Abstract Body (Do not enter title and authors here): Background. Benzene is a ubiquitous environmental pollutant abundant in automobile exhaust, cigarette smoke, forest fires, and present in several household products. It is ranked # 6 on Agency for Toxic Substances and Disease Registry (ATSDR) priority list, however, little is known about its effect on cardiovascular disease, especially atherosclerosis.

Hypothesis. Inhaled benzene induces atherogenesis by increasing vascular inflammation in LDL receptor Knockout (LDLR-KO) mice.

Methods. Male LDLR-KO or apoE-KO mice were exposed to HEPA-filtered air or benzene (1.0 or 10.0 ppm, 6h/day, 5 days/week) for 24 weeks and maintained on Western diet for the last 12 weeks of exposure. Atherosclerotic lesion composition and nature were determined by liquid chromatography-mass spectrometry (LC-MS), single nuclei RNA sequencing (snRNAseq), and Cytometry by time of flight (CyTOF).

Results. Inhaled benzene exposure significantly increased lesions in the aortic valves (>25%, P<0.05). Single cell RNA sequencing of the aortae of benzene-exposed mice revealed 11 major cell types (e.g., vascular smooth muscle cells (VSMCs), endothelial cells, macrophages, T cells, etc.) categorized into 23 sub-populations. snRNAseq and CyTOF analysis showed that benzene exposure robustly increased (P<0.05) CD4⁺ and CD8⁺ T-cells, and B-cells in the lesions. Clustering of T cells showed significantly higher expression (P<0.05) of CD4, CD8, and Th17 marker RAR Related Orphan Receptor C (RORc). snRNAseq data also suggested increased cell proliferation and >1.5-fold increase in VSMC number following benzene exposure. LC-MS analysis revealed that benzene exposure differentially regulated the abundance of 930 proteins (at least 2 two unique peptides) in innominate plaque. Importantly, several of the proteins involved in an extracellular matrix organization and elastic fibre formation (e.g. Elastin, Fibronectin, and Latent transforming growth factor beta binding protein 1) were downregulated in benzene-exposed mice. These data agree with the snRNAseq data. In vitro, benzene metabolite hydroquinone (HQ, 1 μM) induced ROR-gamma, the transcription factor that regulates Th17, in spleen-derived CD4⁺ T cells (P<0.05). HQ (0.1 μM) also increased protein kinase C activity and proliferation of human aortic smooth muscle cells (P<0.001).

Conclusion. Benzene exposure exacerbates atherosclerosis and compromises plaque cellularity and stability.