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Later childhood effects of perinatal exposure to background levels of dioxins in
the Netherlands
ten Tusscher, G.W.
Publication date 2002
Document Version Final published version
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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 the Netherlands.
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* *
*x x
1.. Burning chemicals in the open air is dangerous to
health.. (This thesis).
2.. Perinatal exposure to even background levels of dioxinss leads to lung function sub-optimality. (This
thesis). .
3.. Children exposed perinatally to background levels of dioxinss show a persistent decrease in blood platelets.
(Thiss thesis).
4.. One way of retarding the neurological development of ourr children is to expose them to background levels of dioxinss in the perinatal period. (This thesis).
5.. The increase in aggression and social problems in our societyy is partly the result of perinatal exposure to backgroundd levels of dioxins. (This thesis).
6.. Technology is not the limiting factor in attempting to reducee background dioxin exposure levels.
7.. It is logical that if chlorine production is the predominantt reason for the existence of polychlorinatedd dioxins, we should curtail the productionn of chlorine.
8.. This thesis chronicles the aftermath of a chronic disaster,, a disaster mankind is compelled to repeat with newerr persistent xenobiotics.
9.. The likelihood of a driver being egoistic in the morning trafficc jams exponentially increases with the value of hiss car.
10.. The quality of life is inversely proportional to the standardd of living.
11.. Dutch politics can be summed up in one sentence: sometimess it is right to be left and sometimes the left hass to be left right away.
12.. Children are the highlight of life.
Laterr childhood effects of perinatal exposure
too background levels of dioxins in
Cover:: "Polluting nature in The Netherlands" by
Daniell ten Tusscher (aged 3 years) and Joshua ten Tusscher (aged 15 months) Druk:: Koopmans' drukkerij, Hoorn
Wee are grateful for financial support from:
Universityy of Amsterdam, and especially the Emma Children's Hospital AMC Nationall Institute for Public Health and the Environment (RIVM)
Greenpeacee International Stichtingg Ecobaby
CRII Technology Services BV
Flanderss Environmental and Health Technologies Cambridgee Isotope Laboratories
ISBN:: 90-9016271-2 ©© 2002 G.W. ten Tusscher
Noo part of this thesis may be reproduced or transmitted in any form or by any means, electronicc or mechanical, including photocopy, recording or otherwise without permissionn of the author.
LATERR CHILDHOOD EFFECTS OF
PERINATALL EXPOSURE TO BACKGROUND
LEVELSS OF DIOXINS IN THE NETHERLANDS
ACADEMISCHH PROEFSCHRIFT
terr verkrijging van de graad van doctor aann de Universiteit van Amsterdam opp gezag van de Rector Magnificus
prof.. mr. P.F. van der Heijden
tenn overstaan van een door het college voor promoties ingestelde commissie, in het openbaarr te verdedigen in de Aula der Universiteit
opp vrijdag 25 oktober 2002, te 12:00 uur door r
Gavinn William ten Tusscher
Promotiecommissie e
Promotor r Prof.. Dr. J.G. Koppe
Co-promotoress : Prof. Dr. Ir. H. Spekreijse Dr.. J.W. van der Slikke
Overigee leden Prof.. Dr. H.S.A. Heymans Prof.. Dr. C V . Howard Prof.. Dr. J.G. Vos Dr.. F.H.C, de Jongh Dr.. N.A.M. ScheUart Dr.. T. Vulsma
for... for...
DanielDaniel and Joshua, my children, the highlight of my life; Anna-Leena,Anna-Leena, my ever supportive wife and friend;
Tablee of Contents
Page e
Listt of abbreviations 9
Introductionn 11 Chapterr 1 - Open chemical combustions resulting in a local
increasedd incidence of orofacial clefts
(Chemospheree 2000; 40: 1263-1270) 43 Chapterr 2 - Decreased lung function associated with perinatal
exposuree to Dutch background levels of dioxins
(Actaa Paediatrica 2001; 90: 1292-1298) 61 Chapterr 3 - Persistent haematological and immunological
disturbancess in Dutch eight year old children associatedd with perinatal dioxin exposure
(Submittedd for publication 2002) 79 Chapterr 4.1 - Perinatal dioxin exposure and cytochrome P-450
activity,, thyroid and liver functions at follow-up
afterr 7 - 1 2 years (Submitted for publication 2002) 99 Chapterr 4.2 - Validation of a high-performance liquid
chromatographyy assay for quantification of caffeinee and paraxanthine in human serum in the contextt of CYP1A2 phenotyping (Biomedical
Chapterr 5 - Neurodevelopmental influences of perinatal dioxin exposuree as assessed with
magnetoencephalography,, electroencephalography, psychologicall and neuromotor tests (submitted for
publicationn 2002) 135 Chapterr 6 - Does perinatal exposure to background levels of
dioxinss have a lasting effect on the human dentition?? (Organohalogen Compounds
1999;44:279-281)) 171 Summaryy 177 Samenvattingg 187 Appendicess 197 AA word of thanks 199 Publicationss 203 Curriculumm Vitae 205
LISTT OF ABBREVIATIONS
AHR R ALAT T ASAT T CBCL L CD4+ + CD8+ + CI I CYPP P450 1A2 EEG G FEVj j FT4 4 FVC C HDN N LQ. . kg g MEG G ng g PAH PAH PCB B PCDD D PCDF F SD D SE E T4 4 TCDD D TCDF F TEQ Q TEQcum m TEQdiox x TRF F TSH H WISC-R RAryll hydrocarbon receptor Alaninee aminotransferase Aspartatee aminotransferase Childd Behaviour Check List T-helperr cells
Cytotoxicc T-cells
95%% Confidence interval Cytochromee P-450 1A2 Electroencephalograph h
Forcedd expiratory volume in 1 second Freee T4
Forcedd vital capacity
Haemorrhagicc disease of the newborn Intelligencee quotient
Kilogram m
Magnetoencephalograph h Nanogram m
Polycyclicc aromatic hydrocarbons Polychlorinatedd biphenyls Polychlorinatedd dibenzodioxins Polychlorinatedd dibenzofurans Standardd deviation Standardd error 3,33 * ,5,5' -tetraiodo-L-thyronine 2,3,7,8-tetrachloro-p-dioxin n 2,3,7,8-tetrachlorodibenzofuran n Toxicc equivalency
Cumulativee postnatal dioxin exposure Prenatall dioxin exposure
Teacherr Report Form
Thyroidd stimulating hormone
Introduction n
Whatt are dioxins?
Polychlorinatedd p-dioxins (PCDDs), polychlorinated dibenzo-fiiranss (PCDFs) and some polychlorinated biphenyls (PCBs), such as PCB-77,, -126 and -169 (henceforth jointly referred to as dioxins) belong too the group of most toxic substances known. PCDDs consist of a family off 75 congeners, or structurally similar compounds. They are, in pure form,, colourless crystals or solids. With the exception of small amounts forr research purposes, they are not intentionally manufactured by industryy (1). Dioxins (figure 1) are formed as waste products of combustionn processes and municipal incinerators are amongst the primaryy sources of these compounds in The Netherlands (2). Other sourcess of dioxins include the combustion of fossil fuels and wood, chlorinee bleaching processes in pulp and paper mills, chlorination of drinkingg water and chlorinated organic chemical production. During the Vietnamm War, an extensively used defoliant called Agent Orange, used duringg Operation Ranch Hand, was a major source of dioxin pollution in Southh East Asia (3;4).
PCBss are a family of 209 possible congeners, of which 13 are likely to be similarr to tetrachloro-p-dioxin, TCDD, the most toxic dioxin congener. Finally,, there are 135 possible PCDF congeners. Heating of PCBs is a notablee source of PCDFs.
Whyy are dioxins a human health hazard?
Dioxinss are poorly degradable in nature and persist in the environment, accumulatingg in the human food chain mainly via fish-oils and animal fatss (5;6). They do not dissolve easily in water and hence settle to surfaces,, such as river sediments or grass. Fish eat the plankton and otherr microscopic organisms on river bottoms, herbivores eat the grass. Largerr animals in turn eat these aquatic organisms and animals and eventuallyy the food chain, as it is called, leads to man eating the dioxin
contaminatedd animals or plants, such as dairy products and meat (7). The mainn contributors to dioxin intake in the European Union are milk and
Figuree 1: Structure of dioxin (PCDD = polychlorinated-p-dibenzodioxin), furan (PCDF == polychlorinated dibenzofuran) and PCB (PCB = polychlorinated biphenyl, o = ortho, mm = mono, p = planar) molecules showing their ring positions.
dairyy products (16-39%), meat and meat products (6-32%) and fish and fishh products (2-63%) (8). These chlorinated polyaromatic compounds aree highly lipophilic and are therefore difficult for an organism to metabolise.. The accumulation results in increasing concentrations of dioxinss in each higher step in the food chain. Dioxins in the human are primarilyy stored in adipose tissues and liver (9). Their lipophilicity allows themm to readily pass the placenta, whereupon they are stored in foetal liverr and adipose tissues (10;11). In 1986 relatively high background dioxinn concentrations in the breast milk of Dutch mothers was first reported,, followed by similar findings in other industrialised countries (12-15).. As a result foetuses and breastfed children are exposed to relativelyy high "background" dioxin levels, averaging approximately 30 ng/kgg fat, as measured in breastmilk (12-14;16;17). One nanogram (ng)
iss one billionth of a gram. The half-life, or time it takes to remove half of thee amount, of dioxins in the human body is 7 to 12 years (1).
Twoo Asian incidents of PCB and PCDF contamination of rice oils led to largee numbers of people being poisoned. The 1968 accident in Japan, "Yusho",, and the 1978 accident in Taiwan, "Yucheng", revealed direct toxicityy effects, but also long-term effects and even teratogenic effects (18-20).. In 1976, in the Italian town of Seveso, a chemical plant explodedd releasing a large amount of pure TCDD, or dioxin, into the atmospheree (21;22). The resulting health effects, in many ways similar too those seen in the Asian accidents, demonstrated that the toxic effects off dioxin exposure were varied and largely not understood. Studies done amongstt industrial workers and Agent Orange exposed subjects during thee Vietnam War, exposed in adulthood, have produced conflicting healthh effects (4;23-25).
However,, all these subjects were exposed to high concentrations of dioxinss and PCBs. The shocking finding of dioxins in human breastmilk inn women not living in "contaminated" areas led to growing concern over possiblee teratogenic and long-term health effects in children prenatally exposedd to "background" concentrations. Background concentrations beingg the concentrations found in average, healthy people, not living in areass at extra risk for exposure. It was in this setting that a Dutch longitudinall cohort study was started in 1989, by Koppe and Pluim (16;26),, which later became known as the Amsterdam/Zaandam study. Variouss abnormalities were seen, some of which had normalised by follow-upp at 2Vi years (27), but other abnormalities only presented themselvess later. The number of subjects in the Amsterdam study was limited,, yet the results seen in this group were alarming enough to promptt a larger cohort study, supported by a governmental institution, to re-evaluatee the dioxin and PCB effects of perinatal exposure in children. Thee Rotterdam and Groningen group elicited similar findings to what the Amsterdam/Zaandamm group had found, supporting the validity of the concernss (28-31).
Studiess in various other countries have also shown childhood effects of perinatall exposure (32). Yet, to date very little is known about the effects
off this exposure on the long-term health of children. This was reason enoughh to initiate a further follow-up study in the Amsterdam/Zaandam group,, at pre-adolescent age, to once again assess the influence of perinatall dioxin exposure on the growth and development of children.
Whatt health effects could we expect?
Inn animal studies, dioxin exposure has been demonstrated to have effects
onn various homeostatic systems. In addition, it has become clear that the foetuss and new-born baby are the most vulnerable subjects. Their developingg systems are exposed to the dioxin concentrations of the motherr during pregnancy, and postnatally by their ingestion of contam-inatedd breastmilk. The susceptibility of the foetus and new-born is the resultt of the many imprinting processes that take place in the perinatal periodd (33).
Infantss born after prenatal PCB poisoning in Taiwan (Yucheng-disease) weree characterised by hyperpigmentation, intra-uterine growth retardation,, natal teeth, pigmented dysplasia of the nails, hirsutism, hypertelorism,, conjunctivitis, clinodactyly, widely open fontanels and spottyy calcifications of the skull. Mortality was high, with twenty-five percentt of these hyperpigmented babies dying within four years of birth. Respiratoryy distress and pneumonia during the first six months of life weree common (34). Follow-up revealed shorter stature and musculoskeletall changes (35).
Inn the discussion of what effects may be expected as a result of the backgroundd levels of PCBs and dioxins, congenital anomalies did not havee the highest priority. Animal studies have shown that the dosis of toxicc chemicals has to be rather high in order to cause malformations. In Europee this resulted in studies on this toxicological aspect being neglected,, possibly wrongly so. In certain regions where exposure is high,, clusters of defects can be found. In a literature search, several publicationss indicate congenital defects, for instance, amongst inhabitants
inn the neighbourhood of landfills, or after the spraying of insecticides (36;37). .
Afterr the Seveso incident - the explosion of a chemical plant in Italy, resultingg in extremely high TCDD exposures - no increased incidence of congenitall malformations was detected. However, many women underwentt an abortion after the disaster, for fear of the consequences for theirr unborn baby (38).
Whatt is striking in all the publications relating PCBs, dioxins, insecticidess and herbicides with the incidence of congenital malformations,, is the reported increased incidence of midline defects. Thiss same increased incidence in midline defects is seen with maternal usagee of anticonvulsant medication during pregnancy (39;40). Cleft lip and/orr palate, spina bifida, gastroschisis, heart defects, hypospadias and inguinall hernias are amongst the malformations mentioned: all are exampless of midline defects (41;42). Cleft palate and hydronephrosis weree seen in mice following TCDD exposure (43-45).
Presently,, the incidence of hypospadias, both in Europe and the United States,, is rising. Endocrine disrupting chemicals with estrogenic or anti-androgenicc effects are blamed for this rise (46;47).
Anotherr so far unexplained finding is the fact that parents with high levelss of TCDD after the Seveso-incident gave birth almost exclusively too female offspring (48). This finding, however, was not confirmed in thee offspring of subjects with Yucheng-disease (49). Pure TCDD possiblyy has a different effect than the mixture of PCBs and PCDFs that constitutedd the toxins in Yucheng-disease. Does an anti-estrogenic effect off pure TCDD inhibit nestling in the uterus of especially male embryos? Couldd local exposure lead to an increased incidence of congenital anomaliess in the general population? To answer this question we performedd a retrospective observational study within an area of Amsterdam,, Zeeburg. During the 1960s large scale open chemical combustionss had taken place at the Diemerzee Dyke (50). This waste sitee was situated in close proximity to the residential area, Zeeburg. Whilee researching the incidence of congenital abnormalities following maternall anticonvulsant therapies, Koppe serendipitously found a high
incidencee of specific birth anomalies at a maternity clinic in Zeeburg (39).. Could there be a link between the open chemical combustions and thee apparent high incidence of certain congenital abnormalities in Zeeburg?? This retrospective observational study is the first study, presentedd in chapter one, of this thesis.
Somewhatt alarmed by the outcome of the Zeeburg study, we decided to re-evaluatee the health status of our Amsterdam/Zaandam dioxin cohort at pre-puberty.. The children (and their parents, naturally) were contacted andd asked if they would like to participate in the follow-up study. By the timee approval for the study was obtained from the various Medical Ethics Committees,, and the study could commence, the children were aged betweenn 7 and 12 years. Informed consent from each child and his/her parentss was obtained for the evaluation of the following health items:
Respiratoryy problems
Respiratoryy system effects of dioxin and PCB intoxication have regularly beenn documented (22). Coughing, as an immediate consequence, has been described.. Of the intra-uterine exposed children of the Seveso population, 255 % died in the first four years of life due to respiratory problems. Respiratoryy distress and pneumonia during the first six months of life weree common amongst the children born after the Yucheng incident (34). Thee incidence of broncho-obstructive disease has dramatically increased overr the last twenty years. While a number of hypotheses have been postulated,, no conclusive reason for this rise has been found. Could this risee in incidence be the result of perinatal exposure to dioxins? We hypothesisedd that this is the case, and that there would be a relation betweenn perinatal dioxin exposure and lungfunction during childhood (andd beyond). In order to test our hypothesis we set out to evaluate lung functionn by means of questionnaire and spirometry. The study is reported onn in chapter 2.
Haematologicall and immunological problems
Manyy studies of the thymus and T-cells have been performed, because of thee striking involution of the thymus in exposed animals (45;51;52).
Inn human babies only the Dutch studies have published data of the haematopoieticc system. The Amsterdam/Zaandam group detected a loweringg of blood platelet counts in relation to postnatal dioxin exposure viaa breastmilk at 11 weeks of age (16). Lowering of blood platelet counts wass also seen in Japanese workers with increased dioxin levels (53) and in twoo Austrian women accidentally intoxicated with high concentrations of dioxinss (54).
Onee of the most sensitive systems affected is the immune system (55). Att body burdens of only 5 ng TEQ/kg (toxic equivalency factor per kilogram)) bodyweight effects on the immune-system were elicited in non-humann primates (56). An extensive reduction in lymphocyte stem cellss was seen in the bone marrow of the offspring of maternal rats, treatedd with one dose of dioxins of ten micrograms per kilogram body weightt during pregnancy (57). Another study in non-human primates showedd a lowering of CD4+ lymphocytes and an increase in CD8+ lymphocytess after a single dose of only ten nanograms TCDD per kilogramm body weight (58).
Bothh Dutch dioxin groups (Amsterdam/Zaandam and Rotterdam/ Groningen)) detected a significant lowering of the polynuclear leukocyte andd monocyte counts shortly after birth, in the first weeks to months. Pluimm detected lower concentrations of granulocytes and monocytes on thee seventh day of life, in the prenatally higher dioxin-exposed babies. Att eleven weeks of age the concentration of blood platelets were lower in relationn to the amount of dioxin the babies ingested with their breastmilk (16).. In Rotterdam a similar decrease, in relation to pre- and postnatal exposuree to dioxins, was detected in the concentration of granulocytes at 33 months of age, together with a lower monocyte count (59). The loweringg of white blood cell and blood platelet counts is probably due to ann inhibition of the bone marrow by dioxins. That in the neonatal period bothh thrombocytes and white blood cells were affected, possibly points in
thee direction of damage to stem ceils (60-63). At the follow-up after approximatelyy two years, both studies discovered that the exhibited loweringg of these white blood cells was no longer evident (27;59). Thus, directt effects, though not persistent, on the immune system, also in humans,, are already seen at the current background exposure levels. Will thiss lead to an increase in infectious diseases?
Inn follow-up studies no significant differences in the number or sort of infectiouss diseases were detected, up to the age of about two years (27;59).. Yet, in the Rotterdam study, at the age of 42 months more middle-earr infections were found in relation to the current PCB-levels of thee children (64). This relation to the current PCB-levels, however, mightt not be related to a generally disturbed immune function, but rather couldd be a direct effect of PCBs on the epithelium of the Eustachian tube. Itt is known that PCB-metabolites preferentially accumulate in the Clara cellss of the lung, possibly resulting in hypersecretion (65-67). Due to the factt that the epithelium of the lung is of the same origin as that of the Eustachiann tube (which, for instance, also produces surfactant), it is logicall to offer hypersecretion in the Eustachian tube as an explanation forr the middle-ear infections (67). This hypersecretion could also be an explanationn for the increase in respiratory diseases seen after PCB poisoningg (68) as mentioned above.
Thesee above mentioned studies were conducted amongst normal, healthy, pregnantt mothers and their babies. Pathology in the mothers and babies, orr complications during pregnancy, delivery, or in the neonatal period, ledd to the exclusion of the subjects from the particular study. This approachh may be disadvantageous in that toxic effects resulting in diseasee during pregnancy, delivery or in the neonatal period are not detected.. In other words infections, haemorrhaging and new disease entitiess may not be elicited, due to the exclusion of the non-optimal subjectss from the studies.
AA new disease entity in the new-born is the group B streptococci epidemic.. This epidemic has its unexplained origin in the sixties (69). It cann be hypothesised that this rise in group B streptococci infections is
relatedd to the background dioxin and PCB exposure. A modulation in serumm complement levels following dioxin exposure in mice has been documented.. The dioxins enhanced the susceptibility of the mice to
StreptococcusStreptococcus pneumoniae, a bacterial pathogen. The host defence is
complementt mediated in mice. Humans also rely on their complement systemm to combat this bacterial pathogen (70). The complement system off the human new-born is deficient, rendering the infant susceptible to infections.. This is not so for the mother and it is unknown why the streptococcii infestate her cervix during pregnancy, leading to premature deliveryy and to severely ill babies. However, studies in humans consideringg a relation between this infection and background levels of dioxinss and PCBs have not yet been performed.
AA study in adults revealed a change in serum immunoglobulin M and G3 concentrationss in dioxin exposed workers (71).
Theree are also subtle signs that the leukocytes may be influenced through aa faulty imprinting, during the critical perinatal period. Weisglas-Kuperuss et al. published their findings on background prenatal exposure too dioxins and PCBs in their Rotterdam group: this exposure may influencee the development of particular immune cell populations (29;59). Eighteenn month old children exhibited an increase in T-cells (CD8+, amongstt others) which persisted until the age of 42 months. However, at 422 months the increase was related to the sum of maternal PCBs and not too the I-TEQ dioxin (64). Additionally, amongst the forty-two month olds,, a higher incidence of chickenpox was elicited relative to the sum of thee maternal PCBs, and levels of antibodies to measles were lower in relationn to the sum of the PCB levels in cord blood (64). It remains unclearr why the relations were found with different exposures: some dioxins,, others PCBs. Possibly the TEQ-concept is not a suitable method orr not sensitive (enough) for the endpoints studied. The fact that the relationn was later found with PCBs, and not with dioxins, does not eliminatee the exposure to dioxins as a cause.
Thereforee we hypothesised that the effects seen by Pluim in the perinatal periodd of our cohort would be persistent effects. We assessed the haematologicall parameters of the cohort, with special emphasis on
immunologicall parameters and later on thrombopoiesis. Chapter 3 elaboratess on the parameters tested, the methods used and the outcomes off the study.
Thyroidal,, hepatic and enzymal problems
Anotherr aspect of imprinting is the hormonal and enzymal imprinting. Faultyy imprinting caused by the introduction of certain hormones, or chemicalss mimicking these hormones, during the sensitive period, can causee disturbances in the homeostasis. For instance the setpoint (the optimall concentration point) of the thyroid hormonal system could be set (slightly)) higher or lower. The significance of such an altered setpoint mayy prove detrimental to the long-term, and possibly even the short-term,, health status of the individual.
HO--i HO--i 4 4
Ó Ó
I IY Y
Jl l
PCDD D < < 6 6 9 9 > > S».. 7J J
y^y^ s NH2 2 CH2CHH -I -I -tetraiodothyronine e COO OFiguree 2: Structure of dioxin (PCDD) and thyroxine (T4) molecules
Dioxinss and dibenzofurans have a similar chemical structure to that of thyroidd hormones. Structurally, one of the major differences is that the
chlorinee atom of the former is situated in the position of the iodine atom off thyroxine (figure 2). This structural similarity gave rise to the hypothesiss that dioxins and furans are able to mimic the working of thyroxine,, and while this effect has long been suspected, it has never beenn proven (72;73). Jakobsson and colleagues demonstrated that a (T3-like)) metabolite of polybromodiphenyl ethers, which they synthesised, boundd remarkably to the human thyroid receptor protein. This is a clear demonstrationn of a man-made persistent organic pollutant binding to a humann receptor (74).
Duringg human gestation, the human foetal hypothalamic-pituitary-thyroid systemm is relatively quiescent and foetal thyroid hormone production is limitedd until a gestational age of eighteen to twenty weeks. During the secondd half of gestation, the functioning of the foetal thyroid gland increases,, this under the influence of an increasing TSH concentration. Theree is also a progressive increase in the concentration ratio of free T4 too TSH, suggesting maturation of the hypothalamic-pituitary negative feedbackk control system for TSH secretion (75). 'Thyroid system maturationn during the latter half of gestation can be characterised as a balancee among increasing hypothalamic TRH-secretion, increasing pituitaryy sensitivity to thyroid hormone inhibition of TSH release, and increasingg thyroid follicular cell sensitivity to TSH', according to Fisher (76).. Chemicals mimicking thyroid hormones can disturb this process. Inn this light, studies on the effects of background exposure to dioxins in ourr country also focussed on the thyroid hormone metabolism. In both thee Zaandam study of Pluim et al. and the Rotterdam study of Koopman-Esseboomm et al., abnormalities in thyroid hormone metabolism were detectedd in relation to dioxin levels. Pluim detected a significantly higherr T4 concentration during the first 11 weeks of life in the higher exposedd babies, whom also exhibited a significantly higher TSH concentrationn at 11 weeks of age (77-80).
Koopman-Esseboomm detected lower free thyroxine (FT4) and T4 concentrationss shortly after birth, and also higher TSH concentrations at 22 weeks and 3 months of age, in her higher exposure group (81).
Thyroidd hormones also play a part in brown adipose tissue thermogenesis.. This form of heat production involves the rapid burning off the triglycerides that are packaged around the great vessels, in order to keepp the baby warm. From animal studies it is known that this manner of thermogenesis,, by the burning of brown adipose tissue, is jeopardised by dioxins,, making survival after birth more difficult (82-84).
Itt seems, therefore, that current background concentrations of dioxins disturbb the thyroid hormone metabolism. Can we thus expect more thyroidall diseases and/or thyroidal malignancies in the (near) future? We hypothesisedd that the effects on the thyroid hormone homeostasis seen in thee perinatal period would be persistent. In order to test our hypothesis, wee assessed thyroid stimulating hormone (TSH) and free thyroxine (FT4) concentrationss in relation to prenatal and postnatal dioxin exposure. The resultss of the study are presented in chapter 4, together with the results of thee liver enzyme tests.
Thee liver is the first and primary organ where dioxins and PCBs arrive. Theyy diffuse to the liver cells after transversing the umbilical vein or afterr absorption by the gut. In the Zaandam study, liver size was measuredd by means of ultrasound. As related to the perinatal dioxin exposure,, an increase in hepatic size was expected, contrary to the trend towardss a smaller size found at two weeks of age. This might be due to a relativee growth retardation in utero. Between two and eleven weeks of age,, the liver increased in size, more so in the higher exposed babies than thee lower, resulting in similar sized livers for the two groups by eleven weekss of age (16).
Maruyamaa presented a model to explain dioxin and PCB behaviour after birth,, in relation to breastfeeding, and she came to the conclusion that theree is a sharp rise in dioxin content shortly after birth in the liver (85). Att 2V6 years of age no persistent abnormalities were found in the Zaandam cohortt (27). In the Seveso children, exposed to dioxins following a chemicall plant explosion, a similar rise in ALAT was found amongst the highestt exposed boys, which was decreased to normal levels after about fivee years (86). Although the ALAT had normalised in the Zaandam cohortt by the age of 2Vz years, we hypothesised that signs of hepatic
damagee would still be seen (87), as a result of the long half-life of dioxins. Wee thus determined the ASAT and ALAT levels, which is reported in chapterr 4, together with the thyroid function testing.
Analogouss to the effects of phenobarbital, it was hypothesised that PCBs andd dioxins can disturb vitamin K metabolism in utero and after birth, resultingg in a deficiency of vitamin K (26;88).
Likee dioxins, and dioxin-like PCBs, ortho-substituted non-planar PCBs aree commonly encountered, and can be found in rather high concentrationss in breastmilk - 63% of the total amount of PCBs in human breastmilkk comprises orthosubstituted nonplanar PCB22, 52, 138, -1533 and -180 (89). These PCBs have phenobarbital-like effects. They aree capable of inducing enzymes in the liver, resulting in an increased vitamin-KK metabolism, which may ultimately lead to deficiency of the vitamin,, which is needed for normal coagulation. These effects have beenn shown in animal studies. In addition, vitamin K deficient haemorrhagicc states were more prevalent in male animals - this is counteractedd by oestrogen. Pre-treatment of female rats with androgen decreasedd prothrombin levels, resulting in an increased incidence of the vitaminn K deficiency haemorrhagic state.
Vitaminn K is necessary for the carboxylation of prothrombin in the liver, andd is probably also used for similar reactions elsewhere in the body. Inn human babies a new disease entity has been described by Lane: the latee haemorrhagic disease of the new-born (HDN). HDN is caused by vitaminn K deficiency and is characterised by severe intracranial bleeding betweenn one and three months after birth (90).
Threee forms of HDN have been described: the early, the classic and the latee form. The early form can result in a life-threatening haemorrhage duringg pregnancy, during delivery or within 24 hours after birth. This is knownn to occur in conjunction with maternal usage of anticonvulsant medication,, certain antibiotics, and tuberculostatics - all potent inducers off the hepatic cytochrome P450 enzyme complex.
Thee classic form involves exclusively breastfed infants, occurs during the firstfirst seven days of life and results mostly in a benign gastro-intestinal bleeding,, the so-called 'melaena neonatorum'. Since the discovery of
thee role of vitamin K, the vitamin has been shown to be an effective treatmentt for babies exhibiting melaena during the first neonatal week. Thee late form of HDN, in contrast to the early and classic form, is a new diseasee entity. It is characterised by severe intracranial bleeding between onee and three months after birth. It is exclusively found amongst breastfedd children not receiving vitamin K supplementation.
Epidemicss of this disease have been described. In Japan, in particular, 4255 cases of late vitamin K deficiency bleeding occurred between 1981 andd 1985, including 284 males and 141 females. Once again a preponderancee of males (91). In Germany, Holland and England sporadicc cases of late HDN have been described since 1980 (26). Furthermore,, in a Dutch study about ten percent of breastfed babies had biochemicall abnormalities in their blood indicating a vitamin K deficient statee (92). These babies had not received vitamin K supplementation afterr birth. Vitamin K levels are normally low during pregnancy and afterr birth. As a result of an efficient recycling only nanograms of vitaminn K are needed per day (93). A breastfed infant receives about 5 microgramss per day of the vitamin - therefore 1000 times more than it needs.. However, when the cytochrome P450 enzyme complex is activated,, vitamin K is metabolised and excreted with the gall, and this mayy rapidly lead to a state of deficiency in an exclusively breastfed baby. Inn this respect, activation of this cytochrome P450 enzyme complex in thee perinatal period is thus a toxic phenomenon.
Extraa vitamin K supplementation is therefore used in Europe today in orderr to prevent vitamin K deficiency during breastfeeding. Intramuscularr injections of milligrams of vitamin K are sufficiently effectivee in the prevention of bleeding. However a relation between leukaemiaa and intramuscular vitamin K injections has been elicited, in epidemiologicall studies, so that oral supplementation is now recommendedd (94). Why nature chooses to keep vitamin K levels low in babiess is unknown. An explanation might lie in the endocrine disrupting propertiess of the menadione molecule, a synthetic form of vitamin K, whichh has inherently estrogenic properties (95).
Ass mentioned above, dioxins are known to be potent inducers of cytochromee P450 enzymes (CYP) (96-101), especially 1A2, and these enzymess are involved in a myriad of bodily functions. Perinatal dioxin exposuree may cause a disturbance of the CYP setpoint and this may resultt in an increased or decreased metabolism. Increased metabolism of necessaryy substances, such as hormones, may lead to a relative insufficiencyy (102). Decreased metabolism may lead to a prolonged exposuree to toxins or their metabolites. The setpoint for the induction of enzymess can also be altered, as can be seen in the alteration of the activityy of the cytochrome P450 system. The activity of this enzyme system,, responsible for detoxification, may be altered in a positive or negativee way, in response to prolonged periods of exposure to toxins. In ratt studies, Benzo(a)-pyrene has been shown to change this activity setpointt resulting in a delay of enzyme induction (98). This may be ominouss for the development of malignancies (103).
Ourr hypothesis of a dioxin influence on CYP 1A2 activity was tested usingg a caffeine-loading test (104-106). The test was first validated using volunteers.. Both the validation test and cohort test are reported on in chapterr 4.
Psychologyy and neurology
Behaviourall imprinting is a further example of environmental influences playingg a pertinent role in the perinatal period. Appropriate reactions, suchh as bonding, are dependant on appropriately working hormonal systemss in a baby.
Inn a North Carolina study, hypotonia and hyporeflexia in relation to prenatall exposure to PCBs were already detected in the neonatal period, shortlyy after birth (107). During infancy the higher exposed children exhibitedd developmental delays in gross motor function (19). In the childrenn of mothers ingesting PCB-polluted fish from Lake Michigan a poorerr visual recognition memory (Fagan Test) was associated with increasingg prenatal PCB exposure. The levels of PCBs in the Michigan
studyy were only slightly above U.S. background levels and comparable to Dutchh levels.
Thee Zaandam group was studied at the age of two years and seven months.. Signs of enhanced neuromotor maturation were found and it wass hypothesised that this may be due to the thyroxine-agonistic action off dioxins (27). Enhanced neuromotor maturation is not necessarily a favourablee effect.
Prenatall exposure to PCBs and PCDFs was associated with negative behaviourall effects amongst the offspring of mothers who had ingested thee contaminated rice oil in Yusho, Japan. The children were apathie and uninterestedd (108). Monkeys exposed prenatally to PCBs exhibited hyperactivee behaviour during infancy. This was followed by inactivity at fourr years of age (109; 110). The children of mothers eating fish from Lakee Michigan displayed reduced activity, with current body burdens of PCBs,, at 4 years of age (111). In another neonatal behavioural assessmentt study, neonates of mothers who had ingested Lake Ontario fishfish were tested. Results revealed significant linear relationships between thee most heavily chlorinated PCBs measured in cord blood and performancee impairments for the scores on the Habituation and Autonomicc clusters at 25-48 hours after birth (112).
Inn the Dutch cities of Rotterdam and Groningen a study was performed to investigatee the effects of perinatal exposure to background levels of PCBss and Dioxins on growth and development (31). The total study groupp consisted of 400 healthy mother-infant pairs, of which half the infantss were breast-fed and half bottle-fed. The levels of dioxins (PCDDs andd PCDFs) were comparable with the levels found in Amsterdam. Prenatall PCB exposure was estimated by the PCB-sum (PCB congeners
118,, 138, 153, and 180) in maternal blood and cord blood and the total dioxinn toxic equivalent level (TEQ) in breastmilk (17 dioxin and a total off 8 dioxin like PCB congeners - 3 planar, 3 mono-ortho and 2 di-ortho PCBs).. Postnatal dioxin exposure was calculated as a product of the total dioxinn TEQ level in breastmilk multiplied by the weeks of breastfeeding. Off the measured PCB congeners 118, 138, 153 and 180, the former is dioxin-like,, while the latter three are phenobarbital-like. Effects detected
inn relation to PCBs might then be caused by the non dioxin-like PCBs. Currentt body exposure to the PCB congeners 118, 138, 153 and 180 was measuredd in plasma at 42 months of age.
Follow-upp of brain development was done at the ages of 18 and 42 months.. The study detected hyperactivity and slower mean reaction timess in relation to the current PCB levels in the children at 42 months of agee (113). Irritability and hyperactivity are well known side effects of thee use of phenobarbital in childhood and current PCB levels might act as such.. At the age of 42 months attention during free play behaviour was reducedd in relation to cord and maternal PCB exposure, a persisting effectt on behaviour from damage caused prenatally. These findings are similarr to those seen by Jacobson (114). Prenatal PCB exposure was also adverselyy associated with neurological outcome at 18 months of age (115),, but this was no longer seen at 42 months of age (116). A negative relationn was found between cognitive functioning (from 2 to 6-8 points lowerr IQ) at 42 months, and the sum of the PCBs measured in maternal bloodd collected in the last month of pregnancy (113). Overall cognitive functioningg was negatively influenced, as was the verbal comprehension score.. This finding is in accordance with the study of the Jacobsons', whoo noted a negative effect of prenatal exposure to PCBs on cognitive functioning,, at the age of four years (114). Furthermore, at the age of elevenn years, in the Jacobson study, IQ-test scores were lower in the higherr exposed children. Difficulties in verbal comprehension were elicitedd and the ability to concentrate was reduced in the higher exposed children.. The latter were more than twice as likely to be two years behind inn reading skills and word comprehension (117). Similar attention and verball IQ problems have been detected in children prenatally exposed to anticonvulsants.. Negative effect on psycho-sexual development and reproductivee performance were also detected in the latter group (118). Long-termm follow-up studies, into and through adulthood, are necessary, inn order to detect abnormalities in sensitive endpoints like psycho-sexual developmentt and reproductive performance.
Thee Rotterdam cohort was followed-up at the age of 7 years and fine motorr performances were tested. There were more left-handed children in relationn to higher prenatal PCB exposure as measured in maternal blood in thee last month of pregnancy. Left-handed girls had more lateralisation
betweenn the hands, the dominant hand being better than the non-dominant hand.. Left-handed boys had better co-ordination between the two hands in relationn to higher prenatal PCB-exposure (119).
AA recent study in Germany demonstrated the negative effects on neurodevelopmentt up to 42 months of age arising from prenatal, and also postnatal,, exposure to PCBs. A deficit of 8 and 9 points was seen for mentall and motor development respectively. Congeners 138, 153 and 180 weree used, all non-dioxin-like. At 42 months an intelligence test was performedd to assess higher brain functions. Using this test postnatal exposure,, as measured in blood at 42 months, was related to a significantlyy lower IQ (120).
Severee effects on IQ due to high in utero exposure of a combination of PCBss and PCDFs were found in the Yucheng children (121). Based on thee findings seen in the abovementioned studies, we hypothesised that our cohortt would exhibit a reduced I.Q. and increased behavioural problems inn relation to perinatal dioxin exposure. Furthermore, we hypothesised thatt negative effects in brain functioning could be visualised using state of thee art technology such as MagnetoEncephaloGraphy (MEG) (122), and routinelyy practised ElectroEncephaloGraphy (EEG).
Duringg the critical prenatal and postnatal period, environmental influencess like dioxin and PCB exposure, can disturb hormonal and enzymall activity setpoints or have direct toxic effects on developing organs.. This may result in functional developmental disabilities in later life.. During the first trimester of pregnancy neurons in the brain are formed.. During the second and third trimesters, especially around thirty weeks'' gestational age, when the growth and development of the brain takess place, brain development is characterised by the forming of dendrites,, connecting the neurons, and by the start of glial myelinisation (123). .
Structuress in the brain necessary to process visual and auditory signals, forr instance for language development, are then formed. This process continuess during the first year of life, albeit somewhat slower than prenatally,, and progresses thereafter at a slower pace until adolescence.
Duringg pregnancy, the developmental process depends largely on hormones.. Thyroid hormone is essential for brain development and lowerr levels of maternal thyroid hormone in the first trimester of pregnancyy is associated with lower intelligence in children (124). Testosteronee is also important, as it is necessary around the thirtieth weekk of pregnancy for the typical male brain development, characterised byy good Visio-spatial abilities. Lowering of the testosterone levels, for examplee as a result of an enhanced metabolism induced by medications, causess impairment of the Visio-spatial abilities later in life. This is seen inn the adolescent offspring of mothers using anticonvulsant medication duringg pregnancy (118). Glucocorticosteroids, as used in neonatology forr children with bronchopulmonary dysplasia, have strong negative influencess on brain growth and on growth in general
Dioxinss and PCBs are known to be endocrine disruptors and their influencee on thyroid hormone metabolism has been demonstrated in variouss studies (77-80). Later health effects of these influences are, as yet,, largely unknown.
Iss the premature maturation seen in our cohort at the age of 2Vi years a warningg sign? We hypothesised that the maturation would persist throughoutt childhood, and could be elicited using neuromotor physical examination,, such as the Touwen test (125). Chapter 5 elaborates on the techniquess used and the results seen.
Dentall problems
Finnishh researchers found an increased number of caries in children, in relationn to postnatal dioxin exposure (via breastfeeding) (126). Dental anomaliess were seen in children following the Yusho and Yucheng disasterss (18;34). The children in our cohort breastfed for on average a muchh shorter period than their Finnish counterparts. However, the Dutchh background level of dioxins is higher than that in Finland. We thereforee hypothesised that the children in our cohort would then also havee more caries in their teeth in relation to perinatal dioxin exposure. Thee results of the study are discussed in chapter 6.
Summarising g
Summarisingg then, in the industrialised world dioxins are everywhere to bee found, and we are all exposed to them. Dioxins readily pass the
placentaplacenta and are found in relatively high concentrations in breastmilk. Childrenn are thus exposed to high concentrations of these most highly
toxicc chemicals, prenatally and postnatally. Additionally, the dioxin and PCBB intake of a toddler is more than twice that an adult, and exceeds the acceptablee daily intake with a four-fold factor (127). Effects on various organn systems have been documented for children accidentally exposed too high concentrations of dioxins. Furthermore, effects have been documentedd for babies and toddlers exposed to background concentrations.. However, data on the effects of perinatal exposure to backgroundd levels in later childhood are scarce. We present data on congenitall malformations and follow-up data on respiratory problems, haematologicall and immunological disturbances, thyroid and liver functioning,, cytochrome P450 activity, neuromotor and psychological development,, and dental status.
Aimm of the study
Inn 1998 it was decided to perform a follow-up study of Pluim's original cohort,, born in De Heel Zaans Medical Centre (formerly named "Hospitall De Heel") in 1990/1991. The cohort was expanded with a few childrenn born in 1987 with documented perinatal dioxin exposure and whoo met the selection criteria of Pluim's cohort.
Thee group of children is relatively small (maximally 60), thereby limitingg the power of the outcomes. Therefore, the main purpose of this studyy is to generate hypotheses and elicit indications of developmental deficitss that can be confirmed in other (larger) cohorts.
Wee decided to perform a broad study of several different aspects of development. .
Wee studied:
-- lung function by means of spirometry and medical history taking g
-- haematological and immunological parameters -- hepatic and thyroid function
-- activity of cytochrome P-450 1A2 by determining caffeine metabolitess in venous blood, following a caffeine loading test -- neurological evaluation by means of physical examination -- psychological evaluation by means of I.Q. and behaviour tests -- neurological function imaging by means of MEG and EEG -- dental status by means of dental evaluation
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