doi:10.1093/humrep/dem199
Human Reproduction Vol.23, No.1 pp. 201–210, 2008
Advance Access publication on November 19, 2007
Concentrations of persistent organochlorine compounds
in human milk and placenta are higher in Denmark
than in Finland
Heqing Shen1,2, Katharina M. Main2, Anna-Maria Andersson2,6, Ida N. Damgaard2,
Helena E. Virtanen3,4, Niels E. Skakkebaek2, Jorma Toppari3,4 and
Karl-Werner Schramm1,5
1
Institute of Ecological Chemistry, GSF—National Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764
Neuherberg, Germany; 2University Department of Growth and Reproduction, GR 5064, Rigshospitalet, Blegdamsvej 9, DK-2100
Copenhagen, Denmark; 3Department of Physiology, University of Turku, Kiinamyllynkatu 10, FI-20520 Turku, Finland; 4Department of
Paediatrics, University of Turku, Kiinamyllynkatu 10, FI-20520 Turku, Finland; 5Technische Universität München, Institut für
Ökologische Chemie und Umweltanalytik, Weihenstephaner Steig 23, Freising, Germany
6
Correspondence address. Tel: þ45-35-456054; Fax: þ45-35-456054; E-mail: [email protected]
BACKGROUND: A significantly reduced male reproductive health status, including a higher prevalence of cryptorchidism and hypospadias, has been documented in Danish men compared with Finnish men. Exposure to environmental pollutants with endocrine disrupting activities has been suggested as a possible contributing factor. In this
study, we investigated whether there was a difference in milk and placental concentrations of persistent organohalogen compounds, between the two countries. METHODS: Organohalogens were analysed by high-resolution gas
chromatography–high-resolution mass spectrometry in human milk samples from Finland (n 5 65) and Denmark
(n 5 65) and in placentas from Finland (n 5 112) and Denmark (n 5 168). RESULTS: 1,1-Dichloro-2,2-bis
(4-chlorophenyl)ethane (p,p0 -DDE) was the dominant pollutant. b-Hexa-chloro-cyclohexane (b-HCH), hexachlorobenzene (HCB), endosulfan-I, dieldrin, oxychlordane (OXC), cis-heptachloroepoxide (c-HE) and 1,1,1-trichloro-2,
2-bis(4-chlorophenyl)ethane (p,p0 -DDT) were the other main organochlorines detected. Danish samples had significantly higher concentrations of p,p0 -DDE, p,p0 -DDT, b-HCH, HCB, dieldrin, c-HE and OXC than did the Finnish
samples. Levels of organobrominated compounds were very low and most were undetectable in the majority of
samples. BB-153 and BB-155 were the most abundant polybromobiphenyl congeners. BB-153 was more abundant
in Danish milk samples compared with Finnish samples, whereas BB-155 was more abundant in the Finnish milk.
CONCLUSIONS: The organochlorine levels were higher in Danish, than in Finnish, samples, suggesting a higher
exposure for Danish infants.
Keywords: placenta; breast milk; organochlorine pesticides; organobromine compounds; infants
Introduction
Danish men have a higher prevalence of testicular cancer and a
reduced semen quality compared with Finnish men (Adami
et al., 1994; Jørgensen et al., 2002). Additionally, there is a
higher prevalence of cryptorchidism and hypospadias among
Danish compared with Finnish newborn boys (Virtanen et al.,
2001; Boisen et al., 2004, 2005). It has been speculated that
these differences between Denmark and Finland may be due to
differences in exposures to environmental endocrine disrupting
chemicals, especially during sensitive periods of development.
Several persistent organohalogen compounds have been
shown to have endocrine disrupting activities (Toppari et al.,
1996). Persistent organohalogen compounds bioaccumulate
along the food chain in lipid-rich tissues. Humans are
exposed to these compounds almost exclusively through the
diet. The compounds can be transferred to the fetus across
the placenta during pregnancy (Jacobson et al., 1984; Foster
et al., 2000; Waliszewski et al., 2000) and to the newborn
baby by breastfeeding (Nair et al., 1996; Anderson and
Wolff, 2000). In this study, we investigated the exposure of
Danish and Finnish infants to the organochlorine compounds
pentachlorobenzene (PeCB), hexachlorobenzene (HCB), hexachloro-cyclohexane (HCH) (a-, b-, g-, d- and e-), di-chlorodi-phenyl-trichloro-ethane (DDT)-related compounds [1,1,1trichloro-2,2-bis(4-chlorophenyl)ethane (p,p 0 -DDT), 1,1,1trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane(o,p0 -DDT),
# The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.
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201
Shen et al.
1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (p,p 0 -DDD), 1,1dichloro-2-(2-chlorophenyl)-2,2(4-chloro phenyl)ethane (o,p 0 DDD), 1,1-dichloro-2,2-bis(4-chlorophe nyl)ethane (p,p 0 -DDE)
and 1,1-dichloro-2-(2-chlorophenyl)- 2-(4-chlorophenyl)ethane
(o,p 0 -DDE)], octachlorostyrene (OCS), pentachloroanisole
(PCA), aldrin, dieldrin, cis-chlordance (c-CHL) and trans-CHL
(t-CHL), heptachlor, oxychlordane (OXC), cis-heptachloroepoxide
(c-HE), trans-HE (t-HE), methoxychlor (MOC), mirex, endosulfan
(END)-I and organobromine compounds [polybromobiphenyl
(PBB) congeners, pentabromobenzene (PeBB) and hexabromobenzene (HeBB)], by analysing placenta and human milk
samples from a prospective birth cohort study. The levels of
these compounds in the Finnish placentas have been discussed
elsewhere (Shen et al., 2005). In addition to looking for possible geographical differences in the concentrations of these
contaminants, we also explored some maternal factors, which
have been suggested to affect the prenatal (placental levels)
and post-natal (breast milk levels) exposure of children.
Some of the investigated compounds are the so-called chiral
compounds existing in two enantiomeric forms. For chiral
compounds, the biological activity may be enantiomer selective, e.g. different thalidomide enantiomers contain, respectively, the sedative and the teratogenic activity (Von Blaschke
et al., 1979). Likewise, the interaction of o,p 0 -DDT (Hoekstra
et al., 2001) and MOC metabolites (Hu and Kupfer, 2002)
with estrogen receptors has been shown to be enantiomer
specific (Bocchinfuso and Korach, 1997). In addition, as the
enantiomers of chiral compounds may be differently prone to
biodegradation, the ratio between the enantiomers (enantiomeric ratio, ER) may offer information on the exposure
history. Thus, comparing the ERs of persistent compounds in
human samples with the ERs in the environment may tell if
the exposure is recent or historical. Therefore, we also
measured the concentration of the enantiomers of some of
the chiral compounds and compared ERs between Danish
and Finnish samples.
Materials and Methods
Placentas and breast milk samples were derived from a joint prospective, longitudinal birth cohort study in an urban setting in Finland
(Turku University Hospital) and Denmark (the University Hospital
of Copenhagen) from 1997 to 2001. This study aimed to describe
regional prevalence rates and risk factors (lifestyle and exposure)
for cryptorchidism by means of questionnaires and biological
samples. Recruitment, participation rate and clinical examination
techniques in both countries were completely standardized and
described earlier (Boisen et al., 2004). Human milk samples were collected from 1 to 3 months post-partum in Denmark and from 1 to 2
months in Finland, as successive aliquots. All mothers were given
oral and written instructions to feed the baby first, and then sample
milk aliquots (hind milk) by manual expression into a glass or porcelain container, avoiding the use of mechanical breast pumps. Successive aliquots were frozen in glass bottles [250 ml Pyrex glass bottle
with a Teflon cap (1515/06D), Bibby Sterilin, Staffordshire,
England] and stored in the household freezers. The samples were
delivered frozen to the hospital at the 3 months’ examination and
stored at 2208C. The selected milk samples were thawed at room
temperature for 12 h, heated and shaken for 30 min at 378C to homogenize the samples, then divided into smaller aliquots and refrozen at
202
2208C until further chemical analysis. Placentas were collected at
birth by the midwives and frozen as whole placentas in polyethylene
bags at 2208C. Before analysis, placentas were defrosted, mechanically homogenized and aliquoted into 20 ml glass tubes.
From the total biobank, 65 milk samples from each country were
included for organohalogen measurements. The number of samples
was determined by funding, as exposure measurements were prospectively planned to include persistent and non-persistent chemicals (EU
grant QLK4-2001-00 269). Only breast milk samples with a volume
.125 ml were included to ensure that all prospectively planned chemical analyses could be performed. The samples represent 29 Danish and
33 Finnish boys with cryptorchidism at birth and 36 and 32 healthy
control boys, respectively. In Denmark, all controls were selected randomly from the entire birth cohort of healthy boys. In Finland, the
boys were selected prospectively by a case – control design, in which
the boys with cryptorchidism were matched to controls at birth for
maternal parity, smoking (yes/no), diabetes (yes/no), gestational
age (+7 days) and date of birth (+14 days). This design was
chosen in Finland due to lack of sufficient funding to collect and
store biological samples from all. Placentas were selected from 112
Finnish mother–child pairs (n ¼ 56 boys with cryptorchidism at
birth, n ¼ 56 controls) and from 168 Danish (n ¼ 39 with cryptorchidism, n ¼ 126 controls, n ¼ 3 healthy boys first seen at 3 months of
age). Funding for placenta analysis allowed the inclusion of more
samples from Danish healthy controls. Eighty-six mother– child
pairs (43 Danish and 43 Finnish) were included with both milk and
placenta samples.
The sample preparation, extraction, clean-up and high-resolution
gas chromatography –high-resolution mass spectrometry (HRGC–
HRMS) analyses for organohalogen and chiral compounds of the
Danish and Finnish samples were carried out in the same laboratory.
The methods used have been described in detail previously (Shen
et al., 2005, 2006; Damgaard et al., 2006; Main et al., 2006).
Briefly, Na2SO4 (VWR international GmbH, Germany), sea sand
(Riedel-de Haen, Germany), alumina B (ICN Biomedical GmbH,
Germany), florisil and silica gel (Promochem, UK) were heated at
6508C for at least 6 h before use. Wet samples (10 g placenta tissue
or 10 ml milk) were weighed (to a precision of two decimals) and
homogenized with 30 g Na2SO4 and 15 g sea sand. Extraction was
done in a glass column packed with the homogenized matrix and
spiked with 13C internal standards. The extraction solvent was
250 ml acetone and n-hexane (2:1 v/v). The extracts were collected
in flasks weighed in advance and evaporated using a rotary vacuum
evaporator (water bath at 458C). After evaporation, the flasks were
placed into a desiccator, until stable weight was achieved. Then the
lipid content was calculated on wet weight basis (to a precision of
four decimals). All the results were calculated on the lipid basis
because of the lipophilic properties of the investigated compounds
(Matheson et al., 1990; Needham and Wang, 2002). The lipophilic
residual was redissolved in toluene and the clean-up was done in gel
permeation chromatography (Bio-Beads S-8 column with toluene
eluent at 2 ml/min flow rate) and then in a glass cartridge (packed
with alumina B 0.8 g, Na2SO4 0.3 g, florisil 0.5 g, silica gel 1 g and
Na2SO4 0.5 g from bottom to top). Finally, the sample was condensed
to about 10 ml, and 10 ml 1,2,3,4-tetrachlorodibenzo-p-dioxin was
added as a recovery standard.
The organohalogens were measured by HRGC–HRMS and quantified by an isotope dilution method. HP5890 high-resolution gas
chromatography equipped with 60 m DB-XLB column (0.25 mm
internal diameter 0.25 mm film) was used to separate organochlorines.
MAT95 high-resolution mass spectrometer was used to detect organochlorines. A 30 m DB-XLB column was used to analyse bromobiphenyl (BB) congeners including HeBB and PeBB. 13C labelled
Infant exposure to persistent organohalogen compounds
polychlorobiphenyl (PCB) congeners were used as internal standards
to calibrate PBBs and HeBB in Finnish placenta samples (BB-1, -3, -4
and -15 by PCB28; BB-18, -31 and -37 by PCB 153; BB-52 and -49 by
PCB138; BB-80, -77, -103, -101, PeBB and HeBB by PCB180;
BB-155, -153, -169 and -209 by PCB209). For the analysis of
Danish placenta samples and the milk samples, 13C-BB-77 and
13
C-BB-126 had become available and were used as internal standards
(BB-103, -101, -126, -155, -153, -169 and –209 were calculated based
on 13C-BB-126; all the other PBB congeners including HeBB and
PeBB were according to 13C-BB-77). Calculations were based on
the relative signal responses of different congeners compared with
internal standards, which were stable during the same-instrument
operation conditions. A signal corresponding to 3 times noise was
defined as the limit of detection for all data. All the other organohalogens were calculated by the isotope dilution method. All the results
were calculated on the lipid base because of the lipophilic properties
of the investigated compounds (Matheson et al., 1990; Needham
and Wang, 2002).
The study was conducted according to the Helsinki II declaration
after informed oral and written consent of the parents. It was approved
by the ethics committees (Finland: 7/1996, Denmark: kF01-030/97)
and the Danish Data Protection Agency (1997-1200-074).
Statistics
Lilliefors test was used to check the goodness of fit to a normal distribution of the set of data. Differences in maternal and infant population
characteristics were tested by Mann–Whitney U-test. SPSS (13.0) was
used for multiple regression analyses to examine determinants (including country) of lipid (w/w %) and pesticide concentration (ng/g lipid)
in placenta and milk. Analyses on chemical concentrations were performed on log-transformed data to achieve normal distribution and
values ,LOD were not included. These analyses were only carried
out for the eight most prevalent organochlorines and the two most
abundant PBB congeners. The following factors were included as
covariates: country (Denmark ¼ 1, Finland ¼ 2), parity, maternal
smoking (yes ¼ 1, no ¼ 0), gestational age (days), maternal prepregnancy body mass index (BMI, kg/m2), infant date of birth and
maternal age at delivery (years). Infant date of birth was transformed
into a continuous variable (days): 1 year was defined as 365 days and
the earliest collected samples were assigned 0 for each cohort
separately.
Results
The characteristics of the study subjects are shown in Table I.
There were significant differences between Danish and Finnish
mothers in age, parity and smoking habits, with Danish
mothers being slightly older, more likely to have a first pregnancy, and more likely to be smoking than Finnish mothers.
The BMI of the mothers, the duration of the pregnancy and
the birthweight of the child did not differ between the Danish
and Finnish subjects.
Lipid concentration
The total lipid concentration in placenta and milk data showed
near normal distribution with normal-fitting probabilities of
0.77, 0.53, 0.73 and 0.81 for Danish milk, Finnish milk,
Danish placenta and Finnish placenta, respectively. The lipid
contents were significantly higher in the Finnish samples
than in the Danish for both placenta and breast milk
(P , 0.0001). This country difference in lipid content remained
Table I. Characteristics of the study subjects.
Maternal age (years)
Maternal BMI (kg/m2)
Maternal smoking (n)
Yes
No
Parity
1
2
3
Gestational age (days)
Birthweight (kg)
Denmark
(n ¼ 190)
Finland
(n ¼ 134)
P-value
30.6 (23.6– 38.8)
22.1 (18.0– 35.1)
28.7 (21.4–39.7)
23.0 (18.0–37.7)
0.011
0.132
0.0001
64 (33.7%)
124 (65.3%)
21 (15.7%)
112 (83.6%)
122 (64.2%)
51 (26.8%)
17 (8.9%)
282 (251–297)
3.6 (2.3–4.8)
69 (51.5%)
44 (32.8%)
18 (13.4%)
280 (256–296)
3.6 (2.7–4.5)
0.033
0.267
0.656
Values are given as medians (range) or numbers. Country differences were
tested by Mann– Whitney U-test.
significant after adjustment for parity, maternal age, maternal
BMI, length of pregnancy and maternal smoking (Table II).
In addition, milk lipid content was significantly (P , 0.0001)
correlated to maternal BMI (r ¼20.17), but independent of
parity, maternal smoking and length of pregnancy (gestational
age). Placental lipid content was significantly (P, 0.0001)
associated with length of pregnancy (r ¼ 20.32), but independent of parity, maternal BMI and maternal smoking.
Organochlorine compounds
Heptachlor, aldrin, 1-HCH, END-II and t-HE were undetectable in most samples. The concentrations of the other organochlorines measured in Danish and Finnish human milk and
placenta samples are shown in Table II. Most of these compounds were detectable in all samples from both countries.
There was no difference between the two countries with
respect to the percentage of samples with concentrations
above the detection limit for the individual chemicals.
p,p 0 -DDE, HCB, b-HCH, dieldrin, p,p 0 -DDT, END-I, OXC
and c-HE were the eight major pollutants detected (together
they accounted for 89%, 95%, 98% and 98% of the total pesticide concentration in Finnish placenta, Danish placenta,
Finnish milk and Danish milk, respectively). The unadjusted
concentrations of these eight chemicals were higher in placentas and milk from Denmark than in the samples from Finland.
For p,p 0 -DDE, p,p 0 -DDT, b-HCH, HCB, dieldrin, c-HE and
OXC, the levels remained significantly higher in Danish milk
samples after adjustment for possible confounders (maternal
age, parity, gestational length and date of birth) (Tables II
and III) and for p,p 0 -DDE, p,p 0 -DDT, b-HCH, HCB, dieldrin
and c-HE, the levels remained significantly higher in Danish
placenta samples after adjustment (Tables II and IV).
In a multiple linear regression analysis, lipid concentration,
maternal smoking and maternal BMI did not show any significant correlation to the organochlorine concentrations in milk,
whereas maternal age was positively associated and length of
pregnancy, infant date of birth and parity were negatively
associated with the concentrations of these eight compounds
in milk (Table III). Placenta levels of these eight compounds
were independent of maternal smoking, but positively associated with maternal age and negatively associated with infant
203
Shen et al.
204
Table II. Organochlorine compound concentrations in placenta and breast milk samples from Denmark and Finland (ng/g lipid)a.
Finnish placentab
Danish placenta
Lipid (%)
PeCB
a-HCH
b-HCH
g-HCH
d-HCH
PCA
HCB
OCSb
OXC
c-HE
o,p0 -DDE
p,p0 -DDE
o,p0 -DDD
p,p0 -DDD
o,p0 -DDT
p,p0 -DDT
DDE/
DDT
t-CHL
c-CHL
END-I
Dieldrin
MOC
Mirex
a
Danish milk
Finnish milk
Median (range)
LOD
Nc
Median (range)
LOD
Nc
P-valued
Median (range)
LOD
Nc
Median (range)
LOD
Nc
P-valued
1.1 (0.55–1.5)
0.47 (0.05–3.45)
0.52 (0.04–8.92)
8.73 (2.87–47.76)
0.79 (0.1– 2.04)
0.05 (0.01–0.7)
0.13 (0.03–2.7)
7.68 (2.17–26.49)
0.10 (0.02–0.39)
1.15 (0.15–3.99)
0.91 (0.1– 3.1)
0.02 (0.01–0.2)
40.98 (9.52–269.83)
0.05 (0.01–0.36)
0.67 (0.16–6.35)
0.05 (0.01–0.26)
0.46 (0.07–4.65)
87.14 (11.51– 380.04)
0.33
0.04
0.08
0.04
0.04
0.03
0.03
0.04
0.17
0.06
0.02
0.01–0.1
0.02
0.02
0.03
0.06
168
168
168
168
168
124
168
168
168
168
168
164
168
167
168
167
168
168
1.22 (0.93– 1.52)
0.49 (0.17– 171.71)
0.55 (0.18– 692.79)
4.55 (1.48– 45.54)
0.82 (0.39– 246.87)
0.17 (0.00– 726.74)
0.15 (0.04– 1.7)
4.52 (1.77– 14.59)
0.12 (0.04– 0.55)
0.88 (0.22– 2.76)
0.69 (0.21– 2.01)
0.02 (0.01– 0.6)
18.04 (3.1–79.21)
0.05 (0.01– 0.38)
0.48 (0.2–2.4)
0.05 (0.01– 0.92)
0.27 (0.12– 3.38)
62.54 (2.78– 304.65)
0.02
0.02–0.59
0.02–0.8
0.03–0.68
0.02–0.72
0.11
0.02
0.03
0.1
0.05
0.03
0.01–0.17
0.02
0.04
0.07
0.07
112
112
112
112
112
99
112
112
112
112
112
110
112
112
112
112
112
112
,0.0001
0.24
0.01
,0.0001
0.14
,0.0001
0.49
,0.0001
0.0001
0.02
,0.0001
0.45
,0.0001
0.37
,0.0001
0.65
,0.0001
,0.0001
2.84 (0.36– 7.33)
0.32 (0.13– 1.41)
0.26 (0.05– 3.45)
16.85 (5.97– 66.23)
0.65 (0.23– 3.34)
0.04 (0.01– 0.27)
0.09 (0.02– 0.79)
12.4 (6.01– 24.56)
0.20 (0.05– 0.4)
4.74 (2.26– 12.01)
2.87 (1.25– 10.82)
0.08 (0.02– 0.28)
133.76 (24.59–427.55)
0.02 (0.00– 0.07)
0.36 (0.1– 2.2)
0.46 (0.13– 1.83)
5.68 (1.62– 37.88)
20.49 (4.36– 106.66)
0.02
0.05
0.09
0.04
0.03
0.03
0.04
0.03
0.25
0.14
0.02
0.1
0.03
0.01
0.04
0.04
65
65
65
65
65
41
65
65
65
65
65
65
65
63
65
65
65
65
4.26 (0.95– 10.14)
0.25 (0.08– 1.17)
0.16 (0.04– 0.77)
10.93 (2.74– 30.89)
0.40 (0.08– 4.05)
0.03 (0.01– 0.16)
0.03 (0.01– 0.11)
7.95 (2.94– 18.55)
0.19 (0.04– 0.7)
3.56 (0.91– 9.4)
2.04 (0.63– 17.02)
0.04 (0.01– 0.14)
59.05 (18.95– 331.16)
0.02 (0.00– 0.17)
0.31 (0.1–1.36)
0.27 (0.04– 1.21)
3.43 (1.46– 12.9)
16.58 (8.1–42.51)
0.01
0.02
0.27
0.04
0.1
0.01
0.08
0.02
0.17
0.05
0.05
0.01 –0.11
0.09
0.07
0.33
1.35
65
65
65
65
65
25
64
65
65
65
65
65
65
65
65
64
65
65
,0.0001
,0.0001
,0.0001
,0.0001
0.002
0.002
,0.0001
,0.0001
0.57
0.0001
,0.0001
,0.0001
,0.0001
0.03
0.04
,0.0001
,0.0001
0.001
0.01 (0.00–0.18)
0.01 (0.00–0.19)
1.97 (0.27–6)
2.46 (0.42–19.57)
0.06 (0.02–7.79)
0.14 (0.01–1.08)
0.07
0.07
0.01–0.22
0.1
1.24
0.02
74
55
168
168
168
168
0.05
0.07
0.01–0.15
0.1
0.08
0.02
32
31
112
112
112
112
0.30
0.16
0.56
,0.0001
0.0003
0.002
0.05 (0.01– 0.35)
0.03 (0.01– 0.07)
7.43 (1.92– 18.05)
4.88 (1.74– 35.5)
0.04 (0.00– 0.43)
0.21 (0.03– 0.66)
0.04
0.04
0.01– 0.31
0.17
0.04
0.02
55
32
65
65
65
65
0.03 (0.01– 0.11)
0.02 (0.00– 0.05)
6.40 (1.19– 22.66)
2.37 (0.77– 7.19)
0.06 (0.02– 1.12)
0.26 (0.02– 1.54)
0.04
0.03
0.01 –0.38
0.12
0.17
0.02
53
32
65
65
64
64
0.005
0.2
0.07
,0.0001
0.0002
0.23
0.03
0.03
1.75
1.25
0.04
0.18
(0.00– 0.67)
(0.00– 0.10)
(0.26– 8.78)
(0.44– 4.32)
(0.01– 1.14)
(0.02– 2.08)
Samples with values ,LOD were excluded from the calculation.
For Finnish placenta data see also our previous report (Shen et al., 2005).
c
Number of samples .LOD. Total numbers of Danish and Finnish placenta samples were 168 and 112, respectively, and those of Danish and Finnish milk samples were 65 and 65, respectively.
d
Country differences were tested by multivariate regression controlling for confounders.
b
Infant exposure to persistent organohalogen compounds
Table III. Multivariate analysis of determinants for organochloride concentration in 130 human milk samples [partial regression coefficients are given for
factors that were significantly correlated (P,0.05)].
Compound
r
P-value
Maternal age
(years)
p,p0 -DDE
p,p0 -DDT
b-HCH
HCB
OXC
c-HE
END-I
Dieldrin
0.52
0.40
0.64
0.69
0.63
0.32
0.55
0.59
0.0001
0.0001
0.0001
0.0001
0.0001
0.02
0.0001
0.0001
0.35
Gestational age
(days)
0.42
0.42
0.55
20.18
20.19
20.40
0.47
Infant date of birth
(days)
Parity
Countrya
20.17
20.26
20.36
20.33
20.23
20.21
20.26
20.19
20.37
20.34
20.36
20.45
20.19
20.20
20.38
20.40
20.48
20.41
20.41
r is the correlation coefficient for the overall model. P-value is the P-value for the overall model.
a
A negative partial regression coefficient ¼ levels lower in Finnish samples compared with Danish samples.
date of birth, and parity (Table IV). HCB, c-HE and dieldrin
levels in placenta were positively associated with maternal
BMI, whereas END-I levels were negatively associated with
maternal BMI (Table IV).
Organobrominated compounds
The concentrations of BB congeners, including HeBB and
PeBB, are listed in Table V. Compared with the levels of organochlorines, much lower levels of organobromines were found
and more samples had unmeasurable levels. BB-153 and -155
were the most frequently found congeners. Of these two congeners, BB-153 was generally the more abundant, but in some
placenta samples, the levels of BB-155 were higher than
BB-153, and in some samples only BB-155, but not BB-153
was detectable. The levels of BB-153 were significantly
higher in Danish milk samples compared with Finnish
samples (P , 0.01), whereas the levels of BB-155 were significantly higher in Finnish milk compared with Danish (P ¼
0.016). None of the other confounders (maternal age, parity,
maternal BMI, gestational length and date of birth) was
related to the level of these two congeners in milk.
Most of the samples had a very low BB-155/BB-153 ratio
(Table V). However, 16 Finnish placenta and 11 Danish placenta samples had a BB-155/BB-153 ratio above the average
(.0.3); in these samples, also other congeners could frequently
be found (they were one or more of BB-31, -37, -52, -49, -80,
-77, -103 and -101). In contrast, only two Finnish milk samples
had a BB-155/BB-153 ratio close to 0.3. Only in six paired
milk and placenta samples could BB-155 and BB-153 be
detected in both matrices. In these pairs, there was a strong
linear correlation between the BB-155/BB-153 ratio in placenta and in milk samples (R 2 ¼ 0.99; P,0.0001; N ¼ 6) but
the ratio was 60 times higher in the placenta samples than
in the milk samples.
Enantiomeric ratios of chiral compounds
The ERs with a relative error less than 20% are presented
(Table VI). Only the ERs for a-HCH, c-HE and OXC will be
discussed because the ERs for these three compounds could
be determined for most of the samples.
No significant difference in the ERs between Finnish and
Danish samples for a-HCH, c-HE and OXC was observed,
but for a-HCH and c-HE the ERs changed with the concentration of the isomers. At high concentrations of the
(2)-isomer of a-HCH, the ER was close to the racemic ratio
(ER ¼ 1), whereas at a low concentration the ER ratio
decreased (Fig. 1). For c-HE, the ER increased with lower concentrations of the (þ)-isomer (Fig. 2). There were only a few
samples with extreme ER values (for c-HE values close to 1
and for a-HCH values much larger than 1. In contrast to
a-HCH and c-HE, no changes in ER with changes in the
isomer concentration were found for chiral OXC.
Table IV. Multivariate analysis of determinants for organochloride concentration in 280 placenta homogenates (partial regression coefficients are given for
factors that were significantly correlated (P,0.05)).
Compound
r
P-value
Maternal age
(years)
p,p0 -DDE
p,p0 -DDT
b-HCH
HCB
OXC
c-HE
END-I
Dieldrin
0.52
0.27
0.48
0.66
0.52
0.57
0.47
0.53
0.0001
0.003
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.28
0.15
0.24
0.29
0.48
0.24
0.45
0.15
Maternal
BMI
0.11
Gestational age
(days)
0.15
0.34
20.12
0.21
Infant date of birth
(days)
Parity
Countrya
20.13
20.16
20.15
20.19
20.18
20.33
20.23
20.27
20.42
20.19
20.35
20.53
20.27
20.27
20.24
20.29
20.23
20.27
20.36
20.43
r is the correlation coefficient for the overall model. P-value is the P-value for the overall model.
a
A negative partial regression coefficient ¼ levels lower in Finnish samples compared with Danish samples.
205
Shen et al.
206
Table V. Concentration of PBB congeners, HeBB and PeBB (pg/g lipid) in Danish and Finnish breast milk and placentas samples.
Danish milk
BB-4
BB-31
BB-37
BB-52
BB-49
BB-80
BB-77
BB-103
BB-101
BB-126
BB-155
BB-153
BB-155/
BB-153
BB-169
PeBB
HeBB
Finnish milk
Finnish placentab
Danish placenta
N
Min–Max
Mean (SD)a
N
Min– Max
Mean (SD)a
N
Min– Max
Mean (SD)a
N
Min– Max
Mean (SD)a
0 (0%)
9 (14.1%)
0 (0%)
11 (17.2%)
11 (17.2%)
16 (25.0%)
39 (60.9%)
0 (0%)
19 (29.7%)
0 (0%)
49 (76.6%)
64 (100%)
49 (76.6%)
—
2 –31
—
2 –12
1 –4
1 –7
3 –47
—
2 –66
—
2 –49
41–1499
0.01–0.2
—
8 (9)
—
4 (3)
3 (1)
4 (2)
13 (9)
—
15 (17)
—
10 (10)
200 (197)
0.05 (0.03)
0 (0%)
3 (4.6%)
0 (0%)
2 (3.1%)
1 (1.5%)
4 (6.2%)
27 (41.5%)
0 (0%)
18 (27.7%)
0 (0%)
34 (52.3%)
63 (96.9%)
31 (47.7%)
—
4 –24
—
10–13
2
2 –12
2 –27
—
4 –25
—
3 –60
26–1204
0.02 –0.3
—
12 (10)
—
12 (2)
2
7 (5)
9 (6)
—
13 (7)
—
13 (10)
134 (160)
0.1(0.05)
1 (0.6%)
2 (1.2%)
1 (0.6%)
3 (1.8%)
5 (3.0%)
6 (3.6%)
20 (11.9%)
2 (1.2%)
6 (3.6%)
0 (0%)
17 (10.1%)
63 (37.5%)
10 (6.0%)
6
779– 1067
1919
700– 1005
40– 786
350– 2231
16– 3627
633– 942
42– 5741
—
3– 4569
15– 2628
0.04– 1.7
6
923 (204)
1919
838 (155)
455 (327)
1088 (647)
645 (928)
787 (219)
1240 (2232)
—
448 (1096)
304 (409)
0.4 (0.5)
0 (0%)
11 (9.8%)
1 (0.9%)
2 (1.8%)
2 (1.8%)
6 (5.4%)
3 (2.7%)
1 (0.9%)
5 (4.5%)
0 (0%)
24 (21.4%)
53 (47.3%)
20 (17.8%)
—
2 –149
7
2 –18
3 –16
3 –28
9 –26
12
5 –40
—
3 –374
6 –1610
0.04 –11.2
—
35 (46)
7
10 (11)
10 (9)
15 (11)
20 (9)
12
25 (15)
—
60 (91)
83 (219)
1.4
4
7 (4)
50 (75)
0 (0%)
5 (7.7%)
31 (47.7%)
—
6 –36
12–120
—
14 (13)
37 (24)
1 (0.6%)
4 (2.4%)
31 (18.4%)
548
158 (135)
877 (1959)
0 (0%)
10 (8.9%)
33 (29.5%)
—
10–79
8 –935
1 (1.6%)
27 (42.2%)
49 (76.6%)
4
3 –18
5 –500
548
35– 326
71– 10 260
—
36.8 (22)
122 (194)
N is the number of samples with detectable levels and SD the standard deviation.
a
Samples with values ,LOD were excluded from the calculation. Total numbers of Danish and Finnish placenta samples were 168 and 112, respectively, and those of Danish and Finnish milk samples were 65
and 65, respectively.
b
Finnish placenta and Danish placenta data may not be directly comparable because of different calibration methods.
Infant exposure to persistent organohalogen compounds
Table VI. ERs in Danish and Finnish breast milk and Danish placenta samples.
Danish milk
(2)-a-HCH
(þ)-a-HCH
ER-20%a
(þ)-OXC
(2)-OXC
ER-20%
(þ)-cis-HE
(2)-cis-HE
ER-20%
o,p0 -DDD(I)
o,p0 -DDD(II)
ER-20%
(2)-o,p0 -DDT
(þ)-o,p0 -DDT
ER-20%
Finnish milk
Danish placenta
N
Mean (SD)
Min–Max
N
Mean (SD)
Min–Max
N
Mean (SD)
Min– Max
65
59
64
65
65
65
65
65
65
11
6
6
31
42
25
0.29 (0.37)
0.24 (0.36)
0.58 (0.23)
2.68 (1.35)
2.09 (1.08)
1.33 (0.29)
2.04 (1.08)
1.09 (0.64)
2.02 (0.48)
0.08 (0.05)
0.07 (0.05)
1.73 (0.62)
0.3 (0.25)
0.69 (0.47)
3.29 (1.21)
0.02– 1.76
0.01– 1.72
0.18– 1
0.56– 6.97
0.3– 5.02
0.73– 2.2
0.62– 7.09
0.16– 3.65
1.48– 4.09
0.02– 0.18
0.02– 0.14
1.01– 2.55
0.03-0.9
0.15– 1.69
1.48– 5.78
60
47
63
62
61
63
65
65
65
7
5
2
30
53
31
0.11 (0.08)
0.07 (0.11)
0.47 (0.41)
1.68 (1.12)
1.25 (0.77)
1.38 (0.36)
1.48 (1.36)
0.77 (0.75)
2.05 (0.44)
0.02 (0.01)
0.03 (0.02)
0.41 (0.08)
0.11 (0.09)
0.22 (0.17)
2.74 (0.9)
0.02– 0.42
0.01– 0.58
0.16– 2.8
0.34– 4.99
0.25– 3.34
0.64– 2.53
0.25– 10.68
0.18– 5.59
1.34– 3.25
0.01– 0.04
0.01-0.07
0.35– 0.47
0.03– 0.47
0.03– 1.12
1.68– 5.42
165
160
164
152
141
155
166
159
162
109
129
98
14
2
1
0.62 (0.75)
0.57 (0.73)
0.83 (0.16)
0.81 (0.43)
0.64 (0.34)
1.41 (0.34)
0.64 (0.34)
0.34 (0.2)
2.17 (0.58)
0.06 (0.04)
0.11 (0.08)
0.51 (0.19)
0.07 (0.03)
0.2 (0.04)
–
0.03– 5.36
0.02– 4.82
0.37– 1.28
0.13– 2.24
0.13– 1.82
0.74– 2.56
0.1–2.33
0.05– 1.18
1.19– 4.07
0.01– 0.28
0.03– 0.48
0.14– 0.98
0.02– 0.13
0.17– 0.22
–
SD is the standard deviation.
a
ER-20% means that the relative error of ER was less than 20%, ER ¼ (þ)-isomer concentration/(2)-isomer concentration; relative error of ER ¼ (uncertainty
of ER/ER) 100% (Shen et al., 2006).
Comparison of ER in paired milk and placenta samples
For a-HCH, ER deviated more from the racemic ratio (ER ¼ 1)
in human milk than in placenta (Danish samples: average milk
ER ¼ 0.59, average placenta ER ¼ 0.82, P , 0.0001, N ¼ 41;
Finnish samples: average milk ER ¼ 0.47, average placenta
ER ¼ 0.78, P , 0.0001, N ¼ 43). For c-HE, the ER observed
in the placenta deviated more from the racemic ratio than the
ER observed in milk, although the differences did not reach
statistical significance in the Finnish milk – placenta paired
samples (Danish samples: average milk ER ¼ 1.95, average
placenta ER ¼ 2.24, P ¼ 0.018, N ¼ 40; Finnish samples:
average milk ER ¼ 1.98, average placenta ER ¼ 2.13, P ¼
0.19, N ¼ 31). The ERs of OXC were nearly the same in the
paired placenta and milk samples (Danish samples: average
Figure 1: The ER of (þ)-alpha-HCH and (2)-alpha-HCH in Danish
breast milk (DM), Finnish breast milk (FM) and in Danish placentas
(DP) plotted against the concentration of (2)-alpha-HCH in the
samples.
milk ER ¼ 1.30, average placenta ER ¼ 1.37, P ¼ 0.37, N ¼ 38;
Finnish samples: average milk ER ¼ 1.39, average placenta
ER ¼ 1.23, P ¼ 0.14, N ¼ 28).
Discussion
Our study revealed significant geographical differences in the
levels of organochlorine compounds in human placenta and
milk samples between two Nordic countries. Danish samples
had higher concentrations than Finnish samples for most compounds. This country difference remained highly significant
after adjustment for possible confounders. The findings
suggest that despite close vicinity and comparable lifestyles,
exposure may differ significantly between regions. The levels
of p,p0 -DDE, p,p0 -DDT and HCB in human milk from
Sweden, another Nordic country located between Denmark
and Finland, have been reported (Noren and Meironyte,
Figure 2: The ER of (þ)-c-HE and (2)-c-HE in Danish breast milk
(DM), Finnish breast milk (FM), Danish placentas (DP), and Finnish
placentas (FP) plotted against the concentration of (þ)-c-HE in the
samples.
207
Shen et al.
2000). The levels reported in the Swedish human milk samples
(collected in 1997) were comparable with the levels we
observed in the Danish milk (collected between 1997 and
2001).
Several factors, e.g. maternal age, parity and length of previous lactation, have been reported to affect the exposure of
the infant (Harris et al., 2001). In our study, pollutant levels
in human milk and placenta decreased with increasing parity
for the majority of the eight most prevalent compounds. This
provides additional evidence that previous deliveries and lactation may contribute to the clearance of persistent pollutants
from the mothers’ fatty stores. The concentration of the most
prevalent pollutants in milk and placenta was positively correlated to maternal age, which may reflect differences in
exposure through dietary habits, metabolism or bioaccumulating properties (Brunetto et al., 1996; Czaja et al., 1997; Covaci
et al., 2002).
It has been reported that the clearance rates for DDT, dichloro-di-phenyl-trichloro-ethene (DDE), dieldrin and HCB
were 20.162, 20.113, 20.117 and 20.118 year21, respectively (Noren and Meironyte, 2000). We also observed that
the levels of many of the measured pollutants were negatively
correlated with the date of infant delivery. Additionally, we
found slightly decreasing levels of OCs during the brief study
period, in line with other reports (Smith, 1999; Solomon and
Weiss, 2002). The correlations between maternal BMI and pollutant levels were not consistent and it remains to be determined whether these associations with maternal BMI are
genuine or chance findings due to mass significance.
The observed significant negative correlation of gestational
age with the concentrations of c-HE, END-I and dieldrin in
milk samples suggests that a shorter gestational period may
lead to a higher mobilization of chemicals stored in maternal
fat tissue during breastfeeding instead of during the third trimester when fetal fat accumulation is greatest. For placental,
samples gestational age was significantly and positively associated only with HCB. We suggest that in parallel to the rapid
increase in fetal fat stores from around gestational week 20,
more pollutants may be released from the mother’s adipose
depots and transferred to the fetus via placental regulation of
fatty acid delivery (Haggarty, 2002). Infants born preterm
may be less exposed than mature babies to persistent compounds during pregnancy, but then their exposure during
breastfeeding may be higher. Thus, the overall exposure
appears to be predominantly related to the prenatal maternal
body burden. These results should, however, be interpreted
with caution as only one of the eight most abundant compounds
in placenta correlated with gestational age and thus this may
also be a chance finding.
Although the lipid contents measured were in the normal
range compared with previously reported data on lipid
content in placenta (1 – 1.5%) and milk (1 – 6%) (DeKoning
and Karmaus, 2000), we observed a significantly higher lipid
content in Finnish than in Danish samples. Despite methodological differences in breast milk sample collection periods
and timing between the two countries, the nearly normal
lipid distributions suggest that the selection was random. It
has been reported that maternal nutritional status, including
208
long-term dietary habits and current diet (Harzer et al., 1984;
Specker et al., 1987), as well as obese or lean body composition
(Rocquelin et al., 1998; Anderson et al., 2005; Marin et al.,
2005) affect the lipid content of breast milk. The content and
composition of milk lipids is mainly determined by three
sources of fatty acids: diet, mobilization of body fat depots
and de-novo synthesis of fatty acids by the mammary gland
(Ortiz-Olaya et al., 1996). Thus, our observation may reflect
differences in dietary habits between the two countries.
We have also considered the possible dilution effect of the
fat content on OCs. However, the linear regression models
showed that lipid concentration had no significant correlation
with the OC concentrations in milk. Along with milk
samples, the placenta samples, which have lower fat concentrations, also showed lower OC concentrations for the more
persistent compounds. Furthermore, concerning the most abundant compounds, the OC concentrations in placenta samples
correlated with OC concentrations in milk samples (Shen
et al., 2007). This indicates that lipophilic compounds tend to
be concentrated in tissues that have a higher fat concentration.
In addition, the lipid content of placenta and milk is much
lower than the total lipid burden in the adipose tissue of the
mother, thereby reflecting only a minute fraction of the total
lipid content relevant to the establishment of equilibrium.
Therefore, we think that the country differences in OC concentrations are unlikely to reflect differences in the lipid concentration of the samples.
The fatty acid composition in placenta is determined by the
fatty acid content of maternal liver and plasma (Graham et al.,
2004). The negative correlation between gestational age and
placental lipid concentration found in our study may be the
result of the exponentially increasing fatty acid delivery to
the fetus from week 20, which decreases the storage of lipid
between the microvillous membrane and the basal membrane
of the placenta (Haggarty, 2002).
The ERs of chiral compounds are independent of physical
processes (leaching, volatilization and atmospheric deposition)
and abiotic reactions (hydrolysis and photolysis) (Bidleman
and Falconer, 1999), but are sensitive to biotransformation or
biodegradation because these are enantiomeric selective processes. The present ER-concentration patterns in Danish placental samples as well as in Danish and Finnish milk samples
were similar to previously reported results in Finnish placental
samples (Shen et al., 2006). No significant country differences
in ERs could be observed, indicating similar historical and
recent exposure patterns in the two countries, even though
the absolute levels differed.
Little is known about the variation of environmental ERs
(ERen). Our data suggest that ERen may be close to 1 for
a-HCH and 1.5 for c-HE because ERs measured in samples
with high levels approached these values. ERs close to the presumed ERen at low levels may indicate a recent exposure,
whereas ERs deviating from the presumed ERen may indicate
historical exposure (Shen et al., 2006). The abnormal ER
values for a few samples may reflect a special exposure
source or different enzyme activity of the individual mother.
ERs close to the presumed ERen at low levels may indicate
recent low-level exposure, whereas ERs deviating from the
Infant exposure to persistent organohalogen compounds
presumed ERen may indicate release of historical exposures
stored in adipose depots. The abnormal ER values for a few
samples may reflect a special exposure source or different
enzyme activity of the individual mother.
Tissue-specific ERs for certain chiral pollutants can result
from different enzyme activities (e.g. in kidney and liver) or
enantiomeric selective enrichment (e.g. in the blood – brain
barrier-protected brain) (Kallenborn and Hühnerfuss, 2001).
For c-HE, there was a linear correlation between the absolute
concentrations measured in milk and placenta (Shen et al.,
2007), indicating a balanced distribution of c-HE between the
two tissues. However, the ERs for c-HE deviated more from
the racemic ER in the placenta than in milk indicating that
the placenta contributes to the metabolism of this compound.
This supports the assumption that placenta can act as a filter
towards the fetus by metabolizing foreign compounds. In contrast, ERs for a-HCH found in the placenta was close to the
ERen and it seemed to be metabolized more in breast tissue.
The ERs of OXC may be the mixed results of the metabolism
of c-CHL and t-CHL. Both of them are metabolized to OXC,
but to the racemate in the former and to an optically active
form in the latter case (Buser and Müller, 1992).
PBBs can be transferred across the human placenta (Eyster
et al., 1983; Jacobson et al., 1984). The widely used commercial hexaBBs (HxBB) constitute 87% of the total PBBs and
BB-153 is the principal component (60%) of HxBB,
whereas BB-155 only contributed 0.5% (Hardy, 2002;
EHC 152, 1994). It was therefore expected that BB-153 was
the most abundant PBB congener in our study. In some
samples, the concentrations of BB-155 were higher than
expected. Comparable levels of BB-155 have previously
been reported from Europe, whereas data from USA report
much lower and generally undetectable levels of BB-155
(EHC 152, 1994; de Boer et al., 1998; Luross et al., 2002). It
has been suggested that photochemical debromination of
DBB may be a source for environmental BB-155 (EHC 152,
1994). Thus, the difference in BB-155 levels between Europe
and USA may be due to the more recent cessation of production
of OBB and DBB in Europe compared with the USA (Hardy,
2002). On the basis of our observed ratios of BB-155/
BB-153, we suggest that BB-153 is more readily metabolized
than BB-155 (Hakk and Letcher, 2003) in the human placenta.
In conclusion, multiple factors contribute to the pre- and
post-natal exposure of infants via exposure of the mother to
persistent organochlorines. Our study confirms that maternal
age is positively, and parity negatively, correlated with
higher concentrations of persistent organochlorines. In
addition, in this bi-national Nordic study, we found a significant country difference. This indicates that environmental
and lifestyle differences between these two countries exist,
leading to a higher exposure of the Danish population.
Recently, an association between congenital cryptorchidism
and the combined exposure to the eight most common organochlorines in human milk was indicated (Damgaard et al., 2006).
In the present study, the observed country difference in
exposure levels may have been affected by the selection of
samples from cryptorchid and healthy boys. The prevalence
of cryptorchidism in this subsample is not representative of
the true population prevalence (Boisen et al., 2004), neither
is the distribution of cases and controls exactly 50:50.
However, the observed exposure difference for organochlorines may play a role for the higher prevalence of male reproductive problems such as testicular cancer, decreased sperm
count and cryptorchidism in Denmark when compared with
Finland.
Acknowledgements
We are grateful for the help of Terttu Vartiainen and Hannu Kiviranta
(National Public Health Institute, Kuopio, Finland) for organizing the
homogenization of the placentas and Gerda Krog Mortensen (University Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark) for the homogenization of the milk samples. The
Nordic Cryptorchidism Study Group contributed to the sample collection: Drs Kirsten A. Boisen, Marla Chellakooty and Ida M. Schmidt
from Copenhagen, Denmark and Marko Kaleva, Anne-Maarit
Suomi from Turku, Finland. This work was supported by the European
Commission (QLK4-1999-01 422, QLK4-CT-2001-00 269 and
QLK4-CT-2002-00 603), The Novo Nordisk Foundation, The
Danish Medical Research Council (9700833, 9700909), The Svend
Andersen’s, Lundbeck and Velux Foundations, The Sigrid Juselius
Foundation, The Academy of Finland and Turku University Central
Hospital. The sponsors had no part in study design, data collection,
analysis, interpretation or writing of the manuscript. The article does
not represent the opinion of the European Commission, which is not
responsible for any use that might be made of data appearing
therein. The authors declare that they have no competing financial
interest.
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Submitted on March 7, 2006; resubmitted on February 16, 2007; accepted on
May 31, 2007