Structure-Dependent Cytotoxicity of Environmentally Relevant Benzothiazole Derivatives Across Human Cell Models

Benzothiazoles are widely used rubber additives and common contaminants in urban runoff, yet their toxicity across human tissues remains poorly characterized. We evaluated the cytotoxicity of six benzothiazole derivatives, benzothiazole (BTH), 2-hydroxybenzothiazole (OH-BTH), 2-aminobenzothiazole (NH 2 -BTH), 2-methylbenzothiazole (Me-BTH), 2-mercaptobenzothiazole (SH-BTH), and methyl-thiobenzothiazole (Me-S-BTH), across nine human cell lines representing intestinal, skin, lung, kidney, brain, endothelial, muscle, epithelial, and hepatic tissues. EC 50 values spanned more than nine orders of magnitude (1.5 × 10 -9 M to >1 M), revealing strong compound- and cell-type-specific variability. SH-BTH was the most potent derivative, with EC 50 values of 1.5 × 10 -9 M in Caco-2 (intestinal cells), 1.1 × 10 -8 M in HMC-3 (brain microglia cells), 3.9 × 10 -5 M in MRC-5 (lung fibroblasts), 6.6 × 10 -4 M in HMEC-1 (microvascular endothelial cells), and 1.3 × 10 -3 M in HepaRG (hepatocytes). Me-S-BTH showed similarly high potency in HMC-3 (2.0 × 10 -8 M) and moderate toxicity in endothelial and hepatic cells (2.3 × 10 -3 M and 1.1 × 10 -3 M, respectively). In contrast, BTH and Me-BTH exhibited low cytotoxicity, with EC 50 values typically ≥10 -3 M and no measurable toxicity (>1 M) in several models, including HepaRG. OH-BTH and NH 2 -BTH showed intermediate effects, with micromolar activity in selected cell lines but minimal toxicity in hepatic cells. Caco-2 and HMC-3 cells were the most sensitive models, whereas HepaRG, RMS13 (skeletal muscle), and skin HEK001 (skin keratinocytes) were the least responsive. Overall, toxicity was strongly structure-dependent, with sulfur-containing derivatives showing the highest potency.