Inhibition of Nitrosamine Formation by Ascorbic Acid

Inhibition of nitrosamine formation by ascorbic acid

Steven R Tannenbaum, John S Wishnok, and Cynthia D Leaf

ABSTRACt’ Nitrosation occurs under a wide variety of Chemistry of nitrosation conditions by reaction of most types of amines with any of a Sander and Buerkle (2) first demonstrated that the reaction large number of nitrosating species. Nitrite can be formed in between ingested secondary amines and nitrite could occur in vivo via bacterial reduction of nitrate and by activated macro- vivo and could produce carcinogenic nitrosamines in laboratory phages and endothelial cells. The mechanism ofnitrite formation animals. Many attempts to demonstrate endogenous nitrosation by mammalian cells is via enzymatic oxidation of arginine to in humans were inconclusive, primarily because of inadequate NO followed by oxidation to N203 and N204 . Nitrosatable analytical techniques (3) and lack of proper controls to ensure amines are found in many foods and some, eg, dimethylamine, against the formation of artifacts during collection procedures are synthesized in the body. Precursors ofN-nitroso compounds Downloaded from and during analysis (4). Ohshima and Bartsch (1), however, de- are thus almost constantly present together under favorable re- signed an effective and relatively simple method for demonstrat- action conditions in vivo and there is, consequently, considerable ing the endogenous formation of N-nitrosoproline in humans. interest concerning possible human health risks arising from In a typical example of this technique, sequential oral doses of endogenous formation ofthis class ofcompounds. Among many nitrate and proline are administered to a subject consuming a nitrosation inhibitors, most attention has focused on ascorbic low nitrate diet and the resulting NPRO is measured in a 24-h acid, which reacts with many nitrosating agents and which is ajcn.nutrition.org urine collection. This method is effective because NPRO is not virtually nontoxic. This presentation discusses the chemistry of metabolized, is not carcinogenic, and can be quantitatively ascorbic acid inhibition of nitrosation reactions. Am J C/in measured in urine. Nuir 199 1;53:247S-50S. The Ohshima and Bartsch method (I) has been used by other researchers who have confirmed that nitrosamines are formed KEY WORDS Nitrite, macrophages, nitroso compounds, endogenously (2, 5-7). All studies have demonstrated that un- by guest on October 10, 2012 ascorbic acid nary NPRO levels increase when nitrate and i-proline doses (typically 5 and 4 mmol, respectively) are given and that NPRO Introduction excretion returns to baseline levels of 14-30 nmol/d when a large dose (1 g) of ascorbic acid is administered along with the Much has been written on the endogenous synthesis of nitrate, proline dose. A molar ratio of ascorbic acid to nitrite (2: 1) is nitrite, and N-nitroso compounds, and the many papers dealing sufficient to completely inhibit NPRO formation in vitro, yet, with this subject are a tribute to the current vigor ofthis area of ascorbic acid doses 20-fold larger than the estimated gastric nitrite research. This paper is not intended as a review ofthe field, but [assuming 5% ofthe nitrate dose is reduced to nitrite (8)] do not as the personal comment ofone long-time observer and worker eliminate urinary NPRO. Dietary sources ofpneformed NPRO in the field. do not account for the excess urinary NPRO. In studies where The exposure of people to nitrosating agents occurs through ‘5N-NO was given to the subjects, ‘5N-NPRO formation was multiple pathways ranging from NO2 reactions in the lung to completely inhibited by ascorbic acid, whereas baseline excretion acid catalyzed nitrosation in the stomach to nitrosation mediated of ‘4N-NPRO was unaffected. Similar evidence has been obtained by mammalian cells or bacteria. The use of N-nitrosoproline from studies in the ferret where background NPRO excretion is (NPRO) as an index of endogenous nitrosation ( 1) has proven 2-4 nmol/d (9). Consequently, NPRO may be formed at some to be especially valuable for some of these pathways but may site other than the stomach, most likely one that is inaccessible not be universally indicative. to ascorbic acid, and probably via a mechanism other than that What is increasingly apparent is that a nitrate-nitrite balance of acid-catalyzed nitrosation. sheet does not give an accurate picture of the potential for N- Two possible sources ofendogenous nitrosating agents. in ad- nitrosation in various tissues and body compartments. Large dition to dietary nitrate, are atmospheric nitrogen oxides and quantities of nitrite may have only a small contribution to N- nitrosation if conditions are unfavorable for a reaction. Con- From the Massachusetts Institute of Technology, Cambridge, MA. versely small quantities may play an important role in carci- 2 Supported by The National Institutes ofHealth, PHS grant CA26731. nogenesis in specific tissues. This paper examines some of the CDL supported by NIH grant T32 ES07020. issues related to endogenous nitrosation and the role that ascorbic 3 Address reprint requests to SR Tannenbaum, Massachusetts Institute acid may play in modulating the effects of various nitrosating of Technology, 77 Massachusetts Avenue, 56-31 1, Cambridge. MA agents in the body. 02139.

nitric oxide produced endogenously by cells. Nitric oxide is a rats with LPS and observed a ninefold increase in urinary nitrate labile species that can react rapidly with oxygen yielding NO2, levels and large variations among individuals. They found that which exists in equilibrium with the potent nitrosating agents the enhancement of excreted nitrate correlated with the mag- N2O3 and N204. Subsequent reaction ofthese compounds with nitude of fever induction. Macrophages are directly activated secondary amines would yield nitrosamines; the competing re- by LPS, although other cell types which will similarly convert action with water would yield nitrite and nitrate. L-anginine to nitric oxide may also be stimulated directly or in- The reaction of atmospheric nitrogen oxides, particularly ni- directly by LPS. Endothelial cells, eg, play a role in vasodilation trogen dioxide (NO2). with endogenous amines represents an- as a consequence of fever and could, therefore, make a contri- other possible nitrosation pathway. Cigarettes, eg, are one sig- bution to the increase in excreted nitrate. nificant source of exposure; cigarette smoke contains as much The effects ofa prolonged period ofexercise on excreted nitrate as 1000 ppm nitrogen oxides (10). After administration of di- have been examined (28). In this study, two healthy males con- methylamine to mice followed by exposure to NO2 at levels 20- sumed a defined low-nitrate diet for 8 consecutive days and did 50 times higher than normal atmospheric concentrations, not exercise nor physically exert themselves except on the fifth Mirvish et al ( I 1) found increased levels of N-nitrosodimeth- day, when they ran or bicycled almost continuously for 6 h. An ylamine (NDMA) in the urine. Garland et al (12) reported a increase in excreted nitrate was observed in the I 2-h urine col- positive correlation between atmospheric NO2 levels and NDMA lection period during which the subjects exercised. This is the excretion in human subjects. although it could not be determined first time excreted nitrate has been intentionally increased in whether the increase was due to additional nitrosation of di- humans by a means other than dosing with nitrate. Again, elu- methylamine in vivo or nitrosation in the air and subsequent cidating the cell types involved remains for further investigation. inhalation of NDMA. There was no correlation, however, be- Bacteria are another cell type capable of mediating nitrosation Downloaded from tween urinary NPRO and atmospheric NO2 concentration. This and their role has been considered in hypotheses regarding the could indicate that the urinary NDMA increase was a result of etiology of several cancers. It has been proposed, eg, that the inhalation of higher levels of preformed NDMA in the air or association between a higher risk of gastric cancer and gastric that proline is nitrosated via a different mechanism or at a dif- achlonhydria may be due to increased levels of endogenous ni- ferent site than dimethylamine. trosation via the higher populations of gastric bacteria which

Another pathway for endogenous nitrosation may be linked accompany elevated gastric pH. It was first thought that bacteria ajcn.nutrition.org to the endogenous synthesis of nitrate, a mammalian process facilitated nitrosation primarily by reducing nitrate to nitrite ( 1 3-1 5) estimated to produce 1 mmol nitrate/d in man under (29, 30). Subsequent studies have shown that bacteria act directly normal conditions (7, 16). Immunostimulation increases en- in amine nitrosation (3 1-33) and that this activity is linked to

dogenous nitrate synthesis: eg, a human subject on a nitrate nitrate neductase genes (34-36). E. co/i grown anaerobically in balance study had a ninefold increase in urinary nitrate excretion the presence of nitrate form nitrosation products when nitrite by guest on October 10, 2012 while experiencing nonspecific intestinal diarrhea and fever (17). and a suitable amine are added to the media (34). The reaction Rats treated with Eseherichia co/i Lipopolysacchande endotoxin mechanism was further elucidated by Ji and Hollocher (37),

(LPS) had similarly augmented urinary nitrate levels. Two other who demonstrated that NO is produced from nitrite by E. co/i immunostimulants, carrageenan and turpentine, caused smaller, under anaerobic conditions. Nitrosation occurs only after air is although still significant. increases in urinary nitrate in rats (17). added to the system, indicating that the nitrosating agents are Stuehr and MarIetta ( 1 8) determined that the cell primarily most likely N2O3 and N2O4. responsible for immunostimulated nitrate synthesis is the mac- In summary, studies in humans and animals have clearly rophage. Macrophages and several established macrophage cell demonstrated that endogenous nitrosation occurs intragastrically lines, when stimulated with LPS and/or lymphokines, produce (38) and in at least one other extragastric site. Several types of nitrate and nitrite in vitro via intermediate production of nitric cells including macrophages, endothelial cells, neutrophils, brain oxide ( 19. 20). When suitable secondary amines are added to cells, and bacteria produce nitric oxide in vitro under certain growth media containing stimulated macrophages. the corre- conditions which may be duplicated in the whole animal. Ni- sponding nitrosamines can be detected in the media (2 1). The trosation by macrophages and bacteria has been demonstrated major nitrogen source for these compounds is the terminal in vitro. All of these cells, with the exception ofbacteria, utilize guanido-nitrogen ofL-arginine (22). Endothelial cells (23), neu- nitrogen from L-arginine in the production of nitric oxide. We trophils (24), and brain cells (25) also produce nitric oxide from have demonstrated that L-arginine is a nitrogen source for bio- L-arginine in vitro. This is a general phenomenon occurring synthesized nitrate in rats, ferrets, and humans. These same nitric across species lines and in cells from many sources. oxide-producing cells may be responsible for this novel oxidation We have demonstrated that L-arginine is the precursor of en- of L-arginine to nitrate in humans. Based on known chemistry dogenously synthesized nitrate in rats, ferrets, and humans (26). for nitrosation reactions, it is possible that NO produced en- Following administration of an intraperitoneal dose of ‘5N2-L- dogenously could react with oxygen and, subsequently, nitrosate arginine, rats and ferrets excreted ‘5N-NO . Nitrite is not mea- secondary amines to produce carcinogenic N-nitrosamines in surable in mammalian urine because it reacts rapidly with oxy- vivo. hemoglobin forming methemoglobin and nitrate (27). In the rat, LPS-induced immunostimulation was accompanied by in- Chemistry of ascorbic acid creased excreted nitrate along with a parallel increase in incorporation of ‘5N from ‘5N2-L-arginine into nitrate. The wide range Some relevant aspects of this topic have been discussed in in the LPS-induced increases in excreted nitrate in rats is most greater detail in earlier reports but a summary is appropriate likely attributable to individual variations in response to LPS. here. Ascorbic acid, under anaerobic conditions, can usually In a similar study, Wagner et al (I 5) treated Sprague-Dawley react with N2O3 , H2NO , and NOX with rates higher in each ASCORBIC ACID INHIBITION 2495 case than the corresponding nitrosation rates for amides or di- 10. Neurath GB, Dunger M. Measurement ofnitrogen oxides in tobacco alkyl amines. Ascorbic acid can therefore generally inhibit the smoke by means of the chemiluminescence method. In: Bogovski P. Walker EA. eds. N-nitroso compounds in the environment. Lyon, in vitro nitrosation ofthese classes ofcompounds. This property France: International Agency for Research on Cancer. I974:177-9 has been exploited to prevent nitrosamine formation in foods, (IARC Scientific publication No 9.) and to some extent, to inhibit in vivo nitrosation. I 1. Mirvish SS, Sams IP, Issenberg P. The nitrosating agent in mice Ascorbic acid thus appears to have potential importance as exposed to nitrogen dioxide: improved extraction method and lo- an in vivo nitrite scavenger. This potential, however, has yet to calization in the skin. Cancer Res l983;43:2550-4. be completely realized; not only are the reactions that occur in 12. Garland WA, Kuenzig W, Rubio F, Kornychuk H, Norkus EP, live organisms more complex than might have been expected, Conney AH. Urinary excretion of nitrosodimethylamine and but the in vitro model systems themselves are not straightforward. nitrosoproline in humans: interindividual and intraindividual dif- The equilibria, eg, involve several nitrosating species that can ferences and the effect of administered ascorbic acid and alpha-to- react with ascorbic acid to form NO, which does not nitrosate copherol. Cancer Res l986;46:5392-400. amines. Under anaerobic conditions these reactions can then 13. Green LC, Wagner DA, Ruiz de Luzuriaga K, Istfan N, Young yR. Tannenbaum SR. Nitrate biosynthesis in man. Proc Natl Acad Sci exhaust the nitrosating capacity ofthe system. Oxygen, however, USA 198 1;78:7764-8. can react with NO to form N203 and N2O4 , both of which are 14. Green LC, Tannenbaum SR. Goldman P. Nitrate synthesis in the capable of nitrosation. germfree and conventional rat. Science l98l;2l2:56-8. Under these circumstances, the ascorbic acid may be effectively 15. Wagner DA, Young VR, Tannenbaum SR. Mammalian nitrate bio- removed from the system without significantly affecting the synthesis: incorporation of [1 5N]ammonia into nitrate is enhanced concentration of nitrosating species. In addition to these pro- by endotoxin treatment. Proc NatI Acad Sci USA 1983:80:418-

cesses, ascorbic acid itself can also react directly with oxygen, 21. Downloaded from undergoing conversion to dehydroascorbate (39, 40). 16. Wagner DA, Schultz DS, Deen WM, Young VR, Tannenbaum SR. Evidence now exists that ascorbic acid is a limiting factor in Metabolic fate of an oral dose of ‘5N-labeled nitrate in humans: nitrosation reactions in people. This has recently been demon- effect of diet supplementation with ascorbic acid. Cancer Res l983;43: 1921-S. strated for gastrectomy patients (41) and for patients with chronic 17. Wagner DA. Young VR, Tannenbaum SR. Schultz DS. Deen WM. atrophic gastritis (42). The role that ascorbic acid may play in Mammalian nitrate biochemistry: metabolism and endogenous inhibiting processes of endogenous carcinogenesis has not yet ajcn.nutrition.org synthesis. In: O’Neill 1K. von Borstel RC. eds. N-nitroso compounds: been fully evaluated but future studies should pay close attention occurrence, biological effects and relevance to human cancer. Lyon, to the availability of reduced ascorbic acid in various corn- France: International Agency for Research on Cancer, 1984:247- partments of the human body in populations at high risk for 53 (IARC Scientific publication No 57.). cancer. 13 18. Stuehr DI, Marietta MA. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia by guest on October 10, 2012 co/i lipopolysaccharide. Proc Natl Acad Sci USA l985;82:7738-42. References 19. Stuehr DI, Marietta MA. Induction of nitrite/nitrate synthesis in 1. Ohshima H, Bartsch H. Quantitative estimation ofendogenous ni- murine macrophages by BCG infection lymphokines or interferon- trosation in humans by monitoring N-nitrosoproline excretion in gamma. I Immunol 1987:139:518-25. the urine. Cancer Res 198 l;4l:3658-66. 20. Marletta MA, Yoon PS, Iyengar R, Leaf CD. Wishnok IS. Macro- 2. Sander I, Buerkle G. Induction of malignant tumors in rats by si- phage oxidation of L-arginine to nitrite and nitrate: nitric oxide is multaneous feeding of nitrite and secondary amines. Z Krebsforsch an intermediate. Biochemistry I 988;27:8706- 1 1.

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