HIGHLY SENSITIVE CALUX ASSAY FOR SCREENING DIOXINS IN RETAIL
FISH
Tsutsumi T1*, Amakura Y2, Nakamura M3, Handa H3, Denison MS4, Matsuda R1, Teshima R1
1National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; 2 Matsuyama
University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan; 3 Hiyoshi Corporation, 908 Kitanosho-cho,Omihachiman, Shiga 523-8555, Japan; 4 Department of Environmental Toxicology, Meyer Hall, University ofCalifornia, Davis, CA, USA

Introduction
Fishery products have been identified as the main source of polychlorinated dibenzo-p-dioxins (PCDDs),dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyl (dl-PCBs), collectively referred as to dioxins,in the Japanese diet. It is therefore highly important to develop screening methods for dioxins in retail fish in
Japan. Reporter gene assays, such as the chemical-activated luciferase gene expression (CALUX) assay, arecurrently considered the best screening method for dioxins. A couple of studies have reported on the utility ofthe CALUX assay as a possible alternative for screening dioxins in fish samples 1-3. However, since dioxin
concentrations in fish samples are typically very low, a more sensitive CALUX assay is useful. The CALUX
assay uses a genetically modified cell line that can detect dioxins on the basis of their activation of the arylhydrocarbon receptor (AhR), which in turn increases the expression of the luciferase reporter gene. The
expression of the luciferase gene under the control of the mouse mammary tumor virus promoter is regulated bydioxin responsive elements (DREs, the DNA recognition site of dioxin-bound AhR). Recently, a more sensitive
CALUX assay using a new cell line (H1L7.5) has been developed 4. The H1L7.5 cell line was constructed with anew AhR-inducible reporter plasmid containing an increased numbers of DREs (20 DREs). However, theH1L7.5 cell CALUX bioassay has not yet been used for screening dioxins in fish samples. We have developed asimilar amplified cell line (pGL7.3 cell) by stable transfection of mouse hepatoma (hepa1c1c7) cells with AhRinducible reporter plasmid containing 12 DREs (pGudLuc7.3) 4, and have found that the resulting CALUX assay
using this cell line is highly sensitive 5. Here, we report on the applicability of the new CALUX assay forscreening dioxins in retail fish samples.
Materials and methods
Samples: Fish samples were purchased from supermarkets in Tokyo, Japan. The samples (muscular sections)were homogenized using a food cutter and stored at –20ºC until analysis.
CALUX assay: A 10 g sample of homogenized fish was mixed with 15 ml of acetone and mechanically shaken
for 5 min. The sample was then extracted with 10 ml of dichloromethane/n-hexane (1:2, v/v) by shaking for 2min. The extract was passed through a layered column with (from the bottom) 1.0 g of celite and 7.3 g ofanhydrous sodium sulfate. Each sample was extracted three times, and then passed through the column. Afterelution with an additional 10 ml of n-hexane, the column elute was evaporated to dryness and weighed toestimate the lipid content of the sample. After dissolution in 20 ml of n-hexane, then 16 g of 45% (w/w) sulfuric
acid-silica gel was added, the supernatant was loaded onto a layered sulfuric acid silica gel column with (fromthe bottom) 3.4 g of anhydrous sodium sulfate, 0.7 g of 33% (w/w) sulfuric acid-silica gel, and 4 g of 45% (w/w)
sulfuric acid-silica gel. The sample was eluted with 20 ml of n-hexane, then further loaded onto an XCARB
column. After sample addition, the XCARB column was washed with n-hexane (10 ml); then the first fraction,
containing dl-PCBs, was eluted with n-hexane/toluene/ethyl acetate (8:1:1, v/v) (15 ml), and the second fraction,
containing PCDD/Fs, was eluted with toluene (20 ml). The elutes were concentrated and resuspended in nhexane (4 ml). A portion of the cleaned-up extract (maximum 1.5 ml of n-hexane) was solvent exchanged into
300 µl of MEM culture medium supplemented with 10% (v/v) FBS and 500 µg/ml G418 (the final DMSO
concentration in the culture medium was 1%). In addition, 2,3,7,8-TCDD standard solutions (3 µl of DMSO)
added to 1 ml of n-hexane were prepared using vacuum centrifugation and diluted with the culture medium (300µl). The pGL7.3 cells (7.5×104/well) were cultured in the culture medium in 96-well plates at a temperature of37ºC, in the presence of 5% CO2. After incubation for 20-24 h, the cells were exposed in triplicate (95 µl/well)Organohalogen Compounds

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for samples. The induced luciferase activity was determined with the luciferase assay system using a Centro LB
960 Microplate Luminometer (Berthold Technologies) or an EnSpire® Multimode Plate Readers (PerkinElmer)
and expressed in terms of relative light units (RLUs). Average RLUs were calculated from individual RLUsobtained from triplicate wells for samples and standards. All RLU values were corrected by subtraction of theRLUs from the blanks (standard wells containing no 2,3,7,8-TCDD). Fitting of standard curves and calculationof dioxin contents in fish samples were performed using a four-variable Hill equation.
HRGC/HRMS analysis: Extraction, cleanup, and analysis of dioxins were performed in accordance with apreviously reported protocol using high-resolution gas chromatography/high-resolution mass spectrometry(HRGC/HRMS) 6. The limits of detection (LODs) for PCDD/Fs were 0.01–0.05 pg/g. The LODs for dl-PCBs
were 0.1 pg/g for non-ortho PCBs and 1 pg/g for mono-ortho PCBs. The toxic equivalent (TEQ) concentrations
were calculated using WHO toxic equivalency factors (2005). In calculating the total TEQ concentration in asample, it was assumed that all isomer concentrations lower than the LODs were equal to zero.
Results and discussion
Standard curve of the CALUX assay: Figure 1 shows the standard curve of the pGL7.3 CALUX bioassay,
derived from five individual assays performed on separate days. The curves were highly reproducible, asindicated by the low SDs. The mean concentration, which resulted in 50% of the maximal response for 2,3,7,8TCDD (EC50), was approximately 7.6 pg/ml. The minimum concentration of 2,3,7,8-TCDD detectable by theCALUX assay (defined as 3 SDs above the mean RLU value for the zero standards) was approximately 0.24 pg
2,3,7,8-TCDD/ml (approximately 0.19 pg 2,3,7,8-TCDD/assay). To determine the limit of quantification (LOQ),
8 concentrations of 2,3,7,8-TCDD (0.49 to 31 pg/ml) were assayed in triplicate on 5 different days. From 0.24 to31 pg 2,3,7,8-TCDD/ml, the residual errors were small (-3.3 to 3.2%), and the coefficients of variation (CVs)over the same range were also low (0.8 to 6.0%). Therefore, the assay’s quantification range was determined tobe from 0.49 to 31 pg/ml for the PCDD/Fs and dl-PCBs fractions. The 0.49 pg/ml quantification limit
corresponded to 0.039 pg 2,3,7,8-TCDD/g, when a 10 g fish sample was tested. The quantification limit was 2
times lower than a conventional CALUX assay using the H1L6.1 cell line 2.
Effect of fish matrix on the CALUX assay: To examine the matrix effect for the CALUX assay under thesample preparation procedure, two-fold serial dilutions of the cleaned-up extracts of the fish samples were tested
in the CALUX assay. Three different fish extracts were diluted serially with n-hexane and assayed. As shown inFigure 2, the values observed for the PCDD/Fs and dl-PCBs fractions in these samples tended to increase withdilution. This indicates that the dilution process might eliminate the matrix effects in the CALUX assay. For thisreason, serial dilutions of fish extracts (dilution factors of 1, 2, 4, and 8) were measured in the CALUX assay,
and the maximum concentration was used to reduce the rate of false-negative results.
A recovery test using the fish samples was also carried out to examine the effect of the matrix on the CALUX
assay. Fish samples were spiked with known concentrations of PCDD/Fs and PCB 126, extracted, cleaned-up,and assayed with the serial dilution procedure. Acceptable recoveries (74 to 95%) were obtained over the tested
range for two varieties of fish analyzed (Table 1).
Reproducibility of the CALUX assay: The reproducibility of the CALUX assay using the sample preparation
procedure described above was tested by analyzing replicate samples from the same fish. The samples wereextracted, cleaned-up, and assayed in five separate analyses on different days. The CVs for two varieties of fishsamples were 17 to 21% for PCDD/Fs and 12 to 23% for dl-PCBs (Table 2). The CALUX assay therefore has
acceptable precision for dioxin analysis. However, the ratio of the minimum to maximum concentrations of dlPCBs in mackerel was nearly 1:2. To reduce the impact of variation, the dioxin concentration determinations
reported for the CALUX assay were the mean of two independent sample preparations analyzed in duplicate.
Comparison of the CALUX assay with HRGC/HRMS analysis: The TEQ concentrations of PCDD/Fs and dlPCBs in 32 retail fish samples were determined by both the CALUX assay and HRGC/HRMS analysis (Figure3). The correlation coefficients between the two methods were 0.94 for the PCDD/Fs and 0.96 for the dl-PCBs.
The high correlation between the results obtained by the two methods suggested that the CALUX assay would
be a useful screening method for predicting TEQ concentrations in fish.

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In conclusion, the present study evaluated a new CALUX assay using the pGL7.3 cell line for determination ofdioxin concentrations in retail fish. The assay would be a useful dioxin TEQ screening method prior to
HRGC/HRMS analysis.
Acknowledgements
This work was supported by a Health Sciences Research Grant from the Ministry of Health, Labour, and Welfareof Japan and a grant from the National Institute of Environmental Health Sciences of the United States(ES04699).
References
1. Besselink H, Leonards P, Felzel E, Brouwer B. (2002); Organohalogen Compd. 58: 413-415
2. Tsutsumi T, Amakura Y, Nakamura M, Brown DJ, Clark GC, Sasaki K, Toyoda M, Maitani T. (2003);
Analyst 128(5): 486-492
3. Kojima H, Takeuchi S, Tsutsumi T, Yamaguchi K, Anezaki K, Kubo K, Iida M, Takahashi T, Kobayashi S,Jin K, Nagai T. (2011); Chemosphere 83(6): 753-759
4. He G, Tsutsumi T, Zhao B, Baston DS, Zhao J, Heath-Pagliuso S, Denison MS (2011); Toxicol. Sci. 123(2):511-522
5. Tsutsumi T, Ishizuka N, Matsuda R, Denison MS (2008); The 11th Annual Meeting of Japan Society ofEndocrine Disrupters Research, Program and Abstracts: 72
6. Tsutsumi T, Amakura Y,Sasaki K, Toyoda M,Maitani T. (2003); Anal
Bioanal Chem.
375(6):792-798

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Table 1 Recovery of dioxins from fish samples aSpiked Conc.
(TCDD eq./g)
SalmonPCDD/Fs
0.58
2.5
PCB 126
0.80
2.3
Tuna

PCDD/Fs
PCB 126

Recovery (%)
Mean ± SD
95 ± 684 ± 1295 ± 1386 ± 2

0.57
2.6
0.86
2.4

80 ±74 ±76 ±87 ±591312aFish samples spiked with known quantities of PCDD/Fs or PCB 126 wereextracted, cleaned up, and measured by the CALUX assay (n = 3).

Table 2 Reproducibility of the CALUX assay combined with the sample preparation procedure

Horse mackerel

PCDD/Fs
dl-PCBs

1st0.44
0.23Mackerel

PCDD/Fs
dl-PCBs

0.95
0.91

a2nd0.52
0.27

3rd0.37
0.24

TCDD eq./g
4th
5th0.33
0.32
0.23
0.30

Mean ± SD
0.40 ± 0.084
0.25 ± 0.031

CV(%)
2112

1.32
0.90

0.94
0.77

1.07
0.48

1.03 ± 0.18
0.78 ± 0.1817230.85
0.86

aThe fish contaminated in the natural environment were extracted, cleaned up, and assayed by the CALUX assay
in five separate runs on different days.

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