BIOANALYSISAPPLICATION OF THE CALUX™ ASSAY TO THE ANALYSIS OF DXNSIN FISH (THE SECOND REPORT)
Hisatoshi Yabushita 1, Tomoaki Tsutsumi2, Yoshiaki Amakura2, Masafumi Nakamura 1, Junko
Fujino 1 , Hiroshi Murata 1, Kumiko Sasaki2, Tamio Maitani 2 , Masatake Toyoda 3
1

Hiyoshi Corporation, 908 Kitanoshocho, Omihachiman, Shiga, 523-8555, Japan
National Institute of Health Sciences,1-18-1, Kamiyoga,Setagaya-kuTokyo158-8501,Japan3
Jissen Womaen’s University, 4-1-1, Osakaue, Hino-shi, Tokyo 191-8510,Japan2Introduction
Food has been generally recognized as the main source of human intake of dioxins (DXNs) such aspolychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like coplanar
polychlorinated biphenyls (Co-PCBs), so it is very important to secure the supply of food.
Recently, a total diet study in Japan revealed that main source of the dietary intake of DXNs islikely to come from the intake of fish and shellfish. Therefore, monitoring the levels of DXNs in fishand shellfish would provide important information for risk assessment and management, especially inJapan. Traditionally, DXNs in food and environmental samples have been analyzed by high-resolution
gas chromatography/high-resolution mass spectrometry (HRGC/MS). However, HRGC/MS requires
the use of expensive equipment and the sample preparation procedures are often time consuming andcostly. Thus, the use of HRGC/MS is not entirely suited to the task of rapid and frequent monitoring oflarge numbers of samples. In order to execute the management, it is necessary to develop a rapid andinexpensive screening method.
In DXN20013), we reported preliminary data of CALUX™ (Chemically Activated Luciferase Expressio)
assay for the screening of DXNs retailed fish. In this study, we examined for the application in detail.
Materials and Methods
Fish samples
Twenty two 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 in the comparative studies with HRGC/
MS analysis and CALUXTM assay. A commercially available certified reference sample of carp wasalso analyzed to examine extraction efficiency of DXNs by shaking extract for CALUXTM (Wellington
Laboratories, Guelph, Canada).
Sample extraction and clean-up procedures
1. Grind sample and aliquot 10 grams of sample.
2. Add 15 milliliters of acetone to sample aliquot.
3. Add 10 milliliters of dichloromethane/hexane (1:2) and mix.
4. Centrifuge the mixture at 500 rpm for five minutes to separate the phases.
5. Apply the dichloromethane/hexane layer to the extraction column.
6. Repeat step 3 through 5 two times.
7. Wash the column with 10 milliliters of dichloromethane/hexane (1:2).
8. Following concentration, the sample extract was cleaned up and separated into a fraction
containing PCDDs/DFs and a fraction containing Co-PCBs.

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BIOANALYSISCALUXTM Assay
The CALUXTM assay uses a patented recombinant mouse cell line that contains the luciferase
reporter gene under control of dioxin responsive elements 4) . When these cells are exposed toenvironmental ligands such as DXNs, luciferase protein is synthesized. The amount of light producedby the luciferase protein is directly related to DXNs-TEQ. The CALUXTM assay method used has been
described previously. Briefly, the cells were grown in the 96-well view plates and exposed to fishsample 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 cell culture medium). The plates wereincubated at 37 oC and 5 % CO2 for 20 hours to produce optimal expression of luciferase activity. Andthen, the medium was removed and the cells were lysed. Luciferase activity was determined using aluminometer (Lucy 1 produced by Anthos Corp.). Luciferase activity was determined as relative lightunits (RLU).

Figure 1. Mechanism of CALUX Cell HRGC/MS analysis
The extraction and cleanup of samples for HRGC/MS followed previously published protocols 5).
The analysis of the 17 active PCDDs/Fs and 12 Co-PCBs (non-ortho and mono-ortho PCBs) wereperformed by HRGC/MS using an HP6890 plus gas chromatograph coupled to a JMS-700 mass
spectrometer (JEOL Ltd., Japan). The TEQ concentrations were calculated using the WHO-TEFs(1997).
Results and Discussion
Recovery of DXNs from by shaking extraction in CALUXTM
To examine recovery rate DXNs from fish by shaking extraction in , carp sample was extracted byshaking extraction or alkaline digestion, and then analyzed HRGC/MS. Table1. shows the result ofrecovery of DXNs by the extraction, which was represented as relative values alkaline digestion. The
recovery rate of DXNs was 70-80%, indicating acceptance in screening method.

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BIOANALYSIS
Correlation between CALUX™ Assay and HRGC/MS analysis
Figure 2 shows the correlation of total PCDDs/DFs and Co-PCBs. The correlation coefficient ofPCDDs/DFs was very high (R=0.887) and the value analyzed by CALUX™ Assay was about 1.7 timethat by HRGC/MS. The correlation coefficient of Co-PCBs was also very high (R=0.911) but the value
analyzed by CALUX™ Assay was about 0.22 times that by HRGC/MS.
Table1. Recovery of shaking ext.

PCDD
PCDFs
Co-PCBs

CompoundRecovery(%)

2378-TCDD
12378-PeCDD
23478-PeCDF
33’44’5-PeCB(#126)
Total PCDFs
Total PCDFs
Total PCBs
Total DXNs

78.2
79.6
78.0
74.7
74.0
74.5
75.9
74.8

*recovery was represented as relative values to alkaline digestion

Figure 2. Correlation between CALUX Assay and HRGC/MS (PCDDs/DFs and Co-PCB, x axis ;HRGC/MS(pgTEQ/gfat) y axis ; CALUX (pgTEQ/gfat))
Figure 3 shows the correlation of total DXNs (Separate ; total PCDDs/DFs and total Co-PCBs wereanalyzed separately and sumed) and total DXNs (Non-separate ; total PCDDs/DFs and total Co-PCBs
were mixed and then analyzed). Since CALUX™ Assay was evaluated from biological response, coexistence of Co-PCBs might affect the activity of PCDDs/Fs, but the result was little affected by theco-existence. This might suggest that the distributions of Co-PCBs were restricted to a certain degree inthe case of fish samples.

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Conclusions
The limit of quantification by CALUX™ Assay was 0.16 pgTEQ/g wet. The original extraction andclean-up method can be substituted for alkali extraction. The correlation between HRGC/MS andCALUX™ was very high in any conditions. CALUX™ Assay has a tendency to overestimate total
TEQ compared with HRGC/MS. So it can be concluded that CALUX™ Assay is useful method forscreening and monitoring DXNs in fish samples.

Figure 3. Comparison of CALUX™ Assay and HRGC/MS (Total DXNs;Separate and Non-Separate) (x axis ; HRGC/MS (pgTEQ/gfat) y axis ; CALUX (pgTEQ/gfat))
In order to execute the risk management for DXNs in the foods such as fish, it is indispensable todevelop a rapid and inexpensive screening method to handle a great deal of samples. CALUX™ Assay
meets these conditions (within 5 days and about 1/10 cost) and we confirmed the reliability of thisscreening tool for fish samples. The Belgian Federal Ministries of Public Health and Agriculture has
already analyzed fat samples of chicken, egg and pork by CALUX™ Assay6) . But in Japan, it is very
important to secure fish since people eat many fish. For the risk management, further frequentexamination of DXNs levels in fish are needed and study on the applicability of CALUX™ Assay asscreening and monitoring tool for fish.
References
1. Basic Guidelines of Japan for the Promotion of Measures against Dioxins(1999)
2. Law concerning Special Measures Against Dioxins(1999)
3. APPLICATION OF THE CALUX ASSAY TO THE ANALISIS OF DXNS IN FISH (THE FIRSTREPORT) Junko Fujino, Tomoaki Tsutsumi, Yoshiaki Amakura, Masafumi Nakamura, Hiroko
Kitagawa,Tsukasa Yamamoto, Kumiko Sasaki, Masatake Toyoda (2001) Organohalogen
Compounds51,348
4. Denison,M., Brouwer,A.and Clark,G.(1998). U.S.patent # 5,854,010
5. Masatake Toyoda,Takako Iida,Tsuguhide Hori,Toshihiko Yanagi,Youichi Kono and Hiroyasu
Uchibe:J. Food Hyg. Soc. Japan, 40,111?121(1999)
6. A COMPARATIVE STUDY OF GC-HRMS AND CALUX™ TEQ DETERMINATIONS IN FOODSAMPLES BY THE BELGIAN FEDERAL MINISTRIES OF PUBLIC HEALTH AND
AGRICULTURE, 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 Compounds45,200388ORGANOHALOGEN COMPOUNDSVol. 58 (2002)