Chem. Res. Toricol. 1993,6, 23-27 23

Reactions of the Bioregulatory Agent Nitric in Oxygenated Aqueous Media: Determination of the Kinetics for Oxidation and Nitrosation by Intermediates Generated in the NO/O2 Reaction

David A. Wink,*pt John F. Darbyshire,t Raymond W. Nims,f Joseph E. Saavedra,t and Peter C. Fords Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21 702, Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, and Department of Chemistry, University of California, Santa Barbara, California 97106

Received September 28, 1992

The reaction kinetics of autoxidation in aerobic solutions were investigated by direct observation of the nitrite product and by trapping the strongly oxidizing and nitrosating intermediates formed in this reaction. The rate behavior observed for nitrite formation [rate = k3[0~][NO]~,k3 = (6 f 1.5) X lo6 M-2 s-l at 22 "CI was the same as found for oxidation of Fe(CN)s" and of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonicacid) (ABTS) and as for the nitrosation of sulfanilamide. There was a slight decrease in k3 to (3.5 f 0.7) X lo6 M-2 s-l at 37 "C. The second-order dependency for NO was observed at NO concentrations as low as 3 pM. The results of the competitive kinetics studies suggest that the key oxidizing intermediates, species which are both strong oxidants and nitrosating agents, are not one of those commonly proposed (NOz, Nz03, NO+, or OZNO-)but are one or more as yet uncharacterized NO, species.

Introduction from Fisher Scientific Co. (Fairlawn, NJ), and 2,2'-azinobb(3- ethylbenzthiazoline-6-sulfonicacid) (ABTS) was obtained from A recent discovery in mammalian biology has been the Sigma Chemical Co. (St.Louis, MO). The above-named reagenta important physiological role that nitricoxide plays in blood were used without further purification. Nitric oxide gas was pressure regulation, neurotransmission, macrophage-in- purchased from Potomac AirGas (Frederick, MD) and paseed duced cytostasis and cytotoxicity,and inhibition of platelet through a 10 N KOH solution to remove other NO. species. Nitric aggregation, etc. (1,2). Furthermore, it has been shown oxide solutions were made by degassing aqueous buffer Solutions, that intermediates derived from aerobic solutions of NO followed by introduction of nitric oxide, as described previously (12). Nitric oxide concentrations were determined by the cause mutations in bacterial and mammalian cells, sug- oxyhemoglobin method (13). concentrations were de- gesting a possible involvement of endogenously formed termined with a dissolved oxygen electrode (Orion Reaearch Inc., NO in a genotoxic mechanism (3-6). However, despite Boston, MA). No appreciable effect was observed between evidence that NO has a short half-life in aerobic aqueous aqueous buffers treated or untreated with Chelex 100 (Bio-Rad, systems (1,7-1 l),the chemical intermediates and kinetics Richmond, CA). involved in the aerobic oxidation of this critically important The kinetics of nitrosation by the NO/Oz reaction were species have not been completely elucidated. Clearly, the measured via diazotization of sulfanilamide and subsequent quantitative characterization of the reaction between NO coupling with NEDD to form the azo dye (14). Sulfanilamide and 02 under biologically relevant conditions is essential (25 mM) and NEDD (2.5 mM) were dissolved in 100 mM to understanding the roles of this bioregulatory agent in phosphate buffer (pH 7.4) by stirring for 10 min. The resulting living systems. Described here are kinetics studies of this solutions were filtered with a Nylon-66 syringe filter (Rainin, reaction, which demonstrate that there are fundamental Woburn, MA) to remove any small particulates. Introduction differences between the intermediates generated during of 10-100 pM NO in this aerobic solution yielded an absorbance band at 496 nm which increased with [NO] such that CNO = 950 the oxidation of NO in the gas phase and those generated 130 M-l cm-l. Addition of acid to the solution resulted in an in the same reaction occurring in aqueous solution. absorption band at 546 nm indicative of the corresponding azo dye [lit. 545 nm (14)l. The absorption at 496 nm was not Materials and Methods generated in the presence of as much as 100 mM NaNOa at pH 7.4. Sodium azide, sulfanilamide, N-(1-naphthy1)ethylenediamine Peroxynitrite anion was made as followe: a 25-mL basic dihydrochloride (NEDD),l , and nitroso- solution (0.1 M KOH) was degassed, and the solution was nium tetrafluoroborate were obtained from Aldrich Chemical transferred to a septum vial containing 53 mg of Koa, followed Co. (Milwaukee, WI). Potassium ferrocyanide was purchased by degassing on ice. Nitric oxide was then bubbled through the solution, and after 1min on ice, the headspace was evacuated to To whom correspondence should be addressed. remove excess nitric oxide, which was replaced with argon. The + National Cancer Institute. resulting solution gave a broad band at 300 nm in basic solution t National Heart, Lung, and Blood Institute. characteristic of peroxynitrite anion (OONO-) (15). I University of California. 'Abbreviations: NEDD, N-(1-naphthy1)ethylenediamine dihydro- Kinetic modeling simulations were performed with Stella 2.1 chloride; ABTS, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonicacid). software from High Performance Systems (Hanover, NH).

This article not subject to U.S. Copyright. Published 1993 by the American Chemical Society 24 Chem. Res. Toxicol., Vol. 6, No. 1, 1993 Communications

200

150

100

0.04 50

0.00 5 10 15 20 0 5 10 15 20

kobs 0.12 -1 S

0.00 5 10 15 20 0 5 10 15 20 -v 0 a e Q 0 W 12 1 2 3 4 2? '2- 0 $--jq [N0l2 mM 2 04 Figure 2. Plot of hob (9-1) vs [N0I2(mM2) for the formation of nitrite (A),Fe(CN)e3- (O), and ABTS+ (0)at pH = 7.4 and 22 0 5 10 15 20 0 5 10 15 20 "C when [Oz] = 0.04 mM. The conditions for nitrite formation were 1 mM phosphate buffer (pH 7.4) and [021 = 0.04 mM at 22 "C. The conditions for Fe(CN)e3-andABTS+ formation were 100 mM phosphate buffer (pH 7.4) and [Oz] = 0.04 mM with the 0.04 reductants [Fe(CN)e4-]= 50 mM and [ABTS] = 10 mM, ;pjrespectively. The slope for nitrite formation is 4.78 X 106 M-2 ::::r;/0.02 s-l (IC = 0.996), that for F~(CN)G~-oxidation is 5.08 X lo6M-2 s-l 0.00 (IC = 0.997), and that for ABTS oxidation is 5.23 X 106 M-2 8-1 (IC = 0.997). The inset plot represents the oxygen dependence 0 5 10 15 20 0 5 10 15 20 of ferrocyanide oxidation (O),where [Fe(CN)e4-I= 50 mM and Time (sec) Time (sec) [NO] = 0.04 mM, as well as nitrosation and diazotization of sulfanilamide and NEDD (A),where sulfanilamide was 25 mM, Figure 1. (A) Absorbance changes at 216 nm with time as NEDD was 2.5 mM, and [NO] = 0.26 mM. The slopes of the detected with stopped-flow techniques for a 1.0 mM phosphate lines were 5.6 X 106 M-2 s-l and 5.3 X lo6M-2 s-l for ferrocyanide buffer (pH 7.4) containing 0.1 mM NO and 1 mM 02 at 22 OC. oxidation and sulfanilamide nitrosation, respectively. (B)Absorbance changes at 420 nm [representingthe appearance of Fe(CN)s3-],where the [Fe(CN)64-]= 50 mM in 100 mM phosphate buffer (pH 7.4), [NO] = 0.1 mM, and [02] = 1.0 mM. was linear with a slope of k3 = (4.8 f 1.0) X lo6 M-2 s-l (C) Absorbance changes at 600 nm for the appearance of ABTS+, (Figure 2), effectively within experimental uncertainty of where [ABTS] = 50 mM. (D) Absorbance changes at 500 nm for the value of k2/[021 = (6.3 f 1.2) X lo6 M-2 s-l derived the appearance of the diazotization product, where sulfanilamide was 25 mM and NEDD was 2.5 mM in 100 mM phosphate buffer from the experiment above. Thus, formation of nitrite (pH 7.4), [NO] = 0.1 mM, and [02] = 0.9 mM. Panels E-H are ion in this system is governed by a third-order rate law: plots of l/(A- - A) vs time for each absorbance change. The slopes of the regression lines are 6.23 s-l [linear correlation (IC) d[NO;l/dt = k3[0,1 [NO]' (2) = 0.9181 for panel E, 7.59 s-l (IC = 0.998) for panel F, 0.99 s-l (IC = 0.998) for panel G, and 5.7 s-l (IC = 0.998) for panel H. These data confirm the results of earlier studies conducted at millimolar concentrations of NO and 02 in water (8.8 Stopped-flow experiments were carried out with a Hi-Tech X 106 M-2 s-l; 18) and in CCl, NO^ 2.8 X lo6 M-2 s-l, Scientific Multi-Mixingstopped-flow spectrophotometer, Model d[NOz]/dt = k~o~[O2][No]~;19). SF-51MX (16),with IS 1.0 Rapid Kinetics Software Suite (Hi- The reaction and 02 under these conditions Tech Scientific Limited, Salisbury, Wiltshire, England). of NO generates an intermediate that rapidly oxidizes Fe(CN)64- Rssults and Discussion and ABTS to Fe(CN)s3- and ABTS+,respectively. These reactions can be monitored at 420 nm (Lie420 = LOO0 M-l Nitrite is the product of NO oxidation in aerobic aqueous cm-', Figure 1B,F) and 600 nm (ACW = 6600 M-' cm-l, solution (9, 17, 18). Figure lC,G), respectively. Under limiting [NO], these reactions display second-order kinetics with k2 values of 4N0 + 0, + 2H20- 4H' + 4NO; (1) (7.2 f 1.4) x 103 and (6.5 f 1.2) X lo3M-l s-l, respectively, We have investigated the reaction of NO (100 pM) plus the latter values being within experimental uncertainty 02 (1.0 mM) in pH 7.4 aqueous solution with a stopped- of those observed above for nitrite formation. Similarly, flow spectrophotometer by monitoring absorbance in- when these reactions are carried out under limiting [021, creases at 216 nm (k216 = lo00 M-' cm-') due to the the kobs values again prove to be dependent on [N012, formation of nitrite (Figure 1A). A plot of (Abs, - Abs)-1 with the respective k~ values, (5.1 f 1.0) X lo6 and (5.2 f vs time proved to be linear (Figure lE), consistent with 1.0) X 106 M-, s-l (Figure 2), again being equivalent to the a second-order rate dependence on the limiting reagent, values for nitrite formation. Since the trapping agents NO. The rate constant kp (=slope X Ad so determined are not oxidized by 02, NOz-, or anaerobic NO, the rate was (6.3 f 1.2) X lo3 M-l s-l. When the reaction was data clearly imply that Fe(CN)s4-and ABTS are both being carried out similarly, but with 02 (40 pM) as the limiting oxidized by intermediate species formed subsequent to reagent ([NO] = 0.17-1.7 mM), first-order behavior was the rate-limiting step in the sequence leading to NO observed, and a plot of the resulting kobs values vs [N012 oxidation by 0,in aqueous media. Communications Chem. Res. Toxicol., Vol. 6, No. 1, 1993 26

A8 noted above, the NO/Oz system has been shown to deaminate nucleosides (3, 4) and, therefore, has the potential to cause genotoxicity. This activity has been attributed to nitrosation of the exocyclic amino groups of the nucleosides. In this context, the components of the Greiss reaction [a colorimetric assay for nitrite which involves the nitrosation under acidic conditions of sulf- anilamide by NO+ donors followed by diazotization and coupling with NEDD to form an azo dye (1411 were used to examine the kinetics of nitrosation by intermediate(s) generated during the NO/Oz reaction in aqueous solution at physiological pH. Addition of 100 pM NO to a 0.9 mM 1/[Fe(CN),4-] (mM)-' 02/100mM phosphate solution (pH 7.4) containing 25 200 mM sulfanilamide and 2.5 mM NEDD generated an B absorption at A, = 496 nm, indicative of the characteristic azo product resulting from nitrosation (Figure 1D). Preliminary stopped-flow studies under limiting [NO] show an increase in absorption at 500 nm similar to that of the formation of nitrite and oxidation of Fe(CN)e4-and ABTS observed above in Figure 1. A plot of (A, - A1-l versus time was linear (Figure lH, At~0= 950 f 150), k2 X giving a = (6.0 f 0.7) lo3M-l s-l. As shown in Figure 0- -1 0 123 4 5 6 2 inset, an 1023 dependence was observed such that k3 = (5.3 f 0.5) X 108 M-2 s-l. Thus, it appears that this nitrosation, which may serve as a model for the deami- [N~-I(m~) nation of nucleic acids by NO, also proceeds via the Figure 3. (A) Doublereciprocal plot of the changes in absorbance formation of the same intermediate(s1 as the oxidation at 420 nm with varying Fe(CN)e4-concentration when [NO] = reactions described above. 1.2 mM and [Oz]= 0.04 mM (0)and when [NO] = 0.12 mM and [Og] = 1 mM (H) with a l/Xint= 0.48 mM. (B)The reciprocal It is worthwhile to consider the implications of the of the change in absorbance at 420 nm (appearance of ferricyanide) present observations vis-a-vis the expected reactivities of with varying azide concentration,with an x intercept of 0.437 M. NO under physiologically relevant conditions, where The conditions were 10 mM phosphate buffer (pH 7.4), with maximal concentrations of NO in the cellular microen- [NO] = 0.17 mM and [Oz]= 1 mM. vironment are estimated to be in the range of 0.45-10 pM (18-22). The mechanistic requirement that NO oxidation out using the following two initial [NO], 0.17 and 1.2 mM, be second-order in [NO] allows one to predict half-lives and with initial [021= 1mM and 40 pM, respectively, and of 1-500 s in air-saturated aqueous solution (1021 = 220 the total absorbance change at 420 nm [corresponding to pM, tlp l/(c&)) (23). The validity of this prediction the formation of Fe(cN)e3-] was determined. If NO2 were was tested by carrying out the oxidation of ABTS at low the intermediate intercepted by Fe(CN)&, plots of NO concentrations. When a solution (pH 7.4, 22 "C) [Abs420l-lvs [Fe(CN)e4-1-l would give X, values equal to containing 5 mM ABTS plus 220 pM 02 was rapidly mixed the ratio k~o[NOl/k~,.~However, instead of the predicted with a solution of NO (3-10 pM) by means of stopped- Xm values 62 and 440 mM, both plots (Figure 3A) gave Xm flow techniques, the absorbance increase at 600 nm, values of -2 mM. Hence, we conclude that NO2 is not indicative of ABTS oxidation, followed second-order the species being intercepted by Fe(CN)e4- under these kinetics with a k2 of (5.0 f 0.8) X 106 M-2 s-l, identical to conditions. the above. The first half-life under these conditions ranged From a biological perspective, the most important from 10 to 333 8. At 37 "C,similar second-order behavior feature of the NO/O2 reaction is that the reactive inter- wasobaerved, although the resulting k3 was slightly smaller, mediates generated are capable of both oxidation and (3.5 f 0.5) X 106 M-2 s-~.~In a previous report, little or nitrosation. To examine the relationship between these no difference was detected in the observed rate between pathways, a known NO+ acceptor (28,291, sodium azide, 15 and 30 "C in aqueous solution (18). was used to quench the oxidation of Fe(CN)e4- in a It has been widely speculated, in analogywiththe better competition study. When the azide concentration was characterized gas-phase mechanism, that NO2 is a key varied from 0.1 to 10 mM, with [Fe(CN)&] = 20 mM, reactive intermediate formed in the oxygenation of NO in [NO] = 0.12 mM, and 1021 = 1 mM, a decrease in the aqueous media or in vivo (4, 7,24,25)and, furthermore, amount of Fe(CN)6* formed was observed. A plot of 1/Abs that NO2 is a species responsible for oxidative damage by vs [N3-1 is linear, with Xi = 0.43 f 0.05 mM (Figure 3B).' such systems (26). The role of NO2 in the aqueous These results allow one to evaluate the relative selectivity oxidation may be tested by carrying out competition between different reaction pathways. Since the slopes of kinetics, since the second-order rate constants for the the plot in Figure 3A are independent of [NO], it can be reactions of NO2 with NO (1.1X lo9M-' 8-l) andFe(CN)e4- surmised that the competing pathway is unimolecular (e.g., (3 X lo6 M-' 8-l) have been determined under analogous conditions (17,27). A series of experiments was carried 3X, is equal to the negative reciprocal of the abscissa intercept (Xi.3. In this case, the numerical value of X, is equal to the concentration of *While somewhat surprising, the small negative E. parallels similar Fe(CN)e4-at which half the intermediate is trapped by this quenching behavior in the gas-phase kinetics which is not yet fully explained. In agent to give Fe(CN)sS-. a multistep mechanism, a negative E. is often the result of a reversible 4x1 is equal to the negative of the abscissa intercept (XhJ. In thie equilibrium, with a negative AHo occurring prior to the rate-limiting case, the numerical value of Xi is equal to the concentration of N3- at step. which half the Fe(CN)e3- is quenched and where k.dNs-1 = k~#[Fel. 26 Chem. Res. Toxicol., Vol. 6, No. 1, 1993 Communicatiom involving protonation or hydrolysis). From the X, (Figure the key intermediate is the NO dimer (19,31). However, 3A) and Xi (Figure 3B) values, the relative rate constants it appears that either intermediate would further react to kazida:kFe:kH Can be calculated 88 (2 x 104):500:1. produce a novel reactive intermediate(s). This relative selectivitycan be used to test other possible The chemical reactivity of NO and 02 provides insight intermediates. One possible candidate would be N203 into the bioregulatory functions of NO, as well as its derived from rapid trapping of NO by NO2 via the cytotoxic and genotoxic effects. Similarities between the mechanism described in the following equations: observed rate expressions for protic (this work and ref 18) NO versus nonprotic (19)solutions suggest that, regardless of NO + O2 '02N0 - 2N02 (3) the hydrophilicity or lipophilicity of the biological media, these kinetics will govern the half-life of NO. The second- Hz0 order (NO) dependency of this reaction dictates that the NO2 + NO -+ N203 2NOi + 2H+ (4) - half-life of NO be inversely proportional to its concen- The published rate constant for the reaction of azide with tration, and the total amount of reactive intermediates N2O3 is 2 X lo9M-' s-l (28)and that for the hydrolysis of produced in NO autoxidation be proportionalto the square NzO3 is lo00 s-l (In,a ratio of 2 X lo6 M-l. This ratio is of its concentration. In the course of various bioregulatory 100-fold larger than one observed for the trapping of the activities (such as blood pressure lowering, neurotrans- NO/Oz-reactive intermediate in the experiments described dssion, and inhibition of platelet aggregation)relatively above. This argues against the intermediate being Nz03 lob levels of NO are generated; thus even in the presence and reinforces a similar conclusion drawn from results of of high concentrations of 02, there should be sufficient the deamination of nucleosides by aerobic NO, for which time for NO to reach target sites such as guanylate cyclase it was noted that addition of nitrite for the purpose of without being consumed by oxygen. However, stimulated generating Nz03 dramatically increased the extent of macrophages are estimated to generate as much as lo00 deamination (3). Kinetic modeling of the experiment times higher concentrations of NO (25,321,under which described in Figure 3B,assuming the intermediacy of NO2 conditions a large flux of reactive intermediates must be and Nz03 (formed by rapid reaction of NO and 02) and generated, resulting in cytotoxicity or cytostasis (I). As using published rate constants (1 7,27), predicted that at NO diffuses from these sites, the flux of the reactive 10 mM rN3-1 only 40% of the observed Fe(CN)s3- intermediates would be expected to decrease and the half- formation should be quenched. Instead, complete quench- life of NO would be expected to increase. This increased ing was observed,a discrepencywhich appears to preclude half-life would allow detoxication of this radical species the mechanism described by eqs 3 and 4. by other pathways, possibly via the reaction of NO with Other possible intermediates are the OONO- and the oxyhemoglobin or other oxyhemoproteins to yield nitrate, nitrosyl cation (NO+). Preliminary studies of the reaction a product not otherwise formed in the NO/Oz reaction in of Fe(CN)s4- with OONO- show that the oxidation of aqueous solution. Fe(CN)s4- to F~(CN)G~-is not appreciably quenched in the presence of 15 mM azide. Thus, the peroxynitrite Acknowledgment. P.C.F. acknowledges support by anion does not behave in the manner seen for the key the National Science Foundation (Grant CHE-9024846). oxidant in the NO/O2 reaction, which was intercepted by We thank Dr. Yoichi Osawa for assistance with the ABTS N3-. The nitrosyl cation (NO+) might be a more likely work, Dr. David Stanbury for helpful conversations, and candidate as a nitrosating agent. This species would also Drs. Christopher Michejda, James R. Gillette, Steven be expected to undergo hydrolysis to nitrite, and to Tannenbaum, and Larry Keefer for assistance and com- nitrosate amines or azide and to oxidize Fe(CN)s4- in ments on the manuscript. aqueous solution. The possible role of NO+ was examined by competitionstudies in which solid BFdOwas dissolved References in pH 7.4 aqueous solutions containing varying amounts (1) Moncada, S., Palmer, R. M. J., and Higgs, E. A. 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L., Penman, B. W., Wiehnok, being the key intermediate. J. S., and Tannenbaum, S. R. (1992)DNA damage and mutation in The above experiments provide evidence against NOn, ' human cells exppeed to nitric oxide. Proc. Natl. Acad. Sci. U.S.A. N2O3, ONOz, or NO+ being the key intermediate respon- 89,3030-3034. (5) Arroyo, P. L., Hatch-Pigott, V., Mower, H. F., and Cooney, R. V. sible for the strongly oxidizing and nitrosating properties (1992) Mutagenicity of nitric oxide and ita inhibition by antioxidants. of the intermediate generated in the reaction of NO with Mutat. Res. 281, 193-202. 02. We are thus left with a mechanism involving novel (6) Ieomura, K., Masatsugu, C., Teraniehi, K., and Hamada, K. (1984) N,OYn- intermediates which differ from those interme- Induction of mutatione and chromosome aberrations in lung celln following in vivo exposure of rata to nitrogen . Mutat. Res. diates proposed for the gas-phase NO/O2 reaction mech- 136, 119-125. anism. 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