The Maillard Reaction in Foods and Nutrition - ACS Publications

29

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 25, 2013 | http://pubs.acs.org Publication Date: April 29, 1983 | doi: 10.1021/bk-1983-0215.ch029

Formation of Mutagens by the Maillard Reaction HIROHISA OMURA, NAZMA JAHAN, KAZUKI SHINOHARA, and HIROKI MURAKAMI Kyushu University, Food Chemistry Laboratory, Department of Food Science and Technology, Fukuoka, 812, Japan

Several weak mutagens were produced by Maillard reac tions of 20 different amino acids with sugars at 100 °C. Mutagenicity was studied by Ames test with Salmo nella typhimurium TA 100 and TA 98. Mutagenic a c t i v i ty varied with the nature of amino acids or sugars, the tester strains and the presence or absence of S9 mixture. The mode of mutagenic action was categorized into 5 types. Mutagenicity varied with pH values at the reaction and increased with prolongation of heat ing time. Rec-assay and test on pupal oocytes of s i l k worms also gave positive mutagenicity. Several prin cipal mutagens were identified as 5-hydroxymethyl fur fural, ε-(2-formyl-5-(hydroxymethyl)pyrrol-1-yl)norleucine and 2-methylthiazolidine. Products of triose reductone with amino acids or nucleic acid-related compounds also showed mutagenicity. It i s now w e l l known that mutagenic substances are produced by p y r o l y s i s of food or f o o d s t u f f s which contain p r o t e i n s , amino a c i d s and sugars (1). This f a c t a t t r a c t e d p u b l i c a t t e n t i o n i n view of the p o t e n t i a l mutagenicity and c a r c i n o g e n i c i t y of f o o d s . The o r i g i n a l p y r o l y s i s was c a r r i e d out at unusually high temper atures, over 200 ° C , although these temperatures are involved i n cooking

processes p a r t i c u l a r l y

such as r o a s t i n g

o r f r y i n g . Re

c e n t l y , the c o n t r i b u t i o n of food i n d u s t r i e s to the supply of processed foods has been e x t e n s i v e l y i n c r e a s i n g . T h e r e f o r e , mu tagen formation under milder c o n d i t i o n , at l e a s t below 100 ° C , needed i n v e s t i g a t i o n because of the p o s s i b l e r e l a t i o n to d a i l y life. Many of the r e a c t i o n s between f o o d s t u f f s take place under mild c o n d i t i o n s i n p r o c e s s i n g , s t o r a g e , and cooking of f o o d s . The M a i l l a r d r e a c t i o n appears to be the one to occur most common l y , accompanied not only by browning but a l s o by change of aroma, l o s s of n u t r i t i o n a l v a l u e , and development of a n t i o x i d a t i v e a c 0097-6156/83/0215-0537$07.75/0 © 1983 American Chemical Society

In The Maillard Reaction in Foods and Nutrition; Waller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


538

MAILLARD

REACTIONS

tivity. In t h i s r e a c t i o n , a number of intermediates such as reductones and osones are produced. Since these compounds are q u i t e r e a c t i v e and are expected to have some p h y s i o l o g i c a l e f f e c t s on l i v i n g organisms, we have i n v e s t i g a t e d t h e i r biochemical p r o p e r t i e s and confirmed s e v e r a l a c t i v i t i e s , i n c l u d i n g n u c l e i c a c i d - b r e a k i n g a b i l i t y and mutagenicity of t r i o s e reductone, i t s condensation products with amino a c i d s , and a s c o r b i c a c i d (2, _3, 40 . We then examined the formation of mutagens by M a i l l a r d r e a c tions . As a t y p i c a l model r e a c t i o n , mixtures of 1 M glucose and equimolar amounts of v a r i o u s amino acids were heated at 100 ° C f o r 10 hr under r e f l u x and proper a l i q u o t s of the browned s o l u t i o n were subjected to mutagenic t e s t s (5). By Ames t e s t on Salmonella typhimurium TA 100 and TA 98 with or without S9 mixt u r e , dose-dependent mutagenicity was detected i n s e v e r a l such r e a c t i o n mixtures, although i t was much lower than that of w e l l known chemical mutagens or p y r o l y s a t e s of foods (Table I ) . In a d d i t i o n , the mutagenicity v a r i e d with the amino a c i d s used, which were d i v i d e d t e n t a t i v e l y i n t o 5 c a t e g o r i e s , by c a l l i n g the r e s u l t p o s i t i v e when the number of r e v e r t a n t s formed i n the t e s t was over twice that i n the c o n t r o l (6). No mutagenic a c t i v i t y was detected with the browned s o l u t i o n of glucose and Trp on both s t r a i n s TA 100 and TA 98, with or without S9 mixture, while Trp alone was the most e f f e c t i v e among amino acids i n producing mutagenic products by p y r o l y s i s . Reaction mixtures of glucose and (Cys)2, T y r , Asp, A s n , or Glu were a l s o included i n t h i s group (group E ) . With products from glucose and other amino a c i d s , mutagenic a c t i v i t y was observed on TA 100 without S9 mixture. However, f o r most of them, the a c t i v i t y was decreased by S9 mixture. The r e a c t i o n mixture of glucose and Lys d i d not show any mutag e n i c i t y on TA 98 e i t h e r with or without S9 mixture. Similarly, no mutagenic a c t i v i t y on TA 98 was produced with L e u , S e r , T h r , Met, and Gin (group A ) . However, such a c t i v i t y on TA 98 was o b served with S9 mixture f o r r e a c t i o n mixtures of glucose and A r g , Gly, A l a , V a l , and l i e (group B ) . On the other hand, glucose and Cys products showed mutagenicity on both s t r a i n s , TA 100 and TA 98, without S9 mixture and the a c t i v i t y was enhanced by i t (group C). Reaction between glucose and Phe gave a product with a c t i v i ty on TA 100 without S9 mixture and even more with i t i n lower doses of the sample, but no a c t i v i t y was shown on TA 98 with and without S9 mixture, u n l i k e the case of Cys (group D ) . Instead of g l u c o s e , other sugars were examined f o r the format i o n of mutagens by the M a i l l a r d r e a c t i o n u s i n g a t y p i c a l amino a c i d from each of the 5 groups : L y s , A r g , Cys, Phe, and T r p . Several sugars such as f r u c t o s e , g a l a c t o s e , and xylose showed a b i l i t y to form mutagens with a l l amino a c i d s except T r p , though t h e i r a c t i v i t i e s v a r i e d depending on the sugars as w e l l as the amino a c i d s . The r e s u l t with Lys and sugars i s shown i n F i g . 1. Mutagen formation was then studied at v a r y i n g pH v a l u e s . Browned samples were prepared by heating glucose and L y s , A r g ,



275 530 203 192 199 195 223 153 139 119 208 645 683 200 129 134 369 166 196 163

Glysine Alanine Valine Leucine Isoleucine Serine Threonine Aspartic acid Asparagine Glutamic a c i d Glut amine Lysine Arginine Phenylalanine Tryptophan Tyrosine Cysteine Cystine Methionine Proline

*

; 4-Nitroquinoline

Control 4-NQO* 0.1 pg DEN** 70 jimol

0.1

230 123 218 336 246 262 104 140 162 89 455 152 676 281 118 186 30 134 340 214

-0.3

1-oxide,

129 1800 198

451 566 277 269 319 257 315 173 146 103 309 739 754 253 139 178 213 198 262 183

0.2

** ;

m

187 119 109 400 157 267 48 106 242 68 353 30 559 313 101 246 30 130 416

0.4

0.2

352

136

144 135 148 185 329 138 99 123 178 131 145 119 156 343 155 164 769 131 187 156

0.3

+ 186 134 168 194 332 121 165 144 194 108 147 106 145 260 153 186 396 120 253 214

0.4

Diethylnitrosamine.

188 130 134 133 165 138 150 167 240 224 161 141 164 103 86 91 160 164 165 153 183 138 252 297 218 235 372 365 141 153 15Q 142 763 1669 138 131 165 179 153 129

0.1

TA 100

35 30 26 33 48 15 41 34 27 47 51 15 17 25 24 33 77 27 42 27

0.1 20 18 6 17 19 13 9 30 39 0 39 20 0 11 7 17 48 22 11 35

-0.3

28 130 38

47 23 0 29 30 14 15 41 27 37 60 16 34 21 16 19 76 24 20 30

0.2 13 13 17 28 0 6 7 20 32 0 15 12 0 7 0 13 25 36 5 20

0.4 246 38 51 44 68 39 20 43 36 32 41 52 85 51 17 15 128 51 46 39

0.1

TA 98

MUTAGENICITY OF BROWNED SOLUTION FROM GLUCOSE AND AMINO ACID ON SALMONELLA

Strain S9 Mix. Dose, m l / p l a t e

Table I.

+

70

33

240 36 az 52 36 37 0 56 39 25 49 32 .az 36 9 19 159 82. 26 37

0.2


146 38 33 28 28 38 0 50 30 31 38 34 82 28 0 18 146 53 39 36

0.3

24 0 16 88. 50 49 34

42 24 21 0 38 0 47 31 36 33 39

_

0.4


540

MAILLARD

REACTIONS

Amount of Browning Mixture (ml/plate) Figure 1. Mutagenicity of the browned solutions from varying sugars and Lys. Key: A> fructose; Φ, glucose; O, galactose; A, mannose; arabinose; and M, xylose.



29.

OMURA

E TA L .

Formation

of Mutagens

by Maillard

Reaction

541

Cys or Phe at 100 °C f o r 10 h r at pH 2, 7, and 12. As an example, mutagen formation from glucose and Phe i s shown i n F i g . 2. A browning sample made at pH 7 showed the highest mutagenicity on TA 100, while on TA 98 strong a c t i v i t y was produced at pH 2, i . e . i n s t r o n g l y a c i d i c medium. On the other hand, mutagenic a c t i v i t y on TA 100 was the highest i n the r e a c t i o n mixture of glucose and Lys a t pH 2, whereas those were detected i n the case of glucose and Arg or Cys at pH 7. Furthermore, i t was observed that mutagens were formed p r o p o r t i o n a l l y to heating time and c o l o r a t i o n of the r e a c t i o n mixture of glucose and Lys or Phe. However, a c t i v i ty was sharply produced a f t e r p r e l i m i n a r y heating f o r a few hours of the sample of glucose with Arg or Cys ( F i g . 3 ) . Then, we have confirmed that the a c t i v i t i e s of the mutagens formed between g l u cose and amino a c i d s were decreased by the a c t i o n of s e v e r a l r e ducing substances; a s c o r b i c a c i d and t r i o s e reductone e x h i b i t e d the most s i g n i f i c a n t e f f e c t , while Cys, d i t h i o t h r e i t o l , or peni c i l l a m i n e had lower ones ( F i g . 4 ) . On the contrary, the browned mixture of glucose with Lys o r Phe depressed the a c t i v i t i e s of other mutagens such as n i t r o f u r a n and N - e t h y l - N - n i t r o - N - n i t r o s o guanidine (ENNG), although the sample i t s e l f contains some mutagenic a c t i v i t y ( F i g . 5 ) . By Rec-assay with B a c i l l u s s u b t i l i s H 17 Rec and M 45 Rec , p o s i t i v e a c t i v i t y was a l s o shown by r e a c t i o n mixtures of glucose and amino a c i d s , while the a c t i v i t y v a r i e d depending on amino aci d s , as shown i n Table I I . T h i s suggests that the browned s o l u t i o n s are able to i n j u r e b a c t e r i a l DNA. On the other hand, we have o f t e n demostrated that n u c l e i c a c i d s are degraded i n v i t r o by the a c t i o n of s e v e r a l carcinogens, mutagens, and v i r u s i n ducers as w e l l as antitumor substances by s t u d i e s with v i s c o s i metry, sucrose d e n s i t y gradient c e n t r i f u g a t i o n , and e l e c t r o p h o r e s i s (7). Many reductones brought about strand breaks o f DNA, e s p e c i a l l y i n the presence of C u ^ (2, 3^ 4_) . Therefore, DNA-breaking a b i l i t y of the browned s o l u t i o n s was i n v e s t i g a t e d by means of agarose-gel e l e c t r o p h o r e s i s , and such a c t i v i t y of the samples was observed ( F i g . 6 ) . Thus, i t can be assumed that mutagens formed by the M a i l l a r d r e a c t i o n may have DNA-breaking a c t i v i t y . Attempts were then made to i s o l a t e and i d e n t i f y the p r i n c i p a l mutagens i n the browned samples. Since p r o p e r t i e s of the mutagens vary with amino a c i d s , i t was supposed that those formed by the M a i l l a r d r e a c t i o n may d i f f e r . Therefore, mutagens were sought i n the t y p i c a l r e a c t i o n mixtures of glucose and Phe, Lys, and Cys. A f t e r d i a l y s i s of the browned s o l u t i o n against d i s t i l l ed water at 5 °C overnight, outer and inner f r a c t i o n s were separated and evaporated t o dryness i n vacuo. Only the outer p o r t i o n of the d i a l y s a t e showed both mutagenicity on TA 100 and DNA-breaking a c t i v i t y ( F i g . 7 ) . This observation i n d i c a t e s that the mutagens are of low molecular weight, probably intermediates i n the M a i l l a r d r e a c t i o n between glucose and amino a c i d s . The outer p o r t i o n of the browned s o l u t i o n of glucose and Phe was evaporated t o dryness and the residue extracted with absolute f

+

+



Figure 2.

Effect of pH value on the mutagenic action of the browning mixture of Glc and Phe on TA 100 and TA 98. Key: O , with S9 mix; Φ, without S9 mix.


C/3

δ

H

w > ο

> g

>

to


OMURA E T AL.

Formation

Heating Time (hr)

of Mutagens

by Maillard

Reaction

Heating Time fhr)


543


544

MAILLARD

REACTIONS

C o n c e n t r a t i o n o f the Reducing Agent (mM) Figure 4. Effect of reducing agents on the mutagenic action of the browning mixture of Glc and Lys. Key: · , cysteine; O , ^-acetylcysteine; A , penicillamine; A, dithiothreitol; M, triose reductone; and ascorbic acid.



29.

OMURA E T A L .

Formation

01

of Mutagens by Maillard

0-2

Amounts of Browning Mixture

Reaction

545

0-3 (ml/plate)

Figure 5. Effect of browning solutions on mutagenic action of N-ethyl-N'-nitroN-nitrosoguanidine (ENNG) on TA 100. Key: · , browned sample from Glc and Phe; O , ENNG and browned sample from Glc and Phe; A , browned sample from Glc and Lys; and Δ , ENNG and browned sample from Glc and Lys.



Glutamine

Glutamic a c i d

Asparagine

Aspartic acid

Threonine

Serine

Isoleucine

Leucine

Valine

Alanine

Glycine

980 780 330 415 260 170 136 904 565 608 318 350 245 352 155 148 74 37 715 532 359 179 90 45 480 110

Dose (mg)

3 3 0 1 0 1 0 1 0 5 0 1 0 10 0 2 1 0 6 0 8 8 2 0 2 0

+

8 6 0 4 0 2 0 4 0 2 0 2 0 15 0 5 2 0 10 0 15 13 4 0 3 0 5 3 0 3 0 1 0 3 0 3 0 1 0 5 0 3 1 0 4 0 7 5 2 0 1 0

I n h i b i t i o n zone (mm) Rec Rec" D i f f e r (H-17)(M-45) ence

4-Nitroquinoline-1oxide

Histidine

Cystine Methionine Proline

Cysteine

Tyrosine Tryptophan

Phenylalanine

Arginine

Lysine

Amino a c i d + (Glucose)

760 525 106 354 266 222 58 530 260 980 280 68 251 188 46 375 115 920 744 239 205 170 60 40 20 10 4

Dose (mg)

REC-ASSAY OF BROWNED SUBSTANCE FROM GLUCOSE AND AMINO ACIDS

Amino a c i d + (Glucose)

Table II.

5 4 0 3 2 1 0 2 0 0 2 0 4 3 0 0 0 10 0 3 1 1 0 3 4 0 0

+

12 9 0 10 7 5 0 5 0 0 5 0 10 7 0 0 0 13 0 10 4 2 0 14 6 4 2

7 5 0 7 5 4 0 3 0 0 3 0 6 4 0 0 0 3 0 7 3 1 0 11 2 4 2

I n h i b i t i o n zone (mm) Rec Rec" D i f f e r (H-17)(M-45) ence



29.

OMURA E T A L .

Formation

of Mutagens

400

by Maillard

547

Reaction

*400h

tî 200

»200

-J-

3 10 25 50 100 Amount of D i a l y s a t e F r a c t i o n i_j ι (mg/pj.ate) 0 2 4

12.5

Amount of Retentate (mg/plate)

12.5

25

50 ,

Amount of Brownings(mg/plate)

25

Fraction

Figure 7. Dose-response curve of mutagenic effect of dialyzed fractions from browning mixture of Glc and Lys after heating at 100 °C for 10 h. (Assay with Salmonella t y p h i m u r i u m TA 100 with or without S9 mixture.) Key: ·, dialysate; O, fraction; A , retentate; and Δ , fraction.

Anerican Chemical Society Library 1155 m St. N. w. In The Maillard Reaction in Foods and Nutrition; Waller, G., et al.;

Washington. 0. C. Society: 20038Washington, DC, 1983. ACS Symposium Series; American Chemical


548

MAILLARD

REACTIONS

ethanol. From the s o l u b l e p o r t i o n , the mutagenic substance was p u r i f i e d by successive chromatographic separations on a DEAE-cell u l o s e column, a Dowex-50 column ( F i g . 8), and s i l i c a g e l i n t h i n l a y e r . On the b a s i s of p o s i t i v e r e a c t i o n with 2 , 4 - d i n i t r o p h e n y l hydrazine, Rf value ( F i g . 9 ) , and UV, NMR ( F i g . 10) and mass ( F i g . 11) spectrometries, the mutagen was i d e n t i f i e d as 5-hydroxymethylf u r f u r a l (HMF) by comparing r e s u l t s with those obtained with aut h e n t i c HMF. In a d d i t i o n , i t was found that authentic HMF has mutagenic and DNA-breaking a c t i v i t y and that i t s mutagenic a c t i v i t y c o i n c i d e d with that of the mutagen i s o l a t e d ( F i g . 12). Thus, i t can be concluded that the p r i n c i p a l mutagen formed by the M a i l l a r d r e a c t i o n between glucose and Phe i s HMF. The mutagen i n the browned mixture of glucose and Lys was also examined. From the e t h a n o l - s o l u b l e p o r t i o n of the d i a l y s a t e f r a c t i o n , the mutagen was i s o l a t e d by successive chromatographic separations on a DEAE-cellulose column, a CM-cellulose column ( F i g . 13), and t h i n - l a y e r s i l i c a . From Rf value, p o s i t i v e r e a c t i o n s with 2,4-dinitrophenylhydrazine and n i n h y d r i n reagents ( F i g . 14), NMR ( F i g . 15), and UV spectrum having a shoulder at 265 nm and a peak at 297 nm i n water, i t was suggested to be r e l a t e d to p y r r o l e 2-carbaldehyde as reported by Nakayama, et a l . (8)· They i s o l a t e d the intermediate formed by the M a i l l a r d r e a c t i o n between glucose and Lys and proved i t to be £-(2-formyl-5-(hydroxymethyl)pyrrol-1y l ) n o r l e u c i n e . By comparing the above p r o p e r t i e s and the mutageni c i t y of Nakayama s standard sample with those of ours, i t was confirmed that the samples were the same. Furthermore, t h i n - l a y e r chromatography of the sample gave another p o s i t i v e spot with 2,4d i n i t r o p h e n y l h y d r a z i n e ; t h i s was found to be mutagenically a c t i v e and to agree i n p r o p e r t i e s with HMF. Thus, i t was at l e a s t suggested that the main mutagens formed by the M a i l l a r d r e a c t i o n between glucose and Lys are a p y r r o l e compound, probably £-(2-formyl5 - h y d r o x y m e t h y l ) p y r r o l - l - y l ) n o r l e u c i n e and HMF. 1

F i n a l l y , i s o l a t i o n of the mutagen i n the browned s o l u t i o n of glucose and Cys was c a r r i e d out. From pyrolyzed Cys or (Cys)2, Fujimaki et a l . (9) i d e n t i f i e d 7-8 v o l a t i l e compounds i n c l u d i n g 2 - m e t h y l t h i a z o l i d i n e . Mihara and Shibamoto (10) separated the browning mixture of glucose and cysteamine i n t o 11 f r a c t i o n s having some mutagenicity at c e r t a i n concentrations, 7 v o l a t i l e f r a c t i o n s from the methylene c h l o r i d e e x t r a c t by high-pressure l i q u i d chromatography and 4 from the r e s i d u a l aqueous s o l u t i o n by i o n exchange chromatography. Among them, t h i a z o l i d i n e and 2-methylt h i a z o l i d i n e were found i n the former and 2-(l,2,3,4,5-pentahyd r o x y - n - p e n t y l ) t h i a z o l i d i n e i n the l a t t e r . Since i t was supposed that a s i m i l a r r e a c t i o n may occur when Cys i s employed i n s t e a d of cysteamine, mutagens were surveyed as f o l l o w s . The d i a l y z e d outer part of the dark brown r e a c t i o n mixture from glucose and Cys was d i s t i l l e d i n vacuo to about h a l f i t s volume, and the d i s t i l l a t e was shaken with twice i t s volume of methylene c h l o r i d e . The methlene c h l o r i d e l a y e r was evaporated at room temperature i n vacuo to remove the solvent and the r e s i d u a l aqueous phase was d r i e d over



OMURA

Formation

E TA L .

of Mutagens

by Maillard

Reaction

1.6

0.8

3.3

20

40

60

F r a c t i o n No.

0.1

0.3

0.5

Amounts of Sample (ml/plate)

Figure 8. Chromatograms on DEAE-cellulose column and Dow ex 50 column of browned mixture from Glc and Phe and mutagenicity of fractions eluted.


549


550

MAILLARD

REACTIONS

Rf

0

0.8

4

0

(->

0.6

0*0

0.4

(-)

(+)

0.2

(2)2,4-Dinitriphenylhydrazine pos i t i v e

I HMF Figure 9.

I

(-)

DNA break

TLC of samples from Glc and Phe. Solvent: chloroform-methanol (50:1).



29.

OMURA

Figure 10.

ET AL.

Formation


Reaction

551

Comparison of NMR spectra of (A) the browned sample from Glc and Phe and (B) 5-hydroxy met hy If urfural (HMF).



552

MAILLARD

REACTIONS

Tl3c HMF

20

α α • cd

h 15

a S" > •H 4J • cd

iH .

l«i

nli J

Il

I'" Sample

1*6

290 rvz 1b

Ο

§ 3

h 5

11

se Figure 11.

ibe

r '"'""'r » 2 9 0 Π/2

Comparison of mass spectra of HMF (top) and the browned sample from Glc and Phe (bottom).



29.

OMURA E T AL.

Formation

of Mutagens

by Maillard

Reaction

900 r

Amounts of Sample (pg/plate) Figure 12. Comparative mutagenicities of the browned sample from Glc and Phe, and HMF. Key: A, HMF without S9; Δ, HMF with S9; ·, sample without S9; and O , sample with S9.


553


Figure 13.

Chromatograms on DEAE-cellulose column and CM-cellulose column of the browned sample from Glc and Lys, and mutagenicity of fractions.


M

C/3

δ

Ο Η

>

>

r

>


OMURA E T AL.

Formation

of Mutagens

by Maillard

Reaction

555

Rf

0.75 (5)2,4-Dinitrophenylhydrazine

positive

0.50 (4)Ninhydrin

positive

Ο

0

0.25

HMF

Θ

Sample

Standard

Figure 14. TLC of mutagenic fraction (peak III in figure 13) of the browned sample from Glc and Lys. Solvent: n-BuOH-acetic acid-water (4:1:1).



556

MAILLARD

REACTIONS

anhydrous MgSO^. The r e s u l t a n t o i l y m a t e r i a l was then d i s s o l v e d i n ether and subjected to gas chromatography. Retention time o f the major peak was the same with that of authentic 2-methylthiaz o l i d i n e ( F i g . 16). Furthermore, GC/MS data f o r the main peak co i n c i d e d with that of the a u t h e n t i c compound ( F i g . 17). In addi t i o n , they both showed mutagenicity on TA 100 and TA 98 i n the presence or absence of S9 mixture, j u s t as d i d the browned mixture from glucose and Cys ( F i g . 18). Thus, at l e a s t one o f the v o l a t i l e mutagenic substances formed by the M a i l l a r d r e a c t i o n between glucose and Cys i s 2 - m e t h y l t h i a z o l i d i n e . To t e s t mutagenicity on the animal l e v e l , we used silkworm oocytes (11). Browned s o l u t i o n s were i n j e c t e d i n t o the body cav i t y of the female at mid-pupal stage. Mutagenicity was detected as pink o r red eggs among dark colored wild-type ones. Mutagen i c i t y of the browned r e a c t i o n mixtures between glucose and amino acids was observed on pupal oocytes of the silkworm, but at low er rates than those produced by a t y p i c a l mutagen, mitomycin C, as shown i n Table I I I . T r i o s e reductone c o n t r i b u t e s more r e a d i l y to the browning r e a c t i o n with amino acids than sugars do (12). This r e a c t i o n i s a s p e c i a l one i n the M a i l l a r d r e a c t i o n and i s r e f e r r e d as the "Reductone-Amino Reaction . Amino reductones o r enaminol com pounds are f i r s t produced as the intermediate during the browning by r e a c t i o n of the OH group i n t r i o s e reductone with the NH2 group of amino a c i d s . We have i s o l a t e d such amino reductones from reac t i o n mixtures of t r i o s e reductone and Gly, A l a , Leu, Met, Phe, Trp and l i e , and confirmed that some of them are mutagenic on TA 100 ( F i g . 19). On the other hand, browning reactions o f t r i o s e reduc tone with s e v e r a l n u c l e i c a c i d - r e l a t e d compounds were a l s o ob served (13). Two types of r e d u c t i v e intermediates, l i n e a r and t r i c y c l i c forms, were i s o l a t e d ( F i g . 20). The r e a c t i o n of t r i o s e reductone with guanine produced a brown t r i c y c l i c compound, 1,N^(2-hydroxypropenylidene)guanine ( c y c l i c TR-Gua) and a l a b i l e y e l low intermediate, N^-(3-oxo-2-hydroxypropenyl)guanine (TR-Gua), whereas such r e a c t i o n with guanosine, 2 ( 3 ) - o r 5 - g u a n y l i c a c i d gave N^-(3-oxo-2-hydroxypropenyl)guanosine (TR-Guo), Ν -(3-oxo-2hydroxypropenyl>2 (3 )-guanylic acid (TR-2 (3 )-GMP) o r N -(3-oxo2-hydroxyprpenyl)-5'-guanylic a c i d (TR-5 -GMP), r e s p e c t i v e l y (14). These intermediates are a k i n d of amino reductones too and showed evident mutagenicity on TA 100 without S9 mixture ( F i g . 21), but not on TA 98 on B a c i l l u s s u b t i l i s . H 17 Rec " and M 45 Rec", used f o r Rec-assay (15). Thus, by the M a i l l a r d r e a c t i o n i n d i f f e r e n t browning systems of sugars and amino compounds, some mutagenic substances were formed, although t h e i r a c t i v i t i e s are quite weak compared with those formed by p y r o l y s i s o f amino a c i d s . They were confirmed as intermediates and some o f them were i d e n t i f i e d as furan, p y r r o l e , or t h i a z o l i d i n e d e r i v a t i v e s formed from glucose and amino acids 11

f

1

f

T

T

T

1

f

4


2


OMURA E T A L .

Formation of Mutagens

6

Figure 15.

5

4

(6)

by Maillard

3

Reaction

2

NMR spectrum of browned sample from Glc and Lys.

CM, CM

1

/ A S

NM

Htc—cm

3

3 min

I

3

5 min

Retention Time Figure 16.

Comparison of gas chromatograms of 2-methylthiazolidine and volatile sample from Glc and Cys.


558

MAILLARD

REACTIONS

A

U,


S

NH

—1

ι

1 i

I l 1 1 1 1 I ι ι ι ι ι ι ι .1 1 1 1 1 1 1 1 1 1 1 1

K r>> os *

»2

o —

n r» to

1 1 1 I 1 · · 1 1 1 1 1 1 ·

1 1 1 1

• 1 1

— r-œo> ο ΙΛ r- M

.

Sample

.9 Ε 9 £ 9

Figure 17.

3 ^ ^^

*r»^f*CD»©V/>*-

Ν« 3

Comparison of mass spectra of 2-methylthiazolidine and volatile sample from Glc and Cys.



OMURA ET AL.

Formation

of Mutagens

by Maillard

Reaction

559

Figure 18. Comparison of mutagenicity of 2-methylthiazolidine and volatile sample from Glc and Cys. Key: O , sample with S9; · , sample without S9; Δ , 2-methylthiazolidine with S9; and A, 2-methylthiazolidine without S9.



560

MAILLARD

REACTIONS

Table I I I . MUTAGENICITY OF BROWNED SOLUTIONS FROM GLUCOSE AND AMINO ACIDS ON EGGS OF SILKWORM Amino a c i d

Glycine Alanine Valine Isoleucine Serine Threonine Asparagine Glutamic a c i d Glutamine Lysine Arginine Phenylalanine Tyrosine Tryptophan Cysteine Cystine Methionine Histidine

26 17 16 24 22 12 24 22 18 22 26 28 24 20 31 22 31 28

10,889 6,010 7,015 8,728 6,872 5,167 9,944 7,748 8,140 8,982 9,229 11,361 9,544 8,188 14,415 9,624 11,089 11,173

No. of mutants detected 4 3 2 7 5 7 7 3 7 10 6 10 6 3 11 9 6 9

H0 ^ MNNG , 0.01 /ig Mitomycin C, 6 μg

43 13 10

17,352 5,889 3,146

2 3 617

2

No. of No. of eggs moths observed

* N-Methyl-N'-nitro-N-nitrosoguanidine. sample was i n j e c t e d i n t o a pupa.

Mut. frequency (xlO""^) 3.4 4.7 3.2 10.7 6.9 14.8 6.8 2.8 11.7 10.8 6.1 9.0 6.8 3.6 8.1 10.0 6.8 7.8

( o -•7 .7) ( o -10 • .0) ( o - 8 .1) (6.5--14 .9) ( o -14 • .1) ( o -36 .8) (0.9 -12 .7) ( o - 7 .7) (4.3 -19 .2) (4.6 -16 .9) (0.3 -11 .9) (3.8 -14 .3) (1.5 -12 .1) ( o - 7 .7) (3.5 -12 .7) (1.8 -18 .2) (1.0 -12 .6) (2.6 -13 .0)

1.2 ( o - 2 .8) 7.2 ( 0 -16 .2) 2511.3a, 695-3,327)

An a l i q u o t of 10 μΐ of


29.

OMURA

ET AL.

Formation

of Mutagens

by Maillard

Reaction

561

HC-OH

I

C-OH

I Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on September 25, 2013 | http://pubs.acs.org Publication Date: April 29, 1983 | doi: 10.1021/bk-1983-0215.ch029

HC=0

HC-OH

c-oH

Ο

Ο

3'HC-O

r S ' \ .

H

H N ^ C '

l'HC-HN^

C

X

N^

C

V

-

N

N

Η II

H 0 - C

C

S

N '

I H

C

I

rTr

N

C '

N

\

I

c >

5

\

hr V

H

2-»P''«

m e n l s

c

4

u

III

HC—Ο

Ο

" O H

•

HC —OH

+

3" HC—Ο ί

C*.

Η Ν " NK N

\ Ν

/

H

_ J 1 Ç ] _

a t room

.

Ç - O H ™

Ο f

V

H g

,,,,, 1 . . k . ^ C ^ . ^ C vw. / Γ' C — H N ^

I

(IV) R = /?-D-ribofuranosyl (V) R = 2'(3')-ribotide (VI) R = 5'-ribotide Figure 19.

Reaction between triose reductone and nucleic acid-related bases.



MAILLARD

REACTIONS

Concentration of T R - G l y ( m M ) Figure 20.

Figure 21.

Mutagenicity of the product from triose reductone and Gly.

Mutagenic effects of cyclic TR-Gua (A) or TR-Gua (B) in the presence ofCu on TA 100 or TA 98. 2+


29.

OMURA E T AL.

Formation


Reaction

563


and as combined amino reductones formed from t r i o s e reductone and amino a c i d s or other bases. In a d d i t i o n , i t was found that the mutagens have DNA-breaking a c t i v i t y i n v i t r o . Furthermore, i t i s expected that other mutagens w i l l be s t u d i e d i n s e v e r a l M a i l l a r d reaction mixtures. Acknowledgements The s t u d i e s were supported by a G r a n t - i n - A i d f o r S c i e n t i f i c Research from the Japanease M i n i s t r y of E d u c a t i o n , Science and Culture. The authors thank Dr. E . Kuwano f o r g i v i n g much advice i n i d e n t i f i c a t i o n of the mutagens and are g r a t e f u l to P r o f e s s o r H. Kato of Tokyo U n i v e r s i t y f o r the g i f t of standard sample of £ - (2-f o r m y l - 5 - ( h y d r o x y m e t h y l ) p y r r o l - l - y l ) - n o r l e u c i n e .

Literature Cited

1. Sugimura, T.; Kawachi, T.; Nagao. M.; Yahagi, T.; Seino, Y.; Okamoto, T . ; Shudo, K . ; Kosuge, T . ; Tsuji, K . ; Wakabayashi, K.; Iitaka, Α . ; I t a i , A. Proc. Japan. Acad. 1977, 53, 58. 2. Shinohara, K . ; Fukumoto, Y . ; Tseng, Y - K . ; Inoue, Y.; Omura, H. J . Agr. Chem. Soc. Japan 1974, 48, 499. 3. Omura, H . ; Iiyama, S.; Narazaki, Y . ; Shinohara, K . ; Murakami, H. J . Nutr. S c i . Vitaminol. 1975, 21, 237. 4. Omura, H . ; Tomita, Y . ; F u j i k i , H . ; Shinohara, K . ; Murakami, H. J . Nutr. S c i . Vitaminol. 1978, 24, 263. 5. Shinohara, K . ; Wu, R-T.; Jahan, N . ; Tanaka, M . ; Morinaga, N.; Murakami, H . ; Omura, H. Agric. B i o l . Chem. 1980, 44, 671. 6. Seiler, J . P . ; Mattern, I . E . ; Gree, M.H.L.; Anderson, D. Mutation Res. 1980, 74, 71. 7. Yamafuji, K. "Food, Cancer and Cytodifferentiation" (40th Anniversary of Prof. K. Yamafuji's research), Shukosha Co. Ltd., Fukuoka, 1970. 8. Nakayama, T . ; Hayase, F . ; Kato, H. Agric. B i o l . Chem. 1980, 44, 1201. 9. Fujimaki, M.; Kato, H . ; Kurata, T. Agric. B i o l . Chem. 1969, 33, 1144. 10. Mihara, S.; Shibamoto, T. J. Agric. Food Chem. 1980, 28, 62. 11. Yamashita, Ν . , Shinohara, K . ; Jahan, N . ; Torikai, Y . ; Doira, H . ; Omura, H.J. Japan. Soc. Food Nutr. 1981, 34, 367. 12. Shinohara, K . ; Tseng, Y - K . ; Inoue, Y . ; Sato, M . ; Omura, H. S c i . B u l l . Fac. Agr., Kyushu Univ. 1974, 28, 139. 13. Lee, J - H . ; Shinohara, K . ; Murakami, H . ; Omura, H. J. Agr. Chem. Soc. Japan 1978, 52, 11. 14. Lee, J - H . ; Shinohara, K . ; Murakami, H . ; Omura, H. Agric. B i o l . Chem. 1979, 43, 279. 15. Shinohara, K . ; Lee, J - H . ; Tanaka, M.; Murakami, H . ; Omura,H. Agric. B i o l . Chem. 1980, 44, 1737. RECEIVED November 17, 1982


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