Chem. Res. Toxicol. 1989,2, 325-333
325
Oxidations of the Carcinogen N-Hydroxy-N-( 2-fluoreny1)acetamide by Enzymatically or Chemically Generated Oxidants of Chloride and Bromide Clare L. Ritter and Danuta Malejka-Giganti* Department of Laboratory Medicine and Pathology, University of Minnesota, and Laboratory
for Cancer Research, Veterans Administration Medical Center, Minneapolis, Minnesota 55417 Received February 9, 1989
T h e oxidations of the carcinogen N-hydroxy-N-(2-fluorenyl)acetamide(N-OH-2-FAA) via one-electron (le-) oxidation to equimolar 2-nitrosofluorene (2-NOF) and N-acetoxy-2-FAA and via oxidative cleavage t o 2-NOF by chemically and myeloperoxidase (MPO)/H202generated oxidants of C1- and/or Br- were investigated. 2-NOF was determined spectrophotometrically in the reaction mixtures and by HPLC of their extracts; N-acetoxy-2-FAA was determined by HPLC. In the presence of individual or mixed halides at their physiologic concentrations (0.1 M C1- and/or 0.1 m M Br-) and p H 4-6, MPO/H202-catalyzed oxidation of N-OH-2-FAA to 2-NOF via oxidative cleavage was much greater than le- oxidation. At the respective p H optima, oxidation was much more rapid with Br- and Br- C1- than with C1-. HOBr or HOCl Broxidized N-OH-2-FAA more rapidly than HOC1, also chiefly via oxidative cleavage. This suggested that, in the presence of MPO/H202+ C1- Br-, oxidation was due to HOBr from HOCl oxidation of Br- and/or oxidation of Br- by MPO/H202. In the presence of taurine (1 or 10 mM), a scavenger of hypohalous acids, MPO/H202catalysis of oxidative cleavage was unaffected with Br-, prevented with C1-, and partially prevented with C1- Br-. These results were linked to N-halotaurine formation since it was found that N-bromotaurine, but not N-chlorotaurine, oxidized N-OH-2-FAA chiefly to 2-NOF. With time N-chlorotaurine and N-bromotaurine appeared to undergo a pH-dependent halide exchange with Br- and C1-, respectively. The results led us to conclude that oxidants of Br- may play a role in vivo in the activation of carcinogens.
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Introduction Carcinogenic N-fluorenylhydroxamicacids undergo one electron (le-)l oxidation to nitroxyl free radicals catalyzed by peroxidase/peroxide systems (1-8). Two molecules of the nitroxyl radical then dismutate to equimolar nitrosofluorene and an ester of the N-fluorenylhydroxamic acid (as in reaction 1). The reactivities of the dismutation products derived from N-OH-2-FAA, electrophilicity of N-AcO-2-FAA yielding adducts with DNA (9),and direct mutagenicity of 2-NOF (reviewed in ref 10) established leoxidation as one mechanism of activation of Nfluorenylhydroxamic acids. via le-
K O H-2-FAA
peroxidase
T I :
'I~(N-A~O-Z-FAA +)' / 2 ( ~ (1)- ~ ~ 2-NOF
+ CHjCOOH
(2)
We have also shown that carcinogenic N-2-substituted fluorenylhydroxamic acids were converted largely to 2NOF by haloperoxidase/H202/Br- systems (10-12). We have suggested that this reaction involves oxidative cleavage of the hydroxamic acid resulting from bromination at nitrogen by HOBr/OBr- and decomposition to 2-NOF (as in reaction 2). The Br--dependent oxidation of N-OH-2-FAA to 2-NOF in the presence of H202has been shown with lactoperoxidase and eosinophil peroxidase rich tissue (rat uterus) or cell (rat peritoneal eosinophils) preparations, and also with peroxidase of rat mammary *Address correspondence to this author at the Veterans Administration Medical Center, One Veterans Drive, Building 31, Minneapolis, MN 55417.
gland (10-12). Substantial amounts of 2-NOF were determined even at physiologic Br- concentrations (-0.1 mM) and were increased with increasing Br- concentration. A major source of peroxidative activity in vivo is MPO, which is found in mammalian neutrophils and monocytes and can be released in response to physical or chemical stimuli (reviewed in refs 13 and 14). In the presence of H202and halides, MPO has strong bactericidal activity (13-15), most likely due to generation of HOCl (16-19). Since in vivo C1- and Br- are present at a ratio of 1OOO:l in most tissues (20) and MPO has been reported to prefer C1- (13,21,22), we examined in this study the oxidation of N-OH-2-FAA by MPO in the presence of C1- or Br- as well as their mixture. Since the release of MPO by neu~ trophils is accompanied by acid pH (13,23), we examined oxidation of N-OH-2-FAA in the pH range of 4-6.5. In vivo, hypohalous acids are most likely short-lived due to their reactivities with nitrogen resulting in the formation of N-haloamines, some of which are stable oxidants (16, 19,24-32). In mammalian tissues the p-amino acid taurine is the most abundant intracellular free amino acid (33), and in leukocytes its concentration exceeds 10 mM (31). We, therefore, investigated the effect of taurine (1and 10 mM) on the oxidation of N-OH-2-FAA by MPO/H202in the presence of C1- and/or Br-. To elucidate these oxidations, we chemically generated HOC1, HOBr, and Nchloro- and N-bromotaurine and examined their oxidation Abbreviations: le-, one electron; N-OH-2-FAA, N-hydroxy-N-(2fluoreny1)acetamide;N-AcO-2-FAA,N-acetoxy-N-(2-fluorenyl)acetamide; 2-NOF, 2-nitrosofluorene; 2-NOzF, 2-nitrofluorene; MPO, myeloperoxidase; GU, guaiacol unit; X-, halide (C1- or Br-); IS, internal standard.
0 1989 American Chemical Society
326 Chem. Res. Toxicol., Vol. 2, No. 5, 1989
of N-OH-2-FAA. The product of oxidation 2-NOF was determined spectrophotometrically in the reaction mixtures and in their extracts by HPLC. In addition, NAcO-2-FAA was quantitated by HPLC to assess the extent of le- oxidation in the presence of halides.
Materials and Methods Reagents and Columns. Triple glass-distilled water was used throughout. All solvents used for chromatography were HPLC grade from Matheson Coleman and Bell, Norwood, OH. NaBr (Gold Label) was from Aldrich Chemical Co., Inc., Milwaukee, WI. NaOCl(5% solution) and NaCl (Ultrex Ultrapure) were from J. T. Baker Chemical Co., Phillipsburg, NJ, and Brz was from Fisher Scientific, Pittsburgh, PA. NaOH (5 N) was from VWR Scientific, San Francisco, CA. Sodium phosphate and citric acid were from Mallinkrodt, Inc., Paris, KY. HzOz (30%), taurine, and guaiacol were from Sigma Chemical Co., St. Louis, MO. p-Nitroaniline was from Eastman Kodak Co., Rochester, NY. MPO (human polymorphonuclear leukocytes; >98% purity determined by electrophoresis) was from Calbiochem Corp., San Diego, CA, and was stored at 4 "C. Du Pont Zorbarc CBanalytical and guard columns were from Mac-Mod Analytical, Inc., Chadds Ford, PA, and C18extraction columns (Baker 10SPE) were from J. T. Baker Chemical Co., Phillipsburg, NJ. Fluorenyl Compounds. N-OH-2-FAA, mp 150-151 "C (34), 2-NOF, m p 79-81 "C (35), and N-AcO-2-FAA, mp 111-112 "C (36)were prepared by the published procedures. Compounds were found to be pure by HPLC after repeated recrystallizations. Instruments. Spectrophotometric measurements were performed on a Hitachi llOA UV/vis (Hitachi Instruments, Inc., Mountain View, CA) or a Beckman DU-7 spectrophotometer (Beckman Instruments, Inc., Irvine, CA). HPLC analyses were performed on a Varian 5000 liquid chromatograph (Varian Instruments, Sunnyvale, CA) equipped with a pneumatically activated injector and a 0.01-mL loop (Valco Instruments, Houston, TX), a Hewlett-Packard 1040A high-speed spectrophotometric detector, a DPU multichannel integrator (79882A), a computer (85B), Thinkjet printer (2225A), and a plotter (7470A) (Hewlett-Packard, Palo Alto, CA). Chemical Generation of HOX. NaOCl and Br2 were diluted with 0.1 N NaOH immediately before use as previously described (37). Their concentrations were determined from the absorance maxima by using the c for OC1- at 292 nm of 350 M-' cm-', and for OBr- at 330 nm of 326 M-' cm-' (38). HzOP HzOz (30%) was diluted with HzO and kept on ice in dark bottles. The concentration was calculated from the absorbance a t 230 nm by using c of 81 M-' cm-l (39). Quantitation of MPO Activity Using Guaiacol. Before use MPO was diluted with 0.5 mL of cold sodium phosphate/citric acid buffer, pH 4.5, to give approximately 0.1 mg of protein/mL. This solution was kept on ice throughout the experiment (up to 4 h). Its activity was assayed by adding 10 p L of MPO to 0.1 M sodium phosphate, pH 7.4, containing 13 mM guaiacol (11).After addition of H20zto give a final concentration of 0.12 mM in a total volume of 0.9 mL, the absorbance a t 470 nm was recorded every 10 s for 0.5 min. A GU was the amount of enzyme giving 0.3 absorbance unit/s. Oxidation of N-OH-%-FAAby HOX. Reaction mixtures (0.9 mL) contained sodium phosphate/citric acid buffer, p H 4-6.5, sodium halides a t the concentrations designated, and 0.08 mM N-OH-2-FAA (in 8-10 p L of methanol). HOX (in 15 p L of icecold 0.1 N NaOH) was added with rapid mixing to give 0.04 mM. Oxidation of N-OH-2-FAA by N-X-taurine. Reaction mixtures (0.9 mL) a t designated pH and halide concentrations contained sodium phosphate/citric acid buffer and 10 mM taurine. HOX (in 15 pL of ice-cold 0.1 N NaOH) was added with vortexing to give 0.04 mM. After 10 min to allow N-X-taurine formation, N-OH-2-FAA (in 8-10 pL of methanol) was added to give 0.08 mM. MPO-Catalyzed Oxidation of N-OH-%-FAA. Reaction mixtures (0.9 mL) at the pH and halide concentrations designated contained the following: sodium phosphate/citric acid buffer; 0, 1, or 10 mM taurine; sodium halide; 0.08 mM N-OH-2-FAA (in 8-10 pL of methanol); and 0.0036 or 0.0072 GU MPO (in 10-25 pL of buffer, p H 5 ) . Reaction was started with the addition of
Ritter a n d Malejka-Giganti HzOz in 10 pL to give a final concentration of 0.05 mM. Spectrophotometric Determination of 2-NOF Formation. The absorbances a t 377 nm were recorded every minute for 20 min after initiation of the reactions. References lacked HzOzfor MPO incubations and HOX or N-X-taurine for chemical oxidations. The concentration of 2-NOF was calculated by using its c of 15.8 mM-l cm-'. Incubations longer than 20 min were kept a t room temperature in parafilm-covered test tubes and were protected from the light. Contents were transferred to the cuvette, and the absorbance was recorded at the designated time intervals. Determination of Products of Oxidation of N-OH-%-FAA by HPLC. After spectrophotometric determination of 2-NOF at 3 and 20 min, p-nitroaniline (10 nmol in 10 pL of methanol) was added as IS to the reaction mixture. The mixtures were then extracted with the use of Baker C18 extraction columns. The columns were preactivated and the compounds eluted as described previously (40).The eluates containing IS, N-OH-2-FAA, and products of its oxidation were evaporated to dryness under Nz and the residues dissolved in methanol for HPLC analyses by systems A and B, described by us previously (12). The compounds were quantified as described (12),employing extraction efficiencies of standard compounds relative to that of IS determined at each pH and X- concentration. Determination of Stability of N-AcO-2-FAA in Media Containing Chemical or Enzymatic Oxidants. In a total volume of 0.9 mL of sodium phosphate/citric acid buffer, pH 4-6.5, each incubation mixture contained 0.04 mM N-AcO-2-FAA (in 8-10 pL of methanol) and X- h HOX at concentrations indicated in Table I. Enzymatic oxidant was generated from MPO (0.0036 GU), 0.05 mM HzOz, and X-. After 3 or 20 min, the incubation mixtures were extracted, extracts analyzed by HPLC, and compounds quantified as described above. Statistical Analysis. The data are expressed as means i SE. Statistical differences between control and experimental groups were determined by using Dunnett's test (41). Statistical differences for multiple comparisons were determined by using the Newman-Keuls test (41) and were considered significant at P 5 0.05 or highly significant a t P 5 0.01.
Results Determination of Experimental Conditions for Oxidation of N-OH-2-FAAby MPO/H,O,/X-. Since the pH in the in vivo microenvironment of the MPO may decrease to -4.5 (13, 23), we analyzed the oxidation of N-OH-2-FAA from pH 4 to 6.5. Under the experimental conditions, little X--dependent oxidation occurred at pH greater than 6.5. Quantitation of MPO was based on the le- oxidation of guaiacol and is expressed in GU's. X-dependent oxidation of N-OH-2-FAA increased with GU's of MPO. Except when noted, to each 0.9-mL incubation was added 0.0036 GU of MPO since this amount allowed several incubations per vial of enzyme. An amount of Hz02 that gave measurable initial rates of X--dependent oxidation of N-OH-2-FAA to 2-NOF with the amount of MPO above was experimentally determined. As previously reported for halogenation reactions ( 2 1 , 2 4 , 4 2 ) ,the rate of oxidation of N-OH-2-FAA was decreased if H20z was added before X-. Thus, all reactions were initiated by the addition of HZO2 The pH optima of the MPO-catalyzed oxidation of N-OH-2-FAA,as with other MPO-catalyzed reactions (21, 25, 42), were dependent on the H,O,/Xratio. To evaluate the effect of other variables, the H,Oz was kept constant at 0.05 mM. With the above levels of HzOzand MPO, the rate of 2-NOF formation was independent of N-OH-2-FAA (0.04-0.32 mM) in the presence of 0.1 M C1- at pH 5.5 and was maximal with 0.08 mM N-OH-2-FAA in the presence of 0.1 mM Br- at pH 4. Thus, 0.08 mM N-OH-2-FAA was used throughout. We also considered that in addition t o X--dependent oxidation of N-OH-2-FAA to 2-NOF, le- oxidation of N-OH-2-FAA yielding equimolar N-AcO-2-FAA and 2NOF (0.04 mM each) might occur in the systems above.
Chem. Res. Toxicol., Vol. 2, No. 5, 1989 327
Oxidations of Carcinogen by Halide Oxidants
Table I. Recoveries and Fate of N-AcO-2-FAAfrom Buffers Containing MPO/H,O,/X- or X- f HOX
recovery of N-AcO-2-FAAas,' pM N-AcO-2-FAA 2-FAA N-OH-2-FAA 30.2 0.53 1.25 23.0 0.94 1.08 29.5 0.41 1.36 18.5 0.97 1.28 1.12
