCLM= 72.4±4.8 % (P=0.038), (n=5,mean ± SEM, Tukey?s comparison vs WT). To obtain mechanis-tic insights, we investigated in cell extracts the formation of S-hy-droxymethyl-GSH (HSMGSH), which is the product of the reactionbetween FA and GSH, by ultraperformance liquid chromatographycoupled to high resolution mass spectrometry. ∆ADH5 cells showeda 1.85-fold increase in the ratio HSMGSH/GSH compared to WT(P<0.0001, Mann-Whitney test), indicating GSH reacts with endoge-nous FA forming HSMGSH in cells, and that ADH5 particiates in themetabolization of HSMGSH. In conclusion, GSH might prevent FAtoxicity by forming HSMGSH, and thus limiting the reactivity of thealdehyde. This role of GSH might have an impact for patients withthe human disease Fanconi Anemia and for carriers of mutations inthe tumor suppressor BRCA2, whose cells are more susceptible toFA damage.