Maternal caffeine intake during pregnancy and the risk of delivering a small for gestational age baby: Kuopio Birth Cohort.

PURPOSE: High caffeine intake during pregnancy is associated with restricted fetal growth. We aimed to evaluate the association between maternal caffeine intake during early and late pregnancy and the risk of delivering a small for gestational age (SGA) baby. METHODS: Kuopio Birth Cohort (KuBiCo) is a prospective cohort study including women whose pregnancies and deliveries were treated at the prenatal clinics in outpatient healthcare centers and in Kuopio University Hospital, Finland. Maternal diet and caffeine intake during the first (n = 2007) and third (n = 4362) trimester of pregnancy were assessed using a 160-item food frequency questionnaire (2013-2022). SGA was defined as birth weight corrected for gestational age below - 2 standard deviations from the mean, according to the sex-specific Finnish fetal growth curves. RESULTS: Altogether in 32 and 38% (1st and 3rd trimester) of all women and in 44 and 52% of coffee drinkers, caffeine intake exceeded the recommendation for caffeine intake ( ≤ 200 mg/day) during pregnancy. The women with moderate (51-200 mg/day) (aOR 1.87; 95% CI: 1.16-3.02) and high (> 200 mg/day) (aOR 1.51; 95% CI: 1.08-2.10) caffeine intake during the first trimester were in the highest risk of having an SGA newborn. Caffeine intake in the third trimester of pregnancy was not associated with SGA. CONCLUSIONS: Moderate and high caffeine intake during early pregnancy is associated with SGA. As the results suggest that even moderate caffeine intake during the first trimester may increase the risk of SGA, the intake within recommendation limits does not necessarily appear to be safe for pregnant women and their newborns.

ICH S1 prospective evaluation study: weight of evidence approach to predict outcome and value of 2-year rat carcinogenicity studies. A report from the regulatory authorities subgroup.

Introduction: The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) initiated a process in 2012 to revise the S1B Guideline "Testing for Carcinogenicity of Pharmaceuticals". Previous retrospective analysis indicated the importance of histopathological risk factors in chronic toxicity studies, evidence of endocrine perturbation, and positive genetic toxicology results as potentially predictive indicators of carcinogenic risk. In addition, a relationship between pharmacodynamic activity and carcinogenicity outcome in long-term rodent studies has been reported. It was postulated that these factors could be evaluated in a Weight-of-Evidence (WoE) approach to predict the outcome of a 2-year rat study. Methods: The ICH S1B(R1) Expert Working Group (EWG) conducted a Prospective Evaluation Study (PES) to determine the regulatory feasibility of this WoE approach. Drug Regulatory Authorities (DRAs) evaluated 49 Carcinogenicity Assessment Documents (CADs), which describe the WoE for submitted pharmaceutical compounds. Each compound was categorized into a carcinogenic risk category including a statement of the value of the 2-year rat study. The outcome of the completed 2-year rat studies was evaluated in relation to the prospective CAD to determine the accuracy of predictions. Results: Based on the results of the PES, the EWG concluded that the evaluation process for assessing human carcinogenic risk of pharmaceuticals described in ICH S1B could be expanded to include a WoE approach. Approximately 27% of 2-year rat studies could be avoided in cases where DRAs and sponsors unanimously agreed that such a study would not add value. Discussion: Key factors supporting a WoE assessment were identified: data that inform carcinogenic potential based on drug target biology and the primary pharmacologic mechanism of the parent compound and major human metabolites; results from secondary pharmacology screens for this compound and major human metabolites that inform carcinogenic risk; histopathology data from repeated-dose toxicity studies; evidence for hormonal perturbation; genotoxicity data; and evidence of immune modulation. The outcome of the PES indicates that a WoE approach can be used in place of conducting a 2-year rat study for some pharmaceuticals. These data were used by the ICH S1B(R1) EWG to write the R1 Addendum to the S1B Guideline published in August 2022.

Evaluation of Lippia scaberrima Sond. and Aspalathus linearis (Burm.f.) R. Dahlgren extracts on human CYP enzymes and gold nanoparticle synthesis: implications for drug metabolism and cytotoxicity.

BACKGROUND: Metabolism is an important component of the kinetic characteristics of herbal constituents, and it often determines the internal dose and concentration of these effective constituents at the target site. The metabolic profile of plant extracts and pure compounds need to be determined for any possible herb-drug metabolic interactions that might occur. METHODS: Various concentrations of the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with fermented and unfermented Aspalathus linearis extract were used to determine the inhibitory potential on placental, microsomal and recombinant human hepatic Cytochrome P450 enzymes. Furthermore, the study investigated the synthesis and characterization of gold nanoparticles from the ethanolic extract of Lippia scaberrima as a lead sample. Confirmation and characterization of the synthesized gold nanoparticles were conducted through various methods. Additionally, the cytotoxic properties of the ethanolic extract of Lippia scaberrima were compared with the gold nanoparticles synthesized from Lippia scaberrima using gum arabic as a capping agent. RESULTS: All the samples showed varying levels of CYP inhibition. The most potent inhibition took place for CYP2C19 and CYP1B1 with 50% inhibitory concentration (IC50) values of less than 0.05 µg/L for the essential oil tested and IC50-values between 0.05 µg/L-1 µg/L for all the other combinations and extracts tested, respectively. For both CYP1A2 and CYP2D6 the IC50-values for the essential oil, the extracts and combinations were found in the range of 1 - 10 µg/L. The majority of the IC50 values found were higher than 10 µg/L and, therefore, were found to have no inhibition against the CYP enzymes tested. CONCLUSION: Therefore, the essential oil of Lippia scaberrima, the ethanolic extract of Lippia scaberrima alone and their combinations with Aspalathus linearis do not possess any clinically significant CYP interaction potential and may be further investigated for their adjuvant potential for use in the tuberculosis treatment regimen. Furthermore, it was shown that the cytotoxic potential of the Lippia scaberrima gold nanoparticles was reduced by twofold when compared to the ethanolic extract of Lippia scaberrima.

Maternal caffeine, coffee and cola drink intake and the risk of gestational diabetes - Kuopio Birth Cohort.

AIMS: Coffee intake is associated with a decreased risk of type 2 diabetes among non-pregnant people. We aimed to investigate the association between caffeine, coffee and cola drink intake in early pregnancy and the risk of gestational diabetes (GDM). METHODS: Kuopio Birth Cohort (KuBiCo) is a prospective cohort study including pregnant women who were followed at the prenatal clinics in outpatient healthcare centers and gave birth in Kuopio University Hospital, Finland (n=2214). Maternal diet during the first trimester of pregnancy was assessed using a 160-item food frequency questionnaire. GDM was diagnosed by oral glucose tolerance test according to the Finnish national guidelines mainly between 24 and 28 gestational weeks. RESULTS: Women with moderate coffee intake in the first trimester were less likely diagnosed with GDM than women without coffee intake in an age-adjusted model (OR 0.87; 95% CI 0.76-0.99; p = 0.03), but the association was attenuated in multi-adjusted models (p = 0.11). No association was found between caffeine intake and GDM. One third (32.4%) of pregnant women consumed caffeine over the recommendation (> 200 mg/d). Women who consumed cola drinks more than the median (33.3 mL/d) had an increased risk of GDM (OR 1.29; 95% CI 1.02-1.63, p = 0.037) in multi-adjusted model compared to those who consumed less. CONCLUSIONS: Caffeine intake during the first trimester of pregnancy was not associated with the risk of GDM but a minor non-significant decrease was seen with moderate coffee intake. Although the average consumption of cola drinks was low in the KuBiCo cohort, higher consumption was associated with an increased risk of GDM. Further studies are needed to evaluate the safe amount of coffee during pregnancy, since the recommended caffeine intake was exceeded in almost half of the coffee drinkers.

Longitudinal Analyses of Diet Quality and Maternal Depressive Symptoms During Pregnancy: The Kuopio Birth Cohort Study.

BACKGROUND: Depression and diet quality appear to be associated in the general population. Nevertheless, little is known about their relationship among pregnant females. OBJECTIVE: The aims of this study were first, to investigate longitudinally whether or not diet quality is associated with depressive symptoms during pregnancy; second, to examine whether or not variation in diet quality during pregnancy predicts variation in depressive symptoms; and third, to explore how individual dietary components are associated with depressive symptoms. DESIGN: A longitudinal secondary analysis of the Kuopio Birth Cohort Study in eastern Finland was conducted. Data were collected from pregnant females during the first and third trimesters of pregnancy. PARTICIPANTS/SETTING: The participants were 1,362 pregnant females who entered the study between 2012 and 2017. MAIN OUTCOME MEASURES: Depressive symptoms, as measured with the Edinburgh Postnatal Depressive Scale during the first and third trimesters of pregnancy were used as continuous variables. STATISTICAL ANALYSES PERFORMED: The main analyses consisted of linear mixed model analyses adjusted for potential confounders to longitudinally assess the association between diet quality as measured by the Healthy Eating Index-2015, calculated using data from a food frequency questionnaire completed during the first trimester and third trimester, and depressive symptoms during the study period. An exploratory set of linear mixed models was also used to longitudinally assess the associations between selected individual food frequency questionnaire food groups and depressive symptoms. RESULTS: Descriptive analyses revealed that 12.3% of the participants had clinically relevant levels of depressive symptoms (ie, Edinburgh Postnatal Depressive Scale score ≥10) during either the first or third trimester. Longitudinal modeling suggested that depressive symptoms in pregnant females tend to remain stable throughout pregnancy. Females with a poorer quality diet already displayed higher levels of depressive symptoms during the first trimester of pregnancy (ß = -.038 ± .016; P = 0.022). Variation in diet quality did not predict variation in depressive symptoms over the course of pregnancy (ß = -9.741 × 10-5 ± .001; P = 0.869). CONCLUSIONS: Females entering pregnancy with a poorer quality diet also displayed higher levels of depressive symptoms compared with females with a higher quality diet at the beginning of pregnancy, and this association remained constant throughout pregnancy. Further research is needed to assess the direction and the potential causality of the observed associations between diet quality and depressive symptoms.

Placental ion channels: potential target of chemical exposure.

The placenta is an important organ for the exchange of substances between the fetus and the mother, hormone secretion, and fetoplacental immunological defense. Placenta has an organ-specific distribution of ion channels and trophoblasts, and placental vessels express a large number of ion channels. Several placental housekeeping activities and pregnancy complications are at least partly controlled by ion channels, which are playing an important role in regulating hormone secretion, trophoblastic homeostasis, ion transport, and vasomotor activity. The function of several placental ion channels (Na, Ca, and Cl ion channels, cation channel, nicotinic acetylcholine receptors, and aquaporin-1) is known to be influenced by chemical exposure, i.e., their responses to different chemicals have been tested and confirmed in experimental models. Here, we review the possibility that placental ion channels are targets of toxicological concern in terms of placental function, fetal growth, and development.

Aflatoxin B1 targeted gene expression profiles in human placental primary trophoblast cells.

Aflatoxin B1 (AFB1) is a mycotoxin produced by Aspergillus flavus and A. parasiticus. A high exposure (40 nM and 1 µM AFB1 for 72 h) was used to study mechanistic effects of AFB1 on gene expression patterns in human primary trophoblast cells, isolated from full term placentae after delivery. Gene expression profiling was conducted, and Ingenuity pathway analysis (IPA) software was used to identify AFB1-regulated gene networks and regulatory pathways. In response to 40 nM AFB1, only 7 genes were differentially expressed whereas 1 µM AFB1 significantly dysregulated 170 genes (124 down- and 46 upregulated, ±1.5-fold, p < 0.05) in AFB1-exposed trophoblasts when compared to controls. The top downregulated genes were involved in endocrine signalling and biosynthesis of hormones, and lipid and carbohydrate metabolism. The top upregulated genes were involved in protein synthesis and regulation of cell cycle. The main canonical pathways identified by IPA were associated with endocrine signalling including growth hormone signalling, and corticotropin releasing hormone signalling. Furthermore, genes involved in aryl hydrocarbon receptor (AhR)-mediated estrogen receptor signalling were dysregulated in response to AFB1. Our findings indicate that a high concentration 72 h AFB1 exposure caused relatively moderate number of changes on transcript level to human placental primary trophoblast cells. However, these preliminary results need to be confirmed with human-relevant concentrations of AFB1.

The impact of postpartum depressive symptoms on self-reported infant health and analgesic consumption at the age of 12 months: A prospective cohort study.

The infants of mothers with elevated depressive symptoms (EDS) postpartum appear to be at increased risk of somatic health problems during their first 12 months of life in low- and lower-middle-income countries. However, in higher-income countries, knowledge of this association is scarce. We sought to examine whether maternal reports of infant health problems, adherence to vaccination schedules and analgesic supply to the infant during the first 12 months of life differ between mothers with and without postpartum EDS. Altogether, 969 women who were enrolled in the Kuopio Birth Cohort study (www.kubico.fi) during 2012-2017 were included in this investigation. Depressive symptoms were measured with the Edinburgh Postnatal Depression Scale during pregnancy (1st and/or 3rd trimester) and at eight weeks postpartum. Infant health data were collected as a part of a 12-month online follow-up questionnaire for mothers and were based on self-reports of either maternal observations or physician-determined diagnoses. Postpartum EDS were associated with a 2- to 5-fold increased likelihood of abnormal crying and paroxysmal wheezing (based on parental observations), as well as gastroesophageal reflux and food allergy (based on physician-determined diagnoses). Mothers with postpartum EDS also supplied their infants with analgesic medication for longer periods. Adherence to vaccination schedules was similar between the examined groups. In conclusion, infants of mothers with postpartum EDS may be more likely to experience health problems or to be perceived by their mother as having health problems, and thus receive more medications.

Caffeine content in newborn hair correlates with maternal dietary intake.

PURPOSE: High-maternal caffeine intake during pregnancy may be harmful for perinatal outcomes and future child health, but the level of fetal cumulative exposure has been difficult to measure thus far. Here, we present maternal dietary caffeine intake during the last trimester and its correlation to caffeine content in newborn hair after birth. METHODS: Maternal third trimester diets and dietary caffeine intake were prospectively collected in Kuopio Birth Cohort (KuBiCo) using a 160-item food frequency questionnaire (n = 2840). Newborn hair was collected within 48 h after birth and analyzed by high-resolution mass spectrometry (HRMS) for caffeine (n = 316). Correlation between dietary caffeine intake and neonatal hair caffeine content was evaluated from 203 mother-child pairs. RESULTS: Mean dietary caffeine intake was 167 mg/days (95% CI 162-172  mg/days), of which coffee comprised 81%. Caffeine in the maternal diet and caffeine content in newborn hair correlated significantly (r = 0.50; p < 0.001). Older, multiparous, overweight women, and smokers had the highest caffeine levels in the maternal diet, as well as in their newborn babies' hair. CONCLUSION: Caffeine exposure, estimated from newborn hair samples, reflects maternal third trimester dietary caffeine intake and introduces a new method to assess fetal cumulative caffeine exposure. Further studies to evaluate the effects of caffeine exposure on both perinatal and postnatal outcomes are warranted, since over 40% of pregnant women consume caffeine more than the current suggested recommendations (European Food Safety Association, EFSA recommendations).

Aryl hydrocarbon receptor (AhR) agonist ß-naphthoflavone regulated gene networks in human primary trophoblasts.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in placenta. AhR belongs to a class of transcriptional regulators that control many developmental and physiological events (e.g. xenobiotic metabolism). Our study describes AhR regulated transcriptional responses in human primary trophoblast by using the AhR agonist, ß-naphthoflavone (BNF). Human primary trophoblast cells were isolated from full term placenta after delivery. The trophoblasts were exposed to 25 µM of AhR agonist, BNF, for 72 hours. Gene expression profiling was conducted with Illumina HT-12 expression beadchips. Expression of selected genes was confirmed with RT-qPCR. Ingenuity pathway analysis (IPA) was used to predict functional pathways and upstream regulators of differentially expressed genes in order to identify regulatory networks associated with AhR. In response to BNF exposure, 64 genes were upregulated, and 257 genes were downregulated compared to control trophoblasts (±1.5-fold, p < 0.05). BNF regulated genes included placental hormones and genes implicated in immune- and inflammatory responses in addition to their well-known effects on xenobiotic metabolism, oxidative stress, antioxidant defense. In conclusion, these results show that BNF has wide-ranging effects on placental gene expression beyond xenobiotic metabolism e.g. disruption of inflammatory processes and hormones in the placenta.

In Vitro Human Metabolism and Inhibition Potency of Verbascoside for CYP Enzymes.

Verbascoside is found in many medicinal plant families such as Verbenaceae. Important biological activities have been ascribed to verbascoside. Investigated in this study is the potential of verbascoside as an adjuvant during tuberculosis treatment. The present study reports on the in vitro metabolism in human hepatic microsomes and cytosol incubations as well as the presence and quantity of verbascoside within Lippia scaberrima. Additionally, studied are the inhibitory properties on human hepatic CYP enzymes together with antioxidant and cytotoxic properties. The results yielded no metabolites in the hydrolysis or cytochrome P450 (CYP) oxidation incubations. However, five different methylated conjugates of verbascoside could be found in S-adenosylmethionine incubation, three different sulphate conjugates with 3'-phosphoadenosine 5'-phosphosulfate (PAPS) incubation with human liver samples, and very low levels of glucuronide metabolites after incubation with recombinant human uridine 5'-diphospho-glucuronosyltransferase (UGT) 1A7, UGT1A8, and UGT1A10. Additionally, verbascoside showed weak inhibitory potency against CYP1A2 and CYP1B1 with IC50 values of 83 µM and 86 µM, respectively. Potent antioxidant and low cytotoxic potential were observed. Based on these data, verbascoside does not possess any clinically relevant CYP-mediated interaction potential, but it has effective biological activity. Therefore, verbascoside could be considered as a lead compound for further drug development and as an adjuvant during tuberculosis treatment.

Laparoscopic Roux-en-Y gastric bypass surgery influenced pharmacokinetics of several drugs given as a cocktail with the highest impact observed for CYP1A2, CYP2C8 and CYP2E1 substrates.

There is a lack of information about the changes in drug pharmacokinetics and cytochrome P450 (CYP) metabolism after bariatric surgery. Here, we investigated the effects of laparoscopic Roux-en-Y gastric bypass (LRYGB) surgery on pharmacokinetics of nine drugs given simultaneously which may reveal changes in the activities of the main CYPs. Eight obese subjects undergoing LRYGB received an oral cocktail containing nine drugs, substrates of various CYPs: melatonin (CYP1A2), nicotine (CYP2A6), bupropion (CYP2B6), repaglinide (CYP2C8), losartan (CYP2C9), omeprazole (CYP2C19/CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1) and midazolam (CYP3A). The 6-hours pharmacokinetic profiles in serum and urine of each drug or corresponding metabolite as well as their metabolic ratios were compared before surgery with those at a median 1 year later. LRYGB exerted variable effects on the pharmacokinetics of these drugs. The geometric mean AUC0-6 (90% confidence interval) of melatonin, bupropion, repaglinide, chlorzoxazone and midazolam after LRYGB was 27 (19%-41%), 54 (43%-67%), 44 (29%-66%), 160 (129%-197%) and 74 (62%-90%) of the pre-surgery values, respectively. The pharmacokinetics of losartan, omeprazole and dextromethorphan did not change in response to surgery. Nicotine was not detected in serum, while geometric mean of AUC0-6 of its metabolite, cotinine, increased by 1.7 times after surgery. There were 3.6- and 1.3-fold increases in the AUC ratios of 6-hydroxymelatonin/melatonin and hydroxybupropion/bupropion, respectively. The cocktail revealed multiple pharmacokinetic changes occurring after LRYGB with the greatest effects observed for CYP1A2, CYP2C8 and CYP2E1 substrates. Future studies should be focused on CYP1A2, CYP2A6, CYP2C8 and CYP2B6 to clarify the changes in activities of these enzymes after LRYGB.

The dynamic course of peripartum depression across pregnancy and childbirth.

OBJECTIVE: Peripartum depression (PPD) pertaining to depression in pregnancy and postpartum is one of the most common complications around childbirth with enduring adverse effects on mother and child health. Although psychiatric symptoms may improve or worsen over time, relatively little is known about the course of PPD symptoms and possible fluctuations. METHODS: We applied a person-centered approach to examine PPD symptom patterns across pregnancy and childbirth. 824 women were assessed at three time points: first trimester (T1), third trimester (T2), and again at eight weeks (T3) postpartum. We assessed PPD symptoms, maternal mental health history, and childbirth variables. RESULTS: Growth mixture modeling (GMM) analysis revealed four discrete PPD symptom trajectory classes including chronic PPD (1.1%), delayed (10.2%), recovered (7.2%), and resilient (81.5%). Delivery complications were associated with chronic PPD but also with the recovered PPD trajectory class. History of mental health disorders was associated with chronic PPD and the delayed PPD class. CONCLUSION: The findings underscore that significant changes in a woman's depression level can occur across pregnancy and childbirth. While a minority of women experience chronic PDD, for others depression symptoms appear to significantly alleviate over time, suggesting a form of recovery. Our findings support a personalized medicine approach based on the woman's symptom trajectory. Future research is warranted to identify the mechanisms underlying modifications in PPD symptoms severity and those implicated in recovery.

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.

BACKGROUND: A 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. METHODS: KuBiCo 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. DISCUSSION: This 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.

The association between gestational diabetes mellitus and postpartum depressive symptomatology: A prospective cohort study.

BACKGROUND: The literature suggests an association between type 2 diabetes mellitus and depression, but data on the association between gestational diabetes mellitus (GDM) and postpartum depressive symptomatology (PPDS) are scarce. METHODS: Altogether, 1066 women with no previous mental health issues enrolled in the Kuopio Birth Cohort (KuBiCo, www.kubico.fi) were selected for this study. GDM was diagnosed according to the Finnish Current Care Guidelines. Depressive symptomatology was assessed with the Edinburgh Postnatal Depression Scale (EPDS) during the third trimester of pregnancy and eight weeks after delivery. Additionally, a subgroup of women (n = 505) also completed the EPDS during the first trimester of pregnancy. RESULTS: The prevalence rates of GDM and PPDS in the whole study population were 14.1% and 10.3%, respectively. GDM was associated with an increased likelihood of belonging to the PPDS group (OR 2.23, 95% CI 1.23-4.05; adjusted for maternal age at delivery, BMI in the first trimester, smoking before pregnancy, relationship status, nulliparity, delivery by caesarean section, gestational age at delivery, neonatal intensive care unit admission and third-trimester EPDS scores). A significant association between GDM and PPDS was found in the subgroup of women with available data on first-trimester depression (n = 505). LIMITATIONS: The participation rate of the KuBiCo study was relatively low (37%). CONCLUSIONS: Women with GDM may be at increased risk of PPDS. Future studies should investigate whether these women would benefit from a closer follow-up and possible supportive interventions during pregnancy and the postpartum period to avoid PPDS.

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.

New glutathione conjugate of pyrrolizidine alkaloids produced by human cytosolic enzyme-dependent reactions in vitro.

RATIONALE: The toxic metabolites of pyrrolizidine alkaloids (PAs) are initially formed by cytochrome P450-mediated oxidation reactions and primarily eliminated as glutathione (GSH) conjugates. Although the reaction between the reactive metabolites and GSH can occur spontaneously, the role of the cytosolic enzymes in the process has not been studied. METHODS: The toxic metabolites of selected PAs (retrorsine, monocrotaline, senecionine, lasiocarpine, heliotrine or senkirkine) were generated by incubating them in 100 mM phosphate buffer (pH 7.4) containing liver microsomes of human, pig, rat or sheep, NADPH and reduced GSH in the absence or presence of human, pig, rat or sheep liver cytosolic fraction. The supernatants were analyzed using liquid chromatography connected to Finnigan LTQ ion-trap, Agilent QTOF or Thermo Scientific Q Exactive Focus quadrupole-orbitrap mass spectrometers. RESULTS: Retrorsine, senecionine and lasiocarpine yielded three GSH conjugates producing [M - H]- ions at m/z 439 (7-GSH-DHP (CHO)), m/z 441 (7-GSH-DHP (OH)) and m/z 730 (7,9-diGSH-DHP) in the presence of human liver cytosolic fraction. 7-GSH-DHP (CHO) was a novel metabolite. Monocrotaline, heliotrine and senkirkine did not produce this novel 7-GSH-DHP (CHO) conjugate. 7-GSH-DHP (CHO) disappeared when incubated with hydroxylamine, and a new oxime derivative was formed. This metabolite was formed only by the human liver cytosolic enzymes but not in the presence of rat or sheep liver cytosolic fractions under otherwise identical reaction conditions. CONCLUSIONS: 7-GSH-DHP (CHO) has not been reported before, and thus it was considered as a novel metabolite of PAs. This may clarify the mechanisms involved in PA detoxification and widely observed but less understood species differences in response to PA exposure.

Blocking oestradiol synthesis pathways with potent and selective coumarin derivatives.

A comprehensive set of 3-phenylcoumarin analogues with polar substituents was synthesised for blocking oestradiol synthesis by 17-ß-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-ß-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.