[Frontiers in Bioscience S5, 341-359, January 1, 2013]

Reactive electrophilic metabolites of aromatic amine and amide carcinogens

Philip David Josephy1, Michael Novak2

1Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1, 2Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056 USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Early synthetic chemistry of aromatic amines
4. Monocyclic and polycyclic arylamines
5. Metabolic N-hydroxylation
6. Enzymatic oxidation of aromatic amines
7. Metabolic conjugation of hydroxyarylamines and hydroxyarylamides generates reactive and mutagenic metabolites
8. DNA adducts of aromatic amines: identification of guanine c-8 adducts and postulated role of nitrenium ion
9. Nitrenium ions as postulated reaction intermediates; evidence from early trapping studies
10. Relatively stable arylnitrenium ions
11. Diamines: phenylenediamine and benzidine
12. Rapid reaction studies and mechanistic/product distribution studies of short-lived nitrenium ions; lifetimes; fluorene vs biphenyl systems; effect of n-acetyl group on stability
13. Reactions of nitrenium ions with dG; C-8 vs N-7/C-8 mechanisms of dg adduct formation
14. Heterocyclic arylamines
15. Summary
16. Acknowledgments
17. References

1. ABSTRACT

Aromatic and heterocyclic amines are a major class of chemical mutagens and carcinogens. The toxicity of these compounds is a consequence of their metabolic activation. The best-characterized enzymatic pathways for aromatic amine activation lead to the formation of reactive esters such as N-acetoxyarylamines, which are believed to be precursors of short-lived nitrenium ions. In the 1960s, nitrenium ions were invoked as likely intermediates in the formation of arylamine-derived DNA adducts. More recently, nitrenium ion chemistry has been studied by methods such as trapping with azide ion, laser flash photolysis, and the preparation of highly stabilized of examples (e.g., dianisylnitrenium ion). In this review, we discuss the development of our understanding of nitrenium ion chemistry, with emphasis on their generation in biological systems and their reactions with critical targets such as DNA.

2. INTRODUCTION

In this review, we will focus on the formation and chemical reactivity of the electrophilic species that arise from the metabolism of heterocyclic and aromatic amines. These species - especially, nitrenium ions and related structures - were suggested to account for nucleic acid binding by arylamine carcinogens nearly 50 years ago, but recent advances have provided a clearer understanding of their properties and reactivities.

Exposure to aromatic amine carcinogens may have been an aspect of human life since our species began. The evolution of the large brain of Homo sapiens could have taken place, it has been argued, only because of the taming of fire and the development of cooking (1, 2). Chewing roasted food did not require the ape's large jaw muscles, and cooking made available a more abundant caloric supply, even while reducing the energetic costs of digestion. These changes made possible the massive expansion of the brain case. If this explanation is valid, dietary exposure to the heterocyclic amine carcinogens formed by roasting meat is as old as the human race. However, widespread exposure to the benzenoid arylamines did not take place until the rise of the chemical industry in the 19th Century.

3. EARLY SYNTHETIC CHEMISTRY OF AROMATIC AMINES

In 1856, William Henry Perkin synthesized mauveine, the first successful synthetic dye for silk and cotton, thereby launching the fashion revolution that reached its peak in the "Mauve Decade" of the 1890s. Mauveine was produced by the oxidation of aniline (in the form of an impure preparation that contained substantial amounts of toluidine isomers) with potassium dichromate. The history of Perkins' discovery and its influence on the rise of the chemical and pharmaceutical industries has been recounted in a popular book (3).

Less well-known than the role of mauveine in chemistry and fashion are the toxic consequences of its production and use. As early as the 1860s, industrial production of mauveine and other synthetic dyes was causing serious local groundwater contamination, and the leaching of synthetic dyes from clothing was being blamed for skin inflammations and rashes (3). The multiple constituents of mauveine, amino-substituted phenazines (the full structural characterization has been achieved only with the aid of modern instrumental analysis techniques (4); Figure 1) are polycyclic N-aryl compounds studded with "structural alerts" for mutagenicity and carcinogenicity. However, the hazards due to production of mauveine and other aniline dyes were soon eclipsed by those of the azo dyes, such as Congo red (Boettiger, 1883); see review by Freeman in this series. The occupational epidemic of arylamine-induced bladder cancer was recognized soon after the industrial production of polycyclic precursors of azo dyes (such as benzidine and 2-naphthylamine) began (see review by Dietrich and Golka, and other chapters in this series).

The structures of mauveine components reveal the tendency of oxidized anilines to react with additional monomer units, especially at the para position, to form extended N-aryl chains. Late in the 19th Century, Bamberger discovered the eponymous rearrangement of N-arylhydroxylamines to 4-aminophenols, another reaction that demonstrates the formation of an electrophilic N-aryl species that reacts with a nucleophile (in this case, water) at the para position (5). In 1951, consideration of the mechanism of the Bamberger rearrangement led Heller, Hughes, and Ingold to propose the formation of the nitrenium ion intermediate (see below); (6).

4. MONOCYCLIC AND POLYCYCLIC ARYLAMINES

Polycyclic arylamines (derivatives of fluorene, biphenyl, naphthalene, etc.) are generally more potent mutagens and carcinogens than are the monocyclic arylamines (aniline and derivatives). This greater potency may be due to their greater hydrophobicity (7, 8), although the correlation of hydrophobicity with other chemical properties, such as nitrenium ion stability, complicates interpretation of these trends (9). Nevertheless, aniline derivatives, although typically less toxic than polycyclic arylamines on a molar basis, are prevalent in the environment at much higher levels ((10) and references therein). Some monocyclic arylamines, including ortho-toluidine and 2,6-dimethylaniline, are established rodent carcinogens, and attention has recently been drawn to their potential significance as human environmental carcinogens (11).

5. METABOLIC N-HYDROXYLATION

The study of the biological oxidation of aromatic amines was initiated by the research of Kiese and colleagues on the induction of methemoglobinemia by aniline (12). Nitrosobenzene was detected in the blood of dogs treated with aniline (13); later work implicated N-phenylhydroxylamine as the metabolite that causes haemoglobin oxidation (14). At about the same time (1959), the relevance of aromatic amine N-oxidation in chemical carcinogenesis was recognized, in the laboratory of James and Elizabeth Miller, McArdle Laboratory for Cancer Research, University of Wisconsin. Male rats were fed a diet containing 2-acetylaminofluorene (AAF); urine (1 L) collected from these animals was treated with beta-glucuronidase (to hydrolyze glucuronide conjugates), extracted, and then separated by chromatography on long columns of silica gel. These arduous efforts culminated in the isolation of about 10 mg of a new metabolite and previously unknown substance, the hydroxamic acid N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) (15), which proved to be a more active carcinogen than the parent compound (reviewed in (16)).

6. ENZYMATIC OXIDATION OF AROMATIC AMINES

Since the initial discoveries of arylamine N-hydroxylation, several distinct routes for the enzymatic oxidation of aromatic and heterocyclic amines have been identified, each of which has the potential to generate reactive intermediates of toxicological importance.

The AAF N-hydroxylation paradigm, in which hydroxylamines are recognized as activated metabolites of carcinogenic aromatic amines, was quickly extended to additional primary aromatic amines, such as 2-naphthylamine (17) and 4-aminostilbene. Uehleke demonstrated that rat liver microsomal preparations catalyzed the NADPH-dependent N-oxidation of arylamines, including 2-naphthylamine, 2-aminofluorene, 4-aminostilbene, and 4-aminobiphenyl (18). Cytochrome P450 was discovered in 1964 (19). In the following years, evidence for the role of the P450 enzyme system as a major catalyst of arylamine N-oxidation in the liver (20) was adduced, for example, on the basis of the reaction's sensitivity to P450 inhibitors such as piperonyl butoxide (21) and CO (22). More incisive analysis of the capacities of individual enzymes to catalyze arylamine N-oxidation required their purification from native sources (e.g., (23)). The next step forward was the enabling technology of recombinant protein expression (24) and its combination with sensitive genetic systems for the detection of arylamine mutagenicity (25) and genotoxicity (26), which allowed researchers to study the catalytic properties of specific human enzymes of xenobiotic metabolism with respect to arylamine activation. Human cytochrome P450 1A2, an enzyme that is well expressed in the liver (27, 28), is considered to be the paragon of arylamine-activating P450 enzymes. Nevertheless, many other cytochrome P450 enzymes (e.g., 1A1, 1B1, 3A4, etc.) display detectable activity with one or more aromatic or heterocyclic amines (26, 29-32). Furthermore, one must interpret cautiously the results of studies with recombinant proteins. Enzyme activity in vitro does not translate directly into in vivo significance, since it does not necessarily reflect biological factors governing metabolism in the living person or animal, such as organ-specific expression, level of expression, protein induction and turnover, etc.

In addition to cytochrome P450, flavin monooxygenase (FMO) (33, 34), EC 1.14.13.8, is capable of forming N-arylhydroxylamines. FMO is a family of non-P450 hepatic microsomal enzymes comprising five human forms, two of which are well expressed in the liver (35). The mechanisms of substrate oxidation catalyzed by P450 and FMO are very different; the activated state of P450 enzymes is an oxenoid heme iron-oxygen species (36) whereas that of FMO is an hydroperoxyflavin (37). FMO is much less studied than P450 as a catalyst of arylamine N-oxidation, but both 2-naphthylamine (38, 39) and 2-aminofluorene (40) have been shown to be N-hydroxylated by FMO.

Prostaglandin H synthase (PGHS) is the enzyme catalyzing the first two steps in prostaglandin biosynthesis from polyunsaturated fatty acids: its cyclooxygenase activity converts arachidonic acid to PGG2 and its hydroperoxidase activity reduces the hydroperoxide PGG2 to its corresponding alcohol, PGH2 (41). Two forms of the enzyme are found in humans: PGHS-1, the "housekeeping" enzyme, and PGHS-2, the inducible form that drives inflammation and is the target of "COX-2 inhibitor" drugs, such as celecoxib and rofecoxib (42). Many xenobiotics and carcinogens, including aromatic amines, can undergo oxidative activation catalyzed by PGHS, in a cooxidation process whereby arachidonic acid and the carcinogen are both oxidized (43-49). Cooxidation of aromatic amines can generate free radicals (50, 51), azo dimers (44), and nitro compounds (52), presumably by mechanisms that do not involve hydroxylamine intermediates.

Aminophenol metabolites can be formed from aromatic amines by at least two distinct routes: ring-hydroxylation (e.g. catalysed by cytochrome P450; (40, 53, 54)) and the rearrangement of hydroxylamines (11, 32). Aminophenols may be readily oxidized to reactive quinoneimines. This bioactivation route is reviewed by Murata and Kawanishi, in another paper in this series, and will not be considered further here.

7. METABOLIC CONJUGATION OF HYDROXYARYLAMINES AND HYDROXYARYLAMIDES GENERATES REACTIVE AND MUTAGENIC METABOLITES

The concept that N-hydroxy metabolites can be further activated by conversion to electrophilic esters also arose in the laboratory of Miller and Miller. As they later recounted, "About the same time (1967) Poirier (in our laboratory), unable to synthesize N-hydroxy-MAB (N-methyl-4-aminoazobenzene), synthesized its benzoic acid ester ... N-benzoyloxy-MAB proved to be an electrophilic reactant at neutrality ... Analogous studies with N-acetoxy-AAF showed that it had similar, but stronger, electrophilic reactivity. Thus, N-acetoxy-AAF reacted at neutrality with ... guanine residues in ... polynucleotides; under appropriate conditions nearly all of the guanine residues in DNA or RNA could be modified. ... Further studies emphasized the possible importance of the reactivity of the electrophilic esters of N-hydroxy-AAF ..." (16).

Conjugation of O atoms of xenobiotics with endogenous electrophilic donors, such as UDP-glucuronic acid, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), or acetyl CoA, is an important theme in xenobiotic metabolism (55), and each of these routes was found to generate reactive electrophilic esters of N-hydroxy-AAF, at least in vitro (56-60); reviewed by Kadlubar and Beland (61); Figure 2. Further verification of the role of conjugation to form electrophilic esters was provided by studies in which the conjugating enzymes were either knocked out or overexpressed in (for example) bacterial cells. Thus, the mutagenicity of many aromatic amines (but not all; PhIP is an exception (62)) is greatly reduced in Ames test strains lacking acetyl CoA:arylamine N-acetyltransferase (NAT) activity (63, 64) and is greatly increased in strains that overexpress bacterial (65) or human (66) NAT enzymes. Expressed human sulfotransferase 1A2 enhances the mutagenicity of N-hydroxy-AAF (67, 68). These pathways for conjugation of arylhydroxylamines and arylhydroxylamides are also discussed in the contribution by Neumann in this series and by Turesky and Le Marchand (69). N-Acetylation of arylamines can be a route for their detoxication and elimination, whereas O-acetylation of arylhydroxylamines is a mechanism of bioactivation. Consequently, the net effect on susceptibility to arylamine carcinogenesis - benign or malign - of the highly polymorphic human NAT enzymes is difficult to predict (70, 71).

8. DNA ADDUCTS OF AROMATIC AMINES: IDENTIFICATION OF GUANINE C-8 ADDUCTS AND POSTULATED ROLE OF NITRENIUM ION

Marroquin and Farber observed that when radioactively-labeled AAF was administered to rats, radioactivity became bound - presumably covalently - to nucleic acid (RNA) (72, 73). In a pivotal investigation, Kriek demonstrated that N-hydroxyaminofluorene (N-hydroxy-AF) reacts spontaneously with RNA or DNA in vitro, at a rate which is higher under mildly acidic conditions over the pH range 4-6 (74). He concluded that "the mechanism proposed by Heller, Hughes, and Ingold (1951) may be operative", and included a reaction scheme illustrating a nitrenium ion ("in which the nitrogen atom has only a sextet of valence electrons"), formed by loss of water from the protonated hydroxylamine. He noted that "If this ion is sufficiently resonance-stabilized, as might be the case if Ar = fluorenyl or biphenyl, it could react partly in an electrophilic substitution at C-8 of guanine, which is the most likely to be attacked by an electrophilic agent ..." and he drew a "tentative" structure of a C-8 guanine adduct. The correctness of this assignment was confirmed by subsequent studies, including (Kriek, working in the laboratory of Miller and Miller, 1966) the chromatographic purification and identification of 8- (N-2-fluorenylacetamido)guanosine as the product of the reaction of N-acetoxy-2-AAF with guanosine (75). Kriek later observed that "This was one of the first examples ... in which a carcinogen-DNA adduct was obtained by direct chemical synthesis. In the years that followed the same adducts were found in vivo in tissues of animals treated with AAF. Similar types of adducts were characterized for a number of other aromatic amines and other sites of reaction were identified, e.g. the exocyclic amino groups of guanine and adenine. In almost all instances, however, the major adduct found in vivo was an arylamine derivative in which the amino nitrogen was covalently bound to the C-8 of guanine ..." (76) The structures of arylamine DNA adducts have been reviewed in detail by Beland and Kadlubar (77) and recently by Turesky and Le Marchand (69).

9. NITRENIUM IONS AS POSTULATED REACTION INTERMEDIATES; EVIDENCE FROM EARLY TRAPPING STUDIES

In 1951 Heller, Hughes, and Ingold showed that the rate of the Bamberger rearrangement of N-phenylhydroxylamine is proportional to the concentration of protonated hydroxylamine, reaching a constant value at high acid concentration (6). They also showed that the rate of the reaction is independent of (Cl-) in aqueous HCl under conditions in which 4-chloroaniline and 2-chloroaniline are major reaction products along with the Bamberger rearrangement product, 4-aminophenol. The data are consistent with a two step mechanism involving formation of an "active electrophilic intermediate", which they formulated as a "carbonium ionic intermediate" (Figure 3), recognizing (without drawing it explicitly) the "mesomeric" nitrenium ion structure (Figure 3, the cation resonance structure to the left). The subsequent demonstration that the rearrangement occurs in 18O-H2O with incorporation of 18O into 4-aminophenol, but with no detectable incorporation of 18O into the hydroxylamine, provided the critical evidence for the modern formulation of the mechanism of the Bamberger rearrangement shown in Figure 3 (78, 79). Substituent effects, solvent deuterium isotope effects, and the lack of anchimeric assistance of an intramolecularly positioned nucleophile have provided additional support for the nitrenium ion mechanism of the Bamberger rearrangement (80-82).

Derivatives of N-arylhydroxylamines with better leaving groups than OH- do not require acid conditions to react. Gassman and co-workers showed that the rates of solvolysis of ring-substituted N-tert-butyl-N-chloroanilines in EtOH correlate with the sigma+ value of the substituent, with a rho+ value of -6.4 (83, 84). The Hammett substituent constant, sigma+, is used to correlate rates of reactions in aromatic substrates in which positive charge builds up at a site in resonance contact with the substituent; the substituent constant measures the electron donating (negative sigma) or withdrawing (positive sigma) ability of the substituent. The reaction sensitivity to substituents, rho+, is the slope of a plot of the logarithm of the reaction rate constant vs. sigma+. A negative rho+ indicates a reaction in which electron-donating substituents increase the reaction rate, implying that positive charge builds up at the reaction site in the rate-limiting transition state. The large sensitivity to substituents, as measured by rho+, indicates a transition state in which a full positive charge is developing at a reaction site directly adjacent to the aromatic ring. Solvolysis products in EtOH and MeOH, including rearranged 2-chloro-N-tert-butylanilines, are also consistent with reaction through short-lived nitrenium ion intermediates (84-86). Gassman and Granrud showed that rearrangement of N-methanesulfonatoöxyacetanilides in CDCl3 correlated with sigma+, with a rho+ value of -9.2 (87). Novak and co-workers showed that the hydrolysis of N-sulfonatoöxy-acetanilides in aqueous solution was somewhat less sensitive to substituent effects, with rho+ = -4.5, but products of reaction with water and non-solvent nucleophiles, as well as the lack of dependence of the hydrolysis rate on nucleophile concentration, were consistent with a nitrenium ion mechanism (88). The high yield of intramolecular rearrangement products generated in the presence of solvent and non-solvent nucleophiles suggested that a large proportion of the reaction occurs through an ion pair intermediate generated from the initial heterolysis of the N-Cl or N-O bond, and, therefore, that simple ring-substituted monocyclic N-arylnitrenium ions are short-lived species (£ 1 ns) in aqueous or alcohol solvents.

Lifetimes of reactive cations in water can be determined indirectly by competition between an efficient non-solvent nucleophile such as azide, N3-, that reacts with the cation at the diffusion-controlled limit, and water (89-91). The "azide clock" method uses the change in the yields of azide- and solvent-derived products as a function of (N3-) to determine the ion's N3-/solvent selectivity, the ratio of the second-order rate constant for trapping of the ion by N3- and the pseudo-first-order rate constant for trapping of the ion by solvent: kaz/ks. If kaz is diffusion limited at ca. 5 � 109 M-1 s-1, 1/ks, the lifetime of the ion in water, can be estimated. If kaz is below the diffusion limit, the method provides a lower limit for the cation lifetime. In 1987, Fishbein and McClelland showed that N3- traps the 2,6-dimethylphenylnitrenium ion, generated by the Bamberger rearrangement of the corresponding hydroxylamine, with kaz/ks of 7.5 M-1 (Figure 4) (92). If kaz is diffusion limited, the lifetime of the ion in H2O is ca. 1.5 ns. The ion survives long enough to react inefficiently with non-solvent nucleophiles, but cannot be a selective species.

10. RELATIVELY STABLE ARYLNITRENIUM IONS

Flash photolysis of 4-azido-N,N-dialkylanilines in water generates unusually long-lived cationic N,N-dialkylquinonediimines, detected by UV absorption, that have a nitrenium resonance structure (Figure 5, top) (93). These species can also be produced by oxidation of the corresponding p-phenylenediamines. The N,N-dimethylcation shown in Figure 5 has a lifetime at neutral pH of 400 s at 25 ° C, but it does decompose more rapidly under basic conditions (94). The dianisylnitrenium ion (Figure 5, middle) is generated in CH3CN by two reversible one-electron oxidations of the corresponding amine (95, 96). Dianisylnitrenium ion can be detected by voltammetry or UV spectroscopy, and it has a lifetime of ca. 1 s in neutral CH3CN. These cations are not typical of reactive arylnitrenium ions. They are highly stabilized by the strongly electron donating 4-N,N-dialkylamino or 4,4¢ -dimethoxy substituents, and do not react rapidly with nucleophiles. The bis-amino substituted nitrenium ion 1,3-dimethylbenzotriazolium (Figure 5, bottom) is generated by treatment of 1-methyl-benzotriazole with methyl iodide (97). This cation, and others of similar structure, are indefinitely stable, and their salts can be characterized by x-ray crystallography. Although these examples serve to illustrate that stable nitrenium species can be generated, they do not provide insight into the reactivity of transient nitrenium ions involved in carcinogenesis.

11. DIAMINES: PHENYLENEDIAMINE AND BENZIDINE

Benzidine (synthesized by the rearrangement of hydrazobenzene) and para-phenylenediamine (synthesized by reduction of 4-nitroaniline) are the prototypical aryldiamines, compounds possessing two amino substituents linked in a conjugated aromatic system. As mentioned earlier, and as discussed in several other articles in this series, benzidine and its derivatives are the starting materials for synthesis of a vast range of commercially important azo dyes (98). The relative ease of oxidation of aryldiamines to colored products was noticed immediately, and forms the basis of many analytical applications of these compounds, such as the routine use of diaminobenzidine as a peroxidase substrate in immunohistochemistry (99).

Wurster discovered the stable product of N,N,N′,N′-tetramethyl-p-phenylenediamine (Wurster's blue) in 1879 (100). The oxidation of Wurster's blue was further studied by Michaelis (best-known for his fundamental work in enzyme kinetics), who noted that: "The univalent oxidation products of the aromatic p-diamines, or Wurster's salts, are free radicals which may polymerize in a sufficiently concentrated solution ... it is impossible to obtain any pure solution of these radicals at all. They exist only in equilibrium with one molecular species at a lower level of oxidation, the diamine, and another at a higher level of oxidation, the diimine. Since the latter are very unstable compounds, liable to undergo irreversible changes, such a system may undergo irreversible changes, which are not directly due to any lability of the radical itself ..." (101) Benzidine and its derivatives undergo corresponding chemical oxidations to free radical and diimine products (102-104). Peroxidase enzymes readily catalyze these oxidations (105) and the oxidation can also be performed electrochemically (e.g., cyclic voltammetry (106)).

Holland and colleagues prepared 3,5,3',5'-tetramethylbenzidine (107) (TMB), a compound in which, as in Wurster's blue, the amino group is sterically protected by adjacent methyl substituents on the aromatic rings. The oxidation products of TMB are much less reactive than those of benzidine or dianisidine (3,3-dimethoxybenzidine (108)), and this relative stability made possible the direct detection of the electron paramagnetic resonance (EPR) spectrum of the TMB cation radical in a peroxidase incubation, and measurement of the equilibrium between the radical and the charge-transfer complex of the diamine and diimine (109). Prostaglandin synthase also catalyzes the oxidation of TMB (110). The benzidine cation radical (Figure 6) is also detectable by EPR, although it is much less stable than in the case of TMB (111, 112). The monocation (Figure 6, diimine species with net charge +1) conjugate base of the benzidine diimine dication may be regarded as a stabilized arylnitrenium ion, as noted by McClelland et al. (113). It has an aqueous solution lifetime of ca. 100 s at neutral pH, but decomposes more rapidly under basic conditions (113) and its properties are very similar to those of the N,N-dialkylquinonediimines discussed above. Benzidine diimine (reacting predominately through its monocationic form that is dominant at neutral pH) is an electrophile that reacts to form covalent adducts with nucleophilic biological targets such as N-acetylcysteine (114), glutathione (115, 116), deoxyguanosine (117), and DNA (117-121).

Delocalization of positive charge over two N atoms is also seen in the case of the relatively stable nitrenium ion produced by oxidation of the antipsychotic drug clozapine (Figure 7) (122-125).

12. RAPID REACTION STUDIES AND MECHANISTIC/PRODUCT DISTRIBUTION STUDIES OF SHORT-LIVED NITRENIUM IONS; LIFETIMES; FLUORENE VS BIPHENYL SYSTEMS; EFFECT OF N-ACETYL GROUP ON STABILITY

Early studies on the Bamberger rearrangement and similar reactions of N-chloro, N-acetoxy, or N-sulfonatooxy derivatives had established that these reactions occurred through nitrenium ion intermediates, but the meagre available data on the lifetimes of monocyclic N-arylnitrenium ions suggested that these species had very short lifetimes in an aqueous environment that would not allow them to react selectively in vivo. As of the mid-1980s, the concept that nitrenium ions might be involved in the carcinogenicity of N-arylhydroxylamine derivatives was regarded with skepticism. Other possibilities had been demonstrated. The Boche and Novak groups had shown that aromatic amines can react with esters of hydroxylamines through an SN2 mechanism, even in a polar solvent such as MeOH (126-128) and it was not at all clear that nitrenium ions played a biological role.

In 1993, the Novak group applied the azide clock methodology to the 4-biphenylyl-nitrenium ion and to the N-acetyl-4-biphenylylnitrenium ion, both of which were derived in aqueous solution from ester precursors (Figure 8) (129). The biphenylyl ions exhibit large kaz/ks values: 2.9 � 103 M-1 and 1.0 � 103 M-1 for the NH and NAc ions, respectively. The π-conjugation provided by the distal phenyl ring has an unprecedentedly large effect on the kinetic lability of these ions that is not reproduced in similarly substituted benzyl cations, while the N-acetyl group has remarkably little effect. Assuming that the reaction with N3- is diffusion limited, the lifetimes of the 4-biphenylylnitrenium ion and N-acetyl-4-biphenylylnitrenium ion are ca. 0.6 micros and 0.2 micros, respectively. These relatively long lifetimes imply that N-arylnitrenium ions derived from metabolites of carcinogenic polycyclic arylamines and amides could act as very selective electrophiles in a biological environment.

Shortly afterwards, the Novak and McClelland groups demonstrated that the N-acetyl-4-biphenylyl- and N-acetyl-2-fluorenylnitrenium ions could be detected by fast UV spectroscopy in H2O, following laser flash photolysis (lfp) of esters or N-chloro derivatives of the corresponding hydroxamic acids (130). The transients were identified as nitrenium ions by their second-order reactions with N3-, their insensitivity to O2, and by the equivalence of kaz/ks measured by direct observation and by the azide clock method. McClelland's group also demonstrated that the 4-biphenylyl- and 2-fluorenylnitrenium ions could be generated by lfp of the corresponding azides (131). The lifetimes 1/ks and kaz were measured directly for all four cations (Figure 9). The results verified that the reactions of these ions with N3- is at or near the diffusion-controlled limit, and agreed with the lifetime estimates made by azide clock measurements. The lifetimes of the 2-fluorenyl ions, which are forced into planarity by the methylene bridge, are significantly longer than those of the 4-biphenylyl ions. A lfp study of the acid-base properties of the 4-biphenylyl- and 2-fluorenylnitrenium ions showed that they can be protonated in highly acidic solutions to generate dications, but these dications cannot be physiologically relevant (132).

The parent phenylnitrenium ion is too short-lived to be directly detected in aqueous solution by ns-lfp methods, but it has been estimated to have a lifetime in water of ca. 125-250 ps based on Br- trapping data obtained during the Bamberger rearrangement of N-phenylhydroxylamine (133). A recent direct measurement of its lifetime (110 ps) by ultra-fast lfp of phenyl azide in formic acid is in reasonable agreement with that estimate (134). In general, it appears that the lifetimes of monocyclic N-arylnitrenium ions are too short for these species to be selective electrophiles in biological systems.

13. REACTIONS OF NITRENIUM IONS WITH dG; C-8 VS N-7/C-8 MECHANISMS OF dG ADDUCT FORMATION

Although the identity of the major C-8 adduct formed in the reaction of N-arylhydroxylamine derivatives with guanosine, 2¢ -deoxyguanosine, (dG), and the guanine moiety in DNA/RNA had been known since the mid-1960s, the detailed mechanism of this reaction was not understood until recently. The reaction was not established as involving a nitrenium ion until 1995, when Kennedy and Novak demonstrated that the rate of formation of the dG adduct is independent of dG concentration and equivalent to the rate of disappearance of the nitrenium ion precursor for the N-acetyl-4-biphenylylnitrenium and N-acetyl-2-fluorenylnitrenium ions (135). Trapping experiments with these two ions and, subsequently, with the 4-biphenylylnitrenium ion (136) provided kdG/ks values for each ion, and, since ks was known, the magnitude of the second-order rate constant for the reaction with dG, kdG, was obtained: 1.9 � 109 M-1s-1 for the 4-biphenylylnitrenium and N-acetyl-4-biphenylylnitrenium ions, and 6.2 � 108 M-1s-1 for the N-acetyl-2-fluorenylnitrenium ion. All three ions react efficiently with dG: at (dG) = 10 mM, the yield of the C-8 adduct exceeds 75%. The high selectivity is a direct consequence of the long aqueous solution lifetimes of the cations. McClelland and co-workers verified the magnitudes of kd-G for these and others ions via direct kinetic measurements following lfp generation of the ions (137, 138). Their results confirm that kdG reaches an apparent diffusion-controlled limit of ca. 2.0 � 109 M-1 s-1 for many ions with ks ≥ 106 s-1.

The intermediacy of nitrenium ions in the adduct-forming reactions of monocyclic N-arylhydroxylamine derivatives has not been tested. The N-acetoxy derivatives of 2-, 3-, and 4-tolylhydroxylamines, and 2,3-, 2,4-, 3,5-, and 2,6-dimethylphenylhydroxylamines react inefficiently with monomeric dG or with dG residues in DNA, and show a greater tendency to generate adducts with 2¢ -deoxyadenosine and 2¢ -deoxycytidine than do their 4-biphenylyl or 2-fluorenyl analogues (139-141). Whether these reactions involve the short-lived nitrenium ions that would be generated by these esters remains an open question.

The detailed mechanism for formation of the C-8 adduct has been of considerable interest. Mechanistic investigations have centered around the alternative mechanisms A, B, and C (Figure 10). Humphreys, Kadlubar, and Guengerich favored mechanism A, based on the reported isolation of an 8-methyl product analogous to I from the reaction of 8,9-dimethyl-guanine with N-acetoxy-2-aminofluorene and the isolation of the C-8 adduct from the reaction of 8-bromoguanosine with N-acetoxy-2-aminofluorene (142). It was reasoned that the 8-Br analogue of I would be reduced (by excess hydroxylamine present in the reaction mixture) to the intermediate II, which would then undergo a Stevens rearrangement to the C-8 adduct. Kennedy, Novak, and Kolb were not able to generate a product analogous to I from the reaction of the 4-biphenylyl- or N-acetyl-4-biphenylylnitrenium ions with 8-methylguanosine, but did detect metastable products that appeared to be the 8-methyl analogues of II (136). These metastable products were characterized by their decomposition products and by 1H-NMR spectra taken during the reaction. Although products analogous to I were not detected, mechanism B was proposed on the basis of the reported observations of Humphreys, Kadlubar, and Guengerich, and the observation that the rate of formation of C-8 adducts for a number of purine nucleosides (adenosine, inosine, xanthosine, guanosine, dG) appeared to depend on the basicity of N-7.

McClelland's group observed the kinetics of the reaction of the 2-fluorenylnitrenium ion with dG following lfp to generate the cation (143). They identified the transient intermediate generated during the reaction as III (Ar = 2-fluorenyl, Y = H, R¢ = 2¢ -deoxyribose) based on its absorption spectrum, which extends to 400 nm, indicating a highly conjugated species; a pKa value of 3.9 that is consistent with deprotonation of III; and a rate constant for decomposition of the intermediate that is independent of Ar for Ar = 2-fluorenyl and 4-biphenylyl. The pH dependence of the kinetics of decomposition of the intermediate, and a large H/D kinetic isotope effect of ca. 6-7 for the decomposition of the intermediate derived from 8-deuterated-dG are also consistent with the identification. The rate of appearance of the intermediate was identical to the rate of disappearance of the nitrenium ion and no other intermediate was detected. The data are most readily interpreted in terms of mechanism C, which is analogous to an electrophilic aromatic substitution. The results cannot rule out initial attack by N-7, followed by rapid rearrangement to III, but the N-7 intermediate would have to be very short lived (< 5 ns) to escape detection.

Phillips and coworkers characterized the 2-fluorenylnitrenium ion in CH3CN/H2O mixtures by time-resolved resonance Raman (TR3) spectroscopy (144), and they subsequently used the technique to monitor the reaction of the cation with guanosine (145, 146). They detected an intermediate that appeared to be III (Ar = 2-fluorenyl, Y = H, R¢ = ribose).The TR3 spectrum of the intermediate identified as III is consistent with its calculated (BPW91/cc-PVDZ) vibrational spectrum. Kinetics of formation of III are consistent with its direct formation from the nitrenium ion as concluded by McClelland. A recent DFT computational study employing the B3LYP method applied to 6-31G (d), 6-31G (d,p), 6-31+G (d,p), and cc-pVDZ50 basis sets provided additional evidence in favor of mechanism C (147). The weight of evidence now indicates that the reaction occurs via direct attack of C-8 on the N of the nitrenium ion, for the long-lived ions derived from polycyclic arylamine carcinogens.

14. HETEROCYCLIC ARYLAMINES

In 1939, Widmark demonstrated that extracts of fried horse meat caused cancers when applied to mouse skin (148). However, the identification of carcinogenic components of fried and broiled meats as heterocyclic arylamines (HCAs) took place in the 1970s and 1980s, mainly as a result of the investigations of Sugimura and coworkers (reviewed in (149-151)). More than 20 active HCAs have been identified in grilled meat; a selection is shown in Figure 11. In addition to their presence in cooked meats, HCAs have been detected in commercial food flavorings and sauces, beverages, and tobacco smoke (reviewed in (152)). Initial studies using the Ames (Salmonella typhimurium) mutagenicity assay showed that HCA mutagenicity required the presence of mammalian liver homogenates or purified cytochrome P450 preparations (153, 154). N-Hydroxylation is a necessary step in the metabolic activation of HCAs.

The further metabolic activation of HCAs and the generation of DNA adducts from their metabolites has been reviewed recently (69), and only a few highlights will be mentioned here. Acetyl-CoA enhances the binding of N-hydroxy-IQ to DNA in monkey and rat tissue, while PAPS is ineffective at enhancing the binding of N-hydroxy-IQ to DNA (155). In contrast, PAPS increases the binding of N-hydroxy-PhIP to DNA, particularly in monkey tissues (155). This suggested that the major activation pathways for N-hydroxy-IQ and N-hydroxy-PhIP diverge, with acetyl CoA-dependent NAT responsible for activation of N-hydroxy-IQ and PAPS-dependent sulfotransferase (SULT) responsible for activation of N-hydroxy-PhIP. Subsequent studies using recombinant human NAT and SULT enzymes expressed in S. typhimurium confirmed that N-hydroxy-IQ is specifically activated by NAT and N-hydroxy-PhIP by SULT (67).

Adducts of HCAs with DNA and DNA bases have been extensively investigated since 1979 (69, 156). The major target in monomeric nucleosides, and in DNA in vitro and in vivo, is dG (69, 156). The major adducts are always C-8 adducts, but other structures have been identified in a few instances (69, 156). C-8 adducts of Glu-P-1 and Trp-P-2 were the first to be identified, in 1979 (157, 158). These adducts were isolated from DNA treated with Glu-P-1 or Trp-P-2 in the presence of rat hepatic microsomes (157, 158), from the reactions of the acetic acid esters of N-hydroxy-Glu-P-1 and N-hydroxy-Trp-P-2 with DNA (159, 160), and from hepatic DNA of rats fed the corresponding HCA (161).

The C-8 adducts of IQ, MeIQ, and MeIQx were synthesized by reaction of the N-acetoxy-HCA with dG or DNA (162-164) and were also isolated from in vivo experiments (163-167). Shortly after the C-8 adducts of IQ and MeIQx were discovered, minor N-2 adducts were isolated from the reaction of N-acetoxy-IQ and N-acetoxy-MeIQx with dG or DNA (168). These adducts, reminiscent of the N-2 adduct previously discovered for 2-AAF (169), accounted for 10-15% of the reaction with dG or DNA. The minor IQ adduct was discovered, along with the major C-8 adduct, in tissues of rats and monkeys that were administered single or multiple doses of IQ (170 -172).

The Novak group performed an extensive study of the chemistry of synthetic ester derivatives of a wide variety of N-hydroxy-HCAs (reviewed in 2004 (173)). At neutral pH, all of the ester derivatives investigated generated nitrenium ions that were trappable by N3-, with kaz/ks values that are comparable to those previously measured for nitrenium ions derived from carcinogenic polycyclic aromatic amines. For example, the N-acetyl-IQx and N-acetyl-MeIQx nitrenium ions (Figure 12) were trapped by N3- with kaz/ks values of 5.2 � 104 M-1 and 1.2 � 105 M-1, respectively (174). Both ions were also trapped by dG to yield a mixture of C-8 and N-2 adducts in approximately an 80/20 ratio (174). This is a unique case, because the N-2 adducts of other nitrenium ions have not been generated from the reactions of the nitrenium ions with dG. Typically, these minor N-2 adducts are detected only in reactions with DNA (169)

Because of their heterocyclic structures, some HCA nitrenium ions have acid-base properties that are physiologically relevant. The AC nitrenium ion exhibits pH dependence of its azide/solvent and dG/solvent selectivity that can be traced to ionization of the 9-NH to form a neutral quinonoid (Figure 13) (175). The 9-NMe ion does not exhibit this pH dependence (175). It is interesting that both the cation and its neutral conjugate base are electrophiles that exhibit significant reactivity with both N3- and dG (175). Because the pKa of the cation is 3.0, the neutral quinonoid is responsible for the observed reactions at physiological pH.

15. SUMMARY

Some 50 years after the discovery of the N-hydroxylation of AAF and the initial formulation of the nitrenium ion hypothesis, evidence from a wide variety of experiments has confirmed that N-arylnitrenium ions (or, occasionally, their neutral quinonoid conjugate bases) are responsible for the mutagenic and carcinogenic activity of polycyclic arylamines. Monocyclic arylamines are typically less potent carcinogens. The nitrenium ions derived from monocyclic arylamines have very short lifetimes, and it is less clear that they mediate the biological activity of these compounds. It is now recognized that the deoxyguanosine C-8 adducts of polycyclic arylamines are products of the reaction of nitrenium ions with DNA. The basis of the high regioselectivity of that reaction, and the role that DNA structure plays in determining it, are still not understood. Other types of arylamine-DNA adducts, while quantitatively minor, may nevertheless play significant biological roles. Investigation of the chemistry and biology of carcinogenic N-arylhydroxylamine derivatives has been a very productive area of research for the last fifty years and it promises to remain so for the foreseeable future.

16. ACKNOWLEDGMENTS

We wish to thank the Natural Sciences and Engineering Research Council of Canada (Josephy) and the American Cancer Society and NIH (Novak) for research support. The authors contributed equally to this work.

17. REFERENCES

1. R Wrangham. Catching Fire: How Cooking Made Us Human. Basic Books, New York (2009)

2. C Organ, CL Nunn, Z Machanda, RW Wrangham: Phylogenetic rate shifts in feeding time during the evolution of Homo. Proc Natl Acad Sci USA 108, 14555-14559 (2011)
http://dx.doi.org/10.1073/pnas.1107806108
PMid:21873223    PMCid:3167533

3. S Garfield. Mauve: How One Man Invented a Color that Changed the World. Faber and Faber, London (2000)

4. MM Sousa, MJ Melo, AJ Parola, PJ Morris, HS Rzepa, JS de Melo: A study in mauve: unveiling Perkin's dye in historic samples. Chemistry 14, 8507-8513 (2008)
http://dx.doi.org/10.1002/chem.200800718
PMid:18671308

5. E Bamberger: Ûber das Phenylhydroxylamin. Berichte der deutschen chemischen Gesellschaft 27, 1548-1557 (1894)

6. HE Heller, ED Hughes, CK Ingold: A new view of the arylhydroxylamine rearrangement. Nature 168, 909-910 (1951)
http://dx.doi.org/10.1038/168909a0

7. AK Debnath, G Debnath, AJ Shusterman, C Hansch: A QSAR investigation of the role of hydrophobicity in regulating mutagenicity in the Ames test: 1. Mutagenicity of aromatic and heteroaromatic amines in Salmonella typhimurium TA98 and TA100. Environ Mol Mutagen 19, 37-52 (1992)
http://dx.doi.org/10.1002/em.2850190107
PMid:1732103

8. R Franke, A Gruska, A Giuliani, R Benigni: Prediction of rodent carcinogenicity of aromatic amines: a quantitative structure-activity relationships model. Carcinogenesis 22, 1561-1571 (2001)
http://dx.doi.org/10.1093/carcin/22.9.1561
PMid:11532881

9. GL Borosky: Carcinogenic carbocyclic and heterocyclic aromatic amines: A DFT study concerning their mutagenic potency. J Mol Graphics Model 27, 459-465 (2008)
http://dx.doi.org/10.1016/j.jmgm.2008.08.002
PMid:18799337

10. LS DeBruin, JB Pawliszyn, PD Josephy: Detection of monocyclic aromatic amines, possible mammary carcinogens, in human milk. Chem Res Toxicol 12, 78-82 (1999)
http://dx.doi.org/10.1021/tx980168m
PMid:9894021

11. PL Skipper, MY Kim, H-LP Sun, GN Wogan, SR Tannenbaum: Monocyclic aromatic amines as potential human carcinogens: old is new again. Carcinogenesis 31, 50-58 (2010)
http://dx.doi.org/10.1093/carcin/bgp267
PMid:19887514    PMCid:2802674

12. W Lenk: Obituary: Manfred Kiese. Xenobiotica 13, 197 (1983)
http://dx.doi.org/10.3109/00498258309052254

13. M Kiese: Oxidation of aniline to nitrosobenzene in dogs. Naunyn Schmiedebergs Arch Exp Pathol Pharmakol 235, 354-359 (1959)
PMid:13657190

14. JH Harrison, Jr, DJ Jollow: Contribution of aniline metabolites to aniline-induced methemoglobinemia. Mol Pharmacol 32, 423-431 (1987)
PMid:3670278

15. FF Kadlubar: In memoriam: James A. Miller (1915-2000). Chem Res Toxicol 14, 335-337 (2001)
http://dx.doi.org/10.1021/tx010499q
PMid:11304119

16. JA Miller, EC Miller: Some historical aspects of N-aryl carcinogens and their metabolic activation. Environ Health Perspect 49, 3-12 (1983)
http://dx.doi.org/10.1289/ehp.83493
PMid:6339227    PMCid:1569122

17. E Boyland, CE Dukes, PL Grover: Carcinogenicity of 2-naphthylhydroxylamine and 2-naphthylamine. Br J Cancer 17, 79-84 (1963)
http://dx.doi.org/10.1038/bjc.1963.12
PMid:14014726    PMCid:2071273

18. H Uehleke: N-Hydroxylation of carcinogenic amines in vivo and in vitro with liver microsomes. Biochem Pharmacol 12, 219-221 (1963)
http://dx.doi.org/10.1016/0006-2952(63)90190-4

19. T Omura, R Sato: The carbon monoxide-binding pigment of liver microsomes. J Biol Chem 239, 2370-2378 (1964)
PMid:14209971

20. H Uehleke: The role of cytochrome P-450 in the N-oxidation of individual amines. Drug Metab Dispos 1, 299-313 (1973)
PMid:4149397

21. WG Levine: Metabolism and biliary excretion of N-2-fluorenylacetamide and N-hydroxy-2-fluorenylacetamide. Life Sci 10, 727-735 (1971)
http://dx.doi.org/10.1016/0024-3205(71)90204-9

22. SS Thorgeirsson, DJ Jollow, HA Sasame, I Green, JR Mitchell: The role of cytochrome P-450 in N-hydroxylation of 2-acetylaminofluorene. Mol Pharmacol 9, 398-404 (1973)
PMid:4122821

23. CH Yun, T Shimada, FP Guengerich: Contributions of human liver cytochrome P450 enzymes to the N-oxidation of 4,4'-methylene-bis(2-chloroaniline). Carcinogenesis 13, 217-222 (1992)
http://dx.doi.org/10.1093/carcin/13.2.217
PMid:1740010

24. FP Guengerich, NA Hosea, A Parikh, LC Bell-Parikh, WW Johnson, EMJ Gillam, T Shimada: Twenty years of biochemistry of human P450s - Purification, expression, mechanism, and relevance to drugs. Drug Metab Dispos 26, 1175-1178 (1998)
PMid:9860923

25. PD Josephy, LS DeBruin, HL Lord, JN Oak, DH Evans, Z Guo, MS Dong, FP Guengerich: Bioactivation of aromatic amines by recombinant human cytochrome P4501A2 expressed in Ames tester strain bacteria: a substitute for activation by mammalian tissue preparations. Cancer Res 55, 799-802 (1995)
PMid:7850792

26. Y Oda, P Aryal, T Terashita, EM Gillam, FP Guengerich, T Shimada: Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase. Mutat Res 492, 81-90 (2001)
PMid:11377247

27. D Sesardic, AR Boobis, RJ Edwards, DS Davies: A form of cytochrome P450 in man, orthologous to form d in the rat, catalyses the O-deethylation of phenacetin and is inducible by cigarette smoking. Br J Clin Pharmacol 26, 363-372 (1988)
PMid:3190986    PMCid:1386556

28. T Shimada, H Yamazaki, M Mimura, Y Inui, FP Guengerich: Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270, 414-423 (1994)
PMid:8035341

29. T Aoyama, HV Gelboin, FJ Gonzalez: Mutagenic activation of 2-amino-3-methylimidazo(4,5-f)quinoline by complementary DNA-expressed human liver P-450. Cancer Res 50, 2060-2063 (1990)
PMid:2180561

30. PD Josephy, SM Batty, DR Boverhof: Recombinant human P450 forms 1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens in Escherichia coli lacZ strains. Environ Mol Mutagen 38, 12-18 (2001)
http://dx.doi.org/10.1002/em.1045
PMid:11473383

31. D Kim, FP Guengerich: Cytochrome P450 activation of arylamines and heterocyclic amines. Annu Rev Pharmacol Toxicol 45, 27-49 (2005)
http://dx.doi.org/10.1146/annurev.pharmtox.45.120403.100010
PMid:15822170

32. FP Guengerich: N-Hydroxyarylamines. Drug Metab Rev 34, 607-623 (2002)
http://dx.doi.org/10.1081/DMR-120005663
PMid:12214670

33. JR Cashman, J Zhang: Human flavin-containing monooxygenases. Annu Rev Pharmacol Toxicol 46, 65-100 (2006)
http://dx.doi.org/10.1146/annurev.pharmtox.46.120604.141043
PMid:16402899

34. SC Mitchell: Flavin mono-oxygenase (FMO) - the 'other' oxidase. Curr Drug Metab 9, 280-284 (2008)
http://dx.doi.org/10.2174/138920008784220682
PMid:18473746

35. JR Cashman: Role of flavin-containing monooxygenase in drug development. Expert Opin Drug Metab Toxicol 4, 1507-1521 (2008)
http://dx.doi.org/10.1517/17425250802522188

36. J Rittle, MT Green: Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics. Science 330, 933-937 (2010)
http://dx.doi.org/10.1126/science.1193478
PMid:21071661

37. S Eswaramoorthy, JB Bonanno, SK Burley, S Swaminathan: Mechanism of action of a flavin-containing monooxygenase. Proc Natl Acad Sci USA 103, 9832-9837 (2006)
http://dx.doi.org/10.1073/pnas.0602398103
PMid:16777962    PMCid:1502539

38. DM Ziegler, EM McKee, LL Poulsen: Microsomal flavoprotein-catalyzed N-oxidation of arylamines. Drug Metab Dispos 1, 314-321 (1973)
PMid:4149399

39. LL Poulsen, DM Ziegler: Multisubstrate flavin-containing monooxygenases: applications of mechanism to specificity. Chem Biol Interact 96, 57-73 (1995)
http://dx.doi.org/10.1016/0009-2797(94)03583-T

40. GJ Hammons, FP Guengerich, CC Weis, FA Beland, FF Kadlubar: Metabolic oxidation of carcinogenic arylamines by rat, dog, and human hepatic microsomes and by purified flavin-containing and cytochrome P- 450 monooxygenases. Cancer Res 45, 3578-3585 (1985)
PMid:4016738

41. AL Tsai, RJ Kulmacz: Prostaglandin H synthase: resolved and unresolved mechanistic issues. Arch Biochem Biophys 493, 103-124 (2010)
http://dx.doi.org/10.1016/j.abb.2009.08.019
PMid:19728984    PMCid:2812681

42. RM Botting: Inhibitors of cyclooxygenases: mechanisms, selectivity and uses. J Physiol Pharmacol 57 Suppl 5, 113-124 (2006)

43. JA Boyd, DJ Harvan, TE Eling: The oxidation of 2-aminofluorene by prostaglandin endoperoxide synthetase. Comparison with other peroxidases. J Biol Chem 258, 8246-8254 (1983)
PMid:6408086

44. JA Boyd, TE Eling: Metabolism of aromatic amines by prostaglandin H synthase. Environ Health Perspect 64, 45-51 (1985)
http://dx.doi.org/10.1289/ehp.856445
PMid:3938394    PMCid:1568607

45. JA Boyd, TE Eling: Prostaglandin H synthase-catalyzed metabolism and DNA binding of 2-naphthylamine. Cancer Res 47, 4007-4014 (1987)
PMid:3111680

46. TW Petry, PD Josephy, DA Pagano, E Zeiger, KT Knecht, TE Eling: Prostaglandin hydroperoxidase-dependent activation of heterocyclic aromatic amines. Carcinogenesis 10, 2201-2207 (1989)
http://dx.doi.org/10.1093/carcin/10.12.2201
PMid:2512013

47. PD Josephy: Activation of aromatic amines by prostaglandin H synthase. Free Radic Biol Med 6, 533-540 (1989)
http://dx.doi.org/10.1016/0891-5849(89)90046-4

48. FH Sarkar, G Radcliff, DM Callewaert: Purified prostaglandin synthase activates aromatic amines to derivatives that are mutagenic to Salmonella typhimurium. Mutat Res 282, 273-281 (1992)
http://dx.doi.org/10.1016/0165-7992(92)90134-4

49. P Hlavica, I Golly, M Lehnerer, J Schulze: Primary aromatic amines: their N-oxidative bioactivation. Hum Exp Toxicol 16, 441-448 (1997)
http://dx.doi.org/10.1177/096032719701600805

50. PD Josephy, TE Eling, RP Mason: Co-oxidation of benzidine by prostaglandin synthase and comparison with the action of horseradish peroxidase. J Biol Chem 258, 5561-5569 (1983)
PMid:6304093

51. M Stiborova, M Miksanova, V Havlicek, HH Schmeiser, E Frei: Mechanism of peroxidase-mediated oxidation of carcinogenic o-anisidine and its binding to DNA. Mutat Res 500, 49-66 (2002)
http://dx.doi.org/10.1016/S0027-5107(01)00295-0

52. LD Morrison, TE Eling, PD Josephy: Prostaglandin H synthase-dependent formation of the direct-acting mutagen 2-nitro-3-methylimidazo(4,5-f)quinoline (nitro-IQ) from IQ. Mutat Res 302, 45-52 (1993)
http://dx.doi.org/10.1016/0165-7992(93)90089-E

53. JW Cramer, JA Miller, EC Miller: The hydroxylation of the carcinogen 2-acetylaminofluorene by rat liver: stimulation by pretreatment in vivo with 3-methylcholanthrene. J Biol Chem 235, 250-256 (1960)
PMid:13812622

54. HA Barton, MA Marletta: Comparison of aniline hydroxylation by hemoglobin and microsomal cytochrome P450 using stable isotopes. Toxicol Lett 70, 147-153 (1994)
http://dx.doi.org/10.1016/0378-4274(94)90158-9

55. PD Josephy, B Mannervik. Molecular Toxicology, 2nd ed. Oxford University Press, New York (2006)

56. EC Miller, PD Lotlikar, JA Miller, BW Butler, CC Irving, JT Hill: Reactions in vitro of some tissue nucleophiles with the glucuronide of the carcinogen N-hydroxy-2-acetylaminofluorene. Mol Pharmacol 4, 147-154 (1968)
PMid:5650958

57. JD Scribner, JA Miller, EC Miller: Nucleophilic substitution on carcinogenic N-acetoxy-N-arylacetamides. Cancer Res 30, 1570-1579 (1970)
PMid:5457931

58. JR DeBaun, JY Rowley, EC Miller, JA Miller: Sulfotransferase activation of N-hydroxy-2-acetylaminofluorene in rodent livers susceptible and resistant to this carcinogen. Proc Soc Exp Biol Med 129, 268-273 (1968)
PMid:5697914

59. FF Kadlubar, JA Miller, EC Miller: Hepatic metabolism of N-hydroxy-N-methyl-4-aminoazobenzene and other N-hydroxy arylamines to reactive sulfuric acid esters. Cancer Res 36, 2350-2359 (1976)
PMid:819129

60. EC Miller, U Juhl, JA Miller: Nucleic acid guanine: reaction with the carcinogen N-acetoxy-2-acetylaminofluorene. Science 153, 1125-1127 (1966)
http://dx.doi.org/10.1126/science.153.3740.1125
PMid:5918575

61. FF Kadlubar, FA Beland: Chemical properties of ultimate carcinogenic metabolites of arylamines and arylamides. In: Polycyclic Hydrocarbons and Carcinogenesis. Ed: RG Harvey. American Chemical Society, Washington, DC. (1985)
http://dx.doi.org/10.1021/bk-1985-0283.ch014

62. MH Buonarati, JS Felton: Activation of 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) to mutagenic metabolites. Carcinogenesis 11, 1133-1138 (1990)
http://dx.doi.org/10.1093/carcin/11.7.1133
PMid:2197012

63. K Saito, Y Yamazoe, T Kamataki, R Kato: Mechanism of activation of proximate mutagens in Ames' tester strains: the acetyl-CoA dependent enzyme in Salmonella typhimurium TA98 deficient in TA98/1,8-DNP6 catalyzes DNA-binding as the cause of mutagenicity. Biochem Biophys Res Commun 116, 141-147 (1983)
http://dx.doi.org/10.1016/0006-291X(83)90392-3

64. BF de France, MH Carter, PD Josephy, DW Bryant, DR McCalla: Metabolism and mutagenesis of benzidine in Salmonella typhimurium strains TA98 and TA98/1,8-DNP6. Mutat Res 144, 159-163 (1985)
http://dx.doi.org/10.1016/0165-7992(85)90133-2

65. M Watanabe, T Nohmi, MJ Ishidate: New tester strains of Salmonella typhimurium highly sensitive to mutagenic nitroarenes. Biochem Biophys Res Commun 147, 974-979 (1987)
http://dx.doi.org/10.1016/S0006-291X(87)80165-1

66. DM Grant, PD Josephy, HL Lord, LD Morrison: Salmonella typhimurium strains expressing human arylamine N-acetyltransferases: metabolism and mutagenic activation of aromatic amines. Cancer Res 52, 3961-3964 (1992)
PMid:1617672

67. E Muckel, H Frandsen, HR Glatt: Heterologous expression of human N-acetyltransferases 1 and 2 and sulfotransferase 1A1 in Salmonella typhimurium for mutagenicity testing of heterocyclic amines. Food Chem Toxicol 40, 1063-1068 (2002)
http://dx.doi.org/10.1016/S0278-6915(02)00032-7

68. W Meinl, JH Meerman, H Glatt: Differential activation of promutagens by alloenzymes of human sulfotransferase 1A2 expressed in Salmonella typhimurium. Pharmacogenetics 12, 677-689 (2002)
http://dx.doi.org/10.1097/00008571-200212000-00002
PMid:12464797

69. RJ Turesky, L Le Marchand: Metabolism and biomarkers of heterocyclic aromatic amines in molecular epidemiology studies: lessons learned from aromatic amines. Chem Res Toxicol 24, 1169-1214 (2011)
http://dx.doi.org/10.1021/tx200135s
PMid:21688801

70. DW Hein: Molecular genetics and function of NAT1 and NAT2: role in aromatic amine metabolism and carcinogenesis. Mutat Res 506, 65-77 (2002)
http://dx.doi.org/10.1016/S0027-5107(02)00153-7

71. DW Hein: N-Acetyltransferase SNPs: emerging concepts serve as a paradigm for understanding complexities of personalized medicine. Expert Opin Drug Metab Toxicol 5, 353-366 (2009)
http://dx.doi.org/10.1517/17425250902877698

72. F Marroquin, E Farber: The apparent binding of radioactive 2-acetylaminofluorene to rat-liver ribonucleic acid in vivo. Biochim Biophys Acta 55, 403-405 (1962)
http://dx.doi.org/10.1016/0006-3002(62)90802-8

73. F Marroquin, E Farber: The binding of 2-acetylaminofluorene to rat liver ribonucleic acid in vivo. Cancer Res 25, 1262-1269 (1965)
PMid:5839718

74. E Kriek: On the interaction of N-2-fluorenylhydroxylamine with nucleic acids in vitro. Biochem Biophys Res Commun 20, 793-799 (1965)
http://dx.doi.org/10.1016/0006-291X(65)90088-4

75. E Kriek, JA Miller, U Juhl, EC Miller: 8-(N-2-Fluorenylacetamido)guanosine, an arylamidation reaction product of guanosine and the carcinogen N-acetoxy-N-2-fluorenylacetamide in neutral solution. Biochemistry 6, 177-182 (1967)
http://dx.doi.org/10.1021/bi00853a029
PMid:6030315

76. E Kriek: Fifty years of research on N-acetyl-2-aminofluorene, one of the most versatile compounds in experimental cancer research. J Cancer Res Clin Oncol 118, 481-489 (1992)
http://dx.doi.org/10.1007/BF01225261
PMid:1624539

77. FA Beland, FF Kadlubar: Formation and persistence of arylamine DNA adducts in vivo. Environ Health Perspect 62, 19-30 (1985)
http://dx.doi.org/10.1289/ehp.856219
PMid:4085422    PMCid:1568675

78. II Kukhtenko: Mechanism of N-phenylhydroxylamine rearrangement to para-aminophenol. Zhurnal Organicheskoi Khimii (English translation) 7, 324-326 (1971)

79. S Oae, T Kitao: Elucidation of mechanisms of organic reactions using oxygen-18 as a tracer. Yuki Gosei Kagaku Kyokaishi 19, 899 (1961)
http://dx.doi.org/10.5059/yukigoseikyokaishi.19.880

80. T Sone, Y Tokuda, T Sakai, S Shinkai, O Manabe: Kinetics and mechanisms of the Bamberger rearrangement .3. Rearrangement of phenylhydroxylamines to para-aminophenols in aqueous sulfuric acid solutions. J Chem Soc Perkin Trans 2298-2302 (1981)

81. G Kohnstam, WA Petch, DLH Williams: Kinetic substituent and isotope effects in the acid-catalyzed rearrangement of N-phenylhydroxylamines - are nitrenium ions involved. J Chem Soc Perkin Trans 2423-2427 (1984)

82. LA Sternson, R Chandrasakar: Further evidence for nitrenium ion intermediacy in N-phenylhydroxylamine rearrangement to aminophenol. J Org Chem 49, 4295-4297 (1984)
http://dx.doi.org/10.1021/jo00196a037

83. PG Gassman, GA Campbell: Chemistry of nitrenium ions.16. Mechanism of chlorination of anilines and related aromatic amines - involvement of nitrenium ions. J Am Chem Soc 93, 2567-2569 (1971)
http://dx.doi.org/10.1021/ja00739a053

84. PG Gassman, GA Campbell: Chemistry of nitrenium ions. 22. Thermal rearrangement of n-chloroanilines - evidence for intermediacy of nitrenium ions. J Am Chem Soc 94, 3891-3896 (1972)
http://dx.doi.org/10.1021/ja00766a038

85. PG Gassman, G Campbell, R Frederick: Anilenium ions. Intermediates in nucleophilic substitution of anilines. J Am Chem Soc 90, 7377-7378 (1968)
http://dx.doi.org/10.1021/ja01028a055

86. PG Gassman, GA Campbell: Conversion of anilines into derivatives of cyclohexa-2,5-dienones. J Chem Soc, Chem Commun 427 (1970)

87. PG Gassman, JE Granrud: Synthesis and rearrangement of methanesulfonate esters of N-hydroxyacetanilides - a model for a penultimate carcinogen. J Am Chem Soc 106, 1498-1499 (1984)
http://dx.doi.org/10.1021/ja00317a060

88. M Novak, M Pelecanou, AK Roy, AF Andronico, FM Plourde, TM Olefirowicz, TJ Curtin: Solvolysis of N-sulfonoxyacetanilides in aqueous and alcohol-solutions - generation of electrophilic species. J Am Chem Soc 106, 5623-5631 (1984)
http://dx.doi.org/10.1021/ja00331a036

89. JP Richard, WP Jencks: A simple relationship between carbocation lifetime and reactivity selectivity relationships for the solvolysis of ring-substituted 1-phenylethyl derivatives. J Am Chem Soc 104, 4689-4691 (1982)
http://dx.doi.org/10.1021/ja00381a037

90. JP Richard, WP Jencks: Concerted SN2 displacement reactions of 1-phenylethyl chlorides. J Am Chem Soc 104, 4691-4692 (1982)
http://dx.doi.org/10.1021/ja00381a038

91. JP Richard, WP Jencks: Concerted bimolecular substitution-reactions of 1-phenylethyl derivatives. J Am Chem Soc 106, 1383-1396 (1984)
http://dx.doi.org/10.1021/ja00317a033

92. JC Fishbein, RA McClelland: Azide ion trapping of the intermediate in the Bamberger rearrangement. Lifetime of a free nitrenium ion in aqueous solution. J Am Chem Soc 109, 2824-2825 (1987)
http://dx.doi.org/10.1021/ja00243a045

93. RC Baetzold, LKJ Tong: Kinetics of redox reactions of oxidized para-phenylenediamine derivatives. 1. J Am Chem Soc 93, 1347-1353 (1971)
http://dx.doi.org/10.1021/ja00742a606

94. LKJ Tong: Kinetics of deamination of oxidized N,N-disubstituted para-phenylenediamines. J Phys Chem 58, 1090-1097 (1954)
http://dx.doi.org/10.1021/j150522a012

95. U Svanholm, VD Parker: Chemistry of dianisylnitrenium ion - Observation of stable protonated nitrenium ions. J Am Chem Soc 96, 1234-1236 (1974)
http://dx.doi.org/10.1021/ja00811a053

96. D Serve: Chemistry of electrogenerated diarylnitrenium ions - absorption-spectra of stable protonated nitrenium ions. J Am Chem Soc 97, 432-434 (1975)
http://dx.doi.org/10.1021/ja00835a041

97. G Boche, P Andrews, K Harms, M Marsch, KS Rangappa, M Schimeczek, C Willeke: Crystal and electronic structure of stable nitrenium ions. A comparison with structurally related carbenes. J Am Chem Soc 118, 4925-4930 (1996)
http://dx.doi.org/10.1021/ja9536274

98. KT Chung, CE Cerniglia: Mutagenicity of azo dyes: structure-activity relationships. Mutat Res 277, 201-220 (1992)
PMid:1381050

99. RC Graham, Jr, MJ Karnovsky: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14, 291-302 (1966)
http://dx.doi.org/10.1177/14.4.291

100. C Wurster, R Sendtner: Zur Kenntniss des Dimethylparaphenylendiamins. Berichte der deutschen chemischen Gesellschaft 12, 1803-1807 (1879)

101. S Granick, L Michaelis, MP Schubert: The free radicals of the type of Wurster's salts. J Am Chem Soc 61, 1981-1992 (1939)
http://dx.doi.org/10.1021/ja01877a013

102. R Willstätter, L Kalb: Ûber chinoïde Derivate des Diphenyls. II. Berichte der deutschen chemischen Gesellschaft 38, 1232-1241 (1905)

103. R Adams, RR Holmes: Quinone diimides. XV. Diphenoquinonediimides. J Am Chem Soc 74, 3033-3037 (1952)
http://dx.doi.org/10.1021/ja01132a025

104. S Hünig, P Richters: N,N-Dialkyl-chinon-diimonium-Salze. Berichte der deutschen chemischen Gesellschaft 91, 442-448 (1958)

105. P van Duijn: An improved histochemical benzidine-blue peroxidase method and a note on the composition of the blue reaction product. Rec Trav Chim Pays-Bas 74, 771-778 (1955)
http://dx.doi.org/10.1002/recl.19550740617

106. NA Macías-Ruvalcaba, DH Evans: Oxidation reactions of a series of benzidines: electrochemical detection of dimerization of cation radicals and examples of potential inversion caused by very small steric effects. J Phys Chem C 111, 5811 (2007)

107. VR Holland, BC Saunders, FL Rose, AL Walpole: A safer substitute for benzidine in the detection of blood. Tetrahedron 30, 3299-3302 (1974)
http://dx.doi.org/10.1016/S0040-4020(01)97504-0

108. A Claiborne, I Fridovich: Chemical and enzymatic intermediates in the peroxidation of o-dianisidine by horseradish peroxidase. 1. Spectral properties of the products of dianisidine oxidation. Biochemistry 18, 2324-2329 (1979)
http://dx.doi.org/10.1021/bi00578a029
PMid:221005

109. PD Josephy, T Eling, RP Mason: The horseradish peroxidase-catalyzed oxidation of 3,5,3',5'- tetramethylbenzidine. Free radical and charge-transfer complex intermediates. J Biol Chem 257, 3669-3675 (1982)
PMid:6277943

110. PD Josephy, RP Mason, T Eling: Cooxidation of the clinical reagent 3,5,3',5'-tetramethylbenzidine by prostaglandin synthase. Cancer Res 42, 2567-2570 (1982)
PMid:6805942

111. PD Josephy, TE Eling, RP Mason: An electron spin resonance study of the activation of benzidine by peroxidases. Mol Pharmacol 23, 766-770 (1983)
PMid:6306434

112. RW Wise, TV Zenser, BB Davis: Characterization of benzidinediimine: a product of peroxidase metabolism of benzidine. Carcinogenesis 5, 1499-1503 (1984)
http://dx.doi.org/10.1093/carcin/5.11.1499
PMid:6488474

113. RA McClelland, D Ren, R D'Sa, AR Ahmed: Acidity constants and reactivities of the benzidine and N,N-dimethylbenzidine dications, the two electron oxidation intermediates of benzidine carcinogens. Can J Chem 78, 1178-1185 (2000)

114. PD Josephy, DC Iwaniw: Identification of the N-acetylcysteine conjugate of benzidine formed in the peroxidase activation system. Carcinogenesis 6, 155-158 (1985)
http://dx.doi.org/10.1093/carcin/6.1.155
PMid:3967336

115. JR Rice, PT Kissinger: Cooxidation of benzidine by horseradish peroxidase and subsequent formation of possible thioether conjugates of benzidine. Biochem Biophys Res Commun 104, 1312-1318 (1982)
http://dx.doi.org/10.1016/0006-291X(82)91393-6

116. VM Lakshmi, TV Zenser, BB Davis: Mechanism of 3-(glutathion-S-yl)-benzidine formation. Toxicol Appl Pharmacol 125, 256-263 (1994)
http://dx.doi.org/10.1006/taap.1994.1071
PMid:8171433

117. Y Yamazoe, RW Roth, FF Kadlubar: Reactivity of benzidine diimine with DNA to form N-(deoxyguanosin-8-yl)-benzidine. Carcinogenesis 7, 179-182 (1986)
http://dx.doi.org/10.1093/carcin/7.1.179
PMid:3943140

118. Y Tsuruta, PD Josephy, AD Rahimtula, PJ O'Brien: Peroxidase-catalyzed benzidine binding to DNA and other macromolecules. Chem Biol Interact 54, 143-158 (1985)
http://dx.doi.org/10.1016/S0009-2797(85)80159-9

119. PD Josephy: Benzidine: mechanisms of oxidative activation and mutagenesis. Fed Proc 45, 2465-2470 (1986)
PMid:3091404

120. GH Degen, JH Schlattjan, S Mahler, W Follmann, K Golka: Comparative metabolic activation of benzidine and N-acetylbenzidine by prostaglandin H synthase. Toxicol Lett 151, 135-142 (2004)
http://dx.doi.org/10.1016/j.toxlet.2003.11.015
PMid:15177648

121. KM Gorlewska-Roberts, CH Teitel, JO Lay, Jr., DW Roberts, FF Kadlubar: Lactoperoxidase-catalyzed activation of carcinogenic aromatic and heterocyclic amines. Chem Res Toxicol 17, 1659-1666 (2004)
http://dx.doi.org/10.1021/tx049787n
PMid:15606142

122. ZC Liu, JP Uetrecht: Clozapine is oxidized by activated human neutrophils to a reactive nitrenium ion that irreversibly binds to the cells. J Pharmacol Exp Ther 275, 1476-1483 (1995)
PMid:8531118

123. S Dragovic, JS Boerma, L van Bergen, NPE Vermeulen, JNM Commandeur: Role of human glutathione S-transferases in the inactivation of reactive metabolites of clozapine. Chem Res Toxicol 23, 1467-1476 (2010)
http://dx.doi.org/10.1021/tx100131f
PMid:20849150

124. JS Boerma, NPE Vermeulen, JNM Commandeur: Application of CYP102A1M11H as a tool for the generation of protein adducts of reactive drug metabolites. Chem Res Toxicol 23, 1467-1476 (2010)
PMid:20849150

125. A Sikora, J Adamus, A Marcinek: Disproportionation of clozapine radical: A link between one-electron oxidation of clozapine and formation of its nitrenium cation. Chem Res Toxicol 20, 1093-1098 (2007)
http://dx.doi.org/10.1021/tx700128c
PMid:17630710

126. R Ulbrich, M Famulok, F Bosold, G Boche: SN2 at nitrogen. The reaction of N-(4-cyanophenyl)-O-diphenylphosphinoylhydroxylamine with N-methylaniline - a model for the reactions of ultimate carcinogens of aromatic-amines with (bio)nucleophiles. Tet Lett 31, 1689-1692 (1990)
http://dx.doi.org/10.1016/S0040-4039(00)88855-3

127. M Novak, KA Martin, JL Heinrich: SN2 reactions of a carbon nucleophile with N-aryl-O-pivaloylhydroxylamines - a model for in vivo reactions of carcinogenic metabolites of aromatic amines. J Org Chem 54, 5430-5431 (1989)
http://dx.doi.org/10.1021/jo00284a012

128. JS Helmick, KA Martin, JL Heinrich, M Novak: Mechanism of the reaction of carbon and nitrogen nucleophiles with the model carcinogens O-pivaloyl-N-arylhydroxylamines - competing SN2 substitution and SN1 solvolysis. J Am Chem Soc 113, 3459-3466 (1991)
http://dx.doi.org/10.1021/ja00009a035

129. M Novak, MJ Kahley, E Eiger, JS Helmick, HE Peters: Reactivity and selectivity of nitrenium ions derived from ester derivatives of carcinogenic N-(4-biphenylyl)hydroxylamine and the corresponding hydroxamic acid. J Am Chem Soc 115, 9453-9460 (1993)
http://dx.doi.org/10.1021/ja00074a010

130. PA Davidse, MJ Kahley, RA McClelland, M Novak: Flash-photolysis observation and lifetimes of 2-fluorenyl and 4-biphenylylacetylnitrenium ions in aqueous solution. J Am Chem Soc 116, 4513-4514 (1994)
http://dx.doi.org/10.1021/ja00089a065

131. RA McClelland, PA Davidse, G Hadzialic: Electron-deficient strong bases - generation of the 4-biphenylylnitrenium and 2-fluorenylnitrenium ions by nitrene protonation in water. J Am Chem Soc 117, 4173-4174 (1995)
http://dx.doi.org/10.1021/ja00119a035

132. RA McClelland, MJ Kahley, PA Davidse, G Hadzialic: Acid-base properties of arylnitrenium ions. J Am Chem Soc 118, 4794-4803 (1996)
http://dx.doi.org/10.1021/ja954248d

133. JC Fishbein, RA McClelland: Halide ion trapping of nitrenium ions formed in the Bamberger rearrangement of N-arylhydroxylamines. Lifetime of the parent phenylnitrenium ion in water. Can J Chem 74, 1321-1328 (1996)
http://dx.doi.org/10.1139/v96-147

134. J Wang, J Kubicki, MS Platz: An ultrafast study of phenyl azide: The direct observation of phenylnitrenium ion. Org Lett 9, 3973-3976 (2007)
http://dx.doi.org/10.1021/ol701598m
PMid:17718577

135. M Novak, SA Kennedy: Selective trapping of N-acetyl-N-(4-biphenylyl)nitrenium and N-acetyl-N-(2-fluorenyl)nitrenium ions by 2'-N-deoxyguanosine in aqueous solution. J Am Chem Soc 117, 574-575 (1995)
http://dx.doi.org/10.1021/ja00106a083

136. SA Kennedy, M Novak, BA Kolb: Reactions of ester derivatives of carcinogenic N-(4-biphenylyl)hydroxylamine and the corresponding hydroxamic acid with purine nucleosides. J Am Chem Soc 119, 7654-7664 (1997)
http://dx.doi.org/10.1021/ja970698p

137. RA McClelland, MJ Kahley, PA Davidse: Reactivity of the 4-biphenylyl and 2-fluorenylnitrenium ions with heterocyclic and carbon nucleophiles. J Phys Org Chem 9, 355-360 (1996)
http://dx.doi.org/10.1002/(SICI)1099-1395(199606)9:6<355::AID-POC792>3.0.CO;2-Q

138. RA McClelland, TA Gadosy, D Ren: 1997 Alfred Bader Award Lecture. Reactivities of arylnitrenium ions with guanine derivatives and other nucleophiles. Can J Chem 76, 1327-1337 (1998)
http://dx.doi.org/10.1139/v98-187

139. MM Marques, LL Mourato, MA Santos, FA Beland: Synthesis, characterization, and conformational analysis of DNA adducts from methylated anilines present in tobacco smoke. Chem Res Toxicol 9, 99-108 (1996)
http://dx.doi.org/10.1021/tx950044z
PMid:8924623

140. LL Gonçalves, FA Beland, M Marques: Synthesis, characterization, and comparative 32P-postlabeling efficiencies of 2,6-dimethylaniline-DNA adducts. Chem Res Toxicol 14, 165-174 (2001)
http://dx.doi.org/10.1021/tx0002031

141. L Cui, HL Sun, JS Wishnok, SR Tannenbaum, PL Skipper: Identification of adducts formed by reaction of N-acetoxy-3,5-dimethylaniline with DNA. Chem Res Toxicol 20, 1730-1736 (2007)
http://dx.doi.org/10.1021/tx700306c
PMid:18020398    PMCid:2573389

142. WG Humphreys, FF Kadlubar, FP Guengerich: Mechanism of C8 alkylation of guanine residues by activated arylamines: evidence for initial adduct formation at the N7 position. Proc Natl Acad Sci USA 89, 8278-8282 (1992)
http://dx.doi.org/10.1073/pnas.89.17.8278

143. RA McClelland, A Ahmad, AP Dicks, VE Licence: Spectroscopic characterization of the initial C8 intermediate in the reaction of the 2-fluorenylnitrenium ion with 2'-deoxyguanosine. J Am Chem Soc 121, 3303-3310 (1999)
http://dx.doi.org/10.1021/ja9836702

144. PZ Zhu, SY Ong, PY Chan, YF Poon, KH Leung, DL Phillips: Transient-resonance Raman and density functional theory investigation of 4-biphenylylnitrenium, 2-fluorenylnitrenium, and diphenylnitrenium ions. Chem Eur J 7, 4928-4936 (2001)
http://dx.doi.org/10.1002/1521-3765(20011119)7:22<4928::AID-CHEM4928>3.0.CO;2-C

145. PY Chan, WM Kwok, SK Lam, P Chiu, DL Phillips: Time-resolved resonance Raman observation of the 2-fluorenylnitrenium ion reaction with guanosine to form a C8 intermediate. J Am Chem Soc 127, 8246-8247 (2005)
http://dx.doi.org/10.1021/ja0505651
PMid:15941233

146. JD Xue, PY Chan, Y Du, Z Guo, CWY Chung, PH Toy, DL Phillips: Time-resolved resonance Raman investigation of the 2-fluorenylnitrenium ion reactions with C8 guanosine derivatives. J Phys Chem B 111, 12676-12684 (2007)
http://dx.doi.org/10.1021/jp074778j
PMid:17939708

147. ZZ Yang, SF Qi, DX Zhao, LD Gong: Insight into mechanism of formation of C8 adducts in carcinogenic reactions of arylnitrenium ions with purine nucleosides. J Phys Chem B 113, 254-259 (2009)
http://dx.doi.org/10.1021/jp804128s
PMid:19072693

148. EMP Widmark: Presence of cancer-producing substances in roasted food. Nature 143, 984 (1939)
http://dx.doi.org/10.1038/143984a0

149. T Sugimura: Carcinogenicity of mutagenic heterocyclic amines formed during the cooking process. Mutat Res 150, 33-41 (1985)
http://dx.doi.org/10.1016/0027-5107(85)90098-3

150. T Sugimura: Overview of carcinogenic heterocyclic amines. Mutat Res 376, 211-219 (1997)
http://dx.doi.org/10.1016/S0027-5107(97)00045-6

151. T Sugimura, K Wakabayashi, H Nakagama, M Nagao: Heterocyclic amines: Mutagens/carcinogens produced during cooking of meat and fish. Cancer Sci 95, 290-299 (2004)
http://dx.doi.org/10.1111/j.1349-7006.2004.tb03205.x
PMid:15072585

152. JS Felton, M Jägerstad, MG Knize, K Skog, K Wakabayashi. Contents in foods, beverages, and tobacco. In: Food Borne Carcinogens: Heterocyclic Amines. Eds: M Nagao, T Sugimura. Wiley, New York. (2000)

153. K Wakabayashi, M Nagao, H Esumi, T Sugimura: Food-derived mutagens and carcinogens. Cancer Res 52, 2092s-2098s (1992)
PMid:1544146

154. R Kato, T Kamataki, Y Yamazoe: N-Hydroxylation of carcinogenic and mutagenic aromatic amines. Environ Health Perspect 49, 21-25 (1983)
http://dx.doi.org/10.1289/ehp.834921
PMid:6832094    PMCid:1569125

155. CD Davis, RH Adamson, EG Snyderwine: Studies on the mutagenic activation of heterocyclic amines by cynomolgus monkey, rat and human microsomes show that cynomolgus monkeys have a low capacity to N-oxidize the quinoxaline-type heterocyclic amines. Cancer Lett 73, 95-104 (1993)
http://dx.doi.org/10.1016/0304-3835(93)90250-D

156. EG Snyderwine, KW Turteltaub. Interactions with cellular macromolecules. In: Food Borne Carcinogens: Heterocyclic Amines. Eds: M Nagao, T Sugimura. Wiley, New York. (2000)

157. Y Hashimoto, K Shudo, T Okamoto: Structure of a base in DNA modified by Glu-P-1. Chem Pharm Bull (Tokyo) 27, 2532-2534 (1979)
http://dx.doi.org/10.1248/cpb.27.2532

158. Y Hashimoto, K Shudo, T Okamoto: Structural identification of a modified base in DNA covalently bound with mutagenic 3-amino-1-methyl-5H-pyrido(4,3-b)indole. Chem Pharm Bull (Tokyo) 27, 1058-1060 (1979)
http://dx.doi.org/10.1248/cpb.27.1058

159. Y Hashimoto, K Shudo, T Okamoto: Activation of a mutagen, 3-amino-1-methyl-5H-pyrido(4,3-b)indole. Identification of 3-hydroxyamino-1-methyl-5H-pyrido(4,3-b)indole and its reaction with DNA. Biochem Biophys Res Commun 96, 355-362 (1980)
http://dx.doi.org/10.1016/0006-291X(80)91222-X

160. Y Hashimoto, K Shudo, T Okamoto: Modification of DNA with potent mutacarcinogenic 2-amino-6-methyldipyrido(1,2-a-3',2'-d)imidazole isolated from a glutamic acid pyrolysate - structure of the modified nucleic acid base and initial chemical event caused by the mutagen. J Am Chem Soc 104, 7636-7640 (1982)
http://dx.doi.org/10.1021/ja00390a040

161. Y Hashimoto, K Shudo, T Okamoto: Modification of nucleic acids with muta-carcinogenic heteroaromatic amines in vivo. Identification of modified bases in DNA extracted from rats injected with 3-amino-1-methyl-5H-pyrido(4,3-b)indole and 2-amino-6-methyldipyrido(1,2-a3:3',2'-d)imidazole. Mutat Res 105, 9-13 (1982)
http://dx.doi.org/10.1016/0165-7992(82)90200-7

162. EG Snyderwine, PP Roller, RH Adamson, S Sato, SS Thorgeirsson: Reaction of N-hydroxylamine and N-acetoxy derivatives of 2-amino-3-methylimidazolo(4,5-f)quinoline with DNA. Synthesis and identification of N-(deoxyguanosin-8-yl)-IQ. Carcinogenesis 9, 1061-1065 (1988)
http://dx.doi.org/10.1093/carcin/9.6.1061
PMid:3370750

163. A Tada, M Ochiai, K Wakabayashi, H Nukaya, T Sugimura, M Nagao: Identification of N-(deoxyguanosin-8-yl)-2-amino-3,4- dimethylimidazo(4,5-f)quinoline (dG-C8-MeIQ) as a major adduct formed by MeIQ with nucleotides in vitro with DNA in vivo. Carcinogenesis 15, 1275-1278 (1994)
http://dx.doi.org/10.1093/carcin/15.6.1275
PMid:8020167

164. M Ochiai, H Nagaoka, K Wakabayashi, Y Tanaka, SB Kim, A Tada, H Nukaya, T Sugimura, M Nagao: Identification of N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethyl-imidazo(4,5-f)quinoxaline 3',5'-diphosphate, a major DNA adduct, detected by nuclease P1 modification of the 32P-postlabeling method, in the liver of rats fed MeIQx. Carcinogenesis 14, 2165-2170 (1993)
http://dx.doi.org/10.1093/carcin/14.10.2165
PMid:8222070

165. EG Snyderwine, K Yamashita, RH Adamson, S Sato, M Nagao, T Sugimura, SS Thorgeirsson: Use of the 32P-postlabeling method to detect DNA adducts of 2-amino-3-methylimidazolo(4,5-f)quinoline (IQ) in monkeys fed IQ: identification of the N-(deoxyguanosin-8-yl)-IQ adduct. Carcinogenesis 9, 1739-1743 (1988)
http://dx.doi.org/10.1093/carcin/9.10.1739
PMid:3168152

166. HA Schut, EG Snyderwine, HX Zu, SS Thorgeirsson: Similar patterns of DNA adduct formation of 2-amino-3-methylimidazo (4,5-f)quinoline in the Fischer 344 rat, CDF1 mouse, cynomolgus monkey and Salmonella typhimurium. Carcinogenesis 12, 931-934 (1991)
http://dx.doi.org/10.1093/carcin/12.5.931
PMid:2029759

167. EG Snyderwine, CD Davis, K Nouso, PP Roller, HA Schut: 32P-Postlabeling analysis of IQ, MeIQx and PhIP adducts formed in vitro in DNA and polynucleotides and found in vivo in hepatic DNA from IQ-, MeIQx- and PhIP-treated monkeys. Carcinogenesis 14, 1389-1395 (1993)
http://dx.doi.org/10.1093/carcin/14.7.1389
PMid:8330355

168. RJ Turesky, SC Rossi, DH Welti, JOJ Lay, FF Kadlubar: Characterization of DNA adducts formed in vitro by reaction of N-hydroxy-2-amino-3-methylimidazo(4,5-f)quinoline and N-hydroxy-2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline at the C-8 and N2 atoms of guanine. Chem Res Toxicol 5, 479-490 (1992)
http://dx.doi.org/10.1021/tx00028a005
PMid:1391614

169. JG Westra, E Kriek, H Hittenhausen: Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo. Chem Biol Interact 15, 149-164 (1976)
http://dx.doi.org/10.1016/0009-2797(76)90160-5

170. RJ Turesky, J Markovic: DNA adduct formation of the food carcinogen 2-amino-3-methylimidazo(4,5-f)quinoline at the C-8 and N2 atoms of guanosine, Chem. Res Toxicol 7, 752-761 (1994)
http://dx.doi.org/10.1021/tx00042a007
PMid:7696529

171. RJ Turesky, J Markovic, JM Aeschlimann: Formation and differential removal of C-8 and N2-guanine adducts of the food carcinogen 2-amino-3-methylimidazo(4,5-f)quinoline in the liver, kidney, and colorectum of the rat. Chem Res Toxicol 9, 397-402 (1996)
http://dx.doi.org/10.1021/tx950131r
PMid:8839041

172. RJ Turesky, E Gremaud, J Markovic, EG Snyderwine: DNA adduct formation of the food-derived mutagen 2-amino-3-methylimidazo(4,5-f)quinoline in nonhuman primates undergoing carcinogen bioassay. Chem Res Toxicol 9, 403-408 (1996)
http://dx.doi.org/10.1021/tx950132j
PMid:8839042

173. M Novak, S Rajagopal, LL Xu, S Kazerani, K Toth, M Brooks, TM Nguyen: Chemistry of carcinogenic and mutagenic metabolites of heterocyclic aromatic amines. J Phys Org Chem 17, 615-624 (2004)
http://dx.doi.org/10.1002/poc.765

174. M Novak, K Toth, S Rajagopal, M Brooks, LL Hott, M Moslener: Reactivity and selectivity of the N-acetyl-Glu-P-1, N-acetyl-Glu-P-2, N-acetyl-MeIQx, and N-acetyl-IQx nitrenium ions: Comparison to carbocyclic N-arylnitrenium ions. J Am Chem Soc 124, 7972-7981 (2002)
http://dx.doi.org/10.1021/ja0121944
PMid:12095341

175. M Novak, S Kazerani: Characterization of the 2-(alpha-carbolinyl)nitrenium ion and its conjugate base produced during the decomposition of the model carcinogen 2-N-(pivaloyloxy)-2-amino-alpha-carboline in aqueous solution. J Am Chem Soc 122, 3606-3616 (2000)
http://dx.doi.org/10.1021/ja993433e

Key Words: Aromatic amine; benzidine; nitrenium ion; DNA adduct; laser flash photolysis; heterocyclic amine

Send correspondence to: David Josephy, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1; Tel: 519-824-4120 ext. 53833, Fax: 519-837-1802, E-mail: djosephy@uoguelph.