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.
Partner 1: Accelerator mass spectrometry (AC/MS) studies on DNA-binding did not show a covalent binding of 14C from ochratoxin A to liver and kidney DNA from rats treated in vivo. In addition, mechanistic investigations did not give evidence for formation of DNA-adducts from OTA. Moreover, analysis of possible DNA-modifications induced by OTA by postlabelling did not indicate adduct formation in the lab of P1 and in an external laboratory even in samples from animals treated with high OTA-doses for 2 weeks. Investigation of the biotransformation of OTA did not indicate the formation of metabolites, others then the already known diastereomeres of 4-OH-OTA and 10OH-OTA. Both ochratoxin A and ochratoxin B induced cell proliferation in the kidney, but only limited nephrotoxicity and some DNA-damage. OTB was much less toxic than OTA due to rapid biotransformation. Repeated ochratoxin A administration to rats resulted in high OTA-concentrations in the target organ kidney, but also in the non-target organ liver and induced some DNA-damage both in liver and kidney. Toxicity was, however, restricted to the kidney. Both OTA and OTB caused minor DNA-damage in the non-target organ liver and in the target organ kidney. The spectrum of effects of OTA on the kidney in rats is unusual and not consistent with necrosis as expected from generation of reactive metabolites and their covalent binding. These results suggest a specific mode of action of OTA on the kidney in rats, which is not due to genotoxicity, since no DNA-binding and biotransformation-dependent mutagenicity of OTA was observed. The kidney specific response represents a modulation of cell-adhesion and cell-cell communication involved in tumor formation.
The work performed by partner 2 analysed biomarkers of oxidative stress and showed 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. However, this effect was not confirmed in rat kidney. OTA administration to rats rapidly (after 21 days) decreased the expression of several detoxifying enzymes (GST, UDP-GT etc.) in the kidney but not in the liver. These data stongly suggest that OTA may repress the Nrf2 pathway which has been reported to be responsible for the activation of several detoxifying phase II enzymes and antioxidant proteins. Data from the DNA microarray experiment (Affymetrix) strongly support an inbition of the Nrf2 pathway specifcally in the kidney of rat treated chronically with OTA, resulting in a marked inhibition of the defense mechanism of the kidney cells against oxidative stress.
Partner 3 provided radiolabeled ochratoxin A for AC/MS studies and has produced large amounts of ochratoxin A. In his main contribution, P3 exposed groups of male Fischer rats to OTA in the diet. After 11 months to 0.2 mg/kg OTA daily with feed, OTA did not influence body weight gain and no gross abnormalities were seen. Microscopically, there was only a more prominent karyocytomegaly in renal proximal tubule epithelia than was seen at earlier stages. Giving OTA for up to 2 years induced a dose-dependent increase in the incidence of renal tumors, all tumors were discovered in the last quarter of life of the treated rats. The regime was well tolerated; only at the highest dose there was slight impairment of renal function. Aging Fischer rats have a confounding factor of a particular leukaemia which complicated assessment of tumour incidence. When comparing the present data with that of the NTP (gavage) study of 1989, OTA is app. 5 times less potent when given in diet as compared to oral gavage 5 times a week. Consequently, the threshold dose of the NTP study may be adjusted higher for a dietary exposure regime. Rats given the highest dose of OTA for only the first 10 months developed renal tumours after a further one-year latency, during which circulating ochratoxin A had disappeared with a half-life of about 11 days. Consequently, any mechanism for carcinogenesis in the rat will have to be in place by within at most 10 months' exposure, and must then be able to endure long absence of OTA during which many or all of the mild general toxicologic influences will have probably long been resolved. That mechanism must be shown to apply to the human since OTA has never been shown to cause human morbidity.
Partner 4 incubated OTA with different enzymes in the presence of DNA-oligomers and assessed DNA modifications. OTA induces breaks in the oligomers. DNA modifications on adenine and guanine were induced by OTA in polymeric nucleosides in presence of pig kidney microsomes. Cross links were also formed in poly dG-dC. Pig liver and kidney microsomes formed several OTA-metabolites. Two guanine OTA-DNA adducts (O-C8-dG-OTA & C-C8-dG-OTA) have been synthesized by Pr Manderville (Guelp, CA). In postlabelling analysis, both adducts comigrate with OTA-DNA adducts formed in kidney of rats developing tumours. C-C8-dG-OTA is also formed in kidney of pig fed OTA. The data show that OTA and one of its metabolites is covalently bound on guanine. Some metabolites extracted from cells treated with OTA and from tissues of pigs were characterized by mass spectrometry, notably OP-OA, 4R-OH-OA, 10-OH-OA, OTB and a dechlorinated OTA-derivative different from OTB. Metabolites formation is correlated with DNA adduct formation. Altogether, P4 demonstrated that OTA is a direct genotoxin after metabolic activation. In addition, P4 analysed the mechanism of cell death induced by OTA. OTA, in bronchial epithelial cells, induced necrosis, although at the beginning of the incubation and at low doses, an increase in condensated chromatin was observed. The enzymes responsible of apoptosis were not induced. 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. Thus OTA decreases protection of kidney function due to COX1, and increases risk a cancer link to over-expression of COX2 in male. The main DNA adduct observed in human kidney tumour of patients exposed to OTA as demonstrated by OTA in blood and kidney tissues of these patients, rat treated by OTA and in pig treated by OTA are formed when these enzymes are expressed.
Partner 5: OTA (8 - 80 µM) caused dose- and time-dependent cytotoxicity in cell lines and human cell lines were more sensitive than rodent cells; within the rodent cells the liver epithelial cells were most resistant. Moreover, OTA induced significant increases in mutation frequency in V79 cells in the absence of metabolic activation. The mutagenic effect occurred only in a narrow concentration range. HPRT mutation assays in V79 cells in the absence of metabolic activation and results from the TK assay in mouse lymphoma cells suggest that the weak mutagenicity of OTA is caused by oxidative damage. The molecular nature of OTA-induced mutations at the HPRT locus revealed a similarity with the spontaneous spectrum with an excess of A>C transversions and possibly large deletions. Both events are compatible with oxidative-stress induced mutagenesis. The analysis of the effects of OTA on survival and cell cycle was performed in human lung and kidney epithelial cell lines. OTA induced a decrease in cell growth rate associated with DNA and protein synthesis inhibition. A reversion of this effect was observed after OTA removal. The cell cycle distribution analysis reflected this phenomenon. A transitory accumulation of cells at S/G2 phases was observed during recovery after OTA treatment. OTA treatment did not induce apoptosis.
Partner 6: Exposure of rats to OTA by feed for a period of time 6 months (mean serum OTA 7 ºg/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. Bone marrow, spleen, liver, kidney and whole blood of rats treated for 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. The results obtained showed slight but not significant increases of chromosomal aberrations in animals treated chronically by feed and by oral gavage for two weeks indicating that OTA is not cytogenetically active in vivo. 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 cytogenetic activity we evaluated the possibility whether the induction of endoreduplication was 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 treated with OTA did not show increased frequencies of micronuclei further supportingthat bioactivation is not involved in the DNA-damage.
Partner 7: In aggregating brain cell cultures, 2-20 nM OTA elicited cell type-specific effects, while at higher concentrations general cytotoxicity was observed. Measurements at the mRNA level using real-time RT-PCR showed that 10 nM OTA rapidly and significantly upregulated the inducible nitric oxide synthase, heme oxygenase-1, and peroxisome proliferator-activated receptor-g, while it downregulated two macroglia-specific markers, glial fibrillary acidic protein and myelin basic protein. These findings were confirmed by microarray analysis, in collaboration with P2. In addition, this analysis confirmed that ochratoxin A does not interfere systematically with T3-regulated genes in aggregating brain cell cultures, and showed that 24 h after exposure to 10 nM OTA, alterations in the expression (up- or down-regulation) at a two-fold or greater level (p < 0.0001) occurred for several hundred genes related to different CNS cell types and to a variety of functional classes. Overall, the altered gene expression suggests a predominantly anti-oxidant and anti-apoptotic reaction pattern, and impairments in ras-like GTPase functions, in the cytoskeleton, and in vesicle biosynthesis and trafficking. Our results show that brain cells are highly susceptible to OTA, and suggest that the profound alterations in gene expression caused by nanomolar concentrations of OTA reflect alternative (non-genotoxic) pathways of OTA toxicity.
In summary, the results on the absence of DNA-binding of OTA (P1) are supported by the absence of biotransformation-dependent genotoxicity of OTA (P5 and P6). These data suggest that OTA causes renal tumors by non-genotoxic mechanisms probably involving complex interactions with cell adhesion, reduction of oxidant defense mechanisms and/or alterations in gene expression (P1, 2 and 7). The results by P3 suggest that the use of the tumor incidence data from the NTP-study using oral gavage in oil may not be appropriate for the risk characterisation of OTA since OTA is app. 5-fold less potent when administered to rats with diet as compared to gavage in oil. However, conflicting results on the possible genotoxicity of OTA were obtained by P4 who obtained support for DNA-binding of OTA by postlabelling and showed that DNA-binding of OTA may be related to the formation of metabolites, the structures of some metabolites were confirmed by mass spectrometry.
- 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.