Chronic toxicity mechanisms of 6PPD and 6PPD-Quinone in zebrafish

Abstract

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its oxidation derivative, 6PPD-quinone (6PPDQ), have been extensively detected in environmental and biological samples, raising significant concerns regarding their chronic aquatic toxicity at environmentally relevant concentrations. However, the underlying mechanisms driving this chronic toxicity remain largely unexplored. Here we show that zebrafish exposed to 6PPD and 6PPDQ exhibit distinct toxicokinetic profiles, with 6PPD preferentially accumulating in the liver and 6PPDQ predominantly targeting the brain. Exposure to both compounds impaired zebrafish growth, induced hepatic damage, and disrupted locomotor behavior. Transcriptomic analysis of liver tissue revealed disturbances in lipid and carbohydrate metabolic pathways in both treatment groups, with distinct differences in gene expression patterns and biochemical responses between 6PPD and 6PPDQ. Specifically, both compounds downregulated peroxisome proliferator-activated receptor gamma (PPARγ) and elevated the expression of pro-inflammatory cytokines (TNF-α and IL-6). Molecular dynamics simulations and surface plasmon resonance experiments further demonstrated that hepatotoxicity was associated with direct binding of these compounds to PPARγ, a critical regulator of lipid metabolism and inflammation. Our findings highlight the hepatotoxic risks of 6PPD and 6PPDQ to aquatic life. Importantly, 6PPDQ exhibited greater toxicity compared to 6PPD, emphasizing an urgent need for targeted environmental controls and regulatory actions to mitigate ecological harm and potential public health consequences.

Keywords: 6PPD; 6PPDQ; Hepatotoxicity; PPARγ; zebrafish.

Graphical abstract

Fig. 1

Bioaccumulation and elimination kinetics of 6PPD (a–d) and 6PPDQ (e–h) in brain (a, e), liver (b, f), gonads (c, g), and muscle (d, h) tissues. Values are presented as mean ± standard deviation (n = 3).

Fig. 2

Growth and hepatic morphological changes in zebrafish after three-month exposure to 6PPD or 6PPDQ. a–c, Growth parameters following chemical treatments: body length (a), body weight (b), and hepatosomatic index (c). The three dashed lines within each violin plot represent the 25th percentile, median, and 75th percentile, respectively (n = 3). d, f, Liver sections stained with hematoxylin-eosin (d) and oil red O (f). e, g, Quantification of hematoxylin-eosin-positive areas (e) and oil red O-stained areas (g). Black arrows indicate lipid vacuoles, red arrows indicate pyknotic nuclei, and yellow arrows indicate lipid droplets. Values are presented as mean ± standard deviation (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 indicate statistically significant differences compared to vehicle control (VC).

Fig. 3

Hepatic toxicity biomarker responses in zebrafish after three-month exposure to 6PPD or 6PPDQ. a–c, Liver function indices: alanine aminotransferase (ALT, a), aspartate aminotransferase (AST, b), and alkaline phosphatase (ALP, c) activities. d–g, Antioxidant indicators in the liver: superoxide dismutase (SOD, d), glutathione peroxidase (GSH-Px, e) activity, malondialdehyde (MDA, f) content, and reactive oxygen species (ROS, g) content. The three dashed lines within each violin plot represent the 25th percentile, median, and 75th percentile, respectively (n = 3). h–i, Radar plots of liver indicators following exposure to 6PPD (h) and 6PPDQ (i). j, Integrated biomarker response analysis (IBR) values. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 indicate statistically significant differences compared to vehicle control (VC).

Fig. 4

Transcriptome analysis. a–b, Top 15 enriched KEGG pathway analysis of DEGs in the 6PPD (a) and 6PPDQ (b) groups. P_adj: adjusted P-value. c–d, Protein interaction networks for 6PPD (c) and 6PPDQ (d). The node's size represents the protein's importance or the degree of connectivity with other proteins. e–f, Coregulatory networks of liver glucose and lipid metabolism in 6PPD (e) and 6PPDQ (f). T-CHO: total cholesterol. LDL-C: low-density lipoprotein cholesterol. HDL-C: high-density lipoprotein cholesterol. TBA: total bile acid. TCA: Tricarboxylic acid cycle. ATP: adenosine triphosphate. Red indicates upregulation and blue indicates downregulation, with intensity reflecting log₂(fold change). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 indicate statistically significant differences compared to vehicle control (VC).

Fig. 5

Behavioral responses of zebrafish following three-month exposure to 6PPD or 6PPDQ. a, Representative movement trajectory in the open field test. b, Swimming distance. c, Dwell time in the dark zone. d, Cumulative immobile duration in the light. Values are presented as mean ± standard deviation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 indicate statistically significant differences compared to vehicle control (VC).

Fig. 6

Effects of 6PPD or 6PPDQ on liver inflammation indicators after three-month exposure in zebrafish. a–b, Immunofluorescence staining for interleukin-6 (IL-6), peroxisome proliferator-activated receptor gamma (PPARγ), and tumor necrosis factor-alpha (TNF-α) in the 6PPD (a) and 6PPDQ (b) groups. c, Fluorescence intensity of IL-6, PPARγ, and TNF-α. Values are presented as mean ± standard deviation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 indicate statistically significant differences compared to vehicle control (VC).

Fig. 7

a–b, Western blot analysis of interleukin-6 (IL-6), peroxisome proliferator-activated receptor gamma (PPARγ), and tumor necrosis factor-alpha (TNF-α) in the 6PPD (a) and 6PPDQ (b) groups. c, Relative protein expression levels of IL-6, PPARγ, and TNF-α. Values are presented as mean ± standard deviation. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 indicate statistically significant differences compared to vehicle control (VC).

Fig. 8

Molecular interactions between 6PPD or 6PPDQ and peroxisome proliferator-activated receptor gamma (PPARγ). a, Conformations of 6PPD-zfPPARγ and 6PPDQ-zfPPARγ at 200 ns. Residues highlighted in dark gray circles represent amino acids involved in hydrophobic interactions with the ligand, without forming explicit hydrogen bonds or other specific interactions. b–c, Root mean square deviation (RMSD) changes over time in 6PPD-zfPPARγ (b) and 6PPDQ-zfPPARγ (c). d, Root mean square fluctuation (RMSF) curves. e–g, Surface plasmon resonance sensorgrams for GW9662 (a PPARγ antagonist, e), 6PPD (f), and 6PPDQ (g) binding to hPPARγ. KD: the equilibrium dissociation constant. zfPPARγ: zebrafish PPARγ. hPPARγ: human PPARγ.