Grantee Research Project Results
Final Report: Cultured Fish Cells as Model System for Studying Mechanisms of Dioxin Toxicity
EPA Grant Number: R823889Title: Cultured Fish Cells as Model System for Studying Mechanisms of Dioxin Toxicity
Investigators: Hahn, Mark E. , Stegeman, John J.
Institution: Woods Hole Oceanographic Institution
EPA Project Officer: Hahn, Intaek
Project Period: July 1, 1995 through June 1, 1997
Project Amount: $252,790
RFA: Exploratory Research - Environmental Biology (1995) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Human Health , Aquatic Ecosystems
Objective:
The presence of chlorinated dibenzo-p-dioxins and related compounds (chlorinated dibenzofurans, chlorinated biphenyls) in aquatic environments is a matter of serious concern to scientists, regulators, and the public. Certain of these halogenated aromatic hydrocarbons (HAHs) are known to be highly toxic to fish, and there is general agreement that they may pose an unacceptable risk to the health of aquatic ecosystems. Several uncertainties preclude an adequate assessment of the magnitude and extent of that risk. These include a lack of knowledge concerning the mechanism by which these compounds produce toxicity in fish and other aquatic animals, uncertainty as to the relative contribution of individual HAH congeners to the aggregate HAH risk, a poor understanding of the interactive effects that may occur in HAH mixtures, and an incomplete understanding of the relationship of proximal biochemical changes (such as induction of cytochrome P4501A) to more distal, "toxic" effects. These uncertainties suggest the need for model systems in which to address questions regarding the mechanisms of HAH toxicity to aquatic species. Cultured cells have been used with great success in mammalian toxicology to dissect out important events and pathways involved in responses to environmental contaminants. Earlier, we showed that PLHC-1 fish hepatoma cells possess an AHR and respond to HAH exposure with induction of cytochrome P450 1A (CYP1A) (Hahn et al., 1993). In the work described here, we developed this cell line as a model system to investigate mechanisms of dioxin toxicity in fish.The overall objective of the research conducted under EPA Agreement Assistance I.D. No. R823889-01-0 was to further our understanding of the biochemical response of fish to HAH exposure in relation to cellular physiology, using well-defined model systems: hepatoma cells (PLHC-1) and primary cell cultures. The specific aims of the proposed work were:
- To determine the role of cellular differentiation state in influencing the response to dioxin exposure, and to assess the effect of dioxins on cell proliferation and differentiation in fish cells.
- To determine the mechanism and significance of the altered biochemical responsiveness of cultured cells grown under different culture conditions.
- To identify the nature of interactive effects occurring between selected halogenated aromatic compounds, including possible additive, synergistic, and antagonistic effects.
The responses measured in these studies are ones that have been used or proposed as biochemical markers of HAH presence and effects: induction of cytochrome P4501A (CYP1A) and porphyrin accumulation.
Summary/Accomplishments (Outputs/Outcomes):
Development of bioassay system using PLHC-1 cellsTo develop a bioassay system utilizing PLHC-1 cells, we optimized methods for growth and treatment of these cells in multi-well plates, in situ analysis of ethoxyresorufin O-deethylase (EROD) activity, and measurement of CYP1A protein by immunoblotting of cell lysates (Hahn et al., 1996). EROD activity was undetectable (<1 pmolomin-1omg-1) in untreated or DMSO-treated cells, but was highly induced (up to 150 pmolomin-1omg-1) in cells exposed to Ah receptor agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), or planar chlorobiphenyls (CB). Addition of exogenous NADPH was not required for measurement of EROD activity in PLHC-1 cells. As inducers of EROD activity, TCDD, TCDF, 3,3',4,4',5-pentachlorobiphenyl (CB-126), and 3,3',4,4'-tetrachlorobiphenyl (CB-77) differed both in potency and in apparent efficacy (maximal level of induced activity). In each case, EROD induction was biphasic, with stronger induction at lower concentrations and an attenuated response at higher concentrations. In contrast, the content of immunodetectable CYP1A protein increased monotonically with dose of CB, and the maximum level achieved was similar for all inducers. The discrepancy in results obtained for EROD activity versus CYP1A protein may result from inhibition or inactivation of catalytic function at high concentrations of inducer. By reducing peak EROD values, this inhibition leads to lower apparent EC50 values and thus the overestimation of relative potencies or Toxic Equivalency Factors (TEFs) for many inducers. These studies demonstrated the necessity of measuring both EROD activity and immunodetectable CYP1A protein for the accurate assessment of CYP1A induction and relative potencies in cultured cells. More generally, this work established conditions for using PLHC-1 cells grown in multi-well plates as a bioassay system for use in mechanistic studies as well as in screening of environmental samples for dioxin-like activity (Hahn et al., 1996).
Interaction of Ah responsiveness and cell proliferation/differentiation state
A growing body of evidence indicates that one toxic effect of HAHs is interference with cell proliferation. In turn, there is also some evidence that the proliferation state and cell cycle stage of an individual cell can affect AHR signal transduction. Initially, our research focused on developing methods for synchronizing PLHC-1 cells in order to examine the relationship of the Ah receptor signaling pathway to the cell cycle. Extensive efforts were made to develop synchronous cell populations in an effort to measure possible differential effects of AhR agonists at different stages of the cell cycle. Partially synchronized populations were obtained, but the heterogeneity remaining was sufficient to preclude rigorous examination of the questions posed above. Subsequent efforts focused on the other aims, as described below.
Effect of culture conditions on responsiveness of cultured cells
The effects of chemicals on cellular processes are governed by their ability to enter the cell, which is in turn a function of the composition of the cell's external environment. To examine this relationship, the effect of serum in cell culture medium on the bioavailability of cytochrome P450 1A (CYP1A)-inducing compounds was determined in PLHC-1 cells. The presence of 10% calf serum in the medium increased the EC50 for induction of ethoxyresorufin O-deethylase (EROD) activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 20-fold as compared to treatment in serum-free medium. Measurement of [3[H]TCDD uptake and Ah receptor binding indicated that the apparent difference in potencies was a result of decreased bioavailability in the presence of serum, effectively reducing the concentration of TCDD within the cells. Induction of EROD and CYP1A protein in response to treatment with each of three coplanar polychlorinated biphenyls (PCB congeners 77, 126, and 169) was similarly affected by serum, although the magnitude varied among inducers and assays. Relative potencies (calculated as EC50TCDD / EC50PCB) for EROD induction by the three PCBs were significantly higher in the absence of serum. However, serum showed no significant effect on the relative potencies for CYP1A protein induction. These results demonstrate that measured inducing potencies, and relative potencies for EROD induction, by halogenated aromatic hydrocarbons are strongly dependent on the composition of culture medium, which can lead to artificial differences in comparisons among cell types (Hestermann et al., 1999b).
In additional studies, we found that under some conditions removal of serum from cell culture medium abrogates AHR function in PLHC-1 cells. This is seen only in a limited number of clones, and disappears after the cells have been in culture for longer times (and therefore progressed further from their original state). The mechanisms underlying this phenomenon is not yet known, but is the subject of ongoing studies.
Interactions among HAHs
We have used PLHC-1 cells to define the structure-activity relationships and relative potencies for individual HAHs in fish. Such information is required for application of the dioxin toxic equivalency approach in ecological risk assessment of HAHs (Various authors, 1998). As described above, our research identified a discrepancy between results obtained by measuring CYP1A catalytic activity (EROD)-the most commonly used endpoint in such bioassays-and results obtained by measuring immunodetectable CYP1A protein (Hahn et al., 1993; Hahn et al., 1996). The major factor contributing to this discrepancy is the inhibition of CYP1A activity by residual inducer. This phenomenon is widespread, and results in the overestimation of relative potencies for many HAHs (Hahn, 1994; Hahn, 1996). To overcome this artifact, we proposed the routine measurement of CYP1A protein in such studies; we also proposed alternative methods of data analysis to minimize the influence of enzyme inhibition on the determination of relative potencies (Hahn et al., 1996; Kennedy et al., 1996).
Hepatic uroporphyria is a well known effect of halogenated aromatic hydrocarbons in mammalian and avian systems, including primary cell cultures, but attempts to produce uroporphyria in vertebrate (mammalian) hepatoma lines have been unsuccessful. In studies performed under this grant, we found that PLHC-1 cells are sensitive to uroporphyria, and we used this response in part to further address questions regarding structure-activity relationships and interactive effects of HAHs in fish (Hahn and Chandran, 1996). The ability of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF) and selected chlorobiphenyl congeners to cause uroporphyria was examined. Dose-dependent accumulation of porphyrins was observed in cells treated for 48 hr with TCDD or 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB; IUPAC #77) when the heme precursor d-aminolevulinic acid (ALA) was present during the last 5 hr of treatment. HPLC analysis identified the porphyrins as uroporphyrin (~80%) and heptacarboxylporphyrin (~20%). Uroporphyria did not occur in cells treated with TCDD or 3,3',4,4'-TCB in the absence of added ALA. ALA-dependent porphyrin accumulation was also seen following treatment of PLHC-1 cells with TCDF or with the non-ortho-substituted chlorobiphenyls 3,4,4',5-tetrachlorobiphenyl (IUPAC #81) and 3,3',4,4',5-pentachlorobiphenyl (IUPAC #126). Neither of the mono-ortho-substituted chlorobiphenyls 2,3,3',4,4'-pentachlorobiphenyl (IUPAC #105) and 2,3',4,4',5-pentachlorobiphenyl (IUPAC #118) increased the porphyrin content of PLHC-1 cells. The ability of the PCB congeners to cause porphyria correlated with their ability to induce the CYP1A catalytic activity ethoxyresorufin O-deethylase (EROD) and immunodetectable CYP1A protein in these cells, suggesting direct or indirect regulation of porphyrin accumulation via the Ah receptor and/or the induced CYP1A. Induction of EROD activity by TCDD, TCDF, and the planar PCBs was biphasic, with increases at lower concentrations of inducer followed by decreased induction at higher concentrations, as seen previously. EC50 values for porphyrin accumulation were similar to, or slightly higher than, the concentrations at which peak EROD activities were obtained, suggesting a relationship between the decline in EROD activity and enhanced porphyrin accumulation. Alpha-naphthoflavone inhibited TCDD-induced EROD activity and porphyrin accumulation, providing further evidence for the involvement of a fish CYP1A in the mechanism of this porphyria. Addition of 3,3',4,4'-TCB to TCDD-treated cells also inhibited EROD activity, but enhanced porphyrin accumulation, suggesting that an interaction between the halogenated inducer and the induced CYP1A is necessary for the porphyrogenic response. We concluded that PLHC-1 cells grown in medium supplemented with ALA may be a useful model system for studying mechanisms of chemical uroporphyria induced by Ah receptor agonists, as well as interactive effects of HAHs (Hahn and Chandran, 1996).
Results in PLHC-1 cells (Hahn and Chandran, 1996; Hahn et al., 1996) and other fish assays (Gooch et al., 1989; Walker and Peterson, 1991) consistently showed differences between mammals and fish in the structure-activity relationships for certain HAHs. In particular, mono-ortho substituted PCBs are active in mammals but have little or no capacity to elicit AHR-dependent responses in fish. To investigate the mechanistic basis for such differences, we have taken a rigorous pharmacologic approach to define the nature of the PCB-AHR interactions (Hestermann et al., 1999a). Pharmacological models of receptor action have power for describing properties of the ligand-receptor interaction and are also necessary for mechanism-based risk assessment of receptor-mediated toxic effects. In order to build such a model for the aryl hydrocarbon receptor (AHR), binding affinities and CYP1A induction potencies were measured in PLHC-1 cells for ten halogenated aromatic hydrocarbons: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and polychlorinated biphenyl (PCB) congeners 77, 81, 105, 118, 126, 128, 156 and 169. From these data, an operational model of AHR action was used to determine intrinsic efficacies of these ligands. TCDD, TCDF, and non-ortho-substituted PCBs 77, 81, 126 and 169 behaved as full agonists and displayed high intrinsic efficacy. In contrast, the mono- and di-ortho-substituted PCBs bound to the AHR but displayed decreased or no intrinsic efficacy. PCB 156 was a full agonist, but with lower intrinsic efficacy. PCB 118 was a very weak partial agonist. PCBs 105 and 128 were shown to be competitive antagonists in this system. When the properties of PCBs 126, 128 and 156 were measured in mixtures with TCDD, both PCB 156 (a low intrinsic-efficacy agonist) and PCB 128 (a competitive antagonist) inhibited the response to TCDD. The stimulus-response model predicts that only 1-2% of the receptors in the cell are occupied for 50% CYP1A induction by one of the high intrinsic efficacy agonists, demonstrating the existence of "spare" receptors in this system. These studies have shown that the inactivity of the mono-ortho PCBs in fish is not due to a lack of binding to the AHR. Rather, these PCBs bind to the AHR and act as partial agonists or competitive antagonists. More generally, the results have highlighted the inadequacy of current toxic equivalency models in ignoring variations in "intrinsic efficacy" among AHR ligands. Separation of AHR ligand action into the properties of affinity and intrinsic efficacy allows for improved prediction of the behavior of complex mixtures of ligands, as well as comparison across species and toxic endpoints. This approach has general applicability to the risk assessment of dioxin-like compounds in a variety of species, including humans.
Additional studies assessed possible interactions between AhR and glucocorticoid receptor (GR) pathways. Glucocorticoids are known to affect the response to Ah receptor agonists in mammals, so we performed studies to determine the effects of GR agonists on the response to CYP1A inducers in PLHC-1 fish hepatoma cells. Cells were treated with GR and AhR agonists, alone or in combination, and the amounts of CYP3A (which is inducible by GR agonists in mammals) and CYP1A were measured. A CYP3A protein was detected in PLHC-1 cells, but its expression was not altered by any of the treatments. The GR agonists dexamethasone (DEX), cortisol, and prednisone did not induce CYP1A activity or immunodetectable protein. However, each of the three compounds potentiated the CYP1A induction produced by the AhR agonists 2,3,7,8-TCDD or 3,3',4,4'-TCB. The degree of potentiation was greatest at low concentrations of TCDD (0.1 nM), where CYP1A levels were up to 20-fold greater in the presence of GR agonist. Two antagonists of the mammalian GR receptor-pregnenolone-16a-carbonitrile and RU 38486-blocked the DEX-mediated potentiation of CYP1A induction, suggesting that the potentiation is mediated through a fish GR. These results demonstrate that interactions between AhR and GR pathways are a common feature of vertebrate systems (Celander et al., 1996; Celander et al., 1997).
Cell culture Bioassays
In addition to their use in mechanistic studies as described above, PLHC-1 cells and cells from other species can also serve as a sensitive, mechanism-based, taxon-specific bioassays for detecting AHR-active compounds in environmental samples. We have used the PLHC-1 system to detect the presence of AHR ligands in extracts of water, sediments, and other matrices (Huuskonen et al., 1998a; Huuskonen et al., 1998b; Huuskonen et al., 1999). In other studies, we showed that primary hepatocytes isolated from toadfish (Cornell et al., 1995) and mummichogs (Bello et al., 1999) can be adapted for use as a bioassay system. Other work performed in collaboration with scientists at Environment Canada utilized hepatocytes from avian species to examine species-specific differences in sensitivity to HAHs (Kennedy et al., 1996; Lorenzen et al., 1997). Based on this and similar work in a variety of systems, we proposed that cell culture bioassays might be used as part of the HACCP program for monitoring HAH contamination of seafood (Hahn, 1998).
Journal Articles on this Report : 12 Displayed | Download in RIS Format
Other project views: | All 16 publications | 12 publications in selected types | All 12 journal articles |
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Bello SM, Franks DG, Stegeman JJ, Hahn ME. Acquired resistance to Ah receptor agonists in a population of Atlantic kill fish (Fundulus heteroclitus) inhabiting a marine superfund site: in vivo and in vitro studies on the inducibility of xenobiotic metabolizing enzymes. Toxicological Sciences 2001;60(1):77-91. |
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Celander M, Hahn ME, Stegeman JJ. Cytochromes P450 (CYP) in the Poeciliopsis lucida hepatocellular carcinoma cell line (PLHC-1): dose-and time-dependent glucocorticoid potentiation of CYP1A induction without induction of CYP3A. Archives of Biochemistry and Biophysics 1996;329(1):113-122. |
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Celander M, Bremer J, Hahn ME, Stegeman JJ. Glucocorticoid-xenobiotic interactions: dexamethasone-mediated potentiation of cytochrome P4501A induction by β-naphthoflavone in a fish hepatoma cell line (PLHC-1). Environmental Toxicology and Chemistry 1997;16(5):900-907. |
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Cornell NW, Hahn ME, Martin HA. Characterization and use of isolated toadfish hepatocytes for studies of heme synthesis and utilization. The Biological Bulletin 1995;189(2):227-228. |
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Hahn ME, Woodward BL, Stegeman JJ, Kennedy SW. Rapid assessment of induced cytochrome P4501A protein and catalytic activity in fish hepatoma cells grown in multi-well plates:response to TCDD, TCDF, and two planar PCBs. Environmental Toxicology and Chemistry 1996;15(4):582-591. |
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Hahn ME, Chandran K. Uroporphyrin accumulation associated with cytochrome P4501A induction in fish hepatoma cells exposed to aryl hydrocarbon receptor agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin and planar chlorobiphenyls. Archives of Biochemistry and Biophysics 1996;329(2):163-174. |
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Hahn ME. Biomarkers and bioassays for detecting dioxin-like compounds in the marine environment. Science of the Total Environment 2002;289(1-3):49-69. |
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Hestermann EV, Stegeman JJ, Hahn ME. Serum alters the uptake and relative potencies of halogenated aromatic hydrocarbons in cell culture bioassays. Toxicological Sciences 2000;53(2):316-325. |
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Kennedy SW, Lorenzen A, Jones SP, Hahn ME, Stegeman JJ. Cytochrome P4501A induction in avian hepatocyte cultures: a promising approach for predicting the sensitivity of avian species to toxic effects of halogenated aromatic hydrocarbons. Toxicology and Applied Pharmacology 1996;141(1):214-230. |
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Lorenzen A, Kennedy SW, Bastien LJ, Hahn ME. Halogenated aromatic hydrocarbon-mediated porphyrin accumulation and induction of cytochrome P4501A in chicken embryo hepatocytes. Biochemical Pharmacology 1997;53(3):373-384. |
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Schlezinger JJ, Keller J, Verbrugge LA, Stegeman JJ. 3,3',4,4'-tetrachlorobiphenyl oxidation in fish, bird and reptile species: relationship to cytochrome P450 1A inactivation and reactive oxygen production. Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology 2000;125(3):273-286. |
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White RD, Shea D, Stegeman JJ. Metabolism of the aryl hydrocarbon receptor agonist 3,3',4,4'-tetrachlorobiphenyl by the marine fish scup (Stenotomus chrysops) in vivo and in vitro. Drug Metabolism and Disposition 1997;25(5):564-572. |
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Supplemental Keywords:
RFA, Scientific Discipline, Toxics, Ecosystem Protection/Environmental Exposure & Risk, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Chemical Mixtures - Environmental Exposure & Risk, Environmental Chemistry, HAPS, pesticides, Chemistry, Ecological Effects - Environmental Exposure & Risk, Monitoring/Modeling, Ecological Effects - Human Health, Biology, Ecological Indicators, ecological exposure, ecological risk assessment, aquatic ecosystem, fate and transport, environmental monitoring, dioxin, ecosystem assessment, chemical characteristics, cytochrome P450, hydrocarbon, exposure, cell physiology, HAH, biomonitoring, toxicity, aquatic ecosystems, dioxin toxicity, hydrocarbons, fish , cultured fish cells, dioxin exposureRelevant Websites:
Hahn Laboratory: Research Interests ExitStegeman Laboratory: Research Interests Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.