* 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
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ABSTRACT |
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Key Words: polybrominated diphenyl ethers; cell culture; accumulation; in vitro.
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INTRODUCTION |
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There is little research to date on the mechanisms by which PBDEs produce these toxicities. Upregulation of hepatic UGTs in vivo (Hallgren and Darnerud, 2002; Zhou et al., 2001
; 2002
) is likely responsible for disruption of thyroid hormone homeostasis. In vitro studies have been used to explore a number of other mechanisms. Kodavanti and Derr-Yellin (2002)
demonstrated that PBDEs alter arachadonic acid release in cerebellar primary cell cultures. Several hepatic cell lines have been used to investigate the potential Ah-activity of PBDEs (Behnisch et al., 2003
; Chen et al., 2001
; Chen and Bunce, 2003
; Kuiper et al., 2004
; Villeneuve et al., 2002
). Meerts et al. (2001)
investigated the (anti)estrogenic potencies of PBDEs in three different cell lines using an estrogen receptor (ER)dependent luciferase reporter gene assay. While several of the PBDEs displayed estrogenic activity, they were 104 to 106 as potent as estradiol.
One area of uncertainty in extrapolation of in vitro mechanistic studies to in vivo effects is the lack of understanding of tissue dose. Many in vitro studies employ a range of exposure concentrations to encompass known or probable in vivo blood or target tissue concentration. Thus, a common assumption of in vitro studies is that medium concentration is a predictive marker of tissue concentration. There are very few data available to support or refute this assumption. Preliminary data suggest that ortegam concentration underpredicts tissue concentration by 30-fold to 150-fold in in vitro models of neurotoxicity (Meacham et al., 2003). Tissue dose in in vitro preparations will depend on the interaction of incubation time, uptake by passive or active mechanisms, bioavailability in media, as well as the lipophilicity of the chemical. With the exception of lipophilicity, the contributions of these factors are rarely known for environmental pollutants. Therefore, the purpose of this study was to characterize the relationship between media and tissue dose for 2,2',4,4'-tetrabromodiphenyl ether (PBDE-47) in an in vitro cell culture model.
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MATERIALS AND METHODS |
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Cell culture. Mixed cell cultures (containing neurons and glia) were prepared from the neocortices of newborn Long-Evans rats according to the method of Chandler et al. (1993) with modifications. Briefly, neocortices were collected under sterile conditions in a buffer solution of 137 mM NaCl, 5 mM KCl, 0.17 mM NaH2PO4, 0.2 mM KH2PO4, 58 mM sucrose, 5 mM D-glucose, and penicillin/streptomycin (100 IU/0.1 mg/ml), pH 7.4. The cortices were minced and digested with trypsin (0.25%) for 5 min, followed by digestion with DNase (0.016%) for 5 min at 37°C. After centrifugation, the supernatant was removed and the tissue was resuspended in DMEM containing 25 mM D-glucose, 10 mM HEPES, and 2 mM glutamine, penicillin/streptomycin (100 IU/0.1 mg/ml), and 10% horse serum. The tissue was dissociated by trituration and then filtered through a 100-µm Nitex screen. Cells were plated at a density of 175,000 cells/cm2 in six-well polystyrene plates (Corning, Inc., Corning, NY) that had been precoated with poly-L-lysine. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. After 3 days in vitro (DIV) the medium was replaced by fresh, serum-containing medium plus 5 µM of cytosine arabinoside to limit the growth of non-neuronal cells. On DIV 5 the medium was again replaced by fresh, serum-containing medium. Growth characteristics of these cells have been reported previously (Mundy and Freudenrich, 2000
).
14C-PBDE-47 accumulation. All experiments were performed on DIV 8. Working solutions were prepared immediately before use as described above and kept at 37°C. Cells were exposed to 14C-PBDE-47 by aspiration of the growth medium from the well of a culture plate and addition of 2 ml of the working solution. The cells were then incubated at 37°C with varying concentrations of 14C-PBDE-47, incubation times, media serum concentrations, and medium volumes.
Concentration curves were determined with a 60-min incubation time and exposure concentrations of 0.013.0 µM. Time course determinations were made using 1.0 µM 14C-PBDE-47 at incubation times of 5, 10, 30, 60, 90, and 120 min. The effects of serum concentration were determined using 1.0 µM 14C-PBDE-47 incubated for 60 min in media containing horse serum concentrations of 0%, 0.1%, 0.3%, 1%, 3%, or 10%. The influence of volume of exposure was determined with a 60-min incubation time using 1.0 µM 14C-PBDE-47 in serum-free medium in a total volume of 2.0 or 4.0 ml. In each experiment at the end of the incubation period, 1.0 ml of working solution was sampled from each well; then the reaction was stopped by aspirating the remaining solution and washing the cells twice with 4 ml of room-temperature phosphate-buffered saline. The stop solution was removed and 1.0 ml of 0.1 M NaOH was added to solubilize the cells.
In all experiments 14C-PBDE-47 in the media at the beginning and end of incubation was determined by liquid scintillation counting of aliquots of the working solution in Ultima Gold scintillation cocktail. 14C-PBDE-47 accumulation was determined by liquid scintillation counting of aliquots of the solubilized cells in NaOH in Ultima Gold scintillation cocktail (Packard Bioscience, Meriden, CT). Percent nonspecific binding to the plastic wells of the culture dish was calculated by subtracting the total 14C-PBDE-47 in tissue and media at the end of the incubation period from the total 14C-PBDE-47 added at the start of incubation.
For each experiment, the wet weight of cells per culture well was estimated by harvesting cells from a sister plate followed by centrifugation to obtain a cell pellet. The weight of the cell pellet was then determined after removal of the supernatant. All data are expressed as µM PBDE accumulated in cells (assuming 1 mg of pellet weight was equivalent to 1 ml of volume), and as percentage of total PBDE (added at the beginning of the incubation period) in cells, medium, and bound to plastic. Each well was considered as an "n" of one, and experiments were repeated at least twice using cultures prepared on separate days.
Statistical analysis. Data were analyzed using an unpaired t-test or one-way analysis of variance (ANOVA) followed by Dunnett's test for comparison to control. The level of significance was set at p < 0.05.
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RESULTS |
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DISCUSSION |
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There was a rapid increase in the concentration of PBDE-47 in the cultured cells. PBDE-47 accumulated to levels 100-fold greater than that in the applied medium. PBDEs are highly lipophilic compounds (Kow ranges from 6.5 to 10; European Union (EU), 2001) and do not dissolve readily in aqueous solutions. As would be expected for a compound with a high octanol-water partition coefficient, PBDE-47 partitioned out of the exposure solution and accumulated in cells or bonded to the plastic culture dish. The kinetic experiment showed that there was a redistribution of PBDE-47 out of the medium which did not reach equilibrium. After 120 min only 37% of the original mass of PBDE-47 remained in solution. Thus, the actual concentration of PBDE-47 in the medium was equal to the initial exposure concentration only at time zero. This accumulation of a lipophilic PBDE congener in vitro is similar to that observed after in vitro exposure to PCBs (Meacham et al., 2003
), which are also highly lipophilic (Kow = 5.6 to 8.3; ATSDR, 1997
).
The accumulation of PBDE-47 into cells may be the result of a passive mechanism driven by the high octanol-water partition coefficient. However, there are transporters that can act to move lipophilic compounds across cell membranes. For example, TCDD and other lipophilic chemicals can enter cells through a facilitated transport mechanism bound to hepatic lipoproteins (Souse et al., 1989). At this time there are no data to support or refute an active transport mechanism for PBDEs. In the present study, at every concentration of PBDE-47 tested a similar proportion of the applied concentration accumulated in the cells, indicating that uptake/accumulation was not limited by a saturable, carrier-mediated process. Although in vitro preparations provide a simplified biological system that can be useful for studies of mechanism-of-action, care should be taken when associating the concentrations used in vitro to those observed in vivo. The current data show that the applied concentration greatly underestimates the actual concentration in tissue after in vitro exposure.
We examined the effect of different concentrations of horse serum in the exposure medium on PBDE-47 accumulation. Many in vitro model systems require the addition of growth factors to maintain the health and viability of the tissue. For some in vitro systems, these factors have been identified and can be added as part of a "defined" medium, but in many cases the growth factors are unknown. Thus, it is common to add animal sera (such as fetal bovine sera or horse sera) to the growth medium at concentrations of up to 10%. In the present study, as the proportion of serum in the exposure medium increased, the accumulation of PBDE-47 in cells decreased (although there was still an approximate 20-fold accumulation of PBDE-47 in cells exposed to medium containing 10% serum). The increased amount remaining in the medium suggests that PBDE-47 may be binding to serum proteins. Animal sera contains proteins (including albumins and globulins), polypeptides, and hormones that can provide both specific and nonspecific binding sites. For example, other lipophilic compounds such as PCBs and TCDD (which are structurally similar to PBDEs) show a high rate of binding to blood lipoproteins (Busbee et al., 1985; Henderson and Patterson, 1988
). In addition, horse serum contains the thyroid hormonebinding protein transthyretin, which has been shown to bind PBDEs as well as PCBs (Meerts et al., 2000
; Chauhan et al., 2000
). Thus, it is likely that addition of horse serum to the exposure solution provided a significant number of binding sites for PBDE-47, effectively removing it from the pool available for accumulation into the cells. The addition of calf serum to culture media resulted in a similar decrease in the uptake of TCDD by a hepatocellular carcinoma cell line (Hestermann et al., 2000
). These data highlight the need to monitor the constituents of the exposure solution, because they may influence the bioavailability of a compound in vitro and should be considered when comparing data across studies. The addition of serum to the medium (which may be more analogous to the physiologic situation when tissues are exposed to chemicals via the blood) substantially altered the accumulation of PBDE-47.
The volume of exposure also influenced the accumulation of PBDE-47. Doubling the exposure volume while keeping the concentration constant (and thus doubling the total mass of PBDE-47) resulted in an increased mass of PBDE-47 accumulated in the cells. Similar findings were reported for PCBs (Meacham et al., 2003). These data suggest that for some lipophilic compounds the total mass, rather than the concentration, is the more important determinant for availability and uptake. If, as suggested above, accumulation of PBDE-47 is a passive process driven by the high Kow, then these results are not surprising and could be accounted for, in part, by the laws of mass action. These results also reinforce the fact that care must be taken when comparing results across different in vitro systems.
These results demonstrate that PBDE-47 accumulates up to 100-fold in neuronal cells exposed in vitro. Further, this accumulation in cells is influenced by length of exposure, constituents of the exposure solution, and total media volume applied. In addition, a substantial amount of compound appears to bind to the polystyrene plastic commonly used in the manufacture of tissue culture vessels. These data demonstrate that the use of media concentration is not a good surrogate of in vitro tissue concentrations of PBDE-47 and may drastically underestimate tissue concentration. Accurate information on the tissue concentration for in vitro experiments should be obtained experimentally.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at U.S. Environmental Protection Agency, Neurotoxicology Division, B10506, Research Triangle Park, NC 27711. Fax: (919) 5410700. E-mail: mundy.william{at}epa.gov.
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