Placental transfer of pesticides studied in human placental perfusion.

To investigate the transplacental transport of pesticides, the pyrethroid cypermethrin and the fungicide azoles, propiconazole and bitertanol were tested in the placental perfusion model. Cypermethrin, propiconazole and bitertanol were also tested in the BeWo cell transfer model. The pesticides were chosen with the selection criteria: use in Denmark, significant treated areas and knowledge on hormone-disrupting effects. Propiconazole and bitertanol showed rapid transfer and adsorbance to the system in both placental perfusion and BeWo cell system, whereas cypermethrin had a slower transport across the placental cell layers in the two models. There was no difference between data of the single pesticides and their mixture in either placental perfusion or BeWo cell transfer model. Both the placental perfusion model and the BeWo cell model metabolized the pesticides and released metabolites into both foetal and maternal circulation. Using human exposure models, this study shows the potential exposure of the human foetus to pesticides cypermethrin, propiconazole and bitertanol and their metabolites.

Therapeutic Drug Monitoring of Lithium: A Study of the Accuracy and Analytical Variation Between Laboratories in Denmark.

BACKGROUND: Serum lithium is monitored to ensure levels within the narrow therapeutic window. This study examines the interlaboratory variation and inaccuracy of lithium monitoring in Denmark. METHODS: In 16 samples consisting of (1) control materials (n = 4), (2) pooled patient serum (n = 5), and (3) serum from individual patients (n = 7), lithium was measured in 19 laboratories using 20 different instruments. The lithium concentrations were targeted by a reference laboratory. Ion-selective electrode (n = 5), reflective spectrophotometric (RSM, n = 5), and spectrophotometric (n = 10) methods were used. RESULTS: Acceptable accuracy-interpreted as total differences from target values (bias) less than or equal to ±12%-was generally found in patient samples above 0.7 mmol/L. Below 0.7 mmol/L, 8 instruments had 2 or more patient samples exceeding a difference to the targeted reference value of >12%. Seven of these instruments had a systematic positive or negative bias and more so at lower lithium concentrations. Three poorly calibrated instruments were found in the ion-selective electrode group, 3 in the spectrophotometric group, and 2 in the RSM group. The instruments using reflectance spectrophotometry (RSM) were on average 21% positively biased when measuring control materials. However, this effect was not observed in patient samples. CONCLUSIONS: Large interlaboratory variation was found below 0.7 mmol/L because of 7 instruments with a poor accuracy and 1 with poor precision. Methods should be recalibrated or substituted. Controls below 0.7 mmol/L are recommended.

Kinetics of silica nanoparticles in the human placenta.

The potential medical applications of nanoparticles (NPs) warrant their investigation in terms of biodistribution and safety during pregnancy. The transport of silica NPs across the placenta was investigated using two models of maternal-foetal transfer in human placenta, namely, the BeWo b30 choriocarcinoma cell line and the ex vivo perfused human placenta. Nanotoxicity in BeWo cells was examined by the MTT assay which demonstrated decreased cell viability at concentrations >100 µg/mL. In the placental perfusion experiments, antipyrine crossed the placenta rapidly, with a foetal:maternal ratio of 0.97 ± 0.10 after 2 h. In contrast, the percentage of silica NPs reaching the foetal perfusate after 6 h was limited to 4.2 ± 4.9% and 4.6 ± 2.4% for 25 and 50 nm NPs, respectively. The transport of silica NPs across the BeWo cells was also limited, with an apparent permeability of only 1.54 × 10(-6) ± 1.56 × 10(-6) cm/s. Using confocal microscopy, there was visual confirmation of particle accumulation in both BeWo cells and in perfused placental tissue. Despite the low transfer of silica NPs to the foetal compartment, questions regarding biocompatibility could limit the application of unmodified silica NPs in biomedical imaging or therapy.

Modelling of human transplacental transport as performed in Copenhagen, Denmark.

Placenta perfusion models are very effective when studying the placental mechanisms in order to extrapolate to real-life situations. The models are most often used to investigate the transport of substances between mother and foetus, including the potential metabolism of these. We have studied the relationships between maternal and foetal exposures to various compounds including pollutants such as polychlorinated biphenyls, polybrominated flame retardants, nanoparticles as well as recombinant human antibodies. The compounds have been studied in the human placenta perfusion model and to some extent in vitro with an established human monolayer trophoblast cell culture model. Results from our studies distinguish placental transport of substances by physicochemical properties, adsorption to placental tissue, binding to transport and receptor proteins and metabolism. We have collected data from different classes of chemicals and nanoparticles for comparisons across chemical structures as well as different test systems. Our test systems are based on human material to bypass the extrapolation from animal data. By combining data from our two test systems, we are able to rank and compare the transport of different classes of substances according to their transport ability. Ultimately, human data including measurements in cord blood contribute to the study of placental transport.

Placental transfer of the polybrominated diphenyl ethers BDE-47, BDE-99 and BDE-209 in a human placenta perfusion system: an experimental study.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants in consumer products. PBDEs may affect thyroid hormone homeostasis, which can result in irreversible damage of cognitive performance, motor skills and altered behaviour. Thus, in utero exposure is of very high concern due to critical windows in fetal development. METHODS: A human ex vivo placenta perfusion system was used to study the kinetics and extent of the placental transfer of BDE-47, BDE-99 and BDE-209 during four-hour perfusions. The PBDEs were added to the maternal circulation and monitored in the maternal and fetal compartments. In addition, the perfused cotyledon, the surrounding placental tissue as well as pre-perfusion placental tissue and umbilical cord plasma were also analysed. The PBDE analysis included Soxhlet extraction, clean-up by adsorption chromatography and GC-MS analysis. RESULTS AND DISCUSSION: Placental transfer of BDE-47 was faster and more extensive than for BDE-99. The fetal-maternal ratios (FM-ratio) after four hours of perfusion were 0.47 and 0.25 for BDE-47 and BDE-99, respectively, while the indicative permeability coefficient (IPC) measured after 60 minutes of perfusion was 0.26 h-1 and 0.10 h-1, respectively. The transport of BDE-209 seemed to be limited. These differences between the congeners may be related to the degree of bromination. Significant accumulation was observed for all congeners in the perfused cotyledon as well as in the surrounding placental tissue. CONCLUSION: The transport of BDE-47 and BDE-99 indicates in utero exposure to these congeners. Although the transport of BDE-209 was limited, however, possible metabolic debromination may lead to products which are both more toxic and transportable. Our study demonstrates fetal exposure to PBDEs, which should be included in risk assessment of PBDE exposure of women of child-bearing age.

Dioxin-like exposures and effects on estrogenic and androgenic exposures and micronuclei frequency in mother-newborn pairs.

In utero exposure to environmental dioxin-like, estrogen and androgen compounds can cause adverse health effects. Little is known about potential interactions in vivo between dioxin-like compounds, estrogens and androgens during fetal development in humans. Therefore we explored the potential interactions in vivo between dioxin-like compounds, estrogens, androgens using chemical-activated luciferase expression (CALUX)(R) bioassays in maternal and umbilical cord blood plasma concurrently collected at the time of planned Caesarean section from 98 healthy pregnancies. The dioxin-like activity was also determined after placental transfer of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the ex vivo human placenta perfusion system. Similar dioxin-like activity in maternal and cord blood (37 versus 33pg CALUX(R)-TEQ/g plasma lipids, P>0.05) was detected and it demonstrates transplacental transfer. Increased dioxin-like activity in the perfused placenta tissue after ex vivo TCDD perfusions (from 17 to 280pg CALUX(R)-TEQ/g plasma lipids) suggest that accumulation in the placenta prevents immediate transplacental transfer of TCDD. Androgenic activity were also similar in the paired mother-newborns (0.10 versus 0.18ng CALUX(R)-AEQ/mL plasma), whereas cord blood plasma estrogenic activity was higher than maternal levels (22.6 versus 18.5ng CALUX(R)-EEQ/mL plasma). In cord blood plasma androgenic activity was strongly positively associated with maternal levels (Rs=0.8, P<0.001) whereas dioxin-like and estrogenic activities were modestly associated with maternal levels (Rs

Quality assessment of a placental perfusion protocol.

Validation of in vitro test systems using the modular approach with steps addressing reliability and relevance is an important aim when developing in vitro tests in e.g. reproductive toxicology. The ex vivo human placental perfusion system may be used for such validation, here presenting the placental perfusion model in Copenhagen including control substances. The positive control substance antipyrine shows no difference in transport regardless of perfusion media used or of terms of delivery (n=59, p<0.05). Negative control studies with FITC marked dextran correspond with leakage criteria (<3 ml h(-1) from the fetal reservoir) when adding 2 (n=7) and 20mg (n=9) FITC-dextran/100 ml fetal perfusion media. Success rate of the Copenhagen placental perfusions is provided in this study, including considerations and quality control parameters. Three checkpoints suggested to determine success rate revealed that 15% of the cannulated placentae received in one year (n=202) were successfully perfused.

Modeling placental transport: correlation of in vitro BeWo cell permeability and ex vivo human placental perfusion.

The placental passage of three compounds with different physicochemical properties was recently investigated in ex vivo human placental perfusion experiments (caffeine, benzoic acid, and glyphosate) [Mose, T., Kjaerstad, M.B., Mathiesen, L., Nielsen, J.B., Edelfors, S., Knudsen, L.E., 2008. Placental passage of benzoic acid, caffeine, and glyphosate in an ex vivo human perfusion system. J. Toxicol. Environ. Health, Part A 71, 984-991]. In this work, the transport of these same three compounds, plus the reference compound antipyrine, was investigated using BeWo (b30) cell monolayers. Transport across the BeWo cells was observed in the rank order of caffeine>antipyrine>benzoic acid>glyphosate in terms of both the apparent permeability coefficient and the initial slope, defined as the linear rate of substance transferred to the fetal compartment as percent per time, a parameter used to compare the two experimental models. The results from the in vitro studies were in excellent agreement with the ex vivo results (caffeine approximately antipyrine>benzoic acid>glyphosate). However the transfer rate was much slower in the BeWo cells compared to the perfusion system. The advantages and limitations of each model are discussed in order to assist in the preparation, prediction, and performance of future studies of maternal-fetal transfer.

Transport of benzo[alpha]pyrene in the dually perfused human placenta perfusion model: effect of albumin in the perfusion medium.

Transport of benzo[alpha]pyrene (BaP) across the placenta was examined because it is a ubiquitous and highly carcinogenic substance found in tobacco smoke, polluted air and certain foods. Foetal exposure to this substance is highly relevant but is difficult to estimate. The human placenta is unique compared to other species; since it is available without major ethical obstacles, we have used the human placenta perfusion model to study transport from mother to foetus. Placentas were donated after births at Rigshospitalet in Copenhagen from pregnant mothers who signed an informed consent. BaP is lipophilic and studies using cell culture medium in 6-hr placenta perfusions showed minimal transport through the placenta. To increase the solubility of BaP in perfusion medium and to increase physiological relevance, perfusions were also performed with albumin added to the perfusion medium [2 and 30 mg/ml bovine serum albumin (BSA) and 30 mg/ml human serum albumin (HSA)]. The addition of albumin resulted in increased transfer of BaP from maternal to foetal reservoirs. The transfer was even higher in the presence of an HSA formulation containing acetyltryptophanate and caprylate, resulting in a foetal-maternal concentration (FM) ratio of 0.71 +/- 0.10 after 3 hr and 0.78 +/- 0.11 after 6 hr, whereas the FM ratio in perfusions without albumin was only 0.05 +/- 0.03 after 6 hr of perfusion. Less BaP accumulated in placental tissue in perfusions with added albumin. This shows that transplacental transport of the pro-carcinogenic substance BaP occurs, and emphasizes the importance of adding physiological concentrations of albumin when studying the transport of lipophilic substances.

Placental passage of benzoic acid, caffeine, and glyphosate in an ex vivo human perfusion system.

Ex vivo perfusion of the human term placenta is a method to study placental transfer without extrapolation from animal to human and with no ethical concerns for mother and child. However, ex vivo placenta perfusion has a limited potential within chemical screening and testing as the method is time-consuming. This study was an attempt to construct data needed to develop quantitative structure-activity relationship (QSAR) models that are able to predict placental transfer of new compounds. Placental transfer is a biological activity that statistically may be related to the physiochemical properties of a given group of compounds. Benzoic acid, caffeine, and glyphosate were chosen as model compounds because they are small molecules with large differences in physiochemical properties. Caffeine crossed the placenta by passive diffusion. The initial transfer rate of benzoic acid was more limited in the first part of the perfusion compared to caffeine, but reached the same steady-state level by the end of perfusion. The transfer of glyphosate was restricted throughout perfusion, with a lower permeation rate, and only around 15% glyphosate in maternal circulation crossed to the fetal circulation during the study period.

Participation in environmental health research by placenta donation - a perception study.

BACKGROUND: Much environmental health research depends on human volunteers participating with biological samples. The perception study explores why and how people participate in a placenta perfusion study in Copenhagen. The participation implies donation of the placenta after birth and some background information but no follow up. METHODS: Nineteen semi-structured qualitative interviews were conducted with participants in the placenta perfusion study after donation of placenta. Observation studies were made of recruitment sessions. RESULTS: The interviewed participants are generally in favour of medical research. They participated in the placenta perfusion study due to a belief that societal progress follows medical research. They also felt that participating was a way of giving something back to the Danish health care system. The participants have trust in medical science and scientists, but trust is something which needs to be created through "trust-work". Face-to-face interaction, written information material and informed consent forms play important parts in creating trusting relationships in medical research. CONCLUSION: Medical research ethics do not only amount to specific types of written information material but should also be seen as a number of trust making performances involving researchers as well as research participants.

The human placenta--an alternative for studying foetal exposure.

Pregnant women are daily exposed to a wide selection of foreign substances. Sources are as different as lifestyle factors (smoking, daily care products, alcohol consumption, etc.), maternal medication or occupational/environmental exposures. The placenta provides the link between mother and foetus, and though its main task is to act as a barrier and transport nutrients and oxygen to the foetus, many foreign compounds are transported across the placenta to some degree and may therefore influence the unborn child. Foetal exposures to environmental and medicinal products may have impact on the growth of the foetus (e.g. cigarette smoke) and development of the foetal organs (e.g. methylmercury and thalidomide). The scope of this review is to give insight to the placental anatomy, development and function. Furthermore, the compounds physical properties and the transfer mechanism across the placental barrier are evaluated. In order to determine the actual foetal risk from exposure to a chemical many studies regarding the topic are necessary, including means of transportation, toxicological targets and effects. For this purpose several in vivo and in vitro models including the placental perfusion system are models of choice.

Transplacental transfer of monomethyl phthalate and mono(2-ethylhexyl) phthalate in a human placenta perfusion system.

The transplacental passage of monomethylphtalate (mMP) and mono (2-ethylhexyl) phthalate (mEHP) was studied using an ex vivo placental perfusion model with simultaneous perfusion of fetal and maternal circulation in a single cotyledon. Umbilical cord blood and placental tissue collected both before and after perfusion were also analyzed. Placentas were obtained immediately after elective cesarean section and dually perfused in a recirculation system. mMP or mEHP was added to maternal perfusion medium to obtain concentrations at 10 and 25 microg/L, respectively. The placental transfer was followed analyzing samples from fetal and maternal perfusion media by liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS). Four perfusions with mMP indicated a slow transplacental transfer, with a feto-maternal ratio (FM ratio) of 0.30 +/- 0.03 after 150 min of perfusion. Four perfusions with mEHP indicated a very slow or nonexisting placental transfer. mEHP was only detected in fetal perfusion media from two perfusions, giving rise to FM ratios of 0.088 and 0.20 after 150 min of perfusion. Detectable levels of mMP, mEHP, monoethylphthalate (mEP), and monobutylphthalate were found in tissue. Higher tissue levels of mMP after perfusions with mMP compared to perfusions with mEHP suggest an accumulation of mMP during perfusion. No tendency for accumulation of mEHP was observed during perfusions with mEHP compared to perfusions with mMP. Detectable levels of mEHP and mEP were found in umbilical cord plasma samples. mMP and possibly other short-chained phthalate monoesters in maternal blood can cross the placenta by slow transfer, whereas the results indicate no placental transfer of mEHP. Further studies are recommended.

Phthalate monoesters in perfusate from a dual placenta perfusion system, the placenta tissue and umbilical cord blood.

Fetal exposure to phthalates may be associated with adverse reproductive effects, including cryptorchidism and decreased semen quality. Information about human placental transfer is needed to qualify the hypotheses. A dual recirculating placenta perfusion system to monitor concentrations of eight phthalate monoesters in fetal and maternal perfusates was established. In addition to perfusate background measures of phthalate monoesters, the concentrations in umbilical cord plasma and placenta tissue were measured. Monomethyl phthalate (mMP), monoethyl phthalate (mEP), monobutyl phthalate (mBP), and mono (2-ethyl-hexyl) phthalate (mEHP) were detected in both maternal and fetal perfusate, demonstrating a release of compounds from tissue or blood to perfusates. The distribution of compounds between perfusate, umbilical cord plasma, and tissue was in accordance with the physical-chemical properties of the compounds. Results from the present study of compounds residing in the tissue are essential before studying human transplacental transfer, storage, and metabolism of selected phthalate monoesters.