6PPD quinone induces cytokine production in fish cells via oxidative stress

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) is widely used as an additive in tire manufacturing. 6PPD quinone (6PPDQ), a derivative of 6PPD formed in the environment, is toxic to salmonid fish, particularly to coho salmon (Oncorhynchus kisutch). The sensitivity to 6PPDQ varies among fish species at the cellular level. In this study, we compared the effects of 6PPDQ on cell lines from three salmonid species: coho salmon (CSE-119 cells), rainbow trout (RTG-2 cells), and Chinook salmon (CHSE-214 cells). We also examined the effects of 7PPD and 8PPD and their quinone derivatives, which are structural analogues of 6PPD. 6PPDQ caused significant metabolic and mitochondrial inhibition, as well as cell membrane damage, in CSE-119 cells. 6PPDQ treatment also exhibited cytotoxicity in RTG-2 cells, but the effect was weaker compared with CSE-119 cells. In contrast, 6PPDQ had minimal effects on CHSE-214 cells. 6PPD and 6PPDQ decreased mitochondrial membrane potential and increased oxidative stress in CSE-119 cells. In contrast, mitochondrial membrane potential was increased in RTG-2 and CHSE-214 cells. Additionally, it upregulated the expression of the genes encoding interleukin (IL)-8 and IL-1β in CSE-119 and RTG-2 cells. 6PPDQ also promoted the expression of the gene encoding heme oxygenase-1 (HO-1), an oxidative stress response protein, in RTG-2 cells. 6PPDQ induced mitochondrial dysfunction, oxidative stress, and cytokine gene expression in CSE-119 and RTG-2 cells. In contrast, CHSE-214 cells showed high resistance to oxidative stress. This resistance likely plays a critical role in the species-specific response to 6PPDQ. Furthermore, 7PPDQ and 8PPDQ exhibited lower cytotoxicity in CSE-119 and RTG-2 cells than 6PPDQ, suggesting that the carbon number may be important in the cytotoxicity of 6PPDQ. This study provides valuable data for reducing the toxicity to fish and environmental impact of 6PPDQ as a tire additive.