A Commonly Used Biocide 2-N-octyl-4-isothiazolin-3-oneInduces Blood-Brain Barrier Dysfunction via Cellular Thiol Modification and Mitochondrial Damage

Abstract

Isothiazolinone (IT) biocides are potent antibacterial substances commonly used as preservatives or disinfectants, and 2-n-Octyl-4-isothiazolin-3-one (OIT; octhilinone) is a common IT biocide that is present in leather products, glue, paints, and cleaning products. Although humans are exposed to OIT through personal and industrial use, the potentially deleterious effects of OIT on human health are still unknown. To investigate the effects of OIT on the vascular system, which is continuously exposed to xenobiotics through systemic circulation, we treated brain endothelial cells with OIT. OIT treatment significantly activated caspase-3-mediated apoptosis and reduced the bioenergetic function of mitochondria in a bEnd.3 cell-based in vitro blood-brain barrier (BBB) model. Interestingly, OIT significantly altered the thiol redox status, as evidenced by reduced glutathione levels and protein S-nitrosylation. The endothelial barrier function of bEnd.3 cells was significantly impaired by OIT treatment. OIT affected mitochondrial dynamics through mitophagy and altered mitochondrial morphology in bEnd.3 cells. N-acetyl cysteine significantly reversed the effects of OIT on the metabolic capacity and endothelial function of bEnd.3 cells. Taken together, we demonstrated that the alteration of the thiol redox status and mitochondrial damage contributed to OIT-induced BBB dysfunction, and we hope that our findings will improve our understanding of the potential hazardous health effects of IT biocides.

Keywords: 2-n-Octyl-4-isothiazolin-3-one (OIT); blood–brain barrier (BBB) model; isothiazolinone (IT) biocide; mitochondrial dysfunction; oxidative stress; protein S-nitrosylation (SNO).

Figure 1

Effects of 2-n-Octyl-4-isothiazolin-3-one (OIT) on the apoptosis pathway, metabolic capacity, plasma membrane damage, and endothelial function in bEnd.3 cells. (a) Early-apoptotic, late-apoptotic, and necrotic cells were analyzed at 24 h after treatment with 0, 5, or 25 μM OIT (n = 4). (b) Caspase-3 activity was examined at 3 h after treatment with 0, 5, or 25 μM OIT (n = 4). (c,d) The extents of (c) MTT reduction and (d) LDH release were examined at 0, 1, 3, 6, and 24 h after treatment with 0, 5, or 25 μM OIT (n = 3–6). (e,f) Functional changes in endothelial permeability were analyzed by TEER measurements (e) and an in vitro FITC-dextran (m.w. 4000 Da) permeability assay (f) at 24 h after treatment of OIT (n = 3–4). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 vs. CON (control).

Figure 2

Effects of OIT on tight junction protein expression, protein S-nitrosylation (SNO), reduced glutathione (GSH), and total cellular ROS levels. (a) Immunofluorescence staining with claudin-5 or ZO-1 was performed in the OIT-exposed bEnd.3 cells and visualized by confocal microscopy at 24 h after OIT treatment (n = 3). Scale bar: 20 μm. (b,c) The protein levels of (b) claudin-5 and (c) ZO-1 were determined 24 h after treatment with 5 and 25 μM OIT (n = 3). (d) The level of SNO-modified proteins was detected at 1 h after treatment with 0, 5, or 25 μM OIT by the detection of the TMT-labeled S-nitrosylated proteins in western blot (n = 3). (e) The level of reduced GSH was measured at 24 h after treatment with OIT (n = 3). (f) Total cellular ROS was detected at 0, 1, 3, and 6 h after treatment with OIT (n = 3). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 vs. CON (control).

Figure 3

Changes in mitochondrial membrane potential and bioenergetics after OIT treatment in bEnd.3 cells. (a) Mitochondrial ROS was measured 1 and 3 h after treatment with 25 μM OIT (n = 3–6). Scale bar: 20 μm. (b) JC-1 aggregates (red) and monomers (green) were detected 3 h after OIT treatment by confocal microscopy. Scale bar: 20 μm. (c) Fluorescence intensity of JC-1 aggregates was measured 3 h after OIT treatment by a flow cytometer (n = 3). (d–f) bEnd.3 cells were subjected to Seahorse MitoStress Assay with an acute injection of OIT (n = 3). (d) The profile of the oxygen consumption rate (OCR) was plotted. (e) Parameters of mitochondrial respiration were calculated (f) Extracellular acidification rate (glycolysis) and oxidative phosphorylation (OCR) values were plotted. Arrows indicate metabolic changes between control (vehicle) and OIT-treated cells. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 vs. CON (control).

Figure 4

OIT-induced changes in mitophagy, mitochondrial mass, and mitochondrial morphology in bEND.3 cells. (a) The localization of LC3B (autophagosome) and MitoTracker (mitochondria) was examined 0, 1, and 3 h after treatment with 25 μM OIT by confocal microscopy. Scale bar: 20 μm. Pearson’s correlation coefficient was calculated from three independent experiments using ImageJ software (n = 3). (b) LC3B-II protein levels were detected in the mitochondrial fraction 3 h after treatment with 25 μM OIT (n = 3). (c) Mitochondrial mass was examined 3 h after treatment with OIT by nonyl acridine orange (NAO) staining (n = 3). (d) Changes in mitochondrial morphology and autophagosome formation were detected 3 h after treatment with 5 and 25 μM OIT by TEM (n = 3). M, mitochondria; A, autophagosome; red arrow, swollen mitochondria with distorted cristae; yellow box and yellow arrow, mitochondria-contained vesicle. Scale bar: 200 nm. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 vs. CON (control).

Figure 5

Reversal effects of N-acetyl L-cysteine (NAC) against OIT-mediated effects on GSH level, cell viability, mitochondrial metabolic activity, and endothelial barrier function. (a–c) bEnd.3 cells were pre-treated with NAC for 2 h, and then, the media were replaced by OIT (25 μM)-containing media. (a) The level of reduced GSH was determined in cells 24 h after OIT treatment (n = 3). (b,c) The extent of (b) MTT reduction and (c) LDH release was measured in cells 24 h after OIT (25 μM) treatment (n = 3). (d) bEnd.3 cells were pre-treated with NAC for 1 h and incubated for an additional 1 h with assay media containing NAC during degassing. OIT was then acutely injected into the cells. The profile of the OCR was plotted, and parameters for mitochondrial respiration were calculated (n = 3–4). (e,f) Measurement of (e) TEER and (f) FITC-dextran permeability was conducted in bEnd.3 cells 24 h after OIT (25 μM) treatment with or without NAC pre-treatment. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 vs. CON (control); # p < 0.05, ## p < 0.01 vs. OIT-treated cells.