Fig. 3: Formaldehyde induces oxidative stress.

Oxidative stress determination by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (a, b) and dihydroethidium (DHE) (c) in Wild type (WT), ∆ADH5, and complemented ∆ADH5 (∆ADH5/pA5) HCT116 cells upon 48 h exposure to 0, 60 and 150 μmol L⁻¹ formaldehyde (FA). Data are presented as the % untreated mean ± SEM, n = 3, one-way ANOVA, Tukey´s multiple comparison test, ****P < 0.0001 (b), **P = 0.0017 (c). µM refers to µmol L⁻¹, mM refers to mmol L⁻¹. The positive controls are 100 µmol L⁻¹ L-buthionine sulfoximine (L-BSO) (48 h for H2DCFDA) and 10 µmol L⁻¹ antimycin A (30 min for DHE). d Flow cytometry representative plots obtained from WT and ∆ADH5 HCT116 cells harbouring the cytoplasmic-roGFP reporter and exposed to 150 µmol L⁻¹ FA for 48 h. e Quantification of experiments shown in (d) (n = 3, mean ± SEM, one-way ANOVA, Tukey´s multiple comparison test, **P = 0.0013). Positive control: The signal obtained incubating cells for 48 h with 100 µmol L⁻¹ L-BSO. f Data showing Electron spin resonance (ESR) spectroscopy of reactive oxygen species (ROS) by the spin probe CMH (1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine). Measures are from independent biological replicates exposed to the conditions stated in the figure for 48 h (n = 6 except for ∆ADH5 (−), ∆ADH5 + 150 µmol L⁻¹ FA, ∆ADH5 + 60 µmol L⁻¹ FA + L-BSO, WT + 150 µmol L⁻¹ FA and WT 60 µmol L⁻¹ FA + L-BSO with n = 5), mean ± SEM, one-way ANOVA, Tukey´s multiple comparison test. WT: ***P = 0.0008; ∆ADH5: *P = 0.0485 ((−) vs FA (60)), **P = 0.0065 ((−) vs FA (60) + L-BSO), ****P < 0.0001. g Representative images of transgenic C. elegans expressing the transcriptional reporter Pgst-4::GFP upon 5 h exposure to 0 and 25 mmol L⁻¹ FA. The adh-5 plot refers to same strain carrying Pgst-4::GFP, in which adh-5 expression was silenced by an specific RNAi. h Boxplot with Tukey’s whiskers (1.5 inter-quartile distance, box centre at median, hinges at quartiles Q1 and Q3) depicting the quantification of mean pixel intensities of animals expressing Pgst-4::GFP (n = 25, two-tailed Mann Whitney test ****P < 0.0001, **P = 0.0019, ***P = 0.0002). In the indicated groups, animals were co-incubated with 10 mmol L⁻¹ N-acetyl-L-cysteine (NAC) and 25 mM FA for 5 h.