Growth and Thyroid Function in Children With In Utero Exposure to Dioxin: A 5-Year Follow-Up Study

Transcription

1 /10/ PEDIATRIC RESEARCH Coyright 2010 International Pediatric Research Foundation, Inc. Vol. 67, No. 2, 2010 Printed in U.S.A. Growth and Thyroid Function in Children With In Utero Exosure to Dioxin: A 5-Year Follow-U Study PEN-HUA SU, JIA-YUH CHEN, JEIN-WEN CHEN, AND SHU-LI WANG Deartment of Pediatrics [P.-H.S., J.-Y.C.], Chung Shan Medical University Hosital, School of Medicine [P.-H.S., J.-Y.C.], and Deartment of Public Health [S.-L.W.], Chung Shan Medical University, Taichung 402, Taiwan; Division of Environmental Health and Occuational Medicine [J.-W.C., S.-L.W.], National Health Research Institutes, Zhunan 350, Taiwan ABSTRACT: Because lacental olychlorinated dibenzo--dioxins, dibenzofurans (PCDD/Fs) levels are associated with decreased free thyroxine (FT 4 ) and thyroid stimulating hormone (TSH) in neonates, we assessed develoment by gender and maternal PCDD/Fs exosure at years 2 and 5 in 92 mother and newborn airs. RIA quantified thyroid, sex, and growth hormones. Of 200 subjects followed u from November 2000, 136 and 149 were observed at year 2 and year 5, resectively. PCDD/Fs exosure levels were low (n 35) or high (n 35) in 70 subjects at year 2, low (n 21) or high (n 20) in 41 at year 5. Height, weight, BMI, and head circumference were significantly higher in males, chest girth significantly higher in females at year 2. Significantly, more girls had higher bone age (BA) and chronological age (CA) at both times. Height, weight, FT 4 TSH, and transthyretin (TTR) at year 2; and height, triiodothyronine, and IGF-1 at year 5 differed significantly by PCDD/Fs level. In females, height, weight, CA, BA, and thyroid hormones differed significantly at year 2. In males, FT 4 TSH at year 2 and IGF-1 at year 5 were significantly higher in the high PCDD/Fs grou. In utero exosure to PCDD/Fs differentially affects growth and hormone levels in male and female reschool children. (Pediatr Res 67: , 2010) Dioxins [olychlorinated dibenzo--dioxins, dibenzofurans (PCDD/Fs)] are highly toxic environmental contaminants, which are liohilic and resistant to biodegradation. These comounds enter the human body through the food chain and finally accumulate in adiose tissue (1). A series of deleterious health effects are thought to be associated with organochlorine exosure. These include fetal and infant growth retardation and develomental abnormalities (2,3), altered reroductive caacity (4,5), carcinogenicity (6,7), cognitive deficits (8), and imaired thyroid function (9). It has been confirmed that PCDD/Fs can be transferred from mother to fetus via the lacenta (10) and from mother to infant via breast milk (11). The effects of the endocrine disrution during these critical stages of early develoment might ersist long after birth. Similar to other industrialized and develoing countries, Taiwan is confronted with the roblem of environmental PCDD/Fs contamination. It has been reorted that high concentrations of PCDD/Fs are more revalent in central Taiwan than other areas of Taiwan (12). In a revious study, we examined the association between in utero exosure to PCDD/Fs/olychlorinated bihenyls (PCBs) and altered thyroid and growth hormone concentrations in newborns from central Taiwan (13), we found that after adjusting for other dioxins and PCB congeners in neonates from the general oulation, the multilied concentration of free thyroxine (FT 4 ) thyroid stimulating hormone (TSH) decreased with increasing levels of non-ortho PCBs in the lacenta. In the same study, we also noted a significant and ositive association between thyroxine (T 4 ) and thyroid-binding globulin concentrations with levels of PCDD/Fs (13). Because thyroid hormones are crucial for growth and develoment, thyroid dysfunction in early infancy/childhood may adversely imair these rocesses and have serious longterm consequences. Hence, we conducted follow-u assessments at reschool ages for the cohort of neonates in our revious study (13). Because a revious reort found that schoolchildren (aged y) whose serum level of PCBs was two to four times higher than normal children had no significant imairment of thyroid function (14), the focus of this study was the relationshi between in utero exosure to PCDD/Fs and thyroid function and growth hormones. The objectives of the follow-u study were to examine ossible abnormalities in growth and develoment, bone age (BA) and growth, and hormones related to neurodeveloment in children aged between 2 and 3 y and 5 and 6 y who were exosed to differing levels of PCDD/Fs in utero. Secifically, we made comarisons between children who were exosed to low and high levels of PCDD/Fs. MATERIALS AND METHODS Subjects. A total of 92 mother and newborn airs were recruited from the general oulation in central Taiwan. We consecutively followed u the subjects (n 200) of our revious study (13) from November 2000 to February We studied the subjects at 2 3 y of age (n 188, during 2003) and at 5 6 y (n 149, during ). These grous are referred to as year 2 and year 5, resectively. Children were also classified into low exosure and high exosure grous by the cutoff value 15 g-teq/g liid based on the mother s PCDD/Fs TEQ levels. This study was aroved by the Received June 3, 2009; acceted October 13, Corresondence: Shu-Li Wang, Ph.D., Division of Environmental Health and Occuational Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 350, Taiwan; slwang@nhri.org.tw, jen@csh.org.tw This work was suorted by grants EO-097-PP-05 and EO-098-PP-03 from the National Health Research Institutes. Abbreviations: BA, bone age; CA, chronological age; E 2, estradiol; FT 4, free thyroxine; IGFBP-3, insulin-like growth factor binding rotein 3; PCBs, olychlorinated bihenyls; PCDD/Fs, olychlorinated dibenzo--dioxins, dibenzofurans; T 3, triiodothyronine; T 4, thyroxine; Tg, thyroglobulin; TRAb, TSH recetor autoantibody; TSH, thyroid stimulating hormone; TTR, transthyretin 205

2 206 SU ET AL. ethics review committee of our university hosital, and written arental consent was obtained for all articiants. The study abided by the ethical standards of the Declaration of Helsinki (15). Measurements and blood samling. Demograhic information included maternal age, gestational age, and infants age, gender, height, weight, BMI, head circumference, chest girth, chronological age (CA), BA, and ratio of BA/CA. Body weight and height were comared with the oulation curve (data were obtained from the Taiwanese Deartment of Health) at each visit. Standing height was measured in trilicate using a wall-mounted stadiometer. BMI was calculated by dividing body weight (kg) by the square of height in meters (m 2 ). Left hand radiograhs were taken, and BA was determined using the method of Greulich and Pyle (16). In laboratory analysis, thyroid, sex hormone, and growth factor concentrations with resect to PCDD/F exosure level were collected for evaluation. Venous blood samles were obtained from children whose mothers PCDD/F TEQ levels were available in our database. Blood samles were immediately centrifuged and the serum frozen at 70 C for later analysis of triiodothyronine (T 3 ), T 4,FT 4, TSH, T 3 utake, thyroglobulin (Tg), TSH recetor autoantibody (TRAb) utake, transthyretin (TTR), IGF-1, IGF binding rotein 3 (IGFBP-3), testosterone, and estradiol (E 2 ). T 3,T 4,FT 4, TSH, and T 3 utake analysis. Serum T 3,T 4, free T 4 and TSH concentrations, and T 3 utake were measured using an automated chemiluminescent immunometric assay (Immulite 2000: Diagnostic Products Cor, Los Angeles, CA). Sensitivities were 19 ng/dl for T 3, 0.3 g/dl for T 4, IU/mL for TSH, and 1.6 g/ml for T 3 utake. Intra- and interassay coefficients of variation (CV) were 4.4 and 5.3% for T 3 ; 4.6 and 5.6% for T 4 ; and 5.1 and 6.4% for TSH. FT 4 TSH values were calculated based on the rationale that, for a normal hyothalamic-ituitary axis, a decrease in T 4 feedback inhibition to the hyothalamus resulted leads to TSH secretion by the anterior ituitary. Tg analysis. Serum Tg levels were measured by immunoradiometric assay (RSR Ltd., Cardiff, UK). Sensitivity was 0.7 ng/ml, and the intra- and interassay CVs were 3.32 and 6.36%. TRAb analysis. Serum TRAb levels were determined using a commercially available RIA kit (RSR limited, Cardiff, UK). Sensitivity of this assay was 5 units/l. Result was exressed as inhibition of TSH binding index, % 100 (1 B/B o ). Intra- and interassay CVs were 4.15 and 6.02%. TTR analysis. Serum TTR levels were measured using a kinetic nehelometric method by automated nehelometer (Beckman Coulter, Inc. Array System, Carlsbad, CA). The sensitivity of this assay was 1.17 mg/dl, and the intra- and interassay CV were 2.06 and 1.54%. IGF-1 and IGFBP-3 analysis. Serum total IGF-I and IGFBP-3 levels were measured after acid extraction using commercial RIA kits (Diagnostic Systems Laboratories, Inc., Webster, TX). The sensitivities were 0.9 mg/l and 0.01 ng/ml for IGF-I and IGFBP-3, resectively. The intra- and interassay CV were 7.2 and 9.8% for IGF-I and 5.8 and 8.2% for IGFBP-3. Testosterone analysis. Serum testosterone concentrations were measured by solid-hase, cometitive chemiluminescent enzyme immunoassay (Immulite 2000 Advanced Immunoassay System, Siemens Medical Solutions Diagnostics, Deerfield, IL). The sensitivity of this assay was 15 ng/dl. Intra- and interassay CVs were 9.7 and 12.0%. E 2 analysis. Serum E 2 levels were measured by RIA kit (Diagnostic Systems Laboratories). Sensitivity was 2.2 g/ml, and intra- and interassay CV were 7.5 and 9.3%. Statistical analysis. In general analysis, data were exressed as mean SD for continuous variables and frequency with ercentage for categorical variables. For comarison of difference in means between grous, a t test was erformed for normally distributed data, and the nonarametric Wilcoxon rank sum test was erformed for data that were not normally distributed. A multile regression analysis was erformed with a stewise selection for detecting the factors, which might affect the growth of hormone concentrations by adjusting the maternal age and sex of the infant. All comarisons were considered under a significance level All data were analyzed using SAS version 9.0 (SAS Institute Inc., Cary, NC). RESULTS Summary of anthroomorhic measurements by infant gender is resented in Table 1. Significant increases were aarent for each variable between genders at both ages. At year 2, height, weight, BMI, and head circumference were significantly higher in boys than girls. Chest girth was significantly higher in females at this visit. At year 5, there were no between-grou differences in height, weight, BMI, head circumference, or chest girth. Using BA as an indicator of abnormal growth, we comared BA with CA at both visits. At year 2, children (male and female combined) had a mean BA of y (n 136; note: BA was not assessed for all subjects at year 2), whereas CA was y (n 194). The difference between BA and CA was borderline significant ( 0.056). At year 5, however, mean BA ( y) was significantly lower than mean CA ( y) ( ) (data not shown). BA and CA comarisons with resect to gender were conducted as well; no difference was detected at year 2, but males had a significantly lower mean BA than females at year 5 ( ). Significant differences were detected for the mean of ratio of BA and CA with resect to gender at both year 2 and year 5( for year 2; for year 5) (Table 1). In considering anthroomorhic data and PCDD/Fs exosure levels, high level ( 15 g-teq/g liid) and low level ( 15 g-teq/g liid) are summarized by age and gender. Overall, height and weight were significantly different between the PCDD/Fs exosure levels at year 2 ( for height and for weight). However, only height remained significant at year 5. In subgrous (male and female), it was found that height, weight, CA, and BA were significantly different between the PCDD/Fs exosure levels at year 2 for female ( for height, for weight, for CA, and for BA). In contrast, there were no such differences for males. Similarly, there were no significant differences between exosure levels by gender at year 5 (Table 2). For children who were exosed PCDD/Fs, T 3, and IGF-1 concentrations at year 2 were significantly higher in females than males ( , for T 3 and IGF-1, resec- Table 1. Summary of anthroomorhic demograhics at year 2 and year 5 Year 2 Year 5 Demograhics* Male (n 69) Female (n 67) Male (n 70) Female (n 79) Height (cm) Weight (kg) BMI (kg/m 2 ) Head circumference (cm) Chest girth (cm) CA BA BA/CA * Demograhics are resented as mean SD indicates statistically significant difference between genders.

3 IN UTERO DIOXIN EXPOSURE AND GROWTH 207 Table 2. Summary of anthroomorhic variables with resect to PCDD/F exosure levels (low versus high) as determined at year 2 and year 5 Demograhics* Male Female Overall Year 2 n Height (cm) Weight (kg) BMI (kg/m 2 ) Head circumference (cm) Chest girth (cm) CA BA BA/CA Year 5 n Height (cm) Weight (kg) BMI (kg/m 2 ) Head circumference (cm) Chest girth (cm) CA BA BA/CA * Demograhics are resented as mean SD. Low and high levels for PCDD/Fs levels were categorized as 15, and 15 g-teq/g liid indicates statistically significant difference between the high- and low-exosure levels for a given gender or overall. Table 3. Thyroid and sex hormone, and growth factor concentrations with resect to PCDD/F exosure level at year 2 Variables* Male Female Overall n T 3 (ng/dl) T 4 ( g/dl) TSH (miu/l) T 3 utake (%) FT 4 (ng/dl) FT 4 TSH Tg (ng/dl) TRAb (%) TTR (mg/dl) E 2 (ng/dl) Testosterone (ng/dl) IGF-1 (ng/ml) IGFBP-3 (ng/ml) * Variables including thyroid and sex hormone, and growth factor concentrations were resented as the mean SD. Low and high levels for PCDD/Fs levels were categorized as 15, and 15 g-teq/g liid indicates statistically significant difference between the high and low exosure levels for a given gender or overall. tively). At year 5, FT 4 was significantly higher in males ( ), whereas IGF-1 levels were significantly higher in females ( ; data not shown). The thyroid hormone, sex hormone, and growth factor concentrations with resect to PCDD/Fs exosure levels, high level ( 15 g-teq/g liid) and low level ( 15 g-teq/g liid) were summarized by gender for year 2 and year 5, resectively (Table 3 and Table 4). Overall, FT 4 TSH, TTR levels at year 2 and T 3, IGF-1 at year 5 were significantly higher in the grou with high dioxin/pcb exosure level than the grou with low exosure level ( , for FT 4 TSH and TTR at year 2, resectively; , for T 3 and IGF-1 at year 5, resectively). In females, only T 3 and TTR concentrations at year 2 were significantly higher in the high PCDD/Fs exosure level than the low level ( , for T 3 and TTR, resectively), whereas there was no significant difference detected in concentrations at year 5. In males, FT 4 TSH at year 2 and IGF-1 at year 5 were significantly higher in the high PCDD/Fs exosure level than the low level ( for FT 4 TSH at year 2, and for IGF-1 at year 5). Results of multile linear regression analysis adjusting for maternal age indicated that, overall, only FT 4 TSH at year 2 and IGF-1 at year 5 were significantly higher in the grou

4 208 SU ET AL. Table 4. Thyroid hormone, sex hormone, and growth factor concentrations with resect to PCDD/F exosure level at year 5 Variables* Male Female Overall n T 3 (ng/dl) T 4 ( g/dl) TSH (miu/l) T 3 utake (%) FT 4 (ng/dl) FT 4 TSH Tg (ng/dl) E 2 (ng/dl) Testosterone (ng/dl) IGF-1 (ng/ml) IGFBP-3 (ng/ml) * Variables including thyroid and sex hormone, and growth factor concentrations were resented as the mean SD. Low and high levels for PCDD/Fs levels were categorized as 15 and 15 g-teq/g liid indicated a statistically significant difference between the high and low exosure levels for a given gender or overall. Table 5. Stewise multile linear regression models for selection in thyroid hormone concentrations with resect to PCDD/F exosure level at year 2 Model* Estimate SE -Value T 3, R PCDD/F (PCDD/F) TSH, R Maternal age PCDD/F FT 4 TSH, R Maternal age PCDD/F TTR, R PCDD/F * Model as erformed adjusting infants gender. TSH and FT 4 TSH were log transformed to meet regression assumtions indicates a statistically significant relationshi. with high PCDD/Fs exosure level than the grou with low level exosure ( for FT 4 TSH at year 2; for IGF-1 at year 5). In males, FT 4 TSH at year 2 and IGF-1 at year 5 were still significantly higher in the grou with high PCDD/Fs exosure level than the grou with low exosure level ( for FT 4 TSH at year 2, and for IGF-1 at year 5). In females, TTR was still significantly higher in the high PCDD/Fs exosure level than the low level at year 2; FT 4 TSH (instead of T 3 ) was significantly higher in the high PCDD/Fs exosure level. There was no significant difference in hormone levels at year 5 (data not shown). Table 5 shows the results of the stewise multile linear regression analysis of selection of thyroid hormone concentrations at first follow-u (year 2) with resect to PCDD/Fs exosure level, considering mother s age, arity at first follow-u, and the quadratic form of PCDD/Fs. There were significantly ositive correlations among T 3, TSH, FT 4 TSH, and TTR concentrations and the level of PCDD/Fs exosure. Both TSH and FT 4 TSH concentrations were significantly negatively correlated with maternal age. There was also a negative correlation between T 3 levels and the Figure 1. PCDD/F exosure level in relation to FT 4 TSH level. By simle linear model: (A) for 2 y reschool data, FT 4 TSH (PCDD/F), R , ; (B) for 5-y follow-u, FT 4 TSH (PCDD/F), R , quadratic form of PCDD/Fs. The significant relation between FT 4 TSHlevels and PCDD/Fs exosure level at year 2 was further demonstrated by multile linear model (Fig. 1). DISCUSSION PCDD/Fs are transferred from mother to the fetus through the lacenta and to infants via breast milk (10 12). Therefore, maternal exosure to these endocrine disrutors may adversely imact fetal/infant develoment and functioning. In this study, we erformed follow-u examinations of infants/ children reviously demonstrated to be exosed in utero to

5 IN UTERO DIOXIN EXPOSURE AND GROWTH 209 varying levels of PCDD/F (13). Our findings indicate that children with higher levels of exosure to these environmental contaminants in utero were significantly taller and heavier and had higher serum level of several thyroid hormones (T 3,FT 4 TSH, and TTR) and growth hormone (IGF-1) at the reschool ages. Whether there is a cause-effect relationshi between growth and hormone remains to be studied. Females seem to be more sensitive than males to PCDD/Fs exosure. Females exosed to PCDD/Fs in utero were significantly taller and heavier and had higher BA/CA ratio than male at both follow-u visits. We also noted the intriguing finding of significantly higher T3 and IGF-1 levels in females than males at year 2. IGF-1 remained higher at year 5 visit. Females exosed to high levels of PCDD/Fs were significantly taller and heavier and had higher BA and CA those with low exosure at year 2 follow-u visits. Similarly, we observed a significant correlation between exosure level of PCDD/Fs and thyroid hormones (T 3, TTR) in females at the year 2 follow-u visit. There was no significant correlation between PCDD/Fs exosure level and growth or hormone level in girls at the year 5 follow-u, suggesting that fetal exosure to PCDD/Fs can stimulate growth at early ages but it may become less aarent with increasing age. For males, exosure level did not significantly affect growth, but level of PCDD/Fs exosure in utero had a significant correlation with FT4 TSH at both year 2 and year 5. Correlation between PCDD/Fs exosure level in utero and hormone level during reschool ages was further confirmed using a stewise multile linear regression analysis. The results suggested that there were significantly ositive correlations between T 3, TSH, FT 4 TSH, and TTR concentrations and the level of PCDD/Fs exosure at year 2 follow-u. The finding of higher FT 4 TSH values in the high exosure grou suggested that T 4 feedback inhibition of TSH secretion was imaired in both males and females exosed to high levels of PCDD/Fs. A number of revious studies have noted altered thyroid functioning in dioxin/pcb exosed infants. The major finding of this study was the demonstration of significant ositive correlations between T 3, TSH, FT 4 TSH, and TTR concentrations and the level of PCDD/Fs exosure at year 2 followu. Pluim et al. (9,17) also reorted that term infants exosed to uer-median dioxin levels (as measured in breast milk) had increased lasma T 4 and TBG concentrations. Sauer et al. (18) investigated the growth and develoment of infants in highly industrialized western countries; they found a higher TSH at higher levels of dioxin exosure. We have noted in a revious ublication that FT 4 TSH decreased with increasing concentrations of non-ortho PCB exosure levels in infants, indicating a lack of effective FT 4 feedback to stimulate the hyothalamus (13). It suggested that dioxin/pcb levels in utero may have differential effect in neonates and children 2 3 y of age. The reason for the difference between two develomental stages is unclear and warrants further investigation. Interestingly, there was no significant correlation between PCDD/Fs exosure level and hormone level at year 5 visit after stewise multile linear regression analysis. Mazhitova et al. (14) found that none of the schoolchildren (aged y), whose serum level of PCBs was two to four times higher than healthy Stockholm children, had any imairment of thyroid function, as revealed by the lasma levels of TSH and thyroxine; there was no relation between thyroid hormone status and the blood liid levels of PCBs, hexachlorocyclohexane, and DDT. Findings from our revious study suggest that there is a negative correlation between thyroid hormone levels and maternal age (13). In accordance with that study, we also observed a significant negative correlation between TSH and FT 4 TSH concentrations and maternal age, suggesting that children born to older mothers tend to have lower levels of thyroid hormone. It is aarent that further studies are needed to exlore in more detail how changes in IGF-1, T 3, TTR, and FT 4 TSH correlate with growth and develoment in infants/children following in utero exosure to PCDD/Fs. Exosure level did not affect E 2 and testosterone at the early stage of life. In the future, we lan to closely follow this cohort of children and investigate ossible differences in the levels of hormones involved in regulating sexual develoment (i.e. E 2 and testosterone) with resect to exosure level. The same cohort of children will be continuously followed u though school age and uberty. It will be articularly interesting to know if material exosure level and blood levels of PCDD/Fs have any effect on hormone status and develoment during uberty. In conclusion, the results of this first comlete follow-u study of reschool children exosed to PCDD/Fs in utero indicate that such exosure can significantly affect growth and develoment. Serum levels of several thyroid hormones showed ositive correlation with the level of PCDD/Fs exosure in utero. Age-related changes were also observed. A number of gender-secific differences were detected with regard to growth, basal hormone concentrations, and PCDD/Fs exosure level. These findings emhasize that in utero exosure to PCDD/Fs can affect the growth and develoment of reschool age children. Acknowledgments. We thank Ms Hsiao-Yen Chen for secimen collections. REFERENCES 1. Liem AK, Furst P, Rae C 2000 Exosure of oulations to dioxins and related comounds. Food Addit Contam 17: Yamashita F, Hayashi M 1985 Fetal PCB syndrome: clinical features, intrauterine growth retardation and ossible alteration in calcium metabolism. Environ Health Persect 59: Rylander L, Stromberg U, Dyremark E, Ostman C, Nilsson-Ehle P, Hagmar L 1998 Polychlorinated bihenyls in blood lasma among Swedish female fish consumers in relation to low birth weight. Am J Eidemiol 147: Buck GM, Sever LE, Mendola P, Zielezny M, Vena JE 1997 Consumtion of contaminated sort fish from Lake Ontario and time-to-regnancy. New York State Angler Cohort. Am J Eidemiol 146: Guo YL, Hsu PC, Hsu CC, Lambert GH 2000 Semen quality after renatal exosure to olychlorinated bihenyls and dibenzofurans. Lancet 356: Schecter A, Olson JR 1997 Cancer risk assessment using blood dioxin levels and daily dietary TEQ intake in general oulations of industrial and non-industrial countries. Chemoshere 34: Demers A, Ayotte P, Brisson J, Dodin S, Robert J, Dewailly E 2002 Plasma concentrations of olychlorinated bihenyls and the risk of breast cancer: a congener-secific analysis. Am J Eidemiol 155: Jacobson JL, Jacobson SW 2001 Develomental effects of PCBs in the fish eater cohort studies. In: Robertson LW, Hansen LG (eds) PCBs: Recent Advances in Environmental Toxicology and Health Effects. The University Press of Kentucky, Lexington,

6 210 SU ET AL. 9. Pluim HJ, De Vijlder JJ, Olie K, Kok JH, Vulsma T, van Tijn DA, van der Slikke JW, Koe JG 1993 Effects of re- and ostnatal exosure to chlorinated dioxins and furans on human neonatal thyroid hormone concentrations. Environ Health Persect 101: Wang SL, Lin CY, Guo YL, Lin LY, Chou WL, Chang LW 2004 Infant exosure to olychlorinated dibenzo--dioxins, dibenzofurans and bihenyls (PCDD/Fs, PCBs) correlation between renatal and ostnatal exosure. Chemoshere 54: Chao HR, Wang SL, Lee CC, Yu HY, Lu YK, Pake O 2004 Level of olychlorinated dibenzo--dioxins, dibenzofurans and bihenyls (PCDD/Fs, PCBs) in human milk and the inut to infant body burden. Food Chem Toxicol 42: Ma HW, Lai YL, Chan CC 2002 Transfer of dioxin risk between nine major municial waste incinerators in Taiwan. Environ Int 28: Wang SL, Su PH, Jong SB, Guo YL, Chou WL, Päke O 2005 In utero exosure to dioxins and olychlorinated bihenyls and its relations to thyroid function and growth hormone in newborns. Environ Health Persect 113: Mazhitova Z, Jensen S, Ritzén M, Zetterström R 1998 Chlorinated contaminants, growth and thyroid function in schoolchildren from the Aral Sea region in Kazakhstan. Acta Paediatr 87: World Medical Association Declaration of Helsinki: ethical rinciles for medical research involving human subjects. JAMA 284: Greulich WW, Pyle SI, Waterhouse AM 1971 A radiograhic standard of reference for the growing hand and wrist. Chicago: Case Western Reserve University 17. Pluim HJ, Koe JG, Olie K, Vd Slikke JW, Kok JH, Vulsma T, Van Tijn D, De Vijlder JJ 1992 Effects of dioxins on thyroid function in newborn babies. Lancet 339: Sauer PJ, Huisman M, Kooman-Esseboom C, Morse DC, Smits-van Prooije AE, van de Berg KJ, Tuinstra LG, van der Paauw CG, Boersma ER, Weisglas-Kuerus N, Lammers JH, Kulig BM, Brouwer A 1994 Effects of olychlorinated bihenyls (PCBs) and dioxins on growth and develoment. Hum Ex Toxicol 13: