Maria Kummu

Developmental expression of drug metabolizing enzymes and transporter proteins in human placenta and fetal tissues.

Transporter proteins and xenobiotic metabolizing enzymes have a crucial role in the fate of xenobiotics in human body. The expression in human placenta and fetal tissues of the proteins most commonly participating in pharmaco/toxicokinetics is reviewed. In case human data are not available, relevant animal data are included. Among transporter proteins ABC transporters, monoamine transporters and organic anion transporters are pharmacologically and toxicologically of main interest. From xenobiotic enzymes, both CYP enzymes and transferases are expressed in fetal liver already during pregnancy. In the placenta, the variety of enzymes is much more restricted. During development dynamic changes occur in both xenobiotic metabolizing enzymes and drug transporters. Although the knowledge has increased substantially over the past years it is apparent from the literature that there are uncharacterized areas, especially regarding developmental expression patterns and regulation of transporters in fetal tissues and placenta. Knowledge about tissue-specific distribution and functional significance will aid our understanding of the differences in drug response and risks for adverse events during fetal development.

ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta.

We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of (14)C-PhIP (2 microM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72+/-0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of (14)C-PhIP from maternal to fetal circulation (FM ratio 0.90+/-0.08 at 6 h, p<0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75+/-0.10, p=0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of (14)C-PhIP (R=-0.81, p<0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP (R: -0.11, p=0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of (14)C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarcinoma cells.

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.

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.

Cadmium inhibits ABCG2 transporter function in BeWo choriocarcinoma cells and MCF-7 cells overexpressing ABCG2

Heavy metals such as cadmium, lead and methylmercury are known to be neurotoxic to developing fetus. ABCG2 which is an efflux transporter located in the maternal facing membranes of human placenta protects fetus from xenobiotics by transferring compounds from syncytiotrophoblast to maternal circulation. The aim of this study was to clarify whether heavy metal compounds (CdCl(2), PbCl(2) and MeHgCl) affect the expression and function of ABCG2 transporter in human placental BeWo choriocarcinoma cells. The expression of ABCG2 was determined by immunoblotting and RT-PCR. The functional activity of ABCG2 was evaluated by measuring the efflux of two known ABCG2 substrates: fluorescent mitoxantrone and (14)C-labeled food carcinogen PhIP. According to MTT assay all compounds were cytotoxic as expected (MeHgCl > CdCl(2) > PbCl(2)). CdCl(2) inhibited the efflux of mitoxantrone and (14)C-PhIP suggesting inhibition of ABCG2 transporter function. PbCl(2) had no effect on mitoxantrone efflux. Because of high toxicity, the inhibitory potency of MeHgCl was not tested. According to protein data these heavy metals did not affect ABCG2 transporter protein expression. Also, the expression of ABCC1, ABCC2 or ABCG2 mRNA were not affected by heavy metals. In conclusion, although the studied metal salts did not affect mRNA or protein expression of ABCG2, CdCl(2) inhibited its function. Further studies to evaluate whether this leads to elevated placental transfer of ABCG2 substrates are needed.

ABCB1 and ABCG2 expression in the placenta and fetus: an interspecies comparison

Importance of the field: ABCB1 and ABCG2 are efflux transporters which have a major impact on the pharmacological behavior of numerous drugs. They are expressed, for example, in the intestine, liver, kidney, BBB and placenta. It has become evident that ABCB1 and ABCG2 modify the pharmaco/toxicokinetics in the placenta and fetus and may consequently affect the outcome of pregnancy. Areas covered in this review: Comprehensive literature searches were done using PubMed (until June 2010) to identify publications on ABCB1 and ABCG2 expression in placenta and fetal tissues in human, mouse, rat, guinea-pig and rabbit. What the reader will gain: In this review, we aim to provide an overview of the current knowledge on the ABCB1 and ABCG2 transporter expression profiles in the placenta and fetal tissues in humans relative to other species. Take home message: The available information on ABCB1 and ABCG2 temporal expression profiles in placenta and fetus indicates rather good correlation among human, mouse and rat although some specific differences have been reported. However, at this point no detailed comparisons or comparative functional data are available. Detailed knowledge on the expression patterns and functional activity of ABCB1 and ABCG2 transporters placenta and developing embryo/fetus in different species could possibly help the interspecies extrapolation.

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.

Organic anion transporter 4 (OAT 4) modifies placental transfer of perfluorinated alkyl acids PFOS and PFOA in human placental ex vivo perfusion system

INTRODUCTION Perfluorinated alkyl acids (PFAAs) are widely used in industry and consumer products. Pregnant women are exposed to PFAAs and their presence in umbilical cord blood represents fetal exposure. Interestingly, PFAAs are substrates for organic anion transporters (OAT) of which OAT4 is expressed in human placenta. METHODS To evaluate the contribution of OAT4 and ATP-binding cassette transporter G2 (ABCG2) proteins in the transplacental transfer of perfluoro octane sulfonate (PFOS) and perfluoro octanoate (PFOA) an ex vivo dual recirculating human placental perfusion was used. Altogether 8 placentas from healthy mothers with uncomplicated pregnancies were successfully perfused. RESULTS Both PFOS and PFOA crossed the placenta as suggested by in vivo data in the literature. The expression of OAT4 and ABCG2 proteins were studied by immunoblotting and correlation with the transfer index %(TI %) of PFOS and PFOA at 120 and 240 min (n = 4) was studied. The expression of OAT4 was in negative correlation with TI % of PFOA (R(2) = 0.92, p = 0.043) and PFOS (R(2) = 0.99, p = 0.007) at 120 min while at 240 min the correlation was statistically significant only with PFOA. The expression of ABCG2 did not correlate with TI% of PFOS or PFOA. DISCUSSION Data obtained in this study suggest the involvement of OAT4 in placental passage of PFAAs. Placental passage of PFOS and PFOA is modified by the transporter protein OAT4 but not by ABCG2. This is the first study indicating that OAT4 may decrease the fetal exposure to PFAAs and protect the fetus after maternal exposure to PFAAs but further studies are needed to confirm our findings.

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.