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Later childhood effects of perinatal exposure to background levels of dioxins in theNetherlands

ten Tusscher, G.W.

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Citation for published version (APA):ten Tusscher, G. W. (2002). Later childhood effects of perinatal exposure to background levels of dioxins in theNetherlands.

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Download date: 23 Sep 2020

33PERSISTENTT HAEMATO -LOGICA LL AND IMMUNO -LOGICA LL DISTURBANCES IN DUTCHH EIGHT YEAR OLD CfflLDRE NN ASSOCIATED WIT HH PERINATA L DIOXI N EXPOSURE E

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tenn Tusscher GW, Steerenberg PA, van Loveren H, Vos JG, von dem BorneBorne AEGK, Westra M, van der Slikke JW, Olie K, Pluim HJ, Koppe JG.. Persistent haematological and immunological disturbances in Dutch eightt year old children associated with perinatal dioxin exposure. Submittedd 2002.

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Abstract t

Objectives:Objectives: Perinatal exposure to Dutch "background" dioxin levels in 19900 was rather high, but comparable to other industrialised West Europeann countries. Exposure during the sensitive perinatal period may resultt in permanent disturbances. Therefore we assessed the health status andd various haematological and immunological parameters amongst our longitudinall cohort. StudyStudy design: A medical history was taken, and venipuncture performed, inn the longitudinal cohort of 27 healthy 8 year old children, with documentedd perinatal dioxin exposure. Results:Results: Linear regression revealed a decrease in allergy in relation to prenatall (p=0.02) and postnatal (p=0.03) dioxin exposure. An increase in CD4++ T-helper (p=0.006) and in CD45RA (p=0.02) cells was seen in relationn to postnatal exposure. A persistently decreased platelet count (p=0.04)) and increased thrombopoietin concentration (p=0.03) were seen inn relation to postnatal exposure. Conclusion:Conclusion: This follow-up has shown a decrease in allergy, persistently decreasedd thrombocytes, increased thrombopoietin, increased CD4+ T-helperr and increased CD45RA counts. This study provides indications of effectss at stem cell level of perinatal dioxin exposure, persisting until minimallyy eight years after birth.

Introductio n n

Polychlorinatedd dibenzo-p-dioxins and dibenzomrans (henceforth jointly referredd to as dioxins) belong to the group of most toxic substances known,, and have been associated with malignancy, congenital malformations,, immunosuppression and haematological disturbances (1-6).. Dioxins are formed as waste products of combustion processes and municipall incinerators are amongst the primary sources of these compoundss in The Netherlands. Heating of polychlorinated biphenyls (PCBs)) is a notable source of polychlorinated dibenzofurans. Dioxins, beingg poorly degradable in nature, persist in the environment and accumulatee in the food chain via fish-oils and animal fats (7). These

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polychlorinatedd aromatic compounds are highly lipophilic and are in the humann primarily stored in adipose tissues. Their lipophilicity allows themm to readily pass the placenta, whereupon they are stored in foetal adiposee tissues (8;9). Inn 1979, a PCB and polychlorinated dibenzofuran leakage into rice oil causedd a large-scale human poisoning in Taiwan, called Yucheng Disease.. In the children, born to the poisoned mothers, and thus intra-uterinee exposed to a rather high dose of PCBs and PCDFs, severe respiratoryy problems already developed during the first 6 months of life, andd 25% of these babies died before the age of four years due to lung diseases.. Those who survived had respiratory problem sequelae, such as pneumonias,, which endured until 10 years of age, whereafter a clinical improvementt was seen. Additionally, the incidence of otitis media was increasedd and became a chronic problem in these children, many of whomm developed cholesteatomas until the age of 10 years (10). Inn 1986 relatively high dioxin concentrations in the breast milk of Dutch motherss was reported, followed by similar findings in other industrialised countriess (11-13). As a result foetuses and breastfed children are exposed too relatively high "background" dioxin levels. Pluimm et al. in the same group of children as the current cohort, found thrombocytee counts to be significantly decreased during the perinatal period,, in relation to increasing postnatal dioxin exposure (6). Pluim also foundd a reduced number of granulocytes in relation to prenatal dioxin exposuree (6). This was no longer evident at the 30-month follow-up performedd by Ilsen (14). Similarly, Weisglas-Kuperus found reduced numberss of granulocytes during the perinatal period and an increase in infectiouss diseases, such as otitis media and chickenpox (15; 16). At follow-upp after approximately two years, both the aforementioned Dutch studiess revealed that the previous lowering of these white blood cells was noo longer evident (14; 16). Animal studies have shown similar results (17). .

Presently,, very littl e is known about the effects of perinatal dioxin exposuree on the (longterm) health of children. Perinatal exposure to dioxinss and related compounds may have consequences spanning many years,, possibly due to their long residence time and to their influence on

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homeostaticc setpoints during the various developmental windows (16;; 18). Consequently, we hypothesised that perinatal exposure to backgroundd levels of dioxins in The Netherlands would cause a persistent decreasee in circulating thrombocyte counts. Similarly, the white blood celll counts would also be expected to be influenced in this manner.

Inn our ongoing study of the development of children with documented perinatall dioxin exposure, we thus determined various haematological andd immunological parameters and possible clinical effects of the perinatall exposure.

Thee Medical Ethics Committees of De Heel Zaans Medical Centre (Zaandam,, The Netherlands) and of the Academic Medical Centre (Amsterdam,, The Netherlands) permitted the study. Informed consent wass obtained from the children and their parents/guardians.

Subjects,, Methods and Material s

SubjectsSubjects (table 1) Eightt years after Pluim's Zaandam study (6), the study participants were againn contacted. The perinatal dioxin exposure is documented for all thesee study participants. The children were born to healthy, well-nourishedd Caucasian women, aged between 23 and 38 years (mean 29.2 years),, who intended to breastfeed for a minimum of two months. The motherss were recruited by their obstetrician or midwife. Of the original 355 mother-baby pairs, one child was untraceable, four withdrew from the studyy and three declined venipuncture, leaving 27 children in this follow-upp study. The non-participators in no way introduced a population bias, ass their prenatal and postnatal exposures were spread across the group range.. The children underwent blood withdrawal on one of two Saturday afternoonss in the spring of 1998, at De Heel Zaans Medical Centre, Zaandam.. The dioxin toxic equivalency and cumulative toxic equivalencyy values as determined by Pluim in the breastmilk of their mothers,, were used as such within this follow-up study. Briefly, the concentrationn of dioxins, using the I-TEQ method, in the mother's

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breastmilk,, shortly after having given birth, was taken as the prenatal dioxinn exposure level of the child. The postnatal cumulative dioxin exposuree was calculated as: breastmilk dioxin concentration multiplied byy the amount of breastmilk ingested during the breastfeeding period of thee child. A 2.5% fat concentration in the milk, 700 g daily milk intake duringg the exclusively breastfeeding period and 350 g daily intake during thee transition period to formula feed was assumed (6). Dioxin-like PCBs aree not included in this calculation. In order to approximate the total dioxinn concentration (i.e. dioxins plus dioxin-like PCBs), the dioxin concentrations,, as presented in this study, need to be doubled, according too measurements done by Liem (19). Formula feed has undetectable levelss of dioxins, with the animal fats having been removed and replaced withh plant fats (16). The mothers kept a diary during the breastfeeding periodd for data validation. The average length of breastfeeding was nearlyy 3 months (88 days) with the longest period being six months. For thee 27 participants, the prenatal dioxin exposure ranged from 8.74 to 46.6 (meann 27.8) ng TEQ/kg fat (dioxin toxic equivalency per kilogram fat, measuredd in breastmilk). The children were breastfed for an average of 888 days (ranging from 1 week to 6 months). The postnatal exposure rangedd from 4.34 to 123.72 (mean 43.4) ng TEQ dioxin. These are exclusivelyy dioxins and PCDFs. The exposure is equivalent to the backgroundd exposures in The Netherlands in general during that period (11-13;20).. Subsequent (childhood) dioxin exposures are about 25 times lesss than during the fetal and breastfeeding period, and it can safely be assumedd that all the subjects had similar childhood exposures, seeing as theyy all had similar diets (21).

Urinaryy mercury and blood lead levels were determined. The mercury levelss were all under 1 ug/L, with the exception of one child who had a levell of 5 ug/L, and the lead levels ranged from 16 to 24 ug/L. All mercuryy and lead values were low and within the Dutch acceptable levels,, insinuating that it is unlikely that lead and mercury toxicity might confoundd the results.

MedicalMedical history AA complete medical history was taken of each child and a brief history was takenn of the families of the children. Amongst other complaints, such as

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coughingg spells and dyspnea, a history of asthma, bronchitis, pneumonia, otitis,, chickenpox, measles and allergies were noted. Allergy to milk and chickenn products were the only food allergies we noted. Hayfever, allergy too animals and to house-mite were noted as allergy. .

Genderr (male)

Breastfeedingg period (days)

Motherss smoking

Prenatall dioxin exposure (ng/kg fat)

Postnatall cumulative dioxin exposure (ng)

(nn = 27)

122 (44%)

87.6(7-180) )

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27.88 (8.74 - 46.62)

43.4(4.34-123.72) )

Tablee 1: Characteristics of the cohort

HaematologyHaematology and immunology Thee following parameters were determined in venous blood, collected in EDTA-coatedd tubes, using routine procedures: haemoglobin (Hb), haematocritt (Ht), erythrocytes (RBC), mean volume of RBC (MCV), mean Hbb mass of RBC (MCH), mean Hb concentration of RBC (MCHC), leukocytess (WBC) and differential, and platelets. Additionally, centrifuged plasmaa was saved and frozen. This was later used for measuring the thrombopoietinn concentration, which was performed using a monoclonal antibodyy based sandwich enzyme-linked immunosorbent assay (ELISA), previouslyy published (22). Thrombopoietin, a regulatory protein, continuallyy synthesized at a constant level by the liver, is necessary for regulatingg platelet numbers in the blood. Megakaryocytes bind the proteinn to produce thrombocytes in the bone marrow, and circulating plateletss bind and remove thrombopoietin from the circulation, resulting inn a feedback-mechanism (23).

PhenotypingPhenotyping white blood cells by flowcytometry Thee following monoclonal antibodies were used: monoclonal antibodies includingg the simultest IMK-lymphocyte kit (CD45-FTTaCD14-PE, isotypee control, CD3-HTC/CD19-PE, CD3-FITC/CD4-PE, CD3-HTC/CD8-PE,, CD3-FITC/CD16 and or CD56-PE). In addition, CD45RO-PE,, CD45RA-PE and CD29-PE were used in combination with CD4-FITC.

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Alll materials were obtained from Becton Dickinson (San Jose, CA, USA). Thee staining method was performed in accordance with the manufacture factoryy manual of Becton Dickinson with some modifications. In brief, all incubationss were carried out in V-bottom microliter plates at 4°C. Microtiterr wells were filled with 45uL EDTA blood and lOuL monoclonal antibodiess (combined) and incubated for 30 min at room temperature. Red cellss were lysed with 160 (a L lysing fluid for 10 min. and centrifuged at 300xx g for 5 min. Cells were washed twice in 150 uL 0.1%NaN3 in PBS andd centrifuged at 300x g for 5 min. Finally the stained cell suspension was fixedfixed in 100 pL 1% paraformaldehyde. Cells were stored in the dark, at 4°C.. Immunofluorescence analysis was performed using a single-laser FACScann (Becton Dickinson Immunocytometry, Mountain View, CA, USA). .

Statistics Statistics Withh the exception of the dichotomous outcomes, linear regression (dose-response)) analysis, a two-sided test, was performed using SPSS 10.0®. The dichotomouss outcomes (medical history) were analysed by binary logistic regressionn using SPSS 10.0®. P-values were calculated with standard p=0.055 as criterion for statistical significance. A best-fit line was applied to thee scatter diagrams. None of the children smoked. Correction for mothers whoo smoked during pregnancy and/or at the time of testing did not influencee the statistical outcomes presented here, and it was thus not necessaryy to correct for smoking in this study.

Results Results

ClinicalClinical findings (table 2) Thee medical histories of the subjects revealed a significant decrease in allergyy in relation to prenatal dioxin exposure (p=0.023, slope = -0.141, 95%% CI 0.769 - 0.981), as well as in relation to postnatal exposure (p=0.030,, slope = -0.060, 95% CI 0.892 - 0.994). It must be borne in mindd that the definition of allergy in this study is broad. No relation betweenn perinatal dioxin exposure and diseases such as otitis, pneumonia andd chickenpox was found.

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nn = 27

Allergyy (yes) 8(30%)

Eczemaa (yes) 8(30%)

Asthmaa (yes) 6(22%)

Otitiss (yes) 9 (33%)

Prenatal l exposure e

slopee 95% C.I. p-value

-0.1411 0.769-0.981 0.023*

-0.111 0.785-1.023 0.105

-0.0388 0.876- 1.059 0.436

0.0111 0.923-1.108 0.811

Postnatal l exposure e

slopee 95% C.I. p-value

-0.066 0.892 - 0.994 0.03*

-0.0355 0.919-1.015 0.17

0.0088 0.979-1.038 0.581

-0.0033 0.967-1.029 0.866 Tablee 2: Medical history results. * statistically significant.

Haematology Haematology Thee platelet concentrations ranged from 248 to 449 x 109/L (mean 334 x 109/L,, S.D. 52.6 x 109/L). The normal range is from 150 to 350 xl09/L forr adults, which is generally also used for children, although normal valuess for children have yet to be thoroughly determined. None of the subjectss exhibited thrombocytopenia, whilst nine children had more plateletss than the adult standard. This is commonly seen in children, and withoutt clinical relevance. Linear regression of thrombocytes versus prenatall dioxin exposure revealed an insignificant decrease (p=0.45, with aa slope of -0.79, 95% CI -2.91 to 1.33) in relation to increasing exposure.. Linear regression of platelets versus cumulative postnatal exposuree revealed a statistically significant decrease (p=0.04, with a slopee of-0.67, 95% CI -1.32 to -0.02) in relation to increasing exposure (figuree 1). This is similar to what Pluim et al. found in the same children att 11 weeks of age (6) and hence it was decided to determine the concentrationn of the platelet synthesis regulating protein, thrombopoietin, inn the frozen plasma.

Onee subject was excluded from the thrombopoietin analysis because of a suspectedd cross-reaction with the mouse immunoglobulin antibodies of thee thrombopoietin test. The thrombopoietin concentrations, measured in thee remaining 26 serum samples, ranged from 8 to 42 (mean 17) aU/mL (arbitraryy units per millilitre) . The adult normal values for thrombopoietinn are between 5 and 40 aU/mL. Thrombocytes versus thrombopoietinn revealed a statistically borderline significant inverse correlationn (p=0.07, with a slope of-2.37, 95% CI -4.95 to 0.21). This inferss that interference in the production of thrombocytes is more likely

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thann an enhanced destruction, as explanation for the thrombocyte decrease.. Linear regression of thrombopoietin versus prenatal dioxin exposuree revealed an insignificant increase (p=0.31, with a slope of 0.15, 95%% CI -0.15 to 0.45). Linear regression of thrombopoietin versus cumulativee postnatal dioxin exposure revealed a significant increase (p=0.03,, with a slope of 9.98E-02, 95% CI 0.008 to 0.191) in relation to increasingg exposure.

Thrombocytess vs Postnatal dioxin exposure 8 0 0--

700--

33 600-

pq q OO 500-

ombo

cytes

s (x

OO

O

Ja Ja HH 200

c c

A * *

A A A A

~ ~ ^ ^^ A A

AA 2 Ê ^ _ ^ A_ A

AAA ^ ^ AA A — »

A '' T A

VV T ^ ^ _ _ TT T T ' A Counts at 11 weeks

TT Current munis

200 40 60 80 100 120 140

Cumulativee postnatal dioxin exposure

Figuree 1: Scatter diagram of thrombocytes versus postnatal dioxin exposure, including thee thrombocyte counts at 11 weeks after birth, as determined by Pluim.

Too our knowledge no studies have shown a relation between dioxin exposuree and erythrocyte counts. In the current study, parameters like erythrocytee counts and MCV showed no relation with either the prenatal orr postnatal dioxin exposure.

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Immunology Immunology Wee found a highly significant increase (p=0.006, slope = 0.083, 95% CI 0.0266 - 0.140) in CD4 positive (T-helper) cells (mean 39.2, range 25.27-49.05)) in relation to increasing postnatal dioxin exposure. The T-cell/B-celll ratio (mean 3.89, range 1.5-6.2) was borderline-significantly raised (p=0.086,, slope = 0.042, 95% CI -0.006 - 0.090) in relation to the prenatall dioxin exposure. We found a significant increase in the CD45RAA count (mean 27.3, range 16.1-41.3) in relation to the postnatal dioxinn exposure (p=0.019, slope = 0.078, 95% CI 0.014 - 0.141). Other immunologicall parameters revealed no relation with either the prenatal or postnatall dioxin exposure.

Discussion n

Wee will separately discuss our findings of a decrease in allergy, relative thrombocytopeniaa and immunological disturbances.

/.. Allergy Thee observed decrease in allergy was not altogether unexpected. In anotherr group of Dutch children, born in the same period as our subjects, aa decrease in allergy in relation to current levels of PCBs was also detectedd (16).

AA decrease in allergy sounds beneficial, but this may be deceptive. The decreasee could have its pathogenesis in, for example, a relatively deficientt immune memory system, an altered antigen receptor or in an insufficientt mobilisation of immune cells. Wee are inclined to explain the decrease in allergy as a long-term effect of perinatall dioxin exposure because of the relation seen with the perinatal exposure.. However, it cannot be ruled out that the current levels of dioxinss and/or PCBs play a role, since these levels might be related to the pre-- and postnatal exposures. In four year olds, the levels of PCBs were fourr times higher in breastfed infants than in bottlefed infants (24). The currentt levels of dioxins and PCBs in our study-children were not

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measured.. However, using a model, breastfeeding has an effect of 5% higherr levels of PCBs and dioxins, at the age of eight years (21).

II.II. Laboratory findings A)A) Platelets CurrentCurrent study Thiss study has shown a significant decrease in thrombocytes and borderline increasee in thrombopoietin concentration, both in relation to perinatal dioxinn exposure. The platelet count and thrombopoietin concentration show ann inverse relation. This indicates a suppressed production of platelets, ratherr than increased destruction, as the cause of the relative thrombocyte decreasee (23). The strong inverse correlation between thrombopoietin and platelets,, as well as the fact that the scatter diagrams (not shown) reveal clearr gradients, lends credence to the dioxin influence at the megakaryocyte too thrombocyte differentiation stage, or earlier in the thrombopoiesis. The significantt decrease in the number of granulocytes, as detected by Pluim (6) andd Weisglas-Kuperus (15) in the perinatal period, once again suggest a productionn problem at stem cell level. In this study dioxin effects on red bloodd cells could not be demonstrated.

Thee subjects in this study group all have thrombocyte and thrombopoietinn concentrations falling within the limits of "normal", as is too be expected in an "average, healthy" subject group. However, follow-upp far into adulthood is certainly justified, in order to be vigilant for later clinicall problems. It is important to note that this cohort was selected on pregnancyy and birth-weight optimality. Bearing this in mind, the elicited resultss may be more outspoken in a general population cohort.

Thee persistently decreased thrombocyte count relative to the postnatal dioxinn exposure is remarkable since Pluim et al. who studied the same groupp of children at eleven weeks of age detected the same. Then, too, thee reduced thrombocyte count was related to the postnatal dioxin exposuree (6). This might be caused by a persisting effect of the exposure too dioxins in the perinatal period. This reduction can be induced by either aa reduced production of thrombocytes (inferring dioxin toxicity at the megakaryocytee level in the bone marrow), or by an increased

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sequestrationn of ineffective platelets (inferring toxicity at the gene level). Ann increased destruction is excluded by the finding of the inverse correlationn between thrombopoietin and thrombocyte levels. We must thenn conclude that dioxins may negatively influence the production of platelets,, and thereby necessitating a compensatory increase in thrombopoietinn production. It is not known whether or not the level of thrombocytess is genetically determined, nor if the setpoint for this feed-backk system remains constant throughout life, under healthy circumstances.. Should this be the case, then our findings can be explainedd as a toxic effect on this system in the perinatal period, with effectss persisting into later life. Another explanation for these outcomes iss that we have detected a continuing effect of dioxin exposure, with currentt body levels still able to negatively influence thrombocytogenesis. Thiss was seen in Japanese workers after chronic exposure (25) and in two womenn after acute poisoning (26). The two subjects of the latter study havee current dioxin concentrations a thousand-fold higher than our cohort subjects,, yet the subjects of the former study have levels not much higher thann the mothers of our cohort.

AnimalAnimal studies Zinkll et al. described enduring thrombocytopenia in rather high dosage 2,3,7,8-tetrachloro-para-dioxinn (TCDD, dioxin) treated adult rats and guineaa pigs (27). Kociba et al. reported lower thrombocyte counts for adultt rats given lug TCDD/kg/day though no difference was seen betweenn lower dosage and control groups (28). In a study of TCDD treatedd rhesus monkeys, McConnell et al. described a hypoplastic bone marroww in single high doses (29). Murante and Gasiewicz reported effectss of TCDD exposure on marrow progenitor cells in mice. Their data indicatee that proliferation and/or differentiation processes of hemopoietic stemm cells are influenced by TCDD administered 5-6 weeks postpartum (30). . Bonee marrow and megakaryocyte findings have been inconsistent (27;31;32).. Fine et al. reported a reduction in lymphocyte stem cells in thee offspring after a single dose of 10 ug/kg bodyweight TCDD in maternall mice on gestational day 14 (33).

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Variouss dioxin studies have reported bleeding amongst laboratory animalss exposed to dioxins, albeit far higher doses than the perinatal exposuree of this study group. Viluksela et al. noted bleeding to be the mostt common sign of lethality, apart from wasting syndrome. They noted significantlyy decreased platelet counts amongst the highest exposed groupss of their subchronic/chronic dosage study, with platelet counts particularlyy low in rats moribund as a result of haemorrhage or anaemia. Theyy conclude that it is obvious that haemorrhage as a cause of death is a resultt of thrombocytopenia and prolonged prothrombin times (34;35). Shiverickk and Muther showed that foetuses, of pregnant rats treated with aa daily oral dosage of lug/kg TCDD, had a 66% incidence of visceral lesions,, characterised by intestinal haemorrhage (36). Olson et al. found gastrointestinall bleeding to be the most sensitive indicator of in utero exposuree to TCDD, in the foetuses of maternal rats exposed to 1.5ug/kg. Thee incidence of bleeding was 7.7 5.5% (37). Similarly, Gupta reported bleedingg amongst TCDD treated rats and guinea pigs (32).

HumanHuman studies Inn a letter to the editor of The Lancet in 1977, Laporte focussed attention onn a study of Vietnamese exposed to the dioxin-contaminated defoliant, Agentt Orange, used extensively during the Vietnam War. The study reportedd gastro-intestinal bleeding, caused by thrombocytopenia and prothrombinn deficiency, related to the dioxin exposure (38). Prothrombin deficiencyy might be a result of Vitamin K deficiency caused by PCBs or dioxinss as is hypothesised by Koppe and colleagues (39) and found in the rat-studiess of Bouwman et al. (40).

B)B) T-cells Itt has long been known, from animal and human studies, that the thymus andd T-helper cell (CD4+) are sensitive to dioxin exposure (3;16;27;41). Mostt studies have revealed a decrease in the CD4+ population. In contrast,, we have shown a highly significant tendency towards an increasingg T-helper cell population with increasing postnatal dioxin exposure. . Studiess in TCDD sensitive mice revealed that TCDD may affect polymorphonuclearr cells at the level of hematopoiesis, via a direct

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interactionn with granulocyte precursor cells, or modulate polymorphonuclearr cells at different stages of maturation. Thereby suggestingg an effect on haematopoietic stem cells (17). Totall and dioxin TEQ levels in the breastmilk correlated significantly withh increased T-cell subpopulations in human infants of the Rotterdam study.. Additionally, significantly decreased B-cell markers were observedd following postnatal exposure (16). In a later follow-up study at 422 months of age, prenatal PCB exposure was associated with an increasedd number of peripheral T-cells, as well as with an increased numberr of CD8+ (cytotoxic) and TcRalfa beta+ T-cells. At 42 months of age,, prenatal PCB-exposure was also significantly related with more CD3+HLA-DR++ (activated) T-cells as well as lower antibody levels to measles,, and a higher prevalence of chickenpox. Current PCB levels weree associated with a higher prevalence of recurrent middle ear infectionss and a lower prevalence of allergic reactions as already mentionedd (15). Interestingly, our study has shown an increase in CD45RAA positive cells in relation to cumulative postnatal dioxin exposure.. CD45RA positive cells are characteristic of naive circulating T-cells.. They are essential for T-cell maturation (23). We detected an increasee in CD4+ helper T-cells in relation to perinatal dioxin exposure. Inn the Rotterdam study the number of CD4CD45RA cells correlated positivelyy with the incidence of bronchitis (15). Otitis and pneumonia weree not related to dioxin exposure, in our study, but the limited size of ourr group may play a role here. However, our cohort showed a reduction inn lung function and increase in bronchial obstruction, both relative to increasingg perinatal dioxin exposure (42).

Concluding,, perinatal dioxin exposure, even at Dutch background exposuree levels in 1990, results in a persisting negative effect on thrombocytee numbers, in conjunction with an increased thrombopoietin concentration,, indicating a production problem at stem cell level. A decreasee in allergy, increase in T-helper cells and CD45RA positive cells weree seen in relation to cumulative postnatal dioxin exposure. This persistentt influence in average children with background exposures is a neww finding and warrants further research.

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Acknowledgements s

Wee are grateful to the children of the cohort and their parents; Greenpeacee for sponsoring the study participants' travel expenses; Margalithh van Huiden-ten Brink; L.J.J. de la Fonteyne-Blankestijn; Y.C. Wallerbrink-dee Dreu; Bert Verlaan; Dr. Johannes Oosting; Dr. Marcel Rouwenhorst;; Dr. Taco Kuijpers; De Heel Zaans Medical Centre

Bibliography y

(1)) Ziegler J. Environmental "endocrine disruptors" get a global look (editorial). J Natll Cancer Inst 1997; 89(16): 1184-1187.

(2)) ten Tusscher GW, Stam GA, Koppe JG. Open chemical combustions resulting in aa local increased incidence of orofacial clefts. Chemosphere 2000; 40(9-11):: 1263-1270.

(3)) Vos JG, De Heer C, van Loveren H. Immunotoxic effects of TCDD and toxic equivalencyy factors. Teratog Carcinog Mutagen 1997; 17(4-5):275-284.

(4)) Neubert R, Stahlmann R, Korte M, van Loveren H, Vos JG, Golor G et al. Effectss of small doses of dioxins on the immune system of marmosets and rats. Annn N Y Acad Sci 1993; 685:662-686.

(5)) ten Tusscher GW, Ilsen A, Koppe JG. Background exposure to dioxins and polychlorinatedd biphenyls in Europe and the resulting health effects: a review. Prenatall and Neonatal Medicine 1999; 4(4):261-270.

(6)) Pluim HJ, Koppe JG, Olie K, van der Slikke JW, Slot PC, van Boxtel CJ. Clinicall laboratory manifestations of exposure to background levels of dioxins in thee perinatal period. Acta Paediatr 1994; 83(6):583-587.

(7)) Theelen RMC, Liem AKD, Slob W, van Wijnen J. Intake of 2,3,7,8, chlorine substitutedd dioxins, furans, and planar PCBs from food in The Netherlands: mediann and distribution. Chemosphere 1993; 27:1625-1635.

(8)) van Wijnen J, van Bavel B, Lindström G, Koppe JG, Olie K. Placental transport off PCDDs and PCDFs in humans (extended abstract). Dioxin '90 Congress , 47-50.. 1990.

(9)) Krowke R, Abraham K, Wiesmüller T, Hagenmaier H, Neubert D. Transfer of variouss PCDDs and PCDFs via placenta and mother's milk to marmoset offspring.. Chemosphere 1990; 20(7-9): 1065-1070.

(10)) Guo YL. Human health effects from PCBs and dioxin-like chemicals in the rice-oill poisonings as compared with other exposure episodes. Organohalogen Compoundss 42, 241-242. 1999.

(11)) van den Berg M, van der Wielen FWM, Olie K, van Boxtel CJ. The presence of PCDDss and PCDFs in human breastmilk from The Netherlands. Chemosphere 1986;; 15:693-706.

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(12)) World Health Organisation. Levels of PCBs, PCDDs and PCDFs in breast milk. Copenhagen:: WHO, 1989.

(13)) World Health Organisation. Levels of PCBs, PCDDs and PCDFs in human milk. Bilthoven:: WHO, 1996.

(14)) Ilsen A, Briet JM, Koppe JG, Pluim HJ, Oosting J. Signs of enhanced neuromotorr maturation in children due to perinatal load with background levels off dioxins. Follow-up until age 2 years and 7 months. Chemosphere 1996; 33(7):: 1317-1326.

(15)) Weisglas-Kuperus N, Sas TC, Koopman-Esseboom C, van der Zwan CW, De Ridderr MA, Beishuizen A et al. Immunologic effects of background prenatal and postnatall exposure to dioxins and polychlorinated biphenyls in Dutch infants. Pediatrr Res 1995; 38(3):404-410.

(16)) Weisglas-Kuperus N, Patandin S, Berbers GA, Sas TC, Mulder PG, Sauer PJ et al.. Immunologic effects of background exposure to polychlorinated biphenyls andd dioxins in Dutch preschool children. Environ Health Perspect 2000; 108(12):: 1203-1207.

(17)) Ackermann MF, Gasiewicz TA, Lamm KR, Germolec DR, Luster MI. Selective inhibitionn of polymorphonuclear neutrophil activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.. Toxicol Appl Pharmacol 1989; 101(3):470-480.

(18)) Hurst CH, DeVito MJ, Setzer RW, Birnbaum LS. Acute administration of 2,3,7,8-tetrachlorodibenzo-p-dioxinn (TCDD) in pregnant Long Evans rats: associationn of measured tissue concentrations with developmental effects. Toxicoll Sci 2000; 53(2):411-420.

(19)) Liem AKD, Theelen RMC. Dioxins: chemical analysis, exposure and risk assessment.. University of Utrecht, 1997.

(20)) Koopman-Esseboom C, Huisman M, Weisglas-Kuperus N, Boersma ER, De Ridderr MA, van der Paauw CG et al. Dioxin and PCB levels in blood and human milkk in relation to living areas in The Netherlands. Chemosphere 1994; 29(9-ll):2327-2338. .

(21)) Patandin S, Dagnelie PC, Mulder PG, Op dC, van der Veen JE, Weisglas-Kuperuss N et al. Dietary exposure to polychlorinated biphenyls and dioxins from infancyy until adulthood: A comparison between breast-feeding, toddler, and long-- term exposure. Environ Health Perspect 1999; 107(1):45-51.

(22)) Folman CC, dem Borne AE, Rensink IH, Gerritsen W, van der Schoot CE, de Haass M et al. Sensitive measurement of thrombopoietin by a monoclonal antibodyy based sandwich enzyme-linked immunosorbent assay. Thromb Haemostt 1997; 78(4): 1262-1267.

(23)) Hematology of infancy and childhood. 5 ed. Philadelphia: W.B. Saunders Company,, 1998.

(24)) Patandin S, Weisglas-Kuperus N, De Ridder MA, Koopman-Esseboom C, van Staverenn WA, van der Paauw CG et al. Plasma polychlorinated biphenyl levels inn Dutch preschool children either breast-fed or formula-fed during infancy. Am JJ Public Health 1997; 87(10): 1711-1714.

95 5

(25)) Watanabe S, Kitamura K, Ikida T, Otaki M, Waechter G. Health effects of chronicc exposure to polychlorinated dibenzo-p-dioxins, dibenzofurans and co-planarr PCBs in Japan (extended abstract). Organohalogen Compounds 53, 136-140.2001. .

(26)) Geusau A, Abraham K, Geissler K, Sator MO, Stingl G, Tschachler E. Severe 2,3,7,8-tetrachlorodibenzo-p-dioxinn (TCDD) intoxication: clinical and laboratory effects.. Environ Health Perspect 2001; 109(8):865-869.

(27)) Zinkl JG, Vos JG, Moore JA, Gupta BN. Hematologic and clinical chemistry effectss of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Environ Healthh Perspect 1973; 5:111 -118.

(28)) Kociba RJ, Keeler PA, Park CN, Gehring PJ. 2,3,7,8-tetrachlorodibenzo-p-dioxinn (TCDD): results of a 13-week oral toxicity study in rats. Toxicol Appl Pharmacoll 1976; 35(3):553-574.

(29)) McConnell EE, Moore JA, Dalgard DW. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxinn in rhesus monkeys (Macaca mulatta) following a single oral dose. Toxicoll Appl Pharmacol 1978; 43(1): 175-187.

(30)) Murante FG, Gasiewicz TA. Hemopoietic progenitor cells are sensitive targets of 2,3,7,8-tetrachlorodibenzo-p-dioxinn in C57BL/6J mice. Toxicol Sci 2000; 54(2):374-383. .

(31)) Weissberg JB, Zinkl JG. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin upon hemostasiss and hematologic function in the rat. Environ Health Perspect 1973; 5:119-123. .

(32)) Gupta BN, Vos JG, Moore JA, Zinkl JG, Bullock BC. Pathologic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxinn in laboratory animals. Environ Health Perspectt 1973; 5:125-140.

(33)) Fine JS, Gasiewicz TA, Silverstone AE. Lymphocyte stem cell alterations followingg perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mol Pharmacoll 1989; 35(l):18-25.

(34)) Viluksela M, Stahl BU, Birnbaum LS, Schramm KW, Kettrup A, Rozman KK. Subchronic/chronicc toxicity of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD)) in rats. Part I. Design, general observations, hematology, and liver concentrations.. Toxicol Appl Pharmacol 1997; 146(2):207-216.

(35)) Viluksela M, Stahl BU, Birnbaum LS, Schramm KW, Kettrup A, Rozman KK. Subchronic/chronicc toxicity of a mixture of four chlorinated dibenzo-p-dioxins in rats.. I. Design, general observations, hematology,and liver concentrations. Toxicoll Appl Pharmacol 1998; 151(l):57-69.

(36)) Shiverick KT, Muther TF. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) effects onn hepatic microsomal steroid metabolism and serum estradiol of pregnant rats. Biochemm Pharmacol 1983; 32(6):991-995.

(37)) Olson JR, McGarrigle BP, Tonnuci DA, Schecter A, Eichelberger H. Developmentall toxicity of 2,3,7,8-TCDD in the rat and hamster. Chemosphere 1990;; 20(7-9): 1117-1123.

(38)) Laporte JR. Effects of dioxin exposure. Lancet 1977; 1(8020): 1049-1050.

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(39)) Koppe JG, Pluim HJ, Olie K. Breastmilk, PCBs, dioxins and vitamin K deficiency.. J Royal Soc of Med 1989; 82:416-420.

(40)) Bouwman CA, Seinen W, Koppe JG, van den Berg M. Effects of 2,3,7,8-TCDD orr 2,2',4,4\5,5'-HxCB on vitamin K dependent bloodcoagulation-factors in male andd female germfree WAG/RU-rats (extended abstract). Organohalogen Compoundss 1, 59-62. 1990.

(41)) Vos JG, Moore JA. Suppression of cellular immunity in rats and mice by maternall treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Int Arch Allergy Appll Immunol 1974; 47(5):777-794.

(42)) ten Tusscher GW, de Weerdt J, Roos CM, Griffioen RW, De Jongh FH, Westra MM et al. Decreased lung function associated with perinatal exposure to Dutch backgroundd levels of dioxins. Acta Paediatr 2001; 90(11): 1292-1298.

Data a

I.D. . No. .

22 2 23 3 25 5 26 6 28 8 29 9 32 2 33 3 34 4 35 5 37 7 38 8 39 9 40 0 41 1 43 3 46 6 48 8 49 9 50 0 51 1 52 2 53 3 54 4 55 5 56 6 57 7

TEQDIOX X ng/kgfat ng/kgfat

30.80 0 39.23 3 29.65 5 16.56 6 37.30 0 38.49 9 31.10 0 15.03 3 38.02 2 33.01 1 41.29 9 8.74 4 26.79 9 43.42 2 22.21 1 29.21 1 27.96 6 20.63 3 18.12 2 21.31 1 34.28 8 19.72 2 30.34 4 21.07 7 14.03 3 15.09 9 46.62 2

TEQCUM M

"g "g

44.46 6 123.72 2 80.83 3 10.55 5 21.93 3 109.69 9 59.49 9 27.97 7 86.13 3 45.46 6 50.05 5 5.67 7 39.62 2 45.93 3 32.11 1 8.11 1

64.15 5 46.96 6 25.93 3 6.22 2 4.34 4 13.69 9 38.06 6 45.97 7 25.01 1 40.07 7 68.71 1

SEX X

F F M M M M F F M M M M M M F F F F F F F F M M M M F F F F F F M M M M M M F F M M F F F F F F F F F F M M

ALLERGY Y

Y Y N N N N Y Y N N N N N N Y Y N N N N N N N N N N N N N N Y Y N N N N N N Y Y N N Y Y N N N N Y Y Y Y N N

ECZEMA A

Y Y N N N N N N N N N N N N N N N N Y Y N N Y Y N N N N Y Y N N N N Y Y N N Y Y N N Y Y N N N N N N Y Y N N

ASTHMA A

N N Y Y N N N N N N Y Y N N Y Y N N N N N N Y Y N N N N N N N N N N N N N N N N Y Y N N N N N N Y Y N N N N

OTITIS S EPISODES S

0 0 5 5 2 2 0 0 I I 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 2 2 0 0 1 1 2 2 5 5 2 2 0 0 0 0 0 0 0 0 0 0 0 0

SMOKING G MOTHER R

N N N N N N N N N N N N N N N N Y Y Y Y N N N N N N N N Y Y N N N N N N Y Y Y Y N N Y Y N N N N N N N N N N

Medicall history results. LD. No.: subject identity number; TEQDIOX: prenatal exposure;; TEQCUM: postnatal exposure; SEX: gender; F: female; M: male; Y: yes; N: no. .

97 7

IDD TEQDIOX TEQCUM PLT TPO MCV T- T-sup- Natural TH:TS T:B CD45RA CD45RO CD29

No.. ng/kgfat ng 10*/1 aU/mL ft helper pressor Killer Ratio Ratio CD4+ CD4+ CD4+

85.000 40.99 27.50 7.70 1.50 4.70 27.20 14.20 43.60

76.700 41.44 25.70 7.40 1.60 4.50 31.60 11.00 41.40

78.955 49.05 20.10 12.40 2.40 5.20 41.30 10.10 48.10

81.100 37.69 21.50 10.10 1.80 2.80 22.50 4.60 43.10

79.100 37.84 24.50 4.80 1.50 4.30 24.60 13.80 37.10

81.255 45.12 22.30 3.80 2.00 3.00 33.80 14.50 44.80

85.155 34.90 15.70 13.60 2.20 1.60 25.40 12.70 34.50

84.555 48.18 20.90 6.30 2.30 4.90 37.10 17.10 49.80

88.455 44.33 21.90 19.90 2.00 6.20 32.30 19.10 44.80

76.700 37.78 23.90 14.00 1.60 6.00 18.00 10.30 27.20

80.600 37.66 23.90 11.20 1.60 3.40 23.90 15.80 40.30

77,200 35.85 26.90 7.50 1.30 2.90 26.30 14.40 33.60

81.855 40.10 29.60 4.40 1.40 5.70 28.30 13.30 43.40

85.455 39.90 25.80 11.60 1.50 4.20 24.90 11.50 42.70

79.755 36.33 29.20 8.90 1.20 3.50 25.40 13.30 38.20

83.455 36.60 27.50 10.70 1.30 4.80 23.80 17.20 32.10

80.000 37.33 22.60 12.50 1.60 3.50 24.60 12.50 35.80

78.300 36.01 24.80 6.40 1.50 2.90 25.50 14.50 30.00

84.055 32.51 32.90 8.20 1.00 3.30 24.60 20.30 32.60

81.900 38.31 24.10 11.80 1.60 3.60 29.70 9.10 38.90

79.255 37.49 19.40 9.40 1.90 2.20 27.90 13.30 41.10

79.755 25.27 17.50 16.00 1.40 1.50 16.10 12.30 28.50

80.555 37.21 23.20 12.90 1.60 4.90 22.90 15.20 37.00

82.100 39.91 16.00 14.60 2.50 2.60 30.00 10.30 41.00

87.100 40.34 19.70 13.40 2.00 4.00 29.60 15.00 41.20

79.655 42.85 24.50 6.70 1.80 3.70 30.90 17.20 42.60

83.600 47.05 19.80 9.70 2.40 5.20 29.00 14.40 48.50

48 8

49 9

50 0

51 1

52 2

53 3

54 4

55 5

56 6

57 7

20.63 3

18.12 2

21.31 1

34.28 8

19.72 2

30.34 4

21.07 7

14.03 3

15.09 9

46.62 2

46.96 6

25.93 3

6.22 2

4.34 4

13.69 9

38.06 6

45.97 7

25.01 1

40.07 7

68.71 1

248 8

278 8

371 1

362 2

282 2

321 1

333 3

269 9

379 9

338 8

314 4

330 0

314 4

255 5

347 7

367 7

347 7

270 0

373 3

305 5

449 9

317 7

300 0

394 4

405 5

441 1

42 2

19 9

27 7

13 3

26 6

17 7

14 4

12 2

10 0

25 5

13 3

10 0

21 1

10 0

15 5

16 6

10 0

13 3

20 0

17 7

excl l

18 8

25 5

17 7

12 2

8 8

Haematologyy and immunology results. I.D. No.: subject identity number; TEQDIOX: prenatall exposure; TEQCUM: postnatal exposure; PLT: thrombocytes; TPO: thrombopoietine;; MCV: mean corpuscular volume; TH:TS: T-helper:T-suppressor ratio; T:B:: T-cell:B-cell ratio.

98 8