Evaluation of Aromatic Hydrocarbon Receptor Repressor (AHRR) as a Sensitive Biomarker of Ambient Polyaromatic Hydrocarbons (PAH) Exposure and a Risk Factor for Coronary Artery Disease (CAD).

Abstract

Background: Coronary artery disease (CAD) and morbidities from cigarette smoking and pollution are among the leading causes of deaths world-wide. CAD risk factors include pollution exposure and cigarette smoking. Cigarette smoke contains thousands of chemicals like polyaromatic hydrocarbons (PAHs). PAHs are also bound to particulate matter (PM), a major air pollutant, as a product of incomplete combustion. Studies have shown that methylation on the CpG site (cg05575921) in the aryl hydrocarbon receptor repressor (AHRR) gene is a marker of cigarette smoke exposure as it is hypomethylated among smokers and partially reversible after smoking cessation. Other studies associated this same AHRR CpG site hypomethylation in non-smoking subjects exposed to ambient pollution as measured based on geographic distribution. Furthermore, AHRR cg05575921is hypomethylated in CAD patients.

Aim:

The aim of this study is to evaluate AHRR CpG ROI (cg05575921) methylation status as a sensitive biomarker for ambient PAHs exposure and a risk factor for CAD.

Methods:

Whole peripheral blood and complete data of three urinary PAHs metabolites are available from subjects admitted to American University of Beirut Medical Center (AUBMC) in the Vascular Medicine Program (VMP) for cardiac catheterization, which is considered the gold standard for CAD diagnosis. In addition, data on smoking and CAD status were obtained. DNA was extracted from whole peripheral blood and bisulfite converted. PCR was performed on bisulfite converted samples followed by PCR melting temperature analysis using methylation sensitive high-resolution melting (MS-HRM) and bisulfite sequencing in order to evaluate methylation % and correlate them with urinary PAHs levels and CAD status.

Results:

Data of the MS-HRM and sequencing methylation analyses were highly correlated. There were however no correlations between each of MS-HRM PCR melting temperature, sequencing average methylation and that of CpG site (cg05575921) and the studied urinary OH-PAHs on Pearson’s test. Nevertheless, although the results were not statistically significant, there was a trend of hypomethylation and obstructive CAD among ever and never smokers, with more hypomethylation in smokers. These trends were most apparent with the methylation results based on direct bisulfite sequencing which is the most specific and sensitive method when compared to MS-HRM.

Conclusions:

Preliminary data showed that there was no correlation between AHRR methylation status and three of the analyzed OH-PAHs. This lack of correlation could be due to the fact that the OH-PAHs are based on spot urine collection which may not reflect chronic exposure. In fact, AHRR methylation status is currently an established biomarker for chronic smoking exposure, and may hence be a better marker of PAH exposure from ambient pollution as shown in the trend among the never smokers in this study. It is hoped that with further experiments on the rest of the cohort, there would be enough power to show significant results and adjust for potential confounders. To our knowledge, this is the first study where samples were collected from subjects undergoing cardiac catheterization, the gold standard for CAD diagnosis, and where pollution exposure level was measured based on biomarkers rather than atmospheric measurement, which may not reflect actual exposure.