Benzene is a ubiquitous environmental pollutant abundant in household products, petrochemicals, and cigarette smoke. Benzene is a well-known carcinogen in humans and experimental animals; however, little is known about the cardiovascular toxicity of benzene. Recent population-based studies indicate that benzene exposure is associated with an increased risk for heart failure. Nonetheless, it is unclear whether benzene exposure is sufficient to induce and/or exacerbate heart failure. We examined the effects of benzene (50 ppm, 6 h/day, 5 days/week, 6 weeks) or HEPA-filtered air exposure on transverse aortic constriction (TAC)-induced pressure overload in male C57BL/6J mice. Our data show that benzene exposure had no effect on cardiac function in the Sham group; however, it significantly compromised cardiac function as depicted by a significant decrease in fractional shortening and ejection fraction, as compared with TAC/Air-exposed mice. RNA-seq analysis of the cardiac tissue from the TAC/benzene-exposed mice showed a significant increase in several genes associated with adhesion molecules, cell-cell adhesion, inflammation, and stress response. In particular, neutrophils were implicated in our unbiased analyses. Indeed, immunofluorescence studies showed that TAC/benzene exposure promotes infiltration of CD11b⁺/S100A8⁺/myeloperoxidase⁺-positive neutrophils in the hearts by 3-fold. In vitro, the benzene metabolites, hydroquinone, and catechol, induced the expression of P-selectin in cardiac microvascular endothelial cells by 5-fold and increased the adhesion of neutrophils to these endothelial cells by 1.5-2.0-fold. Benzene metabolite-induced adhesion of neutrophils to the endothelial cells was attenuated by anti-P-selectin antibody. Together, these data suggest that benzene exacerbates heart failure by promoting endothelial activation and neutrophil recruitment.

RNA Isolation and RNAseq analysis: Total RNA was extracted from the hearts of mice using TRIzol kit (Thermo Fisher Scientific, MA, USA), and the purity of RNA was analyzed using NanoDrop One (ThermoScientific, MA, USA). RNA quality was measured by Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA) and samples with high RNA integrity were used for subsequent RNAseq analysis. RNA samples were processed by Novogene using mRNA and small RNA sequencing services (Novogene, Beijing, China). The resultant raw reads of the FASTQ files were processed and quality metrics were visualized using FastQC v 0.11.9. The mRNA differentially regulated genes (DEG) and pathway enrichment analysis were performed using the NGS Data Analysis pipeline.

Animal housing and maintenance: C57BL/6J male mice obtained from Jackson Laboratory (Bar Harbor, ME) were maintained on normal chow in a pathogen-free facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures were approved by the University of Louisville Institutional Animal Care and Use Committee.

Animal Surgeries: For transverse aortic constriction (TAC), male C57BL/6J mice 12 weeks of age were anesthetized (intraperitoneal injections of 50 mg/kg sodium pentobarbital and 50 mg/kg ketamine hydrochloride), antiseptically prepared for surgery, orally intubated, and ventilated (oxygen supplement to the room-air inlet) with a mouse ventilator (Hugo Sachs). Core body temperature was maintained at 36.5–37.5°C with an automatic, electronically regulated heat lamp. The aorta was visualized following an intercostal incision. A 7-0 nylon suture was looped around the aorta between the brachiocephalic and left common carotid arteries. The suture was tied around a 27-gauge needle placed adjacent to the aorta to constrict the aorta to a reproducible diameter. The needle was removed, and the chest was closed in layers. Mice were extubated upon recovery of spontaneous breathing. Analgesia (ketoprofen, 5 mg/kg) was provided prior to recovery and by 24 and 48 h post-surgery. Sham mice were subjected to the same procedure as the TAC cohort except the suture was not tied.

Benzene Exposure: One week after the TAC, mice were exposed to benzene for 6 weeks in bedding-free cages.Benzene atmospheres were generated from liquid benzene (Sigma-Aldrich) in a KIN-TEK Analytical, Inc permeation tube. A carrier gas (N₂) was delivered to the permeation tube at 100 ml/min and diluted with HEPA- and charcoal-filtered room air (3 L/min) and diluted gas directed to an exposure unit. Flow was distributed through a fine mesh screen of a custom cyclone-type top (Teague Enterprises) that distributed air within 10% of the mean concentration at six locations in the cage. Throughout the exposure, benzene concentrations were continuously monitored using an in-line photoionization detector (ppb RAE: Rae Industries) upstream of the exposure unit. Mice were exposed to 50 ppm benzene (6 h/day, 5 days/week) for 6 weeks. Mice exposed to HEPA- and charcoal-filtered room air only were used as controls.