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Chem. Res. Toxicol. 2002, 15, 1581-1588

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Detection and Characterization of a Glutathione Conjugate of Ochratoxin A Jian Dai, Gyungse Park, Marcus W. Wright, Marissa Adams,† Steven A. Akman,† and Richard A. Manderville* Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486, Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157 Received August 2, 2002

The ability of the carcinogenic mycotoxin ochratoxin A (OTA) to react with reduced glutathione (GSH) has been assessed using electrospray ionization (ES)-MS techniques. On the basis of the assumption that OTA undergoes biotransformation into the reactive quinone species OTQ (6), a synthetic sample of the reduced form of OTQ (6), hydroquinone OTHQ (5), was prepared and photoreacted with 6 M equiv of GSH to yield an authentic sample of the conjugate 8 that was definitively identified by mass spectrometry, UV-vis spectroscopy and NMR. With the authentic sample of 8 in hand, it was demonstrated that the same conjugate is produced from reaction of 100 µM OTA (1) in the presence of 5 mM GSH following incubation for 1 h with either horseradish peroxidase (HRP)/H2O2, rat liver microsomes (RLM)/NADPH or free Fe(II). In each of these oxidative systems the conjugate 8 was generated in less than 1% yield and the parent OTA molecule is poorly metabolized. Comparison of the peak area ratio of the conjugate 8 to that for the hydroxyOTA metabolite from the RLM/NADPH system implied that the conjugate was produced at a rate of ∼1-3 pmol min-1 (mg of protein)-1. These studies are the first to demonstrate that OTA undergoes biotransformation to a reactive intermediate [OTQ (6)] that covalently reacts with GSH to yield the conjugate 8. The biological implications of the reactivity of OTA toward GSH are discussed.

Introduction Ochratoxin A (OTA,1 1, Figure 1) is a mycotoxin produced by several strains of Penicillium and Aspergillus fungal species (1, 2). It contaminates a wide range of food and feed (2) and acts as a potent renal carcinogen in rodents (1). In humans, OTA has been associated with Balkan endemic nephropathy (BEN), a degenerative kidney disease in which patients suffer from urinary tract tumors (3). OTA may also be a cause of testicular cancer, as the major features of the descriptive epidemiology of testicular cancer are associated with its exposure (4). OTA is known to facilitate single-strand DNA cleavage (5, 6), and the 32P-postlabeling assay has shown that OTA generates guanine-specific DNA adducts (7, 8) upon oxidative activation by peroxidases and/or certain cytochrome P450 isoforms (7-11). While these observations provide a rationale for the mutagenicity (12) and subsequent carcinogenicity of OTA (1), the chemical structures of the possible adducts have not been defined and nor have the oxidative enzymes been shown to transform

Figure 1. Chemical structures of ochratoxins (OTA, 1) and their analogues.

* To whom correspondence should be addressed. Telephone: (336) 758-5513. Fax: (336) 758-4656. E-mail: [email protected]. † Wake Forest University School of Medicine. 1 Abbreviations: OTA, ochratoxin A (N-{[(3R)-5-chloro-8-hydroxy3-methyl-1-oxo-7-isochromanyl]carbonyl}-3-phenyl-L-alanine); BEN, Balkan endemic nephropathy; OTHQ, ochratoxin hydroquinone (N{[(3R)-5,8-dihydroxy-3-methyl-1-oxo-7-isochromanyl]carbonyl}-3-phenyl-L-alanine); OTQ, ochratoxin quinone (N-{[(3R)-3-methyl 1,5,8-trioxo7-isochromanyl]carbonyl}-3-phenyl-L-alanine); CySH, cysteine; γ-GT, γ-glutamyl transpeptidase; HRP, horseradish peroxidase; RLM, rat liver microsomes; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond connectivity; SCE, saturated calomel electrode; HSA, human serum albumin.

OTA into reactive intermediates capable of binding covalently with biological nucleophiles (9-11). Instead, biotransformation of OTA generates the hydroxylated species 2-4 (Figure 1) (13) which do not react covalently with DNA (14, 15). Recent studies on the metabolism of OTA do not support the premise that OTA or metabolically activated species covalently bind to DNA (16-18). Thus, the mechanism of OTA carcinogenicity in rodents is not known and whether oxidative activation plays a role in OTA-mediated toxicity is still under debate.

10.1021/tx0255929 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/09/2002

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Our laboratory has demonstrated that the hydroquinone (OTHQ, 5)/quinone (OTQ, 6, Figure 1) redox couple is generated from the oxidation of OTA by electrochemical (19) and photochemical processes (20). A biomimetic iron-oxo system and H2O2 converts OTA into OTHQ (5) in the presence of ascorbate (21); the same transformation is also accomplished by replacing the ironoxo system with copper ions.2 Since quinones are both oxidants and electrophiles and can redox-cycle to create an oxidative stress and form covalent adducts with cellular macromolecules (22, 23), the biotransformation of OTA into OTQ (6) would provide a basis for the toxin’s ability to promote oxidative stress (24) and generate DNA adducts (7, 8). To assess the reactivity of OTQ (6) we have been initially studying its interaction with biologically significant sulfur nucleophiles. Here, we recently demonstrated that OTHQ (5) reacts photochemically with cysteine (CySH) to produce the cysteinyl conjugate 7 (Figure 1), probably by the in situ generation of OTQ (6) that reacts with CySH via a Michael addition pathway (25). From the photoreaction of 5 with excess CySH, it was possible to generate sufficient quantities of 7 for NMR characterization. With 7 in hand, we were then able to show that the same species formed from the photochemical reaction of OTA (1) in the presence of excess CySH, providing further evidence for the intermediacy of OTQ (6) in the photooxidation of OTA (20, 25). In the present manuscript we have utilized the photochemical strategy described above to generate an authentic sample of an OTA-GSH conjugate in order to assess bioactivation of OTA in the presence of reduced glutathione (GSH). GSH is the major non-protein sulfhydryl present in cells and while its conjugation with strong electrophiles is considered a mechanism of cellular protection (26), certain conjugates act as nephrotoxicants in tissues rich in γ-glutamyl transpeptidase (γ-GT) (22, 27). Thus, it was anticipated that the demonstrated ability of OTA to react with GSH could have important biological implications for OTA nephrotoxicity and would provide strong evidence that OTA undergoes biotransformation into the quinone species 6 that is capable of reacting covalently with macromolecules.

Experimental Procedures Caution: The work described involves the synthesis and handing of hazardous agents and was therefore conducted in accordance with NIH guidelines for the Laboratory use of Chemical Carcinogens (28). Materials. Ochratoxin A (OTA, 1), reduced glutathione (GSH), horseradish peroxidase Type VI (HRP), β-nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate were purchased from SigmaAldrich and used as received. The hydroquinone form of ochratoxin A (OTHQ, 5, mixtures of diastereomers) was synthesized using a strategy outlined by Sibi and co-workers (29), as previously described (21). Aroclor 1254-induced SpragueDawley rat liver microsomes (RLM) was obtained from IN VITRO Technologies and used without further purification. Acetonitrile (ACN, HPLC grade), methanol (MeOH, HPLC grade) and formic acid (90%, purified) were purchased from Fisher Scientific. Deionized water from a Milli-Q system (Millipore) was used for preparation of all aqueous solutions. LC/MS Analyses. LC/MS analyses were performed on an Agilent 1100 series LC/MSD Trap system with an electrospray 2

Jian Dai and Richard A. Manderville, unpublished results.

Dai et al. ionization (ESI) interface. Samples (20 µL) were injected into the LC/MSD system through an Agilent 1100 series autosampler. Separations were carried out on a 5µm Agilent ZORBAX SB-C18 column (4.6 × 150 mm) at 37 °C, controlled by an Agilent 1100 series thermostat, with a flow rate of 0.75 mL/ min using HPLC method I or II. Two mobile phase solvents were used in these methods. Solvent A was 0.1% formic acid in deionized water. Solvent B was 0.1% formic acid in ACN. HPLC method I, a linear gradient from 80:20 (A:B) to 40:60 in 15 min followed by another linear gradient to 25:75 in 5 min (used for OTA samples). HPLC method II, a linear gradient from 80:20 (A:B) to 40:60 in 15 min followed by isocratic elution for 2 min at 40:60 (employed in analyses of OTHQ samples). The flow was directed to the electrospray source after passing through an Agilent 1100 series diode array multiple wavelength detector (detection at 350 nm). The LC-MSD-Trap-00045 was operated at ESI negative ion mode (ES-) with a capillary voltage of 3500 V. Data were acquired over the m/z range of 100-1000 under standard mode with normal scan resolution (13 000 m/z per s). Data analyses were performed using LC/MSD trap Control 4.0 DataAnalysis Version 2.0 software. NMR Parameters. All NMR spectra were collected on a Bruker 500 DRX equipped with a 5 mm triple resonance broadband inverse probe. Data collection was at 298 K in 99.9% D2O in a 1 mm NMR tube. The 1H 1-D spectra were collected with 64 scans using double-pulsed field gradient water suppression (DPFGSE-WATERGATE) (30). The 2-D gradient selected COSY spectra were collected with 2048 points in the F2 dimension and 512 points in the F1 dimension with 56 scans/ time increment. The 1H 2-D spectral width was 10 ppm centered on the water resonance. Processing 1H 2-D spectra consisted of making both F2 and F1 dimensions 1024 points. After careful inspection the 2-D data was symmetrized. Assignment of the 13C resonances were carried out with gradient selected HMQC and HMBC. The 1H and 13C sweep widths were set to 10 and 250 ppm, respectively, in both experiments. HMQC and HMBC experiments were collected with 1024 points in the F2 dimension and 256 points the F1 dimension with 72 and 192 scans/time increment, respectively. Processing 1H-13C 2-D spectra consisted of making both F2 and F1 dimensions 512 points. HMBC allowed all carbonyl carbons (including carboxylic acids) to be found with confidence; however, the quaternary aromatic carbons were not completely assigned due to the lack of some couplings to 1H neighbors. Autoxidation of OTHQ in the Presence of GSH. A reaction mixture (total volume 500 µL) of OTHQ (5, 10, 100, or 200 µM) in 100 mM phosphate buffer (pH 7.4) was incubated at 37 °C in the presence of 1 mM GSH. After varying lengths of incubation time, a 20 µL aliquot of the reaction mixture was injected on the LC/MS system and analyzed by LC/MS using HPLC method II. Incubation of OTHQ (5, 200 µM) in 10 mM MOPS/100 mM NaCl buffer (pH 7.4) was also carried out and analyzed by LC/MS. Isolation and Characterization of GSH Conjugate 8 from Photoreaction of OTHQ (5) and GSH. A reaction mixture (5 mL total volume) of 1 mM OTHQ (5) and 6 M equiv of GSH in 50 mM phosphate buffer (pH 7.4) was irradiated for 10 min using an ILC Technology 300 W xenon arc lamp through a Pyrex filter (λ > 300 nm). The conjugate 8 was obtained as a diasteroisomeric mixture with the two products almost coeluting at ∼7 min on the Hitachi 7000 HPLC system. Semipreparatory HPLC was performed on this system with a 5 µm Phenomenex Kromasil C8 column (10.00 × 250 mm) at a flow rate of 5.90 mL/min using the following mobile phase: 80:20 (0.l % formic acid in H2O:0.1% formic acid in ACN) to 40:60 in 15 min by a linear gradient followed by isocratic elution for 2 min at 40:60. The photochemical reaction and HPLC separation were repeated four times, and fractions containing the isolated OTA-GSH conjugate (8) were combined. The conjugate 8 was obtained as a white solid after removal of HPLC solvents on a freeze-dry system (Freezon 4.5, LABCONCO). The white solid was dissolved in 99.9% D2O (1 mL), then lyophilized to dryness. The

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Scheme 1. Proposed Pathway for the Autoxidation of OTHQ (5) in the Absence and Presence of GSH

process was repeated twice. Yield was estimated to be 12% based on amount of OTHQ used. 1H NMR (D2O, ppm): 1.37 (3H, H1); 2.00 (2H, H13, 13′); 2.35 (2H, H12, 12′); 2.60 (1H, H3); 3.01 (2H, H9, 9′); 3.04 (1H, H5); 3.05 (1H, H3′); 3.21 (1H, H5′); 3.58 (2H, H11); 3.64 (1H, H14); 4.29 (1H, H10); 4.69 (2H, H2, H4); 7.25 (5H, H6, 7,8) ppm. 13C NMR (D2O, ppm): C)O 169.6, 171.3, 173.8, 174.8, 175.8, 177.4, 178.9; CH: 58.0 (C2); 78.3 (C4); 55.0 (C10); 55.3 (C14); CH2: 29.8 (C3); 38.8 (C5); 37.1 (C9); 44.5 (C11); 32.5 (C12), 27.2 (C13); CH3: 20.9 (C1); proton bearing carbon on phenyl ring: 128.1-130.3; quaternary aromatic carbons (not resolved): 126.4 to 146.2 ppm. UV-vis (λmax, nm): 295; 352. LC/MS (ES-, ion, m/z): [M - H]-, 689; [M H-GSH+SH]-, 416; [M - H-GSH+SH-CO2]-, 372. Bioactivation of OTA (1) in the Presence of GSH. (1) Horseradish Peroxidase Activation. A reaction mixture (500 µL total volume) of 100 µM OTA (1) and 5 mM (or 1 mM) GSH in 100 mM phosphate buffer (pH 7.4) was incubated with horseradish peroxidase Type VI (HRP, 2.6 units/mL) in the absence or presence of H2O2 (100 µM) at 37 °C for at least 60 min (up to 24 h). At various lengths of incubation time, 20 µL of the reaction mixture was analyzed by LC/MS using HPLC method I. Incubations of OTA (1) and HRP in the absence of GSH were also studied. (2) RLM Activation. A reaction mixture (500 µL total volume) of 100 µM OTA (1) and 5 mM GSH in 100 mM phosphate buffer was incubated at 37 °C for 60 min with rat liver microsomes (RLM, 1 mg/mL) in the presence of an NADPH regenerating system (100 µM) (31). The supernatant (20 µL) was analyzed by the LC/MS. Incubations of OTA (1, 500 µM) and GSH (3 mM) with or without RLM were carried out and analyzed in a similar manner. (3) Iron (II) Activation. Reaction mixtures (500 µL total volume) of 100 µM OTA (1), 10 µM Fe(NH3)2(SO4)2 and 5 mM GSH in 100 mM phosphate buffer (pH 7.4) were incubated with or without H2O2 at 37 °C for 60 min. An aliquot (20 µL) was analyzed by LC/MS.

Results Autoxidation of OTHQ in the Absence and Presence of GSH. As presented previously (21), OTHQ (5) is oxidized by O2 in a pH-dependent fashion (t1/2 ) 11.1 h at pH 7.4). The autoxidation is accompanied by a decrease and “blue” shift of the absorbance at ca. 350400 nm (hydroquinone moiety) with an increase in absorbance at 270 nm (21). From previous LC/MS studies (21), the reaction pathway outlined in Scheme 1 was

Figure 2. Base peak chromatograms (BPCs) from incubation of 200 µM OTHQ (5) in (a) 10 mM MOPS/100 mM NaCl buffer, pH 7.4, at 37 °C for 6 h, (b) 100 mM phosphate buffer, pH 7.4, at 37 °C for 6 h in the presence of 1 mM GSH.

proposed for the aqueous decomposition of OTHQ. The autoxidation is believed to generate OTQ (6) that subsequently reacts with H2O/HO- to yield the o-hydroquinone that can undergo oxidation to the catechol. As described by Sawaki and Foote (32), catechols undergo C-C cleavages through reaction with HOO- via an acyclic Baeyer-Villiger type mechanism, which for the catechol would yield the diacid shown in Scheme 1. Since these studies predicted that the autoxidation process would yield OTQ (6) as a reactive intermediate (21), the aqueous decomposition of OTHQ in the absence and presence of GSH was examined by LC/MS. Shown in Figure 2a is the base peak chromatogram from incubation of 200 µM OTHQ (5) in 10 mM MOPS/ 100 mM NaCl buffer, pH 7.4, at 37 °C for 6 h. In Table 1 are given retention times (tR), [M - H]- ions, and λmax values for the species observed in Figure 2a. The proposed structures in the table refer to those given in Scheme 1. The dominant products eluting between 4 and 6 min possess [M - H]- peaks at m/z 432 and exhibited fragment ions at m/z 388 and 370 corresponding to losses of CO2 and H2O from the parent ion. The absence of the

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Table 1. Spectral Data for the Autoxidationof OTHQ (5)a tR (min)b

[M - H]-

λmax (nm)

product

4.7, 5.0, 5.1 7.8, 8.5 9.9, 10.4 10.8 13.2, 13.4 13.6, 13.7 14.2

432 414 400 462 384 428 398

257, 305 231, 277 356 355 352 ndc nd

diacid anhy o-hydro unknown1 OTHQ (5) unknown2 cate

a 200 µM OTHQ in 10 mM MOPS/100mM NaCl buffer, pH 7.4, incubated at 37 °C for 6 h. b HPLC conditions: 80:20 (0.1% formic acid in H2O:0.1% formic acid in ACN) to 40:60 in 15 min using a linear gradient at a flow rate of 0.75 mL/min followed by isocratic elution at 40:60 for 2 min. c Not detected.

Figure 4. 500 MHz 1H NMR spectrum of GSH conjugate 8 acquired in D2O by the double-pulsed field gradient water suppression method.

Figure 3. (a) ES- spectrum of GSH conjugate 8, (b) UV-vis spectrum of GSH conjugate 8 obtained from the diode array detector.

hydroquinone chromophore at 352 nm (λmax ) 257, 305 nm) suggested these species to be the diacid forms of the OTHQ diasteromers (diacid). Broad peaks at 7.8 and 8.5 min had [M - H]- ) 414, which represents a loss of 18 mass units (H2O) from the diacid, indicating the possible formation of an anhydride species (anhy). Peaks eluting at 9.9 and 10.4 min had [M - H]- ) 400, which is 16 mass units heavier than OTHQ. The absorbance of these species was “red” shifted from OTHQ, suggesting formation of the o-hydroquinone shown in Scheme 1. A small peak eluting after OTHQ at 14.2 min, gave a [M - H]peak at m/z 398, indicating the possible presence of a catechol species (cate). Shown in Figure 2b is a base peak chromatogram from incubation of 200 µM OTHQ (5) in the presence of 1 mM GSH in 100 mM phosphate buffer (pH 7.4), at 37 °C for 6 h. Interestingly, peaks ascribed to formation of the diacid and anhy (Scheme 1) were not present and instead a new peak eluting at ∼7.0 min was observed possessing the spectral features shown in Figure 3. The new species had [M - H]- ) 689 corresponding to attachment of GSH to OTHQ with the loss of two protons (Figure 3a). The product showed loss of 273 mass units to form a prominent ion at m/z 416, which is 32 mass units heavier than OTHQ (5) ([M - H]- ) 384). This loss corresponds to

β-elimination of benzoquinol-SH, which is an established fragmentation of S-peptide-benzoquinone adducts (33). The UV spectrum of the product (Figure 3b) indicated two maxima at 295 and 352 nm, which was similar to those observed for the cysteinyl adduct 7 (Figure 1) (25). These results suggested that the new product was the anticipated Michael addition conjugate 8 (Scheme 1) resulting from attachment of GSH to the unsubstituted position of OTQ (6). That addition of 1 mM GSH should inhibit diacid and anhy formation was consistent with the pathways outlined in Scheme 1 where nucleophilic attachment of OTQ (6) by GSH to form 8 would inhibit the aqueous decomposition of OTQ. Isolation and Characterization of GSH Conjugate 8. To obtain sufficient quantities of the GSH conjugate 8 (Scheme 1) for NMR analyses, the photochemical reaction of OTHQ in the presence of 6 equiv of GSH was utilized. As anticipated (25), the photoreaction produced the GSH conjugate 8 in a much higher yield than the autoxidative process and enabled full characterization of the conjugate by NMR methods. Figure 4 shows the 1H NMR spectrum of the GSH conjugate 8. The absence of an aromatic hydroquinone proton resonance confirmed formation of a covalent bond between GSH and OTHQ. The assignments of nonexchangeable protons were made from COSY spectra. All seven carbonyl carbons (including carboxylates) were identified with confidence from the gradient selected HMQC and HMBC spectra (see Experimental Section for NMR assignment of 8). Biotransformation of OTA in the Presence of GSH. With an authentic sample of the conjugate 8 in hand, it was possible to test the prediction that biotransformation of OTA (1) generates OTQ (6) that subsequently reacts with GSH to generate 8. Three oxidative catalysts [HRP, RLM, and free Fe(II)] were utilized, and the products were analyzed by LC/MS with ES- detection. Metabolites were not observed after incubation of OTA (100 µM) and HRP (2.6 units/mL) in the presence of 100 µM H2O2 at 37 °C for 1 h. A trace amount of the lactonering opened form of OTA (LOF) (34), [M - H]- ) 420, was observed after 24 h incubation. The addition of excess GSH (1 or 5 mM) to the OTA/HRP reaction produced a

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Figure 5. Base peak chromatograms (BPCs) from incubations of 100 µM OTA (1) and 5 mM GSH in 100 mM phosphate buffer (pH 7.4) at 37 °C for 1 h in the presence of (a) horseradish peroxidase (HRP, 10 µg/mL) and H2O2 (100 µM), (b) rat liver microsomes (RLM, 1 mM/mL) and NADPH (100µM) [the asterisk (*) indicates 2/3], and (c) Fe2(NH4)2(SO4)2 (10 uM). (d) Base peak chromatogram from incubation of 200 µM OTHQ (5) and 1 mM GSH in 100 mM phosphate buffer (pH 7.4) at 37 °C for 1 h.

small amount of the conjugate 8 after 1 h incubation at 37 °C, as shown in Figure 5a (conjugate is indicated by the arrow for the peak eluting at ∼7 min). The oxidation of OTA (100 µM) by RLM (1 mg/mL) in the presence of the NADPH regenerating system (100 µM) gave a small amount of 4-hydroxyl-OTA, 2/3 (Figure 1, [M - H]- ) 418) as the major metabolite. The addition of 5 mM GSH led to a higher rate of 2/3 formation (17), and generated a small amount of the GSH conjugate 8, as shown in Figure 5b [2/3 are represented with an asterisk (*)]. Many of the peaks eluting between 8 and OTA (7-17 min) in Figure 5b were also present from RLM/NADPH in the absence of OTA. The off-scale peak at ∼11.5 min was ascribed to an oxidized GSH product. Incubation of OTA (100µM) and GSH (5 mM) at 37 °C for 1 h in the presence of 10 µM Fe(NH3)2(SO4)2 produced trace amount of GSH conjugate 8 as the major product (Figure 5c). The addition of H2O2 (1 mM) doubled the rate of GSH conjugate 8 formation. There were no other metabolites detected in this system. The data presented in Figure 5 indicated that OTA (100 µM) was poorly metabolized by HRP/H2O2, RLM/ NADPH, or Fe(II), as noted by others (16-18). In the presence of 1 or 5 mM GSH, the conjugate 8 is detectable by LC/MS, confirmed by spiking the sample with authentic 8, and is generated in less than 1% yield (based on a calibration curve obtained from the isolated sample of 8). It is important to note that no significant differences

in the ability of these catalysts to generate 8 were observed. The yields of adduct remained relatively constant, and only the RLM/NADPH system was able to facilitate formation of the hydroxylated species 2/3 (Figure 1). In this regard, rat liver microsomes fortified with NADPH generates 4(R)-hydroxyochratoxin A (2, Figure 1) at low rates [10-25 pmol min-1 (mg of protein)-1] (17). From the peak areas of the base peak chromatogram presented in Figure 5b [peak for 8 vs peak marked by the asterisk (*)] it is evident that rates of conjugate formation are even lower and are estimated to be ∼1-3 pmol min-1 (mg of protein)-1.

Discussion In the present manuscript we have shown that OTQ (6) reacts covalently with GSH to yield the conjugate 8 (Scheme 1, Figure 4). More importantly, we have demonstrated that the same conjugate is generated from reaction of 100 µM OTA (1) in the presence of excess (1 or 5 mM) GSH following incubation for 1 h with either HRP/H2O2, RLM/NADPH or Fe(II). In each of these systems the conjugate 8 is produced in less than 1% yield and the parent OTA molecule is poorly metabolized. Nevertheless, our studies are the first to show that OTA undergoes biotransformation to a reactive intermediate [OTQ (6)] that covalently reacts with GSH to yield the conjugate 8 that has been definitively identified by mass spectrometry, UV-vis spectroscopy and NMR.

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Scheme 2. Proposed Pathways for the Bioactivation of OTA (1) in the Presence of GSH

That OTA should undergo bioactivation into the quinone OTQ (6) that reacts with GSH to yield 8 was not surprising given our published data on the electrochemical (19), photochemical (20, 25), and model enzyme work (21) on the oxidation of OTA. Structure-activity relationships have demonstrated the importance of the chlorine atom in OTA-mediated toxicity (35) and halogenated phenols are known to undergo bioactivation to yield benzoquinone derivatives that react covalently with macromolecules (36, 37). However, for OTA that is poorly metabolized by oxidative enzymes, detection of products arising from covalent attachment to OTQ (6) is difficult. We have been unable to isolate an authentic sample of the quinone species 6, due to its predicted reactivity and short lifespan, and the reduced precursor OTHQ (5) is not entirely trivial to synthesize. However, with relatively small amounts of OTHQ (5), we have found it possible to utilize its photoreactivity to generate OTQ (6) in situ for reaction with biological nucleophiles, as demonstrated here for GSH. The establishment that OTA can react with GSH to yield 8 has increased our understanding of oxidative activation of OTA (1). The pathways outlined in Scheme 2 represent our model for bioactivation of OTA (1) and subsequent reactions with GSH. As presented previously (19), the phenolic form of OTA undergoes a 2e/1H+ oxidative process in acetonitrile and exhibits a half-peak oxidation potential (Ep/2) of 1.81 V vs the saturated calomel electrode (SCE). The iron-oxo entity in CYP450 is estimated to have a redox potential between 1.7 and 2.0 V vs SCE (38), which is high enough to directly covert protonated OTA into the phenoxonium cation; nucleophilic attachment by H2O with loss of HCl would yield OTQ (6) that reacts with GSH to yield the conjugate 8. The pKa of the phenolic moiety of OTA is ∼7 (33, 39), and recent studies show that binding of OTA to human serum albumin (HSA) lowers the phenolic pKa by more than three pK units (40), suggesting that the dianion may be the dominant form of OTA in a biological system. The phenolate of OTA undergoes a 1e oxidative process to yield the phenolic radical with Ep/2 ≈ 0.8 V (∼1.04 V vs NHE) (19). Horseradish peroxidase is active up to ∼1.16 V vs SCE (41) and so can convert the phenolate of OTA into the phenolic radical, but is predicted to be unable to oxidize the phenolic form into the phenoxonium cation (19). The oxidation potential of GSH is ∼0.9 V vs NHE

(42), and thus the OTA phenolic radical would be expected to cause the oxidation of GSH into the thiyl radical GS•, which would yield the disulfide GSSG + O2•(42). To generate OTQ (6) from the phenolic radical without further oxidation (1e) into the phenoxonium cation requires coupling to an O-centered radical to displace the chlorine atom. In Scheme 2, we propose a radical coupling pathway involving O2•- to yield the intermediate 9. Heterolysis of the C-Cl bond would generate the quinone O-oxide 10, which is the assumed intermediate in the photolysis of chlorinated phenols in the presence of O2 (43). Reaction of this species with H2O would give OTQ (6) following the loss of H2O2 (43). That HRP/H2O2, RLM/NADPH and free Fe generated similar amounts of the conjugate 8 suggested a common route for its formation. As discussed above, the redox properties of the various species favor the phenolic radical route in Scheme 2, as HRP/H2O2 and free Fe are unlikely to oxidize protonated OTA into the phenoxonium cation. Thus, it is not surprising that low levels of the conjugate 8 would be detected, since the OTA phenolic radical is more likely to cause the oxidation of GSH (42) prior to coupling with an oxygen radical for OTQ (6) production. In this regard, Gautier et al. (17) did not detect the GSH conjugate 8 using 100 µM [3H]OTA in the presence of rat liver and kidney and human S-9 preparations following 30 min or 15 min incubation time. Gross-Steinmeyer et al. (18) found no evidence for covalent binding by [3H]OTA following activation by cultured rat and human primary hepatocytes for 8 h, but noted that the cellular ratio of GSH to GSSG was significantly decreased by treatment with 10 µM OTA. It is also worthy to note that OTA has been shown to disrupt Ca2+ homeostasis (40, 44, 45), which is also consistent with generation of the phenolic radical with subsequent oxidation of protein sulfhydryl groups (46). The conjugate 8 may play a role in the toxicity of OTA. De Groene et al. observed a lower mutation frequency of OTA in the presence of the GSH depletor buthionine sulfoximide with recombinant cell lines expressing human P450s (47). The involvement of GSH in the genotoxicity of OTA was also indicated by reduced kidney DNA adduct formation in vivo after pretreatment of mice with phoron which led to GSH depletion (9, 48). Given that other GSH conjugates are potent nephrotoxicants, as a consequence of the relatively high activity of γ-GT

OTA-GSH Conjugate

and dipeptidases within the brush border membrane of renal proximal tubular epithelial cells (22, 27), it is conceivable that 8 and the cysteine conjugate 7 (Figure 1) play roles in the nephrotoxicity, genotoxicity, and carcinogenicity of OTA. Further work in our laboratories is directed toward identifying the biological significance of 7 and 8 in terms of mutagenicity and DNA reactivity.

Acknowledgment. R.A.M. and S.A.A. acknowledge the National Cancer Institute (Grant CA080787) for support of this research. LC/MS measurements were performed on instruments purchased with funds provided by the North Carolina Biotechnology Center (Grant 2001 IDG 1004).

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