抗乙肝病毒新药Bay41-4109的发现毒理学研究

目的:利用发现毒理学中的临床前先导化合物优化技术(preclinical lead optimization technolo-gies,PLOTs)对抗乙肝病毒候选新药Bay41-4109的一般毒性、遗传毒性和生殖毒性进行研究,为早期发现候选新药的毒性提供实验依据.方法:一般毒性研究中分别以MTT比色法和上下法检测Bay41-4109的体外细胞毒性和小鼠LD50;遗传毒性研究中分别以Ames波动试验、SOS显色试验、双核细胞微核试验检测Bay41-4109诱发鼠伤寒沙门菌基因回复突变的能力、诱发大肠杆菌的原发DNA损伤效应以及对CHL细胞的染色体断裂效应;生殖毒性研究中以大鼠胚胎中脑细胞微团培养试验来检测Bay41-4109的致畸性.结果:Bay41-4109对CHL细胞的IC50为54.0 μmol·L-1,对雌性小鼠的LD50大于2 000 mg·kg-1.无论有无S9活化,Bay41-4109均不引起沙门菌回复突变,也不导致DNA损伤和染色体断裂.Bay41-4109对大鼠胚胎中脑细胞亦无致畸作用.结论:早期毒性筛选结果表明:Bay41-4109未表现明显的遗传毒性和生殖毒性.     

NMR-based metabonomic analysis of the hepatotoxicity induced by combined exposure to PCBs and TCDD in rats

A metabonomic approach using ¹H NMR spectroscopy was adopted to investigate the metabonomic pattern of rat urine after oral administration of environmental endocrine disruptors (EDs) polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) alone or in combination and to explore the possible hepatotoxic mechanisms of combined exposure to PCBs and TCDD. ¹H NMR spectra of urines collected 24 h before and after exposure were analyzed via pattern recognition by using principal component analysis (PCA). Serum biochemistry and liver histopathology indicated significant hepatotoxicity in the rats of the combined group. The PCA scores plots of urinary ¹H NMR data showed that all the treatment groups could be easily distinguished from the control group, so could the PCBs or TCDD group and the combined group. The loadings plots of the PCA revealed remarkable increases in the levels of lactate, glucose, taurine, creatine, and 2-hydroxy-isovaleric acid and reductions in the levels of 2-oxoglutarate, citrate, succinate, hippurate, and trimethylamine-N-oxide in rat urine after exposure. These changes were more striking in the combined group. The changed metabolites may be considered possible biomarker for the hepatotoxicity. The present study demonstrates that combined exposure to PCBs and TCDD induced significant hepatotoxicity in rats, and mitochondrial dysfunction and fatty acid metabolism perturbations might contribute to the hepatotoxicity. There was good conformity between changes in the urine metabonomic pattern and those in serum biochemistry and liver histopathology. These results showed that the NMR-based metabonomic approach may provide a promising technique for the evaluation of the combined toxicity of EDs.     

The application of microbore UPLC/oa-TOF-MS and 1H NMR spectroscopy to the metabonomic analysis of rat urine following the intravenous administration of pravastatin

The metabonomic effects of hepatotoxic doses of pravastatin on the urinary metabolic profiles of female rats have been investigated using ultra performance liquid chromatography (UPLC)-oa-TOF-MS and, independently, by (1)H NMR spectroscopy. UPLC was performed using a 1 mm microbore column packed with 1.7 microm particles. Examination of the data obtained from the individual animals, aided by statistical interpretation of the data, made it possible to identify potential markers for toxicological effects, with both NMR and UPLC-MS analysis highlighting distinct changes in the urinary metabolite profiles. These markers, which included elevated taurine and creatine, as well as bile acids, were consistent with hepatotoxicity in some animals, and this hypothesis was supported by histopathological and clinical chemistry findings. The analytical data from both techniques could be used to define a metabolic "trajectory" as toxicity developed and to provide an explanation for the lack of hepatotoxicity for one of the animals. The two analytical approaches (UPLC-MS and NMR) were found to be complementary whilst the use of a 1mm i.d. x 100 mm column reduced the amount of sample required for analysis to 2 microL, compared with 10 microL for a 2.1mm i.d. x 100 mm column. The 1mm i.d. column also provided increased signal-to-noise without loss of chromatographic efficiency.     

H NMR-based metabonomic study on the metabolic changes in the plasma of patients with functional dyspepsia and the effect of acupuncture

Functional dyspepsia (FD) is a common gastrointestinal disorder with multiple pathogenic mechanisms seen in clinical practice, and acupuncture may potentially be an alternative therapy for it. In order to investigate the biological effects of FD and the effect of acupuncture on metabolism, ¹H nuclear magnetic resonance (NMR)-based metabonomic techniques have been used to compare the plasma metabolic profiles of six female FD patients with those of six female healthy control subjects. Plasma metabolic profiles of FD patients treated by acupuncture at the Foot-Yangming Meridian were also collected and compared. Data obtained from NMR spectroscopy were subjected to principal components analysis (PCA). The results show that there are relatively higher levels of glucose, acetate, high-density lipoprotein (HDL), and phosphatidylcholine (PtdCho), and lower levels of lactate, leucine/isoleucine, N-acetyl glycoprotein (NAc), and low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) in FD patients than in healthy controls. Acupuncture treatment of FD patients significantly changed the levels of leucine/isoleucine, lactate and glucose, and slightly changed lipids level towards those of the healthy controls, demonstrating its therapeutic effects on the relief of FD symptoms. Due to the limited number of subjects, the present work is just a proof-of-principle study and further researches with larger number of subjects are needed. Our work shows the potential of an NMR-based metabonomic approach in the study of biological effects of acupuncture.     

Study of a novel indolin-2-ketone compound Z24 induced hepatotoxicity by NMR-spectroscopy-based metabonomics of rat urine, blood plasma, and liver extracts

Antiangiogenic compound has been believed to be an ideal drug in the current cancer biological therapy, but the angiogenesis inhibitors suffer setback for unknown toxicity now. A novel synthetic indolin-s-ketone small molecular compound, 3Z-3-[((1)H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one (Z24) can inhibit angiogenesis in new blood vessels. The hepatotoxicity effects of Z24 oral administration (dosed at 60, 130 and 200 mg/kg) have been investigated in female Wistar rats by using metabonomic analysis of (1)H NMR spectra of urine, plasma and liver extracts, as well as by clinical chemistry analysis, liver histopathology and electron micrographs examination. The (1)H NMR spectra of the biofluids were analyzed visually and via pattern recognition by using principal component analysis. The metabonomic trajectory analysis on the time-related hepatotoxicity of Z24 was carried out based on the (1)H NMR spectra of urine samples, which were collected daily predose and postdose over an 8-day period. Urinary excretion of citrate, lactate, 2-oxo-glutarate and succinate increased following Z24 dosing. Increased plasma levels of lactate, TMAO and lipid were observed, with concomitant decrease in the level of glucose and phosphatidylcholine. Metabolic profiling on aqueous soluble extracts of liver tissues with the high dose level of Z24 showed an increase in lactate and glutamine, together with a decrease in glucose, glycogen and choline. On the other hand, studies on lipid soluble extracts of liver tissues with the high dose level of Z24 showed increased level in lipid triglycerides and decreased level in unsaturated fatty acids and phosphatidylcholine. Moreover, the most notable effect of Z24 on the metabolism was the reduction in the urinary levels of creatinine and TMAO and the increase in acetate, citrate, succinate and 2-oxo-glutamate with time dependence. The results indicate that in rats Z24 inhibits mitochondrial function through altering the energy and lipid metabolism, which results in the accumulation of free fatty acids and lactate because of the lack of aerobic respiration. These data show that the metabonomic approach represents a promising new technology for the toxicological mechanism study.     

Application of LC-NMR and HR-NMR to the characterization of biphenyl impurities in the synthetic route development for vestipitant,a novel NK1 antagonist

Vestipitant (1) is a novel NK1 antagonist currently under investigation for the treatment of CNS disorders and emesis. The first synthetic step comprised a Grignard synthesis. An impurity was identified and initially expected to be a symmetric biphenyl. This paper reports the work to synthesise the supposed structure and the spectroscopic analyses (LC-NMR and HR-NMR) to correctly identify the real structure and understand the chemical pathway of the impurity.     

Chemometric analysis of biofluids following toxicant induced hepatotoxicity: A metabonomic approach to distinguish the effects of 1-naphthylisothiocyanate from its products

Metabonomics using high-resolution ¹H-NMR spectroscopy of biofluids and pattern recognition is highly successful at distinguishing both organ- and sub-organ-specific toxicity. In the current study, this technique was investigated to distinguish the different biological effects caused by 1-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in the rat from that induced by exposure to 1-naphthylisocyanate (NI) and 1-naphthylamine (NA), two products of the metabolism of ANIT. While all three toxicants produced perturbations in similar urinary metabolites, principal components analysis of the temporal progression identified that the rapid initial glycosuria associated with ANIT toxicity was also present with NI but not NA dosing. However, longer-term perturbations in the urinary excretion of succinate, lactate and acetate were common to all three toxicants. The metabolic effects of the three compounds were also followed in blood plasma and liver tissue. Of the three toxicants, the most marked perturbations were induced by ANIT exposure, then NI, thereby indicating the effects of ANIT, NI and NA toxicity were distinct, with ANIT being the most, and NA the least, toxic of the three compounds. This indicates that metabonomics may be useful for following severity and mechanisms of toxicity in a series of related compounds during drug development.     

NMR spectroscopy and surface tension measurements applied to the study of self-association of casopitant mesylate,a novel NK1 antagonist

The aggregation behaviour of casopitant mesylate, a new NK1 antagonist drug, was investigated by means of NMR spectroscopy and surface tension measurements. The critical micelle concentration (CMC) in glycine buffer at pH 3.5 was determined by analyzing the ¹H NMR chemical shifts variation and the surface tension in function of the concentration in a series of solutions. The temperature dependence of the CMC was also evaluated by NMR spectroscopy as well as the thermodynamic parameters contributing to the aggregation discussed. Surface tension measurements were conducted as well in the formulation conditions, e.g. in the presence of sodium chloride.     

BAY 41-2272, a soluble guanylate cyclase agonist, activates human mononuclear phagocytes

BACKGROUND AND PURPOSE

Phagocyte function is critical for host defense against infections. Defects in phagocytic function lead to several primary immunodeficiencies characterized by early onset of recurrent and severe infections. In this work, we further investigated the effects of BAY 41-2272, a soluble guanylate cyclase (sGC) agonist, on the activation of human peripheral blood monocytes (PBM) and THP-1 cells.

EXPERIMENTAL APPROACH

THP-1 cells and PBM viability was evaluated by methylthiazoletetrazolium assay; reactive oxygen species production by lucigenin chemiluminescence; gene and protein expression of NAPDH oxidase components by qRT-PCR and Western blot analysis, respectively; phagocytosis and microbicidal activity by co-incubation, respectively, with zymosan and Escherichia coli; and cytokine release by elisa.

KEY RESULTS

BAY 41-2272, compared with the untreated group, increased spreading of monocytes by at least 35%, superoxide production by at least 50%, and gp91^PHOX and p67^PHOX gene expression 20 to 40 times, in both PBM and THP-1 cells. BAY 41-2272 also augmented phagocytosis of zymosan particles threefold compared with control, doubled microbicidal activity against E. coli and enhanced the release of TNF-α and IL-12p70 by both PBM and THP-1 cells. Finally, by inhibiting sGC with ODQ, we showed that BAY 41-2272-induced superoxide production and phagocytosis is not dependent exclusively on sGC activation.

CONCLUSIONS AND IMPLICATIONS

In addition to its ability to induce vasorelaxation and its potential application for therapy of vascular diseases, BAY 41-2272 was shown to activate human mononuclear phagocytes. Hence, it is a novel pro-inflammatory drug that may be useful for controlling infections in the immunocompromised host.     

Generic ciprofloxacin tablets contain the stated amount of drug and different impurity profiles: A 19F, 1H and DOSY NMR analysis

The objective of this study was to control the purity of 16 commercial formulations of ciprofloxacin tablets purchased in different countries or via the Internet using 19F and 1H nuclear magnetic resonance (NMR). Twelve out of the sixteen commercial formulations of ciprofloxacin measured by 19F NMR contain the active ingredient within 100+/-5% of stated concentration. Three formulations have a lower ciprofloxacin content between 90 and 95% and one shows a higher concentration superior to 105%. The impurity profile was characterised using 19F and 1H NMR, and is characteristic of the manufacturer. Four to twelve fluorinated impurities among them fluoride ion and two already known compounds were detected and quantified in the sixteen formulations analysed by 19F NMR. Two other non-fluorinated impurities were observed in the seven formulations analysed with 1H NMR. The total content of impurities as well as their individual levels are in agreement with those reported previously in the few studies devoted to ciprofloxacin purity. However, all the formulations do not comply with the limits for impurities given in the ciprofloxacin monograph of the European Pharmacopeia. Finally, a "signature" of the formulations was obtained with Diffusion-Ordered SpectroscopY (DOSY) 1H NMR which allowed the characterisation of some excipients present in the formulations studied.     

Influence of kaempferol,a flavonoid compound,on membrane-bound ATPases in streptozotocin-induced diabetic rats

Abstract

Context: Kaempferol is a flavonoid found in many edible plants (e.g. tea, cabbage, beans, tomato, strawberries, and grapes) and in plants or botanical products commonly used in traditional medicine. Numerous preclinical studies have shown that kaempferol have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antidiabetic activities.

Objective: The present study investigates the effect of kaempferol on membrane-bound ATPases in erythrocytes and in liver, kidney, and heart of streptozotocin (STZ)-induced diabetic rats.

Materials and methods: Diabetes was induced into adult male albino rats of the Wistar strain, by intraperitoneal administration of STZ (40?mg/kg body weight (BW)). Kaempferol (100?mg/kg BW) or glibenclamide (600?µg/kg BW) was administered orally once daily for 45?d to normal and STZ-induced diabetic rats. The effects of kaempferol on membrane-bound ATPases (total ATPase, Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase) activity in erythrocytes and in liver, kidney, and heart were determined.

Results: In our study, diabetic rats had significantly (p?<?0.05) decreased activities of total ATPases, Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Mg²⁺-ATPase in erythrocytes and tissues. Oral administration of kaempferol (100?mg/kg BW) or glibenclamide (600?µg/kg BW) for a period of 45?d resulted in significant (p?<?0.05) reversal of these enzymes' activities to near normal in erythrocytes and tissues when compared with diabetic control rats.

Discussion and conclusion: Thus, obtained results indicate that administration of kaempferol has the potential to restore deranged activity of membrane-bound ATPases in STZ-induced diabetic rats. Further detailed investigation is necessary to discover kaempferol’s action mechanism.     

Global metabolic profiling for the study of Rhizoma Paridis saponins‐induced hepatotoxicity in rats

Rhizoma Paridis saponins (RPS) is a traditional Chinese medicine (TCM) from the plant Paris polyphylla var. yunnanensis (Fr.) Hand.‐Mazz. Despite its potentially clinical utility such as anticancer and anti‐inflammation, it has slight side effects and toxicity as previous report. In this work, 90‐day administration of RPS induced liver injury. ¹H‐NMR‐ and GC/MS‐based metabonomic analyses in conjunction with histopathological examinations, blood biochemistry and hepatic phase I and II enzymes assays were performed to evaluate the toxic mechanisms of RPS induced in rats. As a result, oral administration of RPS possessed certain liver toxicity in SD rats. ¹H‐NMR and GC/MS data indicated that RPS inhibited the oxidation of fatty acids, glycolysis, and TCA cycle pathway, and disturbed glycine, serine, and threonine metabolism. Low expression of TG, T‐CHO, and LDL‐C and high levels of ALT and AST indicated that chronic exposure to RPS caused hepatocyte damage, synthesis dysfunction, and transportation failure of lipoproteins. In addition, RPS downregulated the mRNA levels of CYP1A2, CYP2E1, and UGTs. In conclusion, we used metabonomics approach to study the toxicity of RPS for the first time. This research demonstrated that metabonomics method was a promising tool to study and diagnose TCM‐induced toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 99–108, 2017.     

Analysis of illegally manufactured formulations of tadalafil (Cialis) by 1H NMR, 2D DOSY 1H NMR and Raman spectroscopy

Counterfeit and/or imitation medicines are becoming a major health problem not only in developing countries but also in wealthier countries. The need of new and easy analytical methods for quality control of drugs is essential. We describe the use of Raman spectroscopy, 1H nuclear magnetic resonance (NMR) and 2D diffusion-ordered spectroscopy (DOSY) NMR to analyse genuine Cialis and seven illegally manufactured formulations of this drug purchased via the internet. Seven out of the eight commercial formulations of tadalafil contain the active ingredient, measured by high performance liquid chromatography (HPLC), within 100+/-5% of stated concentration. Vardenafil and homosildenafil instead of tadalafil were found in the Chinese imitation. 2D DOSY NMR spectra clearly showed similarities and differences in the composition of the pharmaceutical formulations of tadalafil, thus giving a precise and global "signature" of the manufacturer. Our data show that the quality of the Cialis imitations manufactured in India and Syria is correct, whereas the Chinese formulation is adulterated with active pharmaceutical ingredients.     

Pharmacologic and radioligand binding analysis of the actions of 1,4-dihydropyridine activators related to Bay K 8644 in smooth muscle,cardiac muscle and neuronal preparations

Summary The structure-activity relationships of a series of 1,4-dihydropyridine Ca²⁺ channel activators, including Bay K 8644, have been determined by pharmacologic and radioligand binding techniques. Pharmacologic techniques included tension responses and the measurement of pA₂ values for nifedipine antagonism of Bay K 8644 responses in guinea pig ileal, rat femoral and rat atrial and papillary muscle preparations. Radioligand binding experiments employed competition against [³H]nitrendipine binding in ileal smooth muscle and rat ventricular membranes and rat brain synaptosomal preparations. The series of compounds was employed as the racemates. Binding affinities were not significantly different between smooth muscle, cardiac muscle and brain preparations and the same rank order of pharmacologic activities is observed in smooth and cardiac muscle, where the effects of the 4-phenyl substituents, o m > p, parallel those observed for 1,4-dihydropyridine antagonists. In the ileal and femoral artery smooth muscle preparations a 1:1 correlation is observed between pharmacologic and radioligand binding affinities. However, in the cardiac muscle preparations, left atrium and papillary muscle, there is an approximately 10-fold difference between the binding affinities and the lower pharmacologic affinities. A similar difference between smooth and cardiac muscle is observed with the pA₂ values of 6.97 and 7.06 in atrial and papillary muscle respectively, which are significantly lower than the values of 8.54 and 8.72 measured in ileal and femoral artery respectively. The structure-activity expressions measured for this small series of 1,4-dihydropyridine activators parallel those observed in the larger series of 1,4-dihydropyridine antagonists. This is consistent with proposals that activators and antagonists interact at common binding sites that are components of a voltage-dependent Ca²⁺ channel. Send offprint requests to D. J. Triggle at the above address     

NMR analysis of ion pair formation between timolol and sorbic acid in ophthalmic preparations

Ion pair formation between timolol and sorbic acid was investigated using NMR spectroscopy in order to clarify their interactions within ophthalmic preparation. (13)C and (1)H NMR spectra of timolol, sorbic acid, and a mixture of the two were obtained, and the signal changes induced by pairing were observed. The carbon signals of the butylaminopropanol moiety of timolol were markedly shifted in the mixture, as were the carboxyl and conjugated carbons assigned to sorbic acid. The localizations of the changes in each molecule revealed the binding sites. The profiles of butylaminopropanol carbon chemical shifts plotted against a molar ratio of sorbate were synchronized, which suggested a single type of interaction with sorbic acid. The Job plot showed a typical pattern with a single-maximum at a mole function of 0.5, indicating the presence of a 1:1 complex of timolol and sorbic acid. The stability constants (K) of the timolol-sorbate and timolol-maleate pairs were 1.9x10(1) and 2.2x10(2)M(-1), respectively. The higher K value of the timolol-maleate interaction suggested that it was dominant to the timolol-sorbate interaction when maleate and sorbate coexisted within a timolol solution. Here, we demonstrated evidence of an interaction between timolol and sorbic acid using simple NMR measurements, which suggested the existence of ion pair formation derived from charge neutralization. Our analysis using NMR spectroscopy should advance the understanding and optimization of formulations that are based on ion pair.     

A quantitative NMR protocol for the simultaneous analysis of atropine and obidoxime in parenteral injection devices

A rapid selective and accurate quantitative ¹H NMR method was developed for the simultaneous analysis of obidoxime chloride and atropine sulfate, the active components in parenteral injection devices (PID) used for the emergency treatment of poisoning by toxic organophosphates. The spectra were acquired in 90% H₂O–10% D₂O using sodium 3-(trimethylsilyl)-1-propane sulfonate hydrate as the internal standard. Both synthetic mixtures and dosage forms were assayed. The results were compared with those obtained from a reported HPLC method.     

Urethane,but not pentobarbitone,attenuates presynaptic receptor function in rats: a contribution to the choice of anaesthetic

Background and purpose:

We examined whether cannabinoid CB₁ and histamine H₃ receptors resemble α₂-adrenoceptors in that their presynaptically mediated cardiovascular effects are less marked in urethane- than in pentobarbitone-anaesthetized pithed rats.

Experimental approach:

Effects of the cannabinoid agonist CP-55,940 and the H₃ receptor agonist imetit on electrically induced tachycardic and vasopressor responses, respectively, was compared in pithed rats anaesthetized with urethane or pentobarbitone. The affinity of urethane for the three receptors was measured by radioligand binding studies in rat brain cortex membranes and its potency assessed in superfused mouse tissues preincubated with ³H-noradrenaline.

Key results:

The neurogenic tachycardic response was less markedly inhibited by CP-55,940 in urethane- than in pentobarbitone-anaesthetized pithed rats. Imetit inhibited the neurogenic vasopressor response after pentobarbitone but not after urethane. The catecholamine-induced tachycardic and vasopressor response did not differ between rats anaesthetized with either compound. Urethane 10 mM (plasma concentration reached under anaesthesia) did not affect binding to CB₁ or H₃ receptors and α₂ adrenoceptors, nor did it alter the inhibitory effect of agonists at the three receptors on electrically evoked ³H-noradrenaline release.

Conclusions and implications:

Urethane, but not pentobarbitone, abolished the H₃ receptor-mediated vascular response in pithed rats and attenuated the CB₁ receptor-mediated cardiac response much more than pentobarbitone. The weaker effects of CB₁, H₃ and α₂ receptor agonists cannot be explained by antagonism by urethane at the three receptors in vitro. Pentobarbitone, but not urethane, is suitable as an anaesthetic for investigations of inhibitory presynaptic receptor function in pithed and anaesthetized rats.     

Prospective evaluation of potential toxicity of repeated doses of Thymus vulgaris L. extracts in rats by means of clinical chemistry,histopathology and NMR‐based metabonomic approach

In the field of natural extracts, research generally focuses on the study of their biological activities for food, cosmetic, or pharmacological purposes. The evaluation of their adverse effects is often overlooked. In this study, the extracts of Thymus vulgaris L. were obtained by two different extraction methods. Intraperitoneal injections of both extracts were given daily for four days to male Wistar Han rats, at two different doses for each extract. The evaluation of the potential toxic effects included histopathological examination of liver, kidney, and lung tissues, as well as serum biochemistry of liver and kidney parameters, and ¹H‐NMR‐based metabonomic profiles of urine. The results showed that no histopathological changes were observed in the liver and kidney in rats treated with both extracts of thyme. Serum biochemical investigations revealed significant increases in blood urea nitrogen, creatinine, and uric acid in animals treated with polyphenolic extract at both doses. In these latter groups, metabonomic analysis revealed alterations in a number of urine metabolites involved in the energy metabolism in liver mitochondria. Indeed, the results showed alterations of glycolysis, Krebs cycle, and β‐oxidative pathways as evidenced by increases in lactate and ketone bodies, and decreases in citrate, α‐ketoglutarate, creatinine, hippurate, dimethylglycine, and dimethyalanine. In conclusion, this work showed that i.p. injection of repeated doses of thyme extracts causes some disturbances of intermediary metabolism in rats. The metabonomic study revealed interesting data which could be further used to determine the cellular pathways affected by such treatments. Copyright © 2014 John Wiley & Sons, Ltd.     

Inhibition of [3H]resiniferatoxin binding to rat dorsal root ganglion membranes as a novel approach in evaluating compounds with capsaicin-like activity

Summary We have recently reported the specific binding of [³H]resiniferatoxin to sensory ganglion membranes; this binding appears to represent the postulated vanilloid (capsaicin) receptor. In the present report, we compare the structure/activity relations for binding to rat dorsal root ganglion membranes and for biological responses in the rat, using a series of vanilloids of the capsaicin (homovanilloyl-decylamide, homovanilloyl-dodecylamide, homovanilloyl-cyclododecylamide, homovanilloyl-hexadecylamide, homovanilloyl-piperidine and nonenoyl-homoveratrylamide) and resiniferatoxin (tinyatoxin, 12-deoxyphorbol 13-phenylacetate 20-homovanillate) classes. We find that all the tested biologically active vanilloids, but not the inactive structure analogs, compete for the [³H]resiniferatoxin binding sites in rat dorsal root ganglion membranes, and we conclude that the [³H]resiniferatoxin binding assay may provide an efficient approach for evaluating such compounds. We also provide evidence that the [3H]resiniferatoxin receptor is likely to recognize vanilloids which are inserted into the membranes; and that the apparent activity of capsaicinoids may be significantly influenced by factors other than equilibrium binding affinities. Send offprint requests to P. M. Blumberg at the above address     

Integrated metabolomic analysis of the nano-sized copper particle-induced hepatotoxicity and nephrotoxicity in rats: a rapid in vivo screening method for nanotoxicity

Despite an increasing application of copper nanoparticles, there is a serious lack of information concerning their impact on human health and the environment. In this study, the biochemical compositions of urine, serum, and extracts of liver and kidney tissues of rats treated with nano-copper at the different doses (50, 100, and 200 mg/kg/d for 5 d) were investigated using ¹H NMR techniques with the pattern recognition methods. Serum biochemical analysis and histopathological examinations of the liver and kidney of all the rats were simultaneously performed. All the results indicated that the effects produced by nano-copper at a dose of 100 or 50 mg/kg/d were less than those induced at a higher dose of 200 mg/kg/d. Nano-copper induced overt hepatotoxicity and nephrotoxicity at 200 mg/kg/d for 5 d, which mainly involved scattered dot hepatocytic necrosis and widespread renal proximal tubule necrosis. Increased citrate, succinate, trimethylamine-N-oxide, glucose, and amino acids, accompanied by decreased creatinine levels were observed in the urine; furthermore, elevated levels of lactate, 3-hydroxybutyrate, acetate, creatine, triglycerides, and phosphatide and reduced glucose levels were observed in the serum. The predominant changes identified in the liver tissue aqueous extracts included increased lactate and creatine levels together with reduced glutamine and taurine levels, and the metabolic profile of the kidney tissue aqueous extracts showed an increase in lactate and a drop in glucose. In the chloroform/methanol extracts of the liver and kidney tissues, elevated triglyceride species were identified. These changes suggested that mitochondrial failure, enhanced ketogenesis, fatty acid β-oxidation, and glycolysis contributed to the hepatotoxicity and nephrotoxicity induced by nano-copper at 200 mg/kg/d for 5 d. An increase in triglycerides in the serum, liver and kidney tissues could serve as a potential sensitive biomarker reflecting the lipidosis induced by nano-copper. The data generated from the current study completely supports the fact that an integrated metabolomic approach is promising for the development of a rapid invivo screening method for nanotoxicity.