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1 Available online at Relative Potency Estimation of Dioxin-like Activity by Dioxins, Furans, and Co-planar PCBs Using Chemically Activated Luciferase Gene Expression Assay (H4IIE-luc Assay) Kyu Tae Lee 1, Seung-Kyu Kim 1, Hye Jin Kim 1, Young Ra Kim 1, Kyu Hyuk Jeong 2, Jong Seong Khim 3, J. P. Giesy 4 & Jung Suk Lee 1 1 NeoEnBiz Co., Daewoo Technopark A-136, Bucheon-city, Gyeonggi-do, Korea 2 College of Pharmacy, Sungkyunkwan University, #3, Cheoncheon-dong, Jangan-gu, Suwon, Gyeonggi-do , Korea 3 Division of Environmental Science and Ecological Engineering, Korea University, Seoul , Korea 4 Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Saskatchewan, Canada Correspondence and requests for materials should be addressed to S.-K. Kim (skkim89@gmail.com) Accepted 1 January 21 Abstract Chemical analysis of dioxin-like compounds (DLCs) requires usually very high cost, trained expertise and extended time for the pretreatment and instrumental operation procedure. Alternatively, recently developed in vitro cell-based bioassays offer a rapid, sensitive, and relatively inexpensive solution to estimate the potency of most DLCs including dioxins, furans and co-pcbs. In the present study, we investigated the relative potency (REP) of the cytochrome P45 gene expression through aryl hydrocarbon receptor (AhR) of 21 dioxin-like compounds by using the recombinant cell line (H4IIE-luc), which is stably transfected with firefly luciferase reporter gene to rat hepatoma cell. We exposed the cell system to get a full doseresponse curves of and other representative DLCs to estimate effect concentrations (e.g. EC5) and thereby to compare the REP of each DLC to. The toxic equivalency factor (TEF) of was the highest followed by 2,3,7,8- TCDF, 1,2,3,7,8-PeCDD and PCB 126. The TEFs of dioxins and furans were relatively higher than those of coplanar PCBs. The TEFs quantified using H4IIEluc in this study were not the same with TEFs recommended by WHO, but they were largely comparable in spite of some outliers including PCB 81, 1,2,3,4,6,7,8- HpCDD, 2,3, 7,8-TCDF, PCB 15 and PCB 118 which showed big difference. The difference may be due to the metabolic capacities between in vivo and in vitro system. TCDD toxic equivalent (TEQ) can be estimated as the sum of the concentration multiplied by TEF of each DLC. Therefore, TEQ values for environmental samples estimated by H4IIE-luc bioassay is affected both by TEF value and chemical concentration. Thus, 2,3, 7,8-TCDF, 1,2,3,4,6,7,8-HpCDD and PCB 81 among DLC, which showed big different TEF values may give a big difference in TEQ estimation in environmental sample. In order to improve TEQ predictability more by using H4IIE-luc cell line bioassay, the characterization of the difference between TEF H4IIE and TEF WHO and the distribution pattern of DLC in the samples should be focused more in future study. Keywords: Dioxin, Cell line bioassay, Relative potency, Toxic equivalency factor Introduction There has been growing concerns on the persistent organic pollutants (POPs) in recent years and also increasing regulative activities to reduce the emission/ dispersion of POPs. Among POPs concerned worldwide as well as in Korea, dioxin-like compounds (DLCs) including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls showed the most serious biological adverse effects to cause reproductive, immunological, carcinogenic and endocrine-disrupting toxicity 1. Although the usage of most DLCs was banned under the Stockholm Convention, they are still causing adverse biological effects due to their extreme persistency and resistance to degradation in the environment and their biomagnification through the food chain. The contamination of DLCs has been monitored traditionally using chemical analysis by high resolution gas chromatography-mass spectrometry (HRGC- MS). The HRGC-MS technique requires very special experts, expensive equipment and facilities, and timeconsuming work, which limit the amount of samples to

2 Relative Potency of Dioxin-like Compounds in H4IIE-luc Cell Line Bioassay 13 be monitored and assessed in most researches. Accordingly, a variety of rapid and cost-effective methods for the measurement of DLCs have been developed as alternatives/supportive screening tools for the HRCG- MS analysis. Since the middle of 199s, recombinant cell line bioassays, such as H4IIE-luc applied in this study, estimating aryl hydrocarbon receptor (AhR) mediated response, have been developed and used to determine the mixture dioxin-like activity in the various environmental matrix such as water, soil, sediment, and food for last decade 2-4 More recently, a growing number of AhR-mediated bioassays including recombinant cell assays have been used as a supportive screening tool for the assessment of DLCs in environmental samples such as food and feed in EU, sediments in Netherlands, and soil and fly ash of incinerators in Japan 5,6. Recombinant cell lines screening for DLCs have been constructed from various kind of wild type cells including rat, mouse and fish. H4IIE-luc bioassay, which uses a genetically modified rat hepatoma H4IIE cell line incorporating the firefly luciferase gene coupled to dioxin-responsive elements (DREs) as a reporter gene, is known as one of the most sensitive cell lines due to its responsiveness to at lower than a ppt level 7. However, comparison studies on the REP of DLC in H4IIE-luc are very scarce and limited. The REP information usually calculated as toxic equivalent factor (TEF), the normalized response to of each DLC, can offer very important information on the difference between consensus TEQ recommended by WHO (TEQ WHO ) and bioassayderived TCDD equivalent value (TEQ H4IIE ) in environmental samples. Also the well-defined REP information can be used in the chemical assessment of each dioxin agonist and in the toxicity identification procedure, which evaluate the relative contribution of each chemical or chemical group together with HRGC-MS analytical data. Therefore, the construction of TEF database can be a pre-requisite of application of newly developed and applied bioassay tools for assessing DLCs. In this study, dose-response relationships of 21 representative DLCs covering 6 PCDDs, 3 PCDF and 12 dioxin like PCBs were evaluated and compare the TEF values estimated by H4IIE-luc bioassay (TEF H4IIE ) with TEFs defined by WHO in 25 (TEF WHO ) 8. Results and Discussion H4IIE-luc Cell Line Bioassay Set-up We tested the exposure time of cell line to chemicals and the addition volume of luciferase reagent in each EC5 (fmol/well) μl AVG 75 μlavg 1 μl AVG Exposure time (hr) Figure 1. Change of EC5 depending on exposure time and reagent volume. (a) Bright-Glow EC5 value of TCDD (fmol/well) Exposure time 24 hr 48 hr 72 hr. : :14 :28 :43 :57 1:12 1:26 1:4 1:55 Time (hr:min) (b) Steady-Glow EC5 value of TCDD (fmol/well) Exposure time 24 hr 1 48 hr 72 hr : :14 :28 :43 :57 1:12 1:26 1:4 1:55 Time (hr:min) Figure 2. Variation of TCDD EC5 depending on luminescence reading time after reagent addition. well before full scale dose-response test. Difference in EC5 values depending on the exposure time and reagent volume addition in H4IIE cell line system was illustrated in Figure 1. Largely, there was no big difference in EC5 value depending on exposure time. Difference in EC5 was the smallest in 48 hr exposure case and relative standard deviation was the smallest in the combination of 48 hr exposure time and 1 μl

3 14 Toxicol. Environ. Health. Sci. Vol. 2(1), 12-17, 21 EC5 (fmol/well) Numbers of test Figure 3. Quality control chart of positive standard. (a) PCDDs (b) PCDFs ,2,3,7,8-PeCDD 1,2,3,4,7,8-HxCDD 1,2,3,6,7,8-HxCDD 1,2,3,7,8,9-HxCDD 1,2,3,4,6,7,8-HpCDD 1.E-2 1.E-1 1.E 1.E 1 1.E 2 1.E 3 1.E 4 1.E 5 2,3,7,8-TCDF 1,2,3,7,8-PCDF 1,2,3,4,6,7,8-HpCDF 1.E-2 1.E-1 1.E 1.E 1 1.E 2 1.E 3 1.E 4 1.E 5 Figure 4. Dose-response curve of PCDDs/PCDFs determined by H4IIE-luc cell line system. reagent addition. In this study, 48 hr exposure and 1 μl reagent addition as experimental condition was selected. Change in EC5 value was monitored with 2 min interval for 2 hr after adding flash-typed and glowtyped luciferase reagent, Bright-Glow and Steady- Glow, respectively, in order to check when light emission is stabilized and how long reading time can be delayed without problem (Figure 2). EC5 values were PCB-77 PCB-81 PCB-15 PCB-114 PCB-118 PCB E-2 1.E-1 1.E 1.E 1 1.E 2 1.E 3 1.E 4 1.E 5 1.E 6 1.E 7 1.E 8 5 PCB-126 PCB-156 PCB-157 PCB-167 PCB-169 PCB E-2 1.E-1 1.E 1.E 1 1.E 2 1.E 3 1.E 4 1.E 5 1.E 6 1.E 7 1.E 8 Figure 5. Dose-response curve of dioxin-like PCBs determined by H4IIE-luc cell line system. slightly different with the type of luciferase reagent. EC5 values with elapsed time were very stable in both case, however, in later part of the measurement, the values were more stable in case of Steady-Glow rather than in Bright-Glow. The fact that there was no significant change in EC5 values for 2 hrs, means that there is no need to wait for the stabilization of light emission or that there is no need to worry about the elapsed time for 2 hrs after reagent addition. In this study, Steady-Glow was selected as optimal reagent, and plate reading was run without time delay. Over 3 times of TCDD standard tests were independently carried out for the quality control and the EC5 values were estimated from each full dose-response curve of TCDD (Figure 3). Compared to the results of other s CALUX studies, the EC5 values varied within acceptable range, indicating that the test method was well set-up 3,9. This result was also similar with that in mouse hepatoma cell line bioassay 1. Dose-response Relationship and Relative Potency Full dose-response of DLC including 6 PCDD congeners, 3 PCDF congeners, and 12 PCBs was determined in each 1 dilution series (Figures 4, 5). Full

4 Relative Potency of Dioxin-like Compounds in H4IIE-luc Cell Line Bioassay 15 Table 1. Relative potency of dioxin-like compounds in H4IIE-luc cell line system. This study Standard samples TEF WHO TEF H4IIE REP range Ratio EC5 basis (EC2 to EC5) (TEF H4IIE /TEF WHO ) PCDDs ,2,3,7,8-PeCDD to ,2,3,4,7,8-HxCDD to ,2,3,6,7,8-HxCDD to ,2,3,7,8,9-HxCDD to ,2,3,4,6,7,8-HpCDD to PCDFs 2,3,7,8-TCDF to ,2,3,7,8-PeCDF to ,2,3,4,6,7,8-HpCDF to DL-PCBs PCB to.16.3 PCB to PCB to.22 2 PCB to PCB to PCB to PCB to.6.27 PCB to PCB to.32.3 PCB to PCB to PCB to dose-response curve can be obtained within maximum solubility in hexane for almost all DLC tested in this study, excepting two PCB congeners (PCB 167, PCB 189) which was not reached to maximum response even at the limit of solvent solubility. For 2,3,7,8- TCDD, dose-response relationship was identified between.77 and 23 fmol/well and the linearity was revealed in the range of fmol/well. Maximum response was observed at 233 fmol/well in our dilution series and EC5 of was estimated at 2-5 fmol/well. For the other PCDDs tested in this study, full dose-response curve was expressed with different range of concentration depending on the REP of the chemicals. Linear slopes in dose-response curve were almost similar among PCDD congeners, PCDF congeners and PCB congeners. Maximum response of each chemicals were different more or less but this may be due to the sensitivity of luciferase reagent, not due to its own characteristics. However, irrespective of the maximum response value, REP can be estimated by the comparison with the half of the maximum response of the chemicals, which is defined to EC5 (effect concentration median) in this study. By the comparison of each chemical EC5 value to that of which is the most robust agonist for AhR, TEF H4IIE value of 1,2,3,7,8-PeCDD, 1,2,3,4, 7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,7,8,9-HxCDD and 1,2,3,4,6,7,8-HpCDD was calculated to be.33,.93,.55,.36 and.78, respectively (Table 1). TEF H4IIE of 1,2,3,4,6,7,8-HpCDD was 7.8 times higher than TEF WHO, indicating that it could make dioxin TEQ higher. The other PCDD congeners TEF H4IIE values were lower than TEF WHO, implying that it would let dioxin TEQ lower. With the same way, 2,3, 7,8-TCDF and PCB 81 and 126 can play an important roll in TEQ increase, to the contrary, many of dioxinlike PCB congeners except PCB 81 and 126 will give lower TEQ values. Some compounds such as PCB 81 can be metabolized in whole animal system but H4IIEluc recombinant cell line system may be different with in vivo system in view of metabolic capacity, thus, TEF WHO may be lower than TEF H4IIE for these kinds of chemicals 11. Some compounds can be underestimated in H4IIE-luc cell line system by less bioavailabilty or insufficient exposure time for bioaccumulation in vitro test condition. Comprehensively, TEQ estimation by H4IIE-luc cell line bioassay may be different from chemically-calculated TEQ depending on the composition of DLC in environmental samples. If DLC in a sample evenly distribute, TEQ H4IIE value

5 16 Toxicol. Environ. Health. Sci. Vol. 2(1), 12-17, 21 H4IIE-TEF y=1.45x R 2 = WHO-TEF Figure 6. Relationship between TEF WHO and TEF H4IIE. of the sample will be low as much as 12% relative to TEQ WHO value because the sum of TEF H4IIE is 88% of that of TEF WHO. Since the concentrations of DLC in environmental samples are very different, there would be some disharmony between TEQ H4IIE and TEQ WHO values. In spite of such difference, TEQ predictability of CALUX cell line bioassay including H4IIE-luc is known to be excellent in many validation studies, implying the technique can be used as a semi-quantitative and alternative tool 12,13. Curve fitting result of TEF H4IIE and TEF WHO was shown in Figure 6. The result means that TEF WHO can be accounted for TEF H4IIE by 88%, indicating that there is high possibility of TEQ WHO prediction in environmental sample by H4IIE cell line bioassay. In order to improve TEQ predictability more, understanding of the difference between TEF H4IIE and TEF WHO and distribution characteristics of DLC in the samples should be focused more. Further researches are necessary to evaluate the REP of other AhR agonists and their mixture effect, and also to develop the fractionation method which can be used in the effect identification procedure and risk assessment. Methods Standard operating procedure of H4IIE-luc cell line bioassay was performed as previously described 14. The H4IIE-luc cell line was obtained from Prof. J. P. Giesy at the University of Saskatchewan, Canada. The cell lines were genetically engineered from rat hapatoma cells to produce luciferase by reporter gene when the cells are exposed to DLC. The cells were cultured in Dulbecco s Modified Eagle s Medium (DMEM) without phenol red, supplemented with 1% fetal bovine serum, in 5% CO 2 incubator at 37 C. Harvested cells were plated into a well of 96 well plate at the density of 2*1 4 cells/25 μl/well. Next day (24 hr later), cells were exposed to chemicals for 24 hr, 48 hr, and 72 hr to induce the reporter gene expression. The light of the luciferase induced by the reporter gene was detected by the luminometer. Cell line bioassay test was conducted by triplicate. Positive and negative control was and ethanol, respectively. The final concentration of the carrier solvent was 1%. DLC including 6 dioxins, 3 furans and 12 dioxinlike PCBs were purchased from AccuStandard and the chemicals were dissolved in hexane and the solvent was directly spiked into cell media or exchanged to ethanol for bioassay. Each standard solution was diluted and tested with 1 dilution series to get the full dose-response of the chemicals. Effect concentration median (EC5) of the tested chemicals was defined as the 5% of the maximum response from full dose-response relation. REP of each chemical to induce the dioxin-like activity in cell system was compared to that of which is set to the unit value. Acknowledgements This work was financially supported by Korea Ministry of Land, Transport, and Maritime Affairs and Ministry of Environment. The authors thank the anonymous reviewers for helping in publication of this manuscript. References 1. Reggiani, G. Toxicology of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD): Short review of its formation, occurrence, toxicology, and kinetics, discussing human health effects, safety measures, and disposal. Regul. Toxicol. Pharmacol. 1, (1981). 2. Khim, J. S. et al. Alkylphenols, polycyclic aromatic hydrocarbons, and organochlorines in sediment from Lake Shihwa, Korea: Instrumental and bioanalytical characterization. Environ. Toxicol. Chem. 18, (1999). 3. Yoo, H., Khim, J. S. & Giesy, J. P. Receptor-mediated in vitro bioassay for characterization of Ah-R-active compounds and activities in sediment from Korea. Chemosphere 62, (26). 4. Hoogenboom, L. et al. The CALUX bioassay: Current status of its application to screening food and feed. Trends. Anal. Chem. 25, (26). 5. Japan Ministry of the Environment, Bioassay method manual for dioxin-like compounds (Japanese), method/full. pdf (26).

6 Relative Potency of Dioxin-like Compounds in H4IIE-luc Cell Line Bioassay Behnisch, P. A., Hosoea, K. & Sakai, S. Bioanalytical screening methods for dioxins and dioxin-like compounds - a review of bioassay/biomarker technology. Environ. Int. 27, (21). 7. Sanderson, J. T. et al. Comparison of Ah receptormediated luciferase and ethoxyresorufin-o-deethylase induction in H4IIE cells: implications for their use as bioanalytical tools for the detection of polyhalogenated aromatic hydrocarbons. Toxicol. Appl. Pharmacol. 137, (1996). 8. Van den Berg, M. et al. The 25 World Health Organization reevaluation of human and mammalian toxicity equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. 93, (26). 9. Song, M. et al. Determinations of dioxinlike activity in selected mollusks from the coast of the Bohai Sea, China, using the H4IIE-luc bioassay. Ecotoxicol. Environ. Saf. 67, (27). 1. Windal, I. et al. CALUX as a tool for the estimation of dioxin-like activity in marine biological matrixes. Environ. Sci. Tech. 39, (25). 11. Birnbaum, L. S. The role of structure in the disposition of halogenated aromatic xenobiotics. Environ. Health Perspect. 61, 11-2 (1985). 12. Chou, I. C. et al. Validation of the CALUX bioassay as a screening and semi-quantitative method for PCDD/F levels in cow s milk. J. Hazard Mater. 154, (28) 13. US EPA. Method 4435: Method for toxic equivalents (TEQs) determinations for dioxin-like chemical activity with the CALUX Bioassay. 14. Koh, C. H. et al. Instrumental and bioanalytical measures of dioxin-like and estrogenic compounds and activities associated with sediment from the Korean coast. Ecotoxicol. Environ. Saf. 61, (25).