1 0 0 w 4.f 7 1 1-0 N-Nitroso Compounds 8 9 1 k- b 1/ 2 0 1 0. 1 oi: d 1 | 8 9 1 9, er b m e c e D e: at D n o ati c bli u P 1 0 0 w 4.f 7 1 0 1- 8 9 1 k- b 1/ 2 0 1 0. 1 oi: d 1 | 8 9 1 9, er b m e c e D e: at D n o ati c bli u P N-Nitroso Compounds Richard A. Scanlan, EDITOR Oregon State University Steven R. Tannenbaum, EDITOR 1 Massachusetts Institute of Technology 0 0 w 4.f 7 1 0 1- 8 9 1 bk- Based on a symposium 1/ 2 0 1 0. cosponsored by the Divisions of 1 oi: d 1 | Agricultural and Food Chemistry 8 9 1 9, er and Pesticide Chemistry b m e c e at the 181st Meeting of the D e: at D n American Chemical Society, o ati c bli Atlanta, Georgia, u P March 31-April 1, 1981. ACS SYMPOSIUM SERIES 174 AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C. 1981 1 0 0 w Library of Congress CIP Data 74.f N-nitroso compounds. 1 0 1- (ACS symposium series, ISSN 0097-6156; 174) 98 "Based on a symposium cosponsored by the Divi­ k-1 sions of Agricultural and Food Chemistry and Pesti­ b cide Chemistry at the 181st meeting of the American 21/ Chemical Society, Atlanta, Georgia, March 31-April 10 1, 1981." 0. 1 Includes bibliographies and index. oi: 1. Nitroso-compounds—Congresses. 2. Nitroso- 81 | d aNmitrinoseos—-coCmopnogurnesdsse—s. M3e.t abCoalricsmino—geCnos—ngrCeossnegsr.e sses. 4. 19 I. Scanlan, Richard Α., 1937- . II. Tannenbaum, 9, Steven R., 1937- . III. American Chemical Society. er Division of Agricultural and Food Chemistry. IV. b American Chemical Society. Division of Pesticide m e Chemistry. V. Series. c e D RC268.7.N58N18 616.99'4071 81-19047 e: ISBN 0-8412-0667-8 AACR2 Dat ACSMC8 174 1-400 1981 n o ati c Copyright © 1981 bli Pu American Chemical Society All Rights Reserved. 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Temple, Jr. ati c bli James D. Idol, Jr. Gunter Zweig u P 01 FOREWORD 0 w 4.f 7 01 The ACS SYMPOSIUM SERIES was founded in 1974 to provide 1- 8 a medium for publishing symposia quickly in book form. The 9 1 k- format of the Series parallels that of the continuing ADVANCES b 1/ IN CHEMISTRY SERIES except that in order to save time the 2 0 1 papers are not typeset but are reproduced as they are sub 0. oi: 1 mitted by the authors in camera-ready form. Papers are re d viewed under the supervision of the Editors with the assistance 81 | of the Series Advisory Board and are selected to maintain the 9 9, 1 integrity of the symposia; however, verbatim reproductions of er previously published papers are not accepted. Both reviews b m and reports of research are acceptable since symposia may e c De embrace both types of presentation. e: at D n o ati c bli u P PREFACE It is approximately twenty-five years since John Barnes and Peter Magee discovered that dimethylnitrosamine was a carcinogen for the rat. Since that time N-nitroso compounds have come to be among the most impor tant experimental mutagens and carcinogens for laboratory investigation. Equally, if not more importantly, they have come to be viewed as one of 1 00 the most important classes of environmental carcinogens, and the potential pr 4. for endogenous formation from ubiquitous precursors has been recognized. 7 01 Their presence and the presence of their precursors in foods, water, air, and 1- 8 industrial and agricultural products has led to frantic calls for legislative 9 1 k- and regulatory action, and a never-ending search for more sensitive and b 1/ specific methods of analysis. 2 0 1 The symposium upon which this volume is based was organized at a 0. oi: 1 turning point in nitrosamine research. Almost all types of commercial d products have been tested for volatile nitrosamines, and there have been 81 | a number of outstanding accomplishments of combined university-gov 9 9, 1 ernment-private industry actions to lower or eliminate volatile nitrosa er mines in those products found to be contaminated. However, there is still b m a major gap of knowledge with regard to compounds that are not amenable e c De to analysis by gas chromatography, and this is clearly a frontier of current e: research. There are also many important questions regarding chemistry, at n D mechanism of action, and relation to human disease whose answers lie in o ati the future of research in this field. c bli It is the purpose of this volume to summarize the current state of u P knowledge of nitrosamine research with a chemical orientation, and to help lead the way into the future. RICHARD A. SCANLAN Oregon State University Corvallis, OR 97331 STEVEN R. TANNENBAUM Massachusetts Institute of Technology Cambridge, MA 02139 July 31, 1981 ix 1 Activation of Nitrosamines to Biological Alkylating Agents C. J. MICHEJDA, M. B. KROEGER-KOEPKE, S. R. KOEPKE, and D. H. SIEH Chemical Carcinogenesis Program, Frederick Cancer Research Center, Frederick, MD 21701 1 0 0 h 4.c N-Nitrosamines require metabolic activation to 17 generate the reactive species which result in tumor 0 1- induction. The most commonly accepted hypothesis 8 9 for this activation is the α-hydroxylation hypothe­ 1 k- sis. Nitrosamines which have hydrogens on the alpha b 1/ carbons are hydroxylated in that position by a 2 0 mixed function oxygenase. The resultant α-hydroxy- 1 0. alkyl nitrosamine breaks down to an alkyldiazon- 1 oi: ium ion and the corresponding carbonyl compound. 1 | d The diazonium ion alkylates a variety of nucleophiles 8 and releases molecular nitrogen. The determination 9 9, 1 of the amount of molecular nitrogen provides a er measure of the extent of α-hydroxylation. This con­ mb cept was applied to the in vitro metabolism of ce doubly [15N]-labeled dimethylnitrosamine (DMN) and e D N-nitrosomethylaniline (NMA) by uninduced rat liver ate: S-9 (the postmitochondrial fraction). The amount D of total metabolism was determined by the measurement n o of nitrosamine loss. It was found that measurement cati of formaldehyde formation gave an artificially low ubli value of nitrosamine metabolism. The in vitro re­ P sults indicated that about 34% of the DMN metabolism proceeded by a α-hydroxylation, while only 19% of the theoretical nitrogen was released by NMA. A similar experiment in vivo, using [15N]-labeled DMN showed that about 66% of the metabolism proceeded by α- hydroxylation. Alternative pathways of activation of nitros­ amines, including β-hydroxylation followed by sul­ fate conjugation and the formation of alkoxydiazen- ium ions are discussed. The formation of alkyldiazo- nium ions from trialkyltriazenes is presented to show that the formation of the putative ultimate carcino­ gens from nitrosamines can be studied in a system not requiring metabolic activation. 0097-6156/81/0174-0003$05.00/0 © 1981 American Chemical Society 4 JV-NITROSO COMPOUNDS In 1956 Magee and Barnes (1) reported that dimethylnitros- amine (DMN) was a potent carcinogen in rats* This discovery initiated a mighty outburst of research activity. There are now thousands of papers dealing with the chemistry, metabolism, mutagenicity, teratogenicity, and carcinogenicity of nitros­ amines and other N-nitroso compounds. There are several rea­ sons for this continuing interest. Practically all nitros­ amines are carcinogenic, making them the largest single chemi­ cal group which has that property. In fact, non-carcinogens among them may provide important clues to what makes nitros­ amines carcinogenic. Nitrosamines are remarkably site speci­ fic; essentially all the organs are effected by one or another nitrosamine (JJ). This makes nitrosamines excellent tools for 1 the study of mechanisms of chemical carcinogenesis. Perhaps 0 0 most importantly, nitrosamines can be and are formed in the h 4.c human environment. The reason for this is that the two precur­ 17 sors to nitrosamines, nitrite and amines, are ubiquitous com­ 0 1- ponents of the environmental mix. Moreover, nitrosamines can 8 9 be formed in the stomach by ingesting the precursor amines and 1 k- nitrite. Several chapters in this volume concern themselves b 1/ with environmental and dietary aspects of nitrosamines. 2 0 0.1 The first ten years of nitrosamine research, particularly doi: 1 wrietvhi ewr esbpy ecMta gteoe caarndc inBoagrenneess i(s^,3 )w.e reS isnucmem atrhiazte dt iimne ana n ademniorrambolue s 1 | amount of work has been carried out on those substances. Some 8 9 of this work up to 1974 was reviewed by Magee, Montesano and 1 9, Preussmann (j4). Recently, Lai and Arcos (5) provided a useful er synopsis of contemporary work on the bioactivation of some b m selected dialkylnitrosamines. e c e Nitrosamines, in common with many other organic carcino­ D e: gens, have to be metabolized before their carcinogenic potential Dat can be expressed. Thus, Magee (£) in 1956 showed that dimethyl- n nitrosamine (DMN) was cleared rapidly from the bodies of rats, o ati mice, and rabbits, with very little being excreted in the urine c bli or the feces. Dutton and Heath (J) reported that [^CJ-DMN Pu was metabolized in rats and mice, with a major portion of l^C appearing in expired carbon dioxide, with the rest of the radio­ activity being evenly distributed in the tissues. Only about 7% of the radioactivity was found in the urine. These workers also postulated that the metabolism proceeded by a demethylation reaction and concluded that the lesions produced by DMN were the result of the interaction of metabolites of DMN with cellu­ lar components, rather than with the intact DMN. Since the liver was the principal target of DMN, Magee and Vandekar (J£) used liver slices and subcellular fractions to study the in vitro metabolism of DMN. They found that the metabolizing activ­ ity was localized in the microsomes and in the cytosol and that it required molecular oxygen and the presence of NADPH. The formation of formaldehyde from DMN was demonstrated by Brouwers and Emmelot (Jj)). The principal enzyme system responsible for 1. MICHEJDA ET AL. Biological Alkylating Agents 5 DMN oxidative demethylation has been shown to be a liver mi­ crosome cytochrome P-450 monooxygenase (10)» Lotlikar et al» (11) found that a reconstituted enzyme system, consisting of cytochrome P-450, NADPH-cytοchrome P-450 reductase and phosphatidyl choline was effective in catalyzing the demethy­ lation of DMN. The most commonly accepted mechanism for the oxidative demethylation of DMN and, by extension, of other dialkylnitrosamines is shown in Scheme 1. Scheme 1 0 2 R - Ν - CHo-R1 ». R - Ν - CH - Rf 2 01 NI O NeAnDzPyHme NI O I 0 h c 74. t R»'(CH O 1 0 R - Ν = Ν - OH ^ RNH-NO 81- A 9 k-1 -OH 1/b cellular 2 0 RN+ * R - Nu + N 0.1 2 nucleophiles 2 1 oi: d The α-Hydroxylation Hypothesis 1 | 8 19 The above scheme satisfies much of the metabolic data; ber 9, hploewteev.e r,T hsoem eev iodfe nicte i sf sopre ctuhlea tfiovrem,a tainond iotf itsh cee rαt-ahiyndlyr oxiynlcaotme­d m e intermediate is circumstantial. The acetate ester of a- c De hydroxylated dimethylnitrosamine has been prepared (12.13) e: and has been found to be a potent, directly acting carcinogen at D (14). Other esters of a variety of α-hydroxylated nitros­ on amines have also been prepared (15). While it has been cati shown that DMN acetate is hydrolyzed to hydroxymethylmethyl- bli nitrosamine by an esterase enzyme, it has been pointed out u P that these derivatives of the ot-hydroxylated nitrosamines also dissociate to N-nitrosoimmonium ions (ϋ, 16). Wiessler (15) suggested that the nitrosoimmonium ion could act as the electrophile which alkylates cellular nucleo­ philes. OAc" π HC CH0Ac HC. CH [ Nu;^Nu-CH3 + Ν 3 V / 2 3 ; 2 I ι N-0 NO N=0