ToxSci Advance Access originally published online on July 28, 2004
Toxicological Sciences 2004 82(1):164-169; doi:10.1093/toxsci/kfh239
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Toxicological Sciences vol. 82 no. 1 © Society of Toxicology 2004; all rights reserved.
Accumulation of PBDE-47 in Primary Cultures of Rat Neocortical Cells
* Neurotoxicology Division and Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA
Received June 24, 2004; accepted July 27, 2004
A number of recent studies have examined the neurotoxic actions of polybrominated diphenyl ethers (PBDEs) using in vitro cell culture models. However, there are few data reporting the final concentration of PBDEs in cells after in vitro exposure to these compounds. To address this issue, the present study examined the concentration-dependent and time-dependent accumulation of 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) in primary cultures of rat neocortex. Mixed cultures of neuronal and glial cells were prepared from the neocortex of newborn rats and grown for 7 days in vitro. The cells were then exposed to freshly prepared serum-free culture medium containing 14C-PBDE-47. Radiolabel associated with the cells or remaining in the medium was determined by liquid scintillation spectrometry. Exposure to 0.01–3.0 µM PBDE-47 for 60 min resulted in a concentration-dependent accumulation in cells. At each concentration, approximately 15% of the applied PBDE-47 was associated with the cells, resulting in a 100-fold magnification of the applied concentration (e.g., a 60-min exposure to 1 µM resulted in an approximate 100 µM concentration in the cells); 55% of the PBDE remained in the medium and 30% was associated with the plastic culture dish. Exposure to 1 µM PBDE-47 resulted in a linear increase in PBDE-47 in cells with time for the first 60 min, which began to saturate at 120 min. Addition of serum proteins to the medium decreased accumulation; at 10% serum in the medium, only 3% of the applied PBDE-47 was associated with the cells and 96% remained in the media after 60 min. The total volume of exposure also influenced accumulation of PBDE-47. Doubling the volume of serum-free exposure medium (from 2 ml to 4 ml) but leaving the concentration constant (1 µM) resulted in a 1.5-fold increase in PBDE-47 concentration in the cells. These data show that a number of factors, including duration of exposure, volume of exposure, and concentration of serum proteins in the medium, can influence the accumulation of PBDE-47 in cells in vitro. For this highly lipophilic compound, use of medium concentration underestimates tissue concentration by up to two orders of magnitude. Thus, accurate information on the tissue concentration for in vitro experiments should be determined empirically.
Key Words: polybrominated diphenyl ethers; cell culture; accumulation; in vitro.
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