Anna Wojakowska

Application of Metabolomics in Thyroid Cancer Research

Thyroid cancer is the most common endocrine malignancy with four major types distinguished on the basis of histopathological features: papillary, follicular, medullary, and anaplastic. Classification of thyroid cancer is the primary step in the assessment of prognosis and selection of the treatment. However, in some cases, cytological and histological patterns are inconclusive; hence, classification based on histopathology could be supported by molecular biomarkers, including markers identified with the use of high-throughput “omics” techniques. Beside genomics, transcriptomics, and proteomics, metabolomic approach emerges as the most downstream attitude reflecting phenotypic changes and alterations in pathophysiological states of biological systems. Metabolomics using mass spectrometry and magnetic resonance spectroscopy techniques allows qualitative and quantitative profiling of small molecules present in biological systems. This approach can be applied to reveal metabolic differences between different types of thyroid cancer and to identify new potential candidates for molecular biomarkers. In this review, we consider current results concerning application of metabolomics in the field of thyroid cancer research. Recent studies show that metabolomics can provide significant information about the discrimination between different types of thyroid lesions. In the near future, one could expect a further progress in thyroid cancer metabolomics leading to development of molecular markers and improvement of the tumor types classification and diagnosis.

The dependence between urinary mercury concentration and carotid arterial intima-media thickness in workers occupationally exposed to mercury vapour

OBJECTIVES Mechanisms of the mercury effect on arterial vessel walls include increased free radicals generation, decreased nitric oxide synthesis and increased reactivity to vasoconstrictors, leading to accelerated development of atherosclerosis and arterial hypertension. The aim of this study was to evaluate the correlation between urinary mercury (Hg-U) concentration and carotid intima-media thickness (IMT) or intraventricular septum diastolic diameter (IVSDD) to find the best markers of mercury cardiovascular toxicity. MATERIALS AND METHODS The study included 154 workers of a chemical factory using mercury in chlorine production. Urinary mercury concentration was determined by atomic absorption spectrophotometry. Serum lipids were assessed by routine methods using enzymatic assay. Serum total antioxidant status (TAS) was determined by colorimetry. Measurements of IMT and IVSDD were made by ultrasound imaging using MEDISON SA 9900 PRIME system. RESULTS The mean Hg-U concentration was 1.9+/-2.7 microg/g creatinine in women (n = 29) and 5.6+/-12.2 microg/g creatinine in men (n = 125). In the group of non-smokers (n = 102) there was a positive linear correlation between Hg-U concentration and IMT (r = 0.1728; p < 0.05) and a negative dependence between high density cholesterol (HDL-C) and IMT (r = -0.2109; p < 0.01). The negative linear correlation between serum total antioxidant status (TAS) and carotid IMT (r = -0.2142; p < 0.05), and the positive correlation between HDL-C and TAS (r = 0.1953; p < 0.05) were shown to be valid for the total studied group. Serum lipids in women were normal, but in men the mean triglyceride level was higher than normal. CONCLUSIONS The occupational exposure to mercury vapour remains in a relationship with early, asymptomatic carotid atherosclerosis. The dependence between urinary mercury elimination and carotid intima-media thickness is evidenced in non-smoking workers. Defensive anti-atherosclerotic mechanisms in these workers are strongly related with HDL. In smokers, these protective mechanisms are disturbed.

Characteristics of rose hip (Rosa canina L.) cold-pressed oil and its oxidative stability studied by the differential scanning calorimetry method.

Two new commercially available high linolenic oils, pressed at low temperature from rose hip seeds, were characterised for their composition, quality and DPPH radical scavenging activity. The oxidative stability of oils was assessed using differential scanning calorimetry (DSC). Phytosterols, tocopherols and carotenoids contents were up to 6485.4; 1124.7; and 107.7 mg/kg, respectively. Phenolic compounds determined for the first time in rose hip oil totalled up to 783.55 μg/kg, with a predominant presence of p-coumaric acid methyl ester. Antiradical activity of the oils reached up to 3.00 mM/kg TEAC. The acid, peroxide and p-anisidine values as well as iron and copper contents indicated good quality of the oils. Relatively high protection against oxidative stress in the oils seemed to be a result of their high antioxidant capacity and the level of unsaturation of fatty acids.

Changes of phenolic secondary metabolite profiles in the reaction of narrow leaf lupin (Lupinus angustifolius) plants to infections with Colletotrichum lupini fungus or treatment with its toxin.

Plant interactions with environmental factors cause changes in the metabolism and regulation of biochemical and physiological processes. Plant defense against pathogenic microorganisms depends on an innate immunity system that is activated as a result of infection. There are two mechanisms of triggering this system: basal immunity activated as a result of a perception of microbe-associated molecular patterns through pattern recognition receptors situated on the cell surface and effector-triggered immunity (ETI). An induced biosynthesis of bioactive secondary metabolites, in particular phytoalexins, is one of the mechanisms of plant defense to fungal infection. Results of the study on narrow leaf lupin (Lupinus angustifolius L.) plants infected with the anthracnose fungus Colletotrichum lupini and treated with fungal phytotoxic metabolites are described in the paper. The C. lupini phytotoxins were isolated from liquid cultures, purified and partially characterized with physicochemical methods. Accumulation of secondary metabolites on leaf surface and within the tissues of plants either infected, treated with the fungal phytotoxin or submitted to both treatments was studied using GC-MS and LC-MS, respectively. Substantial differences in isoflavone aglycones and glycoconjugate profiles occurred in response to different ways of plant treatment.

An Optimized Method of Metabolite Extraction from Formalin-Fixed Paraffin-Embedded Tissue for GC/MS Analysis

Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffin embedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material.

Detection of metabolites discriminating subtypes of thyroid cancer: Molecular profiling of FFPE samples using the GC/MS approach.

One of the critical issues in thyroid cancer diagnostic is differentiation between follicular adenoma, follicular carcinoma and the follicular variant of papillary carcinoma, which in some cases is not possible based on histopathological features only. In this paper we performed molecular profiling of thyroid tissue aiming to identify metabolites characteristic for different types of thyroid cancer. FFPE tissue specimens were analysed from 5 different types of thyroid malignancies (follicular, papillary/classical variant, papillary/follicular variant, medullary and anaplastic cancers), benign follicular adenoma and normal thyroid. Extracted metabolites were identified and semi-quantified using the GC/MS approach. There were 28 metabolites identified, whose abundances were significantly different among different types of thyroid tumours, including lipids, carboxylic acids, and saccharides. We concluded, that multi-component metabolome signature could be used for classification of different subtypes of follicular thyroid lesions. Moreover, potential applicability of the GC/MS-based analysis of FFPE tissue samples in diagnostics of thyroid cancer has been proved.

Long-term High Fat Ketogenic Diet Promotes Renal Tumor Growth in a Rat Model of Tuberous Sclerosis

Nutritional imbalance underlies many disease processes but can be very beneficial in certain cases; for instance, the antiepileptic action of a high fat and low carbohydrate ketogenic diet. Besides this therapeutic feature it is not clear how this abundant fat supply may affect homeostasis, leading to side effects. A ketogenic diet is used as anti-seizure therapy i.a. in tuberous sclerosis patients, but its impact on concomitant tumor growth is not known. To examine this we have evaluated the growth of renal lesions in Eker rats (Tsc2+/−) subjected to a ketogenic diet for 4, 6 and 8 months. In spite of existing opinions about the anticancer actions of a ketogenic diet, we have shown that this anti-seizure therapy, especially in its long term usage, leads to excessive tumor growth. Prolonged feeding of a ketogenic diet promotes the growth of renal tumors by recruiting ERK1/2 and mTOR which are associated with the accumulation of oleic acid and the overproduction of growth hormone. Simultaneously, we observed that Nrf2, p53 and 8-oxoguanine glycosylase α dependent antitumor mechanisms were launched by the ketogenic diet. However, the pro-cancerous mechanisms finally took the ascendency by boosting tumor growth.

Identification of serum proteome signatures of locally advanced and metastatic gastric cancer: a pilot study

BackgroundThe gastric cancer is one of the most common and mortal cancer worldwide. The initial asymptomatic development and further nonspecific symptoms result in diagnosis at the advanced stage with poor prognosis. Yet, no clinically useful biomarkers are available for this malignancy, and invasive gastrointestinal endoscopy remains the only reliable option at the moment. Hence, there is a need for discovery of clinically useful noninvasive diagnostic and/or prognostic tool as an alternative (or complement) for current diagnostic tools. Here we aimed to search for serum proteins characteristic for local and invasive gastric cancer.MethodsPre-treatment blood samples were collected from patients with diagnosed gastric adenocarcinoma at the different stage of disease: 35 patients with locally advanced cancer and 18 patients with metastatic cancer; 50 healthy donors were also included as a control group. The low-molecular-weight fraction of serum proteome (i.e., endogenous peptidome) was profiled by the MALDI-ToF mass spectrometry, and the whole proteome components were identified and quantified by the LC–MS/MS shotgun approach.ResultsMulticomponent peptidome signatures were revealed that allowed good discrimination between healthy controls and cancer patients, as well as between patients with locally advanced and metastatic cancer. Moreover, a LC–MS/MS approach revealed 49 serum proteins with different abundances between healthy donors and cancer patients (predominantly proteins associated with inflammation and acute phase response). Furthermore, 19 serum proteins with different abundances between patients with locally advanced and metastatic cancer were identified (including proteins associated with cytokine/chemokine response and metabolism of nucleic acids). However, neither peptidome profiling nor shotgun proteomics approach allowed detecting serum components discriminating between two subgroups of patients with local disease who either developed or did not develop metastases during follow-up.ConclusionsThe molecular differences between locally advanced and metastatic gastric cancer, as well as more obvious differences between healthy individuals and cancer patients, have marked reflection at the level of serum proteome. However, we have no evidence that features of pre-treatment serum proteome could predict a risk of cancer dissemination in patients treated due to local disease. Nevertheless, presented data confirmed potential applicability of a serum proteome signature-based biomarker in diagnostics of gastric cancer.

Mass spectrometric behavior of phenolic acids standards and their analysis in the plant samples with LC/ESI/MS system.

Liquid chromatography coupled to mass spectrometry (MS) with electrospray ionization (ESI) is one of analytical techniques to obtain accurate results of low molecular weight aromatic compounds in biological samples of different origin. The interpretations of mass spectra of these aromatic compounds in the negative spectra registered in the full scan MS mode may be uneasy due to presence of deprotonated molecules [M-H](-) from different co-eluting entities, fragment ions created after the break-up of precursor ions and also ions representing modified molecules clusters. Thus, the first aim of this study was to evaluate general parameters during analysis performed in the full scan MS or MS/MS mode. Secondly, to set general fragmentation rules for aromatic compounds and entities in a complex biological matrix. We established that different groups of low molecular weight phenolic acids form unique adduct ions and additionally registration LC/MS/MS spectra with two different collision energies may allow for differentiating isomeric or isobaric molecules. These findings together with some general fragmentation rules can facilitate identifications of aromatic acids as we outlined in the sample of cold-pressed rose-hip oil and lupine leaves extract.

Association between the Type of Workplace and Lung Function in Copper Miners.

The aim of the analysis was to retrospectively assess changes in lung function in copper miners depending on the type of workplace. In the groups of 225 operators, 188 welders, and 475 representatives of other jobs, spirometry was performed at the start of employment and subsequently after 10, 20, and 25 years of work. Spirometry Longitudinal Data Analysis software was used to estimate changes in group means for FEV1 and FVC. Multiple linear regression analysis was used to assess an association between workplace and lung function. Lung function assessed on the basis of calculation of longitudinal FEV1 (FVC) decline was similar in all studied groups. However, multiple linear regression model used in cross-sectional analysis revealed an association between workplace and lung function. In the group of welders, FEF75 was lower in comparison to operators and other miners as early as after 10 years of work. Simultaneously, in smoking welders, the FEV1/FVC ratio was lower than in nonsmokers (p < 0,05). The interactions between type of workplace and smoking (p < 0,05) in their effect on FVC, FEV1, PEF, and FEF50 were shown. Among underground working copper miners, the group of smoking welders is especially threatened by impairment of lung ventilatory function.