NMR-based metabonomic in hippocampus, nucleus accumbens and prefrontal cortex of methamphetamine-sensitized rats.

(1)H NMR spectroscopy was applied to investigate the changes of cerebral metabolites in brain hippocampus, nucleus accumbens (NAC) and prefrontal cortex (PFC) of the rats subjected to subcutaneous twice-daily injections of 2.5mg/kg methamphetamine (MAP) for 7 days. The results indicated that MAP exposure induced significant behavioral sensitization and altered cerebral metabolites in rats. The neurotransmitters glutamate, glutamine and GABA significantly decreased in hippocampus, NAC and PFC. Specifically, increased succinic acid semialdehyde, a metabolism product of GABA, was observed in hippocampus. Additionally, decreased serotonin was observed in both NAC and PFC, whereas decreased dopamine was only observed in NAC after repeated MAP treatment. Glutathione obviously decreased in above brain regions, whereas acetylcysteine declined in hippocampus and NAC, and taurine declined in NAC and PFC. Homocysteic acid was elevated in hippocampus and NAC by repeated MAP administration. Membrane ingredients like phosphocholine elevated in response to MAP administration in NAC and PFC. N-Acetyl-aspartate, a marker of neuronal viability, decreased in the three regions; however, myo-inositol, a glial cell marker, increased in hippocampus and PFC. Tricarboxylic acid cycle intermediate products, such as α-ketoglutarate, succinate, citrate and the methionine significantly decreased in above three brain regions after MAP administration; however, ADP decreased in hippocampus. These results indicate that repeated MAP treatment causes neurotransmitters disturbance, imbalance between oxidative stress and antioxidants, and gliosis in hippocampus, NAC and PFC. Profound metabolic changes detected across brain regions provide the first evidence of metabonomic changes in MAP-induced sensitized rats.

Transcriptome analysis of long non-coding RNAs of the nucleus accumbens in cocaine-conditioned mice.

Cocaine dependence involves in the brain's reward circuit as well as nucleus accumbens (NAc), a key region of the mesolimbic dopamine pathway. Many studies have documented altered expression of genes and identified transcription factor networks and epigenetic processes that are fundamental to cocaine addiction. However, all these investigations have focused on mRNA of encoding genes, which may not always reflect the involvement of long non-coding RNAs (lncRNAs), which has been implied in a broad range of biological processes and complex diseases including brain development and neuropathological process. To explore the potential involvement of lncRNAs in drug addiction, which is viewed as a form of aberrant neuroplasticity, we used a custom-designed microarray to examine the expression profiles of mRNAs and lncRNAs in brain NAc of cocaine-conditioned mice and identified 764 mRNAs, and 603 lncRNAs were differentially expressed. Candidate lncRNAs were identified for further genomic context characterization as sense-overlap, antisense-overlap, intergenic, bidirection, and ultra-conserved region encoding lncRNAs. We found that 410 candidate lncRNAs which have been reported to act in cis or trans to their targeted loci, providing 48 pair mRNA-lncRNAs. These results suggest that the modification of mRNAs expression by cocaine may be associated with the actions of lncRNAs. Taken together, our results show that cocaine can cause the genome-wide alterations of lncRNAs expressed in NAc, and some of these modified RNA transcripts may to play a role in cocaine-induced neural plasticity and addiction.

(1) H-nuclear magnetic resonance-based metabonomic analysis of brain in rhesus monkeys with morphine treatment and withdrawal intervention.

Comprehensive cerebral metabolites involved in morphine dependence have not been well explored. To gain a better understanding of morphine dependence and withdrawal therapy in a model highly related to humans, metabolic changes in brain hippocampus and prefrontal cortex (PFC) of rhesus monkeys were measured by (1) H-nuclear magnetic resonance spectroscopy, coupled with partial least squares and orthogonal signal correction analysis. The results showed that concentrations of myoinositol (M-Ins) and taurine were significantly reduced, whereas lactic acid was increased in hippocampus and PFC of morphine-dependent monkeys. Phosphocholine and creatine increased in PFC but decreased in hippocampus after chronic treatment of morphine. Moreover, N-acetyl aspartate (NAA), γ-aminobutyric acid, glutamate, glutathione, methionine, and homocysteic acid also changed in these brain regions. These results suggest that chronic morphine exposure causes profound disturbances of neurotransmitters, membrane, and energy metabolism in the brain. Notably, morphine-induced dysregulations in NAA, creatine, lactic acid, taurine, M-Ins, and phosphocholine were clearly reversed after intervention with methadone or clonidine. Our study highlights the potential of metabolic profiling to enhance our understanding of metabolite alteration and neurobiological actions associated with morphine addiction and withdrawal therapy in primates.

Zinc oxide nanoparticles cause nephrotoxicity and kidney metabolism alterations in rats.

Although zinc oxide nanoparticles (ZnO NPs) have been widely used, their potential hazards on mammalian and human remain largely unknown. In this study, the biochemical compositions of urine and kidney from the rats treated with ZnO NPs (100, 300 and 1000 mg/kg, respectively) were investigated using (1)H nuclear magnetic resonance (NMR) technique with the pattern recognition of partial least squares-discriminant analysis. Hematology, clinical biochemistry and kidney histopathological examinations were also performed. Metabolic profiles from rats treated with ZnO NP(S) exhibited increases in the levels of taurine, lactate, acetate, creatine, phosphocholine, trimethylamine-N-oxide, α-glucose, and 3-D-hydroxybutyrate, as well as decreases in lipid, succinate, citrate, α-ketoglutarate, hippurate and 4-hydroxyphenylacetic acid in urine after ZnO NPs treatment for 14 days. A similar alteration pattern was also identified in kidney. Urine choline and phosphocholine increased significantly shortly after ZnO NPs treatment, moreover, some amino acids and glucose also increased during the experimental period. However, succinate, citrate and α-ketoglutarate in urine exhibited a different alteration trend, which showed increases on the first day after ZnO NPs treatment, but decreases gradually until the termination of the study. A similar alteration pattern of urinary (1)H NMR spectra was also detected in kidney. Moreover, ZnO NPs (1000 mg/kg) resulted in significant increases in serum creatine and blood urea nitrogen, decreases in hemoglobin, haematocrit and mean corpuscular hemoglobin concentration, and overt tubular epithelial cell necrosis. These findings show that ZnO NPs can disturb the energy metabolism and cause mitochondria and cell membrane impairment in rat kidney, which may contribute to ZnO NPs-induced nephrotoxicity.

¹H NMR-based metabonomic analysis of brain in rats of morphine dependence and withdrawal intervention.

Metabolic consequences of morphine dependence and withdrawal intervention have not been well explored. In the present study, the metabolic changes in brain hippocampus, nucleus accumbens (NAc), prefrontal cortex (PFC) and striatum of rats with morphine dependence and withdrawal intervention were explored by using ¹H nuclear magnetic resonance coupled with principal component analysis, partial least squares and orthogonal signal correction analysis. We found that the concentrations of neurotransmitters including glutamate, glutamine and gamma-aminobutyric acid changed differentially in hippocampus, NAc, PFC and striatum after repeated morphine treatment. Significant changes were also found in a number of cerebral metabolites including N-acetyl aspartate (NAA), lactic acid, creatine, myo-inositol and taurine. These findings indicate the profound disturbances of energy metabolism, amino acid metabolism and neurotransmitters caused by chronic morphine treatment. Interestingly, morphine-induced changes in lactic acid, creatine and NAA were clearly reversed by intervention of methadone or clonidine. Our study provides a comprehensive understanding of the metabolic alteration associated with morphine addiction and withdrawal therapy, which may help to develop new pharmacotherapies.

Taurine protects methamphetamine-induced developmental angiogenesis defect through antioxidant mechanism.

Investigations have characterized addictive drug-induced developmental cardiovascular malformation in human, non-human primate and rodent. However, the underlying mechanism of malformation caused by drugs during pregnancy is still largely unknown, and preventive and therapeutic measures have been lacking. Using 1H NMR spectroscopy, we profiled the metabolites from human embryo endothelial cells exposed to methamphetamine (METH) and quantified a total of 226 peaks. We identified 11 metabolites modified robustly and found that taurine markedly increased. We then validated the hypothesis that this dramatic increase in taurine could attribute to its effect in inhibiting METH-induced developmental angiogenesis defect. Taurine supplement showed a more significant potential than other metabolites in protecting against METH-induced injury in endothelial cells. Taurine strongly attenuated METH-induced inhibition of proliferation and migration in endothelial cells. Furthermore, death rate and vessel abnormality of zebrafish embryos treated with METH were greatly reversed by taurine. In addition, taurine supplement caused a rapid decrease in reactive oxygen species generation and strongly attenuated the excitable arise of antioxidase activities in the beginning of METH exposure prophase. Dysregulations of NF-κB, p-ERK as well as Bax, which reflect apoptosis, cell cycle arrest and oxidative stress in vascular endothelium, were blocked by taurine. Our results provide the first evidence that taurine prevents METH-caused developmental angiogenesis defect through antioxidant mechanism. Taurine could serve as a potential therapeutic or preventive intervention of developmental vascular malformation for the pregnant women with drug use.

Metabolomics: a revolution for novel cancer marker identification.

The repertoire of small-molecular-weight substances present in cells, tissue and body fluids are known as the metabolites. The global analysis of metabolites, such as by high-resolution ¹H nuclear magnetic resonance spectroscopy and mass spectrometry, is integral to the rapidly expanding field of metabolomics, which is making progress in various diseases. In the area of cancer and metabolic phenotype, the integrated analysis of metabolites may provide a powerful platform for detecting changes related to cancer diagnosis and discovering novel biomarkers. In this review, metabolomics including the technologies in metabolomics research and extracting information from metabolomics datasets are described. Then we discuss the challenges and opportunities in metabolomics for finding metabolic processes in cancer and discovering novel cancer biomarkers. Finally, we assess the clinical applicability of metabolomics.

Proteomic analysis of the nucleus accumbens in rhesus monkeys of morphine dependence and withdrawal intervention.

It has been known that the reinforcing effects and long-term consequences of morphine are closely associated with nucleus accumbens (NAc) in the brain, a key region of the mesolimbic dopamine pathway. However, the proteins involved in neuroadaptive processes and withdrawal symptom in primates of morphine dependence have not been well explored. In the present study, we performed proteomes in the NAc of rhesus monkeys of morphine dependence and withdrawal intervention with clonidine or methadone. Two-dimensional electrophoresis was used to compare changes in cytosolic protein abundance in the NAc. We found a total of 46 proteins differentially expressed, which were further identified by mass spectrometry analysis. The identified proteins can be classified into 6 classes: metabolism and mitochondrial function, synaptic transmission, cytoskeletal proteins, oxidative stress, signal transduction and protein synthesis and degradation. Importantly, we discovered 14 proteins were significantly but similarly altered after withdrawal therapy with clonidine or methadone, revealing potential pharmacological strategies or targets for the treatment of morphine addiction. Our study provides a comprehensive understanding of the neuropathophysiology associated with morphine addiction and withdrawal therapy in primate, which is helpful for the development of opiate withdrawal pharmacotherapies.

Protective role of taurine against morphine-induced neurotoxicity in C6 cells via inhibition of oxidative stress.

This study was carried out to investigate the protective role of taurine (2-aminoethanesulphonicacid) against morphine-induced neurotoxicity in C6 cells. It was found that taurine significantly increased the viability of C6 cells treated by morphine, showing the neuroprotective role against morphine-induced neurotoxicity. However, such neuroprotective effect of taurine could not be blocked by bicuculline, an antagonist of gamma-amino butyrate (GABA) receptor. To determine the oxidative damage induced by morphine, the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured in C6 cells. The decreased activities of SOD, CAT, and GPx in C6 cells were observed after morphine treatment for 48 h. However, taurine administration effectively ameliorated morphine-induced oxidative insult. To estimate anti-apoptosis effect of taurine, flow cytometry analysis as well as detection for caspase-3 and Bcl-2 expressions was performed after morphine exposure for 48 h. It was found that Bcl-2 expression was down regulated by morphine, whereas taurine could reverse morphine-induced decrease in Bcl-2 expression. Taurine showed no effect on caspase-3 expression. Collectively, the results show that taurine possesses the capability to ameliorate morphine-induced oxidative insult and apoptosis in C6 cells, probably due to its antioxidant activity rather than activation of GABA receptors.

Investigation of toxicological effects of Shuanghuanglian injection in Beagle dogs by metabonomic and traditional approaches.

In this study, clinical biochemistry, hematology, histopathology and (1)H nuclear magnetic resonance spectroscopy-based metabonomic approaches were applied to investigate the toxicological effects of Shuanghuanglian (SHL) injection after intravenous administration (dosed at 4, 12 and 36 mL stock/kg) in Beagle dogs for 30 d. Decreases in red blood cells, hemoglobin, mean cell volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were observed in the high-dose group. Elevated reticulocytes, total bilirubin and direct bilirubin were also observed in this group. Moreover, significant hemosiderosis and Prussian blue positivity were detected in the liver, spleen and kidney from high-dose group animals, and transmission electron microscopy examination revealed an appreciable number of acanthrocytes in the liver. These results collectively indicate that SHL injection has the potential to cause hemolytic anemia. Metabonomic analysis showed increases in serum lactate, choline and phosphocholine but a decrease in taurine in treated groups and these findings may underlie the toxicological mechanism of SHL injection. In summary, SHL injection shows hemolytic effects in Beagle dogs; moreover, serum choline and phosphocholine as well as lactate and taurine may be the biomarkers for hemolytic anemia induced by SHL injection.

Mechanism study of Aconitum-induced neurotoxicity in PC12 cells: involvement of dopamine release and oxidative damage.

The Aconitum has been widely used as an important component in traditional Chinese medicine. However, it can cause neurotoxicity, and the mechanism has not been fully elucidated. The present study aimed to investigate the potential dopaminergic neurotoxicity of Aconitum and its mechanism. We found that Aconitum significantly evoked dopamine release from cultured PC12 cells and from the nucleus accubens of mice. These results show that Aconitum can promptly trigger dopamine release both in vitro and in vivo. Aconitum exposure induced reactive oxygen species formation with the decrease of superoxide dismutase and glutathione peroxidase. Moreover, PC12 cells proliferation was inhibited and apoptotic death was detected after Aconitum treatment, but this effect could be attenuated by antioxidants. These findings suggest that Aconitum can damage PC12 cells through oxidative stress mechanism. In conclusion, our results indicate that Aconitum can evoke dopamine release from dopaminergic neurons; excessive extracellular of dopamine can then create stresses on cellular antioxidant systems and induce neuron apoptosis.

NMR-based metabonomic study of the sub-acute toxicity of titanium dioxide nanoparticles in rats after oral administration.

As titanium dioxide nanoparticles (TiO(2) NPs) are widely used commercially, their potential toxicity on human health has attracted particular attention. In the present study, the oral toxicological effects of TiO(2) NPs (dosed at 0.16, 0.4 and 1 g kg( - 1), respectively) were investigated using conventional approaches and metabonomic analysis in Wistar rats. Serum chemistry, hematology and histopathology examinations were performed. The urine and serum were investigated by (1)H nuclear magnetic resonance (NMR) using principal components and partial least squares discriminant analysis. The metabolic signature of urinalysis in TiO(2) NP-treated rats showed increases in the levels of taurine, citrate, hippurate, histidine, trimethylamine-N-oxide (TMAO), citrulline, alpha-ketoglutarate, phenylacetylglycine (PAG) and acetate; moreover, decreases in the levels of lactate, betaine, methionine, threonine, pyruvate, 3-D-hydroxybutyrate (3-D-HB), choline and leucine were observed. The metabonomics analysis of serum showed increases in TMAO, choline, creatine, phosphocholine and 3-D-HB as well as decreases in glutamine, pyruvate, glutamate, acetoacetate, glutathione and methionine after TiO(2) NP treatment. Aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH) were elevated and mitochondrial swelling in heart tissue was observed in TiO(2) NP-treated rats. These findings indicate that disturbances in energy and amino acid metabolism and the gut microflora environment may be attributable to the slight injury to the liver and heart caused by TiO(2) NPs. Moreover, the NMR-based metabolomic approach is a reliable and sensitive method to study the biochemical effects of nanomaterials.

Investigation of the dermal sensitizing potential of traditional medical extracts in local lymph node assays.

Traditional medical extracts are commonly used as complex mixtures, which may contain naturally occurring contact sensitizers. In this investigation, the mice local lymph node assay (LLNA) was performed to evaluate the dermal sensitization potential of Myrrh, Borneolum, Olibanum, Moschus and Cassia Bark, which are widely used in topical traditional medication. In the radioactive LLNA, the stimulation index (SI) values were calculated for each medical extract. Myrrh, Borneolum, Olibanum and Moschus induced dose-dependent cell proliferation and SI was more than 3. Cassia Bark showed no positive response over the range of test concentrations. In the flow cytometry analysis, the total number of CD3(+), CD4(+), and CD8(+) cells in local lymph nodes was increased in Moschus-, Olibanum-, Myrrh- and Borneolum-treated mice. The ratio of the B220(+)/CD3(+) (B/T cell ratio) and the percentage of I-A(k+) cells that was also positive for the CD69 marker (I-A(k+)/ CD69(+)) were increased in the Moschus-, Olibanum- and Myrrh-treated mice. However, no ofbvious change was observed in Borneolum-treated mice. Cassia Bark did not induce changes in the lymphocyte subpopulations. These results indicate that Moschus, Olibanum and Myrrh can be regarded as sensitizers, and Borneolum regarded as an irritant. Cassia Bark is neither a sensitizer nor an irritant. The combination of radioactive and flow cytometric LLNA can be used for the prediction of sensitizing potential of medical extracts which lead to allergic contact dermatitis in humans.

[Toxicity of Caowu on in vitro cultured rat embryos].

OBJECTIVE: To study the developmental toxicity of Caowu and its mechanism on rat embryos. METHODS: Whole embryo culture was applied to test the effects of Caowu on rat embryos during the critical period of organogenesis. All embryos isolated on gestational day 9.5 were exposed to different concentrations of Caowu. The growth and differentiation of the embryos at the end of the 48 h culture period were observed. RESULTS: The embryonic growth and development were adversely affected by Caowu at the concentration of 2.5 mg/mL, which were represented as reduced yolk sac diameters, decreased number of somites, and lower morphological scores. With the increase of the concentrations of Caowu, more severe teratogenic effects appeared, including cardiac defect (undivided cardiac tube and inflated pericardial cavity), irregular somites, and brain malformation such as narrow brain vesicles. CONCLUSION: Caowu has direct embryotoxic effects on rats during the organogenesis period. We suggest that pregnant women in the first trimester use Caowu with cautions before a conclusion has been made.

Study of aconitine toxicity in rat embryos in vitro.

BACKGROUND: Aconitum is widely used in traditional medicine for its anti-inflammatory, analgesic, and cardiotonic properties. Knowledge is limited, however, on its effects on embryonic development. METHODS: Whole embryo culture was applied to explore the effects of aconitine on rat embryos during their critical period of organogenesis. All embryos isolated on gestational day 9.5 were exposed to 0, 1, 2.5, 5, and 10 microg/ml of aconitine with and without S9 mix, and scored for their growth and differentiation at the end of the 48-hr culture period. RESULTS: The embryonic growth and development were adversely affected at the concentration of 2.5 microg/ml aconitine without S9 mix, represented as reduced crown-rump length and head length, decreased number of somites, and lower morphologic score. When the concentration of aconitine was increased to 5 microg/ml, it induced severe dysmorphogenesis effects, including cardiac defect (undivided cardiac tube and inflated pericardial cavity), irregular somites, and brain malformation (e.g., narrow brain vesicles). In the presence of S9 mix, Aconitine toxicity to rat embryos was reduced to a certain extent. CONCLUSIONS: Our study showed that Aconitine had direct embryotoxic effects during the rat organogenetic period. NOAEL was about 1 microg/ml and metabolism in S9 mix could induce the attenuation of Aconitine toxicity. Until more is known about the effects of Aconitine in pregnant women, we suggest its use should be treated with caution.