Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
Received July 2, 1999; accepted October 11, 1999
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ABSTRACT |
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Key Words: bioassay; bioavailability; dioxin; PCB; serum; TEF.
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INTRODUCTION |
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The induction of cytochrome P450 1A (CYP1A) protein and catalytic activity in cultured cells is being used with increasing frequency to compare the sensitivities of a variety of organisms to the effects of halogenated aromatic hydrocarbons (HAH). Cells from mammals (Safe, 1987; Sawyer and Safe, 1982
; Tillitt et al., 1991
), birds (Kennedy et al., 1996a
; Kennedy et al., 1996b
), and fish (Clemons et al., 1996
; Hahn et al., 1996
) have been used to study the mechanisms of HAH toxicity and, in the absence of in vivo data, to establish taxon-specific toxic equivalency factors (TEFs) for these compounds (van den Berg et al., 1998
). Comparisons of these results can reveal mechanistic differences in the induction pathway of CYP1A among taxa. However, differences in CYP1A induction among cell culture systems can also reflect the culture conditions of the cells.
Serum is known to impact the effects of CYP1A inducers and CYP levels in cultured cells. For example, the presence of 10% fetal calf serum reduces the potency of TCDD and PCB126 for inhibiting aromatase (CYP19) activity in JEG-3 human choriocarcinoma cells (Drenth et al., 1998). Serum and other medium components can also alter the detectable levels of cytochromes P450 in rat hepatocytes (Hammond and Fry, 1992
; Turner and Pitot, 1989
) and HepG2 cells (Doostdar et al., 1991
; Doostdar et al., 1988
). Despite these provocative findings, there has been no quantitative study of the effect of serum on CYP1A induction by HAH. Because these compounds are very hydrophobic and have limited aqueous solubilities, it can be expected that serum components, such as proteins and lipids, would have a significant effect on the bioavailability of HAH for cell uptake. Because entry into the cell is the first step in the toxic mechanism of these compounds, effects at this stage will be propagated (and perhaps multiplied) through subsequent cell responses.
Following entry of an inducer into the cell, CYP1A induction is controlled by the ligand-activated transcription factor aryl hydrocarbon receptor (AHR). Binding of HAH to the AHR activates transcription of CYP1A and mediates the toxicity of the inducer. A compound's potency for CYP1A induction in vivo or in cultured cells is a strong predictor of its toxicity (Safe, 1984). Use of cell culture systems for rapid analysis of the potential toxicity of individual compounds and environmental samples has increased with refinements in CYP1A measurement techniques. Levels of both the CYP1A protein (Bruschweiler et al., 1996
; Hahn et al., 1996
) and its ethoxyresorufin O-deethylase (EROD) activity (Kennedy et al., 1993
) can be measured directly in the same multiwell plates used for growth of the cells and exposure to HAH.
The toxic equivalency approach utilizes these induction data to assess the toxic potential of individual compounds or mixtures relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The potency of a compound for eliciting a response can be compared to the potency of TCDD for the same response by calculating the ratio of their respective EC50s (concentration eliciting a 50% maximal effect). Such relative potencies from several systems, including cultured cells, are then used to determine TEFs for individual taxa (van den Berg et al., 1998).
In previous reports we have established the conditions and methods for measuring EROD activity and CYP1A induction in PLHC-1 cells (Hahn and Chandran, 1996; Hahn et al., 1993
; Hahn et al., 1996
), which are derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida (Hightower and Renfro, 1988
). Here we make use of those findings to examine the role of serum in uptake of HAH from the culture medium by cells. This represents a first step in examining the complex interaction between these cells and their chemical milieu. The results provide compelling evidence that serum affects the potency of AHR ligands, and likely other hydrophobic compounds, in cells in culture.
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MATERIALS AND METHODS |
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Phosphate-buffered saline (PBS) is 0.8% NaCl, 0.115% Na2HPO4, 0.02% KCl, 0.02% KH2PO4, pH 7.4. Phosphate buffer is 50 mM Na2HPO4 with pH adjusted to 8.0 with 50 mM NaH2PO4. TCDD, TCDF, and PCB solutions were prepared in dimethyl sulfoxide (DMSO) as described previously (Hahn et al., 1996). Concentrations of [3H]TCDD solutions were verified by liquid scintillation counting (LSC) on a Beckman LS5000TD.
Growth and treatment of cells.
PLHC-1 cells (Hightower and Renfro, 1988) were grown at 30°C in minimum essential medium (MEM) containing Earle's salts, nonessential amino acids, L-glutamine and 10% calf serum (Sigma C6278, lot 106H4628), as described previously (Hahn et al., 1993
). One day prior to dosing, cells were suspended to 0.5 to 1 x 106 per ml and seeded into 48- or 96-well plates (Costar; Cambridge, MA) at 0.5 or 0.2 ml per well, respectively. One day later the medium was removed and replaced with fresh medium. Media used in the experiments include MEM without serum, with 5% serum, with 10% serum, with 10% delipidated, charcoal-stripped calf serum (Sigma C1696), and with 10% fetal bovine serum (FBS; Gibco; Grand Island, NY). Serum-free MEM supplemented with bovine serum albumin (BSA) was also used. The cells were then treated by addition of solutions dissolved in DMSO or DMSO alone (2.5 or 1.0 µl/well). DMSO concentrations were
0.5% (v/v) in all treatments. Following treatment, plates were incubated at 30°C for 24 h unless otherwise indicated. For TCDD- specific binding experiments, cells were seeded into 24-well plates (Corning; Corning, NY) at 2 x 106 cells in 1 ml culture medium per well. With the exception of the delipidated serum and FBS, all serum used was from a single lot. None of the media or HAH treatments reduced cell viability, as assessed by Trypan blue exclusion.
EROD and protein assays.
EROD activity was measured using a multiwell fluorescence plate reader by a modification of the method of Kennedy et al., (1995). Cells were rinsed once with 0.5 ml room-temperature PBS, and the EROD reaction was then initiated with the addition of 2 µM 7-ethoxyresorufin in phosphate buffer (200 µl/well). The reaction was stopped after 8 min (resorufin production is linear with respect to time over this period; Hahn et al., 1996) with the addition of 150 µl ice-cold fluorescamine solution (0.15 mg/ml in acetonitrile). After a 15-min incubation, resorufin and fluorescamine fluorescence was measured. Resorufin and protein concentrations were determined from standard curves prepared in the same plate. BSA was used for the protein standard curve. In some experiments, the EROD reaction was followed kinetically over 8 min, as described previously (Hahn et al., 1996). Protein was measured using fluorescamine as described above.
TCDD uptake.
PLHC-1 cells were seeded in 48-well plates, grown for 1 day, and then fed media as indicated in figure legends. They were treated with [3H]TCDD in DMSO as above and incubated at 30°C. At 0.5, 1, 2, 7, and 24 h post-treatment, the culture medium was transferred from each well to a separate vial. Cells were removed by sequential incubation with two 0.2-ml aliquots of 0.05% (w/v) trypsin, which were then combined in a single vial. Cell removal was verified by microscopy. TCDD retained on well surfaces was extracted with a single 1-ml aliquot of hexane. TCDD associated with each fraction (medium, cells, and well) was determined by LSC. Protein concentrations were determined using fluorescamine in duplicate wells fed each medium and treated with DMSO alone.
TCDD binding.
Specific binding of [3H]TCDD in PLHC-1 cells was measured by a whole-cell filtration assay (Dold and Greenlee, 1990). One day after seeding in 24-well plates, the cells were fed 0.5 ml of the indicated media. Cells were treated with 0.18 nM [3H]TCDD in the presence or absence of 40 nM TCDF and incubated 2 h at 30°C. This time was determined to be sufficient to achieve a steady state of bound radioligand (not shown). Following the incubation, medium was removed, cells were rinsed with 0.5 ml ice-cold PBS, then detached with 0.5 ml trypsin. The trypsin was inactivated by the addition of 0.5 ml ice-cold culture medium (with 10% serum), and cells from each well were collected under vacuum on a 25-mm Whatman GF/F filter that had been prewetted with PBS. Filters were then washed four times with 2.5 ml acetone that had been precooled to 80°C. Replicates were processed in batches of 12 on a Millipore 1225 filter manifold. Radioactivity remaining on the filter was quantified by LSC. Specific binding was measured in triplicate as the difference of each of three total binding (without TCDF) replicates and the average of three nonspecific binding (with TCDF) replicates in each medium. Protein concentrations were determined in duplicate wells fed each medium and treated with DMSO alone.
ELISA assay.
Enzyme-linked immunosorbence assays to detect CYP1A were performed essentially as described (Bruschweiler et al., 1996). One day after treatment in 96-well plates, cells were fixed in 50% ethanol 15 min, in 75% ethanol 15 min, and in 95% ethanol 30 min. After washing three times with PBS, nonspecific antibody binding was blocked with 10% fetal bovine serum and 2% BSA in PBS for 1 h. The primary antibody, mouse anti-scup CYP1A monoclonal antibody 1-12-3 (10 µg/ml; Park et al., 1986), was then added in 100 µl blocking solution for 1 h. After three washing steps with PBS, 100 µl secondary antibody, peroxidase conjugated goat anti-mouse (1:1000 in blocking solution), was added for 1 h. After another three washing steps with PBS, 100 µl substrate solution (100 µM Amplex Red, 100 µM H2O2 in phosphate buffer, pH = 7.0) was added for 30 min. All incubations were performed at room temperature. Resorufin formation was measured in the fluorescence plate reader. For each treatment the background fluorescence, defined as the fluorescence detected in untreated cells, was subtracted, and all values were normalized to the maximum response measured. The assay was also performed on wells without cells or without the addition of primary antibody, and these controls yielded fluorescence values nearly identical to those in untreated cells, consistent with our earlier results detecting no CYP1A protein or EROD activity in untreated cells (Hahn et al., 1996
).
Curve fitting and statistical analysis.
For determination of dose-response relationships, EROD data were fit to a modified Gaussian function, and CYP1A induction data were fit to a logistic function. The rationale for use of these functions has been described previously (Hahn et al., 1996; Kennedy et al., 1993
). The Gaussian function properly reflects the biphasic nature of EROD induction, while a logistic function forms a plateau at higher inducer concentrations, consistent with CYP1A protein induction in these cells. Statistical analyses were performed with the aid of Excel (Microsoft; Redmond, WA) and JMP IN (SAS Institute, Inc.; Cary, NC) software.
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RESULTS |
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Differences in Uptake Affect TCDD Binding by the AH Receptor
The effect of medium composition on specific binding of TCDD by AHR in PLHC-1 cells was measured. Cells were grown in S10, fed one of the four media, treated with 0.18 nM [3H]TCDD in the presence or absence of 40 nM TCDF, and incubated for 2 h. This concentration of TCDD was selected because it is near the value at which cells treated in S0 or S10 showed the greatest difference in EROD response (Figure 1). Binding of TCDD to the AHR was measured by a whole-cell filtration assay (Dold and Greenlee, 1990
). The amount of TCDD bound was 3- to 4-fold higher in cells treated in S0 and showed the same relationship among medium treatments as the fraction of TCDD associated with the cells, i.e., S0>> SDL> S5> S10 (Fig. 3
). Thus, the differences in specific binding reflect the differing concentrations of TCDD within the cells among the treatments, as shown in Figure 2
.
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Culture Medium Composition Alters the Relative Potency of HAH
We wished to determine if serum reduces the uptake of other HAH to the same degree that it does TCDD. PLHC-1 cells were exposed to each of three coplanar PCBs in medium with or without serum, and EROD activity was assayed 24 h later (Fig. 5). The EC50 values for the responses are shown in Table 3
. For each compound, the EC50 in S0 was lower than that in S10. The differences ranged from about 20-fold for TCDD to about 2000-fold for PCB 77, although there is substantial uncertainty in the latter value, because a precise EC50 is difficult to obtain for this compound in S10 (e.g., Hahn et al., 1996).
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The ELISA as performed provides only a relative measure of CYP1A protein content, but the range of values produced and the pattern of induction by TCDD in S10 closely parallel those previously obtained by Western blot (Hahn et al., 1996). This indicates that the response as measured in this assay can be correlated with the values from a more quantitative approach. Furthermore, maximal levels of detected fluorescence from the ELISA assay were similar among all the treatments, indicating that the maximally induced level of CYP1A is similar among the four compounds, regardless of medium used.
The EC50s for the EROD and ELISA assays were used to calculate relative potencies for the four compounds within each medium treatment (Table 4). Relative potencies as determined by EROD assay for the three PCBs were significantly lower in S10 than in S0. In contrast, when CYP1A induction was measured by ELISA, there were neither consistent nor significant differences in relative potencies determined with cells in the two media.
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DISCUSSION |
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HAH Partitioning in a Multiwell Plate
Our measurements of TCDD partitioning demonstrate that the majority of the compound remains in the medium when serum is present. Thus, small changes in medium composition could have significant effects on the amount of compound that enters the cells. The fraction of total TCDD associated with the polystyrene wells was approximately equal to that found in the cells, suggesting that the composition of the chamber used for treatment also could affect the amount of compound that reaches the cells.
The percentage of total TCDD associated with the PLHC-1 cells was lowest at the highest nominal concentration of TCDD (10 nM), regardless of the medium used for treatment (Table 2). Also, at 10 nM TCDD, the fraction associated with the well walls increased, perhaps because at this concentration the cells were saturated with TCDD, and the compound was diffusing through the basal membrane of the cells to the floor of the well. Reduced diffusion at low TCDD concentrations is consistent with the finding of Yu et al., (1997) that H4IIE cells apparently reduced sorption of PCB 77 to the floor of culture plates. That same study also found that a majority of PCB 77 (~75%) remained in the medium, which was supplemented with 15% FBS. They found no effect of carrier (isooctane vs. DMSO) on the fraction of the compound associated with the cells, which was at most 5%. Uptake studies with radiolabeled PCB77 have demonstrated similarly low levels associated with PLHC-1 cells (A. Patel and M. E. Hahn, unpublished results), suggesting that HAH partitioning is consistent between these two cell types and their media. In contrast, Schirmer et al., (1997) found that the presence of 10% FBS in culture medium greatly altered the solubility of fluoranthene but did not significantly change the amount of that compound associated with cells from two fish lines.
AHR Occupancy and CYP1A Induction
The magnitude of the effect of serum on AHR occupancy was nearly identical to the difference in uptake of TCDD by cells (compare Fig. 3 and the 0.1 nM TCDD group in Fig. 2B
). This supports a direct relationship between the amount of compound associated with the cells and the amount bound by the AHR when the concentration of TCDD is sufficiently below the amount required for receptor saturation. The latter condition is satisfied here, as the concentration used was less than the KD for TCDD binding to the AHR (KD = 0.8 nM in S0; Hestermann et al., in preparation).
However, comparison of receptor occupancy and induction of EROD or CYP1A does not reveal a direct relationship like that occurring between TCDD uptake and receptor occupancy. There was a 4-fold increase in receptor occupancy in cells in S0 rather than S10 medium, but a much larger increase in CYP1A content (compare Fig. 3 with the 0.1 nM nominal TCDD concentration in Figs. 1 and 6A
). This is most likely the result of a nonlinear occupancy-response relationship (also known as "spare receptors" or "receptor reserve") (Kenakin, 1999
) for TCDD and the AHR in these cells. Under such conditions, submaximal receptor occupancy will produce maximal cell response, so that small changes in occupancy would produce much larger changes in downstream responses. We are pursuing the precise nature of this relationship in the PLHC-1 cell line.
Relative potencies of the three coplanar PCBs determined in S0 were significantly higher than those determined in S10 for EROD response but not CYP1A protein induction. This suggests that the presence of serum has an effect on CYP1A catalytic activity that is separate from its effect on induction via the AHR. The biphasic dose-response relationships typical of EROD induction are a result of the balance between CYP1A induction and competitive inhibition of catalytic activity by the inducer at higher concentrations (Gooch et al., 1989; Hahn et al., 1993
; Petrulis and Bunce, 1999
). Inhibition lowers EROD induction EC50s relative to EC50s for induction of CYP1A protein, and thereby increases the apparent relative potency for the EROD response (Hahn et al., 1996
). It therefore seems likely that serum influences the inhibitory effect of the inducing compounds. Alternatively, there may be serum components that alter EROD activity in PLHC-1 cells by another mechanism.
Implications of Reduced Uptake
Perhaps the greatest potential for error in interpretation of in vitro bioassay data suggested by our results is in comparison of induction EC50s and relative potencies among cell lines. Cell lines vary widely in culture medium contents. Serum may be absent or present at concentrations of up to 20%, and may come from a variety of animals and different developmental stages. Based on our results, such variations in media composition will affect cellular uptake of HAH and thus measured CYP1A induction potencies. Differences in potencies thus might incorrectly be attributed to mechanistic differences in CYP1A induction among the cell types and lead to false conclusions about relative sensitivities of the cells to the HAH in question.
One solution to this potential problem is to treat different cells in a single medium. Serum-free medium is the best candidate, as variations in composition among the chemically defined basal media (e.g., MEM, DMEM, RPMI-1640, F12) should have a negligible effect on bioavailability. Using serum-free medium also allows the greatest sensitivity in response to inducing compounds. The ability of each cell type to respond to HAH in serum-free medium should be determined, because serum withdrawal greatly reduces AHR content in Swiss 3T3 cells (Vaziri et al., 1996) and can abrogate CYP1A induction in PLHC-1 cells after 48 h (Hestermann et al., unpublished data). As noted previously, the presence of serum also affects the levels of cytochromes P450 in some cultured cells (Doostdar et al., 1991
; Doostdar et al., 1988
; Hammond and Fry, 1992
; Turner and Pitot, 1989
).
PLHC-1 cells have recently been adapted to long-term culture in media with serum replacements (Ultra-Culture, CPSR-1, and TurboDoma; Ackermann and Fent, 1998), providing promise for their future use in a chemically defined medium. Such media should reduce the problems with lot-to-lot variability that can be encountered with serum; however, the serum replacements used still have a high protein and/or lipid content, which can be expected to reduce bioavailability as serum does. In addition, the ability of cells grown in these media to respond to HAH exposure has not been determined.
The effect of serum on bioavailability is also a concern for other assays involving uptake of hydrophobic compounds. The reduction in specific TCDD binding in the presence of 10% serum shown here is an example of such an assay. Serum composition also affects bioavailability of estrogenic compounds (Arnold et al., 1996; Nagel et al., 1997
). This suggests that the effect of serum is a general one, and its magnitude should be determined for individual compounds. Comparisons of apparently anomalous results among assays performed in different laboratories or cell lines should take this factor into account, and previous conclusions regarding extrapolation from cultured cells may require reexamination.
This report continues our work of establishing the utility and optimal conditions for use of PLHC-1 cells in studying the mechanisms of HAH action. It also establishes a framework for measuring other effects of culture medium composition on AHR signal transduction in these cells. Through continued use of this model we hope to gain a better understanding of the molecular mechanisms that ultimately result in HAH toxicity. By comparing the shared and distinct features of AHR signal transduction in a variety of taxa, we can also better approach questions of AHR function and evolution.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Arnold, S. F., Collins, B. M., Robinson, M. K., Guillette, L. J., Jr., and McLachlan, J. A. (1996). Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen. Steroids 61, 642646.[ISI][Medline]
Bruschweiler, B. J., Wurgler, F. E., and Fent, K. (1996). An ELISA assay for cytochrome P4501A in fish liver cells. Environ. Toxicol. Chem. 15, 592596.[ISI]
Clemons, J. H., Lee, L. E. J., Myers, C. R., Dixon, D. G., and Bols, N. C. (1996). Cytochrome P4501A1 induction by polychlorinated biphenyls (PCBs) in liver cell lines from rat and trout and the derivation of toxic equivalency factors (TEFs). Can. J. Fish. Aquat. Sci. 53, 11771185.[ISI]
Dold, K. M., and Greenlee, W. F. (1990). Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture. Anal. Biochem. 184, 6773.[ISI][Medline]
Doostdar, H., Burke, M. D., Melvin, W. T., and Grant, M. H. (1991). The effects of dimethylsulphoxide and 5-aminolaevulinic acid on the activities of cytochrome P450-dependent mixed function oxidase and UDP-glucuronosyl transferase activities in human Hep G2 hepatoma cells. Biochem. Pharmacol. 42, 13071313.[ISI][Medline]
Doostdar, H., Duthie, S. J., Burke, M. D., Melvin, W. T., and Grant, M. H. (1988). The influence of culture medium composition on drug metabolising enzyme activities of the human liver derived Hep G2 cell line. FEBS Lett. 241, 1518.[ISI][Medline]
Drenth, H. J., Bouwman, C. A., Seinen, W., and Van den Berg, M. (1998). Effects of some persistent halogenated environmental contaminants on aromatase (CYP19) activity in the human choriocarcinoma cell line JEG-3. Toxicol. Appl. Pharmacol. 148, 5055.[ISI][Medline]
Gooch, J. W., Elskus, A. A., Kloepper-Sams, P. J., Hahn, M. E., and Stegeman, J. J. (1989). Effects of ortho and non-ortho substituted polychlorinated biphenyl congeners on the hepatic monooxygenase system in scup (Stenotomus chrysops). Toxicol. Appl. Pharmacol. 98, 422433.[ISI][Medline]
Hahn, M. E., and Chandran, K. (1996). Uroporphyrin accumulation associated with cytochrome P4501A induction in fish hepatoma cells exposed to Ah receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and planar chlorobiphenyls. Arch. Biochem. Biophys. 329, 163174.[ISI][Medline]
Hahn, M. E., Lamb, T. M., Schultz, M. E., Smolowitz, R. M., and Stegeman, J. J. (1993). Cytochrome P4501A induction and inhibition by 3,3',4,4'-tetrachlorobiphenyl in an Ah receptor-containing fish hepatoma cell line (PLHC-1). Aquat. Toxicol. 26, 185208.[ISI]
Hahn, M. E., Woodward, B. L., Stegeman, J. J., and Kennedy, S. W. (1996). Rapid assessment of induced cytochrome P4501A (CYP1A) protein and catalytic activity in fish hepatoma cells grown in multi-well plates: Response to TCDD, TCDF, and two planar PCBs. Environ. Toxicol. Chem. 15, 582591.[ISI]
Hammond, A. H., and Fry, J. R. (1992). Effect of serum-free medium on cytochrome P450-dependent metabolism and toxicity in rat cultured hepatocytes. Biochem. Pharmacol. 44, 14611464.[ISI][Medline]
Hightower, L. E., and Renfro, J. L. (1988). Recent applications of fish cell culture to biomedical research. J. Exp. Zool. 248, 290302.[ISI][Medline]
Kenakin, T. (1999). Pharmacologic Analysis of Drug-Receptor Interactions. CRC/Raven Press, New York.
Kennedy, S. W., Jones, S. P., and Bastien, L. J. (1995). Efficient analysis of cytochrome P4501A catalytic activity, porphyrins, and total proteins in chicken embryo hepatocyte cultures with a fluorescence plate reader. Anal. Biochem. 226, 362370.[ISI][Medline]
Kennedy, S. W., Lorenzen, A., James, C. A., and Collins, B. T. (1993). Ethoxyresorufin-O-deethylase and porphyrin analysis in chicken embryo hepatocyte cultures with a fluorescence multi-well plate reader. Anal. Biochem. 211, 102112.[ISI][Medline]
Kennedy, S. W., Lorenzen, A., Jones, S. P., Hahn, M. E., and Stegeman, J. J. (1996a). Cytochrome P4501A induction in avian hepatocyte cultures: a promising approach for predicting the sensitivity of avian species to toxic effects of halogenated aromatic hydrocarbons. Toxicol. Appl. Pharmacol. 141, 214230.[ISI][Medline]
Kennedy, S. W., Lorenzen, A., and Norstrom, R. J. (1996b). Chicken embryo hepatocyte bioassay for measuring cytochrome P4501A-based 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent concentrations in environmental samples. Environ. Sci. Technol. 30, 706715.[ISI]
Nagel, S. C., vom Saal, F. S., Thayer, K. A., Dhar, M. G., Boechler, M., and Welshons, W. V. (1997). Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ. Health Perspect. 105, 7076.[ISI][Medline]
Park, S. S., Miller, H., Klotz, A. V., Kloepper-Sams, P. J., Stegeman, J. J., and Gelboin, H. V. (1986). Monoclonal antibodies to liver microsomal cytochrome P-450E of the marine fish Stenotomus chrysops (scup): Cross-reactivity with 3-methylcholanthrene induced rat cytochrome P-450. Arch. Biochem. Biophys. 249, 339350.[ISI][Medline]
Petrulis, J. R., and Bunce, N. J. (1999). Competitive inhibition by inducer as a confounding factor in the use of the ethoxyresorufin-O-deethylase (EROD) assay to estimate exposure to dioxin-like compounds. Toxicol. Lett. 105, 25160.[ISI][Medline]
Safe, S. (1984). Polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs): biochemistry, toxicology, and mechanism of action. CRC Crit. Rev. Toxicol. 13, 319395.[ISI]
Safe, S. (1987). Determination of 2,3,7,8-TCDD toxic equivalent factors (TEFs): support for the use of the in vitro AHH induction assay. Chemosphere 16, 791802.[ISI]
Sawyer, T., and Safe, S. (1982). PCB isomers and congeners: induction of aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylase enzyme activities in rat hepatoma cells. Toxicol. Lett. 13, 8794.[ISI][Medline]
Schirmer, K., Chan, A. G. J., Greenberg, B. M., Dixon, D. G., and Bols, N. C. (1997). Methodology for demonstrating and measuring the phototoxicity of fluoranthene to fish cells in culture. Toxicol. In Vitro 11, 107119.[ISI]
Tillitt, D. E., Giesy, J. P., and Ankley, G. T. (1991). Characterization of the H4IIE rat hepatoma cell bioassay as a tool for assessing toxic potency of planar halogenated hydrocarbons in environmental samples. Environ. Sci. Technol. 25, 8792.[ISI]
Turner, N. A., and Pitot, H. C. (1989). Dependence of the induction of cytochrome P-450 by phenobarbitol in primary cultures of adult rat hepatocytes on the composition of the culture medium. Biochem. Pharmacol. 38, 22472251.[ISI][Medline]
van den Berg, M., Birnbaum, L., Bosveld, A. T. C., Brunström, B., Cook, P., Feeley, M., Giesy, J. P., Hanberg, A., Hasegawa, R., Kennedy, S. W., Kubiak, T., Larsen, J. C., van Leeuwen, F. X. R. v., Liem, A. K. D., Nolt, C., Peterson, R. E., Poellinger, L., Safe, S., Schrenk, D., Tillitt, D., Tysklind, M., Younes, M., Waern, F., and Zacharewski, T. (1998). Toxic Equivalency Factors (TEFs) for PCBs, PCDDs, and PCDFs for humans and wildlife. Environ. Health Perspect. 106, 775792.[ISI][Medline]
Vaziri, C., Schneider, A., Sherr, D. H., and Faller, D. V. (1996). Expression of the aryl hydrocarbon receptor is regulated by serum and mitogenic growth factors in murine 3T3 fibroblasts. J. Biol. Chem. 271, 2592125927.
Yu, K. O., Fisher, J. W., Burton, G. A., Jr., and Tillitt, D. E. (1997). Carrier effects of dosing the H4IIE cells with 3,3',4,4'-tetrachlorobiphenyl (PCB77) in dimethylsulfoxide or isooctane. Chemosphere 35, 895904.[ISI][Medline]