Glyoxal damages human aortic endothelial cells by perturbing the glutathione, mitochondrial membrane potential, and mitogen-activated protein kinase pathways

Abstract

Background: Exposure to glyoxal, the smallest dialdehyde, is associated with several diseases; humans are routinely exposed to glyoxal because of its ubiquitous presence in foods and the environment. The aim of this study was to examine the damage caused by glyoxal in human aortic endothelial cells.

Methods: Cell survival assays and quantitative fluorescence assays were performed to measure DNA damage; oxidative stress was detected by colorimetric assays and quantitative fluorescence, and the mitogen-activated protein kinase pathways were assessed using western blotting.

Results: Exposure to glyoxal was found to be linked to abnormal glutathione activity, the collapse of mitochondrial membrane potential, and the activation of mitogen-activated protein kinase pathways. However, DNA damage and thioredoxin oxidation were not induced by dialdehydes.

Conclusions: Intracellular glutathione, members of the mitogen-activated protein kinase pathways, and the mitochondrial membrane potential are all critical targets of glyoxal. These findings provide novel insights into the molecular mechanisms perturbed by glyoxal, and may facilitate the development of new therapeutics and diagnostic markers for cardiovascular diseases.

Keywords: Cardiovascular diseases; Glyoxal; Human aortic endothelial cells; Laboratory indexes; Mitogen-activated protein kinase pathways.

Fig. 1

Analysis of DNA damage induced by glyoxal (GX) in human aortic endothelial cells (HAECs) A HAECs were treated with a range of concentrations (50, 100, 150, and 200 µM) of GX for 8 days, and their survival was determined ($\overline{\text{x}}$ ± s, %). Drug lethal dose (LD₉₀) values were confirmed from survival curves and are indicated by an arrow. B Double strand break (DSB) induction (uncropped blots/gels are presented in Additional file 1: Fig. S1). DSBs were detected using static-field gel electrophoresis after treatment with the fraction of DNA released relative to the total DNA. C DNA–protein crosslinks (DPC) induction. DPCs were quantified by the FITC-labelling method. Non-significant differences in the results obtained are denoted by n.s. (p > 0.05)

Fig. 2

Effect of glyoxal (GX) on intracellular glutathione (GSH). A Data are presented as means ± standard deviations of three separate tests. Statistically significant differences are indicated by an asterisk, p < 0.05 indicates statistical significance. B Buthionine sulfoximine (BSO; 1 mM) was used to treat human aortic endothelial cells (HAECs) for different time periods, and cell survival was subsequently presented as means ± standard deviations

Fig. 3

Effect of glyoxal (GX) on the mitochondrial membrane potential. A Human aortic endothelial cells (HAECs) were treated with GX. Statistically significant differences are indicated by an asterisk, p < 0.05 indicates statistical significance. B Survival of cyanide m-chlorophenylhydrazone (CCCP)-treated HAECs presented as means ± standard deviations

Fig. 4

Redox state of Trx1. A Effect of glyoxal (GX) on the redox state of Trx1 (uncropped blots/gels are presented in Additional file 1: Fig. S2). Trx1 was either in a completely reduced state (iodoacetic acid (IAA) band) or in a completely oxidized state (iodoacetamide (IAM) band). Statistical differences are indicated by an asterisk, p < 0.05 indicates statistical significance. B Survival of auranofin-treated human aortic endothelial cells (HAECs) presented as means ± standard deviations

Fig. 5

Effect of glyoxal (GX) on MAP kinase pathways (uncropped blots/gels are presented in Additional file 1: Fig. S3). Human aortic endothelial cells (HAECs) were treated with or without GX, lysed, and subjected to immunoblot assay with anti-phosphorylation or non-phosphorylation- level antibodies of MAP kinase pathways. Statistically significant differences are indicated by an asterisk, p < 0.05 indicates statistical significance