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PURPOSE: Assisted reproduction technologies (ART) are associated with increased risks of pregnancy complications and obstetric interventions. Here, we aimed to determine if ART affects placental inflammation and oxidative stress as a mechanism for unfavorable pregnancy outcomes. METHODS: The levels of six cytokines (IFN-γ, IL-1β, IL-6, IL-8, IL-10, TNFα) were measured using multiplex ELISA. The activity of four antioxidant enzymes (glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase, superoxide dismutase) and levels of two antioxidants (GSH, vitamin E) were measured using commercial/in-house assays. Markers were compared between ART and unassisted pregnancies, and then groups were stratified using ICD9/10 codes to determine differences in specific clinical contexts. RESULTS: In unassisted twin pregnancies, there was a trend of decreased cytokine levels (IL-1β, IL-6, IL-8, TNFα, p

Consumption of sucrose (table sugar) is high in much of the world. The effects of maternal sucrose intake on the placenta and fetal brain remain unknown. In rats, maternal consumption of sucrose at a human-relevant level alters the mother's physiology and steroids, as well as the adult offspring's brain and behavior. Effects in mothers are impaired glucose tolerance, increased liver lipids, increased adipose inflammation, and decreased corticosterone levels in the blood but not in the brain. In contrast, in adult female offspring, maternal sucrose intake increases corticosterone levels in the blood and the brain. In adult male offspring, maternal sucrose intake increases preference for high-sucrose and high-fat diets as well as motivation for sugar rewards in a progressive ratio task. In this study, we investigate the underlying mechanisms of the observed behavioral and endocrine effects in the adult offspring. We examine anti-inflammatory steroids, steroidogenic enzymes and cytokines in the placenta, amniotic fluid, fetal blood and brain. In our model, we feed rat dams either a high-sucrose diet (26% of kCal) or an isocaloric, matched, control diet (1% sucrose) 10 weeks prior to and during gestation. At embryonic day 19 (E19), we collected maternal serum, placenta, amniotic fluid, fetal blood, and fetal brain. We used Palkovits punch to microdissect the placenta and fetal brain. Next, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is highly sensitive and specific, to measure multiple steroids (e. g. corticosterone, estrone, allopregnanolone). We examined multiple regions of the fetal brain (e. g. prefrontal cortex, nucleus accumbens, hypothalamus, hippocampus). We will also examine neuronal proliferation, microglia and tyrosine hydroxylase immunoreactivity in the fetal brain. Maternal high-sucrose diet increased 11-dehydrocorticosterone (inactive metabolite of corticosterone) and aldosterone in maternal serum and amniotic fluid. Testosterone and androstenedione were significantly higher in the amniotic fluid of male fetuses than female fetuses. Placental steroidogenic enzymes 3β-HSD and CYP19 were not affected by maternal diet or fetal sex; however, 3β-HSD activity was higher in the decidua than in the fetal part of the placenta. Steroid and cytokine data from the placenta, fetal blood and fetal brain are currently being analyzed. Ongoing analyses examine how a maternal high-sucrose diet affects brain development possibly by increasing inflammation. Presentation: No date and time listed

Mathematical models that can predict the kinetics of compounds have been increasingly adopted for drug development and risk assessment. Data for these models may be generated from in vitro experimental systems containing enzymes contributing to metabolic clearance, such as subcellular tissue fractions including microsomes and cytosol. Extrapolation from these systems is facilitated by common scaling factors, known as microsomal protein per gram (MPPG) and cytosolic protein per gram (CPPG). Historically, parameterization of MPPG and CPPG has employed the use of recovery factors, commonly benchmarked to cytochromes P450 which work well in some contexts, but could be problematic for other enzymes. Here, we propose absolute quantification of protein content and supplementary assays to evaluate microsomal/cytosolic purity that should be employed. Examples include calculation of microsomal latency by mannose-6-phosphatase activity and immunoblotting of subcellular fractions with fraction-specific markers. Further considerations include tissue source, as disease states can affect enzyme expression and activity, and the methodology used for scalar parameterization. Regional- and organ-specific expression of enzymes, in addition to differences in organ physiology, is another important consideration. Because most efforts have focused on the liver that is, for the most part, homogeneous, derived scalars may not capture the heterogeneity of other major tissues contributing to xenobiotic metabolism including the kidneys and small intestine. Better understanding of these scalars, and how to appropriately derive them from extrahepatic tissues can provide support to the inferences made with physiologically based pharmacokinetic modeling, increase its accuracy in characterizing in vivo drug pharmacokinetics, and improve confidence in go-no-go decisions for clinical trials.

The elimination of numerous endogenous compounds and xenobiotics via glucuronidation by uridine-5'-diphosphate glycosyltransferase enzymes (UGTs) is an essential process of the body's chemical defense system. UGTs have distinct but overlapping substrate preferences, but the molecular basis for their substrate specificity remains poorly understood. Three-dimensional protein structures can greatly enhance our understanding of the interactions between enzymes and their substrates, but because of the inherent difficulties in purifying and crystallizing integral endoplasmic reticulum membrane proteins, no complete mammalian UGT structure has yet been produced. To address this problem, we have created a homology model of UGT1A6 using I-TASSER to explore, in detail, the interactions of human UGT1A6 with its substrates. Ligands were docked into our model in the presence of the cosubstrate uridine-5'-diphosphate-glucuronic acid, interacting residues were examined, and poses were compared to those cocrystallized with various plant and bacterial glycosyltransferases (GTs). Our model structurally resembles other GTs, and docking experiments replicated many of the expected UGT-substrate interactions. Some bias toward the template structures' protein-substrate interactions and binding preferences was evident.

The umbilical cord (UC) connects the fetal blood supply to the placenta, so is exposed to all systemic endo- and xenobiotics. We have extensive experience using UC as an analytical matrix for detecting and/or quantitating drugs, chemicals and endogenous compounds. This technical note describes advantages (large amount available, ease of collection, small sample needed for use, rapid availability) and challenges (clinical relationships, processing difficulties, matrix effects on analytes and detection technologies) of UC as an analytical matrix in ELISA and LC/MS platforms, and provides guidance for successfully working with this tissue.

Highlights • Using umbilical cord, we report a prevalence of NSAID use of ∼3 % close to labour. • A novel UHPLC-MS/MS method for detecting 5 NSAIDs was developed and validated. • NSAIDs are contraindicated in first and third trimesters, but use is increasing. • Determining safe dosages of NSAIDs in pregnancy should be a priority., Background Nonsteroidal anti-inflammatory drugs are contraindicated in the third trimester of pregnancy due to negative effects including alteration of uteroplacental blood flow, premature ductus arteriosus closure, and adverse effects on the fetal kidney. However, many women are unaware of these risks, and commonly report their use in pregnancy. We aimed to determine if umbilical cord was a reliable matrix for detecting NSAID use, determine incidence of use close to labour, and uncover associations with obstetric/neonatal outcomes. Methods We developed a UHPLC-MS/MS method to simultaneously detect diclofenac, ibuprofen, indomethacin, naproxen, and salicylic acid in plasma and umbilical cord lysate. Using this method, we screened 380 lysates to determine the prevalence of NSAID use. Results were compared to the clinical outcomes in pregnancy using ICD9/10 chart codes (n = 21). Results The UHPLC-MS/MS method has excellent linearity, accuracy, and precision in solvent and plasma, but lower sensitivity in umbilical cord lysate. We report a 3 % rate of NSAID ingestion within days of labour – the pharmacokinetically-determined window for active ingestion. There were no significant differences observed for maternal, obstetric, or neonatal outcomes between the NSAID positive group (n = 11) and NSAID negative group (n = 369). Conclusions Because NSAID use in third trimester is contraindicated, even a 3% usage rate is alarmingly high. Based on UHPLC-MS/MS performance of umbilical cord lysate, 3% is likely a conservative estimate. Recent adoption of NSAIDs under clinical supervision to support in vitro fertilisation and prevent pre-eclampsia indicates future work should focus on determining safe dosages of NSAIDs and the correct therapeutic window in pregnancy.

UDP-glucuronosyltransferase (UGT)–mediated metabolism is possibly the most important conjugation reaction for marketed drugs. However, there are currently no generally accepted standard incubation conditions for UGT microsomal assays, and substantial differences in experimental design and methodology between laboratories hinder cross-study comparison of in vitro activities. This study aimed to define optimal experimental conditions to determine glucuronidation activity of multiple UGT isoforms simultaneously using human liver microsomes. Hepatic glucuronidation activities of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17 were determined using cocktail incubations of 10 UGT probe substrates. Buffer components and cosubstrates were assessed over a range of concentrations including magnesium chloride (MgCl2; 0–10 mM) and uridine 5′-diphosphoglucuronic acid (UDPGA; 1–25 mM) with either Tris-HCl or potassium phosphate buffer (100 mM, pH 7.4). Greater microsomal glucuronidation activity by different hepatic UGT isoforms was obtained using 10 mM MgCl2 and 5 mM UDPGA with 100 mM Tris-HCl buffer. The influence of bovine serum albumin (BSA; 0.1%–2% w/v) on glucuronidation activity was also assessed. Enzyme- and substrate-dependent effects of BSA were observed, resulting in decreased total activity of UGT1A1, UGT1A3, and UGT2B17 and increased total UGT1A9 and UGT2B7 activity. The inclusion of BSA did not significantly reduce the between-subject variability of UGT activity. Future in vitro UGT profiling studies under the proposed optimized experimental conditions would allow high-quality positive control data to be generated across laboratories, with effective control of a high degree of between-donor variability for UGT activity and for chemical optimization toward lower-clearance drug molecules in a pharmaceutical drug discovery setting.

The hematological side effects associated with mycophenolic acid (MPA) are relatively common and have severe consequences. The majority of literature data have not shown clear consistency in the MPA exposure-neutropenia relationship. We hypothesized that (i) adult de novo kidney transplant recipients who develop neutropenia have relatively higher dose-normalized MPA exposure than patients without neutropenia, and (ii) the observed neutropenia may be explained by polymorphisms in metabolism and/or transporter genes responsible for MPA disposition. Adult kidney transplant recipients on steady-state tacrolimus and MPA, not receiving a corticosteroid, and with stable renal function were recruited for investigation at three periods post-transplant (1, 3, and 12 months; n = 21, 17, and 13, respectively). Clinical variables (age, weight, MPA daily dose, albumin, serum creatinine, absolute neutrophil count), tacrolimus and MPA concentrations (for exposure calculation), and genotypes (UGT2B7 G211T, UGT2B7 C802T, UGT1A9 T-275A, UGT1A9 T98C, MRP2 C-24T, MRP2 G1249A, OATP1B1 A388G, OATP1B1 C463A) were characterized. A significant inverse association between dose-normalized MPA exposure (a surrogate marker for apparent MPA clearance) and absolute neutrophil count in all three study periods (r2 ~ 0.3–0.7) was observed. No associations between characterized single nucleotide polymorphisms and MPA exposure or absolute neutrophil count were established. However, significant alterations in the minor allele frequencies of UGT2B7*2 C802T, UGT1A9 T275A, and MRP2 G1249A were evident. These findings support the clinical strategy for conducting MPA therapeutic drug monitoring in adult kidney transplant patients on steroid-free immunosuppressant therapy. The novel population genomic analysis data warrant further epidemiological investigations in a larger study sample.

Background Although medications should only be prescribed in pregnancy if benefits to the mother outweigh the risk to the fetus, drug use in pregnancy especially prescribed and over-the-counter analgesics, is very common. Objective The objective of this review is to present an update on known changes in analgesic disposition in pregnancy caused by pharmacokinetic mechanisms. Method Herein, we discuss a wide range of medical, biomedical and scientific literature that includes reports from the fields of dentistry, general medicine, obstetrics and gynecology, pharmacology and toxicology to provide an update on the use (including indications, contraindications and concerns) of major classes of analgesics during human pregnancy. Results Over 50% of analgesics are in pregnancy category C, and even more category D specifically in the third trimester. Changes in renal filtration, cardiac output, plasma protein concentration and plasma volume particularly affect analgesics and dose adjustments may be necessary to maintain therapeutic concentrations in pregnant woman, and/or to protect the developing fetus. Conclusion Analgesics are one of the most frequently used drug classes in pregnancy. More than 60% of women self-report using analgesics while pregnant, both prescribed and by self-medication. For the majority of analgesics available (excepting acetaminophen and the NSAIDs, and to a lesser extent certain opioids), good prospective clinical trials documenting pharmacokinetic changes do not exist. More research is needed in both the scientific and clinical community to understand the risks and benefits of analgesic use in pregnancy, particularly as prevalence is rising.

1. The UDP-glucuronosyltransferase (UGT) enzymes are important in the metabolism, elimination and detoxification of many xenobiotics and endogenous compounds. As extrapolation of in vitro kinetics of drug metabolizing enzymes to predict in vivo clearance rates becomes more sophisticated, it is important to ensure proper optimization of enzyme assays. The luminal location of the enzyme active site (i.e. latency), and the complexity of UGT kinetics, results in consistent under-prediction of clearance of drugs metabolized by glucuronidation.2. We examined inhibition of UGT activity in alamethicin-disrupted human liver microsomes (HLM) by uridine diphosphate (UDP), a UGT reaction product, and its reversal by Mg2+ ions. We also determined whether UDP-sugars other than the co-substrate UDP-glucuronic acid (UDP-GlcA) affected glucuronidation.3. We show that other UDP-sugars do not significantly influence glucuronidation. We also demonstrate that UDP inhibits HLM UGT activity and that this is reversed by ...

Uridine diphosphate glucuronosyltransferases (UGTs) catalyze glucuronidation to facilitate systemic and local clearance of numerous chemicals and drugs. To investigate whether UGT expression is coregulated in human liver, we analyzed the protein expression of UGTs 1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 3A1, and 3A2 using western blots from 164 healthy human liver samples, comparing expression with age and sex. UGT1A6 levels were significantly higher in children than adults, and UGT3A1 and 3A2 expression significantly increased with age from childhood to age65 yearas. In children aged18 years, UGT1A4/1A9 protein expression was significantly correlated, but not for adults aged18 years. UGT1A3 expression was always significantly correlated with other UGT1A isoforms in all adults aged18 years. In individuals aged ≥12 years, expression of UGT1A1/1A4, UGT1A1/1A6, UGT1A1/1A9, and UGT1A4/1A6 significantly correlated, which was not observed in children aged12 years. In contrast, UGT1A4/2B7 showed significant correlation in children aged12 years, but not in individuals aged ≥12 years, and this was observed in female but not male individuals. Expression of UGT1A6/1A9 and UGT3A1/3A2 correlated in the entire sample population, but UGT3As did not correlate with other UGTs. These correlations were sex dependent, as UGT1A3/1A1, UGT1A4/2B7 and UGT3A1/3A2 correlated more highly in male than female individuals, while UGT1A4/1A6 protein correlated more significantly in female than male individuals. This is the first report on the ontogeny of UGT3A isoforms, showing maximal expression in the elderly, and is the first demonstration that UGT isoforms commonly coexpress in vivo, in both age-dependent and sex-dependent manners.

An understanding of the postnatal development of hepatic UDP-glucuronosyltransferase (UGT) enzymes is required for accurate prediction of the age-dependent changes in pharmacokinetics of many drugs used in children. However, the maturation rate of hepatic UGT isoforms remains a major knowledge gap. This study aimed to establish the age-associated changes in glucuronidation activity of 10 major hepatic UGT isoforms in humans, namely, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17. Human liver microsomes from pediatric and adult donors were incubated under optimized incubation conditions to assess the activity rates of hepatic UGT isoforms using a panel of 19 in vitro UGT probe substrates and clinically used drugs. Statistically strong correlations of glucuronidation activities allowed the ontogeny of UGT1A1, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 to be established using multiple selective UGT substrates and matched human liver microsome samples. The postnatal development of hepatic UGTs is isoform-dependent using either individual or cross-correlated selective isoform substrates. Maximal adult activity was reached at different times ranging from within a month (UGT1A1, UGT2B4, UGT2B7, UGT2B10, and UGT2B15), during infancy (UGT1A3, UGT1A4, and UGT1A9), to adolescence (UGT1A6 and UGT2B17). This study provides an extensive characterization of the postnatal ontogeny profiles of hepatic UGT enzymes that are instrumental for predicting drug disposition via in vitro-in vivo extrapolation algorithms and verifying pharmacokinetic predictions against in vivo observations via pediatric physiologically based pharmacokinetic modeling in pediatric patients.

Introduction Cytokines and vascular endothelial growth factors (VEGF) are involved in all aspects of pregnancy: from placentation, through fetal development, parturition and neonatal well-being. Umbilical cord inflammatory cytokines and/or VEGF have not been well studied with respect to dysregulation associated with disorders of pregnancy or maternal/neonatal outcomes. Methods Here we have used multiplex ELISA to screen umbilical cord lysates (comprising cord blood, endothelia and Wharton's jelly, n = 380), for levels of IFN-γ, IL1-β, IL-6, IL-8, IL-10, TNF-α and VEGFs A, C and D and associations with 46 ICD9/10 codes encompassing obstetric, maternal and neonatal variables. Results No significant differences were observed for IFNγ, VEGFC or VEGFD with any clinical outcomes. The cytokines IL1-β, IL-6, IL-8, IL-10, and TNF-α showed varying levels of induction and suppression with primarily fetal-placental and neonatal complications. The largest number of significant differences between umbilical cytokines and clinical outcomes were observed for chorioamnionitis (IL1-β, IL-6, IL-8, TNF-α), and meconium passage during birth (IL1-β, IL-6, IL-8) where significant pro-inflammatory responses occurred and sex differences in IL-8 expression were noted. In contrast, gonococcal infection showed suppressed immune response significantly lowering IL1-β, IL-6, IL-8, IL-10 and TNF-α. For 12/46 negative pregnancy outcomes, strong suppression of VEGFA occurred. Discussion Angiogenic and inflammatory changes in the umbilical cord could be detrimental by increasing vascular permeability in the umbilical artery or vein and/or altering vascular tone, either of which would alter blood flow affecting delivery and removal of compounds. Further elucidation of inflammatory responses in the umbilical cord may provide mechanistic understanding of adverse pregnancy outcomes.

Variants in the SCN1A gene are associated with a wide range of disorders including genetic epilepsy with febrile seizures plus (GEFS+), familial hemiplegic migraine (FHM), and the severe childhood epilepsy Dravet syndrome (DS). Predicting disease outcomes based on variant type remains challenging. Despite thousands of SCN1A variants being reported, only a minority has been functionally assessed. We review the functional SCN1A work performed to date, critically appraise electrophysiological measurements, compare this to in silico predictions, and relate our findings to the clinical phenotype. Our results show, regardless of the underlying phenotype, that conventional in silico software correctly predicted benign from pathogenic variants in nearly 90%, however was unable to differentiate within the disease spectrum (DS vs. GEFS+ vs. FHM). In contrast, patch-clamp data from mammalian expression systems revealed functional differences among missense variants allowing discrimination between disease severities. Those presenting with milder phenotypes retained a degree of channel function measured as residual whole-cell current, whereas those without any whole-cell current were often associated with DS (p = .024). These findings demonstrate that electrophysiological data from mammalian expression systems can serve as useful disease biomarker when evaluating SCN1A variants, particularly in view of new and emerging treatment options in DS.

In pregnancy, opioids may be used medically and also misused. We hypothesized that the umbilical cord (UC) could be a good screening tool for determining opioid exposure and improving medical care. One hundred and one UC, each with 50 associated ICD9/ICD10 codes were used. Using predictive pharmacokinetic analysis we determined that opioids could be detected since last ingestion prior to birth. The UC were lysed and screened using ELISA detecting multiple opioids and their metabolites. Statistical comparisons to obstetric and neonatal outcomes were performed. Although the commercial ELISA was less sensitive in UC than blood or urine, there was perfect method selectivity as compared to a subset of cords designated positive or negative by clinical diagnostics, so our results are accurate and reliable. Absolute quantitation was not possible because the antibody cross reacts with multiple compounds, but ‘low’ or ‘high’ levels of exposure were assigned. Prevalence of opioids was 11%, which reduced to 7% when cesarean-section births were eliminated. For non-cesarean-section infants adjusted for preterm birth, advanced maternal age and smoking (independent risk factors), opioids were significantly associated with intra-uterine growth restriction (p = 0.017) and admission to neonatal intensive care (p = 0.002). UC can be collected noninvasively and rapidly providing a reliable tools for semi-quantitative opioid screening using ELISA. Moreover, as UC are usually discarded collection presents few technical or safety concerns for staff or patients. Further development of this methodology may provide a rapid, noninvasive clinical screening tool to identify NAS and/or opioid use in late pregnancy.

Item does not contain fulltext Limited understanding of drug pharmacokinetics in children is one of the major challenges in paediatric drug development. This is most critical in neonates and infants owing to rapid changes in physiological functions, especially in the activity of drug-metabolising enzymes. Paediatric physiologically based pharmacokinetic models that integrate ontogeny functions for cytochrome P450 enzymes have aided our understanding of drug exposure in children, including those under the age of 2 years. Paediatric physiologically based pharmacokinetic models have consequently been recognised by the European Medicines Agency and the US Food and Drug Administration as innovative tools in paediatric drug development and regulatory decision making. However, little is currently known about age-related changes in UDP-glucuronosyltransferase-mediated metabolism, which represents the most important conjugation reaction for xenobiotics. Therefore, the objective of the review was to conduct a thorough literature survey to summarise our current understanding of age-related changes in UDP-glucuronosyltransferases as well as associated clinical and experimental sources of variance. Our findings indicate that there are distinct differences in UDP-glucuronosyltransferase expression and activity between isoforms for different age groups. In addition, there is substantial variability between individuals and laboratories reported for human liver microsomes, which results in part from a lack of standardised experimental conditions. Therefore, we provide a number of best practice recommendations for experimental conditions, which ultimately may help improve the quality of data used for quantitative clinical pharmacology approaches, and thus for safe and effective pharmacotherapy in children.

Consumption of sucrose (table sugar) is high in much of the world. The effects of a maternal diet high in sucrose on the placenta and fetal brain remain unknown. In rats, maternal consumption of sucrose at a human-relevant level has effects on the mother’s physiology and steroids, as well as long-lasting and sex-specific effects on the adult offspring’s brain and behavior. In the mothers, there are metabolic effects of sucrose intake, such as impaired glucose tolerance, increased liver lipids, and increased adipose inflammation. In rat dams, sucrose intake also decreases corticosterone levels in the blood but not in the brain. In the adult male offspring, preference for a high-sucrose diet and a high-fat diet increases due to maternal sucrose intake. In addition, maternal sucrose intake increases motivation for sugar rewards in a progressive ratio schedule of reinforcement in adult male offspring. In adult female offspring, corticosterone levels increase in the blood and brain as a result of maternal sucrose intake. In this study, we investigated the underlying mechanisms of the observed behavioral and endocrine effects in the adult offspring. Here, we examined cytokines and anti-inflammatory steroids in the placenta, amniotic fluid, and fetal blood and brain. In our model, we feed rat dams either a high-sucrose diet (26% of kCal) or an isocaloric, matched, control diet (1% sucrose) 10 weeks prior to and during gestation. At embryonic day 19 (E19), we collected maternal blood, placenta, amniotic fluid, fetal blood, and fetal brain. We use Palkovits punch to microdissect the placenta and fetal brain. Next, we use a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, which is highly precise and specific, to measure multiple steroids (e.g. corticosterone, progesterone, estradiol, allopregnanolone). The method is highly sensitive, and we can measure neurosteroids in multiple regions of the fetal brain (e.g. prefrontal cortex, nucleus accumbens, hypothalamus, hippocampus). Moreover, we will examine steroidogenic enzymes and cytokines in the fetal brain and placenta. Preliminary data show distinct steroid patterns in amniotic fluid and fetal blood, as well as in different parts of the placenta.

The NADPH dehydrogenase quinone oxido-reductase 1 (NQO1) enzyme is an antioxidant and metabolic enzyme that performs two electron reduction of quinones and other chemicals. Based on the physiologic role(s) of NQO1, we hypothesized that expression and activity of this enzyme would vary with age and other demographic variables. Cytosols from 117 archived human livers were investigated for changes in NQO1 with age, sex, obesity, and ethnicity. Protein expression but not activity of NQO1 was weakly negatively correlated with age (Spearman r = -0.2, P = 0.03). No sex differences were observed for either protein expression or activity and for ethnicity; Caucasians had greater NQO1 activity than Asians (P < 0.05). Overweight children had statistically significantly higher NQO1 activity as compared with ideal weight children (P < 0.05) although this difference was not observed in adults. These findings establish that NQO1 is approximately as active in children as adults. However, modeled NQO1 clearance (both allometric and physiologically based pharmacokinetics) predicted maturation at 23 to 26 years. This is almost certainly an overestimate, with error in the model resulting from a small sample size and inability to scale for age-related changes in hepatic cellularity and/or cytosolic protein content, and indicates a delay in reaching maximum clearance through the NQO1 pathway that is affected by physiologic development as much, or more than, biochemical development. Obesity may increase hepatic NQO1 activity in children, which is likely a protective mechanism in oxidative stress, but may also have significant implications for drug and chemical disposition in obese children.

The hematological side effects associated with mycophenolic acid (MPA) are relatively common and have severe consequences. The majority of literature data have not shown clear consistency in the MPA exposure-neutropenia relationship. We hypothesized that (i) adult de novo kidney transplant recipients who develop neutropenia have relatively higher dose-normalized MPA exposure than patients without neutropenia, and (ii) the observed neutropenia may be explained by polymorphisms in metabolism and/or transporter genes responsible for MPA disposition.Adult kidney transplant recipients on steady-state tacrolimus and MPA, not receiving a corticosteroid, and with stable renal function were recruited for investigation at three periods post-transplant (1, 3, and 12 months; n = 21, 17, and 13, respectively). Clinical variables (age, weight, MPA daily dose, albumin, serum creatinine, absolute neutrophil count), tacrolimus and MPA concentrations (for exposure calculation), and genotypes (UGT2B7 G211T, UGT2B7 C802T, UGT1A9 T-275A, UGT1A9 T98C, MRP2 C-24T, MRP2 G1249A, OATP1B1 A388G, OATP1B1 C463A) were characterized.A significant inverse association between dose-normalized MPA exposure (a surrogate marker for apparent MPA clearance) and absolute neutrophil count in all three study periods (rThese findings support the clinical strategy for conducting MPA therapeutic drug monitoring in adult kidney transplant patients on steroid-free immunosuppressant therapy. The novel population genomic analysis data warrant further epidemiological investigations in a larger study sample.

The impacts of continued degradation of watersheds on coastal coral reefs world-wide is alarming, and action addressing anthropogenic stressors and subsequent rehabilitation of watersheds and adjacent reefs is an urgent priority. The aim of this study is to develop and improve the use of antioxidant enzymes as bioindicators of stress in coral species. In order to fully develop such tools, it is necessary to first understand baseline cycling of these enzymes within coral tissues. Due to inherent links between reproduction and oxidative stress, these aims may be facilitated by sampling coral tissues over reproductively-linked lunar cycles to determine variations from baseline. By developing a greater understanding of biochemical markers of stress in corals, specifically antioxidant defense enzymes catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in HawaiianPocillopora damicornis, we have provided molecular tools that identify thresholds of stress on coral reefs. Our results suggest that the coral reproductive state is a significant factor affecting the activity of antioxidant enzymes. Specifically, CAT and GR display maximum activity during peak reproductive state. Whereas significant maximal Se-independent GPx and SOD activity was measured during off-peak reproductive cycles. Such insight into the cyclical variation of the activity of these enzymes should be applied towards differentiating the influence of natural biological activity cycling in diagnostic tests identifying the effects of different physical environmental factors and chemical pollutants on coral health. Through the development and application of these molecular biomarkers of stress, we look to improve our ability to identify problems at the sub-lethal level, when action can be taken to mitigate a/biotic impacts.

The impacts of continued degradation of watersheds on coastal coral reefs world-wide is alarming, and action addressing anthropogenic stressors and subsequent rehabilitation of watersheds and adjacent reefs is an urgent priority. The aim of this study is to develop and improve the use of antioxidant enzymes as bioindicators of stress in coral species. In order to fully develop such tools, it is necessary to first understand baseline cycling of these enzymes within coral tissues. Due to inherent links between reproduction and oxidative stress, these aims may be facilitated by sampling coral tissues over reproductively-linked lunar cycles to determine variations from baseline. By developing a greater understanding of biochemical markers of stress in corals, specifically antioxidant defense enzymes catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in Hawaiian Pocillopora damicornis, we have provided molecular tools that identify thresholds of stress on coral reefs. Our results suggest that the coral reproductive state is a significant factor affecting the activity of antioxidant enzymes. Specifically, CAT and GR display maximum activity during peak reproductive state. Whereas significant maximal Se-independent GPx and SOD activity was measured during off-peak reproductive cycles. Such insight into the cyclical variation of the activity of these enzymes should be applied towards differentiating the influence of natural biological activity cycling in diagnostic tests identifying the effects of different physical environmental factors and chemical pollutants on coral health. Through the development and application of these molecular biomarkers of stress, we look to improve our ability to identify problems at the sub-lethal level, when action can be taken to mitigate a/biotic impacts.

The hepatic uridine 5′-diphosphate-glucuronosyl transferases (UGTs) are critical for detoxifying endo- and xenobiotics. Since UGTs are also dynamically responsive to endogenous and exogenous stimuli, we examined whether epigenetic DNA methylation can regulate hepatic UGT expression and differential effects of ethnicity, obesity, and sex. The methylation status of UGT isoforms was determined with Illumina Methylation 450 BeadChip arrays, with genotyping confirmed by sequencing and gene expression confirmed with quantitative reverse transcriptase polymerase chain reaction (q-RT-PCR). The UGT1A3 mRNA was 2-fold higher in females than males (p < 0.05), while UGT1A1 and UGT2B7 mRNA were significantly higher in Pacific Islanders than Caucasians (both p < 0.05). Differential mRNA or methylation did not occur with obesity. The methylation of the UGT2B15 locus cg09189601 in Caucasians was significantly lower than the highly methylated locus in Asians (p < 0.001). Three intergenic loci between UGT2B15 and 2B17 (cg07973162, cg10632656, and cg07952421) showed higher rates of methylation in Caucasians than in Asians (p < 0.001). Levels of UGT2B15 and UGT2B17 mRNA were significantly lower in Asians than Caucasians (p = 0.01 and p < 0.001, respectively). Genotyping and sequencing indicated that only UGT2B15 is regulated by methylation, and low UGT2B17 mRNA is due to a deletion genotype common to Asians. Epigenetic regulation of UGT2B15 may predispose Asians to altered drug and hormone metabolism and begin to explain the increased risks for adverse drug reactions and some cancers in this population.

Coral reefs are an indispensible worldwide resource, accounting for billions of dollars in cultural, economic, and ecological services. An understanding of coral reproduction is essential to determining the effects of environmental stressors on coral reef ecosystems and their persistence into the future. Here, we describe the presence of and changes in steroidal hormones along with associated steroidogenic and steroid removal enzymes during the reproductive cycle of the brooding, pan-Pacific, hermaphroditic coral, Pocillopora damicornis. Detectable levels of 17β-estradiol, estrone, progesterone and testosterone were consistently detected over two consecutive lunar reproductive cycles in coral tissue. Intra-colony variation in steroid hormone levels ranged between 1.5- and 2.2-fold and were not statistically different. Activities of the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase and cytochrome P450 (CYP) 17 dehydrogenase were detectable and did not fluctuate over the reproductive cycle. Aromatase-like activity was detected during the lunar reproductive cycle with no significant fluctuations. Activities of regeneration enzymes did not fluctuate over the lunar cycle; however, activity of the clearance enzyme UDP-glucuronosyl transferases increased significantly (ANOVA, post hoc p

Obesity in children is a significant clinical concern. There are many anecdotes and case studies regarding specific reactions of obese children to medications including therapeutic failure, adverse drug reactions and/or requirements for higher weight-adjusted dosing. There isis, however, a lack of basic and clinical data dissecting the mechanisms of these effects on pharmaceutical efficacy and safety. At present it is unknown how much of the difference in drug disposition in obese children can be attributed to obesity, to maturation or to an interaction between the two. Since a major determinant of drug disposition is hepatic metabolism, here we review how obesity alters hepatic drug disposition in children. Basic as well as clinical data summarizing the current knowledge of biochemical, physiological and clinical effects of pediatric obesity on drug disposition are considered. We conclude that there is a dire need for increased research into the direct effects of obesity on absorption, distribution, metabolism and excretion, as well as changes to pharmacokinetic parameters such as bioavailability and clearance. Increased effort in this area may elucidate the effects of obesity on clinical drug disposition with sufficient detail to provide better dosing guidelines where needed for children.

1. The umbilical cord is a direct conduit to the fetus hence transporters could have roles in partitioning substances between the maternal-placental-fetal units. Here we determined the expression and localization of the ATP-Binding Cassette (ABC) transporters BCRP (ABCG2), P-gp (ABCB1) and MRP1 (ABCC1) in human umbilical cords. 2. The mRNA for BCRP and MRP1 was detected in 25/25 samples, but P-gp was detected in only 5/25. ABC transporter mRNA expression relative to 18S was 25.6 ± 0.3, 26.5 ± 0.6 and 22.2 ± 0.2 cycles for BCRP, MRP1 and P-gp respectively. 3. Using a subset of 10 umbilical cords, BCRP protein was present in all samples (immunoblot) with positive correlation between mRNA and proteins (p = 0.07, r = 0.62) and between immunoblotting and immunohistochemistry (IHC) (p = 0.03, r = 0.67). P-gp protein was observed in 4/10 samples by both immunoblot and IHC, with no correlation between mRNA and protein (p = 0.45, r = 0.55) or immunoblotting and IHC (p = 0.2, r = 0.72), likely due to small sample size. MRP1 protein was not observed. 4. Localization of BCRP and P-gp proteins was to Wharton's jelly with no specific staining in arterial or venous endothelia. 5. Understanding ABC transporter expression in the umbilical cord may be useful for determining fetal exposures to xenobiotics if functional properties can be defined.

1. The UDP-glucuronosyltransferase (UGT) enzymes are important in the metabolism, elimination and detoxification of many xenobiotics and endogenous compounds. As extrapolation of in vitro kinetics of drug metabolizing enzymes to predict in vivo clearance rates becomes more sophisticated, it is important to ensure proper optimization of enzyme assays. The luminal location of the enzyme active site (i.e. latency), and the complexity of UGT kinetics, results in consistent under-prediction of clearance of drugs metabolized by glucuronidation. 2. We examined inhibition of UGT activity in alamethicin-disrupted human liver microsomes (HLM) by uridine diphosphate (UDP), a UGT reaction product, and its reversal by Mg

Placental UDP-glucuronosyltransferase (UGT) enzymes have critical roles in hormone, nutrient, chemical balance and fetal exposure during pregnancy. Placental UGT1A isoforms were profiled and differences between preeclamptic (PE) and non-PE placental UGT expression determined. In third trimester villous placenta, UGT1A1, 1A4, 1A6 and 1A9 were expressed and active in all specimens (n = 10), but UGT1A3, 1A5, 1A7, 1A8 and 1A10 were absent. The UGT1A activities were comparable to human liver microsomes per milligram, but placental microsome yields were only 2 % of liver (1 mg/g of tissue vs. 45 mg/g of tissue). For successful PCR, placental collection and processing within 60 min from delivery, including DNAse and ≥300 ng of RNA in reverse transcription were essential and snap freezing in liquid nitrogen immediately was the best preservation method. Although UGT1A6 mRNA was lower in PE (P < 0.001), there were no other significant effects on UGT mRNA, protein or activities. A more comprehensive tissue sample set is required for confirmation of PE interactions with UGT. Placental UGT1A enzyme expression patterns are similar to the liver and a detoxicative role for placental UGT1A is inferred.

The UDP-glucuronosyltransferase (UGT) enzymes are critical for regulating nutrients, hormones, and endobiotics, as well as for detoxifying xenobiotics. Human and murine fetuses are known to express glucuronidation enzymes, but there are currently no data prior to implantation. Here we addressed this gap in knowledge and tested whether Ugt enzymes are already present in preimplantation-stage embryos. Blastocysts were obtained after in vitro fertilization with gametes from B6D2F1 hybrid mice and from embryo culture. Protein expression and localization were determined using pan-specific UGT1A and UGT2B, as well as anti-human isoform-specific antibodies. Immunofluorescence analysis showed that blastocysts expressed Ugt1a globally, in the cytoplasm and nuclei of all of the cells. Western blots demonstrated the presence of Ugt1a6 but not Ugt1a1, Ugt1a3, Ugt1a4, or Ugt1a9. The Ugt2b proteins were not detected by either assay. The level of Ugt activity in murine blastocysts was comparable with that of the adult human liver (per milligram of protein), but the activity of β-glucuronidase, an Ugt-partnering enzyme responsible for substrate regeneration, was lower. Altogether, these data confirm that Ugt1a proteins are present and active in preimplantation murine embryos and point to a potential role for these proteins in implantation and early embryonic and fetal development.

Single blastomere removal from cleavage-stage embryos, a common procedure used in conjunction with preimplantation genetic diagnosis (PGD), may affect reproductive outcomes. We hypothesized that negative pregnancy outcomes associated with PGD may be due to impairment of placental signaling pathways. The goal of this study was to determine the molecular mechanisms through which placental signaling is deregulated by blastomere removal. Four-cell stage murine embryos produced by in vitro fertilization were subjected to removal of a single blastomere (biopsied) or to the same manipulations without the blastomere removal (controls). Placental tissues from term (18.5 day) pregnancies obtained after embryo transfer were tested for levels of nitrosative species, interleukin 6, signal transducers and activators of transcription (STAT) 1 and 3, suppressors of cytokine signaling (SOCS) 1, 2 and 3 and matrix metalloproteinases (MMP) 1, 2, 3 and 9. Significant increases in nitrosative stress (P < 0.05), phosphorylative activation of STAT1 (P < 0.05) but not STAT3, lower levels of the inhibitors SOCS2 (P < 0.01) and SOCS3 (P < 0.001) and activation of MMP9 (P < 0.001) were observed in placentas derived from biopsied embryos, compared with controls. Such effects could contribute to greater levels of premature membrane rupture, incorrect parturition, preterm birth and intrauterine growth restriction associated with PGD. This work has determined signaling mechanisms that may be responsible for blastomere removal effects on placental function, with the potential to become targets for improving obstetric and neonatal outcomes in assisted reproduction.

Small organic molecules have been the pharmaceutical mainstay of the developed world for some time. However, in recent years, advances within the fields of genomics and proteomics have strengthened and given rise to new biologic therapies. Protein therapies, such as monoclonal antibodies and peptide drugs, have provided patients with pharmaceuticals that offer a higher level of selectivity and effectiveness that would be otherwise undeliverable within the realm of small organics. In addition to protein therapies, DNA-based therapy, such as RNA interference (RNAi) and gene therapy, have gained renewed interest within modern medicine and are potentially poised for a comeback within the biotechnology industry. As we discuss here, the advantages of such therapies continue to accumulate and have kept the biologic market strong.

The continued deterioration of coral reefs worldwide demonstrates the need to develop diagnostic tools for corals that go beyond general ecological monitoring and can identify specific stressors at sublethal levels. Cellular diagnostics present an approach to defining indicators (biomarkers) that have the potential to reflect the impact of stress at the cellular level, allowing for the detection of intracellular changes in corals prior to outright mortality. Detoxification enzymes, which may be readily induced or inhibited by environmental stressors, present such a set of indicators. However, in order to apply these diagnostic tools for the detection of stress, a detailed understanding of their normal, homeostatic levels within healthy corals must first be established. Herein, we present molecular and biochemical evidence for the expression and activity of major Phase I detoxification enzymes cytochrome P450 (CYP450), CYP2E1, and CYP450 reductase, as well as the Phase II enzymes UDP, glucuronosyltransferase (UGT), β-glucuronidase, glutathione-S-transferase (GST), and arylsulfatase C (ASC) in the coral Pocillopora damicornis. Additionally, we characterized enzyme expression and activity variations over a reproductive cycle within a coral’s life history to determine natural endogenous changes devoid of stress exposure. Significant changes in enzyme activity over the coral’s natural lunar reproductive cycle were observed for CYP2E1 and CYP450 reductase as well as UGT and GST, while β-glucuronidase and ASC did not fluctuate significantly. The data represent a baseline description of ‘health’ for the expression and activity of these enzymes that can be used toward understanding the impact of environmental stressors on corals. Such knowledge can be applied to address causes of coral reef ecosystem decline and to monitor effectiveness of mitigation strategies. Achieving a better understanding of cause-and-effect relationships between putative stressors and biological responses in corals, and other marine invertebrates, can guide and evaluate mitigation and conservation approaches for marine ecosystem protection.

Obesity and pregnancy both place the liver under metabolic stress, but interactions between obstetric obesity and bilirubin metabolism have not been studied. We determined associations between obesity, maternal/neonatal bilirubin levels, and uridine 5′diphosphate-glucuronosyltransferase 1A1 (UGT1A1) enzyme that eliminates bilirubin. Adult livers were analyzed for UGT1A1 expression, activity, and bilirubin clearance by pharmacokinetic modeling. Then, matched maternal and neonatal sera (N = 450) were assayed for total and unconjugated bilirubin. Associations between obesity, UGT1A1, maternal and neonatal hyperbilirubinemia were determined statistically through correlation analysis (Pearson's test) as well as binned categories (one-way ANOVA). Morbid obesity decreased hepatic UGT1A1 protein levels, activity, and bilirubin clearance (P < .001). Increasing obesity corresponded to elevated maternal unconjugated bilirubin (P < .05). Maternal obesity was also significantly positively correlated with elevated neonatal bilirubin levels (P < .01, N = 450) and this was strongest in Native Hawaiians and Pacific Islander (NHPI) women (P < .01, n = 150). Obstetric obesity is associated with maternal and neonatal hyperbilirubinemia, likely through inhibition of hepatic UGT1A1. The NHPI cohort was the most obese and had the highest levels of maternal and neonatal unconjugated bilirubin. Neonates from obese mothers may be more susceptible to jaundice and side effects from parenteral nutrition.

This article reports on the development of UDP-glucuronosyltransferase 1A9 (UGT1A9) in neonatal and pediatric liver. The substrate 4-methylumbelliferone (4MU) with specific inhibition by niflumic acid was used to define specific UGT1A9 activity. Subsequently, in silico pharmacokinetic (PK) and physiology-based pharmacokinetic (PBPK) modeling was used to determine UGT1A9 maturation and hepatic clearance. Modeled maximal enzyme activity was 27.9 nmol · min(-1) · mg protein(-1) at 4 months of age, which had high concordance with the average V(max) in 45 individual adult (>20 years) livers of 29.0 nmol · min(-1) · mg protein(-1). The activity of UGT1A9 ranged 7.5-fold in the adult population (4.1-54.5 nmol · min(-1) · mg protein(-1)). Expression of UGT1A9 correlated with age only in children younger than 1 year (Spearman r = 0.70). Activity correlated with expression up to 18 years of age (Spearman r = 0.76). Furthermore, scaling intrinsic hepatic clearance of 4MU with an allometric PK model yielded a high clearance at birth and then fell to adult levels (1.3 l · h(-1) · kg(-1) at 18.1 years for well stirred or 1.4 l · h(-1) · kg(-1) at 18.7 years for parallel tube). The Simcyp PBPK models did not converge but showed an increase in clearance at under 1 year of age and then decreased to adult levels at approximately 20 years of age. Allometric scaling may be more accurate in cases of high-extraction drugs. Enzyme activities or hepatic clearances did not differ with gender or ethnicity. The UGT1A9 isoform has higher normalized clearance for 4MU at young ages, which may explain how other UGT1A9 substrates, such as propofol, have higher clearances in children than in adults.

Bamboo leaves are used as a component in traditional Chinese medicine for the anti-inflammatory function. Our previous studies have demonstrated that an ethanol/water extract from Phyllostachys edulis ameliorated obesity-associated chronic systemic inflammation in mice, and therefore relieving the symptoms of type 2 diabetes. The aim of this project was to further investigate the effects of this bamboo extract on hepatic biotransformation enzymes in both lean and obese mice, as an initial step in the toxicological evaluation of using this traditional medicine in obese/diabetic population.Male C57BL/6J mice were randomized to 4 groups and fed standard (10% kcal from fat) diet with or without bamboo extract supplementation at a dose of 10 gram per kilogram diet (n=10 and n=9, respectively), or high fat (45% kcal from fat) diet with or without bamboo extract (n=8 and N=7, respectively). The dietary treatment lasted for 6 months. Subsequently, the activities and expression of the major Phase I and II hepatic biotransformation enzymes were assessed in subcellular fractions from murine livers.Three groups of mice, lean bamboo extract-supplemented, obese/diabetic, and bamboo extract-supplemented obese/diabetic, showed greater activities of cytochromes P450 1a2 and 3a11 compared to control but no changes in the expression level of these proteins. For Phase II enzymes, bamboo extract supplementation in lean mice caused decreased glutathione-S-transferase activity (-12%) and greater uridine diphosphate glucuronosyltransferase activity (+46%), but had no effect on sulfotransferase activity. Conversely, the obese/diabetic condition itself increased glutathione-S-transferase and uridine diphosphate glucuronosyltransferase activities, but decreased total sulfotransferase activity and sulfotransferase 2a1 expression.Bamboo extract and obesity/diabetes show significant independent effects on hepatic biotransformation as well as interaction effects in mice. These changes may alter the clearance of endo- and xenobiotics, including bamboo extract itself, hence this effect should be carefully considered in the medicinal application of bamboo extract as it has potential to alter its own metabolism and that of other medications concurrently administered to obese diabetic patients.