Mutation Research, 264 (1991) 163-170 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-7992/91/$03.50 ADONIS 016579929100099Q
163
MUTLET 0547
Genotoxicity of beryllium, gallium and antimony in short-term assays K o i c h i K u r o d a 1, G i n j i E n d o 2, A k i y o s h i O k a m o t o 1, Y o u n g S. Y o o a a n d Syn'-ichi H o r i g u c h i 2 i Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennofi-ku, Osaka 543, 2Osaka City University Medical School, 1-4-54 Asahi-machi, Abeno-ku, Osaka 545 (Japan) and 3Seoul Health Junior College, 212 Yangji-dong, Soojung-ku, Sungnam-city, Kyunggi-do, 461-250 (Korea)
(Received 19 February 1991) (Revision received 2 June 1991) (Accepted 16 July 1991)
Keywords: Rec assay; SCE; Beryllium;Gallium; Antimony
Summary The genotoxicity of beryllium, gallium and antimony compounds was studied with the rec, Salmonella mutagenicity and SCE assays. In the rec assay, all the salts of the metals, BeC12, Be(NO3)2, GaCl3, Ga(NO3h, SbCl3, SbC15, and an oxide, Sb2Oa, had DNA-damaging activity. None of the compounds was mutagenic to Salmonella. In the SCE assays using V79 cells, 2 antimony(III) compounds, SbCl3 and Sb203, and 2 beryllium compounds, BeCI2 and Be(NO3)2, induced SCEs significantly. Sb203, slightly soluble in water, was positive in both the rec assay and the SCE assay at very low doses.
With the advance of technology, many new metal compounds have been developed and used in industry. The effects of these compounds on workers and on the general population through environmental pollution are of concern (IARC, 1980). Beryllium is considered suspect of being carcinogenic to humans (IARC, 1980), and there are numerous studies on the genotoxicity of beryllium
Correspondence: Dr. Koichi Kuroda, Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennoji-ku, Osaka 543 (Japan).
compounds (reviewed in L6onard and Lauwerys, 1987; Ashby et al., 1990). Gallium and antimony are widely used as materials in alloys; but there are limited data on their genotoxicities (Nishioka, 1975; Kanematsu et al., 1980). We surveyed the genotoxicities of salts, easily soluble in water, and oxides, slightly soluble in water, of beryllium, gallium and antimony with the rec, Salmonella mutagenicity, and SCE assays. The rec assays were done by the spore plate method which is more sensitive than the streak method (Kada et al., 1980). We report here that BeCl2 and Ga(NO3)3 in which Nishioka (1975) and Kanematsu et al. (1980) failed to detect a rec effect had
164 DNA-damaging activity and that an oxide of antimony(Ill), slightly soluble in water, was positive in both the rec assay and the SCE assay at very low doses.
mined by ICP using a Shimadzu ICPS-1000 II (sequential type) inductively coupled plasma emission spectrometer. Although SbCl5 might be hydrolyzed in water, its concentrations in the experiments were described as those of the original compound.
Materials and methods
Metal compounds The test compounds used were 3 beryllium compounds, 3 gallium compounds and 4 antimony compounds. Their purities, sources and solubilities in water are shown in Table 1.50 mg of each compound was dissolved in 1 ml of distilled water, and the solutions were diluted serially 2-fold and used for the assays. All the oxides, BeO, Ga203, S b 2 0 3 , S b 2 0 5 , and a salt, SbCls, were not completely dissolved, and the supernatant fluids of the solutions were used for the experiments. The concentrations of the compounds in the fluids were deter-
Other chemicals and culture medium Negative and positive control compounds in the assays, kanamycin sulfate, mitomycin C, furylfuramide (AF-2) and 2-aminoanthracene, 5-bromodeoxyuridine (BudR) and colcemid were purchased from Wako Pure Chemicals (Osaka, Japan). $9 and cofactors for the $9 mix were obtained from Oriental Yeast Company (Tokyo, Japan). Eagle's MEM and fetal calf serum were obtained from Flow Laboratories (U.S.A.). Short-term assays Rec assays were conducted as described by Kada
TABLE 1 SOLUBILITIES, PURITIES AND SOURCES OF THE METAL COMPOUNDS Compound CAS No.
Source
Purity
Solubility in water a
BeCl2 7787-47-5 Be(NO3)2 13597-99-4 BeO 1304-56-9
High Purity Chem., Saitama, Japan Mitsuwa's Pure Chem., Osaka, Japan Mitsuwa's Pure Chem., Osaka, Japan
99070 min.
easily soluble
52070 rain.
easily soluble
99.5-99.9070
4.3/Lg/ml
GaCI3 13450-90-3 Ga(NO3h- 5H20 13494-90-1 Ga203 12024-21-4
Nacalai Tesque, Kyoto, Japan Nacalai Tesque, Kyoto, Japan Nacalai Tesque, Kyoto, Japan
99.9999070
easily soluble
99070
easily soluble
99.99070
35.2/zg/ml
SbC13 10025-91-9 Sb203 1309-64-4 SbCl5 7647-18-9 Sb205 1314-60-9
Nacalai Tesque, Kyoto, Japan Nacalai Tesque, Kyoto, Japan Nacalai Tesque, Kyoto, Japan Mitsuwa's Pure Chem., Osaka, Japan
980/0
easily soluble
99.999%
17.1 /zg/ml
99.999070
8.64 mg/ml b
99.9070
2.0 mg/ml
a Concentration in supernatant fluid of saturated solution. b Concentrations in the experiments were described as the original compound, although the compound might be hydrolyzed in water.
165 et al. (1980), using the spores of Bacillus subtilis M45(rec-) and H17(rec+). The diameter of the paper disk used was 8 mm.
Salmonella mutagenicity assays were carried out by the preincubation modification (Yahagi et al., 1977) at 37°C, 20 min, using TA100 and TA98.
TABLE 2 Rec ASSAY OF T H E METAL C O M P O U N D S Metal
Dose
Killing zone (mm)
compound
(#g/disk)
M45
BeC12
375 750 1,500
10.5 15.5 22
10.0 14.5 20.5
0.5 1.0 1.5
Be(NO3)2
375 750 1,500
_a 12.5 17.5
_a 10.0 16.0
2.5 1.5
_a
_a
2,350 4,700 9,400
16.5 22.0 29.0
14.5 20.0 25.5
2.0 2.0 3.5
3,200 6,400 12,000
18.5 25.0 31.0
16.5 22.0 28.0
2.0 3.0 3.0
_a
a
BeO c GaCI3
Ga(NO3)3' 5H20
Ga203 c
0.1
1.1
H17
M45 - H 1 7 (mm)
SbC13
6.3 12.5 23
16.5 25.0 25.5
15.0 b 20.5 b 21.0 b
1.5 4.5 4.5
Sb203 c
0.3 0.6 1.1
13.5 15.0 15.5
ll.0 11.0 11.0
2.5 4.0 4.5
SbCI5
65 130 260
21.0 30.0 39.5
16.0 26.0 35.0
5.0 4.0 4.5
5b205 c
60
_a
a
Kanamycln (negative control)
5 10 20
20.0 23.0 24.5
20.0 23.0 24.0
0 0 0.5
0.05 0.1 0.2
24.0 28.0 32.0
16.0 20.0 25.0
8.0 8.0 7.0
Mitomycin C (positive control)
a Killing zone was not observed. b Many microcolonies were observed in killing zones. c Maximal dose was 30/zl o f the supernatant fluids o f the saturated solutions.
166 The assays were conducted in the presence and absence o f rat liver $9 mix (10070). The results were averages o f duplicate plates. Survivals after the preincubation step were counted on glucose minimum medium plates supplemented with histidine and biotin (Maron and Ames, 1983). SCE assays were carried out using V79 Chinese hamster cells obtained f r o m Flow Laboratories (U.S.A.). The cells were grown in Eagle's M E M supplemented with kanamycin sulfate (100 #g/ml) and 7070 fetal calf serum, at 37°C in a 5070 CO2 atmosphere. The assay procedure was as follows (Kuroda et al., 1985). Cells (5 ml) were grown in a plastic petri dish (Terumo, Osaka, Japan, 6 m m diameter) for 24 h. Then 100 or 50/~l of various concentrations of the compounds in solution was added with BudR (1/zg/ml) and incubated for 28 h in the dark. For the last 2 h, colcemid was added. The cells were treated with 0.075 M KCI for 7 min, fixed with ethanol and acetic acid (3:1), and stained by the modified F P G method. SCEs were scored in 20 well-stained metaphases containing 22 chromosomes. The statistical evaluation was done by Student's t-test.
Results
and discussion
Rec assay In the rec assay, a strong positive rec effect (a difference in the diameter o f the killing zones in t h e M45 plate and in the H17 plate larger than 4 mm) was noted with SbCI3, Sb203 and SbCIs. A mild positive rec effect, with a difference of 1 - 4 m m , was noted with BeCI2, Be(NOD2, GaCI3 and Ga(NO3)3 (Table 2). Kanematsu et al. (1980) and Nishioka (1975) failed to detect a rec effect with BeCI2 and Ga(NO3)3 by the streak method using vegetative organisms. The compounds producing a mild rec effect in these experiments must have DNA-damaging effects because their differences were always plus, while those of kanamycin, used as a negative control, were almost zero. As to SbCI3 and SbC15, contradictory results have been reported. Nishioka (1975) observed no rec effect, and Kanematsu et al. (1980) detected only mild rec effects with them. In our experiments, the compounds had a strong rec effect. With SbC13, m a n y microcolonies were observed in the killing zones of H I 7 spore plates. Therefore, B.
TABLE 3 MINIMAL INHIBITORY DOSES OF THE METAL COMPOUNDS Metal c o m p o u n d
IN T H E S A L M O N E L L A M U T A G E N I C I T Y
M i n i m a l i n h i b i t o r y dose Q~g/plate) a T A 100
TA98
- $9
+ $9
- $9
+ $9
BeC12
> 5,000 c
> 5,000 c
>
5,000 c
>
Be(NO3)2
> 5,000 c
> 5,000 c
>
5,000 c
> 5,000 c
BeO b
>
0.43 c
>
>
>
5,000 4,000 3.52c
10,000 8,000 > 3.52c
5,000
>
GaCl3 Ga(NO3)3"5H20 Ga203 b SbCI3
Sb203 b
SbCi5 Sb205b
ASSAY
>
>
1.71 ~
864 200~
>
> >
0.43 c
5,000 4,000 > 3.52c
5,000 ~ 1.71 ~
864~ 200~
0.43 ~
5,000 >
>
1.71 ~
864 200~
>
5,000 c
0.43 ~
10,000 > 8,000c > 3.52~ > >
>
5,000 c 1.71 ~
864 200~
a At these doses, the survivals after the preincubation step were less than 10070 of solvent control (water I00/A/plate). b Maximal dose was 100 ~1 of the supernatant fluids of the saturated solutions per plate. c At the maximal doses, the survivals after the preincubation step were greater than 10070 of solvent control (water I00 ~l/plate).
167 TABLE 4 SALMONELLA MUTAGEN1CITY ASSAY OF METAL C O M P O U N D S Metal
Dose
Revertants per plate a
compound
(~g/plate)
TA 100 - $9 mix
TA98 + $9 mix
- $9 mix
+ $9 mix
BeCI2
313 625 1,250 2,500 5,000
129 127 124 127 131
NT c 169 165 161 141
25 23 27 30 29
NT ~ 22 24 26 24
Be(NO3)2
1,250 2,500 5,000
127 126 !15
166 173 176
22 25 25
33 23 27
BeO b
0.11 0.22 0.44
132 135 137
191 175 171
21 16 18
23 23 28
GaCIs
313 625 1,250 2,500 5,000 10,000
159 136 124 96 88 0
NT ¢ 189 176 150 159 0
30 27 29 13 0 0
NT c 25 25 23 30 1
250
145
NT c
25
NT c
500 1,000 2,000 4,000 8,000
132 99 75 0 0
169 170 156 175 0
33 33 29 0 0
21 26 24 29 19
Ga203
319 638 1,275 2,550 5,100
129 140 135 174 148
NT ¢ 158 147 168 167
NT ¢ 24 22 19 18
NT ¢ 25 20 25 26
SbCI3
625 1,250 2,500 5,000
148 164 120 76
178 181 167 0
23 22 25 7
31 30 23 26
Sb203 b
0.43 0.86 1.71
123 110 128
162 169 159
19 26 28
27 24 31
SbCI5 b
54 108 216 432 864
161 153 104 110 0
NT ¢ 166 176 158 127
20 18 25 23 0
NT c 25 34 32 12
Ga(NO3)3 • 5H20
168 TABLE 4 (continued) Metal
Dose
compound
(#g/plate)
Sb205 b
50 100 200 100#1
Water Furylfuramide
2-Amino-anthracene
Revertants per plate a
0.01 0.1
TA100
TA98
- $9 mix
+ $9 mix
- $9 mix
+ $9 mix
131 134 99
151 169 175
16 26 24
28 30 24
129
177
27
25
510
1 0.5
937 416 357
a The results are averaged of duplicate plates. b Maximal dose was 100/~1 of the supernatant fluids of the saturated solutions per plate. c NT, not tested. The assays were repeated partially 2 or 3 times. A significant increase of revertants was not observed.
subtilis might easily become resistant to the compound, and give contradictory results when tested by the streak method. These results suggest that the spore plate method is much more sensitive than the streak method using vegetative bacteria, and should be used, at least for metal compounds. BeO, Ga203, and Sb205 did not produce killing zones. We could not eliminate their DNA-damaging activity because the oxides were only slightly soluble in water, and their concentrations in the assays might not be enough to produce killing zones. Sb203, however, is also slightly soluble and had a strong rec effect at very low doses. The compound has high DNA-damaging activity. Salmonella mutagenicity assay In the Salmonella mutagenicity assays, it is essential that the survivals in the plates are not reduced too much by the test compounds. The minimal inhibitory doses o f the compounds were determined by counting survivals of TAI00 and TA98 in both the presence and absence of $9 mix after preincubation. At the doses employed, the survivals were less than 10°70 of solvent control (water 100 ~d/plate). The minimal inhibitory doses were determined with only 4 compounds, GaCI3,
Ga(NO3h, SbCI3 and SbCI5 (Table 3). Toxicity of Ga and Sb compounds tended to decrease in the presence of $9 mix. The assays were done through the range from the maximal dose in Table 3, to 1/8 or 1/32 diluted dose. None of the compounds increased the number of revertants significantly. The results of a single typical experiment are shown in Table 4. The assays were repeated partially 2 or 3 times. Be(NO3)2 (Tso and Fung, 1981), SbCI3 and Sb203 (Kanematsu et al., 1980) were not mutagenic to Salmonella. Why do the compounds which have strong or mild rec effects not cause mutation in Salmonella? There are 2 possibilities. One is that the cell wall of Salmonella, Gram-negative, is less permeable to these compounds than that of Bacillus, Grampositive, even though TA100 and TA98 have the rfa mutation (Ames et al., 1973). The compounds were more toxic to the Bacillus than tO the Salmonella, bacause the water-soluble compounds produced large killing zones in the rec assays but were not or only slightly toxic in the Salmonella assays (Tables 2, 3). It is well known that some kinds of substances do not permeate through the cell walls of Gram-negative bacteria, but do through those of Gram-positive bacteria (Nikaido and Vaara, 1985).
169 The other possibility is that the mutagenic range of these metal compounds may be very close to the toxic range. The range of Cr(VI) causing mutations in Salmonella was very close to the range kiliing the organisms (Petrilli and De Flora, 1977). Nishioka (1975) found mutagenicity of NaAsO2, K2Cr207, and (NH4)6Mo7024 in E. coli at the concentration in which the survival was about 40% of the control. Marzin and Phi (1985) studied the mutagenicity of several metal compounds with TA102 which is much more sensitive to Cr(VI) compounds than TA98 or TA100. The strain, however, gave the
same qualitative results as other Salmonella strains. It may be true that the metal compounds positive in the rec assays did not or only slightly permeate through the cell walls and did not really cause mutation to Salmonella, even though the latter possibility is not eliminated. S C E assay
In the SCE assays the Sb(III) compounds, SbCI3 and Sb203, and the Be compounds, BeCI2 and Be(NO3)2, induced SCEs significantly (Table 5).
TABLE 6 TABLE 5
METAL COMPOUNDS NEGATIVE IN T H E SCE ASSAY
METAL C O M P O U N D S POSITIVE IN T H E SCE ASSAY
Metal compound
Metal compound
BeCI2
Be(NO3)2
SbC13
56203 c
Water Mitomycin C
Concentration
SCEs/metaphase
(#g/ml)
Expt. 1 (Mean a +_SD)
Expt. 2 (Mean a +_SD)
31 63 125 250
6.2+1.8"* 7.1+_2.5"* 7.2+-2.3** Toxic b
7.8+_3.4 8.5+_3.7* 9.1+_2.8"* 10.2 _+2.8**
31 63 125 250 500
7.0+-3.2* 7.4+2.7** 8.7+_3.2** 7.3+-3.0** Toxic b
6.3_+2.2 8.2+_2.2* 8.0+- 1.8" 7.6+_2.9 Toxic b
5.3_+2.1 7.5+4.3* 8.0:1:4.0"* 11.8-+4.0"* Toxic b
7.3___3.0 6.2+ 1.6 8.6+-2.9** 9.7_+4.4** Toxic b
6.0_+2.3 8.2-+5.8* 12.4-+6.6"*
10.6_+3.7"* 9.0-+3.7** 14.6+_6.3"*
1.3 2.5 5 10 20 0.09 0.17 0.34 100 ttl 0.01
4.5 + 2.2
6.3 + 2.5
46.8+_8.6**
56.0+-9.3**
a Mean of SCEs in 20 metaphases. b Metaphases were very few. c Maximal concentration was 20 t~l of the supernatant fluids of the saturated solutions per ml of culture medium. *'**Significantly different from the value of solvent control (water 100 p.l/ml), p < 0.05 and p < 0.1, respectively. The assays were repeated partially 3 or 4 times.
BeO c
Dose (ttg/ml)
SCEs/metaphase (Mean" _+SD)
0.02
4.5 +__1.7
0.04 0.09
4.8 + 2.5 4.7 +- 2.0
GaCI3
12.5 25 50 100
5.6_+2.0 4.4 + 2.5 4.9 +2.2 Toxic b
Ga(NO3)3.5H20
50 100 200 400
4.5_+2.2 5.0_+2.5 4.5 _+2.0 Toxic b
Ga203 ~
0.18 0.35 0.7
5.0_+2.4 4.7_+2.2 4.6_+2.4
SbCIs
8.6 17.3 35 70
4.3 _+ 1.7 3.4_+ 1.3 4.4 _+ 1.3 Toxic b
Sb205 c
10 20 40
4.9+_2.6 5.1_+2.3 4.0_+2.1
Water
50 #1
4.9+ 1.6
a Mean of SCEs in 20 metaphases. b Metaphases were very few. c Maximal concentration was 20/~l of the supernatant fluids of the saturated solutions per ml of culture medium. The assays were repeated partially 2 or 3 times. A significant increase of SCEs was not observed.
170
The SCE-inducing ability of the compounds was confirmed by repeating the assays partially 3 or 4 times. SbzO~ induced the most SCEs among them, more than twice the number of the spontaneous SCEs, at the highest doses. They were, however, weaker SCE inducers and had less dose dependence than organic mutagens and/or carcinogens (Perry and Evans, 1975; Wolff, 1977). BeSO4 is also a weak inducer in human lymphocytes and in Syrian hamster cells (Larramendy et al., 1981). Ashby et al. (1990) studied the genotoxicity of BeSO4 by several assays in vivo and in vitro, and concluded that the compound was negative in 3 standard assays, the Salmonella mutation assay, the chromosome aberration assay, and the micronucleus assay. SCE gives few false-positive results for mutagenic carcinogens (Latt et al., 1981). Antimony(III) compounds and beryllium compounds gave positive results in both the rec assay and the SCE assay. Further investigations of genotoxicity and carcinogenicity should be done on these compounds, especially on Sb203 which is slightly water-soluble and was positive at very low doses. Data for SCE-negative compounds are shown in Table 6. The slightly soluble compounds BeO, Ga203 and 5b205 did not show toxicity at the highest concentrations.
Acknowledgements We are grateful to T. Tsujinoue and I. Kubo, students at Osaka Pharmacological College, for help. We also greatly appreciate the critical reviewing of the manuscript and suggestions offered by Dr. D.M. Shankel, University of Kansas. This study was partly supported by a Grant-in-Aid for Scientific Research, No. 63304038, from the Ministry of Education, Science and Culture of Japan.
References Ames, B.N., F.D. Lee and W.E. Durston (1973) An improved bacterial test system for the detection and classification of mutagens and carcinogens, Proc. Natl. Acad. Sci. (U.S.A.), 70, 782-786.
Ashby, J.M., M. lshidate Jr., G.D. Stoner, M.A. Morgan, F. Ratpan and R.D. Callander (1990) Studies of the genotoxicity of beryllium sulfate in vitro and in vivo, Mutation Res., 240, 217-225. IARC (1980) Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 23, International Agency for Research on Cancer, Lyon, pp. 33-35, 143-204. Kada, T., K. Hirano and Y. Shirasu (1980) Screening of environmental chemical mutagens by the rec-assay system with Bacillus subtilis, in: F.J. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Vol. 6, Plenum, New York, pp. 149-173. Kanematsu, N., M. Hara and T. Kada 0980) Rec assay and mutagenicity studies on metal compounds, Mutation Res., 77, 109-116. Kuroda, K., K. Haruki, Y.S. Yoo and M. Oka (1985) Airborne particulate; cytotoxicity in HeLa cells and the induction of sister chromatid exchange in Chinese hamster Don cells, J. Japan Soc. Air Pollut., 20, 342-348. Larramendy, M.L., N.C. Popescu and J.A. DiPaolo (1981) Induction by inorganic metal salts of sister chromatid exchanges and chromosome aberrations in human and Syrian hamster strains, Environ. Mutagen., 3, 597-606. Latt, S.A., J. Allen, S.E. Bloom, A. Carrano, E. Falke, D. Kram, E. Schneider, R. Schreck, R. Tice, B. Whitfield and S. Wolff (1981) Sister-chromatid exchanges: a report of the Gene-Tox Program, Mutation Res., 87, 17-62. L6onard, A., and R. Lauwerys (1987) Mutagenicity, carcinogenicity and teratogenicity of beryllium, Mutation Res., 186, 35-42. Maron, D.M., and B.N. Ames (1983) Revised method for the Salmonella mutagenicity test, Mutation Res., 54, 139-147. Marzin, D.R., and H.O. Phi (1985) Study of the mutagenicity of metal derivatives with Salmonella typhimurium TA102, Mutation Res., 155, 49-51. Nikaido, H., and M. Vaara (1985) Molecular basis of bacterial outer membrane permeability, Microbiol. Rev., 49, 1-32. Nishioka, H. (1975) Mutagenic activities of metal compounds in bacteria, Mutation Res., 31, 185-189. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange, Nature (London), 258, 121-125. Petrilli, F.L., and S. De Flora (1977) Toxicity and mutagenicity of hexavalent chromium on Salmonella typhimurium, Appl. Environ. Microbiol., 33,805-809. Tso, W., and W. Fung (1981) Mutagenicity of metal cations, Toxicol. Lett., 8, 195-200. Wolff, S. (1977) Sister chromatid exchange, Annu. Rev. Genet., 11, 183-201. Yahagi, T., M. Nagao, Y. Seino, T. Matsushima, T. Sugimura and M. Okada (1977) Mutagenicities of N-nitrosoamines on Salmonella, Mutation Res., 48, 121-130. Communicated by D.M. Shankel
163
MUTLET 0547
Genotoxicity of beryllium, gallium and antimony in short-term assays K o i c h i K u r o d a 1, G i n j i E n d o 2, A k i y o s h i O k a m o t o 1, Y o u n g S. Y o o a a n d Syn'-ichi H o r i g u c h i 2 i Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennofi-ku, Osaka 543, 2Osaka City University Medical School, 1-4-54 Asahi-machi, Abeno-ku, Osaka 545 (Japan) and 3Seoul Health Junior College, 212 Yangji-dong, Soojung-ku, Sungnam-city, Kyunggi-do, 461-250 (Korea)
(Received 19 February 1991) (Revision received 2 June 1991) (Accepted 16 July 1991)
Keywords: Rec assay; SCE; Beryllium;Gallium; Antimony
Summary The genotoxicity of beryllium, gallium and antimony compounds was studied with the rec, Salmonella mutagenicity and SCE assays. In the rec assay, all the salts of the metals, BeC12, Be(NO3)2, GaCl3, Ga(NO3h, SbCl3, SbC15, and an oxide, Sb2Oa, had DNA-damaging activity. None of the compounds was mutagenic to Salmonella. In the SCE assays using V79 cells, 2 antimony(III) compounds, SbCl3 and Sb203, and 2 beryllium compounds, BeCI2 and Be(NO3)2, induced SCEs significantly. Sb203, slightly soluble in water, was positive in both the rec assay and the SCE assay at very low doses.
With the advance of technology, many new metal compounds have been developed and used in industry. The effects of these compounds on workers and on the general population through environmental pollution are of concern (IARC, 1980). Beryllium is considered suspect of being carcinogenic to humans (IARC, 1980), and there are numerous studies on the genotoxicity of beryllium
Correspondence: Dr. Koichi Kuroda, Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennoji-ku, Osaka 543 (Japan).
compounds (reviewed in L6onard and Lauwerys, 1987; Ashby et al., 1990). Gallium and antimony are widely used as materials in alloys; but there are limited data on their genotoxicities (Nishioka, 1975; Kanematsu et al., 1980). We surveyed the genotoxicities of salts, easily soluble in water, and oxides, slightly soluble in water, of beryllium, gallium and antimony with the rec, Salmonella mutagenicity, and SCE assays. The rec assays were done by the spore plate method which is more sensitive than the streak method (Kada et al., 1980). We report here that BeCl2 and Ga(NO3)3 in which Nishioka (1975) and Kanematsu et al. (1980) failed to detect a rec effect had
164 DNA-damaging activity and that an oxide of antimony(Ill), slightly soluble in water, was positive in both the rec assay and the SCE assay at very low doses.
mined by ICP using a Shimadzu ICPS-1000 II (sequential type) inductively coupled plasma emission spectrometer. Although SbCl5 might be hydrolyzed in water, its concentrations in the experiments were described as those of the original compound.
Materials and methods
Metal compounds The test compounds used were 3 beryllium compounds, 3 gallium compounds and 4 antimony compounds. Their purities, sources and solubilities in water are shown in Table 1.50 mg of each compound was dissolved in 1 ml of distilled water, and the solutions were diluted serially 2-fold and used for the assays. All the oxides, BeO, Ga203, S b 2 0 3 , S b 2 0 5 , and a salt, SbCls, were not completely dissolved, and the supernatant fluids of the solutions were used for the experiments. The concentrations of the compounds in the fluids were deter-
Other chemicals and culture medium Negative and positive control compounds in the assays, kanamycin sulfate, mitomycin C, furylfuramide (AF-2) and 2-aminoanthracene, 5-bromodeoxyuridine (BudR) and colcemid were purchased from Wako Pure Chemicals (Osaka, Japan). $9 and cofactors for the $9 mix were obtained from Oriental Yeast Company (Tokyo, Japan). Eagle's MEM and fetal calf serum were obtained from Flow Laboratories (U.S.A.). Short-term assays Rec assays were conducted as described by Kada
TABLE 1 SOLUBILITIES, PURITIES AND SOURCES OF THE METAL COMPOUNDS Compound CAS No.
Source
Purity
Solubility in water a
BeCl2 7787-47-5 Be(NO3)2 13597-99-4 BeO 1304-56-9
High Purity Chem., Saitama, Japan Mitsuwa's Pure Chem., Osaka, Japan Mitsuwa's Pure Chem., Osaka, Japan
99070 min.
easily soluble
52070 rain.
easily soluble
99.5-99.9070
4.3/Lg/ml
GaCI3 13450-90-3 Ga(NO3h- 5H20 13494-90-1 Ga203 12024-21-4
Nacalai Tesque, Kyoto, Japan Nacalai Tesque, Kyoto, Japan Nacalai Tesque, Kyoto, Japan
99.9999070
easily soluble
99070
easily soluble
99.99070
35.2/zg/ml
SbC13 10025-91-9 Sb203 1309-64-4 SbCl5 7647-18-9 Sb205 1314-60-9
Nacalai Tesque, Kyoto, Japan Nacalai Tesque, Kyoto, Japan Nacalai Tesque, Kyoto, Japan Mitsuwa's Pure Chem., Osaka, Japan
980/0
easily soluble
99.999%
17.1 /zg/ml
99.999070
8.64 mg/ml b
99.9070
2.0 mg/ml
a Concentration in supernatant fluid of saturated solution. b Concentrations in the experiments were described as the original compound, although the compound might be hydrolyzed in water.
165 et al. (1980), using the spores of Bacillus subtilis M45(rec-) and H17(rec+). The diameter of the paper disk used was 8 mm.
Salmonella mutagenicity assays were carried out by the preincubation modification (Yahagi et al., 1977) at 37°C, 20 min, using TA100 and TA98.
TABLE 2 Rec ASSAY OF T H E METAL C O M P O U N D S Metal
Dose
Killing zone (mm)
compound
(#g/disk)
M45
BeC12
375 750 1,500
10.5 15.5 22
10.0 14.5 20.5
0.5 1.0 1.5
Be(NO3)2
375 750 1,500
_a 12.5 17.5
_a 10.0 16.0
2.5 1.5
_a
_a
2,350 4,700 9,400
16.5 22.0 29.0
14.5 20.0 25.5
2.0 2.0 3.5
3,200 6,400 12,000
18.5 25.0 31.0
16.5 22.0 28.0
2.0 3.0 3.0
_a
a
BeO c GaCI3
Ga(NO3)3' 5H20
Ga203 c
0.1
1.1
H17
M45 - H 1 7 (mm)
SbC13
6.3 12.5 23
16.5 25.0 25.5
15.0 b 20.5 b 21.0 b
1.5 4.5 4.5
Sb203 c
0.3 0.6 1.1
13.5 15.0 15.5
ll.0 11.0 11.0
2.5 4.0 4.5
SbCI5
65 130 260
21.0 30.0 39.5
16.0 26.0 35.0
5.0 4.0 4.5
5b205 c
60
_a
a
Kanamycln (negative control)
5 10 20
20.0 23.0 24.5
20.0 23.0 24.0
0 0 0.5
0.05 0.1 0.2
24.0 28.0 32.0
16.0 20.0 25.0
8.0 8.0 7.0
Mitomycin C (positive control)
a Killing zone was not observed. b Many microcolonies were observed in killing zones. c Maximal dose was 30/zl o f the supernatant fluids o f the saturated solutions.
166 The assays were conducted in the presence and absence o f rat liver $9 mix (10070). The results were averages o f duplicate plates. Survivals after the preincubation step were counted on glucose minimum medium plates supplemented with histidine and biotin (Maron and Ames, 1983). SCE assays were carried out using V79 Chinese hamster cells obtained f r o m Flow Laboratories (U.S.A.). The cells were grown in Eagle's M E M supplemented with kanamycin sulfate (100 #g/ml) and 7070 fetal calf serum, at 37°C in a 5070 CO2 atmosphere. The assay procedure was as follows (Kuroda et al., 1985). Cells (5 ml) were grown in a plastic petri dish (Terumo, Osaka, Japan, 6 m m diameter) for 24 h. Then 100 or 50/~l of various concentrations of the compounds in solution was added with BudR (1/zg/ml) and incubated for 28 h in the dark. For the last 2 h, colcemid was added. The cells were treated with 0.075 M KCI for 7 min, fixed with ethanol and acetic acid (3:1), and stained by the modified F P G method. SCEs were scored in 20 well-stained metaphases containing 22 chromosomes. The statistical evaluation was done by Student's t-test.
Results
and discussion
Rec assay In the rec assay, a strong positive rec effect (a difference in the diameter o f the killing zones in t h e M45 plate and in the H17 plate larger than 4 mm) was noted with SbCI3, Sb203 and SbCIs. A mild positive rec effect, with a difference of 1 - 4 m m , was noted with BeCI2, Be(NOD2, GaCI3 and Ga(NO3)3 (Table 2). Kanematsu et al. (1980) and Nishioka (1975) failed to detect a rec effect with BeCI2 and Ga(NO3)3 by the streak method using vegetative organisms. The compounds producing a mild rec effect in these experiments must have DNA-damaging effects because their differences were always plus, while those of kanamycin, used as a negative control, were almost zero. As to SbCI3 and SbC15, contradictory results have been reported. Nishioka (1975) observed no rec effect, and Kanematsu et al. (1980) detected only mild rec effects with them. In our experiments, the compounds had a strong rec effect. With SbC13, m a n y microcolonies were observed in the killing zones of H I 7 spore plates. Therefore, B.
TABLE 3 MINIMAL INHIBITORY DOSES OF THE METAL COMPOUNDS Metal c o m p o u n d
IN T H E S A L M O N E L L A M U T A G E N I C I T Y
M i n i m a l i n h i b i t o r y dose Q~g/plate) a T A 100
TA98
- $9
+ $9
- $9
+ $9
BeC12
> 5,000 c
> 5,000 c
>
5,000 c
>
Be(NO3)2
> 5,000 c
> 5,000 c
>
5,000 c
> 5,000 c
BeO b
>
0.43 c
>
>
>
5,000 4,000 3.52c
10,000 8,000 > 3.52c
5,000
>
GaCl3 Ga(NO3)3"5H20 Ga203 b SbCI3
Sb203 b
SbCi5 Sb205b
ASSAY
>
>
1.71 ~
864 200~
>
> >
0.43 c
5,000 4,000 > 3.52c
5,000 ~ 1.71 ~
864~ 200~
0.43 ~
5,000 >
>
1.71 ~
864 200~
>
5,000 c
0.43 ~
10,000 > 8,000c > 3.52~ > >
>
5,000 c 1.71 ~
864 200~
a At these doses, the survivals after the preincubation step were less than 10070 of solvent control (water I00/A/plate). b Maximal dose was 100 ~1 of the supernatant fluids of the saturated solutions per plate. c At the maximal doses, the survivals after the preincubation step were greater than 10070 of solvent control (water I00 ~l/plate).
167 TABLE 4 SALMONELLA MUTAGEN1CITY ASSAY OF METAL C O M P O U N D S Metal
Dose
Revertants per plate a
compound
(~g/plate)
TA 100 - $9 mix
TA98 + $9 mix
- $9 mix
+ $9 mix
BeCI2
313 625 1,250 2,500 5,000
129 127 124 127 131
NT c 169 165 161 141
25 23 27 30 29
NT ~ 22 24 26 24
Be(NO3)2
1,250 2,500 5,000
127 126 !15
166 173 176
22 25 25
33 23 27
BeO b
0.11 0.22 0.44
132 135 137
191 175 171
21 16 18
23 23 28
GaCIs
313 625 1,250 2,500 5,000 10,000
159 136 124 96 88 0
NT ¢ 189 176 150 159 0
30 27 29 13 0 0
NT c 25 25 23 30 1
250
145
NT c
25
NT c
500 1,000 2,000 4,000 8,000
132 99 75 0 0
169 170 156 175 0
33 33 29 0 0
21 26 24 29 19
Ga203
319 638 1,275 2,550 5,100
129 140 135 174 148
NT ¢ 158 147 168 167
NT ¢ 24 22 19 18
NT ¢ 25 20 25 26
SbCI3
625 1,250 2,500 5,000
148 164 120 76
178 181 167 0
23 22 25 7
31 30 23 26
Sb203 b
0.43 0.86 1.71
123 110 128
162 169 159
19 26 28
27 24 31
SbCI5 b
54 108 216 432 864
161 153 104 110 0
NT ¢ 166 176 158 127
20 18 25 23 0
NT c 25 34 32 12
Ga(NO3)3 • 5H20
168 TABLE 4 (continued) Metal
Dose
compound
(#g/plate)
Sb205 b
50 100 200 100#1
Water Furylfuramide
2-Amino-anthracene
Revertants per plate a
0.01 0.1
TA100
TA98
- $9 mix
+ $9 mix
- $9 mix
+ $9 mix
131 134 99
151 169 175
16 26 24
28 30 24
129
177
27
25
510
1 0.5
937 416 357
a The results are averaged of duplicate plates. b Maximal dose was 100/~1 of the supernatant fluids of the saturated solutions per plate. c NT, not tested. The assays were repeated partially 2 or 3 times. A significant increase of revertants was not observed.
subtilis might easily become resistant to the compound, and give contradictory results when tested by the streak method. These results suggest that the spore plate method is much more sensitive than the streak method using vegetative bacteria, and should be used, at least for metal compounds. BeO, Ga203, and Sb205 did not produce killing zones. We could not eliminate their DNA-damaging activity because the oxides were only slightly soluble in water, and their concentrations in the assays might not be enough to produce killing zones. Sb203, however, is also slightly soluble and had a strong rec effect at very low doses. The compound has high DNA-damaging activity. Salmonella mutagenicity assay In the Salmonella mutagenicity assays, it is essential that the survivals in the plates are not reduced too much by the test compounds. The minimal inhibitory doses o f the compounds were determined by counting survivals of TAI00 and TA98 in both the presence and absence of $9 mix after preincubation. At the doses employed, the survivals were less than 10°70 of solvent control (water 100 ~d/plate). The minimal inhibitory doses were determined with only 4 compounds, GaCI3,
Ga(NO3h, SbCI3 and SbCI5 (Table 3). Toxicity of Ga and Sb compounds tended to decrease in the presence of $9 mix. The assays were done through the range from the maximal dose in Table 3, to 1/8 or 1/32 diluted dose. None of the compounds increased the number of revertants significantly. The results of a single typical experiment are shown in Table 4. The assays were repeated partially 2 or 3 times. Be(NO3)2 (Tso and Fung, 1981), SbCI3 and Sb203 (Kanematsu et al., 1980) were not mutagenic to Salmonella. Why do the compounds which have strong or mild rec effects not cause mutation in Salmonella? There are 2 possibilities. One is that the cell wall of Salmonella, Gram-negative, is less permeable to these compounds than that of Bacillus, Grampositive, even though TA100 and TA98 have the rfa mutation (Ames et al., 1973). The compounds were more toxic to the Bacillus than tO the Salmonella, bacause the water-soluble compounds produced large killing zones in the rec assays but were not or only slightly toxic in the Salmonella assays (Tables 2, 3). It is well known that some kinds of substances do not permeate through the cell walls of Gram-negative bacteria, but do through those of Gram-positive bacteria (Nikaido and Vaara, 1985).
169 The other possibility is that the mutagenic range of these metal compounds may be very close to the toxic range. The range of Cr(VI) causing mutations in Salmonella was very close to the range kiliing the organisms (Petrilli and De Flora, 1977). Nishioka (1975) found mutagenicity of NaAsO2, K2Cr207, and (NH4)6Mo7024 in E. coli at the concentration in which the survival was about 40% of the control. Marzin and Phi (1985) studied the mutagenicity of several metal compounds with TA102 which is much more sensitive to Cr(VI) compounds than TA98 or TA100. The strain, however, gave the
same qualitative results as other Salmonella strains. It may be true that the metal compounds positive in the rec assays did not or only slightly permeate through the cell walls and did not really cause mutation to Salmonella, even though the latter possibility is not eliminated. S C E assay
In the SCE assays the Sb(III) compounds, SbCI3 and Sb203, and the Be compounds, BeCI2 and Be(NO3)2, induced SCEs significantly (Table 5).
TABLE 6 TABLE 5
METAL COMPOUNDS NEGATIVE IN T H E SCE ASSAY
METAL C O M P O U N D S POSITIVE IN T H E SCE ASSAY
Metal compound
Metal compound
BeCI2
Be(NO3)2
SbC13
56203 c
Water Mitomycin C
Concentration
SCEs/metaphase
(#g/ml)
Expt. 1 (Mean a +_SD)
Expt. 2 (Mean a +_SD)
31 63 125 250
6.2+1.8"* 7.1+_2.5"* 7.2+-2.3** Toxic b
7.8+_3.4 8.5+_3.7* 9.1+_2.8"* 10.2 _+2.8**
31 63 125 250 500
7.0+-3.2* 7.4+2.7** 8.7+_3.2** 7.3+-3.0** Toxic b
6.3_+2.2 8.2+_2.2* 8.0+- 1.8" 7.6+_2.9 Toxic b
5.3_+2.1 7.5+4.3* 8.0:1:4.0"* 11.8-+4.0"* Toxic b
7.3___3.0 6.2+ 1.6 8.6+-2.9** 9.7_+4.4** Toxic b
6.0_+2.3 8.2-+5.8* 12.4-+6.6"*
10.6_+3.7"* 9.0-+3.7** 14.6+_6.3"*
1.3 2.5 5 10 20 0.09 0.17 0.34 100 ttl 0.01
4.5 + 2.2
6.3 + 2.5
46.8+_8.6**
56.0+-9.3**
a Mean of SCEs in 20 metaphases. b Metaphases were very few. c Maximal concentration was 20 t~l of the supernatant fluids of the saturated solutions per ml of culture medium. *'**Significantly different from the value of solvent control (water 100 p.l/ml), p < 0.05 and p < 0.1, respectively. The assays were repeated partially 3 or 4 times.
BeO c
Dose (ttg/ml)
SCEs/metaphase (Mean" _+SD)
0.02
4.5 +__1.7
0.04 0.09
4.8 + 2.5 4.7 +- 2.0
GaCI3
12.5 25 50 100
5.6_+2.0 4.4 + 2.5 4.9 +2.2 Toxic b
Ga(NO3)3.5H20
50 100 200 400
4.5_+2.2 5.0_+2.5 4.5 _+2.0 Toxic b
Ga203 ~
0.18 0.35 0.7
5.0_+2.4 4.7_+2.2 4.6_+2.4
SbCIs
8.6 17.3 35 70
4.3 _+ 1.7 3.4_+ 1.3 4.4 _+ 1.3 Toxic b
Sb205 c
10 20 40
4.9+_2.6 5.1_+2.3 4.0_+2.1
Water
50 #1
4.9+ 1.6
a Mean of SCEs in 20 metaphases. b Metaphases were very few. c Maximal concentration was 20/~l of the supernatant fluids of the saturated solutions per ml of culture medium. The assays were repeated partially 2 or 3 times. A significant increase of SCEs was not observed.
170
The SCE-inducing ability of the compounds was confirmed by repeating the assays partially 3 or 4 times. SbzO~ induced the most SCEs among them, more than twice the number of the spontaneous SCEs, at the highest doses. They were, however, weaker SCE inducers and had less dose dependence than organic mutagens and/or carcinogens (Perry and Evans, 1975; Wolff, 1977). BeSO4 is also a weak inducer in human lymphocytes and in Syrian hamster cells (Larramendy et al., 1981). Ashby et al. (1990) studied the genotoxicity of BeSO4 by several assays in vivo and in vitro, and concluded that the compound was negative in 3 standard assays, the Salmonella mutation assay, the chromosome aberration assay, and the micronucleus assay. SCE gives few false-positive results for mutagenic carcinogens (Latt et al., 1981). Antimony(III) compounds and beryllium compounds gave positive results in both the rec assay and the SCE assay. Further investigations of genotoxicity and carcinogenicity should be done on these compounds, especially on Sb203 which is slightly water-soluble and was positive at very low doses. Data for SCE-negative compounds are shown in Table 6. The slightly soluble compounds BeO, Ga203 and 5b205 did not show toxicity at the highest concentrations.
Acknowledgements We are grateful to T. Tsujinoue and I. Kubo, students at Osaka Pharmacological College, for help. We also greatly appreciate the critical reviewing of the manuscript and suggestions offered by Dr. D.M. Shankel, University of Kansas. This study was partly supported by a Grant-in-Aid for Scientific Research, No. 63304038, from the Ministry of Education, Science and Culture of Japan.
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