Conformation of <i>CD40LG</i> and <i>ANXA5</i> as Key Events of 2,7-Dibromocarbazole-Induced Cardiotoxicity Using <i>in Vivo</i> and <i>in Vitro</i> Models

Polyhalogenated carbazoles (PHCZs) are emerging dioxin-like compounds (DLCs) with uncertain toxicological effects. Cardiotoxicity caused by DLCs has been well-characterized within the framework of an adverse outcome pathway, demonstrating consistent aryl hydrocarbon receptor (AhR) activation as the molecular initiating event. However, the downstream key events (KEs) contributing to PHCZ-induced cardiotoxicity have been less characterized. Herein, PHCZ-induced cardiotoxicity was evaluated by <i>in vivo</i> (Sprague-Dawley rats) and <i>in vitro</i> (rat cardiomyocytes) models, and the underlying mechanisms were confirmed using rat cardiomyocytes (H9c2 cells). As a model PHCZ, 2,7-dibromocarbazole (2,7-DBCZ) induced significant rat cardiac tissue apoptosis following exposure to 100 μg/kg/d via intragastric infusion. In addition, 2,7-DBCZ also induced apoptosis and suppressed the mitochondrial membrane potential in H9c2 cells (at 10^-5 M). <i>CD40LG</i> promotes apoptosis and inhibits cell growth through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, and <i>ANXA5</i> plays a vital role in apoptosis by possessing Ca²⁺ channel activity. Apoptosis-focused PCR array and Ingenuity Pathway Analysis further indicated <i>CD40LG</i> and <i>ANXA5</i> as upstream regulators for 2,7-DBCZ-induced apoptosis. 2,7-DBCZ increased Akt phosphorylation and intercellular Ca²⁺ levels, which were reversed by pretreatment with an Akt agonist and a Ca²⁺ inhibitor. Silencing of <i>Ppp2ca</i> further confirmed Ca²⁺ as a key intercellular messenger in regulating induced apoptosis. Finally, rat cardiac tissue and H9c2 cell gene profiles and AhR antagonist cotreatment demonstrated that <i>CD40LG</i> and <i>ANXA5</i> are potential downstream KEs in regulating 2,7-DBCZ-induced cardiotoxicity, as they exhibited the same transcription patterns with the AhR activation indicators <i>CYP1A1</i> and <i>CYP1B1</i>. These findings offer crucial new insight into the mechanisms underlying PHCZ-induced cardiotoxicity, providing valuable mode of action data that can be used in the Weight of Evidence components of risk assessments and regulation decisions regarding these emerging contaminants.