Regioselective In Vitro Toxicity of Aromatic Hydroxylated Metabolites of 6PPD-Quinone

Abstract

6PPD-Q undergoes rapid enzymatic reactions within salmonids to form two major hydroxylated metabolites, one alkyl and one aryl. While alkyl–OH-6PPD-Q has been confirmed to be nontoxic, the toxicity of aryl–OH-6PPD-Q remains unknown. We herein synthesized three aryl–OH-6PPD-Q isomers and tested their in vitro toxicity in a coho salmon (O. kisutch) embryo (CSE-119) cell line. para-aryl–OH-6PPD-Q was determined to be the isomer formed in vivo and was marginally more toxic (EC50 = 4.72 μg/L) than the parent 6PPD-Q. The toxicity of aryl–OH-6PPD-Q isomers is highly regioselective, with reduced toxicity observed for meta-aryl–OH-6PPD-Q (EC50 = 32.8 μg/L), while ortho-aryl–OH-6PPD-Q did not exhibit toxicity up to 167 μg/L. The structure-related toxicity trend was also conserved for their methoxy analogues, demonstrating that the regioselective toxicity should not be attributed to the desolvation energy of −OH. Instead, DFT calculations suggest that the reduced toxicity of ortho-aryl–OH-6PPD-Q could be due to its relatively high torsional energy (7.1 kcal/mol) compared to other isomers. In summary, this study highlighted the regioselective toxicity of aryl–OH-6PPD-Q isomers, where steric repulsion and rotational restrictions could possibly play an important role.