Oxidation of Ochratoxin A by an FeâPorphyrin System: Model for

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Oxidation of Ochratoxin A by an Fe-Porphyrin System: Model for Enzymatic Activation and DNA Cleavage Ivan G. Gillman, T. Nicole Clark, and Richard A. Manderville* Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109-7486 Received June 16, 1999

Ochratoxin A (OTA, 1) is a fungal toxin that facilitates single-strand DNA cleavage, DNA adduction, and lipid peroxidation when metabolically activated. To model the enzymatic activation of OTA, we have employed the water-soluble iron(III) meso-tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS) oxidation system. In its presence, OTA has been found to facilitate single-strand cleavage of supercoiled plasmid DNA through production of reactive oxygen species (ROS) (i.e., the hydroxyl radical, HO•). The reaction of OTA with the FeTPPS oxidation system also generated three hydroxylated products (chlorine atom still attached), which was taken as evidence for production of the known hydroxylated metabolites (2-4) of OTA. This result suggested that the FeTPPS system served as a reasonable model for the enzymatic activation of OTA. When the reaction of OTA with FeTPPS was carried out in the presence of excess hydrogen peroxide (H2O2) and sodium ascorbate, a hydroquinone species (OTHQ, 5) was detected in which an OH group has replaced the chlorine atom of OTA. The production of OTHQ (5) was dependent on the presence of the reducing agent, sodium ascorbate, which suggested that the oxidation catalyst furnished the quinone derivative OTQ (6) that was subsequently reduced to OTHQ (5) by ascorbate. Utilizing a synthetic sample of OTHQ (5), the hydroquinone was found to undergo autoxidation with a t1/2 of 11.1 h at pH 7.4, and to possess a pKa value of 8.03 for the phenolic oxygen ortho to the carbonyl groups. Our findings imply that the hydroquinone (OTHQ) and quinone (OTQ) metabolites of OTA have the ability to cause alkylation/redox damage and have allowed us to propose a viable pathway for oxidative damage by OTA.

Introduction Ochratoxin A (OTA,1 1, Figure 1) is a mycotoxin produced by several species of Aspergillus and Penicillium fungi (1). It colonizes a wide range of foodstuffs and has been implicated in numerous mycotoxicoses in farm animals, especially in pigs (2, 3). Of greatest concern to humans is its implication in a fatal kidney disease called Balkan endemic nephropathy in which patients suffer from high incidences of kidney, pelvic, ureter, and urinary bladder carcinomas (4, 5). In terms of OTA genotoxicity, the toxin promotes single-strand DNA cleavage in mouse spleen cells in vitro and in the kidneys, liver, and spleen of mice in vivo (6, 7). Using the 32P-postlabeling method (8), OTA has been shown to induce DNA adduct formation in the kidneys, testicles, liver, spleen (9, 10), and urinary bladder (11) of mice. Although these results establish a basis for its genotoxicity, the mechanisms of OTA-induced DNA damage and carcinogenicity are currently not known. * To whom correspondence should be addressed: Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109-7486. Telephone: (336) 758-5513. Fax: (336) 758-4656. E-mail: [email protected]. 1 Abbreviations: OTA, ochratoxin A; OTB, ochratoxin B; OTHQ, ochratoxin hydroquinone; OTQ, ochratoxin quinone; SOD, superoxide dismutase; TCP, 2,4,6-trichlorophenol; PCP, pentachlorophenol; FeTPPS, iron(III) meso-tetrakis(4-sulfonatophenyl)porphyrin; TPPS, 5,10,15,20-tetraphenyl-21H,23H-porphine-P,P′,P′′,P′′′-tetrasulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; TMEDA, N,N,N′,N′-tetramethylethylenediamine; LDA, lithium diisopropylamide; DPPA, diphenylphosphoryl azide; TEA, triethylamine; MeCN, acetonitrile; SCE, saturated calomel electrode.

Figure 1. Structure of ochratoxin A (OTA, 1) and its analogues.

Despite this, structure-activity studies on OTA have shown that the chlorine atom is essential for its genotoxicity, as the nonchlorinated derivative ochratoxin B

10.1021/tx9901074 CCC: $18.00 © 1999 American Chemical Society Published on Web 10/01/1999

Model for Oxidative Damage by OTA

(OTB) is not genotoxic (12). DNA adduct formation is also effectively inhibited by antioxidants (11), i.e., R-tocopherol (vitamin E) and ascorbic acid, and by the enzymes catalase and superoxide dismutase (SOD) (13). These findings suggest that an oxidative pathway, which may be activated by cytochrome P450 (14) and/or enzymes with peroxidase activity (11), is responsible for OTAinduced DNA damage and carcinogenicity. However, studies aimed at elucidating the nature of OTA oxidation (15, 16) have shown that OTA is metabolized into hydroxylated products 2-4 (Figure 1), which are not toxic (17). While literature data on the oxidation of OTA are lacking, we speculated that its deleterious activities could be rationalized by oxidation of its para chlorophenolic group to a quinone species. This hypothesis stemmed from our recent finding that photoirradiation of the molecule generates the hydroquinone derivative OTHQ (5, Figure 1) in the presence of sodium ascorbate (18). This observation suggested that OTHQ may have originated from the quinone precursor OTQ (6, Figure 1) in analogy to the photooxidation of other halogenated phenols that yield benzoquinone derivatives (19). Chlorinated phenols, such as 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP), have also been shown to undergo bioactivation to benzoquinones (20-23). Because quinones can redox cycle, creating oxidative stress, and form covalent adducts with a variety of cellular macromolecules (24), the oxidation of OTA to OTQ would provide a rationale for its in vivo genotoxicity and would be consistent with the importance of the chlorine atom derived from structure-activity relationships (12). To model enzymatic activation of OTA, we have employed the water-soluble iron(III) meso-tetrakis(4sulfonatophenyl)porphyrin (FeTPPS) complex, which was recently used by Meunier to study the oxidation of TCP (22). In its presence, OTA has been found to facilitate single-strand cleavage of supercoiled plasmid DNA through production of reactive oxygen species (ROS) (i.e., the hydroxyl radical, HO•). Our studies also demonstrate, for the first time, that an Fe-porphyrin oxidation system converts OTA into OTHQ (5) in the presence of a suitable reducing agent (sodium ascorbate). This observation suggested strongly that the oxidation of OTA generated the quinone derivative, OTQ (6). This finding, together with the DNA cleavage results, has allowed us to propose a model for oxidative damage by OTA. Our model is consistent with the in vivo results of Malaveille and coworkers (12) that implicated the chlorine atom as an important determinant of OTA genotoxicity.

Experimental Procedures Caution: The work described herein involves the synthesis and handling of hazardous agents and was therefore conducted in accordance with the NIH Guidelines for the Laboratory Use of Chemical Carcinogens. Materials. Ochratoxin A (OTA, 1, Figure 1) was purchased from R. R. Marquardt (Department of Animal Sciences, University of Manitoba, Winnipeg, MB) and was used without further purification. Supercoiled plasmid DNA (form I) was a gift from F. W. Perrino (Department of Biochemistry, Wake Forest University). The plasmid was a derivative of pOXO4 containing the dnaQ gene (25). The following enzymes and reagents were obtained commercially and used without further purification: 5,10,15,20-tetraphenyl-21H,23H-porphineP,P′,P′′,P′′′-tetrasulfonic acid (TPPS), ferrous sulfate heptahy-

Chem. Res. Toxicol., Vol. 12, No. 11, 1999 1067 drate (FeSO4‚7H2O), 4-morpholinepropanesulfonic acid (MOPS), 2-(cyclohexylamino)ethanesulfonic acid (CHES), N,N,N′,N′-tetramethylethylenediamine (TMEDA), sec-butyllithium (secBuLi), lithium diisopropylamide (LDA), diethylcarbamyl chloride, iodomethane, diphenylphosphoryl azide (DPPA), triethylamine (TEA), and nitrosonium tetrafluoroborate (NOBF4) (all from Aldrich Chemical Co.), L-phenylalanine tert-butyl ester hydrochloride (ICN), and catalase (Sigma). Other inorganic reagents and solvents were obtained from Fisher Scientific. Methods. Elemental analyses were carried out by Atlantic Microlab Inc. High-resolution mass spectral analyses were carried out by either the Nebraska Center for Mass Spectrometry (Lincoln, NE) or Mass Consortium (San Diego, CA). 1H NMR spectra were recorded on either a Varian VXR-200 (200 MHz) or a Bruker AVANCE 300DMX (300 MHz) spectrometer. Chemical shifts are given in parts per million relative to tetramethylsilane (TMS), and coupling constants (J) are reported in hertz. 13C NMR spectra were recorded on a VXR-200 spectrometer operating at 50.3 MHz. Spectra were referenced to the residual solvent peak. UV spectra were obtained on a Hewlett-Packard 8452A diode array spectrometer equipped with a temperature-controlled cell. IR spectra were recorded on a Mattson 4020 Galaxy FT-IR system. Thin-layer chromatography was carried out on Analtech 250 µM layer, UV254 silica gel plates with glass backing, while silica gel chromatography was performed using Kieselgel 60, 230-400 mesh. Distilled, deionized water from a Milli-Q system was used for all aqueous solutions and manipulations. Other solvents were purified and dried according to standard procedures. FeTPPS was prepared from the reaction of FeSO4‚7H2O with 1 equiv of TPPS (26). The concentration of the FeTPPS solution was quantified by UV-vis and was ca. 80% FeIII(H2O)(TPPS)3- (393 ) 1.5 × 105 M-1 cm-1). Agarose gel electrophoresis was carried out in 40 mM Tris-acetate buffer (pH 8.0), containing 5 mM EDTA. Agarose gel loading buffer consisted of 40 mM Tris-acetate (pH 8.0), 5 mM EDTA, 40% glycerol, and 0.3% bromophenol blue. HPLC. Reverse-phase HPLC was performed using a Hitachi 7400 pump and a Waters 991 photodiode array detector set to acquire data from 220 to 800 nm. Analytical separations were carried out using a Waters symmetry shield C-18 column (3.9 mm × 150 mm) at a flow rate of 1 mL/min using the following mobile phase: 20/80 methanol/50 mM phosphate buffer (pH 7.0) followed by a linear gradient to 80/20 methanol/50 mM phosphate buffer (pH 7.0) over the course of 30 min. LC/MS. Electrospray mass spectra were acquired using a Micromass Quattro II instrument operating in the negative ion spray mode (ES-). The system acquired signals over a m/z range of 200-1000 at a rate of 8 s/scan. Separations were carried out using a Waters symmetry shield C-18 column (2.1 mm × 150 mm) at a flow rate of 0.1 mL/min using the following mobile phase: 20/80 methanol/10 mM ammonium bicarbonate followed by a linear gradient to 80/20 methanol/10 mM ammonium bicarbonate over the course of 30 min. A postcolumn split was used which generated a 10 µL flow to the electrospray source with 90 µL diverted to a Hewlett-Packard photodiode array. FeTPPS Oxidation of OTA. Under one set of conditions, 100 µM OTA in 10 mM phosphate buffer (pH 7.0) was allowed to react with 0.1% FeTPPS and 6 molar equiv of H2O2 and sodium ascorbate. The reaction mixture (1 mL total volume) was incubated at 37 °C for varying lengths of time, and then 20 µL was injected on an analytical scale reverse-phase HPLC system. In separate experiments, 100 µM OTA solutions in 10 mM phosphate buffer (pH 7.0) were reacted with 0.1% FeTPPS and 6 molar equiv of H2O2. After the lengths of time had been varied, the reactions were quenched by addition of 10 µL of 0.2 M sodium ascorbate (final concentration of 2 mM). The quenched solutions were then analyzed by reverse-phase HPLC. Synthesis of OTHQ (5). The synthesis of OTHQ (5) was carried out starting from the N,N-diethylcarbamate 7 (Scheme 1), which was prepared from 4-methoxyphenol (27). N,N-Diethyl-2,5-dimethoxy-3-(diethylcarbamoyl)benzamide (8). To a stirred solution of 7 (2.71 g, 12.14 mmol) in

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Scheme 1. Synthesis of OTHQ (5), with Isolated Yields in Parentheses

tetrahydrofuran (THF, 200 mL) at -78 °C under argon were added via syringe TMEDA (1.99 mL, 14.57 mmol) and then secBuLi (11.2 mL, 14.57 mmol from a 1.3 M solution in cyclohexanes) over the course of 10 min. The resulting solution was stirred for 1 h at -78 °C, and then via syringe was added diethylcarbamyl chloride (1.65 g, 12.14 mmol). The resulting solution was allowed to warm to room temperature and then stirred for an additional 20 h, at which point the solution was again cooled to -78 °C and treatment with TMEDA and secBuLi was repeated. After 20 h at room temperature, the resulting black solution was neutralized with 100 mL of brine followed by 1 M HCl. The solution was concentrated under reduced pressure and extracted with CH2Cl2 (3 × 100 mL). Following the standard workup, concentration of the CH2Cl2 solution afforded a dark yellow oil. The crude product was dissolved in 200 mL of acetone, and then excess K2CO3 followed by iodomethane (10 g, 70.45 mmol) was added and the resulting suspension heated at reflux for 24 h. The suspension was then concentrated under reduced pressure and extracted with CH2Cl2. The organic layer was separated, dried over MgSO4, and then concentrated under reduced pressure, followed by purification by silica gel chromatography (ethyl acetate) to afford 8 as a light yellow oil: Rf ) 0.27; yield 2.11 g (52%); 1H NMR (CDCl3) δ 6.68 (s, 2H), 3.70 (s, 6H), 3.6-3.0 (br, 6H), 1.17 (t, 3H, J ) 7.14 Hz), 0.982 (t, 3H, J ) 7.15 Hz); 13C NMR (DMSO-d6) δ 169.0, 165.2, 156.0, 146.8, 135.1, 114.8, 111.7, 74.5, 56.1, 28.6, 20.3; IR 2976, 2940, 1634, 1477, 1234, 1052 cm-1. Anal. Calcd for C18H28O4N2: C, 64.26; H, 8.39; N, 8.33. Found: C, 64.18; H, 8.48; N, 8.37. N,N-Diethyl-2,5-dimethoxy-3-(diethylcarbamoyl)-6methylbenzamide (9). To a stirred solution of 8 (2.00 g, 5.95 mmol) in THF at -78 °C under argon were added via syringe TMEDA (0.97 mL, 7.13 mmol) and then sec-BuLi (5.5 mL, 7.13 mmol from a 1.3 M solution in cyclohexanes) over the course of 10 min. The resulting solution was allowed to stir at -78 °C for 1 h, and then iodomethane (5 g, 35.23 mmol) was added via syringe. The solution was allowed to warm to room temperature, and after 20 h, the reaction mixture was neutralized with 100 mL of brine followed by 1 M HCl. The solution was concentrated under reduced pressure and extracted with CH2Cl2 (3 × 75 mL). Following the standard workup and concentration under reduced pressure, the resulting oil was purified by silica gel chromatography (ethyl acetate) to afford 9 as a light yellow oil: Rf ) 0.24; yield 1.60 g (77%); 1H NMR (CDCl3) δ 6.65 (s, 1H), 3.78 (s, 3H), 3.73 (s, 3H), 3.6-3.0 (br, 6H), 2.08 (s, 3H), 1.23 (t, 3H, J ) 7.20 Hz), 1.03 (m, 3H); 13C NMR (CDCl3) δ 169.5, 167.0, 151.0, 147.8, 127.9, 124.8, 117.4, 109.0, 76.1, 56.2, 42.9, 39.1, 28.2, 20.8, 14.1, 12.8; IR 2981, 2940, 2878, 1638, 1472, 1436, 1275, 1088 cm-1. Anal. Calcd for C19H30O4N2(H2O)1/4: C, 64.29; H, 8.66; N, 7.89. Found: C, 64.53; H, 8.60; N, 7.74. 3-Methyl-5,8-dihydroxy-7-carboxy-2,3-dihydroisocoumarin (10). To a stirred solution of 9 (2.3 g, 6.81 mmol) in THF

at -78 °C under argon was added via syringe LDA (5.10 mL, 10.21 mmol from a 2.0 M solution). The reaction mixture was stirred for 1 h, and then acetaldehyde (1 mL, 17.9 mmol) was added via syringe. The solution was allowed to warm to room temperature, and after 20 h, the mixture was neutralized with 50 mL of brine and 1 M HCl. The solution was concentrated under reduced pressure and then extracted with CH2Cl2 (3 × 50 mL). Following the standard workup and concentration under reduced pressure, the resulting crude oil was dissolved in 100 mL of N2-purged 6 N HCl and the reaction mixture was refluxed under argon for 6 days. The mixture was cooled and filtered to yield a yellow powder. Recrystallization from CH2Cl2/MeOH afforded 10 as a white powder: yield 0.795 g (49%); 1H NMR (DMSO-d6) δ 9.80 (s, 1H), 7.56 (s, 1H), 4.63 (m, 2H), 3.10 (dd, 2H, J ) 17, 3 Hz), 2.60 (dd, 1H, J ) 11.6, 5.6 Hz), 1.40 (d, 3H, J ) 6.2 Hz); 13C NMR (DMSO-d6) δ 168.6, 165.8, 155.0, 144.9, 132.8, 122.3, 115.1, 111.1, 74.7, 28.8, 20.4; IR 3284, 3121, 1699, 1618, 1454, 1402, 1221 cm-1. Anal. Calcd for C11H10O6(H2O)1/4: C, 54.44; H, 4.36. Found: C, 54.47; H, 4.32. OTHQ tert-Butyl Ester (11). To a stirred solution of 10 (100 mg, 0.420 mmol) in 10 mL of dry DMSO were added under argon 2.2 equiv of TEA (0.093 g, 0.924 mmol) and DPPA (254 mg, 0.924 mmol) followed by 1 equiv of L-phenylalanine tert-butyl ester hydrochloride (108.3 mg, 0.420 mmol). The solution was stirred for 12 h and then concentrated under reduced pressure. The resulting residue was resuspended in 30 mL of CH2Cl2 and washed with 1 M phosphate buffer (pH 7.0). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to yield an oil that was purified by silica gel chromatography (80/20 CH2Cl2/ethyl acetate) to afford 11 as a light yellow oil: Rf ) 0.57; yield 70.7 mg (40%); 1H NMR (CDCl3) δ 12.03 (s, 1H), 9.80 (s, 1H), 8.51 (d, 1H, J ) 4.0 Hz), 7.71 (s, 1H), 7.24 (m, 5H), 4.80 (m, 1H), 4.68 (d, 1H, J ) 6.9 Hz), 3.14 (m, 2H), 2.73 (m, 2H), 1.36 (br, 12H); 13C NMR (CDCl3) δ 170.4, 170.3, 152.6, 146.1, 137.0, 130.7, 130.5, 129.6, 128.6, 127.1, 123.8, 118.7, 109.7, 81.6, 76.5, 54.7, 37.4, 28.4, 27.9, 20.6; IR 3468, 1735, 1660, 1637, 1595, 1534, 1367, 1158 cm-1; MS (HRFAB) m/z 442.1878 (MH+) (C24H27NO7 requires 441.1866). OTHQ (5). To a round-bottom flask containing 11 (50 mg, 0.113 mmol) was added 10 mL of dry HCl (40 mmol in dioxane) under argon. The solution was allowed to stir at room temperature for 12 h, and then the solvent was removed under reduced pressure. To the flask was then added 20 mL of water, and the suspension was filtered to give an off-white solid. The material was dissolved in 10 mL of saturated sodium carbonate and precipitated with 1 N HCl. This was filtered to give pure 5: yield 34.7 mg (80%); 1H NMR (DMSO-d6) δ 13.08 (s, 1H, COOH), 12.04 (s, 1H, OH), 9.88 (s, 1H, OH), 8.95 (d, 1H, J ) 6.0 Hz, NH), 7.72 (s, 1H), 7.29 (m, 5H), 4.29 (m, 2H), 3.12 (m, 3H), 2.66 (dd, 1H, J ) 11.9, 11.6 Hz), 1.43 (d, 3H, J ) 6.2 Hz); 13C NMR (DMSO-d6) δ 172.5, 169.9, 613.1, 155.2, 145.8, 136.9, 130.2, 129.3, 128.4, 126.7, 123.4, 118.3, 109.4, 76.2, 53.9, 36.7, 28.1,


Figure 2. DNA cleavage by OTA (25 µM) in the presence of Fe(III)TPPS (5 µM). Cleavage was carried out at 37 °C for 30 min in 10 mM MOPS (pH 7.4) buffer solution: lane 1, DNA alone; lane 2, DNA and Fe(III)TPPS; lane 3, DNA and OTA; lane 4, DNA and OTA and Fe(III)TPPS; lane 5, DNA and 1 µL of DMSO; lane 6, DNA and 1 µL of t-BuOH; and lane 7, DNA and 1000 units/mL catalase. 20.3; IR 2927, 2854, 1731, 1582, 1442, 1148, 1021 cm-1; MS (HRFAB) m/z 386.1251 (MH+) (C20H19NO7 requires 385.1161). Autoxidation of OTHQ (5). Autoxidation reactions for OTHQ (5) were followed spectrophotometrically using a HewlettPackard (HP-8452) UV-vis spectrometer at 350 or 396 nm. Reactions were initiated by adding 10 µL of a stock solution of 5 (2.6 mM) in MeOH to a 3 mL cuvette containing 2 mL of buffer (10 mM) and 100 mM NaCl equilibrated at 37 °C. Mixing could be achieved within 2 or 3 s, and the data, which were analyzed using the ENZFITTER program, gave good first-order rate constants for at least two half-lives. Proton Affinity. The pH measurements were obtained at 25 °C on a Fisher Scientific Accumet 910 pH meter using standard glass electrodes. Calibration was carried out using commercial buffers (BDH, pH 4.00, 7.00, and 10.00, all (0.01). Over the pH range of 6.6-9.5, the proton affinity of 5 was determined spectrophotometrically (28) at 25 °C using a 1 mL cuvette containing 1 mL of a 10 mM solution of buffer with 100 mM NaCl. To the sample cell was added 10 µL of a 2.6 mM stock solution of 5 in MeOH. UV-vis spectra were recorded by the overlay method in the wavelength range of 250-500 nm. Oxidation of OTHQ (5) by Nitrosonium Tetrafluoroborate. Reactions of OTHQ (5) with nitrosonium tetrafluoroborate (NOBF4) were followed by UV-vis using a Hewlett-Packard (HP-8452) spectrometer set to acquire data over the range of 220-600 nm. The oxidation was carried out in dry distilled acetonitrile (MeCN) and oven-dried quartz cuvettes. A stock solution of OTHQ (13 mM) was prepared, and 10 µL was added to a 1 mL cuvette containing 990 µL of MeCN. To this solution was added 10 molar equiv of NOBF4. The resulting yellow solution was passed through a strong ion exchange cartridge to remove excess NOBF4. Relaxation of Supercoiled Plasmid DNA by OTA/FeTPPS. Reaction mixtures (20 µL total volume) contained 400 ng of supercoiled plasmid DNA (form I), 10 mM MOPS (pH 7.4), 25 µM OTA, and 5 µM FeTPPS. Reaction mixtures were incubated at 37 °C for 30 min, and then reactions were quenched by the addition of 4 µL of loading buffer. Samples were loaded onto a 1% agarose gel containing ethidium bromide (1 µg/mL). The gel was run at 110 V for 2 h and visualized by UV illumination. In separate experiments, the effects of catalase (1000 units/mL), Me2SO (1 µL), tert-butyl alcohol (1 µL), and NaCl (10-500 mM) were examined.

Results DNA Cleavage by OTA. The ability of OTA to effect DNA cleavage in the presence of FeTPPS was determined using supercoiled plasmid DNA (form I) and agarose gel electrophoresis. As shown in Figure 2, cleavage by OTA did not result in the absence of FeTPPS (lane 3), nor could FeTPPS effect strand scission alone (lane 2). However,

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OTA (25 µM) in the presence of FeTPPS (5 µM) was able to effect single-strand breaks (lane 4) and convert form I into nicked circular DNA (form II). These results were in direct contrast to our DNA cleavage studies with free Fe, where no role for the toxin could be established (25). The oxidative nature of the strand scission was confirmed by the finding that the enzyme catalase, which lowers solution concentrations of H2O2, almost completely inhibited cleavage (lane 7). Hydroxyl radical scavengers, Me2SO (lane 5) and tert-butyl alcohol (t-BuOH, lane 6), also provided partial protection against strand scission. Additional experiments showed that the cleavage was insensitive to the ionic strength of the medium (10-500 mM NaCl, data not shown). Taken together, these results strongly suggested that the neutral HO• mediated the DNA cleavage by the FeTPPS/OTA mixture (29). The results also suggested that OTA provided the reducing equivalents to convert Fe(III)TPPS to Fe(II)TPPS. HPLC combined with electrospray mass spectrometry was then utilized to examine the reaction of OTA with FeTPPS and H2O2 in more detail. Reaction of OTA with FeTPPS/H2O2/Sodium Ascorbate. As described by Meunier (22), the FeTPPS/H2O2 system efficiently catalyzes the oxidation of chlorinated phenols (ortho- or para-substituted) to their respective benzoquinones. Our goal was to utilize the FeTPPS/H2O2 system to carry out the oxidation of OTA in the presence of a reducing agent (sodium ascorbate), which would reduce the anticipated quinone, OTQ (6), into OTHQ (5), as outlined in eq 1 FeTPPS

sodium ascorbate

OTA (1) 9 8 OTQ (6) 98 OTHQ (5) (1) H O 2

2

In this way, we could monitor for the presence of OTHQ through comparison to our authentic sample, which was generated from the photoreaction of OTA in N2-flushed phosphate buffer (18). Under one set of conditions, 100 µM OTA in 10 mM phosphate buffer (pH 7.0) was allowed to react with 0.1% FeTPPS and 6 molar equiv of both H2O2 and sodium ascorbate. The reaction mixture was incubated at 37 °C for varying lengths of time and then analyzed by reversephase HPLC (detection at 330 nm). Representative HPLC results are depicted in Figure 3. The HPLC profile exhibited seven products (a-g); UV spectra (250-500 nm) for the four major products (d-g), including OTA, are also shown. Note that OTA shows two absorbances at ca. 330 and 380 nm. The phenolic pKa of OTA is 7.0 (25), and so both the protonated (λmax ) 333 nm) and deprotonated (λmax ) 380 nm) forms of the toxin are present. Compounds d-f exhibited UV spectra similar to that of OTA, while compound g exhibited a single absorbance at 350 nm. Insight into the nature of products d-g was achieved using electrospray mass spectrometry with negative ionization (ES-). As shown in Figure 4, products d-f had identical masses with an [M - H]- ion at 418 (Figure 4B), which is 16 mass units heavier than OTA (Figure 4A, [M - H]- ion at 402). The ES- spectrum of these products also exhibited the characteristic chlorine isotope splitting pattern (Figure 4B). While we lack authentic standards to unambiguously characterize d-f, the UVvis characteristics and the ES- data were consistent with formation of the three hydroxylated metabolites of OTA (2-4, Figure 1) that have been previously characterized using liver microsomes to oxidize the toxin (16). In

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Figure 3. HPLC elution profile and accompanying UV-vis spectra of peaks d-g and OTA from the reaction of OTA with FeTPPS/ H2O2/sodium ascorbate at 37 °C and pH 7.0.

contrast, product g had an [M - H]- ion at 384 with the loss of the chlorine isotope pattern (Figure 4C), which was consistent with OTHQ (5). Product g also comigrated with our authentic sample of OTHQ (verified by sample spiking) and had an identical UV spectrum at pH 7.0 (λmax ) 350 nm). These results firmly established product g as the hydroquinone of OTA, i.e., OTHQ (5). In the presence of sodium ascorbate, the hydroquinone 5 was generated in roughly 5% yield from the reaction of OTA with FeTPPS/H2O2. Also, only ca. 15% of the starting OTA was consumed over a 14 h period (data not shown). In a separate experiment, OTA was incubated with the FeTPPS/H2O2 mixture under physiological conditions and excess sodium ascorbate was added at various times. Figure 5 shows that under these conditions the % concentration of OTA was reduced to ca. 30% after 30 min. The hydroquinone OTHQ (5) and the hydroxylated products could still be detected, but their concentration remained at ca. 5%. In the absence of sodium ascorbate, the hydroxylated products were noted, but no OTHQ could be detected in the HPLC traces from the reaction of OTA with FeTPPS/H2O2 (data not shown). These results were consistent with our hypothesis that OTA oxidation generates the quinone OTQ (6) that was reduced to OTHQ in the presence of sodium ascorbate (eq 1). Synthesis of OTHQ (5). To study the characteristics of the hydroquinone OTHQ (5) in more detail, a sample was prepared using total synthesis. Scheme 1 outlines the procedures used for the preparation of 5 that were based on strategies described by Snieckus and co-workers for the synthesis of OTA (30). From 10, the peptide

coupling reagent, diphenylphosphoryl azide (DPPA) (31) was used to attach the L-phenylalanine tert-butyl ester moiety to afford 11, which was subsequently treated with acid (dry HCl/dioxane) to yield a sample of OTHQ (5). The sample of 5 was obtained as a mixture of diastereomers. Examination by reverse-phase HPLC indicated the presence of two equivalent peaks eluting at 17.6 and 18.7 min (data not shown). Confirmation that the peak eluting at 18.7 min was the 3R-epimer was established through comparison to our sample of 5 generated from the photoreaction of OTA (18), which generates the 3Repimer as the photoreaction does not alter the stereochemistry of the dihydroisocoumarin. Aqueous Reactivity of OTHQ/OTQ. Experiments were performed to determine the rate of OTHQ autoxidation at 37 °C and at different pH values. Representative UV-vis data for the decomposition of OTHQ are shown in Figure 6. At pH 8.0, the autoxidation was accompanied by a decrease and “blue” shift of the absorbance at ca. 350-400 nm (at pH 8.0, the phenolate of OTHQ is also present, with a pKa of 8.03; see below), along with an increase in absorbance at 270 nm. The fact that the decomposition was due to autoxidation was confirmed by the finding that an N2-purged solution of OTHQ remained relatively stable at pH 8.0 (monitored over a 12 h period). First-order rate constants for the autoxidation of OTHQ are given in Table 1, which shows that the autoxidation was pH-dependent between pH 7.0 and 9.0. As noted for other hydroquinone systems, this observation indicates that proton removal is involved in the rate-determining step, which is consistent with semiquinone and subsequently quinone formation (32).



Figure 5. Time course of the loss of OTA in the reaction with 0.1% Fe(III)TPPS and 6 molar equiv of H2O2. Aliquots were combined with 2.0 mM sodium ascorbate at various times and analyzed by HPLC.

Figure 6. UV-vis data for the autoxidation of OTHQ (5) in 10 mM MOPS buffer (pH 8.0) containing 100 mM NaCl at 37 °C. Scans were taken every 100 min. Table 1. Summary of the Rate Constants for the Autoxidation of OTHQ (5)a pH

kobs × 105 (s-1)b

t1/2 (h)

7.0 7.4 8.0 8.5 9.0

1.13 1.74 3.50 4.72 8.92

16.98 11.09 5.49 4.08 2.16

a All experiments were conducted in 10 mM buffer and 100 mM NaCl at 37 °C. b Estimated error in rate constants was (5%.

Figure 4. Negative ion electrospray mass spectra (ES-) of peaks d-g and OTA from Figure 3. (A) OTA, with an [M - H]ion at 402. (B) d-f, with an [M - H]- ion at 418. (C) g, with an [M - H]- ion at 384 [OTHQ (5)].

This work demonstrated that OTHQ is oxidized by O2 alone (t1/2 ) 11.1 h at pH 7.4; Table 1), which is not the case for OTA (33). In the pH range of 7.0-9.0, OTHQ also underwent a change in UV-vis characteristics exhibiting a red shift in λmax from 350 nm at pH 7.0 to 396 nm at pH 9.0. This change was consistent with deprotonation of the phenolic oxygen ortho to the carbonyl groups. At 25 °C, a pKa value of 8.03 ( 0.06 (95% confidence interval) was established

for this phenolic group of OTHQ (5), which is ca. 1.0 pH unit above the corresponding pKa of OTA (25). Further insight into the aqueous decomposition of OTHQ (5) was obtained through MS analyses. Figure 7 shows the ES- spectrum obtained from the reaction of OTHQ with aqueous NH4HCO3 (10 mM, pH 8.0). Neither the starting hydroquinone ([M - H]- ) 384) nor the anticipated quinone product OTQ (6) ([M - H]- ) 382) was detected. Instead, a series of peaks were noted with major species at 387, 432, and 449. Collision-induced dissociation indicated that peaks at 449 and 387 were derived from the peak at 432. On the basis of the known chemistry of quinone systems (22, 34), we speculated that the decomposition product with an [M - H]- ion at 432, which is 48 mass units (three oxygen atoms) heavier than OTHQ, may have originated from the reaction of OTQ (6) with nucleophilic species (H2O/HO- or HOO-) in solution (22, 34). To test this hypothesis, the oxidation of OTHQ using excess nitrosonium tetrafluoroborate (NOBF4) in a non-nucleophilic “dry” acetonitrile (MeCN) solution was examined, and the subsequent product ascribed to OTQ (6) was reacted with water (NH4HCO3, pH 8.0). Our choice to utilize NOBF4 as the oxidant stemmed from the fact that the formal potential of NO+

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Figure 7. Negative ion electrospray mass spectrum (ES-) taken following the aqueous decomposition of OTHQ (5) from the reaction in NH4HCO3 (10 mM, pH 8.0).

Figure 8. UV-vis spectra of OTHQ (5) in the absence (spectrum a) and presence of 10 equiv of NOBF4 (spectrum b) in acetonitrile.

is 1.27 V versus SCE in MeCN (35), which is more than sufficient to oxidize OTHQ.2 Figure 8 shows the UV-vis changes in OTHQ (spectrum a) upon addition of 10 equiv of NOBF4 (spectrum b). The OTHQ solution immediately turned yellow, and its absorption maxima at 350 nm (350 ) 3400 M-1 cm-1) and 240 nm were replaced by a broad absorbance band in the visible region along with an intense absorbance at 260 nm. While attempts to isolate the oxidized product failed, these spectral changes were in good agreement with those reported for the oxidation of TCP to its respective benzoquinone (23). Addition of 10 mM NH4HCO3 (100 µL) to the yellow solution ascribed to OTQ (6) (Figure 7) led to the immediate disappearance of the intense absorbance at 260 nm. Inspection of the solution by ES- again showed the presence of the species in Figure 7. A possible reaction sequence for the aqueous decomposition of OTHQ (5) is outlined in the Discussion.

Discussion The ability of OTA to induce single-strand DNA cleavage (6, 7) and DNA adduction (8-11) in vivo, coupled with its implication in human kidney carcinogenesis (4, 5), has stimulated numerous investigations into its mode of action (12-17). While these studies have established that an oxidative mechanism is involved in OTA-medi2 In MeCN, the electrochemical oxidation of OTA exhibits behavior almost identical with that of p-chlorophenol. The half-wave potential of OTA is 1.45 V versus SCE, while the half-wave potential of OTHQ is 0.92 V in MeCN. Upon successive scans of OTA, a secondary oxidation peak with a half-wave potential of 0.92 V versus SCE was observed which corresponds to OTHQ (M. W. Calcutt, R. E. Noftle, and R. A. Manderville, unpublished results).

Gillman et al.

ated DNA damage (8-12) and lipid peroxidation (32), the exact pathway by which OTA initiates oxidative damage has remained elusive. One scenario put forward is that OTA forms a complex with Fe(III) and the resulting Fe(III)‚OTA chelate is reduced by NADPH-cytochrome P450 reductase to an Fe(II)‚OTA complex, which activates molecular O2 to initiate oxidative damage (36). However, Hoehler has recently shown that the phenoxy O-methylated derivative of OTA, which does not chelate Fe(III) effectively, facilitates HO• production in hepatocytes, mitrochondria, and microsomes from rats just as efficiently as OTA (37). Our recent studies also show that OTA facilitates DNA cleavage by the Cu(II) complex of 1,10-phenanthroline [Cu(OP)2], a prototypical Cu-mediated nuclease that requires an external reducing agent for activation (25). In the presence of free Fe(III) or Cu(II) with an equimolar amount of or excess OTA, no role for the toxin could be established. Like Hoehler’s results (37), these studies also implied that oxidative damage by OTA can be attributed to its redox properties, instead of to its ability to coordinate redox active transition metals (25). The studies presented here on the ability of OTA to facilitate DNA cleavage in the presence of FeTPPS also support this notion. Iron-porphyrin systems have been shown to cause DNA cleavage in the presence of O2 and a reducing agent, usually a thiol such as dithiothreitol or 2-mercaptoethanol (38, 39). A mechanism for DNA cleavage by these systems involves production of HO• through a Fenton type of reaction between Fe(II) and H2O2 (39). The ability of the FeTPPS system to facilitate strand scission in the presence of OTA (Figure 2) suggested that OTA provides the reducing equivalents to convert Fe(III)TPPS to Fe(II)TPPS. The fact that the enzyme catalase inhibited the cleavage provided a direct role for H2O2. The cleavage was also found to be insensitive to the ionic strength of the medium, and the hydroxyl radical scavengers, Me2SO and tert-butyl alcohol, provide protection (Figure 2). Taken together, these results strongly suggested that HO• initiated cleavage (29). This hypothesis was also consistent with the demonstrated ability of the toxin to facilitate HO• production in cellular systems (33, 40). Once it was established that the FeTPPS/OTA system facilitates DNA cleavage, the reaction of OTA with FeTPPS in the presence of H2O2 was investigated. We anticipated that FeTPPS/H2O2 would oxidize OTA to the quinone derivative OTQ (6), in analogy to the oxidation of other chlorinated phenols (20-23). This prediction was also based on our recent findings regarding the ability of OTA to act as a photoactivatable DNA cleaving agent (18). Here, the photocleavage was found to stem from initial C-Cl bond homolysis, and product analysis of the photoreaction showed a direct correlation with the known photochemistry of p-chlorophenol (19). For example, photoirradiation of OTA under anaerobic conditions produced the hydroquinone derivative, OTHQ (5). Interestingly, OTHQ was also detected in the presence of O2 when a reducing agent (sodium ascorbate) was added to the photoreaction mixture. This observation suggested that OTHQ originated from its oxidized precursor OTQ (6), in analogy to photooxidation of halogenated phenols in the presence of O2 (19). Given the fact that the photochemical behavior of OTA mimicked that established for p-chlorophenol, we speculated that the oxidation of OTA would similarly involve the quinone deriva-



Scheme 2. Model Pathway for the Oxidation of OTA to OTQ (6)

tive OTQ (6). To provide evidence for the intermediacy of OTQ in OTA oxidation, we carried out the oxidation with FeTPPS/H2O2 in the presence of sodium ascorbate, which was used as a trapping agent to convert OTQ (6) to its reduced form OTHQ (5). As predicted, the hydroquinone OTHQ (5) was only detected when sodium ascorbate was added to the reaction mixture, which was consistent with direct formation of OTQ (6) with subsequent reduction to OTHQ. The reaction of OTA with the FeTPPS system also generated three hydroxylated products (chlorine atom still attached, Figure 4B), which was taken as evidence of the production of 2-4 (Figure 1), i.e., the known hydroxylated metabolites of OTA (16). This finding suggested that the FeTPPS system served as a reasonable model for the bioactivation of OTA. Further insight into the nature of OTA oxidation was derived from the finding that production of the hydroxylated products did not depend on the presence of sodium ascorbate. This result was in direct contrast to the production of OTHQ (5), and suggested that like the quinone OTQ (6), the hydroxylated products are the direct result of OTA oxidation. This finding allows one to speculate that the inability of others to detect OTHQ (5) in reactions of OTA with microsomal/sodium ascorbate mixtures could be that OTQ (6) reacts with adventitious protein prior to reduction by sodium ascorbate (41). This factor, coupled with the lack of authentic standards, could make detection of OTHQ in a microsomal assay problematic. Scheme 2 outlines a possible pathway for the oxidation of OTA by the FeTPPS/H2O2 system. The first step involves production of the OTA phenoxyl radical (12). This species may result from electron abstraction by a high-valent Fe-oxoporphyrin generated from the reaction of FeTPPS with H2O2 (22), or by electron transfer to Fe(III)TPPS to produce Fe(II)TPPS, as suggested in the DNA cleavage results. Further oxidation of the phenoxy radical by a second molecule of FeTPPS or molecular O2 would then yield the phenoxonium cation. The presence of the chlorine leaving group provides a viable pathway for OTQ (6) production through nucleophilic attachment of H2O with the loss of HCl (22). The fact that we detect low levels of OTHQ in the oxidation of OTA by FeTPPS/ H2O2 in the presence of sodium ascorbate is attributed to two factors. First, sodium ascorbate would be expected to reduce the OTA phenoxy radical to the OTA phenoxy anion (42). This would make the oxidation process

inefficient, as was noted when sodium ascorbate was added to the reaction mixture (long incubation times and a slow rate of turnover of the toxin). This observation is also consistent with the ability of sodium ascorbate to quench the genotoxicity of OTA (13). Second, the anticipated electrophilicity of the quinone OTQ would make trapping by sodium ascorbate difficult. For example, when the incubation of OTA with FeTPPS/H2O2 was carried out in the absence of sodium ascorbate, the toxin was consumed over the course of approximately 50 min (Figure 5). Upon quenching the reaction with sodium ascorbate, we could still only detect ca. 5% OTHQ. We attribute this to the fact that the quinone product OTQ reacts with nucleophilic species in solution [H2O/HO- or HOO- (34)] to form decomposition products (Figure 7) prior to being reduced by sodium ascorbate. This factor makes it difficult to determine the yield of OTQ from the oxidation. The aqueous reactivity of OTHQ (5) also suggests that if it were formed in vivo, then it would make a significant contribution to the toxicity of OTA. Like other hydroquinones (32, 43), OTHQ undergoes autoxidation (Figure 6 and Table 1), a process that generates superoxide and subsequently H2O2 (32). This information demonstrates that OTHQ is more reactive than OTA, which is not oxidized by O2 alone (33). Autoxidation of OTHQ was also anticipated to yield the quinone OTQ (6). While we did not detect OTQ, we did find evidence for formation of a species with an [M - H]- ion at 432, which was accompanied by other species with [M - H]- ions at 449 and 387 (Figure 7). While we currently lack unambiguous evidence regarding the identity of these decomposition products, a possible decomposition pathway for OTHQ is outlined in Scheme 3. The proposed pathway is based on the available literature information on the chemistry of quinone systems and is consistent with the MS data. Thus, autoxidation of OTHQ 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 Foote (34), catechols undergo C-C cleavages through reaction with HOO- via an acyclic Baeyer-Villiger type mechanism. This type of reaction for the catechol would yield the diacid derivative (both tautomers are shown), which has an [M - H]- ion at 432 (Figure 7). Treatment of the diacid with NH4HCO3 would yield the ammonium salt with an [M - H]- ion at 449, while decarboxylation would generate a species with an

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Gillman et al.

Scheme 3. Proposed Pathway for the Aqueous Decomposition of OTHQ (5)

Scheme 4. Model for the Enzymatic Activation of OTA

[M - H]- ion at 388 and an [M - 2H]2- ion at 387. The fact that the chemical oxidation of OTHQ by NOBF4 in dry acetonitrile (Figure 8) also generated the same species upon reaction with aqueous NH4HCO3 provides additional evidence that the decomposition products originated from the reaction of OTQ (6) with nucleophilic species in solution. Hence, the anticipated reactivity of the quinone OTQ makes it a likely candidate for covalent attachment to biopolymers (24, 41) and provides a rationale for the ability of OTA to generate DNA adducts in vivo. On the basis of the results from these studies, we propose the model outlined in Scheme 4 for the enzymatic activation of OTA. The reaction of OTA with an Feporphyrin system, such as cytochrome P450 (14, 15), is envisioned to follow two pathways. The CH-oxidation pathway generates hydroxylated metabolites 2-4 (16). These species are readily eliminated in the bile of rats (possibly through glucuronide formation) and consequently are far less toxic than OTA (17). Thus, the CHoxidation route is regarded as a detoxification pathway. The phenol-oxidation route converts OTA into the quinone OTQ (6). This pathway generates reactive oxygen species (ROS) (H2O2, HO•, or O2•-) that can lead to DNA cleavage (6, 7) and lipid peroxidation (36). The quinone OTQ can undergo either a two-electron reduction to form OTHQ or a one-electron reduction to yield a semiquinone anion radical. These events establish futile redox cycles in which formation of ROS is amplified (32, 41). Finally,

the quinone OTQ may react with DNA-derived nucleophiles to produce DNA adducts. In addition to the pathways outlined in Scheme 4, we have also demonstrated that OTA can activate DNA cleavage by the Cu(OP)2 complex (25). New evidence from our laboratory3 also shows that the Cu(II) complex of OTA (25, 44) can facilitate DNA cleavage by Cu(II) bound to the DNA surface. Since copper activation represents a nonenzymatic pathway by which certain phenolic compounds (45-47), including hydroquinone metabolites of PCP (47), facilitate DNA damage, we also expect that the presence of Cu(II)/Cu(I) will contribute to the genotoxicity of OTA. In fact, copper activation may be more efficient than the enzymatic pathway proposed in Scheme 4, as copper coordination by OTA facilitates DNA binding, since by itself OTA is electrostatically repelled by the DNA helix. Current efforts in our laboratory are focused on determining whether the quinone OTQ, or its electrophilic precursors depicted in Scheme 2, react with DNA bases, as anticipated.

Acknowledgment. We thank Dr. Fred W. Perrino (Department of Biochemistry, Wake Forest University School of Medicine) for the sample of plasmid DNA. This work was partially supported by Grant IRG from the American Cancer Society and Wake Forest University. NMR spectra were recorded on instruments purchased 3

I. G. Gillman and R. A. Manderville, unpublished results.


with the partial support of the NSF (CHE-9708077) and NCBC (9703-IDG-1007).

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