Genotoxic activity of caramel on Salmonella and cultured mammalian cells

Mutation Research, 139 (1984) 161-165

161

Elsevier MRLett 0530

Genotoxic activity of caramel on Salmonella and cultured mammalian cells Y i n g - n i a n Y u 1, X i n g - r u o

C h e n 1, C h e n D i n g 1, Z h u - n a n

Cai 1 and Qian-gao

Li E

~Department of Pathophysiology, Zhejiang Medical University, Hangzhou (People's Republic of China) and 2Zhejiang College of Traditional Chinese Medicine, Hangzhou (People's Republic of China)

(Accepted 23 November 1983)

Summary The genetic activity of 2 commercial caramel preparations, manufactured either by heating the malt sugar solution directly (non-ammoniated caramel) or by heating it with ammonia (ammoniated caramel) was studied in the Salmonella mutagenicity test and UDS assay in cultured mammalian ceils. The nonammoniated caramel was found to be mutagenic to S. typhimurium TA100, while the ammoniated one was genetically active in all the tester strains used, namely TA100, TA97 and TA98. It was also demonstrated that non-ammoniated caramel was capable of inducing UDS in cultured human amnion FL cells, but for the ammoniated one, no such activity was observed. Furthermore, based on the results obtained in the DNA synthesis inhibition assay, it was suggested that the DNA synthesis inhibition seen in our experiments with the ammoniated caramel was probably not of DNA damage in origin. These data indicate that the mutagenic fractions formed during ammoniated and non-ammoniated caramelization were quite different.

Caramel, a highly viscous material with a very deep brown hue, is formed after heating pentose or hexose sugar in the absence of water or in concentrated solutions. It is widely used as a brown colouring material in soy bean sauce, a daily condiment in China, Japan and other countries in FarEast Asia, as well as in food products such as beverages, beer and meat products. Because of its wide use in food products and cooking, the genotoxic action of caramel naturally draws great interest and particular concern in food safety control. In this paper, we present our results obtained in both prokaryotic and eukaryotic systems on the genotoxic activities of two Chinese-made caramel preparations. 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.

Materials and methods Materials

Two commercially used caramel preparations were assayed. They were manufactured by heating the malt sugar solution directly (as nonammoniated caramel) or by heating it with ammonia (as ammoniated caramel). Mutagenesis assay

The mutagenic activity was assayed with his(idine-requiring strains of Salmonella typhimurium by the Ames' standard plate-incorporation method (Maron and Ames, 1983). Tester strains of TAI00, TA97 and TA98, kindly provided by B.N. Ames

162 were used. Caramel preparations, diluted 1:1 (v/v) with double distilled water, were added to the plates with a volume of 0.1-0.4 ml. The results were defined as positive, if the number of revertants/dish on the test plates approximated or exceeded twice the number of spontaneous revertants/dish on the control plates, and a concentration-response relationship could be confirmed as well. UDS assay in cultured mammalian cells The method used was one modified recently in the authors' laboratory reported in detail elsewhere (Yu et al., 1983). Briefly, the human amnion cell line FL proliferated in the Eagle's Minimal Essential Medium (supplemented with 15 070 bovine calf serum) was prelabelled with 14CTdR (0.01 /~Ci/ml, 54 mCi/mmole) for 72 h and then synchronized by the combined treatment of arginine starvation with arginine-deficient MEM medium supplemented with 0.5°70 bovine calf serum and hydroxyurea (HU, 10 ttmoles/ml). Cells thus prepared were exposed to the test caramels for 5 h in the presence of 3H-TdR (2 #Ci/ml, 34 Ci/mmole), and the HU-resistant-incorporation of radioactive label into the acid insoluble fraction of the target ceils was measured by liquid scintillation counting. As the cells had previously incorporated laC-TdR into their parent DNA, the final counts recorded as the ratio of 3H/14C radioactivities was a measure of the specific activity of the 3H, or the level o f UDS. The weighed test caramel was dissolved directly in the arginine-deficient medium. In testing the influence of the $9 preparation on the UDS-inducing activity of the caramels, incorporated in the medium was also the $9 preparation prepared from livers from polychlorinated biphenyl-induced rats (Maron and Ames, 1983) (4°70 v/v in final concentration) and cofactors composed at a final concentration of Mg 2 ÷ (3 /~moles/ml), HPO42- (20 /zmoles/ml), glucose 6-phosphate (1 #mole/ml) and N A D P (0.8 #moles/ ml). A positive result was obtained when a concentration-response relationship could be established in addition to a reproducibly-induced significant increase in the HU-resistant 3H-TdR incorporation.

DNA synthesis inhibition test The test system used was one developed by Painter (1981). H u m a n amnion FL cells prelabelled with lnC-TdR as described above was exposed to caramel for 1-3 h. At the moment of the removal of the test compound and after an incubation in the test substance-free medium (Eagle's MEM medium supplemented with 15070 of bovine calf serum) for 0.5 and 1.5 h following the removal of caramel, the DNA synthesis rates were determined by a 20-min-pulse labelling with 3H-TdR (4 /xCi/ml, 34 Ci/mmole). The 3H/14C ratios, the measures of the rate of DNA synthesis for caramel-treated cells, were expressed as percentage of the control incorporation into DNA as a function of time of incubation by the end of the treatment. The DNA synthesis inhibition observed was defined as DNA-damage in origin, if the rate of DNA synthesis continued to decrease after the retreat of the cells from exposure to the test substances.

Results and discussion

All of the assays have been performed at least twice, and the results obtained demonstrate high reproducibility. The data presented are the representative ones. Shown in Tables 1 and 2, respectively, are the numbers of histidine-independent (revertant) colonies in the S. typhimurium tester strains and HUresistant unscheduled 3H-TdR incorporations in cultured FL cells exposed to non-ammoniated and ammoniated caramel, and their concentrationresponse curves are plotted in Figs. 1 and 2 respectively. It has been concluded that at a concentration of 0.20 ml/dish, the non-ammoniated caramel is mutagenic to S. typhimurium TAI00 only, while the ammoniated one is genetically active in all the tester strains TA100, TA97 and TA98, and the active concentration ranges are 0.10-0.20 ml/dish, 0.05-0.20 ml/dish and 0.025-0.10 ml/dish respectively. It has also been demonstrated that at a concentration of 0.60-1.0 mg/ml, non-ammoniated caramel is capable of inducing UDS in the cultured

163 TABLE 1

human

RESPONSE OF S. typhirnurium TESTER STRAINS TO NON-AMMON1ATED AND AMMONIATED CARAMEL IN STANDARD PLATE-INCORPORATION ASSAY

the medium

But concerning the ammoniated

one, no UDS in-

Sample

Mean His + revertants per plate

d u c i n g a c t i v i t y is d e m o n s t r a t e d

w h e t h e r t h e $ 9 is

TAI~

Amount per plate (ml)

a m n i o n F L cells a n d t h e a d d i t i o n o f $ 9 t o could significantly reduce the UDS-

inducing activity of the non-ammoniated

caramel.

present or not. These data on the genetic activity of

TA97

TA98

the non-ammoniated

caramel are in good agree-

Solvent control

162

173

32

ment with those reported by Kawachi (1979) of the

Non-ammoniated 0.05 caramel 0.10 0.15 0.20

199 182 192 200

33 31 29

m u t a g e n i c a c t i v i t y i n S. typhimurium T A 1 0 0 o f t h e

256 281 300

Ammoniated caramel

MNNG

0.0125 0.025 0.05 0.10 0.15 0.20 1 10

NaN3

1.5

371 388 386

342 422 446 457

caramelized glucose, arabinose, bitol and

those

clastogenic 55 60 75 77

arabinose,

activity glucose,

cultured

CHO

fructose and sor-

b y S t i c h et al.

cells.

of

maltose As

(1981b) of the

caramelized and

regards

sucrose,

fructose

in

ammoniated

c a r a m e l , b e s i d e s T A 1 0 0 , it is a l s o a c t i v e i n T A 9 8 a n d T A 9 7 . It is t h e f i r s t t i m e t h a t it h a s b e e n s h o w n t h a t a c a r a m e l p r e p a r a t i o n is a b l e t o i n d u c e f r a m e -

#g /zg

462 > 4000

shift mutagenesis in a bacterial mutagenic

#g

> 1210

ammoniated

On

the

other

hand,

counterpart,

in

contrast

to

its

assay. non-

it g i v e s a n e g a t i v e r e s u l t

TABLE 2 UNSCHEDULED 3H-TdR UPTAKE IN THE ACID-INSOLUBLE FRACTION OF 14C-TdR-PRELABELLED AND SYNCHRONIZED HUMAN AMNION FL CELLS EXPOSED TO THE NON-AMMONIATED AND AMMONIATED CARAMELS IN VITRO Sample

Concentration (mg/ml)

Solvent control Non-ammoniated caramel

0.01 0.10 0.12 0.50 0.60 1.00 1.20 5.00

MMS

10 -4 M

Cyclophosphamide

10 -5 M

Solvent control

Relative 3H/14C ratio

- $9

-

1.00 +_0.07

1.00 +_0.05

1.00 + 0.04

1.11 +0.02*

0.86+0.08*

1.47 +_0.12"*

1.00 +_0.03*

1.19+_0.04" 1.23 +_0.09*

1.01 +_0.05* 0.93 +_0.14"

$9

$9

0.98+__0.14' 1.13+0.05" 1.22+_0.12" 1.28 + 0.03** 1.05 + 0.07* 2.00+_0.11"** 1.48+-0.12"* 1.00 +-0.05

1.00 + 0.02 1.05 + 0.13" 1.19+_0.09" 1.08+0.03' 0.80+_0.11"

Ammoniated caramel

0.01 0.10 1.00 10.00

1.05 _+0.03* 0.98+_0.01" 1.12+_0.13" 0.53 +0.15 a

MMS

10 .3 M

4.35+0.31"**

Cyclophosphamide

10 -5 M

M + S.E. * p>0.05, ** p<0.05, *** p<0.001. a Harmful to the target cells.

+

1.46+0.11"*

164

6°fs

TA 915

10C

4O

20,

"6

i

0~

O TA 97 .__.O_._----- - - 4

~o o

2O0

/ks i

*~ 100 ,

0

f

/-

< i

U-3Y-

-G

Tkme after" the end of trectment (h) TA 100

Fig. 3. Inhibition of DNA synthesis in FL cells by ammoniated caramel (10 mg/ml, O, o), methyl methanesulfonate (0.5 x 10 -3 M, zx, A) and hydroxyurea (3 × 10 -3 M, o, m). The target cells were incubated with the chemicals for 1 h (open symbols) or 3 h (closed symbols).

.

g

Y_

200

IO0

~'o

0

loo

~o

2oo

Test compounds ( ~1/ plate)

Fig. 1. Concentration-response effects of non-ammoniated (open symbols) and ammoniated (closed symbols) caramel with S. typhimurium TA100, TA97 and TA98 in the absence of $9. Spontaneous revertants have been subtracted. in U D S assay in m a m m a l i a n ceils, a n d is thus cont r a d i c t o r y to the r e p o r t b y Stich et al. (1981b), which d e m o n s t r a t e d t h a t a c a r a m e l p o w d e r prepared commercially by heating a sugar-amm o n i u m s o l u t i o n (not the s a m e p r e p a r a t i o n used in this s t u d y b u t a p r o d u c t o f h e a t e d a m m o n i a - s u g a r solution) also e x h i b i t e d clastogenic activity in

50

~

L

001

010 1 O0 Test compounds (rag/ rql)

~000

Fig. 2. Mean relative unscheduled 3H-TdR incorporation of FL cells expressed as percentages of mean control incorporation ( - - - ) vs. concentration of non-ammoniated (open symbols) and ammoniated (closed symbols) caramel in the absence (circular symbols) or presence (triangular symbols) of $9. Each data point represents the mean of triplicate samples.

c u l t u r e d C H O cells. T h e d i f f e r e n t reactions o f S. typhimurium tester strains to the n o n - a m m o n i a t e d a n d a m m o n i a t e d c a r a m e l s used in this s t u d y as well as the d i f f e r e n t b e h a v i o r in U D S i n d u c t i o n in c u l t u r e d m a m m a l i a n cells e x p o s e d to them, s t r o n g ly suggest t h a t the m u t a g e n i c f r a c t i o n s f o r m e d d u r ing a m m o n i a t e d (with a m m o n i a ) a n d n o n a m m o n i a t e d c a r a m e l i z a t i o n processes are quite different. T h e D N A synthesis i n h i b i t i o n test is p e r f o r m e d in o r d e r to rule out the p o s s i b i l i t y o f the cop r e s e n t a t i o n o f D N A - r e p a i r inhibitor(s) with the D N A - d a m a g i n g c o m p o n e n t in the a m m o n i a t e d c a r a m e l , or the D N A d a m a g e ( s ) p r o d u c e d b y a m m o n i a t e d c a r a m e l being m a i n l y i n t e r c a l a t i o n which m i g h t give a f r a m e - s h i f t m u t a g e n e s i s in b a c t e r i a l m u t a g e n i c assays a n d / o r negative results in U D S assay. T h e results are s h o w n in Fig. 3. A s seen f r o m the curve, the D N A synthesis rate o f the t a r g e t cells increases i m m e d i a t e l y , as s o o n as the a m m o n i a t e d c a r a m e l is r e m o v e d . It is in s h a r p contrast to the curve o b t a i n e d s i m u l t a n e o u s l y with a k n o w n D N A - d a m a g i n g agent, m e t h y l m e t h a n e s u l f o n a t e , a n d in g o o d a g r e e m e n t with t h a t o b t a i n e d with the n o n - D N A d a m a g i n g D N A synthesis inhib i t o r , h y d r o x y u r e a . Thus the D N A synthesis inh i b i t i o n seen in o u r e x p e r i m e n t s with the a m m o n i a t e d c a r a m e l was p r o b a b l y not o f D N A d a m a g e

165 in origin. T h e r e f o r e , the d i s a g r e e m e n t b e t w e e n the results o b t a i n e d f r o m a m m o n i a t e d c a r a m e l in Salm o n e l l a m u t a g e n i c i t y test a n d the U D S a s s a y in m a m m a l i a n cells m i g h t be due to the d i f f e r e n c e in the v u l n e r a b i l i t y o f D N A s , in m e t a b o l i c p a t t e r n s among prokaryotes and eukaryotes (Garner, 1980), or to the R f a c t o r - d e p e n d e n t m u t a g e n i c i t y in these S. typhimurium tester strains (Rinkus a n d L e g a t o r , 1979). A n o t h e r e x p l a n a t i o n m i g h t be the g e n e r a l l y lower sensitivity in m a m m a l i a n shortt e r m assay systems ( M a r t i n et al., 1978). It is o f g r e a t significance to i d e n t i f y which o f the p r o d u c t s f o r m e d d u r i n g c a r a m e l i z a t i o n is responsible f o r the g e n o t o x i c a c t i o n o f the h e a t e d sugar. T h e clastogenic activities in C H O cells o f f u r a n a n d its derivatives (Stich et al., 1981a) a n d the m u t a g e n i c effect on S. typhimurium T A 1 0 0 b u t n o t o n T A 9 8 o f m a l t o l (Bjeldanes et al., 1979), b o t h f o r m e d d u r i n g p y r o l y t i c d e g r a d a t i o n o f carb o h y d r a t e s , c o u l d explain, in p a r t , the g e n o t o x i c i ty o f n o n - a m m o n i a t e d c a r a m e l r e p o r t e d . W h i l e m o s t o f the tested pyrazines a n d i m i d a z o l e s which a r e f o r m e d d u r i n g a m m o n i a t e d c a r a m e l i z a t i o n to give a c o n c e n t r a t i o n o f up to 20°7o a n d 10070 respectively in a m m o n i a t e d c a r a m e l p r e p a r a t i o n s ( W H O , 1978), are inert in b a c t e r i a l m u t a g e n i c i t y tests (Stich et al., 1980; V o o g d et al,, 1979). Thus, the m u t a g e n i c f r a c t i o n s in the c a r a m e l s , especially t h o s e in the a m m o n i a t e d ones, r e m a i n to be further identified.

Addendum In g o o d a g r e e m e n t with the p r e s e n t results o b t a i n e d f r o m the in vitro U D S - a s s a y s in m a m m a l i a n cells, an in vitro assay testing the s a m e c a r a m e l p r e p a r a t i o n s was c a r r i e d o u t in a n i n d e p e n d e n t l a b o r a t o r y in C h i n a a n d the results have revealed t h a t the n o n - a m m o n i a t e d c a r a m e l , b u t n o t the a m m o n i a t e d one, is a b l e to induce m i c r o n u c l e u s o f b o n e m a r r o w cells in mice (Yao, p e r s o n a l c o m m u n i c a t i o n , 1983).

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