Mutation Research, 262 (1991) 171-176 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0165-7992/91/$ 03.50 ADONIS 016579929100017U

MUTLET 0465

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Investigation of soy sauce treated with nitrite in the chromosomal aberration test in vitro and the micronucleus test in vivo

A. Nagahara, K. Ohshita and S. Nasuno

Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba 278 (Japan)

(Received 2 June 1989) (Revision received 10 October 1990)

(Accepted 10 October 1990)

Keywords: Soy sauce; Nitrate pretreatment; Chromosome aberrations

Summo_ry

Soy sauce pretreated with 2300 ppm nitrite caused no more aberrations than did untreated soy sauce in the chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line with or without $9 mix- ture. The aberration induction by soy sauce is likely to be caused by the 17% sodium chloride it contains. Soy sauce with or without pretreatment with 2300 ppm nitrite was orally given to ICR mice at a dose of 14 ml/kg body weight once or 6 ml/kg body weight/day for 5 consecutive days. This oral administration did not induce any significant increase in micronuclei in the micronucleus test in vivo.

A number of reports have been published on the mutagenicity of a variety of foods since the development of the simple and convenient Ames test. Soy sauce, a condiment commonly used in Asia, is often tested with this method. For exam- ple, soy sauce pyrolysed at 400°C for 30 min show- ed mutagenicity to Salmonella typhimurium TA98 with $9 mixture (Shimizu et al., 1980). When treated with 2000 ppm nitrite, soy bean sauce is reported to produce mutagenic substances as demonstrated using the Salmonella mutagenicity

Correspondence: Ayumu Nagahara, Food Research Institute, University of Wisconsin, Department of Food Microbiology and Toxicology, 1925 Willow Drive, Madison, WI 53706 (U.S.A.).

test (Lin et al., 1979). Tyramine, (-)-(1S,3S)-l-me- thyl- 1,2,3,4- tetrahydro -/3- carboline- 3- carboxylic acid ((-)-(lS,3S)-MTCA) and its stereoisomer, (-)- (1R,3S)-MTCA, have been identified as mutagen precursors when treated with a high concentration (50 raM: 2300 ppm) of nitrite at the acidic condi- tions found in soy sauce (Wakabayashi et al., 1983; Ochiai et al., 1984).

On the other hand, one author reported that the mutagenicity of soy sauce for Salmonella typhi- murium TA98, TA100 and Escherichia coli WP2uvrA was a false-positive result due to the r- histidine and L-tryptophan present in the condi- ment (Nagahara, 1985). Moreover, soy sauce with the amount of nitrite taken in daily meals added was not mutagenic and its mutagen precursors did

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not turn into mutagens under physiological condi- tions (Nagahara et al., 1986).

In a different approach, Inshidate et al. (1980) reported positive results for soy sauce without nitrite treatment using the chromosomal aberration test in vitro with a Chinese hamster fibroblast cell line. Therefore, in vitro chromosomal aberration and in vivo micronucleus tests were carried out to re-examine the mutagenicity of soy sauce with or without nitrite treatment. This report deals with the results of these tests.

Materials and methods

Cell line Chromosomal aberration tests were carried out

on soy sauce treated with nitrite using a Chinese hamster fibroblast cell line (CHL) according to the method of Ishidate and Odashima (1977). The cell line was originally established from the lungs of a newborn female hamster by Koyama et al. (1970) at the Cancer Research Institute, Tokyo. It has been maintained by 4-day passages in minimum essential medium (MEM; Gibco) containing 10°70 calf serum. The fibroblast cell has a model number of 25 chromosomes after serial transfers and its doubling time is approximately 15 h.

Animals The animals used in the micronucleus test were

male ICR mice about 8 weeks of age and were ob- tained from Charles River Japan Co.

Test substances The soy sauce sample (Kikkoman-brand dark-

colored soy sauce) was obtained at a local market in Japan.

Nitrite treatment l-ml samples of soy sauce, adjusted to pH 3.0

with 12 M HCI to simulate conditions in the stomach, were mixed with 1 ml of nitrite at dif- ferent concentrations (0, 100, 500, 4600 ppm). The high concentration of nitrite (2300 ppm) was chosen to be comparable with published articles. The mixtures were incubated in the dark at 37°C

for 60 min and 1 ml 0.1 M ammonium sulfamate was added to stop the reaction. The reaction mix- tures were passed through a Millex-HA filter (0.45 /~m, Millipore) for sterilization and used for test materials.

Preparation of $9 mixture Liver microsome fraction ($9) was prepared

from male SD rats (Crj:CD, Charles River Japan Co.), 7 weeks of age. To induce the microsomal en- zymes, sodium phenobarbital was injected in- traperitoneally at doses of 30 mg/kg 4 days prior to killing and 60 mg/kg 3, 2 and 1 days prior to kill- ing. The rats were also given an i.p. injection of 5,6-benzoflavone at a dose of 80 mg/kg 2 days before they were killed. Following decapitation, the livers were carefully removed and homogenized in ice-cold 1.15°70 KCI solution with a teflon homogenizer. The homogenate was centrifuged at 9000×g for 10 min at 0-4°C and the supernatant fraction ($9) was stored in a deepfreezer ( - 80°C) before use.

The reaction mixture ($9 mixture) contained 3 ml of $9, 2 ml of 20 mM HEPES buffer (pH 7.2), 1 ml of 50 mM MgCI2, 1 ml of 330 mM KCI, 1 ml of 50 mM G-6-P, 1 ml of 40 mM NADP and 1 ml of distilled water (Matsuoka et al., 1979). All the ingredients of the $9 mixture except the $9 fraction were sterilized through a Millex-HA filter (0.45 #m, Millipore) and were prepared just prior to use.

Chromosomal aberration tests in vitro Chromosomal aberration tests using CHL were

carried out as described by Ishidate and Odashima (1977). In the absence of the metabolic activation system, 3-day-old cultures (about 105 cells/6-cm dish) in 5 ml medium were exposed to 50/~1 of soy sauce or 50/~l equivalent soy sauce treated with various concentrations of sodium nitrite in petri dishes for 24 and 48 h. The final concentration of samples was 10 ~l/ml in the cultures. This concen- tration of test sample showed less than 50~/0 growth inhibition. In the presence of the metabolic activa- tion system, 0.2 ml of trypsinized cells (1.2x I07/ml) were incubated with the test ma- terials and 1.0 ml of $9 mixture in each 10-ml test

t ube . The f inal vo lume was a b o u t 1.2 ml and the

f ina l concen t r a t i on o f sample was 10 ~ l / m l in the

cul tures . The tubes were shaken con t inuous ly at

37°C for 3 h. A f t e r cen t r i fuga t ion , the cells were

cu l tu red in pet r i dishes for an add i t i ona l 21 h. 17°70

s o d i u m ch lor ide so lu t ion (the same concen t ra t ion

as in soy sauce) with o r wi thou t ni t r i te t r ea tmen t

was a lso tested. The f inal concen t ra t ion o f 17070

s o d i u m ch lor ide so lu t ion was 10 # l / m l and the f inal

m o l a r i t y was a b o u t 30 m M in the cul tures . Cells

were t r ea ted with Co lcemid (0.2 # g / m l ) for 2 h and

a f t e r t ryps in i za t ion they were incuba ted in 0.075 M

KCI h y p o t o n i c so lu t ion for 13 min at r o o m

t e m pe ra tu r e . A f t e r cen t r i fuga t ion o f the incuba-

t ion mix ture , the cells were f ixed with f ixa t ive

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(acetic a c i d : m e t h a n o l , 1:3, v / v ) and sp read on

c lean glass slides. A f t e r a i r -dry ing , the slides were

s t a ined with 1.5070 G i e m s a so lu t ion (E. Merck) for

12-15 min . The n u m b e r o f cells with c h r o m o s o m a l

a b e r r a t i o n s was coun ted in 100 wel l - spread

m e t a p h a s e cells wi th wel l - spread c h r o m o s o m e s .

Micronucleus test in vivo

The micronuc leus test was car r ied ou t as descr ib-

ed b y Schmid (1975). The an imals used in this test

were ma le I C R mice, a b o u t 8 weeks o f age. The test

s amples o f soy sauce or soy sauce t rea ted with

va r ious concen t ra t ions o f ni t r i te were given by

gavage tube into the s tomach at a dose o f 14 m l / k g

equiva len t o f soy sauce (the LDso value o f soy

TABLE 1

RESULTS OF CHROMOSOMAL ABERRATION TEST OF SOY SAUCE WITHOUT NITRITE AND 17% SODIUM CHLORIDE SOLUTION

Sample Final dose O,1/ml)

Incidence of chromosome aberrations

Exp. time % Ab) Type and number of Ab . b Judge c (h)

Without $9 mixture Soy sauce 10 24 15 g(8), b(7), t(6) + Soy sauce 10 48 24 g(11), b(8), t(9) + +

NaCI (17%) 10 (30 mM) 24 14 g(9), b(6), t(4), r(l) + NaCI (17%) 10 (30 mM) 48 23 g(14), b(7), t(7) + +

Saline d 10 24 1 g Saline 10 48 2 g, b m

MMC e 0.2 (ng/ml) 24 54 g, b, t, r + + + MMC 0.2 (ng/ml) 48 78 g, b, t, r + + +

With $9 mixture Soy sauce I0 24 25 gOD, bOO), t(7) + + NaCI (17%o) 10 (30 mM) 24 24 gOD, b(12), t(5) + + Saline d 10 24 1 g

D M N f 2.0 (mg/ml) 24 61 g, b, t, r + + +

" Percentages of cells with aberrations. b Types of aberration: g, gap; b, break; t, exchange; r, ring. Breaks less than the width of a sister chromatid were designated as gaps in our criteria. Number of aberrations: Number/100 cells. c The judgment was made as follows: - , negative (less than 4.9%o); ± , suspicious (5.0-9.9%0); +, positive (10.0-19.9%0); + +, 20.0-49.9%; + + +, more than 50%e. d Negative control (physiological saline). • Positive control (without $9 mix: mitomycin C). f Positive control (with $9 mix: dimethyl nitrosamine).

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sauce in mice is about 28 ml/kg body weight) once or 6 ml/kg/day equivalent of soy sauce for 5 con- secutive days at 24-h intervals. Five animals were used for each test sample. The positive control animals received mitomycin C and the negative control animals received physiological saline.

After killing the animals (24 h/48 h for single- dose animals, 6 h after the last dose for 5-dose animals), the marrow in the femurs was expelled with 0.6 ml of fetal bovine serum. This suspension was then centrifuged at 1000 rpm for 5 min. The supernatant was removed, then the cells in the bone marrow were resuspended in a small drop of serum and slides were prepared. After drying at room temperature, the slides were fixed in absolute methanol and stained in 3O7o Giemsa (E. Merck) for 25-30 min. For each mouse 5-6 slides were prepared and for each of the 2 best slides 1000 polychromatic erythrocytes were counted for

micronuclei.

Results

Chromosomal aberration test in vitro

Table 1 shows the results for chromosomal aber- ration tests of soy sauce without nitrite, 17070 sodium chloride solution and negative and positive controls. Soy sauce showed weak mutagenicity with or without $9 mix. Similar results were obtain- ed for 17070 sodium chloride solution.

Fig. 1 shows the results for chromosomal aber- ration tests of soy sauce and 17070 sodium chloride solution treated with various concentrations of nitrite for 24 h without $9 mixture. Soy sauce and 17070 sodium chloride solution showed weak mutagenicity without or with nitrite at various con- centrations. The results obtained for soy sauce were quite similar to those for 17070 sodium chloride solution (Table 1). The dotted line (Fig. 1) indicates the range of aberrations that is considered to be negative (less than 4.9070 cells with aberra- tions) (Ishidate et al., 1984).

Even soy sauce treated with as much as 2300 ppm nitrite did not cause more aberrations than soy sauce alone in the chromosomal aberration test without $9 mixture, harvested at 24 h. Similar

~ 15 ~ : ~ / / - - . ~ _ f

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

~---//" 50 250 2, 301]

Ni trite concentroti on(ppm)

Fig. 1. Mutagenicity of soy sauce (O) and 17070 sodium chloride solution (O) treated with nitrite at various concentrations deter- mined by the chromosomal aberration test without $9 mixture. Expression time 24 h. The dotted line indicates the range of aberrations that is regarded as negative (less than 4.9070 aber-

rations).

results were also obtained for 48-h-expressed samples without $9 mixture and 24-h-expressed samples with $9 mixture.

Micronucleus test in vivo Table 2 shows the results of micronucleus tests

24 h after single oral doses (14 mg/kg) of test

TABLE 2

RESULTS OF THE MICRONUCLEUS TEST OF SOY SAUCE TREATED WITH NITRITE AT VARIOUS CON- CENTRATIONS GIVEN BY GAVAGE IN A SINGLE DOSE

TO MALE ICR MICE

Nitrite (ppm) Number of Number of micronuclei/ mice 1000 PCEs a

Mean b SD c

2300 5 1.4 0.55

250 5 1.6 0.55

50 5 1.8 0.84

0 5 1.4 0.55

Saline d 5 1.6 I. 14

MMC e 5 20.0 2.70

Expression time 24 h. a PCE, polychromatic erythrocytes. e 1000 PCEs of duplicate preparations were evaluated for every animal. c Standard deviation. d Negative control (physiological saline). c Positive control (mitomycin C, 2 mg/kg i.p.).

TABLE 3

RESULTS OF THE MICRONUCLEUS TEST OF SOY SAUCE TREATED WITH VARIOUS CONCENTRATIONS OF NITRITE GIVEN BY GAVAGE ON 5 CONSECUTIVE DAYS TO MALE ICR MICE

Nitrite (ppm) Number of Number of micronuclei/ mice 1000 PCEs*

Mean b SD c

2300 5 1.8 0.84

250 5 1.6 0.89

50 5 1.6 0.55

0 5 2.0 0.71

Saline d 5 1.8 0.84

MMC • 5 21.8 3.01

a PCE, polychromatic erythrocytes. b 1000 PCEs of duplicate preparations were evaluated for every animal. c Standard deviation. d Negative control (physiological saline). • Positive control (mitomycin C, 2 mg/kg i.p.).

samples. In this assay no significant difference was

found between the soy sauce sample treated with even 2300 ppm nitrite and the negative control. Similar results were also obtained for mice 48 h after single oral doses (14 mg/kg) (data not shown) and for mice given 5 consecutive daily oral doses of test samples (6 ml /kg /day ) at 24-h intervals (Table 3).

Discusgon

When soy sauce treated with various concentra- tions of nitrite was examined with the chromo-

somal aberrat ion test in vitro, even the soy sauce treated with 2300 p p m nitrite did not increase the number of chromosomal aberrations, although it has been reported to be mutagenic to bacteria (Lin et al., 1979). However, soy sauce itself has been shown to have a weak mutagenic effect in the chromosomal aberrat ion test in vitro (Table 1, and Ishidate et al., 1984). Similar results were obtained with 17o70 sodium chloride solution 10 #l /ml in the cultures with or without nitrite t reatment in this assay system. Since soy sauce also contains 17070 sodium chloride, these results suggest that the

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chromosomal aberrat ion induction by soy sauce is mainly caused by the sodium chloride in the sauce. Furthermore, when oral doses of soy sauce or soy sauce treated with various concentrations of nitrite

were given to ICR mice (14 ml /kg once or 6 m l / k g / d a y for 5 consecutive days), no significant

differences were found between test samples and the negative control.

There are a few reports about the mutagenicity of soy sauce treated with a high concentration of

nitrite in Salmonel la /mammal ian microsome tests (Lin et al., 1983; Shibamoto, 1983). However, no report was found about the mutagenicity of soy sauce treated with nitrite in the chromosomal test in vitro or the micronucleus test in vivo. In these well-recognized mutagenicity test systems, we show that soy sauce treated with nitrite does not have a greater effect than soy sauce alone. Thus soy sauce treated with nitrite shows mutagenic activity in the

bacterial test system, but not in the mammal ian test systems. Coffee is another example of a substance that has been shown to be mutagenic in bacterial

test systems but does not cause aberrations in mam- malian cells (Aeschbacher et al., 1984).

The lack of mutagenicity of soy sauce treated with various concentrations of nitrite in the mam- malian test systems is important for the risk assess- ment of the Japanese diet.

Acknowledgements

The authors wish to thank Mr. S. Nagano and Miss K. Fukushima for their technical assistance.

The authors are also indebted to Ms. J. Storkson, Food Research Institute/University of Wisconsin- Madison, for her advice.

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Communicated by S.M. Galloway