CMIT/MIT produces mitochondrial ROS via inhibiting mitochondrial complex I and II

A 3:1 mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT) is an isothiazolinone biocide commonly used as preservative in various consumer products. It is also the active ingredient in humidifier disinfectants that were widely used in South Korea, and its toxicity on the respiratory system have been investigated. CMIT/MIT exhibits strong electrophilicity and easily binds to sulfhydryl groups in biological molecules. Although oxidative stress is considered a key mechanism underlying CMIT/MIT-induced toxicity, the specific molecular targets responsible for ROS generation remain unclear. In this study, we aimed to investigate the effect of CMIT/MIT on redox regulation within mitochondria, a major site of ROS production. Our findings indicate that CMIT/MIT induces mitochondrial bioenergetic perturbation by inhibiting mitochondrial Complexes I and II in in vitro experiments using H441 cells. Notably, CMIT/MIT showed high binding affinity for succinate dehydrogenase complex subunit B (SDHB), which contains the iron-sulfur cluster (ISC) binding site. Furthermore, inhibition of SDHA amplified CMIT/MIT-induced ROS production, an effect that was reversed by mitochondrial uncoupling agents. Finally, we observed that CMIT/MIT-induced mitochondrial dysfunction led to dysregulation of mitochondrial dynamics and promoted cellular senescence. Taken together, our results suggest that mitochondrial complexes I and II serve as molecular initiating events in CMIT/MIT-induced cytotoxicity, ultimately contributing to lung injury.