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Mold Toxins Impact on the Kidneys:
Another Cause of Renal Failure?
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]
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]
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]
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]
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]
[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]
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.
Publication Types:
PMID: 17130176 [PubMed - indexed for MEDLINE]
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.
Publication Types:
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, Bezenon 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]
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.
Publication Types:
PMID: 16978030 [PubMed - indexed for MEDLINE]
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