Pasi Huuskonen

Aflatoxin B1--a potential endocrine disruptor--up-regulates CYP19A1 in JEG-3 cells.

Previous studies have indicated that aromatase (CYP19A1) is involved in the metabolism of aflatoxin B1 (AFB1). We hypothesized that exposure to AFB1 contaminated food during pregnancy could disrupt the normal production of steroid hormones in placenta. We examined the capability of AFB1 exposure to disrupt CYP19A1 expression as a putative endocrine disrupter, and to investigate the metabolism of AFB1 by CYP19A1. JEG-3 cells, as model for placental cells, were exposed alone and in combination to AFB1 and estrogen receptor ligands for 24-96 h. AFB1 (0.3-1.0 μM) induced the expression of CYP19A1 by 163%-339% compared to control at the 96 h time point, although no induction was observed at 24 h. AFB1 concentrations higher than 1 μM were cytotoxic to JEG-3 cells, and the cytotoxicity was inhibited by the aromatase inhibitor, finrozole. AFB1 was metabolized to aflatoxicol (AFL) by JEG-3 cells and CYP19A1 recombinant protein. AFL formation was partially inhibited by addition of tamoxifen and finrozole to the JEG-3 cells. AFB1 had no effect on the expression of CYP1A2 and CYP3A4 in JEG-3 cells. These results reveal that AFB1 can affect the expression of aromatase enzyme, indicating that chronic exposure to AFB1 may cause endocrine disruption in the foetoplacental unit.

The effects of aflatoxin B1 on transporters and steroid metabolizing enzymes in JEG-3 cells

Effects of 96 h aflatoxin B1 (AFB1) exposure at concentrations from 0.2 μM to 6 μM on the mRNA and protein expression levels of the following transporters ABCB1/B4, ABCC1, ABCC2, ABCG2, OAT4 and the mRNA expression of steroid-metabolizing enzymes CYP1A1, CYP19A1, HSD3B1 and HSD17B1, and conjugating enzyme family UGT1A were evaluated in trophoblastic JEG-3 cells. Statistically significant dose-dependent five-fold increases in the expression levels with ABCC2 and OAT4 were recorded at 2 and 6μM AFB1. Protein expression of ABCG2 was decreased dose-dependently with 0.2-6 μM AFB1. With the other transporters, only a trend of increased expression was observed. Analogously, a three-fold increase in the expressions of CYP19A1, HSD3B1, HSD17B1 and UGT1A-family were observed at 0.3 μM AFB1. When an inhibitor of CYP19A1, finrozole, was dosed simultaneously with AFB1, no increases in the transcripts of transporters or steroid hydroxylases or CYP19A1 were observed. This delayed increase in the expression levels - only after 96h incubations - may indicate that the response is due to a secondary metabolite of AFB1 or other secondary controlling cascades rather than the parent compound itself. In conclusion, AFB1 affected the placental steroid synthesizing, metabolizing and conjugating enzymes as well as the expression levels of several transporter proteins in JEG-3 cells. These alterations may lead to anomalies in the foetoplacental hormonal homeostasis.

The human placental proteome is affected by maternal smoking

Highlights • The effects of maternal smoking on the term placental proteome was studied.• Maternal smoking significantly affected 18% of protein spots.• Maternal smoking affects systems controlling the development and function of placenta.• The observed placental changes may contribute to the lowered birth weights.

The unique characteristics of the placental transcriptome and the hormonal metabolism enzymes in placenta

The placenta regulates the passage of both endogenous and xenobiotic compounds to the fetus during pregnancy. A small number of xenobiotic metabolizing CYP enzymes are constantly expressed in placenta, those include CYP19A1 which mainly converts androgens to estrogens, and CYP1A1 whose substrates include steroid hormones and xenobiotics. We performed an analysis of co-expression of xenobiotic metabolizing enzyme-coding genes and transcription factors in placenta and in 86 other tissues to discern the unique characteristics of the placental transcriptome. Transcription factors (TFs) driving the expression of proteins involved in phase I and II xenobiotic metabolism in the liver were not expressed in the placenta, nor were the bulk of xenobiotic metabolizing hepatic CYP enzymes. In contrast, TFs whose co-expression correlated with CYP1A1, i.e. AHR, PPARG, and CEBPB were highly expressed in placenta. The placenta is a hormonal tissue, and one needs to maintain the tissue-specific focus by removing the hepatic spectacles.

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.

Structure-Activity Relationship Analysis of 3-phenylcoumarin-Based Monoamine Oxidase B Inhibitors

Monoamine oxidase B (MAO-B) catalyzes deamination of monoamines such as neurotransmitters dopamine and norepinephrine. Accordingly, small-molecule MAO-B inhibitors potentially alleviate the symptoms of dopamine-linked neuropathologies such as depression or Parkinsons disease. Coumarin with a functionalized 3-phenyl ring system is a promising scaffold for building potent MAO-B inhibitors. Here, a vast set of 3-phenylcoumarin derivatives was designed using virtual combinatorial chemistry or rationally de novo and synthesized using microwave chemistry. The derivatives inhibited the MAO-B at 100 nM−1 μM. The IC50 value of the most potent derivative 1 was 56 nM. A docking-based structure-activity relationship analysis summarizes the atom-level determinants of the MAO-B inhibition by the derivatives. Finally, the cross-reactivity of the derivatives was tested against monoamine oxidase A and a specific subset of enzymes linked to estradiol metabolism, known to have coumarin-based inhibitors. Overall, the results indicate that the 3-phenylcoumarins, especially derivative 1, present unique pharmacological features worth considering in future drug development.

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.

Peroxisome proliferator activated receptor gamma (PPAR-γ) ligand pioglitazone regulated gene networks in term human primary trophoblast cells

Peroxisome proliferator-activated receptor (PPAR-γ) is a nuclear receptor that is highly expressed in placenta. In this study, the PPAR-γ regulated gene networks were characterized in human primary trophoblast cells in vitro. The trophoblasts were isolated from full term placenta after delivery and exposed to 20 μM of the PPAR-γ agonist, pioglitazone, for 72 h and gene expression profiles were examined. Differential expression of selected genes was confirmed with RT-qPCR. Ingenuity pathway analysis was performed to identify PPAR-γ induced biological functions and downstream signaling pathways. In response to pioglitazone treatment, 37 genes were upregulated and 42 genes were downregulated as compared to control cells. The upregulated genes included those involved in metabolic pathways, whereas the expressions of many cytokines and chemokines were downregulated. Our data indicate that PPAR-γ possesses pleiotropic functions also in term trophoblasts regulating genes especially involved in cellular growth and proliferation, inflammatory and immunomodulatory responses and lipid metabolism.

Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives

Abstract A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-β-hydroxysteroid dehydrogenase 1 (HSD1) in the latter part of the sulphatase pathway. Five analogues produced ≥62% HSD1 inhibition at 5 µM and, furthermore, three of them produced ≥68% inhibition at 1 µM. A docking-based structure-activity relationship analysis was done to determine the molecular basis of the inhibition and the cross-reactivity of the analogues was tested against oestrogen receptor, aromatase, cytochrome P450 1A2, and monoamine oxidases. Most of the analogues are only modestly active with 17-β-hydroxysteroid dehydrogenase 2 – a requirement for lowering effective oestradiol levels in vivo. Moreover, the analysis led to the synthesis and discovery of 3-imidazolecoumarin as a potent aromatase inhibitor. In short, coumarin core can be tailored with specific ring and polar moiety substitutions to block either the sulphatase pathway or the aromatase pathway for treating breast cancer and endometriosis.

Kuopio birth cohort - design of a Finnish joint research effort for identification of environmental and lifestyle risk factors for the wellbeing of the mother and the newborn child

BackgroundA Finnish joint research effort Kuopio Birth Cohort (KuBiCo) seeks to evaluate the effects of genetics, epigenetics and different risk factors (medication, nutrition, lifestyle factors and environmental aspects) during pregnancy on the somatic and psychological health status of the mother and the child.MethodsKuBiCo will ultimately include information on 10,000 mother-child pairs who have given their informed consent to participate in this cohort. Identification of foetal health risk factors that can potentially later manifest as disease requires a repository of relevant biological samples and a flexible open up-to-date data handling system to register, store and analyse biological, clinical and questionnaire-based data. KuBiCo includes coded questionnaire-based maternal background data gathered before, during and after the pregnancy and bio-banking of maternal and foetal samples that will be stored in deep freezers. Data from the questionnaires and biological samples will be collected into one electronic database. KuBiCo consists of several work packages which are complementary to each other: Maternal, foetal and placental metabolism and omics; Paediatrics; Mental wellbeing; Prenatal period and delivery; Analgesics and anaesthetics during peripartum period; Environmental effects; Nutrition; and Research ethics.DiscussionThis report describes the set-up of the KuBiCo and descriptive analysis from 3532 parturients on response frequencies and feedback to KuBiCo questionnaires gathered from June 2012 to April 2016. Additionally, we describe basic demographic data of the participants (n = 1172). Based on the comparison of demographic data between official national statistics and our descriptive analysis, KuBiCo represents a cross-section of Finnish pregnant women.