Hypochlorous Acid-Mediated Activation of N-acetylbenzidine to Form N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine

Vijaya M. Lakshmi*, Fong Fu Hsu{dagger}, Alaine E. McGarry*, Bernard B. Davis* and Terry V. Zenser*,1

* VA Medical Center, Division of Geriatric Medicine and Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri; and {dagger} Department of Medicine, Washington University, St. Louis, Missouri

Received March 3, 1999; accepted October 19, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hypochlorous acid (HOCl), a chemically reactive oxidant, is an important component of the inflammatory response and may contribute to carcinogenesis. This study assessed the possible activation of N-acetylbenzidine (ABZ) by HOCl to form a specific DNA adduct, N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. HOCl was incubated with 0.06 mM 3H-ABZ, and transformation assessed by HPLC. Similar results were observed at pH 5.5 or 7.4. A linear increase in transformation was observed from 0.025 to 0.1 mM HOCl with up to 80% of ABZ changed. Approximately, 2 nmoles of HOCl oxidized 1 nmole of ABZ. N-oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected. Oxidation of ABZ was prevented by taurine, DMPO, glutathione, and ascorbic acid, whereas mannitol was without effect. Results are consistent with a radical mechanism. In the presence of 2'-deoxyguanosine 3'-monophosphate (dGp), a new product (dGp-ABZ) was observed. The same adduct was observed with DNA. dGp-ABZ was found to be quite stable (>80% remaining) at 70°C in pH 5.5 (60 min) and 7.4 (240 min). Electrospray mass spectrometry indicated that dGp-ABZ was N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine, and this was confirmed by NMR. 32P-postlabeling in combination with TLC and HPLC determined that the adduct made by either HOCl or prostaglandin H synthase oxidation of ABZ in the presence of dGp or DNA was dGp-ABZ. Thus, HOCl activates ABZ to form dGp-ABZ and may be responsible for the presence of this adduct in peripheral white blood cells from workers exposed to benzidine. Reaction of ABZ with HOCl provides an easy, convenient method for preparing dGp-ABZ.

Key Words: hypochlorous acid; aromatic amines; DNA adducts; N-acetylbenzidine; benzidine..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neutrophile/monocyte-derived oxidant hypochlorous acid (HOCl) is an important part of the inflammatory response and may contribute to carcinogenesis. Stimulation of phagocytosis triggers a membrane-associated NADPH oxidase, which reduces molecular oxygen to superoxide. The latter dismutates to hydrogen peroxide. Phagocytic cell myeloperoxidase catalyzes the formation of HOCl from hydrogen peroxide and chloride ions. Depending on the stimulus, HOCl can account for up to 70% of the hydrogen peroxide liberated by inflammatory cells. The pKa of HOCl is 7.46. During the course of phagocytosis, the pH of phagosomes decreases from neutral to acidic pH (<pH 6) (for review see Kettle and Winterbourn, 1997).

HOCl is microbicidal and induces oxidative injury during phagocytosis. When exposed to bacteria, HOCl kills cells within seconds via mechanisms involving ATP depletion and inhibition of membrane transporters and respiratory enzymes (Albrich et al., 1981Go; Barrette et al., 1989Go). With mammalian cells, HOCl can oxidize many important biologic constituents such as proteins, fatty acids, cholesterol, and DNA. Reactions demonstrated include conversion of primary and secondary amines to both mono- and di-N-chloramines (Thomas et al., 1986Go), oxidation of free amino groups to aldehydes (Hazen et al., 1998Go), and chlorination of fatty acids and cholesterol (Hazen et al., 1996Go; Carr et al., 1996Go). These modifications have been implicated in the pathogenesis of conditions ranging from aging to atherosclerosis (Berlett and Stadtman, 1997Go; Steinberg, 1997Go). Although treatment of DNA with HOCl causes large increases in pyrimidine oxidation (i.e., thymine glycols), purine oxidation (8-hydroxyguanine) was not detected (Whiteman et al., 1997Go). HOCl oxidizes a variety of aromatic amines to dications or nitrenium ions that react with DNA forming adducts (Kozumbo et al., 1992Go; Uetrecht et al., 1995Go; Liu and Uetrecht, 1995Go; Ritter and Malejka-Giganti, 1989Go).

Animal and epidemiologic studies indicate that urinary tract infection is a significant risk factor for the development of bladder cancer. Using the heterotopically transplanted rat urinary bladder, heat-killed Escherichia coli or its endotoxin lipopolysaccharide increased the number of tumors in rats receiving a single dose of N-methyl-N-nitrosourea (Yamamoto et al., 1992Go). In this model, migration of polymorphonuclear leukocytes into the urothelium and of other inflammatory cells into the lamina propria was observed (Kawai et al., 1993Go; Yamamoto et al., 1992Go). 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 (Bejany et al., 1987Go) and individuals who are infected with Schistosomal haematobium (El-Sebai, 1981Go). Smoking was found to significantly increase the odds ratio for bladder cancer in individuals with a history of Schistosomal haematobium infections (Bedwani, 1998Go).

Aromatic amines are prevalent in cigarette smoke (Patrianakos and Hoffmann, 1979Go) and are thought to contribute to a 2- to 3-fold increase in the relative risk of developing bladder cancer (Ross et al., 1988Go). 4-Aminobiphenyl is an aromatic amine constituent of cigarette smoke (Patrianakos and Hoffmann, 1979Go). Relationships have been established between both 4-aminobiphenyl-hemoglobin and 4-aminobiphenyl-DNA adduct levels in urothelial cells and the occurrence of bladder cancer, suggesting a causal role for tobacco smoking (Bartsch et al., 1993Go). Peripheral white blood cells or their polymorphonuclear leukocyte, monocyte, and lymphocyte subpopulations are used as surrogate tissue to assess environmental exposure and have demonstrated increased levels of 4-aminobiphenyl-DNA adduct in smokers compared to nonsmokers (Dallinga et al., 1998Go).

Peripheral white blood cells (Zhou et al., 1997Go) and exfoliated urothelial cells (Rothman et al., 1996Go) from workers exposed to benzidine contain the same major DNA adduct. Levels of adduct in peripheral white blood cells correlated with levels in exfoliated bladder cells. This adduct N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is mutagenic and thought to be responsible for the initiation of bladder carcinogenesis by benzidine (Beland et al., 1983Go; Fox et al., 1990Go; Heflich et al., 1986Go; Melchior, Jr. et al., 1994Go). N-Acetylbenzidine (ABZ) is the major metabolite observed in urine (Hsu et al., 1996Go) and plasma of workers exposed to benzidine. Therefore, this N-acetylated arylamine is the likely substrate involved in adduct formation. ABZ activation by white blood cells may contribute to the formation of dGp-ABZ in these cells and bladder cells. This study is designed to assess the formation and characterization of dGp-ABZ derived from the reaction of HOCl with ABZ. Results from this study are expected to provide insight into the possible metabolism and activation of ABZ by neutrophile/monocyte-derived HOCl to form dGp-ABZ. An easy, convenient method is described for preparing dGp-ABZ, and other 2'-deoxyguanosine analogues for use as standards in qualitative and quantitative assessment of adduct levels.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
3H-Benzidine (180 mCi/mmol) was purchased from Chemsyn (Lenexa, KS). ABZ and 3H-ABZ were synthesized by acetylation of benzidine using glacial acetic acid with the final product purity greater than 98% (Lakshmi et al., 1990Go). Benzidine-free base and hydrochloride salt, taurine, NaOCl, H2O2, glutathione, ascorbic acid, methionine, 2-methyl-2-nitropropane (tNB), diethylenetriaminepentaacetic acid (DETAPAC), proteinase K, DNA (calf thymus, Type I), 2'-deoxyguanosine 3'-monophosphate (dGp), 2'-deoxyguanosine 5'-monophosphate (dpG), 2'-deoxyguanosine (dG), micrococcal nuclease (EC 3.1.31.1), and potato apyrase (grade I; EC 3.6.1.5) were purchased from Sigma Chemical Co., St. Louis, MO. Nuclease P1 (EC 3.1.30.1), spleen exonuclease (EC 3.1.16.1), and T4 polynucleotide kinase (EC 2.7.1.78) were from Boehringer Mannheim, Indianapolis, IN. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was obtained from Aldrich Chemical Co., Milwaukee, WI. The dGp-ABZ standard was prepared as previously described (Lakshmi et al., 1995Go). N-oxidation standards of ABZ including N'-hydroxy-N-acetylbenzidine, N-hydroxy-N-acetylbenzidine, and 4'-nitro-4-acetylaminobiphenyl were synthesized by Dr. Shu Wen Li using 4'-nitro-4-aminobiphenyl (TCI America, Portland, OR) as starting material (Babu et al., 1995Go) and were identified by mass spectrometry. Ultima-Flo AP was purchased from Packard Instruments, Meriden, CT.

Reaction of ABZ with HOCl.
3H-ABZ (0.06 mM) was added to 100 mM phosphate buffer pH 5.5 or 7.4, containing 0.1 mM DETAPAC in a total volume of 0.1 ml. The reaction was started by the addition of NaOCl to a final concentration of 0.1 mM and incubated at 37°C for 1 min. A 1 mM stock solution of NaOCl was made in 0.025 N NaOH. Where indicated, 1 mg/ml DNA or 3 mg/ml nucleosides/nucleotides were present at the beginning of the incubation. Similar results were observed in the presence or absence of DETAPAC. Blank values were obtained in the absence of NaOCl. The reaction was stopped by adding 0.01 ml of 10 mM methionine, 0.2 ml of dimethylformamide, and frozen. Transformation was assessed by HPLC as described below. Samples containing DNA or dGp were extracted twice with two volumes of ethyl acetate, and the aqueous fraction was processed as described below.

HPLC analysis of products.
Products were assessed using a Beckman HPLC with System Gold software, which consisted of a 5-µm, 4.6 x 150 mm C-18 ultrasphere column attached to a guard column. For solvent system 1, the mobile phase contained 20 mM phosphate buffer (pH 5.0) in 50% methanol, 0–2 min; 50–60%, 2–7 min; 60–90%, 7–27 min; 90–50%, 35–40 min; flow rate 1 ml/min. For solvent system 2, the mobile phase contained 20 mM 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. Radioactivity in HPLC eluents was measured using a FLO-ONE radioactive flow detector. Data are expressed as a % of total radioactivity recovered by HPLC. The amount of ABZ transformed was determined by subtracting the % of ABZ recovered (unchanged) from 100%. Some fractions were also collected for 32P-postlabeling.

Preparation of DNA adduct.
DNA was prepared as previously described (Lakshmi et al., 1998Go). Following treatment with proteinase K and ribonuclease A and T1, the purity of DNA was evaluated by the ratio of A260/A280 to be approximately 1.7 for each sample. DNA samples were enzymatically hydrolyzed to dGp adducts by digestion with micrococcal nuclease and spleen phosphodiesterase (Gupta et al., 1982Go). Enrichment of the adduct from unmodified nucleotides was achieved with n-butanol extraction (Gupta, 1985Go). After ethyl acetate extraction, tetrabutylammonium chloride (1 mM) was added to the aqueous fraction along with an equal volume of n-butanol. Following three extractions, the n-butanol layers were pooled, back-extracted with water twice, evaporated using a speed vac, and dissolved in 0.05 ml distilled water.

Nuclease P1 hydrolysis of dGp adduct.
The enriched adduct sample was added to a reaction mixture containing 0.1 M sodium phosphate, pH 5.5, and 0.15 mM ZnCl2 at 37°C (Reddy and Randerath, 1986Go). One aliquot was incubated with 2 µg nuclease P1, while another aliquot was incubated without nuclease for the indicated time. Sensitivity is indicated by cleavage of the 3'-phosphate of dGp-ABZ to dG-ABZ. Samples were analyzed by HPLC using solvent system 2.

Adduct analysis by 32P postlabeling.
The dGp adduct was analyzed by 32P-postlabeling as previously described (Gupta et al., 1982Go; Lakshmi et al., 1995Go). Labeled adduct was separated on PEI-cellulose sheets using the following multicomponent solvent systems: D-1 = 1.7 M sodium phosphate (pH 5.5); D-3 = 4 M ammonium hydroxide; D-4 = 0.6 M lithium formate/0.5 M Tris–HCl/7 M urea (pH 8.0). 32P-labeled adduct was observed by autoradiography. The adduct was extracted from plates with 14 M ammonium hydroxide:methanol (1:1), evaporated, redissolved in methanol, and further analyzed by HPLC using solvent system 2 described above. In addition to synthetic dGp-ABZ (Lakshmi et al., 1995Go), an identical standard was prepared by the incubation of N'-hydroxy-N-acetylbenzidine with either calf thymus DNA or dGp (Lakshmi et al., 1998Go).

Preparation of dG-ABZ and dGp-ABZ for NMR and MS analysis.
To prepare a sufficient amount of dG-ABZ adduct for NMR analysis, the ratio of HOCl to ABZ was kept constant while the concentration of ABZ and HOCl was increased to 0.24 and 0.4 mM, respectively, at pH 5.5. Following a 10-ml incubation with 3 mg/ml dG, the HOCl/ABZ reaction mixture was applied to a 500-mg C-18 Bakerbond spe column. After washing with water (20 ml), the adduct was eluted with 4 ml of 100% methanol. The adduct was further purified with the HPLC system described above using a mobile phase consisting of 20 mM phosphate buffer (pH 5.0) in 35% methanol, 0–2 min; 35–41%, 2–8 min; 41–80%, 8–14 min; 80–35%, 19–25 min; flow rate 1 ml/min. With this solvent system, the elution times of ABZ and dG-ABZ were 10.2 and 15.9 min, respectively.

Following a 10-ml incubation with 3 mg/ml dGp, the reaction mixture was extracted four times with three volumes of ethyl acetate. dGp-ABZ was recovered by adding an equal volume of n-butanol and extracting three times. The pooled n-butanol extracts were further purified by HPLC using solvent system 2 and then 1.

Mass spectral analysis.
Electrospray ionization mass spectrometry (ESI/MS) analyses were performed on a Finnigan (San Jose, CA) TSQ-7000 triple-stage quadrupole spectrometer 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 electrospray needle and the skimmer were at ground potential and the electrospray chamber and the entrance of the glass capillary were at 4.4 kV. The heated capillary temperature was 200°C. For collisionally activated dissociation (CAD) tandem mass spectra, the collision gas was argon (2.2–2.5 mtorr), and collision energy was set at 50 eV. Product ion spectra were acquired in the profile mode at the scan rate of one scan per 3 sec.

NMR analysis.
Samples were prepared in 100% d6-DMSO (Aldrich gold label) under dry nitrogen purge. About 30 µg of the sample was dissolved in 1 ml of DMSO and transferred into high quality 5-mm tubes and capped under nitrogen. NMR spectra were acquired on a Varian Inova-500 instrument with proton resonance frequency at 499.97 MHz with an INVERSE probe (1H 90 pulse of 9 microsec). 128 transients were signal averaged with a recycle delay of 5 s and a 45-degree tip. The time domain data was processed on a SUN work station using the VNMR software. A line broadening parameter of 0.5 Hz was used for the Fourier transformation. Chemical shifts were referenced to TMS at 0.0 ppm.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The reaction of 0.06 mM ABZ with 0.1 mM HOCl at pH 5.5 and 7.4 was monitored by HPLC. HOCl elicited substantial transformation, with only 30% and 43% ABZ remaining unchanged at pH 5.5 and 7.4, respectively. Major product peaks at 11.7, 15.2, and 19.8 min appear to be present in incubations at both pHs. Because the products were unstable during purification, they were not identified. N-oxidation products of ABZ were prepared as standards and used to assess the reaction of ABZ with HOCl. N'-hydroxy-N-acetylbenzidine, N-hydroxy-N-acetylbenzidine, and 4'-nitro-4-acetylaminobiphenyl were not detected using a variety of incubation conditions.

To better understand the ABZ and HOCl reaction, the concentration of 3H-ABZ was kept constant at 0.06 mM and the concentration of HOCl was varied from 0.025 to 0.2 mM (Fig. 1Go). From 0.025 to 0.1 mM HOCl, a rather linear increase in ABZ transformation and product formation was observed (Fig. 1Go, panel A). At 0.1 mM HOCl, less than 20% of ABZ remains unchanged. Because 80% of ABZ was already transformed with 0.1 mM HOCl, little additional transformation and product formation was observed when the concentration of HOCl was increased to 0.2 mM. The amount of ABZ transformed per nanomole of HOCl present was relatively constant from 2.5 to 10 nmoles of HOCl (Fig. 1Go, panel B). Over this linear range of transformation, the average ratio of oxidant HOCl to reductant ABZ transformed was about 2. Similar results were observed at pH 7.4.



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 1. Relationship between reductant ABZ and oxidant HOCl at pH 5.5. The concentration of 3H-ABZ was maintained at 0.06 mM; the concentration of HOCl was varied from 0 to 200 µM. Panel A illustrates the relationship between HOCl concentration and the distribution and amount of radioactive products, using HPLC solvent system 1. Times indicate when the product eluted from the HPLC. Panel B assesses the relationship between the amount of ABZ transformed versus HOCl concentration. Also illustrated is the amount of oxidant HOCl (Ox) required to transform a specific amount of reductant ABZ (Red) expressed as the ratio of Ox/Red versus HOCl concentration.

 
Test agents were selected to further evaluate the ABZ and HOCl reaction (Table 1Go). Taurine is known to selectively react with HOCl, forming an N-chloramine (Thomas et al., 1986Go). At 10 mM, taurine completely inhibited ABZ oxidation. Both DMPO and tNB are radical trapping agents (Mottley and Mason, 1989Go). Whereas DMPO caused complete inhibition of ABZ transformation, tNB elicited only a modest effect. The antioxidants glutathione and ascorbic acid are known to react with HOCl (Winterbourn, 1985Go) and 1 mM of each completely inhibited oxidation. Mannitol, which reacts with hydroxyl radicals, was without a significant effect on metabolism. Although all of the test agents discussed above had similar effects at both pH 5.5 and 7.4, the effects of dG were different. At the acidic pH, more transformation of ABZ was observed. However at both pHs, a new product was detected when dG was present.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Effect of Various Test Agents on the Reaction of HOCl with N-acetylbenzidine at pH 5.5 or 7.4a
 
The ability of HOCl to activate 3H-ABZ to react with a nucleoside, nucleotide, and DNA was further evaluated (Fig. 2Go). A distinct new radioactive product peak was observed by HPLC at 21.2 and 23.4 min when dGp and dG, respectively, were present. Nuclease P1 treatment of the dGp adduct demonstrated dephosphorylation to the dG adduct (not shown). Following reaction with calf thymus DNA, the incubation mixture was treated to hydrolyze this macromolecule to its nucleotide residues. The n-butanol extract of this nucleotide mixture exhibited a peak (Fig. 2Go, panel D) identical to that observed when dGp was present (Fig. 2Go, panel C). Partial hydrolysis of the adducted DNA nucleotide mixture with nuclease P1 gave the dG adduct (23.4 min) (Fig. 2Go, panel E). Identical peaks at 21.2 and 23.4 min were observed when incubations with dG, dGp, or DNA were conducted at pH 7.4 (not shown). With dpG, a new peak was observed at 20.9 min that was converted to the dG adduct (23.4 min) by alkaline phosphatase treatment (not shown). Thus, ABZ can form the same adduct whether HOCl-activated ABZ reacts with dG, dGp, dpG, or DNA.



View larger version (36K):
[in this window]
[in a new window]
 
FIG. 2. HPLC profiles of the reaction of HOCl with 3H-ABZ at pH 5.5 in the absence (panel A) and presence of dG (panel B), dGp (panel C), calf thymus DNA (panel D), or calf thymus DNA followed by nuclease P1 hydrolysis (panel E). Samples were analyzed using solvent system 2.

 
Formation of adduct with nucleoside, nucleotides, and DNA was quantitated (Table 2Go). More adduct was detected with dG at pH 5.5 than any of the other conditions. For dG, about 30% less adduct was detected at pH 7.4 compared to 5.5. In contrast, pH had a much more dramatic effect on the amount and proportion of adduct formed with either the 3'- or 5'-monophosphate. For dGp, 4-fold more adduct was formed at pH 5.5 than 7.4. The opposite pH preference was observed for dpG, with 1.7-fold more adduct formed at pH 7.4 than 5.5. Similar amounts of DNA adduct were formed at both pHs.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Adduct Formation during Reaction of HOCl with N-acetylbenzidine at pH 5.5 or 7.4a
 
Selected test agents were used to characterize the formation of the dG adduct (Table 3Go). Results were similar with respect to ABZ transformation as reported in Table 1Go. Taurine, DMPO, glutathione, and ascorbic acid elicited nearly complete inhibition of ABZ transformation and formation of the dG-ABZ adduct. Mannitol and tNB had little or no effect on ABZ transformation or adduct formation. Similar results were observed at pH 5.5 or 7.4. Consistent with Tables 1 and 2GoGo, considerably more ABZ was transformed at pH 5.5 than 7.4, even though the amount of adduct present at either pH was similar.


View this table:
[in this window]
[in a new window]
 
TABLE 3 Effect of Various Test Agents on the Reaction of HOCl with N-acetylbenzidine in the Presence of 2'-Deoxyguanosine at pH 5.5 or 7.4a
 
The pH stability of the dGp-ABZ adduct was assessed (Table 4Go). The n-butanol purified adduct was incubated in acidic (pH 1.5), mildly acidic (pH 5.5), neutral (pH 7.4), or basic (pH 13) media at 70°C for 5 to 240 min. The adduct was quite sensitive to strong acid treatment, with no adduct observed after 5 min. The major product appeared to be dG-ABZ. At pH 5.5, little degradation of adduct was detected until 60 min of incubation. After 240 min, 44% of the adduct still remained. dGp-ABZ was quite stable at neutral pH, with no detectable change observed for 4 h at 70°C. Strong base treatment results in only 16% of dGp-ABZ remaining after 60 min. dG-ABZ is not a breakdown product of base hydrolysis.


View this table:
[in this window]
[in a new window]
 
TABLE 4 pH Stability of N'-(3'-monophospho-deoxyguanosin-8-yl)- N-acetylbenzidinea
 
The ESI mass spectrum of dGp-ABZ gives [M – H] ion at m/z 570 in the negative ion mode (Fig. 3Go). The product ion spectrum resulting from CAD of m/z 570 yields a major fragment ion at m/z 374, representing N-(guanin-8-yl)-acetylbenzidine. The fragmentation pathway to ion formation is shown in scheme 1 and is consistent with the structure being N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. Results in the positive ion mode yielded data corresponding to the suggested structure.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 3. ESI CAD tandem mass spectra of N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine in the negative ion mode at m/z 570. Scheme illustrates the fragmentation of the adduct.

 
NMR analysis was used to further determine the structure of the dG-ABZ adduct. 1H-NMR spectral parameters for dG, ABZ, and dG-ABZ are given in Tables 5, 6, and 7GoGoGo, respectively. All 8 aromatic protons of ABZ were detected in the adduct, which suggests that substitution occurred through the nitrogen of the amino group and not through the aromatic carbon atoms. Furthermore, the NH2 signal observed at 5.138 ppm with ABZ was not detected in the adduct spectrum. A new D2O-exchangeable resonance that integrated to 1 proton was detected at 8.717 ppm and tentatively assigned to NH at position 9 in the adduct. The adduct signal observed at 6.338 ppm was assigned to 2-NH2 (G). A single-proton D2O exchangeable resonance detected at 10.507 ppm was tentatively assigned to 1-NH (G) in the adduct. The H-8 (G) proton, normally seen at 7.898 ppm (Table 5Go), was missing, and its absence demonstrates that adduction occurred through the C-8 position of dG. The amide NH proton at position 14 was detected in the adduct (9.964 ppm) along with the CH3 protons (2.040 ppm) at position 15. All of the deoxyribose protons were accounted for in the adduct spectrum. Thus, the 1H-NMR spectrum of the adduct is consistent with that expected for N'-(deoxyguanosin-8-yl)-N-acetylbenzidine.


View this table:
[in this window]
[in a new window]
 
TABLE 5 H NMR Spectral Parameters of 2`-Deoxyguanosine
 

View this table:
[in this window]
[in a new window]
 
TABLE 6 H NMR Spectral Parameters of N-Acetylbenzidine
 

View this table:
[in this window]
[in a new window]
 
TABLE 7 1H NMR Spectral Parameters of N`-(deoxyguanosin-8-yl)-N-Acetylbenzidine
 
dGp-ABZ derived from HOCl oxidation of ABZ was compared to synthetic dGp-ABZ standard by 32P-postlabeling (Fig. 4Go). Following postlabeling, adducts were eluted from TLC plates and further analyzed by HPLC with and without nuclease P1 treatment (not shown). This analysis allowed further comparison of the adduct derived from either HOCl or prostaglandin H synthase oxidation of ABZ to synthetic dGp-ABZ. Results demonstrate that the HOCl-derived adduct (Fig. 4Go, panel B) was identical to the synthetic standard (Fig. 4Go, panel A). Furthermore, the DNA adduct derived from prostaglandin H synthase oxidation of ABZ (Fig. 4Go, panel C) was also identical to the synthetic dGp-ABZ standard (Lakshmi et al., 1998Go). Similar results were observed with HOCl when DNA was substituted for dGp or with prostaglandin H synthase when dGp was substituted for DNA (not shown). In addition, HPLC fractions corresponding to the dGp-ABZ adduct (Fig. 2Go, panel C) were collected and used for 32P-postlabeling. Fractions collected from HOCl or prostaglandin H synthase incubations or from the synthetic standard exhibited the same 32P-postlabeling profile. Blank incubations in the absence of either HOCl or prostaglandin H synthase did not contain the adduct (Fig. 4Go, panel D).



View larger version (8K):
[in this window]
[in a new window]
 
FIG. 4. Analysis of HOCl and prostaglandin H synthase oxidation of ABZ by 32P-postlabeling. 32P postlabeled material was separated on PEI-cellulose. The following samples are represented: panel A, synthetic dGp-ABZ; panel B, HOCl dGp adduct; panel C, PHS DNA adduct; panel D, blank samples incubated in the absence of PHS or HOCl.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Substantial HOCl oxidation of ABZ was demonstrated. With similar ABZ transformation observed at either pH 5.5 or 7.4, the reaction does not seem to be influenced by the 7.46 pKa of HOCl (Fig. 1Go and Table 1Go). The oxidant to reductant ratio of 2 observed for ABZ transformation is consistent with a 2-electron oxidation. A ratio of 2 has been reported for HOCl oxidation of alanine (Thomas et al., 1986Go). N-oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected using the different incubation conditions used in this study. Test agent taurine inhibition was attributed to N-chloramine formation (Thomas et al., 1986Go; Winterbourn, 1985Go). Glutathione and ascorbic acid are also known to react with HOCl (Winterbourn, 1985Go) and completely inhibited transformation of ABZ. HOCl has been shown to react under a variety of conditions to generate hydroxyl radicals (Candeias et al., 1993Go; Candeias et al., 1994Go). The lack of effect of mannitol (50 mM) suggests that hydroxyl radicals are not involved in ABZ oxidation. The radical scavenger DMPO completely inhibited ABZ transformation, whereas another scavenger tNB was much less effective (Mottley and Mason, 1989Go).

Although examination of the mechanism of ABZ activation is beyond the focus of this study, information gathered from this and other studies can address this issue. Horseradish peroxidase and prostaglandin H synthase peroxidatically metabolize benzidine to a radical cation and then a diimine (Josephy et al., 1983Go; Wise et al., 1985Go). Although HOCl oxidation of ABZ may be by electron withdrawal, spectral studies have not indicated the presence of a diimine (not shown). Oxidation of ABZ may result in the formation of a less ring-activated intermediate, such as a diimine monocation, which is a resonance structure of the ABZ nitrenium ion. This intermediate has been proposed recently as the reactive intermediate responsible for dGp-ABZ formation (Dicks et al., 1999Go). Alternatively, HOCl reacts with amines to form chloramines (Thomas et al., 1986Go), which could lose HCl, forming a diimine or imidoimine. However, HOCl oxidation of 4-aminobiphenyl does not result in formation of an N-chloro intermediate (Scarborough and Waters, 1926Go). Ongoing experiments are directed at identification of labile reaction products, which will assist in assessing the mechanism of ABZ activation to form dGp-ABZ.

Both pH 5.5 and 7.4 were selected to assess transformation, because the pH involved in HOCl's possible carcinogenic effect is not known. Whereas the pH inside a phagosome can be quite acidic, other parts of the cell exhibit a more neutral pH, and HOCl released from cells would be exposed to a varied environment. The major pH effect observed was the reaction of activated ABZ with 2'-deoxyguanosine 3'- and 5'-monophosphates (Table 2Go). A higher rate of adduct formation was observed with the former at the acidic pH, whereas the latter exhibited a higher rate at the neutral pH. Previous studies with aromatic amines have demonstrated a higher rate of adduct formation at acidic pH and attributed this to stabilization of a nitrenium ion or increased electrophilicity of a iminoquinone due to protonation of the imine nitrogen (Yamazoe et al., 1985Go). This does not seem likely in the present case because neither intermediate has been detected and these intermediates would not be expected to be more reactive with dpG at pH 7.4 than 5.5. However, although the general overall charge may be similar for both nucleotides at each pH, their relative conformations may be different and that may alter their reactivity. The 7.46 pKa for HOCl may also contribute to the pH effect observed in Table 2Go. As pH did not alter the formation of adduct in the presence of DNA, it is unlikely that pH would be a factor in adduct formation in vivo.

HOCl activated ABZ to a reactive intermediate that reacted with dGp to form dGp-ABZ. This was verified by ESI/MS/MS and 1H-NMR analyses. Aromatic amines are known to form reactive intermediates that reacted with dG to form C-8 adducts. Previous studies have demonstrated the formation of dGp-ABZ adduct and its presence in animals and humans exposed to benzidine (Kennelly et al., 1984Go; Lakshmi et al., 1995Go; Martin et al., 1982Go; Rothman et al., 1996Go). This DNA adduct produces genotoxic lesions, causing mutations in various bacterial and mammalian test systems in vitro (Beland et al., 1983Go; Heflich et al., 1986Go; Melchior, Jr. et al., 1994Go) and mutations in oncogenes of tumors induced in vivo by benzidine (Fox et al., 1990Go). This suggests that dGp-ABZ could be involved in the initiation of benzidine-induced bladder cancer. A recent study has demonstrated dGp-ABZ formation following prostaglandin H synthase activation of ABZ (Lakshmi et al., 1998Go). 32P-postlabeling in combination with HPLC confirmed that both HOCl and prostaglandin H synthase produce the same dGp-ABZ adduct. This is of interest because concentrations of DMPO that completely inhibit HOCl-mediated ABZ adduct formation are not effective in preventing prostaglandin H synthase activation (Zenser et al., 1999Go). The latter involves a peroxygenation reaction, which was not observed with HOCl. Thus, although ABZ appears to be activated by two different mechanisms, the same adduct is formed. dGp-ABZ was quite stable at 70°C from pH 5.5 to 7.4, and only exhibited lability at extremely acidic or basic pHs. The stability of this adduct suggests that its recovery from biologic systems should be efficient.

The glycosidic linkage conformation of adducted DNA is an important factor in adduct persistence in vivo and may influence the biologic properties of the adduct. The two preferential conformations of the deoxynucleoside are termed anti and syn and are in rapid exchange. The relative proportion of these conformations depends upon substituents on either the sugar or the base. The chemical shift of the sugar 2' proton is a gauge of the preferred conformation of the glycosidic linkage because of the deshielding effect of the guanine N-3 atom in the syn conformation (Evans et al., 1980Go). The H-2' deoxyribose signals for dG-ABZ are found at 3.307 ppm; the shift for dG is at 2.456 ppm. This chemical shift is consistent with the preferred conformation of dG-ABZ being syn. Additional evidence that supports this postulated syn geometry comes from the ribose ring conformation, where marked downfield shifts of the OH-5' and OH-3' signals are observed. These results and conclusions are similar to that reported for N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (Turesky et al., 1992Go). The relative conformations of 2'-deoxyguanosine 3'- and 5'-monophosphates may be affected by pH in a different manner and that may be, in part, responsible for the pH effect reported in Table 2Go.

This study characterized the reaction of ABZ with HOCl to provide further insight into in vivo ABZ activation. HOCl can activate ABZ to form dGp-ABZ and may initiate adduct formation in peripheral white blood cells. Additional experiments are needed to determine whether inflammatory cells contribute to adduct formation by bladder epithelium. dGp-ABZ formation by the reaction of ABZ with HOCl provides an easy, convenient method for preparing this adduct standard.


    ACKNOWLEDGMENTS
 
This work was supported by the Department of Veterans Affairs (T.V.Z.) and National Cancer Institute grant CA72613 (T.V.Z.). Mass spectrometry was performed at the Mass Spectrometry Resource Center, Washington University School of Medicine, through NIH grants RR-00954 and AM-20579. 1H-NMR analysis was performed by Dr. Narayana Mysore, Shell Chemicals, subsidiary of Shell Oil Company, Houston, Texas. The authors wish to thank Cindee Rettke and Priscilla DeHaven for excellent technical assistance.


    NOTES
 
1 To whom correspondence should be addressed at VA Medical Center (GRECC/11G-JB), St. Louis, MO 63125–4199. Fax: (314) 894–6614. E-mail: zensertv{at}slu.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Albrich, J. M., McCarthy, C. A., and Hurst, J. K. (1981). Biological reactivity of hypochlorous acid: Implications for microbicidal mechanisms of leukocytic myeloperoxidase. Proc. Natl. Acad. Sci. USA 78, 210–214.[Abstract]

Babu, S. R., Lakshmi, V. M., Hsu, F. F., Zenser, T. V., and Davis, B. B. (1995). Glucuronidation of N-hydroxy metabolites of N-acetylbenzidine. Carcinogenesis 16, 3069–3074.[Abstract]

Barrette, W. C., Hannum, D. M., Wheeler, W. D., and Hurst, J. K. (1989). General mechanism for the bacterial toxicity of hypochlorous acid: Abolition of ATP production. Biochemistry 28, 9172–9178.[ISI][Medline]

Bartsch, H., Malaveille, C., Friesen, M., Kadlubar, F. F., and Vineis, P. (1993). Black (air-cured) and blond (flue-cured) tobacco cancer risk. IV: Molecular dosimetry studies implicate aromatic amines as bladder carcinogens. Eur. J. Cancer 29A, 1199–1207.

Bedwani, R. (1998). Schistosomiasis and the risk of bladder cancer. Br. J. Cancer 7, 1186–1189.

Bejany, D. E., Lockhart, J. L., and Rhamy, R. K. (1987). Malignant vesical tumors following spinal cord injury. J. Urol. 138, 1390–1392.[ISI][Medline]

Beland, F. A., Beranek, D. T., Dooley, K. L., Heflich, R. H., and Kadlubar, F. F. (1983). Arylamine-DNA adducts in vitro and in vivo: their role in bacterial mutagenesis and urinary bladder carcinogenesis. Environ. Health Perspect. 49, 125–134.[ISI][Medline]

Berlett, B. S., and Stadtman, E. R. (1997). Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272, 20313–20316.[Free Full Text]

Candeias, L. P., Patel, K. B., Stratford, M. R., and Wardman, P. (1993). Free hydroxyl radicals are formed on reaction between the neurophil-derived species superoxide anion and hypochlorous acid. FEBS Lett. 333, 151–153.[ISI][Medline]

Candeias, L. P., Stratford, M. R., and Wardman, P. (1994). Formation of hydroxyl radicals on reaction of hypochlorous acid with ferrocyanide, a model iron (II) complex. Free Radic. Res. 20, 241–249.[ISI][Medline]

Carr, A., van den Berg, J. J. M., and Winterbourn, C. (1996). Chlorination of cholesterol in cell membranes by hypochlorous acid. Arch. Biochem. Biophys. 332, 63–69.[ISI][Medline]

Dallinga, J. W., Pachen, D. M. F. A., Wijnhoven, S. W. P., Breedijk, A., van't Veer, L., Wigbout, G., van Zandwijk, N., Maas, L. M., van Agen, E., Kleinjans, J. C. S., and van Schooten, F.-J. (1998). The use of 4-aminobiphenyl hemoglobin adducts and aromatic DNA adducts in lymphocytes of smokers as biomarkers of exposure. Cancer Epidemiol., Biomarkers Prev. 7, 571–577.[Abstract]

Dicks, A. P., Ahmad, A. R., D'Sa, R., and McClelland R. A. (1999). Tautomers and conjugate base of the nitrenium ion derived from N-acetylbenzidine. J. Chem. Soc. Perkin II. 1–3.

El-Sebai, I. (1981). Carcinoma of urinary bladder in Egypt: current clinical experience. In Detection of Bladder Cancer Associated with Schistosomiasis (M.N.El-Bolkainy and E.W.Chu, Eds.), pp. 9–18. The National Cancer Institute, Cairo University and Al-Ahram Press, Cairo.

Evans, F. E., Miller, D. W., and Beland, F. A. (1980). Sensitivity of the conformation of deoxyguanosine to binding at the C-8 position by N-acetylated and unacetylated 2-aminofluorene. Carcinogenesis 1, 955–959.[ISI][Medline]

Fox, T. R., Schumann, A. M., Watanabe, P. G., Yano, B. L., Maher, V. M., and McCormick, J. J. (1990). Mutational analysis of the H-ras oncogene in spontaneous C57BL/6xC3H/He mouse liver tumors and tumors induced with genotoxic and nongenotoxic hepatocarcinogens. Cancer Res. 50, 4014–4019.[Abstract]

Gupta, R. C. (1985). Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen-DNA adducts. Cancer Res. 45, 5656–5662.[Abstract]

Gupta, R. C., Reddy, M. V., and Randerath, K. (1982). 32P-Postlabeling analysis of non-radioactive aromatic carcinogen-DNA adducts. Carcinogenesis (Lond.). 3, 1081–1092.[ISI][Medline]

Hazen, S. L., d'Avignon, A., Anderson, M. M., Hsu, F. F., and Heinecke, J. W. (1998). Human neutrophils employ the myeloperoxidase-hydrogen peroxide-chloride system to oxidize {alpha}-amino acids to a family of reactive aldehydes. Mechanistic studies identifying labile intermediates along the reaction pathway. J. Biol. Chem. 273, 4997–5005.[Abstract/Free Full Text]

Hazen, S. L., Hsu, F. F., Duffin, K., and Heinecke, J. W. (1996). Molecular chlorine generated by the myeloperoxidase-hydrogen peroxide-chloride system of phagocytes converts low density lipoprotein cholesterol into a family of chlorinated sterols. J. Biol. Chem. 271, 23080–23088.[Abstract/Free Full Text]

Heflich, R. H., Morris, S. M., Beranek, D. T., McGarrity, L. J., Chen, J. J., and Beland, F. A. (1986). Relationships between the DNA adducts and the mutations and sister-chromatid exchanges produced in Chinese hamster ovary cells by N-hydroxy-2-aminofluorene, N-hydroxy-N'-acetylbenzidine and 1-nitrosopyrene. Mutagenesis 1, 201–206.[Abstract]

Hsu, F.-F., Lakshmi, V., Rothman, N., Bhatnager, V. K., Hayes, R. B., Kashyap, R., Parikh, D. J., Kashyap, S. K., Turk, J., Zenser, T., and Davis, B. (1996). Determination of benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine in human urine by capillary gas chromatography/negative ion chemical ionization mass spectrometry. Anal. Biochem. 234, 183–189.[ISI][Medline]

Josephy, P. D., Eling, T. E., and Mason, R. P. (1983). Co-oxidation of benzidine by prostaglandin synthase and comparison with the action of horseradish peroxidase. J. Biol. Chem. 258, 5561–5569.[Free Full Text]

Kawai, K., Yamamoto, M., Kameyama, S., Kawamata, H., Rademaker, A., and Oyasu, R. (1993). Enhancement of rat urinary bladder tumorigenesis by lipopolysaccharide-induced inflammation. Cancer Res. 53, 5172–5175.[Abstract]

Kennelly, J. C., Beland, F. A., Kadlubar, F. F., and Martin, C. N. (1984). Binding of N-acetylbenzidine and N,N' -diacetylbenzidine to hepatic DNA of rat and hamster in vivo and in vitro. Carcinogenesis 5, 407–412.[Abstract]

Kettle, A. J., and Winterbourn, C. C. (1997). Myeloperoxidase: a key regulator of neutrophil oxidant production. Redox Rprt. 3, 3–15.

Kozumbo, W. J., Agarwal, S., and Koren, H. S. (1992). Breakage and binding of DNA by reaction products of hypochlorous acid with aniline, 1-naphthylamine, or 1-naphthol. Toxicol. Appl. Pharmacol. 115, 107–115.[ISI][Medline]

Lakshmi, V. M., Mattammal, M. B., Spry, L. A., Kadlubar, F. F., Zenser, T. V., and Davis, B. B. (1990). Metabolism and disposition of benzidine in the dog. Carcinogenesis 11, 139–144.[Abstract]

Lakshmi, V. M., Zenser, T. V., and Davis, B. B. (1998). N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine formation by peroxidative metabolism. Carcinogenesis 19, 911–917.[Abstract]

Lakshmi, V. M., Zenser, T. V., Goldman, H. D., Spencer, G. G., Gupta, R. C., Hsu, F. F., and Davis, B. B. (1995). The role of acetylation in benzidine metabolism and DNA adduct formation in dog and rat liver. Chem. Res. Toxicol. 8, 711–720.[ISI][Medline]

Liu, Z. C., and Uetrecht, J. P. (1995). Clozapine is oxidized by activated human neutrophils to a reactive nitrenium ion that irreversibly binds to the cells. J. Pharmacol. Exp. Ther. 275, 1476–1483.[Abstract]

Martin, C. N., Beland, F. A., Roth, R. W., and Kadlubar, F. F. (1982). Covalent binding of benzidine and N-acetylbenzidine to DNA at the C-8 atom of deoxyguanosine in vivo and in vitro. Cancer Res. 42, 2678–2686.[Abstract]

Melchior, W. B., Jr., Marques, M. M., and Beland, F. A. (1994). Mutations induced by aromatic amine DNA adducts in pBR322. Carcinogenesis 15, 889–899.[Abstract]

Mottley, C., and Mason, R. P. (1989). Nitroxide radical adducts in biology: Chemistry, applications, and pitfalls. Biol. Magn. Reson. 8, 489–546.

Patrianakos, C., and Hoffmann, D. (1979). Chemical studies of tobacco smoke. LXIV. On the analysis of aromatic amines in cigarette smoke. J. Anal. Chem. 3, 150–154.

Reddy, M. V., and Randerath, K. (1986). Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543–1551.[Abstract]

Ritter, C. L., and Malejka-Giganti, D. (1989). Oxidations of the carcinogen N-hydroxy-N-(2-fluorenyl)acetamide by enzymatically or chemically generated oxidants of chloride and bromide. Chem. Res. Toxicol. 2, 325–333.[ISI][Medline]

Ross, R. K., Paganini-Hill, A., and Henderson, B. E. (1988). Epidemiology of bladder cancer. In Diagnosis and Management of Genitourinary Cancer (D. G.Skinner and G. Lieskovsky, Eds.), pp. 23–31. W.B. Saunders Co., Philadelphia, PA.

Rothman, N., Bhatnagar, V. K., Hayes, R. B., Zenser, T. V., Kashyap, S. K., Butler, M. A., Bell, D. A., Lakshmi, V., Jaeger, M., Kashyap, R., Hirvonen, A., Schulte, P. A., Dosemeci, M., Hsu, F., Parikh, D. J., Davis, B. B., and Talaska, G. (1996). The impact of interindividual variation in NAT2 activity on benzidine urinary metabolites and urothelial DNA adducts in exposed workers. Proc. Natl. Acad. Sci. USA 93, 5084–5089.[Abstract/Free Full Text]

Scarborough, H. A., and Waters, W. A. (1926). The chlorination and bromination of 4-aminodiphenyl. J. Chem. Soc. 557–562.

Steinberg, D. (1997). Low density lipoprotein oxidation and its pathobiological significance. J. Biol. Chem. 272, 20963–20966.[Free Full Text]

Thomas, E. L., Grisham, M. B., and Jefferson, M. M. (1986). Preparation and characterization of chloramines. Methods Enzymol. 132, 569–585.[Medline]

Turesky, R. J., Rossi, S. C., Welti, D. H., Lay, J. O., Jr., and Kadlubar, F. F. (1992). Characterization of DNA adducts formed in vitro by reaction of N-hydroxy-2-amino-3-methylimidazo[4,5- f]quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline at the C-8 and N2 atoms of guanine. Chem. Res. Toxicol. 5, 479–490.[ISI][Medline]

Uetrecht, J. P., Ma, H. M., MacKnight, E., and McClelland, R. (1995). Oxidation of aminopyrine by hypochlorite to a reactive dication: possible implications for aminopyrine-induced agranulocytosis. Chem. Res. Toxicol. 8, 226–233.[ISI][Medline]

Whiteman, M., Jenner, A., and Halliwell, B. (1997). Hypochlorous acid-induced base modifications in isolated calf thymus DNA. Chem. Res. Toxicol. 10, 1240–1246.[ISI][Medline]

Winterbourn, C. C. (1985). Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite. Biochim. Biophys. Acta 840, 204–210.[ISI][Medline]

Wise, R. W., Zenser, T. V., and Davis, B. B. (1985). Prostaglandin H synthase oxidation of benzidine and o-dianisidine: Reduction and conjugation of activated amines by thiols. Carcinogenesis 6, 579–583.[Abstract]

Yamamoto, M., Wu, H., Momose, H., Rademaker, A., and Oyasu, R. (1992). Marked enhancement of rat urinary bladder carcinogenesis by heat-killed Escherichia coli. Cancer Res. 52, 5329–5333.[Abstract]

Yamazoe, Y., Miller, D. W., Weis, C. C., Dooley, K. L., Zenser, T. V., Beland, F. A., and Kadlubar, F. F. (1985). DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo. Carcinogenesis 6, 1379–1387.[Abstract]

Zenser, T. V., Lakshmi, V. M., Hsu, F. F., and Davis, B. B. (1999). Peroxygenase metabolism of N-acetylbenzidine by prostaglandin H synthase. J. Biol. Chem. 274, 14850–14856.[Abstract/Free Full Text]

Zhou, Q., Talaska, G., Jaeger, M., Bhatnagar, V. K., Hayes, R. B., Zenser, T. V., Kashyap, S. K., Lakshmi, V. M., Kashyap, R., Dosemeci, M., Hsu, F. F., Parikh, D. J., Davis, B., and Rothman, N. (1997). Benzidine-DNA adduct levels in human peripheral white blood cells significantly correlate with levels in exfoliated urothelial cells. Mutat. Res. 393, 199–205.[ISI][Medline]





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (5)
Disclaimer
Request Permissions
Google Scholar
Articles by Lakshmi, V. M.
Articles by Zenser, T. V.
PubMed
PubMed Citation
Articles by Lakshmi, V. M.
Articles by Zenser, T. V.