Stem Cells Derived from Human Exfoliated Deciduous Teeth Functional Assessment: Exploring the Changes of Free Fatty Acids Composition during Cultivation.

The metabolic regulation of stemness is widely recognized as a crucial factor in determining the fate of stem cells. When transferred to a stimulating and nutrient-rich environment, mesenchymal stem cells (MSCs) undergo rapid proliferation, accompanied by a change in protein expression and a significant reconfiguration of central energy metabolism. This metabolic shift, from quiescence to metabolically active cells, can lead to an increase in the proportion of senescent cells and limit their regenerative potential. In this study, MSCs from human exfoliated deciduous teeth (SHEDs) were isolated and expanded in vitro for up to 10 passages. Immunophenotypic analysis, growth kinetics, in vitro plasticity, fatty acid content, and autophagic capacity were assessed throughout cultivation to evaluate the functional characteristics of SHEDs. Our findings revealed that SHEDs exhibit distinctive patterns of cell surface marker expression, possess high self-renewal capacity, and have a unique potential for neurogenic differentiation. Aged SHEDs exhibited lower proliferation rates, reduced potential for chondrogenic and osteogenic differentiation, an increasing capacity for adipogenic differentiation, and decreased autophagic potential. Prolonged cultivation of SHEDs resulted in changes in fatty acid composition, signaling a transition from anti-inflammatory to proinflammatory pathways. This underscores the intricate connection between metabolic regulation, stemness, and aging, crucial for optimizing therapeutic applications.

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.

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.

Dissolution and Phosphate-Induced Transformation of ZnO Nanoparticles in Synthetic Saliva Probed by AGNES without Previous Solid-Liquid Separation. Comparison with UF-ICP-MS.

The variation over time of free Zn2+ ion concentration in stirred dispersions of ZnO nanoparticles (ZnO NPs) prepared in synthetic saliva at pH 6.80 and 37 °C was followed in situ (without solid-liquid separation step) with the electroanalytical technique AGNES (Absence of Gradients and Nernstian Equilibrium Stripping). Under these conditions, ZnO NPs are chemically unstable due to their reaction with phosphates. The initial stage of transformation (around 5-10 h) involves the formation of a metastable solid (presumably ZnHPO4), which later evolves into the more stable hopeite phase. The overall decay rate of ZnO NPs is significantly reduced in comparison with phosphate-free background solutions of the same ionic strength and pH. The effective equilibrium solubilities of ZnO (0.29-0.47 mg·L-1), as well as conditional excess-ligand stability constants and fractional distributions of soluble Zn species, were determined in the absence and presence of organic components. The results were compared with the conventional ultrafiltration and inductively coupled plasma-mass spectrometry (UF-ICP-MS) methodology. AGNES proves to be advantageous in terms of speed, reproducibility, and access to speciation information.

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.

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.

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.

Suitability of analytical methods to measure solubility for the purpose of nanoregulation.

Solubility is an important physicochemical parameter in nanoregulation. If nanomaterial is completely soluble, then from a risk assessment point of view, its disposal can be treated much in the same way as "ordinary" chemicals, which will simplify testing and characterisation regimes. This review assesses potential techniques for the measurement of nanomaterial solubility and evaluates the performance against a set of analytical criteria (based on satisfying the requirements as governed by the cosmetic regulation as well as the need to quantify the concentration of free (hydrated) ions). Our findings show that no universal method exists. A complementary approach is thus recommended, to comprise an atomic spectrometry-based method in conjunction with an electrochemical (or colorimetric) method. This article shows that although some techniques are more commonly used than others, a huge research gap remains, related with the need to ensure data reliability.

Fronto-parietal connectivity changes following noxious stimulation during anesthesia.

RATIONALE: The aim of our study was to assess the changes in the fronto-parietal connectivity estimated by the cross approximate entropy (XAppEn) during noxious stimulation while under chloral hydrate anaesthesia, in rats. METHOD: A group of 11 Wistar rats chronically implanted with Ni-Cr electrodes, which were placed on the dura mater of the right hemisphere (over the olfactory cortex, the frontal and the parietal lobes), were used in the present study. Noxious stimuli of a mechanical and thermal nature were applied on the left hindpaw during chloral hydrate anesthesia. The anesthetic depth was estimated through median frequency computation, which in that instance was of 2-3 Hz. Fronto-parietal functional cortical connectivity was assessed by using XAppEn. RESULTS: After data processing and analysis we observed an increase of fronto-parietal functional connectivity during mechanical and thermal noxious stimulation. In addition, MEF increased both in frontal and parietal areas during the mechanical and thermal stimulation compared to baseline. CONCLUSION: Mechanical and thermal stimulation induces an increase in the fronto-parietal connectivity during chloral hydrate anesthesia in rats.

Measurement of metals using DGT: impact of ionic strength and kinetics of dissociation of complexes in the resin domain.

As the measurement of metals by DGT (diffusion gradients in thin films) in low salinity media has been controversial, a thorough study of the impact of ionic strength (I) is timely. DGT accumulations of Cd, Co, and Ni in the presence of NTA at pH 7.5 with I in the range from 10(-4) to 0.5 M were obtained. An observed decrease in the metal accumulation as the ionic strength of the system decreased is partially explained by the electrostatic repulsion between the negatively charged resin domain and the dominant negatively charged complex species M-NTA. This electrostatic effect reduces the complex penetration into the resin domain, especially for nonlabile complexes, which do not fully dissociate in the gel domain. Analytical expressions, based on the Donnan model, were able to quantify these electrostatic effects. Additionally, the data indicate that the kinetic dissociation constant of M-NTA complexes in the resin layer is higher than Eigen predictions, suggesting a ligand-assisted dissociation mechanism. As the ionic strength decreases, the rate of reaction in the resin layer decreases due to the repulsion between the negatively charged resin sites and the complex species. This decrease contributes to the decrease in metal accumulation. These novel, previously unconsidered, effects of ionic strength and the ligand-assisted dissociation mechanism in the resin domain will affect DGT measurements made in freshwaters and soils.

Systematic investigation of the physicochemical factors that contribute to the toxicity of ZnO nanoparticles.

ZnO nanoparticles (NPs) are prone to dissolution, and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn(2+) or whether the NPs themselves have additional toxic effects. We address this by establishing ZnO NP solubility in dispersion media (Dulbecco's modified Eagle's medium, DMEM) held under conditions identical to those employed for cell culture (37 °C, 5% CO2, and pH 7.68) and by systematic comparison of cell-NP interaction for three different ZnO NP preparations. For NPs at concentrations up to 5.5 µg ZnO/mL, dissolution is complete (with the majority of the soluble zinc complexed to dissolved ligands in the medium), taking ca. 1 h for uncoated and ca. 6 h for polymer coated ones. Above 5.5 µg/mL, the results are consistent with the formation of zinc carbonate, keeping the solubilized zinc fixed to 67 µM of which only 0.45 µM is as free Zn(2+), i.e., not complexed to dissolved ligands. At these relatively high concentrations, NPs with an aliphatic polyether-coating show slower dissolution (i.e., slower free Zn(2+) release) and reprecipitation kinetics compared to those of uncoated NPs, requiring more than 48 h to reach thermodynamic equilibrium. Cytotoxicity (MTT) and DNA damage (Comet) assay dose-response curves for three epithelial cell lines suggest that dissolution and reprecipitation dominate for uncoated ZnO NPs. Transmission electron microscopy combined with the monitoring of intracellular Zn(2+) concentrations and ZnO-NP interactions with model lipid membranes indicate that an aliphatic polyether coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn(2+). Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by apparently frustrating cellular uptake. To limit toxicity, ZnO NPs with nonacicular morphologies and coatings that only weakly interact with cellular membranes are recommended.

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.

Intraoperative electron beam radiotherapy and extended surgical resection for gynecological pelvic recurrent malignancies with and without external beam radiation therapy: long-term outcomes.

OBJECTIVE: To analyze prognostic factors in patients treated with intraoperative electrons containing resective surgical rescue of locally recurrent gynecological cancer (LRGC). METHODS: From January 1995 to December 2012, 35 patients with LRGC [uterine cervix (57%), endometrial (20%), ovarian (17%), vagina (6%)] underwent extended [multiorgan (54%), bone (9%), soft tissue (54%), vascular (14%)] surgery and intraoperative electron-beam radiation therapy [IOERT (10-15 Gy)] to the pelvic recurrence tumor bed. Sixteen (46%) patients also received external beam radiation therapy [EBRT (30.6-50.4 Gy)]. Survival outcomes were estimated using the Kaplan-Meier method, and risk factors were identified by univariate and multivariate analyses. RESULTS: Median follow-up time for the entire cohort of patients was 46 months (range, 3-169). Ten-year rates for locoregional control (LRC) and overall survival (OS) were 58 and 16%, respectively. On multivariate analysis non-EBRT at the time of pelvic re-recurrence [HR 4.15; p = 0.02], no tumor fragmentation [HR 0.13; p=0.05] and time interval from primary tumor to LRR < 24 months [HR 5.16; p=0.01], retained significance with regard to LRR. Non-EBRT at the time of pelvic re-recurrence [HR 4.18; p=0.02] and time interval from primary tumor to LRR < 24 months [HR 6.67; p=0.02] showed a significant association with OS after adjustment for other covariates. CONCLUSIONS: EBRT treatment integrated for rescue, time interval for relapse ≥ 24 months, and not multi-involved fragmented resection specimens are associated with improved LRC in patients with LRGC in the pelvis. Present results suggest that a significant group of patients may benefit from EBRT treatment integrated with extended surgery and IOERT.

Peripheral motor axons of SOD1(G127X) mutant mice are susceptible to activity-dependent degeneration.

Motor neuron disorders may be associated with mitochondrial dysfunction, and repetitive electrical impulse conduction during energy restriction has been found to cause neuronal degeneration. The aim of this study was to investigate the vulnerability of motor axons of a presymptomatic late-onset, fast-progression SOD1(G127X) mouse model of amyotrophic lateral sclerosis to long-lasting, high-frequency repetitive activity. Tibial nerves were stimulated at ankle in 7 to 8-month-old SOD1(G127X) mice when they were clinically indistinguishable from wild-type (WT) mice. The evoked compound muscle action potentials and ascending compound nerve action potentials were recorded from plantar muscles and from the sciatic nerve, respectively. Repetitive stimulation (RS) was carried out in interrupted trains of 200-Hz for 3h. During the stimulation-sequence there was progressive conduction failure in WT and, to a lesser extent, in the SOD1(G127X). By contrast, 3 days after RS the electrophysiological responses remained reduced in the SOD1(G127X) but recovered completely in WT. Additionally, morphological studies showed Wallerian degeneration in the disease model. Nerve excitability testing by "threshold-tracking" showed that axons recovering from RS had changes in excitability suggestive of membrane hyperpolarization, which was smaller in the SOD1(G127X) than in WT. Our data provide proof-of-principle that SOD1(G127X) axons are less resistant to activity-induced changes in ion-concentrations. It is possible that in SOD1(G127X) there is inadequate energy-dependent Na(+)/K(+) pumping, which may lead to a lethal Na(+) overload.

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.

Aortic regurgitation due to quadricuspid aortic valve--case report and literature review.

Quadricuspid aortic valve is a rare variant of aortic semilunar valve, often being an unexpected discovery during cardiac surgery. We present the case of a 59 years old patient, who was admitted for dyspnea on light exertion and palpitations. The transthoracic echocardiography revealed severe aortic valve regurgitation due to a quadricuspid aortic valve, the result being confirmed by the transesophageal echo examination. The patient had a first class indication for aortic valve replacement and the surgical intervention was uneventful. The operative technique and case particularities are discussed in view of the literature published so far regarding this uncommon condition.

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.

Acute embolic events--myocardial infarction and stroke, in the presence of an interatrial septal aneurysm.

We will present the case of a 52 year old patient, admitted to our Department for exertional angina, with 2 recent acute events--inferior myocardial infarction and stroke. The coronary angiography revealed patent coronary arteries, without atherosclerotic lesions. The transthoracic echocardiography established the presence of an interatrial septal aneurysm with interatrial shunt. Under these circumstances, we have considered the presence of paradoxical embolism as a potential pathophysiological mechanism of the acute ischemic events. The percutaneous closure of the interatrial shunt to prevent the recurrence of embolic events will be discussed.

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.