Chicken Embryo Cardiomyocyte Cultures—A New Approach for Studying Effects of Halogenated Aromatic Hydrocarbons in the Avian Heart

Abstract

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and other halogenated aromatic hydrocarbons (HAHs) elicit a variety of adverse biological effects on the cardiovascular systems of mammalian, piscine and avian species. Many of the cardiotoxic effects of HAHs are mediated by the aryl hydrocarbon receptor (AHR). Induction of cytochrome P4501A (CYP1A) is a well-known AHR-dependent response to HAHs in the liver, but there are a limited number of studies on CYP1A induction by these compounds in the heart. We used an in vitro approach to examine effects of TCDD and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) on CYP1A in the avian heart. The responses of primary cultures of chicken embryo cardiomyocytes (CEC) and chicken embryo hepatocytes (CEH) to TCDD and PCB 77 were compared using immunofluorescence staining for CYP1A, the ethoxyresorufin-O-deethylase (EROD) assay, and real-time RT-PCR analysis of CYP1A4 mRNA and CYP1A5 mRNA. Immunofluorescent detection of CYP1A indicated that induction of CYP1A by TCDD was localized within the cytoplasm of CEC cells. EROD activity and CYP1A4/5 mRNA levels were strongly induced in CEC and CEH cultures by TCDD and PCB 77, and the shapes of the concentration-response curves in CEC and CEH cultures were similar. The studies provide clear evidence that the AHR signaling pathway is induced by TCDD and PCB 77 in CEC, and establish a new in vitro approach for studying the effects of HAHs in the avian heart. Induction of CYP1A5 by TCDD in avian cardiomyocytes is a novel finding, and might help direct future studies on mechanisms of action of HAHs in the heart.