Relative stabilities of nitrenium ions derived from heterocyclic amine food carcinogens: Relationship to mutagenicity

Chem.-Biol. Interactions, 81 (1992) 19-33 Elsevier Scientific Publishers Ireland Ltd.

19

RELATIVE STABILITIES OF NITRENIUM IONS DERIVED FROM HETEROCYCLIC AMINE FOOD CARCINOGENS: RELATIONSHIP TO MUTAGENICITY

GEORGE P. FORD and GALEN R. GRIFFIN

Department of Chemistry, Southern Methodist University, Dallas, TX 75275 (U.S.A.) {Received May 3rd, 1991) (Revision received August 23rd, 1991) {Accepted August 24th, 1991)

SUMMARY

The bacterial mutagenicities of a wide variety of complex heteroaromatic amine mutagens and carcinogens present in cooked foods are approximately related to the stabilities of the corresponding nitrenium ions through equations of the kind: log(m) = aAAH + b. The stabilities of the nitrenium ions (ASJ/) were computed using the semiempirical AM1 molecular orbital procedure. Parallel calculations of the energies, charge distributions and geometries of simple model compounds provides a qualitative framework within which the stabilities of the nitrenium ions derived from the food carcinogens can be easily understood.

Key words: Mutagen -- Nitrenium ion -- Molecular orbital -- Structure-activity relationship

INTRODUCTION

In recent years a large number of complex heterocyclic amines have been identified in foods such as beef and fish when cooked at high temperatures [1- 3]. In many cases these substances, formed in what is sometimes referred to as the Malliard reaction [4], have been shown to be potent mutagens. Subsequent studies showed the same compounds to be powerful inducers of hemoangioendothelial sarcomas, hepatocarcinomas and adenocarcinomas in rodents [5].

Correspondence to: George P. Ford, Department of Chemistry, Southern Methodist University, Dallas, TX 75275, U.S.A. 0009-2797/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

20

1

2

Among the best characterized of these complex heterocyclic amines are the aza analogs of 2- (1) and 3-aminofluorene (2). The former is a well known carcinogen [6] and mutagen [7]. Indeed its N-acetyl analog has played a pivotal role in the development of the entire field of chemical carcinogenesis [8]. Carcinogenic activity has also been demonstrated for the N-acetyl derivative of 2 [9]. The amine itself, although known to be a bacterial mutagen [7], has received relatively little attention. Heterocyclic analogs of 2-aminofluorene (1) found in foods cooked at high temperatures include 3 and 4, first isolated from L-tryptophane pyrolysates [10] and 5 and 6, isolated from soybean pyrolysate [11]. The phenylalanine pyrolysis product 7 [10], although not an aza fluorene, is an obvious structural relative, as is the tetracyclic L-ornithine pyrolysis product 8 [12]. Analogs of 3-aminofluorene include compounds isolated from the pyrolysis of L-tryptophane (9,10) [13] and the glutamic acid (11,12) [14]. A second and clearly distinct class of heterocyclic amines formed in protein and amino acid pyrolysates are the fused 2-aminoimidazole derivatives 1 3 - 1 8 [15- 191. The general patterns of metabolic activation and subsequent DNA binding of these complex heteroaromatic amines seem to be similar to those of their carbocyclic analogs [20]. In both cases N-oxidation mediated by the cytochrome P-450 family of enzymes is followed by O-acylation or sulfonation [21]. Under suitable conditions decomposition of the unstable arylhydroxylamine ester then leads directly to covalent bond formation between the arylamine and one of the DNA base sites. DNA adducts analogous to the well characterized guanine C8-1inked adduct with 2-aminofluorene [22] have been identified for both the GluP-1 and Trp-P-2 [23]. The key reactive intermediate in this sequence (Fig. 1) is generally believed to be the highly electrophilic aryl nitrenium ion, ArNH * [8,20]. Unlike the amines,

CH3

~

N

H H

R

2 R

I

NH2

H

3: R = CH3 (Trp-P-1)

4: R = H

~

(Trp-P-2)

5: R = CH3 (MeActC) 6: R = H (AaC)

~

NH2 7 (Phe-P-1)

21

H3

~

NH2

t~'~

N ..__..~ N~V

NH2

NH 2

9" R = CH 3 (3AH) 1 0 : R = H (3AN)

8 (Orn-V-1)

11: 12:

R = CH 3 (GIu-P-1) R =H (Glu-P-2)

or their neutral derivatives, the relative energies of these ions are very sensitive to the nature of the aryl group [24,25]. This, in turn, should have a significant effect on the ease of NO heterolysis and nitrenium ion formation and therefore on their propensities for DNA damage and perhaps, on the mutagenicities of their precursors. Although far from general, qualitative parallels between bacterial mutagenicity and DNA damage, have indeed been identified for several of the compounds of interest here [26-29]. We have previously found that for a small group of polycyclic aromatic amines for which consistent mutagenicities towards the Salmonella TA98 and TA100 tester strains had been reported, the logarithms of the mutagenicities increased linearly with the relative stabilities of the corresponding nitrenium ions computed using AM1 semiempirical molecular orbital calculations [24]. In the present paper the same semiempirical molecular orbital theory is used to calculate the relative stabilities of the nitrenium ions derived from 3 - 18. These are discussed in terms of the specific aza substitution patterns and compared with the mutagenicities of the parent amines in the same way. METHODS

Semiempirical calculations were carried out in the AM1 approximation [31] using version 5 of the MOPAC package [31] adapted for a Harris Corporation

18: 13:

X=CH;

R I=H;

R2=H;

R3=H

14:

X = CH; R 1 = CH3; R 2 = H ; R 3 = H

15:

X=N;

R l=H;

R2=H;

(IQ) (MeIQ)

R 3 = C H 3 (McIQx)

16: x = N; R 1 = CH3; R 2 = H; R 3 = CH 3 (4,8-DiMeIQx) 17:

X = N; R 1 = H; R 2 = CH3; R 3 = CH 3 (7,8-DiMeIQx)

(Ph-I-P)

22 P-450 mediated N-hydroxylation Ar-NH2

O-acctyltransferase

~

Ar-NH-OH

non-enzymatic loss of acetate

DNA binding

ArNH +

~

O I|

Ar-NH--O-C-CH 3

Fig. 1. Activation of aromatic amines to arylnitrenium ions via loss of acetate from the N-acetoxy amino precursors.

HS00 minicomputer [32]. Optimized geometries were obtained by refinement of trial structures using the 'PRECISE' option without imposing geometrical constraints of any kind. Trial geometries for the amines were generated from standard bond lengths and angles. The optimized geometries were then used to generate trial structures for the nitrenium ions by deleting the appropriate hydrogen atom. In a few cases exploratory calculations were carried out on the N-hydroxy and N-acetoxy amines. The global minima for the former were located through complete searches of the rotational hypersurfaces. In the Nacetoxy derivatives the O-acyl groups were assumed to adopt conformations in which the NO and C = O bonds were approximately eclipsed as in their carbocyclic analogs [25]. RESULTS AND DISCUSSION

The calculated heats of formation of 3 - 18 and the corresponding nitrenium ions are summarized in Table I. This Table also includes the relative energetics of nitrenium ion formation defined by the enthalpies of the processes described by Eqn. 1 (AAH). The latter quantify the thermodynamic stabilities of the aryl nitrenium ions ArNH ÷ relative to the simple monocyclic nitrenium ion derived from aniline. A negative value of h ~ J / t h e r e f o r e indicates the amount by which ArNH ÷ is more stable than PhNH +. These quantities are discussed below in terms of the structural features of the individual aryl groups followed by an attempt to assess the extent, if any, to which the nitrenium ion stabilities are related to the bacterial mutagenicities of the amines. For the latter purpose the relative energies of the N-O heterolyses (ArNHOAc - ArNH +), rather than those of the composite processes (Eqn. 1), might have seemed more directly relevant to the in vivo formation of nitrenium ions. The calculations were performed on the amines, rather than the acetoxy derivatives, as a matter of computational expediency in order to avoid the conformational complexities of the latter [25]. Given that any relationship that might exist between nitrenium ion stability and biological activity would be approximate at best, together with the expectation that the relative energies of the composite processes (Eqn. 1) would not differ

23 TABLE I CALCULATED H E A T S OF FORMATION AND R E L A T I V E E N E R G E T I C S OF HETEROCYCLIC A M I N E S AND R E L A T E D N I T R E N I U M IONS a Energies in kcal m o l - 1. hA/-/is the calculated energy for the process shown in Eqn. 1. In 3-12 the conformation in which the NH group is directed towards the adjacent aza substituent is designated syn. In 1 3 - 1 7 the conformation in which the N + H bond is directed towards the > N - C H 3 substituent is designated syn. Structure

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Amine

Syn nitrenium ion

Anti nitrenium ion

z:k/-/f

~J--/f

A ~ It

A/-/f

A~t/

52.8 53.1 66.3 73.3 73.4 80.8 58.3 112.9 75.4 80.8 107.4 115.0 102.3 96.3 110.3 104.0 104.7 113.4

262.6 a 270.4 b 277.0 288.1 287.7 298.5 280.4 343.7 278.9 286.8 311.2 320.3 308.6 301.7 318.4 311.5 311.1 327.8

- 16.6 -9.1 - 15.7 - 11.6 - 12.1 - 8.7 - 4.3 4.4 - 22.9 - 20.4 -22.6 - 21.1 - 20.1 -21.0 - 18.3 - 18.9 -20.0 - 12.0

262.8 270.9 284.7 295.7 294.2 304.8 287.1 352.1 286.9 293.7 316.3 325.4 305.7 298.9 315.3 308.4 308.0 324.4

- 16.4 -8.6 - 8.0 - 4.0 - 5.6 - 2.4 2.4 12.8 - 14.9 - 13.5 - 17.5 - 16.0 - 23.0 -23.8 - 21.4 - 22.0 -23.1 - 15.4

aN +H bond directed t o w a r d s the methylene bridge. DN + H bond directed away from the methylene bridge.

ArNH2 + Phl~H - -

Arl~H + PhNH2

(1)

markedly from those of the NO heterolyses, this strategy seemed entirely permissible.* *Only the final (NO heterolysis) step in the sequence: A r N H 2 - A r N H O H - ArNHOAc -- A r N H ÷ varies significantly with the nature of Ar. F o r the diverse series of primary aromatic amines studied in the preceding paper, only when the substituted carbon was in a peri or bay region, were the relative energies of the N-hydroxylation reaction, quantified by Eqn. i, calculated to differ from zero by more than 0.2 kcal m o l - 1 [24]. ArNH 2 + PhNHOH ~ A r N H O H + PhNHOAc

ArNHOH + PhNH 2 .-

ArNHOAc + P h N H O H

(i) (ii)

A comparable situation w a s found (this work) for the heteroaromatic amines 6, 12 and 13 chosen as representative examples of compounds of the p r e s e n t type. Here the corresponding energies of Eqn. i were 0.4, 0.3 and - 0 . 2 kcal m o l - 1, respectively. The acylation reactions (ArNHOH - ArNHOAc) were even less sensitive to the n a t u r e of the aryl group. F o r the hydroxylamines corresponding to 6, 12 and 13 the overall enthalpies of Eqn. ii were computed to be 0.3, 0.2 and 0.2 kcal tool-1, respectively.

24

In all cases the nitrenium ions were calculated to be essentially planar with the N-H bonds in one of two alternative conformations. As before [24,33] these conformations are designated syn and anti. In the former the NH bond is directed towards and in the latter away from, the ring atom of highest priority on the basis of the Kahn-Ingold-Prelog sequence rules (Fig. 2). In the absence of major steric considerations, energetic differences between the conformers arise from the electrostatic interaction between the group moment of the NH substituent and the unsymmetrical charge distribution within the aryl moiety [33]. These differences are especially marked in the nitrenium ions derived from the azafluorenes (3 - 12). Here the syn conformers, in which the positive ends of the substituent dipoles are closer to the electronegative ring nitrogens (Fig. 2), are invariably the more stable. The energies of the two conformers differ less in the 2-aminoimidazole derivatives ( 1 3 - 18), but again the lower energy conformations are those in which the NH bonds are directed towards the regions of higher electron density which in these cases correspond to the methyl bearing nitrogens. From the calculated nitrenium ion stabilities in Table I several general trends are apparent. Aza substitution at positions directly conjugated with the electron deficient nitrenium ion center tend to destabilize the ion. This effect is easily seen in the ion derived from 7. Here resonance of the kind: 19a ~ 19b, places a partial positive charge on the electronegative nitrogen atom -- an inherently unfavorable situation. An effect of this kind finds quantitative expression in even simple Hfickel molecular orbital theory where the perturbation associated with substituting a ring carbon for a nitrogen atom is approximately proportional to the charge at that position [34]. For odd alternant hydrocarbons (with which 19 is isoconjugate [24]) the Hfickel charges are easily deduced through simple pencil and paper computations [35]. Indeed these ideas were used by both Lowe [36] and Lehr [37] and their coworkers, to estimate the effects of aza-substitution on the energetics of conversion of condensed heteroaromatics to triol carbenium ion metabolites. The extension of these very simple perturbational ideas to arylnitrenium ions was demonstrated in a previous paper [33]. Unfortunately, this simple approach is not applicable to the compounds of interest here since, apart from 19, the nitrenium ions are non-alternant. Nevertheless the major

N

syn

anti

Fig. 2. Approximate orientation of the NH group moment and designation of syn and anti orientations. In the syn conformer the H atom is directed towards the ring atom of the highest priority.

25

19a

19b

trends can be qualitatively understood using simple resonance arguments. Unlike aza substitution in the six-membered rings, substitution in the five membered ring is generally stabilizing, especially where the aza substituent is para to the nitrenium ion center. This is most easily seen when the computed stabilities of ions 20 - 22 (Table II) are compared with those of their carbocyclic analogs. These are shown below as resonance hybrids involving the key iminoforms implied by the calculated charge and geometrical data. In 20 the stabilizing effect of the pyrrole-like nitrogen, which is formally meta to the exocyclic substituent, is only 0.5 kcal mol-1 more effective than the methylene bridge in the related 2-fluorenyl ion derived from 1. On the other hand, in 21 the para relationship between the the aza-substituent and the N +H group contributes significantly to the stability of this ion through the iminoquinone-resonance shown in the lower structure. The same is true in 22. The ions 21 and 22 are 18.2 and 19.2 kcal mol-1 more stable than the 3-aminofluorenyl nitrenium ion, respectively. The relative stabilities of the nitrenium ions derived from the more complex amino heterocycles found in cooked foods can be qualitatively understood in terms of a superposition of these ideas. As already mentioned, the ion derived from 7 (19) is significantly destabilized by the electronegative aza substituent at an electron-deficient ring position. In 3 - 6 the two six-membered rings are bridged by an - NH - group. This contributes little to the stability of the system through direct conjugation with the - N +H group (cf. discussion surrounding

T A B L E II C A L C U L A T E D H E A T S OF F O R M A T I O N AND R E L A T I V E E N E R G E T I C S OF A D D I T I O N A L N I T R E N I U M IONS E n e r g i e s in kcal mol - 1. A~ur/is the calculated e n e r g y for the process shown in Eqn. 1. In 23 - 25 th e confo rmation in which the N ÷ H bond is dire c t e d t o w a r d s the n i t r o g e n a t om b e a r i n g t he me t hyl g r o u p is d e s i g n a t e d syn. Nitrenium ion s t r u c t u r e

20 21 22 23 24 25

Amine

Syn n i t r e n i u m ion

A nt i n i t r e n i u m ion

~r~/f

~/f

Az~r~

~/f

A~kH

66.4 67.2 100.6 57.2 73.4 90.8

275.7 266.5 300.1 265.8 286.9 291.9

-

275.2 266.3 298.7 262.4 283.9 289.1

-

17.1 27.1 26.9 17.8 12.9 25.3

17.6 27.3 28.3 21.2 15.9 28.1

26

I

I

H

H

I H

I H

20

21

22

20) although v-electron donation from this group to the aza substituent tends to offset the destabilizing effect of the latter. The stabilities of these ions are therefore intermediate between that of 19 and that derived from 1. The nitrenium ion from 8 is computed to be very unstable indeed. Here, charge delocalization in the ring skeleton places a partial positive charge on the nitrogen of the dihydropyrimidine ring. The ions derived from 9 - 12 are of similar, although somewhat lower stability than 21 and 22 due to the destabilizing effect of the aza substituent in the six-membered ring. The tricyclic imidazole type nitrenium ions (13 - 17) are generally more stable than those related to the aminofluorene analogs. The special stability of the 2-imidazolyl nitrenium ion moiety has been discussed at length by Bolton and McClelland in connection with their theoretical and experimental studies on the reductive activation of 2-nitroimidazole radiation sensitizers [38]. According to these authors the special stability of ions of this kind is related to the involvement of the electrons of the 1-nitrogen which results in a structure (23b) in which (unlike those in the six-membered ions) all atoms achieve full octets. Significant participation of structures like 23b is indicated by the extensive transfer of positive charge to the imidazole ring, as well as the short exocyclic CN, and long C 4 - C 5, bond lengths.* Interestingly, the fusion of a benzo ring in 24 destabilizes the nitrenium ion. This curious effect is manifest in the unfavorable energetics of nitrenium ion formation calculated for Ph-I-P (18) where the nitrenium ion is further destabilized by aza substitution in the six-membered ring. In contrast, fusion of a second benzo ring, as in 25 does lead to the expected stabilizing effect. In the nitrenium ions derived from the related food carcinogens 13 - 17 this is partially offset by aza

*The computed exocyclic CN bond lengths (/k) in the anti conformations of 2 3 - 2 5 and (parent amines) were: 1.276 (1.420); 1.281 (1.409); 1.281 (1.413). The corresponding C 4- C 5 bond lengths were:l.520 (1.407); 1.532 (1.452); 1.533 (1.438).

27

I

I

CH3

CH3

23a

23b

substitution in the naphtho ring. Nevertheless, in all cases nitrenium ion formation is predicted to be as, or more energetically favorable, than for the aminofluorene analogs 3 - 12. The charge on the exocyclic NH group in the ions 23 - 25 becomes progressively more delocalized with increasing benzo substitution (qNlq = 0.168, 0.150 and 0.119, respectively) as would have been expected and as would have suggested a parallel stabilization of the ion. The stability order actually calculated, 25 > 23 > 24 seems to reflect the energetic penalty associated with the incorporation of the unusually long imidazole C 4 - C ~ bond into the benzo ring [38]. This destabilizing effect is more than offset by the added charge delocalizing effect of the naphtho, but not the benzo substituent. Bacterial mutagenicity data for many heteroaromatic amines are available through the work of Sugimura and Sato [5] and Sugimura [39] using the Salmonella TA98, TA100 and Felton et al. [40] using the TA 1538 tester strains. These are summarized in the units of revertants per nmole in Table III in order of decreasing mutagenicities in the TA98 strain. With only few exceptions, the same order holds for TA100 and TA1538. For comparison we have also included the data for 2-aminofluorene based on averages of those reported by many different laboratories [24]. Without exception the fused aminoimidazole compounds (13 - 16) are the most mutagenic in all strains and frequently two or three orders of magnitude more mutagenic than 2-aminofluorene. These compounds are also among those for which nitrenium ion formation is predicted to be the more favorable. At the other end of the scale, Phe-P-1 (7), was a compound for which nitrenium ion formation was predicted to be among the least favorable and is only very weakly mutagenic. Two other compounds for which the nitrenium ions are predicted to be less stable than that corresponding to 2-aminofluorene (1) were also less mutagenic than the latter. In order to more quantitatively assess the role of nitrenium ion stability on the mutagenicities of these compounds the quantities log(m), where m is the

NII I CH3

I

CH3 24

25

28 TABLE III M U T A G E N I C I T I E S O F A R O M A T I C A N D H E T E R O A R O M A T I C A M I N E S IN S. TYPHIMURIUM Compound

Mutagenicity (revertants/nmol)

MeIQ (14) IQ (13) 4,8-DiMeIQx (16) 7,8-DiMeIQx (17) M e I Q x (15) Trp-P-2 (4) Orn-P-1 (8) Glu-P-1 {11) Trp-P-1 (3) Ph-I-P (18) Glu-P-2 (12) 2-Aminofluorene (1) A~C (6) M e A a C (5) Phe-P-1 (7)

TA98 a

TA100 a

TA1538 b

140132 85734 41541 37001 30885 20488 13509 9702 8229 403 c 350 55 d 55 39 7

6360 1386 1816 2247 2982 355 -634 359 -221 28 d 4 24 4

148400 39600 72640 c 42903 c 14910 ----896 ------

aData f r o m Refs. 5 or 39 u n l e s s noted o t h e r w i s e . bRef. 40. CRef. 1. d A v e r a g e d d a t a f r o m m a n y i n d e p e n d e n t studies [24].

m u t a g e n i c i t y in r e v e r t a n t s / n m o l e , w e r e plotted against the predicted values of AAH for the m o r e stable nitrenium ion conformer. The results are shown for each of the Salmonella strains in Figs. 3 - 5 and s u m m a r i z e d in the regression Eqns. 2 - 4. (Since Orn-P-1 (8) was a clear outlier, it was excluded in the derivation of Eqn. 2) TA98: log(m) = ( - 0 . 1 8 1 ± 0.043)AAH + 0.227 ± 0.2792

(2)

o = 0.966, r 2 = 0.593, n = 14 TA100: log(m) = ( - 0 . 1 4 7 ± 0 . 0 2 4 ) A A H - 0.1619 ± 0.450 = 0.540,

r 2 =

0.770, n

=

(3)

13

TA1538: log(m) = ( - 0 . 2 4 1 7 ± 0 . 0 3 5 3 ) ~ A H -

0.801 ± 0.765

(4)

= 0.245, r 2 = 0.922, n = 6. In each case the slope of the r e g r e s s i o n line is negative indicating t h a t increasing stability of the nitrenium ion is indeed associated with a higher degree of mutagenicity in the p r e c u r s o r amine as intimated above and as seemed to be true

29

®

©

©

n

~ £../.. "3 ~. r,.,-

(m)~oI

i

i

i

t

i

{ ® ©

~ ,~

~ ~,

"~O 0

l

~

•

~~

~ o

o~ ~..

~-

-

J

_

I

I

I

I

©

30 6

~MelQ

7,8-DiMelQx M e I : ~

AAH 2

-30

I

I

t

-25

-20

-15

-10

Fig. 5. Relationship between the relative nitrenium ion stabilities (AA//) and the mutagenicities of the corresponding amines towards S. typhimurium tester strain TA1538.

for the polycyclic arylamine data presented in the preceding paper [24]. The precision of these relationships vary with the particular strain. The higher r 2 in the TA1538 correlation is due, at least in part, to the absence of many of the aminofluorene analogs. In fact it is interesting that the TA98 mutagenicities for the 2-aminoimidazole compounds are particularly well correlated with the nitrenium ion stabilities and it may well not be a coincidence that it is for these compounds, in this strain, that the relationship between mutagenicity and DNA damage seems to be most clear-cut [28,29]. On the other hand it would be surprising if a biological response as complex as bacterial mutagenicity could be wholly explained in these terms. Differences in enzymatic activation efficiencies appear to play a major role in the different mutagenic activities of the polycyclic aromatic amines [42]. A similar situation would be expected for the present compounds [44]. Differences in activity associated with the presence or absence of methyl substituents may be related to their effects on the activation process(es). In every case methyl substitution stabilizes the nitrenium ion by amounts varying from 0 . 6 - 4.1 kcal mo|- 1. How-

31

ever the effects of methylation on the mutagenic activities vary with both the structure of the amine and the bacterial strain. The genetic consequences of DNA adduct formation may vary considerably with the precise structures of the heteroaryl moieties, even if bound at similar levels. Significant differences in the transport properties of the amines, or those of their metabolites, are likely to be of importance. Structure activity correlations involving octanol/water partition coefficients have been presented by Trieff et al. for the polycyclic aromatic amines [43], although the significance of these is currently unclear [22]. Nevertheless the general trend relating the mutagenicities of the parent amines and the stabilities of the related nitrenium ions seems clear and seems to provide a useful framework for the qualitative discussion of the varying potencies of this interesting class of heteroaromatic amines. Indeed a re-examination of the most obvious outliers to these correlations may throw into clearer perspective other complex factors that no doubt contribute to the overall pattern of activities. ACKNOWLEDGEMENT

This work was supported by grants from the Robert A. Welch Foundation (Grant N-1080) and the SMU Research Council. The Harris Corporation H800 minicomputer used in this work was a gift from the Harris Corporation. We are grateful to Dr. P.S. Herman for technical assistance. REFERENCES 1 C. deMeester, Bacterial mutagenicity of heterocyclic amines found in heat-processed food, Mutat. Res., 221 (1989) 235-262. 2 F.T. Hatch, M.G. Knize, S.K. Healy, T. Slezak and J.S. Felton, Cooked-food mutagen reference list and index, Environ. Mol. Mutagen., Suppl. 14, 12 (1988) 1-85. 3 C. Furihata and T. Matsushima, Mutagens and carcinogens in foods, Annu. Rev. Nutr., 6 (1986) 67-94. 4 S. Kawamura, Seventy years of the Maillard reaction, in: G.R. Waller and M.S. Feather (Eds.), The Maillard Reaction in Foods and Nutrition, A.C.S. Monograph 215, American Chemical Society, Washington, D.C., 1983, p. 3; T. Nyhamar, K. Olsson, and P.-~,. Pernemalm, Strecker degradation products from (1-13C)-D-glucose and glycine, ibid. p. 71; M. J~igerstad, A.L. Reutersw~ird, R. Oste, A. Dahlqvist, S. Grivas, K. Olsson and T. Nyhammar, ibid., p. 507. 5 T. Sugimura and S. Sato, Bacterial mutagenicty of natural materials, pyrolysis products and additives in foodstuffs and their association with genotoxic effects in mammals, in: A.W. Hayes, R.C. Schnell and T.S. Miya (Eds.), Developments in the Science and Practice of Toxicology, Elsevier, Amsterdam, 1983, pp. 115-133. 6 R.C. Garner, C.N. Martin and D.B. Clayson, Carcinogenic aromatic amines and related compounds, in: C.E. Searle (Ed.), Chemical Carcinogenesis, A.C.S. Monograph 182, American Chemical Society, Washington, D.C., 1984, pp. 175-276. 7 D.W. Later, R.A. Pelroy, D.L. Stewart, T. McFall, G.M. Booth, M.L. Lee, M. Tedjamulia and R.N. Castle, Microbial mutagenicity of isomeric two-, three and four-ring amino polycyclic aromatic hydrocarbons, Environ. Mutagen., 6 (1984) 497-515. 8 J.A. Miller and E.C. Miller, Some historical aspects of N-aryl carcinogens and their metabolic activation, Environ. Hlth. Perspect., 49 (1983) 3-12. 9 H.R. Gutmann, D.S. Leaf, Y. Yost, R.E. Rydell and C.C. Chen, Structure-activity relationships of N-acylarylhydroxylamines in the rat, Cancer Res., 30 (1970) 1485-1498.

32 10

11

12

13 14 15

16

17

18

19

20

21

22

23

24 25

26

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