DIOXIN AND ORGANOCHLORINE PESTICIDE CONCENTRATIONS OF
BREAST MILK IN CHINA AND JAPAN.

Fujio Kayama1, Hyogo Horiguchi1, Yi-he Jin 2, Tatsuya Kunisue3 Shinsuke Tanabe3, Hideo Fukatsu4,Naoki Yamazaki4, Masafumi Nakamura5, Hisatoshi Yabushita5, Junko Fujino5, George Clark6

1

Jichi Medical School, Tochigi 329-0498 JAPAN, and CREST/JST, Kawaguchi 332-0012, JAPAN

2

Dept. Hygienic Toxicology, College of Public Health, China Medical University.

3

Center for Marine Environmental Studies, Ehime University, Matsuyama 790-8577, Japan4

SRL, Inc, 2-41-19 Akebono-cho, Tachikawa-shi Tokyo 190-8567 JAPAN

5

Hiyoshi Corporation, Omihachiman, Shiga 523-8555 JAPAN,

6

Xenobiotic Detection System International, Inc. (XDS) Durham, NC 27704 USA.

Introduction
Breast milk contains many lipid soluble compounds that are present in mother’s adipose tissue.
It means that the levels of dioxins and dioxin-like PCBs in breast milk are representing for those in serum
lipid and adipose tissue as body burdens. Dioxins and other persistent contaminants in breast milk areapparently a great concern for the health of the neonates. To elucidate body burden of dioxins andorganochlorine pesticides in China and Japan, we have collected breast milk specimens in these countries.
But the sample volume collected and the funding were limited to measure dioxin concentrations in breast
milk by HRGC-MS. For large-scale epidemiological studies, quick and accurate measurements of dioxins
are requested. We previously reported a validation study of the CALUX assay in biological samples1,2,3. In
this paper, we demonstrate that CALUX assay for dioxin was applied for breast milk, and we validated
measurement of breast milk by Dioxin and PCB specific (DIPS)-CALUX cell assay in comparison with
HRGC/MS method. From our best knowledge, it will be the first report on contaminations of dioxins andorganochlorine pesticide in breast milk among Chinese in northern region.

Material and Methods.
We have collected breast milk samples from 47 breast-feeding mothers in Dalian and 32 in
Shenyang, Liaoling Province, China, and 20 samples in Japan after collecting informed approval on
dioxin measurements from the subjects. In China, the specimens were collected within a week after theOrganohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MAdelivery. In Japan, collection time ranged from 3 to 12 weeks after delivery. We measured in total 99
samples by DIPS-CALUX assay at Hiyoshi Corporation. Results for test samples measure by CALUX
bioassay for dioxins were compared in a double blind study to HRGC/MS results measured at SRL, Inc..
Persistent organochlorine pesticide concentrations in the Chinese milk were also measured by GC-ECD.

Results and Discussion:
Two samples, one from Japan, one from China, with exceptionally low lipid percentage (0.5%, 0.6%) wereexcluded

fromthestatisticalanalysis.

Correlation

coefficiency

(R)

of

PCDD+PCDF

and

PCDD+PCDF+coPCB per g fat were 0.920 and 0.763, respectively. As CALUX assay is slightly lesssensitive to coPCB, we adjust low responsiveness by multiplication of 1.8 to the coPCB values, the Rvalue for PCDD+PCDF+coPCB per gram fat was 0.917. We concluded that DIPS-CALUX assay wassensitive enough to apply for human breast milk specimens.
Figure 1 Correlation between HRGC-MS and DIPS-CALUX assay(After adjustment for low responsiveness to coPCB by ratio 1.8.)
Total PCDDs+PCDF（ gfat)Total PCB
(gfat)

4040

Total DXN
(gfat)

80

y = 0.8559x + 3
R = 0.920

30306020204010100200010203040010203040

y = 1.0709x + 7
R = 0.917

0020406080

Horizontal axis is pgTEQ/g fat by HRGC-MS and vertical axis is pgTEQ/g Fat by CALUX assay

Talbe 1 GM, median, and range of dioxins concentrations in breast milk in Japan
HRGC/MSCALUXPCDD+PCDF0.52, 0.48, (0.25-1.3)

0.52, 0.49. (0.28-1.3)

coPCB

0.19, 0.18, (0.08-1.10)

0.13, 0.18, (0.05-0.46)

Total

0.72, 0.67, (0.36-2.40)

0.69, 0.66, (0.4-1.4)

PCDD+PCDF

15.5, 19.0, (7-31)

17.0, 18.0, (7.7-29)

coPCB

5.8, 5.9, (2.2-22)

4.3, 4.5, (1.7-17)

Total

21.6, 24.0, (10-49)

22.5, 20.0, (13-37)

pgTEQ/gwet

pgTEQ/gFat

Data analyzed in 19 samples of breast milk from Japanese mothers.
Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MA

In Table 1, geometric means of dioxin concentrations of Japanese breast milk (n-19) by HRGC/MS were
15.5 pgTEQ/gFat for PCDD+PCDF, 5.8 for coPCB, and those by CALUX were 17.0 for PCDD+PCDF,
4.3 for coPCB, respectively. The values were almost the similar levels of dioxin concentration previously
reported in other districts in Japan.

Talbe 2 Geometric mean, median and range of dioxins in breast milk
Shenyang

DalianJapann32

47

19

PCDD+PCDF

8.4, 7.2, (1.6-48.7)

14.5, 14.7, (3.5-148)

17.0, 18.0, (7.7-29)

coPCB

1.8, 1.6, (0.7-11.3)

3.9, 3.5, (1.1-17.0)

4.3, 4.5, (1.7-17)

Total

10.6, 8.8, (3.2-54.1)

19.4 , 18.0, (6.1-158)

22.5, 20.0, (13-37)

The values are measured by CALUX Assay
The values by CALUX were 10.6 pgTEQ/gFat in Shenyang.and 19.4 pgTEQ/gFat in Dalian
Both the values of PCD+PCDF and coPCB fractions in Dalian were higher than those in Shenyang. It isnoteworthy that the levels of the contaminations of Dalian were close to those of Japan. It depicts thatdioxin body burden in Dalian may be higher than that in Shenyang. This difference might be due topeople in Dalian eat more marine products than those in Shenyang.
Table 3 Geometric mean, median and range of dioxins in breast milk among age groups
Shenyang

DalianJapanAgen

pgTEQ/g fatn

pgTEQ/g fat

n

20-25 yo910.8, 9.4, (3.2-54.1)

5

16.0, 17.6, (6.1-71.6)

026-30 yo1 6 11.8, 9.2, (4.5-48.4)

3 6 19.4, 18.5, (7.4-158.9)

473

1 5 25.7, 20.0, (13-78)

older than 31
The values

8.0, 7.6, (5.1-13.5)

20.7, 17.4, (12.6-40.5)

pgTEQ/g fat

18.2, 18.5, (16-20)

are measured by CALUX Assay

It was also reported previously that dioxin concentrations elevated when mothers are older. But
a distinctive difference from Japanese data was that no age-dependent elevations of dioxin concentrations
were observed in both cities of China (Table 3). PCB, p,p’DDE, ß-hexachlorohexane (ß-HCH), andhexachlorebenzen (HCB) were also measured in remaining 20 samples from Dalian and 20 fromShenyang. It revealed that these contaminants were significantly higher in the specimens from Dalianthan those from Shenyang (Table 4). There was also no age-dependent elevation of these contaminants inbreast milk in China which is also distinct in other developed countries. There might exist heterogeneous
sources and routes of the contaminant exposure among these subjects, even if they live in the same city.

Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MATable 4 Geometric mean, median and range of POPs in Chinese breast milkPOPsShenyangDalianｎ20

20

PCB

16.0, 17.6, (6.1-71.6)

36, 39, (11-91)*

pp'DDE

586, 629, (110-3100)

1685,1800,

ß-HCH

396, 370, (110-2200)

961,905

HCB

50, 48, (25-120)

The values were expressd by ng/g Fat,(710-5300)*(130-7200)*

71, 78, (31-220)** ; statistically significant(p< 0.05)

Conclusions
1. DIPS-CALUX assay is suitable tools to assess dioxin concentrations in human breast milk samples inlarge-scale epidemiological studies.
2. Two cities in Liaoning Province, China showed distinct difference in dioxin concentrations and PCBs
and organochlorine pesticides in breast milk. The values of all the contaminants measured weresignificantly higher in Dalian, a port city in Liaodong Peninsula, than those in Shenyang, an inland
capital of the province.
3. There were no age-dependent elevations of the contaminants in Chinese breast milk.

Acknowledgement
This study is supported by grants from CREST-JST, and ministry of Health, Labor, and Welfare
and Ministry 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. Fujio Kayama, Hyogo Horiguchi, Etsuko Oguma, Junko Fujino, Hisatoshi Yabushita, David Brownand George Clark. Regional differences of blood dioxin and organochlorine pesticides concentrations ofJapanese female farmers. (2002) Organohalogen Compounds 55: 275-278

Organohalogen Compounds, Volumes 60-65, Dioxin 2003 Boston, MA