Line Mathiesen

Urinary 1-hydroxypyrene (1-HP) in environmental and occupational studies--a review.

A total of 132 studies were identified, of which 25 studies addressed environmental exposure to polycyclic aromatic hydrocarbons (PAHs) in the general population. Of these 9 studies included children. Of 101 studies among workers in various occupations, 32 studies were in petrochemical industries, 29 studies in foundries, 14 studies in asphalt work, 21 studies working in urban air, 6 studies in combustion, 3 studies in soil remediation, and 2 studies in printing. Environmental and occupational studies were identified through an extensive search of the PubMed database up to November 2006. The aim of this review was to provide an overview of studies using urinary 1-hydroxypyrene (1-HP) as a biomarker of environmental and occupational exposure to PAHs. Hereby, we aim to support the current validation of 1-HP by summarizing the evidence of specific PAH exposure and1-HP associations in humans. Urine from more than 7000 children from 8 countries and 3400 adults from 9 countries were included in the 25 studies assessing environmental exposure to PAH. Among the occupational studies 7500 men and women gave urine samples to assess occupational PAH exposure. When measuring biomarkers in urine, volume (and time) or concentrations of creatinine are often used to standardize for diuresis. To be able to compare the included studies we recalculated the given concentrations to mumol 1-HP/mol creatinine. In conclusion, the highest concentrations of urinary 1-HP are found among workers in petrochemical industries among coke-oven workers. Occupational PAH exposure was the major factor determining high urinary concentration of 1-HP. The highest concentrations were observed among workers in the petrochemical industry at work places in Taiwan and China. To evaluate environmental and low-level occupational exposure to PAH it is crucial to provide knowledge on intra- and inter-individual variation in the evaluation of 1-HP. The highest contribution originates from environmental tobacco smoke, but also different country, cooking culture, and behavior influences urinary of 1-HP.

Placental transport and in vitro effects of Bisphenol A

Bisphenol A (BPA), an estrogen-like chemical, leaches from consumer products potentially causing human exposure. To examine the effects of BPA exposure during pregnancy, we performed studies using the BeWo trophoblast cell line, placental explant cultures, placental perfusions and skin diffusion models, all of human origin. Results showed BPA cytotoxicity in BeWo cells with an apparent EC50 at 100-125 microM. BPA exposure significantly increased beta-hCG secretion and caspase-3 expression in placental explants at an environmentally relevant concentration of 1 nM. In the transport studies, a rapid transfer of BPA was observed across the term placentae and the BeWo cell monolayer. Further, transdermal transport of BPA was observed. These results indicate that fetal BPA exposure through placental exchange occurs with potential adverse implications for placental and fetal development. This battery of test systems within the realm of human implantation and fetal development represents important elements in risk assessment of reproductive toxicity.

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.

Adhesion of Plasmodium falciparum infected erythrocytes in ex vivo perfused placental tissue: a novel model of placental malaria.

BackgroundPlacental malaria occurs when Plasmodium falciparum infected erythrocytes sequester in the placenta. Placental parasite isolates bind to chondroitin sulphate A (CSA) by expression of VAR2CSA on the surface of infected erythrocytes, but may sequester by other VAR2CSA mediated mechanisms, such as binding to immunoglobulins. Furthermore, other parasite antigens have been associated with placental malaria. These findings have important implications for placental malaria vaccine design. The objective of this study was to adapt and describe a biologically relevant model of parasite adhesion in intact placental tissue.ResultsThe ex vivo placental perfusion model was modified to study adhesion of infected erythrocytes binding to CSA, endothelial protein C receptor (EPCR) or a transgenic parasite where P. falciparum erythrocyte membrane protein 1 expression had been shut down. Infected erythrocytes expressing VAR2CSA accumulated in perfused placental tissue whereas the EPCR binding and the transgenic parasite did not. Soluble CSA and antibodies specific against VAR2CSA inhibited binding of infected erythrocytes.ConclusionThe ex vivo model provides a novel way of studying receptor-ligand interactions and antibody mediated inhibition of binding in placental malaria.

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

BackgroundPolybrominated 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.MethodsA 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 DiscussionPlacental 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.ConclusionThe 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.

Transport of Benzo[α]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.

Meta-analysis of data from human ex vivo placental perfusion studies on genotoxic and immunotoxic agents within the integrated European project NewGeneris

In the E.U. integrated project NewGeneris, we studied placental transport of thirteen immunotoxic and genotoxic agents in three ex vivo placental perfusion laboratories. In the present publication, all placental perfusion data have been re-analyzed and normalized to make them directly comparable and rankable. Antipyrine transfer data differed significantly between the studies and laboratories, and therefore normalization of data was necessary. An antipyrine normalization factor was introduced making the variance significantly smaller within and between the studies using the same compound but performed in different laboratories. Non-normalized (regular) and normalized data showed a good correlation. The compounds were ranked according to their transplacental transfer rate using either antipyrine normalized AUC120 or transfer index (TI120(%)). Normalization generated a division of compounds in slow, medium and high transfer rate groups. The transfer rate differed slightly depending on the parameter used. However, compounds with passage similar to antipyrine which goes through the placenta by passive diffusion, and good recovery in media (no accumulation in the tissue or adherence to equipment) were highly ranked no matter which parameter was used. Antipyrine normalization resulted in the following ranking order of compounds according to AUC(120NORM) values: NDMA ≥ EtOH ≥ BPA ≥ IQ ≥AA ≥ GA ≥ PCB180 ≥ PhIP ≥ AFB1 > DON ≥ BP ≥ PCB52 ≥ TCDD. As the variance in all parameters within a study decreased after antipyrine normalization, we conclude that this normalization approach at least partially corrects the bias caused by the small methodological differences between studies.

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.

Kinetics of silica nanoparticles in the human placenta

Abstract 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.

Preliminary interlaboratory comparison of the ex vivo dual human placental perfusion system

As a part of EU-project ReProTect, a comparison of the dual re-circulating human placental perfusion system was carried out, by two independent research groups. The detailed placental transfer data of model compounds [antipyrine, benzo(a)pyrene, PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine) and IQ (2-amino-3-methylimidazo(4,5-f)quinoline] has been/will be published separately. For this project, a comparative re-analysis was done, by curve fitting the data and calculating two endpoints: AUC(120), defined as the area under the curve between time 0 and time 120 min and as t(0.5), defined as the time when the fetal to maternal concentration ratio is expected to be 0.5. The transport of the compounds from maternal to fetal circulation across the perfused placenta could be ranked in the order of antipyrine>IQ>PhIP in terms of both t(0.5) and AUC(120) by both partners. For benzo(a)pyrene the curve fitting failed. These prevalidation results give confidence for harmonization of the placental perfusion system to be used as one of the test methods in a panel for reproductive toxicology to model placental transfer in humans.