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.

Analysis of Polycyclic Aromatic Hydrocarbons and Phthalate Esters in Soil and Food Grains from the Balkan Peninsula: Implication on DNA Adduct Formation by Aristolochic Acid I and Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephropathy affecting residents of rural farming areas in many Balkan countries. Although it is generally believed that BEN is an environmental disease caused by multiple geochemical factors with much attention on aristolochic acids (AAs), its etiology remains controversial. In this study, we tested the hypothesis that environmental contamination and subsequent food contamination by polycyclic aromatic hydrocarbons (PAHs) and phthalate esters are AA toxicity factors and important to BEN development. We identified significantly higher concentrations of phenanthrene, anthracene, diethyl phthalate (DEP), dibutyl phthalate (DBP), and benzyl butyl phthalate (BBP) in both maize and wheat grain samples collected from endemic villages than from nonendemic villages. Other PAHs and phthalate esters were also detected at higher concentrations in the soil samples from endemic villages. Subsequent genotoxicity testing of cultured human kidney cells showed an alarming phenomenon that phenanthrene, DEP, BBP, and DBP can interact synergistically with AAs to form elevated levels of AA-DNA adducts, which are associated with both the nephrotoxicity and carcinogenicity of AAs, further increasing their disease risks. This study provides direct evidence that prolonged coexposure to these environmental contaminants via dietary intake may lead to greater toxicity and accelerated development of BEN.

Aristolochic acid I: an investigation into the role of food crops contamination, as a potential natural exposure pathway.

Aristolochic acid I (AAI) is a potent nephrotoxic and carcinogenic compound produced by plants of the Aristolochiaceae family and thoroughly investigated as a main culprit in the etiology of Balkan endemic nephropathy (BEN). So far, the AAI exposure was demonstrated to occur through the consumption of Aristolochia clematitis plants as traditional remedies, and through the contamination of the surrounding environment in endemic areas: soil, food and water contamination. Our study investigated for the first time the level of AAI contamination in 141 soil and vegetable samples from two cultivated gardens in non-endemic areas, A. clematitis being present in only one of the gardens. We developed and validated a simple and sensitive ultra-high-performance liquid chromatography-ion trap mass spectrometry method for qualitative and quantitative AAI analysis. The results confirmed the presence of AAI at nanogram levels in soil and vegetable samples collected from the non-endemic garden, where A. clematitis grows. These findings provide additional evidence that the presence of A. clematitis can cause food crops and soil contamination and unveil the pathway through which AAI could move from A. clematitis to other plant species via a common matrix: the soil. Another issue regarding the presence of AAI, in a non-endemic BEN area from Romania, could underlie a more widespread environmental exposure to AAI and explain certain BEN-like cases in areas where BEN has not been initially described.

Botanical Therapeutics (Part II): Antimicrobial and In Vitro Anticancer Activity against MCF7 Human Breast Cancer Cells of Chamomile, Parsley and Celery Alcoholic Extracts.

BACKGROUND: This study was designed as a continuation of a complex investigation about the phytochemical composition and biological activity of chamomile, parsley, and celery extracts against A375 human melanoma and dendritic cells. OBJECTIVE: The main aim was the evaluation of the antimicrobial potential of selected extracts as well as the in vitro anticancer activity against MCF7 human breast cancer cells. METHODS: In order to complete the picture regarding the phytochemical composition, molecular fingerprint was sketched out by the help of FTIR spectroscopy. The activity of two enzymes (acetylcholinesterase and butyrylcholinesterase) after incubation with the three extracts was spectrophotometrically assessed. The antimicrobial potential was evaluated by disk diffusion method. The in vitro anticancer potential against MCF7 human breast cancer cells was appraised by MTT, LDH, wound healing, cell cycle, DAPI, Annexin-V-PI assays. RESULTS: The results showed variations between the investigated extracts in terms of inhibitory activity against enzymes, such as acetyl- and butyrilcholinesterase. Chamomile and parsley extracts were active only against tested Gram-positive cocci, while all tested extracts displayed antifungal effects. Among the screened samples at the highest tested concentration, namely 60µg/mL, parsley was the most active extract in terms of reducing the viability of MCF7 - human breast adenocarcinoma cell line and inducing the release of lactate dehydrogenase. On the other hand, chamomile and celery extracts manifested potent anti-migratory effects. Furthermore, celery extract was the most active in terms of total apoptotic events, while chamomile extract induced the highest necrosis rate. CONCLUSION: The screened samples containing phytochemicals belonging in majority to the class of flavonoids and polyphenols can represent candidates for antimicrobial and anticancer agents.

Aristolochic Acids as Persistent Soil Pollutants: Determination of Risk for Human Exposure and Nephropathy from Plant Uptake.

Exposure to aristolochic acids (AAs) from Aristolochia plants is one of the major global causes of nephropathy, including Balkan endemic nephropathy (BEN); renal failure; and urothelial cancer. The high incidence of BEN on the Balkan Peninsula is assumed to result from consumption of Aristolochia clematitis L. seeds coharvested with crops. Here, we show that AAs are long-lived soil contaminants that enter wheat and maize plants by root uptake with strong pH dependence. Soil and crops from Serbian farms in areas endemic for A. clematitis were found to be extensively contaminated with AAs, with contamination strongly correlated with local incidence of BEN. The persistence of AAs as soil contaminants suggests that weed control for A. clematitis plants is needed to reduce the incidence of BEN and aristolochic acid nephropathy, systematic surveys of soil and crop AA levels would identify high-risk regions, and it is imperative to research soil-remediation methods.

Design, Synthesis and Biological Activity Evaluation of S-Substituted 1H-5-Mercapto-1,2,4-Triazole Derivatives as Antiproliferative Agents in Colorectal Cancer.

Colon cancer is a widespread pathology with complex biochemical etiology based on a significant number of intracellular signaling pathways that play important roles in carcinogenesis, tumor proliferation and metastasis. These pathways function due to the action of key enzymes that can be used as targets for new anticancer drug development. Herein we report the synthesis and biological antiproliferative evaluation of a series of novel S-substituted 1H-3-R-5-mercapto-1,2,4-triazoles, on a colorectal cancer cell line, HT-29. Synthesized compounds were designed by docking based virtual screening (DBVS) of a previous constructed compound library against protein targets, known for their important role in colorectal cancer signaling: MEK1, ERK2, PDK1, VEGFR2. Among all synthesized structures, TZ55.7, which was retained as a possible PDK1 (phospholipid-dependent kinase 1) inhibitor, exhibited the most significant cytotoxic activity against HT-29 tumor cell line. The same compound alongside other two, TZ53.7 and TZ3a.7, led to a significant cell cycle arrest in both sub G0/G1 and G0/G1 phase. This study provides future perspectives for the development of new agents containing the 1,2,4-mercapto triazole scaffold with antiproliferative activities in colorectal cancer.

Toxicological and chemical studies of wastewater from hydraulic fracture and conventional shale gas wells.

New technology has enabled recovery of inaccessible natural gas shale deposits; however, the potential impacts to human health from the migration of brines into drinking water or surface spills are unknown. To provide information that can inform these potential impacts, chemical characterization and in vitro toxicologic testing were conducted using pre- and postinjection waters from conventional and unconventional oil and gas wells. Wastewater concentrations may be diluted or reduced by fate and transport processes when released into the environment by unknown amounts, and laboratory studies only imply potential effects. In acute cytotoxicity and wound healing assays, there was dose-dependent toxicity in human and rat cells with growth promotion at low concentrations. Lethality was measured in time studies up to 10 d postinjection. Produced water samples from both well types were equally toxic to human cells and were corrosive at high concentrations. Measurement of protein and gene expression identified metabolic pathways responding to both well types as NADPH quinone oxidoreductase oxidative stress-responsive enzyme and tight junction protein genes. A KCl sample of matched ionic strength showed a different toxicity profile from produced waters, indicating that salts alone were not the cause of toxicity. Organic chemicals and branched alkanes were present in hydraulic fracture wells, and mainly branched alkanes were present in conventional wells. One organic substance was still present after 240 d. The known properties of these chemicals include potential toxicity to multiple human organs, sensitization, irritation, developmental effects, and tumor promotion, depending on the concentrations and synergistic effects of chemicals during exposure. Environ Toxicol Chem 2018;37:2098-2111. © 2018 SETAC.

Selective in vitro anti-melanoma activity of ursolic and oleanolic acids.

Products of natural origin have become important agents in the treatment of cancer, and the active principles of natural sources could be used in combination with chemotherapeutic agents to increase their effects and to minimize their toxicity. Oleanolic (OA) and ursolic (UA) acids are intensely studied for their promising anticancer potential. The aim of this study was focused on the in vitro toxicological effects induced by UA and OA human mesenchymal stem cells and on melanoma, one of the most frequent cancers whose incidence is increasing every year. The two compounds were tested for their cytotoxic, cell cycle arrest and pro-apoptotic effects on melanoma cells (A375 and B164A5) and mesenchymal stem cells. UA exerted a cytotoxic effect in a dose-dependent manner on melanoma cells, while OA's activity has been shown to be low or moderate. Both compounds produced alterations of the cell cycle, arresting cells in the G0/G1 phase. Furthermore, UA induced significant apoptosis through the bcl-2 genes family pathway, with the decrease of the bcl-2 gene expression. The two compounds exerted selective effects on melanoma cells with no effects on human mesenchymal stem cells. The presented results reveal the anticancer potential of UA on melanoma cells, with no detectable toxicity on the mesenchymal stem cells.

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.

Effects of ursolic and oleanolic on SK­MEL­2 melanoma cells: In vitro and in vivo assays.

Among the triterpenoids, oleanolic acid (OA) and its isomer, ursolic acid (UA) are promising therapeutic candidates, with potential benefits in the management of melanoma. In this study, we aimed to examine the in vitro and in vivo anti­invasive and anti­metastatic activity of OA and UA to determine their possible usefulness as chemopreventive or chemotherapeutic agents in melanoma. For the in vitro experiments, the anti­proliferative activity of the triterpenic compounds on SK­MEL­2 melanoma cells was examined. The anti­invasive potential was assessed by testing the effects of the active compound on vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) adhesion to melanoma cells. Normal and tumor angiogenesis were evaluated in vivo by chicken embryo chorioallantoic membrane (CAM) assay. The two test triterpenoid acids, UA and OA, exerted differential effects in vitro and in vivo on the SK­MEL­2 melanoma cells. UA exerted a significant and dose­dependent anti­proliferative effect in vitro, compared to OA. The cytotoxic effects in vitro on the melanoma cells were determined by the examining alterations in the cell cycle phases induced by UA that lead to cell arrest in the S phase. Moreover, UA was found to affect SK­MEL­2 melanoma cell invasiveness by limiting the cell adhesion capacity to ICAM molecules, but not influencing their adhesion to VCAM molecules. On the whole, in this study, by assessing the effects of the two triterpenoids in vivo, our results revealed that OA had a greater potential to impair the invasive capacity and tumor angiogenesis compared with UA.

Improvement of ursolic and oleanolic acids' antitumor activity by complexation with hydrophilic cyclodextrins.

Ursolic and oleanolic acids have been brought into the spotlight of research due to their chemopreventive, anti-inflammatory and immunomodulatory properties. The most important disadvantage of ursolic and oleanolic acids is their weak water solubility which limits their bioavailability. Pentacyclic triterpenes can form inclusion complexes with different types of cyclodextrins which provide the hydrophilic matrix requested for the molecular dispersion of drugs in order to become more water soluble. The aim of the current study is the complexation of ursolic and oleanolic acids with hydrophilic cyclodextrins in order to achieve an improvement of their pharmacological effect. After the virtual screening of the binding affinities between ursolic and oleanolic acids and various cyclodextrins, 2-hydroxypropyl-ß-cyclodextrin and 2-hydroxypropil-γ-cyclodextrin were selected as host-molecules for the inclusion complexation. Using the scanning electron microscopy, differential scanning calorimetry and X-ray diffraction the formation of real inclusion complexes between ursolic and oleanolic acids and the two cyclodextrins was confirmed. The anti-proliferative potential of the complexes was tested in vitro on several melanoma cell lines, using the pure compounds as reference. The complexes exhibited higher in vitro anti-proliferative activity as compared to the pure compounds; this improvement was significant for ursolic acid complexes, the highest activity being reported for the 2-hydroxypropil-γ-cyclodextrin complex. Weaker results were recorded for the oleanolic acid complexes where 2-hydroxypropyl-ß-cyclodextrin proved to be the most fitted inclusion partner. The entrapment of the two active compounds inside ramified hydrophilic cyclodextrins proved to be a suitable option to increase their anti-proliferative activity.

In Vivo Biological Evaluation of Polyurethane Nanostructures with Ursolic and Oleanolic Acids on Chemically-induced Skin Carcinogenesis.

BACKGROUND/AIM: Oleanolic and ursolic acids (OA and UA) are two pentacyclic triterpenes, ubiquitously spread in plants, previously known for their chemopreventive capacity on different types of cancer. The major pharmacological disadvantage of these phytocompounds is their poor water solubility, which often limits their applicability. MATERIALS AND METHODS: Using the interfacial polycondensation combined with spontaneous emulsification technique, polyurethane nanostructures (PU) were synthetized in order to improve this problem. In order to test the in vivo chemopreventive potential of the two pure compounds, as well as the encapsulated compounds in PU used as drug carriers, a chemically-induced skin carcinogenesis model was constructed. RESULTS: UA and OA have a moderate chemopreventive activity against tumors induced by 7,12-dimethylbenzantracene (DMBA) and 12-O-tetradecanoilphorbol-13-acetate (TPA) application. Incorporation of active agents in PU did not lead to increased chemopreventive effect. CONCLUSION: PU is not a suitable formulation of UA and OA.

Revealing a Pre-neoplastic Renal Tubular Lesion by p-S6 Protein Immunohistochemistry after Rat Exposure to Aristolochic Acid.

Aristolochic acid (AA) has, in the last decade, become widely promoted as the cause of the Balkan endemic nephropathy and associated renal or urothelial tumours, although without substantial focal evidence of the quantitative dietary exposure via bread in specific households in hyperendemic villages. Occasional ethnobotanical use of Aristolochia clematitis might be a source of AA, and Pliocene lignite contamination of well-water is also a putative health risk factor. The aim of this study was two-fold: to verify if extracts of A. clematitis and Pliocene, or AA by itself, could induce the development of renal or urothelial tumours, and to test the utility of the ribosomal protein p-S6 to identify preneoplastic transformation. Rats were given extracts of A. clematitis in drinking water or AA I, by gavage. After seven months, renal morphology was studied using conventional haematoxylin and eosin and immunohistochemistry for ribosomal p-S6 protein. Plant extracts (cumulative AA approximately 1.8 g/kg b.w.) were tolerated and caused no gross pathology or renal histopathological change, with only faint diffuse p-S6 protein (except in the papilla) as in controls. Cumulative AA I (150 mg/kg b.w. given over 3 days) was also tolerated for seven months by all recipients, without gross pathology or kidney tumours. However, p-S6 protein over-expression was consistent particularly within the renal papilla. In one case given AA I, intense p-S6 protein staining of a proximal tubule fragment crucially matched the pre-neoplastic histology in an adjacent kidney section. We briefly discuss these findings, which compound uncertainty concerning the cause of the renal or upper urinary tract tumours of the Balkan endemic nephropathy.

Atmospheric particulate matter in proximity to mountaintop coal mines: sources and potential environmental and human health impacts.

Mountaintop removal mining (MTM) is a widely used approach to surface coal mining in the US Appalachian region whereby large volumes of coal overburden are excavated using explosives, removed, and transferred to nearby drainages below MTM operations. To investigate the air quality impact of MTM, the geochemical characteristics of atmospheric particulate matter (PM) from five surface mining sites in south central West Virginia, USA, and five in-state study control sites having only underground coal mining or no coal mining whatsoever were determined and compared. Epidemiologic studies show increased rates of cancer, respiratory disease, cardiovascular disease, and overall mortality in Appalachian surface mining areas compared to Appalachian non-mining areas. In the present study, 24-h coarse (>2.5 µm) and fine (≤2.5 µm) PM samples were collected from two surface mining sites in June 2011 showed pronounced enrichment in elements having a crustal affinity (Ga, Al, Ge, Rb, La, Ce) contributed by local sources, relative to controls. Follow-up sampling in August 2011 lacked this enrichment, suggesting that PM input from local sources is intermittent. Using passive samplers, dry deposition total PM elemental fluxes calculated for three surface mining sites over multi-day intervals between May and August 2012 were 5.8 ± 1.5 times higher for crustal elements than at controls. Scanning microscopy of 2,249 particles showed that primary aluminosilicate PM was prevalent at surface mining sites compared to secondary PM at controls. Additional testing is needed to establish any link between input of lithogenic PM and disease rates in the study area.

Enucleation: a possible mechanism of cancer cell death.

There are few major morphologies of cell death that have been described so far: apoptosis (type I), cell death associated with autophagy (type II), necrosis (type III) and anchorage-dependent mechanisms-anoikis. Here, we show for the first time a possibly novel mechanism inducing tumour cell death under in vitro conditions-enucleation. We pursued the influence of colloidal suspensions of Fe3 O4 nanoparticles on tumour cell lines (SK-BR-3 and MCF-7 breast cancer cell lines) grown according to standard cell culture protocols. Magnetite nanoparticles were prepared by combustion synthesis and double layer coated with oleic acid. Scanning and transmission electron microscopy revealed that tumour cells developed a network of intracytoplasmic stress fibres, which induce extrusion of nuclei, and enucleated cells die. Normal adult mesenchymal stem cells, used as control, did not exhibit the same behaviour. Intact nuclei were found in culture supernatant of tumour cells, and were visualized by immunofluorescence. Enucleation as a potential mechanism of tumour cell death might open new horizons in cancer biology research and development of therapeutic agents capable of exploiting this behaviour.

Possible health impacts of naturally occurring uptake of aristolochic acids by maize and cucumber roots: links to the etiology of endemic (Balkan) nephropathy.

Aristolochic acids (AAs) are nephrotoxic and carcinogenic derivatives found in several Aristolochia species. To date, the toxicity of AAs has been inferred only from the effects observed in patients suffering from a kidney disease called "aristolochic acid nephropathy" (AAN, formerly known as "Chinese herbs nephropathy"). More recently, the chronic poisoning with Aristolochia seeds has been considered to be the main cause of Balkan endemic nephropathy, another form of chronic renal failure resembling AAN. So far, it was assumed that AAs can enter the human food chain only through ethnobotanical use (intentional or accidental) of herbs containing self-produced AAs. We hypothesized that the roots of some crops growing in fields where Aristolochia species grew over several seasons may take up certain amounts of AAs from the soil, and thus become a secondary source of food poisoning. To verify this possibility, maize plant (Zea mays) and cucumber (Cucumis sativus) were used as a model to substantiate the possible significance of naturally occurring AAs' root uptake in food chain contamination. This study showed that the roots of maize plant and cucumber are capable of absorbing AAs from nutrient solution, consequently producing strong peaks on ultraviolet HPLC chromatograms of plant extracts. This uptake resulted in even higher concentrations of AAs in the roots compared to the nutrient solutions. To further validate the measurement of AA content in the root material, we also measured their concentrations in nutrient solutions before and after the plant treatment. Decreased concentrations of both AAI and AAII were found in nutrient solutions after plant growth. During this short-term experiment, there were much lower concentrations of AAs in the leaves than in the roots. The question is whether these plants are capable of transferring significant amounts of AAs from the roots into edible parts of the plant during prolonged experiments.

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.

A pilot study of nuclear instability in archived renal and upper urinary tract tumours with putative ochratoxin aetiology.

DNA ploidy measurement has been applied uniquely to wax-embedded tissue of primary renal cell and metastatic tumours of a key experimental researcher on porcine ochratoxicosis, a control, and four transitional cell carcinomas from cases of Balkan endemic nephropathy. Primary renal tumour was diploid, and hyperdiploid metastasis was within the lower ploidy range for typical renal cell carcinoma. Three Balkan primary tumours showed extensive aneuploidy indicating marked nuclear instability, similar to model rat renal carcinoma caused by ochratoxin A. In contrast, much less nuclear instability in the putative occupational ochratoxicosis case fitted poorly with the ochratoxin A model.

Evaluating nephrotoxicity of high-molecular-weight organic compounds in drinking water from lignite aquifers.

High-molecular-weight organic compounds such as humic acids and/or fulvic acids that are naturally mobilized from lignite beds into untreated drinking-water supplies were suggested as one possible cause of Balkan endemic nephropathy (BEN) and cancer of the renal pelvis. A lab investigation was undertaken in order to assess the nephrotoxic potential of such organic compounds using an in vitro tissue culture model. Because of the infeasibility of exposing kidney tissue to low concentrations of organics for years in the lab, tangential flow ultrafiltration was employed to hyperconcentrate samples suitable for discerning effects in the short time frames necessitated by tissue culture systems. Effects on HK-2 kidney cells were measured using two different cell proliferation assays (MTT and alamarBlue). Results demonstrated that exposure of kidney tissue to high-molecular-weight organics produced excess cell death or proliferation depending on concentration and duration of exposure.

In vitro differentiation of human mesenchymal stem cells to epithelial lineage.

Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.