Occurrence, identification, and bacterial mutagenicity of heterocyclic amines in cooked food

Mutation Research, 259 (1991) 205-217 © 1991 Elsevier Science Publishers B.V. 0165-1218/91/$03.50 ADONIS 016512189100059F

205

MUTGEN 00032

Occurrence, identification, and bacterial mutagenicity of heterocyclic amines in cooked food J a m e s S. F e l t o n a n d M a r k G. Knize Biomedical Sciences Division, Lawrence Livermore National Laboratory, University of California, Lioermore, CA 94550 (U.S.A.)

(Received 23 March 1990) (Accepted 1 August 1990)

Keywords: Heterocyclic amine; Amino-imidazoazaarenes; Mutagens; Ames/Salmonella assay; Cooked foods; Structure/activity; Analytical chemistry

Summary Potent mutagenic activity in Salmonella bacteria has been reported in cooked foods in numerous laboratories worldwide. Determining the human risk from exposure to these biologically active compounds in our diet requires genotoxic and carcinogenic evaluation of the chemicals coupled with determination of the dose consumed. Thus, knowledge of the exact structure of the mutagens present in the food and enough synthesized material for biological assessment are essential for this evaluation. To reach this goal, isolation of these compounds requires the Ames/Salmonella assay to guide the purification and identification process. Mass and N M R spectrometry are used to identify the isolated compounds. Finally, these findings are followed by synthesis of the exact isomer. The predominant class of mutagens found in evoked foods of the western diet are amino-imidazo-quinoxalines, amino-imidazo-pyridines and amino-imidazo-quinolines, collectively called amino-imidazoazaarenes (AIAs). Mass amounts of these specific compounds range from less than 1 to 70 n g / g of meat. The mutagens are formed from the heating of natural precursors (creatine, amino acids, and possibly sugars) in the food. These AIAs are some of the most potent mutagens ever tested in Salmonella bacteria with the number and position of methyl groups having an important influence on the mutagenic activity.

Correspondence: Dr. James S. Felton, Biomedical Sciences Division, Lawrence Livermore National Laboratory, University of California, P.O. Box 5507, Livermore, CA 94550

(U.S.A.). Abbreviations: AIA, amino-imid~_Taarene; 1,6-DMIP, 2-

amino-l,6-dimethylimidazo[4,5-b]pyridine; MeIQ, 2-amino3,4-dimethylimidazo{4,5-f]quinoline; 4-MeIQx, 2-amino-3,4dimethylimidazo[4,5-f]quinoxaline; 7-MeIQx, 2-amino-3,7-dimethylimidazo[4,5-f]quinoxaline; 8-MeIQx, 2-amino-3,8-dimethylimidazo{4,5-f]quinoxaline; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; IQx, 2-amino-3-methylimidazo[4,5-f]-

quinoxaline; PhlP, 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine; 3-MePhlP, 2-amino-3-methyl-6-phenylimidaz0[4,5-b]pyridine; 4,7,8-TriMelQx, 2-amino-3,4,7,8-tetramethyfimidazo[4,5-f]quinoxaline; 4,7-DiMelQx, 2-amino-3,4,7-trimethylimidazo[4,5-f]qninoxaline; 4,8-DiMelQx, 2-amino-3,4,8-trimethylimidazo[4,5-f]qninoxaline; 5,7-DiMelQx, 2-amino3,5,7-trimethyFmfidazo[4,5-f]quinoxaline; 5,8-DiMeIQx, 2amino-3,5,8-trimethylimidazo[4,5-f ]quinoxaline; 5,7,8-TriMeIQx, 2-anfino-3,5,7,8-tetramethylimidazo[4,5-f]quinoxaline; 7,8-DiMelQx, 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline; B[a]P, benzo[a]pyrene.

206

Interest in aromatic amine mutagens in foods began in 1977 when Sugimura et al. reported that the mutagenic activity of both smoke condensate and the charred surface of broiled fish and beef was much higher than could be accounted for by the polyaromatic hydrocarbon (PAH) content of the products. Soon after this discovery, Commoner et al. (1978) showed that similar mutagen formation occurred under household cooking conditions typically used for frying beef. Since then, the finding of potent mutagenicity in cooked meats has been confirmed in laboratories around the world. Studies in our laboratory in conjunction with the Department of Nutritional Science, UC Berkeley, revealed that several of the major sources of cooked protein in the American diet showed significant mutagen content (Bjeldanes et al., 1982a). A positive mutagenic response ( A m e s / Salmonella assay) was found for cooked beef, pork, ham, bacon, lamb, chicken, fish, and eggs after broiling, frying, and barbecuing. Other foods having high protein content, but not derived from vertebrate muscle including tofu, beans, and cheese when cooked under similar conditions, produced very low or negligible mutagenic activity (Bjeldanes et al., 1982b). T o assess the importance to human health of these mutagens from cooked meat, identification, quantitation of the dose and toxicological evaluation of the biologically active molecules needs to be determined. T h i s work has become a focus of research in many laboratories worldwide, including our own.

(I) Mutagen formation in foods J~igerstad et al. suggested in 1983 that creatine, amino acids and sugars derived from muscle are important precursors in the production of mutagens i n cooked meats. The involvement of these precursors has been confirmed in many laboratories. As its structural analogy suggests, creatine has proven to be an essential precursor for A I A (amino-imidazoazaarene) mutagen formation. Creatine (or the cyclized form creatinine) when combined w i t h amino acids and, in some cases, sugars successfully produces AIA mutagens in model reactions. These are reviewed in this issue by J~igerstad et al. Creatine has a mutagen-en-

TABLE

1

MUTAGENIC

ACTIVITY

OF FRIED

MEAT

MIXTURES

Meat type

Milk

Creatine

Starch

TA1538

+30%

+4%

+4.4%

rev/g 16700

Pork and veal

+

+

+

Pork and veal

+

-

+

1 151

Pork and veal

-

+

+

2984

Beef

+

+

-

12 7 0 0

Beef

+

+

+

12100

Beef

-

+

-

5 018

Beef

-

+

+

6 925

Beef

+

-

-

1082

Beef

+ (evaporated)

+

-

10412

Beef

+ (evaporated)

-

-

702

Beef

+ (powdered)

+

-

4 784

Beef

+ (powdered)

-

-

Beef

-

-

236 1861

hancing effect when added to meat (Knize et al., 1988b) or meat mixtures (Nes, 1986; Becher et al., 1988) before cooking. Other amine mutagens notably the pyridoindoles and pyridoimidazoles have been isolated from pyrolyzed amino acids and found in some foods. The AIA mutagens have been shown to be the predominant structural type found in cooked foods of the western diet, and are the focus of our work and this review. The structures and occurrences of the non-AIA mutagen types have been reviewed (Felton and Knize, 1990b; Sugimura et al., 1988). A recent detailed investigation in our laboratory of mutagen production in meat mixtures which is similiar to the mutagenic recipe used by both Nes (1986) and Becher et al. (1988) is shown in Table 1. Meat patties were cooked for 10 rain per side at 220°C and extracted in aqueous acid and purified on an XAD-2 resin column according to Felton et al. (1984). The original pork and veal mixture with milk, creatine and starch gave the highest mutagenic activity (16700 TA1538 revertants/g of original uncooked meat). Eliminating the milk (source of sugars and amino acids) or creatine greatly reduced the mutagen formation. In fried beef, high activity was seen with the milk and creatine, but the starch had little effect. Eliminating the milk lowered the activity by half. Again the presence of the starch had little effect.

207 Finally, the use of evaporated milk (a more concentrated source of milk components) and powdered (dehydrated) milk increased mutagenic activity over the beef alone, but only in combination with added creatine. These recent results, together with those cited above, suggest that creatine is rate limiting since added milk alone does not increase activity. Liquid milk increases the mutagenic activity more than the powdered. Possibly the water in the milk may be aiding precursor movement to the hot meat surface where the mutagen-forming reactions occur. Free amino acids and sugars in the milk may also contribute to mutagen formation. But these results reinforce the conclusion (Table 1) that creatine is rate limiting. Overvik et al. (1989) showed that the individual addition of 15 different amino acids to ground pork before frying increased mutagenic activity from 1.5- to 43-fold. These results sharply contrast with the report of Ashoor et al. (1980) in which 17 amino acids showed no effect when individually added to ground beef before frying. Only the addition of proline caused an increase (8-fold) in mutagenic activity. There are a number of differences in experimental procedure that may explain these contradictory results. Ashoor et al. used ground beef containing 19% fat and cooked the patties for 2 rain per side at 191°C, adding 0.5 or 1.0 mmole of amino acid dissolved in 2 ml water, to a 35-g patty (approximately 0.4 g/100 g meat). Overvik et al. used ground pork containing 4% fat cooked for 6 min on the first and 4 min on the second side at 200°C. Amino acids were added neat, at 1 g/100 g of meat. The discrepancy in the effect of added amino acids can be explained by the cooking time and water content differences. For cooking time, our laboratory (Bjeldanes et al., 1983) and others (Spingarn and Weisburger, 1979), showed that mutagen formation increases dramatically after an initial lag of 2-4 min. The cooking times in the linear range of mutagen formation used by 0~,ervik et al. would be more useful for studying the effects of additives. Taylor et al. (1986a,b) showed that water is an inhibitor of mutagen formation. It appears that the addition of water with amino acids and the very short cooking time used by

Ashoor et al. affected the mutagen-forming reactions, negating the effects of the added amino acids. The work by Overvik et al. supports the idea that free amino acids are involved in the production of mutagens in meat and, like creatine, appear to be limiting mutagen formation. The role of sugars in mutagen formation in meats is unclear. Laser-Reutersw~d et al. (1987) examined the content of creatine, creatinine, monosaccharides and free amino acids in bovine tissues before and after cooking. The amounts of creatine and creatinine correlated best with the mutagen production. In mutagen-modeling systems glucose and fructose have been shown to be necessary for mutagen production in aqueous-ethylene glycol refluxing experiments (reviewed by J~igerstad et al., this issue). Many of the mutagens found in foods have also been formed from dry heating reactions without sugars (see Felton and Knize, 1990b for review). But the addition of glucose to the dry heated reaction of glutamic acid and creatine increased the mutagenic activity 7fold (Knize et al., 1991). These data suggest some involvement of sugars or their breakdown products although they are not necessary for mutagen formation. Determining if atoms from sugar molecules are incorporated into the mutagenic molecules would be an important step in clarifying their role in mutagen formation. It is now apparent why cooked non-muscle foods such as tofu, beans, and cheese have little or no mutagenic activity. They lack the creatine that is present at about 0.5% by weight in muscle meats. Removing the creatine and water-soluble precursors before cooking is a way to sharply reduce the mutagenic activity produced during cooking. A scheme to do this utilizing brief microwave cooking and separation of the mutagen precursors from the meat before frying was devised by Taylor et al. (1986b). The physical reaction conditions of mutagen formation are beginning to be understood. Mutagens produced by dry heating (Taylor et al., 1986a; Knize et al. 1988a) or water-evaporated diethylene glycol (Skog and J~igerstad, 1989) appear to be predominantly the AIA type, indicating that water is not necessary to form mutagens. It was also shown that mutagen precursors are water soluble (Taylor et al., 1986b). The water solubility may

208 explain why the outside surface of the meat has the most mutagenic activity (Dolara et al., 1979). Mutagen precursors are easily dissolved and transported to the. surface in water where they are exposed to the high temperatures required for the mutagen-forming reactions. Also, thick or thin meat patties have the same mutagenic activity per g of meat and not per amount of surface area (Knize et al., 1985). This result may be explained by the movement of water-soluble mutagen precursors that can move to the surface as water evaporates even in a thick piece of ground meat. The high mutagenic activity of the pan residue after cooking meat seen by Overvik et al. (1987, 1989) and Knize et al. (1988b) could arise from the water-soluble precursors that drain from the meat upon heating. They might evaporate and then be dry heated on the pan surface. It is also possible that grinding the meat helps to release precursors and increase mutagenic activity over the activity found with meat steaks, but the comparative study has not been made.

water and concentrated on 'blue cotton' (Hayatsu et al., 1983). Mutagenicity chromatograms which are the result of Ames/Salmonella determinations for all fractions eluting from a standardized chromatographic separation have been compared for several meat types, recipes, cooking times, and cooking temperatures. A number of these have been published (Gry et al., 1986; Becher et al., 1988; Knize et al., 1985, 1988a,b; Felton et al., 1984) and the positions and amounts of the mutagenic peaks are all remarkably similiar, but not identical. These results suggest that there are similar precursors and pathways for the mutagen-forming reactions regardless of meat type and cooking conditions.

2500

2000 [

(II) Extraction of mutagens Since the mutagenic molecules are present at low concentrations, a number of approaches have been used to maximize the amount at the start of the purification procedures. They include the use of: (1) large amounts of meat (10-100 kg) cooked to produce large mass amounts of mutagen; (2) higher temperatures than those commonly used in household cooking practices to produce a greater mutagenic response; and (3) spiking of the meat material with creatine, a 'natural' rate-limiting precursor of the mutagen-forming reactions (Becher et al., 1988). It was shown in our laboratory that, in general, the same set of mutagenic compounds is produced at high and low temperatures (Knize et al., 1985). In experiments by Becher et al. added creatine increased the mutagen yields 17-fold, and thus increased their concentration per kg of meat. Extractions are done to solubilize the mutagens from the solid meat matrix. Either the mutagens are extracted with methanol (Kasai et al., 1981b), mixed solvents (Felton et al., 1981), water at low p H followed by concentration on XAD-2 resin (Bjeldanes et al., 1982c), or solubilized in boiling

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1200

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~)" I'o" ¥ o" i ~" ~'~" 's'o" 'a'~ '; o" i ~" '~ ~" i ~'o "i ;~' ~'2o

1000 a

800

=:

Fish ,oo

10

...............

:10

30

40

M

50

.........

60

70

00

............

,...-..

90 100 110

120

1200

~

'>~

~:

000 ,,,iIf, II ,,,II,,II 800

Chicken

ooo

_04oo ,ooo

Ll.'llli 10

ill, ..I 20

lind 30

40

50 Time

60

70

80

t,

I mn,,, 90 100 110 120

(mln)

Fig. 1. Mutagenic activity profiles of the HPLC separation of ground-fried beef, fish and chicken with equal amounts of mutagenic activity injected onto each c~l~mm.

209 Fig. 1 shows HPLC separations of the mutagenic activity for beef, fish, and chicken; ground, formed into patties, and fried at 220°C for 10 rain per side. The mutagenic activity in Salmonella strain TA1538 per 100 g of uncooked meat was 117000 revertants for the beef, 14900 revertants for the fish, and 72 300 revertants for the chicken. The complex pattern of mutagenic activity of each meat shows 3 broad peaks, from 30-40 rain, 55-65 rain, and 85-105 rain. They differ in proportion, most notably with the 55-65-rain peak being smaller in fish, but larger in chicken when compared to the beef sample. Although the preparation and cooking of the fish and chicken is not typical for these foods, these meat types contain the precursors necessary to form a set of mutagenic compounds like those found in beef. Since each broad peak in Fig. 1 may be composed of several mutagens in varying amounts, determining the exact structures of the many mutagens in the meats and quantifying their amounts is critical for future toxicology and risk assessment studies. The reason for low mutagenic activity in fish is not known. The amounts of free amino acids and creatine in fish are very similar to beef and chicken (Knize et al., 1988a), but the mutagenic activity produced in fish is much lower even when it is prepared and cooked under conditions identical to the chicken and beef. Understanding the reasons for the low mutagenic activity of fish may lead to ideas for inhibition of mutagen production in other muscle meats. (III) Identification of the mutagens in cooked meats Many laboratories using a variety of analysis and separation techniques have isolated and identified mutagens from cooked foods. The first AIA mutagen was isolated from cooked fish by Kasal et al. (1981a) and continues to the present. The work through mid-1988 was recently reviewed (Felton and Knize, 1990a,b). Purification has been based on HPLC fractionation of extracts of cooked meats with detection using the Ames/Salmonella assay (Knize et al., 1987b). For known mutagens, identification is made using mass or UV spectra, or chromatographic retention times. For unknown mutagens usually larger sample amounts, NMR spectra and

synthesis of the proposed structure are also necessary. It must be pointed out that all currently characterized mutagens were able to be isolated because they were potent genotoxins in the Ames/Salmonella assay. Weak mutagens not present in high mass concentration in the meat may be missed using the standard methods described above. Weaker Ames/Salmonella mutagens present in cooked meats may be genotoxic in mammalian cells and possibly carcinogenic in rodents and humans. To help identify these compounds immunoaffinity column separations are being investigated at LLNL as a method to isolate these compounds (Vanderlaan et al., 1988, 1989). The identification of a new mutagen starts with a sample of a pure compound. In our laboratory, a photodiode-array HPLC detector is useful for assessing peak purity. UV absorbance spectra can be useful to determine a structural type. The quinoline-based AIA mutagens, IQ and MeIQ have an absorbance maximum at 264 nm. The quinoxalines (8-MeIQx, 4,8-DiMeIQx, IQx and 4-MeIQx) all have an absorbance maximum in the 273-277 nm range. PhIP and 1,6-DMIP have maximum absorbance maxima at 316 and 305 nm, respectively. Mass spectra for AIA mutagens show a similar ion fragmentation pattern. A large molecular ion and small fragment ions denoting a loss of 1 (H), 15 ( C H 3 ) , 28 (CH2N) and 42 (CH2N2) are typical features. NMR spectra give information about atom position which can be very useful for showing a general structural type and for determining some exact structures, but for PhlP (Felton et al., 1986b) and 4,8-DiMelQx (Knize et al., 1987a) synthesis and chromatographic comparisons of positional isomers were necessary to determine the exact isomeric structure of the food mutagen even when enough of the natural product was available for an NMR spectrum. Table 2 lists the NMR spectral peak shifts of a family of 2-amino-3-methylimidazo[4,5-f]quinoxaline mutagens differing in the number and position of methyl groups. Spectra were acquired on the same instrument with methylsulfoxide-d6 as solvent showing a reference signal (residual methylsulfoxide-d5 at 2.501 ppm relative to TMS).

210 TABLE 2 NMR SPECIAL PEAK POSITIONS (ppm) OF QUINOXALINE MUTAGENS IN

DMSO-d 6

NH 2

3-Me

4-H

4--Me

5-H

5-Me

7-H

7-Me

8-H

8-Me

IQx

6.66

3.68

7.61

-

7.84

-

8.75

-

8.80

-

4-MelQx 7-MelQx 8-MelQx

6.55 6.56 6.55

3.85 3.65 3.66

7.50 7.55

2.82 -

7.32 7.75 7.72

-

8.69 8.65

2.65 -

8.72 8.71 -

2.69

4,7-DiMelQx 4,8-DiMelQx 5,7-DiMelQx 5,8-DiMelQx 7,8-DiMelQx

6.89 6.42 6.79 6.39 6.60

3.85 3.84 3.70 3.62 3.64

7.82 7.61 7.65

2.81 2.79 -

7.31 7.26 7.47

2.72 2.72 -

8.59 8.66 -

2.65 2.74 2.65

8.68 8.82 -

2.65 2.68 2.62

4,7,8-TriMelQx 5,7,8-TriMelQx

6.41 6.33

3.83 3.60

7.53

2.77 -

7.19 -

2.70

-

2.63 2.70

-

2.61 2.66

T h e s e d a t a s h o u l d b e useful in c o n f i r m i n g structures of k n o w n a n d u n k n o w n q u i n o x a l i n e m u t a gens a n d their m e t a b o l i t e s . F i v e o f these m u t a g e n s h a v e b e e n r e p o r t e d in c o o k e d meats, 8 - M e l Q x ( K a s a i et al., 1981a), 4 , 8 - D i M e l Q x ( K n i z e et al., 1987a), 7 , 8 - D i M e l Q x ( T u r e s k y et al., 1988), I Q x (Becher et al., 1988), a n d 4 - M e l Q x (M. Vahl, p e r s o n a l c o m m u n i c a t i o n ; K n i z e et al., 1989). F o r the 4 a n d 5 p o s i t i o n s o f these q u i n o x a l i n e s t y p i c a l A B p a t t e r n s are seen for a d j a c e n t C - H p r o t o n s (J = 8.8 Hz). P r o t o n s a d j a c e n t to m e t h y l g r o u p s are also c o u p l e d (J = 1.2 Hz). F o r the 7 a n d 8 positions, n o c o u p l i n g is o b s e r v e d b e t w e e n C - H p r o t o n s a n d a d j a c e n t m e t h y l groups. F o r a d j a c e n t p r o t o n s ortho c o u p l i n g s are small (J = 1.9 Hz). Peak a s s i g n m e n t s for the q u i n o x a l i n e s with b o t h 7- a n d 8 - p o s i t i o n p r o t o n s ( I Q x a n d 4 - M e l Q x ) o r 7- a n d 8 - m e t h y l g r o u p s ( 7 , 8 - D i M e l Q x a n d 4,7,8a n d 5 , 7 , 8 - T r i M e l Q x ) are assigned b a s e d on c o m p a r i s o n with the relative p e a k p o s i t i o n s o f the o t h e r m u t a g e n s a n d are assigned with s o m e uncertainty. C h e m i c a l syntheses a r e essential for p r o o f o f s t r u c t u r e a n d to m a k e sufficient q u a n t i t i e s for toxicological testing. E s p e c i a l l y for new m o l e c u l e s these syntheses c a n b e very difficult a n d t i m e - c o n suming. It s h o u l d b e e m p h a s i z e d t h a t the identific a t i o n o f n e w m u t a g e n s is a l o n g - t e r m process. I n s o m e f o r t u n a t e cases though, the synthesis is a s t r a i g h t f o r w a r d a d a p t a t i o n of a p r e v i o u s l y des c r i b e d synthesis route. A d a p t a t i o n s o f previous syntheses were used for m a k i n g the n e w m u t a g e n s

I Q x ( a d a p t e d f r o m the synthesis o f 8 - M e l Q x ) , 4-MelQx (from 4,8-DiMelQx), and 1,6-DMIP ( f r o m PhlP). C o m p a r i s o n s s h o w e d the s y n t h e t i c p r o d u c t s to b e i d e n t i c a l w i t h the n a t u r a l p r o d u c t s for IQx, 1 , 6 - D M I P (Becher et al., 1988, 1989) a n d 4 - M e l Q x (M. Vahl, p e r s o n a l c o m m u n i c a t i o n ; K n i z e et al., 1989).

(IV) Occurrence T h e r e are 2 classes o f h e t e r o c y c l i c a m i n e s rep o r t e d in foods, a m i n o - i m i d a z o a z a a r e n e s ( A I A s ) a n d n o n - a m i n o - i m i d a z o heterocycles, i n c l u d i n g p y r i d o i m i d a z o l e s , p y r i d o i n d o l e s a n d others. T h e c r e a t i n e - d e r i v e d A I A m u t a g e n s are the m o s t c o m m o n l y found, especially in the western diet. Because o f the d i f f i c u l t y in the c h e m i c a l a n a l y s i s d u e to b o t h the c o m p l e x i t y o f m e a t e x t r a c t s a n d low m a s s a m o u n t s of m u t a g e n present, the r e p o r t s on the a m o u n t s a n d types o f m u t a g e n s p r e s e n t are f r a g m e n t e d a m o n g several f o o d s a n d c o o k i n g conditions. U s u a l l y o n l y 1 o r 2 m u t a g e n s a r e i d e n t i fied a n d f r e q u e n t l y n o q u a n t i t a t i v e a m o u n t s a r e determined. T a b l e 3 shows a c o m p a r i s o n o f the m a s s a m o u n t s a n d relative p e r c e n t a g e for 4 k n o w n m u t a g e n s f r o m c o o k e d m u s c l e m e a t s a n d a beefd e r i v e d d r y - h e a t i n g reaction. O n l y studies that q u a n t i f i e d at least 3 o f the m u t a g e n s a r e i n c l u d e d . T h e r e are several o t h e r m u t a g e n s p r e s e n t in each o f these foods, b u t they a r e n o t i n c l u d e d in this comparison.

211 TABLE 3 MASS OF MUTAGEN IN COOKED-MUSCLE-DERIVEDMIXTURES (ng/g)

Fried beef, 10 rain, 300 oC Fish Dry-heated beef supernatant Beef, 15 rain, 200°C Beef, 13 rain, 190°C

IQ 0.02 (0.12) 0.16 (0.21) 0.36 (3.2) 1.9 nd

8-MelQx 1.0 (6.2) 6.44 (8.5) 0.91 (8.0) 12.3 8.3

4,8-DiMelQx 0.6 (0.37) 0.10 (0.13) 0.37 (3.2) 3.9 2

PhlP 15 (93) 69.2 (91) 9.7 (86) nd 48.5

(Felton et al., 1986a) (Zhanget al., 1988) (Taylor et al., 1989) (Tureskyet al., 1988a) (Gross et al., 1990)

(), percent of total of the 4 cpds. nd, not determined.

Fried beef cooked at 300°C, fish, and dry heated beef supernatant are all similar in their relative amounts of the 4 mutagens listed, with PhIP having 86-91% of the mutagen mass. 8-MeIQx is the next most abundant with 6.2-8.5% of the total mass, or about 10% of the PhIP amount. There is more variation with IQ and 4,8-DiMeIQx having less than 0.1 to 3.2% of the total mass of the mutagenic compounds. The beef cooked at 200°C was not assayed for PhIP, but 8-MeIQx is present in greater amounts than either IQ or 4,8-DiMeIQx. For the cooked pork, PhIP is more abundant than 8-MeIQx, which is more abundant than 4,8-DiMeIQx. Beef cooked at 1 9 0 ° C also showed PhIP to be the most abundant with less MeIQx and 4,8-DiMeIQx for the 3 mutagens detected. A recent analysis of fried beef by Murray et al. (1988) showed about twice as much 8-MeIQx present as 4,8-DiMeIQx. These relative amounts agree in general with those in Table 3. Gross et al. (1989) examined fried beef finding almost equal amounts of MeIQx and PhIP, and a trace of 4,8-DiMeIQx. Although the total mutagenic activity varies greatly with cooking temperature, time and type of meat, the relative proportions are similar for the known mutagens quantified. As was noted in the above discussion of Fig. 1, the mutagenic ratios do not appear dramatically different between beef and fish, and the finding of similar amounts in the dry heated beef supernatant suggests that the mutagen-forming reactions are similiar to those in the ground beef. Several improved methods of sample preparation suitable for the analysis of cooked meats have

recently been published. G o o d recovery of specific mutagens was reported by Murray et al. (1988) using liquid-liquid extraction, by Turesky et al. (1988b) using monoclonal antibody affinity chromatography, and by Gross et al. (1989) using Kieselgur adsorption followed by medium-pressure liquid chromatography and by Gross (1990) using coupled cartridges of diatomaceous earth and copper phthalocyanine-derived silica. These processes are important steps towards the practical goal of quantifying mutagens in cooked meats and need to be further evaluated.

(V) Future needs in analytical chemistry of food mutagens The small amount of quantitative data available describing mutagen content in foods makes the dose estimation of even the known mutagens difficult. For reasonable risk estimates, accurate quantitation is essential. Once the compounds have been identified, simpler and more sensitive methods need to be employed to confirm or accurately quantify the mutagens in other foods and cooking conditions. These include chromatography, either HPLC or GC, with sensitive and specific detection for the mutagens of interest. Offline techniques such as measurement of mutagenic activity using the Ames/Salmonella test (quantitative response of Salmonella strains and sensitivity to nitrite under acid conditions) (Tsuda et al., 1985) or detection of specific molecules by antibody-ELISA assays would not require chemically pure fractions, but currently these methods lack adequate compound specificity.

212 Recently published on-line analysis techniques such as H P L C or G C coupled to a mass spectrometer (Yamaizumi et al., 1986; Turesky et al., 1988a; Murray et al., 1988) appear to be sensitive and specific enough for accurate mutagen quantitation using deuterium-labeled internal standards. The fact that several other mutagens are present in meat, either partly characterized (see Felton and Knize, 1990b, for review) or unknown, makes their identification a high priority. The success using model heating reactions to form the same mutagens that are found in cooked meats suggests

N'CH3

N

the model reactions are potentially useful for generating large amounts of mutagenic compounds for structural studies. Once identified, the mutagens can then be quantified in various cooked foods. Our laboratory is currently pursuing this approach. (VI) Bacterial

mutagenicity

(A) Structure-activity relationships The mutagens found in cooked foods have mutagenic activity in Salmonella that spans 5 N NH2 / T--2_~ 3N~ C

~N /

10 6 --

Quinolines

Quinoxalines

4-MelQ

4-MelQx

~N

Pyridines

R=C6Hs=PhlP R=Me=-DMIP

7-MelQx 4,7-DiMelQx 5,7-DiMelQx 10 5 --

IQ

4,8-DiMelQx 7,8-DiMelQx 8-MelQx IQx

5,8-DiMelQx

10 4 --

4,7,8-TriMelQx =

PhlP

lO 3 -

B[a]P

10 2 --

10 1--

3-MePhlP 1,6-DMIP

0

Fig. 2. Logarithmicscale plot showingmutagenic activity of quinoline, quinoxaline and pyridine mutagens. Mutagens reported found in at least 1 cooked food are shown in bold type. Ames/Salmonella plate incorporation assay was used with Aroclor-treated rat liver $9. All values are derived from the linear portion of the dose-response curves.

213 orders of magnitude in response. Synthesized isomers can also be compared and features that affect the relative Salmonella mutagenic activity can be identified. Fig. 2 shows mutagenic activity data from our laboratory on a logarithmic scale for a series of quinoline-, quinoxaline- and pyridine-based AIA mutagens differing in the number and position of their methyl groups. Benzo[a]pyrene, a mutagen and carcinogen, of a different class is included for reference. Nine of the compounds compared, 4MelQ, IQ, 4-MelQx, 4,8-DiMelQx, 7,8-DiMelQx, IQx, 8-MelQx, PhlP, and 1,6-DMIP have been reported in at least one cooked meat (Felton and Knize, 1990a) and are shown in Fig. 2 in bold type. Several factors affecting mutagenic activity can be seen from this group of mutagens. The 3fused-ring quinoline and quinoxaline mutagens are much more potent than the pyridine-based mutagens. The extra nitrogen atom in the quinoxaline ring system makes little difference in the mutagenic activity (compare IQ to IQx). The number and position of methyl groups affect mutagenic aotivity, as was first shown by Nagao et al. (1981). Methyl groups at the 4 posi5000

tion increase activity (compare IQ to 4-MelQ, IQx to 4-MelQx, and 8-MelQx to 4,8-DiMelQx). Methyl groups at the 5 position lower activity (compare 8-MelQx to 5,8-DiMelQx and 7-MelQx to 5,7-DiMelQx). A methyl group in the 7 position is more mutagenic than the 8 position (compare 7- to 8-MelQx and 5,7- to 5,8-DiMelQx). A methyl group at the 8 position decreases activity (compare 4-MelQx to 4,8-DiMelQx, 7-MelQx to 7,8-DiMelQx and 4,7-DiMelQx to 4,7,8-TriMelQx). For the pyridine-based mutagens, PhlP (1-Me) is 100-fold more mutagenic than its 3-methyl isomer. The substitution of a methyl group (1,6DMIP) for the phenyl group of PhlP lowers activity 200-fold. These large differences in SalmoneUa-mutagenic activity based on seemingly small changes in the structure of the molecule demonstrate the importance in determining the exact structures of the mutagens in cooked foods. A more detailed treatment of the structure/activity relationships of heteroeyclic amine mutagens has been submitted for publication by Hatch et al. (1990). Whether the Salmonella mutagenicity differences related to structure are the result of a

~

5000

A 4000'

m 13.

1:: Q > Q

•m-e4•.e-o. -O-

TA1538 TA98 TAg7 TA96 TA100 TA102

/ / ~

/ /

"~ -e-=+ -I-o-it-

/

/

/

B

4000

/ /

3000'

3000

2000

2000 -

1000

1000

TA1538 TA98 TA97 TA96 TAIO0 TA102 TA104

-

o

10-2

. . . . . . . . . . . .

#

10"1

100

........

Dose, ~g

, 101

........

0 102

r

100

........ 101

u 102.

_

.

_

. ~.. . . . . . 103

Dose, I~g

Fig. 3. Dose-responsecurvesfor 1-MePhIP(A) and 3-MePhlP(B) in 7 Ames/Salmonellastrains. The plate incorporationassaywas used withAroclor1254-inducedrat (Sprague-Dawley)liver $9 (2 mg protein/plate). All pointsare the meanof duplicateplatings.

214

8000 -

5000

4000

m

O 3000

TA1538 TA98 TAg7 TA96 TA100 TA102 T A

•

TA1538 TA98 TA97

,,

TA0,

A t

l 6000 -

1

/I fl 1

0

B

/,,"

•

/f

II

?

~

r

4000 "E

2000

>

2000 1000

0 ~l-----.. . . . . . .

10 0

10 1

........

a ........ I ........ 10 2 10 3 10 4

0 •

10 "1

10 0

Dose, pg

10 1

10 2

10 3

Dose, ng

Fig. 4. D o s e - r e s p o n s e curves for 4-MelQx (A) and 4,8-DiMelQx (B) in 7 A m e s / S a l m o n e l l a strains. The conditions are the same as in Fig. 3.

better fit with the catalytic P-450-activating enzymes, the lifetime of reactive intermediates, or the repair ability of the bulky adduct formed is unclear.

(B) Bacterial mutagenicity in different Salmonella strains The mutagenic response of 2 PhlP isomers is compared in 7 Ames/Salmonella strains in Fig. 3. Both isomers showed mutagenic responses in the 2 strains sensitive to G C D N A frameshift changes (strains TA1538 and TA98). In the case of 1MePhlP, induced changes have been sequenced and were shown to occur only in the G C run upstream from the hisD3052 mutation (Fuscoe et al., 1988). A lesser response is seen at doses over 1 #g for TA100, TA97 and TA104. Only TA97 is a frameshift-sensitive strain in this group, the others are base-substitution sensitive. TA96, an AT frameshift-sensitive strain shows only a small, but significant response over background, at the highest dose tested. In contrast, 3-MePhlP showed a response at a 2 orders of magnitude higher dose and only with strains TA1538 and TA98. It is possible that at doses over 1 m g / p l a t e the other strains may also show a response• They are negative up to 0.5 mg/plate. It is interesting that a change in the position of one methyl group can affect the muta-

genicity by 2 orders of magnitude and not just in 1 tester strain, but in all 7 used for this analysis. A second comparison can be made between the most mutagenic AlAs, 4-MelQx, and 4,8-DiMelQx (Fig. 4). These compounds only differ by an additional methyl group on the 8 position, but differ almost 2 orders of magnitude in the frameshift-sensitive strains TA1538, TA98, and TA97. In contrast to the 1-MePhlP, these quinoxaline compounds show little response with the other tester strains. It is interesting that TA97, a frameshift strain, is positive for all compounds except 3-MePhlP. Finally, this data shows clearly the utility of the Ames/Salmonella assay for structure-activity analysis, as the responses described in Figs. 3 and 4 scan 5 orders of magnitude in dose. It is also worth noting that all four of the compounds described are closely related structurally (they all contain the amino-imidazo moiety), but show a wide range of mutagenic response•

(VII) Conclusions The risk to humans of the consumption of these mutagens is the product of the toxicity of the specific mutagens and the exposure dose. A prerequisite to risk estimates is the identification of the

215

compounds and their availability for study of genotoxic and carcinogenic effects. There are many mutagenic chemicals in cooked meats. We estimate 15-20 separable mutagenic compounds. There should be a finite number of mutagens and, importantly, their relative amounts will probably not vary greatly among the cooked meat types. This conclusion is based on the fact that the relative amounts of the precursors (amino acids, sugars and creatine) are similar among muscle types. Moreover, data comparing diverse muscle types such as beef, chicken and fish produce a similiar set of mutagenic compounds. The total mass amount of each appears to be influenced primarily by cooking temperature and time. Many of the mutagen isolation schemes currently being used require elevated temperatures or added creatine to increase the mutagen yields for identification purposes. Once the structures are known, the amount of the mutagens in the typically prepared foods cooked under household conditions needs to be determined for risk estimates on humans. The mutagenic chemicals currently fall into 4 groups, quinoline, quinoxaline, pyridine, and the proposed 'oxygen-containing' compounds (Felton and Knize, 1990b). It may not be necessary to thoroughly investigate each individual mutagenic compound but members of each group should be thoroughly, evaluated for genotoxicity since mutagenic potencies of the known food mutagens differ depending on the test system used (Thompson et al., 1987). Acknowledgements The authors would like to thank Prof. Kjell Olsson and Dr. S. Grivas, Swedish University of Agricultural Sciences, Uppsala, Sweden for the samples of 4,7-, 5,7-, 5,8- and 7,8-DiMeIQx and 4,7,8- and 5,7,8-TriMeIQx. Work performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48 and supported by the National Institute of Environmental Health Sciences/National Toxicology Program under IAG NIEHS 222YO1-ES-10063 and NCI Grant 40811.

References

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