Antimutagenic action of cobaltous chloride on radiation-induced mutations in cultured Chinese hamster cells

Mutation Research, 245 (1990) 99-105

99

Elsevier MUTLET 0404

Antimutagenic action of cobaltous chloride on radiation-induced mutations in cultured Chinese hamster cells A k i k o Y o k o i y a m a 1, T s u n e o K a d a I a n d Y u k i a k i K u r o d a 2 XDepartment of lnduced Mutation and 2Department of Ontogenetics, National Institute of Genetics, Yata 1, 111, Mishima, Shizuoka 411 (Japan)

(Received 1 September 1987) (Revision received 31 May 1990) (Accepted 1 June 1990)

Keywords: Antimutagenic action; Cobaltous chloride; 8AG-resistant mutations; Chinese hamster cells; Gamma-rays; UV light

Summa The effects of cobaltous chloride on 8-azaguanine (8AG)-resistant mutations induced by ~/-rays or ultraviolet (UV) light in cultured Chinese hamster V79 ceils were examined. Cobaltous chloride alone had no significant effects on survival and mutations of V79 cells at concentrations less than 1 x 10 -5 M. Cobaltous chloride at a concentration o f 3 x 10 - 6 M had a marked effect in reducing 8AG-resistant mutations induced by 'r-rays o f 2-6 Gy, when cells were incubated for 6-7 days in the presence o f cobaltous chloride after "r-ray irradiation (posttreatment). The pretreatment of cells with cobaltous chloride for 6 days before "r-ray irradiation reduced 8AG-resistant mutations induced by -r-rays. Pre- or post-treatment with cobaltous chloride had no such effect on UV-induced mutations, however. The difference in responsiveness to cobaltous chloride between bacterial and m a m m a l i a n cell systems is discussed.

We previously reported that cobaltous chloride works as an antimutagen in different bacterial systems. The frequencies of mutations induced by N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNNG), Correspondence: Yukiaki Kuroda, Department of Ontogenetics, National Institute of Genetics, 1, 111, Yata, Mishima, Shizuoka 411 (Japan). Abbreviations: SAG, 8-azaguanine; UV, ultraviolet; MNNG, N-methyl-N'-nitro-N-nitrosoguankfine; Trp-P-l, 3-amino-l,4-

dimethyl-5H-pyrido[4,3-b]indole; HGPRT, guanine-phosphoribosyl transferase.

hypoxanthine-

3-amino-l,4-dimethyl-5H-pyrido-[4,3-b]indole (Trp-P-1), UV or 'r-rays were markedly lower in the presence of cobaltous chloride than in its absence, during the course o f incubation o f treated cells (Kada and Kanematsu, 1978; Kada et al., 1979; Mochizuki and Kada, 1982a,b). The high frequency o f spontaneous mutations in a m u t a t o r strain o f Bacillus subtilis possessing an altered D N A polymerase III was also reduced noticeably by the presence of cobaltous chloride (Inoue et al., 1981). Cobalt is a bio-element whose existence is universally essential for life in m a m m a l s . One o f the

0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

10o essential elements of the antimutagenic principle in the human placenta was proved to be cobaltous ions (Kada and Mochizuki, 1981; Mochizuki and Kada, 1982b; Komura et al., 1981). It is interesting to examine its antimutagenic activity in the mammalian system. We studied the effects of cobaltous chloride on -r-ray- and UV light-induced mutations in cultured Chinese hamster V79 cells, in which the fundamental aspects of mutagenesis have been well established (Chu and Mailing, 1968; Bridges and Huckle, 1970; Arlett and Potter, 1971; Arlett and Harcourt, 1972; Kuroda, 1975a,b; Kuroda et al., 1985). We found that 8-azaguanine (8AG)-resistant mutations induced by 7-rays were markedly reduced by the presence of cobaltous chloride after and before exposure to 7-rays. Materials and methods

Cells and culture The cell line used was the V79 strain of Chinese hamster lung cells, isolated by Ford and Yerganian (1958) from a normal young adult male Chinese hamster. The cells were grown in large mass, distributed in many small ampoules, and frozen at - 8 0 ° C . Before use, ceils were thawed at 37°C, suspended in fresh medium, and cultured in GHAT medium (3x 10 -6 M glycine, 10 -4 M hypoxanthine, 4 x 10 -7 M amethopterine, and 1.6x 10 -5 M thymidine) at 37°C for 24 h, to remove the preexisting 8AG-resistant cells from the population. The culture medium was Eagle's minimal essential medium (Nissui Seiyaku Co., Tokyo) supplemented with 10070 fetal bovine serum (Gibco Laboratory, Grand Island, NY). The cells were cultured in 60-mm plastic petri dishes (Falcon; catalog No. 3002) in 5 ml of medium under 5°70 CO2 and 95070 air at 37°C. Chemical and irradiation Cobaltous chloride (COC12.6H20) was purchased from Wako Chemicals Co., Ltd. 8AG was obtained from Sigma Chemicals Co., Ltd. The 7-ray source used was 6000 Ci of 137Cs and the exposure was carried out with Toshiba RI-423 irradiation

equipment at a dose rate of 1.2 Gy/min. The UV source was a 4-W National germicidal lamp with a dose rate of 5 erg/mm2/s.

Cytotoxicity assay Cells were dissociated from monolayer culture by treatment with 0.25°70 trypsin (Difco, 1:250) solution for 10 min at 37°C. The cytotoxicity assays of -r-rays and UV irradiation were carried out by determining colony formation of ceils. Triplicate inocula of 102 cells in 5 ml of culture medium in 60-mm petri dishes were incubated for 24 h at 37°C, during which time most cells became attached to the surface of the dishes. Then cells were washed twice with Hanks' salt solution, and exposed to -r-rays or UV light. The irradiated cells were incubated in fresh medium for 7 days at 37°C, in the presence (post treatment) or absence of cobaltous chloride. The cell colonies formed in petri dishes were fixed with ethyl alcohol and stained with Giemsa solution. Colonies containing more than 50 cells were scored under a binocular microscope, and the colonyforming activity was calculated as the average number of colonies found as a percentage of the number of cells initially inoculated. The effects of "r-rays or UV irradiation are expressed as fractions (surviving fractions) of the colony-forming activity of unirradiated control cells. Mutagenicity assay The mutagenicity of 7-rays of UV irradiation was determined by the 'replating method' (Kuroda, 1975a,b; Kuroda et al., 1985). The inocula of 105 cells in 5 ml of culture medium in 60-mm petri dishes were incubated for 24 h at 37°C and then exposed to "r-rays or UV light. Then the ceils were incubated in culture medium for a mutation expression time of 6 days in the presence (post-treatment) or absence of cobaltous chloride. The ceils were then treated with trypsin, and 104 cells were replated each in 20 60-mm petri dishes in medium containing 10/~g/ml of 8AG. After incubation for 14 days, the cell colonies formed were fixed and stained and the number of 8AG-resistant colonies was scored. In parallel experiments, triplicate in-

101 ocula o f 10 2 cells in 60-mm petri dishes were replated and incubated in normal medium for 7 days. The colony-forming activity of replated cells was determined as described above. The observed numbers of 8AG-resistant mutant colonies were corrected for the decrease in the colony-forming activity of cells in normal medium. The number of induced mutants was calculated by subtracting the number of colonies in untreated control cultures from those in treated cultures. The induced mutation frequency is expressed as the number of induced mutants per 103 colony-forming cells.

In experiments with posttreatment of cobaltous chloride, cells were incubated in normal medium for 24 h, then washed with H a n k s ' salt solution, and exposed to y-rays or UV light. The irradiated cells were incubated in medium containing cobaltous chloride for 7 days for cytotoxicity assays or for an expression time of 6 days for mutagenicity assays.

Treatment with cobaltous chloride

Cobaltous chloride alone had no significant effects on survival of V79 cells at concentrations less than 3 × 10-6 M (Fig. 1). At higher concentration as 1 × 10-5 M cobaltous chloride showed cytotoxicity. The frequency of 8AG-resistant mutations was also not affected by cobaltous chloride at concentrations less than 9 × 10-3 M (Fig. 1). Because o f this, in further experiments to examine the effects o f cobaltous chloride on cell survival and

In experiments with pretreatment o f cobaltous chloride, inocula of 103 cells were cultured in medium containing cobaltous chloride at various concentrations for 6 days. Then the cells were treated with trypsin, and 103 cells were replated each in 60-ram petri dishes in normal medium and incubated for 24 h at 37°C. The cells were washed with H a n k s ' salt solution and exposed to y-rays or UV light.

Results Effects o f cobaltous chloride alone on cell survival and mutations

1.0

50 m

1.Oi

5O ,=4

0.5 0.5

40

e~

40

r~

e.

.o

.o

o

s0 ~ ao

•~

0.1

0.1

20

m 0.05

2o

r~ 0.05

oo

10 o"

0.01 0.01

I Ixl0 -e

I 3x10 -6

Concentration of COC12, 6 H 2 0

0 9x10-6 Ix10-5 (M)

Fig. 1. Effect of cobaltous chloride at different concentrations on survival (o) and the frequency of 8AG-resistant mutations (i) in Chinese hamster V79 cells.

I 2 Dose

of gamma

I 4 rays

0 (Gy)

Fig. 2. Effect of posttreatment of cobaltous chloride at a concentration of 3 x 10- ~M on survival (o,©) and the frequency of 8AG-resistant mutations (-, o) induced by various doses of 7-rays in Chinese hamster V79 cells, o, i, n o C o C l 2 • 6HzO; ©, o, 3 × 10-4 M COC12-6H20.

102

mutations induced by 3,-rays and UV light, cobaltous chloride at a concentration of 3 × 10- 6 M was used.

0

..> m 1.0i

Effects o f cobaitous chloride on .y-ray-irradiated cells

0.5 10

.o

O

Posttreatment with cobaltous chloride 3,-Rays at doses less than 6 Gy showed a weak cytotoxicity in Chinese hamster V79 cells. In the posttreatment with 3 x 10-6 M COC12.6H20, the cytotoxicity of .y-rays was slightly enhanced (Fig. 2). 3,-Rays effectively induced 8AG-resistant mutations in V79 cells. The induced mutation frequency increased with increasing doses o f -y-rays. At a dose o f 6 Gy the ~/-ray-induced mutation frequency was 38 per 105 survivors (Fig. 2). In the posttreatment with 3 x 10- 6 M COC12.6H20, the 3,-ray-induced mutation frequency was maintained at a lower level, less than half of that in cells not treated with cobaltous chloride. The inhibitory effect of cobaltous chloride on ~ray-induced mutations was further examined in cells treated with COC12 • 6H20 at different concentrations. Fig. 3 indicates that 8AG-resistant mutations induced by 4 Gy 3,-rays decreased with increases in the concentration of cobaltous chloride. At a concentration of 1 x 10- 5 M COC12 • 6H20, -yray-induced mutations were almost completely inhibited. At this concentration of cobaltous chlo-

0.1

~6 0.05

o~

0.01

i lxlO-e

! 3x10 -a

0 lx10 -5

Concentration of COC12. 0 H 2 0 ( M )

Fig. 3. Effect of posttreatment of cobaltous chloride at different concentrations on survival (o) and the frequency of 8AGresistant mutations (w) induced by 4 Gy of "r-rays in Chinese hamster V79 cells.

ride, 4 Gy -/-rays showed only a slight decrease in the survival of V79 cells (Fig. 3).

Pretreatment with cobaitous chloride When cells were incubated in medium containing cobaltous chloride for 6 days before irradiation with -r-rays, the survival of cells was decreased

TABLE 1 EFFECTS OF PRETREATMENT WITH COBALTOUS CHLORIDE ON THE FREQUENCIES OF 8AG-RESISTANT MUTATIONS INDUCED BY ,y-RAYS OR ULTRAVIOLET LIGHT IN CHINESE HAMSTER V79 CELLS Concentration of COC12.6H20 ( x 10-6 M)

Dose of -~-rays or UV

Mean number of surviving colonies per dish

Surviving fraction

Total number of mutant colonies (number of dishes)

Mutation frequency per lOs survivors

0 0 3 3

0 4 0 4

108 73 69 58

1.00 0.68 0.64 0.54

6 (19) 14 (15) 4 (20) 9 (20)

3.2 13.7 3.1 8.3

0 0 3 3

0 100 0 100

87 75 80 62

1.00 0.86 0.92 0.71

0 63 0 46

0 43.0 0 40.8

(19) (20) (17) (16)

103 more than that in irradiated cells without preincubation with cobaltous chloride. The frequency o f 8AG-resistant mutations induced by 7-rays was significantly inhibited by pretreatment with cobaltous chloride (Table 1).

Effects o f cobaltous chloride on UV-irradiated cells Posttreatment with cobaltous chloride Posttreatment with 3 × 10 -~ M COC12.6H20 had no significant effects on survival and frequency of mutations in cells irradiated with UV light. Fig. 4 shows that UV effectively induced 8AGresistant mutations. With 200 e r g / m m 2 of UV, the frequency o f induced mutations was 40 per 10~ survivors, which was similar to the value in cells without posttreatment with cobaltous chloride. The effects o f posttreatment with cobaltous chloride at different concentrations on UV-induced mutations were examined. The survival of cells irradiated with 100 e r g / m m 2 of UV was not

significantly affected by posttreatment with cobaltous chloride up to concentrations of 9 x 10- ~ M (Fig. 5). However, the frequency of UV-induced mutations was slightly enhanced by posttreatment with cobaltous chloride.

Pretreatment with cobaltous chloride Pretreatment with cobaltous chloride at a concentration of 3 x 10-6 M had no significant effects on either cell survival or 8AG-resistant mutations induced by 100 e r g / m m 2 of UV (Table 1).

Discussion Effects of cobaltous chloride were studied in a forward mutation system o f mammalian cells, where genetic changes in the sensitivity to 8AG were examined (Chu and Mailing, 1968; Bridges and Huckle, 1970; Kuroda, 1975a,b). It has been established that v-rays or UV light induce mutations at a locus of hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), whose enzyme

O 1.0 ¸

50

E

1.2

0.~

,to ~

1.0i

t~

.o

a0 .o

.o

60

O

40

.~

0.5

0.1

..

0.05

20

ta

6

r,~

0.1 !

10 ~d

2O 0.05

~.01

g 0 Dose

50 of UV

light

100 (erg/mm

150

200

2)

Fig. 4. Effect of posttreatment of cobaltous chloride at a concentration of 3 x 10-6 M on survival (e,O) and the frequency of 8AG-resistant mutations (i,ta) induced by various doses of UV light in Chinese hamster V79 cells, e,m, no CoCl2.6H20; O,~, 3 x 10-6 M COC12'6H20.

I

I

I

lxl0-O

3xl0-O

6xl0-e

0

9xl0-e

Concentration of CoCI2- 6H20 (M) Fig. 5. Effect of posttreatment of cobaltous chloride at different concentrations on survival (O) and the frequency of 8AGresistant mutations (,,) induced by 100 erg/mm2 of UV light in Chinese hamster V79 cells.

104

activity loss results in resistance to 8AG (Arlett and Potter, 1971; Arlett and Harcourt, 1972). The present studies revealed that cobaltous chloride had an activity as an antimutagen in -y-rayinduced 8AG-resistant mutations in Chinese hamster V79 cells cultured in vitro. This antimutagenic effect was not found in cells irradiated with UV light. With bacteria, antimutagenic effects of cobaltous chloride were found with irradiation both with 7-rays and with UV light (Kada and Kanematsu, 1978; Kada et al., 1979). Results obtained in the present study and other bacterial systems (Inoue et al., 1981) indicate that this metal compound is effective in reducing errors produced in the course of inducible SOS repair (Witkin, 1976; Radman et al., 1977). However, it is not certain whether similar repair processes exist in Chinese hamster cells (Cleaver, 1978) or not. A greater contrast to the findings in bacterial systems is that, in Chinese hamster ceils, the antimutagenicity of cobaltous chloride was observed in cells irradiated with 3~-rays, but not in cells irradiated with UV light. There is evidence to support the idea of SOS repair in X- or 7-ray-induced base-change-type mutations in bacteria (Kada et al., 1966; Bridges and Mottershead, 1978; Bridges and Southworth, 1982). In the present study with mammalian cells, the 8AG-resistant mutations are forward mutations produced by DNA damages leading to the inactivation of HGPRT activity. That the inducibility of mutations in the present mammalian cell system is very dependent on the growth phase of cells (Arlett and Potter, 1971) may indicate the importance of certain factors involved in DNA metabolism such as replication or postreplication repair. Little is known about interference and action sites of cobaltous ions in mammalian ceils. Such ions may certainly modify the specificities of some DNA enzymes by competing with other bi-cationic metal ions. It is known that cobaltous ions form chelations with certain peptides or proteins and modify their functions. Research on the effects of DNA metabolism may help in the elucidation of the antimutagenicity of cobaltous ions in -y-ray-induced mutations in mammalian cells.

Acknowledgements This work was supported in part by grants for basic sciences from the Ministry of Education, the Ministry Of Health and Welfare and the Science and Technology Agency of Japan. Contribution No. 1900 from the National Institute of Genetics, Mishima, Shizuoka 411 (Japan).

References Arlett, C.F., and J. Potter (1971) Mutation to 8-azaguanine resistance induced by -r-radiation in a Chinese hamster cell line, Mutation Res., 13, 59-65. Arlett, C.F., and S.A. Harcourt (1972) Expression time and spontaneous mutability in the estimation of induced mutation frequency following treatment of Chinese hamster cells by ultraviolet light, Mutation Res., 16, 301-306. Bridges, B.A., and J. Huckle (1970) Mutagenesis of cultured mammalian ceils by radiation and ultraviolet light, Mutation Res., 10, 141-151. Bridges, B.A., and R.P. Mottershead (1978) Mutagenic DNA repair in Escherichia coil VIII. Involvement of DNA polymerase III in constitutive and inducible mutagenic repair after ultraviolet and gamma irradiation, Mol. Gen. Genet., 163, 35-41. Bridges, B.A., and M. Southworth (1982) Constitutive errorprone repair at sites of excision repair in Escherichia coli: a re-examination, Biochemie, 64, 655-659. Chu, E.H., and H.Y. Mailing (1968) Mammalian cell genetics. II. Chemical induction of specific locus mutations in Chinese hamster cells in vitro, Proc. Natl. Acad. Sci. (U.S.A.), 61, 1306-1312. Cleaver, J.E. (1978) Absence of interaction between X-rays and UV light in inducing ouabain- and thioguanine-resistant mutants in Chinese hamster cells, Mutation Res., 52, 247-253. Ford, D.K., and G. Yerganian (1958) Observations on the chromosomes of Chinese hamster ceils in tissue culture, J. Natl. Cancer Inst., 21,393-425. Inoue, T., Y. Ohta, Y. Sadaie and T. Kada (1981) Effect of cobaltous chloride on spontaneous mutation induction in a Bacillus subtilis mutator strain, Mutation Res., 91, 41-45. Kada, T., and N. Kanematsu (1978) Reduction of N-methylN'-nitro-N-nitrosoguanidine-induced mutations by cobalt chloride in Escherichia coli, Proc. Jpn. Acad., 54, 234-237. Kada, T., and H. Mochizuki (1981) Antimutagenic activities of human placental extract on ultraviolet light- and gamma-rayinduced mutation in Escherichia coil WP2 B/r trp, J. Radiat. Res., 22, 297-302. Kada, T., C.D. Doudney and F.L. Hass (1966) Mutation frequency response of irradiated bacteria to a second radiation exposure, Mutation Res., 3, 118-128.

105

Kada, T., T. Inoue, A. Yokoiyama and L.B. Russell (1979) Combined genetic effects of chemicals and radiation, in: S. Okada, M. Imamura, T. Terashima and H. Yamaguchi (Eds.), Radiation Research, Proc. 6th Internat. Cong. Radiat. Res., pp. 711-720. Komura, H., H. Minakata, K. Nakanishi, H. Mochizuki and T. Kada (1981) Chemical features of antimutagenic factor in human placenta, 3rd Int. Conf. Environ. Mutagens, 3B18, p. 83 (Abstract). Kuroda, Y. (1975a) Mutagenesis in cultured human diploid ceils. III. Induction of 8-azaguanine-resistant mutations by furylfuramide, Mutation Res., 30, 229-248. Kuroda, Y. (1975b) Mutagenesis in cultured human diploid cells. IV. Induction of 8-azaguanine-resistant mutations by phloxine, a mutagenic red dye, Mutation Res., 30, 239-248. Kuroda, Y., A. Yokoiyama and T. Kada (1985) Assays for the induction of mutations to 6-thioguanine resistance in Chinese hamster V79 cells in culture, in: J. Ashby, F.J. de Serres, M. Ishidate Jr., B.H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, Elsevier, Amsterdam, Oxford, New York, pp. 537-542.

Mochizuki, H., and T. Kada (1982a) Antimutagenic action of cobaltous chloride on Trp-P-l-induced mutations in Salmonella typhimurium TA98 and TA1538, Mutation Res., 95, 145-157. Mochizuki, H., and T. Kada (1982b) Antimutagenic action of mammalian placental extracts on mutations induced in Escherichia coli by UV radiation, y-rays and N-methylN'-nitro-N-nitrosoguanidine, Mutation Res., 95,457-474. Radman, M., G. Villani, S. Boiteux, M. Defais, P. CailletFauquet and S. Spadari (1977) On the mechanism and genetic control of mutagenesis induced by carcinogenic mutagens, in: H.H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Cold Spring Harbor Conferences on Cell Proliferation, Vol. 4, Cold Spring Harbor, NY, pp. 903-922. Witkin, E.M. (1976) Ultraviolet mutagenesis and inducible D N A repair in Escherichia coli, Bacteriol. Rev., 40, 869-907. Communicated by F.H. Sobels