TRANSFER OF PHTHALATE MONOESTERS ACROSS THE HUMAN PLACENTA

1
TRANSFER OF PHTHALATE MONOESTERS ACROSS THE HUMAN
PLACENTA

• Tina Mose (1), Gerda K. Mortensen (2) and Lisbeth
  E. Knudsen (1).
• 1. Institute of Public Health, Department of
  Environmental Health, Copenhagen University,
  Denmark 2. University Department of Growth and
  Reproduction, Rigshospitalet, Copenhagen.

Fig 3. Schematic drawing of the human placenta.
It consist of cotyledons with closed fetal
circulations surrounded by intervillous space.
Maternal arteries from supplies the intervillous
space with blood.
Introduction. Some phthalates and their
metabolites are reproductive and fetal
developmental toxicants in animals. However, the
adverse effects in humans are still not fully
documented. Phthalates are widely used in
consumer products from where they transmit to the
surrounding environment leading to everyday
exposure. Phthalates are rapidly hydrolysed to
phthalate monoesters increasing the water
solubility and urinary excretion (Fig 1). In
several studies phthalates, phthalate monoesters
and oxidized metabolites are used as biomarkers
of internal exposure of phthalates. These
biomarkers are found in human urine, serum,
umbilical cord blood, breast milk, amniotic fluid
and their distribution have provided knowledge
about the internal exposure and metabolic
pathways involved in the excretion of phthalates.
As most concern regards the developing (male)
fetus, the objective of this study was to use our
human placental perfusion system to study the
placental transfer of selected phthalate
monoesters monomethyl phthalate (mMP) and
mono(2-ethylhexyl) phthalate (mEHP).
Fig 2. The dual
re-circulating human
placental perfusion system.
Maternal side
Fetal side
Human placental perfusion method. Immediately
after birth, the fetal circulation in a single
cotyledon was re-established by cannulation of
the fetal vein and artery (Fig 23). The
cotyledon was placed in the perfusion chamber,
with maternal side up. Maternal arteries were
connected to the intervillous space. Perfusion
media was Krebs Ringer-buffer added Heparin and
Dextran. Antipyrine (100µg/mL) and mMP (10µg/L)
or mEHP (25µg/L) were added to the maternal
reservoir and samples collected from both
circulations during 2.5 hour perfusions.
Antipyrine was used as a positive control of
proper connection between the established
maternal and fetal circulation. To limit
contamination, all utensils were made of glass
and were flushed with MeOH before use. Tubes and
dispenser tibs were made of polypropylene,
PVC-free, and latex or nitril gloves were used.
Analysis of phthalate monoesters. Proteins were
precipitated by centrifugation (4000 g, 5min) and
degradation of phthalate diesters were avoided by
addition of 1.2 M H3PO4 (110). Sample
preparation consisted of  two solid phase
extractions.  Analysis of phthalate monoesters
was accomplished using high pressure liquid
chromatography with triple quadrupole mass
spectrometer detection (LC-MS-MS).
Fig 1.
Hydrolysis of phthalates to
phthalte monoesters. A.
Dimethyl
phthalate (DMP) to monomethyl
phthalate (mMP), B. Di
(2-ethylhexyl)

phthalate (DEHP) to mono(2-
ethylhexyl) phthalate
(mEHP). An
alcohol is also formed
during the
hydolysis (not shown)
A
Hydrolysis
B
Hydrolysis
Fig 6. mMP and mEHP in placental tissue
µg/kg). Tissue samples from before perfusion
(semi-white, n8) and perfused cotyledon (grey
and black) is illustrated. The grey columns are
without perfusion with actual phthalate monoester
(mMP n4, mEHP n4) while the black columns
represent the level after perfusion with actual
phthalate monoester (n4). mMP is only detectable
(LOD0.05µg/kg) in one sample before and one
sample after perfusion without mMP.
Results. All together 14 placentas were perfused
each for 150 minutes. Eight perfusions succeeded
according to our criteria constant venous
outflow, leakage from fetal circulation lt 20 mL,
FM ratio of antipyrine diffusion gt 0.75, pH
7.2-7.5, time from birth to laboratory lt 30
minutes, uptake of oxygen in the placenta,
temperature 35-39ºC. The eight reported
perfusions consisted of four perfusion with mMP
and four with mEHP. Placental transfer. The
mean fetal/maternal concentration ratio of
antipyrine (FM ratio) was 0.85 0.07 and 0.30
0.03 for mMP (Fig. 4). The mean recovery of mMP
in fetal and maternal perfusion media after
perfusion compared to added to maternal perfusion
media in the beginning of the perfusion was 8.1
0.37µg/L (80.9 3.7). mEHP (25 µg/L) was not
detectable (LOD0.5µg/L) in all fetal perfusion
media and therefore, the maternal and fetal
concentrations are illustrated separately instead
of the FM ratio (Fig 5). In two perfusions, 1.8
µg/L and 3.0 µg/L mEHP was found after end
perfusion given rise to FM ratios of 0.088 and
0.20, respectively. These two perfusions had
higher FM ratios of antipyrine transfer and
larger leak of perfusion media from fetal to
maternal circulation.
Cord plasma and placenta tissue Seven plasma
samples from umbilical cord blood were analysed.
A mean level of 9.4 4.8 µg/L mEHP was found.
mEHP was detcetable in all placenta samples
whereas mMP only was detected in one before
perfusion (Fig. 6). The level of mMP increased by
nearly 10-fold in the perfusions where mMP was
added compared to the other four perfusions where
mEHP was added indicating an accumulation of mMP
in placental tissue. The accumulated level in
tissue represents 42 of the added mMP. After end
perfusion, mEHP was increased from 8.5 to 15.3
µg/kg even though the compound was not added to
the system The accumulated level of mEHP was
around 15µg/kg in the two different spike studies
signifying that the tissue is saturated with
mEHP. No association was found between levels of
mEHP placenta tissue and umbilical cord
plasma. Affinity and exchange to the perfusion
system. The affinity of mMP (10 µg/L) and mEHP
(25µg/L) to the perfusion system was tested using
the maternal circulation through an empty
perfusion chamber. The affinity was insignificant
because the concentration was vaguely increasing
for mEHP (y 0.0048x 22.1) and vaguely
decreasing for mMP (y 0.0063x 8.2) during the
150 minutes test (Fig. 7).
Fig 4. The mean
feto-maternal
concentration ratio (FM ratio) of antipyrine
(black, n8) and mMP
(grey, n4) during
perfusions lasting 150 minutes. When the
ratio is one equal amount of
compound is found in
fetal and maternal circulation.
Fig 5. The mean
concentration of mEHP
from maternal circulation (grey) and fetal
circulation (black) during
150 minutes of human
placental perfusion. The initial
concentration in maternal perfusion
medium is 25µg/L
Fig 7. Time-dependent
exchange of mMP (grey) and
mEHP (black) with an empty
perfusion system during 150 minutes
circulation in maternal system connected
to perfusion chamber.The
initial concen-
tration in maternal perfusion medium is
25µg/L mEHP and 10µg/L mMP.
Conclusion. Our data suggest a slow transfer of
mMP across the placenta, no detectable levels in
umbilical cord blood, and very low levels in
placental tissue. However, mMP is accumulated in
placental tissue during perfusions with mMP.
For mEHP, our data suggest no placental transfer
because the levels found in fetal perfusion
medium was close to LOD. Detectable level was
found in umbilical cord blood, and in placental
tissue. No different accumulation tendencies were
observed in the mEHP spike studies compared to
the mMP spike studies.
Acknowledgements Financially support from
EU-project CHILDRENGENONETWORK
(QLK4-CT-2202-02198) and Danish Ministry of the
Interior and Health Research Centre for
Environmental Health (ISMF, J.nr 0302-02-9/5).
The authors gratefully acknowledge collaboration
with the Maternity Unit at the Danish National
Hospital and Dr. Morten Hedegaard.