Vesa Karttunen

Transplacental transfer of acrylamide and glycidamide are comparable to that of antipyrine in perfused human placenta

Most drugs can penetrate the placenta but there are only a few studies on placental transfer of environmental toxic compounds. In this study, we used dual recirculating human placental perfusion to determine the transfer rate through the placenta of a neurotoxic and carcinogenic compound found in food, acrylamide and its genotoxic metabolite glycidamide. Putative acrylamide metabolism into glycidamide during the 4-h perfusions and acrylamide-derived DNA adducts in placental DNA after perfusions were also analyzed. Placentas were collected immediately after delivery and kept physiologically functional as confirmed by antipyrine kinetics, glucose consumption and leak from fetal to maternal circulation. Acrylamide (5 or 10 microg/ml) or glycidamide (5 microg/ml), both with antipyrine (100 microg/ml), was added to maternal circulation. Acrylamide and glycidamide were analyzed in the perfusion medium by liquid chromatography/mass spectrometry. Acrylamide and glycidamide crossed the placenta from maternal to fetal circulation with similar kinetics to antipyrine, suggesting fetal exposure if the mother is exposed. The concentrations in maternal and fetal circulations equilibrated within 2h for both studied compounds and with both concentrations. Acrylamide metabolism into glycidamide was not detected during the 4-h perfusions. Moreover, DNA adducts were undetectable in the placentas after perfusions. However, fetuses may be exposed to glycidamide after maternal metabolism. Although not found in placental tissue after 4h of perfusion, it is possible that glycidamide adducts are formed in fetal DNA.

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 transfer and DNA binding of benzo(a)pyrene in human placental perfusion

Benzo(a)pyrene (BP) is the best studied polycyclic aromatic hydrocarbon, classified as carcinogenic to humans. The carcinogenic metabolite, benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), binds covalently to DNA. The key enzyme in this metabolic reaction is CYP1A1, which has also been found in placenta and human trophoblastic cells. By using human placental perfusion we confirmed that BP added to the maternal circulation in concentrations of 0.1 and 1 microM reaches fetal compartment but somewhat slower than the freely diffusible reference substance antipyrine. A well-known P-glycoprotein (ABCB1/P-gp) antagonist verapamil did not affect the transfer more than it did in the case of antipyrine, indicating that ABCB1/P-gp does not have a role in BP transfer. In one of the two placentas perfused for 6 h with the higher concentration of BP (1 microM) BPDE specific DNA adducts were found in placental tissue after the perfusion, but not before. The ability of human trophoblastic cells to activate BP to BPDE-DNA adducts was confirmed in human trophoblastic BeWo cells. This study shows that maternal exposure to BP leads to the exposure of the fetus to BP and/or its metabolites and that placenta itself can activate BP to DNA adducts.

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.

Human placental cell and tissue uptake of doxorubicin and its liposomal formulations.

The anticancer drug doxorubicin and its liposomal formulations are in clinical use, doxorubicin also during pregnancy. However, little is known about how doxorubicin and its liposomal formulations are taken up by placental cells and whether they can cross human placenta. We therefore investigated quantitative cellular uptake and toxicity of doxorubicin and its two liposomal formulations, pH-sensitive liposomal doxorubicin (L-DOX) and commercially available pegylated liposomal doxorubicin (PL-DOX), in human placental choriocarcinoma (BeWo) cells. PL-DOX showed significantly lower cellular uptake and toxicity compared with doxorubicin and L-DOX. In preliminary studies with human placental perfusion, PL-DOX did not cross the placenta at all in 4h, whereas doxorubicin and L-DOX crossed the placenta at low levels (max 12% of the dose). Furthermore, PL-DOX did not accumulate in placental tissue while doxorubicin did (up to 70% of the dose). Surface pegylation probably explains the low placental cell and tissue uptake of PL-DOX. Formulation of doxorubicin thus seems to enable a decrease of fetal exposure.

The significance of ABC transporters in human placenta for the exposure of the fetus to xenobiotics

The placenta has an important role in transporting nutrients and oxygen to the fetus. Transplacental transport is, however, not restricted to physiological exchange of agents, but includes the majority, if not all, of xenobiotics in maternal blood. Although passive diffusion is the most common transfer mechanism, transporter proteins play a significant role in transplacental transfer. The largest superfamily of transporters and one of the most interesting groups regarding xenobiotics is the ATP-binding cassette (ABC) transporters. The efflux ABC transporters from families ABCB, ABCC and ABCG play a role in transporting xenobiotics and their conjugates in addition to endogenous compounds. Many of them probably play a role in the protection of the fetus, as is implicated in animal studies. Both genetic polymorphisms, endogenous regulation, e.g. by hormones, and xenobiotics can modify the expression and function of transporters. Interference in the function of transporters (e.g., inhibition) may significantly increase the fetal exposure to xenobiotics or drugs. Many different models can be used to study human placental ABC transporters. These include human placental perfusion, tissue preparations, tissue explants, primary cell cultures, immortalized cell lines, cancer cell lines, placental membrane vesicles and cloning. When estimating the contribution of an ABC transporter for fetal exposure localization has to be taken into account in addition to the level of expression, functional status and substrate specificity.

Acute effects of ethanol on the transfer of nicotine and two dietary carcinogens in human placental perfusion.

Many mothers use, against instructions, alcohol during pregnancy. Simultaneously mothers are exposed to a wide range of other environmental chemicals. These chemicals may also harm the developing fetus, because almost all toxic compounds can go through human placenta. Toxicokinetic effects of ethanol on the transfer of other environmental compounds through human placenta have not been studied before. It is known that ethanol has lytic properties and increases the permeability and fluidity of cell membranes. We studied the effects of ethanol on the transfer of three different environmental toxins: nicotine, PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine) and NDMA (N-nitrosodimethylamine) in placental perfusion. We tested in human breast cancer adenocarcinoma cell line MCF-7 whether ethanol affects ABCG2/BCRP, which is also the major transporter in human placenta. We found that the transfer of ethanol is comparable to that of antipyrine, which points to passive diffusion as the transfer mechanism. Unexpectedly, ethanol had no statistically significant effect on the transfer of the other studied compounds. Neither did ethanol inhibit the function of ABCG2/BCRP. These experiments represent only the effects of acute exposure to ethanol and chronic exposure remains to be studied.

Criteria and challenges of the human placental perfusion – Data from a large series of perfusions

Perfusion of human placental cotyledon has been used extensively to study transplacental transfer of endogenous and exogenous compounds. However, many challenges in the use of the method exist, including availability of placentas and complexity of the method itself. In Kuopio, Finland we have carried out human placental perfusions since 2005 using the same method with data now from over one hundred perfusions. This has allowed us to study whether the way of delivery, placental weight, and/or the length of pregnancy affect the two major criteria of a successful perfusion: volume loss (leak) from fetal to maternal circulation, and transplacental transfer of the reference compound antipyrine. The only statistically significant result was the reduction of the fetomaternal ratio of antipyrine by the placental age over 40 weeks (p=0.0004). The success criteria were not affected by the weight of the placenta or the way of delivery. There was no effect by the antipyrine concentration on antipyrine transfer. In vitro incubation with different concentrations of study compounds and different tubing materials could offer an easy way to study potentially reduced recovery due to binding to perfusion system.

Biomarkers of toxicity in human placenta

Nutrient and gas exchange between mother and fetus and production of hormones sustaining fetal development are important placental functions carried out by a rich selection of transporter proteins and a variety of metabolizing enzymes. The same proteins handle many xenobiotics which in placenta may pass, accumulate, and change placental functions, causing toxicity. Because of structural and functional variation between species, human placental tissue and human trophoblastic cell lines are the most used systems in the search for placental biomarkers of toxicity. They would be helpful in evaluation of drug toxicity in placenta, and in toxicological risk assessment. Only a few exist so far, e.g. changes in CYP19A1/aromatase by hormonally active compounds, metallothioneins in metal exposure, and level of PAH-DNA adducts associated with fetotoxicity. New possibilities are provided, e.g. by the emerging field of placental epigenetics. Use of placenta and placental biomarkers in regulatory toxicology also awaits further data.

Transplacental transfer and metabolism of diuron in human placenta

Diuron is a broad-spectrum phenylurea derived herbicide which is commonly used across the globe. Diuron is toxic to the reproductive system of animals and carcinogenic to rat urothelium, and recently found to be genotoxic in human cells. In in vivo, it is metabolized predominately into 3-(3,4-dichlorophenyl)-1-methyl urea (DCPMU) in humans and 3-(3, 4-dichlorophenyl)urea (DCPU) in animals. Information on diuron toxicokinetics and related toxicity in human placenta is absent. We have investigated the toxicokinetics of diuron in ex vivo human placental perfusion and in in vitro human placental microsomes and human trophoblastic cancer cells (BeWo). Diuron crossed human placenta readily in placental perfusion. Furthermore, diuron was metabolized into DCPMU in perfused placenta and in in vitro incubations using microsomes from placentas of smokers. In incubations with placental microsomes from non-smokers, and in BeWo cells, metabolism to DCPMU was detected but only with the highest used diuron concentration (100 μM). Diuron metabolism was inhibited upon addition of α-naphthoflavone, a CYP1A1 inhibitor, underscoring the role of CYP1A1 in the metabolism. In conclusion, it is evident that diuron crosses human placenta and diuron can be metabolized in the placenta to a toxic metabolite via CYP1A1. This implicates in vivo fetal exposure to diuron if pregnant women are exposed to diuron, which may result in fetotoxicity.