Institut für Toxikologie, Universität Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany
Received June 30, 2000; accepted October 5, 2000
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ABSTRACT |
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Key Words: mycotoxin; biotransformation; cytochromes P450; renal disease; rodents; Ames test.
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INTRODUCTION |
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Both genotoxic and non-genotoxic mechanisms may contribute to tumor formation by ochratoxin A and discrepant data have been published. A genotoxic potential for ochratoxin A has been indicated in vitro (sister chromatid exchange in Chinese hamster ovary [CHO] cells and the induction of SOS-DNA repair in E. coli: Kuiper-Goodman and Scott, 1989; Malaveille et al., 1994, respectively) and in vivo (DNA single-strand breaks in liver and kidney of rats, Kane et al., 1986). Mutagenicity testing in the Ames test showed mostly negative results (Hennig et al., 1991), but a positive response was reported with media from ochratoxin A-exposed hepatocytes, and in the presence of mouse kidney microsomes and arachidonic acid (Obrecht-Pflumio et al., 1999
). In one study, a mutagenic response was observed in mammalian cells transfected with human cytochrome P450 enzymes (de Groene et al., 1996).
The nature of DNA-damage and/or mutations caused by ochratoxin A is unknown (Degen et al., 1997; De Groene et al., 1996; Flieger et al., 1998
; Grosse et al., 1995
, 1997
; Pfohl-Leszkowicz et al., 1991
; Würgler et al., 1991
). The formation of spots interpreted as ochratoxin A-derived DNA adducts was observed in the target tissues of ochratoxin A toxicity in rodents by the very sensitive 32P-postlabeling assay. However, the chemical structures of the possible adducts have not been defined. A role of biotransformation of ochratoxin A in DNA-binding and renal tumorigenicity has been postulated and a variety of enzymes have been suggested to catalyze transformation of ochratoxin A to reactive intermediates (Fink-Gremmels et al., 1995
; Hennig et al., 1991
; Hietanen et al., 1991
; Malaveille et al., 1994
; Obrecht-Pflumio et al., 1996
, 1999
; Pfohl-Leszkowicz et al., 1998
; Würgler et al., 1991
). However, none of these studies assessed the capacity of the respective enzymes to transform ochratoxin A to metabolites and suggested structure(s) for a reactive metabolite (Bartsch et al., 1995
; Castegnaro et al., 1998
). The known biotransformation reactions of ochratoxin A should not result in intermediates capable of binding to DNA (Omar et al., 1996
; Xiao et al., 1996b
). In a recent study, an ochratoxin A derived quinone was demonstrated as a possible reactive metabolite formed in a system mimicking cytochromes P450 (Gillman et al., 1999
).
In this study, we have investigated the capacity of subcellular fractions and recombinant or purified enzymes to transform ochratoxin A, in order to devise an optimal activation system for mutagenicity testing, which was performed in Salmonella typhimurium with subcellular fractions containing various cofactors.
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MATERIAL AND METHODS |
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Incubations of ochratoxin A with microsomal proteins.
For detection and quantitation of metabolites, ochratoxin A was incubated with liver and kidney microsomes of male and female Wistar rats (Harlan-Winkelmann, Borchen, Germany). Complete incubation systems (final volume 500 or 1000 µl) contained liver or kidney microsomal protein (0.52 mg/ml), 0.1 M phosphate buffer (pH 6.5) (Omar et al., 1996; Omar and Rahimtula, 1993
), NADPH (5 mM) and glutathione (5 mM). When kinetic studies were performed, ochratoxin A (6 concentrations and a blank, concentration of stock solution was 10 mM in C2H5OH) concentrations varied from 20 to 250 µM. Due to problems with solubility, higher concentrations of ochratoxin A were not applied. The reaction was started by addition of ochratoxin A and terminated after various times (time-dependence) or after 10 min (determination of kinetic parameters) at 37°C by addition of an equivalent volume of ice cold ethanol. After centrifugation, the supernatants were analyzed by HPLC. Incubations with troleandomycin or
-naphthoflavone (50 µM) were preincubated at 37°C for 15 min in the presence of microsomes and NADPH (Werner et al., 1995b
), then ochratoxin A (20 µM) was added and incubations carried out as described above. Enzyme kinetics were determined by a Lineweaver-Burk plot (computerized fit) in Microsoft Excel worksheets from data generated with samples from one microsome preparation.
Incubations of ochratoxin A with liver and kidney cytosol and glutathione.
Incubation mixtures contained liver or kidney cytosolic proteins (0.55 mg/ml) from rats or mice and glutathione (5 mM) in phosphate buffer, pH 7.4. Reactions were stopped after 30 min at 37°C and analyzed by HPLC. Incubations of ochratoxin A with semi-purified rat liver glutathione S-transferases (Sigma-Aldrich) were performed in phosphate buffer at pH 7.4 at 37°C for 30 min and contained 3 U of glutathione S-transferase (Lot-No. 44H 8040) and 40 µM ochratoxin A (Vamvakas et al., 1988b, 1989b
). Some incubations were also performed with rat liver S-9 mix (5 mg/ml) in the presence of NADPH (5 mM) and/or glutathione (5 mM) and ochratoxin A (20 µM). Reactions were stopped by adding equivalent volumes of ethanol and analyzed by HPLC with fluorescence detection.
Mutagenicity of ochratoxin A in the Ames test.
These experiments were performed using Salmonella typhimurium TA 100 and TA 2638 in the presence of subcellular fractions from livers and kidneys of male and female rats and mice using various cofactors or with semipurified enzymes (Vamvakas et al., 1987, 1988a
,b
, 1989a
,b
). Benzo[a]pyrene was used as positive control for activity of cytochromes P450. Mutagenicity testing in the presence of peroxidase was performed in analogy to published studies (Glatt et al., 1979
). Bacteria were preincubated in the presence of horseradish peroxidase (0.01 mg/ml) and the required cofactors. A doubling of spontaneous revertant frequencies was considered as indicative of a mutagenic response. Concentrations of ochratoxin A ranging from 0.01 to 0.2 mg/plate were tested in triplicate.
Animals and treatment.
Adult male and female Wistar rats were used for all studies (Zentralinstitut für Versuchstierkunde, Hannover, Gerrmany); they had free access to water and a standard diet (Altromin). Cytochrome P450 induction experiments were performed according to literature protocols with dexamethasone, phenobarbital (Benoit et al., 1992) and 3-methylcholanthrene. Animals (organs from 3 animals were pooled for microsome preparation) were sacrificed by cervical dislocation and liver microsomes prepared as described 24 h after the last administration of the inducers (Dekant et al., 1987
; Dohn and Anders, 1982
; Wolf et al., 1984
). The liver and kidney 9000-g supernatant was prepared using published procedures (Dekant et al., 1987
).
Incubation of ochratoxin A with human cytochromes P450.
Microsomes from baculovirus-infected insect cells expressing human cytochrome P450 3A4 (Lot-No. 19), cytochrome P450 1A2 (Lot-No. 11) and cytochrome P450 2C9-1 (Lot-No. 6) were purchased from Gentest (Woburn, MA). Activity was verified using the substrates given by Gentest. Incubations were performed in 0.1 M phosphate buffer at pH 6.5 with ochratoxin A concentrations of 100 µM, 50, or 100 pmoles of cytochrome P450, NADPH (5 mM) and glutathione (5 mM) in a final volume of 1 ml. Incubations were stopped at various times as described above and ochratoxin A metabolite concentrations were determined by HPLC with fluorescence detection.
Ochratoxin A biotransformation by other enzyme systems.
Horseradish peroxidase, soybean lipoxygenase and glutathione S-transferases were obtained from Sigma-Aldrich. Incubations with semi-purified glutathione S-transferases from rat liver were performed as described (Vamvakas et al., 1988b, 1989b
; Dekant et al., 1998
). Incubations with horseradish peroxidase contained 1 mM H2O2 and 0.01 mg enzyme in acetate buffer, pH 5.5, using ochratoxin A concentrations between 10 and 20 µM. Incubations were performed at 25°C in a final volume of 200 µl for up to 30 min. Incubation with soybean lipoxygenase were performed as described by Roy and Kulkarni (1999), the ochratoxin A concentration was 20 µM. Aliquots of the incubation mixtures (50 µl) were directly injected into the HPLC system for metabolite detection.
Liver and kidney 9000-g supernatant was prepared from untreated or pretreated male rats (Dekant et al., 1987). Incubations contained up to 10 mg/ml of protein, NADPH (5 mM), glutathione (5 mM), and ochratoxin A (520 µM) in a final volume of 200 µl.
General methods.
Cytochrome P450 content was measured according to the method of Omura and Sato (1964), and protein concentrations were determined by the method of Bradford (Bradford, 1976) using the Bio-Rad protein kit with bovine gamma globulin as the standard.
Instrumental analyses.
HPLC-analyses were performed with a Hewlett-Packard HP 1090 series HPLC pump and a Hewlett-Packard 1046 fluorescence detector. For HPLC-separations, a steel column (250 x 4 mm) filled with Hypersil ODS, 5 µm, was used. Material was eluted by gradient elution, solvent A was water containing 5% methanol adjusted to pH = 2 with trifluoroacetic acid, and solvent B was methanol. A linear gradient from 0 to 100% B in 40 min at a flow rate of 1 ml/min was applied. Eluting material was monitored by fluorescence detection (excitation at 236 nm and emission at 456 nm). Photomultipier voltage was set at 14 and a response time of 2 s was used. Metabolite formation was quantified by comparison of the peak areas with calibration curves with authentic 4R- and 4S-hydroxyochratoxin A. The eluents from the HPLC columns were also examined using a Hewlett-Packard 1040 diode array detector set at 320 nm. Characterization of metabolites, purified from incubation mixtures by HPLC, was performed by mass spectrometry. MS/MS-spectra were recorded on a Finnigan TSQ7000 triple quadrupole mass spectrometer equipped with a nanospray interface (Protano, Odense, Denmark). HPLC fractions containing fluorescent metabolites were lyophilised and resuspended in 10 µl of 1% acetic acid in water. The sample was loaded onto a custom-made pipette tip filled with C18 material (Pros2, Perseptive Biosystems) equilibrated with MeOH followed by water. After washing with 200 µl of water for removal of salts, the sample was directly eluted with 5 µl of a mixture of MeOH/water/acetic acid (60:40:1). This eluent was placed in a fused silica nanospray tip (Protano, Odense, Denmark). Nanospray MS was performed with a capillary voltage of 600 V. The heated capillary was set at 250°C, and spectra were acquired in negative ionization mode.
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RESULTS |
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In liver microsomes from non-induced rats, rates of metabolite formation were very low (Table 1); determined vmax-values were higher in microsomes from female animals than in microsomes from male animals, suggesting an involvement of different P450s in male and female rats. In liver microsomes from both sexes of mice, rates of formation of 4R- and 4S-hydroxyochratoxin A were clearly higher than in rat liver microsomes (Table 1
). No sex-differences in the rates of ochratoxin A oxidation were apparent in mice. In kidney microsomes of both rats and mice, rates of ochratoxin A oxidation were below the limit of detection. The kidney microsome system had the capacity to oxidize marker substrates (testosterone, chlorozoxazone) (Urban et al., 1994
; Werner et al., 1995a
). When liver microsomes from rats, pretreated with cytochrome P450 inducers, were used to study ochratoxin A biotransformation, large increases in the rates of oxidation were seen with phenobarbital, methylcholanthrene, and dexamethasone as inducers (Table 2
). Both methylcholanthrene and dexamethasone predominantly increased the rates of formation of 4R-hydroxyochratoxin A. Oxidation of ochratoxin A in liver microsomes from dexamethasone-pretreated rats could be inhibited by troleandomycin (50 µM), a mechanism-based inhibitor of cytochrome P450 3A1/2 in rats by 70%. Rates of ochratoxin A hydroxylation were also increased in microsomes from methylcholanthrene-induced rats and could be inhibited by low concentrations of
-napthoflavone (data not shown). These observations suggest a participation of cytochrome P450 1A1/2 and 3A1/2 enzymes in ochratoxin A biotransformation in rodents.
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DISCUSSION |
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The DNA damage induced by ochratoxin A may be caused by oxidative stress. Several experimental observations support this hypothesis. An unusually large number of DNA adducts (up to 30 individual adducts) from ochratoxin A in low yields is formed in different experimental systems (Castegnaro et al., 1998; Pfohl-Leszkowicz et al., 1998
). Similar patterns of modification, as observed with ochratoxin A by postlabeling, were observed in kidney DNA of rodents exposed to iron(III)nitrilotriacetate (Randerath et al., 1995
), a renal carcinogen active through oxidative stress, or in DNA exposed to hydrogen peroxide (Randerath et al., 1996
).
The absence of intermediates capable of covalent interactions with macromolecules is also supported by the results of mutagenicity studies performed by us and by others (IARC-Scientific-Publications, 1991). An unequivocal mutagenic response of ochratoxin A has not been demonstrated in well defined and characterized experimental systems. A positive response in the Ames-test was only reported with cultured media from ochratoxin A-exposed hepatocytes (Hennig et al., 1991
) and in the presence of mouse kidney microsomes and arachidonic acid as cofactor for prostaglandin H synthase (Obrecht-Pflumio et al., 1999
). Our results on the biotransformation of ochratoxin A and mutagenicity testing do not support these observations. Moreover, the observation of a positive response of ochratoxin A in mouse kidney microsomes (Obrecht-Pflumio et al., 1999
) is not consistent with the known species differences in sensitivity to renal tumor induction by ochratoxin A, i.e., that rats are much more sensitive than mice. In the experiments cited above (Obrecht-Pflumio et al., 1999
), ochratoxin A did not induce a positive response when rat kidney subcellular fractions were used for activation. Moreover, a positive response was observed only in the Salmonella typhimurium strain TA 1535, which is much less sensitive to mutagens than the strain TA 100, where ochratoxin A was not mutagenic under the conditions used (Obrecht-Pflumio et al., 1999
). Mechanisms of mutagenicity are identical in TA 100 and TA 1535.
In summary, results of this study and others support the assumption that ochratoxin A induces renal tumors not by covalent interactions of a reactive metabolite with DNA, but by other mechanisms.
Induction of renal toxicity, oxidative stress by mitochondrial dysfunction, and chronic cell proliferation represents an alternative mechanism to explain the renal carcinogenicity of ochratoxin A, which is known to induce oxidative stress (Aleo et al., 1991) and the formation of hydroperoxides (Omar et al., 1990
). In addition, mechanisms linked to chronic renal toxicity and oxidative stress seem to play an important role in tumor induction in the kidneys of rats (Dietrich and Swenberg, 1993
; Hard, 1998
; Swenberg and Maronpot, 1991
). Several non-genotoxic chemicals not subjected to bioactivation reactions induce renal tumors in rodents. For example, DNA-damage and cellular toxicity mediated by oxidative stress seem to be involved in the renal carcinogenicity of iron(III)nitrilotriacetate or potassium bromate in rodents. These compounds are potent renal carcinogens and induce renal tumors in rodents in high yields after short exposure times (Li et al., 1987
; Wolf et al., 1998
). Sex-differences in tumor incidences are also seen with these compounds. For example, as seen with ochratoxin A, male rats are more susceptible to renal tumor induction by potassium bromate (Kurokawa et al., 1983
; Kurokawa et al., 1990
; Umemura et al., 1998
).
In conclusion, our results suggest that bioactivation reactions to give reactive ochratoxin A-derived metabolites do not play a role in the renal tumorigenicity of ochratoxin A in rats.
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NOTES |
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