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Chronic exposure is associated with increased risk for cardiovascular disease; however, the underlying mechanisms remain unknown. We examined the effect of bioactive benzene metabolites on endothelial integrity. In vitro, highly reactive benzene metabolites, specifically trans, trans-muconaldehyde (MA, 10\u2009\u00b5M), decreased the impedance of murine cardiac microvascular endothelial cells (MCMVEC) in a time- and dose-dependent manner and increased the endothelial permeability to 70\u2009kDa dextran. Intradermal injection of MA (400\u00a0pmol) increased the vascular leakage by 54% (P\u2009&amp;lt;\u20090.0001) in adult male C57BL\/6J mice. This was accompanied by increased levels of endothelial microparticles in the circulation. RNA sequencing of MA-treated MCMVEC and human aortic endothelial cells revealed the robust induction of heat shock proteins (HSPs), particularly members of the HSP70 and HSP90 families. Reactome pathway enrichment analyses suggested that MA dysregulates pathways associated with G protein-coupled receptor and heat shock factor-1-dependent transactivation. Pharmacological inhibition of HSP70s and HSP90s prevented an MA-induced increase in MCMVEC monolayer permeability. Similarly, pharmacological inhibition of Rho-associated coiled-coil-containing protein kinase (ROCK) attenuated MA-induced endothelial permeability in MCMVEC, accompanied by a dose-dependent activation of Rac1 GTPase. To assess the contribution of HSPs to MA-induced endothelial function impairment, we generated a transgenic mouse overexpressing HSPA1B (a member of the HSP70 family; HSPA1B-TGEC). MA exposure increased the vascular leakage by 15% (P\u2009&amp;lt;\u20090.05) in HSPA1B-TGEC mice as compared with the littermate controls. Collectively, our data suggest that MA increases vascular permeability by activating HSP and GTPase signaling pathways.<\/jats:p>","DOI":"10.1093\/toxsci\/kfaf055","type":"journal-article","created":{"date-parts":[[2025,4,29]],"date-time":"2025-04-29T07:55:44Z","timestamp":1745913344000},"page":"111-122","source":"Crossref","is-referenced-by-count":5,"title":["Benzene metabolites increase vascular permeability by activating heat shock proteins and Rho GTPases"],"prefix":"10.1093","volume":"206","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3976-3884","authenticated-orcid":false,"given":"Igor N","family":"Zelko","sequence":"first","affiliation":[{"name":"Superfund Research Center, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Envirome Institute, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Division of Environmental Medicine, Department of Medicine, University of Louisville , Louisville, KY 40202,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahtesham","family":"Hussain","sequence":"additional","affiliation":[{"name":"Superfund Research Center, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Envirome Institute, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Division of Environmental Medicine, Department of Medicine, University of Louisville , Louisville, KY 40202,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina V","family":"Malovichko","sequence":"additional","affiliation":[{"name":"Superfund Research Center, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Envirome Institute, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Division of Environmental Medicine, Department of Medicine, University of Louisville , Louisville, KY 40202,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nalinie S","family":"Wickramasinghe","sequence":"additional","affiliation":[{"name":"Superfund Research Center, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Envirome Institute, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Division of Environmental Medicine, Department of Medicine, University of Louisville , Louisville, KY 40202,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanjay","family":"Srivastava","sequence":"additional","affiliation":[{"name":"Superfund Research Center, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Envirome Institute, University of Louisville , Louisville, KY 40202,","place":["United States"]},{"name":"Division of Environmental Medicine, Department of Medicine, University of Louisville , Louisville, KY 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