Accumulation of PBDE-47 in Primary Cultures of Rat Neocortical Cells

William R. Mundy*,1, Theresa M. Freudenrich*, Kevin M. Crofton* and Michael J. DeVito{dagger}

* Neurotoxicology Division and {dagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polybrominated diphenyl ethers (PBDEs), produced commercially as mixtures, are commonly used as flame-retardants for various consumer products including electronic equipment. Increasing concentrations in environmental samples and human breast milk (Meironyté et al., 1999Go; Sellstrom et al., 1993Go; Stern and Ikonomou, 2000Go) has focused worldwide attention on the potential health effects of PBDEs (Darnerud et al., 2001Go; Hooper and McDonald, 2000Go; McDonald, 2001Go). Current evidence for health effects based on data from animal models includes endocrine disruption (Darnerud and Sinjari, 1996Go; Fowles et al., 1994Go; Zhou et al., 2001Go; 2002Go), developmental neurotoxicity (Branchi et al., 2002Go; Eriksson et al., 2002Go), and reproductive toxicity (Stoker et al., 2004Go).

There is little research to date on the mechanisms by which PBDEs produce these toxicities. Upregulation of hepatic UGTs in vivo (Hallgren and Darnerud, 2002Go; Zhou et al., 2001Go; 2002Go) 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)Go 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., 2003Go; Chen et al., 2001Go; Chen and Bunce, 2003Go; Kuiper et al., 2004Go; Villeneuve et al., 2002Go). Meerts et al. (2001)Go 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 10–4 to 10–6 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., 2003Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and solutions. 2,2',4,4'-tetrabromo[14C]diphenyl ether (14C-PBDE-47; 55 µCi/mg, stated purity > 96%) was a generous gift from Great Lakes Chemical Corp. (Indianapolis, IN). All media and reagents for use in tissue culture were purchased from GIBCO/Invitrogen (Carlsbad, CA). All other reagents were purchased from Sigma Chemical Company (St. Louis, MO). A 10-mM stock solution of 14C-PBDE-47 was prepared in DMSO and stored in a polypropylene tube at 4°C. On the day of use, working solutions were prepared by dilution of the stock solution into serum-free medium (DMEM containing 25 mM D-glucose, 10 mM HEPES, and 2.0 mM glutamine; pH 7.4) in a 15-ml polystyrene tube. To confirm the concentration a 1.0-ml aliquot was taken immediately after preparation and counted using liquid scintillation spectrometry. If the working solutions were allowed to sit at 37°C for periods longer than 15 min, it was noted that the concentration of 14C-PBDE-47 in solution would progressively decrease (data not shown), presumably by binding to nonspecific sites on the plastic tube. Thus, in all experiments the working solutions were prepared fresh and applied to the cells within 10 min. The final concentration of DMSO in the working solutions was <0.1%.

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)Go 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, 2000Go).

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 0.01–3.0 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure of neocortical cell cultures to 14C-PBDE-47 (0.01–3.0 µM) for 60 min resulted in a concentration-dependent accumulation in cells (Fig. 1A). There was an ~100-fold increase in the tissue concentration relative to all applied media concentrations. For example, exposure to 1 µM 14C-PBDE-47 in the medium for 60 min resulted in a cellular concentration of 100 µM. The relationship between medium and tissue concentration was best described with a linear regression model (y = 100.1x + 0.01, r2 = 0.96). Data in the graph (Fig. 1) are plotted on a log-log scale for ease of visual inspection. The percentage of the applied dose associated with cells, medium, and the plastic tissue culture plate after 60 min is shown in Figure 1B. For all concentrations tested, approximately 15% of the applied dose was associated with the cells. The 14C-PBDE-47 remaining in the medium ranged from 45% to 65%. The remaining 14C-PBDE-47 that was not recovered was assumed to be associated with the plastic tissue culture plate and ranged from 20% to 40%. The mass of PBDE-47 associated with cells, remaining in the medium, or associated with the plastic is shown in Figure 1C. There is a linear relationship between exposure and mass of PBDE-47 associated with each fraction up to the highest exposure level examined.



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FIG. 1. Concentration-dependent accumulation of PBDE in cultures of neocortical cells exposed to increasing concentrations of 14C-PBDE-47 for 60 min. (A) PBDE accumulation in µM based on wet weight of cells. Data are mean ± SEM (n = 3). (B) Average distribution of PBDE in cells, remaining in medium, and bound to the plastic tissue culture plate as a percent of the total applied dose. (C) Mass of PBDE in cells, remaining in medium, and bound to the plastic tissue culture plate as a function of total applied mass of PBDE.

 
The time-course of 14C-PBDE-47 accumulation in cells is shown in Figure 2A. Accumulation was linear up to approximately 90 min. The rate of accumulation decreased between 90 min and 120 min. The time-dependent accumulation of 14C-PBDE-47 in cells is also reflected in the percentage of the applied dose associated with cells, medium, and the plastic tissue culture plate (Fig. 2B). These data demonstrated that the recovery of 14C-PBDE-47decreased with increasing time (i.e., the percentage of the applied dose assumed to be associated with the plastic tissue culture plate increased). The percentage of the applied dose associated with cells, medium, and plastic at 120 min was approximately 25%, 37%, and 38%, respectively.



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FIG. 2. Time course of PBDE accumulation in cultures of neocortical cells exposed to 1.0 µM 14C-PBDE-47 for 5–120 min. (A) PBDE accumulation in µM based on wet weight of cells. Data are mean ± SEM (n = 3). (B) Average distribution of PBDE in cells, remaining in medium, and bound to the plastic tissue culture plate as a percentage of the total applied dose.

 
The data presented above were obtained by exposing cells to PBDE in serum-free medium. In some cases cultured cells may be exposed to xenobiotics in growth media that contain animal sera. Thus, we examined the influence of serum-containing medium on the accumulation of 14C-PBDE-47. Cells were exposed for 60 min to 1 µM 14C-PBDE-47 prepared in medium containing 0–10% horse serum. There was a decrease in cellular accumulation of 14C-PBDE-47 as the percentage of horse serum in the medium increased (Fig. 3A). This decrease was significant at serum concentrations of 3% and greater. Examination of the percentage of the applied dose associated with cells, medium, and plastic shows that as the serum concentration increased, an increasing proportion of the 14C-PBDE-47 remained in the medium (Fig. 3B).



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FIG. 3. Effect of addition of horse serum to the medium on the accumulation of PBDE. Cells were exposed for 60 min to 1.0 µM 14C-PBDE-47 prepared in medium containing 0–10% serum. (A) PBDE accumulation in µM based on wet weight of cells. Data are mean ± SEM (n = 3). (B) Average distribution of PBDE in cells, remaining in medium, and bound to the plastic tissue culture plate as a percentage of the total applied dose. *Significantly different from serum-free (0%) medium (Dunnett's test for comparison to control following significant ANOVA, p < 0.05).

 
The influence of the volume of exposure on PBDE accumulation in cells was also examined. Cells were exposed to either 2 ml or 4 ml of 1 µM 14C-PBDE-47 (prepared in serum-free medium) for 60 min. Thus, while the exposure concentration and number of cells remained constant, the total amount of 14C-PBDE-47 in the well was doubled. Cells exposed to 4 ml accumulated a significantly greater amount of 14C-PBDE-47 (150.7 ± 4.6 µM, n = 3) compared to cells exposed to 2 ml (104.6 ± 5.4 µM, n = 3) (unpaired t-test, p < 0.05).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, PBDE-47 rapidly accumulated in cells in culture to levels that are orders of magnitude above the applied media concentration. This accumulation was dependent on incubation time, media constituents, and medium volume. In addition, a substantial mass of the PBDE-47 was not recovered and assumed to be bound to the polystyrene plastic used in tissue culture plates.

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), 2001Go) 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., 2003Go), which are also highly lipophilic (Kow = 5.6 to 8.3; ATSDR, 1997Go).

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., 1989Go). 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., 1985Go; Henderson and Patterson, 1988Go). In addition, horse serum contains the thyroid hormone–binding protein transthyretin, which has been shown to bind PBDEs as well as PCBs (Meerts et al., 2000Go; Chauhan et al., 2000Go). 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., 2000Go). 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., 2003Go). 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.


    ACKNOWLEDGMENTS
 
The authors thank Brian Robinette for his assistance in preparation of the cell cultures. The critical suggestions of Dr. Michael Hughes and Dr. Timothy Shafer on a previous draft of this manuscript are much appreciated. This document has been reviewed by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


    NOTES
 

1 To whom correspondence should be addressed at U.S. Environmental Protection Agency, Neurotoxicology Division, B105–06, Research Triangle Park, NC 27711. Fax: (919) 541–0700. E-mail: mundy.william{at}epa.gov.


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 DISCUSSION
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