Metabolomics study on the toxicity of aconite root and its processed products using ultraperformance liquid-chromatography/electrospray-ionization synapt high-definition mass spectrometry coupled with pattern recognition approach and ingenuity pathways analysis

The mother and lateral root of Aconitum carmichaelii Debx, named "Chuanwu" (CW) and "Fuzi", respectively, has been used to relieve joint pain and treat rheumatic diseases for over 2000 years. However, it has a very narrow therapeutic range, and the toxicological risk of its usage remains very high. The traditional Chinese processing approach, Paozhi (detoxifying measure),can decompose poisonous Aconitum alkaloids into less or nontoxic derivatives and plays an important role in detoxification. The difference in metabolomic characters among the crude and processed preparations is still unclear, limited by the lack of sensitive and reliable biomarkers. Therefore, this paper was designed to investigate comprehensive metabolomic characters of the crude and its processed products by UPLC-Q-TOF-HDMS combined with pattern recognition methods and ingenuity pathway analysis (IPA). The significant difference in metabolic profiles and changes of metabolite biomarkers of interest between the crude and processed preparations were well observed. The underlying regulations of Paozhi-perturbed metabolic pathways are discussed according to the identified metabolites, and four metabolic pathways are identified using IPA. The present study demonstrates that metabolomic analysis could greatly facilitate and provide useful information to further comprehensively understand the pharmacological activity and potential toxicity of processed Aconite roots in the clinic.     

Therapeutic approaches of melatonin in microwave radiations-induced oxidative stress-mediated toxicity on male fertility pattern of Wistar rats

Microwave (MW) radiation produced by wireless telecommunications and a number of electrical devices used in household or in healthcare institutions may adversely affects the reproductive pattern. Present study aimed to investigate the protective effects of melatonin (is well known antioxidant that protects DNA, lipids and proteins from free radical damage) against oxidative stress-mediated testicular impairment due to long-term exposure of MWs. For this, 70-day-old male Wistar rats were divided into four groups (n?=?6/group): Sham exposed, Melatonin (Mel) treated (2?mg/kg), 2.45?GHz MWs exposed and MWs?+?Mel treated. Exposure took place in Plexiglas cages for 2?h a day for 45 days where, power density (0.21?mW/cm²) and specific absorption rate (SAR 0.14?W/Kg) were estimated. After the completion of exposure period, rats were sacrificed and various stress related parameters, that is LDH-X (lactate dehydrogenase isoenzyme) activity, xanthine oxidase (XO), ROS (reactive oxygen species), protein carbonyl content, DNA damage and MDA (malondialdehyde) were performed. Result shows that melatonin prevent oxidative damage biochemically by significant increase (p?0.001) in the levels of testicular LDH-X, decreased (p?0.001) levels of MDA and ROS in testis (p?0.01). Meanwhile, it reversed the effects of MWs on XO, protein carbonyl content, sperm count, testosterone level and DNA fragmentation in testicular cells. These results concluded that the melatonin has strong antioxidative potential against MW induced oxidative stress mediated DNA damage in testicular cells.     

Maternal and developmental toxicity of ayahuasca in Wistar rats

INTRODUCTION: Ayahuasca is a psychotropic plant beverage initially used by shamans throughout the Amazon region during traditional religious cult. In recent years, ayahuasca has also been used in ceremonies of a number of modern syncretic religious groups, including pregnant women. However, no documented study has been performed to evaluate the risk of developmental toxicity of ayahuasca. METHODS: In the present work, maternal and developmental toxicity was evaluated in Wistar rats. Ayahuasca was administered to pregnant rats in three different doses [the equivalent typical dose (TD) administered to humans, five‐fold TD and 10‐fold TD] during the gestational period (6–20 days). RESULTS: Dams treated with the highest ayahuasca dose showed maternal toxicity with decrease of weight gain and food intake. Visceral fetal findings were observed in all treatment groups. Skeletal findings were observed in the intermediate‐ and high‐dose groups. The fetuses deriving from the highest dose group also presented a decrease in body weight. CONCLUSIONS: From these results, it is possible to conclude that there is a risk of maternal and developmental toxicity following ayahuasca exposure and that the level of toxicity appears to be dose‐dependent. Birth Defects Res (Part B) 89:207–212, 2010. © 2010 Wiley‐Liss, Inc.     

Metabolomics study of type 2 diabetes using ultra-performance LC-ESI/quadrupole-TOF high-definition MS coupled with pattern recognition methods

Type 2 diabetes (T2D), called the burden of the twenty-first century, is growing with an epidemic rate. Here, we explored the differences in metabolite concentrations between T2D patients and healthy volunteers. Metabolomics represents an emerging discipline concerned with comprehensive analysis of small molecule metabolites and provides a powerful approach to discover biomarkers in biological systems. The acquired data were analyzed by ultra-performance liquid chromatography–electrospray ionization/quadrupole time-of-flight high-definition mass spectrometry coupled with pattern recognition approach [principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA)] to identify potential disease-specific biomarkers. PCA showed satisfactory clustering between patients and healthy volunteers. Biomarkers reflected the biochemical events associated with early stages of T2D which were observed in PLS-DA loading plots. These urinary differential metabolites, such as adiponectin, acylcarnitines, citric acid, kynurenic acid, 3-indoxyl sulfate, urate, and glucose, were identified involving several key metabolic pathways such as taurine and hypotaurine metabolism; cysteine and methionine metabolism; valine, leucine, and isoleucine biosynthesis metabolism, etc. Our data suggest that robust metabolomics has the potential as a noninvasive strategy to evaluate the early diagnosis of T2D patients and provides new insight into pathophysiologic mechanisms and may enhance the understanding of its cause of disease.     

Effects of folic acid on testicular toxicity induced by bisphenol-A in male Wistar rats

We investigated the protective effect of the folic acid (FA) against bisphenol-A (BPA) induced toxicity in rat testis. We used four groups of seven adult male Wistar albino rats. The control group was fed corn oil, the BPA group was given BPA, the FA group was given FA and the FA + BPA group was given FA initially followed by BPA 1 h later. The BPA, FA and corn oil were administered by oral gavage for 14 days. At the end of the experiment, testis sections were examined for histological and histomorphometric characteristics. The TUNEL method was used to detect apoptosis and immunohistochemistry was used to examine the distribution of spermatogonial stem cells. Levels of serum testosterone were measured, and sperm viability and morphology were determined. The histological structure of the testis was normal in the control and FA groups. Although the number of TUNEL positive cells/tubule increased, the seminiferous epithelium height (SEH) at stages VII?VIII decreased in the BPA group compared to the control, FA and FA + BPA groups. The number of TUNEL positive cells/tubule decreased and the SEH at stages VII?VIII increased in the FA + BPA group compared to the BPA group. No significant difference in spermatogonial stem cells was found among groups. The level of serum testosterone and percentage of viable sperm was significantly lower, while the head, midpiece and total sperm abnormalities were significantly higher in the BPA treated group compared to control, FA, FA + BPA groups. It appears that the toxic effects of BPA on testis might be minimized by FA treatment.     

Hesperidin ameliorates heavy metal induced toxicity mediated by oxidative stress in brain of Wistar rats

Cadmium (Cd) induces neurotoxicity owing to its highly deleterious capacity to cross the blood brain barrier (BBB). Recent studies have provided insights on antioxidant properties of bioflavonoids which have emerged as potential therapeutic and nutraceutical agents. The aim of our study was to examine the hypothesis that hesperidin (HP) ameliorates oxidative stress and may have mitigatory effects in the extent of heavy metal-induced neurotoxicity. Cd (3 mg/kg body weight) was administered subcutaneously for 21 days while HP (40 mg/kg body weight) was administered orally once every day. The results of the current investigation demonstrate significant elevated levels of oxidative stress markers such as lipid peroxidation (LPO) and protein carbonyl (PC) along with significant depletion in the activity of non-enzymatic antioxidants like glutathione (GSH) and non-protein thiol (NP-SH) and enzymatic antioxidants in the Cd treated rats’ brain. Activity of neurotoxicity biomarkers such as acetylcholinesterase (AchE), monoamine oxidase (MAO) and total ATPase were also altered significantly and HP treatment significantly attenuated the altered levels of oxidative stress and neurotoxicity biomarkers while salvaging the antioxidant sentinels of cells to near normal levels thus exhibiting potent antioxidant and neuroprotective effects on the brain tissue against oxidative damage in Cd treated rodent model.     

Quantitative analysis of heterocyclic amines in urine by liquid chromatography coupled with tandem mass spectrometry

A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid–liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.     

Forskolin ameliorates mancozeb‐induced testicular and epididymal toxicity in Wistar rats by reducing oxidative toxicity and by stimulating steroidogenesis

In the present study, we have tested the beneficial effects of forskolin in protecting the mancozeb‐induced reproductive toxicity in rats. Adult male Wistar rats were exposed to either mancozeb (500 mg/kg body weight/day) or forskolin (5 mg/kg body weight/day) or both for 65 days and analyzed for spermatogenesis and steroidogenesis and testicular and epididymal oxidative toxicity. A significant decrease in daily sperm production, epididymal sperm count, motile, viable, and hypo‐osmotic swelling‐tail swelled sperm was observed in mancozeb‐treated rats. The activity levels of testicular 3β‐hydroxysteroid dehydrogenase and 17β‐hydroxysteroid dehydrogenase and circulatory testosterone levels were significantly decreased in mancozeb‐treated rats. Exposure to mancozeb resulted in a significant decrease in glutathione levels and superoxide dismutase and catalase activity levels with an increase in lipid peroxidation levels in the testes and epididymis. Coadministration of forskolin mitigated the mancozeb‐induced oxidative toxicity and suppressed steroidogenesis and spermatogenesis.     

Proteomics analysis of cerebral cortex in Wistar rats

To analyze the protein expression pattern of the cerebral cortex in Wistar rats using the proteomics approach, proteins were separated by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue and digested with trypsin. Then, we analyzed the peptide section using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and identified the protein by indexing special database (SwissProt) according to the finger printing of the peptide quality. Eighty-four protein spots were identified, including metabolic enzymes, skeleton proteins, heat shock proteins, antioxidant proteins, signaling proteins, proteasome related proteins, neuron and glial specific proteins and serum associated proteins. The result of this study enriches the database of the proteome in the cerebral cortex of rats and lays a foundation for further research of neurological disorders in rat models. __________ Translated from Acta Biophysica Sinica, 2007, 23 (1): 151–156 [译自: 生物物理学报]     

Proteomics analysis of cerebral cortex in Wistar rats

To analyze the protein expression pattern of the cerebral cortex in Wistar rats using the proteomics approach, proteins were separated by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue and digested with trypsin. Then, we analyzed the peptide section using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and identified the protein by indexing special database (SwissProt) according to the finger printing of the peptide quality. Eighty-four protein spots were identified, includ-ing metabolic enzymes, skeleton proteins, heat shock pro-teins, antioxidant proteins, signaling proteins, proteasome related proteins, neuron and glial specific proteins and serum associated proteins. The result of this study enriches the database of the proteome in the cerebral cortex of rats and lays a foundation for further research of neurological disorders in rat models.     

Hepatoprotective effect of Aloe vera against cartap- and malathion-induced toxicity in Wistar rats

Exposure to mixture of pesticides in agricultural practices pose a serious threat to the nontarget animals. In present work, we have evaluated the synergistic effect of cartap and malathion on rat liver followed by impact of Aloe vera leaves aqueous extract, which is not known. The animals in eight groups were used; each containing six rats: Group 1 acted as a control, Group 2—control with A. vera leaves aqueous extract, Group 3—with cartap, Group 4—with malathion, Group 5—with mixture of cartap and malathion, Group 6—cartap with the pretreatment of A. vera leaf extract, Group 7—malathion with the pretreatment of A. vera leaf extract, Group 8—mixture of cartap and malathion with the pretreatment of A. vera leaf extract . The animals treated for 15 days were killed after 24hr of last treatment. The biochemical studies in the rat liver demonstrated significant perturbations in the levels of nonenzymatic (glutathione and malondialdehyde) and enzymatic (superoxide dismutase, catalase, and glutathione- S-transferase) antioxidative indices. The histopathological examination of liver revealed serious congestion in central vein and the disorganization of hepatic cords due to pesticide treatment. The administration of A. vera leaves aqueous extract was able to markedly protect rat liver from the pesticides-induced toxicity. The data indicated that pesticides were able to significantly induce oxidative stress which was substantially reduced by the application of plant extract .     

Protective effect of Abutilon indicum against lead-induced reproductive toxicity in male Wistar rats

Despite ample literature on the toxic impact of lead on the environment and health, the exact mechanism of pathogenesis/toxicity is not clearly known. Because it is well established that lead induces oxidative stress, it is assumed that exposure to antioxidants may reduce the toxic impact of lead. In this study, we evaluated the impact of coadministration of the methanolic root extract of a plant Abutilon indicum (50, 100, 200 mg kg ⁻¹b.wt.) in mitigating the toxic impact of lead on the reproductive system of rats. In brief, Wistar rats were exposed to lead acetate in drinking water with or without coadministration of plant root extract and compared with that of control animals. After 45 days of exposure as outlined above, the animals were killed and the reproductive toxicity was assessed by sperm parameters, hormone and antioxidant enzyme assays, and testis histopathology. Significant reduction in testis weight, sperm count, testosterone levels, and antioxidant enzymes levels such as Superoxide Dismutase, Catalase, and Glutathione peroxidase was seen in lead-treated animals, confirming the toxic impact. The coadministration of A. Indicum (100 and 200 mg kg ⁻¹b.wt.) was found to bring the studied parameters close to the levels seen in untreated (control) animals. Our findings are indicative of the protective nature of A. Indicum against lead-induced reproductive toxicity in a dose-dependent manner. However, further characterization of the root extract is required to elucidate the probable mechanism of protection.     

Effect of short- and medium-term toxicity of doxorubicin on spermatogenesis in adult Wistar rats

Doxorubicin (DXR) is a widely used chemotherapeutic anticancer agent that has potent activity against several solid and non-solid human malignant tumors, including childhood malignancies. However, DXR has serious toxic effects on tissues with rapid cell cycles, such as myeloid and lymphatic tissues, intestinal mucosa, testes and ovaries. In the present study, the short- and medium-term toxic effects of DXR on the reproductive system of male Wistar rats were evaluated using morphometric and stereological tools to quantify damage to the seminiferous epithelium. Adult male Wistar rats were treated with dose of 7.5?mg/kg of DXR and were sacrificed at seven, 14, 21 and 28?days after treatment. The testes were fixed in glutaraldehyde solution, routinely processed and embedded in plastic for evaluation under a light microscope. A significant reduction in testis weight was found as a result of massive germ cell apoptosis. Differences in comparison to the control group were found in the relative frequency of all stages of the seminiferous epithelium cycle, with significant differences for stages VIII–XI. Apoptosis significantly decreased the number of pachytene spermatocytes in the stages evaluated (I, II–III and VIII) at seven and 14?days. At 21 and 28?days after treatment, the testes exhibited the massive loss of germ cells that resulted in a missing cell layer. Moreover, reductions in the height of seminiferous tubules, tubular diameter and tubular compartment as well as an increase in the intertubular compartment were found in the period studied.     

Systematic variations associated with renal disease uncovered by parallel metabolomics of urine and serum

Background

Membranous nephropathy is an important glomerular disease characterized by podocyte injury and proteinuria, but no metabolomics research was reported as yet. Here, we performed a parallel metabolomics study, based on human urine and serum, to comprehensively profile systematic metabolic variations, identify differential metabolites, and understand the pathogenic mechanism of membranous nephropathy.

Results

There were obvious metabolic distinctions between the membranous nephropathy patients with urine protein lower than 3.5 g/24 h (LUPM) and those higher than 3.5 g/24 h (HUPM) by Partial Least Squares Discriminant Analysis (PLS-DA) model analysis. In total, 26 urine metabolites and 9 serum metabolites were identified to account for such differences, and the majority of metabolites were significantly increased in HUPM patients for both urines and serums. Combining the results of urine with serum, all differential metabolites were classified to 5 classes. This classification helps globally probe the systematic metabolic alterations before and after blood flowing through kidney. Citric acid and 4 amino acids were markedly increased only in the serum samples of HUPM patients, implying more impaired filtration function of kidneys of HUPM patients than LUPM patients. The dicarboxylic acids, phenolic acids, and cholesterol were significantly elevated only in urines of HUPM patients, suggesting more severe oxidative attacks than LUPM patients.

Conclusions

Parallel metabolomics of urine and serum revealed the systematic metabolic variations associated with LUPM and HUPM patients, where HUPM patients suffered more severe injury of kidney function and oxidative stresses than LUPM patients. This research exhibited a promising application of parallel metabolomics in renal diseases.

     

Global metabolic profiling analysis on human urine by UPLC-TOFMS: issues and method validation in nutritional metabolomics

Optimisation and method validation was assessed here for metabolic profiling analysis of urine samples using UPLC-TOFMS. A longer run time of 31 min revealed greater reproducibility, and the higher number of variables was identified as compared to shortened run times (10 and 26 min). We have also implemented two QC urine samples enabling the assessment of the quality and reproducibility of the data generated during the whole analytical workflow (retention time drift, mass precision and fluctuation of the ion responses over time). Based on the QC data, suitable standards for ensuring consistent analytical results for metabolomics applications using the UPLC-MS techniques are recommended.     

Systems toxicology study of doxorubicin on rats using ultra performance liquid chromatography coupled with mass spectrometry based metabolomics

A metabolomics-based systems toxicology approach was used to profile the urinary metabolites for the toxicity related processes and pathogenesis induced by doxorubicin (DOX) to rats. Endogenous metabolite profiles were obtained with ultra performance liquid chromatography-mass spectrometry (UPLC-MS) for rats receiving different single dosages of DOX (5, 10 or 20 mg/kg) prior and at three time points after dosage. Principal components analysis (PCA) allowed detection of two major systemic metabolic changes with the time due to the induced toxicity. Furthermore, Analysis of variance (ANOVA) Simultaneous Component Analysis (ASCA) was applied to reveal the variation caused by time and dose, and their interaction in a multivariate way. Finally, various metabolites involved in the toxic processes could be identified using their accurate mass and MSⁿ experiments, and possible mechanisms of the toxicity of DOX were postulated. In conclusion, metabolomics as a systems toxicology approach was able to provide comprehensive information on the dynamic process of drug induced toxicity. In addition, detection of the systemic toxic effects could be obtained with metabolomics at an earlier stage compared to the clinical chemistry and histopathological assessment.     

Fractionation of vanadium in urine of Wistar rats as a function of time after intraperitoneal injection

[(48)V]Vanadium was intraperitoneally injected into Wistar rats. Urine and feces were collected at regular intervals (n=19) between 1 and 144 h after injection. In case of urine, maximal excretion (V activity/ml urine) of vanadium was seen 3 h after injection. In case of feces, a maximum appeared 32 h after injection. Urine samples were fractionated on two types of gel filtration column (Superose 12 HR 10/30 and Superdex Peptide 10/30). We found that vanadium in urine exists as both high (protein-bound) and low molecular mass species and that the partition about these forms depends on the time elapsed after injection. After 1 h, respectively, four (one high molecular and three low molecular mass species) and five (one high molecular and four low molecular mass species) vanadium peaks were present in the chromatograms of the Superose 12 and the Superdex Peptide columns. Then 3 h after injection, a different high molecular species showed up in the chromatograms, while the first high molecular and some low molecular mass species disappeared. Vanadium in urine after 8 h occurred as one high (slightly different from the high molecular complex after 3 h) and one low molecular mass complex. However, after 48 h the pattern changed again and vanadium in urine was excreted largely as one low molecular mass species, presumably one of the species that also occurred 1 h after injection but was not present in the period 6-24 h.     

Intravenous repeated-dose toxicity study of ZnPcS2P2-based-photodynamic therapy in Wistar rats.

The purpose of the current study was to investigate the potential repeated-dose toxicity of ZnPcP2S2-based photodynamic therapy (ZnPc-PDT) in Wistar rats. The animals were administered ZnPcS2P2 intravenously ten times successively every 4 d and irradiated with a 670 nm laser light for 6 min at subsequent 48 h and 72 h. At the end of the treatment period, 10 rats/sex/group were sacrificed, while 5 rats/sex/group were sacrificed after a two-week recovery period. During the test period, clinical signs, mortality, body weights, food and water consumption, ophthalmoscopy, hematology, serum biochemistry, urinalysis, organ weights, gross findings and histopathology were examined. The association between the increased liver weight and hepatic spotty and lytic necrosis seen in high dose females corroborates the conclusion that high dose ZnPc-PDT could induce hepatic injury in Wistar rats and they are probably related to the abnormality of certain biochemical parameters of females in the high dose group. Furthermore, microscopic examination for the ZnPc-PDT groups shows the presence of some Kelly and khaki granules in Kupffer cells and endothelia of the livers, epithelia of the renal tubules, marginal sinus and medulla of the spleens, alveolar walls of the lungs, reticular cells and macrophages of the mesenteric lymph nodes, testicular Leydig cells, epididymal epithelial cells, endometrial stromal cells, and interstitial cells and corpora lutea of the ovaries from all or most of the animals. There were no adverse effects on mortality, clinical signs, food and water consumption, ophthalmoscopy, uranalysis, hematology, serum biochemistry, body weights and necropsy findings in control, low and mid dose groups. Based on these results, it was concluded that the intravenous repeated-dose of ZnPcP2S2-PDT induced the abnormalities of liver weights, hepatic biochemistry and histopathology, and pigmentation in the several important organs in Wistar rats at 4 mg kg(-1) d(-1). The target organ was determined to be liver (and spleen perhaps), but this was not so obvious in males. The no-observed-adverse-effect level (NOAEL) was considered to be 1.0 mg kg(-1) for both sexes.     

Specific antibody levels and antigenic recognition of Wistar rats inoculated with distinct isolates of Trypanosoma evansi.

"Mal de Cadeiras", an enzootic disease caused by Trypanosoma evansi, is one of the most important trypanosomiases in the Brazilian Pantanal region. The disease affects mainly horses, which are widely used in extensive cattle production, an activity of greatest economical significance for the region. The parasite also infects sylvan (coatis and capybaras) and domestic (dogs) animals, respectively considered wild and domestic reservoirs of T. evansi. For a better understanding of the interaction of T. evansi with its rodent host, we evaluated the differences in the specific antibody level patterns and in the parasitic peptides recognition patterns of experimentally infected Wistar rats. The rats experimentally infected with T. evansi isolates obtained from coatis, dogs and horses were submitted to indirect immunofluorescence test (IgM e IgG) and Western blotting. The serological titers for IgM and IgG ranged between 1:40 and 1:160. The most recognized polypeptide profiles were in a range of 17 and 74 kDa. Our data suggest that the humoral immune response in Wistar rats is not sufficient for granting an effective control of T. evansi infections.