Detection and Proinflammatory Effects of BTEX within the Human Atherosclerotic Plaque

Blood levels of the pollutants benzene, toluene, xylene (<i>i.e</i>., the sum of <i>ortho</i>-, <i>meta</i>-, and <i>para</i>-xylene isomers), and ethylbenzene (BTEX) are associated with an increased incidence of cardiovascular diseases. However, the underlying mechanisms are unknown. Here, we tested the hypothesis that BTEX accumulate in human atherosclerotic plaque, promoting local proinflammatory responses within atheroma tissues. To this aim, we retrospectively analyzed carotid plaque samples from 147 individuals from a cohort of people with asymptomatic carotid stenosis undergoing carotid endarterectomy. In atheroma specimens, we quantified BTEX and 1,4-benzoquinone, a benzene metabolite, through flame ionization detector gas chromatography and by high-performance liquid chromatography, respectively. We categorized individuals as BTEX-positive or BTEX-negative and compared the expression of plaque inflammatory markers in these two groups. To explore the mechanistic basis for our clinical observations, we also treated human monocytes with relevant concentrations of the four BTEX and evaluated their possible proinflammatory effect. Among the 147 plaques analyzed, 93 had evidence of at least one BTEX pollutant. Benzene was the most abundant, and its levels were positively correlated to those of 1,4-benzoquinone. Patients with evidence of BTEX within the atheroma, compared with those without, showed higher levels of CD68, MMP9, NLRP3, IL-1β, and TNF-α within the plaque but also of systemic markers of inflammation such as the white blood cell count and neutrophil-to-lymphocyte ratio. BTEX levels were positively correlated with NLRP3 and IL-1β levels as well as with triglyceride. BTEX levels were higher in people with a prior diagnosis of dyslipidemia and with a subsequent major cardiovascular event. Treatment of monocytes for 5 days with benzene, xylene, and ethylbenzene, but not toluene, fostered a consistent proinflammatory response, an effect observed in the absence of cytotoxicity and phenocopied by the treatment with 1,4-benzoquinone. BTEX and 1,4-benzoquinone also promoted foam cell formation in monocytes differentiated into macrophages. These data represent the first evidence that BTEX pollutants accumulate within the carotid plaque tissue. This phenomenon is associated with increased local inflammation, possibly suggesting the activation of proatherogenic pathways at the local level.