Urine Metabolomic Signature of People Diagnosed with Balkan Endemic Nephropathy and Other Types of Chronic Kidney Disease Compared with Healthy Subjects in Romania.

Metabolomic analysis methods were employed to determine biomarkers for various chronic kidney diseases (CKDs). Modern analytical methods were developed and applied successfully to find a specific metabolomic profile in urine samples from CKD and Balkan endemic nephropathy (BEN) patients. The aim was to explore a specific metabolomic profile defined by feasible/easy-to-identify molecular markers. Urine samples were collected from patients with CKDs and BEN, and from healthy subjects from endemic and nonendemic areas in Romania. Metabolomic analysis of urine samples, extracted by the liquid-liquid extraction (LLE) method, was performed by gas chromatography-mass spectrometry (GC-MS). The statistical exploration of the results was performed through a principal component analysis (PCA) evaluation. Urine samples were statistically analyzed using a classification based on six types of metabolites. Most urinary metabolites are distributed in the center of a loading plot, meaning that these compounds do not represent significant markers for BEN. One of the most frequent and higher-concentration urinary metabolites in BEN patients was p-Cresol, a phenolic compound that implies a severe injury of the renal filtration function. The presence of p-Cresol was associated with protein-bound uremic toxins, which have specific functional groups such as indole and phenyl. In prospective studies for future investigation, prevention, and disease treatment, we suggest a larger sample size, sample extraction using other methods, and analysis using other chromatography techniques coupled with mass spectrometry, which can generate a more significant data set for statistical analysis.

Aristolochic acid I as an emerging biogenic contaminant involved in chronic kidney diseases: A comprehensive review on exposure pathways, environmental health issues and future challenges.

Described in the 1950s, Balkan Endemic Nephropathy (BEN) has been recognized as a chronic kidney disease (CKD) with clinical peculiarities and multiple etiological factors. Environmental contaminants - aromatic compounds, mycotoxins and phytotoxins like aristolochic acids (AAs) - polluting food and drinking water sources, were incriminated in BEN, due to their nephrotoxic and carcinogenic properties. The implication of AAs in BEN etiology is currently a highly debated topic due to the fact that they are found within the Aristolochiaceae plants family, used around the globe as traditional medicine and they were also incriminated in Aristolochic Acid Nephropathy (AAN). Exposure pathways have been investigated, but it is unclear to what extent AAs are acting alone or in synergy with other cofactors (environmental, genetics) in triggering kidney damage. Experimental studies strengthen the hypothesis that AAI, the most studied compound in the AAs class, is a significant environmental contaminant and a most important causative factor of BEN. The aim of this review is to compile information about the natural exposure pathways to AAI, via traditional medicinal plants, soil, crop plants, water, food, air. Data that either supports or contradicts the AAI theory concerning BEN etiology was consolidated and available solutions to reduce human exposure were discussed. Because AAI is a phytotoxin with physicochemical properties that allow its transportation in environmental matrices from different types of areas (endemic, nonendemic), and induce CKDs (BEN, AAN) and urinary cancers through bioaccumulation, this review aims to shed a new light on this compound as a biogenic emerging pollutant.

Complexation of Danube common nase (Chondrostoma nasus L.) oil by ß-cyclodextrin and 2-hydroxypropyl-ß-cyclodextrin.

ß-Cyclodextrin- and 2-hydroxypropyl-ß-cyclodextrin/Danube common nase (Chondrostoma nasus L.) oil complexes (ß-CD- and HP-ß-CD/CNO) have been obtained for the first time. The fatty acid (FA) profile of the CNO indicates an important content of polyunsaturated fatty acids, the most important being eicosapentaenoic acid (EPA, 6.3%) and docosahexaenoic acid (DHA, 1.6%), both ω-3 FAs. The complexes have been obtained by kneading method. The moisture content and successful of molecular encapsulation have been evaluated by thermal and spectroscopic techniques. Thermogravimetry and differential scanning calorimetry analyses reveals that the moisture content of CD/CNO complexes significantly decreased, compared to starting CDs. On the other hand, the crystallinity index was for the first time determined for such type of complexes, the ß-CD/CNO complex having values of 43.9(±18.3)%, according to X-ray diffractometry. FA profile and CD/CNO characteristics sustain the use of these ω-3 based complexes for food supplements or functional food products, but further studies are needed.

Fatty acid profile of Romanian's common bean (Phaseolus vulgaris L.) lipid fractions and their complexation ability by ß-cyclodextrin.

The goal of the present study was the evaluation of the fatty acid (FA) profile of lipid fraction from dry common beans (Phaseolus vulgaris L.) (CBO) harvested from North-East (NE) and South-West (SW) of Romania and to protect against thermal and oxidative degradation of the contained omega-3 and omega-6 polyunsaturated fatty acid (PUFA) glycerides by ß-cyclodextrin (ß-CD) nanoencapsulation, using kneading method. The most abundant FAs in the CBO samples were PUFAs, according to gas chromatography-mass spectrometry (GC-MS) analysis. Linoleic acid (methyl ester) was the main constituent, having relative concentrations of 43.4 (±1.95) % and 35.23 (±0.68) % for the lipid fractions separated from the common beans harvested from the NE and SW of Romania, respectively. Higher relative concentrations were obtained for the omega-3 α-linolenic acid methyl ester at values of 13.13 (±0.59) % and 15.72 (±0.30) % for NE and SW Romanian samples, respectively. The omega-3/omega-6 ratio consistently exceeds the lower limit value of 0.2, from where the PUFA glyceride mixture is valuable for the human health. This value was 0.32 (±0.02) for the NE samples and significantly higher for the CBO-SW samples, 0.51 (±0.01). These highly hydrophobic mixtures especially consisting of PUFA triglycerides provide ß-CD complexes having higher thermal and oxidative stability. Kneading method allowed obtaining ß-CD/CBO powder-like complexes with higher recovery yields of >70%. Thermal analyses of complexes revealed a lower content of hydration water (3.3-5.8% up to 110°C in thermogravimetry (TG) analysis and 154-347 J/g endothermal effect in differential scanning calorimetry (DSC) analysis) in comparison with the ß-CD hydrate (12.1% and 479.5-480 J/g, respectively). These findings support the molecular inclusion process of FA moieties into the ß-CD cavity. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) analysis reveals the formation of the ß-CD/CBO inclusion complexes by restricting the vibration and bending of some bonds from the host and guest molecules. Moreover, powder X-ray diffractometry (PXRD) analysis confirm the formation of the host-guest complexes by modifying the diffractograms for ß-CD/CBO complexes in comparison with the ß-CD and ß-CD + CBO physical mixtures. A significant reduction of the level of crystallinity from 93.3 (±5.3) % for ß-CD to 60-60.9% for the corresponding ß-CD/CBO complexes have been determined. The encapsulation efficiency (EE), the profile of FAs, as well as the controlled release of the encapsulated oil have also been evaluated. The EE was >40% in all cases, the highest value being obtained for ß-CD/CBO-SW complex. The SFA content increased, while the unsaturated FA glycerides had lower relative concentrations in the encapsulated CBO samples. It can be emphasized that the main omega-3 FA (namely α-linolenic acid glycerides) had close concentrations in the encapsulated and raw CBOs (13.13 (±0.59) % and 14.04 (±1.54) % for non-encapsulated and encapsulated CBO-NE samples, 15.72 (±0.30) % and 12.41 (±1.95) % for the corresponding CBO-SW samples, respectively). The overall unsaturated FA content significantly decreased after complexation (from 19.03-19.16% for the raw CBOs to 17.3-17.7% for encapsulated oils in the case of MUFAs, and from 55.7-58.8% to 35.13-43.36% for PUFAs). On the other hand, the omega-3/omega-6 ratio increased by ß-CD nanoencapsulation to 0.51 (±0.07) and 0.76 (0.26) for ß-CD/CBO-NE and ß-CD/CBO-SW complexes, respectively. As a conclusion, the lipid fractions of the Romanian common beans are good candidates for ß-CD complexation and they can be protected against thermal and oxidative degradation in common beans based food products such as functional foods or food supplements using natural CDs.

A Theoretical Study of the Molecular Coupled Structures of Aristolochic Acids and Humic Acid, Potential Environmental Contaminants.

An understanding of the fate of organic compounds originating from plants in soil is crucial for determining their persistence and concentrations in the environment. Aristolochic acids are believed to be the causal agents that induce Balkan endemic nephropathy by food contamination through soil adsorption of humic acids, major components of soil. Aristolochic acids are active chemicals in Aristolochia plant species found in endemic villages. In this article, molecular structure interactions between 18 structures of aristolochic acids with an inserted humic acid structure were studied. These structures were optimized in vacuo and by periodic box simulation with water solvate using the computational molecular mechanics MM+ method with HyperChem software. The QSPR models were used for correlation of the relationship between the hydrophobicity values of 18 AA structures coupled with a HA structure by MM+ and QSAR+ properties. Computational hydrophobicity values were considered dependent variables and were related to the structural features obtained by molecular and quantum mechanics calculations by multiple linear regression approaches. The obtained model was validated, and the results indicated differing hydrophobicity between the MM+ and QSAR+ properties.

Balkan endemic nephropathy and aristolochic acid I: an investigation into the role of soil and soil organic matter contamination, as a potential natural exposure pathway.

Aristolochic acids (AAs) are carcinogenic and nephrotoxic plant alkaloids present in Aristolochia species, used in traditional medicine. Recent biomolecular and environmental studies have incriminated these toxins as an etiological agent in Balkan endemic nephropathy (BEN), a severe kidney disease occurring in the Balkan Peninsula. The questions on how the susceptible populations are exposed to these toxins have not yet been clearly answered. Exposure to AAs through the food chain, and environmental pollution (soil/dust), could provide an explanation for the presence of BEN in the countries where no folkloric use of the plant has been documented (Bulgaria, Croatia). Additional exposure pathways are likely to occur, and we have shown previously that AAs can contaminate crop plants through absorption from soil, under controlled laboratory environment. Here, we attempt to provide additional support to this potential exposure pathway, by revealing the presence of AAI in soil and soil organic matter samples collected from BEN and non-BEN areas. The samples were processed in order to be analyzed by high-pressure liquid chromatography, and ion trap mass spectrometry. Our results showed the presence of AAI in small concentrations, both in BEN and non-BEN soils, especially where Aristolochia plants and seeds were present.

Moisture evaluation of ß-cyclodextrin/fish oils complexes by thermal analyses: A data review on common barbel (Barbus barbus L.), Pontic shad (Alosa immaculata Bennett), European wels catfish (Silurus glanis L.), and common bleak (Alburnus alburnus L.) living in Danube river.

The moisture content of ß-cyclodextrin/Danube fish oils complexes (common barbel, Pontic shad, European wels catfish, common bleak) was evaluated by thermal methods. Saturated and monounsaturated fatty acids were the most concentrated in fish oils (25.3-30.8% and 36.1-45.0%). ω-3 And ω-6 fatty acids were identified in low concentrations of 2.8-12.1% and 4.1-7.1%. The moisture content was significantly lowered after ß-CD complexation, as revealed by thermogravimetric (TG) analysis (13.3% for ß-CD, 2.5-6.5% for complexes). These results are consistent with the differential scanning calorimetry (DSC) data for the peaks corresponding to dissociation of water (calorimetric effect of 536Jg-1 for ß-cyclodextrin and 304-422.5Jg-1 for complexes). Furthermore, both TG and DSC results support the formation of inclusion complexes. This is the first study on the nanoencapsulation of Danube fish oils in ß-cyclodextrin.

Data on expression of lipoxygenases-5 and -12 in the normal and acetaminophen-damaged liver.

Here we present additional data on the expression of lipoxygenases -5 and -12 in the normal and acetaminophen-damaged liver, which are associated with our manuscript recently published in Chemico-Biological Interactions on lipid metabolism and eicosanoid signaling pathways involved in acetaminophen-induced liver damage in a mouse model (http://dx.doi.org/10.1016/j.cbi.2015.10.019 [1]). It has been demonstrated that the expression of lipoxygenase-5 and leukotriene formation are increased in the livers of rats with carbon tetrachloride (CCl4)-induced cirrhosis (http://dx.doi.org/10.1053/gast.2000.17831 [2]). In addition, the lipoxygenase-12 is known to be expressed in the resident macrophage population of the liver (http://dx.doi.org/10.1016/S0014-5793(99)00396-8 [3]). Mice were injected with acetaminophen, and at 48 h their livers were processed for immunohistochemistry with anti-mouse lipoxygenase-5 and -12 antibodies. At the same time point, the RNA was also extracted from the liver to assess the expression of lipoxygenase-5 and -12 genes via qPCR analysis. Our results show that lipoxygenase-5 expression, but not that of lipoxygenase-12, changes significantly in the acetominophen-damaged liver.

Thermal and oxidative stability of Atlantic salmon oil (Salmo salar L.) and complexation with ß-cyclodextrin.

The thermal and oxidative stability of Atlantic salmon oil (Salmo salar L.) as well as its ß-cyclodextrin (ß-CD) complexation ability has been verified for the first time. The main omega-3 fatty acids, EPA and DHA, were significantly degraded, even at 50 °C. Their relative concentrations decrease from 6.1% for EPA and 4.1% for DHA to 1.7% and 1.5% after degradation at 150 °C, respectively. On the other hand, the relative concentrations of monounsaturated and saturated fatty acids remained constant or slightly increased by a few percent after degradation (e.g., from 10.7% to 12.9% for palmitic acid). Co-crystallization of ASO with ß-CD at a host-guest ratio of 1:1 and 3:1 from an ethanol-water mixture and kneading methods has been used for the preparation of ß-CD/ASO complexes. The analysis of the complexes by thermogravimetry, differential scanning calorimetry (DSC), and Karl Fischer titration (KFT) as well as the decrease of the "strongly-retained" water content confirm the formation of the inclusion compound. Furthermore, the DSC parameters correlate well with the KFT kinetic data for ß-CD/ASO complexes.

Mesenchymal Stromal Cells Differentiating to Adipocytes Accumulate Autophagic Vesicles Instead of Functional Lipid Droplets.

Adult bone marrow mesenchymal stromal cells (BMSCs) can easily be differentiated into a variety of cells. In vivo transplantation of BMSCs-differentiated cells has had limited success, suggesting that these cells may not be fully compatible with the cells they are intended to replace in vivo. We investigated the structural and functional features of BMSCs-derived adipocytes as compared with adipocytes from adipose tissue, and the structure and functionality of lipid vesicles formed during BMSCs differentiation to adipocytes. Gas chromatography-mass spectrometry showed fatty acid composition of BMSCs-derived adipocytes and adipocytes from the adipose tissue to be very different, as is the lipid rafts composition, caveolin-1 expression, caveolae distribution in their membranes, and the pattern of expression of fatty acid elongases. Confocal microscopy confirmed the absence from BMSCs-derived adipocytes of markers of lipid droplets. BMSCs-derived adipocytes cannot convert deuterated glucose into deuterated species of fatty acids and cannot uptake the deuterated fatty acid-bovine serum albumin complexes from the culture medium, suggesting that intra-cellular accumulation of lipids does not occur by lipogenesis. We noted that BMSCs differentiation to adipocytes is accompanied by an increase in autophagy. Autophagic vesicles accumulate in the cytoplasm of BMSCs-derived adipocytes and their size and distribution resembles that of Nile Red-stained lipid vesicles. Stimulation of autophagy in BMSCs triggers the intra-cellular accumulation of lipids, while inhibition of autophagy prevents this accumulation. In conclusion, differentiation of BMSCs-derived adipocytes leads to intra-cellular accumulation of autophagic vesicles rather than functional lipid droplets, suggesting that these cells are not authentic adipocytes. J. Cell. Physiol. 231: 863-875, 2016. © 2015 Wiley Periodicals, Inc.

Acetaminophen-induced liver injury: Implications for temporal homeostasis of lipid metabolism and eicosanoid signaling pathway.

Acetaminophen is a commonly used drug that induces serious hepatotoxicity when overdosed, leading to increased levels of serum aminotransferases. However, little knowledge exists linking acetaminophen to liver free fatty acids and the eicosanoid-mediated signaling pathway. To this end, adult NMRI mice injected with a dose of 400 mg/kg acetaminophen were monitored for one week post-treatment. Consistent changes were observed in serum transaminases, profile of hepatic free fatty acids, expression of cyclooxygenase, elongase, lipogenesis, and lipolysis genes; as well as in expression patterns of cyclooxygenase-1 and -2 in the liver. Both linoleic acid and arachidonic acid--substrates in eicosanoid biosynthesis--were significantly influenced by overdose, and the latter peaked first among the free fatty acids examined here. There was a close similarity between the temporal dynamics of linoleic acid and aspartate aminotransferases. Moreover, serum transaminases were reduced by cyclooxygenase-2 inhibitors, but not by cyclooxygenase-1 inhibitors. Our results hence attest to the hazard of acetaminophen overdose on the temporal homeostasis of hepatic concentrations of free fatty acids and expression of key genes underlying liver lipid metabolism. There is also evidence for activation of a cyclooxygenase-mediated signaling pathway, especially the cyclooxygenase 2-prostanoid pathway, during acetaminophen-induced liver injury. Therefore, the results of the present study should provide valuable information to a wide audience, working to understand the health hazard of this drug and the implications of the eicosanoid signaling pathway in liver pathophysiology.

Thermal and oxidative stability of the Ocimum basilicum L. essential oil/ß-cyclodextrin supramolecular system.

Ocimum basilicum L. essential oil and its ß-cyclodextrin (ß-CD) complex have been investigated with respect to their stability against the degradative action of air/oxygen and temperature. This supramolecular system was obtained by a crystallization method in order to achieve the equilibrium of complexed-uncomplexed volatile compounds in an ethanol/water solution at 50 °C. Both the raw essential oil and its ß-CD complex have been subjected to thermal and oxidative degradation conditions in order to evaluate the protective capacity of ß-CD. The relative concentration of the O. basilicum L. essential oil compounds, as determined by GC-MS, varies accordingly with their sensitivity to the thermal and/or oxidative degradation conditions imposed. Furthermore, the relative concentration of the volatile O. basilicum L. compounds found in the ß-CD complex is quite different in comparison with the raw material. An increase of the relative concentration of linalool oxide from 0.3% to 1.1%, in addition to many sesquiterpene oxides, has been observed. ß-CD complexation of the O. basilicum essential oil modifies the relative concentration of the encapsulated volatile compounds. Thus, linalool was better encapsulated in ß-CD, while methylchavicol (estragole) was encapsulated in ß-CD at a concentration close to that of the raw essential oil. Higher relative concentrations from the degradation of the oxygenated compounds such as linalool oxide and aromadendren oxide were determined in the raw O. basilicum L. essential oil in comparison with the corresponding ß-CD complex. For the first time, the protective capability of natural ß-CD for labile basil essential oil compounds has been demonstrated.

Arachidonic acid accumulates in the stromal macrophages during thymus involution in diabetes.

Diabetes is a debilitating disease with chronic evolution that affects many tissues and organs over its course. Thymus is an organ that is affected early after the onset of diabetes, gradually involuting until it loses most of its thymocyte populations. We show evidence of accumulating free fatty acids with generation of eicosanoids in the diabetic thymus and we present a possible mechanism for the involution of the organ during the disease. Young rats were injected with streptozotocin and their thymuses examined for cell death by flow cytometry and TUNEL reaction. Accumulation of lipids in the diabetic thymus was investigated by histology and electron microscopy. The identity and quantitation of accumulating lipids was done with gas chromatography-mass spectrometry and liquid chromatography. The expression and dynamics of the enzymes were monitored via immunohistochemistry. Diabetes causes thymus involution by elevating the thymocyte apoptosis. Exposure of thymocytes to elevated concentration of glucose causes apoptosis. After the onset of diabetes, there is a gradual accumulation of free fatty acids in the stromal macrophages including arachidonic acid, the substrate for eicosanoids. The eicosanoids do not cause thymocyte apoptosis but administration of a cyclooxygenase inhibitor reduces the staining for ED1, a macrophage marker whose intensity correlates with phagocytic activity. Diabetes causes thymus involution that is accompanied by accumulation of free fatty acids in the thymic macrophages. Excess glucose is able to induce thymocyte apoptosis but eicosanoids are involved in the chemoattraction of macrophage to remove the dead thymocytes.

Tumour-associated fibroblasts and mesenchymal stem cells: more similarities than differences.

Tumour-associated fibroblasts (TAFs) are part of the tumour stroma, providing functional and structural support for tumour progression and development. The origin and biology of TAFs are poorly understood, but within the tumour environment, TAFs become activated and secrete different paracrine and autocrine factors involved in tumorigenesis. It has been shown that bone marrow mesenchymal stem cells (MSCs) can be recruited into the tumours, where they proliferate and acquire a TAF-like phenotype. We attempted to determine to what extent TAFs characteristics in vitro juxtapose to MSCs' definition, and we showed that TAFs and MSCs share immunophenotypic similarities, including the presence of certain cell surface molecules [human leukocyte antigen-DR subregion (HLA-DR), CD29, CD44, CD73, CD90, CD106 and CD117]; the expression of cytoskeleton and extracellular matrix proteins, such as vimentin, α-smooth muscle actin, nestin and trilineage differentiation potential (to adipocytes, chondrocytes and osteoblasts). When compared to MSCs, production of cytokines, chemokines and growth factors showed a significant increase in TAFs for vascular endothelial growth factor, transforming growth factor-ß1, interleukins (IL-4, IL-10) and tumour necrosis factor α. Proliferation rate was highly increased in TAFs and fibroblast cell lines used in our study, compared to MSCs, whereas ultrastructural details differentiated the two cell types by the presence of cytoplasmic elongations, lamellar content lysosomes and intermediate filaments. Our results provide supportive evidence to the fact that TAFs derive from MSCs and could be a subset of 'specialized' MSCs.