Ochratoxin A: Lack of Formation of Covalent DNA Adducts - Chemical

The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rodents. However, the mechanism of OTA-induced tumor formation is ...
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Chem. Res. Toxicol. 2004, 17, 234-242

Ochratoxin A: Lack of Formation of Covalent DNA Adducts Angela Mally,† Herbert Zepnik,† Paul Wanek,† Erwin Eder,† Karen Dingley,‡ Heiko Ihmels,§ Wolfgang Vo¨lkel,† and Wolfgang Dekant*,† Institut fu¨ r Toxikologie, Universita¨ t Wu¨ rzburg, Versbacher Strasse 9, 97078 Wu¨ rzburg, Germany, Biology & Biotechnology Research Program, Lawrence Livermore National Laboratory, L452, 7000 East Avenue, Livermore, California 94550, and Institut fu¨ r Organische Chemie, Universita¨ t Wu¨ rzburg, Am Hubland, 97074 Wu¨ rzburg, Germany Received September 17, 2003

The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rodents. However, the mechanism of OTA-induced tumor formation is unknown and conflicting results have been obtained regarding the potential of OTA to bind to DNA. OTA is poorly metabolized, and no reactive intermediates capable of interacting with DNA have been detected in vitro or in vivo. Recently, a hydroquinone/quinone redox couple and a carbon-bonded OTA-deoxyguanosine (OTA-dG) adduct formed by electrochemical oxidation and photoreaction of OTA have been reported and suggested to be involved in OTA carcinogenicity. This study was designed to characterize the role of DNA binding and to determine if formation of these derivatives occurs in vivo and in relevant activation systems in vitro using specific and sensitive methods. Horseradish peroxidase activation of OTA and its dechlorinated analogue ochratoxin B (OTB) yielded ochratoxin A-hydroquinone (OTHQ), but the postulated OTA-dG adduct was not detectable using LC-MS/MS. In support of this, no OTA-related DNA adducts were observed by 32P-postlabeling. In vivo, only traces of OTHQ were found in the urine of male F344 rats treated with high doses of OTA (2 mg/kg body wt) for 2 weeks, suggesting that this metabolite is not formed to a relevant extent. In agreement with the in vitro data, OTA-dG was not detected by LC-MS/MS in liver and kidney DNA extracted from treated animals. In addition, DNA binding of OTA and OTB was assessed in male rats given a single dose of 14C-OTA or 14C-OTB using accelerator mass spectrometry, a highly sensitive method for quantifying extremely low concentrations of radiocarbon. The 14C content in liver and kidney DNA from treated animals was not significantly different from controls, indicating that OTA does not form covalent DNA adducts in high yields. In summary, the results presented here demonstrate that DNA binding of OTA is not detectable with sensitive analytical methods and is unlikely to represent a mechanism for OTA-induced tumor formation.

Introduction OTA1 (Scheme 1a), a mycotoxin produced by several species of Penicillium and Aspergillus fungi, is a widespread food contaminant, resulting in chronic human exposure. In rodents, OTA is a potent nephrotoxin and induces renal tumors, with a tumor incidence of up to 74% in male rats (1). However, the mechanism of OTA carcinogenicity is unknown. OTA has been shown to be not mutagenic in several strains of Salmonella typhimurium (2, 3), but weak genotoxic effects have been observed in some mammalian cell systems (5-7). Putative DNA adducts have been detected using 32P-postlabeling (8), * To whom correspondence should be addressed. Tel: +49-931-20148449. Fax: +49-931-201-48865. E-mail: [email protected]. † Institut fu ¨ r Toxikologie, Universita¨t Wu¨rzburg. ‡ Biology & Biotechnology Research Program, Lawrence Livermore National Laboratory. § Institut fu ¨ r Organische Chemie, Universita¨t Wu¨rzburg. 1 Abbreviations: OTA, ochratoxin A; OTR, ochratoxin alpha; OTHQ, ochratoxin A-hydroquinone; OTQ, ochratoxin A-quinone; OTA-GSH, glutathione conjugate of ochratoxin A; OTA-dG, carbon-bonded ochratoxin A-deoxyguanosine adduct; dG, 2′-deoxyguanosine; AMS, accelerator mass spectrometry; OTB, ochratoxin B; dGMP, 2′-deoxyguanosine-3′-monophosphate; HRP, horseradish peroxidase; 4-OH-TAM, 4-hydroxytamoxifen.

although the chemical structure of these has not been elucidated and it remains unclear whether the spots observed by 32P-postlabeling are covalent DNA adducts containing the OTA molecule. Bioactivation of OTA to reactive intermediates with the potential to bind to DNA has been suggested to be involved in OTA-induced tumor formation, but so far, no stable metabolites indicative of reactive metabolite formation have been characterized. On the contrary, a number of studies on biotransformation in vitro and in vivo have shown that OTA is poorly metabolized and the metabolites formed unlikely involve electrophilic intermediates in their mechanisms of formation (3, 9, 10). Using a panel of subcellular fractions and enzymatic activation systems, no metabolites were detected in vitro other than the hydroxylated derivatives 4(R)- and 4(S)hydroxyochratoxin A (9). In vivo, a pentose and a hexose conjugate linked by an aminoacylic bond have recently been identified in urine of OTA-treated animals (11) in addition to OTR (Scheme 1a), which is considered to be less toxic than OTA (12). Recently, Calcutt et al. (13) reported the formation of a hydroquinone/quinone redox couple generated by elec-

10.1021/tx034188m CCC: $27.50 © 2004 American Chemical Society Published on Web 01/23/2004

DNA Adducts in Ochratoxin A Carcinogenicity Scheme 1

trochemical and photochemical oxidation of OTA. It has been suggested that biotransformation of OTA to OTHQ/ OTQ could lead to the formation of reactive oxygen species due to redox cycling and therefore promote oxidative DNA damage. In addition, the electrophilic quinone may react with tissue nucleophiles to form covalently bound adducts as shown for some other quinones (14). In support of this, autoxidation of OTHQ (Scheme 1a) in the presence of GSH resulted in the formation of OTA-GSH (Scheme 1b) (15). The same conjugate was formed in low yields when OTA was incubated with HRP. Using photoactivation of OTA, Dai et al. (16) demonstrated the formation of a carbon-bonded C8-dG adduct of OTA (OTA-dG) (Scheme 1c). This adduct is formed by dechlorination and radical attack of the OTA phenoxyl radical at the C8 position of dG. Because both bioactivation of OTA to OTHQ/OTQ and formation of the postulated OTA-dG adduct have been speculated to be involved in OTA carcinogenicity, it is crucial to determine if these structures are formed in vivo and in systems representing enzymatic activation in vitro. Furthermore, we applied the extremely sensitive method of AMS to support or reject the hypothesis that direct binding of OTA or OTA metabolites to DNA is the initiating step in OTA-induced tumor formation in rats.

Materials and Methods Chemicals. OTA and OTB were purchased from SigmaAldrich Chemie GmbH, Deisenhofen, Germany, and were >99%

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 235 pure as assessed by HPLC with fluorescence detection. 14C-OTA (0.25 mCi/mmol) and 14C-OTB (0.1mCi/mmol) were obtained from Prof. Peter Mantle, Imperial College, London, and were produced by feeding [U]-14C-acetate (100-120 mCi/mmol) to Aspergillus ochraceus. Trifluoroacetic acid was purchased from Merck, Darmstadt, Germany. HPLC grade acetonitrile, methanol, and water were from Roth, Germany. HRP (type VI) and all other chemicals were obtained from Sigma-Aldrich Chemie GmbH. Animals and Animal Treatment. 1. Binding of OTA or OTB to DNA by AMS. Male F344 Fisher rats (8-9 weeks old) were purchased from Harlan-Winkelmann, Borchen, Germany. Animals were housed in Macrolon cages and allowed free access to standard laboratory chow (Altromin) and tap water. Room temperatures were maintained at 21 ( 2 °C with a relative humidity of 55 ( 10% and a day/night cycle of 12 h. Following a week of acclimatization, rats (3/group) were treated with a single dose of 500 µg/kg body wt 14C-OTA or 14C-OTB in corn oil by oral gavage. Control rats received equal volumes of corn oil. Rats were sacrificed by CO2 asphyxiation and cervical dislocation 72 h postdosing. Livers and kidneys were removed, flash-frozen in liquid nitrogen, and stored at -80 °C until further analysis. 2. Formation of Metabolites and OTA-dG in Vivo. Male F344 Fisher rats (7-8 weeks old, n ) 3) obtained from HarlanWinkelmann were treated with OTA (2 mg/kg body wt) dissolved in corn oil by gavage for 2 weeks (5 days/week). Animals were housed in metabolic cages with tap water and powdered diet (Altromin) ad libitum. Urine was collected every 24 h on ice and stored at -20°C until further analysis. Animals were sacrificed by CO2 asphyxiation and cervical dislocation 72 h after the final dose. Blood samples were obtained by cardiac puncture, and plasma was separated by centrifugation and stored at -80 °C. Livers and kidneys were removed, flash-frozen in liquid nitrogen, and stored at -80 °C until further analysis. Preparation of Tissue, Plasma, and Urine Samples for LC-MS/MS. Tissue homogenates were prepared by homogenizing 200-400 mg of tissue in 4 volumes of ice-cold 50 mM potassium phosphate buffer, pH 6.5, using an ultra-turrax. Proteins were precipitated by the addition of an equal volume of ice-cold ethanol and centrifugation at 14 000 rpm at 4 °C for 30 min. Plasma and urine samples were mixed with 1 volume of ice-cold ethanol and centrifuged at 14 000 rpm at 4 °C for 30 min to precipitate proteins. Of the resulting supernatant, 10 µL was injected into the LC-MS/MS system. DNA Isolation and 14C-AMS. DNA was isolated from livers and kidneys by the Nucleobond method (Macherey-Nagel, Dueren, Germany) according to the manufacturers instructions with minor modifications. Briefly, 300-400 mg of tissue was homogenized by an ultra-turrax, treated with proteinase K and RNase, and loaded onto a Nucleobond AX G 500 ion exchange cartridge. After it was washed, DNA was eluted from the cartridge using a modified elution buffer (1.5 M NaCl, 0.05 M Tris, 15% ethanol, pH 7.0). DNA was precipitated by the addition of 0.7 volumes of 2-propanol. After they were washed with 70% ethanol, DNA pellets were either freeze-dried and stored at -80 °C until further analysis by AMS or dissolved in H2O before postlabeling or hydrolysis by nuclease P1 and alkaline phosphatase for LC-MS/MS. 14C-AMS was performed at the Lawrence Livermore National Laboratory, Livermore, CA, with graphitized DNA samples as described elsewhere (17). In Vitro Incubations. In vitro incubations were carried out for up to 24 h at 25 °C in 0.1 M potassium phosphate buffer, pH 7.4. Reaction mixtures contained 100-250 µM OTA or OTB, 1 µg/µL calf thymus DNA or 0.5 µg/µL dGMP or 0.5 µg/µL dG, 0.5 µg/µL HRP, and 1 mM cumene hydroperoxide or 1 mM H2O2. Control reactions were performed in which OTA and OTB were omitted. As a positive control, 100 µM 4-OH-TAM was used. Further incubations investigating iron(II) activation contained OTA or OTB (100 µM), 5 mM GSH, 1 µg/µL DNA, 0.5 µg/µL dGMP or 0.5 µg/µL dG, 10 µM Fe(NH3)2(SO4) 2, and 1 mM H2O2. These mixtures were incubated at 37 °C for 60 min.

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DNA Hydrolysis. Solutions containing 100 µg of DNA in 100 µL were incubated with 11 units of nuclease P1 (Calbiochem) and 2.5 µL of 1 M sodium acetate/45 mM zinc chloride buffer, pH 4.8, for 60 min at 37 °C. Ten microliters of 100 mM Tris, pH 8.0, and 7.5 units of alkaline phosphatase from calf intestine (Sigma) were then added for an additional 30 min of incubation at 37 °C. Proteins were precipitated by the addition of an equal volume of chloroform and centrifugation at 1000g for 5 min. From the resulting aqueous layer, 50 µL was injected into the LC-MS/MS system. Photoirradiation of OTA. Under aerobic conditions, a solution of OTA (500 mM) and dG (20 mM) in a mixture (3:1) of potassium phosphate buffer (0.1 M, pH 7.4) and DMSO was irradiated for 8 min with a mercury high-pressure lamp (Heraeus TQ 150) through a cutoff filter (Schott WG 305, l > 305 nm). Before injection into the LC-MS/MS (10 µL), reaction mixtures were cleared by centrifugation at 14 000 rpm for 10 min to sediment nonsoluble precipitates. The OTA adduct standard for 32P-postlabeling was prepared by irradiation of OTA in the presence of dGMP (7 mM) instead of dG. 32P-Postlabeling Analysis. 32P-postlabeling analysis was performed as described previously (18). In the case of DNA, DNA was precipitated from the reaction mixture by addition of 0.1 volumes of 5 M NaCl and 0.7 volumes of 2-propanol. DNA (10 µg) was digested at 37 °C for 4 h with micrococcal nuclease (375 mU; Sigma) and spleen phosphodiesterase (10 mU; Calbiochem, Darmstadt, Germany) in a reaction mixture containing 20 mM sodium succinate and 10 mM calcium chloride, pH 6.0. In the case of dGMP, aliquots containing 10 µg of dGMP were used. For postlabeling of the OTA adduct standard generated by irradiation, the reaction mixture was diluted 1:100 and 10 µL of this solution was used. Digested DNA or dGMP was treated with nuclease P1 (8 U) for 45 min at 37 °C before postlabeling with T4 polynucleotide kinase (Amersham, Braunschweig, Germany) and [γ-32P]ATP (ICN, Eschwege, Germany). Nucleotides were resolved on polyethyleneimine cellulose by multidimensional TLC. After normal nucleotides, pyrophosphate and excess ATP, were removed by overnight chromatography in 2.3 M NaH2PO4, pH 6.8 (D1), origins were cut out, contact transferred to another PEI/cellulose TLC plate, and resolved in 4.8 M lithium formiate and 7.8 M urea, pH 3.5 (D2). Chromatography in 0.6 M NaH2PO4 and 5.95 M urea, pH 3.5 (D3), was performed perpendicular to D2. A final washing step was run in the same direction in 1.7 M NaH2PO4, pH 6.0. TLC plates were analyzed using an Instant Imager (Packard, Meriden, CT). Instrumental Analyses. LC-MS/MS analysis was performed on a Agilent 1100 series LC coupled to an API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Darmstadt, Germany). Samples were injected into the LC-MS/MS system through an Agilent 1100 series autosampler. Separations were carried out on a 15 mm × 2 mm Nucleosil 5 µm/100 Å column fitted with a security guard precolumn system (Phenomenex, Aschaffenburg, Germany) with a flow rate of 0.3 mL/min using the following mobile phase: 100% A (water) for 2 min, followed by a linear gradient to 60% A/40% B (acetonitrile) in 5 min and a second gradient to 50% A/50% B in 5 min. This was held for 3 min before increasing to 95% B in 2 min. The API 3000 mass spectrometer was operated with a Turbo Ion Spray source in the negative ion mode with a voltage of -4000 V. Spectral data were recorded with N2 as the heater gas at 400 °C and as the collision gas (CAD ) 4) in the multiple reaction monitoring mode (MRM). The following m/z transitions were analyzed: m/z 402.0 w 357.8 and 402.0 w 166.8 (OTA); m/z 254.8 w 210.7 and 254.8 w 166.8 (OTR); m/z 367.8 w 323.8 and 367.8 w 132.9 (OTB); m/z 220.9 w 176.9 and 220.9 w 132.9 (OTβ); m/z 384.0 w 192.0 (OTHQ); 418.0 w 166.8 (4-OH-OTA); m/z 689.0 w 416.0 (OTAGSH); and m/z 633 w 517.0 (OTA-dG). Enhanced product ion spectra for m/z 384 (OTHQ) and m/z 633 (OTA-dG) were recorded over the range of m/z 100-383 and m/z 100-632, respectively, on a API Q-Trap mass spec-

Mally et al.

Figure 1. Radiocarbon content of DNA isolated from livers and kidneys of male rats treated with a single dose of 500 µg/kg body wt 14C-OTA (a) or 14C-OTB (b). No significant differences were observed in the group mean data as compared to controls (ANOVA, p > 0.05). trometer (Applied Biosystems) operating at negative ion mode. A Turbo Ion Spray source was used with N2 as the heater gas at 400 °C and a capillary voltage of -4500 V. The collision gas was N2 at CAD ) 4, and the collision energy was -30 V. The Q-Trap was coupled to an Agilent 1100 series HPLC/autosampler system, and LC conditions were as described above.

Results DNA Adducts of OTA and OTB as Studied by AMS. The 14C-labeled material used in this study was generated by Prof. Peter Mantle, Imperial College, by feeding 14C-acetate to an efficient fungus, resulting in 14COTA and 14C-OTB labeled in all carbon atoms of the isocumarine moiety. This procedure yielded 14C-OTA and 14 C-OTB in high purity (99% as determined by HPLC with UV detection) with a specific activity of 0.25 and 0.1 mCi/mmol, respectively. DNA isolated from livers and kidneys of animals treated with 14C-OTA or 14C-OTB was converted to graphite, and the 14C/13C ratios were determined by AMS. Sample sizes ranged from 0.3 to 8.85 mg of DNA. Samples that provided less than 0.5 mg of carbon (1.75 mg DNA) had tributyrin carrier added to provide sufficient carbon for efficient graphitization. Determined 14 C/13C ratios were then converted to fraction modern values, where 1 modern ) 13.56 dpm/g carbon. In all samples analyzed, fraction modern values ranged from 0.86 to 1.49 and the 14C content in DNA from livers and kidneys of treated animals was not significantly different from controls (Figure 1). Therefore, it was concluded that DNA from animals treated with 14C-OTA and 14C-OTB did not contain increased concentrations of 14C and thus no DNA adducts. On the basis of the specific activity of the compounds and a signal-to-noise ratio of 1.2 using the smallest DNA sample in the set, detection limits for these samples were calculated to be 3 adducts/109 nucleotides for OTA and 10 adducts/109 nucleotides for OTB. Formation of Metabolites and DNA Adducts in Vitro. LC-MS/MS analysis of the photoreaction of OTA

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Figure 2. LC-MS/MS analysis of the photoreaction of OTA (MRM m/z 402 to 358) in the presence of dG demonstrating formation of the carbon-bonded OTA-dG adduct (MRM m/z 633 to 517), OTHQ (MRM m/z 384 to 192), and OTB (MRM m/z 368 to 324) (a); enhanced product ion spectrum for m/z 633 (OTA-dG) (b) and m/z 384 (OTHQ) (c).

in the presence of dG revealed the formation of the carbon-bonded OTA-dG adduct (m/z 633 w 517.0) as described in the literature (Figure 2a) (16). The product was further characterized by enhanced product ion scans (Figure 2b). Beside OTA-dG, photoirradiation also yielded OTHQ (m/z 384.0 w 192.0) and OTB (m/z 367.8 w 323.8 and 367.8 w 132.9) (Figure 2a,c). In incubations of OTA with HRP and cumene hydroperoxide or H2O2, no metabolites were detected using HPLC with fluorescence detection (11) (data not shown). However, LC-MS/MS analysis revealed the presence of OTR, OTB, and OTHQ in incubations of OTA with HRP and 1 mM cumene hydroperoxide (Figure 3a). Interestingly, OTHQ was also

formed when OTB was incubated with peroxidase (Figure 3b). Using activation of OTA by Fe/H2O2 in the presence of GSH, both the hydroquinone and the GSH conjugate were detected by LC-MS/MS (Figure 4a). However, formation of the C8-guanosine adduct was not observed by LC-MS/MS using peroxidase or Fe/H2O2 activation of OTA in the presence of dG or DNA (Figure 4b). In agreement with the LC-MS/MS data, adducts were also not detected by 32P-postlabeling when either dGMP or DNA was incubated with OTA and various activation systems. In contrast, control incubations using peroxidase activation of 4-OH-TAM did result in formation of ad-

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Figure 3. LC-MS/MS analysis of in vitro incubations of OTA (a) and OTB (b) in the presence of HRP and cumene hydroperoxide (1 mM) for 60 min. Peroxidase activation of OTA (MRM m/z 402 to 358) leads to the formation of OTHQ (MRM m/z 384 to 192) and OTB (MRM m/z 368 to 324). OTHQ is also formed by HRP activation of OTB in the presence of 1 mM cumene hydroperoxide.

ducts as described in the literature (19). Activation of 4-OH-TAM was also evident by a color change of the reaction mixture due to polymerization of the substrate and loss of 4-OH-TAM as determined by HPLC as described previously by Davies et al. (20). In addition to 4-OH-TAM, a benz[a]pyrene adduct standard was used to ensure chromatographic conditions and efficiency of the 32P-postlabeling procedure. Representative chromatogramms of 32P-postlabeling analyses are shown in Figure 5.

Analyses of Metabolites and DNA Adducts in Vivo. To study formation of metabolites and DNA adducts in vivo, male F344 Fischer rats were administered high doses of OTA (2 mg/kg body wt) by gavage for 2 weeks. Animals showed clear signs of renal toxicity as evidenced by histopathology, reduced body wt gain, and an increase in urinary volume (data not shown, manuscript in preparation). In plasma, liver, and kidney homogenates of treated animals, OTA but no metabolites of OTA were detected. In contrast, analysis of urine

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Figure 4. (a) LC-MS/MS analysis showing oxidation of OTA to OTHQ (MRM m/z 384 to 192) and formation of a OTA-GSH conjugate (MRM m/z 689 to 416) in the presence of Fe/H2O2 and GSH. OTB is also formed (MRM m/z 368 to 324). (b) The carbon-bonded OTA-dG adduct (MRM m/z 633 to 517) is not detectable in incubations using peroxidase activation of OTA.

revealed the presence of OTR and the recently identified pentose and hexose conjugates (11). In addition, trace amounts of OTHQ and OTB were detected in urine collected over 24 h after the final dose (Figure 6). However, the postulated OTA-dG adduct could not be detected in DNA extracted from treated animals using LC-MS/MS. Moreover, no other OTA-related DNA adducts were evident in liver or kidney DNA by postlabeling (Figure 7).

Discussion The objectives of the work described here were to determine if OTA or metabolites formed from OTA bind to DNA in rat kidney, the target tissue for OTA carcinogenicity. In previous studies, conflicting results have been obtained regarding the potential of OTA to react with DNA. While experiments using 3H-labeled OTA in

vivo and in vitro indicate lack of formation of covalent DNA adducts (9, 10, 21), spots detected by 32P-postlabeling have been attributed to treatment with OTA (8, 22). However, these putative DNA adducts have not been shown to contain OTA or part of the OTA molecule and so far no structural information has been provided. Studies on biotransformation of OTA in vivo and in vitro suggest that OTA does not form reactive intermediates capable of interacting with DNA (3, 9-11). Alternatively, the involvement of cytotoxicity and oxidative stress in the carcinogenicity of OTA has been suggested as a potential mechanism to explain OTA-induced DNA damage (23, 24). Recently, formation of a carbon-bonded C8-dG adduct of OTA and a OTHQ/OTQ redox couple capable of reacting with GSH has been reported using electrochemical and photochemical activation (15, 16). This has

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Figure 5. 32P-postlabeling of DNA incubated with OTA in the presence of HRP or Fe/H2O2. No OTA related spots are observed. In contrast, DNA adducts are evident following peroxidase activation of 4-OH-TAM.

Figure 6. Detection of traces of OTHQ (MRM m/z 384 to 192, tR 10.4) and OTB (MRM m/z 368 to 324) in urine of rats treated with 2 mg/kg body wt OTA for 2 weeks.

prompted us to investigate if these OTA derivatives are also formed in vivo and in relevant, more physiological systems in vitro. Although small amounts of OTHQ and the derived GSH conjugate were formed in vitro using peroxidase and/or Fe/H2O2 activation, the postulated OTA-dG adduct was not detected. Lack of formation of OTA-derived DNA adducts was also confirmed by using the sensitive 32P-postlabeling method. In support of this, DNA adducts were also not observed using liquid scintil-

lation counting when [3H]OTA was incubated with various activation systems including HRP/H2O2 (9). Trace amounts of OTHQ and OTB were seen in urine of rats exposed to very high doses of OTA using a highly sensitive LC-MS/MS method. We have previously studied biotransformation of OTA in rats treated with a single dose of 500 µg /kg body wt. In these animals, we did not observe formation of OTHQ (11). While levels of OTHQ may have been below the limit of detection in urine of

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Figure 7. 32P-postlabeling analysis of synthetic OTA-dGMP as standard formed by photoirradiation of OTA (A) and DNA extracted from kidney of a rat treated with high doses of OTA (2 mg/kg body wt) for 2 weeks (B) showing lack of formation of DNA adducts by OTA using identical conditions.

animals given the lower dose, it is also possible that formation of the hydroquinone in high dose animals may be a result of the reaction of OTA with radicals generated by oxidative stress or inflammatory processes due to the severe cytotoxicity observed in the kidneys of these rats. In addition, the OTHQ-derived GSH conjugate was not detectable in plasma, liver, and kidney homogenates of treated animals and lack of detection of this conjugate despite the use of a sensitive method also indicates that the OTHQ/OTQ may only be formed to a very small extent in vivo. Interestingly, incubation of HRP with the nonchlorinated OTA analogue OTB yielded higher levels of OTHQ than incubation with OTA. Only few data are available regarding toxicity and genotoxic effects of OTB, but it has been shown to be nonmutagenic and also less toxic than OTA (25-28). Because biotransformation of OTB may also result in formation of OTHQ as suggested by

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our in vitro data, it is unlikely that the hydroquinone/ quinone redox couple plays a significant role in OTAmediated toxicity. Although the presence of the chlorine atom at C-5 of the OTA molecule has often been considered a structural requirement for OTA-induced toxic effects, differences in toxicity between OTA and OTB may rather be due to differences in toxicokinetics and the available data suggest that OTB is eliminated much faster (29). It is also important to note that OTR, which contains the chlorine atom and may also form the respective hydroquinone, is relatively nontoxic (12). Taken together, it appears that OTA-induced toxic effects are not associated with biotransformation to potentially reactive metabolites such as OTHQ. Metabolism of OTA has been extensively studied in vitro and in vivo, but no reactive intermediates capable of binding to DNA have been identified (3, 11). Therefore, until recently, no chemical structures of potential OTADNA adducts have been proposed. The carbon-bonded OTA-dG adduct formed by photoirradiation as reported by Dai et al. (16) is the first structurally characterized synthetic OTA-DNA adduct and was used as a standard for both LC-MS/MS and postlabeling analysis in this study. However, OTA-dG could not be detected in liver and kidney DNA of OTA-treated animals by either method, and together with the in vitro data, this suggests that this adduct is not formed in relevant concentrations under physiological conditions. Consistent with these findings and results from Gautier et al. (9), no OTArelated adducts were observed in DNA extracted from OTA-treated animals using the sensitive 32P-postlabeling technique. The AMS studies also suggest that DNA binding of OTA or OTB metabolites containing the isocoumarine ring does not occur. AMS is an extremely sensitive method to detect trace amounts of 14C in biological samples, and the sensitivity of this method permitted the detection of OTA-derived adducts in concentrations approximately 2 orders of magnitude below those estimated by the evaluation of spots obtained after 32P-postlabeling of DNA isolated from mice after treatment with OTA (8). In contrast to DNA binding, only few data are available regarding the potential of OTA to bind to proteins. However, no reactive intermediates have been detected so far and this implies that covalent binding to proteins is unlikely to play a major role in OTA toxicity. Although Gautier et al. (9) reported weak binding of OTA to microsomal protein of ram seminal vesicles enriched with prostaglandin-H-synthetase, the levels were approximately 100-fold lower than those observed after activation of pentachlorophenol or IQ (2-amino-3-methylimidazo[4,5-f]quinoline). Schwerdt et al. (30) investigated OTA protein binding in subcellular fractions of a number of kidney cell lines, rat intestine, liver, kidney, and spleen using OTA coupled to HRP to detect OTA binding proteins following separation by SDS-polyacrylamide electrophorhesis. OTA was shown to bind to several cytosolic and organelle proteins with high affinity. These data suggest that binding of OTA to specific proteins may occur independent of metabolic activation. Identification and characterization of OTA binding proteins may help identify specific cellular targets and provide important insight into the mechanism of OTA carcinogenicity. In conclusion, DNA binding of OTA, as postulated by others, could not be confirmed using a panel of independent methods. Furthermore, results from this study are

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consistent with and support findings of several groups who have applied methods other than postlabeling, such as the use of 3H-labeled OTA (9-10, 21). Our data clearly show that formation of DNA adducts through covalent interaction of OTA with DNA constituents does not occur to a relevant extent and is unlikely to represent a mechanism of OTA-induced renal tumor formation.

Acknowledgment. Parts of this work were supported by the Fifth RTD Framework Program of the European Union, Project No. QLK1-2001-01614. Other parts were supported by the Physiological Effects of Coffee Committee (PEC) in Paris, France. The AMS analyses were conducted at the Research Resource for Biomedical AMS, which is operated at LLNL under the auspices of the U.S. Department of Energy under Contract No. W-7405-ENG-48. The Research Resource is supported by the National Institutes of Health, National Center for Research Resources, Biomedical Technology Program. LC-MS/MS systems used were purchased with grants from the State of Bavaria and the Deutsche Forschungsgemeinschaft. We would also like to thank Prof. Peter Mantle for the preparation of 14C-OTA and 14 C-OTB.

References (1) Boorman, G. A., McDonald, M. R., Imoto, S., and Persing, R. (1992) Renal lesions induced by ochratoxin A exposure in the F344 rat. Toxicol. Pathol. 20, 236-245. (2) Fo¨llmann, W., and Lucas, S. (2003) Effects of the mycotoxin ochratoxin A in a bacterial and a mammalian in vitro mutagenicity test system. Arch. Toxicol. 77, 298-304. (3) Zepnik, H., Pa¨hler, A., Schauer, U., and Dekant, W. (2001) Ochratoxin A-induced tumor formation: is there a role of reactive ochratoxin A metabolites? Toxicol. Sci. 59, 59-67. (4) Bendele, A. M., Neal, S. B., Oberly, T. J., Thompson, C. Z., Bewsey, B. J., Hill, L. E., Rexroat, M. A., Carlton, W. W., and Probst, G. S. (1985) Evaluation of ochratoxin A for mutagenicity in a battery of bacterial and mammalian cell assays. Food Chem. Toxicol. 23, 911-918. (5) Dopp, E., Mu¨ller, J., Hahnel, C., and Schiffmann, D. (1999) Induction of genotoxic effects and modulation of the intracellular calcium level in syrian hamster embryo (SHE) fibroblasts caused by ochratoxin A. Food Chem. Toxicol. 37, 713-721. (6) Ehrlich, V., Darroudi, F., Uhl, M., Steinkellner, H., Gann, M., Majer, B. J., Eisenbauer, M., and Knasmu¨ller, S. (2002) Genotoxic effects of ochratoxin A in human-derived hepatoma (HepG2) cells. Food Chem. Toxicol. 40, 1085-1090. (7) Degen, G. H., Gerber, M. M., Obrecht-Pflumio, S., and Dirheimer, G. (1997) Induction of micronuclei with ochratoxin A in ovine seminal vesicle cell cultures. Arch. Toxicol. 71, 365-371. (8) Pfohl-Leszkowicz, A., Grosse, Y., Kane, A., Creppy, E. E., and Dirheimer, G. (1993) Differential DNA adduct formation and disappearance in three mouse tissues after treatment with the mycotoxin ochratoxin A. Mutat. Res. 289, 265-273. (9) Gautier, J., Richoz, J., Welti, D. H., Markovic, J., Gremaud, E., Guengerich, F. P., and Turesky, R. J. (2001) Metabolism of ochratoxin A: absence of formation of genotoxic derivatives by human and rat enzymes. Chem. Res. Toxicol. 14, 34-45. (10) Gross-Steinmeyer, K., Weymann, J., Hege, H. G., and Metzler, M. (2002) Metabolism and lack of DNA reactivity of the mycotoxin ochratoxin a in cultured rat and human primary hepatocytes. J. Agric. Food. Chem. 50, 938-945. (11) Zepnik, H., Voelkel, W., and Dekant, W. (2003) Toxicokinetics of the mycotoxin ochratoxin A in F344 rats after oral administration. Toxicol. Appl. Pharmacol. 192, 36-44.

Mally et al. (12) Xiao, H., Madhyastha, S., Marquardt, R. R., Li, S., Vodela, J. K., Frohlich, A. A., and Kemppainen, B. W. (1996) Toxicity of ochratoxin A, its opened lactone form and several of its analogues: structure-activity relationships. Toxicol. Appl. Pharmacol. 137, 182-192. (13) Calcutt, M. W., Gillman, I. G., Noftle, R. E., and Manderville, R. A. (2001) Electrochemical oxidation of ochratoxin A: correlation with 4-chlorophenol. Chem. Res. Toxicol. 14, 1266-1272. (14) Bodell, W. J., Pathak, D. N., Levay, G., Ye, Q., and Pongracz, K. (1996) Investigation of the DNA adducts formed in B6C3F1 mice treated with benzene: implications for molecular dosimetry. Environ. Health. Perspect. 104 (Suppl. 6), 1189-1193. (15) Dai, J., Park, G., Wright, M. W., Adams, M., Akman, S. A., and Manderville, R. A. (2002) Detection and characterization of a glutathione conjugate of ochratoxin A. Chem. Res. Toxicol. 15, 1581-1588. (16) Dai, J., Wright, M. W., and Manderville, R. A. (2003) Ochratoxin a forms a carbon-bonded C8-deoxyguanosine nucleoside adduct: implications for C8 reactivity by a phenolic radical. J. Am. Chem. Soc. 125, 3716-3717. (17) Turteltaub, K. W., Vogel, J. S., Frantz, C. E., and Fultz, E. (1993) Studies on DNA adduction with heterocyclic amines by accelerator mass spectrometry: a new technique for tracing isotope-labeled DNA adduction. IARC Sci. Publ. 124, 293-301. (18) Obrecht-Pflumio, S., and Dirheimer, G. (2000) In vitro DNA and dGMP adducts formation caused by ochratoxin A. Chem.-Biol. Interact. 127, 29-44. (19) Pathak, D. N., Pongracz, K., and Bodell, W. J. (1995) Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in SpragueDawley rats treated with tamoxifen. Carcinogenesis 16, 11-15. (20) Davies, A. M., Malone, M. E., Martin, E. A., Jones, R. M., Jukes, R., Lim, C. K., Smith, L. L., and White, I. N. (1997) Peroxidase activation of 4-hydroxytamoxifen to free radicals detected by EPR spectroscopy. Free Radical Biol. Med. 22, 423-431. (21) Schlatter, C., Studer, R. J., and Rasonyi, T. (1996) Carcinogenicity and kinetic aspects of ochratoxin A. Food Addit. Contam. 43-44. (22) Obrecht-Pflumio, S., and Dirheimer, G. (2001) Horseradish peroxidase mediates DNA and deoxyguanosine 3′-monophosphate adduct formation in the presence of ochratoxin A. Arch. Toxicol. 75, 583-590. (23) Gautier, J. C., Holzhaeuser, D., Markovic, J., Gremaud, E., Schilter, B., and Turesky, R. J. (2001) Oxidative damage and stress response from ochratoxin a exposure in rats. Free Radical Biol. Med. 30, 1089-1098. (24) Schaaf, G. J., Nijmeijer, S. M., Maas, R. F., Roestenberg, P., de Groene, E. M., and Fink-Gremmels, J. (2002) The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells. Biochim. Biophys. Acta 1588, 149-158. (25) Wurgler, F. E., Friederich, U., and Schlatter, J. (1991) Lack of mutagenicity of ochratoxin A and B, citrinin, patulin and cnestine in Salmonella typhimurium TA102. Mutat. Res. 261, 209-216. (26) O’Brien, E., Heussner, A. H., and Dietrich, D. R. (2001) Species-, sex-, and cell type-specific effects of ochratoxin A and B. Toxicol. Sci. 63, 256-264. (27) Malaveille, C., Brun, G., and Bartsch, H. (1991) Genotoxicity of ochratoxin A and structurally related compounds in Escherichia coli strains: studies on their mode of action. IARC Sci. Publ. 115, 261-266. (28) Dietrich, D. R., O’Brien, E., Stack, M. E., and Heussner, A. H. (2001) Species- and sex-specific renal cytotoxicity of ochratoxin A and B in vitro. Exp. Toxicol. Pathol. 53, 215-225. (29) Breitholtz-Emanuelsson, A., Fuchs, R., Hult, K., and Appelgren, L. E. (1992) Synthesis of 14C-ochratoxin A and 14C-ochratoxin B and a comparative study of their distribution in rats using whole body autoradiography. Pharmacol. Toxicol. 70, 255-261. (30) Schwerdt, G., Freudinger, R., Silbernagl, S., and Gekle, M. (1999) Ochratoxin A-binding proteins in rat organs and plasma and in different cell lines of the kidney. Toxicology 135, 1-10.

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