Horseradish Peroxidase/Hydrogen Peroxide-Catalyzed Oxidation of the Carcinogen N-Hydroxy-N-Acetyl-2-Aminofluorene As Effected by Cyanide and Ascorbate1

[CANCER RESEARCH 36, 1510-1519, April 1976]

Horseradish Peroxidase/Hydrogen Peroxide-catalyzed Oxidation of the Carcinogen N-Hydroxy-N-acetyl-2-aminofluorene as Effected by Cyanide and Ascorbate1

Robert A. Floyd,2 Lailing M. Soong, and Peggy L. Culver

Oklahoma Medical Research Foundation, Biomembrane Research Laboratory, Oklahoma City, Oklahoma 73104

SUMMARY these tissues. The initial observations of Fonnestenet a!. (8) and Bartsch et a!. (4) laid the foundation for this possibility. Horseradish penoxidase and H202 mediate N-hydmoxy-N Independently, they observed that, in an organic solvent acetyl-2-aminofluorene (N-OH-AAF) conversion into two system, 1-electron oxidants oxidized N-hydmoxyalkylarylam more potent carcinogens, 2-nitrosofluonene and N-acetoxy me carcinogens into the nitmoxyl free radical form of the N-acetyl-2-aminofluomene. Optical studies of this system in carcinogen which then dismutated, presumably (4), into 2 dicate that horseradish pemoxidase is operating as a pemoxi observed products, the nitrosoamene and N-alkoxylalkylary dase with N-OH-AAF as the electron donor. Our studies lamine. Bartsch et a!. (2, 3) extended this observation to a confirm the earlier finding that 2-nitrosofluomene and N- more biologically relevant system by demonstrating that the acetoxy-N-acetyl-2-aminofluorene are the products of the penoxidases, HAP, myelopenoxidase, and lactopemoxidase, type II enzyme-mediated oxidation of N-OH-AAF, but sum were capable of activating N-OH-AAF into NOF and N- pnisingly, the results with the type VI enzyme indicate that OAC-AAF. The following mechanism was postulated to more 2-nitrosofluorene was formed and, in addition, an explainthe results:N-OH-AAF acted as the 1-electron other product absorbing at 245 nm was formed. If ascombate donor in the normal cycling of H2O2-oxidized pemoxidase, is present in the N-OH-AAF/honsemadish peroxidase/H202 thus forming the nitroxyl radical of the carcinogen, which system, ascorbate is oxidized preferentially. Cyanide, a complexed with another similar radical and then dismutated known inhibitor of the pemoxidase, does not inhibit when into the nitmoso and acetoxy ester carcinogens (2). N-OH-AAF is the electron donor. The reaction products Very little is known concerning the action of N-OH-AAF are the same in the presence or absence of cyanide. in the HRP/H202 system. In fact, the carcinogen was pro posed to act as an electron donor to the cycling peroxidase only on the basis of the observed electron spin resonance INTRODUCTION spectra of the nitroxyl radical of the carcinogen in a steady state system containing a high excess of H2O2(2). The lack The acetylarylamine carcinogens are activated to more of understanding of the system is underscored by the recent potent carcinogens by microsomal hydnoxylation of the observation of King et a!. (13) that the HAP/N-OH-AAF/ amine nitrogen, termed N-hydmoxylation (6, 17, 18, 21). Fol H2O2system continued to produce N-OAC-AAF in the pres lowing N-hydroxylation, additional metabolic activation is ence of cyanide or sulfide, very potent inhibitors of HAP. required to form the ultimate carcinogen-reactive form, the Observations such as this, and, in addition, the very real nature of which is still uncertain (17, 18, 21). Themeis good possibility that the HAP system may prove to be a model for evidence that the sulfate ester of N-OH-AAF3 is the ultimate a proposed in vivo penoxidase activation system has reactive form in male matliven (21). However, tissues other prompted us to investigate the HAP/N-OH-AAF/H2O2 sys than liven, such as mammary gland and Zymbal's gland, de tem in greater detail. We present here the first absonbance velop tumors other than liver tumors as a result of feeding spectnoscopy results on the peroxidase/carcinogen system. AAF to mats, yet these tissues are not capable of forming the We have obtained data on the effects of added asconbate, a sulfate ester form (11). Therefore, other activation mecha known peroxidase electron donor, and cyanide which help nisms must be operating in these tissues and perhaps in one to understand the moleof the carcinogen and function liventissuesalso. ing of the pemoxidase in the system. Preliminary reports of It is possible that a penoxidase-mediated on what can be some of this work has been presented previously (7). termed a â€œfreeradical-activationâ€•route is operating in

â€˜This research was in part supported by Grant 1-R01-CA18591-01 from the MATERIALS AND METHODS National Cancer Institute. 2 To whom reprint requests should be addressed, at the Oklahoma Medi HAP, either type II (AZ = 1 to 1.5) ontype VI (AZ = 3.0), was cal Research Foundation, Biomembrane Research Laboratory, 825 Northeast 13 Street, Oklahoma City, OkIa. 73104. obtained from Sigma Chemical Co. (St. Louis, Mo.). Type II 3 The abbreviations used are: N-OH-AAF, N-hydroxy-N-acetyl-2-amino was the type used in the original reports of the HAP/N- fluorene; HRP, horseradish peroxidase; NOF, 2-nitrosofluorene; N-OAC AAF, N-acetoxy-N-acetyl-2-aminofluorene; TLC, thin-layer chromatography. OH-AAF/H202 system (2). Ascorbic acid and Lubnol WX Received August 1, 1975; accepted January 9, 1976. were purchased from Sigma Chemical Co. and H2O2was

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from Fisher Chemical Co. (Houston, Texas). 2-Nitrofluorene and N-acetyl-2-aminofluonene were purchased from AId rich Chemical Co. (Milwaukee, Wis.). N-OH-AAF was synthe sized from 2-nitrofluomene and recrystallized from benzene according tothe procedure of Poiniereta!. (18). The melting point of the synthesized N-OH-AAF was 146Â°which is within the desirable range (145 to 147Â°),according to Poimier et a!. (18). The extinction coefficient of N-OH-AAF in the buffer system used here was â‚¬= 18,900, 18,500, and 16,400 for 281, 289, and 301 nm, respectively. The absorbances are slightly lower than in methanol (4) but absorbance peak positions are the same in the buffer as in methanol. The carcinogen was solubilized in methanol (1 mg/mI) and added as such to the solutions. N-Hydroxy-2-aminofluorene was prepared according to the procedure of Lotlikar et a!. (15). 2-Nitmosofluomene was prepared by oxidation of the N- hydroxy-2-aminofluomene with diethylazod icarboxylate (1). The reaction mixture was purified by silicic acid chromatog I raphy using n-hexane/benzene (10/3, v/v) as the solvent 0.1 A system. The first green band after crystallization and solu bility in 95% ethanol gave a maximum at 362 nm with a peak at 244 nm and a shoulder at 260 nm and minima at 278 and 224 nm, as described by Lotlikar et a!. (15). The absorbance at 362 nm as given by Lotlikar et a!. (15) was used as a measure of the amount of compound present. N-OAC

AAF was synthesized from N-OH-AAF by the procedure of Gutmann and Erickson (10). Incubation of the HRP/N OH-AAF/H2O2 system for TLC of the products was carried out according to the methods of Bartsch and Hecker (2). The incubation mixture was extracted with cold dichloro methane by the procedure as described by Bartsch and Heckem(2). TLC of the extract was performed with Silica Gel 60 F-254 absorbed on aluminum sheets (Merck, Darmstadt, West Germany). The solvent system used was dichlomo methane/acetone (85/5, v/v). @ â€˜,,1''''T@-@ â€˜@ The concentration of HAP used was determined, using 300 400 500 600 the absomptivity at 403 nm of 102.2 mM' cm â€˜asgiven by Schonbaum (19). The buffer system used was 0.05 M, pH 7.4, A (am) potassium phosphate to which, in some cases, 1 mg of Chart 1. Difference absorption spectra of type II HAP-catalyzed oxidation Lubrol WX per ml was added. Lubrol WX only altered the of N-OH-AAF by small pulses of H202. Both sample and reference cuvets contained 2.78 nmoles oftype IIHAP in the phosphate buffer containing I mg rate of the reaction, which was useful in kinetic experi of Lubrol WX per ml, as described in the text. Trace 1 is the resultant spectra ments, the results of which will be presented later. Absorb obtained when 119 nmoles of N-OH-AAF were added to the sample cuvet and an equivalentamount of methanolwas addedto the referencecuvet. ance spectroscopy was performed on a Camy14 recording Trace 2 is the spectra obtained after approximately 20 nmoles of H202 were spectrophotometer. All reactions were run at room temper added to the sample cuvet and an equivalent amount of H2Owas added to the ature, which average 25Â°. reference cuvet. Traces 3, 4, and 5 are the spectra after successive H,O, and H20 additions to the sample and reference cuvets as shown in Trace 2. The volume added to the cuvet per H202 pulse was 0.20% of the total volume (1.5 ml). RESULTS obtained after N-OH-AAF was added to the sample side Chart 1 presents results demonstrating the absombance containing HAP, and an equivalent amount of methanol was changes observed when small amounts of H2O2were added added to the reference side, which contained the same to a solution containing HRP and N-OH-AAF. The con amount of HAP. Theme is a large increase in absorbance at centrations of H2O2 and N-OH-AAF used here are much 301, 289, and 281 nm as expected from the known absorb less than that used by Bamtschet a!. (2) and, in fact, on a ance of N-OH-AAF. There is also a slight decrease at 403 molar basis, the amount of N-OH-AAF is about 42 times nm, which is apparently due to the binding of the cancino the amount of HAP present. The amount of H2O2added per gen to HAP. Trace 2 shows that the addition of 1 pulse of pulse is about 8 times the amount of HAP present. This H2O2caused a decrease of 0.19 absorbance unit at 301 nm amount of H202is less than that used by Keilin and Hartmee and an increase of 0.25 absorbance unit at 370 nm. The 2nd (12) to form Compound II, so therefore, very little if any and 3rd pulses of H202(Traces 3 and 4) caused absorbance Compound Ill would be formed (12). Trace 1 is the spectra changes similar to that observed with Pulse 1. The absorb

APRIL 1976 1511

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1976 American Association for Cancer Research. A. A. Floyd et a!. ance increase at 370 nm was 0.25 and 0.18 absorbance unit for Pulses 2 and 3, respectively. The 4th pulse (Trace 5) of H202 caused no apparent increase at 370 nm and only a small decrease at 301 nm (0.08 absorbance unit). The 4th pulse of H202also caused the appearance of a peak at about 425 nm, which is due to the appearance of Compound II of HAP (12, 19). The lack of increase in absorbance at 370 nm as a result of the 4th pulse of H2O,is only apparent, for there is a Soret band shift to the medas a result of Compound II formation and a concomitant decrease in the 370 nm region that offsets the real but small increase at 370 nm. The peak at 370 nm is slightly shifted from 370 to 365 nm by the 4th pulse of H202which is due to the medshift of the Sonet band. Subsequent pulses of H202 caused no difference in the spectrumfrom thatshown inTrace5. According to Bartsch et a!. (2, 3), the products of the HAP/ H202-catalyzed N-OH-AAF oxidation are NOF and N-OAC AAF, which occur in equal amounts. NOF absorbs maxi mally in the 362 to 368 nm region (2, 3) with approximately equal absonbance at 362 to 368 nm, as does N-OH-AAF in the 281 to 301 nm region. N-OAC-AAF absorbs maxi mally in the 301 to 271 nm region (301, 289, 275, and 271 nm) with an extinction coefficient of a = 19.6, 20.0, 28.5, and 28.0 mM' cm@, respectively (4). After careful compami son of Trace 1 with Trace 5 of Chart 1 (also see Chart 5, Trace 3 compared with Trace 1), the addition of H2O2caused 01A a massive absorbance increase in the 365 to 370 nm region and also a shift from a maximum of 281 nm for N-OH-AAF to a maximum of 271 nm, which is characteristic of N- OAC-AAF. In addition, the decrease in absombance at 301 nm is approximately equal to the absombance increase at 365 to 370 nm. AfterTrace5,the additionoffurtherH202 caused no further decrease at 301 nm. All of the above observations of these absorbance studies tend to commobo rate the results of Bartsch et a!. (2, 3), namely, 1 NOF and 1 N-OAC-AAF molecule is formed for every 2 N-OH-AAF molecules oxidized in the HAP/N-OH-AAF/H2O2 system (Fig. 1). The results presented in Chart 1 were obtained with type II HAP, whereas those shown in Chart 2 were obtained with type VI HAP. There are many similarities as to the absorb 300 400 500 600 ance changes observed between the 2 enzyme pmepama tions, yet there are distinct differences. For instance, the Chart 2. Difference absorption spectra of type VI HRP-catalyzed oxida type VI enzyme caused a greater fraction of N-OH-AAF to tion of Nâ€”OHâ€”AAFbysmall pulses of H,02. Both sample and references cuvets contained 5.1 nmoles of type VI HAP in the buffer system described in be transformed into NOF. That is, the ratio of the final peak Chart 1. Traces 1 through 5 are exactly as described in Chart 1, except in height at 370 nm to the initial N-OH-AAF peak at 301 nm Trace3,whereapproximately50nmolesof H,02wereadded,comparedwith was 0.52 for type II enzyme but was 0.68 for type VI enzyme. Traces2,4,and5,to which20nmolesofH202wereadded. The H202-induced spectral changes in the 240 to 300 nm region are quite different for type VI, compared with type II for type VI enzyme and, Chart 4, for type II enzyme. Both enzyme. Comparing Charts 1 and 2, it can be seen that a enzymes behaved in a similar manner with respect to the peak occurs in the 240- to 250-nm region for type VI enzyme action of asconbate in the presence of N-OH-AAF. The when an excess of H202 is present, but this is not the case results of 2 different experiments are shown in Chart 3, for type II enzyme. N-OAC-AAF does not have an absonp where the absolute absorbance change at 301, 370, and 265 tion shoulder in the 240 to 250 nm region (4). In independ nm is used as a measure of N-OH-AAF, NOF, and ascor ent studies using type VI HAP, we have shown that the bate concentration, respectively. Chart 3A indicates a large appearance of the 245 nm peak is a time-dependent process 301 nm absonbanceincreasewhen N-OH-AAF was added. and accompanies a decrease in absombance at 271 nm (Fig. There was a stepwise decrease at 301 nm and a con 1). comitant 370 nm absonbance increase when successive Charts 3 and 4 illustrate that ascombate is oxidized before pulses of H202were added to the solution. The 2nd pulse of N-OH-AAF in the HAP/H2O2 system. Chart 3 presents data H202 consumed all of the N-OH-AAF since there was no

1512 CANCER RESEARCH VOL.36

1.0 as a decrease at 301 nm and a concomitant increase at 370 A 0.9 nm. This indicates that this pulse of H2O2oxidized all of the ascorbate and, in addition, some of the N-OH-AAF was 0.8 oxidized to NOF. The equivalence of 265 and 301 nm ab 0.7 sorbance shown after the last H2O2 pulse is as expected. 0.6 That is, utilizing the spectra from which the data presented A in Chart 3A were obtained, the ratio of 265 to 301 nm 0.5 absorbance was 0.90 for N-OH-AAF plus HRP only, and 0.4 0@ was 1.78 when all of the N-OH-AAF was oxidized by H2O2. 0.3 Chart 4 presents additional information that helps to clam ify the ascorbate effect. Trace 3 shows the spectra that were 0.2 obtained after a slight excess of H2O2 was added to the 0.1 sample cuvet containing HRP plus N-OH-AAF and an equiv 1@I7r alent amount of H2O was added to the reference cuvet HIP Only N-OH-AM H202 h1202 H 02 Add.d Add.d Add.d [email protected] containing HRP only. A sharp 301 nm peak is apparent, and LI 370am there is a 270 nm peak maximum, which is as would be 13 B expected from N-OAC-AAF. Also there is a 360 to 370 nm @ 12 301am peak due to NOF. Compound II is apparent (425 nm), mdi cating that an excess of H2O2was added. Trace 4 shows the 11 H 265nm results obtained when the same amount of H2O2was added 1.0 to the sample cuvet, but in addition to HAP and N-OH

0.9 AAF, ascorbate was present in an amount approximately A equivalent to one-half that of the N-OH-AAF. The pres 0.8 ence of NOF is apparent, in an amount approximately equal 0.7 to one-half that obtained when no ascorbate was present (Trace 3). The 301 nm peak is not decreased as much as in 0.6 / Trace 1 and this indicates N-OH-AAF is still present (see 0.5 / below). Trace 2 shows the results obtained when the same 0.4 amount of H202 as in Traces 4 and 2 was added to a sample a3 ,- in which more ascorbate, approximately equivalent in / amount to the N-OH-AAF content, was present. Very little, 0.2 / / if any, NOF was formed by the addition of H2O2. The 301 nm / 0.1 / I absombance, when compared with Trace 1 showing the [ / spectra of N-OH-AAF plus HAP versus HRP, indicates mmr r Onlyescâ€¢rmavâ€¢Add.dm-uM-A*pAddedM2U2 Add.dH202 Add.d that very little, if any, N-OH-AAF had reacted as a result of H2O2addition. These results, combined with those of Chart Chart 3. The absolute absorption at 370, 301 , and 265 nm of type VI HAP catalyzed oxidation of N-OH-AAF by pulses of H202, with ascorbate either 3, indicate that when H2O2is added to a solution containing present or absent. In Chart 3A, the absorption was obtained when a total of HAP, N-OH-AAF, and ascorbate, the N-OH-AAF is not 3.3 nmoles of HAP in the buffer system described in Chart 1 were compared with the buffer system only. In the 2nd column is the absorption obtained oxidized until after all of the ascorbate is oxidized. when 54 nmoles of N-OH-AAF were added to the sample cuvet of Column 1 The results presented in Chart 5 demonstrate that: (a) and an equivalent amount of methanol (a total of 0.8%) was added to the CN did not prevent H2O2/HAP-catalyzed oxidation of N-OH reference cuvet. Column 3 is the spectra obtained when about 27 nmoles of H2O2were added to the HAP-carcinogen sample cuvet. Columns 4 and 5 are AAF and (b) the products of the reaction were the same in spectra obtained after additional H2O2additions as described for Column 3. the presence as in the absence of CN (Fig. 1). The data In Chart 38, the absorption spectra of type VI HAP were obtained as de shown in Chart 5 were obtained with type VI enzyme, but scribed in Chart 3A. Column 1 data were obtained from the spectra of the HAP solution only. Column 2 shows the absorption obtained after 150 nmoles similar results were obtained with type II enzyme. Trace 1 of ascorbate were added to the sample cuvet. The amount of ascorbate was shows the absolute spectra of the enzyme per se, Trace 2 determined by the absorptivity of 7 m@' cm' at 265 nm. Column 3 is the shows the spectra obtained after N-OH-AAF was added to absorption obtained after 54 nmoles of N-OH-AAF were added to the sample cuvet and an appropriate amount of methanol was added to the the HAP solution, and Trace 3 shows the spectra after reference cuvet. Column 4 is the absorption obtained after about 160 nmoles NaCN to a final concentration of 10@ M was added to the of H202 were added to the sample cuvet, and Column 5 is the absorption spectra obtained after another pulse of H202 (160 nmoles) was again added to solution shown in Trace 2. The appearance of the 425 to the sample cuvet. 430 nm-absorbing CN enzyme complex (12) is apparent. Trace 5 shows the resultant spectra after an excess of H2O2 further decrease at 301 nm and no further increase at 370 was added to the solution shown in Trace 3. Trace 4 gives nm. Chart 3B shows there was a large absorbance increase the comparison spectra of a solution exactly as in Trace 5, at 265 nm but no increase at 301 nm when ascorbate was except CN was not present. The spectra is the same added to the solution. N-OH-AAF addition then caused an whether CN is present or not. The shoulder at 425 nm in increase at 265 nm, since the carcinogen absorbs strongly Trace 4 is due to Compound II formation. at that wavelength. H2O2addition caused a decrease at 265 Chart 6 demonstrates that HAP will form the CN com nm due to ascorbate oxidation, but no decrease at 301 nm. plex even after the enzyme in combination with H2O2has Addition of H2O2caused a further decrease at 265 nm as well converted N-OH-AAF to NOF and N-OAC-AAF and then

APRIL 1976 1513

The data presented in Chart 7 demonstrate that if the type II peroxidase is first converted to compound II by an excess of H202 and then mixed with N-OH-AAF, (a) the enzyme reacts with N-OH-AAF and (b) the products of the reac tion are the same as when N-OH-AAF is mixed with the enzyme first and then reacted with H2O2.Trace 1 shows the difference spectra of HAP versus HAP plus H2O2.Trace 2 is the spectra of the same solutions as Trace 1, except N- OH-AAF was added to the H2O2/HRP mixture. The 365 to 370 nm peak isdue toNOF, and themaximum at270 nm is indicative of N-OAC-AAF. This spectra can be compared with Trace 4 of Chart 1 which is a spectra of the same reac tion, except that N-OH-AAF was added before H2O2. We have also determined that the Compound II formed as

I 0.1*

-i@ 0.1A

Ill I 300 400 300 600 X)nm) Chart 4. The effect of ascorbate on H202/HAP-catalyzed oxidation of N- OH-AAF. Type II HAP at a concentration of 2.78 nmoles dissolved in 1.5 ml of the buffer system described in Chart 1 was placed in the sample and refer ence cuvets. Trace 1 is the spectra after 103 nmoles of N-OH-AAF were added to the sample cuvet and an equivalent amount of methanol was added to the reference cuvet. Trace 2 is the spectra resultant from solutions made up exactly as described in Trace 1, except that 60 nmoles of ascorbate were added, and then approximately 66 nmoles of H202 were added to the sample cuvet. The appropriate dilutions were maintained in the reference cuvet. Trace 3 is the spectra of HAP solution versus HAP solution to which 103 nmoles of N-OH-AAF and then 66 nmoles of H202 were added to the sample cuvet; appropriate dilutions were added to the reference cuvet. Trace 4 is as in Trace 3 except that, in addition to N-OH-AAF, the sample cuvet 300 400 500 600 contained 30 nmoles of ascorbate to which H2O2(66 nmoles) was added.

remained in contact with the reaction products for 10 mm. Trace 1 is the absolute spectra of type II HAP in the pres Chart 5. Type VI HAP-catalyzed N-OH-AAF oxidation by H202 as influ enced by cyanide. Trace 1 is the spectra where the sample cuvet contained ence of N-OH-AAF to which just enough H2O2 had been 2.64 nmoles of HAP in the buffer system described in Chart 1 and the added to completely oxidize N-OH-AAF, yet not enough reference cuvet contained the buffer system. In Trace 2, 65 nmoles of N- to form Compound II. Trace 2 is the same solution as in OH-AAF were added to the HAP solution and, in Trace 3, NaCN was then added to the HAP/carcinogen solution to a final concentration of 10@ H. In Trace 1 to which NaCN to a final concentration of 10@M had Trace 5, about 70 nmoles of H2O2were then added to the HAP/carcinogen/ been added.The appearanceoftheshoulderat425 to430 CN solution. In all the operations described above, appropriate dilutions were made to the reference cuvet solution. Trace 4 is the spectra of a solu nm is indicative of CN/enzyme complex. The time elapsed tion containingall of the componentsin exactlythe sameconcentrationas between the completion of Trace 1 and Trace 2 was 10 mm. in Trace 5, except that CN was not present.

1514 CANCERRESEARCHVOL.36

of H2O22 mm later caused approximately a 0.1 absombance unit increase. Compound II decreased when ascorbate was then added after 2 mm (bottom trace) or after 30 mm (top trace). In both cases, ascorbate addition caused a 425-nm absorbance decrease to within 80 to 90% of the level before H2O2addition. The reason for the delay in the 425 nm ab sorbance decrease in the bottom trace after ascorbate addi tion is not known. It perhaps is due to the time required for the cycling peroxidase in the presence of ascorbate to T consume the excess H2O2. 0.1 A Fig. 1 shows a TLC demonstration of the major points presented here and corroborates the deductions arrived at from the absorbance studies.Columns 6,7,8,and 9 of Fig. 1A are the patterns obtained from the standard compounds. The AF'sobtained are close to that obtained by Bartsch et a!. (4). In Column 7, the top spot is N-OAC-AAF and the 2 lower spots are degradation products. The compound tended to decompose with time in the solvent systems used. Freshly prepared and dissolved N-OAC-AAF gave essen tially only the top spot which gave an RFthat agreed closely with that given by Bartsch et a!. (4). Columns3, 4, and 5 shows that types VI and II HAP plus H2O2 and N-OH-AAF yielded NOF and N-OAC-AAF as the major products. There also appeared to be some AAF present and also a prod 2 uct, unidentified as yet, running between NOF and N-

I 300 400 500 600 Xnm) Chart 6. The formation of the cyanide/HAP complex with the enzyme that has reacted to form NOF and N-OAC-AAF from N-OH-AAF and remained in contact with the products for 10 mm. Trace 1 is the spectra of 2.79 nmoles of type II HAP plus 69 nmoles of N-OH-AAF to which had been added a -1@ near saturating amount (35 to 40 nmoles) of H202.Trace 2 is the spectra of the above-described solution to which NaCN to a total concentration of 10@ M 0.1A had been added. The solution in the reference cuvet was the buffer system to which appropriate dilutions were made. a result of the reaction of a small amount of N-OH-AAF and a larger amount of H2O2will then react with more N- OH-AAF (Chart 8). Trace 1 is the difference spectra of HAP versus HAP plus N-OH-AAF. Trace 2 is the spectra ob tamed after addition of an excess H2O2to the cuvet contain ing the peroxidase and carcinogen. NOF formation is appar ent by the increase in absorbance at 365 nm. Compound II @1 formation is apparent by the 425-nm increase and 403-nm decrease. After the addition of N-OH-AAF, Compound II disappeared and more NOF was formed, as illustrated by Trace 3. The data in Chart 8 were obtained with type VI peroxidase, but similar results were obtained with type II pemoxidase. . . 5@0 Chart 9 shows that Compound II, formed as a result of the . 3 400 600 reaction of a small amount of N-OH-AAF and a larger amount of H202, will then react with ascorbate, even if A(nm) Compound II has been present in the reaction mixture for Chart 7. The reactivity of N-OH-AAF with Compound II of HAP. Trace 1 either 2 or 30 mm. The amount of Compound II present Is the difference spectra of 2.78 nmoles of type II HAP in both the sample and reference cuvets; 35 to 40 nmoles of H202 were added to the sample cuvet was monitored at 425 nm. Addition of N-OH-AAF did not only. In Trace 2, 69 nmoles N-OH-AAF were then added to the HRP/H20, cause an increase at 425 nm, but the addition of an excess solution shown in Trace 1.

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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1976 American Association for Cancer Research. A. A. Floyd et a!. The results shown in Fig. lB illustrate that N-OH-AAF oxidation by HAP and an excess of H2O2yielded a different product at the apparent expense of N-OAC-AAF as reac tion time proceeded. This was true for type VI pemoxidase but not for type II pemoxidase. The top spot of Figure lB corresponds to NOF and the next lower major spot come sponds to N-OAC-AAF. The next lowest faint spot corre spondstoAAF. Itcanbe seenthatfortypeVIbutnotfortype II pemoxidase there is another compound occurring be tween NOF and N-OAC-AAF but closer to NOF. Visual com panison of spot intensity reveals that this new compound occurs at the expense of N-OAC-AAF. This conforms with the results of the absonbance studies presented earlier (Charts1and 2).

DISCUSSION

The normal cycling behavior of HAP as first elucidated by Chance (5) and George (9) and later corroborated by the electron spin resonance experiments of Yamazaki et a!. (22) can be summarized by the following equations, in which HAP refers to the normal ferriheme oxidation state of the enzyme and OH to an electron donor. HAP-I and HAP-Il are Compound Iand Compound IIofhorseradish.

HAP + H2O2 â€”*HAP-I + 2H20 (A) HAP-I + OH -* HAP-Il + b (B) , I HAP-Il+ OH â€”@HAP+ b 300 400 500 600 (C) )jnm) Thus the enzyme oxidizes 2 electron donors into 2 free radicals (b) for every turn of the cycle. Bartsch and Chart 8. The reactivity of N-OH-AAF with HAP-Il formed by the addition of saturating amounts of H2O2to the HAP solution containing small amounts Hecken (2) postulated that N-OH-AAF would act as an elec of N-OH-AAF. The sample as well as the reference cuvet contained 5.1 tron donor to the cycling enzyme. On the basis of electron nmoles of type VI HAP in the buffer system described in Chart 1. Trace I is the spectra resulting after 23 nmoles of N-OH-AAF were added to the sample spin resonanceexperiments,they(2)postulatedthatthe cuvet. Trace 2 is the spectra obtained when about 20 nmoles of H,O, were electron was removed from the easily oxidized amine hy added to the solution described in Trace 1 and, in Trace 3, another 23 nmoles dnoxyl yielding the nitroxyl radical. Two molecules of N-OH of N-OH-AAF were then added to the cuvet. AAF were consumed per enzyme cycle, and the 2 nitmoxyl 425am radicals formed then dismutated (4) to form NOF and N- Ascorbot. OAC-AAF. These demonstrations laid the foundation for the very real possibility that a peroxidase-type activation mechanism for @ !iE;@@,@ arylamine carcinogens occurs in vivo. However, the impor tance of the theoretical possibility of a peroxidase-type acti H2O@ â€˜1 vation mechanism is overshadowed by doubts as to whether Ascorbot. @, N-OH-AM HAP actually opelates as a pemoxidase on the carcinogen N-OH-AAF. For instance, it has been demonstrated that CN did not prevent HAP/H2O2-catalyzed conversion of N- OH-AAF into products, presumably N-OAC-AAF, that me acted with nucleic acids (13). Also, in the experiments Chart 9. The reactivity of ascorbate with HAP-Il formed by the addition of reported by Bartsch and Hecker (2), the ratio of H202to HAP saturating amounts of H202 to a solution containing small amounts of N- was greater than 50,000 (based on our measurements of the OH-AAF. The sample and reference cuvet solutions were exactly as de scnbed in Chart 8. The absorptivity was monitored at 425 nm and, at the point HAP content, duplicating their conditions exactly). H2O2in indicated, 23 nmoles of N-OH-AAF were added to the sample cuvet. At the high concentrations, actually in H2O2-to-HAPratios of 20 or point indicated, about 60 nmoles of H,O, were added to the sample cuvet. At more, is known to form Compound Ill (9, 12). This form of the time indicated, i.e., 30 to 31 mm in the top trace and 3 to 4 mm in the lower trace, about 60 nmoles of ascorbate were added. the enzyme would be expected to remove the enzyme from the normal cycling events. Thus, it seemed necessary to OAC-AAF but closer to NOF. Column 2 shows that the study the N-OH-AAF/HAP/H202 system in much greater products obtained when CN was pmesentare the same as in detail under conditions such that it would be possible to its absence. Column 1 shows that no products were formed make more valid judgements as to the molecular events when an excess of asconbate was present. occurring.

1516 CANCERRESEARCHVOL.36

The results of the absorbance studies presented here help enzyme, which then cycles through Compound I, Com clarify the molecular events occurring in the N-OH-AAF/ pound II, and then back to the original enzyme mediated by HAP/H202 system. With type II enzyme, the absorbance me electrons from N-OH-AAF in a manner that Bartsch and suIts tend to corroborate the previous findings in that, for Hecker (2) suggest yields NOF and N-OAC-AAF. These every 2 N-OH-AAF molecules consumed, 2 product mole observations then provide evidence that Compound II will cules, 1 NOF and 1 N-OAC-AAF, are formed. The absorb accept electrons from N-OH-AAF. This is an essential ance studies with the type VI enzyme indicate there are step in the Bartsch and Hecker proposal (2). differences in amount of products formed in the reaction The data presented here also demonstrate that Com when compared with the type II enzyme; i.e., when the type pound II, formed by the addition of an excess of H2O2and a VI enzyme is compared to the type II enzyme, more N-OH small amount of N-OH-AAF, will then either react with AAF is converted to NOF, and, in addition, a product ab N-OH-AAF to form mome NOF and N-OAC-AAF or be sonbing in the 245 nm region is present. The 245-nm absorb come reduced to the original enzyme by ascorbate. In the ing product appears to be formed from N-OAC-AAF. The latter case, ascorbate reduced Compound II after being in nature of the 245 nm product is being determined now. The contact with NOF and N-OAC-AAF for 2 mm or as long as results presented here do demonstrate that the enzyme 30 mm. We have also shown that the enzyme that reacted to purity and/or the peroxidase isoenzyme makes a difference form NOF and N-OAC-AAF and then remained in contact in the eventual products obtained in the reaction. with these compounds will combine with CN to form the The results presented here demonstrate that, if the 2 enzyme/CN complex. These observations have some in electron donors, ascorbate and N-OH-AAF, are present, teresting implications in terms of carcinogenesis by AAF; then H2O2additions result first in HAP-catalyzed ascombate that is, even though NOF and N-OAC-AAF are more reactive oxidation and then in N-OH-AAF oxidation. This observa toward biological molecules then N-OH-AAF, HAP in the tion has some obvious important implications in terms of Compound II state is not inactivated with respect to its ascorbate prevention of peroxidase-catalyzed activation of ability to accept electrons from ascorbate or N-OH-AAF, N-hydroxy-alkylarylammne carcinogens, if such an activation by incubation in the presence of these 2 more potent carcin mechanism is operating in vivo. From the results presented ogens. here, it is impossible to determine the exact molecular N-OAC-AAF is considered a very reactive molecule (20). events occurring in the ascorbate competition effect. For Yet, on the basis of the absorbance studies presented instance, it is possible to explain the results by assuming here, it appears that N-OAC-AAF remains in the solution that ascombatereacts more rapidly with Compound I and/or containing the type II HAP which catalyzed its formation. Compound II than does N-OH-AAF, or, alternatively, that This appears not to be true for the type VI enzyme, for, on ascorbate reduces the nitroxyl radical of N-OH-AAF formed the basis of our results, the final products of the type VI by Compound I and/or Compound II oxidation of N-OH-AAF. enzyme-catalyzed reaction is not entirely N-OAC-AAF The results presented here do help clarify the action of and NOF, for a product having an absorption band at 245 the peroxidase on N-OH-AAF when CN is present. Sum nm is present also. It appears that the 245-nm absorbing pnisingly, our results demonstrate that H2O2/HRP in the compound is formed from N-OAC-AAF. This implies that presence of CN did catalyze the conversion of N-OH-AAF the N-OAC-AAF in the type VI enzyme environment is into the same products, based on the absorbance spectra, much more unstable than in the type II environment. The as those observed in the absence of CN . These results, in a results have importance to arylamine carcinogenesis, and sense, confirm the observations made by King et a!. (13). the positive identification of the new compound is now in Cyanide, a known inhibitor of the peroxidase (12), presum progress. ably binds to the heme iron and prevents H2O2reduction. However, it is not clear why the peroxidase continues to operate in the presence of CN when N-OH-AAF is pres ACKNOWLEDGMENTS ent. The reason(s) may be due to the specificity, affinity, and location of N-OH-AAF binding to the peroxidase. For We would like to thank Dr. Paul B. McCay and Dr. J. L. Poyer for stimulat ing and helpful discussion and encouragement, and Dr. Helmut Bartsch for instance, Schonbaum (19) has shown that certain hydrox suggesting the use of diethylazodicarboxylate as an oxidizing agent to form amic acids bind to HAP, but this does not prevent the nitrosofluorene. binding of cyanide. However, the peroxidase activity of the hydroxamic acid/CN/HRP complex was not reported (19) and, also, it is not known whether N-OH-AAF binds in a REFERENCES manner similar to the tested hydroxamic acids, although it has some structural similarities to these acids. Low-temper 1. Ames, B. N., Gurney, E. G., Miller, J. A., and Bartsch, H. Carcinogens as atune electron spin resonance investigation of N-OH Frameshift Mutagens: Metabolites and Derivatives of 2-Acetylaminoflu orene and Other Aromatic Amine Carcinogens. Proc. NatI. Acad. Sci. U. AAF-HAP complexes, as compared to the spectra of other S., 69: 3128-3132, 1972. HAP/donor complexes (14), may provide answers. 2. Bartsch, H., and Hecker, E. On the Metabolic Activation of the Carcino gen N-Hydroxy-N-2-acetylaminofluorene. Ill. Oxidation with Horseradish The data presented here demonstrate that the peroxidase Peroxidase to Yield 2-Nitrosofluorene and N-Acetoxy-N-2-acetylamino yields the same products when H2O2 is added before N- fluorene. Biochim. Biophys. Acta, 237: 567-578, 1971. OH-AAF or as it does when the addition sequence is me 3. Bartsch, H., Miller, J. A., and Miller, E. C. N-Acetoxy-N-Ac@tylaminofluo rene and Nitrosoarenes, One-Electron Non-Enzymatic and Enzymatic versed. This observation can be interpreted as indicating Oxidation Products of various Carcinogenic Aromatic Acethyroxamic that Compound II oxidized N-OH-AAF to form the original Acids.Biochim.Biophys.Acta,273:40-51, 1972.

APRIL1976 1517

4. Bartsch, H., Traut, M., and Hecker, E. On the Metabolic Activation of N- 13. King, C. M., Bednar, T. W., and Linsmaier-Bednar, E. M. Activation of the Hydroxy-N-2-acetylaminofluorene. I. Simultaneous Formation of 2- Carcinogen N-Hydroxy-2-fluorenylacetamide: Insensitivity to Cyanide Nitrosofluorene and N-Acetoxy-N-2-acetylaminofluorene from N-Hy and Sulfide of the Peroxidase-H202 Induced Formation of Nucleic Acid droxy-N-2-acetylaminofluorene via a Free Radical Intermediate. Biochim. Adducts.Chem.-Biol.Interactions,7: 185-188,1973. Biophys. Acta, 237: 556-566, 1971. 14. Leigh, J. S., Maltempo, M. M., Ohlsson, P. I., and Paul, K. G. Optical, NMR, 5. Chance, B. The Kinetics and Stoichiometry of the Transition from the and EPA Properties of Horseradish Peroxidase and Its Donor Complexes. Primary to the Secondary Peroxidase Peroxide Complexes. Arch. Bio Federation European Biochem. Soc. Letters, 51: 304-308, 1975. chem. Biophys., 41: 416â€”424,1952. 15. Lotlikar, P. D., Miller, E. C., Miller, J. A., and Margreth, A. The Enzymatic 6. Cramer, J. W., Miller, J. A., and Miller, E. C. N-Hydroxylation: A New Reduction of the N-Hydroxy Derivatives of 2-Acetylaminofluorene and Metabolic Reaction Observed in the Rat with the Carcinogen 2-Acetyl Related Carcinogens by Tissue Preparations. Cancer Aes., 25: 1743- aminofluorene. J. Biol. Chem., 235: 885-888, 1960. 1752, 1965. 7. Floyd, A. A. Carcinogen Free Radicals: Observationson the Horseradish 16. Miller, J. A. Carcinogenesis by Chemicals: An Overviewâ€”G. H. A. Clowes Peroxidase Hydroperoxide Generation of the Nitroxyl Aadical of N-Hy Memorial Lecture. Cancer Res., 30: 559-576, 1970. droxyacetylaminofluorene. Biophys. J., 15: 135a, 1975. 17. Miller, J. A., and Miller, E. C. Guest Editorial. Chemical Carcinogenesis: 8. Forrester, A. A., Ogilvy, M. M., and Thomson, A. H. Mode of Action of Mechanisms and Approaches to Its Control. J. NatI. Cancer Inst., 47: V Carcinogenic Amines. Part I. Oxidation of N-Aryl-hydroxamic Acids. J. XIV,1971. Chem. Soc. Sec. C, 1081-1083, 1970. 18. Poirier, L. A., Miller, J. A., and Miller, E. C. The N- and Ring-hydroxyla 9. George, P. The Chemical Nature of the Second Hydrogen Peroxide tion of 2-Aminofluorene in the Dog. Cancer Res. , 23: 790-800, 1963. Compound Formed by Cytochrome C Peroxidase and Horseradish Per 19. Schonbaum, G. A. New Complexes of Peroxidases with Hydroxamic oxidase. Biochem. J.. 54: 267-276, 1953. Acids, Hydrazines, and Amides. J. Biol. Chem., 248: 502-51 1, 1973. 10. Gutmann, H. A., and Erickson, A. A. The Conversion ofthe Carcinogen N- 20. Scribner, J. D., Miller, J. A., and Miller, E. C. Nucleophilic Substitution on Hydroxy-2-fluorenylacetamide to o-Amidophenols by Rat Liver in Vitro. Carcinogenic N-Acetoxy-N-arylacetamides. Cancer Res., 30: 1570-1579, An Inducible Enzymatic Reaction. J. Biol. Chem., 244: 1729-1740, 1969. 1970. 11. Irving, C. C., Janss, D. H., and Russell, L T. Lack of N-Hydroxy-2-acetyl 21. Weisburger, J. A., and Weisburger, E. K. Biochemical Formation and aminofluorene Sulfotransferase Activity in the Mammary Gland and zym Pharmacological, Toxicological, and Pathological Properties of Hydrox bal's Gland of the Rat. Cancer Aes., 31 : 387-391 , 1971. ylamines and Hydroxamic Acids. Pharmacol. Rev., 25: 1-66, 1973. 12. Keilin, D., and Hartree, E. F. Purification of Horseradish Peroxidase and 22. Yamazaki, I., Mason, H. S., and Piette, L. Identification, by Electron Comparison of Its Properties with Those of Catalyse and Methaemoglo Paramagnetic Resonance Spectroscopy, of Free Radicals Generated bin. Biochem. J., 49: 88-104, 1951. from Substrates by Peroxidase. J. Biol. Chem., 235: 2444-2449, 1960.

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1234 56789 1234 Fig. 1. TLC of the HAP/H202-catalyzed oxidation of Nâ€”OHâ€”AAFaseffected by cyanide and ascorbate. The conditions of incubation and extraction of the reaction systems are described in the text. In A, the conditions for each of the columns are as follows: Column 1, type II HAP, N-OH-AAF, ascorbate, H20,; Column2, type II, HAP,N-OH-AAF,NaCN,H,02; Column 3, type VI HAP, N-OH-AAF,H202reactiontime, 30 mm; Column 4, type VI HAP, N-OH-AAF, H,02 reaction time, 9@mm; Column 5, type II HAP, N-OH-AAF, H2O2 reaction time, 9 mm; Column 6, NOF standard; Column 7, N-OAC-AAF standard; Column8, AAFstandard;Column9, N-OH-AAFstandard.In B, Column 1, type II HAP,N-OH-AAF,H202reactiontime, 9 mm; Column2, type II HAP,N- OH-AAF, H,02 reaction time, 30 mm; Column 3, type vI HAP, N-OH-AAF, H502 reaction time, 9 mm; Column 4, type â€˜/lHAP, N-OH-AAF, H302 reaction time, 30 mm. The approximate amount of each component when present in the incubation system at the start of the reactions is as follows: HAP, 1.7 @M;N- OH-AAF, 45 @.tH;H202,50 @M;NaCN,0.1 mM; ascorbate, 85 @M.Theprints are actual photographic prints of the TLC plates illuminated with uv (254 nm) excitation light which caused the green background fluorescence to develop the film.

APRIL1976 1519

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1976 American Association for Cancer Research. Horseradish Peroxidase/Hydrogen Peroxide-catalyzed Oxidation of the Carcinogen N-Hydroxy-N-acetyl-2-aminofluorene as Effected by Cyanide and Ascorbate

Robert A. Floyd, Lailing M. Soong and Peggy L. Culver

Cancer Res 1976;36:1510-1519.

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