1 Division of Ambulatory Pediatrics and Adolescent Medicine, Department of Pediatrics and 2 Laboratory of Toxicology, University of Liège, C.H.U. Sart Tilman, Liège, Belgium and Departments of Pediatrics, 3 Universities of Ghent, 4 Antwerp, 5 Brussels and 6 Centre Hospitalier de Luxembourg, in collaboration with the Belgian Study Group for Pediatric Endocrinology
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Abstract |
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Key words: adopted children/organochlorine pesticides/p,p'-DDE/precocious puberty
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Introduction |
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Materials and methods |
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Height was measured using a Harpenden or wall-mounted stadiometer. Height was calculated as Standard Deviation Scores (SDS) (Tanner et al., 1966). Weight was measured and expressed as BMI SDS calculated according to a published method (Rolland-Cachera et al., 1982
). Data on auxology and pubertal development were obtained at 6-month intervals during therapy.
Investigations
Two studies were performed. In a retrospective epidemiological and auxological study, the aetiology of PP and country of origin of patients as well as data (age, height SDS and BMI SDS) at immigration and at diagnosis of PP were evaluated in a group of 145 patients seen in a 9-year period. In a toxicological study, the blood samples obtained in 41 patients on the occasion of a routine follow-up visit were used with the informed consent of the family to measure pesticides including p,p'-DDE [(1,1-dichloro-2,2-bis(4-chlorophenyl) ethylene)], a main metabolite of the insecticide DDT [1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane].
The serum concentration of p,p'-DDE was measured using a gas chromatographic analyser coupled to a Tandem mass spectrometer detector. Seven other pesticides were also measured: DDT, lindane arachlor, heptachlor, aldrin, endrin and hexachlorobenzene (HCB). Sample preparation included a liquidliquid extraction (petroleum ether:diethyl ether; 98:2) followed by a solid-phase extraction (Bond Elut Certify; Varian, Walnut Creek, CA, USA). The eluate was evaporated to dryness and reconstituted with the derivatization mixture (N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS) 10% in n-hexane) and then injected into the gas chromatograph (Saturn 2000; Varian). The column was a HP-5 Trace (30 mx0.25 mm internal diameter) from Hewlett Packard (Wilmington, Delaware, USA). Ionization by electronic impact occurred at 70 eV. All solvents were pesticide-grade quality. Reference standards were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). The calibration curve was constructed from 0 to 40 ppb, and linearity applied for this concentration range. Endosulphan-d4 (0.5 ppb) was used as internal standard. The recovery of p,p'-DDE was 96%. The limit of detection (0.1 ppb) was defined as three times the standard deviation (SD) of the results from the lowest quality-control serum pool over the course of the analyses (n = 15). For p,p'-DDE, this was consistent with a detection limit of 0.1 ng/ml serum. The coefficients of variation were between 4.6 and 7.8%
Data analysis
Results were expressed as mean ± SD. For the serum concentrations of p,p'-DDE, the median concentration was calculated after log transformation of the data. The comparison of mean height and BMI SDS between the patient groups was made using the unpaired Student's t-test. The comparison of p,p'-DDE concentrations was made using one-way analysis of variance (ANOVA). All results were considered to be significant at the 5% critical level (P < 0.05).
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Results |
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Discussion |
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Observations made in the present study involving many different countries worldwide do not support the concept that a particular ethnic group is at increased risk of PP due to genetic factors. Though Afro-American girls have relatively early menarche (1 year before the white Americans; Herman-Giddens et al., 1997), the African girls have normal or late menarche depending on the nutritional conditions (Burgess and Burgess, 1964), such as in the Indian girls (Proos et al., 1991
). It is therefore unlikely that the present findings result from genetic factors linked to the ethnic origin, though their role cannot be excluded.
Many children adopted from developing countries have delayed growth and development due to unfavourable effects of malnutrition and infectious diseases (Miller et al., 1995; Albers et al., 1997
; Rutter, 1998
). The growth consequences of deprivation were obvious in the Swedish study reporting a mean height SDS of 2.8 in Indian girls on arrival in Sweden. Rapid catch-up growth in the foster country resulted in a recovery of 2 SD in height before the onset of pubertal growth (Proos et al., 1993
). In the present study, as in the Italian and French reports (Virdis et al., 1998
; Baron et al., 2000
), the adopted children showed less retarded growth, though mean age at immigration was not different from the Swedish experience with Indian girls. In addition, some patients entered PP without any evidence of former deprivation. Height and BMI status were often normal at immigration, though national standards from the country of origin were not available. In this study, the existence of PP was highlighted in foreign children moving to Belgium with their original families without history of psychosocial deprivation. It is possible that patients with PP commonly classified as idiopathic in other studies belong to this particular group. On arrival, these children were not height-retarded and some showed obvious weight excess.
Because PP occurred in children moving from developing countries to Belgium irrespective of the ethnic or country background and independent of the height and weight status and the context of adoption, the possible role of environmental factors was pointed out. In developed countries, pubertal signs were observed following increased exposure to xeno-oestrogens such as plastics or insecticides (Marshall, 1993). The present study was focused on the organochlorine pesticide DDT and its derivative p,p'-DDE for several reasons: (i) Among eight organochlorines which were sought in serum from foreign adopted children in a pilot study, p,p'-DDE appeared to be present in several samples while no other compounds were detected; (ii) Some DDT isomers such as o,p'-DDT, o,p'-DDE and p,p'-DDT were shown to exhibit weak but obvious oestrogenic activity in many systems such as the rat uterus (Bitman et al., 1968
; Gellert et al., 1972
; Bustos et al., 1988
), the seagull embryo (Fry and Toone, 1981
), the MCF-7 breast cancer cell line (Soto et al., 1992
), transfected human embryonal kidney cells or yeast expressing the oestrogen receptor (Kuiper et al., 1998
; Sheeler et al., 2000
) and vitellogenin induction in rainbow trout liver slices (Shilling and Williams, 2000
); (iii) The use of DDT has been banned in western European countries and the USA since the 1960s/early 1970s (Key and Reeves, 1994
), whereas it is still used extensively and in large amounts in the developing countries; (iv) In the environment, the breakdown and elimination of DDT takes several decades (Calabrese, 1982
), making possible an assessment of contamination several years after stopping chronic exposure to this compound; (v) While the o,p'-isomers are not stable and found in only small amounts in nature (Lamont et al., 1970
), the p,p'-isomer derivatives such as p,p'-DDE are more stable and can be used as markers of DDT exposure, though p,p'-DDE does not show obvious oestrogenic activity (Gellert et al., 1972
) and does not displace 17ß-oestradiol from oestrogen receptor (ER)
or ERß (Kuiper et al., 1998
). However, some oestrogenic effect of p,p'-DDE was reported recently in the salamander (Clark et al., 1998
), as well as an anti-androgen action (Kelce et al., 1995
).
The mean serum concentration of p,p'-DDE was 10-fold higher in foreign children with PP than in Belgian native patients with PP in whom the organochlorine derivative was usually undetectable. Though this expected finding might be incidental, it may also be related to the occurrence of PP. Obviously, evidence of pesticide effects should require measurements of p,p'-DDE in foreign adopted girls showing no PP. It could also be helpful to elucidate the reason why children from some countries such as China have not presented with PP so far (age at immigration, early feeding habits, etc.). The contribution of intrauterine exposure to pesticides also warrants further study. Among the foreign non-adopted girls with PP, three were born in Belgium but showed detectable concentrations of p,p'-DDE (data not shown), suggesting possible contamination by the mother during intrauterine life or breast-feeding. An intrauterine effect is particularly interesting since there is some experimental and clinical evidence that the timing of onset of puberty can be affected by sex steroids prenatally (Zachmann et al., 1986; Wood et al., 1991
; Plant, 1994
).
In Figure 4, a putative mechanism is proposed of organochlorine involvement in the pathogenesis of PP. There are two possible pathways to account for PP following chronic exposure to oestrogenic DDT-related compounds. A weak stimulation of oestrogen-sensitive tissues may occur peripherally, as seen in an epidemic of PP, possibly due to xeno-oestrogens (Saenz de Rodriguez et al., 1985
). A stimulation of oestrogen-sensitive tissues may also occur centrally (Klein et al., 1994
); these authors explained the physiologically earlier onset of puberty in girls than in boys by the maturation-promoting effects of oestrogens in the female, prepubertally. A similar mechanism was advocated to account for central PP following peripheral isosexual precocity (Pasquino et al., 1987
; Kukuvitis et al., 1995
). Some evidence against a potentiation of central maturation by environmental oestrogens comes from the absence of early sexual maturation in the developing countries. This issue however may not have been addressed carefully enough so far, and nutritional deprivation may be a confounding factor. It is also possible that pesticides contributed to the advancing menarcheal age in industrialized countries, since the menarcheal age has remained unchanged after the ban on pesticides in the 1960s. An alternative or complementary mechanism (Figure 4
) may lie in the negative feedback inhibition of the gonadotrophins, which is a well-established central effect of oestrogen in the prepubertal subject (Kulin and Reiter, 1976
; Mauras et al., 1991
). Immigration to a developed country may then result in removal of the suppressing effect of oestrogens on the gonadotrophins and subsequent expression of accelerated hypothalaemic maturation. Such a mechanism could also explain the occurrence of central PP following treatment of peripheral PP (Pasquino et al., 1987
; Kukuvitis et al., 1995
). In children remaining chronically exposed to xeno-oestrogens in the developing countries, the central inhibition may last until it is overcome by endogenous developmental activation, resulting in normal or delayed hypothalamicpituitary maturation. The absence of increased incidence of PP in Western countries while the use of organochlorine pesticides was prohibited in the 1960s could be explained by the progressive suppression of those pesticides, which is different from the acute withdrawal resulting from a move to Belgium.
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Acknowledgements |
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Notes |
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8 To whom correspondence should be addressed at: Division of Ambulatory Pediatrics and Adolescent Medicine, University of Liège, C.H.U. Sart Tilman, B-4000 Liege, Belgium. E-mail: jpbourguignon{at}ulg.ac.be
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References |
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Submitted on September 19, 2000; accepted on February 12, 2001.