Mutagenic activity of glycine upon nitrosation in the presence of chloride and human gastric juice: A possible role in gastric carcinogenesis

Teratogenesis, Carcinogenesis, and Mutagenesis 16:275-286 (1996)

Mutagenic Activity of Glycine Upon Nitrosation in the Presence of Chloride and Human Gastric Juice: A Possible Role in Gastric Carcinogened

J. Gaspar, A. Laires, S. Va, S. Pereira, A. Mariano, M. Quina, and J. Rueff Department of Genetics, Faculty of Medical Sciences (J.G., A. L., S.P., A.M., J. R.), and Faculty of Sciences and Technology (A.L.) New University of Lisbon, ISMAG (J. G.), and Universitary Clinic of Internal Medicine and Gastroenterology, Hospital Pulido Valente (S. V., M. C?.), Lisbon, Portugal

The mutagenic activity of glycine upon nitrosation was studied in the Ames tester strains TA98, TA100, TA102, and TA104. The results obtained show that glycine at acidic pH values and in the presence of C1- can react with nitrite giving rise to geno- toxic compounds to the tester strains used. When these experiments were carried out in the presence of gastric juice the genotoxicity observed was associated with the C1- concentration in the different gastric juice samples. The nature and the mechanism of genetic lesion induced by the ultimate genotoxicant arising from the nitrosation of glycine are not fully understood. Primary amines (e.g., amino acids) have been de- scribed as potential alkylating agents after nitrosation. However, in our experimental conditions these alkylating activities were not detected, suggesting that other mecha- nisms could be involved in the genetic lesion induced by nitrosated glycine. The influence of C1- in the genotoxic activity of glycine and other primary amines upon nitrosation and its possible involvement in the etiology of gastric cancer are dis- cussed. 0 1997 Wiley-Liss, Inc.

Key words: glycine nitrosation, nitroso compounds, human gastric juice, chloride, sodium chloride

INTRODUCTION

Stomach cancer still remains the most common cancer in both sexes on a world- wide basis. Epidemiological studies unraveled, among other factors, a salted-rich diet and exposure to nitrate and nitrite as possible etiological factors for stomach cancer [ 11.

Address reprint requests to J. Rueff, Department of Genetics, Faculty of Medical Sciences, New Uni- versity of Lisbon, Rua da Junqueira 96, P-1300 Lisbon, Portugal.

'Dedicated to Professor Luis Archer on the occasion of his 70th birthday.

0 1997 Wiley-Liss, Inc.

276 Gaspar et al.

Consumption of nitrite and dietary nitrogen containing compounds may result in the formation of nitrosoamines and nitrosoamides under the acidic conditions of the stomach. These latter compounds can form adducts with DNA and are carcino- genic representing a potential risk for human gastrointestinal cancer [2]. In areas of high risk for gastric cancer it has been found that food items often consumed are ren- dered directly genotoxic after nitrosation leading to the formation of several nitrosated amines and phenolic compounds that are themselves also direct mutagens upon nitrosation [34]. On the other hand, nitrate, which is consumed in much higher amounts than nitrite (54 vs. 4 mg daily), can be reduced to nitrite by salivary flora thus representing an in- creased source of nitrite [7]. The nitrosatable mutagenic compounds found in foodstuffs were so far recognized as being essentially aromatic compounds such as phenol and indol derivatives (e.g., tyramine, tryptophan, and flavonoids), but not alkylamides [6,8- lo]. On assessing the risk of formation of carcinogenic N-nitroso compounds from di- etary precursors in the stomach, secondary and aromatic arnines among others were ranked as potentially important risk factors in gastric cancer [2].

Both secondary and tertiary amines, under the conditions found in human stom- ach, can react with nitrite to form dialkylnitrosoamines, eventually leading to tu- mors. In contrast, there is a relative scarcity of data for the mutagenicity of nitrosated primary amines and its putative role in carcinogenesis. The primary aromatic amines amino antipyrine and aniline were shown to be mutagenic upon nitrosation and the resultant mutagenic compounds seem to be unstable diazonium salts [ l l] . Several peptides [12], methylamine and ethylamine, were also shown to be mutagenic upon nitrosation but the low stability of the products formed would play a potential role in carcinogenesis only when the reaction takes place close to the site of formation [ 131. In vivo experiments [14] show that the administration by gavage of radiolabeled methylamine and nitrate to rats leads to an increase of the 7-methyl guanine in the DNA isolated from stomach and from the first 15 cm of small intestine. Methionine was also shown to be genotoxic upon nitrosation, but this effect was only observed in the presence of NaCl [15].

However, as far as physiologic primary amines are concerned, there is a lack of data on its possible mutagenicity upon nitrosation and of the mechanisms involved.

In the present work we demonstrate that glycine is highly mutagenic upon nit- rosation and its genotoxicity upon nitrosation is dependent on the presence of C1- and Br- in the reaction mixture. This effect is also observed when nitrosation reac- tions are carried out in the presence of human gastric juices with high C1- contents.

MATERIALS AND METHODS Chemicals

N-( 1 -naphthyl)-1 -ethylenediamine dihydrochloride was from Ridel-de Haen (Seeize, Germany), 4-@-nitrobenzyl)pyridine (NBP), deoxyribose, and ethylenediaminetetraacetic acid (EDTA) were from Merck (Darmstadt, Germany), thiobarbituric acid (TBA) was from Fluka (Buchs, Switzerland), and sulfanilamide was from Sigma (St. Louis, MO). All other chemicals were reagent grade.

Gastric Juice Samples

Fasting gastric juice samples were collected by upper gastrointestinal endos- copy. The gastric content was aspirated with a 20 ml he r sterile disposable syringe

CI- and Mutagenicity of Glycine Upon Nitrosation 277

attached to a sterile Teflon catheter passed down the suction-biopsy channel of the endoscope. After collecting the gastric juice the samples were stored at -20°C. After thawing, subsamples were taken for pH, protein, NO;, and C1- determinations.

Determination of Concentrations of CI- in Gastric Juice Samples The determination of C1- in the gastric juice samples was carried out by the

Mohr reaction as described in Harris [16]. Briefly, 2 drops of potassium chromate 5% (p/v) were added to 0.1 ml of gastric juice samples and the mixture was titulated with AgN03 (0.01 M) until the formation of a red precipitate indicating the endpoint of the reaction. Each value is the mean of two independent determinations.

Determination of Concentrations of NO; in Gastric Juice Samples The determination of NO; in the gastric juice samples was carried out using

the Griess reaction as described by Niphadkar et al. [17]. Gastric juice samples were centrifuged ( 5 min, 2,00Og, 4°C) and 0.1 ml of centrifuged gastric juice sample was diluted to 1 ml with H20 and 200 p1 of HC10.5 M, 200 pl of sulfanilamide 0.2% (p/ v), 40 pl of ammonium sulfamate 0.044 M and 200 pl of N-( 1 -naphthyl)- 1 -ethylene- diamine dihydrochloride 0.012% (p/v). The absorbance was measured at 546 nm and the concentration of nitrite was calculated using a standard curve for sodium nitrite. Each value is the mean of two independent determinations.

Determination of Gastric Juice pH The pH of the gastric juice samples was measured using a combined glass elec-

trode, calibrated at pH 4.0 and 7.0. The pH values for each sample are the mean of two independent determinations.

Nitrosation Reactions Nitrosation reactions were carried out essentially as described by Ochiai et al.

[8]. Briefly, 1.5 ml of a solution of glycine (5 mM) was added to 1.5 ml of NaN02 (0.1 M) The pH was adjusted to 3 with HC1 (H2S04, HC104 in some experiments in order to establish the influence of C1-, Br-, I-, and F on the nitrosation reactions) and the mixture was incubated for 60 min at 37°C. The reaction was stopped by the addition of 1.5 ml of ammonium sulfamate (0.1 M).

When the experiments were carried out in the presence of gastric juice the reac- tion mixture (in a final volume of 1 ml) contained 0.5 ml of gastric juice, 0.1 ml of NaNO, (0.5 M), and 0.4 ml of glycine (6.25 mM). The reaction was stopped by the addition of 0.5 ml of ammonium sulfamate (0.1 M). Control experiments were car- ried out in the absence of glycine and also in the absence of nitrate.

Rat Liver Enzymes Preparation of S9 (microsomal fraction of rat liver, induced with Aroclor 1254)

was carried out as described by Maron and Ames [ 181. The protein content in the S9 fractions was determined according to Lowry et al. [19].

Ames Test Mutagenic activity was assayed in the Ames assay, as described by Maron and

Ames [18], using the strains TA98, TA100, TA102, and TA104, kindly provided by Professor Bruce Ames. Dose-response curves were generated for each nitrosated

278 Gaspar et al.

sample using the plate incorporation assay, and in the presence or absence of 500 p1 of S9 Mix. At least two independent experiments were carried out for each dose level. The mutagenic activity (revertants/nmol) was determined from the slope of the least-square line of the dose-response curves. The results presented are the mean of at least three independent experiments.

Detection of Alkylating Activity

Detection of alkylating activity of glycine upon nitrosation was carried out as described by Meier et al. [20], using NBP as a nucleophilic scavenger of alkylating agents. Briefly, at different times 1 ml of nitrosation mixture, prepared as described previously, was taken and added to 3 ml of alkylating mixture composed of 0.05 g of NBP, 0.5 ml of acetone, 2 ml of ethylene glycol, and 0.5 ml of phosphate buffer pH 6.60 (0.4 M). This mixture was incubated at 37°C with shaking. From this reaction mixture at different times 0.8 ml was taken, added to 0.2 ml of triethylamine, and the absorbance at 580 nm was determined after 30 sec. In these experiments iodomethane was used as a positive control.

Detection of Hydroxyl Radicals by Deoxyribose Degradation

Hydroxyl radicals were measured by the deoxyribose assay according to Laughton et al. [21] by incubating for 0, 30, 60, 90, and 120 min at 37"C, 1.2 ml of a reaction mixture composed of potassium phosphate buffer (6.7 mM) pH 4.5, 300 yl of the reaction mixture containing glycine after nitrosation, deoxyribose (2.8 mM), FeCI, (20 pM), and EDTA (100 pM). Hydrogen peroxide was used as a positive control (1.42 mM) for hydroxyl radical generation. Deoxyribose degradation by hy- droxyl radicals was measured by the TBA method using 1 ml of trichloroacetic acid (2.8%) and 1 ml of TBA (1%) in 0.05 M NaOH. The mixture was incubated at 100°C for 15 min, cooled, and the absorbance measured at 532 nm. For each assay three independent experiments were performed. Negative controls (iron plus EDTA and no nitrosated glycine or nitrosated glycine without iron and EDTA) were per- formed and the values of the controls without nitrosated glycine were subtracted in each experiment.

RESULTS

The results obtained for the genotoxic activity of glycine upon nitrosation show that in our experimental conditions it exhibits significant genotoxic activities after nitrosation in the Ames tester strains TA98, TA100, TA102, and TA104 (Fig. 1). The genotoxic activity of glycine upon nitrosation is not increased by the presence of S9 in strain TAlO0, but is abolished in the strains TA98, TA102, and TA104. The differ- ent S9 effects on the genotoxicity of nitrosated glycine in the different Ames tester strains strongly suggest that several genetic lesion mechanisms could arise from the nitrosation of glycine, or the reaction produce different compounds partially respon- sible for the genotoxicity observed in the different tester strains. The preferential response of nitrosated glycine in TA102 and TA104 in the absence of S9 Mix points to a possible oxidative mechanism in its mutagenicity. However, it was not possible to detect the production of OH' radicals by the products arising from glycine nitrosa- tion using the deoxyribose degradation (data not shown), suggesting that other oxi- dative compounds arising from the reaction could be involved in the mechanisms of

CI- and Mutagenicity of Glycine Upon Nitrosation 279 80

70

60

50

a2 - 5 4c

3c

2C

I (

(

TA98 a)

0 200 400 600 800 1000 1200

nmol Glycine

Figure la.

Fig. 1. Genotoxic activity of glycine in the Ames tester strains TA98 (a), TAlOO (b), TA102 (c) and TA104 (d) upon nitrosation and in the absence of S9 Mix (+), upon nitrosation and in the presence of S9 Mix (m), and without nitrosation (A), Each point is the average of three independent experiments f standard deviation.

mutagenicity on these tester strains. Nitrosated glycine did not show any alkylating activity in our experimental conditions (data not shown).

The genotoxic activity of glycine in strain TAlOO is dependent on the pH, giving rise to genotoxic products only at pH values of 2.0 and 3.0 (Fig. 2) and is also dependent on the presence of C1- and Br- in the reaction mixture. In fact, in strain TA100, when these experiments were carried out using H2S04 or HClO, to adjust the pH of the reac- tion mixture, instead of HC1, no genotoxic effect was observed. However, when the pHs of the reaction mixture were adjusted with HC104, and C1- or Br- was simultaneously added to the reaction mixture, this led to the formation of ge.notoxic compounds arising from glycine nitrosation with a clear dependence on the C1- and Bt concentration (Fig. 3). Since F and I- are toxic to the tester strains, no conclusion can be drawn on its possible role in the formations of mutagens from glycine upon nitrosation.

280 Gaspar et al.

700

600

500

400

3

>’ 5 d

300

200

100

0

0 200 400 600 800 1000 1200

nmol Glycine

Figure lb.

Human gastric juices used in this study were obtained from dyspeptic patients submitted to upper endoscopy. The gastric juice samples were studied as a function of their pH and protein, C1-, and NO2- concentrations (Table I).

The results obtained for the nitrite contents of gastric juice samples are in agree- ment with results previously published by Tricker et al. [22], who found a nitrite concentration in gastric juice from non-detectable values up to 8 pg/ml (0.43 pg/ml mean value). Protein content of different gastric juice samples ranked between 2.42 and 7.47 mg/ml.

Gastric juice pHs ranged between 1.36 and 3.34, and were considered acidic (pH<4) according to Tricker et al. [22]. The genotoxicity of nitrosated glycine in the presence of gastric juice was not associated with the pH values and protein and nitrite contents of the gastric juice samples used, but was dependent on the concen-

3000

2500

2000

a, i 7 1500

d

1000

500

0

CI- and Mutagenicity of Glycine Upon Nitrosation 281

c, 1 TA102

600 800 1000 1200 0 2 w 400

nmol Glycine

Figure Ic.

tration of C1- in the different samples used (Table I). In fact, when the nitrosation reactions were carried out in the presence of gastric juice, the samples with a C1- content higher than 82 mM (21%) showed a clear genotoxic activity arising from the nitrosation of glycine (Table I).

DISCUSSION

The results obtained show that glycine, after reaction with nitrite at pH values between 2 and 3, and in the presence of C1- or Br- give rise to genotoxic compounds to the Ames tester strains TA98, TA100, TA102, and TA104. It has been reported previously [23] that glycine is not genotoxic upon nitrosation in the Ames tester strain TAl 00. This discrepancy could be related with different experimental condi-

282 Gaspar et al.

600C

5000

4000

W - 5 3000 ,. a"

2000

1000

0

d) TA104

0 200 400 600 800 1000 1200

nmol Glycine

Figure Id.

tions used (pH 4 and glycine and nitrite concentrations of 1.5 and 0.5 M, respec- tively). In fact, in our experimental conditions, at pH 4 no genotoxic activity was observed arising from glycine nitrosation.

The genotoxicity observed is strictly dependent on the C1- concentration on the reaction mixture, suggesting that this ion could react with HN02 giving rise to CINO, which could be the nitrosating agent [24], or that C1- itself could react with nitrite and glycine giving rise to the ultimate genotoxicant. The nature of the ultimate genotoxicant arising from glycine nitrosation is not clear. Primary amines can react with nitrite giving rise to unstable diazonium compounds. Considering these reac- tions we cannot exclude that glycine, after reaction with nitrite giving rise to an unstable diazonium compound, can rearrange leading to the formation of the ulti- mate genotoxicant arising from glycine nitrosation.

It was reported that some amino acids after reaction with nitrite at acidic pH

CI- and Mutagenicity of Glycine Upon Nitrosation 283

Fig. 2. Genotoxic activity of glycine upon nitrosation in the Ames tester strain TAlOO at differ- ent pH values. Nitrosation reactions were carried out as described in Materials and Methods and the pH value adjusted to the values indicated with HCI.

can give rise to alkylating agents, as observed for nitrosoureas [25]. In fact, the strain TAlOO used in this study is very sensitive to alkylating agents, but the relationship between the alkylating activity of glycine upon nitrosation and its genotoxic effects is not clear. Some other amino acids studied and also reported as behaving as alky- lating agents, and with an alkylating activity comparable to glycine [25], are not genotoxic in our experimental conditions (e.g., lysine, alanine). However, it was not possible to detect any alkylating activity arising from the nitrosation of glycine in our experimental conditions.

A wide variety of primary amines is usually found in significant amounts in human foodstuffs, particularly those that are prepared by microbial fermentation. Free amino acids are also present in sigmficant amounts in food products (e.g., meat, mushrooms) and some of those can react with nitrite, at acidic pH values, giving rise to mutagens. As these are derivatives of endogenous compounds there is also the possibility that the cell membrane carrier systems might help the nitroso amino acids to gain access to cell com- ponents. The reconition of the strong influence of C1- on the genotoxicity of methionine [ 151 and glycine could help to clarify the possible involvement of Cl- in gastric carcino- genesis. Kinetic studies for glycine nitrosation show that the nitrosation reaction is de- pendent on the quadratic concentration of nitrite

-- dNoC - K2[HN02]2[glycine] dt

where the K2 value is dependent on the pH [20]. However, this equation does not consider the participation of Cl- in the nitrosation reaction which is relevant for some primary amines (glycine and methionine) [this paper, 151.

284 Gaspar et al.

120c

looC

800

e - 5 600

a

400

200

0 0 200 400 600 800 1000 1200

nmol Glycine

Fig. 3. Genotoxic activity of glycine upon nitrosation in the Ames tester strain TAlOO in the presence of different concentrations of C1-. These experiments were carried out in the presence of different concentrations of CI- and the pH of the reaction mixture was adjusted up to 3 with HC104.

The intake of salted food has been reported as a risk factor for gastric carcino- genesis in several epidemiological studies [26]. However, the molecular mechanisms underlying these effects are not fully understood. The results obtained show that the concentration of C1- in gastric juice increases the genotoxicity of glycine in the pres- ence of nitrite. When comparing the results obtained using exogenous C1- in the reaction mixture (Fig. 2) and the results obtained in the presence of gastric juice (Fig. 3, Table I) it seems that in gastric juice C1- is the main factor involved in the mutagenic activity of glycine upon nitrosation. The concentration of C1- in the hu- man gastric juice samples used in this study ranged between 0.05 and 0.09 M. This range of C1- concentrations was shown to give rise to significant genotoxic activity

C1- and Mutagenicity of Glycine Upon Nitrosation 285

TABLE I. Characterization of Gastric Juice Samples and Genotoxic Activity of Glycine Upon Nitrosation in the Presence of Gastric Juice*

Genotoxic activity

Sample diagnosis PH (mg/ml) (mM) (pg/ml) (revertants/pmol) Histological Protein c1- Nitrite of glycine

1 - a 1.74 f 0.14 4.26 f 0.06 58.0 f 0.0 ND Neg. 2 Gastritis 3.34 f 0.05 4.86 f 0.48 54.0 f 2.8 0.810 f 0.041 Neg. 3 Gastritis 1.44 f 0.08 3.05 f 0.13 68.0 f 0.0 ND Neg. 4 Gastritis 1.36 f 0.06 4.25 f 0.10 88.0 f 2.8 ND 272.7 f 42.3 5 Gastritis 2.18 f 0.03 5.95 f 0.01 66.0 f 1.4 ND Neg. 6 Gastritis 1.39 f 0.02 2.50 f 0.06 82.0 f 2.8 0.963 f 0.067 151.2 f 49.9 7 Gastritis 2.08 f 0.03 4.04 f 0.06 56.0 f 0.0 ND Neg. 8 Gastritis 1.65 f 0.02 3.21 f 0.06 58.0 f 4.2 ND Neg. 9 Gastritis 3.28 f 0.02 2.99 f 0.25 79.0 f 2.8 0.296 f 0.014 Neg. 10 - a 2.98 f 0.01 7.26 f 0.02 90.0 f 0.0 0.362 f 0.027 244.6 +_ 17.5 1 1 Gastritis 1.86 f 0.01 3.12 f 0.21 47.0 f 5.7 0.210 f 0.054 Neg. 12 Gastritis 2.31 f 0.00 2.42 f 0.07 65.0 f 1.4 ND Neg. 13 Gastritis 1.72 * 0.10 7.47 f 0.07 60.0 f 2.8 ND Neg. 14 Gastritis 1.85 f 0.03 3.41 f 0.10 50.0 f 5.7 ND Neg.

*ND = not detectable; Neg. = no dose-response curve or no doubling of spontaneous revertants for any of the doses used. ‘Not done.

of glycine upon nitrosation (Fig. 3). Rojas-Campos et al. [27] reported that the geno- toxic activity of nitrosated black beans is increased in the presence of NaCl, but the mechanisms involved in the increase of mutagenicity are not understood. The recog- nition of the involvement of C1- in nitrosation reactions, and the knowledge of di- etary potentially nitrosable molecules in the presence of this ion could provide a tool to evaluate the human risk associated with the consumption of salted food. These results suggest that the increase of gastric C1- concentration could be involved in the formation of genotoxic compounds in the stomach and possibly have a role in the etiology of gastric cancer.

ACKNOWLEDGMENTS

Our current research is supported by the European Commission and the PRAXIS XXI Programme. We also acknowledge the collaboration of J. Romiio and the CMDT for the use of equipment. J.G. is supported by a postdoctoral fellowship from the PRAXIS XXI Programme.

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