Deutsch Intern
    Department of Toxicology

    Recent Results

    Summary: The experiments are progressing as planned and changes to the technical annex were made to follow up some of the observations made during the first 2 years. The major discrepancy in the results between the partners when using different techniques are the conclusions regarding covalent binding of OTA to DNA. Consistent with the results from other groups using similar approaches and the absence of mutagenicity of OTA as seen in this project, radioactivity from labelled OTA was not found bound to DNA. Postlabelling by P4 suggests the formation of DNA-modifications in kidney after OTA-administration. The results of the postlabelling exercise show that differences in the DNA-extraction procedures do not account for the different results using radiolablelled OTA and the postlabelling. This point will be adressed again in the 3rd year and P4 will attempt to isolate the DNA-modifications indicated for structure elucidation.

    Partner 1: Accelerator mass spectrometry studies on DNA-binding has been completed and do not show a covalent binding of 14C from ochratoxin A to liver and kidney DNA from rats treated in vivo. The stability of potential adducts and the role of DNA-isolation procedures was investigated The results show that adduct recovery from a positive control is not different when the two procedures are compared. Analytical methods to detect ochratoxin A metabolites were developed. In samples from ochratoxin A treated these metabolites could not be detected. Both ochratoxin A and ochratoxin B treated animals induced cell proliferation in the kidney, only limited nephrotoxicity and some DNA-damage in the kidney after ochratoxin A treatment., but OTB was mcuh less toxic than OTA due to rapid biotransformation.


    The work performed by partner 2 has established procedures to analyse biomarkers of oxidative stress and shoiwed an induction of the inducible nitric oxide synthase (INOS), however, the stress-response was inconsistent with regard to the different parameters determined and a possible delay in the response in vivo is indicated. The liver of animals treated for 7 and 12 months with OTA showed a significant induction of 4-HNE adduct formation. Results form a DNA-array expeiment will be upcoming in the 3rd reporting period.

    In the laboratory of partner 3, groups of rats was exposed to ochratoxin A in the diet and samples from animals sacrificed at different time-points werde evaluated. P3 provided radiolabeled ochratoxin A for AC/MS studies and has produced large amounts of ochratoxin A. Samples were taken at 11 months in the main group in which treated animals received 100 micrograms OTA daily in feed. Body weight of the treated group closely matched (95%) that of controls. No gross abnormalities were seen. Microscopically, there was only a more prominent karyocytomegaly in renal proximal tubule epithelia than was seen at earlier stages..

    Partner 4 has incubated ochratoxin A with different enzymes in the presence of DNA-oligomers and assessed DNA modifications. Preliminary results indicate that OTA induces break in the oligomers. Polymeric nucleosides were incubated in presence of pig kidney microsomes and DNA modifications were indicated with adenine and guanine. Cross link were also formed in poly dG-dC. Pig liver and kidney microsomes form several OTA-metabolites. In pigs, OTA ­modulation of the expression of COX1, COX2 and 5-LIPOX is different in genders. COX1 was inhibited in male cortex, an over-expression is observed in females. Expression of COX2 is only modulated in male kidney (cortex and medulla) where an over-expression is observed in cortex. Expression of 5-LIPOX was only modulated in males by OTA. No basal expression of 5-LIPOX was observed in male cortex, whereas high expression is observed in cortex and medulla of female independently of OTA treatment. CYP 2C9 is not expressed in cortex in both sexes. OTA induced slightly the expression of this CYP in cortex. In medulla, CYP 2C9 is expressed, but is not modulated by OTA.

    Partner 5: Cytotoxicity experiments showed dose- and time-dependent cytotoxicity between 8 and 80 µM of OTA and human cell lines were more sensitive than rodent cells; within the rodent cells the liver epithelial cells were most resistant. Moreover, a cell system (V79 cells in the presence of kidney S9) which showed a significant increases in mutation frequency by OTA, was identified. The mutagenic effect occurred only in a narrow range. Results from HPRT mutation assays in V79 cells in the absence of metabolic activation and results from the TK assay in LY5178 mouse lymphoma cells.suggest that OTA is mutagenic by oxidative damage. OTA-induced HPRT mutant clones were expanded to mass culture, RNAs were isolated and the sequencing of cDNAs is under progress to identify the type of mutations and and DNA-damage. Experiments are in progress where the plasmid DNA is treated with OTA and Fpg-sensitive sites were not detected after 30 min incubation of the plasmid with OTA doses up to 1mM.

    Partner 6: Exposure of rats to OTA by feed for a period of time 6 months (mean serum OTA 7 mg/ml) resulted in slight genetic damage in some tissues and organs. Increases in the "tail moment values" of "comets" observed in the liver and bone marrow reflect the induction of primary DNA lesions. The presence of micronuclei and chromosomal aberrations in bone marrow erythrocytes indicate that "primary DNA lesions" have been processed by DNA repair activities, thus generating chromosomal damage. In vitro studies show that OTA does not induce chromosomal aberrations and SCE's, but an increase in the frequencies of both endoreduplicated and polyploid cells. Since OTA in vitro does not show any cytogenetic activity we evaluated the possibility whether the induction of endoreduplication could have been caused by a possible activity of OTA on the cell cycle. Endoreduplication is not induced via inhibition of mictrotubuli and can be caused by interference of OTA with cell cycle progression. Metabolically competent cell lines were also treated with OTA and did not show an increase in the frequency of micronuclei indicating no role of bioactivation.
    Bone marrow, spleen, liver, kidney and whole blood of rats chronically treated for approximately 15 month with OTA in food only showed marked increases in DNA breakage in kidney. The effect was amplified by Fpg indicating unrepaired oxidative damage which is not converted into permanent cytogenetic damage.

    Partner 7: In serum-free aggregating brain cell cultures, nanomolar concentrations of ochtatoxin A decreased the levels of the two T3-dependent parameters cyclic nucleotide phosphohydrolase (CNP), an oligodendroglia-specific enzyme, and glial fibrillary acidic protein (GFAP), an astrocyte-specific intermediate filament protein. Increasing the levels of T3 during prolonged OTA exposure showed partial protection. Similar results were obtained in experiments with shorter exposure times. However, increased T3 also somewhat raised the basal levels of CNP activity and GFAP content. In subsequent studies, OTA interference with T3-dependent gene expression was examined by real-time RT-PCR using the Taqman system. Expression of several genes were examined after treatment of early aggregate cultures (DIV 5) with OTA for 4 days and for 9 days.
    The effects of ochratoxin A on the expression of one specifically T3-regulated gene have been tested and OTA increased Na+, K+-ATPase mRNA level in cultured liver cells and kidney cells. The protection by T3 of the effects of ochratoxin A has been tested in aggregating brain cell cultures. The experiments at the protein level did not allow to distinguish between a potential protection by T3 of OTA-effects or a simple stimulatory effect of the increased concentration of T3. In aggregates treated with ochratoxin A in the presence of increased levels of T3 a slightl attenuation in the decrease in GFAP mRNA, without increasing GFAP mRNA was seen. It had no effect on ochratoxin A-induced decrease in MBP mRNA, but increased MBP mRNA by itself. No inhibition by ochratoxin A of T3-mediated transcription of MBP has been found.

    Benefits and Beneficiaries: Regulators in Europe are currently considering to introduce maximum limits on ochratoxin A. The overall economic impact on farmers of these measures is most likely significant, since a significant part of a harvest may have to be rejected. It is important that sound scientific data are produced to enable a proper risk assessment. The knowledge of the effects of exposure to ochratoxin A will improve the protection of general population and provide a more effective prevention of diseases. Food standards based on genotoxicity of OTA, which are likely lower than present ones, will result in increased protection if genotoxic mechanisms are involved in ochratoxin A tumorigenesis to the kidney. However, if ochratoxin A does not act through genotoxic mechanisms, higher levels of contaminations would likely be permissible and lower limits would not result in increased health protection, but in increased expenses to meet the standards.

    Statement: By end of project, these problems can be readdressed based on sound and reliable scientific data.


    Publications

    • Zepnik H, Völkel W, Dekant W (2003) Toxicokinetics of the mycotoxin ochratoxin A in F 344 rats after oral administration. Toxiol. Appl. Pharmacol. 192(1), 36-44.
    • Mally, A., Zepnik, H., Wanek, P., Eder, E., Dingley, K., Ihmels, H., Volkel, W. and Dekant, W.: Ochratoxin A: lack of formation of covalent DNA adducts. Chem. Res. Toxicol. 17, 234-42 (2004).
    • Mally, A., Keim-Heusler, H.; Zepnik, H., Mantle, P., Völkel, W., and Dekant, W.:Biotransformation and Toxicokinetics of Ochratoxin B in Rats. Toxicol. Appl. Pharmacol. (in press, available online).
    • Mally, A., Völkel, W., Amberg, A., Kurz, M., Wanek, P., Eder, E. Hard, G. and Dekant, W.: Functional, biochemical and pathologic effects of repeated administration of ochratoxin A to rats. Chem. Res. Toxicol. (in press)
    • Mally, A., Pepe, G., Ravoor, S., Fiore, M., Gupta, R., Dekant, W. and Mosesso, P.: Ochratoxin A causes DNA-damage and cytogenetic effects, but not DNA-adducts in rats. Chem. Res. Toxicol. (in press)
    • Delatour T, Richoz J, Vouros P, Turesky RJ (2002) Simultaneous determination of 3-nitrotyrosine and tyrosine in plasma proteins of rats and assessment of artifactual tyrosine nitration. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 779(2), 189-199.
    • Delatour T, Richoz J, Vuichoud J, Stadler RH (2002) Artifactual nitration controlled measurement of protein-bound 3-nitro-L-tyrosine in biological fluids and tissues by isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry. Chem. Res. Toxicol. 15(10), 1209-1217.
    • Delatour T, Guy PA, Stadler RH, Turesky RJ (2002) 3-Nitrotyrosine butyl ester: a novel derivative to assess tyrosine nitration in rat plasma by liquid chromatography-tandem mass spectrometry detection. Anal. Biochem. 302(1), 10-18.
    • Mottier P, Gremaud E, Guy PA, Turesky RJ (2002) Comparison of gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry methods to quantify alpha-tocopherol and alpha-tocopherolquinone levels in human plasma. Anal. Biochem. 301(1), 128-135.
    • Pfohl-Leszkowicz A, Bartsch H, Azémar B, Mohr U, Estève J, Castegnaro M (2002) MESNA protects rats against nephrotoxicity but carcinogenicity induced by ochratoxin A, implicating two separate pathways. Facta universitatis, series Medicine & Biology, 9 (1), 57-63.
    • Arlt,V.M, Ferluga, D., Stiborova, M., Pfohl-Leszkowicz, A., Vukelic, M., Ceovic, S., Schmeiser, H.H. & Jean-Pierre Cosyns, J-P. (2002) Is aristolochic acid a risk factor for Balkan endemic nephropathy-associated urothelial cancer? Int. J. Cancer, 101, 500-502.
    • Miljkovic, A., Pfohl-Leszkowicz, A., Dobrota, M. & Mantle, P.G. (2002) Comparative responses to mode of oral administration and dose of ochratoxin A or nephrotoxic extract of Penicillium polonicum in rats. Exp Toxic Pathol., 54, 305-312.
    • Petkova-Bocharova, T., Castegnaro, M., Pfohl-Leszkowicz, A., Garren, L., Grosso, F., Nikolov, I., Vrabcheva, T. , Dragacci, S. & Chernozemsky, I., (2003) Analysis of ochratoxin A in serum and urine of inhabitants from an area with Balkan Endemic Nephropathy: A one month follow up study. Facta Universitatis, 10 (2), 62-68.
    • Faucet, V., Pfohl-Leszkowicz, A., Dai, J., Castegnaro, M. & Manderville, R. (2004) Evidence for Covalent DNA Adduction by Ochratoxin A Following Chronic Exposure to Rat and Subacute Exposure to Pig . Chem. Res Toxicol, 17, 1289-1296.
    • Dai, J., Park, G, Perry, J.L., Ill'ichev Y.V., Bow, D.A.J., Pritchard, J.B., Faucet, V., Pfohl-Leszkowicz, A., Manderville, R. & Simon,J.D. (2004) Molecular aspects of the transport and toxicity of ochratoxin A. Account Chem Res, 37 (11) 874-881.
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