6PPD, Not 6PPD-Quinone, Induced Serious Zebrafish Eye Damage by Disrupting the Thyroid Signaling Pathway

N-(1,3-Dimethylbutyl)-<i>N</i>'-phenyl-1,4-phenylenediamine (6PPD) and its oxidation product 6PPD-quinone (6PPDQ) showed different acute toxicities and bioaccumulation potencies in fish. In this study, we compared the thyroid disrupting effects of 6PPD and 6PPDQ through <i>in vitro</i>, <i>in silico</i>, and <i>in vivo</i> assays. Interestingly, although 6PPD and 6PPDQ showed similar docking affinities with thyroid hormone receptor (TR) isoforms and GH3 cell inhibition effects, the thyroid signaling pathway, eye development, phototactic behaviors, and cell density in the retinal layer in the larval zebrafish were significantly affected only following 6PPD exposure. Further investigation demonstrates that 6PPD can act as a TR antagonist to reduce the opsin protein abundance and inhibit the cone photoreceptor cell proliferation, which finally alters the retinal layer structure and causes microphthalmus in zebrafish. Especially, under environmental relevant concentration exposure, 6PPD induced alterations of <i>trβ</i>, <i>opn1lw1</i>, <i>opn1mw1</i>, <i>rpe65a</i>, <i>nr2e3</i> gene expressions although no significant eye histopathological change was observed. This study illustrates for the first time the more serious visual system impairment of 6PPD compared to 6PPDQ, with thyroid signaling disruption being a contributing factor, while other important toxic targets still require further research.