I. Mullaney

Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM).

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

Quantitative assay of urinary hepcidin using MALDI-TOF mass spectrometry.

Hepcidin has been identified as the principle regulatory hormone essential for iron homeostasis. Quantitative analysis of hepcidin in bodily fluids provides an insight into the pathogenesis of disorders of iron metabolism such as hereditary hemochromatosis and anemia of chronic disease. This study describes the use of solid phase extraction (SPE) as a preparative step followed by matrix assisted laser desorption/ionization-orthogonal-time-of-flight mass spectrometry (MALDI-TOF MS) with internal standard for the quantitative analysis of unlabelled urinary hepcidin. More than 70% extraction recovery of hepcidin (hepcidin-25) with monoisotopic resolution was achieved. Urinary creatinine was analyzed using HPLC-UV/Vis with hepcidin-25 levels of 2.2 to 2.7 nmol/mmol of creatinine observed in healthy controls. Spot-to-spot variation of hepcidin standard additions was less than 3.5%. Intra- and inter-day precision assay of less than 9.5% relative standard deviation was achieved with less than 0.5% variation between the intra-day assay data. In summary, a validated non-invasive method has been developed for the quantification of unlabelled urinary hepcidin-25 which can be used to screen for iron-related disorders such as hemochromatosis in iron-overloaded patients.

Experimental design and reporting standards for metabolomics studies of mammalian cell lines

Metabolomics is an analytical technique that investigates the small biochemical molecules present within a biological sample isolated from a plant, animal, or cultured cells. It can be an extremely powerful tool in elucidating the specific metabolic changes within a biological system in response to an environmental challenge such as disease, infection, drugs, or toxins. A historically difficult step in the metabolomics pipeline is in data interpretation to a meaningful biological context, for such high-variability biological samples and in untargeted metabolomics studies that are hypothesis-generating by design. One way to achieve stronger biological context of metabolomic data is via the use of cultured cell models, particularly for mammalian biological systems. The benefits of in vitro metabolomics include a much greater control of external variables and no ethical concerns. The current concerns are with inconsistencies in experimental procedures and level of reporting standards between different studies. This review discusses some of these discrepancies between recent studies, such as metabolite extraction and data normalisation. The aim of this review is to highlight the importance of a standardised experimental approach to any cultured cell metabolomics study and suggests an example procedure fully inclusive of information that should be disclosed in regard to the cell type/s used and their culture conditions. Metabolomics of cultured cells has the potential to uncover previously unknown information about cell biology, functions and response mechanisms, and so the accurate biological interpretation of the data produced and its ability to be compared to other studies should be considered vitally important.

Untargeted metabolomics of neuronal cell culture: A model system for the toxicity testing of insecticide chemical exposure

Toxicity testing is essential for the protection of human health from exposure to toxic environmental chemicals. As traditional toxicity testing is carried out using animal models, mammalian cell culture models are becoming an increasingly attractive alternative to animal testing. Combining the use of mammalian cell culture models with screening‐style molecular profiling technologies, such as metabolomics, can uncover previously unknown biochemical bases of toxicity. We have used a mass spectrometry‐based untargeted metabolomics approach to characterize for the first time the changes in the metabolome of the B50 cell line, an immortalised rat neuronal cell line, following acute exposure to two known neurotoxic chemicals that are common environmental contaminants; the pyrethroid insecticide permethrin and the organophosphate insecticide malathion. B50 cells were exposed to either the dosing vehicle (methanol) or an acute dose of either permethrin or malathion for 6 and 24 hours. Intracellular metabolites were profiled by gas chromatography–mass spectrometry. Using principal components analysis, we selected the key metabolites whose abundance was altered by chemical exposure. By considering the major fold changes in abundance (>2.0 or <0.5 from control) across these metabolites, we were able to elucidate important cellular events associated with toxic exposure including disrupted energy metabolism and attempted protective mechanisms from excitotoxicity. Our findings illustrate the ability of mammalian cell culture metabolomics to detect finer metabolic effects of acute exposure to known toxic chemicals, and validate the need for further development of this process in the application of trace‐level dose and chronic toxicity studies, and toxicity testing of unknown chemicals.

Repetitive low intensity magnetic field stimulation in a neuronal cell line: a metabolomics study

Low intensity repetitive magnetic stimulation of neural tissue modulates neuronal excitability and has promising therapeutic potential in the treatment of neurological disorders. However, the underpinning cellular and biochemical mechanisms remain poorly understood. This study investigates the behavioural effects of low intensity repetitive magnetic stimulation (LI-rMS) at a cellular and biochemical level. We delivered LI-rMS (10 mT) at 1 Hz and 10 Hz to B50 rat neuroblastoma cells in vitro for 10 minutes and measured levels of selected metabolites immediately after stimulation. LI-rMS at both frequencies depleted selected tricarboxylic acid (TCA) cycle metabolites without affecting the main energy supplies. Furthermore, LI-rMS effects were frequency-specific with 1 Hz stimulation having stronger effects than 10 Hz. The observed depletion of metabolites suggested that higher spontaneous activity may have led to an increase in GABA release. Although the absence of organised neural circuits and other cellular contributors (e.g., excitatory neurons and glia) in the B50 cell line limits the degree to which our results can be extrapolated to the human brain, the changes we describe provide novel insights into how LI-rMS modulates neural tissue.

The application of metabolomics for herbal medicine pharmacovigilance: a case study on ginseng

Herbal medicines are growing in popularity, use and commercial value; however, there remain problems with the quality and consequently safety of these products. Adulterated, contaminated and fraudulent products are often found on the market, a risk compounded by the fact that these products are available to consumers with little or no medical advice. Current regulations and quality control methods are lacking in their ability to combat these serious problems. Metabolomics is a biochemical profiling tool that may help address these issues if applied to quality control of both raw ingredients and final products. Using the example of the popular herbal medicine, ginseng, this essay offers an overview of the potential use of metabolomics for quality control in herbal medicines and also highlights where more research is needed.

Untargeted Metabolomic Analysis of Rat Neuroblastoma Cells as a Model System to Study the Biochemical Effects of the Acute Administration of Methamphetamine

Methamphetamine is an illicit psychostimulant drug that is linked to a number of diseases of the nervous system. The downstream biochemical effects of its primary mechanisms are not well understood, and the objective of this study was to investigate whether untargeted metabolomic analysis of an in vitro model could generate data relevant to what is already known about this drug. Rat B50 neuroblastoma cells were treated with 1 mM methamphetamine for 48 h, and both intracellular and extracellular metabolites were profiled using gas chromatography–mass spectrometry. Principal component analysis of the data identified 35 metabolites that contributed most to the difference in metabolite profiles. Of these metabolites, the most notable changes were in amino acids, with significant increases observed in glutamate, aspartate and methionine, and decreases in phenylalanine and serine. The data demonstrated that glutamate release and, subsequently, excitotoxicity and oxidative stress were important in the response of the neuronal cell to methamphetamine. Following this, the cells appeared to engage amino acid-based mechanisms to reduce glutamate levels. The potential of untargeted metabolomic analysis has been highlighted, as it has generated biochemically relevant data and identified pathways significantly affected by methamphetamine. This combination of technologies has clear uses as a model for the study of neuronal toxicology.

Oxidative stress does not predispose neuronal cells to changes in G protein coupled (opioid) receptor gene expression in cortical B50 neurons in culture

Abstract Oxidative stress adversely affects neuronal cells in which they may die when oxygen supply is reduced or eliminated and opioid receptor agonists elicit several central nervous system effects. The aim of this study was to evaluate the effect of oxidative stress on opioid receptor gene expression in cortical B50 cells. The cells were cultured in normoxia, hypoxia and treated with opioid agonists; DAMGO(μ) DSLET (δ) and ICI—199,441 (κ) for 48 hours after 48 hours of initial culture at dose of 10μM, 50μM and 100μM. The level of mu opioid receptor mRNA was assessed using RT-PCR. The results show that oxidative stress induced changes in B50 cells in hypoxia while mu opioid mRNA levels showed no change. The results show that B50 cells are susceptible to damage by oxidative stress and opioid agonist treatments showed no change in the level of mu opioid receptor gene expression in B50 cells.