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Mold Toxins Impact on the Kidneys:
Another Cause of Renal Failure?

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Cytotoxicity, metabolism and cellular uptake of the mycotoxin deoxynivalenol in human proximal tubule cells and lung fibroblasts in primary culture.

Kšnigs M, Lenczyk M, Schwerdt G, Holzinger H, Gekle M, Humpf HU.

Institut fŸr Lebensmittelchemie, WestfŠlische Wilhelms-UniversitŠt MŸnster, Corrensstrasse 45, 48149 MŸnster, Germany.

At the level of the whole animal, the toxic effects of the mycotoxin deoxynivalenol (DON) range from causing diarrhoea, vomiting, gastro-intestinal inflammation to necrosis of several tissues. It also affects the immune system and leads to kidney lesions. Although DON has been tested in different human and animal cell lines for its cytotoxicity, these tests might be limited due to the disadvantages of cell lines (e.g. immortalization, tumour derivation, longtime cultivation) and do not necessarily reflect the response of normal cells. In order to overcome this problem and to be closer to the human situation, we studied the effect of DON in human kidney epithelial cells (renal proximal tubule epithelial cells, RPTEC) and human lung fibroblasts (normal human lung fibroblast, NHLF) in primary culture. Cell viability, apoptotic and necrotic cell death, collagens I, III and IV as well as fibronectin secretion were determined. It could be demonstrated that DON has a distinct cytotoxic effect on human primary cells. A reduction in viability can be observed in both cell types, with fibroblasts reacting more sensitive. Furthermore, DON caused mainly necrotic cell death in kidney cells whereas mainly apoptotic cell death in fibroblasts. DON had no effect on collagen secretion in RPTEC cells. Collagen secretion was partially decreased in NHLF. In both cells, fibronectin secretion was reduced after 5 days of exposure. We also studied the metabolism and the cellular uptake of DON using LC-MS/MS. DON was neither metabolized by proximal tubule cells nor by fibroblasts. DON is incorporated into the cells whereas the intracellular amount of DON in kidney cells is higher than in fibroblasts. No accumulation of DON occurred in the cells.

Publication Types:
PMID: 17825972 [PubMed - indexed for MEDLINE]

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New molecular and field evidences for the implication of mycotoxins but not aristolochic acid in human nephropathy and urinary tract tumor.

Pfohl-Leszkowicz A, Tozlovanu M, Manderville R, Peraica M, Castegnaro M, Stefanovic V.

Laboratoire GŽnie chimique, UMR CNRS/INPT/UPS 5503, INP/ENSA Toulouse, Auzeville-Tolosane, France. leszkowicz@ensat.fr

To find out whether ochratoxin A (OTA), citrinin (CIT), aristolochic acids (AA) are etiologic agents of Balkan endemic nephropathy (BEN) or Chinese herbal nephrotoxicity, and associated urinary tract tumor (UTT), we have compared (i) in human kidney cell culture, the DNA adduct formation and persistence of OTA/CIT and AA adducts (ii) analyzed DNA adduct in several tumors from human kidney suspected to be exposed to either OTA and CIT, or AAs (iii) analyzed OTA, CIT, and AA in food. In kidney cell cultures, formation of specific OTA-DNA adduct and AA-DNA adduct were detected in the same range (around 10 adducts/10(9) nucleotides) and were time- and dose-dependent. After 2 days all disappeared. DNA adduct related to OTA and CIT are found in human kidney tissues from Balkans, France, and Belgium whereas no DNA adducts related to AA could be found in any tumors of BEN patients from Croatia, Bulgaria, or Serbia. No DNA adduct was found in kidney biopsy or necropsy of the French women suspected to be exposed to AA. OTA and CIT are more frequently found in rural area. AA was never detected. All these plead for implication of mycotoxins, especially OTA, in BEN and UTT.

Publication Types:
PMID: 17729220 [PubMed - in process]

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Growth factor induction of Cripto-1 shedding by glycosylphosphatidylinositol-phospholipase D and enhancement of endothelial cell migration.

Watanabe K, Bianco C, Strizzi L, Hamada S, Mancino M, Bailly V, Mo W, Wen D, Miatkowski K, Gonzales M, Sanicola M, Seno M, Salomon DS.

Tumor Growth Factor Section, Mammary Biology & Tumorigenesis Laboratory, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.

Publication Types:
PMID: 17720976 [PubMed - indexed for MEDLINE]

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Comment on:
Cereals consumption and risk for renal cell carcinoma: Can be hypothesized a role of mycotoxins?

Galvano F, Ritieni A, La Fauci L, Li Volti G, Di Giacomo C, Vanella L, Marcantoni C, Peraica M.

Publication Types:
PMID: 17640056 [PubMed - indexed for MEDLINE]

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Ochratoxin A as a potential etiologic factor in endemic nephropathy: lessons from toxicity studies in rats.

Mally A, Hard GC, Dekant W.

Department of Toxicology, University of WŸrzburg, Versbacher Str. 9, 97078 WŸrzburg, Germany. mally@toxi.uni-wuerzburg.de

Various reports suggest that chronic dietary exposure to ochratoxin A (OTA), a mycotoxin frequently detected in various food items may be linked to the pathogenesis of endemic nephropathy, a chronic tubulointerstitial kidney disease which occurs in geographically limited areas of the Balkan region. OTA is a potent nephrotoxin and renal carcinogen. However, the pathological lesions observed in kidneys of rats treated with OTA appear be rather different from the clinical and pathological characteristics of endemic nephropathy. Moreover, increasing evidence suggests that OTA does not bind to DNA but induces tumors by an epigenetic, thresholded mechanism. This implies that there is a dose below which no adverse health effects are expected to occur. Based on food consumption data and OTA serum concentrations, it appears that human exposure - even in areas with relatively high dietary exposure to OTA such as endemic villages - is several orders of magnitude below doses known to cause nephrotoxicity and tumor formation in laboratory animals. While it is undoubtedly important to encourage prevention of food contamination by OTA and other mycotoxins, these observations suggest that OTA is not likely to be an etiological factor involved in BEN and indicate a need to search for new clues for the etiology of this endemic kidney disease.

PMID: 17629386 [PubMed - indexed for MEDLINE]

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TRPM7 channel is sensitive to osmotic gradients in human kidney cells.

Bessac BF, Fleig A.

Laboratory of Cell and Molecular Signalling, Center for Biomedical Research at The Queen's Medical Center and John A. Burns School of Medicine at the University of Hawaii, Honolulu, Hawaii 96813, USA.

TRPM7 (transient-receptor-potential melastatin 7) is an ion channel with alpha-kinase function. TRPM7 is divalent-selective and regulated by a range of receptor-stimulated second messenger pathways, intracellular Mg-nucleotides, divalent and polyvalent cations and pH. TRPM7 is ubiquitously found in mammalian cells, including kidney, the responsible organ for osmolyte regulation, posing the question whether the channel is osmosensitive. Recent reports investigated the sensitivity of native TRPM7-like currents to cell swelling with contradictory results. Here, we assess the sensitivity of TRPM7 to both hypo- and hyperosmotic conditions and explored the involvement of the channel's kinase domain. We find that hypotonicity facilitates TRPM7 at elevated intracellular magnesium and Mg.ATP (3-4 mm), but has no effect in the absence of these solutes. Hypertonic conditions, in contrast, inhibit TRPM7 with an IC(50) of 430 mosmol l(-1). This inhibitory effect is maintained in the complete absence of intra- and extracellular divalent ions, although shifted to higher osmolarities (IC(50) = 510 mosmol l(-1)). TRPM7 senses osmotic gradients rather than ionic strength and this is independent of cAMP or not affected by cytochalasin D treatment. Furthermore, the kinase-domain deletion mutant of TRPM7 shows a similar behaviour to osmolarity as the wild-type protein, both in the presence and absence of divalent ions. This indicates that at least part of the osmosensitivity resides in the channel domain. Physiologically, TRPM7 channels do not seem to play an active role in regulatory volume changes, but rather those volume changes modulate TRPM7 activity through changes in the cytosolic concentrations of free Mg, Mg-nucleotides and a further unidentified factor. We conclude that TRPM7 senses osmotically induced changes primarily through molecular crowding of solutes that affect channel activity.

Publication Types:
PMID: 17510191 [PubMed - indexed for MEDLINE]

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Carry-over of Fusarium toxins (deoxynivalenol and zearalenone) from naturally contaminated wheat to pigs.

Goyarts T, DŠnicke S, Valenta H, UeberschŠr KH.

Institute of Animal Nutrition, Federal Agricultural Research Centre, Braunschweig, Germany. tanja.goyarts@fal.de

The frequent contamination of grain with the Fusarium toxins, deoxynivalenol (DON) and zearalenone (ZON), is an important issue in animal and human nutrition. However, data on the exposure of humans to these toxins through consumption of animal tissues exposed to Fusarium toxins (carry-over) are fragmentary. Therefore, residues of DON, ZON and their metabolites were determined in tissues and body fluids of pigs (female and castrated male) from a fattening trial. Pigs were fed a control (n = 6, 0.24 mg DON and 0.009 mg ZON per kg diet as fed) or a Fusarium toxin-contaminated diet (n = 12, 6.68 mg DON and 0.056 mg ZON per kg diet as fed) either ad libitum or for restrictive consumption for 12 weeks. After slaughter (96.3 +/- 11.6 kg live weight), the concentrations of DON and its metabolite, de-epoxy-DON, were measured in serum, bile, liver, kidney, musculus longissimus and back fat, while ZON and its metabolites, alpha- and beta-zearalenol (alpha-/beta-ZOL), were determined in serum, bile and liver. The mean carry-over factor of DON + de-epoxy-DON, defined as the concentration of both substances in the tissue/fluid divided by the DON concentration in the diet, for all pigs decreased from bile (0.1046 +/- 0.0653) >> kidney (0.0151 +/- 0.0070) > liver (0.0057 +/- 0.0043) > serum (0.0023 +/- 0.0018) > muscle (0.0016 +/- 0.0016) >> back fat (0.0002 +/- 0.0004). The time interval between the end of feeding and slaughter had no consistent effect on DON + de-epoxy-DON concentrations in the analysed specimen of Fusarium toxin-exposed pigs fed restrictively. No transfer of ZON and its metabolites could be observed into serum of pigs, while the mean carry-over factors of ZON + alpha-ZOL + beta-ZOL were 0.0094 +/- 0.0123 and 4.0 +/- 2.2 for liver and bile, respectively. Therefore, it can be concluded that serum is a reliable indicator for DON exposure, but an inappropriate parameter to deduce ZON exposure, which is better represented by bile concentration of ZON + alpha-ZOL + beta-ZOL. However, the exposure risk to humans by consumption of edible tissues of animals exposed to Fusarium toxins is negligible compared to the direct consumption of grain-based food.

Publication Types:
PMID: 17454110 [PubMed - indexed for MEDLINE]

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Citrinin and endosulfan induced maternal toxicity in pregnant Wistar rats: pathomorphological study.

Singh ND, Sharma AK, Dwivedi P, Patil RD, Kumar M.

Division of Pathology, Indian Veterinary Research Institute, Izatnagar- 243122, India.

Dietary exposures to environmental food pollutants such as mycotoxin(s) or pesticide(s) have gained immense significance due to their adverse effects on production and reproduction in animal and human populations. The present investigation was conducted to evaluate the maternal toxicity of citrinin (CIT) and endosulfan administered per os either alone or in combination in pregnant rats during gestational days 6-20. CIT (group I, 10 mg kg(-1) feed, through diet) and endosulfan (group II, 1 mg kg(-1) body weight, by oral intubation) when administered either alone or in combination (group III) in Wistar rats caused clinical signs of toxicity and pathomorphological changes in all the toxin treated groups, the severity being more pronounced in the combination treatment compared with that observed in the control (group IV). The rate of fetal resorptions was highest (22.22%) in the combination treatment followed by endosulfan (16.48%) and CIT (12.50%) treatment groups compared with the control group (3.86%). The histopathological changes such as engorged vasculature, vacuolar degeneration and karyomegaly in liver; congestion, tubular degeneration and cast formation in kidneys; vascular changes and hemosiderosis in uterus and lymphocytic depletion and apoptosis in the lymphoid organs were recorded in the animals of the toxin treated groups. The lesions were consistent and more severe in the combination treatment group compared with the individual treatment groups, suggesting an additive interaction of CIT and endosulfan in inducing maternal toxicity in Wistar rats.

PMID: 17429798 [PubMed - indexed for MEDLINE]

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The chloride dependence of the human organic anion transporter 1 (hOAT1) is blunted by mutation of a single amino acid.

Rizwan AN, Krick W, Burckhardt G.

Abteilung Vegetative Physiologie und Pathophysiologie, Zentrum Physiologie und Pathophysiologie, Georg-August-UniversitŠt Gšttingen, Humboldtallee 23, 37073 Gšttingen, Germany.

Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.

Publication Types:
PMID: 17353191 [PubMed - indexed for MEDLINE]

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In vitro gene expression data supporting a DNA non-reactive genotoxic mechanism for ochratoxin A.

Arbillaga L, Azqueta A, van Delft JH, L—pez de Cerain A.

Department of Food Sciences and Toxicology, Faculty of Pharmacy, University of Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain.

Ochratoxin A (OTA) is a mycotoxin often found in cereals and agricultural products. There is unequivocal evidence of renal carcinogenicity of OTA in male rats, although the mechanism of action is unknown. At present, available data support an epigenetic mechanism (DNA non-reactive) resulting from oxidative stress and cytotoxicity, because a direct OTA interaction with DNA has not been demonstrated. Genotoxic mechanism (DNA-reactive vs. DNA non-reactive) may have implications on human risk assessment. Therefore, the aim of the present work was to identify biological pathways modulated by OTA in vitro in a human renal cell line (HK-2) to contribute to the elucidation of the mechanism of OTA toxicity. For that purpose, cells were exposed to 50 microM OTA during 6 and 24 h, and gene expression profiles were analyzed using Affymetrix Human Genome U133 A 2.0 Gene Chips. Under the same experimental conditions, genotoxicity was evaluated by the modified comet assay using FPG and Endo III to detect oxidative DNA damage, and intracellular ROS level by the H(2)DCF assay. After 6 h, with slight cytotoxicity (83% survival), genes involved in mitochondrial electron transport chain were up-regulated; and after 24 h, with a more pronounced cytotoxicity (51% survival), genes implicated in oxidative stress response were also up-regulated. Increase in intracellular ROS level and oxidative DNA damage was evident at both exposure times being more pronounced with high cytotoxicity. On the contrary, up-regulation of genes implicated in DNA damage response, as cell cycle control or apoptosis, was not detected at any exposure time. In conclusion, these results support a DNA non-reactive mechanism of OTA genotoxicity.

Publication Types:
PMID: 17316727 [PubMed - indexed for MEDLINE]

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Fumonisin B(1): oxidative status and DNA damage in rats.

Domijan AM, Zeljezi D, Mili M, Peraica M.

Unit of Toxicology, Institute for Medical Research and Occupational Health, Ksaverska c. 2, 10000 Zagreb, Croatia. adomijan@imi.hr

Fumonisin B(1) (FB(1)) is a carcinogenic mycotoxin involved in several animal diseases and assumed to be involved in the etiology of some human tumors. FB(1) disturbs the metabolism of sphinganine (Sa) and sphingosine (So), increasing the ratio of their concentrations (Sa/So). FB(1) is mutagenic in cell cultures, but the mechanism of its genotoxicity is not understood. The aim of this study was to see whether DNA lesions in kidney and liver cells of rats treated with FB(1) were related to the changes in the oxidative status or to the disturbance of the sphingolipid metabolism. Male Wistar rats were receiving either FB(1) (0.5 mg/kg b.w./day, i.p. for 2 or 7 days) or solvent only and were sacrificed 24 h after the last treatment. The ratio of Sa and So concentrations and parameters of oxidative status (catalytic activity of catalase and the concentrations of protein carbonyls and malondialdehyde, MDA) were measured in plasma and liver and kidney homogenates, while DNA damage was measured in liver and kidney using the comet assay. In plasma and liver and kidney homogenates catalase activity and the concentrations of protein carbonyls and MDA were not affected by the 2-day treatment with FB(1), but the ratio of Sa and So in plasma and liver and kidney homogenates was significantly higher than in controls (0.99+/-0.27 versus 0.38+/-0.08, 1.05+/-0.12 versus 0.59+/-0.09 and 4.51+/-0.51 versus 0.54+/-0.17, respectively) (p<0.05). After the 2-day treatment, the tail length and tail intensity measured with the comet assay in the liver homogenate did not change, while in the kidney homogenate, the difference between the treated and control animals was significant in both the tail length (26.4+/-0.7 microm versus 14.6+/-0.1 microm) and tail intensity (8.0+/-0.4% versus 1.7+/-0.02% DNA) (p<0.05). After the 7-day treatment all measured parameters significantly differed from controls (p<0.05). This study showed that FB(1) causes DNA lesions in the kidney of experimental animals before affecting the catalytic activity of catalase and the concentration of protein carbonyls and MDA. The ratio of Sa and So significantly increases in all tissues already after 2-day treatment thus indicating that the metabolism of sphingolipids may have an important role in the DNA damage caused by FB(1).

Publication Types:
PMID: 17291664 [PubMed - indexed for MEDLINE]

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Long-term effects of ochratoxin A on fibrosis and cell death in human proximal tubule or fibroblast cells in primary culture.

Schwerdt G, Holzinger H, Sauvant C, Kšnigs M, Humpf HU, Gekle M.

Physiologisches Institut, UniversitŠt WŸrzburg, Ršntgenring 9, D-97070 WŸrzburg, Germany. gerald.schwerdt@mail.uni-wuerzburg.de

Ochratoxin A (OTA) is a mycotoxin produced by several fungi which grow on human food source material. Consumption of OTA is almost unavoidable. The consumption leads to low but detectable amounts of OTA in human blood. Risk assessment of OTA is based on studies performed either in animals or cultured cells. So far, mainly cell lines of different origin were used. To be as close as possible to the situation in humans with respect to the experimental setup, we studied the effect of OTA in human proximal tubule cells (RPTEC) and human fibroblasts in primary culture. OTA was administered at concentrations ranging from 0.3 nmol/l up to 10 micromol/l for time periods up to 14 days. Apoptotic and necrotic cell death, collagen I, III, IV and fibronectin secretion as well as NF-kappaB activation were studied. Under our experimental conditions OTA exerted comparable effects on caspase-3 activity and necrosis in both cell types, however RPTEC were more sensitive (order of 10). Surprisingly, very low concentrations of OTA (0.3-10nM) led to cell hypertrophy during prolonged exposure (14 days). RPTEC but not fibroblasts responded with an increase of NF-kappaB activity and collagen III as well as fibronectin secretion underlining the profibrotic action of OTA in the kidney. Collagen I and IV secretion was only slightly changed. The results presented here give good reasons to re-asses the risk of OTA consumption leading to low blood concentrations which have so far been considered harmless.

Publication Types:
PMID: 17218050 [PubMed - indexed for MEDLINE]

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Ochratoxin A in nephropathic patients from two cities of central zone in Portugal.

Dinis AM, Lino CM, Pena AS.

Group of Bromatology-CEF, Faculty of Pharmacy, University of Coimbra, 3000 Coimbra, Portugal.

Ochratoxin A (OTA) produced by Aspergillus and Penicillium genera contaminates several foods. OTA is nephrotoxic to all animal species studied so far, and most likely to humans, who show the longest half-life for elimination of this toxin among all examined species. OTA has other toxic effects such as teratogenicity, immunotoxicity, genotoxicity, and is also mutagenic and carcinogenic, all of which lead to life-threatening pathologies through several molecular pathways. A sensitive, specific and rapid method applying high performance liquid chromatography coupled to a spectrofluorimeter for the determination of ochratoxin A in human serum was validated. Serum samples were extracted with chloroform-orthophosphoric acid, and cleaned-up through immunoaffinity column (IAC). The separation and identification was performed by HPLC coupled to a spectrofluorimeter, and, after OTA methylation, the confirmation was achieved. Chromatographic separation of the analyte was performed on a reverse phase column with a mobile phase of water:acetonitrile:glacial acetic acid (49.5:49.5:1.0). Linearity was established between the range of 1 and 10 ng/ml. Under the optimized conditions, the recoveries were higher than 83.0% for all fortification levels. The intra-day precision oscillated between 8.0 and 5.0% at levels of 0.25 and 0.5 microg/l, while the inter-day precision was in the range of 10.7-16.0%. The limit of quantification of the method was 0.05 microg/l. The method is appropriate for quantitative determination of OTA in human serum and has been successfully applied to the analysis of OTA in haemodialysis patients from two principal cities of Portugal, in order to evaluate its exposure degree. Levels of OTA in Coimbra were higher than in Aveiro, 0.50 microg/l versus 0.49 microg/l. In respect to gender, levels of OTA were higher in males from Aveiro than in females, 0.52 microg/l versus 0.44 microg/l, and in Coimbra were similar, 0.50 microg/l versus 0.51 microg/l. However, in none of the cases, significant statistical differences were found.

Publication Types:
PMID: 17208405 [PubMed - indexed for MEDLINE]

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Erratum in:
  • Mol Nutr Food Res. 2007 Sep;51(9):1192.

Comment in:
Ochratoxin A: An overview on toxicity and carcinogenicity in animals and humans.

Pfohl-Leszkowicz A, Manderville RA.

Laboratoire de GŽnie Chimique, UMR CNRS/INPT/UPS 5503, INP/ENSA Toulouse, Auzeville-Tolosane, France.

Ochratoxin A (OTA) is a ubiquitous mycotoxin produced by fungi of improperly stored food products. OTA is nephrotoxic and is suspected of being the main etiological agent responsible for human Balkan endemic nephropathy (BEN) and associated urinary tract tumours. Striking similarities between OTA-induced porcine nephropathy in pigs and BEN in humans are observed. International Agency for Research on Cancer (IARC) has classified OTA as a possible human carcinogen (group 2B). Currently, the mode of carcinogenic action by OTA is unknown. OTA is genotoxic following oxidative metabolism. This activity is thought to play a central role in OTA-mediated carcinogenesis and may be divided into direct (covalent DNA adduction) and indirect (oxidative DNA damage) mechanisms of action. Evidence for a direct mode of genotoxicity has been derived from the sensitive 32P-postlabelling assay. OTA facilitates guanine-specific DNA adducts in vitro and in rat and pig kidney orally dosed, one adduct comigrates with a synthetic carbon (C)-bonded C8-dG OTA adduct standard. In this paper, our current understanding of OTA toxicity and carcinogenicity are reviewed. The available evidence suggests that OTA is a genotoxic carcinogen by induction of oxidative DNA lesions coupled with direct DNA adducts via quinone formation. This mechanism of action should be used to establish acceptable intake levels of OTA from human food sources.

Publication Types:
PMID: 17195275 [PubMed - indexed for MEDLINE]

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Citrinin and endosulfan induced teratogenic effects in Wistar rats.

Singh ND, Sharma AK, Dwivedi P, Patil RD, Kumar M.

Division of Pathology, Indian Veterinary Research Institute, Izatnagar- 243122, India.

Dietary exposures to food pollutants such as mycotoxin(s) or pesticide(s) are most significant due to their adverse effects on the production and reproduction in animals and the human population. The present investigation was conducted to evaluate the teratogenic potential of citrinin (CIT) and endosulfan either alone or in combination in pregnant rats during gestational days 6-20. Endosulfan (1 mg kg(-1) body weight, by oral intubation) and CIT (10 mg kg(-1) feed, through diet) when administered either alone or in combination in pregnant rats caused significant teratogenic effects in the developing fetuses. There was no maternal mortality, however, reduced maternal weight gain and number of live fetuses and increased fetal resorptions were recorded in all the treated groups. The fetal body weights and crown to rump lengths were significantly decreased and the per cent gross, visceral and skeletal anomalies were significantly increased in the fetuses of dams of all the treated groups. The internal hydrocephalus, cerebellar hypoplasia, microphthalmia, contracted and notched kidneys, multilobulated liver, dilated renal pelvis, incomplete ossification of skull bones, rib anomalies and sacral and caudal vertebrae agenesis were the important fetal malformations. The occurrence of fetal gross, skeletal and visceral malformations was more severe in the combination group, suggesting an additive interaction of CIT and endosulfan in inducing developmental toxicity in Wistar rats.

Publication Types:
PMID: 17186572 [PubMed - indexed for MEDLINE]

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[Acute liver failure--medical viewpoints]

[Article in German]

Schattenberg JM, Galle PR, Schuchmann M.

Medizinische Klinik und Poliklinik, Johannes-Gutenberg-UniversitŠt, Mainz.

Acute liver failure is a rare disease that can cause death in the majority of untreated cases. Sudden loss of liver function in the absence of a preexisting liver disease is considered the true form and has to be distinguished from impaired function following exacerbation of an underlying liver disease (acute or chronic failure). Common causes include acute viral hepatitis, drug induced liver injury (DILI) and toxins. The loss of the excretory and synthetic function of the liver marks the clinical presentation and results in icterus, coagulopathy and encephalopathy. Additionally impairment of renal function and sepsis occur and contribute to the high mortality of this disease. The activation of cell death mechanisms (apoptosis) leading to a reductio of viable, functional liver tissue is considered to be an important pathophysiologic mechanism. Curative therapy of this disease includes liver transplantation that has been performed in Germany for the first time in 1969. In the year 2004 a total of 91 liver transplantation were performed for acute liver failure (10.3% of all transplants) in German transplant centers.

Publication Types:
PMID: 17176926 [PubMed - indexed for MEDLINE]

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Oxidative DNA damage induced by Ochratoxin A in the HK-2 human kidney cell line: evidence of the relationship with cytotoxicity.

Arbillaga L, Azqueta A, Ezpeleta O, L—pez de Cerain A.

Department of Food Sciences and Toxicology, Faculty of Pharmacy, University of Navarra C/ Irunlarrea 1, 31008 Pamplona, Spain.

Ochratoxin A (OTA) is a mycotoxin produced by species of the genera Aspergillus and Penicillium. The kidneys are the target organ of this mycotoxin and it is considered a potent renal carcinogen in male rats. The mechanisms of its genotoxicity and carcinogenicity have been studied thoroughly, but controversial results have been published. The aim of this study was to evaluate the ability of OTA to produce single-strand DNA breaks and oxidative DNA damage in the human renal proximal tubular epithelial cell line (HK-2), due to the fact that there is no study on human kidney cells as the toxic target. In addition, we attempted to determine if biotransformation processes mediate OTA genotoxicity. Therefore, single-cell gel electrophoresis assay (comet assay) was performed after 3h- and 6h-treatments using different OTA concentrations, both cytotoxic and non-cytotoxic, in order to be able to distinguish a genotoxic effect of the mycotoxin from an indirect effect derived from its general cellular toxicity. No effect was shown where no cytotoxicity was found, both in the presence and in the absence of metabolic activation (10% rat liver S9-mix). However, oxidative DNA damage was shown at cytotoxic concentrations when formamidopyrimidine DNA glycosylase (FPG) and endonucleaseIII (EndoIII) were introduced in the assay with or without metabolic activation. Furthermore, at these concentrations, an elevation of reactive oxygen species was measured and pre-incubation with N-acetyl-L-cysteine was able to produce a slight protective effect on OTA-induced oxidative DNA damage as well as cytotoxicity. These data suggest that OTA is not acting as a direct genotoxic carcinogen and that oxidative stress is implicated in the genotoxicity and cytotoxicity observed in these human renal cells.

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PMID: 17130176 [PubMed - indexed for MEDLINE]

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Survey of pigs' kidneys with lesions consistent with PMWS and PDNS and ochratoxicosis. Part 1: concentrations and prevalence of ochratoxin A.

Gresham A, Done S, Livesey C, Macdonald S, Chan D, Sayers R, Clark C, Kemp P.

Veterinary Laboratories Agency, Bury St Edmunds, Rougham Hill, Bury St Edmunds, Suffolk.

One thousand condemned pigs' kidneys were collected in February 2002 from two pig abattoirs in England to assess the possible contribution of ochratoxicosis to postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS); 250 of the kidneys with macroscopic lesions consistent with nephrosis/nephritis (pale or white cortical lesions) were selected, and the concentration of ochratoxin A was measured in samples of renal cortex by high-performance liquid chromatography (HPLC). Low concentrations were detected in 230 (92 per cent) of the kidneys tested, and in 41 (16.4 per cent) of them the concentration was below the limit of quantification of 0.2 microg/kg. In 187 (74.8 per cent) of the kidneys, the concentration was more than 0.2 microg/kg, and the highest concentration detected was 2.3 microg/kg. The mean (sd) concentration was 0.31 (0.33) microg/kg. The identification of ochratoxin A was confirmed by mass spectrometry. The concentrations of ochratoxin A did not exceed the threshold assessed by the Food Standards Agency to be safe for human food.

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PMID: 17127757 [PubMed - indexed for MEDLINE]

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Reduction in antioxidant defenses may contribute to ochratoxin A toxicity and carcinogenicity.

Cavin C, Delatour T, Marin-Kuan M, HolzhŠuser D, Higgins L, Bezenon C, Guignard G, Junod S, Richoz-Payot J, Gremaud E, Hayes JD, Nestler S, Mantle P, Schilter B.

Quality and Safety Department, NestlŽ Research Center, CH-1000 Lausanne 26, Switzerland. christophe.cavin@rdls.nestle.com

Ochratoxin A (OTA) is a renal carcinogen in rodents. Its human health significance is unclear. It likely depends upon the mechanism of carcinogenesis. In a previous microarray study a reduction in nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-dependent gene expression was observed in the kidney but not in the liver of rats fed OTA up to 12 months. Nrf2 regulates detoxification and antioxidant gene expression. The present report shows that OTA decreased the protein expression of several markers of the Nrf2-regulated gene battery in kidney in vivo indicating that the effects observed at mRNA level may be of biological significance. The OTA-mediated Nrf2 response could be reproduced in an NRK renal cell line and in primary hepatocyte cultures. In in vitro systems, an OTA-mediated inhibition of Nrf2 activity was demonstrated by electrophoretic mobility shift and Antioxidant Regulatory Element-driven luciferase reporter assays. The reduction of Nrf2-regulated gene expression resulted in oxidative DNA damage as evidenced by formation of abasic sites in vitro and confirmed in kidney in vivo. All OTA-mediated effects observed were prevented by pretreatment of cell cultures with inducers of Nrf2 activity. Our data suggest that reduction of cellular defense against oxidative stress by Nrf2 inhibition may be a plausible mechanism of OTA nephrotoxicity and carcinogenicity.

PMID: 17110534 [PubMed - indexed for MEDLINE]

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Caspase-dependent and -independent induction of phosphatidylserine externalization during apoptosis in human renal carcinoma Cak(1)-1 and A-498 cells.

Lock EA, Reed CJ, Kinsey GR, Schnellmann RG.

Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC 29425, USA. e.lock@ljmu.ac.uk

Renal cell carcinoma is the most common neoplasm occurring in the kidney and is largely resistant to current chemotherapy. Understanding the mechanisms involved in renal carcinoma cell death may lead to novel and more effective therapies. In Cak(i)-1 renal cancer cells, using phosphatidylserine externalization as a marker of apoptosis, the anti-cancer drugs 5-fluorouracil (5-FU), and its pro-drugs, doxifluridine (Dox) and floxuridine (Flox) proceeds via a caspase-dependent mechanism. In contrast, phosphatidylserine externalization produced by staurosporine in the renal cancer cell lines Cak(i)-1 and A-498 proceeds via a caspase-independent mechanism. That is, the pan caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) did not ameliorate annexin V binding, cell shrinkage or changes in nuclear morphology. Subsequent experiments were conducted to determine mediators of phosphatidylserine externalization, using annexin V binding, when caspases were inhibited. Prior treatment of A-498 cells with cathepsin B (CA74 methyl ester), cathespsin D (pepstatin A) or calpain inhibitors (calpeptin, E64d) in the presence or absence of ZVAD did not ameliorate annexin V binding. The endonuclease inhibitor aurintricarboxylic acid (ATA), phospholipase A(2) inhibitor bromoenol lactone (BEL), protein synthesis inhibitor cycloheximide (CH) and chloride channel blockers niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) all had no effect on staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. We also modulated sphingomyelin and the de novo pathways of ceramide synthesis and found no amelioration of staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. These results indicate that 5-FU, Dox and Flox induce externalization of phosphatidylserine during apoptosis in Cak(i)-1 renal cancer cells primarily through a caspase-dependent mechanism and that externalization of phosphatidylserine during apoptosis produced by staurosporine in the renal cancer cell line A-498 is independent of many of the common signaling pathways known to be involved in this process.

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PMID: 17097791 [PubMed - indexed for MEDLINE]

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Effects of repeated ochratoxin exposure on renal cells in vitro.

Heussner AH, O'Brien E, Dietrich DR.

Environmental Toxicology, University of Konstanz, P.O. Box X-918, 78457 Konstanz, Germany.

In the present study an in vitro model of subchronic repeated exposure to OTA and OTB was employed to generate ochratoxin-derived subpopulations of human and porcine proximal tubular cells (HKC, IHKE, PKC, LLC-PK1). These cell subpopulations were subsequently used to investigate effects on cell proliferation rates, expression of marker proteins (cytokeratins, vimentin) and the acute cytotoxicity of OTA and OTB (MTT reduction, neutral red uptake, cell number). The hypothesis was tested whether repeated exposure at moderate concentrations of these toxins could provide for a reduced sensitivity of selected cell subpopulations to subsequent toxin exposure. Despite the observed increased cell population doubling times and the reduced sensitivity toward OTA and OTB exposure of some cell types, with the exception of the primary human epithelial cells, no overt changes in the expression of cytokeratin and vimentin could be determined. The presented data, however suggest that repeated exposure of renal epithelial cells to ochratoxins A or B will provide for a subpopulation of cells with reduced ochratoxin-sensitivity and alterations in growth characteristics.

PMID: 17045452 [PubMed - indexed for MEDLINE]

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Genotoxicity of the hydroquinone metabolite of ochratoxin A: structure-activity relationships for covalent DNA adduction.

Tozlovanu M, Faucet-Marquis V, Pfohl-Leszkowicz A, Manderville RA.

Ecole Nationale SupŽrieure Agronomique de Toulouse, Lab. Genie Chimique, UMR-CNRS 5503, Department Toxicology & Food Safety, Agrobiopole-BP 107-F31326 Castanet-Tolosan Cedex, France.

Ochratoxin A (OTA) is a mycotoxin that shows potent nephrotoxicity and renal carcinogenicity in rodents. One hypothesis for OTA-induced tumor formation is based on its genotoxic properties that are promoted by oxidative metabolism. Like other chlorinated phenols, OTA undergoes an oxidative dechlorination process to generate a quinone (OTQ)/hydroquinone (OTHQ) redox couple that may play a role in OTA-mediated genotoxicity. To determine whether the OTQ/OTHQ redox couple of OTA contributes to genotoxicity, the DNA adduction properties, as evidenced by the (32)P-postlabeling technique, of the hydroquinone analogue (OTHQ) have been compared to OTA in the absence and presence of metabolic activation (pig kidney microsomes) and within human bronchial epithelial (WI26) and human kidney (HK2) cells. Our experiments show that OTHQ generates DNA adduct spots in the absence of metabolic activation. These adducts are ascribed to covalent DNA adduction by OTQ generated through autoxidation of the hydroquinone precursor, OTHQ. Although OTA does not interact with DNA in the absence of metabolism, the OTQ-mediated DNA adduct spots noted with OTHQ are also observed with OTA following treatment with pig kidney microsomes and NADPH, suggesting that OTA undergoes oxidative activation to OTQ by cytochrome P450 or enzymes with peroxidase activity. Comparison of DNA adduction by OTHQ and OTA in human cell lines shows that OTQ-mediated adduct spots form in a dose- and time-dependent manner. The adduct spots form at a faster rate with OTHQ, which is consistent with more facile generation of OTQ from its hydroquinone precursor. These results establish structure-activity relationships for OTA-mediated DNA adduction and provide new evidence for the potential role of the OTQ/OTHQ redox couple in OTA-induced genotoxicity.

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PMID: 16978030 [PubMed - indexed for MEDLINE]

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Three controversial issues in extracorporeal toxin removal.

Feinfeld DA, Rosenberg JW, Winchester JF.

Division of Nephrology and Hypertension, Beth Israel Medical Center, New York, New York 10003, USA. dfeinfel@chpnet.org

The optimal method of extracorporeal removal of many toxic compounds is often a matter of debate. Due to the lack of well-designed studies, we are often left with circumstantial evidence, and we must exercise our best clinical judgment as to whether extracorporeal drug removal is beneficial and if so, by what method. It is clear, however, that rapidity in toxin removal is beneficial. We present three issues dealing with extracorporeal removal of toxins for which there is no definitive answer but which may arise in clinical practice. The first is whether continuous renal replacement therapy (CRRT) is better at removing dialyzable toxins than classic hemodialysis. The second is whether charcoal hemoperfusion is at all useful in treating paraquat poisoning. Finally, is any modality of extracorporeal treatment useful in the treatment of amatoxin poisoning? After a thorough literature review, it is evident that definitive answers are not strikingly apparent. However, extracorporeal treatment in the latter two instances may have potential benefit and may be the only hope for patient survival. Due to the urgent nature of treatment for poisoning, as well as the somewhat obscure nature of these issues, there may never be well-designed evidence-based studies to help guide us. In the meantime, we must continue to use less than ideal evidence and our own experience in dealing with these controversial issues to guide our decision-making process.

Publication Types:
PMID: 16970731 [PubMed - indexed for MEDLINE]

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Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra-Golgi pH.

L‡zaro-DiŽguez F, JimŽnez N, Barth H, Koster AJ, Renau-Piqueras J, Llopis JL, Burger KN, Egea G.

Departament de Biologia Cellular i Anatomia Patol˜gica, Facultat de Medicina, Universitat de Barcelona, 08036 Barcelona, Spain.

Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis. Copyright 2006 Wiley-Liss, Inc.

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PMID: 16960891 [PubMed - indexed for MEDLINE]

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Thermal degradation of the Fusarium mycotoxin deoxynivalenol.

Bretz M, Beyer M, Cramer B, Knecht A, Humpf HU.

Institut fŸr Lebensmittelchemie, WestfŠlische Wilhelms-UniversitŠt MŸnster, Corrensstrasse 45, 48149 MŸnster, Germany.

Deoxynivalenol (DON) is a toxic secondary metabolite produced by molds of the Fusarium genus, which are able to infect cereal crops in the field. Concerning its rate of occurrence and mean concentration, DON is one of the most important mycotoxins in cereal commodities. Its toxic effects range from causing diarrhea, vomiting, and gastro-intestinal inflammation to noncompetitive inhibition of the biosynthesis of proteins in eukaryotic cells. To study the stability of DON under food-processing conditions such as cooking or baking, we performed model heating experiments and screened the residue for degradation products. Heating of DON and 3-acetyldeoxynivalenol (3-AcDON), especially under alkaline conditions, gave a mixture of compounds, which were isolated and structurally elucidated by NMR and MS experiments. Three of these compounds were already known (norDON A, norDON B, and norDON C), while four were new and named 9-hydroxymethyl DON lactone, norDON D, norDON E, and norDON F. The significance of the DON degradation products was checked by analyzing commercially available food samples. norDON A, B, and C were detected in 29-66% of the samples in mean concentrations ranging from 3 to 15 microg/kg. Furthermore, cell culture experiments using IHKE cells showed that the compounds that were detected in food samples are less cytotoxic in the formazan dye cytotoxicity assay compared to DON. Whereas DON revealed a median effective concentration (EC50) at 1.1 micromol/L, all other compounds did not show any significant effect up to 100 micromol/L. These findings indicate that the degradation of DON under thermal treatment might reduce the toxicity of DON contaminated food.

PMID: 16910743 [PubMed - indexed for MEDLINE]

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Does aflatoxin as an environmental mycotoxin adversely affect the renal and hepatic functions of Egyptian lactating mothers and their infants? A preliminary report.

Hassan AM, Sheashaa HA, Abdel Fatah MF, Ibrahim AZ, Gaber OA.

Pediatric Department, Zagazig University, Egypt.

BACKGROUNDS/AIMS: Aflatoxin as a mycotoxin constitutes a real human threat. Its presence in human milk was previously reported in different countries. This work is the first Egyptian report that aimed to assess the presence of aflatoxin in both mothers' milk and the infants' sera and studied its correlation with infants' kidney functions. METHODS: Fifty healthy breast lactating mothers and their infants who were exclusively breast fed for at least 4 months were included. All of them were subjected to thorough laboratory evaluation including determination of aflatoxin concentration by high performance liquid chromatography. RESULTS: Twenty-four mothers (48%) and their infants had been contaminated with aflatoxin with the following mean contamination levels (ng/ml); mothers' serum of 8.9+/-4.2, mothers' milk of 1.9+/-0.6 and infants' serum of 1.8+/-0.9. The presence of this contamination level is not associated with renal or hepatic dysfunction. CONCLUSION: Mothers and their infants in our locality showed a relatively high aflatoxin contamination rate. We did not find a correlation of this contamination level and either renal or hepatic dysfunction.

PMID: 16868707 [PubMed - indexed for MEDLINE]

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Enhanced expression of glucose transporter 1 on erythrocyte membrane in hemodialysis patients: the possible role in erythrocyte ascorbate recycling.

Wann JG, Lin CS, Chang LC, Hsu YH, Chien CT, Tai DW, Chen JS, Yang CC, Yeung LK, Chen CF.

Department of Physiology, College of Medicine, National Taiwan University Hospital, Taipei, Taiwan.

BACKGROUND: Human erythrocytes can take up dehydroascorbate on the glucose transporter 1 (GLUT 1) and reduce it to ascorbate. Intraerythrocyte ascorbate was proved to be directly responsible for decreased oxidation of extraerythrocytic ascorbate. In addition to spontaneous and irreversible loss of ascorbate in plasma, the hemodialysis (HD) process itself consumes plasma ascorbate. However, intraerythrocyte ascorbate status in uremic patients during HD has yet to be reported. METHODS: Plasma and intraerythrocyte ascorbate, dehydroascorbate, GLUT 1 expression on erythrocyte membranes, and in vitro studies of "erythrocyte ascorbate recycling" were investigated in age- and sex-matched healthy subjects (control group) and HD patients (HD group). RESULTS: Intraerythrocyte ascorbate concentrations decreased after 1 HD session compared with pre-HD and recovered to pre-HD values 2 days later, whereas plasma ascorbate concentrations did not recover. In vitro studies suggested that erythrocytes of HD patients have a stronger ability to maintain intracellular ascorbate concentrations compared with healthy subjects. This ability could be inhibited by cytochalasin B (GLUT 1 inhibitor). We also found increased GLUT 1 expression (P = 0.002) on erythrocyte membranes in the HD group compared with the control group. CONCLUSION: Erythrocytes of uremic patients lost large amounts of ascorbate during HD, but regained it to the pre-HD level 2 days later. Enhanced GLUT 1 expression on erythrocyte membranes for HD patients may contribute to better preservation of intracellular ascorbate compared with healthy subjects.

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PMID: 16731301 [PubMed - indexed for MEDLINE]

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Balkan endemic nephropathy: role of ochratoxins A through biomarkers.

Castegnaro M, Canadas D, Vrabcheva T, Petkova-Bocharova T, Chernozemsky IN, Pfohl-Leszkowicz A.

International Agency of Research on Cancer, Lyon, France. castegnaro.marcel@free.fr

Several studies implicated mycotoxins, in endemic kidney disease geographically limited to Balkan region (Balkan endemic nephropathy (BEN)). In Bulgaria, much higher prevalence of ochratoxin A (OTA), exceeding 2 microg/L, was observed in the blood of affected population. OTA is found more often in the urine of people living in BEN-endemic villages. To confirm and quantify exposure to OTA in Vratza district, we followed up OTA intake for 1 month, OTA in blood and urine from healthy (20-30 years old) volunteers, from two villages with high risk for BEN disease. Food samples were collected daily, blood and urine at the beginning of each week. Relations between increasing OTA intake, blood concentration and elimination of OTA in urine have been studied in rats. Average weekly intake of OTA varies from 1.9 to 206 ng/kg body weight, twice tolerable weekly intake recommended by JECFA. OTA blood concentrations are in the same range as previously reported in this region with concentrations reaching 10 microg/L. Weekly OTA food intake is not directly correlated with blood and urine concentrations. Biomarkers of biological effects such as DNA adducts were detected in patients affected by urinary tract tumours (UTT) and in rat study. All these plead for the implication of OTA, in BEN and UTT.

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PMID: 16715544 [PubMed - indexed for MEDLINE]

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How much should we involve genetic and environmental factors in the risk assessment of mycotoxins in humans?

Creppy EE, Moukha S, Bacha H, Carratu MR.

Dept of Toxicology, University of Bordeaux 2, 146, rue L—o-Saignat, 33076 Bordeaux, France. edmond.creppy@tox.u-bordeaux2.fr

Despite consented efforts in prevention, mycotoxins remain a problem of human health concern in several parts of the world including developed countries. Within the same range of toxins concentrations in the blood some people develop a disease while others do not. Could this inequality in front of mycotoxins effects be explained by environment factors and/or genetic predisposition? Among recent advances in environmental health research Correlation between chronic diseases and mycotoxins in humans deserves attention through several questions: Are genetic factors involved in disease causation of mycotoxins? How much are these factors currently taken into account for mycotoxins risk assessment and how much should we involve them? Answers are still to come. Genetic and environment factors deserve therefore more attention when dealing with regulatory limits, since among the general population, those who are at risk and will develop specific diseases are likely those bearing genetic predispositions. We have addressed these questions for the specific case of ochratoxin A in humans by investigating in Tunisia, county of Jelma, in four rural families forming a household of 21 persons all exposed to ochratoxin A in diet. Our results confirm that ochratoxin A induces chronic tubular nephropathy in humans and mainly point at those having the HLA haplotype A3, B27/35, DR7 to be more sensitive to the disease for quantitatively similar or lower exposure. Persons with such haplotype were found to bear chronic interstitial nephropathy with tubular karyomegalic cells while others were apparently healthy. Godin et al. (1996) in France have also found in sibling (a sister and her brother from urban area) that have similar HLA haplotype B35-patern, OTA-related renal tubulopathy with mild proteinuria including beta2-microglobulinuria. Several mechanisms are discussed that could be put ahead to explain how the HLA haplotype could lead to tubular cells lyses and renal failure. In the mean time it is urgent to search for mass screening biomarkers for mycotoxins in humans and related genetic factors to set-up more appropriate regulation.

PMID: 16705817 [PubMed - indexed for MEDLINE]

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Evidence of a new dechlorinated ochratoxin A derivative formed in opossum kidney cell cultures after pretreatment by modulators of glutathione pathways: correlation with DNA-adduct formation.

Faucet-Marquis V, Pont F, St¿rmer FC, Rizk T, Castegnaro M, Pfohl-Leszkowicz A.

Department BioSyM, Laboratoire de GŽnie Chimique, UMR CNRS/INPT/UPS5503, Auzeville-Tolosane, France.

Ochratoxin A (OTA), a nephrotoxic mycotoxin probably implicated in human Balkan endemic nephropathy and associated urothelial tumors, induces renal carcinomas in rodents and nephrotoxicity in pigs. OTA induces DNA-adduct formation, but the structure of the adducts and their role in nephrotoxicity and carcinogenicity have only partly been elucidated. In vivo, 2-mercaptoethane sulfonate (MESNA) protects rats against OTA-induced nephrotoxicity but not against carcinogenicity, indicating two different mechanisms leading to nephrotoxicity or carcinogenicity. To better understand how DNA-adduct could be generated, opossum kidney cells (OK) have been treated by OTA alone or in presence of several compounds such as MESNA or N-acetylcysteine (another agent that, like MESNA, reduces oxidative stress by increasing of free thiols in kidney), buthionine sulfoximine (BSO) (an inhibitor of glutathione-synthase), and alpha amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid (ACIVICIN) (an inhibitor of gamma glutamyl transpeptidase). Cytotoxicity of OTA on OK cells was evaluated by applying the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. None of the listed agents diminished OTA cytotoxicity significantly; ACIVICIN even increases OTA cytotoxicity. In contrast, analysis of the HPLC profiles of OTA metabolites produced during these incubations indicated that the pattern, the quantity of metabolites, and the nature of the derivatives were modulated by these agents. Ochratoxin B (OTB), open-ring ochratoxin A (OP-OA), 4 hydroxylated OTA, 10 hydroxylated OTA, OTA without phenylalanine, OTB without phenylalanine, and a dechlorinated OTA metabolite could be identified by nano-ESI-IT-MS.

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PMID: 16671059 [PubMed - indexed for MEDLINE]

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Ochratoxin A: apoptosis and aberrant exit from mitosis due to perturbation of microtubule dynamics?

Rached E, Pfeiffer E, Dekant W, Mally A.

Department of Toxicology, University of WŸrzburg, Versbacher Strasse 9, 97078 WŸrzburg, Germany.

Ochratoxin A (OTA) is a potent nephrotoxin and causes high incidences of renal tumors in rodents. The molecular events leading to tumor formation by OTA are not well defined. Early pathological changes observed in kidneys of rats treated with OTA in vivo include frequent mitotic and abnormally enlarged cells, detachment of tubule cells, and apoptosis within the S3 segment of the proximal tubule, suggesting that OTA may interfere with molecules involved in the regulation of cell division and apoptosis. In this study, treatment of immortalized human kidney epithelial (IHKE) cells with OTA (0-50 microM) resulted in a time- and dose-dependent increase in apoptosis and activation of c-Jun N-terminal kinase. At the same time, OTA blocked metaphase/anaphase transition and led to the formation of aberrant mitotic figures and giant cells with abnormally enlarged and/or multiple nuclei, sometimes still connected by chromatin bridges. Immunostaining of the mitotic apparatus using an alpha-tubulin antibody revealed defects in spindle formation. In addition, OTA inhibited microtubule assembly in a concentration-dependent manner in a cell-free, in vitro assay. Interestingly, treatment with OTA also resulted in activation of the transcription factor nuclear factor kappa B (NFkappaB), which has recently been shown to promote cell survival during mitotic cell cycle arrest. Based on these observations, we hypothesize that the mechanism by which OTA promotes tumor formation involves interference with microtubuli dynamics and mitotic spindle formation, resulting in apoptosis or-in the presence of survival signals such as stimulation of the NFkappaB pathway-premature exit from mitosis. Aberrant exit from mitosis resulting in blocked or asymmetric cell division may favor the occurrence of cytogenetic abnormalities and may therefore play a critical role in renal tumor formation by OTA.

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PMID: 16641321 [PubMed - indexed for MEDLINE]

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Ochratoxin A in human blood in Abidjan, C™te d'Ivoire.

Sangare-Tigori B, Moukha S, Kouadio JH, Dano DS, Betbeder AM, Achour A, Creppy EE.

Department of Toxicology, University of Bordeaux 2, 146, rue LŽo-Saignat, 33076 Bordeaux, France.

Ochratoxin A (OTA) produced by Aspergillus and Penicillium genera contaminates a diversity of foods including cereals; cereals-derived foods; dry fruits; beans; cocoa; coffee; beer; wine; and foodstuffs of animal origin mainly poultry, eggs, pork and milk, including human breast milk. OTA is nephrotoxic to all animal species studied so far and most likely to humans, who show the longest half-life for elimination of this toxin among all species examined. Among other toxic effects, OTA is teratogenic, immunotoxic, genotoxic, mutagenic and carcinogenic, all of which lead to life-threatening pathologies through several molecular pathways. In C™te d'Ivoire, preliminary surveys conducted by us have proven from 1998 to 2004 the reality of ochratoxin A-contamination of foodstuffs. To assess OTA in human blood, the immunoaffinity columns were used along with HPLC for separation and fluorimetric quantification of blood samples collected in Abidjan from two categories of people: apparently healthy donors (n=63) and nephropathy patients undergoing dialysis (n=39). Among healthy donors, 34.9% show OTA concentrations ranging from 0.01 - 5.81 microg/l with a mean value of 0.83 microg/l, whereas, among nephropathy patients undergoing dialysis 20.5% are OTA positive in a range of 0.167-2.42 microg/l and a mean value of 1.05. Although the sex ratio is 0.82 (46 females for 56 males) ochratoxin A contamination is equally distributed in both sexes. Nephropathy patients undergoing dialysis appear, however, less frequently contaminated than healthy donors (20.5 versus 34.9%) and show higher OTA concentrations (higher mean value, p=0.01). Ochratoxin A concentrations found in human blood reflect concentrations previously detected in cereals and peanuts according to the eating habits and diets of people in C™te d'Ivoire. But, the prevalence of ochratoxin A in blood of nephropathy people undergoing dialysis appears lower than expected from the frequency of OTA contamination in cereals and peanuts. Pearson chi(2)-test indicates that among OTA-positive individuals renal dialysis and age are important modalities for consideration.

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PMID: 16626769 [PubMed - indexed for MEDLINE]

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Contribution of Src-FAK signaling to the induction of connective tissue growth factor in renal fibroblasts.

Graness A, Cicha I, Goppelt-Struebe M.

Department of Nephrology, University of Erlangen-Nuremberg, Erlangen, Germany.

Expression of connective tissue growth factor (CTGF) is sensitive to reorganization of the actin cytoskeleton, but also to alterations in cell morphology due to extracellular forces, for example, cyclic stretching or mechanical loading. Dynamic alterations of focal adhesion proteins were thus proposed to modulate CTGF induction. Immortalized human renal fibroblasts were cultured in or on top of preformed collagen-1 gels. Proteins were detected by immunofluorescence and quantified by Western blotting. Fibroblasts cultured in/on collagen gels resembled cells in vivo by their spindle-like morphology, absence of actin stress fibers, small punctiform focal contacts, and low levels of CTGF expression. Disassembly of microtubules by short-term treatment with colchicine induced cell rounding, cortical recruitment of patchy F-actin, reorganization of focal contacts into strong clusters, and upregulation of CTGF, all of which were dependent on RhoA-Rho-kinase signaling. Clustering of focal adhesion sites activated Src-family kinases and focal adhesion kinase (FAK). Interference with Src activity by PP2 had no effect on the morphological alterations but decreased tyrosine phosphorylation of focal adhesion proteins and almost completely prevented upregulation of CTGF. Furthermore, inhibition of phosphatidylinositol 3-kinase reduced CTGF expression. On the other hand, when the fibroblasts were cultured on a rigid matrix, that is collagen-coated plates, strong focal complexes prevented the dynamic alterations, and RhoA-mediated upregulation of CTGF expression was independent of Src-FAK signaling. Assembly of focal adhesion proteins regulates CTGF expression, providing a link between actin network, adhesion receptors, and CTGF-mediated functions such as synthesis of extracellular matrix proteins.

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PMID: 16531982 [PubMed - indexed for MEDLINE]

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Attenuation of mycotoxin-induced IgA nephropathy by eicosapentaenoic acid in the mouse: dose response and relation to IL-6 expression.

Shi Y, Pestka JJ.

Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824-1224, USA.

Clinical trials have revealed that progression of immunoglobulin A nephropathy (IgAN), the most common form of human glomerulonephritis, is inhibited by dietary (n-3) polyunsaturated fatty acid (PUFA) supplementation. The early stages of IgAN can be mimicked by feeding mice the mycotoxin deoxynivalenol (DON). Here, the effects of consuming the (n-3) PUFA eicosapentaenoic acid (EPA) on DON-induced IgAN were assessed relative to dose dependency and to expression of interleukin (IL-6). In the dose-response study, weight gain and feed intake did not differ among mice consuming 20 ppm DON supplemented with 0%, 0.1%, 0.5% and 3% EPA for 16 weeks. Mice fed the two highest EPA concentrations exhibited markedly increased splenic EPA, docosapentaenoic acid and docosahexaenoic acid, whereas arachidonic acid was decreased in all three EPA fed groups. Deoxynivalenol consumption significantly increased serum IgA and IgA immune complexes as well as kidney mesangial IgA deposition. All three IgAN markers were attenuated in mice fed 3% EPA diet but not in those fed 0.1% or 0.5% EPA. Elevated IgA production was observed in spleen and Peyer's patch (PP) cell cultures derived from mice fed DON in control diets, but this was reduced in cultures from mice fed 0.1%, 0.5% and 3% EPA. Acute DON exposure increased serum levels of IL-6, a cytokine that drives differentiation of IgA-committed B cells to IgA secretion. Relatedly, expression of IL-6 mRNA and IL-6 heteronuclear RNA, a marker of IL-6 transcription, was increased in spleen and PP. All three indicators of IL-6 expression were suppressed in mice consuming 3% EPA. Suppressed IL-6 corresponded to decreased binding activity of two factors that regulate transcription of this cytokine, cyclic AMP response element-binding protein and activator protein-1. The results indicate that a threshold existed for EPA relative to suppression of experimental IgAN and that the threshold dose was effective at inhibiting IL-6 transcription.

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PMID: 16524712 [PubMed - indexed for MEDLINE]

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Standard and Fpg-modified comet assay in kidney cells of ochratoxin A- and fumonisin B(1)-treated rats.

Domijan AM, Zeljezi D, Kopjar N, Peraica M.

Unit of Toxicology, Institute for Medical Research and Occupational Health, Ksaverska c. 2, 10000 Zagreb, Croatia. adomijan@imi.hr

The effect of ochratoxin A (OTA), fumonisin B(1) (FB(1)), and their combinations on DNA damage was studied using the standard alkaline comet assay and the Fpg-modified comet assay. Rats were orally receiving OTA (5 ng/kg b.w., 0.05 mg/kg b.w., and 0.5mg/kg b.w., respectively) for 15 days, FB(1) (200 ng/kg b.w., 0.05 mg/kg b.w., and 0.5mg/kg b.w., respectively) for 5 days, and the combinations of two lower OTA and FB(1) doses. The tail length, tail intensity, and Olive tail moment (OTM) obtained with the standard comet assay and Fpg-modified comet assay were significantly higher in treated animals than in controls, even at the lowest dose of OTA or FB(1) (p<0.01). The Fpg-modified comet assay showed significantly greater tail length, tail intensity, and OTM in all treated animal than did the standard comet assay (p<0.05), which suggests that oxidative stress is likely to be responsible for DNA damage. DNA damage detected by the standard comet assay at all OTA or FB(1) doses indicates that some other mechanism is also involved. Combined OTA+FB(1) treatment measured either by the standard comet or the Fpg-modified comet assay showed a synergistic increase in the tail intensity and OTM in kidney cells, even at doses that correspond to the daily human exposure in Europe.

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PMID: 16497426 [PubMed - indexed for MEDLINE]

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Ochratoxin A: potential epigenetic mechanisms of toxicity and carcinogenicity.

Schilter B, Marin-Kuan M, Delatour T, Nestler S, Mantle P, Cavin C.

Department of Quality and Safety, NestlŽ Research Center, Lausanne, Switzerland. benoit.schilter@rdls.nestle.com

Assessment of the significance to human health of ochratoxin A (OTA) in food is limited by a lack of human toxicity data. Therefore, OTA risk evaluation relies mainly on the use of animal data, with renal carcinogenicity in rat being considered as the pivotal effect. The elucidation of the mechanism of action would improve the use of the carcinogenicity data for risk assessment. Direct genotoxicity versus epigenetic mechanisms appears to be a key question. In this presentation, new biochemical and toxicogenomic results obtained in a recent European project (EU-Grant # QLK1-CT-2001-011614) will be summarized in the context of previously reported mechanisms of action including inhibition of protein synthesis, production of oxidative stress and alteration of cell signalling. Amongst others, the new data indicate that chronic administration of a carcinogenic dose of OTA affected cell-signalling pathways resulting in a significantly reduced renal antioxidant defence and increased oxidative DNA damage. These data confirm previous hypotheses involving oxidative stress as a possible key epigenetic mechanism of OTA toxicity and carcinogenicity.

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PMID: 16332626 [PubMed - indexed for MEDLINE]

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Renal tumourigenesis in male rats in response to chronic dietary ochratoxin A.

Mantle P, Kulinskaya E, Nestler S.

Department of Environmental Science and Technology, Imperial College London, UK. p.mantle@imperial.ac.uk

The potency of ochratoxin A (OTA) as a renal carcinogen in the rat in response to lifetime administration by oral gavage is a basis of current concern about possible human risk from dietary exposure to the mycotoxin. In this study, dietary delivery of OTA was chosen as the mode of administration, since this mimics human intake of OTA-contaminated food more accurately than gastric intubation. Young male Fischer rats were given approximately 300 microg OTA/kg body weight (bwt) daily until they reached 333 g; thereafter their daily intake was held at about 100 microg. Renal tumours, mostly unilateral carcinomas, were first discovered at week 75 and total incidence reached 25%. Statistical comparison of total carcinoma incidence (20%) in this study with that of the classic US NTP study suggested that OTA was significantly less carcinogenic when administered in feed than when given by oral gavage. The finding may moderate perceptions of a putative risk of trace amounts of OTA in some foodstuffs to human health.

Publication Types:
PMID: 16332623 [PubMed - indexed for MEDLINE]

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Ochratoxin A in human kidney diseases.

Fuchs R, Peraica M.

Institute for Medical Research and Occupational Health, Zagreb, Croatia.

Ochratoxin A (OTA) is an ubiquitous nephrotoxic and carcinogenic mycotoxin considered to be involved in the aetiology of Balkan endemic nephropathy (BEN). The occurrence of this human fatal disease that appears in regions of Bosnia and Herzegovina, Bulgaria, Croatia, Rumania, and Serbia and Monte Negro correlates with very high incidence of otherwise rare urothelial tumours of the renal pelvis and ureters. Although OTA was found more frequently and/or in higher concentration in food and blood of inhabitants in regions with BEN than in other regions, the involvement of OTA in the development of BEN is still open. Patients with chronic renal insufficiency treated with dialysis have higher blood OTA concentrations than healthy persons, and OTA concentration does not decrease with such treatment.

Publication Types:
PMID: 16332622 [PubMed - indexed for MEDLINE]

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Ochratoxin A: previous risk assessments and issues arising.

Walker R, Larsen JC.

School of Biomedical and Molecular Sciences, University of Surrey, Guildford, UK. profronwalker@aol.com

Ochratoxin A (OTA) causes nephropathy in all species tested with large sex and species differences in potency, pigs being most sensitive. It has been linked to Balkan endemic nephropathy (BEN) in humans. Embryotoxicity, teratogenicity, and immunotoxicity occur only at doses higher than those causing nephrotoxicity. OTA has long serum half-lives in various species including humans. OTA produced renal tumours in mice and rats. The male rat was most sensitive, renal carcinomas occurring after 70 microg/kg bw per day but not 21 microg/kg bw per day. OTA was not mutagenic in most studies in bacteria and mammalian cells, but produced DNA damage and chromosomal aberrations in mammalian cells in vitro, and in mice in vivo. DNA adducts found in the kidneys of mice and rats dosed with OTA, did not contain fragments of OTA. OTA in food has been evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), and by the EC Scientific Committee on Food (SCF). JECFA established a provisional tolerable weekly intake (PTWI) of 100 ng/kg bw based on the LOEL for renal effects in pigs. Conversely, SCF recommended reducing exposure to OTA as much as possible, e.g. below 5 ng/kg bw per day. Both committees recommended further studies to clarify the mechanism by which OTA induces nephrotoxicity and carcinogenicity.

Publication Types:
PMID: 16332615 [PubMed - indexed for MEDLINE]

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Mycotoxin patulin activates the p38 kinase and JNK signaling pathways in human embryonic kidney cells.

Liu BH, Wu TS, Yu FY, Wang CH.

Department of Life Sciences, Chung Shan Medical University, Taichung, Taiwan. bingliu@csmu.edu.tw

Patulin (PAT), a mycotoxin mainly produced by Penicillium and Aspergillus, is frequently detected in moldy fruits and fruit products. Exposure of human embryonic kidney (HEK293) cells to PAT led to a dose- and time-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), p38 kinase and c-Jun N-terminal kinase (JNK). The phosphorylated forms of MAPK kinase 4 (MKK4), c-Jun, and ATF-2 were also seen in PAT-treated cultures. The cell death caused by PAT was significantly reduced by the p38 kinase inhibitor, SB203580, but not by the JNK inhibitor, SP600125. Neither p38 kinase nor JNK played a role in the PAT-induced DNA damage. In PAT-treated cells, inactivation of double-stranded RNA-activated protein kinase R (PKR) by the inhibitor, adenine, markedly suppressed JNK and ERK phosphorylation. Treatment of HEK293 cells with PAT-cysteine adduct, a chemical derivative of PAT, showed no effect on MAPK signaling pathways, cell viability, or DNA integrity. These results indicate that PAT causes rapid activation of p38 kinase and JNK in HEK293 cells, but only the p38 kinase signaling pathway contributes to the PAT-induced cell death. PKR also plays a role in PAT-mediated MAPK activation.

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PMID: 16306151 [PubMed - indexed for MEDLINE]

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Ochratoxin A induces oxidative DNA damage in liver and kidney after oral dosing to rats.

Kamp HG, Eisenbrand G, Janzowski C, Kiossev J, Latendresse JR, Schlatter J, Turesky RJ.

Department of Chemistry, Division of Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Erwin-Schroedinger-Strasse 52, D-67663 Kaiserslautern, Germany. hennicke.kamp@basf-ag.de

The nephrotoxic/carcinogenic mycotoxin ochratoxin A (OTA) occurs as a contaminant in food and feed and may be linked to human endemic Balkan nephropathy. The mechanism of OTA-derived carcinogenicity is still under debate, since reactive metabolites of OTA and DNA adducts have not been unambiguously identified. Oxidative DNA damage, however, has been observed in vitro after incubation of mammalian cells with OTA. In this study, we investigated whether OTA induces oxidative DNA damage in vivo as well. Male F344 rats were dosed with 0, 0.03, 0.1, 0.3 mg/kg bw per day OTA for 4 wk (gavage, 7 days/wk, five animals per dose group). Subsequently, oxidative DNA damage was determined in liver and kidney by the comet assay (single cell gel electrophoresis) with/without use of the repair enzyme formamido-pyrimidine-DNA-glycosylase (FPG). The administration of OTA had no effect on basic DNA damage (determined without FPG); however, OTA-mediated oxidative damage was detected with FPG treatment in kidney and liver DNA of all dose groups. Since the doses were in a range that had caused kidney tumors in a 2-year carcinogenicity study with rats, the oxidative DNA damage induced by OTA may help to explain its mechanism of carcinogenicity. For the selective induction of tumors in the kidney, increased oxidative stress in connection with severe cytotoxicity and increased cell proliferation might represent driving factors.

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PMID: 16302199 [PubMed - indexed for MEDLINE]

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A toxicogenomics approach to identify new plausible epigenetic mechanisms of ochratoxin a carcinogenicity in rat.

Marin-Kuan M, Nestler S, Verguet C, Bezenon C, Piguet D, Mansourian R, Holzwarth J, Grigorov M, Delatour T, Mantle P, Cavin C, Schilter B.

NestlŽ Research Center, PO Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland. maricel.marin-kuan@rdls.nestle.com

Ochratoxin A (OTA) is a mycotoxin occurring naturally in a wide range of food commodities. In animals, it has been shown to cause a variety of adverse effects, nephrocarcinogenicity being the most prominent. Because of its high toxic potency and the continuous exposure of the human population, OTA has raised public health concerns. There is significant debate on how to use the rat carcinogenicity data to assess the potential risk to humans. In this context, the question of the mechanism of action of OTA appears of key importance and was studied through the application of a toxicogenomics approach. Male Fischer rats were fed OTA for up to 2 years. Renal tumors were discovered during the last 6 months of the study. The total tumor incidence reached 25% at the end of the study. Gene expression profile was analyzed in groups of animals taken in intervals from 7 days to 12 months. Tissue-specific responses were observed in kidney versus liver. For selected genes, microarray data were confirmed at both mRNA and protein levels. In kidney, several genes known as markers of kidney injury and cell regeneration were significantly modulated by OTA. The expression of genes known to be involved in DNA synthesis and repair, or genes induced as a result of DNA damage, was only marginally modulated. Very little or no effect was found amongst genes associated with apoptosis. Alterations of gene expression indicating effects on calcium homeostasis and a disruption of pathways regulated by the transcription factors hepatocyte nuclear factor 4 alpha (HNF4alpha) and nuclear factor-erythroid 2-related factor 2 (Nrf2) were observed in the kidney but not in the liver. Previous data have suggested that a reduction in HNF4alpha may be associated with nephrocarcinogenicity. Many Nrf2-regulated genes are involved in chemical detoxication and antioxidant defense. The depletion of these genes is likely to impair the defense potential of the cells, resulting in chronic elevation of oxidative stress in the kidney. The inhibition of defense mechanism appears as a highly plausible new mechanism, which could contribute to OTA carcinogenicity.

Publication Types:
PMID: 16251485 [PubMed - indexed for MEDLINE]

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Ochratoxin A secretion by ATP-dependent membrane transporters in Caco-2 cells.

Schrickx J, Lektarau Y, Fink-Gremmels J.

Department of Veterinary Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 16, 3584 CM, Utrecht, The Netherlands, J.A.Schrickx@vet.uu.nl

The ATP-dependent membrane transporters, P-gp, MRP2 and BCRP, localized in the luminal membranes of the intestines, liver and kidney, counteract absorption and increase excretion of xenobiotics and drugs. Previously, it has been suggested that the mycotoxin ochratoxin A (OTA) is a substrate for ATP-dependent transporters, and hence the absorption and secretion of OTA in the Caco-2 cell model was investigated. To this end, Caco-2 cells were cultured as confluent monolayers in bicameral inserts and the transepithelial transport of the mycotoxin was assessed. Caco-2 cells secreted OTA to the luminal side in a concentration-dependent manner. This secretory permeability was higher than the absorptive permeability, while the absorptive permeability remained constant for all OTA concentrations tested. The secretion decreased and absorption increased in the presence of the MRP-inhibitor MK571, the P-gp and BCRP inhibitor GF120918, and the BCRP-inhibitor Ko143, suggesting that the secretion of OTA is mediated by MRP2 and BCRP. Cyclosporine A also decreased the secretory permeability, but did not affect absorptive permeability, while PSC833 did neither change absorption nor secretion of OTA. Hence it can be suggested that OTA is a substrate for MRP2 as well as BCRP. These findings are of interest in evaluating mycotoxin absorption after oral ingestion, tissue distribution and particularly excretion pathways, including renal, biliary and mammary gland excretion.

PMID: 16244858 [PubMed - indexed for MEDLINE]

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Study of ochratoxin A as an environmental risk that causes renal injury in breast-fed Egyptian infants.

Hassan AM, Sheashaa HA, Fattah MF, Ibrahim AZ, Gaber OA, Sobh MA.

Pediatric Department, Zagazig University, Zagazig, Egypt.

Ochratoxin A (OTA) constitutes a real human threat. Its presence in human milk has previously been reported in different countries. This study is the first Egyptian report on the presence of OTA in both mothers' milk and infants' sera, addressing its correlation with infants' kidney functions, which was not previously addressed in the literature. Fifty healthy breast-lactating mothers and their infants who were exclusively breast-fed for at least 4 months were included. All of them were subjected to a thorough laboratory evaluation including determination of OTA concentration by high-performance liquid chromatography. Thirty-six mothers (72%) and their infants had been contaminated with OTA. Univariate analysis showed that the presence of OTA was associated with significantly higher levels of urinary beta2 microglobulin and microalbuminuria. Multivariate logistic regression analysis showed that there was a significant correlation between a higher OTA level in infants' sera and the degree of microalbuminuria. Mothers and their infants in our locality are exposed to a high OTA contamination rate (72%). To establish the role of OTA in causation of future renal dysfunction for infants, large controlled studies are warranted.

PMID: 16235098 [PubMed - indexed for MEDLINE]

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The impact of plasma protein binding on the renal transport of organic anions.

Bow DA, Perry JL, Simon JD, Pritchard JB.

Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Mail Drop F1-03, P.O. Box 12233, Research Triangle Park, NC 27709, USA.

Drugs and xenobiotics bind to plasma proteins with varying degrees of affinity, and the amount of binding has a direct effect on free drug concentration and subsequent pharmacokinetics. Multiple active and facilitative transport systems regulate the excretion of anionic compounds from the blood in excretory and barrier tissues. Assumptions are made about in vivo substrate affinity and route of elimination based on data from plasma protein-free in vitro assays, particularly following expression of cloned transporters. Ochratoxin A (OTA), a fungal mycotoxin, is a high-affinity substrate for several renal secretory organic anion transporters (OATs), and literature suggests that this elimination pathway is the route of entry leading to proximal tubule-targeted toxicity. However, OTA is known to bind to several plasma proteins with a high affinity, particularly serum albumin, which may impact elimination. In this study, we have systematically examined the handling of OTA and other organic anions, estrone sulfate (ES) and methotrexate (MTX), by OATs in the presence of serum albumin. Increasing concentrations of albumin markedly reduced uptake of OTA by both Xenopus laevis oocytes expressing OATs 1, 3, and 4 and organic anion-transporting polypeptide 1. For all transporters tested, virtually all mediated OTA uptake was eliminated by an albumin concentration equivalent to 10% of that present in the blood plasma. Thus, OTA uptake is dependent on the free substrate concentration and severely limited by binding to human serum albumin. MTX and ES uptake were likewise dependent on free concentration.

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PMID: 16195420 [PubMed - indexed for MEDLINE]

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In vitro investigation of individual and combined cytotoxic effects of ochratoxin A and other selected mycotoxins on renal cells.

Heussner AH, Dietrich DR, O'Brien E.

Department of Environmental Toxicology, University of Konstanz, Jakob-Burkhardt-St. 25, P.O. Box X-918, 78457 Konstanz, Germany.

Hundreds of mycotoxins are known to date and many of them are of great interest with regard to human and animal health since they are detected frequently in plant-derived products. Various mycotoxins may occur simultaneously, depending on the environmental and substrate conditions. Considering this coincident production, it is very likely, that humans and animals are always exposed to mixtures rather than to individual compounds. Therefore, future risk assessments should consider mixture toxicity data. This is particularly true for ochratoxin A (OTA), ochratoxin B (OTB), citrinin (CIT) and occasionally for patulin (PAT) as they are all produced by a number of Penicillium and Aspergillus species. Therefore, these four toxins were chosen to study the interactive effects in vitro, using the well-established porcine renal cell line LLC-PK1 and the MTT reduction test as a cytotoxicity endpoint. By application of a step-wise approach to test combination toxicity, using various full factorial as well as a central composite experimental designs, the interactive (synergistic) cytotoxic effects of the these four toxins were assessed. The results obtained in this study confirm a potential for interactive (synergistic) effects of CIT and OTA and possibly other mycotoxins in cells of renal origin.

PMID: 16140496 [PubMed - indexed for MEDLINE]

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Role of membrane microdomains in PTH-mediated down-regulation of NaPi-IIa in opossum kidney cells.

Nashiki K, Taketani Y, Takeichi T, Sawada N, Yamamoto H, Ichikawa M, Arai H, Miyamoto K, Takeda E.

Department of Clinical Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan.

BACKGROUND: Parathyroid hormone (PTH) rapidly down-regulates type IIa sodium-dependent phosphate transporter (NaPi-IIa) via an endocytic pathway. Since the relationship between PTH signaling and NaPi-IIa endocytosis has not been explored, we investigated the role of membrane microdomains in this process. METHODS: We examined the submembrane localization of NaPi-IIa in opossum kidney (OK-N2) cells that stably expressed human NaPi-IIa, and searched for a PTH-induced specific phosphorylating substrate on their membrane microdomains by immunoblotting with specific antibody against phospho substrates of protein kinases. RESULTS: We found that NaPi-IIa was primarily localized in low-density membrane (LDM) domains of the plasma membrane; PTH reduced the levels of immunoreactive NaPi-IIa in these domains. Furthermore, PTH activated both protein kinase A (PKA) and protein kinase Calpha (PKCa) and increased the phosphorylation of 250 kD and 80 kD substrates; this latter substrate was identified as ezrin, which a member of the ezrin-radixin-moesin (ERM) protein family. In response to PTH, ezrin was phosphorylated by both PKA and PKC. Dominant negative ezrin blocked the reduction in NaPi-IIa expression in the LDM domains that was induced by PTH. CONCLUSION: These data suggest that NaPi-IIa and PTH-induced phosphorylated proteins that include ezrin are compartmentalized in LDM microdomains. This compartmentalization may play an important role in the down-regulation of NaPi-IIa via endocytosis.

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PMID: 16105044 [PubMed - indexed for MEDLINE]

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Functional, biochemical, and pathological effects of repeated oral administration of ochratoxin A to rats.

Mally A, Všlkel W, Amberg A, Kurz M, Wanek P, Eder E, Hard G, Dekant W.

Department of Toxicology, University of WŸrzburg, Germany.

Ochratoxin A (OTA), a mycotoxin produced by several fungi of Aspergillus and Penicillium species, may contaminate agricultural products, resulting in chronic human exposure. In rats, OTA is a potent nephrotoxin, and repeated administration of OTA for 2 years to rats in doses up to 0.21 mg/kg of body wt resulted in high incidences of renal tumors arising from the proximal tubular epithelial cells. The mechanism of tumor formation by OTA in the kidney is not well-defined, and controversial results regarding mode of action have been published. The aim of this study was to characterize dose-dependent changes induced by OTA by application of clinical chemistry, biochemical markers, and toxicokinetics for a better conclusion on modes of action. Administration of OTA (0, 0.25, 0.5, 1, and 2 mg/kg of body wt) to male F344 rats (n = 3 per group) by oral gavage for 2 weeks resulted in a dose-dependent increase in OTA plasma concentrations and concentrations of OTA in both liver and kidney. Although oxidative stress has been implicated in OTA carcinogenicity, treatment with OTA did not induce overt lipid peroxidation or an increase in 8-oxo-7,8-dihydro-2'deoxyguanosine (8-OH-dG) in kidney. In the kidney, OTA-induced pathology was present at all dose levels administered, with a clear increase in severity related to dose. Pathology was restricted to the outer stripe of the outer medulla and consisted of disorganization of the tubule arrangement, frequent apoptotic cells, and abnormally enlarged nuclei scattered through the S3 tubules. Consistent with the histopathology, a dose-dependent increase in the expression of proliferating cell nuclear antigen (PCNA), indicative of cell proliferation, was observed in kidneys, but not in livers of treated animals. The most prominent change in the composition of urine induced by OTA analyzed by 1H NMR and principal component analysis consisted of a major increase in the excretion of trimethylamine N-oxide. However, typical changes observed with other proximal tubular toxins such as increased excretion of glucose were not observed at any of the doses administered. Similarly, treatment with OTA had no clear effects on clinical chemical parameters indicative of nephrotoxicity, although urinary volume was increased at the higher-dose groups. Taken together, the uncommon changes induced by OTA suggest that a unique mechanism may be involved in OTA nephrotoxicity and carcinogenicity.

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PMID: 16097797 [PubMed - indexed for MEDLINE]

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Effect of hydro-osmotic pressure on polycystin-2 channel function in the human syncytiotrophoblast.

Montalbetti N, Li Q, Gonz‡lez-Perrett S, Semprine J, Chen XZ, Cantiello HF.

Laboratorio de Canales I—nicos, Departamento de Fisicoqu’mica y Qu’mica Anal’tica, Facultad de Farmacia y Bioqu’mica, Buenos Aires, Argentina.

Polycystin-2 (PC2), one of the gene products whose mutations cause autosomal dominant polycystic kidney disease is a transient receptor potential (TRP)-type (TRPP2) Ca(2+)-permeable, non-selective cation channel. PC2 is localized in the plasma membrane, the primary cilium, and other cellular organelles of renal epithelial and other cells. Recent studies indicate that PC2 is involved in signal transduction events associated with the transient increase in cytosolic Ca(2+). Proof of evidence now hinges on involvement of the PC2 channel in the transduction of environmental signals. PC2 is abundantly expressed in the apical membrane of human syncytiotrophoblast (hST), a highly intricate epithelial tissue, which is essential for the maternal-fetal transfer of solutes, including ions. Physical forces such as hydrostatic (H) and osmotic (Pi) pressure play important roles in placenta homeostasis. In this study, we provide new information on PC2 channel regulation in the hST by these environmental factors, and propose a model as to how they may trigger the activation of PC2. Using apical hST vesicles reconstituted in a lipid bilayer system, we found that a change in either H or Pi modified PC2 channel activity. This stimulatory effect was no longer observed in hST vesicles pre-treated with the actin cytoskeleton disrupter cytochalasin D. As shown by immunofluorescence analysis PC2 co-localized with actin filaments in the vicinity of the plasma membrane. This co-localization was disrupted by cytochalasin D. Taken together, our findings indicate that physical forces exerted on cells regulate PC2 channel activity by a sensory mechanism involving the actin cytoskeleton.

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PMID: 16025301 [PubMed - indexed for MEDLINE]

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Breast cancer resistance protein (Bcrp1/Abcg2) reduces systemic exposure of the dietary carcinogens aflatoxin B1, IQ and Trp-P-1 but also mediates their secretion into breast milk.

van Herwaarden AE, Wagenaar E, Karnekamp B, Merino G, Jonker JW, Schinkel AH.

Division of Experimental Therapy, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

The breast cancer resistance protein (BCRP/ABCG2) usually protects the body from a wide variety of environmental and dietary xenotoxins by reducing their net uptake from intestine and by increasing their hepatobiliary, intestinal and renal elimination. BCRP is also highly expressed in lactating mammary glands in mice, and this expression is conserved in cows and humans. As a result, BCRP substrates can be secreted into milk. We investigated whether different classes of dietary carcinogens are substrates of Bcrp1/BCRP and the implications for systemic exposure and breast milk contamination. Using polarized cell lines, we found that Bcrp1 transports the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and the potent human hepatocarcinogen aflatoxin B1, and decreases their cellular accumulation up to 10-fold. In vivo pharmacokinetic studies showed that [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin B1 plasma levels were substantially lower in wild-type compared with Bcrp1-/- mice, after both oral and intravenous administration, demonstrating that Bcrp1 restricts systemic exposure to these carcinogens. Moreover, Bcrp1 mediates transfer of [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin into milk, with 3.4+/-0.6, 2.6+/-0.3 and 3.8+/-0.5-fold higher milk to plasma ratios, respectively, in lactating wild-type versus Bcrp1-/- mice. We have thus identified Bcrp1/BCRP as one of the molecular mechanisms by which heterocyclic amines and aflatoxin are transferred into milk, thereby posing a health risk to breast-fed infants and dairy consumers. Paradoxically, Bcrp1/BCRP appears to have both protective and adverse roles with respect to exposure to dietary carcinogens.

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PMID: 16000399 [PubMed - indexed for MEDLINE]


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