- Email: [email protected]
Quantitative determination of carcinogens and mutagens as alkylating agents following chemical activation
Chem.-BioL Interactions, 33 (1981) 207--214
207
© Elsevier/North-HollandScientific Publishers Ltd.
QUANTITATIVE DETERMINATION OF CARCINOGENS AND MUTAGENS AS ALKYLATING AGENTS FOLLOWING CHEMICAL ACTIVATION
M.C. A R C H E R
and V.W.S. E N G
Department of Medical Biophysics, Universityof Toronto, 500 Sherbourne Street, Toronto M 4 X 1K9, Ontario (Canada)
(Received March 5th, 1980) (Revision received August 3rd, 1980) (Accepted August 7th, 1980)
SUMMARY
Reaction of nitrosodiethylamine (NDEA) in a non-enzymatic, chemical activation system consisting of ascorbic acid, ferrous ions, EDTA and molecular oxygen yielded an alkylating intermediate that was detected by its reaction with 4-(p-nitrobenzyl)pyridine (PNBP) to yield a colored product. Reaction conditions ~or the chemical activation-detection system were optimized, allowing detection of approx. 10 umol NDEA. The method detected a variety of acyclic and cyclic nitrosamines and other compounds that become alkylating agents only following oxidative metabolism.
INTRODUCTION In 1955, Epstein et al. [1] described the use of PNBP as an analytical reagent for ethylenimines and various direct-acting alkylating agents. Friedman and Boger [2] and Agree and Meeker [3] used the same reagent for the quantitative estimation of nitrogen mustards in aqueous media and acylating agents in non-aqueous media respectively. More recently, PNBP has been used to detect trichothecene mycotoxins [4] and nitrosoureas [5] by thin-layer chromatography, to examine the effect of vegetable juices and milk on the alkylating activity of methylnitrosourea [6], to correlate the mutagenicity of nitrosoureas with their alkylating activity [7], to detect epoxides [8] and to correlate their mutagenicity with their alkylating activity [9]. In all of these studies, PNBP was used to detect only directacting alkylating agents. Abbreviations: NDEA, nitrosodiethylamine;NDMA, nitrosodimethylamine;NMPA, nitrosomethylpropylamine;PNBP, 4-(p-nitrobenzyl)pyridine.
208 Bartsch et al. [10] have described detection of volatile alkylating metabolites by their reaction with excess PNBP in a trap when a mixture of vinyl chloride/oxygen or vinyl bromide/air was passed through a mouse liver microsomal system. Sladek [11] and Vogel et al. [12] have also described the use of PNBP to detect biological conversion of cyclophosphamide and imidazole mustards to alkylating metabolites. In general, however, alkylating metabolites of carcinogens and mutagens are neither volatile nor stable enough to be isolated and reacted with a nucleophilic trapping reagent. In 1954, Udenfriend et al. [14] developed a system consisting of ascorbic acid, ferrous ions, EDTA and molecular oxygen for hydroxylation of aromatic compounds as a model for the enzymatic hydroxylation process. When nitrosodimethylamine (NDMA) was incubated in this chemical activation system, Malling [15] was able to demonstrate the formation of mutagenic products for a reverse mutation strain of Neurospora crassa. Mayer [16] using the same chemical activation system, was also able to induce cytoplasmic petite mutants and genic, canavanine-resistant mutants in Saccharomyces cerevisiae with NDMA and nitrosodiethylamine (NDEA). When we investigated the nature of the products of reaction of nitrosamines in the chemical activation system [17], we found that NDEA yielded N-nitroso-2-(ethylamino)-ethanol while nitrosopiperidine yielded N-nitroso4-piperidone. These products, however, did not represent ultimate mutagenic forms of the nitrosamines, and were only mutagenic for Salmonella typhimurium TA-1535 following further activation by a rat liver microsomal preparation. The rationale for the current study then, was to determine if the same chemical activation system that made nitrosamines mutagenic also produced alkylating agents, as detected by reaction with PNBP. In addition, we wanted to optimize the activating reaction and PNBP detection system in order to develop a simple chemical test for newly discovered synthetic or environmental agents that may produce alkylating species only following activation. We chose to use NDEA to develop the test system and have shown that the test can also be applied to other compounds. MATERIALS AND METHODS Chemicals were obtained from the following sources: NDMA, NDEA and nitrosodipropylamine, Eastman Organic Chemicals, Rochester, NY; nitrosodibutylamine, nitrosodipentylamine and nitrosomethylbenzylamine by synthesis according to Druckrey et al. [18]; nitrosomorpholine, Nutrition Biochemical Corp., Cleveland, OH; nitrosopyrrolidine, nitrosopiperidine and trimethylphosphate, Chemical Procurement Laboratories Inc., College Park, NY; nitrosoproline by synthesis according to Hansen et al. [19]; nitrosomethylurethane and 1,1-dimethyhydrazine, K & K Laboratories Inc., Plainsview, NY; nitrosomethylurea, methylmethanesulfonate, ~-propiolactone, and caffeine, Sigma Chemical Co., St. Louis, MO; nitrosopropylurea by synthesis according to Park et al. [ 20] ; BCNU, Bristol
209 Laboratories of Canada, Quebec, Canada; Mustagen, Merck Sharp and Dohme Canada Ltd., Kirkland, Quebec; glycidol, Aldrich Chemical Corp. Inc., Milwaukee, WI; acetylsalicylate, glucose, phenobarbitone, urethane, codeine phosphate, and chloral hydrate, British Drug Houses (Canada) Ltd., Toronto, Ontario; glutamate, Fisher Scientific, Fairlawn, NJ; cyclophosphamide, Homer, Montreal, Quebec.
Detection o f alkylating agents in the presence o f activation system To 3 ml 0.2 M sodium acetate (pH 4.0), containing 10 mM EDTA, were added 0.1 ml of 0.06 M ferrous sulfate, 0.1 ml of 1.2 M ascorbic acid and 2 ml water. Oxygen was bubbled through the solution for 2 rain. Then the test compound (usually in the range 0.1 -1.0 mmol) dissolved in 1 ml water (or up td 0.2 ml acetone) was added to start the reaction, the head-space of the reaction flask was immediately flushed with oxygen and sealed with a ground glass stopper. The mixture was incubated at 37°C in a metabolic shaker. After 60 rain, 1 ml of a 10% solution of PNBP in acetone was added to the reaction mixture which was then placed in a bath of boiling water. After 20 min, the solution was chilled on ice and 7 ml ethylacetate/acetone (5 : 2) added followed by 1.5 ml 5N NaOH. After mixing in a vortex mixer for about 1/2 rain, the organic phase was removed and its absorbancy measured at 540 nm. After the addition of base, the manipulations were carried out rapidly (within 2--3 rain) without lights because of the instability of the alkylated PNBP following alkalization. Blank solutions contained all components except the test compound. The positive control was NDEA. Detection o f alkylating agents in the absence o f activation system To 3 ml 0.2 M sodium acetate (pH 4.0) was added to the test compound dissolved in 3 ml water and 1 ml of 10% PNBP in acetone. The reaction mixture was placed in a bath of boiling water. After 20 min, the alkylated PNBP was extracted and measured as described above. All the reactions were run at least in duplicate. RESULTS AND DISCUSSION
Preliminary experiments demonstrated that NDEA, following reaction in the chemical activation system first developed by Udenfriend [14] yielded a colored addition product with PNBP. No color was formed in the absence of the activation system. Experiments were therefore carried out to determine the optimum conditions for production of alkylated PNBP with NDEA as the test compound. Repetition of the experiments of Friedman and Boger [2] confirmed that even in the presence of the chemical activation system, acetate buffer was preferred over citrate or lactate, and that the pH optimum was still about 4. Ferrous sulphate, EDTA, and ascorbic acid gave maximum production of alkylated PNBP at final concentrations of 1.5 mM and 20 mM, respectively. In each case, higher or lower concentrations of these components resulted in a decreased production of alkylated PNBP.
210 Lack of any one of the cofactors resulted in no formation of alkylated PNBP. There was also an absolute requirement for the presence of oxygen. One milliliter of 10% PNBP gave optimum yield of alkylated product. The production of alkylated PNBP was linear with incubation time at 37°C up to about 60 min. Sixty minutes was therefore chosen for all subsequent tests. Direct addition of PNBP to the reaction solution prior to incubation at 37°C produced slightly less alkylated product than the normal procedure of adding the PNBP after the incubation. We also showed that there was no advantage to removing 1-ml aliquots of the reaction mixture every 10 min and adding them sequentially to boiling PNBP solution. The optimum boiling time was about 20 min, considerably lower yields of alkylated PNBP being obtained with either a 10-min or 30-min boiling time. Figure 1 illustrates the calibration curve for NDEA in the range 0--240 mM. The minimum detectable level of NDEA is about 10 gmol per incubation (1.6 mM). This value could undoubtedly be improved either by scaling down the incubation mixtures or by analyzing the amount of alkylated PNBP formed by high performance liquid chromatography. Table I illustrates the application of the chemical oxidation method coupled to reaction with PNBP for a variety of different compounds. For comparison, reaction of the test compounds with PNBP is recorded in the absence of the chemical activation system. The symmetrical dialkylnitrosamines (1--5) all formed alkylated PNBP only in the presence of the activation system. The result was anticipated since these compounds are known to require metabolic activation in order for them to exert their carcinogenic or mutagenic activity [21]. It is not clear at this time why NDMA gives a much weaker response in this test system compared to the other dialkylnitrosamines, i
i
r
i
0
120
160
i
r
ZOO
240
3.2 Z.8
~ 2.4 ~5 z ol 1.6'
/
0,8 i
O-4i i 0
! '40
Nitrosodiethylomine
(rnM)
~j
Fig. 1. C a l i b r a t i o n c u r v e f o r n i t r o s o d i e t h y l a m i n e . 1 m M n i t r o s o d i e t h y l a m i n e is e q u i v a l e n t to 6 . 2 ~ m o l per i n c u b a t i o n .
211 TABLE I ALKYLATION OF PNBP BY VARIOUS AGENTS WITH AND WITHOUT CHEMICAL ACTIVATION SYSTEM Agent (1) Nitrosodimethylamine (2) Nit~osodiethylamine (3) Nitrosodipropylamine (4) Nitrosodibutylamine (5) Nitrosodipentylamine (6) Nitrosomethylbenzylamine (7) Nitrosomethylpropylamine (8) Nitrosomorpholine (9) Nitmsopyrrolidine (10 ) Nitrosopiperidine (11 ) Nitrosoproline (12) Nitrosomethylurethane (13) Nitrosomethylurea (14) Nitrosopropylurea (15) BCNUd (16) Methylmethanesulfonate (17) Mustagen e (18) Trimethylphosphate (19) Glycidol f (20) ~-propiolactone (21) Acetylsalicylate (22) Caffeine (23) Glucose (24) Glutamate (25) Phenobarbitone (26) Urethane (27) Cyclophosphamide (28) Codeine phosphate (29) Chloral hydrate (30) 1,1-Dimethylhydrazine
A/mmol a with CASb
A/mmol a
0.07 4.0 1.7 0.84 0.33 0.26 2.6 0.30 0.35 0.31 3.2 6.7 40 22 556 113 1340 331 152 34 0 0 0 0 0 0 98 1.1 1.0 0.95
0 0 0 0 0 0 0.4 0 0 0 ND c 10 64 23 478 114 325 34 152 52 0 0 0 0 0 0 105 0 1.8 0
without CAS b
a Absorbance at 540 nm/mmol of test substance in reaction systems described under Materials and Methods. Values were estimated for comparative purposes by linear extrapolation from experimental measurements. b CAS, chemical activation system. c ND, not determined. d N,N-Bis(2-chloroethyl)-N-nitrosourea.
e 2-chloro-N-(2-chloroethyl )-N-methylethanamine. f 2,3 -Epoxy-l-propanol. The two unsymmetrical nitrosamines, nitrosomethylbenzylamine and n i t r o s o m e t h y l p r o p y l a m i n e ( N M P A ) w e r e b o t h active in t h e p r e s e n c e o f t h e c h e m i c a l a c t i v a t i o n s y s t e m , b u t N M P A also gave a positive r e s p o n s e in t h e a b s e n c e o f t h e c h e m i c a l a c t i v a t i o n s y s t e m . T h e l a t t e r result was u n e x p e c t e d , a n d we s u s p e c t e d t h a t o u r N M P A s t a n d a r d was c o n t a m i n a t e d . G C - - M S analysis o f this c o m p o u n d , h o w e v e r , w h i c h h a d b e e n p u r i f i e d b y distillation
212 under reduced pressure, revealed no impurities. An explanation for the reactivity of NMPA must await further work, particularly isolation and complete characterization of the alkylated PNBP, and investigation of the activity of other unsymmetrical dialkylnitrosamines. The heterocyclic nitrosamines (8--11) also gave alkylated PNBP, but again only in the presence of the activation system. Nitrosoproline also gave a strong response, although this c o m p o u n d is n o t a carcinogen [22]. It can, however, form an alkylating agent via oxidation at the a-carbon atom in the same manner as other nitrosamines [20], and the reasons for its lack of carcinogenic activity are not known. For comparative purposes Table I also shows the strong positive activity of a number of direct acting alkylating agents (12--20) in the absence of the activation system. Some of these c o m p o u n d s show a decrease in formation of alkylated PNBP in the presence of the activation system while several show increased activity in an effect that appears to be unrelated to c o m p o u n d class. The table also shows a number of inactive c o m p o u n d s (21--26) that were not expected to possess alkylating activity. These compounds all gave zero absorbance at 540 nm. Finally, Table I shows several miscellaneous c o m p o u n d s (27--30} that gave positive results. Cyclophosphamide gave a positive response both in the presence and absence o f the chemical activation system. This result was unexpected since cyclophosphamide is known to act as an alkylating agent in vivo only following enzymatic activation [11]. Sladek, however, also found that cyclophosphamide reacted with PNBP reagent in the absence of an activation system [11]. It is likely that the cyclic phosphate ester ring undergoes chemical hydrolysis to produce the alkylating nitrogen mustard moiety when cyclophosphamide is heated to 100°C at pH 4 in the test system. Codeine phosphate reacted with PNBP in the presence of the activation system. Codeine phosphate is not a mutagen for Salmonella typhimurium even in the presence of rat liver preparations [23], and the reason for its reactivity in the current test is obscure. Chloral hydrate was also active both with and without activation. This result, however, reflects the mutagenic activity of chloral hydrate in the absence of rat liver S-9 [23]. 1,1-Dimethylhydrazine is mutagenic only in the presence of rat liver S-9 [23], and in the current test it alkylated PNBP only in the presence of the chemical activation system. In summary, we have demonstrated a sensitive, quantitative system for the detection of agents such as nitrosamines that become alkylating agents only following oxidative metabolism. The test generally gave positive results and negative results where expected, although there was not a quantitative correlation with mutagenic or carcinogenic p o t e n c y of the compounds. The method may prove to be useful in elucidating the mechanism of action of carcinogenic and mutagenic alkylating agents and for detecting alkylating agents in environmental samples.
213 ACKNOWLEDGEMENTS
This research was supported by Grant No. MA-7025 from the Medical Research Council of Canada and a grant from the National Cancer Institute of Canada. REFERENCES 1 J. Epstein, R.W. Rosenthal and R.J. Ess, Use of 7-(4-nitrobenzyl)pyridine as ana]ytica] reagent for ethylenimines and alkylating agents, Anal. Chem., 27 (1955) 1435. 2 0 . M . Friedman and E. Boger, Colorimetric estimation of nitrogen mustards in aqueous media, Anal. Chem., 33 (1961) 906. 3 A.M. Agree and R.L. Meeker, Quantitative determination of micro and semi-micro concentrations of acylating agents by 4-(p-nitrobenzyl)pyridine reagent, Ta]anta, 13 (1966) 1151. 4 S. Takitani, Y. Asabe, T. Kato, M. Suzuki and Y. Ueno, Spectrodensitometric determination of triehothecene mycotoxins with 4-(p-nitrobenzyl)pyridine on silica gel thin layer chromatograms, J. Chromatogr., 172 (1979) 335. 5 M. Asami, K. Nakamura, K. Kawada and M. Tanaka, Evaluation of the a]kylating activity of nitrosoureas by thin layer chromatography, J. Chromatogr., 174 (1979) 216. 6 K. Yano, Effect of vegetable juices and milk on alkylating activity of N-methyl-Nnitrosourea, J. Agric. Food Chem., 27 (1979) 456. 7 K. Yano and M. Isobe, Mutagenieity of N-methyl-N'-aryl-N-nitrosoureasand N-methylN'-aryl-N'-methyl-N-nitrosoureas in relation to their alkylating activity, Cancer Res., 39 (1979) 5147. 8 S.C. Agarwa], B.L. Van Duuren and T.J. Kneip, Detection of epoxides with 4-(p-nitrobenzyl)pyridine, Bull. Environ. Contain. Toxicol., 23 (1979) 825. 9 K. Hemminki and K. Fa]ck, Correlation of mutagenicity and 4-(p-nitrobenzyl)pyridine a]kylation by epoxides, Toxicol. Lett., 4 (1979) 103. 10 H. Bartsch, C. Malaveille, A. Barbin and G. Planche, Mutagenic and a]kylating metabolites of ha]o-ethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human liver tissues, Arch. Toxicol., 41 (1979) 249. 11 N.E. Sladek, Metabolism of cyclophosphamide by rat hepatic microsomes, Cancer Res., 31 (1971) 901. 12 C.L. Vogel, C. Denham, T.P. Waalkes and V.T. DeVita, The physiological disposition of the carcinostatic imidazole-4-(or 5)-carboxamide, 5(or 4)-[3,3-bis (2-chloroethyl)1-triazeno] (NSC82196) (imidazole mustard) in mice and dogs, Cancer Res., 30 (1970) 1651. 13 R. Preussmann, K.N. Arjungi and G. Ebers, In vitro detection of nitrosamines and other indirect a]kylating agents by reaction with 3,4-dichlorothiophenol in the presence of rat liver microsomes, Cancer Res., 36 (1976) 2459. 14 S. Udenfriend, C.T. Clark, J. Axelrod and B.B. Brodie, Ascorbie acid in aromatic hydroxylation. 1. A model system for aromatic hydroxylation, J. Biol. Chem., 208 (1954) 731. 15 H.V. Malling, Mutagenicity of two potent carcinogens, dimethylnitrosamine and diethylnitrosamine, in Neurospora crassa, Mutat. Res., 3 (1966) 537. 16 V.W. Mayer, Mutagenicity of dimethylnitrosamine and diethylnitrosamine for Saccharomyces in an in vitro hydroxylating system, Mol. Gen. Genet., 112 (1971) 289. 17 S. Hsieh, P.L. Kraft, M.C. Archer and S.R. Tannenbaum, Reaction of nitrosamines in
214
18 19 20
21 22 23
the Udenfriend system: principal products and biological activity, Mutat. Res., 35 (1976) 23. H. Druekrey, R. Prcussmann, S. Ivankovic and D. Schmahl, Organotrope carcinogen Wirkungen bie 65 verschiedenen N-Nitroso-Verbindungen an BD-Ratten, Z. Krebsforsch., 69, (1976), 103. T. Hansen, W.T. Iwaoka and M.C. Archer, A high-yield synthesis of 14C-labelled nitrosoproline and nitrososarcosine, J. Label. Compounds, 10 (1974) 689. K.K. Park, J.S. Wishnok and M.C. Archer, Mechanism of alkylation by N-nitroso compounds: detection of rearranged alcohol in the microsomal metabolism of N-nitrosodi-n-propylamine and base-catalyzed decomposition of N-n-propyl-Nnitrosourea, Chem.-BioL Interact., 18 (1977) 349. P.N. Magee and J.M. Barnes, Carcinogenic nitroso compounds, Adv. Cancer Res., 10 (1967) 164. J.E. Nixon, J.H. Wales, R.A. Scanlan, D.D. Bills and R.O. Sinnhuber, Null carcinogenic effect of large doses of nitrosoproline and nitrosohydroxyproline in Wistar rats, Fd. Cosmet. Toxieol., 14 (1976) 133. W.R. Bruce and J.A. Heddle, The mutagenic activity of 61 agents as determined by the micronucleus, Salmonella, and sperm abnormality assays, Can. J. Genet. Cytol. 21 (1979) 319.
207
© Elsevier/North-HollandScientific Publishers Ltd.
QUANTITATIVE DETERMINATION OF CARCINOGENS AND MUTAGENS AS ALKYLATING AGENTS FOLLOWING CHEMICAL ACTIVATION
M.C. A R C H E R
and V.W.S. E N G
Department of Medical Biophysics, Universityof Toronto, 500 Sherbourne Street, Toronto M 4 X 1K9, Ontario (Canada)
(Received March 5th, 1980) (Revision received August 3rd, 1980) (Accepted August 7th, 1980)
SUMMARY
Reaction of nitrosodiethylamine (NDEA) in a non-enzymatic, chemical activation system consisting of ascorbic acid, ferrous ions, EDTA and molecular oxygen yielded an alkylating intermediate that was detected by its reaction with 4-(p-nitrobenzyl)pyridine (PNBP) to yield a colored product. Reaction conditions ~or the chemical activation-detection system were optimized, allowing detection of approx. 10 umol NDEA. The method detected a variety of acyclic and cyclic nitrosamines and other compounds that become alkylating agents only following oxidative metabolism.
INTRODUCTION In 1955, Epstein et al. [1] described the use of PNBP as an analytical reagent for ethylenimines and various direct-acting alkylating agents. Friedman and Boger [2] and Agree and Meeker [3] used the same reagent for the quantitative estimation of nitrogen mustards in aqueous media and acylating agents in non-aqueous media respectively. More recently, PNBP has been used to detect trichothecene mycotoxins [4] and nitrosoureas [5] by thin-layer chromatography, to examine the effect of vegetable juices and milk on the alkylating activity of methylnitrosourea [6], to correlate the mutagenicity of nitrosoureas with their alkylating activity [7], to detect epoxides [8] and to correlate their mutagenicity with their alkylating activity [9]. In all of these studies, PNBP was used to detect only directacting alkylating agents. Abbreviations: NDEA, nitrosodiethylamine;NDMA, nitrosodimethylamine;NMPA, nitrosomethylpropylamine;PNBP, 4-(p-nitrobenzyl)pyridine.
208 Bartsch et al. [10] have described detection of volatile alkylating metabolites by their reaction with excess PNBP in a trap when a mixture of vinyl chloride/oxygen or vinyl bromide/air was passed through a mouse liver microsomal system. Sladek [11] and Vogel et al. [12] have also described the use of PNBP to detect biological conversion of cyclophosphamide and imidazole mustards to alkylating metabolites. In general, however, alkylating metabolites of carcinogens and mutagens are neither volatile nor stable enough to be isolated and reacted with a nucleophilic trapping reagent. In 1954, Udenfriend et al. [14] developed a system consisting of ascorbic acid, ferrous ions, EDTA and molecular oxygen for hydroxylation of aromatic compounds as a model for the enzymatic hydroxylation process. When nitrosodimethylamine (NDMA) was incubated in this chemical activation system, Malling [15] was able to demonstrate the formation of mutagenic products for a reverse mutation strain of Neurospora crassa. Mayer [16] using the same chemical activation system, was also able to induce cytoplasmic petite mutants and genic, canavanine-resistant mutants in Saccharomyces cerevisiae with NDMA and nitrosodiethylamine (NDEA). When we investigated the nature of the products of reaction of nitrosamines in the chemical activation system [17], we found that NDEA yielded N-nitroso-2-(ethylamino)-ethanol while nitrosopiperidine yielded N-nitroso4-piperidone. These products, however, did not represent ultimate mutagenic forms of the nitrosamines, and were only mutagenic for Salmonella typhimurium TA-1535 following further activation by a rat liver microsomal preparation. The rationale for the current study then, was to determine if the same chemical activation system that made nitrosamines mutagenic also produced alkylating agents, as detected by reaction with PNBP. In addition, we wanted to optimize the activating reaction and PNBP detection system in order to develop a simple chemical test for newly discovered synthetic or environmental agents that may produce alkylating species only following activation. We chose to use NDEA to develop the test system and have shown that the test can also be applied to other compounds. MATERIALS AND METHODS Chemicals were obtained from the following sources: NDMA, NDEA and nitrosodipropylamine, Eastman Organic Chemicals, Rochester, NY; nitrosodibutylamine, nitrosodipentylamine and nitrosomethylbenzylamine by synthesis according to Druckrey et al. [18]; nitrosomorpholine, Nutrition Biochemical Corp., Cleveland, OH; nitrosopyrrolidine, nitrosopiperidine and trimethylphosphate, Chemical Procurement Laboratories Inc., College Park, NY; nitrosoproline by synthesis according to Hansen et al. [19]; nitrosomethylurethane and 1,1-dimethyhydrazine, K & K Laboratories Inc., Plainsview, NY; nitrosomethylurea, methylmethanesulfonate, ~-propiolactone, and caffeine, Sigma Chemical Co., St. Louis, MO; nitrosopropylurea by synthesis according to Park et al. [ 20] ; BCNU, Bristol
209 Laboratories of Canada, Quebec, Canada; Mustagen, Merck Sharp and Dohme Canada Ltd., Kirkland, Quebec; glycidol, Aldrich Chemical Corp. Inc., Milwaukee, WI; acetylsalicylate, glucose, phenobarbitone, urethane, codeine phosphate, and chloral hydrate, British Drug Houses (Canada) Ltd., Toronto, Ontario; glutamate, Fisher Scientific, Fairlawn, NJ; cyclophosphamide, Homer, Montreal, Quebec.
Detection o f alkylating agents in the presence o f activation system To 3 ml 0.2 M sodium acetate (pH 4.0), containing 10 mM EDTA, were added 0.1 ml of 0.06 M ferrous sulfate, 0.1 ml of 1.2 M ascorbic acid and 2 ml water. Oxygen was bubbled through the solution for 2 rain. Then the test compound (usually in the range 0.1 -1.0 mmol) dissolved in 1 ml water (or up td 0.2 ml acetone) was added to start the reaction, the head-space of the reaction flask was immediately flushed with oxygen and sealed with a ground glass stopper. The mixture was incubated at 37°C in a metabolic shaker. After 60 rain, 1 ml of a 10% solution of PNBP in acetone was added to the reaction mixture which was then placed in a bath of boiling water. After 20 min, the solution was chilled on ice and 7 ml ethylacetate/acetone (5 : 2) added followed by 1.5 ml 5N NaOH. After mixing in a vortex mixer for about 1/2 rain, the organic phase was removed and its absorbancy measured at 540 nm. After the addition of base, the manipulations were carried out rapidly (within 2--3 rain) without lights because of the instability of the alkylated PNBP following alkalization. Blank solutions contained all components except the test compound. The positive control was NDEA. Detection o f alkylating agents in the absence o f activation system To 3 ml 0.2 M sodium acetate (pH 4.0) was added to the test compound dissolved in 3 ml water and 1 ml of 10% PNBP in acetone. The reaction mixture was placed in a bath of boiling water. After 20 min, the alkylated PNBP was extracted and measured as described above. All the reactions were run at least in duplicate. RESULTS AND DISCUSSION
Preliminary experiments demonstrated that NDEA, following reaction in the chemical activation system first developed by Udenfriend [14] yielded a colored addition product with PNBP. No color was formed in the absence of the activation system. Experiments were therefore carried out to determine the optimum conditions for production of alkylated PNBP with NDEA as the test compound. Repetition of the experiments of Friedman and Boger [2] confirmed that even in the presence of the chemical activation system, acetate buffer was preferred over citrate or lactate, and that the pH optimum was still about 4. Ferrous sulphate, EDTA, and ascorbic acid gave maximum production of alkylated PNBP at final concentrations of 1.5 mM and 20 mM, respectively. In each case, higher or lower concentrations of these components resulted in a decreased production of alkylated PNBP.
210 Lack of any one of the cofactors resulted in no formation of alkylated PNBP. There was also an absolute requirement for the presence of oxygen. One milliliter of 10% PNBP gave optimum yield of alkylated product. The production of alkylated PNBP was linear with incubation time at 37°C up to about 60 min. Sixty minutes was therefore chosen for all subsequent tests. Direct addition of PNBP to the reaction solution prior to incubation at 37°C produced slightly less alkylated product than the normal procedure of adding the PNBP after the incubation. We also showed that there was no advantage to removing 1-ml aliquots of the reaction mixture every 10 min and adding them sequentially to boiling PNBP solution. The optimum boiling time was about 20 min, considerably lower yields of alkylated PNBP being obtained with either a 10-min or 30-min boiling time. Figure 1 illustrates the calibration curve for NDEA in the range 0--240 mM. The minimum detectable level of NDEA is about 10 gmol per incubation (1.6 mM). This value could undoubtedly be improved either by scaling down the incubation mixtures or by analyzing the amount of alkylated PNBP formed by high performance liquid chromatography. Table I illustrates the application of the chemical oxidation method coupled to reaction with PNBP for a variety of different compounds. For comparison, reaction of the test compounds with PNBP is recorded in the absence of the chemical activation system. The symmetrical dialkylnitrosamines (1--5) all formed alkylated PNBP only in the presence of the activation system. The result was anticipated since these compounds are known to require metabolic activation in order for them to exert their carcinogenic or mutagenic activity [21]. It is not clear at this time why NDMA gives a much weaker response in this test system compared to the other dialkylnitrosamines, i
i
r
i
0
120
160
i
r
ZOO
240
3.2 Z.8
~ 2.4 ~5 z ol 1.6'
/
0,8 i
O-4i i 0
! '40
Nitrosodiethylomine
(rnM)
~j
Fig. 1. C a l i b r a t i o n c u r v e f o r n i t r o s o d i e t h y l a m i n e . 1 m M n i t r o s o d i e t h y l a m i n e is e q u i v a l e n t to 6 . 2 ~ m o l per i n c u b a t i o n .
211 TABLE I ALKYLATION OF PNBP BY VARIOUS AGENTS WITH AND WITHOUT CHEMICAL ACTIVATION SYSTEM Agent (1) Nitrosodimethylamine (2) Nit~osodiethylamine (3) Nitrosodipropylamine (4) Nitrosodibutylamine (5) Nitrosodipentylamine (6) Nitrosomethylbenzylamine (7) Nitrosomethylpropylamine (8) Nitrosomorpholine (9) Nitmsopyrrolidine (10 ) Nitrosopiperidine (11 ) Nitrosoproline (12) Nitrosomethylurethane (13) Nitrosomethylurea (14) Nitrosopropylurea (15) BCNUd (16) Methylmethanesulfonate (17) Mustagen e (18) Trimethylphosphate (19) Glycidol f (20) ~-propiolactone (21) Acetylsalicylate (22) Caffeine (23) Glucose (24) Glutamate (25) Phenobarbitone (26) Urethane (27) Cyclophosphamide (28) Codeine phosphate (29) Chloral hydrate (30) 1,1-Dimethylhydrazine
A/mmol a with CASb
A/mmol a
0.07 4.0 1.7 0.84 0.33 0.26 2.6 0.30 0.35 0.31 3.2 6.7 40 22 556 113 1340 331 152 34 0 0 0 0 0 0 98 1.1 1.0 0.95
0 0 0 0 0 0 0.4 0 0 0 ND c 10 64 23 478 114 325 34 152 52 0 0 0 0 0 0 105 0 1.8 0
without CAS b
a Absorbance at 540 nm/mmol of test substance in reaction systems described under Materials and Methods. Values were estimated for comparative purposes by linear extrapolation from experimental measurements. b CAS, chemical activation system. c ND, not determined. d N,N-Bis(2-chloroethyl)-N-nitrosourea.
e 2-chloro-N-(2-chloroethyl )-N-methylethanamine. f 2,3 -Epoxy-l-propanol. The two unsymmetrical nitrosamines, nitrosomethylbenzylamine and n i t r o s o m e t h y l p r o p y l a m i n e ( N M P A ) w e r e b o t h active in t h e p r e s e n c e o f t h e c h e m i c a l a c t i v a t i o n s y s t e m , b u t N M P A also gave a positive r e s p o n s e in t h e a b s e n c e o f t h e c h e m i c a l a c t i v a t i o n s y s t e m . T h e l a t t e r result was u n e x p e c t e d , a n d we s u s p e c t e d t h a t o u r N M P A s t a n d a r d was c o n t a m i n a t e d . G C - - M S analysis o f this c o m p o u n d , h o w e v e r , w h i c h h a d b e e n p u r i f i e d b y distillation
212 under reduced pressure, revealed no impurities. An explanation for the reactivity of NMPA must await further work, particularly isolation and complete characterization of the alkylated PNBP, and investigation of the activity of other unsymmetrical dialkylnitrosamines. The heterocyclic nitrosamines (8--11) also gave alkylated PNBP, but again only in the presence of the activation system. Nitrosoproline also gave a strong response, although this c o m p o u n d is n o t a carcinogen [22]. It can, however, form an alkylating agent via oxidation at the a-carbon atom in the same manner as other nitrosamines [20], and the reasons for its lack of carcinogenic activity are not known. For comparative purposes Table I also shows the strong positive activity of a number of direct acting alkylating agents (12--20) in the absence of the activation system. Some of these c o m p o u n d s show a decrease in formation of alkylated PNBP in the presence of the activation system while several show increased activity in an effect that appears to be unrelated to c o m p o u n d class. The table also shows a number of inactive c o m p o u n d s (21--26) that were not expected to possess alkylating activity. These compounds all gave zero absorbance at 540 nm. Finally, Table I shows several miscellaneous c o m p o u n d s (27--30} that gave positive results. Cyclophosphamide gave a positive response both in the presence and absence o f the chemical activation system. This result was unexpected since cyclophosphamide is known to act as an alkylating agent in vivo only following enzymatic activation [11]. Sladek, however, also found that cyclophosphamide reacted with PNBP reagent in the absence of an activation system [11]. It is likely that the cyclic phosphate ester ring undergoes chemical hydrolysis to produce the alkylating nitrogen mustard moiety when cyclophosphamide is heated to 100°C at pH 4 in the test system. Codeine phosphate reacted with PNBP in the presence of the activation system. Codeine phosphate is not a mutagen for Salmonella typhimurium even in the presence of rat liver preparations [23], and the reason for its reactivity in the current test is obscure. Chloral hydrate was also active both with and without activation. This result, however, reflects the mutagenic activity of chloral hydrate in the absence of rat liver S-9 [23]. 1,1-Dimethylhydrazine is mutagenic only in the presence of rat liver S-9 [23], and in the current test it alkylated PNBP only in the presence of the chemical activation system. In summary, we have demonstrated a sensitive, quantitative system for the detection of agents such as nitrosamines that become alkylating agents only following oxidative metabolism. The test generally gave positive results and negative results where expected, although there was not a quantitative correlation with mutagenic or carcinogenic p o t e n c y of the compounds. The method may prove to be useful in elucidating the mechanism of action of carcinogenic and mutagenic alkylating agents and for detecting alkylating agents in environmental samples.
213 ACKNOWLEDGEMENTS
This research was supported by Grant No. MA-7025 from the Medical Research Council of Canada and a grant from the National Cancer Institute of Canada. REFERENCES 1 J. Epstein, R.W. Rosenthal and R.J. Ess, Use of 7-(4-nitrobenzyl)pyridine as ana]ytica] reagent for ethylenimines and alkylating agents, Anal. Chem., 27 (1955) 1435. 2 0 . M . Friedman and E. Boger, Colorimetric estimation of nitrogen mustards in aqueous media, Anal. Chem., 33 (1961) 906. 3 A.M. Agree and R.L. Meeker, Quantitative determination of micro and semi-micro concentrations of acylating agents by 4-(p-nitrobenzyl)pyridine reagent, Ta]anta, 13 (1966) 1151. 4 S. Takitani, Y. Asabe, T. Kato, M. Suzuki and Y. Ueno, Spectrodensitometric determination of triehothecene mycotoxins with 4-(p-nitrobenzyl)pyridine on silica gel thin layer chromatograms, J. Chromatogr., 172 (1979) 335. 5 M. Asami, K. Nakamura, K. Kawada and M. Tanaka, Evaluation of the a]kylating activity of nitrosoureas by thin layer chromatography, J. Chromatogr., 174 (1979) 216. 6 K. Yano, Effect of vegetable juices and milk on alkylating activity of N-methyl-Nnitrosourea, J. Agric. Food Chem., 27 (1979) 456. 7 K. Yano and M. Isobe, Mutagenieity of N-methyl-N'-aryl-N-nitrosoureasand N-methylN'-aryl-N'-methyl-N-nitrosoureas in relation to their alkylating activity, Cancer Res., 39 (1979) 5147. 8 S.C. Agarwa], B.L. Van Duuren and T.J. Kneip, Detection of epoxides with 4-(p-nitrobenzyl)pyridine, Bull. Environ. Contain. Toxicol., 23 (1979) 825. 9 K. Hemminki and K. Fa]ck, Correlation of mutagenicity and 4-(p-nitrobenzyl)pyridine a]kylation by epoxides, Toxicol. Lett., 4 (1979) 103. 10 H. Bartsch, C. Malaveille, A. Barbin and G. Planche, Mutagenic and a]kylating metabolites of ha]o-ethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human liver tissues, Arch. Toxicol., 41 (1979) 249. 11 N.E. Sladek, Metabolism of cyclophosphamide by rat hepatic microsomes, Cancer Res., 31 (1971) 901. 12 C.L. Vogel, C. Denham, T.P. Waalkes and V.T. DeVita, The physiological disposition of the carcinostatic imidazole-4-(or 5)-carboxamide, 5(or 4)-[3,3-bis (2-chloroethyl)1-triazeno] (NSC82196) (imidazole mustard) in mice and dogs, Cancer Res., 30 (1970) 1651. 13 R. Preussmann, K.N. Arjungi and G. Ebers, In vitro detection of nitrosamines and other indirect a]kylating agents by reaction with 3,4-dichlorothiophenol in the presence of rat liver microsomes, Cancer Res., 36 (1976) 2459. 14 S. Udenfriend, C.T. Clark, J. Axelrod and B.B. Brodie, Ascorbie acid in aromatic hydroxylation. 1. A model system for aromatic hydroxylation, J. Biol. Chem., 208 (1954) 731. 15 H.V. Malling, Mutagenicity of two potent carcinogens, dimethylnitrosamine and diethylnitrosamine, in Neurospora crassa, Mutat. Res., 3 (1966) 537. 16 V.W. Mayer, Mutagenicity of dimethylnitrosamine and diethylnitrosamine for Saccharomyces in an in vitro hydroxylating system, Mol. Gen. Genet., 112 (1971) 289. 17 S. Hsieh, P.L. Kraft, M.C. Archer and S.R. Tannenbaum, Reaction of nitrosamines in
214
18 19 20
21 22 23
the Udenfriend system: principal products and biological activity, Mutat. Res., 35 (1976) 23. H. Druekrey, R. Prcussmann, S. Ivankovic and D. Schmahl, Organotrope carcinogen Wirkungen bie 65 verschiedenen N-Nitroso-Verbindungen an BD-Ratten, Z. Krebsforsch., 69, (1976), 103. T. Hansen, W.T. Iwaoka and M.C. Archer, A high-yield synthesis of 14C-labelled nitrosoproline and nitrososarcosine, J. Label. Compounds, 10 (1974) 689. K.K. Park, J.S. Wishnok and M.C. Archer, Mechanism of alkylation by N-nitroso compounds: detection of rearranged alcohol in the microsomal metabolism of N-nitrosodi-n-propylamine and base-catalyzed decomposition of N-n-propyl-Nnitrosourea, Chem.-BioL Interact., 18 (1977) 349. P.N. Magee and J.M. Barnes, Carcinogenic nitroso compounds, Adv. Cancer Res., 10 (1967) 164. J.E. Nixon, J.H. Wales, R.A. Scanlan, D.D. Bills and R.O. Sinnhuber, Null carcinogenic effect of large doses of nitrosoproline and nitrosohydroxyproline in Wistar rats, Fd. Cosmet. Toxieol., 14 (1976) 133. W.R. Bruce and J.A. Heddle, The mutagenic activity of 61 agents as determined by the micronucleus, Salmonella, and sperm abnormality assays, Can. J. Genet. Cytol. 21 (1979) 319.