Environ. Sci. Technol. 2000, 34, 3568-3573
Vertical Profiles of Dioxin-like and Estrogenic Activities Associated with a Sediment Core from Tokyo Bay, Japan K U R U N T H A C H A L A M K A N N A N , * ,† DANIEL L. VILLENEUVE,† NOBUYOSHI YAMASHITA,‡ TAKASHI IMAGAWA,‡ SHINYA HASHIMOTO,§ AKIRA MIYAZAKI,‡ AND JOHN P. GIESY† National Food Safety and Toxicology Center, Department of Zoology, Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824, National Institute for Resources and Environment, 16-3 Onogawa, Tsukuba 305-8569, Japan, and Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108, Japan
In vitro bioassays were used to measure dioxin-like and estrogenic activities associated with florisil fractions of extracts from a sediment core collected from Tokyo Bay, Japan. Florisil fractions 2 (F2) and 3 (F3) elicited significant dioxin-like responses in vitro. Dioxin-like activities of F2 samples were correlated with the vertical profile of PAH concentrations (R 2 ) 0.85). Contribution of PAHs to Ah receptor-mediated activities in sediments was greater than those by PCDDs/DFs, PCBs, and PCNs. The dioxin-like activity of F3 samples suggests the presence of relatively polar, Ah receptor-active compounds in the Tokyo Bay sediment core. Significant estrogenic activities, which may be related to the presence of certain estrogenic PAHs, were observed for F2 samples. Estrogen equivalents (E2EQs) calculated from the concentrations and relative potencies of known estrogenic compounds in F2 were greater than bioassay-derived E2-EQs. This suggests that complex interactions between estrogenic and antiestrogenic compounds (PAHs, PCDD/DFs, and PCNs) may have modulated the activity. F3 samples were toxic to MVLN cells; therefore, their estrogenic activities could not be estimated.
Introduction Environmental matrixes such as sediments contain complex mixtures of residues of organic compounds of both natural and anthropogenic origin. The concentrations and toxic potencies of compounds present in such complex mixtures can range over several orders of magnitude and can be modulated by interactions (synergism, antagonism, etc.) among chemicals. This complicates hazard evaluation for complex mixtures of xenobiotics present in environmental matrixes. Instrumental analytical techniques are available to identify and quantify some compounds in complex * Corresponding author phone: (517)432-6321; fax: (517)432-2310; e-mail:
[email protected]. † Michigan State University. ‡ National Institute for Resources and Environment. § Tokyo University of Fisheries. 3568
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mixtures, but there are many compounds for which neither methods nor standards are available. Furthermore, instrumental analyses provide little information on the biological effects of complex mixtures and do not account for possible interactions among individual chemicals. In vitro bioassays are useful tools for characterizing complex mixtures of contaminants, which act through a known mechanism of action. In vitro bioassays provide a biologically relevant, integrated measure of the combined potency of all compounds in a sample (1, 2). When combined with instrumental analysis, sample fractionation techniques, and mass-balance analysis, in vitro bioassays can be used to identify specific compounds or classes of compounds associated with observed biological activity (3-6). In this study, florisil fractions of extracts from a sediment core collected from Tokyo Bay, Japan, were analyzed using in vitro bioassays in order to evaluate vertical profiles of dioxin-like and estrogenic activities. Two in vitro bioassays were used. In vitro luciferase assay with recombinant rat hepatoma cells (H4IIE-luc; 7) was used to screen for compounds capable of modulating aryl hydrocarbon receptor (AhR)-mediated gene expression. In vitro luciferase assay with recombinant MCF-7 human breast carcinoma cells (MVLN; 8) was used to screen extracts for compounds that can modulate gene expression through an estrogen receptor (ER)-mediated mechanism. Where possible, bioassay-derived potencies, relative to a 2,3,7,8-tetrachlorodibenzo-p-dioxin or 17-β-estradiol standard (TCDD-EQ or E2-EQ), were compared to relative potency estimates calculated by multiplying the concentrations of known dioxin-like or estrogenic compounds (reported elsewhere: 9) by assay-specific relative potencies and summing the total (TEQ or EEQ). This type of mass-balance analysis (3) was used to evaluate whether the known composition of the extracts could account for the magnitude of dioxin-like or estrogenic activity observed. TCDD-EQ or E2-EQ estimates significantly greater than TEQ or EEQ estimates would suggest either the presence of unidentified dioxin-like or estrogenic compounds or the synergistic interactions between components of the extract. TCDD-EQ or E2-EQ estimates significantly less than TEQ or EEQ estimates would suggest the presence of antagonists or interfering compounds in the extracts. This type of massbalance analysis has been applied in the identification and characterization of dioxin-like and estrogenic compounds in both surficial sediment and surface waters (6, 10). This study applied the same mass-balance principles to help characterize the historical profile of dioxin-like and estrogenic activities associated with a dated sediment core.
Materials and Methods Samples and Fractionation. A sediment core was collected in May 1995 from the northern part of Tokyo Bay (35′35′′ N and 139′55′′ E) using an acrylic tube (120 cm long and 11 cm i.d.). The core was sliced at 2-cm intervals for up to 20 cm and then at 5-cm intervals for up to 93 cm using a clean stainless steel slicer. Each section was freeze-dried and stored in a refrigerator until analysis. A detailed description of the sample collection, extraction, and fractionation procedure has been provided elsewhere (9). Briefly, sediments were Soxhlet extracted using dichloromethane (DCM) and hexane (3:1, 400 mL). Extracts were treated with acid-activated copper granules to remove sulfur. Concentrated extracts were passed through 10 g of activated Florisil packed in a glass column (10 mm i.d.) for fractionation. The first fraction (F1; nonpolar), eluted with 100 mL of hexane, contained PCBs. PAHs and certain organochlorine pesticides were eluted in the second 10.1021/es001044a CCC: $19.00
2000 American Chemical Society Published on Web 07/27/2000
FIGURE 1. Luciferase induction in H4IIE-luc (dioxin-responsive) cell bioassay elicited by Tokyo Bay sediment core extract fractions 1 (F1), 2 (F2), and 3 (F3) and procedural blank. Response magnitude presented as percentage of the maximum response observed for a 3130 pM 2,3,7,8-tetrachlorodibenzo-p-dioxin (% TCDD max). Horizontal line equals 3 SD above the mean solvent control response (set to 0% TCDD max). fraction (F2; midpolar) with 100 mL of 20% DCM in hexane. The APs such as nonylphenol (NP) and octylphenol (OP) were eluted in the third fraction (F3; polar) with 100 mL of 50% DCM in methanol. PCNs were eluted in F1 and F2, while PCDDs/DFs were eluted in all the three fractions, with a predominant proportion in F2. Cell Culture and Bioassay. H4IIE-luc cells are rat hepatoma cells, which were stably transfected with a luciferase gene under control of dioxin-responsive elements (DREs) (7). MVLN cells are human breast carcinoma cells stably transfected with a luciferase reporter gene under control of estrogen-responsive elements (EREs) of the Xenopus vitellogenin A2 gene (8, 11). Culturing conditions for both cell lines have been described previously (6). MVLN and H4IIE-luc cells were cultured in 100-mm disposable Petri plates and incubated at 37 °C in a humidified 95:5 air:CO2 atmosphere. Cells for bioassay were plated into the 60 interior wells of 96-well culture plates (250 µL/well) at a density of approximately 18 000 cells/well. Cells were incubated overnight prior to dosing. Test wells were dosed with 2.5 µL of the appropriate florisil fraction. Samples were tested using three replicate wells. Control wells received appropriate solvents. Sample responses, expressed as mean relative luminescence units (RLU), from three replicate wells were converted to a percentage of the mean maximum response observed for standard curves generated on the same day (% E2 max and % TCDD max for 17-β-estradiol and TCDD standards, respectively). Significant responses were defined as those outside the range defined by three times the standard deviation (expressed in % standard max) of the mean solvent control response (0% standard max). Dose-response relationships were examined for sediment core extracts from selected depths to estimate potencies relative to 17-βestradiol (E2) and TCDD. Dose-responses consisted of six concentrations prepared by 3-fold dilution from the final extract. Details regarding the derivation of relative potency estimates from bioassay results have been described elsewhere (1, 5, 6, 12, 13).
Results and Discussion Dioxin-like Activity. None of the F1 extracts elicited a significant increase in luciferase expression in H4IIE-luc cells (Figure 1). On the basis of the detection limit for TCDD in
the H4IIE-luc bioassay, approximately 5.0 pg of TEQ/g dry wt would be needed to produce a significant response. Thus, these results suggest that the total concentration of TEQs in F1 samples was less than 5.0 pg/g dry wt. Tri- and tetrachloronaphthalenes elute in F1, but they have not been shown to elicit significant activity in H4IIE-luc cells (12). PCBs were the only AhR agonists known to be present in F1. PCB congeners that elicit dioxin-like activity, including non- and mono-ortho-PCBs, were detected in the order of, on average, CB118 (0.006-8.4 ng/g) > CB105 (0.004-3.8 ng/g) > CB77 (0.003-3.2) > CB156 CB126 (0.00010.05 ng/g) > CB169 (
