Contribution of AhR-mediated activation of the AC/cAMP/PKA cascade to 6PPDQ-induced cardiac defects in zebrafish.

The ecological and health risks of 6PPDQ (N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone) have raised significant concerns. We recently demonstrated that the Aryl Hydrocarbon Receptor (AhR) mediates 6PPDQ-caused extrinsic apoptosis and heart defects; however, the underlying molecular pathways remain elucidated. In this study, we observed that inhibition of Protein Kinase A (PKA) effectively mitigated 6PPDQ-caused apoptosis and cardiac dysfunction in zebrafish larvae. Subsequent experiments revealed significant increases in both PKA levels and cyclic adenosine monophosphate (cAMP) concentrations within the hearts of zebrafish embryos exposed to 6PPDQ. Notably, pharmacological inhibition or genetic knockdown of AhR abolished 6PPDQ-induced PKA overexpression. Dual-luciferase reporter assays indicated that AhR activation by 6PPDQ directly enhance the transcription of adcy6a, which encodes an adenylate cyclase (AC) isoform predominately expressed in the heart of zebrafish. Inhibition of β-adrenergic receptors did not significantly influence the AC/cAMP/PKA cascade. Furthermore, exposure to 6PPDQ induced phosphorylation of cAMP response element-binding protein (CREB), an effect that was mitigated by PKA inhibition. Activated CREB subsequently promoted the expression of Fos, a component of the activator protein-1 (AP-1) transcription factor family. Inhibition of Fos (AP-1) counteracted the 6PPDQ-induced overexpression of fas and faslg, as well as the subsequent extrinsic apoptosis. In conclusion, our findings indicate that AhR activation by 6PPDQ triggers AC/cAMP/PKA cascade through direct transcriptional upregulation of adcy6a. PKA-mediated phosphorylation of CREB then facilitates the overexpression of fas and faslg via AP-1, resulting in extrinsic apoptosis and cardiac defects.