CALUX ASSAY IS APPLICABLE TO HUMANEPIDEMIOLOGICAL STUDIES.
Kayama F1,4, Hamamatsu A2, Sagisaka K2, Brown D3, Clark G3, and Suzuki T4
1Jichi Medical School, Tochigi 329-0498 JAPAN, 2Tokyo Medical Examiner's Office, Otsuka,

Tokyo 112-0012 JAPAN, 3Xenobiotic Detection System International, Inc. (XDS) Duhram, NC
27704 USA. 4Hiyoshi Corporation, Omihachiman, 523-8555 JAPAN, 4CREST/JST, Kawaguchi
332-0012, JAPANIntroduction
There are growing concerns regarding human health effects of dioxins and dioxin-like
polychlorinated biphenyls ( coPCBs ) to groups at higher risk for exposure as well as the general
population. Additionally, in utero exposure to the fetus may cause alteration of development ofimmune and nerve systems as well as endocrine systems. We need to establish prospective cohort
studies to evaluate risks of low-dose exposures over an extended period. For these studies it isnecessary to carry out large-scale measurement of dioxin exposures.
However, current methods of detection using high resolution gas chromatography/mass
spectrometer (HRGC/MS) require more than 50 ml of blood. This sampling volume is too much forordinary volunteers in epidemiological studies. Additionally, the measurement is expensive andrequires several weeks. These conditions make it difficult to conduct large-scale epidemiologicalstudies. In this paper, we have tried to validate a less expensive and quicker bioassay, CALUX assay,
for application to human epidemiological studies. CALUX assay uses a genetically modified
mammalian cell line to contain the firefly luciferase gene as a reporter for dioxin exposure.
Material and Methods.
To validate CALUX assay, results for test samples were compared in a double blind study toHRGC/MS results from at a laboratory certified for dioxin measurement. Test samples included 21
fat tissue samples collected from autopsy cases in the Tokyo Medical Examiner's Office, for which
we obtained informed consent of the family of the deceased. In Addition, we collected 70 ml blood
samples from volunteers in the medical school. The blood samples were divided into 20 ml and 50
ml aliquots, used for CALUX assay and for HRGC-MS measurement, respectively.

Result and Discussion
Themeanconcentration

polychlorinateddibenzodioxins(PCDD)

andpolychlorinateddibenzofurans (PCDF) in 21 human fat tissue by HRGC/MS was 37 pg-TEQ/g fat, ( median: 33pg-TEQ/g fat, range 16.7-72 pg-TEQ/g fat). The mean concentration of PCDD+PCDF+coPCB was
42.5 pg TEQ/g fat (median: 44 pg-TEQ/g fat, range: 18.5 - 83.8 pg-TEQ/g fat). When the results forthese samples from both GC-MS and the CALUX bioassay were compared, the correlation
coefficient was as high as 0.8675 (Figure1). In addition, we measured dioxin concentrations in 17blood samples from volunteers by both methods. Most of the samples had low dioxin concentrations.
Correlation coefficient for PCDD+PCDF per wet weight and per gram fat was 0.604, and 0.543,
respectively. However, when the results for samples with greater than 20 pg/g fat were compared,
coefficients on PCDD+PCDF per gram wet weight and per gram fat were 0.763 (Fig.2), and 0.702,
respectively. The correlation coefficient for PCDD+PCDF+coPCB per gram wet weigh of blood
was 0.746. These results suggest that CALUX assay is applicable as an economical and quick
method for fat tissue or fat rich samples such as breast milk. However, at low level concentration ofdioxins such as in serum and blood the measurement was difficult These results indicate that theCALUX bioassay for dioxins can be utilized in epidemiological studies of dioxin burden among
workers at risk for exposure and can be applicable to fat tissues and fat rich samples for individuals
from the general population.
References
1. Kayama F, Hamamatsu A, Sagisaka K, Brown D, Clark G, Suzuki T (2000), The 3rd Annuarl
Meeting of Japan Society of Endocrine Disruptoers Research.
2. Brwon D. Kishimoto Y, Ikeno O, Chu M, Nomura J, Murakami T, and Murata H. (2000)
Organohologen Compounds 45: 200-203

Colationship between HRGC/M S andCALUXAssay forHum an FatSam ples
(PCDD+PCDF+Co-PCB)

10080

R = 0.8675

60402000204060

80

100

CALUXAssay(pgTEQ/gFat)

FIGURE 1

C orelation betw een H R G C /M S
and C ALU X Assay forH um anBlood Sam ples(PC D D +PC D F)
0.16

R = 0.764
0.120.080.04

0.00
0.00

0.04

0.08

0.12

0.16

C ALU X Assay(pgTEQ /g W et)
FIGURE 2120