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Transformation and Activation of Benzidine by Oxidants of the Inflammatory Response Vijaya M. Lakshmi,† Fong Fu Hsu,‡ and Terry V. Zenser*,†,§ Division of Geriatric Medicine, VA Medical Center, St. Louis, Missouri, Department of Medicine, Washington University, St. Louis, Missouri 63110, and Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63125 Received October 21, 2002
Aromatic amines, such as benzidine (BZ), initiate bladder cancer in humans. Inflammation/ infection play an important role in this cancer. This study was designed to assess the influence of inflammatory oxidants, including reactive nitrogen oxygen species (RNOS), on BZ transformation and activation. RNOS were generated under various conditions and reacted with BZ, and the products were examined by HPLC. Conditions that generate nitrogen dioxide radical, NO2- + myeloperoxidase + H2O2 and ONOO-, produced primarily a single new product, which was identified by MS as azobenzidine (AZO-BZ). The myeloperoxidase-catalyzed reaction was inhibited by 1 mM cyanide and did not require NO2-. Chloride (100 mM) reduced the myeloperoxidase reaction by 30% with taurine having little effect. In contrast, conditions that generate N2O3, i.e., NO donor diethylamine (DEA) NONOate, produced two products, which were identified by MS as 4′-OH-4-aminobiphenyl (4′-OH-ABP) and 4-aminobiphenyl (ABP). 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an oxidant of NO thought to produce NO2•, had a biphasic effect on product formation. At a concentration equal to DEA NONOate, a 5-fold increase in BZ nitrosation was observed, while at higher concentrations nitrosation was greatly diminished and formation of AZO-BZ occurred. Glutathione prevented RNOS transformation of BZ. With MPO and ONOO-, a new product was formed that cochromatographed with 3-(glutathione-S-yl)BZ. Glutathione also prevented nitrosation of BZ but did not form additional BZ products. HOCl-mediated activation of BZ, 4′-OH-ABP, and ABP to bind DNA was assessed. A higher level of binding was observed at pH 5.5 than pH 7.4. BZ elicited the most binding. More binding was observed at both pH values with 4′-OH-ABP than ABP. Thus, components of the inflammatory response are capable of BZ transformation and activation.
Introduction Bladder cancer is diagnosed in about 53 000 people each year and results in over 11 700 deaths (1). It is the third most prevalent cancer type in men 60 years and older and represents approximately 7% of human malignancies (1, 2). A little over 100 years ago, aromatic amines were first suspected of causing bladder cancer (3). It is now recognized that several aromatic amines used in the dye, rubber, and chemical industries and present in cigarette smoke and vehicle exhaust contribute to the increased incidence of bladder cancer in exposed individuals (4, 5). BZ,1 an aromatic amine, can elicit a 100-fold increased risk for bladder cancer in workers exposed to high levels of this compound (6). Metabolism of BZ involves multiple metabolic pathways. Human liver slices acetylate BZ to N-acetylbenzi* To whom correspondence should be addressed. Tel: 314/894-6510. Fax: 314/894-6614. E-mail:
[email protected]. † VA Medical Center. ‡ Washington University. § St. Louis University School of Medicine. 1 Abbreviations: BZ, benzidine; CAD, collisionally activated dissociation; DEA, diethylamine; DETAPAC, diethylenetriaminepentaacetic acid; ESI, electrospray ionization; iNOS, inducible nitric oxide synthase; MPO, myeloperoxidase; NO, nitric oxide; ONOO-, peroxynitrite anion; PHS, prostaglandin H synthase; RNOS, reactive nitrogen oxygen species.
dine and N-acetylbenzidine to N,N′-diacetylbenzidine (7, 8). Both BZ and N-acetylbenzidine are preferred substrates for N-acetyltransferase 1 rather than Nacetyltransferase 2 (8). The N-glucuronides of BZ and N-acetylbenzidine are acid labile, while O-glucuronides of their metabolites are acid stable (9-11). The human recombinant UDP-glucuronosyltransferases (UGT) most effective in N-glucuronidation of BZ are UGT1A9 and UGT1A4 (11). BZ can be activated by a variety of peroxidases, including PHS, to a diimine that reacts with DNA to form adducts (12-15) or, in the absence of a nucleophile, azobenzidine (AZO-BZ) (16). In contrast, cytochrome P-450 metabolism of BZ forms 3-hydroxybenzidine, which has not been shown to be reactive (17). Chronic inflammation/infection and injury play an important role in several types of cancer (e.g., bladder and colon) (18), which can be initiated by aromatic and heterocyclic amines (19, 20). A close association has been reported for chronic urinary tract infections and vulnerability to bladder cancer in patients who are paraplegic secondary to spinal cord injury (21) and individuals who are infected with Schistosoma haematobium (22). Smoking was found to significantly increase the odds ratio for bladder cancer in individuals with a history of S. haematobium infections (23). Chronic inflammation caused by chronic infections may cause about 21% of new cancer
10.1021/tx0200966 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/15/2003
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cases in developing countries as compared with 9% in developed countries (24). RNOS, components of the inflammatory response, are bactericidal and tumoricidal and contribute to the deleterious effects attributed to inflammation on normal tissues (25). Upregulation of iNOS during inflammation produces high levels of NO. NO interacts with either oxygen or superoxide to produce RNOS, including ONOO-, dinitrogen trioxide (N2O3), and nitrogen dioxide radical (NO2•) (25). Peroxynitrite-derived carbonate radical anion and NO2• play an important role in pathophysiologic processes with their combined presence increasing the efficiency of protein-tyrosine nitration (26). Additional oxidants released during an inflammatory response can be derived from infiltrating neutrophils. These cells produce superoxide, H2O2, and other reactive oxygen species. MPO present in neutrophils uses H2O2 to produce cytotoxic oxidants, such as HOCl in the presence of Cl- (27). RNOS catalyze nitrosation, oxidation, and nitration reactions with numerous biological targets representing lipids, proteins, and DNA (25, 28-30). This can cause lipid peroxidation, inhibition of enzymes, and deamination of DNA. RNOS have the potential for oxidizing primary aromatic amines to a variety of products, which could cause cell DNA damage/mutations, contributing to the multistep carcinogenic process. This study assesses the transformation of BZ by RNOS, identifies products of transformation, and evaluates the ability of these transformation products to be activated and bind DNA.
Experimental Procedures Materials. [2,2′-3H]BZ (250 mCi/mmol) was purchased from Chemsyn, Lenexa, KS. BZ-2HCl, 4-aminobiphenyl (ABP), NaNO2, NaOCl, calf thymus DNA, H2O2, catalase (bovine liver), ascorbic acid, DETAPAC, and NaCN were purchased from Sigma Chemical Co., St. Louis, MO. DEA NONOate, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), and MPO from human polymorphonuclear leukocytes (180-220 U/mg protein) were purchased from Calbiochem, San Diego, CA. PHS (ovine) was obtained from Oxford Biochemical Research, Oxford, MI. Alkaline solutions of ONOO- were prepared from acidified NO2- and H2O2 and quantitated spectrophotometrically (302 ) 1.67 mM-1 cm-1) as described (31). Stock solutions were kept at -70 °C. Ultima-Flo AP was purchased from Packard Instruments, Meriden, CT. 4′-OH-4-Aminobiphenyl (4′-OH-ABP) was prepared from N-acetylbenzidine following diazotization and acid hydrolysis. Bakerbond spe columns (C-18, 500 mg) were purchased from J. T. Baker, Phillipsberg, NJ. Caution: BZ and ABP are carcinogenic and should be handled in accordance with NIH Guidelines for the Laboratory Use of Chemical Carcinogens (32). RNOS Transformation of BZ. 3H-BZ (0.06 mM) was incubated in 100 mM potassium phosphate buffer, pH 7.4, containing 0.1 mM DETAPAC in a total volume of 0.1 mL at 37 °C. For incubation with MPO, 1 µg/mL of peroxidase was added in the presence or absence of 0.3 mM NO2-, and the reaction started by the addition of 0.05 mM H2O2. Incubations with DEA NONOate or ONOO- were started immediately following their addition, and the tubes were capped. The pH was checked at the conclusion of these incubations and did not change by more than (0.1 pH unit. Incubation times were 10 min for MPO and DEA NONOate and 1 min for ONOO-. Blank values were obtained in the absence of either RNOS generating agent or H2O2. The reactions were stopped by adding 0.05 mL of dimethylformamide containing 10 mM ascorbic acid and immediately frozen. BZ transformation was assessed by HPLC as described below. DNA Binding. Activation of aromatic amines was assessed by their binding to DNA. Reaction mixtures were as described
Lakshmi et al. above, except that 1 mg/mL DNA was present and 5 min incubations were conducted at either pH 5.5 or pH 7.4. Activation was accomplished by addition of 0.1 mM HOCl. Blank values were obtained in the absence of HOCl. Calf thymus DNA was purified before use by extracting with phenol and a 24:1 chloroform/isoamyl alcohol mixture. After addition of methionine (1 mM) to stop the reaction, DNA was precipitated by adjusting the concentration of NaCl to 0.25 M, adding 2 vol of ethanol, and leaving overnight at -20 °C (33). The DNA pellet was washed twice with 70% ethanol and dissolved in water, and the process of precipitation and washing was repeated. The radioactivity of the water-dissolved DNA was determined. The purity and quantity of DNA were assessed by the absorbance at 260 and 280 nm. A A260/A280 ratio of approximately 1.7 was achieved for each sample. Binding is expressed as nmol/mg DNA. HPLC Analysis of Metabolites. Metabolites were analyzed using a Beckman HPLC with System Gold software and a 5 µm, 4.6 mm × 150 mm C-18 ultrasphere column attached to a guard column. Mobile phase 1 contained 20 mM potassium phosphate buffer (pH 5.0) in 20% methanol, 0-2 min; 20-33%, 2-8 min; 33-40%, 8-15 min; 40-80%, 15-22 min; 80-20%, 32-37 min; flow rate, 1 mL/min. In mobile phase 2, the potassium phosphate buffer of mobile phase 1 was replaced by 20 mM ammonium formate buffer (pH 3.1). Mobile phase 3 contained 20 mM ammonium formate buffer (pH 3.1) in 45% methanol, 0-1 min; 45-80%, 1-15 min; 80-90%, 20-25 min; 90-45%, 25-30 min; flow rate, 1 mL/min. Radioactivity in HPLC eluents was measured using a FLO-ONE radioactive flow detector. Product formation is expressed as a percent of total radioactivity or nanomoles recovered by HPLC. The amount of BZ transformed was calculated by subtracting the percent of BZ recovered by HPLC from 100%. ESI Mass Spectral Analysis. ESI/MS analyses were performed on a Finnigan TSQ-7000 triple stage quadrupole mass spectrometer (San Jose, CA) equipped with a Finnigan ESI source and controlled by Finnigan ICIS software operated on a DEC alpha workstation. Samples were loop-injected onto the ESI source with a Harvard syringe pump at a flow rate of 5 µL/min. The entrance of the heated capillary and the skimmer were at ground potential, and the electrospray needle was at 4.5 kV. The mass spectrometer was adjusted to unit mass resolution, and the heated capillary temperature was set at 200 °C. For CAD tandem mass spectra, the collision gas was argon (2.3 mTorr), and the collision energy was set at 22 eV. Production spectra were acquired in the profile mode at the scan rate of 3 s per scan.
Results To assess RNOS chemical transformation, 3H-BZ was incubated with 0.2 mM DEA NONOate, which releases free NO into solution with a half-life of 2.5 min at neutral pH and 37 °C. With HPLC mobile phase 1 (Figure 1, top panel), new peaks were observed at 14.2 min (4′-OHABP) and 23.9 min (ABP). Under this condition, 4′-OHABP and ABP represent 7 and 26%, respectively, of the total radioactivity recovered by HPLC. When BZ was incubated with MPO + H2O2 + NO2-, these products were not observed (Figure 1, middle panel). The major product observed with MPO eluted at 29 min (AZO-BZ) and represented 17% of the total recovered radioactivity. The latter product was also observed during ONOOtransformation of BZ (not shown). These results suggest BZ transformation by different RNOS to different products. To further identify the products of DEA NONOatetransformed BZ, HPLC purified material was analyzed by ESI/MS. In the positive ion mode, the 23.9 min product yields a prominent protonated molecular ion (MH+) at m/z 170 (Figure 2A). Ions at m/z 153 and 152 represent
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Figure 2. Positive mode CAD tandem mass spectra of ABP (panel A), 4′-OH-ABP (panel B), and AZO-BZ (panel C).
Figure 1. HPLC analysis of BZ oxidation by DEA NONOate or MPO. 3H-BZ (0.06 mM) was incubated with DEA NONOate (0.2 mM) for 10 min (top panel), MPO + 0.05 mM H2O2 + 0.3 mM NO2- for 10 min (middle panel), or MPO + 0.05 mM H2O2 + 0.3 mM NO2- + 0.1 GSH for 10 min (bottom panel).
consecutive losses of NH3 and H, respectively; m/z 169 and 143 consecutive losses of H and C2H2, respectively; and m/z 93 and 92 consecutive losses of C6H5 and H, respectively (see Scheme 1). This is consistent with the 23.9 min product (ABP; Figure 1, top panel) being ABP. In addition, this product exhibits identical chromatographic and spectral characteristics as the commercially available material. For the 14.2 min product in the
positive mode, a prominent protonated (MH+) molecular ion at m/z 186 was observed (Figure 2B). Ions at m/z 169 and 143 represent consecutive losses of NH3 and C2H2, respectively; m/z 168 and 167 consecutive losses of H2O and H, respectively; and m/z 93 loss of C6H5NH2. This is consistent with the 14.2 min product (4′-OH-ABP; Figure 1, top panel) being 4′-OH-ABP. This assignment is supported with an identical compound being made by direct chemical synthesis involving a diazotization reaction with either nitrobenzene and aniline or BZ. Thus, nitrosation products of BZ are ABP and 4′-OH-ABP. The stability of the nitrosation products 4′-OH-ABP and ABP was assessed at 37 °C. After an 18 h incubation at pH 5.5, 7.4, and 9.0 in phosphate buffer, each compound was analyzed by HPLC. Little loss of either compound was detected (
