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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: 15 Aug 2019

PERINATA LL DIOXI N EXPOSUREE AND CYTO-CHROMEE P-450 ACTIVITY , THYROI DD AND LIVE R FUNCTIONSS AT FOLLOW -UPP AFTER 7 -12 YEARS EVIDENCEE FOR TRANSIENT EFFECTS ONN THYROI D AND LIVE R FUNCTIONS

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tenn Tusscher GW, Guchelaar H-J, Koch J, Ilsen A, Vulsma T, Westra M, vann der Slikke JW, de Wolff FA, Olie K, Koppe JG. Perinatal dioxin exposuree and cytochrome P-450 activity, thyroid and liver functions at follow-upp after 7 - 12 years. Submitted 2002.

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Abstract t

Objectives:Objectives: Perinatal exposure to Dutch "background" dioxin levels is ratherr high. Health effects may span many years. Therefore we determinedd plasma TSH, FT4, ALAT and ASAT levels amongst our longitudinall cohort, as was done perinatally and at 2V£ years. The childrenn underwent a caffeine loading test to determine CYP1A2 activity. StudyStudy design: The longitudinal cohort of 37 healthy children (7 - 12, meann 8.2 years), with documented perinatal dioxin exposure, ingested 3 mgg caffeine/kg BW 6 hours prior to blood withdrawal. Paraxanthine/ caffeinee molar ratio, TSH, FT4, ALAT and ASAT were determined in venouss blood. Results:Results: Linear regression of TSH and FT4 revealed no relation with prenatall and postnatal dioxin exposure. No relation was found between ASATT and ALAT and prenatal and postnatal exposure. No correlation wass found between the paraxanthine/caffeine molar ratio and prenatal andd postnatal dioxin exposure.. Conclusion:Conclusion: This follow-up has shown a normalisation of previously abnormall thyroid hormone homeostasis and ALAT levels, indicating a transientt effect. CYP1A2 activity, measured by means of a caffeine-loadingg test, revealed no correlation with the pre- and postnatal exposures.. This study provides new data on longterm follow-up after perinatall dioxin exposure to background levels of dioxins.

Introductio n n

Polychlorinatedd dibenzo-p-dioxins and dibenzofurans and some PCBs, suchh as PCB-77, -126 and -169 (henceforth jointly referred to as dioxins) belongg to the group of most toxic substances known, and have been associatedd with malignancy, congenital malformations, immuno-suppression,, enzyme induction and endocrine disruption (1-6). Dioxins aree formed as waste products of combustion processes and municipal incineratorss are amongst the primary sources of these compounds in The Netherlands.. Heating of polychlorinated biphenyls (PCBs) is a notable sourcee of polychlorinated dibenzofurans. Dioxins, being poorly

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degradablee in nature, persist in the environment, accumulating in the humann food chain via fish-oils and animal fats (7). These chlorinated polyaromaticc compounds are highly lipophilic and are in the human primarilyy stored in adipose tissues and liver. Their lipophilicity allows themm to readily pass the placenta, whereupon they are stored in fetal adiposee tissues (8;9). In 1986 relatively high background dioxin concentrationss in the breast milk of Dutch mothers was first reported, followedd by similar findings in other industrialised countries (10-12). As aa result fetuses and breastfed children are exposed to relatively high "background"" dioxin levels of averaging approximately 30 ng/kg fat, as measuredd in breastmilk (10-14). Yet, very littl e is known about the effectss of this exposure on the (longterm) health of children.

Dioxinss influence thyroid and liver determinants in humans (6;13;14), andd induce cytochrome P-450 1A2 (CYP1A2) activity in animals (15). Pluim,, in the same group of children as the current cohort, found a tendencyy toward higher thyroxine (T4) concentrations at birth in relation too increasing prenatal dioxin exposure, and increased mean T4 concentrationss and increased T4/thyroxine-binding globulin ratio, at one andd eleven weeks of age, related to dioxin exposure. TSH levels, while beingg similar in the higher and lower exposure groups at birth and one weekk post partum, were significantly higher in the higher exposure group att eleven weeks of age (13; 16). Furthermore, plasma alanine (ALAT) andd aspartate (ASAT) aminotransferase activity was found to be significantlyy increased at eleven weeks of age, in relation to increasing cumulativee (postnatal) dioxin exposure (14). In contrast, Ilsen, investigatingg the same children at two and a half years of age, found no correlationss between the perinatal dioxin exposures and thyroid hormones.. Neither did she find a correlation between perinatal dioxin exposuress and ASAT or ALAT concentration (17). Increasedd CYP1A2 activity in humans has been associated with an increasedd risk of developing colorectal neoplasms (18), and dioxin exposuree has been linked to sigmoid cancer in an 11-year-old boy (19). Perinatall exposure to dioxins and related compounds may have consequencess spanning many years, due to their long residence time in thee body. Based on the reported half lives, it may take up to a few

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decadess to attain steady state levels. In practice this means that accumulationn will continue through childhood and puberty. Our working hypothesiss is that perinatal exposure to background levels of dioxins in Thee Netherlands could:

1)) have a life-long effect on thyroid hormone homeostasis (13; 16); 2)) cause persistent liver damage (seen in abnormal plasma

aminotransferasee levels) and thereby negatively affecting variouss metabolic reactions (14);

3)) alter CYP1A2 activity resulting in a life-long reduced activity byy a lowering of the setpoint, in accordance with Csaba's study inn laboratory animals (20;21).

Inn our ongoing study of the development of children with documented perinatall dioxin exposure, we therefore assessed the thyroid and liver determinantss and the CYP1A2 activity in the study participants, using routinee blood investigations and a caffeine-loading test, respectively. Thee children had previously been studied during the perinatal period and att the age of two and a half years.

Methodss and materials

Thee institutional Medical Ethics Committees of the De Heel Zaans Medicall Center (Zaandam, The Netherlands) and of the Academic Medicall Center (Amsterdam, The Netherlands) approved the study. Writtenn informed consent was obtained from parents/guardians and children. .

Subjects Subjects Eightt years after Pluim's Zaandam study (13; 14), the cohort was again contacted,contacted, as was the group from his first study (up to twelve years ago) (22).. The perinatal dioxin exposure is documented for all these study participants.. Out of the original cohort of 61 subjects, those born prematurely,, born out of suboptimal pregnancies necessitating medical intervention,, or who were twins, were not included in this follow-up studyy (9 children). Two children had moved abroad, 5 children chose not

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too participate in the follow-up study and 5 children were untraceable. The prenatall and postnatal dioxin exposures of the excluded subjects were variedd and in no way did their exclusion introduce a population bias. The elderr sister (2 years older) of a subject requested to take part in the study andd was included in the cohort for this reason. Her breastfeeding period wass known and she was assigned the same prenatal dioxin exposure (25.44 ng/kg fat) as her younger sister. [In reality, being the firstborn and thee first child to be breastfed, her prenatal exposure would have been higherr than that of her younger sister, caused by the fact that dioxins are soo readily excreted in breastmilk].

Thee 37 study subjects were 7 to 12 years old (mean 8.0 years, SD 1.5 years).. The children underwent the test and blood withdrawal on one of threee Saturday afternoons in the spring of 1998, at De Heel Zaans Medicall Centre, Zaandam. The dioxin toxic equivalency and cumulative toxicc equivalency values as determined by Pluim in the breastmilk of theirr mothers, were used as such within this follow-up study. Briefly, the concentrationn of dioxins, using the I-TEQ method, in the mother's breastmilk,, shortly after having given birth, was taken as the prenatal dioxinn exposure level of the child (teqdiox), a reliable approximation of thee prenatal exposure (23) . The postnatal cumulative dioxin exposure (teqcum)) was calculated as: teqdiox multiplied by the amount of breastmilkk ingested during the breastfeeding period of the child. A 2.5% fatt concentration in the milk, 700 g daily milk intake during the exclusivelyy breastfeeding period and 350 g daily intake during the transitionn period to formula feed was assumed (22). Formula feed has undetectablee levels of dioxins, with the animal fats having been removed andd replaced with plant fats (24). The prenatal exposure ranged from 8.74 too 88.80 (mean 34.6) ng TEQ dioxin/kg milk fat. The postnatal exposure rangedd from 4.34 to 384.51 (mean 75.4) ng TEQ dioxin. These exposures aree indicative of the background exposures in The Netherlands, and similarr studies in other parts of the country have found similar backgroundd concentrations (11;12;25;26). Subsequent (childhood) dioxin exposuress are about 25 times less than during the fetal and breastfeeding periodd (27), and it can safely be assumed that all the subjects had similar childhoodd exposures, seeing as they all had similar diets.

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ThyroidThyroid f unction Thyroidd stimulating hormone (TSH) and free thyroxine (FT4) were measuredd in plasma immediately after venapuncture, on a routinely used Beekmann Access® HYPERsensitive hTSH and FREE T4 immunoassay systemm respectively. TSH was quantified using a standard paramagnetic particle,, chemiluminescent immunoassay, with a functional sensitivity of 0.01-0.022 mlU/L (product no. 33820). Similarly, FT4 was quantified usingg a standard paramagnetic particle, chemiluminescent immunoassay, usingg a two-step enzyme immunoassay and monoclonal anti-thyroxine antibodiess (product no.33880).

ALATandASAT ALATandASAT Standardd aspartate aminotransferase (ASAT) (product no. 07 3737 2) and alaninee aminotransferase (ALAT) (product no. 07 3734 8) measurements inn plasma, using a Roche Cobas Integra 700 system®, were performed immediatelyy after venapuncture. The standard pyridoxal phosphate activatedd test (product no. 07 3902 2) was used for both ASAT and ALAT . .

CaffeineCaffeine loading test Thee subjects refrained from caffeine intake for 48 hours prior to the ingestionn of caffeine, in a single dosage of 3 mg/kg body weight, dissolvedd in 'AA'® soda drink, which contains no caffeine, but to which thee caffeine was added. Six hours later blood was drawn and later analysedd in the laboratory of the AMC Clinical Pharmacy Department (Amsterdam,, The Netherlands).

Thee venapuncture was performed by an experienced clinical laboratory worker.. The blood was centrifuged within two hours and thereafter cooledd at 4° C until the following Monday, whereupon it was analysed. Inn these blood samples paraxanthine and caffeine concentrations were determined,, and the paraxanthine/caffeine molar ratio was calculated. Methodss of analysis for simultaneous determination of paraxanthine and caffeinee have been described (28;29), however, to our knowledge, only in aa single, recent publication the necessary validation of the assay is describedd (29).

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Wee therefore developed a HPLC assay for simultaneous determination of paraxanthinee and caffeine and validated this assay before the above mentionedd study was performed (30).

Caffeinee and paraxanthine in plasma was quantitated with a 'reversed-phase'-HPLCC using UV-detection. Only analytical grade chemicals were used,, and included paraxanthine (Sigma, Zwijndrecht, batch no. 117H4066),, caffeine (OPG, Utrecht, batch no. 92K10AL-WP04732), sulfadioxine,, theophylline, methanol (Merck, Amsterdam), trichloric aceticc acid, tri-ethylamine (Merck, Amsterdam, batch no. 40613917), and sodiumm dihydrogenphosphate monohydrate (Merck, Amsterdam, batch no.. A858546).

Thee analysis was performed using a HPLC-system consisting of a Waters M455 pump, Applied Biosystems 757 UV-detection system, set to a wave lengthh detection of 273 nm, Shimadzu-CR3A integrator, Supelco LC-18-DB-087311ACC column, Rheodyne 7125 injector. The pH of the mobile phasee was adjusted using a Radiometer Copenhagen PHM83 Autocal and filtratedfiltrated over a Millipore GVHP04700 filter (poresize 0,22 urn). During samplee preparation a Hettich Rotanta/P centrifuge was used.

AA stock solution of 15 mg caffeine and 15 mg paraxanthine in eluent was usedd for the preparation of standard solutions of caffeine and paraxanthine.. All standard solutions were prepared by diluting this stock solution.. The validation and calibration samples were prepared by spikingg human blank serum with the above-mentioned stock solution. Serumm proteins were precipitated before the sample was injected onto the chromatographicc system. Protein precipitation was achieved using 50 uL serumm + 20 uL internal standard (sulfadioxine 5 mg/L) + 50 uL methanol ++ 50 uL 10% trichloric acetic acid, mixed on a Vortex for 1 minute and centrifugedd at 4000 rpm for 10 minutes. 15 uL of supernatant was injectedd onto the HPLC system. Quantificationn of caffeine and paraxanthine was achieved by comparing peakk areas, using sulfadioxine as an internal standard.

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Statistics Statistics Linearr regression, which is a dose-response analysis and two-sided test, wass performed using SPSS 10.0®. P-values were calculated and P-values lesss than 0.05 were considered statistically significant. Smoking (mothers andd children) was considered a confounder as smoking may induce CYP1A22 activity. Gender was also considered as a confounder, as metabolismm and hormone effects may be different between males and females. .

Results s

Subjects Subjects Off the 42 children taking part in the study, 4 declined to allow vena-puncture,, and 1 was not available on the planned Saturdays due to his residingg outside the country. Therefore 37 blood samples were obtained forr analysis. None of the children smoked at the time of the study. Statisticall correction for mothers who smoked at the time of the study, or whoo smoked during pregnancy, had no effect on the results. Smoking wass therefore not considered to be a confounder in this study.

ThyroidThyroid hormones Fourr of the 37 children (11 %) had a TSH value above the upper limit of thee normal values (4.4 mU/L) with the highest value being 4.64 mU/L. Onee value fell below the lower limit of normal (0.7 mU/L), being 0.62 mU/L.. The average TSH value amongst the 37 samples was 2.14 (SD 1.20)) mU/L. All the FT4 results fell within the normal range (13 -27 pmol/LL for 2 - 7 year olds, and 10 - 25 pmol/L for 8 - 20 year olds), with thee exception of one 7 year old child with a FT4 value of 12.6 pmol/L. Thee lowest FT4 value was 12.5 pmol/L and the highest was 23.1 pmol/L. Thee average FT4 value was 17.4 (SD 2.69) pmol/L. It must be stressed thatt the normal values used are based on American children, as European normall values are not available (31). None of the children exhibited clearlyy pathological thyroid hormone levels. Linearr regression revealed no correlation between TSH and prenatal dioxinn exposure (p=0.74), TSH and postnatal dioxin exposure (p=0.17),

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FT44 and prenatal exposure (p=0.65), nor with FT4 and postnatal exposuree (p=0.84). Finally there was no correlation between either TSH (p=0.90)) or FT4 (p=0.29) levels and CYP1A2 activity, although the scatterr diagram of FT4 against CYP1A2 suggests an inverse gradient (figuree 1).

Freee Thyroxine versus 24--

22--

20--

18--

33 16-O O

a a o, , aa 14" H H D D O O

HHH 1 2.

0 0

A A

A A

A A

A A

AA A

A A

— ^ ^^ A A A

44 r——_̂^ A A

AA A

A A A A

AA A A A

A A A

00 .5 1.0 1.5

Ratioo paraxantine/caffeine

CYP1A22 Activity

A A

A A

A A

** A

2.00 2.5 3.0 0

Figuree 1: Free thyroxine (FT4) versus cytochrome P-450 1A2 activity, measured by the ratioo of paraxanthine to caffeine.

ASAT/ALAT ASAT/ALAT Thee AS AT measurements ranged from 16 to 33 U/L with a mean of 20.4 (SDD 3.28) U/L. The normal ASAT range for children from 3 to 15 years iss 0 to 40 U/L, which means that all the children had normal aspartate aminotransferasee levels. Linear regression showed no correlation betweenn ASAT and prenatal dioxin exposure (p=0.22), nor with ASAT andd postnatal exposure (p=0.49).

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Thee ALAT measurements ranged from 8 to 24 U/L with a mean of 12.6 (SDD 3.65) U/L. The normal ALAT range for children from 1 to 15 years iss 0 to 45 U/L, which means that all the children had normal alanine aminotransferasee levels. Linear regression showed no correlation betweenn ALAT and prenatal dioxin exposure (p=0.19), nor with ALAT andd postnatal dioxin exposure (p=0.30).

3 3

U U X> >

1 1 3 3

z z

Histogramm of Paraxanthine/Caffeine ratio 141 1

Std.. Dev = .48

Meann = 1.35

N== 37.00

1.000 1.25 2.255 2.50

Ratioo paraxanthine/caffeine

Figuree 2: Histogram of the paraxanthine/caffeine ratio; the ratio is an indication of the cytochromee P-450 1A2 activity.

CaffeineCaffeine loading test Thee distribution of the paraxanthine/caffeine molar ratio within the groupp of children is displayed in a histogram (figure 2). The ratio ranged fromm 0.41 to 2.48, with a mean of 1.35 (SD 0.48). Linear regression of thee paraxanthine/caffeine molar ratio, indicative of the CYP1A2 activity, versuss prenatal dioxin exposure revealed no significant linear correlation

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(p=0.27).. In the same manner there was no correlation between the molarr ratio and postnatal dioxin exposure (p=1.00).

Discussion n

ThyroidThyroid hormones Duringg the neonatal period of our cohort, Pluim detected a trend towards aa higher T4 and T4/TBG ratio (as indicator of the FT4 concentration) in thee cord blood of the higher dioxin exposure group. This trend attained statisticall significance at one and eleven weeks post partum. The TSH levell was also increased at eleven weeks post partum (13; 16). The thyroidd parameters of the mothers of the higher and lower exposure groupp were comparable and all the infants were breastfed. The hypothesis wass that dioxins ultimately decrease the nuclear occupancy of T3 receptors,, leading to a stimulation of TSH secretion, and a higher thyroxinee synthesis. In this manner the FT4 setpoint, or optimal FT4 level,, is increased. From a physiological point of view, for optimal metabolisationn in children a certain level of occupation of T3 receptors is needed.. Peripheral thyroid hormone problems, such as thyroid hormone hypo-responsivenesss (an hereditary illness), lead to supraphysiological levelss of FT4 with only slightly increased TSH levels. In this case the setpointt for the thyroid hormone is set higher in order to compensate. The setpointt is the optimal thyroid hormone concentration, being a characteristicc of the whole body, with the hypophysis as custodian. While thee setpoint certainly has a genetic basis, it is also susceptible to (transient)) external influences, such as is seen in non-thyroidal illnesses, orr shortly after birth when there is an increased need. Fourr different thyroxine receptors have been identified (alpha 1 & 2 and betaa 1 & 2). This heterogenicity could explain why dioxins seem to have variedd effects in different tissues. An example of this may be the enhancedd maturation of the neuromotor system (in relation to perinatal dioxinn exposure), as documented by Ilsen (17), which she explained as an effectt of FT4 agonism.

Untill such time as it is clear precisely how dioxins influence the thyroidal system,, it is probably safest to assume that the different effects seen are a

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resultt of receptor differences and binding differences. In other tissues, withh a (slightly) different receptor for T4, dioxins might not bind as well, andd this imperfect binding may result in a blockade of the function, with aa decrease in nuclear thyroxine occupancy in the hypophysis and hypothalamus.. In other words, in such a setting the regulatory system is affected. . Takingg this into consideration, it is interesting to note that Koopman-Esseboomm et al. noted a lower T3 and T4 level in relation to total dioxin andd PCB exposure, in their study of mothers before and after delivery (32).. This may be ominous, bearing in mind that women with low normal T44 levels have been shown to produce children with impaired psychomotorr development (33;34). Furthermore, Koopman-Esseboom, in aa group of babies comparable to our cohort, found a significantly lower T44 and FT4, and an increased TSH level, in the second week after birth. Inn umbilical cord plasma and at the age of three months the TSH level wass slightly increased (32). The contradictory findings between her lowerr T4 and that of Pluim, with his higher T4, might be explained by a populationn bias or methodological error in the determinations. The latter iss unlikely, seeing as the determination of thyroidal system hormone concentrationss in plasma are standard determinations. Thee fact that both the Amsterdam and Rotterdam groups, working independentlyy of each other, detected increased TSH levels strengthens thiss finding, and makes it unlikely that the finding is accidental. The supposedlyy contradictory results from both studies in the neonatal period cann only be explained by an influence of dioxins on both the thyroid hormonee regulating systems and peripheral tissues. In our cohort, both at twoo and a half years (17) and now at the age of eight years, no abnormalitiess were detected in thyroid hormone regulation. The external influencee of dioxins on the setpoint then seems transient. Very young childrenn have a relatively greater thyroxine need than older children. This iss especially evident shortly after birth when the neonate's energy supply comess from free fatty acids, with the glucose transport from the mother havingg ceased, and gluconeogenesis not yet operational. Furthermore, non-shiveringg thermogenesis is necessary for temperature control, and a rapidd breakdown of triglycerides in the brown adipose tissue is used for this.. In 1984, Rozman already hypothesised that brown adipose tissue is a

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mainn target for dioxin exposure (35;36). The mobilisation of fat after birthh is dependent on thyroxine, and this probably explains why the abnormalitiess in T4 and TSH levels attain significance, in relation to dioxinn exposure, during the first few weeks after birth. After the neonatal period,, the T4 need diminishes, from 10 micrograms/kg in the neonate to 22 micrograms/kg in the adult. This is a result of a physiological lowering off the setpoint, as a result of the reduced T4 need, and probably also as a resultt of the relative decrease of dioxin exposure, including a greater dilutionall effect. This would result in the normalisation of the setpoint of T44 seen at the age of two and eight years. Further follow-up is necessary inn order to establish that the apparent transient influence has no later effectss on the thyroid and its regulatory system.

Thee FT4 concentration and CYP1A2 activity seem to show an inverse correlationn in the scatter diagram (figure 1), yet statistically show no significantt relation. The latter might be due to the limited size of the group.. Cytochrome P450 1A2 possibly plays a role in FT4 metabolism.

ALATandASAT ALATandASAT Inn contrast to ALAT , a literature search failed to uncover an effect of dioxinn exposure on ASAT. Acute dioxin exposure is known to increase plasmaa ALAT levels in laboratory animals, such as mice (37) and rhesus monkeyss (38), and in children(14;39), yet these changes seem to be transient.. Seefeld's study of adult rhesus monkeys, exposed to various concentrationss of dioxin, revealed increased ALAT levels during the coursee of intoxication, indicating liver damage. The maximum effect seenn after two to three weeks was again normalised at about 6 weeks, indicatingg a transient effect (38). Mocarelli's six year follow-up study, of thee Seveso children exposed to dioxins following a chemical plant explosion,, revealed a similar increase in ALAT amongst the highest exposedd boys, which was decreased to levels comparable to the control populationn after about five years (39). This again indicates a transient effectt of dioxin on ALAT levels, even at high exposure concentrations. Follow-upp of industrially exposed workers in the U.S. showed no increasee in ALAT activity (40). In the light of these studies, it is then not surprisingg that our current study has no longer found the relation between

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thee perinatal dioxin concentration and the ALAT levels, seen by Pluim in thiss same cohort shortly after birth (14). It is during the prenatal (in utero)) and breastfeeding period that children get exposed to the highest dioxinn concentrations (11;12;27). The later exposure, being predominantlyy determined by dietary exposure, is much less (27). It is thenn to be expected that the acute rise in ALAT levels, the reaction to the acutee high dioxin exposure, will again normalise as was seen in our two follow-upp studies of the same children, namely this study and that of Ilsenn (17). We, too, are then inclined to interpret these findings as indicatingg that dioxin causes reversible damage to the liver. Whether the explanationn for this lies in the rapid distribution of dioxin to adipose tissue,, to the liver's regenerating capacity, to an (as yet) unknown mechanism,, or to a combination of factors remains to be elucidated.

CytochromeCytochrome P-450 1A2 Measuringg paraxanthine/caffeine molar ratios in plasma, after a caffeine-loadingg test, has been shown to be an accurate method of estimating CYP1A22 activity (28;29;41). Thee mean (1.35, range 0.41-2.48) of the paraxanthine/caffeine molar ratioo in the cohort differs (though not statistically significantly) from the adultt mean of 0.80 (range 0.16-1.7) [28,41]. However, it has long been assumedd (although never demonstrated) that children show a higher CYP1A22 activity than do adults. Itt is commonly known that CYP activity is altered by dioxin exposure [e.g.. (42;43)] and the supportive literature is exhaustive. Yet, long term effectss of perinatal exposure are still not clearly known, and this is especiallyy so in humans. The degree of enzyme induction is seldom, if ever,, in correlation to the dosage. In other words, should dioxins indeed causee induction, this induction need not be correlated to the level of exposuree perinatally. We can therefore only conclude that we have found noo visible decreased nor increased CYP1A2 activity, relative to perinatal dioxinn exposure, seven to twelve years post exposure. It is possible that thee activity was increased in the perinatal period (yet without permanentlyy influencing the setpoint) due to the relatively far higher toxicity,, and that the activity has normalised with the relative decrease in thee toxin. Halperin also failed to show an association between industrial

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exposuree to TCDD and CYP1A2 induction in a larger adult group (44). Likee CYP1A2, CYP1A1 also metabolises caffeine. However, CYP1A1 iss expressed at very low levels in the liver, and caffeine metabolism is exclusivelyy hepatic. It is therefore very unlikely that CYP1A1 colours thee CYP1A2 results seen. There are no other human enzymes known to metabolisee caffeine with high affinity and a similar pattern of metabolites ass CYP1A2 (29). The results of the loading test then accurately represent thee CYP1A2 activity in the children. We therefore could not confirm our hypothesiss that dioxins decrease CYP1A2 activity in later childhood. Thee activity was not determined in the previous studies of the cohort.

Whilee dioxins are widely known to be endocrine disrupters, it has been difficultt to confirm their influence on humans. The cohort in this study is aa group that was recruited antenatally, and in which the prenatal and postnatall dioxin exposures were determined perinatally. The children havee since been studied at regular intervals and therefore form a usable longitudinall cohort, whereby the dioxin influence on the various developmentall windows can be accurately followed over time. However, aa longitudinal study often means losing subjects during the follow-up periods.. Such is the case in this study, where the original group of 61 has beenn reduced to the current 37 children participating in this pre-pubertal follow-up.. Yet, the grouping of values, without a directional tendency, seenn in the scatter diagrams shows that the size of the group probably in noo way limits the outcome. It is unlikely that a larger group would have producedd a different outcome. The absence of statistically significant resultss at this age might be the effect of (partial) reversibility and/or recoveryy of the abnormalities seen in the perinatal period.

Concluding,, follow-up of 7 to 12 year old children with documented prenatall and postnatal dioxin exposure has shown a normalisation of previouslyy increased setpoint of thyroid hormone homeostasis. Similarly thee relation between perinatal dioxin exposure and ALAT levels, seen shortlyy after birth, is no longer evident. These results point to a transient effectt of the toxic influences of background levels of dioxins in The Netherlandss on these determinants. This may be a result of the combinationn of body composition (less adipose tissue) in the perinatal

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periodd and decreasing exposure from the relatively high perinatal to the muchh lower later childhood background exposure, and from a dilutional effectt (more adipose tissue). The cytochrome P-450 1A2 activity, measuredd by means of a caffeine-loading test revealed no correlation withh the pre- and postnatal dioxin exposures. Knowledge about the influencee of perinatal background dioxin exposure on children is limited. Thiss study provides new data.

Acknowledgements s

Wee are grateful to: -- Greenpeace for sponsoring the travel expenses of the study

participants,, and some of the laboratory investigations; -- Prof. Dr. Martin van den Berg and Dr. Marcel Rouwenhorst for

theirr critical appraisal of the manuscript; -- Mrs. Margalith van Huiden-ten Brink for invaluable organisational

andd secretarial support; -- Mrs. Gerda Stam and Mrs. Anna-Leena Pentikainen for their

organisationall support; -- De Heel Zaans Medical Centre for their graciously allowing this

studyy to be performed under their auspices; and,, of course, our greatest indebtedness is to the children, who participatedd in this study, and their parents, for their time, effort and enthusiasm. .

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