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Mechanisms of ochratoxin a induced carcinogenicity as a basis for an improved risk assessment

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Radiolabeled ochratoxin A for AC/MS studies have been provided and large amounts of ochratoxin a produced. In this part of the project, groups of male Fischer rats were exposed to OTA in the diet. After 11 months at 0.2mg/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. More information on the project can be found at: http://www.uni-wuerzburg.de/toxikologie/EU-OTA/OchratoxinA.html
OTA (8 - 80µM) 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 kidney S9. The mutagenic effect occurred only in a narrow concentration 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. A low, but significant increase in mutation frequency was detected in two rodent cell lines (V79 and L5178Y) by using two target genes (HPRT and TK). 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 two human lung and kidney epithelial cell lines. OTA induced a decrease in cell growth rate associated with DNA and protein synthesis inhibition in both cell lines. 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 in these human cell lines. More information on the project can be found at: http://www.uni-wuerzburg.de/toxikologie/EU-OTA/OchratoxinA.html
Biomarkers of oxidative stress have been analysed. The stress response was inconsistent regarding the different parameters however, a striking effect on the inducible nitric oxide synthase (iNOS) was observed in cell cultures correlated with a significant increase in protein nitration (3-nytrotyrosine). In addition, a major effect of OTA was observed in vitro cultures on the basal expression of several genes (GST, UDP-GT, GCLC, GCLM) regulated by the transcription factor Nrf2. Nrf2 is known to interact with the Antioxidant Response Element (ARE)-promoter controlling the expression of many genes involved in the cellular antioxidant defence. Using rats treated chronically with 300mg OTA/ kg bw/day for 7 days up to 12 months, no confirmation of the effect of OTA on iNOS expression and 3-nytrotyrosine formation could be found in the kidney. In contrast, a significant inhibition of several detoxifying enzymes (e.g. glutathione S-transferases, UDP-glucuronosyltransferase) and enzymes involved in the production of the strong antioxidant glutathione was found at the mRNA and protein levels in the kidney but not in the liver of OTA treated rats. Interestingly, further investigation in in vitro systems showed that OTA-mediated cytotoxicity (LDH release), inhibition of protein synthesis and DNA damage were prevented by pre-treatment of the cells with agents (e.g. coumarin) known to activate Nrf2. Our data suggest that a reduction of the cellular defence against oxidative stress may be a plausible mechanism of OTA nephrocarcinogenicity. More information on the project can be found at: http://www.uni-wuerzburg.de/toxikologie/EU-OTA/OchratoxinA.html
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 10nM 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 addition, this analysis confirmed the results obtained during the first two years indicating that ochratoxin A does not interfere systematically with T3-regulated genes in aggregating brain cell cultures, and showed that 24 h after exposure to 10nM 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. More information on the project can be found at: http://www.uni-wuerzburg.de/toxikologie/EU-OTA/OchratoxinA.html
OTA was incubated 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 metabolite 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, it has been demonstrated that OTA is a direct genotoxin after metabolic activation. In addition, the mechanism of cell death induced by OTA was analysed. OTA in epithelial bronchial cells induced necrosis, although at the beginning of the incubation and for low dose an increase of condensate 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 of 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, in rat treated by OTA and in pig fed OTA are formed when enzymes are expressed. DNA-binding of OTA is related to the formation of some metabolites, some of them being already identified by mass spectrometry. More information on the project can be found at: http://www.uni-wuerzburg.de/toxikologie/EU-OTA/OchratoxinA.html
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 post labelling did not indicate adduct formation in the project lab or in an external laboratory even in samples from animal treated with high OTA-doses for 2 weeks. In samples from ochratoxin A treated animals, conceivable metabolites of OTA were not detected. 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 bio transformation. 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. In summary, the results suggest a specific mode of action of OTA on the kidney in rats, which is not due to genotoxicity due to the absence of DNA-binding and absence of biotransformation-dependent mutagenicity of OTA. The kidney specific response represents a modulation of cell-adhesion and cell-cell communication involved in tumor formation. One partner 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 strongly 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 ambition of the Nrf2 pathway specifically in the kidney of rat treated chronically with OTA, resulting in a marked inhibition of the defence mechanism of the kidney cells against oxidative stress. More information on the project can be found at: http://www.uni-wuerzburg.de/toxikologie/EU-OTA/OchratoxinA.html
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. 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 unprepared oxidative damage which is not converted into permanent cytogenetic damage as evinced by the absence of clastogenicity in G0 resting splenocytes from spleen of treated rats and stimulated with concanavalin-A (Con-A) to grow in vitro. Negative results in terms of clastogenicity were also obtained in animals treated by oral gavage at 250, 500, 1000 and 2000ug/kg for 5 days per week for 2 weeks consecutively, though the spectra of induced primary DNA lesions was differ in the different organ/tissues analysed. 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 indicating no role of bioactivation. More information on the project can be found at: http://www.uni-wuerzburg.de/toxikologie/EU-OTA/OchratoxinA.html

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