Mutation Research, 259 (1991) 219-233 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 0165121891000603

219

M U T G E N 00033

Formation of heterocyclic amines using model systems M a r g a r e t h a J~igerstad 1, Kerstin S k o g 1, Spiros G r i v a s 2 a n d Kjell O l s s o n 2 l Department of Applied Nutrition and Food Chemistry, Chemical Center, University of Lurid, Lurid (Sweden) and 2 Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala (Sweden) (Received 16 January 1990) (Accepted 12 June 1990)

Contents Summary

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pyrolysates of amino acids and proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Imidazoquinolines, imidazoquinoxalines and imidazopyridines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reaction mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors affecting the yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical synthesis of mutagenic heterocyclic amines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -. . . . . . . . . . . . . . ........................

219 220 220 220 226 228 229 230

Keyworcb: Heterocyclic amines; Mutagens; Formation; Model systems; Creatin(in)e; A m i n o acids; Sugars; Maillard reactions

Summary Initially, modeling was used to identify the mutagenic heterocyclic amines and their precursors. Major precursors have been shown to be single amino acids or amino acids together with creatine or creatinine. There is also evidence that MaiUard reactions are involved since heating sugar and amino acids together with creatine or creatinine has been shown to produce several of the mutagenic heterocyclic amines, especially the aminoimidazoazaarenes (AIA compounds), e.g., IQ, MeIQ, MeIQx, DiMeIQx and PhIP. Due to a low yield in the model systems, the mechanisms behind the formation of the mutagenic heterocyclic amines are still unclear and need further substantiation. The fact that some AIA compounds are also produced in the absence of sugar casts some doubts on an obligatory participation of the Maillard reaction; alternative routes might exist. Further work using isotopicaUy labeled precursors needs to be done and so far such work has only been performed for PhiP. The formation of mutagenic heterocyclic amines is dependent on time, temperature, pH, concentration of the precursors, type of amino acid, and the presence of certain divalent ions. Water may have an impact both as a temperature regulator and as a solvent medium for the reactants.

Abbreviations: IQ, 2-amino-3-methylimidazo[4,5-f]quinofine; Correspondence: Dr. Margaretha J~gerstad, Department of Food Chemistry, Chemical Center, University of Lund, P.O. Box 124, S-22100 L u n d (Sweden).

MelQ, 2-amino-3,4--dimethylimidazo[4,5-f]quinoline; IQx, 2. amino-3-methylimidazo[4,5-f]quinoxaline; MeIQx, 2 - a m i n o 3,8-dimethylimidazo[4,5-f]quinoxaline; DiMeIQx, 2-amino3,n,8-trimethyfimidazo[4,5-f]quinoxaline; PhIP, 2-amino-1. methyl-6-phenylimidazo[4,5-b]pyridine; T M I P , ' 2-amino. n, n, n-trimethylimidazo[4,5-b ]pyridine.

220

Introduction Modeling studies on the formation of mutagenic heterocyclic amines have been conducted either by dry heating of the reactants using ovenbaking at moderate cooking temperatures (150250 ° C) or by pyrolysing them at temperatures far exceeding 300 ° C. Liquid systems that have been used include reflux boiling of reactants in water, usually aminocarbonyl model reactions (Maillard reactions). However, the formation of heterocyclic amines is almost negligible at 100 ° C. To raise the reaction temperature above 100°C and keep the reactants in solution, a model system in which the reactants are dissolved in a mixture of diethylene glycol and water has been developed. The proportion of water to diethylene glycol determines the reaction temperature. Model systems are valuable tools for our understanding of the conditions that result in the formation of food mutagens. Besides, inhibitors or promoters can be evaluated by such systems. Further, modeling of the mutagen formation might also contribute to the identification of new food mutagens that perhaps are present in very small amounts in cooked foods. Thus, with the purpose of designing strategies for reduction or prevention of mutagen formation, model studies may be useful in providing basic knowledge about precursors, reaction conditions, kinetics and mechanisms.

Pyrolysates of amino acids and proteins The pyrolysis model was the first model system used to study the formation of mutagenic heterocyclic amines. The observation that smoke condensates and extracts of the charred parts of broiled or grilled meat and fish contained high mutagenic activity (Nagao et al., 1977a; Sugimura et al., 1977) raised the question regarding its origin. On pyrolysing pure proteins, DNA, RNA, starch, and vegetable oil, only proteins produced considerable mutagenicity of the same kind as that found in broiled or grilled meat and fish (Nagao et al., 1977b). By pyrolysing single amino acids, a series of mutagenic heterocyclic amines were isolated and identified in the smoke condensates of tryptophan, glutamic acid, lysine, phenylalanine, and ornithine (Table 1). From smoke condensate of

soybean globulin pyrolysate, two a-carbolines (AaC and MeAaC) were isolated (Yoshida et al., 1978). Many of these mutagenic pyrolysates have later been demonstrated in grilled foods (Yoshida et al., 1986; Sugimura and Sato, 1983).

Imidazoquinolines, idazopyridines

imidazoquinoxalines, and im-

Pyrolysing of dry material is performed at high temperatures, usually well above 300°C. The identification of a new class of mutagenic heterocyclic amines constituting imidazoquinolines (IQ, MelQ) and imidazoquinoxalines (MeIQx) in the early eighties (Kasai et al., 1980a,b,c, 1981) drew attention to mutagen formation during moderate cooking conditions, between 100 and 300°C. These food mutagens were isolated from the crust of broiled or fried meat and fish cooked under normal heating conditions (100-300 ° C), and also from meat extracts manufactured by long-term boiling until all the water had evaporated. Nonenzymatic browning (Maillard reactions) was suggested to be involved in their formation (Spingarn and Garvie, 1979; Shibamoto et al., 1981; Wei et al., 1981; Powrie et al., 1982; J~igerstad et al., 1983a; Aeschbacher, 1986). The Maillard reaction takes place in foods through the reaction of carbonyl compounds (aldehydes and ketones), notably reducing sugars, such as glucose, fructose, etc., with compounds possessing free amino groups, such as amino acids, peptides, and proteins. The reaction is of great importance for the development of flavors, texture, and brown pigments during heat treatment of foodstuffs, thereby contributing to the palatability of cooked foods. The number of different compounds that are formed during the reaction is overwhelming. After an initial condensation between reducing sugar and amino groups, rearrangements produce Amadori compounds. These react further by various pathways (enolization, dehydration, retro-aldol condensation, Strecker degradation) to produce hundreds of reaction products, viz., furans, pyrones, pyrazines, pyrroles, thiophenes, thiazolidines, pyridines, imidazoles, etc. It is, however, beyond the scope of this paper to describe the Maillard reaction in detail (for

221 TABLE 1 MUTAGENS ISOLATED FROM PYROLYSATES(NAGAO ET AL., 1983) Compound Trp-P-l* 3-amino-1,4-dimethyl5H-pyrido[4,3-b]indole

Structure

Precursor Tryptophan

CH3 H ~~

H NH 2 CH3

Trp-P-2* 3-amino-l-methyl-5Hpyrido[4,3-b]indole

Tryptophan

CH3 N NH 2

H

GIu-P-1 2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole

N

N.~. NH 2

Glutamic acid

CH3 Glu-P-2* 2-amino-dipyrido[1,2-a: 3',2 '-d ]imidazole

Glutamic acid

Lys-P-1 3,4-cyclopentenopyrido[3,2-a]carbazole

Lysine

Orn-P-1 4-amino-6-methyl-1H-2,5, 10,10b-tetraaza-fluoranthene

Ornithine NH 2 Phenylalanine

Phe-P-1 2-amino-5-phenylpyridine AaC * 2-amino-9H-pyrido-[2,3b]indole MeAaC* 2-amino-3-methyl-9H-pyrido[2,3-b]indole

@NH Soybeanglobulin ~

N

H

2

~ ~ N ~

CH3

H

H

The asterisk indicates occurrencein grilled foods.

NH 2

Soybean globulin

222 reviews, see Eriksson, 1981; Waller and Feather, 1983; Fujimaki et al., 1986). To assess the role of browning reactions in mutagen formation, both sugar alone and sugaramino acid model systems have been investigated after reflux boiling in water at various pHs. Although many of these binary systems produced mutagenicity towards Salmonella typhimurium strain TA98 in the absence of $9, only a few mutagenic principles have been identified, such as 5-(hydroxymethyl)-2-furaldehyde (HMF) and de-

HN NH2 HOOC "T"" ~ N,,. / Me creatine - H20

,,. NH~RCH " CO2 amino acid

06H1206

hexose y

rivatives of pyrrole and thiazolidine (Omura et al., 1983). They are all weak mutagens and none belongs to the group of heterocyclic amines (for references, see Powrie et al., 1986; Namiki, 1988). A possible involvement of the Maillard reaction in the formation of the IQ compounds was proposed more precisely by J~igerstad et al. (1983a) at the Second International Maillard Meeting in Las Vegas. Three precursors, all naturally occurring in muscle meat, were assumed to participate, namely, creatine, certain amino acids, and sugars (Fig.

==(,NH2

Y Z + X'~(~)N~" Me

OHCxR + O ~ I ~ ' M 7 aldehyde creatinine

pyridine or pyrazine

Y8 Z

1 N~---~NH2 N~Me

IQ compound , , NH2

Y

Z

N~-----~NH2

X ZC)N~]~ Me

+

O ~

pyridine or pyrazine

Y- Z ~ .N~ 8 ~ O ' # O " ~ 3 Me X" 7" N" ~'~""4" R IQ compound

N\Me CH N

R creatininealdehyde l

OHC ~ R I - H20

/

HN i NH2 HOOC "'/ B

"Me

creatine

-

O

\

creatinine

Me

Fig. 1. Postulated reaction route (A) (Jiigerstad et al., 1983a) and alternativeroute (B) (Nyhammar, 1986) for the formation of IQ compounds. R, X, and Y may be H or Me; Z may be CH or N.

223 1A). Creatine was postulated to form the 2aminoimidazo part by cyclization and water elimination, a reaction that takes place spontaneously when the temperature is raised above 100°C. The 2-aminoimidazo part is a common moiety of all IQ compounds. It is also responsible for the mutagenicity of the IQ compounds. Without this part, and especially its 2-amino group, the mutagenicity of the IQ compounds becomes almost negligible (Grivas and J~igerstad, 1984). The quinoline or quinoxaline part of the molecule was postulated to arise through condensation reactions between pyrazines or pyridines and aldehydes - - all typical Maillard reaction products formed by Strecker degradation. A new model system was introduced to verify the suggested route. Creatine or creatinine, glycine or alanine, and glucose or fructose were boiled under reflux in a molar ratio of 1:1:0.5 in a solvent mixture of diethylene glycol containing 14% water (DEG-H20) (J~igerstad et al., 1983a,b). Mixing diethylene glycol and water in this proportion produced a solvent that boiled around 130 o C. This system produced high mutagenicity for TA98 in the presence of $9. The mixture of diethylene glycol and water itself produced no mutagenicity, and heating the reactants two by two produced only weak if any significant mutagenicity (J~igerstad et al., 1983a,b). The first evidence in support of the proposed reaction route came from a collaborative study between groups in Sweden and Japan. Reflux boiling a mixture of creatine, glycine, and glucose dissolved in diethylene glycol-water at 128 °C for 2 h produced 2 mutagenic compounds. These were isolated and identified as MelQx (J~igerstad et al., 1984) and 7,8-DiMelQx (Negishi et al., 1984). The latter product was a new heterocyclic amine, and has to date been demonstrated in cooked foods only once (Turesky et al., 1988). Using threonine instead of glycine produced MelQx and a methyl derivative of MelQx, namely 4,8-DiMelQx (J~igerstad et al., 1986a,b). This methyl derivative has also been demonstrated in fried hamburgers (Felton et al., 1986; Turesky et al., 1988; Becher et al., 1988). Almost simultaneously, Grivas et al. (1985) isolated traces of MelQ together with 4,8DiMelQx using the same model system and alanine as the amino acid. Because of the similar

spectral characteristics of 4,8-DiMelQx and its isomer 5,8-DiMelQx, the 2 compounds were compared by 1H-NMR spectrometry, HPLC retention data and specific mutagenic activity. It was then clearly shown that 4,8-DiMelQx, and not 5,8-DiMelQx, was produced when heating fructose, creatinine, and alanine (Nyhammar et al., 1986). If alanine was replaced with glycine, MelQx and small amounts of IQ were produced (Grivas et al., 1986). Muramatsu and Matsushima (1985), using the same model system, reported that alanine, but also lysine, produced MelQx and 4,8-DiMelQx irrespective of whether glucose or ribose was used as the sugar. In a modification of this system, the reactants creatine, glycine, and glucose were heated in the same proportion as before (1 : 1 : 0.5, molar basis) in the D E G - H 2 0 system in open glass tubes at 180 °C for 15 min (Skog et al., 1990). The water was allowed to evaporate, and the mutagenicity produced was separated into 4-5 fractions on HPLC. By LC-MS and comparison with synthetic references, MelQx was identified as a major form together with small amounts of 4,8-DiMelQx and 7,8-DiMelQx. The yield of MelQx after this short heating time was of the same magnitude, approximately 4 nmole/mmole creatin(in)e, as obtained after reflux boiling for 2 h at 128 ° C. Work is in progress to identify the remaining mutagenic peaks. Although the D E G - H 2 0 model system needs sugar to produce IQ compounds in appreciable amounts, several of the IQ compounds have been produced by heating only creatin(in)e and amino acids. In a series of experiments, Yoshida et al. (1980a,b, 1982) reported that creatine heated either with glucose, fatty acids, or various amino acids in the temperature range 100-200°C produced mutagenic activity towards Salmonella typhimurium TA98. Based on these results, he suggested creatine, glucose, and free amino acids to be precursors of the AIA compounds. To verify this hypothesis, IQ was looked for in dry-heated mixtures of either creatine and glucose or creatine and oleic acid, or finally creatine and various amino acids. The reactants were heated two by two in equimolar amounts in an electric furnace at 180 ° C atmospheric air for I h. IQ was demonstrated only in the heated mixture of creatine and proline, but

224 TABLE 2 MUTAGENIC HETEROCYCLIC AMINES PRODUCED IN MODEL SYSTEMS FROM CREATIN(IN)E, AND AMINO ACIDS WITH OR WITHOUT SUGAR Compound

IQ

Yield (nmole/ mmole creatin(in)e

Precursors: creatin(in)e, amino sugar acid

Heating conditions

Methods a of identification

References

0.4

pro

-

MS, UV, AS

Yoshida et al. (1984)

1.0

gly

fructose

phe -

NMR, MS, RT, AS MS, RT, AS

Grivas et al. (1986)

3.0

I h/180°C dh 2 h/128 o C DEG-H20 1 h/200 o C dh 1 h/200 ° C dh 1 h/200 ° C dh

MS, RT, AS

Felton et al. (1990)

UV, MS, RT

Knize et al. (1988)

Felton et al. (1990)

13.5

phe

glucose

3.7

ser

-

traces

ala

fructose

2 h/128 ° C DEG-H20

MS, UV, RT AS

Grivas et al. (1985)

IQx

2.7

set

-

I h/200 ° C dh

NMR, MS, UV, AS

Knize et al. (1988a)

MeIQx

4.4

gly

glucose

ala

glucose

1.8

ala

ribose

4.2

lys

ribose

thr

glucose

6-7

gly

fructose

4

gly

glucose

NMR, MS, UV, AS NMR, MS, UV, AS NMR, MS, UV, AS NM1L MS, UV, AS NMR, MS, UV, AS NMtL MS, UV, AS LC-MS, AS

Jligerstad et al. (1984)

0.9

set

-

LC-MS, AS

Overvik et al. (1989)

ala

-

LC-MS, AS

Overvik et al. (1989)

tyr

-

2 h/128 o C DEG-H20 3 h/125 o C DEG-H20 3 h/125 o C DEG-H20 3 h/125 o C DEG-H20 2 h/128 o C DEG-H20 2 h/128°C DEG-H20 10'/180 o C DEG-H20 1 h/200 o C dh 1 h/200 o C dh 1 h/200 o C dh

LC-MS, AS

0vervik et al. (1989)

thr

glucose

ala

fructose

4.2

ala

glucose

1.5

ala

ribose

26.1

lys

ribose

gly

glucose

NMR, MS, UV, AS NMR, MS, UV, AS NMR, MS, UV, AS NMR, MS, UV, AS NMR, MS, UV, AS LC-MS, AS

Negishi et aL (1985)

1.9-2.6

2 h/128 ° C DEG-H20 2 h/128 ° C DEG-H20 3 h/125 o C DEG-H20 3 h/125 o C DEG-H20 3 h/125 o C DEG-H20 10'/180 o C DEG-H20

MelQ

4,8-Di MeIQx

Muramatsu and Masushima (1985) Muramatsu and Masushima (1985) Muramatsu and Masushima (1985) Negishi et al. (1985) Grivas et al. (1986) Skog et al. (1990)

Grivas et al. (1985) Muramatsu and Matsushima (1985) Muramatsu and Matsushima (1985) Muramatsu and Matsushima (1985) Skog et al. (1990)

225 TABLE2(continu~) Cornpound

Yield (nmole/ mmole creatin(in)e

Precursors: creatin(in)e, amino sugar acid

Heating conditions

Methods a of identification

References

7,8-Di MelQx

1.1

gly glucose

2 h/128°C DEG-H20 10'/180°C DEG-H20

NMR, MS, UV, AS LC-MS, AS

Neglshi et ai. (1984)

2 h/128 ° C DEG-H20 1 h/200° C dh 1 h/200 ° C dh 1 h/200 ° C dh 1 h/200 o C dh 10'/180°C DEG-H20

MS, UV, RT, AS MS, RT, AS

Shioya et al. (1987)

MS, RT, AS

Felton et al. (1990)

LC-MS, AS

Overvik et al. (1989)

LC-MS, AS

()vervik et al. (1989)

LC-MS, AS

J~igerstad et ai. (1989)

gly glucose PhlP

3.6

phe glucose

735

phe -

560

phe glucose leu phe phe glucose

Skog et al. (1990)

Felton et ai. (1990)

The modeling has been performed either by dry-heating (d h) or by reflux boiling of the reactants in diethylene glycol containing 14% water (DEG-H20). UV, ultraviolet light spectrum; MS, mass spectrum; NMR, nuclear magnetic resonance; RT, retention time on HPLC; AS, authentic sample (synthetic sample).

not in any of the other binary combinations. A ternary system consisting of creatine, glucose, and amino acid was never tried. IQ has also been isolated by dry-heating (oven-baking) equimolar amounts of creatinine and phenylalanine in both the absence and the presence of glucose (Felton et al., 1990). The yield of IQ was about 3 times higher in the presence of glucose. Knize et al. (1988a) have isolated IQ and IQx in dry-heated mixtures of creatinine and serine. This is the first report of the formation of IQx in a model system, but IQx has also been demonstrated in fried meat products enriched with creatine (Becher et al., 1988). ()vervik et al. (1989) reported the formation of MeIQx by dry-heating of creatine and various amino acids, e.g., serine, alanine, and tyrosine without sugar. A few years ago, a new class of mutagenic heterocyclic amines consisting of 2 imidazopyridines (PhIP and TMIP), was isolated by Felton et al. (1986). Shortly thereafter, PhIP was isolated from dry-heated mixtures of creatine and phenyl-

alanine both with and without sugar (Felton et al., 1990). The yield of PhlP was higher without sugar, 735 nmole versus 560 nmoles per mmole creatin(in)e. PhlP has also been isolated from dry-heated mixtures of creatine and leucine (Overvik et al., 1989). Also the model system based on D E G - H 2 0 has produced PhlP from creatinine, phenylalanine, and glucose (Shioya et al., 1987; J~igerstad et al., 1989). The precursors of TMIP have not yet been determined, mainly because the exact positions of its methyl groups are not known, and because synthetic references are lacking. A mutagenic compound with the same molecular weight (176) has been isolated from a dry-heated mixture of threonine and creatine (0vervik et al., 1989). Table 2 shows all the mutagenic AIA compounds, i.e., IQ, MeIQ, IQx, MeIQx, DiMeIQx and PhIP, that to date have been produced and isolated from model systems. Only data supported by spectral similarities (UV, MS, NMR, or LC-MS) with synthetic references are included.

226 Reaction mechanisms

The majority of the model system studies have been focused on the isolation and identification of mutagenic compounds based on various combinations of reactants in either binary or ternary systems. Thus, the precursors and the mutagenic products have received most attention, whereas the underlying mechanisms are less well understood. The mutagenic heterocyclic amines arising from pyrolysing proteins or amino acids have been proposed to be formed through free radical reactions (Sugimura and Sato, 1983; Yoshida et al., 1986). Quinones are known to act as free radicals when oxidized; and according to experiments reported by Yoshida et al. (1986), addition of quinone enhanced the formation of mutagenicity when single amino acids were heated at 250 ° C for lh. The suggested route for formation of imidazoquinolines and imidazoquinoxalines needs more substantiation. First, it must, however, be emphasized that the suggested route for formation comprises only the imidazoquinolines and the imidazoquinoxalines, i.e., IQ, MelQ, IQx, MelQx and DiMelQx, not PhlP and TMIP. These 2 latter compounds were identified several years later (Felton et al., 1986) and found to be imidazopyridines. The postulated reaction route for the imidazoquinolines and imidazoquinoxalines shown in Fig. 1A has been outlined in detail in Nyhammar's doctoral thesis (1986). He assumed that there is an aldol-type condensation between pyridines or pyrazines and aldehydes originating from amino acids after Strecker degradation. The resulting vinylpyridines or vinylpyrazines may then undergo conjugated addition with the enol form of creatinine. Ring closure, elimination of water, and aromatization would yield the imidazoquinolines or imidazoquinoxalines. However, another tentative route was also discussed by Nyhammar (1986). He proposed that creatinine first undergoes an aldol condensation with aldehyde before subsequent conjugated addition of a pyridine or pyrazine according to Fig. lB. Such a route has recently also been suggested by Jones and Weisburger (1989). They have reported on the formation of new mutagenic compounds, other than the

IQ compounds, after heating ( 1 5 0 ° C / 2 h) threonine and acetaldehyde without sugar in D E G - H 2 0 (Jones and Weisburger, 1989). Indirect support for the outlined reaction route is, of course, that the used precursors actually produce the predicted heterocyclic amines. Unequivocal evidence that the precursors are correctly identified is offered by using isotopically labeled precursors. The very low yield, only a few nmole per mmole creatin(in)e or amino acid is, however, a serious drawback for such studies. The relatively efficient formation of PhlP in dry-heating reactions has made it possible for Taylor et al. (for ref. see Felton et al., 1990) to examine the incorporation of specific atoms of [1-15N]creatine, [methyl-13C]creatine, or [15NH2]creatine into PhlP when these compounds were heated one by one together with phenylalanine. Each purified PhlP product was analyzed by mass spectrometry and found to be one mass unit greater than the reference PhlP. This shows that the methyl-carbon, the adjacent nitrogen and the amino-nitrogen from creatine are each incorporated into PhlP. ~3C- and ~SN-NMR spectrometry of the isotopically labeled PhlP molecules is under way in order to show the exact position of each isotope (Felton et al., 1990). Because the 2-aminoimidazo moiety is common to many of the mutagenic heterocyclic amines, creatinine might be expected to play the same role in the formation of them all. This is supported by observations that cooking of meat products to which extra creatine was added increased all the mutagenic HPLC fractions to a similar extent (Becher et al., 1988; Knize et al., 1988b). Although creatinine is an essential precursor, its concentration does not seem to be the yieldlimiting factor. Both in model systems and in cooked foods, creatinine is still present after heating. Only a small part, perhaps a few percent, is used up in the reaction (Laser Reutersw~ird et al., 1987; Skog et al., 1990). According to the suggested route, the amino acids, together with sugars, produce pyrazines, pyridines, and aldehydes by Maillard reactions. When various vinylpyrazines or vinylpyridines have been added to the model system, the amount of mutagenicity has at most doubled (J~igerstad et al., 1983a,b) or not increased at all (Taylor et al., 1986; Jones et al., 1989). The observation that

227

many of the AIA compounds are produced in the absence of sugar also casts some doubt on the outlined reaction route. However, it has been reported by Wang and Odell (1973) that dry-heating of amino acids without sugar produced several derivatives of pyrazines. Especially the hydroxy amino acids, such as threonine and serine, but none of the others examined, were able to produce about 10 different pyrazines after dry-heating at 200 °C for 4 h. Obviously, there are routes other than the Maillard reaction that may produce pyrazines. A consistent finding in the model systems is that each amino acid whe.a heated together with creatine in the absence or presence of sugar might produce at least 2, but l~robably 3-5, different mutagenic compounds ~[gtultaneously (Tables 2 and 3). On the other haad~ ~.ach mutagenic heterocyclic amine might be l ~ u c e d from several different amino acids; MelQx, for instance, has been

produced from threonine, alanine, glycine, serine, and tyrosine (Table 2). A possible explanation is that several derivatives of pyrazines and pyridines might be formed simultaneously by each amino acid. PhIP has, to date, only been produced from phenylalanine or leucine. Although tyrosine differs from phenylalanine only by possessing a phydroxyl group, it seems not to be able to produce PhIP in any significant amounts (Knize et al., personal communication; Skog et al., unpublished observations). That phenylalanine is a precursor of PhIP has been convincingly demonstrated by using heavy-isotope-labeled phenylalanine (Felton et al., 1990). Separate batches of PhIP were generated by heating creatine (for 2 h at 200 o C) with L-[ring- 13C]phenylalanine; DL-[3-13C]phenylala nine and L-[15NH:]phenylalanine; or DL-[1-13C]phenylalanine; and after the purification of PhIP, mass spectra were obtained. The first reaction

TABLE 3 A M I N O ACIDS AS P R E C U R S O R S OF M U T A G E N I C I T Y A N D FOI~MATION O F H E T E R O C Y C L I C A M I N E S W H E N H E A T E D T O G E T H E R W I T H C R E A T I N ( I N ) E IN T H E ABSENCE O R P R E S E N C E OF D I F F E R E N T M O N O S A C C H A R I D E S A m i n o acid

Threonine Glycine Lysine Alanine Serine Leucine Histidine Arginine Valine Isoleucine Asparaglne Tyrosine Aspartic acid Phenylalanine Tryptophan Cysteine Methionine Glutamine Proline Glutamicacid Cyst±he

R e v / # m o l e creatin(in)e I a

II b

1068 ± 281 410 ± 59 246+108 199± 32 1 9 7 ± 85 161 ± 22 1264- 33 101 ± 29 9 1 + 29 75 ± 26 63 ± 23 56 ± 23 55 + 17 5 0 + 26 5 0 ± 27 40_+ 13 3 4 ± 17 33_+ 12 3 1 + 22 3 0 ± 13 19 + 8

108 ± 10 17 ± 1 5 ± 1.5 176 + 2 2 1.5 ± 0.4 5 ± 1.5 0.8 ± 0.3 5 ± 0.9 18 + 3.5 4 ± 1.0 7 ± 0,8 40 ± 5 12 + 1.5 -

Mutagenic heter0cyclic amines (for refs. see Table 2) MelQ~i 4~8-DiMelQx, IQ; MelQx; 4,8-DiMelQx; 7,8-DjMelQx MelQx; 4,8-DiI~¢IQx MelQ; MelQx; 4,8-DiMelQx IQ; IQx; MelQx; 4,8-DiMelQx PhlP

MelQx IQ; PhIP

IQ

a Creatine, amino acid and glucose ( 1 : 1 : 0 . 5 molar ratio) boiled under reflux in D E G - H 2 0 for 2 h at 1 2 8 ° C (J~igerstad et al., 1983b). b Creatine and amino acid (1 : 1 molar ratio) dry-heated for 1 h at 200 o C ({~vervik et al., 1989).

228 yielded a molecule with a mass 5-6 units higher than reference PhlP, showing that the phenyl ring from phenylalanine was incorporated intact. The other 2 reactions each produced a product 1 mass unit higher than reference PhIP, thus showing that the 3-carbon atom and the amino nitrogen from phenylalanine were incorporated into PhIP (Felton et al., 1990). The model system based on dry-heating of creatine and amino acids produces the same mutagenic heterocyclic amines in similar amounts without sugar. It rules out the involvement of the Maillard reaction as an obligatory route for their formation. However, it does not exclude the possibility that sugar might enhance the formation of pyrazines, pyridines, and aldehydes. Wang and Odell (1973), for instance, concluded that sugar obviously favored the formation of pyrazines in their systems without being necessary. Although sugar might not be an obligatory precursor of mutagenic heterocyclic amines, there are some data supporting the fact that sugar markedly enhances the yield of mutagen formation. When fried beef patties were prepared from a glucose-deficient meat, very low mutagenicity was produced when compared with beef patties prepared from meat containing normal amounts of sugar (J~igerstad et al., 1983a). Usually, the concentration of free and phosphate-bound monosaccharides in muscle meat is nearly half that of creatine (Laser-Reutersw~ird et al., 1987; Sulser, 1978). The short-time model system developed by Skog et al. (1990) points to the role of sugar as a catalytic reagent in the formation of mutagenicity, at least in certain concentrations.

Factors affecting the yield Conditions that affect the yield of mutagenic heterocyclic amines are of great interest, because they might contribute to the understanding of the reaction mechanism and become of practical importance as a way to limit their formation. Studies in both model systems and cooking show the great influence of time and temperature on formation of mutagenic heterocyclic amines. Generally, the mutagenicity as measured in the Ames test increases with increasing temperature.

The time effect is linear within limited time intervals, but then reaches a plateau. For the dry-heating model systems, the time/temperature dependence seems to have been very little studied. Usually a temperature between 150 and 2 0 0 ° C and 1 - 2 h have been used. The formation of PhIP by dry-heating of creatine and phenylalanine with or without sugar increased between 160 and 200 ° C and remained constant between 200 and 2 7 5 ° C (Felton et al.,, 1990). At 2 0 0 ° C the mutagenicity appeared within 10 min and reached a plateau 1-2 h later. In the D E G - H 2 0 system reflux boiling around 1 3 0 ° C resulted in a rise in mutagenicity for 4 h, although the rate seemed to become slower after 2 h (J~igerstad et al., 1983a). In the modified D E G - H 2 0 system, significant mutagenicity appeared as early as 5 min after heating at 180 °C. No mutagenicity was detected at 1 4 0 ° C after 15 min (Skog et al., 1990). At 1 6 0 ° C significant mutagenicity was seen after 15 rain, but not earlier. Interestingly, in analogy with meat-cooking experiments, a plateau in mutagenicity is generally seen after only 10 rnin. There may be several explanations for that, viz., lack of reactants or intermediates, formation of blocking agents, volatilization, or decomposition of the produced mutagenic compounds. The observation that the rate of mutagen formation initially is much higher at temperatures between 200 and 3 0 0 ° C compared with 100 and 200 ° C raises doubt that water is necessary. The dry-heating experiments show that water is not obligatory. However, in meat systems the mutagen formation is water-dependent (Bjeldanes et al., 1983; Taylor et al., 1986; 0vervik et al., 1989). The most likely explanation is that water acts as a vehicle to transport the water-soluble precursors to the crust area during cooking. Water or water activity is known to affect the rate of the Maillard reaction (Powrie et al., 1986). Thus, if the Maillard reaction favors mutagen formation, the water concentration may also have a stimulating effect. But the effect of water on the Maillard reaction rate in foods might as well be as a solvent medium and as a vehicle for transportation of low-molecular precursors. The concentration, as well as the proportion of the precursors, affects the yield of mutagenic heterocyclic amines. Jagerstad et al. (1983b) and Skog

229 et ai. (1990) have shown that creatinine produces more mutagenicity than creatine. The effect of various monosaccharides and disaccharides has been investigated only in the D E G - H 2 0 system. Muramatsu and Matsushima (1985) found that ribose produces more mutagenicity than glucose, and according to data presented by Skog et al. (1990) fructose is more effective than glucose, and sucrose is more effective than lactose. Monosaccharides and disaccharides show an optimum effect on the mutagenicity yield when present in amounts around half that of creatin(in)e or amino acid. By increasing the concentration of glucose or fructose, the formation of mutagenic compounds decreased. Heating mixtures of creatin(in)e and glycine or phenylalanine with increasing amounts of glucose or fructose resulted in the formation of 3-4 different peaks corresponding to mutagenic products. All the peaks decreased when the monosaccharides were added in increasing amounts. At equimolar levels or in excess of those of the other reactants, the formation of mutagenic compounds was almost completely inhibited (Skog et al., 1990). The mechanism behind the inhibitory effect of hexoses is unknown. It can only be speculated that by increasing the concentration of reducing sugar the Maillard reaction might favor other Maillard reaction products, which might interfere or compete with the reaction route resulting in the formation of mutagemc heterocyclic amines. By moni. toring the recovery of creatinine during this reaction, Skog et al. (1990) found more than 90% unreacted creatine when glucose was added in half the molar concentration of creatine. With increasing amounts of glucose added, the recovery of creatin(in)e decreased. The decrease in the creatin(in)e recovery was seen only in the presence of amino acid, which indicates that the Maillard reaction was involved and that some Maillard reaction product might have reacted with creatin(in)e to non-mutagenic products. Using different amino acids results in considerably different values of total mutagenicity (Table 3). One possible explanation is that each amino acid might affect not only the concentration of each mutagenic heterocyclic amine produced, but also the relative proportion between them. Because the mutagenic heterocyclic amines differ markedly in specific mutagenicity, various

amounts as well as proportions have a great influence on the total mutagenicity. There are also reports that certain amino acids enhance or inhibit the mutagenicity when added to model systems or meat before frying. Proline, for instance, produced IQ when dry-heated together with creatine (Yoshida et al., 1984). Accordingly, Ashoor et al. (1980) found that proline, but none of the other amino acids, added to ground-beef patties before frying enhanced mutagen formation. Taylor et al. (1985) upon adding tryptophan to their meat extract system observed an increase in the formation of IQ. Overvik et al. (1989) reported dramatically enhanced mutagenicity in the crust and pan-residue after adding 1% of each amino acid to beef patties, especially after adding threonine, serine, proline and alanine. On the other hand, Jones and Weisburger (1988a,b,c) have reported that both proline and tryptophan act as inhibitors in model systems. The inhibitory effect of tryptophan was also studied in a meatcooking experiment (Jones and Weisburger, 1988b), whereas tryptophan was found to be more inhibitory when spread directly on the surface of the beef patty compared with blending with the other ingredients before frying or broiling.

Chemical synthesis of mutagenic heterocyclic amines For a more detailed review of the synthesis of mutagenic heterocyclic amines and their analogues or derivatives the interested reader should refer to the book Heterocyclic Amines - Mutagens and Carcinogens in Cooked Food and Pyrolysed Amino Acids, edited by T. Sugimura and S. Takayama, published by Cambridge University Press. The imidazoquinolines IQ and MelQ have been synthesized by a number of research groups in Japan, U.S.A. and Sweden (Kasai et al., 1980a; Kasai and Nishimura, 1982; Lee et al., 1982; Adolfsson and Olsson, 1983; Waterhouse and Rapoport, 1985; Ziv et al., 1988). The method of Adolfsson and Olsson (1983) outlined below is the most efficient one since it is short, can be performed on a large scale, involves no difficult separations, affords isomerically pure products and can be adapted to the preparations of laC-labeled compounds.

230 NH 2 N------@ ~ . @ N - Me

NO 2 ~ N H M e

NO 2

)

)

R

R = H , Me

R = H , IQ R = Me, MeIQ

The related quinoxalines IQx, MelQx, its homologues (4,8-, 5,8- and 7,8-DiMelQx) and their 14C-labeled analogues are most efficiently synthesized by using a suitable 2,1,3-benzoselenadiazole as outlined below (Grivas, 1986; Nyhammar and Grivas, 1986 and Becher et al., 1988).

Synthetic methods for the mutagenic di- and tri-methylimidazopyrines (DMIP and TMIP) isomers have not been published.

NH 2

NO 2 ~ . _ _ ~ j . NHMe

N---
--NH2

iN

5,8-DiMelQ x 7,8-DiMelQ x

NH 2 PhIP

231 T h a t creatine is one of the obligatory precursors of the A I A c o m p o u n d s explains why the m u t a g e n i c heterocyclic amines are p r o d u c e d only in a n i m a l foods, or more precisely, muscle foods. A meat organ like liver c o n t a i n s hardly a n y creatine, a n d c o n s e q u e n t l y fried liver c o n t a i n s low m u t a g e n i c i t y (Laser Reuterswiird et al., 1987). Creatine b o u n d to p h o s p h a t e participates in the energy m e t a b o l i s m of muscle cells. Creatine, as well as creatinine, is excreted via urine. However, I Q has b e e n isolated from the smoke c o n d e n s a t e of cigarettes ( Y a m a s h i t a et al., 1986). A possible e x p l a n a t i o n could b e that a n i m a l urine, c o n t a i n i n g creatin(in)e, has b e e n spread over the leaves of the tobacco p l a n t through fertilization. Still m u c h work r e m a i n s to be d o n e o n precursors, kinetics, yield optimization, a n d reaction mechanisms, as well as o n factors that might enh a n c e or i n h i b i t the f o r m a t i o n of heterocyclic amines. D a t a o b t a i n e d from model systems, although a little premature, are encouraging regarding the possibility of limiting the f o r m a t i o n of heterocyclic a m i n e s b y m a n i p u l a t i n g the precursors or b y affecting the presence or absence of i n h i b i t o r y or s t i m u l a t i n g c o m p o u n d s . T h e simplest p r e c a u t i o n is, of course, to cook food at temperatures as low as possible or shorten the cooking time at high temperatures.

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