Placental Toxicity Research Articles

Protective effects of Zingiber officinale extract on myocardium and placenta against labetalol-induced histopathological, immune-histochemical, and ultrastructural alterations in pregnant rats

BackgroundLabetalol is an antihypertensive drug commonly used in obstetrics for both long-term treatment and the acute management of severe maternal hypertension. However, there have not been published articles about the effects of labetalol on the myocardium and the placenta. This study aimed to estimate the histological, immune-histochemical, and ultrastructural cardio- and placental-toxicity of labetalol administration and the effectiveness of ginger against this toxicity in pregnant rats. Labetalol was daily administrated orally with or without ginger at a dose of 300 mg/kg and 200 mg/kg, respectively, during the gestation days 6 to 20.ResultsIn the labetalol-administrated group, the myocardium displayed histological and ultrastructure destructive changes and a significant increase in caspase-3 expression. Labetalol also decreased the placental weight compared with the control group, caused marked degeneration and disorganization of their architecture, and increased caspase-3 expression. Co-administration of ginger after labetalol highly ameliorates the adverse effect of labetalol on both cardiac and placental tissues.ConclusionsIt is concluded that ginger can mitigate cardiac and placental toxicity induced by labetalol administration into pregnant rats.

PFOS Inhibited Normal Functional Development of Placenta Cells via PPARγ Signaling.

Perfluorooctane sulfonic acid (PFOS), a persistent environmental pollutant, has adverse effects on gestation pregnancy. Peroxisome proliferator-activated receptor γ (PPARγ) is involved in angiogenesis, metabolic processes, anti-inflammatory, and reproductive development. However, the function of PPARγ in PFOS evoked disadvantageous effects on the placenta remain uncertain. Here, we explored the role of PPARγ in PFOS-induced placental toxicity. Cell viability, cell migration, angiogenesis, and mRNA expression were monitored by CCK-8 assay, wound healing assay, tube formation assay, and real-time PCR, respectively. Activation and overexpression of PPARγ were conducted by rosiglitazone or pcDNA-PPARγ, and inhibition and knockdown of PPARγ were performed by GW9662 or si-PPARγ. Results revealed that PFOS decreased cell growth, migration, angiogenesis, and increased inflammation in human HTR-8/SVneo and JEG-3 cells. Placenta diameter and fetal weight decreased in mice treated with PFOS (12.5 mg/kg). In addition, rosiglitazone or pcDNA-PPARγ rescued cell proliferation, migration, angiogenesis, and decreased inflammation induced by PFOS in HTR8/SVneo and JEG-3 cells. Furthermore, GW9662 or si-PPARγ exacerbated the inhibition of cell viability, migration, angiogenesis, and aggravated inflammation induced by PFOS in HTR-8/SVneo and JEG-3 cells. Meanwhile, the results of mRNA expression level were consistent with the cell representation. In conclusion, our findings revealed that PFOS induced placenta cell toxicity and functional damage through PPARγ pathway.

Efficacy of vitamin E in protection against methotrexate induced placental injury in albino rats

Methotrexate (MXT) is a chemotherapeutic drug that has been used in a wide range of clinical practices. Unfortunately, the administration of MXT during pregnancy may induce abortion, fetal deformities, and intrauterine growth retardation. Vitamin E is an antioxidant agent that can ameliorate free radical damage. The current work aimed to shed more light on the possible protective effect of vitamin E against MXT induced placental toxicity and to determine the possible mechanisms; biochemically, histologically, and immunohistochemically. Four groups were used: control pregnant, Vitamin E (VIT E) pregnant, Methotrexate (MXT) pregnant, and Vitamin E Methotrexate (VIT E-MXT) pregnant. The placental tissues were processed for light, immunohistochemical, and electron microscopic study. Other samples were obtained for biochemical study; the placental oxidant/antioxidant status was evaluated. The results showed that MXT caused various placental morphological changes in the form of distorted chorionic projection with an accumulation of hemosiderin granules in the trophoblastic cells. Maternal blood vessels showed a homogenous acidophilic material Edema of the extra-embryonic fetal membranes was noticed. A significant decreased in placental weight as well as increase in the oxidative and inflammatory markers were detected. Increased COX2 and decreased eNOS expressions were observed in the MXT group if compared to the control group. VIT E significantly restored the normal histological and immunohistochemical appearance, placental weight, and oxidant/antioxidant balance. It could be concluded the biochemical, morphological, and morphometric findings suggested that vitamin E coadministration is promising in attenuating the placental toxic effect of methotrexate. In this study, VIT E decreased the inflammatory and oxidative stress effect of methotrexate on the placental tissue by enhancing the level of eNOS.

Triphenyl phosphate disturbs the lipidome and induces endoplasmic reticulum stress and apoptosis in JEG-3 cells

Triphenyl phosphate (TPP) is a frequently used aryl organophosphate flame retardant. Epidemiological studies have shown that TPP and its metabolite diphenyl phosphate (DPP) can accumulate in the placenta, and positively correlated with abnormal birth outcomes. TPP can disturb placental hormone secretion through the peroxisome proliferator-activated receptor γ (PPARγ) pathway. However, the extent and mechanism of placental toxicity mediation by TPP remains unknown. In this study, we used JEG-3 cells to investigate the role of PPARγ-regulated lipid metabolism in TPP-mediated placental toxicity. The results of lipidomic analysis showed that TPP increased the production of triglycerides (TG), fatty acids (FAs), and phosphatidic acid (PA), but decreased the levels of phosphatidylethanol (PE), phosphatidylserine (PS), and sphingomyelin (SM). TG accumulation was accompanied by increased levels of sterol regulatory element binding transcription factor 1 (SREBP1), acetyl-coA carboxylase (ACC), and fatty acid transport protein (CD36). Although PPARγ and its target CCAAT/enhancer binding proteins (C/EBPα) was decreased, the TG content and gene expression of SREBP1, ACC, and CD36 decreased when TPP was co-exposed to the PPARγ antagonist GW9662. TPP also induced inflammatory responses, endoplasmic reticulum stress (ERS), and cell apoptosis. Expression of genes related to ERS and apoptosis were attenuated by GW9662. Together, these results show that TPP can disturb lipid metabolism and cause lipid accumulation through PPARγ, induce ERS, and cell apoptosis. Our findings reveal that the developmental toxicity of TPP through placental toxicity should not be ignored.

Cadmium triggers oxidative stress and mitochondrial injury mediated apoptosis in human extravillous trophoblast HTR-8/SVneo cells

Cadmium (Cd) is a bioaccumulative heavy metal element with potential placental toxicity during pregnancy. Up to now, however, the precise toxic effects of Cd on human placentae, particularly as they pertain to trophoblast cells remain obscure. We therefore sought to investigate the cytotoxic effects of Cd on human extravillous trophoblast HTR-8/SVneo cells and the mechanisms involved in the processes. Results in this present study showed that CdCl2 treatment significantly suppressed cell viability and induced noticeable oxidative stress in HTR-8/SVneo cells. Further studies showed that CdCl2 treatment caused distortion of mitochondrial structure, reduction of mitochondrial membrane potential (Δψm), DNA damage and G0/G1 phase arrest. Under the same condition, CdCl2 treatment increased Bax/Bcl-2 ratios by up-regulating Bax expression and down-regulating Bcl-2 expression, and activated apoptotic executive molecule caspase-3, which irreversibly induced HTR-8/SVneo cell apoptosis. N-acetyl-l-cysteine (NAC), ROS scavenger, significantly attenuated CdCl2-caused mitochondrial injury, DNA damage, G0/G1 phase arrest and apoptosis. In addition, in vivo assay suggested that CdCl2 induced trophoblast cells apoptosis but not other cells in mice placental tissue. Taken together, these data suggest that Cd selectively triggers oxidative stress and mitochondrial injury mediated apoptosis in trophoblast cells, which might contribute to placentae impairment and placental-related disorders after Cd exposure. These findings may provide new insights to understand adverse effects of Cd on placentae during pregnancy.

648 Utilizing primary human tissue/cell models to assess for pesticide-induced placental toxicity

Chorionic villi serve as the primary interface between mother and fetus. Dysfunction at this interface is implicated in pregnancy complications. Pesticides are widely used in agriculture and vector control, but the full impact on human pregnancy is unknown. Specific pesticides such as organophosphate pesticides (OPs) are suspected to target the developing central nervous system and placenta. We aimed to leverage ex vivo models of the human placenta (primary chorionic villous explants and isolated cytotrophoblasts (CTBs)) for in vitro studies of toxicological agents. Isolated CTBs from 2nd trimester placentas were exposed to 9 diverse pesticides at 5 concentrations and compared to vehicle (0.1% DMSO) and media-only controls. At 24h, viability and cell death were assayed by neutral red lysosomal uptake or lactate dehydrogenase activity, respectively. Benchmark concentrations (BMC10) of a 10% change in cytotoxicity were identified to assess relative potencies. Human chorionic villi explants dissected from placentas were exposed to naled or vehicle for 24h. After exposure, 1st trimester villi were evaluated by gross morphology and 2nd trimester villi by histopathology and immunofluorescence. Sections were scored by 2 blind evaluators for syncytiotrophoblast (STB) denudation, quantified by ImageJ analysis. We used ANOVA and Tukey’s test to determine significant changes between groups. We observed concentration-dependent cytotoxicity with the three OPs (naled, dichlorovos and malathion) in CTBs (p<0.05; Figure 1). Naled was the most toxic pesticide. Gross morphologic evaluation of naled-exposed villi showed frayed appearance and impaired vascularity compared to the control groups. Naled-exposed explants displayed a ∼4-fold increase in STB denudation compared with media-only or vehicle controls (p<0.001, Figure 2). Our results suggest that naled, an OP commonly-used in agriculture and mosquito control is particularly toxic in two human placental models. We demonstrate that primary cell/tissue models can be used to evaluate placental toxicity of pesticide compounds.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Toxicity of anticancer drugs in human placental tissue explants and trophoblast cell lines

The application of anticancer drugs during pregnancy is associated with placenta-related adverse pregnancy outcomes. Therefore, it is important to study placental toxicity of anticancer drugs. The aim of this study was to compare effects on viability and steroidogenesis in placental tissue explants and trophoblast cell lines. Third trimester placental tissue explants were exposed for 72 h (culture day 4–7) to a concentration range of doxorubicin, paclitaxel, cisplatin, carboplatin, crizotinib, gefitinib, imatinib, or sunitinib. JEG-3, undifferentiated BeWo, and syncytialised BeWo cells were exposed for 48 h to the same drugs and concentrations. After exposure, tissue and cell viability were assessed and progesterone and estrone levels were quantified in culture medium. Apart from paclitaxel, all compounds affected both cell and tissue viability at clinically relevant concentrations. Paclitaxel affected explant viability moderately, while it reduced cell viability by 50% or more in all cell lines, at 3–10 nM. Doxorubicin (1 µM) reduced viability in explants to 83 ± 7% of control values, whereas it fully inhibited viability in all cell types. Interference with steroid release in explants was difficult to study due to large variability in measurements, but syncytialised BeWo cells proved suitable for this purpose. We found that 1 µM sunitinib reduced progesterone release to 76 ± 6% of control values, without affecting cell viability. While we observed differences between the models for paclitaxel and doxorubicin, most anticancer drugs affected viability significantly in both placental explants and trophoblast cell lines. Taken together, the placenta should be recognized as a potential target organ for toxicity of anticancer drugs.

PFOS-induced placental cell growth inhibition is partially mediated by lncRNA H19 through interacting with miR-19a and miR-19b

Perfluorooctane sulfonic acid (PFOS), a persistent environmental pollutant, has been associated with decreased birth weight. The dysregulation of long non-coding RNA (lncRNA) H19 has been implicated in pregnancy complications such as intra-uterine growth retardation (IUGR), preeclampsia (PE), however, the expression and function of H19 in PFOS-exerted detrimental effects in the placenta remains to be unveiled. Here, we explored the role of H19 in PFOS-induced placental toxicity. Results showed that PFOS caused decreased cell growth in human HTR-8/SVneo cells. Expression of H19 was increased, while miR-19a and miR-19b expression were decreased in mice placenta tissues and in HTR-8/SVneo cells exposed to PFOS. A significant hypomethylation was observed at the H19 promoter in the placentas of mice that were gestational exposed to high dose of PFOS. H19 was confirmed to bind with miR-19a and miR-19b, targeting SMAD4. Furthermore, H19 appeared to partially improve the cell growth of HTR-8/SVneo cells exposed to PFOS via upregulation of miR-19a and miR-19b. In summary, our findings revealed that H19/miR-19a and miR-19b/SMAD4 axis exerted important functions in PFOS-induced placenta cell toxicity.

Cancer During Pregnancy: The Role of Vascular Toxicity in Chemotherapy-Induced Placental Toxicity.

Breast cancer is diagnosed in ~0.3% of pregnant women. Studies that have addressed gestational and neonatal outcomes of chemotherapy during pregnancy have demonstrated increased gestational complications including preeclampsia and intrauterine growth retardation. We hypothesized that anthracycline-induced gestational complications could be derived from direct toxicity on the placenta vasculature. Pregnant ICR mice (day E12.5) were treated with doxorubicin (DXR; 8 mg/kg) or saline, while their umbilical cord blood flow was imaged by pulse-wave (PW) Doppler. Mice were euthanized on day E18.5, and their embryos and placentae were collected for further analysis. Unlike control mice, the DXR-treated mice presented an acute change in the umbilical cord’s blood flow parameters (velocity time integral and heart rate interval), reduced embryos’ weight, reduced placenta efficiency, and modulation in vascular-related pathways of treated placenta proteomics. Apoptosis and proliferation were also enhanced, as demonstrated by TUNEL and proliferating cell nuclear antigen (PCNA) analysis. We further examined the placentae of patients treated with epirubicin (EPI), who had been diagnosed with breast cancer during pregnancy (weeks 27–35). The immunohistochemistry of the EPI-treated human placentae showed enhanced proliferation and apoptosis as compared with matched chemo-naïve placentae, as well as reduced neovascularization (CD34). Our findings suggest that anthracycline-induced vascular insult promotes placental toxicity, and could point to potential agents designated to offset the damage and to reduce gestational complications in pregnant cancer patients.

Activities of metabolizing enzymes in human placenta

In addition to the transfer across the placenta, placenta displays hormonal and xenobiotic metabolism, as well as enzymatic defense against oxidative stress. We analyzed aromatase (CYP19A1), uridine 5′-diphospho-glucuronyltransferase (UGT), glutathione-S-transferase (GST) and catalase (CAT) activities in over 70 placentas from nonsmokers stored at -80 °C from former perfusion studies. A wide interindividual variation in all activities was found. Longterm storage at -80 °C did not affect the activities. Ethoxyresorufin-O-deethylase (EROD, CYP1A1) was not detected in any of the studied placentas perfused with chemicals. Several compounds in placental perfusion changed statistically significantly the enzyme activities in placental tissue. Melamine and nicotine increased CYP19A1, melamine increased UGT and GST, PhIP with ethanol decreased CYP19A1 and increased GST, and PhIP with buprenorphine decreased CAT. Antipyrine in 100 μg/ml also changed the studied enzyme activities, but not statistically significantly. Because antipyrine is a reference compound in placental perfusions, its potential effects must be taken into account in human placental perfusion. Enzyme activities deserve further studies as biomarkers of placental toxicity.Finally, enzyme activities deserve further studies as biomarkers of placental toxicity.

The trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine induces progressive mitochondrial dysfunction in HTR-8/SVneo trophoblasts

Trichloroethylene is an industrial solvent and common environmental pollutant. Despite efforts to ban trichloroethylene, its availability and usage persist globally, constituting a hazard to human health. Recent studies reported associations between maternal trichloroethylene exposure and increased risk for low birth weight. Despite these associations, the toxicological mechanism underlying trichloroethylene adverse effects on pregnancy remains largely unknown. The trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC) induces mitochondrial-mediated apoptosis in a trophoblast cell line. To gain further understanding of mitochondrial-mediated DCVC placental toxicity, this study investigated the effects of DCVC exposure on mitochondrial function using non-cytolethal concentrations in placental cells. Human trophoblasts, HTR-8/SVneo, were exposed in vitro to a maximum of 20 μM DCVC for up to 12 h. Cell-based oxygen consumption and extracellular acidification assays were used to evaluate key aspects of mitochondrial function. Following 6 h of exposure to 20 μM DCVC, elevated oxygen consumption, mitochondrial proton leak and sustained energy coupling deficiency were observed. Similarly, 12 h of exposure to 20 μM DCVC decreased mitochondrial-dependent basal, ATP-linked and maximum oxygen consumption rates. Using the fluorochrome TMRE, dissipation of mitochondrial membrane potential was detected after a 12-h exposure to 20 μM DCVC, and (±)-α-tocopherol, a known suppressor of lipid peroxidation, attenuated DCVC-stimulated mitochondrial membrane depolarization but failed to rescue oxygen consumption perturbations. Together, these results suggest that DCVC caused progressive mitochondrial dysfunction, resulting in lipid peroxidation-associated mitochondrial membrane depolarization. Our findings contribute to the biological plausibility of DCVC-induced placental impairment and provide new insights into the role of the mitochondria in DCVC-induced toxicity.

Molecular mechanisms of Cisplatin- induced placental toxicity and teratogenicity in rats and the ameliorating role of N-acetyl-cysteine

The aim of the present study is to investigate the molecular mechanisms of Cisplatin- induced placental toxicity and teratogenicity in rats and the ameliorating role of N-acetyl-cysteine (NAC). Cisplatin was administrated intraperitoneally at 5 mg/kg.b.wt as a single dose on the 12th day of gestation while NAC was administered orally throughout gestation either alone or in concomitant injection of Cisplatin at 200 mg/kg.b.wt. Cisplatin + NAC group showed reduction in the elevated morphological, visceral and skeletal abnormalities as well as the morphological and histopathological changes in placenta compared to Cisplatin - treated rats. Importantly, NAC attenuated Cisplatin-induced placental apoptosis through down-regulation of Fas and Caspase-3 genes expression. In conclusion, induction of placental apoptosis by overexpression of Fas and Caspase-3 genes gives a new insight into the mechanism of Cisplatin teratogenicity. The protective role of NAC, on the other hand, was characterized by attenuation of Fas and Caspase-3 genes- mediated apoptosis.

Smoking and pregnancy: Epigenetics and developmental origins of the metabolic syndrome.

Maternal smoking causes lower birth weight, birth defects, and other adverse pregnancy outcomes. Epidemiological evidence over the past four decades has grown stronger and the adverse outcomes attributed to maternal smoking and secondhand smoke exposure have expanded. This review presents findings of latent and persistent metabolic effects in offspring of smoking mothers like those observed in studies of maternal undernutrition during pregnancy. The phenotype of offspring of smoking mothers is like that associated with maternal undernutrition. Born smaller than offspring of nonsmokers, these children have increased risk of being overweight or obese later. Plausible mechanisms include in utero hypoxia, nicotine-induced reductions in uteroplacental blood flow, placental toxicity, or toxic growth restriction from the many toxicants in tobacco smoke. Studies have reported increased risk of insulin resistance, type 2 diabetes and hypertension although the evidence here is weaker than for overweight/obesity. Altered DNA methylation has been consistently documented in smoking mothers' offspring, and these epigenetic alterations are extensive and postnatally durable. A causal link between altered DNA methylation and the phenotypic changes observed in offspring remains to be firmly established, yet the association is strong, and mediation analyses suggest a causal link. Studies examining expression patterns of affected genes during childhood development and associated health outcomes should be instructive in this regard. The adverse effects of exposure to tobacco smoke during pregnancy now clearly include permanent metabolic derangements in offspring that can adversely affect life-long health.

Developing novel in vitro methods for the risk assessment of developmental and placental toxicants in the environment

During pregnancy, the placenta is critical for the regulation of maternal homeostasis and fetal growth and development. Exposures to environmental chemicals during pregnancy can be detrimental to the health of the placenta and therefore adversely impact maternal and fetal health. Though research on placental-derived developmental toxicity is expanding, testing is limited by the resources required for traditional test methods based on whole animal experimentation. Alternative strategies utilizing in vitro methods are well suited to contribute to more efficient screening of chemical toxicity and identification of biological mechanisms underlying toxicity outcomes. This review aims to summarize methods that can be used to evaluate toxicity resulting from exposures during the prenatal period, with a focus on newer in vitro methods centered on placental toxicity. The following key aspects are reviewed: (i) traditional test methods based on animal developmental toxicity testing, (ii) in vitro methods using monocultures and explant models, as well as more recently developed methods, including co-cultures, placenta-on-a-chip, and 3-dimensional (3D) cell models, (iii) endpoints that are commonly measured using in vitro designs, and (iv) the translation of in vitro methods into chemical evaluations and risk assessment applications. We conclude that findings from in vitro placental models can contribute to the screening of potentially hazardous chemicals, elucidation of chemical mechanism of action, incorporation into adverse outcome pathways, estimation of doses eliciting toxicity, derivation of extrapolation factors, and characterization of overall risk of adverse outcomes, representing key components of chemical regulation in the 21st century.

Morphology and physiology of rat placenta for toxicological evaluation.

The placenta plays a pivotal role in fetal growth, and placental dysfunction and injury are associated with embryo/fetal toxicity. Histological examination of the rat placenta for safety evaluation provides valuable clues to the mechanisms of this toxicity. However, the placenta has specific and complex biological features unlike those of other organs, and placental structure dramatically changes depending on the time during the gestation period. Thus, time-dependent histopathological examination of the rat placenta should be performed based on the understanding of normal developmental changes in morphology and function. The placentas of rats and humans are both anatomically classified as discoid and hemochorial types. However, there are differences between rats and humans in terms of placental histological structure, the fetal-maternal interface, and the function of the yolk sac. Therefore, extrapolation of placental toxicity from rats to humans should be done cautiously in the evaluation of risk factors. This review describes the development, morphology, physiology, and toxicological features of the rat placenta and the differences between the rat and human placenta to enable accurate evaluation of reproductive and developmental toxicity in studies.

Genomic Profiling of BDE-47 Effects on Human Placental Cytotrophoblasts.

Despite gradual legislative efforts to phase out flame retardants (FRs) from the marketplace, polybrominated diphenyl ethers (PBDEs) are still widely detected in human maternal and fetal tissues, eg, placenta, due to their continued global application in consumer goods and inherent biological persistence. Recent studies in rodents and human placental cell lines suggest that PBDEs directly cause placental toxicity. During pregnancy, trophoblasts play key roles in uterine invasion, vascular remodeling, and anchoring of the placenta-fetal unit to the mother. Thus, to study the potential consequences of PBDE exposures on human placental development, we used an in vitro model: primary villous cytotrophoblasts (CTBs). Following exposures, the endpoints that were evaluated included cytotoxicity, function (migration, invasion), the transcriptome, and the methylome. In a concentration-dependent manner, common PBDE congeners, BDE-47 and -99, significantly reduced cell viability and increased death. Upon exposures to sub-cytotoxic concentrations (≤ 5 µM), we observed BDE-47 accumulation in CTBs with limited evidence of metabolism. At a functional level, BDE-47 hindered the ability of CTBs to migrate and invade. Transcriptomic analyses of BDE-47 effects suggested concentration-dependent changes in gene expression, involving stress pathways, eg, inflammation and lipid/cholesterol metabolism as well as processes underlying trophoblast fate, eg, differentiation, migration, and vascular morphogenesis. In parallel assessments, BDE-47 induced low-level global increases in methylation of CpG islands, including a subset that were proximal to genes with roles in cell adhesion/migration. Thus, using a primary human CTB model, we showed that PBDEs induced alterations at cellular and molecular levels, which could adversely impact placental development.

Chemotherapy-induced placental toxicity: The role of vascular toxicity.

e13097Background: Several studies have addressed gestational and neonatal outcomes of chemotherapy during pregnancy (CDP) and demonstrated increased complications as pre-eclampsia and intrauterine ...

Histomorphologic Analysis of the Late-term Rat Fetus and Placenta.

Histological examination of the rat placenta and fetus is uncommon. Toxicological studies mainly rely on gross examination of the fetus and on fetal and placental weights. These are often insufficient to assess the fetal and placental toxicity of xenobiotics. The small size of the fetus makes its dissection labor-intensive. Thus, our objective was to develop a simple and accurate technique to evaluate the rat fetus and placenta. Sprague-Dawley rat fetuses at gestational day 19.5 ( n = 18) and their placentas ( n = 32) were fixed in formalin. Placentas were cut transversally in the center. Fetuses were cut following a freehand whole-body serial sectioning diagram adapted from Wilson's method. Sections were stained with hematoxylin-eosin-phloxine-saffron, and histomorphometry was used to measure the area of the fetal placental region (27.2 ± 1.7 mm2), including the labyrinth (22.2 ± 1.0 mm2) and the basal zone (4.8 ± 0.8 mm2). Our whole-fetus serial sectioning technique resulted in 12 precise cutting planes that fit on 3 histological slides, enabling the examination of most organs without labor-intensive dissection. Quantitative analysis of placental areas improves the understanding of the pathogenesis of treatment-related changes. This technique provides a standardized method for future research in pertinent fields such as developmental biology and toxicology.

O-28 Placental transfer of the immunosuppressive drug tacrolimus and its effects on placental function; relevance for renal transplant recipients?

Background The number of pregnancies in the kid-ney transplant patient population is high. During gesta-tion these women continue using immunosuppressant drugs, but knowledge about their placental disposition and toxicity is scarce. We now investigated placental transfer of the immunosuppressive drug tacrolimus (TAC) as well as the potential effects on trophoblast cell viability and barrier function. Methods Isolated dual side perfusions of human pla-cental cotyledons were performed to study disposition of TAC. Additionally, clinical data on TAC concentrations in placental tissue of kidney transplant recipients and ma-ternal whole blood concentrations were gathered. BeWo choriocarcinoma cells were used to evaluate effects on trophoblast cell viability, while interaction with placental ATP-binding cassette transporters was studied in mem-brane vesicles derived from HEK293 cells recombinantly overexpressing human Breast Cancer Resistance Protein (BCRP) or P-glycoprotein (P-gp). Results We found that maternal perfusate levels de-creased during 180 min of perfusion, while being unde-tectable in the fetal circulation. At t=180 min a concen-tration of 220±50 nM was measured in placental tissue, which is almost 100-fold higher than the maternal per-fusate concentration. Analysis of placental tissue of renal transplant recipients revealed a 13-fold higher tacrolimus concentration compared to the maternal blood concen-tration (88±7 nM and 6.8±1.1 nM, respectively). TAC did not affect BeWo cell viability up to the maximum concen-tration of 1 µM tested. In transporter studies we did find stimulation of P-gp-mediated transport and inhibition of BCRP-mediated transport, at 1 and 10 µM, respectively. Conclusion TAC demonstrates strong accumulation in placental tissue and distribution across the tissue was not homongenously. However, the tissue concentrations reached are unlikely to affect trophoblast cell viability or BCRP and P-gp transport function.

Editor's Highlight: PlacentalDisposition and Effects of Crizotinib: An Ex Vivo Study in the Isolated Dual-Side Perfused Human Cotyledon.

Tyrosine kinase inhibitors (TKIs) play an important role in cancer pharmacotherapy, yet there is limited data on their use during pregnancy. We studied placental disposition and placental toxicity of crizotinib, a TKI used to treat nonsmall cell lung cancer. Term placentas were perfused for 3 h with crizotinib (1 µM) using the ex vivo dual-side cotyledon perfusion technique. Interference of TKIs with trophoblast viability was studied using BeWo cells. Expression of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) in placental tissue was assessed by immunohistochemistry and inhibition of these transporters was determined in vitro by transport studies with membrane vesicles overexpressing human P-gp or BCRP. We found that crizotinib rapidly and strongly accumulates in cotyledon perfusion experiments, reaching a concentration of 3.1 ± 0.4 µM in placental tissue. Final drug concentrations in the maternal and foetal reservoirs were 0.2 ± 0.05 and 0.08 ± 0.01 µM, respectively. Furthermore, crizotinib inhibited BeWo cell viability (IC50: 234 nM, 95% CI: 167-328 nM) 10 times more potently than other TKIs tested. In vitro transport studies revealed that crizotinib is a potent inhibitor of the transport activities of BCRP (IC50: 5.7 µM, 95% CI: 2.7-11.8 µM) and P-gp (IC50: 7.8 µM, 95% CI: 3.4-18.0 µM). In conclusion, crizotinib strongly accumulated in placental tissue at clinically relevant concentrations. IC50 values for transporter inhibition and trophoblast cell viability were similar to the tissue concentrations reached, suggesting that crizotinib can inhibit placental BCRP and P-gp function and possibly affect trophoblast viability.