Rec assay and mutagenicity studies on metal compounds

109

Mutation Research, 77 (1980) 109--116 © Elsevier/North-Holland Biomedical Press

REC ASSAY AND MUTAGENICITY STUDIES ON METAL COMPOUNDS

NOBUTAKE KANEMATSU 1, MASAKO HARA and TSUNEO KADA

1 Department of Oral Surgery, Gifu College of Dentistry, Gifu-ken 501-02, and Department of Induced Mutation, National Institute of Genetics, Misima, Sizuoka-ken 411 (Japan)

(Received 6 November 1978) (Revision received 16 August 1979) (Accepted 28 August 1979)

Summary We carried out tee assays on 127 metal compounds with Bacillus subtilis to check their DNA-damaging capacity and mutagenicity. Certain compounds of beryllium, cobalt, cesium, iridium, osmium, platinum, rhodium, antimony, tellurium, thallium and vanadium were newly found to be positive in addition to those of known positive metals such as arsenic, cadmium, chromium, mercury, molybdenum and selenium. Reverse mutation assays with Escherichia coli and Salmonella strains showed that compounds of rhodium (RhC13), tellurium (Na:H4TeO6, Na2TeO3) and platinum (PtC14, (NH4)2PtC16) are potent mutagens.

Studies on genetic toxicity of metal compounds are relatively limited. In the bacterial system, Demerec and his collaborators [4] reported, some 30 years ago, that certain compounds of iron, manganese and mercury might induce point mutations. Nishioka [ 24] showed that potassium dichromate, ammonium molybdate, and sodium arsenite are mutagens among the rec-assay-positive metal compounds. In a similar way, mutagenic activities of arsenic, chromium and selenium compounds were established in bacteria [18,23]. When inhibition of cellular growth by a chemical is more pronounced with recombination-repair-deficient (rec-) than with wild bacteria (rec*), it is supposed that this chemical is damaging cellular DNA. Use of the rec assay with Bacillus subtilis in mutagenicity screening has been successful and enabled, in combination with other microbial systems, various authors to detect a number of environmental mutagens [10"12,27]: Recently the rec-assay procedures with Bacillus subtilis have been improved in several respects; in particular, the insertion of a cold incubation before incubation of plates at 37°C [13] increased considerably the assay sensitivity by prolonging the contact period of the drug with non-growing cells. We have tested 127 metal compounds with the above coId incubation proce-

110 dures. Metal c o m p o u n d s showing positive results in this assay were then examined as to their capacity to induce reversions in E. coli WP2 [31] as well as Salmonella TA [1] strains. We found that certain compounds of beryllium, cobalt, cesium, iridium, osmium, platinum, rhodium, antimony, tellurium, thallium and vanadium were DNA-damaging agents. Certain compounds of rhodium (RhC13), tellurium (Na2H4TeO6, NA2TeO3) and platinum (PtC14, (NH4)2PtC16) were p o t e n t mutagens in bacteria. Preliminary results have already been summarized [16]. Materials and methods Rec assay Experimental procedures of the rec assay with cold incubation were mainly based on those already described b y Kada et al. [10]. Two strains o f B . subtilis H17 (Rec ÷, arg- try-) and M45 (Rec-, arg- try-) [26] were used. These strains were grown overnight in B-2 broth {meat extract 10 g, p o l y p e p t o n e p o w d e r 10 g and NaC1 5 g in 1 1, pH adjusted to 7.0) and conserved at --80°C with 12.5% glycerol supplementation. On the day of the experiments, each stock was melted and streaked radially from small pipettes o n t o B-2 agar. A 0.05-ml portion o f each metal solution (0.005--0.5 M) was dropped onto a filter paper disk (diameter 10 mm), and the disk was placed on the starting point of the streak. The plates were kept at 4°C for 24 h and then incubated at 37°C overnight [13]. The above "cold incubation" inserted between drug administration and 37°C incubation increases the assay sensitivity a b o u t 20--50 times for many drugs. When the DNA damage is produced by a chemical and subjected to cellular recombination-repair function, the growth of recombinationdeficient cells is usually inhibited much more than that of wild cells. Many mutagens were efficiently detected among the rec-assay positive chemicals [10-12,24,27]. Reversion assay A spot test, first described by Iyer and Sybalsky [8] and later by Ames et al. [1], was carried o u t with 2 strains of E. coli as well as 5 strains of Salmonella. E. coli strains (B/r WP2 try [31] and WP2 hcr try [6]) were spread on broth-enriched minimal agar (MB agar containing 10 ml of Difco liquid broth, 15 g of Difco agar and 4 g of glucose in 1 1 of a modified Vogel-Bonner's medium [31]). Salmonella strains were also spread on the above MB medium supplemented with biotin at 0.1 #g/ml. The filter-paper disks containing each metal sample were placed on the above plates on the surface o f which bacteria had been spread. When a metal c o m p o u n d induces reverse mutations, revertant bacterial colonies appear around the disk. The 5 strains of Salmonella (Ames) used in the present assay were TA98, TA100, TA1535, TA1537 and T A 1 5 3 8 in which histidine reversions were studied [1,19]. Reversion assays with drug treatment in liquid were also carried o u t with t w o strains of E. coli. Bacteria harvested from exponential cultures in B-2 broth were washed with cold phosphate buffer (M/15, pH 7.0), resuspended in the same solution containing different concentrations of each drug, and kept at 37°C for 20 min under slow shaking. The treated cells were then washed with

111 cold phosphate buffer, remspended in phosphate buffer, appropriately diluted, and spread on MB agar. Plates were incubated at 37°C for 3 days. Plates, on each of which 0.1 ml of bacterial suspension without dilution was spread, gave colonies of revertants. Plates that were spread with highly diluted bacterial cultures give colonies of surviving cells. Frequencies of mutations were calculated in a similar way to that already described [9]. Chemicals All metal compounds used in this study were those of the highest purity commercially available in this country and purchased through Maruichi Chemical Ltd., Misima (Japan). They were all dissolved in distilled water at concentrations of 0.001--10 M as described in the results.

Remits Results of the rec-assay screening of 127 metal compounds are shown in Table 1 for negative samples and in Table 2 for positive ones. When a metal compound has the capacity to damage DNA, growth of the Rec- strain of B. subtilis must be more inhibited than that of the wild strain. So we can easily predict its DNA-damaging capacity by measuring and comparing the inhibition zones.

TABLE 1 METAL COMPOUNDS THAT GAVE NEGATIVE RESULTS IN THE REC ASSAY A12SO 4 A1CI3 a A1203 A1PO 4

K4Fe(CN) 6 K3Fe(CN) 6 Ga(NO3 )3 In(OH) 2

PbO2 Pb304 Pb(CH3 COO)2

AgC1 AgNO 3 a Ag 2 SO 4 HAuCI4

In2 (SO4)3 inC13 In(OH )3 In(NO3 )3

RbC1 RuC13 Na2 SiO3 Na2 SiF6

Ba(NO3)2 BaCI 2 BiCl 3 a Bi203

KNO3 KCI La(NO3)3 a LaCl 3 a

SiO2 Si3N 4 SnCI 2 a SnCl4 a

Bi(NO3 )2 4BiN O 3 (OH)2 Cs2 (SO4)3

LiC1 MgCI2 MoS 2 H 2 MoO 4

SnSO 4 Na 2 SnO 3 SrCl2 TIC14

Cs(NO3)3 CsCI3 CuCI CuCI 2

MoO3 MnCI 2 a Mn(NO3)2 Mn(CH3COO) 2

TiB2 TiC TIC13 TiF 4

K2 Cr2 (SO4)4 Cr2(SO4) 3 a EuCl 3 a FeCI 2 a FeCI 3 a

NbCI5 NiCI2 a NiO Ni203 PbC12

MgTiO3 NiTiO3 TiO 2 TaC15 Y(NO3) 3

CaCl 2

a Highly toxic samples on bacteria.

PbO

ZrOCl 2 ZnCl 2

112

TABLE 2 RESULTS OF THE REC ASSAY ON POSITIVE METAL COMPOUNDS A 0.05 ml portion of aqueous solution containing each metal compound ( 0 . 0 0 5 - - 0 . 5 M) w a s u s e d . Metal compound

Concentration of solution (M)

Inhibition length (mm) H 1 7 ( R e c +)

M45 (Rec-)

4 3 8 15 27

10 15 15 21 41.5

of different concentrations

Difference

Conclusion b

6 12 7 6 13.5

++ ++ ++ ++ ++

As 2 05 As203 AsCI 3 3AsO 5 Na 2 HAsO 4

0.05 0.05 a 0.05 0.1

BeSO 4

0.01

6

10

4

+

CdC12 Cd(N03)2 CdSO 4

0.005 0.005 0.005

18 15 17

23 20 21

5 5 4

+ + +

COC12 Co(OH) 3 COSO4 2COCO 3 • 3 C o ( O H ) 2

0.05 0.25 0.05 0.05

6 3.1 16.5 1.5

10 6.4 20.2 4.0

4 3.4 3.7 2.5

+ -+ -+ -+

CrO 3 K2CrO 4 K 2 Cr 2 0 ?

0.01 0.005 0.005

CsC1 Cs 2 SO 4 Cs2CO 3 CsNO 3 CHCsO 2

5 5 5 5 5

HgC1 H g C I2 CH3HgCI C6H 5HgOCOCH 3

0.05 0.05 0.005 0.001

H 2 IrCl6

0.1

(NH4)6Mo7024

0.1

OsO 4

0.005

( N H 4 ) 2 PtCI6 H 2 PtCI 6 PtCI 4

0.1 0.01 0.001

RhCI 3 RhNO 3

0.005 0.005

9 0 3

29 21 20

20 12 17

++ ++ ++

8.5 16 13 0 2

13.5 28.1 20 7.5 6

5 12.1 7 7.5 4

+ ++ ++ ++ +

8 5 32 6

12 8 37 14

4 3 5 8

+ ++ ++

5

11

6

+

3

+

3

6

11

22

II

++

6.8 3 0

16.1 10 11

9.3 7 11

++ ++ ++

0 1

12 5

12 4

++ +

SeO 2

0.01

18.8

27

8.2

++

SbCI s SbC13 Sb 2 0 3

a 0.01 0.05

14 17 0

20 24 5

6 7 5

++ ++ +

Na2H4TeO 6 Na 2 TeO 3 TeCI4

0.01 0.01 0.001

4 30 0

18 39 4

14 9 4

++ ++ +

4

+

5.5 5 5

+ + +

TINO 3

0.001

2

6

VOCI2 V 205 NH4VO 3

0.4 0.5 0.3

6 0 0

11.5 5 5

a T h e s e m e t a l c o m p o u n d s a r e o r i g i n a l l y H q u i d : 0 . 0 3 m l w a s a p p l i e d to a f i l t e r p a p e r a n d a s s a y e d . h ++ M o r e t h a n 6 m m o f d i f f e r e n c e b e t w e e n g r o w t h i n h i b i t i o n s f o r R e c + a n d R e c - s t r a i n s . + Less than 6 mm and more than 4 mm of difference.

113

Strong positive rec effects were noted with compounds of arsenic (As2Os, As203, AsC13, 3AsOs, Na2HAsO4), chromium (CrO3, K2CrO4, K2Cr2Ov), cesium (Cs2SO4, Cs2CO3, CsNO~), osmium (OsC14), platinum ((NH4)2PtC16, H2PtC16; PtC14), rhodium (RhC13), selenium (SeO2), antimony (SbCls, SbCI3), tellurium (Na2H4TeOe, Na2TeO3) and vanadium (VOC12). Mild positive rec effects were noted with compounds of beryllium (BeSO4), cadmium (CdCI2, Cd(NO3)2, CdSO4), cobalt (CoC12, Co(OH)3, COSO4, 2CoCO3 • 3C0(OH)2), mercury (HgC1, HgC12, CH3HgC1, C6Hs • HgOCOCH3), iridium (H2IrC16), molybdenum ((NH4)6Mo7024) and thallium (T1NO3). The spot mutation induction tests with E. coli and Salmonella strains were carried out with some typical compounds showing positive rec effects such as those of arsenic (As2Os, As203) cadmium (CdC12), cobalt (CoC12, Co(OH)3), chromium (CrO3, K2CrO4, K2Cr2OT), cesium (CsC1), mercury (CH3HgC1), iridium (H2IrC16), platinum ((NH,)2PtC16), rhodium (RhC13), selenium (SeO2), antimony (SbC13, Sb,O3), tellurium (Na2H4TeO6, Na2TeO3), thallium (T1NO3) and vanadium (V2Os, NH4VO3). The results are summarized in Table 3. Among the samples assayed, 6 metal compounds induced reverse mutations. Both rhodium trichloride (RhC13 • 3H20) and ammonium hexachloroplatinate ((NH4)2PtC16) induced base-change mutations in strains of E. coli WP2. Platin u m tetrachloride (PtC14) and sodium tellurous acids (Na2TeOQ induced frameshift mutations in the strain Salmonella TA98 (Fig. 1). Sodium telluric acid (Na2H,TeO6) induced base-change mutations in TA1535 and TA100. Potassium dichromate (K2Cr2OT) induced base-change mutations in E. coli WP2 hcr. Some organic platinum compounds known as anti-cancer agents have mutagenic capacities [2,17,21]. It would be necessary to carry out other different assays before absence of mutagenicity is assumed for compounds that are not listed in Table 3. Induction of reverse mutations by rhodium trichloride (RhCI3 • 3H20) was studied quantitatively by treating cells in liquid suspension with the drug (Table 4). When the cells of E. coli B/r WP2 try- were treated with this chemical at

TABLE

3

RESULTS

OF SPOT TESTS

ON THE MUTAGENICITY

OF METAL

COMPOUNDS

M e t h o d s axe d e s c r i b e d in t h e t e x t , A n t h e rec-assay-POsitive m e t a l c o m p o u n d s ( T a b l e 2 ) w e r e a s s a y e d w i t h t h e s e t e s t s and o n l y t h o s e g i v i n g p o s i t i v e results in s o m e o f 7 s t r a i n s a r e s h o w n h e r e , Metal

S a | m o n e l l a typhimurlurn

E s c h e r f c h i a coli

compound B/r WP2

WP2

TA1535

TA100

TA98

TA1537

TA1538

try-

hcr- try-

his-

his-

his-

his-

hls-

RhC13 • 3H20

+

+

--

_

+

--

a

a

+

--

(NH4)2 PtCI6

--

PtCl4

.

.

Na2TeO 3

.

.

Na2H4TeO 6

_

_

+

K 2 Cr 207

--

+

.

a Hot conclusive.

+ .

.

.

.

+ + + .

a .

.

--

114

Fig. 1. S p o t t e s t o f PtCI 4 on S a l m o n e l l a T A 9 8 . T h e m e t a l c o m p o u n d (0.1 p g p e r d i s k ) w a s s p o t t e d b y a p a p e r d i s k o n b a c t e r i a l a y e r e d o n t h e s u r f a c e o f agar m e d i u m as d e s c r i b e d i n t h e t e x t .

a concentration of 1000 pg/ml for 20 min at 37°C, a net mutation induction was observed. The strain WP2 t r y - hcr- was also mutated at lower drug concentrations (10 or 100 pg per ml for 20 rain at 37°C). The presence of hcr- greatly increased the sensitivities both for lethality and mutation induction. Discussion We detected DNA-damaging and/or mutagenic metal compounds by the rec assay with cold-incubation as well as reversion-assay procedures with bacteria. In mutagenicity screening with the B. subtilis rec assay in combination with

TABLE 4 I N D U C T I O N O F M U T A T I O N BY R h C I 3 • 3 H 2 0 I N E. coli B a c t e r i a i n s u s p e n s i o n w e r e t r e a t e d w i t h t h e c h e m i c a l f o r 2 0 m i n at 3 7 ° C a n d P l a t e d , a f t e r w a s h i n g a n d a p p r o p r i a t e d i l u t i o n s , o n M B agar. D e t a i l s a r e d e s c r i b e d in t h e t e x t . Strain

B~ WP2 try-

WP2 hcr- try-

Concentration (~g/ml) of RhCI3 • 3H20

Survivals per ml (X 1 0 8 )

Try + colonies per plate

0

28.7

6.0

100 1000

29.0 19.0

19.8 49.3

0

5.7

9.9

10 100

3.9 0.4

47.8 30.4

Try+.induced r e v e r t a n t s p e r 108 survivals -4.8 23.4 -10.5 74.3

115 - - Salmonella reversion assays, usually more than 70% of the rec-assaypositive samples were found to be real mutagens in bacterial systems in food additives or pesticides [11,27]. In the present metal-screening studies, only a small proportion (less than 30%) of the rec-assay-positive samples showed positive effects in the reversion assays. The reason for this may not be due to problems of the interference by media components with metal compounds, because the rec assay utilizes complex and natural media such as broth. It is more likely that each strain may have some specificities as to its membrane permeability, metabolic modification of metals, etc. Therefore, further trials should be continued to detect mutagenic compounds among the rec-assay-positive samples. In any case, the B. subtilis rec assay may be one of the most sensitive systems for detection of metal mutagens. Despite a positive correlation between mutagenicity and carcinogenicity of metal compounds, there are exceptions in our data. Some metal compounds whose epidemiological studies on their carcinogenic effects have been reported were negative in the present assays. For example in the case of nickel [5,22, 30], it is possible that certain ingredients might be involved in inducing the actual effects. Iron, lead and titanium are reported to induce tumors in rats [3], but they gave negative results in the present studies. It is probable that certain metals may not act directly with DNA but cause DNA alterations because of infidelity of DNA-polymerase owing to metal cofactors [20,28]. In addition, some metal compounds, such as cobaltous chloride, are antimutagens [14,15]. Arsenic is carcinogenic only in animals [7] and not in humans [7,25] ; this metal is positive in the present assay. It is probably important to study roles of cellular metabolic capacities that modify the valence of metals by oxidation or reduction [29]. It is also possible that bacterial systems themselves have such capacities. Therefore, relationships between carcinogenicity and mutagenicity of metal compounds may be complex.

E. coli

Acknowledgements We thank Dr. K. Kawai of the National Institute of Industrial Health for helpful information for present research. This work was supported in part by grants from the Ministry of Welfare, the Ministry of Education, the Science and Technology Agency of Japan, the Takamatsunomiya Cancer Foundation and the Nissan Science Foundation. Contribution No. 1275 from the National Institute of Genetics, Misima (Japan). References 1 Ames, B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s for detecting carcinogens a n d m u t a g e n s w i t h the S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 Beck, D.J., a n d R.R. B~ubaker, Mutagenie properties o f c/s-platinum(II) d i a m m l n o d i c h l o l , ide i n E. coli, M u t a t i o n Res., 27 ( 1 9 7 5 ) 1 8 1 - - 1 9 0 . 3 Beliles, R.P., Metals i n T o x i c o l o g y , in: L.J. C a s a r e t t a n d J. D o u n (Ed.), The Basic Science o f Poisons, MacMillan, New Y o r k , 1 9 7 5 , p. 4 5 4 . 4 D e m e r e c , M., G. B e r t a l n a n d H. Flint, A survey o f chemicals for m u t a g e n i c action o f E. coli, A m . Nat., 8 5 ( 1 9 5 1 ) 1 1 9 - - 1 3 6 . 5 Doll, R., C a n c e r o f the lung a n d nose in nickel w o r k e r s , Br. J. Ind. Med., 15 ( 1 9 5 8 ) 217---223.

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