Association of Hepatic Nuclear Factor 4 Alpha Gene Polymorphisms With Free Imatinib Plasma Levels and Adverse Reactions in Chinese Gastrointestinal Stromal Tumor Patients.

BACKGROUND: As the first-line treatment of gastrointestinal stromal tumor (GIST), the pharmacokinetic and pharmacodynamic of imatinib (IM) were characterized by marked interindividual variability. Pharmacogenetics of IM involved metabolic enzymes and transporters have been extensively reported, but the results remained inconsistent. This study investigated the effect of genetic variants in hepatocyte nuclear factor 4 alpha (HNF4α, encoded by gene NR2A1), a pivotal transcriptional regulator of drug disposition genes, on dose-adjusted IM-free plasma levels and related adverse reactions in Chinese GIST patients. METHODS: Five common polymorphisms of NR2A1 (rs3818247, rs1884613, rs2071197, rs2425640, and rs736824) were genotyped in 70 Chinese GIST patients who had been administered IM 300-600 mg/d. The free IM trough plasma levels were determined based on a method of ultrafiltration coupled with high performance liquid chromatography-tandem mass spectrometry. RESULTS: There were wide interpatient variations in free plasma levels of IM (range, 9.50-67.50 ng/mL), in which significant sex differences were observed (P < 0.01). The dose-adjusted IM-free plasma levels showed a significant negative correlation with body surface area (r = -0.302, P = 0.012). Although there were no significant effects of NR2A1 polymorphisms on dose-adjusted IM-free plasma levels among the study population, polymorphism in rs736824 was found to be significantly associated with dose-adjusted IM-free plasma levels in male subjects (P = 0.031). For the IM-related adverse reaction, polymorphisms in rs3818247 were found to be significantly associated with periorbital edema (P = 0.032). In addition, no significant correlations were found between IM-free plasma levels and IM-related adverse reactions, except for the correlation of IM-free plasma levels with periorbital edema among male patients (P = 0.013). CONCLUSIONS: The research demonstrated that NR2A1 polymorphisms may act as contributors of IM pharmacokinetics and responses in Chinese GIST patients. This represents an attractive opportunity for IM therapy optimization, worth testing in clinical trials.

Establishment and assessment of a novel in vitro bio-PK/PD system in predicting the in vivo pharmacokinetics and pharmacodynamics of cyclophosphamide.

1. A novel bio-pharmacokinetic/pharmacodynamic (PK/PD) system was established and assessed in predicting the PK parameters and PD effects of the model drug cyclophosphamide (CP) considering the interrelationships between drug metabolism, pharmacological effects and dynamic blood circulation processes in vitro. 2. The system contains a peristaltic pump, a reaction chamber with rat liver microsomes (RLMs) encapsulated in pluronic F127-acrylamide-bisacrylamide (FAB) hydrogels, an effector cell chamber and a recirculating pipeline. The metabolism and pharmacological effects of CP (5, 10 and 20 mM) were measured by HPLC and MTT assay. A mathematical model based on mass balance was used to predict the in vitro clearance of CP. In vivo clearance of CP was estimated by in vitro to in vivo extrapolations (IVIVE) and simulations using Simcyp® software. 3. The predicted in vivo clearance of CP at concentrations of 5, 10 and 20 mM was 11.36, 10.12 and 10.68 mL/min/kg, respectively, within two-fold differences compared with the reported 11.1 mL/min/kg. The survival ratio of effector cells during the metabolism and circulation of CP was significantly enhanced. 4. This system may serve as an alternative approach to predict in vivo metabolism, pharmacological effects and toxicity of drugs, ensuring an efficient drug screening process.

Genetic Polymorphisms Contribute to the Individual Variations of Imatinib Mesylate Plasma Levels and Adverse Reactions in Chinese GIST Patients.

Imatinib mesylate (IM) has dramatically improved the outcomes of gastrointestinal stromal tumor (GIST) patients. However, the clinical responses of IM may considerably vary among single individuals. This study aimed to investigate the influences of genetic polymorphisms of drug-metabolizing enzyme (CYP3A4), transporters (ABCB1, ABCG2), and nuclear receptor (Pregnane X Receptor (PXR, encoded by NR1I2)) on IM plasma levels and related adverse reactions in Chinese GIST patients. A total of 68 Chinese GIST patients who have received IM 300-600 mg/day were genotyped for six single nucleotide polymorphisms (SNPs) (CYP3A4 rs2242480; ABCB1 rs1045642; ABCG2 rs2231137; NRI12 rs3814055, rs6785049, rs2276706), and the steady-state IM trough plasma concentrations were measured by a validated HPLC method. There were statistically significant variances in the steady-state IM trough plasma concentrations (from 272.22 to 4365.96 ng/mL). Subjects of GG in rs2242480, T allele carriers in rs1045642 and CC in rs3814055 had significantly higher steady-state IM dose-adjusted trough plasma concentrations. Subjects of CC in rs3814055 had significantly higher incidence rate of edema. The genetic polymorphisms of rs2242480, rs1045642, rs3814055 were significantly associated with IM plasma levels, and the genetic variations of rs3814055 were significantly associated with the incidence rate of edema in Chinese GIST patients. The current results may serve as valuable fundamental knowledge for IM therapy in Chinese GIST patients.

Application of a New Dynamic Model to Predict the In Vitro Intrinsic Clearance of Tolbutamide Using Rat Microsomes Encapsulated in a Fab Hydrogel.

Currently used in vitro models for estimating liver metabolism do not take into account the physiologic structure and blood circulation process of liver tissue. The Bio-PK metabolic system was established as an alternative approach to determine the in vitro intrinsic clearance of the model drug tolbutamide. The system contained a peristaltic pump, recirculating pipeline, reaction chamber, and rat liver microsomes (RLMs) encapsulated in pluronic F127-acrylamide-bisacrylamide (FAB) hydrogel. The metabolism of tolbutamide at initial concentrations of 100, 150, and 200 µM was measured in both the FAB hydrogel and the circular medium. The data from the FAB hydrogel and the circular medium were fitted to a mathematical model to obtain the predicted intrinsic clearance of tolbutamide after different periods of preincubation. The in vitro clearance value for tolbutamide was incorporated into Simcyp software and used to predict both the in vivo clearance value and the dynamic process of elimination. The predicted in vivo clearance of tolbutamide was 0.107, 0.087, and 0.095 L/h/kg for i.v. injection and 0.113, 0.095, and 0.107 L/h/kg for oral administration. Compared with the reported in vivo clearance of 0.09 L/h/kg (i.v.) and 0.10 L/h/kg (oral), all the predicted values differed by less than twofold. Thus, the Bio-PK metabolic system is a reliable and general in vitro model, characterized by three-dimensional structured RLM and circulation and perfusion processes for predicting the in vivo intrinsic clearance of low-extraction compounds, making the system more analogous with the rat in terms of both morphology and physiology.

Metabolomic analysis reveals the impact of ketoprofen on carbon and nitrogen metabolism in rice (Oryza sativa L.) seedling leaves.

Pharmacologically active compounds (PACs) are becoming common pollutants in the natural environment, posing potential risks to crop quality; however, the toxic effects and metabolic changes that they cause in agricultural plants remain unclear. Here, we investigated the effects of ketoprofen on respiration rate, ATP synthesis, carbon and nitrogen metabolism, and metabolomics in rice seedling leaves. The results showed that ketoprofen treatment adversely affected the respiration rate, ATP content, H+-ATPase activity and induced changes in the contents of carbon assimilation products (soluble sugar, reducing sugar, sucrose, and starch) and the activities of key enzymes in carbon metabolism (sucrose synthase (SS), sucrose phosphate synthase (SPS), and sucrose invertase (InV)). The contents of nitrate, ammonium, and free amino acids, and the activities of key enzymes involved in nitrogen metabolism (nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH)) were also affected in a concentration-dependent manner. Metabolomics analysis showed that ketoprofen disturbed the type and content of metabolites (amino acids, carbohydrates, and secondary metabolites) to varying degrees and perturbed key metabolic pathways (substance synthesis and energy metabolism), ultimately resulting in the reduction of rice seedling biomass. This study provides important information and a useful reference for the accurate assessment of the environmental risks of PACs.

Metabolomics and proteomics reveal the toxicological mechanisms of florfenicol stress on wheat (Triticum aestivum L.) seedlings.

Although the ecological impacts of antibiotics have received attention worldwide, research on the toxicity of florfenicol is still limited. We conducted a metabolomic and proteomic study on wheat (Triticum aestivum L.) seedlings to reveal the toxicological mechanism of florfenicol. The growth of the wheat seedlings was found to be inhibited by florfenicol. Antioxidant enzyme activities (superoxide dismutase, peroxidase and catalase), malondialdehyde content and membrane permeability increased with increasing florfenicol concentration. The contents of chlorophyll and chlorophyll synthesis precursor substances (Proto IX, Mg-proto IX and Pchlide), photosynthetic and respiration rates, and chlorophyll fluorescence parameters decreased, indicating that photosynthesis was inhibited. The ultrastructure of chloroplasts was destroyed, as evidenced by the blurred membrane surface, irregular grana arrangement, irregular thylakoid lamella structure, and increased plastoglobuli number. Proteome analysis revealed that up-regulated proteins were highly involved in protein refolding, translation, oxidation-reduction, tricarboxylic acid cycle (TCA cycle), reactive oxygen species metabolic process, cellular oxidant detoxification, and response to oxidative stress. The down-regulated proteins were mainly enriched in photosynthesis-related pathways. In the metabolome analysis, the content of most of the metabolites in wheat leaves, such as carbohydrates and amino acids increased significantly (p < 0.05). Combined pathway analysis showed that florfenicol stress stimulated the TCA cycle pathway and downregulated the photosynthesis pathway.

Metabolomic analysis of Scenedesmus obliquus reveals new insights into the phytotoxicity of imidazolium nitrate ionic liquids.

Due to the persistence of ionic liquids (ILs) in aquatic environments, it is necessary to reveal their ecological risk to aquatic organisms. Herein, the biotoxicity of two alkyl-methylimidazolium nitrate ILs ([C10mim]NO3 and [C12mim]NO3) against Scenedesmus obliquus were studied. Results showed that the growth inhibition of S. obliquus increased with increasing concentrations of ILs, maximum values of 94.61% at 4 mg/L of [C10mim]NO3 and 97.34% at 0.8 mg/L of [C12min]NO3 were observed. The fluorescence parameters of photosystem II, such as light quantum yield and electron transfer rate, showed a negative relationship with the exposure dose. [C12mim]NO3 had a more significant effect than [C10mim]NO3. Moreover, the redox homeostasis of algae was disrupted; the accumulation of reactive oxygen species, leading to obvious inhibition of superoxide dismutase and catalase activities was observed. A metabolomic analysis indicated that the contents of most metabolites were reduced significantly, and fructose and galactose decreased significantly by 42.3% and 88.6%, respectively, in the [C10mim]NO3 treatment compared to those in the control. The inhibition of amino acid biosynthesis and glyoxylate and dicarboxylate metabolism explained the more serious biotoxicity of [C12mim]NO3 than that of [C10mim]NO3. This study facilitates a better understanding of the environmental safety and ecological risks of ILs.

ROS as a key player in quinolone antibiotic stress on Arabidopsis thaliana: From the perspective of photosystem function, oxidative stress and phyllosphere microbiome.

With the increasing use of antibiotics, their ecological impacts have received widespread attention. However, research on the toxicity of quinolone antibiotics is still limited, especially regarding the oxidative stress and phyllosphere of plants. In this study, the toxic effects of enrofloxacin, norfloxacin, and levofloxacin on Arabidopsis thaliana and their underlying mechanisms were investigated. The toxicity of the three quinolone antibiotics decreased in the following order: enrofloxacin > norfloxacin > levofloxacin. Physiological cellular changes, such as plasmolysis and chloroplast swelling, were observed using electron microscopy. Photosynthetic efficiency was inhibited with a decline in the effective photochemical quantum yield of photosystem II (Y(II)) and non-photochemical quenching (NPQ), indicating that quinolone antibiotics might reduce light energy conversion efficiency and excess light energy dissipation. Oxidative stress occurred in A. thaliana after quinolone antibiotic treatment, with an increase in reactive oxygen species (ROS) levels and malondialdehyde (MDA) content. High ROS levels stimulated the over-expression of superoxide-responsive genes for self-protection. Structural equation modeling (SEM) analysis showed that photosynthesis inhibition and cellular damage caused by oxidative stress were critical factors for growth inhibition, suggesting that the antioxidant response activated by ROS might be a potential mechanism. Furthermore, the diversity of the phyllospheric microbial communities decreased after enrofloxacin exposure. Additionally, specific microbes were preferentially recruited to the phyllosphere because of the higher ROS levels.

HTR1A/1B DNA methylation may predict escitalopram treatment response in depressed Chinese Han patients.

BACKGROUND: The serotonin receptor 1A and 1B (HTR1A/1B) gene have been suggested to be involved in the pathogenesis of major depressive disorder (MDD) and the antidepressant treatment response. Gene expression differences were partly mediated by genetic polymorphism and DNA methylation which might be affected by environmental factors. In the present study, we attempt to identify whether HTR1A/1B DNA methylation and genetic polymorphism could predict antidepressant treatment response. METHODS: 85 Chinese Han MDD patients were clinically assessed 8 weeks after of initiating escitalopram treatment for the first time. Antidepressant treatment response was assessed by changes in the Hamilton Depression Rating Scale-17 items (HAMD-17) score. The Life Events Scale (LES) and the Childhood Trauma Questionnaire (CTQ) were utilized as the assessment of previous life stress. The Illumina HiSeq platform was used to assess DNA methylation at 96 CpG sites located in HTR1A and HTR1B gene promoter regions. Six single nucleotide polymorphisms (SNPs) (HTR1A rs6294, rs116985176; HTR1B rs6296, rs6298, rs1228814, rs1778258) were genotype by using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) or PCR sequencing. Regression analyses were used to explore the relationship between DNA methylation and SNP and antidepressant response. RESULTS: We identified two CpG sites predictor of antidepressant treatment response (CpG 668, amplicon HTR1A_1, NC_000005.10, P = 0.025; CpG 1401, amplicon HTR1B_4, NC_000006.12, P = 0.033). The interaction of four CpG sites hypomethylation of HTR1A/1B with high recent stress might result in impaired antidepressant treatment response. What's more, the present data indicated that age, environments and antidepressant treatment might affect DNA methylation status. It was found that DNA methylation status could be influenced by antidepressant treatment in turn. However, HTR1A and HTR1B genotypes did not influence antidepressant response and DNA methylation status. CONCLUSIONS: The results suggest that HTR1A/1B DNA hypomethylation and its interaction with recent life stress might drive impaired antidepressant treatment response. Meanwhile, DNA methylation, in turn, was modified by antidepressant treatment and environments. Our results offer new evidence for the role of epigenetic and genetic polymorphism in pharmacological response to antidepressants.

Mineralized growth of Janus membrane with asymmetric wetting property for fast separation of a trace of blood.

Separation of plasma from whole blood is requisite for the accurate measurement of glucose levels. From the wettability point of view, in this study, we report the fabrication of a mineralized Janus membrane with an asymmetric wetting property; this membrane can transport fast microliter-quantity blood and separate out the red blood cells. The membrane is composed of a hydroxyapatite (HAP)-mineralized polyvinylidene fluoride (PVDF) membrane prepared via an interface diffusion-controlled chemical precipitation method. Due to gradient distribution of the HAP nanocrystals across the PVDF membrane, the composite membrane exhibits an asymmetric wetting property where a tiny whole blood droplet (13 µL) can spontaneously permeate across the membrane within 20 s and red blood cells in the whole blood are successfully blocked by the membrane. The membrane is then integrated into blood glucometers for accurate measurements of the glucose levels. The results show that the porous membrane can successfully prevent red blood cells in whole blood from entering the enzyme layer; moreover, the negative effect of hematocrit levels on the blood glucose measurements is effectively minimized and an obviously high and stable glucose current signal is achieved.

Microbial transformation of androst-4-ene-3,17-dione by Beauveria bassiana.

The microbial transformation of androst-4-ene-3,17-dione (I) by the fungus Beauveria bassiana CCTCC AF206001 has been investigated using pH 6.0 and 7.0 media. Two hydroxylated metabolites were obtained with the pH 6.0 medium. The major product was 11alpha-hydroxyandrost-4-ene-3,17-dione (II) whereas the minor product was 6beta,11alpha-dihydroxyandrost-4-ene-3,17-dione (III). On the other hand, four hydroxylated and/or reduced metabolites were obtained with the pH 7.0 medium. The major product was 11alpha,17beta-dihydroxyandrost-ene-3-one (V) and the minor products were 17beta-hydroxyandrost-ene-3-one (IV), 6beta,11alpha,17beta-trihydroxyandrost-ene-3-one (VI) and 3alpha,11alpha,17beta-trihydroxy-5alpha-androstane (VII). The products were purified by chromatographic methods, and were identified on the basis of spectroscopic methods. This fungus strain is clearly an efficient biocatalyst for 11alpha-hydroxylation and reduction of the 17-carbonyl group.

[Morphology and function of platelets stored in modified platelet additive solution at low temperature].

The aim of study was to evaluate the function of modified platelet additive solution (PAS-IIIM) with trehalose as a substitute of plasma for the storage of platelet concentrates at low temperature (10 degrees C). Apheresis platelets from 6 donors were divided and added with different media (group A: 100% plasma; group B: 70% PAS-IIIM/30% plasma; group C: 100% plasma/trehalose). Groups A, B, C were stored at 10 degrees C, 22 degrees C and -85 degrees C separately. In addition, group D (platelet concentrates stored with 100% plasma at 4 degrees C) was set up as control group for scan electronmicroscopy. The samples of each platelets were collected on day 0, 1, 5, 7 and 9 after storage respectively, while samples of platelets stored at -85 degrees C (group C) were collected on day 20 after storage. CD62p, hypotonic shock response (HSR), platelet aggregation, lactic dehydrogenase (LDH) and morphology of platelets were evaluated. The results showed that the expressions of CD62p in groups A and B increased in a time-dependent manner, but HSR and platelet aggregations decreased. The expression of CD62p, LDH release, and platelet aggregation in group A were significant higher than that in group B (p < 0.05). HSR in group A was significant lower than that in group B (p < 0.05). LDH release was significant high in samples of group C and the expression of CD62p was lower than that in other two groups (p < 0.05). It is concluded that the protective effects of 70% PAS-IIIM/30% plasma (10 degrees C) and plasma platelets (22 degrees C) on morphology of platelets are similar, but better than those of plasma platelets (4 degrees C) and plasma/trehalose (-85 degrees C). In short, PAS-IIIM serves as a good substitute of plasma for platelet storage, and protects the chilled platelets.

[Report on difficulty in blood group identification due to anti-H antibody in three cases].

Anti-H antibody belongs to IgM type cold antibody, which often induces the unconformity of positive and reverse typing and leads to the difficulty in clinical blood typing. Anti-H antibody was found during identification of the counter blood group in 3 cases. The antibody was found to be active at 37 degrees C, room temperature and 4 degrees C when determined by blood group serology, and was finally analyzed to be IgM. It is suggested that not to give erythrocytes of O group unreasoningly to blood recipient of AB group during emergent moment, but instead, to give same type of blood. If there was no same type of blood during urgent events, O type erythrocytes could be employed after being matched by saline centrifuging with host side coincidence and screened by incomplete method. In this case, anti-H antibody leading to adverse-reaction in blood transfusion should be prevented.