MINIREVIEW; DIALKYLNITROSAMINE BIOACTIVATION AND CARCINOGENESIS David Y. Lai and Joseph C. Arcos Prepared for the Chemical Hazard Identification Branch "Current Awareness" Program Invited review, Appeared in Life Sciences Vol 27, 2149-2165 (1980) November 1980 ------- -Table of Contents: Introduction Dime thy Initr'osamine Diethylnitrosamine Methylethylnitrosamine and higher nitrosamines Mechanism of carcinogenic action Conclusions References ------- Life Sciences, Vol. 27, pp. 2149-2165 Pergamon Press Printed in the U.S.A. MINIREVIEW DIALKYLNITROSAMINE BIOACTIVATION AND CARCINOGENESIS * David Y. Lai2 and Joseph C. Arcos Environmental Protection Agency, Office of Toxic Substances (TS- 792), 401 M St. S.W., Washington, D.C. 20460; and Department of Medicine, Tulane University Medical Ctr., New Orleans, LA. 70112 Since the initial discovery of Magee and Barnes (1,2,3) that dimethylnitrosamine (DMN) which had originally been used as an industrial solvent is hepatotoxic and carcinogenic to a variety of animal species, the metabolism and carcinogenic action of nitrosamines have been extensively investigated. In addition to DMN, several higher nitrosamines and their precursors are present in the environment. Nitrosamines have been detected in processed meats (4,5), tobacco and its smoke (6), agricultural chemicals and cosmetics (7), in urban air (8), and in drinking water (9).. Thus, they represent an important class of chemical carcinogens and mutagens potentially hazardous to human health (10). The aim of this minireview is to present a synopsis of studies on the mechanisms of metabolic activation and carcino- genesis of dialkylnitrosamines. A better understanding of the mechanisms may be helpful in assessing the potential environmen- tal risk that these chemical agents represent. For more exten- sive details on the biological effects and environmental hazards of nitrosamines, the reader is referred to the following articles (11-14). The views expressed in this article are those of the authors and do not necessarily represent those of the U.S. Environmental Protection Agency or of JRB Associates. 2Under EPA Contract 68-01-4839 with JRB Associates, a Subsidiary of Science Applications Inc., McLean, Va. 0024-3205/80/492149-17$02.00/0 ------- 2150 Nitrosamine Bioactivation Vol. 27, No. 23, 1980 Dimethylnitrosamine The simplest and most common dialkylnitrosamine is DMN; its metabolism was first studied by Magee (2) in rats, mice and rabbits. Rapid fall in total body content of DMN after injection led to the conclusion that its metabolism is fast. Using 14C- labeled DMN, Dutton and Heath (15) demonstrated that the major radioactive metabolic product is 14C°;> and concluded that deme- thylation of DMN takes place and the removed methyl group(s) is then further oxidized. They also suggested that the biological effects of DMN may be due to a'metabolite rather than the compound itself. Studies on the rate of decline of total body content of DMN in hepatectomized rats, and in rats with induced hepatic dysfunction, indicated that the liver is the. major organ that metabolizes DMN (14). . . • " H3C N-N=0 AcCX^c' LJ r* u r* fee] |H2C-NsNJ 1 ' tin iznT ~ Vi rj n a-hydroxylation 3N • - /N N ° NADPH,02 N N 0 H3C • i HO.H2C - -HCHO i ;• . : n :;/ ' fu ^el ••••- -N2 [;, /. § J -HO® , L^c J ' • r^c ^-NJ * -.- • • 3ZE \. 3 -Hip H C ' 3/N N— n H :M H3C-N=N-OH I W Mocromolecular binding FIG'.r I • Activating:metabolic pathway'of DMN according to the d^-hydro- xylation hypothesis. " • ------- Vol. 27, No. 23, 1980 Nitrosamine Bioactivation 2151 In vitro studies of DMN metabolism by Magee and Vandekar (16), using liver subcellular fractions and tissue slices showed that the metabolic activity requires C>2 and is localized entirely in the microsomes plus cytosol fraction. For optimal metabolism NADPH is required (17,18). These observations were confirmed by Brouwers and Emmelot (19) who established that formaldehyde is the main product of in vitro metabolism. Extensive research in various laboratories led to the currently accepted o(-hydroxyla- tion hypothesis as the mechanism of metabolic activation of DMN. According to this hypothesis (FIG. 1) hydroxylation at the o(-carbon is the critical, rate-limiting first step. The putative C^-hydroxylated DMN (II) is extremely unstable and yields, upon hydrolysis, formaldehyde and monomethylnitrosamine (III); the overall reaction is N-demethylation. Monomethylnitro- samine (III) is also highly unstable and readily undergoes a nonenzymatic spontaneous rearrangement or "breakdown" to a methylating intermediate, which is presumably responsible for the biological actions of DMN. The evidence that a methylating intermediate has been formed is the presence, in the carcinogen- esis target tissues, of proteins and nucleic acids that were methylated by incorporation of one of the methyl groups derived from DMN (20-25). However, the chemical nature of the methylat- ing intermediate is not clearly understood. It has been suggested at various times that it may represent monomethylnitro- samine (III) itself (26,27), its tautomeric form, methyldiazonium hydroxide (IV) (27,28), diazomethane (VII) (29,30) or methyl- carbonium ion (VI) (21,31,32). Studies by Lijinsky et^ a±. (33) using deuterated DMN have shown that alkylation of DNA and RNA involves methylcarbonium ion (VI), without the involvement of diazomethane (VII). The o(-hydroxylation hypothesis is supported by the findings that oC-acetoxydimethylnitrosamine (VIII) (which gives rise to C-hydroxylated DMN upon hydrolysis) is more carcinogenic, mutagenic and toxic than the parent compound itself (34,35). The existence of the enzyme system responsible for the oxidative demethylation of DMN was reported first by Brouwers and ------- 2152 Nitrosamine Bioactivation Vol. 27, No. 23, 1980 Emmelot (19). The properties of DMN-demethylase have since been extensively studied. It is, in several respects, a typical cyto- chrome P-450-dependent microsomal mixed-function oxidase in that it requires NADPH and 02 (16,19,36), and can be markedly inhibi- ted by carbon monoxide (37,38) and repressed by pretreatment with cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450 (38). Recent investigations by Lake et_ ja_l_. (39), Arcos et a.l. (40) and Sipes et al. (41) have revealed that at least two DMN-demethylase enzyme systems are responsible for the demethyla- tion of DMN in the liver. One is a low Kj^ enzyme (DMN-demethy- lase I) and the other a high KJJ, enzyme (DMN-demethylase II). DMN- demethylase I is regarded to be the enzymic form actually respon- sible for the in vivo metabolic activation of DMN (40); the properties of DMN-demethylase I have been extensively investi- gated and reviewed (38,40-44). Consistent with the concept that demethylation is the rate- limiting step for the production from DMN of the toxic "methylat- ing intermediate", several agents that bring about a lowering of DMN-demethylase I activity also decrease the carcinogenic activ- ity and toxicity of DMN (36,45-51). However, recent reports in- dicated discrepancies in the correlation. For example, Friedman and Sanders (52) showed that piperonyl butoxide significantly in- hibits the demethylation of DMN, but does not affect its acute toxicity or covalent binding to nucleic acids. Similarly, nitro- sosarcosine, DEN or dibutylnitrosamine decrease the demethylation of DMN, but without affecting its LD50 in the rat (53,54). We have shown that in vivo pretreatment with |3-naphthoflavone (3-NF) pregnenolone -16 0(-carbonitrile (PCN) and 3-methylcholanthrene (3-MC) decrease the microsome-catalyzed in vitro binding of DMN to DNA, and this is consistent with their repression of DMN- demethylase in the rat liver (43,55). However, the p-NF effect is not consistent with the observation that this compound strong- ly potentiates DMN hepatocarcinogenesis (TABLE I). In the mouse, [i -NF increases DMN-demethylase activity, cytochrome P-450 level and DMN mutagenicity, without affecting the LD5Q (55). PCN in- creases the P-450 level but decreases DMN-demethylase activity and DMN toxicity, and has no effect on mutagenicity (55). ------- TABLE I Effect of Treatment of Rats with Modifiers on DMM-demethylase Activity, in vitro Binding and on DMN-induced Hepatocarcinogenicity* Effect of modi- § fiers'on DMN - In Vitro Binding of DMH to DNA induced hepato- DMN-demethylase Hicrosomes Microsomes + cytospl carcinogenicity activity (% tumor inci- Treatment (% of control) pmoles/mg DNA % control pmoles/mg DNA % control dence) Control 100 172.3 + 6.5 100 412.5 + 12.3 100 32.6 % — — n o Cfi . » p-NF 52.3 104.3 +_ 5.9 60.5 221.5 +_ 10.9 53.7 74.4 H ID FCIN jy./ luy.J -t- 5.1 bJ.I 1Mb. 5 + 1U.1 4b.^ Ib.b — ^_ 3-MC 54.0 79.8 + 2.0 46.3 128.5 + 9.2 31.1 13.8** o tu o rt H- ^ rt H- 0 * Compiled from Refs. 45, 55, 56 **Corrected value based on the difference between control tumor incidences in Refs. 45 and 56 Ul u> ------- 2154 Nitrosamine Bioactivation Vol. 27, No. 23, 1980 These discrepancies may be partially explained by the existence of the two enzymic forms of DMN-demethylase. Of these DMN-demethylase I is repressed and DMN-demethylase II is induced by pretreatment of the animals with enzyme inducers (40). There are also speculations that the cytochrome P-450-dependent micro- somal mixed-function oxidase system might not be exclusively responsible for the metabolism of DMN, and that possibly alternative mechanism(s) for the metabolic activation of this nitrosamine exist. Lake _et_ a^- (39,57-59) suggested that DMN may be metabolized in part by N-oxidation involving an amine oxidase, unrelated to cytochrome P-450-dependent mixed-function oxidases. Their conclusion was based on the findings that: (i) the storage stability of DMN-demethylase is different from that of ethylmorphine N-demethylase or 4-chloro-N-methylaniline N- demethylase, (ii) interaction with DMN produces unique cytochrome P-450 binding spectra, different from those produced by typical substrates of mixed-function oxidases, (iii) the demethylation of DMN is inhibited by benzylamine, a typical substrate of monoamine oxidase. Argus et al. (56) suggested the possible presence of a DMN-activating enzyme system in the nuclear membrane with a differential response to certain inducers, as compared to the microsomal enzyme. Olah et al. (60) proposed the possible existence of a protolytic cleavage mechanism in biological systems leading to the generation of reactive amino-alkylating intermediate(s). Extensive studies by Lai et al. (55), however, failed to obtain any experimental evidence for the metabolism of DMN by either microsomal mixed-function amine oxidase or mono- amine oxidase. They were also unable to detect any DMN-deme- thylase or diethylnitrosamine-deethylase activity in the nuclei or nuclear membrane preparations (55). Lake ^t_ a^. (58,59) showed that addition of cytosol to the microsomal preparation markedly increases DMN demethylase activity and suggested that the cytosol contains substance(s) that may act as activator(s) of DMN-demethylase, similar to the effect of cytosol on other microsomal N-demethylases (57,61- 63). Recently, Kroeger-Koepke and Michejda (64) reported the presence in the so-called "pH 5 enzyme" fraction of the post- ------- Vol. 27, No. 23, ,1980 Nitrosamine Bioactivation 2155 raicrosomal supernatant some DMN-demethylase activity as deter- mined by the deuterium isotope effect. The enzyme.in the postmicrosornal supernatant was, shown to be different from that in the rnicrosomal pellet and has different characteristics in two different strains of rats. The increase in the binding of DMN to DNA by the addition of cytosol, observed by Chin and Bosman (65) and by Lai _e_t_ al_* (55), appears to be consistent with these find- ings. However, the effect of mixed-function .oxidase modifiers on this soluble enzyme and its metabolic relationship to the two endoplasmic reticulum-localized DMN-demethylases remain to be investigated. :Diethylnitrosamine • In contrast to the considerable number of studies on DMN, there has been until recently lesser interest in the metabolic activation of diethylnitrosamine (DEN) and other dialkylnitrosa- mines. For DEN,;monodeethylation followed by the production of a reactive ethonium ion from the remainder of the molecule, monoe- thylnitrosamine, appears to be the pathway of bioactivation. The properties of DEN-deethylase have been studied- by several inves- tigators (17, 18, 66-68). Magour and Nievel (66) have shown that deethylase activity is enhanced by pretreatment of animals with 3-MC, phenobarbital (PB), butylhydroxytoluene and DDT [1,1,1-tri- chloro-2,2-bis(j>rchlorophenyl) ethane] . Arcos^eJ^ al_. (67) confirm- ed the inducing effect.of PB, but found 3-MC pretreatment to have an inhibitory effect, and they suggested the possible existence of more than one form of DEN-deethylase responding differently to 3-MC treatment. Chau,j2t_aK (68) reported that the Km for DEN- deethylation is an order of magnitude smaller than that of DMN. Diethylnitrosamine metabolism does not lead to the formation of any formaldehyde, suggesting that in vitro metabolism of DEN occurs exclusively by iX-oxidation, yielding acetaldehyde (67). The relevance of deethylase activity to hepatocarcinogenesis has been studied by Rao and Vesselinovitch (69) who described a positive correlation between the degree of DEN-deethylation and susceptibility to hepatocarcinogenesis in mice, as a function of age. Montesano and Magee" (70,71) have studied the organ distri- ------- 2156 Nitrosamine Bioactivation Vol. 27, No. 23, 1980 bution of DEN-deethylase activity in the rat and the hamster, and observed a positive correlation between deethylase activity and the relative susceptibility of the various organs to DEN carcino- genesis. The finding that PB significantly increases, whereas 3- MC decreases the microsome-catalyzed binding of DEN to DNA (61) are consistent with these drug effects on the metabolism of DEN (67). The observation (61) that addition of cytosol enhances the binding of DEN suggests that activating substance(s) of microso- mal DEN-deethylase may be present in the cytosol. The possibility that DEN may be ^3-oxidized and dehydrated to vinylethylnitrosamine or oxidized to an unknown bifunctional derivative, which would interact with cellular macromolecules by cross-linking, has been hypothesized (72,73); however, there is as yet no experimental evidence for this. The observation (61) that addition of mitochondria stimulates the microsome-mediated binding of DEN and N-nitrosopiperidine and, furthermore, that mitochondria alone can catalyze the binding of N-nitrosopiperi- dine support the possibility that nitrosamines may be metabo- lized, at least in part, by enzymes unrelated to cytochrome P- 450-dependent mixed-function oxidases. The mitochondrial enhancement of binding appears to be a function of the alkyl chain length of the nitrosamine, since the mitochondrial stimu- lation of binding is nil with DMN, small (1.4-fold) with DEN, but considerable (4-5 fold) with N-nitrosopiperidine (61). The bind- ing of N-nitrosopiperidine was further shown to be strongly re- duced by benzylamine, a typical substrate of mitochondrial monoa- mine oxidase (61). This suggests that amine oxidase pathway(s) probably play a role in the metabolism of DEN and other higher nitrosamines, but DMN does not appear to be a substrate for either microsomal or mitochondrial amine oxidase (55,74). Methylethylnitrosamine and Higher Nitrosamines. Chau et^ &!_. (68) have studied the kinetics of oxidative dealkylation of methylethylnitrosamine (MEN) by rat liver microsomes using a colorimetric assay for the simultaneous analysis of formaldehyde and acetaldehyde. Both MEN-demethylase and -deethylase are stimulated by PB (68,75) and inhibited by ------- Vol. 27, No. 23, 1980 . Nitrosamine Bioactivation 3-MC (75) pretreatment. The Km for MEN-demethylase and -deethyl- ase are 48mM and 75mM, respectively (68). Upon testing the carcinogenicity of a series of MEN that are deuterated at 'different sites, Lijinsky and Reuber (76) showed that the carcinogenicity of these compounds increases consider- ably with the increasing deuteration of the ethyl group. Since the C-D bond is much stronger than the C-H bond, they concluded that oxidation of the ethyl group at either carbon is unrelated to tumor induction by MEN. More recently, Lai ^t_ al^. (75) showed that rat liver microsomes catalyze substantially the covalent binding of C-MEN to DNA. Converging lines of evidence in the study suggest that the reactive intermediate which binds to DNA is ethylcarbonium ion derived from monoethylnitrosamine. This is consistent with the view of Lijinsky and Reuber (76) that ethylation rather than methylation is the critical molecular event in liver carcino- genesis by MEN. The biotransformation and metabolic fate of dialkylnitrosa- mines higher than DEN is considerably more complex and is being extensively studied presently. Studies by Kruger (77) suggest that higher dialkylnitrosamines are metabolically degraded by /3- -oxidation (analogous to fatty acid metabolisms) to methyl-alkyl or dimethylnitrosamine, which then react as the methylating agent. Investigations by other workers indicate that hydroxy- lation of higher dialkylnitrosamines at carbon atoms other than the c( and ft positions also occurs. The reader is referred to the papers of Okada (78) and Blattmann and Preussmann (79,80) for details on this study area. MECHANISM OF CARCINOGENIC ACTION Covalent binding of reactive intermediates of carcinogens to cellular macromolecules is generally considered to be the mecha- . nism initiating carcinogenesis by most chemical agents. The consensus is that infidelity of DNA replication as a result of modification of DNA by the chemical agents is the basis of carcinogenesis. ------- 2158 Nitrosamine Bioactivation Vol. 27, No. 23, 1980 In vivo covalent binding to DNA of a methyl group of DMN was first demonstrated by Magee and Farber (23). Since then a large number of reports have appeared showing covalent binding of various alkylcarbonium ions originating from dialkylnitrosamines, under both in vivo and in vitro conditions (24,61,65,81-34). The predominance of methylation of guanine at the N7-position has led to the suggestion early that this reaction may have potential significance in carcinogenesis. However, the degree of alkyla- tion at the N -position of guanine showed no correlation with the carcinogenic activities (85-87). Moreover, in a study of the properties of 7-methylguanine-containing templates, Ludlum (88) showed that the methylated base can still pair normally with cytosine. Moreover, the coding properties of DNA-containing guanine residues substituted in the 7-position were not lost, indicating that alkylation of the nucleic acid at this site may be of little biological significance. There is increasing evidence that alkylation at the 0 - position of guanine may be responsible for the carcinogenic activity of nitrosamines (25,89-91). The formation and persis- tence of 0 -alkyl derivatives in DNA after treatment with N- nitroso carcinogens have recently been reviewed by Pegg and Nicoll (92) and by Singer (93). An important piece of evidence for the biological importance of 0 -alkylation as opposed to N •- alkylation was provided by Loveless (94): deoxyguanine was methylated in vitro at the 0 -position by nitrosourea, but not by , j the non-carcinogenic methylmethanesulfonate. Similarly, evidence for the formation of 0 -methylguanine was found in cells treated with N-methyl N'-nitro N-nitrosoguanidine, but not in cells treated with dimethylsulfate, which is relatively non-carcinogen- ic under these conditions (95). A positive correlation between the formation of 0 -alkylguanine and the ability to induce muta- tion in phage has also been observed (94,96,97). In the studies of the properties of 0 -methylguanine-containing template, Gerchman and Ludlum (98) showed the incorporation of "incorrect nucleosides" by bacterial RNA polymerase. It was suggested that 06-alkylation leads to the inability of the guanosine residue to undergo normal base-pairing with cytosine and, thus, may lead to transition mispairing resulting in mutation (94,98). ------- Vol. 27, No. 23, 1980 • Nitrosamine Bioactivation 2159 Studies by various investigators have recently established that tissues which readily develop tumors after a single dose of carcinogen, have repair mechanisms which are much less capable of removing the 0 -alkylguanine from their DNA, than other tissues. For instance, after a single large dose of DMN, which produces kidney tumors but not liver tumors, 0 -methylguanine was found to have a much longer half-life in kidney DNA than in liver DNA (89-91). Similarly, after treatment of rats with low doses of ethyl or methylnitrosourea, 0 -alkylguanine was found to be more persistent in the DNA of the brain, in which tumors are obtained (99,100). Thus, it appears that the properties of the repair system capable of removing 0 -alkylguanine from DMA before cell division is important in determining organ susceptibility to the carcinogenic stimulus of nitrosamines and other alkylating carcinogens. An enzyme that catalyzes the release of 0 -methylguanine as free base from alkylated DNA has been isolated from J2. coli (101) as well as from the liver and kidney of the rat (102). Recently, evidence has been reported that cells which become malignant after treatment with nitrosamines are deficient in repair enzyme activity (90,91,103,104). The report by Pegg (105) on an enzyme that excises 0 -alkylguanine but not N -alkylguanine emphasizes the biological significance of 0 -alkylation. Pegg and Hui (102) observed that the removal of 0 -methyl- guanine from DNA was much more efficient after low doses of DMN than higher doses. Thus, it was suggested that the repair enzyme system itself may be the target for inactivation by large doses of the carcinogen. Although little is known about how the modification of enzymes and other proteins by alkylating agents affect transcription and translation, the modification could lead to: (i) alteration or inactivation of repair enzymes; (ii) infidelity of DNA replication as a result of the alteration or inactivation of DNA polymerase; (iii) changes in gene expres- sion; and (iv) derepression of part or all of integrated tumor virus genomes or oncogenes. ------- 2160 Nitrosamine Bioactivation Vol. 27, No. 23, 1980 CONCLUSIONS It is widely accepted that prior metabolic activation of nitrosamines by various oxidases to alkylating agents which react with cellular macromolecules (DNA and/or RNA) is required for their carcinogenic or mutagenic activity. In vitro studies (14,16) Have demonstrated that they are primarily metabolized by cytochrome P-450-dependent microsomal mixed-function oxidases in the liver. At least two distinct forms of DMN-demethylase have been shown to demethylate DMN (40). The possible existence of more than one form of DEN- deethylase has also been suggested (67). Despite earlier reports to the contrary, the postmicrosomal soluble cytosol itself appears to contain DMN-demethylase activ- ity, present in the "pH 5 enzyme" fraction (64), as well as activating substance(s) of DEN-deethylase.. Moreover, mitochon- drial enzyme system(s) appear to be involved in the bioactivation of nitrosamines higher than DMN (61). These mixed-function oxidases are subject to induction and repression by various agents and, thus, they may influence the bioactivation and carcinogenesis by nitrosamines. Moreover, these modifiers may influence nitrosamine carcinogenesis by mechanism(s) entirely unrelated to the enhancement or inhibition of nitrosamine metabolism. The mechanisms could include: effect on the rate of cytoplasmlc transport and stabilization of the reactive intermediate(s), the rendering of specific macromole- cular sites more or less available to alkylation by acting on DNA conformation-modifying enzymes, inhibiting or enhancing DNA repair activity, etc. The evidence so far available suggest that p-NF may be one of the agents which enhances DMN carcino- genesis by a mechanism unrelated to its effect on DMN metabolism (43,56). The presently perceived interrelationship of factors influencing nitrosamine metabolism and binding to DNA is illustrated in FIG. 2. ------- Vol. 27, No. 23, 1980 Nitrosamine Bioactivation 2161 L activation mechanisms Modifiers Miciosomul « muiagenesis ^- dealkylases \ carcinogenes 1 1 Mitochondrial =C2 activation (enzymatic nature: unknown) 1 1 Cytoplasmic DMN-demethylase ("pH5 enzyme" group) \ / ^ Cytoplosmic /stabilization of and is a • 0 D % / ^ \ _ / * * In vivo ^ binding ^ toONA A nuclear membrane i i 1 o X n z 1 1 ONA conformation modifying enzymes FIG. 2 The quantitatively predominant site of alkylation of (and RNA) by nitrosamines is the N -position of guanine. For this reason, the formation of this product was believed for some time to be the key molecular event in nitrosamine carcinogenesis. However, recent experimental data do not support this view. 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