Accelerating Effect of Ascorbic Acid on A/-Nitrosamine Formation and Nitrosation by Oxyhyponitrite1

[CANCER RESEARCH 39, 3871-3874, October 1979] 0008-5472/79/0039-OOOOS02.00 Accelerating Effect of Ascorbic Acid on A/-Nitrosamine Formation and Nitrosation by Oxyhyponitrite1

Shaw-Kong Chang,2 George W. Harrington,5 Marc Rothstein,3 William A. Shergalis,4 Daniel Swern, and Saroj K. Vohra.

Department of Chemistry, Temple University. Philadelphia. Pennsylvania 19122, and the Fels Research Institute. Temple University, Philadelphia. Pennsylvania 19140

ABSTRACT ments to be made readily during the initial and intermediate stages of reaction. DPP as used in this study avoids this The reaction of nitrite ion with ascorbic acid and its effect on difficulty and allows reactions to be studied in situ, yielding the rate of nitrosation of secondary amines have been investi interesting results. The use of DPP as an analytical method for gated by differential pulse polarography in aqueous acidic A/-nitrosamines and as a technique to study some of the chem solution. Ascorbic acid shows nonuniform behavior: it accel istry of W-nitrosamines has been previously reported by us (6- erates the nitrosation of N-methylaniline between pH 1.00 and 8, 31, 32). 1.95, allows the nitrosation of diphenylamine and iminodiacetonitrile, but inhibits the nitrosation of secondary amines, such MATERIALS AND METHODS as dimethylamine, diethylamine, proline, hydroxyproline, N- methylaminoacetonitrile, N-methylaminopropionitrile, and sar- Instrumentation. The instrumentation, cells, and conditions cosine. The nitrosating agent generated by the reaction be used for DPP and spectral studies have been previously de tween ascorbic acid and nitrite ion appears to be oxyhyponitrite scribed (6, 7). The pH was measured with a Leeds & Northrup ion (N2CV2). expanded-scale pH meter. Constant temperature studies were made by using an Exacal 300 temperature controller. Reagents. Diphenylamine and iminodiacetonitrile were re- INTRODUCTION crystallized twice from methanohwater and benzene, respec The importance of A/-nitrosamines as carcinogens (16) and tively. NMA was purified by vacuum distillation. The A/-nitros- the ready availability of the necessary precursors, nitrite and amines were prepared in the usual way (30). The sodium salt amines, in the environment and in food products have prompted of oxyhyponitrous acid was prepared by the method of Naik et many investigators to study the kinetics of nitrosamine forma al. (23). These workers obtained the salt as Na2N2O3; our tion (11 -13, 15, 17, 20, 24, 25, 29). Of particular interest has results show it to be the monohydrate Na2N2O3-H2O. The been the search for inhibitors to block the formation of N- hydrate can be converted into the anhydrous form by heating in a vacuum at 120Â°. nitrosamines (14,21). Ascorbic acid and ascorbates have been among the most popular compounds studied probably because Na2N2O3-H2O of their presumed safety in various food and drug products. Calculated: N 20.01, Na 32.84 The effect of ascorbic acid on the formation of /v-nitrosocom- Found: N 20.04, Na 33.34 pounds in vitro and in vivo has been examined (2, 10, 18, 19), The water used was triple distilled, and the nitric oxide was and the kinetics of the reaction between ascorbic acid and Matheson CP. All inorganic reagents were reagent grade and nitrous acid have been studied (9). Ascorbic acid effectively were used without further purification. inhibits the formation of many nitrosamines, including dimeth- Procedures for Rate Studies. A stock solution of 5.0 mM ylnitrosamine, methylnitrosourea, nitrosomorpholine, and mon- NMA was prepared in an appropriate mixture of NaCIO4 and onitrosopiperazine, but it is not completely effective in blocking HCIO4 so that the solution was 0.10 M in CIO4~ after pH the formation of NMNA6 (18). adjustment. A portion (20.0 ml) of this solution was placed in Because of the current interest in vitamin C and its suggested the polarographic cell and deaerated with N2. A stock solution role in cancer prevention and nitrosamine formation (3), we are of NaNO2 was prepared in the appropriate mixture of NaCIO4 reporting preliminary observations on the interaction of nitrite and HCIO4, and a small aliquot was delivered to the cell, so ion with secondary amines in the presence and absence of that the solution was 0.10 mwi in NO2~. The solution was then ascorbic acid. stirred magnetically for 10 to 15 sec. Nitrite was the limiting Kinetic studies on nitrosamine formation performed to date reagent in all cases. When ascorbic acid was used, it was usually use spectroscopic or Chromatographie techniques re prepared in the corresponding NaCIO4:HCIO4 mixture, and an quiring special sampling techniques that do not allow measure- aliquot was delivered to the cell prior to nitrite addition to make 1 This investigation was supported by Grants CA-18618 and 12227 awarded the solution 0.80 mM in ascorbic acid. All solutions were freshly made before each run. The pH's studied were 1.00, 1.20, by the National Cancer Institute, HEW. 2 Present address: Johnson & Johnson Co., New Brunswick. N. J. 3 Present address: Leeds & Northup Co.. North Wales. Pa. 1.50, 1.70, and 1.95. The reduction peak potential of the ' Present address: Widener College, Chester, Pa. NMNA versus the saturated calomel electrode was found to be 5 To whom requests for reprints should be addressed at Department of -0.61 V at pH 1.95. Three different solutions were studied at Chemistry. Temple University, Philadelphia, Pa. 19122. each pH: Solution 1, a control in which only NMA and NO2~ 6 The abbreviations used are: NMNA, N-methyl-/V-nitrosoaniline; DPP. differ were present; Solution 2, a solution containing NMA, NO2~, ential pulse polarography; NMA. W-methylaniline, Ep, peak potential. E, potential and ascorbic acid, with the polarographic cell "open" to the Received April 5, 1979; accepted June 25, 1979.

OCTOBER 1979 3871

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1979 American Association for Cancer Research. S-K. Chang et al. atmosphere; and Solution 3, Solution 2 with the polarographic cell "closed" with Parafilm to retard the loss of any gaseous products from the cell. The peak current (/â€ž)dueto the NMNA was monitored as a function of time. "Time zero" was taken as the moment of addition of nitrite.

RESULTS AND DISCUSSION

Chart 1 shows the results of the polarographic study of the nitrite ion-ascorbic acid system. Curve A is that of the back ground solution free of NO2~ and ascorbic acid at pH 1.0. Upon the addition of the nitrite stock solution to the background solution such that nitrite concentration was 0.10 mM, Curve B, characteristic of nitrite, was obtained (8). When more than an equivalent amount of ascorbic acid was then added, Curve C with Ep = â€”0.87 V appeared along with the complete disap Q. pearance of Curve B due to the NO2~. On addition of excess E O diphenylamine to this solution, Curve D resulted with the dis i. appearance of Curve C. Curve D is identical with the polaro- gram of diphenylnitrosamine (8). This was verified by the ad dition of authentic diphenylnitrosamine to the solution that yielded Curve D; Curve D increased in height with no change in peak potential or bandwidth. The species giving rise to Curve C has interesting properties. If nitrogen is bubbled through the solution yielding Curve C for only a few moments, the curve disappears. If, instead, the solution is blanketed with nitrogen and allowed to remain open to the atmosphere, Curve C gradually diminishes with time. If NMA or iminodiacetonitrile is added in place of diphenylamine, the peak due to the corresponding N-nitrosamine appears and Volts vs SCE j 1 Curve C disappears. If, however, dimethylamine, diethylamine, -0.6 - I.O -1.4 sarcosine, proline, hydroxyproline, A/-methylaminoacetonitrile, or /V-methylaminopropionitrile is added, no nitrosamine peak appears. Solutions containing the species giving rise to Curve Chart 1. DPPs: Curve A. background solution, NaCIO4:HCIO4 (pH 1.0); Curve B, background solution + 1.0 mM NaNOzi Curve C, solution yielding Curve B + C were sealed under N2 with the latter amines for 3 to 4 days 8.0 mM ascorbic acid; Curve D, solution yielding Curve C + excess diphenyla and then scanned for the presence of the W-nitrosamines. No mine. SCE, saturated calomel electrode. nitrosamines were found, and the species yielding Curve C was still present. salt of oxyhyponitrous acid was added to the solution yielding The species giving rise to Curve C was also generated from Curve C, an increase in peak height with no shift in peak nitrite ion by reagents other than ascorbic acid, such as cys- potential was produced. Aqueous solutions containing only Na2N2O3 at pH 1.0 also yielded a curve with a peak at -0.87 teine, pyrogallol, and iodide ion. (The iodide solution yielded free iodine.) The reaction of iodide ion with nitrous acid is a V. The peak disappeared rapidly when nitrogen was bubbled standard laboratory preparation for nitric oxide. NO, however, through the solution. does not yield a curve with a peak at â€”0.87 V when passed When NMA, diphenylamine, or iminodiacetonitrile was added through an aqueous solution at pH 1.0 to 2.0. It produces a to aqueous solutions of oxyhyponitrite at pH 1.0, the corre curve similar to that of nitrite ion, i.e.. Curve B. This is probably sponding nitroso compound was rapidly formed as evidenced due to reaction of NO with trace amounts of oxygen, remaining by the appearance of the respective nitrosamine peak and the in solution, generating NO? which produces nitrite and nitrate disappearance of Curve C. Other amines, such as sarcosine, proline, hydroxyproline, /V-methylaminopropionitrile, /V-meth- ions in acidic water. It has also been shown that NO is not an effective nitrosating agent (4). On the basis of these observa ylaminoacetonitrile, dimethylamine, and diethylamine, were not tions, the possibility that NO was the species giving rise to nitrosated under identical conditions. Oxyhyponitrous acid is not a well-characterized chemical species, and its structure is Curve C was ruled out. It has been suggested that dinitrogen trioxide (N2O3) is not known. However, aqueous solutions of the compound are involved in the reaction between ascorbic acid and nitrite ion unstable and decompose to yield nitric oxide and water (23). It is quite possible that oxidation-reduction reactions between (9). This species, however, is the anhydride of nitrous acid (22) and is unlikely to exist at concentrations large enough to give nitrite ion and a reducing agent, such as ascorbic acid, at low rise to a peak such as that seen in Chart 1. Its formation does, pH, proceed via oxyhyponitrite as an intermediate. however, agree with the observed stoichiometry (9), which is 2HNO2 + C6H8O6 H2N203 + C6H6O6 + H2O 2:1, nitrite:ascorbic acid. (ascorbic acid) (dehydroascorbic acid) Accordingly, oxyhyponitrous acid (H2N2O3) was studied as I (A) the possible species giving rise to Curve C. When the sodium 2ND + H2O

3872 CANCER RESEARCH VOL. 39

Dehydroascorbic acid had been previously suggested (9) as IOO one of the products of the ascorbic acid-nitrite ion reaction. Its pH =195 presence in the solution in this study was shown by the ap 90 pearance of a small kinetic peak at about â€”0.4V characteristic of dehydroascorbic acid (27). In addition, after thorough de 8C gassing to remove all N2O3~2, the ascorbic acid could be regenerated by bubbling H2S through the solution. H2S is known to reduce dehydroascorbic acid to ascorbic acid (1). If the reaction proceeds as suggested by Equation A, one 60 would expect to see NO2 appear over the solution when the gaseous product comes in contact with air. The concentrations Â£50 of reactants used in the polarographic study are, however, too small to permit the observation of NO2. However, if the concen 40 trations are increased to 0.5 M ascorbic acid and 1.0 M nitrite ion, a brown gas (presumably NO2) is seen over the solution. 30 Since the species generated by the reaction between ascor bic acid and nitrite ion can nitrosate certain secondary amines, it was important to study the rate of nitrosamine formation. Among the 3 amines examined, NMA was chosen for the rate study since its nitroso compound is an established carcinogen. Results are shown in Chart 2. All data points were obtained in 100 quadruplicate; reproducibility was Â±3%. Although not shown pH =l 50 90 here, the results at pH 1.20 and 1.70 fit the trend shown in Chart 2. In Chart 2, the yield was calculated from the ratio of lp, measured at any given time, to the lp of the control reaction 80 when no further nitrosation was occurring. Chart 2 shows that ascorbic acid accelerates the rate of formation of NMNA even though at certain pH levels the final 60 yield of nitrosamine is reduced. The effect differs slightly de pending on whether the cell is open or closed; the closed cell y so gives the higher yield. This result is consistent with the hypoth esis that the nitrosating species is volatile or decomposes yielding a volatile product. The effect is also very dependent on pH, as seen by comparing the results in Chart 2. These show that if one used an analytical technique that examined the solution after only 5 or 10 min, at pH 1.0, one might conclude that partial inhibition had occurred without any evi dence of acceleration. The reason for the increase in the reaction rate is not immediately clear from these preliminary studies. As shown in Equation A and Chart 1, ascorbic acid converts all nitrite into pH=100 oxyhyponitrite which can nitrosate certain secondary amines. The increase in rate may simply be due to the increase in the concentration of the nitrosating species, or be due to a change in the reaction mechanism, or a combination of both of these effects. Aqueous solutions of nitrous acid contain many spe cies, only a few of which can act as nitrosating agents. The reason only certain amines are nitrosated by the oxy hyponitrite may be related to the base strength of the amines. The 3 amines that are nitrosated are relatively weak bases (pKa = 0.9 to 4.8), whereas those that are not are relatively strong ( pKa = 10 to 11 ). These strong bases should be largely in the form of their salts at pH 1 to 2, thus reducing their nucleophilicity substantially and slowing nitrosation by electro- philic nitrosating species. Rapid formation of nitrosamines in aqueous systems is of concern not only because some of them are known carcino gens but also due to their ability to transnitrosate under appro 300 600 900 'ZOO iSOO I8OO priate conditions (5, 26, 28). The addition of ascorbic acid to time - sec Chart 2. Rate of formation of NMNA at 3 pH values. â€¢,no ascorbic acid a commercial product containing a variety of unknown amines (control); â€¢with excess ascorbic acid, cell closed; x, with excess ascorbic acid, might slow down the formation of some nitrosamines, but in cell open.

OCTOBER 1979 3873

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1979 American Association for Cancer Research. S-K. Chang et al. contrast, it might accelerate the formation of others which, 15. Keefer. L. K.. and Roller, P. P. N-Nitrosation by nitrite ion in neutral and basic medium. Science, 181: 1245-1246, 1973 although less potent carcinogens, might generate more potent 16. Magee, P. N.. Montesano. R.. and Preussmann, R. N-Nitroso compounds ones by transnitrosation. and related carcinogens. ACS Monogr. ; 73. 491-625, 1976. 17. Mirvish, S. S. Kinetics of dimethylamine nitrosation in relation to nitrosamine carcinogenisis. J. Nati. Cancer Inst., 44: 633-639, 1970. REFERENCES 18. Mirvish. S. S. Formation of N-nitroso compounds: chemistry, kinetics and in vivo occurrence. Toxicol. Appi. Pharmacol.. 31: 325-351. 1975. 1. Abdoh, Y., and Nejat. N. Estimation of vitamin C. Acta Biochim. Irano, 3(1). 19. Mirvish, S. S., Cardesa, A.. Wallace, L., and Shubik. P. 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3874 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1979 American Association for Cancer Research. Accelerating Effect of Ascorbic Acid on N-Nitrosamine Formation and Nitrosation by Oxyhyponitrite

Shaw-Kong Chang, George W. Harrington, Marc Rothstein, et al.

Cancer Res 1979;39:3871-3874.

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