EXPLORING THE METABOLISM AND MIXTURE EFFECTS OF THE ISOTHIAZOLINONE BIOCIDES BIT AND OIT IN PLHC-1 CELLS

The powerful antimicrobial properties of isothiazolinones make them a common preservative in both industrial and consumer products. However, methylated derivatives of isothiazolinones have been associated with concerning allergenic effects leading to stricter regulations. As a consequence, industries have turned to more lipophilic isothiazolinones, such as benzisothiazolinone (BIT) and octylisothiazolinone (OIT), which are less regulated. Similar effects have however been reported for exposures of BIT and OIT, highlighting the importance of expanding the knowledge about these compounds, to enforce stricter regulation and potentially controlling the isothiazolinones as a group. This study aimed to investigate the toxicodynamic and toxicokinetic responses in a fish liver cell model system, Poeciliopsis lucida hepatocellular carcinoma (PLHC-1) following exposure to BIT and OIT, alone and in mixture. Results from the ethoxyresorufin-O-deethylase (EROD) assay revealed no apparent induction of the common detoxification enzyme CYP1A after exposure to BIT and OIT. However, more data is needed for a more robust evaluation of whether the enzyme is involved in their metabolism. Analysis of cultured exposure media in high performance liquid chromatography (HPLC) demonstrated cellular biotransformation of BIT into a more polar metabolite. A rapid cellular uptake of OIT was observed, however no potential OIT metabolite could be detected, neither extracellularly nor intracellularly. A cytotoxic assessment was also conducted on exposed PLHC-1 cells investigating the mitochondrial activity and membrane integrity, common markers for cellular viability. Significant toxic effects were observed on both markers after exposure to 10 µM OIT, while no toxic effect was observed for the same concentration of BIT. However, co-exposure to these concentrations of OIT and BIT, displayed an even greater toxicity, indicating a synergistic mixture effect between the two. While this study provides valuable insights into the toxicodynamic and toxicokinetic profiles of BIT and OIT, highlighting the importance of considering mixture effects in assessing their potential health and environmental impacts, additional research is needed to fully understand their specific metabolic pathways and interaction mechanisms, to assess their long-term exposure effects.