DOI: 10.5061/dryad.12jm63xz6
title: Differentially regulated genes in the heart of benzene exposed TAC-instrumented mice

description: 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.
description: <b>RNA Isolation and RNAseq analysis:</b> 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.
 <b>Animal housing and maintenance: </b>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. <b>Animal Surgeries:
 </b>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. <b>Benzene Exposure:
 </b>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 (N<sub>2</sub>) 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.