ABSTRACT
The majority of metabolomic studies used in ecotoxicology have implemented (1)H NMR analysis. Despite constant improvement, major limitations of NMR-based techniques include relatively low sensitivity that results in an examination of a limited number of metabolites. An alternative approach is the use of liquid or gas chromatography (GC) for separation of metabolites and mass spectrometry (MS) for their quantification and identification. The objective of our study was to develop a two dimensional GC coupled with time of flight MS (GCxGC/TOF-MS) coupled with multivariate analysis to compare metabolite profiles of Diporeia under different environmental conditions. We compared metabolite profiles between Diporeia collected from Lake Michigan (declining populations) to those residing in Lake Superior (stable populations), and also between Diporeia exposed to a chemical stressor (atrazine) and controls. Overall, 76 and 302 total metabolites were detected from the lake comparison and atrazine studies, respectively. Many of the identified metabolites included fatty acids, amino acids, and hydrocarbons. Furthermore, we observed unique and almost non-overlapping metabolite profiles in both studies. In conclusion, we established the feasibility of using GCxGC/TOF-MS for detecting metabolites as well as developed software to align and merge chromatographic peaks to compare metabolite differences between invertebrate groups sampled under different environmental conditions. This ability to detect unique metabolite profiles under different environmental conditions will increase our undertsanding on the physiological processes and whole-organism reponses occuring as a result of exposure to different environmental stressors.
Subject(s)
Atrazine/toxicity , Ecotoxicology/methods , Herbicides/toxicity , Invertebrates/drug effects , Invertebrates/metabolism , Water Pollutants, Chemical/toxicity , Amphipoda/drug effects , Amphipoda/metabolism , Animals , Chromatography, Gas , Fresh Water , Mass Spectrometry , Metabolism/drug effects , Principal Component AnalysisABSTRACT
Catechins were subjected to in vitro gastric and small intestinal digestion. EGCG, EGC, and ECG were significantly degraded at all concentrations tested, with losses of 71-91, 72-100, and 60-61%, respectively. EC and C were comparatively stable, with losses of 8-11 and 7-8%, respectively. HLPC-ESI-MS/MS indicated that EGCG degradation under simulated digestion resulted in production of theasinensins (THSNs) A and D (m/z 913) and P-2 (m/z 883), its autoxidation homodimers. EGC dimerization produced the homodimers THSN C and E (m/z 609) and homodimers analogous to P-2 (m/z 579). ECG homodimers were not observed. EGCG and EGC formed heterodimers analogous to the THSNs (m/z 761) and P-2 (m/z 731). EGCG and ECG formed homodimers analogous to the THSNs (m/z 897). This study provides an expanded profile of catechin dimers of digestive origin that may potentially form following consumption of catechins. These data provide a logical basis for initial screening to detect catechin digestive products in vivo.
Subject(s)
Catechin/metabolism , Digestion , Catechin/analogs & derivatives , Dimerization , In Vitro Techniques , Lipase/metabolism , Oxidation-Reduction , Pancreatin/metabolism , Pepsin A/metabolismABSTRACT
Epidemiological studies suggest that the intake of natural/nutrient products is inversely related to cancer risk. While oxidative stress, generating reactive oxygen species, has been linked to cancer initiation and progression, dietary antioxidants have reduced the risk of certain cancers. Experimental studies have demonstrated that antioxidants and phytochemicals could prevent cancer metastasis, and antioxidants were suggested as adjuvants in cancer therapy. Ganoderma lucidum is an Asian medicinal mushroom that has been used for the past two thousand years for the treatment of various diseases, including cancer. G. lucidum is currently popular as a dietary supplement in the form of tea, powder or extract. We have previously demonstrated that G. lucidum suppresses growth, angiogenesis and invasiveness of highly invasive and metastatic breast cancer cells. The present study was undertaken to evaluate the effect of G. lucidum on oxidative stress-induced metastatic behavior of poorly-invasive MCF-7 breast cancer cells. We show that G. lucidum inhibits oxidative stress-induced migration of MCF-7 cells by the down-regulation of MAPK signaling. G. lucidum suppressed oxidative stress stimulated phosphorylation of extracellular signal-regulated protein kinases (Erk1/2), which resulted in the down-regulation of expression of c-fos, and in the inhibition of transcription factors AP-1 and NF-kappaB. The biological effect of G. lucidum on cell migration was mediated by the suppression of secretion of interleukin-8 from MCF-7 cells exposed to oxidative stress. In summary, our results suggest that G. lucidum inhibits the oxidative stress-induced invasive behavior of breast cancer cells by modulating Erk1/2 signaling and can be potentially considered as an antioxidant in adjuvant cancer therapy.
Subject(s)
Antioxidants/pharmacology , Interleukin-8/metabolism , Oxidative Stress/physiology , Reishi/chemistry , Antioxidants/isolation & purification , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/physiopathology , Cell Line, Tumor , Cell Movement/drug effects , Chromatography, Liquid/methods , Enzyme Activation/drug effects , Heptanoic Acids/analysis , Heptanoic Acids/pharmacology , Humans , Hydrogen Peroxide/pharmacology , Lanosterol/analogs & derivatives , Lanosterol/analysis , Lanosterol/pharmacology , Lipid Peroxidation/drug effects , Luciferases/genetics , Luciferases/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Polysaccharides/analysis , Polysaccharides/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , TransfectionABSTRACT
Progress is being made in understanding the biochemical and molecular basis of nickel (Ni)/zinc (Zn) hyperaccumulation in Thlaspi; however, the molecular signaling pathways that control these mechanisms are not understood. We observed that elevated concentrations of salicylic acid (SA), a molecule known to be involved in signaling induced pathogen defense responses in plants, is a strong predictor of Ni hyperaccumulation in the six diverse Thlaspi species investigated, including the hyperaccumulators Thlaspi goesingense, Thlaspi rosulare, Thlaspi oxyceras, and Thlaspi caerulescens and the nonaccumulators Thlaspi arvense and Thlaspi perfoliatum. Furthermore, the SA metabolites phenylalanine, cinnamic acid, salicyloyl-glucose, and catechol are also elevated in the hyperaccumulator T. goesingense when compared to the nonaccumulators Arabidopsis (Arabidopsis thaliana) and T. arvense. Elevation of free SA levels in Arabidopsis, both genetically and by exogenous feeding, enhances the specific activity of serine acetyltransferase, leading to elevated glutathione and increased Ni resistance. Such SA-mediated Ni resistance in Arabidopsis phenocopies the glutathione-based Ni tolerance previously observed in Thlaspi, suggesting a biochemical linkage between SA and Ni tolerance in this genus. Intriguingly, the hyperaccumulator T. goesingense also shows enhanced sensitivity to the pathogen powdery mildew (Erysiphe cruciferarum) and fails to induce SA biosynthesis after infection. Nickel hyperaccumulation reverses this pathogen hypersensitivity, suggesting that the interaction between pathogen resistance and Ni tolerance and hyperaccumulation may have played a critical role in the evolution of metal hyperaccumulation in the Thlaspi genus.
