CONTRIBUTION OF ENVIRONMENTAL POLLUTANTS TO EPIGENETIC MODIFICATION IN RAT VASCULAR SMOOTH MUSCLE CELLS

Abstract

Objective: More than a quarter of human diseases have been linked to exposure to environmental pollutants. These pollutants can exacerbate disease conditions through labile epigenetic modifications and directly result in changes in gene transcription. The most commonly reported epigenetic changes are acetylation and methylation of histone lysine. Contribution of acrolein, an environmental pollutant and a major component of cigarette smoke, to cardiovascular diseases and several neurological disorders has been reported. We have previously reported toxicity of acrolein in rat's vascular smooth muscle cells (VSMCs) and the effect of precursor of glutathione transferase, N-acetyl cysteine (NAC), in prevention of acrolein toxicity. In the present study, modification of histones and their effect on protein expression was further investigated in rat's VSMCs in the present/absent of acrolein and NAC. Design and method: VSMCs were treated with 3 μg/ml of acrolein for 6 and 24 hours in the present/absent of 0.2 mM NAC. At the end of the treatment, MTS essay, ELISA, western blot analysis, immunofluorescence, and LC/MS/MS analysis were used to check cells viability, superoxide dismutase (SOD) activity/expression, reactive oxygen species (ROS) generation, expression and cellular localization of H3K9 tri-methylation and acetylation, and identification of proteins affected by these changes. Results: Acrolein treated VSMCs exhibited the highest toxicity after 6 hours. Acrolein increased generation of ROS, reduced SOD activity and expression. There was 52% induction of acetylation and 62% in tri-methylation. Addition of N-acetyl cysteine reduced ROS, induced SOD (45%), reduced H3K9 acetylation by 102% and tri-methylation by 120%. These changes were confirmed with immunofluorescence staining. LC/MS/MS analysis revealed increased in actin protein and a significant decreased in annexin, heat shock cognate, and myosin 9 proteins. Conclusions: Based on our data we are concluding that effect of acrolein on VSCMs is partially due to alterations of H3K9 methylation/acetylation, resulting in overexpression of actin and significant reduction of annexin, heat shock cognate, and myosin 9 proteins. Addition of 0.2 mM NAC resulted in prevention of toxicity by acrolein by reducing ROS, improving SOD and preventing changes in H3K9 acetylation and methylation.