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[CANCER RESEARCH 36, 2761-2767, August 1976] Lipid Hydroperoxide Activation of N-Hydroxy-N acetylaminofluorene via a Free Radical Route1 Robert A. Floyd,2 Lailing M. Soong, Robert N. Walker, and Melissa Stuart Oklahoma Medical Research Foundation, Biomembrane Research Laboratory, Oklahoma City, Oklahoma 73104 SUMMARY The data presented here demonstrate that linoleic acid hydroperoxide in the presence of methemoglobin or hema tin activated the carcinogen N-hyd roxy-N-acetyl-2-amino fluorene via the nitroxyl free radical intermediate into 2nitrosofluorene and N-acetoxy-N-acetyl-2-aminofluorene. Ascorbate inhibited the activation, in which case the free radical intermediate was replaced by the ascorbate free radical. On the basis of optical kinetics,we have established that the rate of linoleic acid hydroperoxide decrease paral leled the rate of N-hydroxy-N-acetyl-2-aminofluorene de crease and also the rate of 2-nitrosofluorene increase. The stoichiometry of the reaction was such that, for every 2 linoleic acid hydroperoxide molecules consumed, 2 N-hy d roxy-N-acetyl-2-aminofluorene molecules were oxidized and 1 2-nitrosofluorene and 1 N-acetoxy-N-acetyl-2 amino fluorenemolecule was formed. INTRODUCTION The exact chemical events that result in the activation of the arylamine carcinogens to the ultimate reactive chemical form are still an open question. However, much has been learned in recent years (see Refs. 15, 16, and 21 for re views). For example, it was discovered in 1960 (5) that the N-hydroxylation of AAF3 into N-OH-AAF considerably en hanced the potency of this carcinogen; thus, this step is now considered an essential step in the activation of the arylamine carcinogens. N-OH-AAF is apparently not the ulti mate reaction form because it does not react with proteins or nucleic acids directly (21); thus, further activation is apparently required. There are many observations that sug gest that the sulfate ester of N-OH-AAF is the ultimate reac tive form in liver (21), but AAF feeding causes tumors of the ear duct gland and mammary gland of rat. Yet, both of these tissues are devoid of the enzymes necessary to activate NOH-AAF to the sulfate ester form (12). One mechanism that may be of importance in N-OH-AAF 1 This research was in part supported by Grant 1-RO1-CA18591-01 from the National Cancer Institute. °Towhom requests for reprints should be addressed, at the Oklahoma Medical Research Foundation, Biomembrane Research Laboratory, 825 Northeast 13 Street, Oklahoma City, OkIa. 73104. 3 The abbreviations used are: AAF, N-acetyl-2-aminofluorene; N-OH-AAF, N-hydroxy-N-acetyl-2-aminofluorene; ESR, electron spin resonance; LAHP, linoleic acid hydroperoxide; NOF, 2-nitrosofluorene; N-OAC-AAF, N-acetoxy N-acetyl-2-aminofluorene; TLC , thin-layer chromatography. Received December 9, 1975; accepted April 12, 1976. AUGUST activation is the peroxidase or free radical activation route (7). The initial observations that provided a basis for consid ering this mechanism were made independently by Bartsch et a!. (4) and Forrester et a!. (8), first in a purely organic chemical system and laterextrapolatedto in vitroperoxi dase enzyme systems by Bartsch et al. (2, 3). The molecular model for this activation system is discussed at length by Bartsch and Hecker (2). Briefly, it postulates that N-OH-AAF acts as a 1-electron donor to the hydrogen peroxide-in duced cycling peroxidase. The nitroxyl free radicals formed dismutate to form the nitrosofluorene and N-acetoxyace tylaminofluorene carcinogens . The horserad ish peroxidase system has been studied in greater detail, and we (7) have concluded that the model proposed by Bartsch and Hecker (2) is correct; however, the peroxidase has some peculiar properties in the presence of the Carcinogen, cyanide does not inhibit the reaction (7), as was observed by King et al. (13). The free radical activation route is an attractive possibil ity, and the observation of Stier et al. (20), that the chloro form/methanol extract of microsomes metabolizing arylam me carcinogens yields nitroxyl free radical ESR signals, emphasizes the need to consider it as a possible in vivo activation mechanism. It is possible that lipid peroxides and an endogenous peroxidase would act in a manner that would activate N-OH-AAF. Preliminary work using heme proteins and various hydroperoxides was encouraging; therefore, we have tested these ideas in a strictly in vitro system using LAHP as the peroxide and methemoglobin or hematin as the catalyst. The results reported here do mdi cate that this system will activate N-OH-AAF through a ni troxylfree radicalintermediateto form the carcinogens nitrosofluorene and N-acetoxyacetylaminofluorene . Ascor bate inhibits thisactivationmechanism. MATERIALS AND METHODS Hematin (prepared from bovine blood), methemoglobmn Grade I (prepared from crystallized bovine blood hemoglo bin), lipoxygenase Grade I (prepared from soybeans), and linoleic acid Grade I (99% purity) were purchased from Sigma Chemical Co. , St. Louis, Mo. AAF and 2-nitrofluo rene were purchased from Aldrich Chemical Co. , Milwau kee, Wis. LAHP was prepared from linoleic acid using soy bean lipoxygenase according to the procedure of Hamberg and Gotthammar (10). The prepared LAHP was actually the 13-L-hydroperoxy-cis-9,trans-i 1-octadecadienoic acid iso mer (10). The prepared LAHP was stored in methanol under 1976 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research. 2761 A. A. Floyd et a!. nitrogen at —20°. An absorbance ofe = 25.25 m@i1 cm1 at 233 nm in 95% ethanol was used to assay the amount of LAHP present. N-OH-AAF was synthesized from 2-nitrofluorene and re crystallized from benzene according to the procedure of Poirier et a!. (17). The melting point of the synthesized N-OH-AAF was 146°,which is within the desirable range (146-147°) according to the results of Poirieret al. (17). The absorptivity in buffer was used as a measure of the amount of N-OH-AAF present as we have described previously (7). The carcinogen was solubilized in methanol (1 mg/mI) and was added as such to the reactant solutions. N-Hydroxy-2aminofluorene was prepared according to the procedure of Lotlikar et a!. (14). NOF was prepared by oxidation of the Nhydroxy-2-aminofluorene with diethyl azodicarboxylate (1). The reaction mixture was purified by silicic acid chromatog raphy using N-hexane/benzene (10/3, v/v) as the solvent system. The 1st green band emerging from the silicic acid column was crystallized. The absorption spectrum in 95% ethanol demonstrated a maximum at 362 nm, a peak of 244 nm, a shoulder at 260 nm, and minima at 278 and 224 nm. This spectrum agrees with that reported for NOF by Lotlikar et al. (14). The absorptivity at 362 nm as given by Lotlikar et al. (14) was used as a measure of the amount of NOF present. N-OAC-AAF was synthesized according to the pro cedure of Gutmann and Erickson (9). The absorption peaks and their ratios matched those reported by Gutmann and Erickson (9). Also, our synthesized N-OAC-AAF had absorp tivities and TLC patterns identical to those of a sample of N-OAC-AAF generously supplied by Dr. E. K. Weisburger (National Cancer Institute, Bethesda, Md.). Incubation of the horserad ish peroxidase/N-OH-AAF/ H2O2system for TLC of the products was carried out accord ing to the method of Bartsch and Hecker (2). Extraction of the incubation mixture was with cold dichloromethane as described by Bartsch and Hecker (2). TLC ofthe extract was carried out using Silica Gel 60 F-254 absorbed on aluminum sheets (Merck, Darmstadt, Germany) as we have described previously (7). The solvent system used was dichlorometh ane/acetone (85/5, v/v). In the hematin/N-OH-AAF/LAHP system, incubations were the same as those described for the optical and ESR studies, except that larger volumes were used. Extraction and TLC of this system were con ducted as with the peroxidase system (7). Hematin stock solution was made up by dissolving ox blood hematin in glass-distilled H20 to which had been added a trace of alkali as NH4OHor NaOH. The hematin stock solution was stored at 4°or —20° until used. The catalytic capacity of the hema tin decreased slightly with time, apparently due to the p0lymerization of the hematin (11). This affected only the rate of reaction, not the pattern of the kinetics or the nature of the products. An absorptivity of 122 mM1 cm1 at 398 nm was used as a measure of the amount of hematin present (ii). Optical studies were carried out with a Gary Model 14 recording spectrophotometer. The reactions (ESR) were conducted at room temperature, which averaged 25°.ESR spectra were taken with a Varian E-9 X-band spectrometer operating at 100 kHz modulation. A 3-port stop-cock di rectly coupled 2 syringes and directly connected the sy 2762 ringes to the liquid sample cuvet positioned in the ESR cavity. This system made it possible to inject a sample and start an ESR sweep in less than 20 sec after adding the reactants. RESULTS Chart 1 demonstrates the free radical signal obtained with methemoglobin- and hematin-catalyzed oxidation of N-OH AAF by LAHP. We compute a g value of 2.0063 forthe free radical observed in both reactions. This value is the same as the one we have obtained in the horseradish peroxidase/N OH-AAF/H202 system and the same as that reported by Bartsch and Hecker (2) for the latter system. The g value of the lower spectrum in Chart 1 appears to be slightly lower, but this is due to a slight shift in frequency between the 2 samples. Many separate experiments in which potassium nitrosodisülfonate was used as a standard have shown that the g values of the free radical intermediates in both reac tions are the same. The 14Nhyperfine splitting is 7.7 gauss. This value agrees with that reported by Bartsch and Hecker (2) in the horseradish peroxidase system. Thus, for the above reasons, we conclude that the free radical signal we obtain in either the methemoglobin- or hematmn-catalyzed oxidation of N-OH-AAF by LAHP is the nitroxyl free radical of N-OH-AAF. The peak heights decrease from low field to high field (left to right) because the amount of free radical is decreasing with time (see below). Ifeitherthe carcinogen or the LAHP is left out of the reaction mixture, no signal is obtained. The LAHP must be added last in order to obtain a nitroxyl free radical signal. Chart 2 shows the optical difference spectrum of methe moglobin and of hematin plus N-OH-AAF before and after Chart 1. ESR spectra of the hematin- and methemoglobin-catalyzed oxi dation of N-OH-AAF by LAHP. Top spectrum, methemoglobin-catalyzed reac tion, middle spectrum, hematin-catalyzed reaction. The concentrations of methemoglobin, N-OH-AAF, and LAHP were 3.6, 81, and 45 MM, respectively, and the concentrations of hematin, N-OH-AAF, and LAHP were 4.0, 41, and 25 @M, respectively. The temperature was approximately 25°.The spectra were recorded at 25 gauss/mm with a filter constant of 3 sec. The microwave frequency was 9.529 GHz and the modulation amplitude was 5 gauss at a frequency of 100 KHz. Bottom spectrum, potassium nitrosodisulfonate standard. CANCER RESEARCH VOL. 36 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research. @ @- Lipid Peroxide Activation of Carcinogens ment are shown in Chart 3. There is a large and rapid monotonic absorbance decrease at 233 nm which is accom panied by a monotonic 368 nm absorbance increase. As the reaction proceeds, there is an absorbance decrease at 301 nm, but this decrease has 2 phases. The results of another kinetic experiment are presented in Chart 4. The semiloga rithmic plot of the absorbance changes illustrates these results clearly. The 233 nm absorbance decrease and 368 A 368nm 5.01 260 @Ju @ juu 4W 560 360 4@lOmn ôôo lóOnm Chart 2. The optical spectra of methemoglobin or hematin plus N-OH-AAF before and after LAHP addition. A. Trace I, difference spectrum of 3.6 @zM of methemoglobin in 0.05 M potassium phosphate buffer, pH 7.4, to which NOH-AAF was added to the sample cuvet to a final concentration of 40 @M; Trace II, spectrum after LAHP to a final concentration of 42 .tMwas added to the sample cuvet. B. Trace I, difference spectrum of [email protected] hematin in potas sium phosphate buffer to which N-OH-AAF was added to the sample cuvet to a final concentration of 34 @; Trace II, spectrum after LAHP (30 @M) was added to the sample cuvet. LAHP addition. Carcinogen addition to the solutions con taming the heme compounds yields a spectrum similar to that obtained after carcinogen addition to buffer solution only. There is a sharp 301 nm peak, a shoulder at 289 nm, a slight shoulder at 281 nm, and a maximum at 273 nm. This spectrum is very similar to that obtained after N-OH-AAF addition to methanol, exceptthatthe maximum in methanol is 281 nm (7). In separate experiments we have found that the phosphate buffer shifts the maximum to 273 nm. LAHP addition to the solutions containing the heme compounds plus carcinogen caused a decrease in absorbance at 301, 289, and 273 nm, but there is a concomitant 30@nm 0 4 ;3 2 1 0 mm Chart 3. The kinetics of the 233, 301, and 368 nm absorbance changes of LAHP added to a solution containing hematin plus N-OH-AAF. The hematin and N-OH-AAF were added to potassium phosphate buffer (0.05 M, pH 7.4) to a final concentration of 5 and 35 @M, respectively. The carcinogen was added to the sample cuvet only, but hematin was in both sample and reference cuvets. LAHP to a final concentration of 30 MMwas then added to the sample cuvet only, and the time course was recorded at the appropriate wavelength starting 20 sec after LAHP addition. The slit width was no more than 1 nm in any of the measurements, and the temperature was approximately 27*. increase of a broad absorbance centered at 368 nm. This absorbance increase is due to nitrosofluorene accumulation (see below) which absorbs maximally in this region (4). LAHP addition also causes absorbance alterations at about 410 and 420 nm as seen in the methemoglobin solutions (Chart 2A). How ever, in separate experiments (not shown here), these ab sorbance changes can be accounted for by the action of LAHP only on the heme compound. Similar changes of absorbance were observed aftercumene hydroperoxidead dition to microsomes (6). LAHP absorbs maximally at 233 nm, and this occurs at a position such that absorbance from either the heme com pounds or N-OH-AAF does not interfere greatly (see Chart 2). We have conducted kinetic experiments on the LAHP/ hematin/N-OH-AAF system where LAHP disappearance and NOF appearance were determined by monitoring absorb ance changes at233 and 368 nm, respectively. Absorbance changes at 301 nm were also determined as the reaction proceeded, but the interpretation of these absorbance changes is compounded in the sense that, in addition to NOH-AAF, N-OAC-AAF also absorbs strongly at this wave length (9). We have conducted these kinetic experiments and the remainder of the studies reported herein using hematin as a catalyst. The results of a typical kinetic experi AUGUST 1976 tim.(s.c) Chart 4. The semilogarithmic plot of the kinetics of the relative absorb ance changes at 233, 301, and 368 nm following LAHP addition to a solution containing hematin plus N-OH-AAF. The experiments were carried out ex actly as described in Chart 3 except that the concentrations of hematin, NOH-AAF, and LAHP were 5, 37. and 30 @M, respectively. The scale for the 301 nm change is 10 times more sensitive than that for the 233 and 368 nm absorbance changes. 2763 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research. A. A. Floyd et al. nm absorbance increase are kinetically equivalent, with a half-time in this particular reaction of 25 and 26 sec, respec tively. The 30i nm decrease can be broken into 2 phases. The fast phase is kinetically equivalent to the 233 and 368 nm absorbance changes, with a half-time of 24 sec. How ever, there is a slow phase that continues after the fast phase is complete and that has a half-time of 186 sec in the experiment presented in Chart 4. We have conducted many similar experiments, and they all have exhibited this same behavior kinetically. Thus, it is clear that the 233 nm absorb ance decrease corresponds kinetically both to the 368 nm absorbance increase and to the fast phase of the 301 nm absorbance decrease. The total absorbance changes at I .1 233, 301, and 368 nm in the 1st 2 mm of the reaction presented in Chart 3 are 0.13, 0.023, and 0.052, respec tively. After 2 mm of reaction, there was a slight 233 nm absorbance increase at the same time that there was a slow but steady 301 nm decrease which continued for as long as 15 to 20 mm. These slow absorbance changes were ex cluded from the calculations presented above and will be discussed later. Chart 5 shows that ascorbate inhibits the LAHP-induced 301 and 368 nm absorbance changes in solutions contain ing hematin plus N-OH-AAF. The lowest trace is the spec trum of hematin plus N-OH-AAF versus hematin. The upper trace is the spectrum after ascorbate addition to the hema tin plus N-OH-AAF solution only. Ascorbate addition re suIted in an increased absorbance at 265 nm as would be expected. After LAHP addition (middle trace) there was a large decrease in ascorbate, but N-OH-AAF was not con sumed and, in addition, nitrosofluorene was not formed (see below). There was a disproportionate amount of ascor bate consumed to LAHP added (approximately 12/i). In separate experiments (not shown) in which we monitored the kinetics of these components, ascorbate oxidation did not follow kinetically LAHP consumption except for only a small initial component. After the 1st 2 mm, ascorbate oxi dation proceeded in a somewhat linear fashion for 8 to 10 mm after LAHP consumption had ceased. It appears as if the catalytic decomposition of LAHP triggered ascorbate oxidation. The ascorbate radical was present ratherthan the nitroxyl radical during ascorbate prevention of N-OH-AAF oxidation. This is shown clearly in Chart 6. Trace A shows the presence of the nitroxyl free radical of N-OH-AAF during the hematin calalyzed, LAHP-induced oxidation of N-OH-AAF. When ascorbate was present, the nitroxyl radical was not present, but there was a radical present with a g value of 2.005 (Chart 6, Trace C) which split into a doublet (Chart 6, Trace B) when the modulation amplitude was decreased in order to resolve the hyperfine structure. The g value and splitting constant (1.7 to 2.0 gauss) fit the properties of the ascor bate radical (18). The nitroxyl radical of N-OH-AAF was monitored on a kinetic basis (Chart 6, Trace D) in the LAHP/ hematin/N-OH-AAF systems. Analysis of the time course of the nitroxyl radical disappearance in these reactions re vealed that in most cases the decay curves were usually biphasic, the kinetics of which did not correlate rigidly with the optical absorption changes discussed previously (Charts 3 and 4). Chart 6, Traces E and F illustrates that, 2764 2@t 360 4ô0 5ô0 6ôOnm Chart 5. Optical difference spectra of the effect of LAHP addition to a solution of hematin plus N-OH-AAF containing ascorbate. The sample and reference cuvets contained 5 MM hematin in potassium phosphate buffer. Bottom trace, spectrum after N-OH-AAF was added to a final concentration of 36 MMto the sample cuvet. Top trace, spectrum after ascorbate addition (61 @.tM);middle trace, spectrum after LAHP (5 @M)was then added to the sample cuvet. when ascorbate was present in the system, the ascorbate radical was present in the early phases of the reaction at which time the nitroxyl radical was absent. However, after the ascorbate radical disappeared, the nitroxyl radical ap peared. Trace G shows that a small amount of nitroxyl radical was present when as small a concentration of LAHP as 1.8 j.@M was added to the system. Fig. 1 presents the results of TLC of the hematin-cata lyzed LAHP oxidation of N-OH-AAF. The results show that NOF and N-OAC-AAF are produced in the hematin-catalyzed reaction as is true for the horseradish peroxidase/H2O2catalyzed oxidation of N-OH-AAF. The horseradish peroxi dase reaction system yielded 2 major products correspond ing to N-OAC-AAF and NOF (Fig. 1A, Lane 5). This result agrees with that observed by Bartsch and Hecker (2) and ourselves (7). NOF and N-OAC-AAF are also the products of the hematin-catalyzed reaction (Fig. 1A, Lane 6). Ascorbate prevents the formation of these prod ucts as is shown in Fig. iB , Lane 2. Thus, the TLC experiments corroborate what we deduced from the optical studies presented earlier (Chart 5). In the hematin-catalyzed reaction, there was some N-OH AAF remaining at the termination of the reaction, but this was not true of the peroxidase-catalyzed reaction. There was a small amount of AAF formed in both the hematin- and peroxidase-catalyzed reactions. On the basis of visual com parison of spot intensity there is less N-OAC-AAF than NOF formed in the hematin-catalyzed reaction. The reaction shown in Fig. 1 was stopped after 9 mm, but if the reaction CANCER RESEARCH VOL. 36 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research. Lipid Peroxide Activation of Carcinogens A was stopped after only 2.5 mm (experiments not shown), then the N-OAC-AAF spot was nearly as intense as the NOF spot. Thus, we conclude that the continued slow decrease at 301 nm (see Chart 4) is due to a decrease in the amount of N-OAC-AAF present. The products B . of N-OAC-AAF decompo sition are not known under our conditions. r DISCUSSION The data presented in this paper demonstrate that a lipid hydroperoxide in the presence of a heme compound acti vated N-OH-AAF via the nitroxyl free radical intermediate of N-OH-AAF into the products nitrosofluorene F. and N-acetoxy acetylaminofluorene. The reaction was inhibited by ascor bate, in which case the free radical of N-OH-AAF was re placed by the ascorbate free radical. We do not know the detailed mechanism of the reaction p. i .. and, in fact,we are studying itingreaterdetailnow. How @ ever, there are certain characteristics that the present study does make clear, namely, that the rate of LAHP disappear ance parallels the rate of NOF accumulation and that the 2 3456 123 Fig. 1. TLC of the hematin/LAHP-catalyzed N-OH-AAF oxidation reaction as effected by ascorbate. A. The conditions for each ofthe lanes are: Lane 1, AAF standard; Lane 2, N-OH-AAF standard; Lane 3, NOF standard; Lane 4, NOAC-AAF standard (the large spot corresponds to N-OAC-AAF, the smaller spots are breakdown products as described in Ref. 7; Lane 5, horseradish peroxidase/H202/N-OH-AAF system as described previously (7); Lane 6, hem atin/LAHP/N-OH-AAF system similar to that described in Chart 3. B. Lane 1 corresponds to AAF standard, Lane 3 corresponds to the NOF, and Lane 2 is as described in Lane 6 of A , but ascorbate was present as described in Chart 5. rate of LAHP disappearance parallels the rate of the fast phase of the 301 nm absorbance decrease, which in large part is due to N-OH-AAF disappearance. An examination of the stoichiometry of the reaction also allows certain conclu sions. In Chart 3 it was noted that the absorbance changes D at233, 301 ,and 368 nm were 0.13,0.023,and 0.052 absorb Chart 6. ESR spectra and kinetics of the hematin plus N-OH-AAF solution with or without ascorbate present after LAHP addition. Trace A, ESR spec trum started 20 sec after the addition of LAHP (10 @.tM) to a phosphate buffer solution containing 5 @M hematin and 40 @M N-OH-AAF. The modulation amplitude was 5 gauss, microwave frequency 9.529 GHz, scan rate 20 gauss/ mm, filter constant 3 sec. and the receiver gain 4 x 10g. Trace B, ESR spectrum of a similar solution as in Trace A except that 170 @M ascorbate was also present. The conditions are the same as in Trace A except that the modulation amplitude was 2 gauss. Trace C, ESR spectrum of a solution exactly as in Trace B except that the modulation amplitude was 5 gauss. Trace 0, time course of the decay of the nitroxyl free radical monitored by setting the field at the maximum of the low field transition. The solution and the conditions used were as in Trace A. Trace E, time course of the decay of the ascorbate free radical monitored by setting the field at the maximum of the over-modulated ascorbate radical transition (Trace C). The conditions otherwise are as in Trace A. The solutions used are as in Traces B and C. Trace F, time course of the nitroxyl radical in the solutions to which ascor bate was present. The nitroxyl radical was monitored at the field position as described in Trace D. The solutions used were exactly as in Traces B, C, and E. Trace G, time course of the nitroxyl free radical in a phosphate buffer solution containing 10 @M hematin and 40 @M N-OH-AAF to which LAHP was added to a final concentration of 1.8 @M. The free radical was monitored as described in Trace 0; otherwise, the conditions were as described for Trace A. Downward arrows of Traces 0, E, F, and G, time zero in the reaction; upward arrows, time when reaction mixture was injected into the cavity. AUGUST 1976 ance units, respectively. On the basis of the known absorb ance of the compounds, we calculated that for every 2 LAHP molecules consumed 1 NOF molecule accumulated. The absorbance change at 301 nm is more complicated since NOAC-AAF absorbs stronger than N-OH-AAF at this wave length and since NOF would contribute some absorbance at this wavelength. If we presume that for every 2 LAHP mole cules decomposed, 2 N-OH-AAF molecules are oxidized which yield 1 N-OAC-AAF and 1 NOF molecule, then using the known absorptivities of N-OH-AAF, N-OAC-AAF, and NOF at 301 nm we can account for0.0189 ofthe observed 0.023 (or 82%) of the absorbance change at 301 nm, by the above mechanism. As was noted previously, N-OAC-AAF is decreasing with time and, since the absorbance changes were calculated at 2 mm, we would have overestimated the N-OAC-AAF contribution; thus, this would increase the cal culated 82% value closer to the expected 100%. On the basis of the above considerations, we postulate that the heme compound causes LAHP to decompose; either the decomposition products or the decomposition process per se oxidizes N-OH-AAF into the nitroxyl free radical of N-OH AAF. Two of these radicals then form NOF and N-OAC-AAF, perhaps by a dismutation process. The postulated reaction is presented in Chart 7. The reaction as written is not com plete, for another product, perhaps a lipid alcohol, should 2765 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1976 American Association for Cancer Research. A. A. Floyd et al. @-H H H c5@' C—C—'C—(CH2)7—COOH ‘I 2 CH3—(CH2)4—C—C H @ @ CH@1 1 I NOF LAHP + @— hematin ,. 2L@J@J3@. _J —@ + 0 I 1 2(@i―@T'@T'@ —C—CH3 0 @@@yN—C—CH3 O—C—CH3 0 N—OH—AAF N —OAC—AAF Chart 7. Proposed reaction scheme for the LAHP/hematin-catalyzed activation of N-OH-AAF into N-OAC-AAF and NOF. also be represented. We are currently investigating the re action mechanism in greaterdetail. The important point, as far as carciiogenesis is concerned, is that more active carcinogens are formed in the reaction. It cannot be stated unequivocally on the basis of the present results that the reaction is proceeding via an obliga tory nitroxyl free radical intermediate; however, all the ob servations presented here certainly indicate strongly that this is true. The lack of rigid correlation between the rate of nitroxyl radical decrease and the rate of N-OH-AAF disap pearance and NOF appearance certainly does not rule out the obligatory free radical intermediate concept. The amount of the nitroxyl free radical present at any time would depend on the ratio of its rate of formation to its rate of decay. It would be expected that the ratio of these 2 rates would not necessarily remain constant as the reaction pro ceeded and the substrates became depleted. Also, it is possible that N-OAC-AAF may decompose via the nitroxyl free radical form. Bartsch and Hecker (2) discovered that horseradish per oxidase plus H2O2would activate N-OH-AAF into nitrosoflu orene and N-acetoxyacetylaminofluorene. However, in an attempt to extrapolate their horseradish peroxidase obser vations, Bartsch and Hecker (2) were unable to recover Nacetoxyacetylaminofluorene or were unable to observe the nitroxyl free radical in lipid peroxidizing microsomes; yet Stier et al. (20) did obtain nitroxyl free radical spectra in chloroform/methanol extracts of microsomes metabolizing arylamine carcinogens. The fact that we have observed the nitroxyl free radical with as small a concentration of lipid hydroperoxide as 1.8 @M does force a consideration of the activation of N-OH-AAF by lipid peroxidation products when a heme protein such as hematin or methemoglobin is pres ent. We have also observed that ascorbate inhibits the lipid hydroperoxide plus heme compound activation of N-OH AAF. This was also true of the horseradish peroxidase/H202 activation of N-OH-AAF (7). From our results alone, it is not possible to determine the action of ascorbate in the system. We speculate that ascorbate is reducing the nitroxyl free radical back to N-OH-AAF, since N-OH-AAF was not oxi dized when ascorbate was present (see Chart 5). However, ascorbate involvement in the reduction state of the heme cannot be ruled out. Ascorbate radical was not present when ascorbate was added to hematin solution per se. This observation would tend to exclude the oxygen scavenger action of ascorbate here. On the basis of our results, a protective effect for ascor 2766 bate against AAF carcinogenesis is implicated. Perhaps other free radical scavengers such as vitamin E and other antioxidants would inhibit the reaction described herein. Many observations indicate that antioxidants do exert an inhibiting influence on carcinogenesis; conversely, lipid peroxidation may enhance carcinogenesis (see Ref. 19 and references therein). The reaction we describe here does provide a theoretical model to support these observations, especially in AAF carcinogenesis. More detailed studies, now underway, are necessary before generalizations can be drawn. ACKNOWLEDGMENTS I would like to thank Dr. Helmut Bartsch for suggesting the use of diethyl azodicarboxylate as an oxidizing agent to form nitrosofluorene and Dr. E. K. 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