HUMAN EXPOSURE I
REGIONAL DIFFERENCES OF BLOOD DIOXIN AND
ORGANOCHLORINE PESTICIDES CONCENTRATIONS OF JAPANESEFEMALE FARMERS. - APPLICATION OF CALUX ASSAY FOR
EPIDEMIOLOGICAL STUDY Fujio Kayama1, Hyogo Horiguchi1, Etsuko Oguma1, Junko Fujino2, Hisatoshi Yabushita2, DavidBrown3 and George Clark3
1

Jichi Medical School, Tochigi 329-0498 JAPAN, and CREST/JST, Kawaguchi 332-0012, JAPANHiyoshi Corporation, Omihachiman, Shiga 523-8555 JAPAN3
Xenobiotic Detection System International, Inc. (XDS) Durham, NC 27704 USA2Introduction
We hypothesized that farmers who used pesticides and herbicides frequently may have higher
dioxin body burden due to impurities in these chemicals. To elucidate body burden of dioxins andorganochlorine pesticides, and relationships between health status of female farmers and environmental
contaminants including dioxins, we designed epidemiological studies in 5 distant districts in Japan. In
this paper, we demonstrate that CALUX assay for dioxin is applicable in epidemiology and a usefuldioxins screening method for identifying higher exposure groups. In addition, the current analysis of
dioxin contamination among female farmers in Japan revealed a distinct difference of distribution ofblood dioxin concentration in the studied districts in Japan.
Material and Methods.
Female farmers in five districts scattered in the Japan Archipelago were asked through regional
agricultural organizations to cooperate in a nation-wide health survey studying relationships among
health, nutrition and contaminants. The Committee on Medical Ethics in Jichi Medical School
approved this research protocol and we obtained informed consents from all volunteer examinees.
Health examinations included serum chemistry and urinalysis, bone metabolic markers, and wrist bonedensity measured by Dual Energy X-ray Absorption (DEXA) scanner as well as life, medical and diethistory questionnaires double-checked by nurses and nutritionists at interview.
Result and Discussion
We previously reported a validation study of the CALUX assay in biological samples 1,2. Wecollected 70 ml blood samples from volunteers in the medical school. Ten-ml and 50-ml whole blood
samples were used for CALUX assay and HRGC-MS measurement, respectively. Results for test
samples measure by CALUX bioassay for dioxins at Hiyoshi Corporation were compared in a double
blind study to HRGC/MS results in blood measured at Shimazu Techno-Research (Kyoto, Japan).
When we started to apply CALUX assay for human biological samples, correlation coefficient (Rvalues) for PCDD+PCDF+coPCB per gram fat was 0.4758. As the test blood samples were taken fromyoung volunteers with lower dioxin body burden, most of the samples had low dioxin concentrations.
In addition, as CALUX assay is slightly less sensitive to coPCB, we excluded coPCB fractions formeasurement. The R-values on PCDD+PCDF per gram wet weight and per gram fat were 0.7445 and0.6417, respectively. When we limit applications of CALUX assay for blood specimens to evaluate

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PCDD+PCDF fraction and set detection limit of 20 pg/g fat, the R-values of PCDD+PCDF per gram
wet weight and per gram fat is 0.9161 and 0.8297 respectively. This showed that CALUX assay ispractically sensitive enough for screening moderately high exposure groups for dioxins (Table1).
Table 1. Correlation coefficients (R) of Blood Dioxin Concentrations between HRGC/MS and CALUXAssay

PCDD+PCDF+co-PCB
All sample
Samples > 20pgTEQ/gFat
PCDD+PCDF
All sample
Samples > 20pgTEQ/gFat

pg/g wet weight

pg/g fat(n=21)(n=14)***
0.82180.47580.7305(n=20)(n=13)

0.74450.91610.64170.8297In the current study we measured blood dioxin (PCDD+PCDF) concentrations in 1160 female
farmers out of 1407 volunteers in five districts. The average (±SD) of examinees’ age was 55.5±10.3.
The average (±SD) blood dioxin concentration was 32.3±12.1 pg TEQ/g fat. When results for these
five districts were compared, the average dioxin concentration in District A was distinctively high.
Seventy percent of samples were higher than the detection limit (20 pg TEQ/g fat) in District A, whilein the other districts approximately 25 % were higher. This indicates that there may be some dioxin
contamination in district A, and it should be reexamined by HRGC-MS to determine the isomerpattern. HRGC-MS results may help to identify the character and origin of the contamination in thisdistrict. Figure 1 showed distribution of incidence is shifted to the right in District A and there are someindividuals whose blood dioxin concentrations are more than 100 pg TEQ/g fat.

Table 2. Blood Dioxin Concentrations of Female Farmers in Five Districts of Japan

Total No. of Examinees
Age of the Examinees
No. of Blood Samples
No. of Detected
No. of Not Detected% of >20pgTEQ/g Fat
RangeMedian
Average ±S.D.

District A

District B

District C District D

District E

Total59660.3 ± 11.4
449
313
136
70 %
20.0-102.730.730.0±12.7

203
55.5 ± 9.5
177
43
134
24 %
20.0-50.0
25.2
27.9±7.0

202
204
56.9 ± 9.2 51.6 ± 8.0
155
191
4047
115
144
26 %
25 %
20.6-46.7 20.7-71.1
25.6
23.6
28.9±7.6 26.6±11.3

20257.8 ± 9.3
188
38
150
20 %
20.3-66.9
25.3
31.7±13.0

1407
55.5 ± 10.3
1160
481
679
41 %
20.0-102.729.432.3±12.1

Female middle-aged farmers were asked to participate in nation-wide health check regarding
osteoporosis, nutrition and contaminants. Dioxins (PCDD+PCDF) in 10 ml of heparinized whole blood
were determined by CALUX assay. Detection limit was 20 pg TEQ/g fat.

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Figure 1. Regional Difference of Blood Dioxin Concentrations of Female Farmers

Figure 2. Data lower than detection limit (20 pg TEQ/g fat) are treated as half of detection limit (10 pgTEQ/g fat) and plotted in the figure.

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Figure 2 shows correlation between age and blood dioxin level. The analysis of dietary history
revealed moderate correlation to fish consumption. These data show quite similar results to HRGC-MS
measurements recently conducted by Japan’s Ministry of Environment.
Acknowledgement
This study is supported by grants from CREST-JST, and ministry of Health, Labor, and Welfare andMinistry of Agriculture, Forestry and Fishery, JAPAN.
References
1. Kayama F., Hamamatsu A., Sagisaka K., Brown D., Clark G., Suzuki T. (2000) The 3rd Annual
Meeting of Japan Society of Endocrine Disruptoers Research.
2. Kayama F., Hamamatsu A., Sagisaka K., Brown D., Clark G., Suzuki T. (2001) Organohalogen
Compounds 54: 48-50
3. Kayama F., Hamamatsu A., Sagisaka K., Brown D., Clark G. and Nakamura M. Vietnam-UnitedStates Scientific Conference on Human Health and Environmental Effects of Agent Orange/Dioxin
p-114, (March 2002, Hanoi Vietnam).

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