APPLICATION OF THE CALUXTM ASSAY TO THE ANALYSISOF DXNs IN FISH (THE FIRST REPORT)
Junko Fujino 1, Tomoaki Tsutsumi2, Yoshiaki Amakura2Masafumi Nakamura 1,
Hiroko Kitagawa 1, Tsukasa Yamamoto 1, Kumiko Sasaki2, Masatake Toyoda 221

National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501,JapanHiyoshi Corporation, 908 Kitanoshocho, Omihachiman, Shiga, 523-8555, Japan

Introduction
Food has been generally recognized as the main source of human intake of polychlorinateddibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like coplanar polychlorinatedbiphenyls (Co-PCBs); more than 90% of the total daily intake of these contaminants generally
derives from food1. Recently, a total diet study in Japan revealed that about 60% of the dietaryintake of these compounds, collectively know as dioxins (DXNs), is likely to come from the intake
of fish and shellfish2. Therefore, monitoring the levels of DXNs in fish and shellfish would
provide important information for risk assessment.
Traditionally, DXNs in food andenvironmental
sampleshave
been
analyzedbyhigh-resolutiongaschromatography/high-resolution mass spectrometry (HRGC/MS). However, HRGC/MS requires
the use of expensive equipment and the sample preparation procedures are often time consumingand costly. Thus, the use of HRGC/MS is not entirely suited to the task of rapid and frequentmonitoring of large numbers of samples.
One possible alternative to HRGC/MS is a bioassay for DXNs such as the CALUXTM assay.
We have previously applied the CALUXTM assay to dioxin analysis in Japanese fat samples withsuccess3. In this study, we describe the application of the CALUXTM assay for the monitoring ofDXNs in fish samples
Materials and Methods
Fish samples
Nineteen fish samples (3 yellow tail, 3 mackerel, 2 cod, 4 tuna, 2 salmon. 2 bonito, 2 sea
bass and a flatfish) purchased at the market in Japan were analyzed by the CALUXTM assay. A
commercially available certified reference sample of carp was also analyzed (Wellington
Laboratories, Guelph, Canada).
Sample extraction and clean-up procedures
The treatment of samples for the CALUXTM assay was conducted in original way describedas follows.
1.
2.
3.
4.
5.
6.
7.
8.

Grind sample and aliquot 10 grams of sample.
Add 15 milliliters of acetone to sample aliquot.
Add 10 milliliters of dichloromethane/hexane (1:2) and mix.
Centrifuge the mixture at 500 rpm for five minutes to separate the phases.
Apply the dichloromethane/hexane layer to the extraction column.
Repeat step 3 through 5 two times.
Wash the column with 10 milliliters of dichloromethane/hexane (1:2).
Following concentration, the sample extract was cleaned up and separated into a fraction
containing PCDDs/PCDFs and a fraction containing Co-PCBs.

CALUXTM Assay
The CALUXTM assay uses a patented recombinant mouse cell line that contains the luciferase
reporter gene under control of dioxin responsive elements4. When these cells are exposed toenvironmental ligands such as DXNs, luciferase protein is synthesized. The amount of light
produced by the luciferase protein is directly related to DXNs-TEQ. The CALUXTM assay
method used has been described previously5. Briefly, the cells were grown in the 96-well viewplates and exposed to fish sample extracts and 2,3,7,8-TCDD standards (250, 125, 62.5, 31.2, 15.6,7.8, 3.9, 1.9, 0.9, 0.5 ppt), using DMSO as the vehicle (final DMSO concentration 1% in cellculture medium). The plates were incubated at 37o C and 5% CO2 for 20 hours to produce
optimal expression of luciferase activity. Following incubation, the medium was removed andthe cells were lysed. Luciferase activity was determined using a luminometer (Lucy 1 producedby Anthos Corp.). Luciferase activity was reported as relative light units (RLU).

Figure 1. Method of the CALUXTM analysis of the samples

AhR
XRE

Luciferase

Determine the RLU

RLU

Cell

Concentration of 2,3,7,8-TCDD

HRGC/MS analysis
The extraction and cleanup of samples for HRGC/MS followed previously publishedprotocols 6. The analysis of the 17 active PCDDs/Fs and 12 Co-PCBs (non-ortho andmono-ortho PCBs) were performed by HRGC/MS using a HP6890 plus gas chromatographcoupled to a JMS-700 mass spectrometer (JEOL Ltd., Japan). The TEQ concentrations werecalculated using the WHO-TEFs (1997).

Results and Discussion
Table 1 shows the data of the concentration of DXNs in fish analyzed by the CALUXTM assay.
The averages of TEQ of salmon, tuna, bonito, candlefish, flatfish, mackerel, sea bass andyellowtail were 1.948, 1.368, 0.496, 1.432, 0.759, 1.110, 1.292 and 3.697 pg-TEQ/g wet weight(wg) respectively. We previously reported the data of the concentration of DXNs in Japanesefishes. From that study the averages of TEQ of flatfishes, mackerels and sea basses were 0.404,0.992 and 10.397 pg-TEQ/wg respectively5. These data indicate that TEQ levels of flatfishes ormackerels analyzed by the CALUXTM assay are approximately equal to the data by HRGC/MS.
Furthermore the samples of commercially available carp whose DXNs concentration wascertified were analyzed by the CALUXTM assay and these results were compared with the dataanalyzed by HRGC/MS (Table 1). PCDDs/Fs-TEQ analyzed by the CALUXTM assay and by
HRGC/MS were 23.300 and 19.681 pg-TEQ/wg respectively. Co-PCBs-TEQ analyzed by theCALUXTM assay and by HRGC/MS were 9.345 and 69.404 pg-TEQ/wg respectively. TEQ
analyzed by the CALUXTM assay and analyzed by HRGC/MS was 32.645 pg-TEQ/wg and 89.085
pg-TEQ/wg respectively. The CALUXTM assay results and HRGC/MS data were very similar
but there are small differences that might be dependent on the characteristic of low sensitivity toCo-PCBs of the CALUXTM assay.
Table 1. Concentration of PCDDs/Fs and Co-PCBs in fish
Samplepg TEQ/g on wet weight basis
Dioxin-like PCBs
PCDD/Fs+dioxin-like PCBs
0.311
1.067
0.7732.829
0.542
1.948

Salmon

No.1
No.2
Mean

PCDD/Fs
0.756
2.056
1.406

Tuna

No.1
No.2
No.3
No.4
Mean

ND
2.375
2.000
ND
1.094

ND
0.385
0.710
ND
0.274

ND
2.760
2.700
ND
1.368

Bonito

No.1
No.2
Mean

0.435
0.240
0.338

0.120
0.197
0.159

0.555
0.437
0.496

Cod

No.1
No.2
Mean

0.968
0.693
0.831

0.579
0.623
0.601

1.547
1.316
1.432

Flat fish

No.1

0.524

0.235

0.759

Mackerel

No.1
No.2
No.3
Mean

1.638
0.498
0.340
0.825

0.754
ND0.100
0.285

2.392
0.498
0.440
1.110

Sea bass

No.1
No.2
Mean

1.505
0.526
1.016

0.553
ND
0.277

2.058
0.526
1.292

Yellowtail

No.1
No.2
No.3
Mean

2.511
3.501
1.666
2.559

1.038
1.646
0.729
1.138

3.549
5.147
2.395
3.697

Carp
No.1
23.300 (19.681)
9.345 (69.404)
The values in parentheses were determined by HRGC/HRMS.

32.645 (89.085)

Conclusions
Considering Japanese lifestyle of eating, it should be necessary for its safety to screen morekinds of fishes and to monitor the DXNs in fish more frequently and continuously. But now inJapan few extensive surveys of the concentrations of DXNs in foods such as fishes wereTMconducted. In this study we applied CALUX assay to the analysis of DXNs in fishes. This
result confirmed its similarity to HRGC/MS, thereby from the standpoint of its rapidity and costs
CALUXTM assay will be useful for monitoring and screening. Belgium government's Scientific
Institute of Public Health has already reported DXNs in foods such as a chicken fat sample,
porcine fat samples and samples of eggs analyzed by the CALUXTM assay6. That report also
showed the usefulness of the CALUXTM assay as an alternative method for HRGC/MS todetermine TEQ levels in foods. More data of the concentration of DXNs in fish are required toverify the correlation of CALUXTM assay with HRGC/MS method.
References
1. Dioxins (1999), Ministries and Agencies that are members of the Ministerial Council on
Dioxin Policy Japan.
2. Denison, M., Brouwer, A. and Clark, G. (1998). U.S.patent # 5,854,010.
3. Kayama F, Hamamatsu A, Sagisaka K, Brown D, Clark G, Suzuki T (2000), The 3rd Annual
Meeting of Japan Society of Endocrine Disrupters Research.
4. Brown, D., Kishimoto, Y., Ikeno, O., Chu, M., Nomura, J., Murakami, T., and Murata, H.
(2000) Organohalogen Compounds 45: 200-203.
5. Masatake Toyoda, Takako Iida, Tsuguhide Hori, Toshihiko Yanagi, Youichi Kono andHiroyasu Uchibe : Journal of the Food Hygienic Society of Japan, (1999) 40, 111～121.
6. Ilse Van Overmeire, Leo Goeyens, Hedwig Beernaert, Sarah Srebrnik, Geert De Poorter,Willy Baeyens, George Clark, Michael Chu, Andrew Chu, David Chu, Richard Morris andDavid Brown, (2000) Organohalogen Compounds 45: 196-199.