Chem. Res. Toxicol. 1992,5,431-439 study. N . Engl. J. Med. 286, 853-857. (44) Koppenol, W. H., Moreno, J. J., Pryor, W. A,, Ischiropoulos, H., and Beckman, J. S. (1992)Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide (submitted for publication). (45) Brot, N.,and Weissbach, H. (1982)The biochemistry of methionine sulfoxide residues in proteins. Trends Biochem. Sci. 7, 137-139. (46) Brot, N.,and Weissbach, H. (1983)Biochemistry and physio-
431
logical role of methionine sulfoxide residues in proteins. Arch. Biochem. Biophys. 223, 271-281. (47) Buxton, G. V., Greenstock, C. L., Helman, W. P., and Ross, A. B. (1988)Critical review of rate constanb for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals in aqueous solution. J. Phys. Chem. Ref. Data 17,513-886. (48) Nomenclature of Inorganic Chemistry (1990)Blackwell Scientific Publications, Oxford.
Mutagenic Activation of Benzidine Requires Prior Bacterial Acetylation and Subsequent Conversion by Prostaglandin H Synthase to 4-Nitro-4’-(acetylamino)biphenyl Bill J. Smith,+>’Lillian DeBruin,s P. David Josephy,§ and T h o m a s E. Eling*tt
Eicosanoid Biochemistry Section, Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O.Box 12233, Research Triangle Park, North Carolina 27709, and Guelph- Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N l G 2 W l Received January 21, 1992 We have used the Ames test in combination with prostaglandin H synthase (PHS) to study the bioactivation of benzidine as well as other aromatic amines. Previous invest*igationsestablished that the formation of benzidine mutagens by P H S is dramatically enhanced in Salmonella t y p h i m u r i u m strains with high levels of acetyl CoA-dependent arylamine N-acetyltransferase/arylhydroxylamine0-acetyltransferase activity despite the fact that acetylation of aromatic amines decreases their susceptibility to oxidation by peroxidases. In this study, we used a new strain (YG1012)that has very high acetylation capability to investigate the metabolism and mutagenicity of benzidine and N-acetylbenzidine catalyzed by PHS (from r a m seminal vesicle microsomes) and horseradish peroxidase (HRP). YG1012 bacteria rapidly acetylated benzidine to N-acetylbenzidine and N,”-diacetylbenzidine. Preincubation of the bacteria with benzidine before addition of P H S increased the mutagenicity. Under conditions identical to those used to assess mutagenicity, PHS metabolized benzidine rapidly, but the substrate was not totally consumed, with about 40% of the original concentration remaining intact. These data suggest that conversion to N-acetylbenzidine may be the initial step in the bioactivation of benzidine in the PHS-mediated Ames assay. N-Acetylbenzidine is a coeubstrate for PHS peroxidase activity as measured by 5-phenyl-4-pentenyl hydroperoxide reduction, spectral changes, and formation of protein adducts. N-Acetylbenzidine was converted to mutagens by P H S but not HRP, with enhanced mutagenicity observed in bacteria with high acetylation activity. We used reverse-phase HPLC to characterize the metabolites of N-acetylbenzidine formed by PHS and HRP. On the basis of UV/vis spectral evidence we suggest that HRP converted N-acetylbenzidine to dimers or polymers while the major product of P H S oxidations was identified as 4-nitro-4’-(acetylamino)biphenyl by cochromatography with an authentic standard and by UV/vis spectrophotometry. 4-Nitro-4’-(acetylamino)biphenylwas a direct-acting mutagen in YG1012. The results indicate that benzidine must be converted by the bacteria to N-acetylbenzidine prior to metabolism by PHS. P H S peroxidase then converts N-acetylbenzidine to 4-nitro-4’-(acetylamino)biphenyl, a potent mutagen. The data suggest that extracellular generation of free radical metabolites of benzidine does not play a role in bacterial mutagenesis.
Introduction Benzidine (4,4’-diaminobiphenyl) and several structurally related aromatic amines are recognized as human urinarv bladder 2 ) . ~~~~~~i~ amine . , . bioactivation in the urothelium may be dependent on *To whom correspondence and requests for reprints should be addressed. ‘National Institute of Environmental Health Sciences. *Presentaddress: Procter and Gamble, Co., Miami Valley Laboratories, P.O. Box 398707,Cincinnati, OH 45239. 5 University of Guelph.
peroxidative oxidation by prostaglandin H synthase (PHS)’during the reduction of peroxides (3). Benzidine is oxidized both by PHs peroxidase and horseradish peroxidase to a cation free radical and benzidine diimine Abbreviations: prostaglandin H synthase, PHS; acetyl CoA-dependent arylamine N-acetyltransferase/arylhydroxylamineO-acetyltransferase activity, NAT/OAT; ram seminal vesicle microsomes,RSVM; high-pressure liquid chromatography,HPLC; ultraviolet/visible,W/vis; 5-phenyl-4-pentenylhydroperoxide, PPHP 5-phenyl-4-pentenylalcohol, PPA; horseradish peroxidase, HRP; thin-layer chromatography, TLC; ethylenediaminetetraacetic acid, EDTA; dimethyl sulfoxide, DMSO; 2amino-3-methylimidazo[4,5-flquinoline, IQ.
This article not subject to U.S. Copyright. Published 1992 by the American Chemical Society
432 Chem. Res. Toxicol., Vol. 5, No. 3, 1992 ( 4 ) . Unstable electrophilic intermediates of benzidine produced by peroxidase metabolism have been trapped through the formation of adducts with glutathione (5), N-acetylcysteine (6),protein (7), and DNA (8). The formation of benzidine-DNA adducts is particularly important because they are the molecular lesions which lead to mutations and ultimately the induction of neoplasia (9). Benzidine was converted to mutagenic metaboliteb) by PHS as measured in the Ames assay in a number of Salmonella typhimurium strains (8, 10, 11). Further studies suggested a possible role for bacterial acetyl CoAdependent arylamine N-acetyltransferase/arylhydroxylamine O-acetyltransferase activity in the PHS-dependent mutagenicity of benzidine. In NAT/OAT-deficient TA98/ 1,B-DNP bacteria, the formation of a benzidine mutagen was not observed (8). Watanabe and co-workers have developed a series of S. typhimurium derivativeswith elevated levels of NAT/OAT (12-14). In one of these derivatives,YG1006, a dramatic enhancement of sensitivity to PHS-dependent benzidine mutagenicity was observed (11). Acetylation of aromatic amines, in general, decreases their ability to serve as a reducing substrate for peroxidases and hence their potential for oxidation by the peroxidase. The objective of these studies was to elucidate the mechanisms responsible for the dependence on bacterial acetylation in the PHS-dependent mutagenic activation of benzidine. We have selected a new S. typhimurium derivative, YG1012, which has even higher NAT/OAT than used in previous studies. We have also compared the formation of benzidine mutagens by PHS to that produced by HRP. The results of these studies indicate that a critical initial step in the PHS-dependent mutagenicity of benzidine is conversion to its N-acetyl derivative by S. typhimurium bacteria. Subsequently, N-acetylbenzidine is metabolized by the peroxidase activity of PHS to 4nitro-4'-(acetylamino)biphenyl,a direct-acting mutagen in YG1012.
Materials and Methods Chemicals. [14C]Benzidine(21.7 mCi/mmol) was obtained from Pathfinder Laboratories (St. Louis, MO). Caution: Benzidirze and its metabolites are known carcinogens. Prior to use, the compound was repurified by reverse-phase HPLC to a radiochemical purity of >99%. Methods for chromatographic purification of [14C]benzidineare described by Petry et al. (8). [ 14C]Benzidinewas diluted with unlabeled benzidine to a lower specific activity as indicated for each experiment. [ 14C]-NAcetylbenzidine was synthesized as described by Petry et al. (8) and was purified by preparative TLC (Whatman PLF5K, 20 cm x 20 cm x 1000 pm, Clifton, NJ) using benzene/acetone/ethanol (8:l:l) as the solvent. N-Acetylbenzidine was obtained from ICN Pharmaceuticals (Plainview, NY)and was repurified as described above for the radiolabeled material or by normal-phase HPLC (8). Na'-Diacetylbenzidine was obtained from Pfaltz and Bauer (Samford, CT). Phenol and hydrogen peroxide (30%) were purchased from Fisher Chemical Co. (Fairlawn, NJ). Arachidonic acid was from Nu-Check Prep (Elysian, MN). The following items were obtained from Sigma Chemical Co. (St. Louis, MO): benzidine, fatty acid free bovine serum albumin, and p-nitrobenzyl alcohol. Oxford Biomedical (Oxford, MI) was the source of 5phenyl-4-pentenyl hydroperoxide. All other chemicals and solvents were reagent grade or better. Preparation a n d Storage of Ram Seminal Vesicle Microsomes. Ram seminal vesicle microsomes are a rich source of prostaglandin H synthase activity ( 1 5 ) and were used to demonstrate PHS-dependent benzidine metabolism and mutagenicity. Ram seminal vesicles were obtained from Dr. Solomon, Case Western Reserve University (Cleveland, OH), and were stored at -70 "C. The ram seminal vesicle microsomal subcellular fraction were prepared by differential ultracentrifugation using a modification of the method of Marnett and Wilcox (15). Tween-20solubilized RSVM were prepared for use in spectrophotometric
Smith et al. studies. Following preparation, the microsome aliquots were stored at -70 "C until use. Protein concentration was determined by the method of Lowry et al. (16) using bovine serum albumin as the standard. Microsomes for use in mutagenicity assays were sterilized with y irradiation as described by Petry et al. (8). Cyclooxygenase activity was confirmed by measuring oxygen incorporation [Clark-type oxygen electrode (Gilson 5/6 Oxygraph, Middleton, WI)] into arachidonic acid (100 pM) using 0.5 mg/mL protein concentration. Typically this resulted in -40% of oxygen consumed in 30 s. Mutagenicity Assays. Mutagenicity experiments with RSVM were performed using the Ames S. typhimurium preincubation protocol as described (8,11, 17). The bacterial strains used were graciously provided by Dr. D. Bryant, McMaster University, Ontario, Canada (TA1538), and Dr. M. Watanabe, National Institutes of Hygienic Sciences, Tokyo, Japan [YG1006;TA1538/ 1,8-DNP(pYG121) and YGlOl2;TA1538/1,8-DNP(pYG213)]. TA1538/1,&DNP was isolated as previously described (11). The RSVM protein concentration was 1 mg/plate (650-pL total incubation volume). For the RSVM-mediated test (14),mutagen, RSVM, and bacteria were incubated a t 37 "C for 3 min; arachidonic acid was added, if so indicated, to give a concentration of 100 pM, and the system was incubated a further 30 min before plating. Where indicated, arachidonic acid or hydrogen peroxide was added to a final concentration of 100 pM. Determination of Bacterial N-Acetyltransferase Activity. The assay of 2-aminofluorene N-acetyltransferase activity in crude extracts of bacteria was based on the method of Hein et al. (18). Crude extracts were prepared from overnight cultures of TA1538/ 1,8-DNP, TA1538, YG1006, and YG1012. Overnight cultures (40 mL) were centrifuged and resuspended in 1 mL of sonication buffer (66 mM sodium phosphate buffer, pH 7.2, containing 1 mM EDTA, 2 mM dithiothreitol, and 50 pM phenylmethanesulfonyl fluoride). The suspensions were sonicated and centrifuged, and the supernatants (crude extract) were used in the assay. Assay incubations contained 2-aminofluorene (0.22 mM, added in 5 pL of DMSO), acetyl CoA (2.22 mM), and crude extract (65 pL), in a total volume of 90 pL. In the case of strain YG1012,5 pL of crude extract was diluted to 65 pL with sonication buffer. Incubation times were 1 h (TA1538/1,8-DNP and TA1538), 5 min (YG1006), and 3 min (YG1012). The incubations were stopped by the addition of 10% trichloroacetic acid (50 pL), and the remaining 2-aminofluorene was measured colorimetrically, as described (18). Protein concentrations of extracts were measured by the method of Peterson (19). Metabolic Experiments with Benzidine. The metabolism of [14C]benzidineby YG1012 and RSVM was performed under conditions identical to those used for mutagenicity testing. De France et al. (20) showed that S. typhimurium strain TA98 converted small amounts of benzidine to N-acetylbenzidine and N,N'-diacetylbenzidine. Therefore, we similarly measured the conversion of benzidine to acetylated derivatives by strain YG1012 using reverse-phase HPLC. To culture tubes on ice were added YG1012 culture (100 pL, AsSOnm = 1.0) and 500 pL of 100 mM sodium phosphate buffer (pH 7.4)/60 mM potassium chloride. The tubes were preincubated at 37 "C for 3 min, and the reaction was started by the addition of [14C]benzidine (50 nmol, 1.0 X lo5 dpm). At selected times after benzidine addition the reactions were terminated by filtration through C18solid-phase extraction columns (Fisher Chemical Co.). The columns were washed with water (1 mL) and eluted twice with methanol (0.5 mL). The recovery of radioactivity in the methanol eluate was >95%. A 100-pL aliquot was analyzed by reverse-phase HPLC. A Waters component HPLC system (Milford, MA) consisting of two Model 6000A pumps, a Model 721 solvent controller, a WISP 710B autosampler, and a pBondapak CL8column (3.9 mm X 300 mm) was used for all HPLC analyses. The initial solvent composition was 60% 10 mM sodium citrate (pH 6.0)/40% methanol with a 10-min linear gradient to 100% methanol at 15 min. The flow rate was 2 mL/min. The UV absorbance a t 280 nm (Spectra Physics Model 770 variable-wavelength detector, San Jose, CA) and radioactivity (Flow-one p , Packard Instruments, Chicago, IL) were used to monitor the elution of benzidine and its acetylated derivatives from the column. The retention times of benzidine, N-acetylbenzidine, and N,"-diacetylbenzidine were 7.1,10.9, and 17 min, respectively. The integrated [14C]peak area percentage
PHS and 4-Nitro-4'- (acety1amino)biphenylFormation
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 433
Table I. Bacterial Strains and N-Acetyltransferase Activity N-acetyltransferase bacterial strain activitf ref or source
determined using the method of Boyd and Eling (23). To culture tubes on ice were added RSVM (1mg/mL) in 100 mM sodium phosphate buffer (pH 7.4) and [14C]-N-acetylbenzidine(25 pM, 1 X lo6 dpm). The tubes were preincubated at 37 OC for 3 min, and the reaction was started with hydrogen peroxide (100 pM). The total volume was 1.0 mL. At selected times the reaction was terminated and processed as described (23). The extent of protein binding at time zero was determined after the 3-min preincubation. The metabolites of [ 14C]-N-acetylbenzidinecatalyzed by PHS were analyzed by HPLC. The reactions were identical to those described above for protein arylation with the exception that the N-acetylbenzidine concentration was 50 pM. Separate reactions were also performed with horseradish peroxidase (1 pg/mL) and hydrogen peroxide (100 pM). The reaction was terminated after 5 min using solid-phase extraction. The columns were washed with 1mL of water and eluted with 1mL of methanol, and then the eluant was evaporated under argon and reconstituted with 200 pL of methanol. One hundred microliters was analyzed using the HPLC system described above with a Waters 990 diode array UV/vis detector (Milford, MA) in tandem with a radioflow detector. The initial solvent conditions were 5% citrate buffer/95% methanol with a linear gradient to 40% citrate buffer/60% methanol at 12.5 min followed by a second linear gradient to a final composition of 10% citrate buffer/90% methanol at 30 min. The retention time of N-acetylbenzidine was 15 min in this system. Synthesis a n d Characterization of 4-Nitro-4'-(acetylamino)biphenyl. 4-Nitro-4'-(acetylamino)biphenyl was synthesized by oxidizing N-acetylbenzidine with m-chloroperoxybenzoic acid using the method of Martin et al. (24). The UV/vis spectrum of the yellow product in alxsolute ethanol showed a broad absorbance band from 280 to 400 nm with a maximum at 327 nm. The product was further analyzed by mass spectrometry on a Kratos CONCEPT I SQ double-focusing high-resolution mass spectrometer (Manchester, England). The sample was analyzed in the electron impact mode using a fast-response direct probe (IGT, Munich, Germany) fitted with an aluminum sample cup. The probe was programmed from 40 to 300 "C at 1 OC/s. The data were analyzed on a Sun 3/60 Workstation (Mountain View, CA) using Mach 3 software (Kratos Analytical). Three major ions were observed 256 m/z (M+), 214 m / z (M+ - C,H,O), and 168 m / z (214 m / z - NOJ. These ions are consistent with the structure of 4-nitro-4'-(acetylamino)biphenyl. The crude material was repurified by reverse-phase HPLC to a purity of >99% using the system described above in the study of N-acetylbenzidine metabolism. The highly purified 4-nitro-4'-(acetylamino)biphenyl was subsequently used for mutagenicity studies.
TA1538/ 1.8-DNP TA1538' ' YGlW,TA1538/ 1,8-DNP(pYG121) YG1012;TA1538/ 1,8-DNP(pYG213)
0 0 6.5 259
12
B. N. Ames 12 12
In units of nmol of 2-aminofluorene acetylated/(min.mg of crude extract protein). was used to calculate the product yield (nmol). The PHS-dependent metabolism of [l%]benzidine was assessed by determining the disappearance of parent compound by reverse-phase HPLC. To culture tubes on ice were added sodium phosphate/KCl buffer (500 pL), RSVM (1 mg of protein), and [14C]benzidine(50 nmol, 1 X lo6 dpm). The reactions were preincubated for 3 min at 37 "C prior to the addition of hydrogen peroxide (100 pM) or arachidonic acid (100 pM) where indicated. The total volume was 650 pL. At selected times 100-pL aliquots were withdrawn and the reaction was terminated by filtration through CISsolid-phase extraction columns. The samples were eluted and analyzed by HPLC as described above. Assessment of N-Acetylbenzidine Metabolism. The ability of N-acetylbenzidine to serve as a reducing cosubstrate for PHS was assessed by determining the extent of reduction of 5phenyl-Cpentenyl hydroperoxide to 5-phenyl-4-pentenylalcohol as described by Weller et al. (21) and modified by Petry and Eling (22). RSVM (0.175 mg/mL) in 100 mM sodium phosphate buffer (pH 7.4) and the cosubstrate (200 pM) were added to culture tubes on ice. Reactions (performed in triplicate) contained either N-acetylbenzidine, phenol, or no cosubstrate (control). The tubes were preincubated for 3 min at 37 "C, and the reaction was started by the addition of 5-phenyl-4-pentenyl hydroperoxide (100 pM). The total volume was 1.0 mL. The reaction was terminated 3 min after &phenyl-4pentenyl hydroperoxide addition by fdtration through C18solid-phaseextraction columns. 5-Phenyl-4-pentenyl hydroperoxide and PPA were eluted with methanol (1.0 mL), p-nitrobenzyl alcohol was added as the internal standard, and 80 pL was analyzed by HPLC with UV detection at 254 nm. All other procedures used were as described (22). Changes in the UV/vis spectra which occurred during Nacetylbenzidine oxidation by solubilized RSVM were recorded using a Hewlett Packard 8450A diode array spectrophotometer equipped with a magnetic stirring temperature-controlled cuvette holder. Solubilized RSVM (0.5 mg/mL) in 100 mM sodium phosphate buffer (pH 7.4) were combined with N-acetylbenzidine (25 pM) and preincubated for 3 min at 37 OC. The reaction was started by the addition of hydrogen peroxide (100 pM) or arachidonic acid (100 pM). Spectra from 250 to 800 nm were recorded every 2 s for 1min. Control experiments showed that the spectral changes observed were dependent on hydrogen peroxide or arachidonic acid, active PHS, and N-acetylbenzidine. The ability of PHS to catalyze the bioactivation of ['*C]-Nacetylbenzidine to a species capable of protein arylation was
Results PHS-Dependent Mutagenicity of Benzidine in Bacterial Strain YG1012. Previous studies demonstrated that the PHS-dependent mutagenicity of benzidine was enhanced in S. typhimurium strain YG1006 (II), a strain with elevated NAT/OAT (12,13).Shown in Table I is a comparison of the N-acetyltransferase activity (using 2-aminofluorene as the substrate) in four isogenic derivatives of TA1538. YG1012 expresses even higher levels of
Table 11. Mutagenic Activation of Benzidine by Horseradish Peroxidase and Prostaglandin H Synthase in S.typhimurium Strain YG1012" revertants/plate dose direct RSVM + AA RSVM + H202 H202 HRP + H202 acting (nmol) RSVM (100 PM) (77 rM) HRP (77 rM) (100rM) 54 f 7 50 f 6 51 f 6 59 f 13 49 f 9 61 f 12 0 58 f 8 10 20 30 50 100 500 lo00
58 f 7
ndb nd 68 f 8 98 f 17 175 f 32 231 f 23
598 f 59 1411 f 247 1735 f 505 2236 f 490
toxic nd nd
552 f 64 1779 f 111 2242 f 322 2226 f 239
toxic nd nd
705 f 41 1670 f 169 2243 f 206 2491 f 219
toxic nd nd
59 f 4
78 f 22
73 f 10
nd nd
nd nd
nd nd
75 90 181 275
f5 f 13 f 43
f 42
158 f 15 248 f 37 463 f 47 702 f 55
170 f 27 292 f 48 513 f 104 719 f 80
a Benzidine, bacteria, and horseradish peroxidase (HRP: 10 pg/plate) or ram seminal vesicle microsomes (RSVM: 1 mg of microsomal protein/plate) were incubated at 37 "C for 3 min prior to the addition of arachidonic acid (AA) or hydrogen peroxide (H202).This was followed by a 30-min incubation prior to plating. Data are expressed as the mean f standard deviation (n = 9). *nd = not determined.
434
Smith et al.
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 1700 H Benzidine E
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* Acetylbenzidine A Diacelylbenzidine
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1500
1400
al
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1300 0
15
30
45
60
75
90
,
105
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Figure 1. Time course of benzidine acetylation by YG1012. [14C]Benzidine(50 nmol/incubation) was incubated with YG1012 in 100 mM sodium phosphate/W mM potassium chloride buffer (pH 7.4) at 37 O C for 0,5, 10,15,30,60,and 120 min. Each point represents the mean f standard deviation (n = 3; except 15 min, n = 2).
acetyltransferase activity compared with YG1006. As shown in Table 11, YG1012 bacteria were extremely sensitive to the mutagenic effecta of benzidine in the presence of PHS. A dose-response in the number of revertants/ plate was observed between 0 and 30 nmol of benzidine/ plate, with a maximal mutagenic response at 30-50 nmol of benzidine/plate. Above 50 nmol of benzidinejplate, toxicity was observed. There were no dramatic differences in the mutagenicity of benzidine in the presence or absence of either hydrogen peroxide or arachidonic acid. This is not surprising, in light of previous studies which suggest that S. typhimurium bacteria produce enough hydrogen peroxide to support the PHS-dependent mutagenicity of benzidine (21). Therefore, PHS peroxidase-dependent activation can continue to occur over a time course of many minutes, driven by the continuing metabolic production of H202by the respiring bacteria. However, it should be emphasized that benzidine is not mutagenic unless the peroxidase activity of PHS remains active (8,11). We also examined the mutagenicity of benzidine following activation by horseradish peroxidase (HRP), a commonly used model to study the peroxidative metabolism of aromatic amines. In the presence of HRP a slight enhancement in benzidine mutagenicity was observed compared to benzidine alone or benzidine with hydrogen peroxide. However, in YG1012, HRP could not produce mutagenicity as high as observed with PHS, even at doses of benzidine 20-fold greater. This suggests that differences exist between PHS and HRP in the mutagenic pathway for benzidine activation or in the extent of conversion of benzidine to mutagenic products. Time-Dependent Acetylation of Benzidine to N Acetylbenzidine by YG1012. One possible role for the bacterial acetyltransferase in the enhancement of PHSdependent benzidine mutagenicity could be the conversion of benzidine to N-acetylbenzidine. This was assessed by incubating YG1012 bacteria with [ 14C]benzidinefor selected times and determining the extent of conversion to [14C]-N-acetylbenzidineby HPLC under the same conditions as the mutagenicity assay. A dose of 50 nmol of benzidine /incubation was selected because it produced a maximal mutagenic response in the presence of PHS. The time course of [14C]benzidineN-acetylation of YG1012 is shown in Figure 1. YG1012 bacteria rapidly N-acetylated benzidine as indicated by the loss of benzidine and the appearance of N-acetylbenzidine. The concentration of N-acetylbenzidine was maximal at 60 min and declined slightly thereafter. The N-acetylation of both amine
1 zoo
0 0
15
30
45
60
Preincubation Time (min)
Figure 2. Enhancement of PHS-dependent benzidine mutagenicity with extended preincubation of benzidine and YG1012. Bacteria (0.1 mL) were added to benzidine (20 nmol) in 0.5 mL of buffer (0.1 M phosphate/bO mM KCl) and incubated at 37 "C. Ram seminal vesicle microsomes (1mg of microsomal protein) were added at the indicated times, and the samples were incubated for an additional 30 min prior to plating. Only background mutagenicity was observed in the absence of RSVM. Each point represents the mean f standard deviation (n = 3). groups of benzidine produced N,"-diacetylbenzidine, which accumulated at a slower rate and was not detectable until after 15 min of incubation. The rapid acetylation of benzidine by YG1012 is important for two reasons. First, in many strains the PHS-dependent mutagenicity of Nacetylbenzidine is greater than that of benzidine (8). Second, during the mutagenicity test, the bacteria, mutagen, and PHS are incubated for 30 min before plating, allowing sufficient time for substantial benzidine Nacetylation. Preincubation of YG1012 Bacteria with Benzidine. In order to determine the importance of benzidine acetylation in the mutagenic response of YG1012, bacteria and benzidine were preincubated for selected times prior to the addition of RSVM. This procedure would allow time for benzidine N-acetylation by YG1012 prior to the addition of RSVM. The results in Figure 2 show that the mutagenicity of benzidine was enhanced by preincubating the bacteria with benzidine. Mutagenicity only occurred in incubations which received RSVM after the preincubation (data not shown). In light of these results and those presented in Figure 1,this enhancement is probably due to bacterial metabolism of benzidine to N-acetylbenzidine. Incomplete Benzidine Metabolism by PHS Peroxidase. Since benzidine is one of the best cosubstrates for the peroxidase of PHS (25),it was important to determine the amount of benzidine remaining after PHS oxidation. The results are shown in Figure 3. In reactions with RSVM only, no loss of benzidine was observed over the 30-min incubation period. However, when hydrogen peroxide or arachidonic acid was added, the initial amount of benzidine was reduced from 50 nmol/incubation to approximately 20 nmol/incubation in 1min, and no further oxidation occurred during the remainder of the 30-min incubation period. This is consistent with the rapid self-catalyzed inactivation of PHS which occurs during arachidonic acid or peroxide turnover (26). The loss of benzidine from the incubations represents the formation of protein adducts and polymeric species, characteristic of the formation of free radical intermediates during peroxidative oxidation by PHS (4). These results indicate
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 435
PHS and 4-Nitro-4'- (acety1amino)biphenylFormation
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e a RSYMIAA RSvM Only
A
10
0
5
0
10
1
I
I
I
15
20
25
30
Incubation Time (min)
Figure 3. Disappearance of benzidine during PHS-mediated oxidation. [14C]Benzidine(50 nmol/incubation) was incubated with RSVM (1mg/incubation) in 100 mM sodium phosphate/60 mM potassium chloride buffer (pH 7.4) at 37 O C . The reactions were preincubated for 3 min prior to the addition of 100 pM of either arachidonic acid (AA) or hydrogen peroxide (H,O,) where indicated. Aliquots were withdrawn and analyzed for benzidine by HPLC at 0, 1,5, and 30 min. Each point represents the mean f standard deviation (n = 3).
2000
1500 1000 500UI
a
200015001000-
I 0
25
50
75
100 0
25
50
,
75
,I
100
MutageniPlate (nmol)
Figure 4. Comparison of the PHS-dependentmutagenicity of benzidine and N-acetylbenzidine in TA1538 derivatives with different acetyltransferase levels. No direct-actingmutagenicity was observed with either compound except in YG1012 where N-acetylbenzidine (100nmol/plate) produced 314 f 140 revertants/plate. In the absence of mutagen and enzyme the background mutagenicity in YG1012 was 63 f 6 revertanta/plate. Each point represents the mean f standard deviation (n = 6-9). = benzidine; A = N-acetylbenzidine.
that in the presence of hydrogen peroxide or arachidonic acid the PHS-dependent oxidation of benzidine is rapid but incomplete. Enough unmetabolized benzidine would remain to allow for bacterial formation of N-acetylbenzidine. Mutagenicity of Benzidine and N-Acetylbenzidine in Isogenic Derivatives of TA1538 with Differing Acetylation Capability. The PHS-dependent mutagenicities of benzidine and N-acetylbenzidine were compared in selected isogenic derivatives of TA1538 with differing acetylation capability. The results in Table I showed that the rank order of N-acetyltransferase activity was YG1012 >> YG1006 > TA1538 = TA1538/1,8-DNP. [Using isoniazid as a substrate for bacterial N-acetylation, other investigators have shown that NAT/OAT is present in TA1538 and undetectable in TA1538/1,8-DNP (12).] These four TA1538 derivatives were used to compare the PHS-dependent mutagenicity of benzidine and Nacetylbenzidine, and the results are shown in Figure 4.
N-Acetylbenzidine log (1 + nmollplate)
Figure 5. Comparison of N-acetylbenzidine mutagenicity catalyzed by PHS and HRP. N-Acetylbenzidine, bacteria, and horseradish peroxidase (HRP: 10 pg/plate) were incubated at 37 O C for 30 min prior to plating (n = 6/point). The open circles represent direct-actingmutagenicity of N-acetylbenzidine. The filled triangles represent HRP-mediated N-acetylbenzidine mutagenicity. Addition of hydrogen peroxide (77 rM) to incubations with N-acetylbenzidine in the presence or absence of HRP gave results which were essentially superimposable and hence are not shown. The filled circles represent PHS-mediated (RSVM) N-acetylbenzidinemutagenicity. The data for PHS were replotted from Figure 4 for comparison.
The PHS-dependent mutagenicity of both benzidine and N-acetylbenzidine increased with increasing acetylation capability of the four strains. The greatest mutagenicity of these two compounds was observed in YG1012, the derivative with the greatest N-acetyltransferase activity. The response was greater for benzidine than N-acetylbenzidine in YG1012. At first glance this is surprising; however, N-acetylbenzidine is a substrate for immediate conversion to N,"-diacetylbenzidine by YG1012. This may represent a detoxication reaction, as it reduces the amount of N-acetylbenzidine available for PHS activation. In the case of incubations with benzidine, N-acetylbenzidine is formed by the bacterial NAT activity. At least at early times, during the incubation, benzidine remains the preferred substrate for acetylation, rather than Nacetylbenzidine, due to mass action. Thus, the presence of benzidine may prolong the exposure of the bacteria to the metabolically generated N-acetylbenzidine. Further, kinetic analysis of this situation would require characterization of the enzyme kinetic constants for benzidine and N-acetylbenzidine acetylation, which is beyond the scope of the present investigation. The enhanced PHSdependent mutagenicity of N-acetylbenzidine in bacteria with elevated NAT/OAT suggests that following PHS activation additional acetylation reactions (for example, 0-acetylation) occur to form the ultimate mutagen. Lack of Mutagenicity of N-Acetylbenzidine with HRP Activation. We were also interested in comparing the mutagenicity of N-acetylbenzidine following HRP activation. The results of this study are shown in Figure 5. HRP in the presence or absence of hydrogen peroxide did not enhance the mutagenicity of N-acetylbenzidine. An unexpected fmding in this experiment was a significant direct-acting mutagenic effect of N-acetylbenzidine in YG1012. We also performed additional studies where the concentrations of HRP and peroxide were varied, but in no case could we detect HRP-dependent mutagen forma-
Smith et al.
436 Chem. Res. Toxicol., Vol. 5, No. 3, 1992 tion (data not shown). This suggests that, as with benzidine, N-acetylbenzidine is converted to mutagenic products following activation by PHS but not HRP. Evidence for PHS-Dependent Metabolism of N Acetylbenzidine. Thus far, the studies have focused on the conversion of benzidine to N-acetylbenzidine as a requirement for the PHS-dependent mutagenicity of benzidine. The peroxidase activity of PHS is required to support the mutagenicity of N-acetylbenzidine (8),but acetylation of benzidine dramatically reduces the ability to serve as a substrate for the peroxidase. Therefore, it was important to determine whether N-acetylbenzidine was a cosubstrate for the peroxidase. A convenient assay used to rank PHS cosubstrates is their ability to support the reduction of 5-phenyl-4-pentenyl hydroperoxide (PPHP) to 5-phenyl-4-pentenyl alcohol (PPA) (21). This assay was used to determine whether N-acetylbenzidine was a cosubstrate for PHS. A reduction index of 0.5 indicates that 50% of PPHP was converted to PPA. Initially, the RSVM concentrations were varied to determine the amount of enzyme needed to convert 50% of PPHP to PPA in the presence of phenol (200 pM).Under these conditions, the reduction index of N-acetylbenzidine was 0.30 f 0.02. This is only slightly greater than the reduction index in the absence of a cosubstrate (0.22 f 0.01) and shows that N-acetylbenzidine is a poor cosubstrate for the peroxidase activity of PHS. Nevertheless, it suggests that a small amount of Nacetylbenzidine is metabolized by PHS during the reduction of peroxides. Changes in the UV/vis spectra of N-acetylbenzidine were used to estimate PHS-dependent metabolism. The spectrophotometric changes observed were dependent on PHS, hydrogen peroxide or arachidonic acid, and Nacetylbenzidine and also suggested that PHS can metabolize N-acetylbenzidine. Representative spectrophotometric changes which occurred during the oxidation of N-acetylbenzidine by the PHS/hydrogen peroxide system are shown in Figure 6A. The decrease in absorbance at 290 nm represents loss of parent compound during the reaction. The increase in absorbance from 320 to 400 nm probably represents the formation of metaboliteb) or protein adducts. The metabolism of N-acetylbenzidine by PHS was also assessed by measuring the formation of covalent adducts to RSVM protein. Covalent binding of N-acetylbenzidine to RSVM protein was nearly maximal 1 min after the addition of hydrogen peroxide (Figure 6B). Only a slight increase was observed in protein adduct formation from 1 to 30 min. These results suggest not only that Nacetylbenzidine is metabolized by PHS but also that reactive intermediates are produced in the process. This is in agreement with the resulta of Petry et al. which showed that PHS catalyzes the covalent binding of N-acetylbenzidine to DNA in vitro (8). HPLC Analysis of N -Acetylbenzidine Metabolism by Peroxidases. The studies with PPHP reduction, UV/vis spectral changes, and protein adduct formation suggested that PHS could metabolize N-acetylbenzidine despite the fact that N-acetylbenzidine is a poor reducing cosubstrate. Therefore, we investigated the nature of the stable metabolites of N-acetylbenzidine formed by PHS as well as HRP oxidation. The objective of these studies was to search for stable mutagenic metabolite(s). Since N-acetylbenzidine is not mutagenic in YG1012 when activated by HRP, we compared the HPLC metabolite profile of HRP-catalyzed or PHS-catalyzed N-acetylbenzidine oxidation to search for mutagenic metabolites
A
250
300
450
400
350
WAVELENGTH (nm)
B 4 ,
0
0
”
5
”
10
”
15
~
M
25
30
Incubation time iminl
Figure 6. Oxidation of N-acetylbenzidine by PHS: Evidence from spectrophotometric and protein binding studies. (A) Solubilized RSVM (0.5 mg/mL) and N-acetylbenzidine(25 pM) in 100 mM sodium phosphate (pH 7.4) were preincubated for 3 min at 37 OC in a stirred cuvette. Changes in absorbancewere recorded from 250 to 800 nm every 2 s. Spectra were recorded for 10 s before adding hydrogen peroxide (100 pM). The spectral changes shown were identical if arachidonic acid was substituted for hydrogen peroxide. Spectral changes were dependent on active enzyme, arachidonic acid or hydrogen peroxide, and N-acetylbenzidine. (B)PHS-dependent covalent binding of [“CI-Nacetylbenzidineto ram seminal vesicle microsomalprotein. RSVM (1 mg) and [l4C]-N-acety1benzidine(25 pM) in 100 mM sodium phosphate (pH 7.4) were preincubated for 3 min at 37 OC (total volume 1.0 mL), after which the time zero samples were removed. The reaction was initiated with hydrogen peroxide (100 pM) and terminated after 1,5,10, and 30 min. Each point representsthe mean standard deviation ( n = 3).
*
unique to the PHS system. The HPLC profiles of radiolabeled N-acetylbenzidines metabolites formed by HRP or PHS are shown in Figure 7. Both peroxidases oxidized N-acetylbenzidine to one major and one minor metabolite. Metabolite I1 for both peroxidases eluted at the same retention time and had the same UV/vis spectrum as shown in the inserts to Figure 7. This spectrum was characteristic of an azo dimer, but the metabolite was not further characterized. The major HRP metabolite had a UV/vis spectrum characteristic of a dimeric coupling product of N-acetylbenzidine but was not further characterized. In the PHS system a radiolabeled metabolite was observed which eluted at 19 min (PHS metabolite I) and was not observed in the HRP system. This metabolite cochromatographed with chemically synthesized 4-nitro4’-(acety1amino)biphenyl and had an identical UV/vis absorbance spectrum as determined using the incline photodiode array detector, We concluded that PHS metabolite I was 4-nitro-4’-(acetylamino)biphenyl.
PHS and 4-Nitro-4’-(acetylamino)biphenyl Formation
Chem. Res. Toxicol., Vol. 5, No.3, 1992 437
o “
j i, 00
T
, 05
,
,
,
,
,
10
15
20
25
30
nmoliPlate
Figure 8. Mutagenicity of 4-nitro-4’-(acetylamino)biphenylin YG1012. 4-Nitro-4’-(acetylamino)biphenylwas added t o bacteria
HRP
(0.1 mL) in 0.5 mL buffer (0.1 M phosphate/60 mM KCl), incubated for 30 min, and plated. Each point represents the mean i standard deviation (n = 3).
II
I
I
”m
\
Elution Time (min)
Figure 7. Comparison of the reverse-phase HPLC metabolite profile of PHS- and HRP-catalyzed [14C]-N-acetylbenzidineoxidation. RSVM (1mg) or HRP (1pg) and [‘%]-N-acetylbenzidine (50 pM) in 100 mM sodium phosphate (pH 7.4) was preincubated for 3 min at 37 “C (totalvolume 1.0 mL) and the reaction initiated with hydrogen peroxide (100 pM). After 5 min, reactions were terminated and analyzed by HPLC as described in Materials and Methods. Metabolites were not observed with PHS or HRP in the absence of hydrogen peroxide. Substitution of arachidonic acid (100 pM) for hydrogen peroxide with PHS gave similar results.
Mutagenicity of 4-Nitro-4’-(acetylamino)biphenyl in YG1012. Since 4-nitro-4’-(acetylamino)biphenyl was detected as a PHS-dependent metabolite of N-acetylbenzidine, it was tested for direct-acting mutagenicity in YG1012, and the results are shown in Figure 8. 4Nitro-4’-(acetylamino)biphenyl was mutagenic when tested at doses from 0.1 to 3.0 nmol/plate. The specific mutagenicity at doses of 1and 3 nmol/plate was approximately 300 revertants/nmol. We have estimated the quantity of 4-nitro-4’-(acetylamino) biphenyl formed by PHS under identical condition used for mutagenicity. HPLC analyses indicated that approximately 3-3.5 nmol of 4-nitro-4’(acety1amino)biphenyl was formed from 50 pM acetylbenzidine. Thus, it appears that PHS-dependent oxidation of N-acetylbenzidine produces 4-nitro-4’-(acetylamino)biphenyl, which is a stable direct-acting mutagen in YG1012 and most likely is the major mutagenic metabolite of N-acetylbenzidine. The formation of 4-nitro-4’-(acetylamino)biphenyl may also account for the PHS-dependent mutagenicity of benzidine itself, but we cannot exclude the possibility that other mutagenic pathways may also occur. Discussion Robertson et al. (10)reported that PHS can activate benzidine and other aromatic amines to mutagenic species
in the Ames assay (strain TA98). In subsequent studies with sensitive tester strains which produce high levels of NAT/OAT, we have shown that this activation requires PHS peroxidase activity, but does not require exogenous PHS substrate (arachidonic acid), since S. typhimurium bacteria produce low levels of hydrogen peroxide (8). Both PHS peroxide activity and HRP readily metabolize benzidine by one-electron oxidation to reactive free radical and diimine products (27). Other aromatic amines are also oxidized to radical intermediates by peroxidases (26). At the onset of our studies, we anticipated that such reactive species would prove to be responsible for the PHS-catalyzed mutagenicity of benzidine. However, this hypothesis was not consistent with the observed correlation between tester strain sensitivity to PHS-dependent benzidine mutagenicity and bacterial NAT/OAT activity (8, 1I). If the benzidine radical or diimine were the mutagenic species, there would be no apparent role for acetylation in mutagenic activation. Indeed, since N-acetylbenzidine is a much poorer peroxidase reducing substrate than is benzidine, acetylation should simply divert substrate away from the activation pathway. Synthetic benzidine diimine, the product of peroxidative metabolism of benzidine, is not mutagenic in strains TA98 (27) or YG1012.2 Furthermore, HRP cannot activate benzidine to a mutagenic species, despite facile substrate oxidation (28). Our present results suggest an explanation for these findings. The critical activation step is PHS-dependent oxidation of N-acetylbenzidine, produced by bacterial metabolism of benzidine. PHS-dependent benzidine oxidation is unrelated to the mutagenic activation process. As outlined in Figure 9, PHS-dependent oxidation of N-acetylbenzidine (but not HRP-dependent oxidation) generates 4nitre4’-(acetylamino) biphenyl, which is further activated, presumably, by bacterial nitroreduction and O-acetylation. In contrast, HRP metabolizes N-acetylbenzidine to dimers and polymers derived from free radical intermediates. However, when formed extracellularly, these free radical intermediates of N-acetylbenzidine are not mutagenic to S. typhimurium. Thus, in the case of N-acetylbenzidine, PHS but not HRP has the ability to metabolize the arylamine to a nitro derivative which is a direct-acting mutagen. In the model shown in Figure 9 we propose that 4-nitro-4’-(acety1amino)biphenylis further metabolized in P. D. Josephy, unpublished data.
438 Chem. Res. Toxicol., Vol. 5, No. 3, 1992
-1 PHS
H2NW -
1
Benzidine Cation Free Radical Benzidine Diimine UnidentifiedProducts
1-
Smith et al. Polymer Products
PHS
0
n
+
Bacterial Nitroreductase
\ Bacterial Cell Figure 9. Schematic representation of the proposed PHS-dependent bioactivation pathway of benzidine in S. typhimurium.
bacteria by nitroreductase to N-hydroxy-”-acetylbenzidine followed by conversion by OAT to the N-acetoxy ester intermediate (29). This unstable intermediate is probably responsible for the genotoxic reactions with DNA which lead to mutagenesis. Evidence to support this conclusion was the observation that the mutagenic response of bacteria to N-acetylbenzidine activated by PHS increased along with the NAT/OAT activity of the tester strain. Furthermore, there is evidence that N-acetoxy ester intermediates of aromatic amines are potent mutagens (30). The N-acetoxy ester is unstable and would be expected to undergo spontaneous decomposition to the nitrenium ion which could react with DNA and account for mutagenesis in S. typhimurium. There is increasing evidence in the Ames assay that the mutagenic activation of several arylamine compounds by PHS is mediated by conversion to nitro derivatives or oxygenated metabolites. In addition to N-acetylbenzidine, 2-aminofluorene and 2-amino-3-methylimidazo[4,5-flquinoline (IQ) are also converted by PHS peroxidase to nitro metabolites (31). Similar to 4-nitro-4’-(acetylamino)biphenyl, 2-nitrofluorene and nitro-IQ are stable direct-acting mutagens in S. typhimurium (32, 33). Moreover, the PHS-dependent mutagenicity of 2-aminofluorene, I&, and N-acetylbenzidine are all dependent on the level of bacterial NAT/OAT present in the test strain (8, 11, 34, 35). This is consistent with a role of OAT to convert the N-hydroxy compounds to N-acetoxy esters following reduction of the nitro metabolites by bacterial nitroreductase. Bacterial nitroreductase activity has been demonstrated with numerous substrates (14).However, the presence or properties of analogous enzymes in target tissues for benzidine carcinogenesis is not as well characterized; l-nitropyrene reductase activity may be inferred from the mutagenicity and DNA adduct formation by l-nitropyrene in Chinese hamster ovary cells (36). The importance of mutagenic nitro metabolites of aromatic
amines, formed by PHS, has been established by this and other studies using the Ames assay; the possible significance of such activation in mammalian target tissues such as the bladder requires further study. HRP has long been used as a model for PHS peroxidase to study the metabolism of aromatic amines to reactive metabolites. The metabolites of aromatic amines formed through conversion to free radical metabolites are similar with PHS and HRP. However, our data indicate that HRP and PHS can also form different metabolites of aromatic amines. A clear difference exists between HRP and PHS in the conversion of aromatic amines to nitro derivatives or oxygenated metabolites. The mechanism of PHS-dependent conversion of aromatic amines to nitro metabolites is unknown; however, it appears to occur only with amines that are poor cosubstrates for the peroxidase (25). PHS-dependent benzidine mutagenicity depends upon a multistep pathway of‘metabolic activation and requires both PHS and the bacterial NAT/OAT enzyme. The facile oxidation of benzidine to free radical metabolites, which we and others have explored previously, appears to be irrelevant to the process of PHS-dependent mutagenic activation. On the other hand, the endogenous bacterial peroxidase, hydroperoxidase I, can catalyze mutagenic activation of aromatic amines (37). Apparently, the radical metabolites of aromatic amines, while electrophilic and reactive with macromolecules, are too short-lived to reach the genome and cause bacterial mutations in the Ames away, unless they are formed within the bacterial cell itself. Extracellular formation of these species, or direct treatment of bacteria with the synthetic diimine, does not lead to mutation. 4-Nitro-4’-(acetylamino)biphenylis a stable metabolite formed in the N-acetylbenzidinelPHS system and is mutagenic to S. typhimurium, presumably following metabolism by bacterial nitroreductase and NAT/OAT, as has been observed for other nitroaromatic compounds.
PHS and 4-Nitro-4‘- (acety1amino)biphenylFormation
Acknowledgment. Research in P.D.J.’s laboratory is supported by a grant from the National Cancer Institute of Canada. We thank Mr. Steve McGown for conducting the mass spectral analysis.
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