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Organic anion transporting polypeptide 2B1 (OATP2B1, gene SLCO2B1) is an uptake transporter that is thought to determine drug disposition and in particular, the oral absorption of medications. At present, the clinical relevance of SLCO2B1 genetic variation on pharmacokinetics is poorly understood. We sought to determine the functional activity of 5 of the most common missense OATP2B1 variants (c.76_84del, c.601G>A, c.917G>A, c.935G>A, and c.1457C>T) and a predicted dysfunctional variant (c.332G>A) in vitro. Furthermore, we measured the basal plasma concentrations of endogenous OATP2B1 substrates, namely estrone sulfate, dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate, coproporphyrin I (CPI), and CPIII, and assessed their relationships with SLCO2B1 genotypes in 93 healthy participants. Compared to reference OATP2B1, the transport activities of the c.332G>A, c.601G>A and c.1457C>T variants were reduced among the substrates examined (estrone sulfate, DHEAS, CPI, CPIII and rosuvastatin), although there were substrate-dependent effects. Lower transport function of OATP2B1 variants could be explained by diminished cell surface expression. Other OATP2B1 variants (c.76-84del, c.917G>A and c.935G>A) had similar activity to the reference transporter. In the clinical cohort, the SLCO2B1 c.935G>A allele was associated with both higher plasma CPI (42%) and CPIII (31%) concentrations, while SLCO2B1 c.917G>A was linked to lower plasma CPIII by 28% after accounting for the effects of age, sex, and SLCO1B1 genotypes. No association was observed between SLCO2B1 variant alleles and estrone sulfate or DHEAS plasma concentrations, however 45% higher plasma pregnenolone sulfate level was associated with SLCO2B1 c.1457C>T. Taken together, we found that the impacts of OATP2B1 variants on transport activities in vitro were not fully aligned with their associations to plasma concentrations of endogenous substrates in vivo. Additional studies are required to determine whether circulating endogenous substrates reflect OATP2B1 activity. [ABSTRACT FROM AUTHOR]

Organic anion transporting polypeptide 2B1 (OATP2B1, gene SLCO2B1) is an uptake transporter that is thought to determine drug disposition and in particular, the oral absorption of medications. At present, the clinical relevance of SLCO2B1 genetic variation on pharmacokinetics is poorly understood. We sought to determine the functional activity of 5 of the most common missense OATP2B1 variants (c.76_84del, c.601G>A, c.917G>A, c.935G>A, and c.1457C>T) and a predicted dysfunctional variant (c.332G>A) in vitro. Furthermore, we measured the basal plasma concentrations of endogenous OATP2B1 substrates, namely estrone sulfate, dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate, coproporphyrin I (CPI), and CPIII, and assessed their relationships with SLCO2B1 genotypes in 93 healthy participants. Compared to reference OATP2B1, the transport activities of the c.332G>A, c.601G>A and c.1457C>T variants were reduced among the substrates examined (estrone sulfate, DHEAS, CPI, CPIII and rosuvastatin), although there were substrate-dependent effects. Lower transport function of OATP2B1 variants could be explained by diminished cell surface expression. Other OATP2B1 variants (c.76-84del, c.917G>A and c.935G>A) had similar activity to the reference transporter. In the clinical cohort, the SLCO2B1 c.935G>A allele was associated with both higher plasma CPI (42%) and CPIII (31%) concentrations, while SLCO2B1 c.917G>A was linked to lower plasma CPIII by 28% after accounting for the effects of age, sex, and SLCO1B1 genotypes. No association was observed between SLCO2B1 variant alleles and estrone sulfate or DHEAS plasma concentrations, however 45% higher plasma pregnenolone sulfate level was associated with SLCO2B1 c.1457C>T. Taken together, we found that the impacts of OATP2B1 variants on transport activities in vitro were not fully aligned with their associations to plasma concentrations of endogenous substrates in vivo. Additional studies are required to determine whether circulating endogenous substrates reflect OATP2B1 activity.

Phytohormones are a group of small chemical molecules that play vital roles in plant development, metabolism, and stress responses. Phytohormones often have distinct biosynthesis and signaling perception sites, requiring long- or short-distance transportation. Unlike biosynthesis and signal transduction, phytohormone transport across cells and organs is poorly understood. The transporter activity assay is a bottleneck for the functional characterization of novel phytohormone transporters. In the present study, we report a tobacco syringe agroinfiltration and liquid chromatography tandem mass spectrometry (TSAL)-based method for performing a phytohormone transporter activity assay using endogenous hormones present in tobacco (Nicotiana benthamiana) leaves. A transporter activity assay using this method does not require isotope-labeled substrates and can be conveniently performed for screening multiple substrates by using endogenous hormones in tobacco leaves. The transporter activities of three known hormone transporters, namely AtABCG25 for abscisic acid, AtABCG16 for jasmonic acid, and AtPUP14 for cytokinin, were all successfully validated using this method. Using this method, cytokinins were found to be the preferred substrates of an unknown maize (Zea mays) transporter ZmABCG43. ZmABCG43 transporter activities toward cytokinins were confirmed in a cytokinin long-distance transport mutant atabcg14 through gene complementation. Thus, the TSAL method has the potential to be used for basic substrate characterization of novel phytohormone transporters or for the screening of novel transporters for a specific phytohormone on a large scale.

Phytohormones are a group of small chemical molecules that play vital roles in plant development, metabolism, and stress responses. Phytohormones often have distinct biosynthesis and signaling perception sites, requiring long- or short-distance transportation. Unlike biosynthesis and signal transduction, phytohormone transport across cells and organs is poorly understood. The transporter activity assay is a bottleneck for the functional characterization of novel phytohormone transporters. In the present study, we report a tobacco syringe agroinfiltration and liquid chromatography tandem mass spectrometry (TSAL)-based method for performing a phytohormone transporter activity assay using endogenous hormones present in tobacco (Nicotiana benthamiana) leaves. A transporter activity assay using this method does not require isotope-labeled substrates and can be conveniently performed for screening multiple substrates by using endogenous hormones in tobacco leaves. The transporter activities of three known hormone transporters, namely AtABCG25 for abscisic acid, AtABCG16 for jasmonic acid, and AtPUP14 for cytokinin, were all successfully validated using this method. Using this method, cytokinins were found to be the preferred substrates of an unknown maize (Zea mays) transporter ZmABCG43. ZmABCG43 transporter activities toward cytokinins were confirmed in a cytokinin long-distance transport mutant atabcg14 through gene complementation. Thus, the TSAL method has the potential to be used for basic substrate characterization of novel phytohormone transporters or for the screening of novel transporters for a specific phytohormone on a large scale. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette family transporter MRP2 (multidrug resistance-associated protein 2), encoded by the ABCC2 gene, is involved in the renal excretion of numerous xenobiotics and it is likely that it also transports many endogenous molecules arising from not only normal essential metabolic processes but also from environmental toxins or food intake. We used a targeted gas chromatography-mass spectrometry metabolomics analysis to study whether endogenous organic anions are differentially excreted in urines of healthy volunteers according to their genotype for three functional single nucleotide polymorphisms (SNPs) in ABCC2 . This was the case for 35 of the 108 metabolites analyzed. Eight of them are most likely substrates of MRP2 since they are the most contributive to the difference between carriers of a decreasing function allele vs those carrying an increasing function one. Seven out of 8 metabolites are fatty acids (dodecanoic acid; 3-hydroxypropanoic acid) or metabolites of polyphenols (caffeine; resorcinol; caffeic acid; 2-(3,4-dihydroxyphenyl) acetic acid; and 4-hydroxyhippuric acid). Most of them were structurally similar to a series of substances previously shown to interact with MRP2 function in vitro. Interestingly, coproporphyrin isomer I, a prototypical substrate of MRP2, also belonged to our final list although it was not significantly discriminant on its own. This suggests that the simultaneous measurement of a set of endogenous metabolites in urine, rather than that of unique metabolites, has the potential to provide a phenotypic measure of MRP2 function in vivo . This would represent an innovative tool to study the variability of the transport activity of MRP2 under a physiological or pathological condition, especially in pharmacokinetic studies of its substrates. [ABSTRACT FROM AUTHOR]

Purpose: To assess the use of glycochenodeoxycholate-3-sulfate (GCDCA-S) and chenodeoxycholate 3- or 24-glucuronide (CDCA-3G or -24G) as surrogate endogenous substrates in the investigation of drug interactions involving OATP1B1 and OATP1B3. Methods: Uptake of GCDCA-S and CDCA-24G was examined in HEK293 cells transfected with cDNA for OATP1B1, OATP1B3, and NTCP and in cryopreserved human hepatocytes. Plasma concentrations of bile acids and their metabolites (GCDCA-S, CDCA-3G, and CDCA-24G) were determined by LC-MS/MS in eight healthy volunteers with or without administration of rifampicin (600 mg, po). Results: GCDCA-S and CDCA-24G were substrates for OATP1B1, OATP1B3, and NTCP. The uptake of [H]atorvastatin, GCDCA-S, and CDCA-24G by human hepatocytes was significantly inhibited by both rifampicin and pioglitazone, whereas that of taurocholate was inhibited only by pioglitazone. Rifampicin elevated plasma concentrations of GCDCA-S more than those of other bile acids. The area under the plasma concentration-time curve for GCDCA-S was 20.3 times higher in rifampicin-treated samples. CDCA-24G could be detected only in plasma from the rifampicin-treatment phase, and CDCA-3G was undetectable in both phases. Conclusions: We identified GCDCA-S and CDCA-24G as substrates of NTCP, OATP1B1, and OATP1B3. GCDCA-S is a surrogate endogenous probe for the assessment of drug interactions involving hepatic OATP1B1 and OATP1B3. [ABSTRACT FROM AUTHOR]

The extrusion of anticancer drugs by members of the ATP-binding cassette (ABC) transporter family is one of the most widely recognized mechanisms of multidrug resistance, and can be considered a hijacking of their normal roles in the transport of xenobiotics, metabolites and signaling molecules across cell membranes. While roles in cancer multidrug resistance have been clearly demonstrated for P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP) and Multidrug Resistance Protein 1 (MRP1), direct evidence for a role in multidrug resistance in vivo is lacking for other family members. A less well understood but emerging theme is the drug efflux-independent contributions of ABC transporters to cancer biology, supported by a growing body of evidence that their loss or inhibition impacts on the malignant potential of cancer cells in vitro and in vivo . As with multidrug resistance, these contributions likely represent a hijacking of normal ABC transporter functions in the efflux of endogenous metabolites and signaling molecules, however they may expand the clinical relevance of ABC transporters beyond P-gp, BCRP and MRP1. This review summarizes established and emerging roles for ABC transporters in cancer, with a focus on neuroblastoma and ovarian cancer, and considers approaches to validate and better understand these roles. [ABSTRACT FROM AUTHOR]

Abstract: CYP2D6, one of the major cytochrome P450 isoforms present in the human brain, is associated with the incidence and prevalence of central nervous system (CNS) diseases. Human CYP2D6 and rat CYP2D are involved in the metabolism of various neurotransmitters and neurosteroids. Brain CYP2D can be regulated by endogenous steroids, including sex hormones. The alteration of CYP2D-mediated metabolism induced by endogenous steroids may cause changes in sensitivity to environmental and industrial toxins and carcinogens as well as physiological and pathophysiological processes controlled by biologically active compounds. This review summarizes the current knowledge regarding the distribution, endogenous substrates, and regulation of brain CYP2D. [Copyright &y& Elsevier]

The catalytic oxidation of phenolic substrates by polyphenoloxidase (PPO) causes pericarp browning of postharvest rambutan fruit. In the present study, PPO and its endogenous substrates were extracted from rambutan pericarp tissues (RPT). The substrate extracts were sequentially partitioned with ethyl acetate and n-butanol. The analysis of total phenolic content showed that the most phenolic compounds were distributed in ethyl acetate fraction. By high-performance liquid chromatography (HPLC), (−)-epicatechin (EC) and proanthocyanidin A2 (PA2) were identified from this fraction. After reacting with rambutan PPO, EC turned brown rapidly within 10 min, indicating that it was a significant endogenous substrate. Although PA2 could also be oxidized by the PPO, it turned brown very slowly. In addition, because EC and PA2 were continually catalyzed into browning products by PPO during storage of the fruit at 4 and 25 °C, their contents in RPT gradually declined with the extended storage time. It was further observed that both substrate contents in rambutan fruit storing at 25 °C decreased more rapidly than that storing at 4 °C, suggesting that low temperature inhibited the catalytic oxidation of substrates so as to slow down pericarp browning. Practical Application: Pericarp browning is a serious problem to storage and transport of harvested rambutan fruit. A generally accepted opinion on the browning mechanism is the oxidation of phenolic substrates by PPO. Ascertaining PPO substrates will effectively help us to control enzymatic reaction by chemical methods so as to delay or even prevent pericarp browning of harvested rambutan fruit. [ABSTRACT FROM AUTHOR]

Abstract: The metabolism of xenobiotics in human brain constitutes a field of recent intensive research in relation to the potential implications in the pharmacological effect of drugs acting on the central nervous system. Cytochrome P450 enzymes (CYPs) play a crucial role in these metabolic pathways and the existence of functional CYP monooxygenases in brain is now well established. These enzymes are preferentially localized in the neuronal cells within the microsomal fraction and the inner membrane of mitochondria. Although low, the metabolism in situ could influence individual response to xenobiotics or produce reactive, toxic metabolites causing irreversible damage in the neuronal cells. The abundant presence of CYPs in selective cell populations within different regions of the brain has also suggested a role for these enzymes in brain physiology thus not restricted to xenobiotic-induced neurotoxicity. For instance, CYPs participate in the regulation of neurotransmitters and steroids and brain maintenance of cholesterol homeostasis. Recent advances support an additional role for these enzymes in the pathogenesis of psychiatric and neurodegenerative disorders such as depression, schizophrenia, and Alzheimer''s and Parkinson''s diseases. The characterization of brain CYP isoforms and their localization, the identification of their substrates and metabolic end-products will allow better understanding of the role of these enzymes in brain physiology, development and diseases. [Copyright &y& Elsevier]

Integration of genomic data from pharmacokinetic pathways and drug targets is an emerging trend in bioinformatics, but is there a clear separation of pharmacokinetic pathways and drug targets? Should we also consider the potential interactions of endogenous substrates of drug-metabolizing enzymes with receptors and other molecular drug targets as we combine pharmacogenomic datasets? We discuss these overarching questions through a specific pharmacogenomic case study of the cytochrome P450 (CYP)2D6, serotonin and dopamine triad. Importantly, CYP2D6 may contribute to the regeneration of serotonin from 5-methoxytryptamine by virtue of its catalytic function as a 5-methoxyindolethylamine O-demethylase. Furthermore, serotonergic neurons provide a regulatory feedback on dopaminergic neurotransmission. Hence, we hypothesize that independent of its role as a pharmacokinetic gene, CYP2D6 may nuance the regulation of serotonergic and dopaminergic neurophysiology. Additionally, we reflect upon the contribution of hyperspecialization in biomedicine to the present disconnect between research on pharmacokinetics and drug targets, and the potential for remedying this important gap through informed dialogue among clinical pharmacologists, human geneticists, bioethicists and applied social scientists. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) play important roles in regulating mitochondrial function, as well as in ischemia-reperfusion injury and cardioprotection. Here we show that, in the absence of exogenous substrates, cardiac mitochondria have a surprisingly large capacity to phosphorylate ADP by oxidizing endogenous substrates, provided that H2O2 is removed from the extramitochondrial environment and a reduced environment is maintained in the matrix. In isolated mitochondria without exogenous substrates, addition of catalase and the membrane-permeant reducing agent N-acetylcysteine (Nac) or the ROS scavenger mercaptopropionyl glycine significantly increased the ability to phosphorylate added ADP, as demonstrated by 1) full recovery of membrane potential (ΔΨ) and matrix volume from ADP-induced dissipation and shrinkage, 2) ADP-dependent increase in O2 consumption, and 3) enhanced rate of ATP synthesis. Removal of extramitochondrial H2O2 by cataiase was required to stimulate endogenous substrate oxidation, as shown by the increase in O2 consumption and ΔΨ. This effect was greatly enhanced by addition of Nac or mercaptopropionyl glycine to suppress oxidation-induced ROS increases in the matrix. Theoretical considerations, as well as reversible inhibition of O2 consumption with 3-mercaptopropionic acid and pyruvate in state 3, indicate that these substrates are fatty acids. Under in vivo conditions in which powerful antioxidant conditions are maintained, this mechanism may be important in stimulation of β-oxidation and ATP production at low levels of extramitochondrial fatty acids. Incapacitation of this mechanism may potentially contribute to mitochondrial dysfunction during oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: Polygalacturonase (PG) from mango pulp revealed three isoforms (I, II, III) upon ion exchange and gel filtration chromatography, each having an abundance of 68%, 6% and 26%, and molecular weights (M r) 40, 51 and 45kDa, respectively. The pH optimum for the isoforms was between 3 and 4. PG-I was stable over a wide pH range (4–7.5) unlike PG II and III, which were stable at pH 4 and 5, respectively. The optimum temperature was around 40°C for all the three isoforms. Their apparent K m for pectic acid was in the range 0.22–0.25mgml−1. The V max for PG I, II and III was 5.7, 3.6 and 4.4μmolGalAequivalenth−1, respectively. Cd2+, Cu2+ and Fe2+ and EDTA inhibited whereas GalA, Gal, Fuc, Rha and Ara stimulated PG-I activity, in particular. The major endogenous substrates for mango PG were identified to be two rhamnogalacturonans varying in their sugar ratio. These results are discussed in the light of pectin dissolution in vivo in ripening mango. [Copyright &y& Elsevier]

Toxicologic and physiologic roles of CYP1A enzyme induction, the major biochemical effect of aryl hydrocarbon receptor activation by TCDD and other receptor ligands, are unknown. Evidence is presented that CYP1A exerts biologic effects via metabolism of endogenous substrates (i.e., arachidonic acid, other eicosanoids, estrogens, bilirubin, and melatonin), production of reactive oxygen, and effects on K + and Ca 2+ channels. These interrelated pathways may connect CYP1A induction to TCDD toxicities, including cardiotoxicity, vascular dysfunction, and wasting. They may also underlie homeostatic roles for CYP1A, especially when transiently induced by common chemical exposures and environmental conditions (i.e., tryptophan photoproducts, dietary indoles, and changes in oxygen tension). [ABSTRACT FROM AUTHOR]