Transplacental action of sodium nitrite on embryonic cells of Syrian golden hamster

149

Mutation Research, 66 (1979) 149--158

© Elsevier/North-HollandBiomedicalPress

TRANSPLACENTAL ACTION OF SODIUM NITRITE ON EMBRYONIC CELLS OF SYRIAN GOLDEN HAMSTER

N A O M I C H I INUI 1,2,Y O S H I S U K E NISHI I M A S A K O T A K E T O M I I and M A K I K O M O R I I I

Section of CellBiology and Cytogenetics,BiologicalResearch Center, Japan Tobacco and Salt Public Corporation, Hatano, Kanagawa 257, and 2 Department of Experimental Pathology, Cancer Institute,Tokyo, Toshima-ku, Tokyo 170 (Japan) (Received 29 March 1978) (Revisionreceived5 September 1978) (Accepted 15 September 1978)

Summary Hamster embryos were treated with various doses of NaNO2 in utero, by its oral administration to the mothers, and then the embryonic cells were examined for micronucleus formation, chromosomal aberrations, morphological or malignant transformation and drug-resistant mutations. For induction of resistant mutations, the cells were cultured in normal medium for 72 h, and then selected in media containing 8-azaguanine (10 or 20 #g/ml) or 1 mM ouabain. This treatment with NaNO2 caused marked dose~lependent induction of 8-azaguanine- and ouabain-resistant mutations. Cultured embryonic fibroblasts in the resting state also showed a marked dose-dependent increase in micronucleus formation but not an increase in chromosomal aberrations. This treatment also caused morphological and neoplastic transformation of the cells. Transplacental oral treatment with DMN, as a positive control, caused changes of similar extent in biological effects of embryonic fibroblasts, and in addition it caused chromosomal aberrations in metaphase plates. On the contrary, transplacental oral application of NaNOa did not induce any biological change in cultured embryonic fibroblasts.

Introduction The mutagenic action of sodium nitrite (NaNO2) or nitrous acid have been demonstrated in fungi, phages, tobacco mosaic virus and Escherichia coli [7,17,19,20,23,25]. Morphological and neoplastic transformation of cultured A b b r e v i a t i o n s : 8AG, 8-azaguanine; DMEM, Dulbecco's modified Eagle's medium; DMN, dimethyl-

nit~osamine; FCS, fetal-calf serum; MEM, Eagle's minimal essential medium; Oua, ouabain.

150 hamster fibroblastic cells have also been observed when NaNO2 is added directly to cell cultures [21,22]. In contrast, nitrous acid itself has not been found to have any carcinogenic effect in a variety of tests in vivo. Nakahara and Fukuoka [13] reported that neither nitrous acid nor NaNO2 had any carcinogenic action on mice when injected subcutaneously. Druckrey et al. [1] and other authors [3,12,18] also reported that oral administration of NaNO2 did not induce malignant tumors in mice or rats. However, NaNO2 has attracted attention as an environmental carcinogenic precursor, rather than as a strong carcinogen itself, because it reacts with amines under physiological conditions to form various nitrosamines, and especially the strong carcinogen, dimethylnitrosamine (DMN) [11,15]. This paper reports studies on the transplacental biological action of NaNO2 on cells of hamster embryos in mothers treated with NaNO2 orally by stomach tube. The transplacental effects of NaNO2 have not been studied previously, because NaNO2 causes acute toxicity in animals. Materials and methods

The experimental procedures for transplacental administration of chemicals and assay of mutants were described in detail previously [6--8].

Transplacental application of NAN02 Syrian golden hamsters, from a closed colony, weighing 150 + 30 g (Matsumoto Experimental Animal Lab., Chiba, Japan) on the l l t h or 12th day of pregnancy were given 0.5--1 ml of physiological saline solution containing 125, 250 or 500 mg of NaNO2 per kg body weight by stomach tube. The dose of NaNO2, 500 mg/kg, proved to be over the LDs0 dose within 24 h for pregnant hamsters. The hamsters were carefully kept in a barrier-system animal room and given sterilized standard laboratory chow (Clea Japan Ltd., Tokyo) and water ad libitum just before and for 24 h after administration of NaNO2. Their fetuses were then excised. The babies from mothers treated with NaNO3 (500 mg/kg) and with DMN at 50, 100 or 200 mg/kg served as negative and positive controls. Primary cultures The fetuses were chopped up finely with scissors and digested with 0.25% trypsin (Difco Lab., Detroit, U.S.A.) at room temperature for 45 min. Primary cultures of trypsinized cells were initiated by seeding 5--10 × l 0 s cells into 10 ml of medium in 75~m 2 plastic flasks (Falcon Plastic, Oxnard, Calif., U.S.A.). Most cells were grown in Eagle's minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS, GIBCO, Grand Island, NY, U.S.A.), but some other cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 20% FCS at 37°C under 5% CO2 in air. Examination of chromosomes and micronuclei For examination of chromosomes within the first 24 h of primary culture, cells grown in MEM were treated with colcemid (GIBCO) at 0.3 #g/ml for 3 h. Mitotic cells in the first cell cycle in culture were then examined. Chromosome preparations were made by the usual air-drying method with a slight modifica-

151

tion [14] and stained with Giemsa (Merck, Darmstadt, West-Germany). The numbers and types of chromosomal abnormalities were assessed by examining 200 well-spread metaphase plates. For examination of micronuclei, samples were made by a modification of the method of Schmid [16] : after culture for 30 h, the cells were collected with 0.1% trypsin, smeared on slideglasses,fixed with methanol and stained with Giemsa. In each experiment, over 5000 resting nuclei were examined for micronuclei.

Selection of resistantmutants For induction of resistant mutations, cells that had been grown in primary culture in standard M E M plus 10% FCS for 72 h were treated with 0.1% trypsin; then 5 × 10 s cells were inoculated into m e d i u m containing 8 A G at 10 or 20 #g/ml or 1 m M Oua in 60-ram plastic dishes (Falcon). Experiments were repeated, and a total of over 20 dishes was used for each dose. For selection of 8AG-resistant mutants, the m e d i u m containing 8 A G was changed every day for the firstweek and then, when cells that could not produce colonies had died, the m e d i u m was changed once every 3 days. For selection of Oua-resistant mutants, the m e d i u m containing Oua was changed after 1 week. After total cultivation periods of 15--20 days (SAG) and 30 days (Oua), the contents of the dishes were fixed and stained, and the numbers of resistant colonies were scored. Embryonic cells from mothers treated with DMN or NaNO3 and from untreated mothers were used as controls. These control cells were cultured and selected in the same way as those in the experimental groups. The effect of transplacental oral application of NaNO2 on cell survival was determined by examining the colony formation by cells from controls and NaNO2-treated mothers. The cells from some 8AG-resistant colonies were transferred into HAT medium and cultured for over 20 days, to test for revertant colonies. In this study, 4 or 5 independent repeated experiments were made on each chemical and each dose. For examination of resistance to Oua, Oua-resistant cells were transferred to normal MEM medium and cultured for 4--6 weeks, and then their ability to grow in medium containing 1 mM Oua was examined again.

Induction of morphological transformation For determination of transformation, the cells were cultured in D M E M plus 2 0 % F C S for 3--5 days from primary culture and then seeded in inocula of 5 × 103/60-mm dish (Falcon) for studies on colony formation and transformation [5,22]. The dishes were incubated for 8 days without change of medium, then fixed and stained with Giemsa solution. The number of colonies was scored, and the percentage of morphologically transformed colonies was determined. S o m e transformed colonies were cloned by the cylinder-cup method and cultured in D M E M m e d i u m for 7--9 weeks. Then 5--10 × 106 cells from the transformed colonies were implanted into the cheek pouches of young golden Syrian hamsters weighing 80--110 g to test for malignant transformation. Results

Micronucleus formation Results on micronucleus formation are summarized in Table 1. Cells were

152 TABLE 1 MICRONUCLEUS FORMATION APPLICATION OF NaNO 2

Chemical

Dose (mg/kg)

IN HAMSTER

Number of cells o b s e r v e d

EMBRYONIC

CELLS AFTER

Number of cells w i t h M.N. a

Control

0

5000

21

NaNO 2

125 250 500

5000 5000 5000

65 122 259

NaNO 3

500

5000

36

DMN

50 100 200

5002 5055 5021

68 175 311

TRANSPLACENTAL

Cells w i t h M.N. a (%) 0.42 1.30 2.44 c 5.18 c 0.72 1.36 3.46 c 6.19 e

Induced ratio b

-X3.1 X0.8 ×12.3 X1.7 X3.2 X8.3 X14.8

a Micronucleus. b R a t i o o f the number o f m i e r o n u c l e i i n d u c e d t o t h a t in c o n t r o l cells. c p .: 0.01.

examined 30 h after initiation of primary cultures. Micronuclei were f o u n d in only 0.42% of the control cells, b u t in 0.72% of the cells in NaNO3-treated samples. Thus the induction ratio was 1.4. Treatment with NaNO2 or DMN caused a marked increase in cells with micronuclei: among cells from embryos after transplacental oral treatment with NaNO2 at 125 mg per kg, 1.30% had micronuclei, representing about 3 times the control value. After treatments with NaNO: at 250 and 500 mg/kg, 2.44 and 5.18% of the cells formed a micronucleus and the induced ratios of aberrant cells were thus 5.8 and 12.3 times that of the control. Transplacental oral application of DMN also induced 3.2--14.8 times as many micronuclei as in control cells. In this experiment, NaNO2 at 125 and 500 mg/kg induced almost the same percentages of cells with micronuclei as DMN at 50 and 200 mg/kg, respectively.

Induction of chromosomal aberrations As shown in Table 2, transplacental oral administration of NaNO2 at doses of 125 to 500 mg/kg did not induce chromosomal aberrations. In contrast, DMN at doses of 100 and 200 mg/kg produced chromosomal aberrations markedly (9 and 15%) in cultured embryonic cells.

Mutation frequencies after transplacental application of NaN02 Tables 3 and 4, and Fig. 1, give results of 4 or 5 independent experiments on the induction of 8AG- and Oua-resistant mutations after transplacental oral treatment with NaNO2. Under the experimental conditions used, NaNO2 was not appreciably toxic to culture cells selected with medium containing 8AG or Oua; the lowest observed percentage survival was 66.9%, seen in cells treated with NaNO2 (500 mg/kg) and selected with 1 mM Oua. NaNO2 caused a marked dose-dependent increase in 8AG-resistant mutants (Fig. 1). On transplacental oral application, doses of NaNO2 at 125, 250 and 500 mg/kg produced, respectively, 3.3- to 10-fold increases in mutants selected with 8AG at 10 #g/ml, compared with those among control cells. Only 11.8 and 2.2 mutants

50 100 200

NaNO3

DMN

200 200 200

200

200 200 200

200

N u m b e r of metaphase cells examined

4 8 10

5

3 3 4

3

SG a

1 3 27

0

0 0 1

1

SB b

0 3 3

0

0 1 2

0

IG c

Chromosomal aberrat/ons

2 0 1

0

0 0 1

0

IB d

0 2 7

0

0 0 0

0

E e

0 0 0

0

0 0 1

0

M f

Single e b x o m a t i d gap. Single c h r o m a t i d b r e a k . I s o c h r o m a t i d gap. Isochromatid break. Single o r i s o e h r o m a t i d e x c h a n g e . Minute chromosome, Dicentrtes, acentric c h r o m o s o m e s , c h r o m o s o m e f r a g m e n t a t i o n and other c h r o m o s o m a l aberrations. N u m b e r o f ceils w i t h c h r o m o s o m a l a b e r r a t i o n s . N o r m a l d i p l o i d eelts (2 n = 4 4 ) w i t h o r w i t h o u t s m a l l c h r o m o s o m a l a b n o r m a l i t / e s . P ~ 0~)5. p ~ 0 . 0 1 ; c o m p a r e d w i t h c o n t r o l b y X2 t e s t .

500

NaNO 2

a b c d e f g h i J k

0

125 250 500

Control

Dose (mg/kg)

0 0 30

0

0 0 0

0

Others g

4 J 18 k 30 k

5 }

SJ 4 J 9 J

4

Cells w i t h aberrations h

4.0 7.5 9.0 10.0

:i.o 10.0 10.5 11.0

89.0 87.0 86.5 93.0 8O.O 76.5 80.5

(%)

Cells w i t h o v e r 44 chromosomes

89.0

Normal diploid (%) i

OF C H R O M O S O M A L A B E R R A T I O N S IN H A M S T E R E M B R Y O N I C CELLS A F T E R O R A L A P P L I C A T I O N OF NaNO 2

Chemical

FREQUENCY

TABLE 2

154

TABLE 3 I N D U C T I O N O F 8 A G - R E S I S T A N T M U T A T I O N S IN S Y R I A N H A M S T E R C E L L S BY T R A N S P L A C E N TAL APPLICATION OF NaNO 2 Chemical and dose (mg/kg)

Selection dose (/~g/ml)

Relative survival a (%)

Control

I0 20

100

NaNO 2 (125)

10 20

NaNO 2 (250)

Mutant colonies Observed colonies p e r 107 p l a t i n g cells b

Induced colonies

Induced ratio d

Revertants p e r 107 cells

Corrected value c

1 1 . 8 -+ 8.2 2.2 -+ 1.2

11.8 2.2

---

95.6

3 6 . 7 -+ 5.0 2.1 + 1.2

38.5 2.4

26.7 0.2

X3.3 X1.1

N.D. N.D.

10 20

88.3

4 3 . 4 -+ 12.8 4 . 4 -+ 3.9

50.3 4.8

38.5 2.6

X4.3 X2.2

3, 2, 0 1, 0, 0

NaNO 2 (500)

I0 20

79.5

9 1 . 2 + 23.9 1 1 . 5 -+ 9.1

122.3 15.8

110.5 13.6

XI0.4 X7.2

2, 0, 1 0, 0 , - -

NaNO 3 (500)

10 20

95.4

10.7 -+ 2.1 3.3 -+ 4.8

11.3 3.5

0.5 1.3

DMN (I00)

10 20

92.6

4 8 . 5 + 11.2 . .

DMN (200)

I0 20

86.6

174.1 + 25.7 2 5 . 4 -+ 4.9

52.4 . 200.2 28.9

---

40.6 .

N.D. e N.D.

X0.95 ×1.6

N.D. 0, 0 , - -

×4.4

N.D.

X16.9 X12.1

N.D. N.D.

. 188.3 26.7

a A b s o l u t e survival o f c o n t r o l cells ( a c t u a l P.E.) w a s 1 . 2 1 % w h e n cells h a d b e e n p l a t e d i n t o 6 0 - m m dishes in i n o c u l a o f 5 X 103 cells p e r dish. b M e a n s o f at least 4 i n d e p e n d e n t r e p l i c a e x p e r i m e n t s w i t h s t a n d a r d d e v i a t i o n s . c V a l u e s w e r e c o r r e c t e d f o r t h e survival r a t e o f t h e c o n t r o l . d Ratio of induced m u t a t i o n frequency to spontaneous m u t a t i o n frequency. e Not determined.

TABLE 4 I N D U C T I O N O F O U A B A I N - R E S I S T A N T M U T A T I O N S IN S Y R I A N H A M S T E R C E L L S BY T R A N S P L A C E N T A L A P P L I C A T I O N OF NaNO 2 Chemical and dose (mg/kg)

Relative survival (%) a

Control NaNO 2 NaNO 2 NaNO 2 NaNO 3 DMN DMN

100 71.6 68.7 66.9 93.2 92.3 86.6

(0) (125) (250) (500) (500) (100) (200)

Mutation colonies

Induced colonies

Observed c.lorries p e r 107 p l a t e d cells b

Corrected value c

10.6 37.8 54.4 78.4 13.5 40.6 54.1

10.6 52.4 78.9 116.8 14.5 43.8 62.2

+ 9.7 + 18.1 + 11.8 -+ 10.9 -+ 2.3 + 9.5 -+ 1 5 . 5

-41.8 68.3 106.2 3.9 33.2 51.6

Induced ratio d

-×4.9 X7.4 Xll.0 X1.4 X4.1 X5.8

Resist a n c e to Oua

N.D. e + f + + + + +

a A b s o l u t e survival (P.E.) o f c o n t r o l cells was 1 . 9 8 % w h e n cells w e r e t r a n s f e r r e d i n t o 5 X 103 c e l l s / 6 0 - m m dish. b Means o f a t least 4 i n d e p e n d e n t r e p l i c a e x p e r i m e n t s w i t h s t a n d a r d d e v i a t i o n s . c Values c o r r e c t e d f o r survival r a t e o f t h e c o n t r o l . d Ratio of induced m u t a t i o n frequency to spontaneous m u t a t i o n frequency. e Not determined. f A b i l i t y t o g r o w in m e d i u m c o n t a i n i n g 1 m M O u a .

155

~. 10001

0

125 250 500 NaNO~(mg/kg)

F i g . 1. I n d u c t i o n o f m u t a n t c o l o n i e s s e l e c t e d w i t h S A G a f t e r t r a n s p l a c e n t a l o r a l application of N a N O 2. A marked dose-dependent i n c r e a s e i n m u t a t i o n s w a s o b s e r v e d . V a l u e s a r e t a k e n f r o m T a b l e 3. o, Selection w i t h S A G a t 1 0 ~zg/ml. o, S e l e c t i o n w i t h S A G a t 2 0 / ~ g / m l .

were observed in cultured cells from untreated mothers, and 11.3 and 3.5 mutants were found among cells treated with NaNO2 at 500 mg/kg and selected with 8AG at 10 and 20/~g/ml. DMN at doses of 100 and 200 mg/kg also caused a 4.4- to 16.9-fold increase in mutants when selected with 8AG at 10 or 20 ug/ml, compared with those among control cells. As shown in Table 3, 13 colonies were cloned from SAG-resistant mutants. In this experiment to confirm the induction of mutations, a few revertant colonies were obtained in H A T medium: 0--3 revertant colonies per 107 cells were obtained in 13 indep e n d e n t experiments on the growth ability of mutants in H A T medium. From these results, it may be concluded t h a t the cells that grew in selection medium were HGPRT
Induction of morphological transformation Morphological transformation of hamster embryonic cells in vitro was observed after transplacental oral administrations of N a N O 2 and D M N . The results are summarized in Table 5. The transplacental application of N a N O 2 did not

156 TABLE 5 MORPHOLOGICAL NaNO 2 Chemical

Control NaNO 2 NaNO 2 NaNO 2 NaNO 3 DMN DMN a b e d e f

Dose (mg/kg)

0 125 250 500 500 100 200

TRANSFORMATION

Plating e f f i c i e n c y (%)

1.15 1.02 0.87 1.16 1.12 1.13 1.09

-+ 0 . 3 6 + 0.17 + 0.11 -+ 0 . 3 3 -+ 0 . 2 8 ¢ 0.04 ¢ 0.12

INDUCED

BY T R A N S P L A C E N T A L

Transformation r a t e (%) a

0.12 0.66 1.45 1.94 0.15 0.55 1.10

+- 0 . 1 5 + 0.25 +- 0 . 6 6 ¢ 1.41 2 0.13 ¢ 0.10 2 0.20

Transformed colonies/dish

0.11 0.33 0.50 0.79 0.10 0.25 0.49

+ 0.13 + 0.12 + 0.21 2 0.12 -+ 0 . 0 6 + 0.05 + 0.12

APPLICATION OF

B a c k tran.sPlantation 45d. b

60d. c

0/3 0/2, 0/2 1/1 e 0 / 3 3/3 f N.D. 0/3 N.D.

N.D. d 0/3 2/2 e 0/2 N.D. N.D. N.D. N.D.

P e r c e n t a g e o f m o r p h o l o g i c a l l y t r a n s f o r m e d c o l o n i e s a m o n g the t o t a l surviving colonies. Cells 4 5 d a y s after i n i t i a t i o n o f c u l t u r e s . Cells 6 0 d a y s after i n i t i a t i o n o f c u l t u r e s . Not determined. Animal died from the turlor. T u m o r s r e g r e s s e d w i t h i n 4 m o n t h s after i n o c u l a t i o n .

have any apparent cytotoxic effect on the embryonic cells in culture, the plating efficiencies of NaNO2-, NaNO3- or DMN-treated cells being similar to that of cells from untreated embryos. The transformation rates of the cells after transplacental application of NaNO2 were 5.5- to 16.2-fold that of untreated control cells. DMN-treated cells also showed higher rates of transformation than control cells. The transformation rates of treated cells showed only a slight correlation with the dose of NaNO2 or DMN. Treatment with NaNO~ induced no more transformed colonies than those of control cells. In this study, transformed colonies had the following characters: colonies showed marked piling up and a criss-cross arrangement, with many layers of randomly orientated criss-crossing cells in central parts, and many mitoses in peripheral regions. In normal colonies, cells were regularly arranged with no criss-crossing in peripheral parts, but cells were piled up in the central parts of a few colonies. (For photographs see Inui and Taketomi [5]). Some of the transformed colonies were isolated by the cylinder-cup method, and the cells from the transformed colonies were transferred to 75-cm 2 tissue culture flasks (Falcon) and cultured for 45--60 days in standard medium (DMEM + 20% FCS). The cloning efficiency of transformed colonies was 18.9% (7/27) in this study. Then 5--10 × 106 cells from cloned lines were inoculated into the cheek pouches of non-immunized young Syrian golden hamsters obtained from the same closed colony, and weighing 80--110 g. The cheek pouches of the animals were examined every week for at least 5 months. The cells from 2 of 7 transformed colonies, 45 days after cloning produced tumors at the site of inoculation within 4 - 6 weeks, and the cells from one transformed colony actually killed the host animal 50 days after inoculation. With cells from another clone, however, the tumors regressed within 3 months after inoculation. Results on cells 60 days after cloning were similar to those on cells 45 days after cloning. The tumors that formed in the cheek pouch were

157 identified histologically as pleomorphic fibrosarcomas. No granuloma or abscess was observed in this back-transplantation study.

Discussion The present work showed that transplacental oral administration of sodium nitrite, applied into pregnant hamsters, caused marked micronucleus formation, SAG- or Oua-resistant mutations and morphological or neoplastic transformation of cells from the embryos. No marked chromosomal aberrations, however, were observed in the cells. This discrepancy of data between chromosomal aberrations and the micronucleus test could be explained as follows. NaNO2 might act not only on nucleic acids themselves [23,24], but also on proteins or - S H compounds, so that mitotic apparatus, i.e. spindle-fiber formation, was also affected and damaged. As the result, the large number of cells with micronucleus were observable in this study, as compared with chromosomal aberrations. The main biological activity of NaNO2 in the living cells might be considered to attack both proteins and nucleic acids. On the other hand, DMN may have reacted only with nucleic acids and, as the result, methylation of nucleic acids occurred [10]. These biological effects of NaNO2 might be the result of a direct transplacental action of NaNO2. The total amount of N-nitroso compounds in the stomach after treatment with NaNO2 was slightly higher than after treatment with NaNO3 or after consumption of the control diet. (Results are not presented here; see Inui et al., ref. 7.) The total amount of N-nitroso compounds, probably DMN, formed corresponded to a dose of 2.7 mg/kg. However, in this experiment, treatment with DMN at 100 mg/kg induced only 4.4 times the control number of 8AG-resistant mutations. From these results it may be concluded that NaNO2 itself acted transplacentally on the embryonic tissues, inducing SAG- and Oua-resistant mutations, micronucleus formation and transformation of the cells. In 1939, Thorn and Steinberg [20] first reported that NaNO2 induced genetic changes in fungi, and later the mutagenic action of nitrite was demonstrated in a wide range of organisms [7,17,19,20,23,25]. The chief action of nitrite is thought to be oxidative deamination of nucleic acid, resulting in conversion of guanine to xanthine, adenine to hypoxanthine and cytosine to uracil [23,24]. One of the authors has also shown that NaNO2 caused malignant transformation of newborn hamster fibroblasts [21,22], suggesting that NaNO2 itself may be carcinogenic. Sodium nitrite is widely used as a food additive. Moreover it is known that NaNO3 is metabolized to NaNO2 in saliva [2]. Most of our intake of nitrate is from vegetables, water and food additives; and it is reported that tap water with a high nitrate content is related to a high incidence of human hepatomas in England [4]. The present study suggests that nitrite is not only a mutagen or precursor of carcinogens but also a transplacental mutagen or carcinogen. This conclusion indicates the necessity for studies on the direct and transplacental carcinogenic effects of NaNO2.

158

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