Bioaccumulation of N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) and its potential cardiotoxicity in larval zebrafish (Danio rerio)

Abstract

N-(1,3-dimethylbutyl)-N-phenyl-p-phenylenediamine (6PPD) is a type of p-phenylenediamine (PPD)-based additive widely used in the manufacture of rubber tires towing to its excellent antiozonant properties. This study investigated the bioaccumulation hazards of 6PPD. We noted bioaccumulation of up to 2,657 ng/g 6PPD in zebrafish larvae on being exposed to 100 μg/L 6PPD for 12 h. While the LC50 of 6PPD for larval zebrafish at 96 hpf was approximately 737 μg/L. Treatment with 100μg/L 6PPD for 7 days could induce oxidative stress and cell apoptosis in zebrafish in their early developmental stages. Moreover, transcriptome analysis showed that 6PPD exposure could potentially cause cardiotoxicity in larval zebrafish by affecting the transcription of the genes related to the calcium signal pathway, cardiac muscle contraction, etc. qRT-PCR verification of the genes related to calcium signaling pathway ( slc8a2b, cacna1ab, cacna1da, and pln) revealed that these genes were significantly downregulated in larval zebrafish after exposure to 6PPD. Simultaneously, the mRNA levels of the genes related to cardiac functions (myl7, sox9, bmp10, and myh71) also respond accordingly. H&E staining and heart morphology investigation indicated cardiac malformation in the larvae exposed to 6PPD. Furthermore, the phenotypic observation of transgenic Tg (myl7: EGFP) zebrafish also confirmed that 6PPD exposure could change the distance of atria and ventricles of the heart and inhibit some key genes related to cardiac function in larval zebrafish. Overall, our study has revealed the toxic effects of 6PPD exposure on the cardiac system of zebrafish.

Keywords: 6PPD, zebrafish larvae, cardiac toxicity, Oxidative stress, apoptosis