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Isothiazolinone (IT) biocides are potent antibacterial substances used as preservatives and disinfectants. These biocides exert differing biocidal effects and display environmental stability based upon chemical structure. In agreement with our recent study reporting that 2-n-octyl-4-isothiazolin-3-one (OIT) induced dysfunction of the blood-brain barrier (BBB), the potential adverse health effects of two IT biocides 1,2-benzisothiazolin-3-one (BIT) and 4,5-dichloro-2-n-octyl-isothiazolin-3-one (DCOIT) were compared using brain endothelial cells (ECs) derived from murine brain endothelial cell line (bEND.3). BIT possesses an unchlorinated IT ring structure and used as a preservative in cleaning products. DCOIT contains a chlorinated IT ring structure and employed as an antifouling agent in paints. Data demonstrated that DCOIT altered cellular metabolism at a lower concentration than BIT. Both BIT and DCOIT increased reactive oxygen species (ROS) generation at the mitochondrial and cellular levels. However, the effect of DCOIT on glutathione (GSH) levels appeared to be greater than BIT. While mitochondrial membrane potential (MMP) was decreased in both BIT- and DCOIT-exposed cells, direct disturbance in mitochondrial bioenergetic flux was only observed in BIT-treated ECs. Taken together, IT biocides produced toxicity in brain EC and barrier dysfunction, but at different concentration ranges suggesting distinct differing mechanisms related to chemical structure. [ABSTRACT FROM AUTHOR]