Conformation of CD40LG and ANXA5 as Key Events of 2,7-Dibromocarbazole-Induced Cardiotoxicity Using in Vivo and in Vitro 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 in vivo (Sprague-Dawley rats) and in vitro (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). CD40LG promotes apoptosis and inhibits cell growth through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, and ANXA5 plays a vital role in apoptosis by possessing Ca2+ channel activity. Apoptosis-focused PCR array and Ingenuity Pathway Analysis further indicated CD40LG and ANXA5 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 Ppp2ca 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 CD40LG and ANXA5 are potential downstream KEs in regulating 2,7-DBCZ-induced cardiotoxicity, as they exhibited the same transcription patterns with the AhR activation indicators CYP1A1 and CYP1B1. 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.