Expression of UDP-glucuronosyltransferase 1A4 in human placenta at term

The placenta contains a large variety of metabolizing enzymes, among them UDP-glucuronosyltransferase (UGT). Several UGT2B isozymes have so far been detected in human placenta, but little is known on placental expression of UGT1A isozymes. The antiepileptic drug lamotrigine (LTG) is a UGT1A4-substrate, and its serum concentration falls by over 50% during pregnancy, leading to impaired seizure control. The placenta may be involved in this. Microsomes from term placentas of 4 LTG-users and 10 healthy control subjects were prepared. Western blot analysis detected UGT1A proteins in all placentas. The presence of UGT1A4 in placenta from LTG users was confirmed with UGT1A4 commercial standard and a specific UGT1A4 primary antibody. Since LTG is primarily metabolized by UGT1A4 and this isozyme is shown to be present in placenta at term, it may be hypothesized that the placenta is involved in the fall of LTG serum concentrations during pregnancy

Activation of epidermal growth factor receptor is required for Chlamydia trachomatis development

Background Chlamydia trachomatis (C. trachomatis) is a clinically significant human pathogen and one of the leading causative agents of sexually transmitted diseases. As obligate intracellular bacteria, C. trachomatis has evolved strategies to redirect the host’s signaling and resources for its own survival and propagation. Despite the clinical notoriety of Chlamydia infections, the molecular interactions between C. trachomatis and its host cell proteins remain elusive. Results In this study, we focused on the involvement of the host cell epidermal growth factor receptor (EGFR) in C. trachomatis attachment and development. A combination of molecular approaches, pharmacological agents and cell lines were used to demonstrate distinct functional requirements of EGFR in C. trachomatisinfection. We show that C. trachomatis increases the phosphorylation of EGFR and of its downstream effectors PLCγ1, Akt and STAT5. While both EGFR and platelet-derived growth factor receptor-β (PDGFRβ) are partially involved in bacterial attachment to the host cell surface, it is only the knockdown of EGFR and not PDGFRβ that affects the formation of C. trachomatis inclusions in the host cells. Inhibition of EGFR results in small immature inclusions, and prevents C. trachomatis-induced intracellular calcium mobilization and the assembly of the characteristic F-actin ring at the inclusion periphery. By using complementary approaches, we demonstrate that the coordinated regulation of both calcium mobilization and F-actin assembly by EGFR are necessary for maturation of chlamydial inclusion within the host cells. A particularly important finding of this study is the co-localization of EGFR with the F-actin at the periphery of C. trachomatis inclusion where it may function to nucleate the assembly of signaling protein complexes for cytoskeletal remodeling required for C. trachomatisdevelopment. Conclusion Cumulatively, the data reported here connect the function of EGFR to C. trachomatis attachment and development in the host cells, and this could lead to new venues for targeting C. trachomatis infections and associated diseases

In search of models for hepatic and placental pharmacokinetics

Abstract Several in vitro methods using both human and animal tissues have been developed to study hepatic metabolism and placental transfer. The pressure to minimize animal studies has increased during the past few decades due to the public opinion and ethical considerations. However, these methods need further evaluation of their predictive power when applied in vivo. The aim of this work was to produce new information of the metabolism and transplacental passage of several anticonvulsants as well as to evaluate the usefulness of the placental perfusion method and several in vitro methods for analyzing metabolism in the prediction of clinical pharmacokinetics. Carbamazepine (CBZ) metabolism was studied using human and mouse liver microsomes, human hepatocytes, human liver slices and yeast cells expressing recombinant enzymes. All test systems predicted well the major metabolite carbamazepine-10,11-epoxide (CBZ-E). Also, minor metabolites were produced in slightly variable amounts in all systems except cells with recombinant enzymes. All human liver systems demonstrated that CYP3A4 is the principal CBZ metabolising enzyme. However, our results on CBZ-treated mice suggested that the metabolism of CBZ to CBZ-E is mainly mediated by CYP1A1 in C57/BL6 mice. Autoinduction of CBZ metabolism was seen in hepatocytes and in incubations using microsomes from CBZ-treated mice. Human liver and mouse liver microsomes metabolized oxcarbazepine (OCBZ) mainly to its active metabolite, 10-hydroxy-10,11-dihydro-carbamazepine (10-OH-CBZ). Also, 10,11-trans-dihydroxy-10,11-dihydro-carbamazepine (10,11-D) and an unknown metabolite were detected. Placental transfer of lamotrigine (LTG) and diazepam (DZP) was considerable in the human placental perfusion system, indicating marked fetal exposure in vivo. The OCBZ, 10-OH-CBZ and 10,11-D analyzed from maternal venous and cord blood also suggested significant fetal exposure. The placental perfusion system predicts well the transplacental passage of LTG and OCBZ and its major metabolite. However, in vivo cord blood concentrations of DZP are higher than maternal concentrations. Placental perfusion studies did not predict this. Still, even with its limitations, the human placental perfusion method provides information that can be used to evaluate the risk factors associated with drug use during pregnancy because understanding of specific transport characteristics is a good basis for rational risk assessment. In conclusion, all of the tested in vitro systems were useful in the prediction of at least some aspects of in vivo pharmacokinetics and metabolism, but validation and refinement are still essential, as is also the need to keep in mind the limitations characteristic of each in vitro method

Fate of the teratogenic and carcinogenic ochratoxin A in human perfused placenta

Ochratoxin A (OTA) is one of the most frequent mycotoxins detected in human blood worldwide. Apart from its well known nephrotoxicity, OTA-induced teratogenicity and carcinogenicity proven in animals are potential effects also in humans. Pregnant women have been exposed to this food contaminant via dietary exposure in a continuous and widespread manner. Although the transplacental transfer of OTA has been demonstrated in laboratory animals and the presence of OTA in human fetal samples has been reported, little is known about the role of human placenta in OTA toxicokinetics. In this study, human perfused placenta was used to reveal the actual placental toxicokinetics of OTA using concentrations found in serum of pregnant women. Moreover, the effect of protein concentration and biological significance of placental transporters on the OTA transfer in human placenta were also determined. Our study is the first to pursue the transfer of OTA through perfused human placenta. The transfer of OTA through term human placenta was barely detectable in all perfusions. Inhibitors of neither ABCG2 nor ABCC2 increased the transport of OTA to fetal circulation in placental perfusion, and thus these transporters apparently do not have biological significance in inhibiting transplacental transfer of OTA. Human albumin has inhibited OTA transfer through a tight monolayer of BeWo b30 cells. Finding from this study clearly contradict the existing epidemiological studies reporting higher OTA levels in fetal than in maternal circulation in vivo. © 2011 Elsevier Ireland Ltd.link_to_subscribed_fulltex

The xenoestrogens, bisphenol A and para-nonylphenol, decrease the expression of the ABCG2 transporter protein in human term placental explant cultures.

Many endogenous and xenobiotic compounds are substrates and regulators of human placental ABC transporters. ABCG2 is protecting fetus against foreign chemicals. Environmental xenoestrogens, like bisphenol A (BPA) and p-nonylphenol (p-NP), mimic natural estrogens and can affect hormonal systems. Effects of BPA, p-NP, DES (diethylstilbestrol) and estradiol (E2), on ABCG2 expression were studied using human first trimester and term placental explants. Role of estrogen receptors (ER) in the effects of chemicals was studied by ER antagonist. Term placenta expressed less ABCG2 protein. In term placentas BPA (p < 0.05), p-NP (p < 0.01) and E2 (p < 0.05) decreased the ABCG2 protein expression after 48 h exposure while after 24 h exposure, only E2 decreased the expression (p < 0.05). The chemicals did not affect ABCG2 in first trimester placentas. The ER antagonist affected differently the responses of chemicals. In conclusion, environmental xenoestrogens downregulate placental ABCG2 protein expression depending on gestational age

Quantitative estimation of mercury intake by toxicokinetic modelling based on total mercury levels in humans

Mercury is a toxic metal that can be disseminated into the environment from both natural and anthropogenic sources. Human exposure to the metal stems mainly from food, and more particularly from the consumption of fish and other seafoods. Examining dietary exposure and measuring mercury levels in body tissues are two ways of estimating exposure to mercury. In this study, we utilized a modelling system consisting of three linear toxicokinetic models for describing the fate of methyl mercury, inorganic mercury, and metallic mercury in the body, in order to estimate daily intake of mercury as measured through total mercury concentrations in the blood. We then compared the results stemming from our modelling system to those of the detailed semi-quantitative food frequency questionnaire (FFQ) of the Norwegian Fish and Game (NFG) Study, a project that focused on dietary mercury exposure. The results indicate that toxicokinetic modelling based on blood levels gave higher daily intake values of mercury compared to those of the FFQ. Furthermore, the former had a wider range of estimates than the latter. The properties of the toxicokinetic model or limitations in the dietary exposure assessment could be posited as reasons for the differences between the respective methods. Moreover, the results may have been influenced by sources of mercury exposure that cannot be described as dietary, such as amalgam filling

Transplacental transfer of melamine

Objective: To characterize transplacental transfer of melamine and related mechanisms as well as toxicity using human placental perfusion and cultured cells. Methods: Transfer and toxicity were analyzed in 4-h perfusions with 10 μM or 1 mM melamine, or 10 μM melamine with 10 nM cyanuric acid (CYA). Efflux transporters were studied in accumulation assay and toxicity in BeWo cells by MTT assay. Results: Of added melamine 34-45% was transferred to fetal circulation and CYA made no difference. Histology, hCG production, and PLAP activity indicated functionality of placental tissue with no grave toxicity. Highest concentration of melamine used (2 mM) with CYA and long treatment time decreased viability of BeWo cells. Inhibitors of ABCB1, ABCG2, ABCC2 did not affect the accumulation of melamine in cells. Conclusion: Melamine goes through human term placenta with no contribution of efflux transporters. Toxicity of melamine is low in placental tissue and BeWo cells. © 2011 Elsevier Ltd. All rights reserved.link_to_subscribed_fulltex