PW09 Exposure and Effect Endpoints
Exhibit Hall
8:00 AM - Wednesday, 12 November 2003
(PW168) Characterization of the hypoxic functional response using a fish model system.
Billiard, S1, Rau, M1, Rees, B2, Di Giulio, R1, 1 Duke University, Durham, NC, USA2 University of New Orleans, New Orleans, LA, USA
ABSTRACT- Both the aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor-1
(HIF-1a) require the AHR nuclear translocator protein (ARNT) for AHR- and hypoxia-mediated signaling, respectively. In mammalian systems, activated AHR has been shown to compete for and sequester ARNT from the HIF homologous pathway in vitro. Because cardiovascular development and survival during vertebrate embryogenesis depends on the cell's ability to respond to hypoxia, we hypothesize that AHR agonists may interfere with normal cardiovascular development and function in fish, especially at early life stages, through disruption of the HIF transcriptional pathway. Current studies are investigating the potential for cross talk between AHR and hypoxia pathways in vitro using a fish-specific hypoxia-responsive reporter gene assay. Preliminary experiments were designed to characterize the hypoxic functional response in the PLHC-1 cell line, derived from a hepatocellular carcinoma in the topminnow, using the pN400 plasmid construct. This plasmid contains approximately 400 bases of the Fundulus heteroclitus lactate dehydrogenase-B (LDH-B) promoter cloned upstream of the firefly luciferase gene and includes both the hypoxia responsive element (HRE) and promoter functions of the gene. PLHC-1 cells were co-transfected in duplicate with a range of pN400 and Renilla plasmid DNA for either 5 or 24 hours. These studies were used to optimize transfection time (5h) and experimental:control reporter DNA ratios for further study. In hypoxic experiments, transfected cells were exposed for 24h to cobalt chloride in order to mimic hypoxia. Preliminary results suggest that chemical hypoxia significantly increased HRE-reporter expression (∼1.5 fold) relative to transfected controls. Future studies will investigate the time course of maximal firefly luciferase expression induced by cobalt exposure and determine whether this expression is altered in PLHC-1 cells dosed with AHR agonists.
Key words: HIF, AHR , cross talk, PLHC-1