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Genotoxicity of zinc in 4 short-term mutagenicity assays
Mutation Research, 223 (1989) 267-272 Elsevier
267
MUTGEN 01419
Genotoxicity of zinc in 4 short-term mutagenicity assays E.D. Thompson,
J.A. M c D e r m o t t ,
T.B. Zerkle, J.A. Skare, B.L.B. Evans and D.B. Cody
The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, OH 45239-8707 (U.S.A.)
(Received 3 February 1987) (Revisio~a received 8 December 1988) (Accepted 5 January 1989)
Keywords: Zinc, genotoxicity; Short-term mutagenicity assays, zinc; Zinc-2,4-pentanedione; UDS; Rat hepatocytes; L5178Y mouse lymphoma
Summary The genotoxicity of zinc was examined in 4 short-term mutagenicity assays. Zinc acetate produced dose-related positive responses in the L5178Y mouse lymphoma assay and an in vitro cytogenetic assay with Chinese hamster ovary cells, but was negative in the Salmonella mutation assay and did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. Zinc-2,4-pentanedione produced frameshift mutations in Salmonella tester strains TA1538 and TA98, but did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. The effect of ligand binding of zinc in the in vitro test systems is discussed.
Zinc is an essential metal which functions as a cofactor for more than 70 enzymes (Sandstead and Evans, 1984). In addition to the cofactor role, zinc is involved in stabilizing D N A by binding to the phosphate portions (Eichhorn and Shin, 1968) and also functions in the regulation of D N A synthesis and cell division in mammalian cells (Rubin, 1975). The mutagenicity of zinc has recently been reviewed by Hansen and Stern (1984). In vitro genotoxicity test results for zinc are mixed.
CAS numbers: Zinc acetate, 557-34-6; 2,4-Pentanedione, 12354-6; Zinc 2,4-pentanedione, 14024-63-6. Correspondence: Dr. E.D. Thompson, The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, OH 45239-8707 (U.S.A.).
Kalinina et al. (1977) reported ZnC12 to be a frameshift mutagen in Salmonella, whereas Gocke et al. (1981) reported ZnSO 4 to be negative in the Salmonella assay. ZnC12 has been shown to be inactive in the rec assay with Bacillus subtilis (Kanematsu et al., 1980; Nishioka, 1975). In mammalian cells, ZnC12 has been shown to be a weak clastogen in human lymphocytes (Deknudt and Deminatti, 1978), but did not induce mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells (Amacher and Paillet, 1980). Zinc salts do not appear to be genotoxic when fed to whole animals. ZnSO 4 was negative in a Drosophila sex-linked recessive lethal assay and a bone-marrow micronucleus assay in mice (Gocke et al., 1981). Deknudt and Gerber (1979) reported that zinc chloride induced chromosome aberrations in bone-marrow cells of calcium-deficient mice, but not mice fed a standard diet.
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
268
Materials and methods
Zinc acetate was purchased from Fisher Scientific, Springfield, NJ. Zinc 2,4-pentanedionate was purchased from Alfa Products, Danvers, MA. 2,4-pentanedione was purchased from Aldrich, Milwaukee, WI. All other chemicals were purchased from commercial sources.
Mutagenicity assays
Results
This paper describes the genotoxicity of zinc acetate in 4 short-term mutagenicity assays. A rationale for the conflicting genotoxicity data for zinc is proposed.
Salmonella / mammafian microsome assay. The S a l m o n e l l a / m a m m a l i a n microsome plate-incorporation assay was performed with strains TA1535, TA100, TA1537, TA1538 and TA98 as described by Maron and Ames (1983). Toxicity was estimated by measuring the extent of reduction of the bacterial lawn in the overlay agar with strain TA100. L 5 1 7 8 Y T K + / - mouse lymphoma assay. L5178Y mouse l y m p h o m a cells were received from D. Clive, Burroughs, Welcome Co., Research Triangle, NC. Zinc acetate was tested according to the procedures of Clive and Spector (1975) and Clive et al., 1979). In vitro cvtogenetic C H O assay. The methods used to measure the induction of chromosome aberrations in vitro were a modification of those described by Natarajan et al. (1976). The method has been described in detail by T h o m p s o n et al. (1984).
The results of the S a l m o n e l l a / m a m m a l i a n microsome plate-incorporation assay for zinc acetate were uniformly negative. Zinc acetate was neither toxic (determined by reduction of bacterial lawn) nor mutagenic to any of the 5 strains tested over a dose range of 5 0 - 7 2 0 0 / ~ g / p l a t e (data not shown). However, when zinc was complexed with an organic ligand, 2,4-pentanedione, mutagenic results were obtained with strains TA1538 and TA98, and toxicity was obtained with both strains at 400 /~g/plate (an approx. 75% reduction in bacterial lawn). The results for TA1538 are shown in Fig. 1. As shown in Fig. 1, the addition of $9 diminished the response in a dose-dependent manner. The response obtained with strain TA98 was dose-related, but relatively weak (approx. 4-fold over background at 400/~g/ml). 2,4-Pentanedione was neither toxic nor mutagenic to any of the 5 tester strains at 800 /ag/plate, indicating the mutagenic response and toxicity was due to zinc. 200o)
Unscheduled D N A synthesis in rat hepatocytes. The unscheduled D N A synthesis assay was conducted by a modified procedure originally described by Williams et al. (1977) and Williams (1978). The modifications were recently described in detail by Skare et al. (1986). Materials Aroclor-induced rat liver $9 from SpragueDawley rats was purchased from Microbiological Associates, Bethesda, M D 20816. Aroclor 1254-induced rat liver $9 was prepared in 0.15 M KC1 for the Salmonella and cytogenetic assays. A 2 : 1 mixture of Aroclor 1242:1254 was used to induce rat livers for the mouse lymphoma assay. This $9 was prepared in 0.25 M sucrose.
1130¢r
I
J
1oo
20o
I
400 Dose ~g/plate)
l
800
Fig. 1. Mutagenic response of strain TA1538 to zinc 2,4-pentanedione and the effects of Aroclor-induced rat liver $9 on that response. Zinc 2,4-pentanedione and 2,4-pentanedione were dissolved in DMSO. o, zinc 2,4-pentanedione without $9. X, zinc 2,4-pentanedione with 1% $9 mix. zx, zinc 2,4-pentanedione with 4% $9 mix. D, zinc 2,4-pentanedione with 10% $9 mix. ©, 2,4-pentanedione.
269 TABLE 1 RESULTS OF THE L5178Y TK +/ MOUSE LYMPHOMA ASSAY ON ZINC ACETATE With $9 activation
Without $9 activation Concentration (/~g/ml)
Colonies per TFT plate
Colonies per VC plate
H2Ocontrol
61_+ 1 a
146_+ 9
0.8
-
H2Ocontrol
H 20 control
68_+ 7
127_+15
1.1
-
H 20 control
59_+ 1 61_+ 6 62_+ 6 59-+ 1 59_+ 1 60+ 3 54-+14 117-+14 194-+12
138_+ 5 113_+ 3 152_+ 5 151-+11 133-+ 5 152_+ 5 144_+14 127_+ 5 92-+ 2
0.9 1.1 0.8 0.8 0.9 0.8 0.8 1.8 4.2
79 61 113 119 82 130 82 60 31
1.3 1.8 2.4 3.2 4.2 5.6 7.5 10 13
Pos. control EMS (0.5 ~l/ml) 251_+19 (1.0~l/ml) 142_+ 7
80_+ 3 17_+ 2
Mutation frequency per 104 surviving cells
6.3 16.7
% Concentration total (/zg/plate) growth
35 4
Colonies per TFT plate
Colonies per VC plate
Mutation % frequency total per 104 growth surviving cells
62_+ 4
143+ 8
0.9
-
38_+ 1
145_+11
0.5
-
4.2 5.6 7.5 10 13 18 24 32 42
63+ 4 92_+ 7 87_+ 7 78+ 3 94_+ 4 108+ 8 194_+ 7 275-+12 230_+ 2
162___ 5 169_+ 6 151+ 6 132+21 159+10 130_+14 138_+10 135_+ 8 62-+ 5
0.8 1.1 1.2 1.2 1.2 1.7 2.8 4.1 7.4
111 112 79 74 79 49 33 19 8
Pos. control DMBA (5.0 ~g/ml) (7.5 ~g/ml)
145_+ 1 194_+ 6
132-+ 4 2.2 125_+13 3.1
64 57
a Standard deviation. The results of the T K +/- m o u s e l y m p h o m a assay with zinc acetate are shown i n T a b l e 2. D o s e - d e p e n d e n t positive responses were o b t a i n e d in the presence a n d absence of the $9 m e t a b o l i c activation system with a d o u b l i n g of the m u t a t i o n frequency occurring at 10 / ~ g / m l for b o t h portions of the assay. The results of the C H O cell in vitro cytogenetic assay with zinc acetate are s h o w n in T a b l e 2. D o s e - d e p e n d e n t positive responses were o b t a i n e d in the presence a n d absence of the $9 activation system, although the $9 reduced b o t h the clastogenic response a n d the toxicity. Both zinc acetate a n d zinc 2 , 4 - p e n t a n e d i o n e were tested for i n d u c t i o n of U D S in p r i m a r y cultures of rat hepatocytes. N e i t h e r form of zinc i n d u c e d U D S or p r o d u c e d toxicity i n these cells over a range of 1 0 - 1 0 0 0 / ~ g / m l (data n o t shown).
Discussion As p o i n t e d out i n the i n t r o d u c t i o n , the results of short-term m u t a g e n i c i t y assays for zinc are
mixed. T h e reason for the conflicting results m a y be the form of zinc tested a n d the possible interactions b e t w e e n m e d i u m c o m p o u n d s a n d the zinc forms. F o r instance, w h e n i n o r g a n i c zinc (ZnC12 or Z n S O 4) is a d d e d to growth m e d i u m , the zinc is c o m p l e t e l y dissociated a n d n u m e r o u s c o n s t i t u e n t s in the m e d i u m (especially serum proteins) are c a p a b l e of b i n d i n g the free zinc (Parisi a n d Vallee, 1970). Thus, i n o r g a n i c zinc m a y be b o u n d to m e d i u m c o m p o n e n t s , a n d be u n a v a i l a b l e for t r a n s p o r t into the cell. O n the other h a n d , when zinc is tested as a n organic complex, there exists a c o m p e t i t i o n b e t w e e n that complex a n d m e d i u m c o n s t i t u e n t s for the zinc. If the organic c o m p o u n d b i n d s zinc with a higher affinity t h a n the m e d i u m c o m p o n e n t s , the zinc m a y be t r a n s p o r t e d as the organic complex. S u b s e q u e n t m e t a b o l i s m within the cell w o u l d then release the zinc. Thus, the presence or absence of a m u t a g e n i c response in a s h o r t - t e r m assay w o u l d d e p e n d o n the type of m e d i u m , the type of cell, a n d the b i n d i n g c o n s t a n t of the m e t a l complex. T h e b u l k of the in vitro m u t a g e n i c i t y d a t a appears to be c o n s i s t e n t with this proposal, as discussed below.
270 TABLE 2 RESULTS OF IN VITRO C Y T O G E N E T I C ASSAY W I T H CHO CELLS ON ZINC ACETATE Dose
Metabolic
Relative
Aberration types
(g g / m l )
activation
cloning efficiency
Chromatid b
Chromatid
gaps
break
Neg. Control (water)
-
100
25 34 45
-
Pos. control ( T E M - 0.5 g g / m l )
-
Neg. control (water)
+
45 60 80
+ + +
Pos. control (cyclophosphamide 35 g g / m l )
+
Fragments
Exchanges
Rings
> 10 Aberrations
% Polyploidy
Aber rations c per cell
2
0
0
0
0
0
2
0
100 36 53
2 1 3
0 5 10
0 1 5
0 15 22
0 0 0
0 0 5
4 4 4
0 0.42 1.74
2
4
23
26
42
1
8
4
3.44
0
0
0
0
0
0
0
0
73 76 58
3 2 1
7 17 7
0 2 0
10 21 5
0 0 0
1 3 5
4 6 0
0.54 1.40 1.24
9
9
28
17
45
0
4
4
2.60
(58)
a
100 (33) a
The cloning efficiency for water is arbitrarily set at 100. The number in parentheses is the number of clones formed when 200 cells (was determined by hemacytomer counts) were plated. The relative cloning efficiency of each dose equals the number of viable clones for that dose divided by the viable clones in the water control, times 100. b Counted, but not used in the aberrations per cell calculation. c Aberrations per cell is the total number of aberrations (excluding gaps) divided by the number of cells scored (50 for all doses). Cells with > 10 aberrations are counted as 10 aberrations. a
4 types of zinc have been tested in the Salmonella assay, 2 inorganic and 2 organic forms. Kalinina et al. (1977) and Gocke et al. (1981) tested ZnC12 and ZnSO4 respectively. The negative response by Gocke et al. is consistent with inorganic zinc binding to agar constituents. McCann (1978) has also demonstrated that inorganic metals are not transported through procaryotic cell walls. The positive response reported by Kalinina et al. appears to be inconsistent with the data presented here as well as those of Gocke et al. (1981). However, a close examination of the methods used by Kalininia et al. (1977) indicate their data are probably consistent with those reported here. We employed a standard plate-incorporation assay for the Salmonella studies in which the inorganic salts and loosely bound forms of zinc are negative. Kalinina et al. performed a suspension-type assay in which they suspended
the cells in a phosphate buffer which contained 0.2% sodium citrate, then added ZnC12 and incubated the mixture for 30 min. Under these conditions, the citrate will rapidly exchange Z n 2+ for N a +. Thus, Kalinina et al. were most likely testing a mixture of zinc citrate and ZnC1 z- Citrate has a log binding constant for zinc of 4 - 5 (Martell and Smith, 1977) which is close to that of the zinc 2,4-pentanedione complex (log binding constant of 5.06). The negative response with zinc acetate in the Salmonella assay suggests that binding of zinc by acetate (log of binding constant = 1.57, Martell and Smith, 1977) is either not sufficiently strong to compete with the agar medium or that zinc acetate does not cross the bacterial cell wall. The zinc 2,4-pentanedione complex (log of binding constant = 5.06) appears to be sufficiently stable to retain the zinc in the agar medium and is transported across the bacterial cell wall. The fact
271
that the mutagenic responses obtained by Kalinina et al. (1977) and those reported here agree, i.e. zinc-induced frameshift type mutations, supports the contention that the mutagenicity of zinc 2,4pentanedione is due to zinc. The disparity with the mouse lymphoma results reported by Amacher and Paillet (1980) and those reported here is also consistent with testing different forms of zinc. The ZnC12 tested by Amacher and Paillet will dissociate and likely be bound to serum proteins, whereas a significant portion of the zinc acetate may be available for transport. Deknudt and Deminatti (1978) reported ZnC12 to be "a weak clastogen in cultured human lymphocytes, whereas Gasiorek and Bauchinger (1981) reported zinc acetate to be negative in cultured human lymphocytes. However, Gasiorek and Bauchinger treated freshly isolated lymphocytes for 3 h, then induced replication. Thus the cells were in G o during treatment. Since cytogenetic events occur most readily in dividing cells, it is not surprising that a negative response was obtained. It is also possible that the 3 h exposure induced metallothionein which could bind zinc before cell replication occurred (Enger et al., 1983). Our data show zinc acetate to be a potent clastogen in CHO cells at concentrations equivalent to those tested by Deknudt and Deminatti (1978) and Gasiorck and Bauchinger (1981). The differences in the responses reported here and those reported by Deknudt and Deminatti may be differences in transport of the two forms of zinc tested. The differences in our results and those of Gasiorek and Bauchinger (1981) are most likely due to the cell-cycle stage during treatment, although metallothionein levels cannot be ruled out since CHO cells normally have very low levels of this protein (Enger et al., 1986). The results of the UDS assay in primary cultures of rat hepatocytes suggest that these cell are not susceptible to D N A damage by zinc. Whether this is due to lack of transport of the organic ligands by the hepatocytes or unique metabolic capabilities such as constitutive production of the metal detoxifying enzyme metallothionein (Enger et al., 1986) is not known. In vivo genotoxicity studies with zinc are generally negative (Gocke et al., 1981). A single report of clastogenicity in bone marrow of calcium-deft-
cient mice has appeared (Deknudt and Gerber, 1979), but the exposure conditions were so exaggerated the data are nearly uninterpretable e.g. the mice were maintained for 30 days on a diet which resulted in average body weights of 30 g in the controls and 12 g in the treated group. The same authors reported no effects by zinc on mice fed a standard diet. Thus, zinc fed to animals under realistic conditions is not likely to be genotoxic because of the homeostatic controls of zinc absorption (Underwood, 1977) and the fact that zinc is almost entirely bound to serum proteins or metalloproteins in the cells (Parisi and Vallee, 1970). The results presented here indicate zinc is an effective mutagen and clastogen when presented to a susceptible cell population in an appropriate form. While mutagenic responses were obtained in cultured mammalian fibroblasts at levels lower than may be found in many tissues in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.
Acknowledgements We thank Drs. R.L. Anderson and R.A. LeBoeuf for critical review of this manuscript. We are indebted to Ms. L.R. Schroer for assistance in preparation of the manuscript.
References Amacher, D.E., and S.C. Paillet (1980) Induction of trifluorothymidine-resistant mutants by metal ions in L5178Y TK +/- cells, Mutation Res., 78, 279-288. Clive, D., and J.F.S. Spector (1975) Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells, Mutation Res., 31, 17-29. Clive, D., K.O. Johnson, J.F.S. Spector, A.G. Batson and M.M.M. Brown (1979) Validation and characterization of the L5178Y TK +! mouse lymphoma mutation assay system, Mutation Res., 59, 61-108. Deknudt, Gh., and M. Deminatti (1978) Chromosome studies in human lymphocytes after in vitro exposure to metal salts, Toxicology, 10, 67-75. Deknudt, Gh., and G.B. Gerber (1979) Chromosomal aberrations in bone marrow cells of mice given a normal or a calcium-deficient diet supplemented with various heavy metals, Mutation Res., 68, 163-168.
272 Eichhorn, E.L., and Y.A. Shin (1968) Interaction of metal ions with polynucleotides and related compounds, XII. The relative effect of various metal ions on DNA helicity, J. Am. Chem. Soc., 90, 7323-7328. Enger, M.D., C.E. Hildebrand and C.C. Stewart (1983) Cd 2+ responses of cultured human blood cells, Toxicol. Appl. Pharmacol. 69, 214-224. Enger, M.D., C.E. Hildebrand, J. Seagrave and R.A. Tobey (1986) Cellular resistance to cadmium, in: E.C. Foulkes (Ed.), Cadmium, Springer, New York, pp. 363-396. Gasiorek, K., and M.Bauchinger (1981) Chromosome changes in human lymphocytes after separate and combined treatment with divalent salts of lead, cadmium, and zinc, Environ. Mutagen., 3, 513-518. Gocke, E., M.T. King, K. Echardt and D. Wild (1981) Mutagenicity of cosmetics ingredients licensed by the European Communities, Mutation Res., 90, 91-109. Hansen, K., and R.M. Stern (1984) A survey of metal-induced mutagenicity in vitro and in vivo, J. Am. Coll. Toxicol., 3, 381-430. Kanematsu, N., M. Hara and T. Kada (1980) Rec assay and mutagenicity studies on metal compounds, Mutation Res., 77, 109-116. Kalinina, L.M. and G.H. Polukhina and L.I. Lukasheva (1977) A test system for the detection of the mutagenic activity of environmental pollutants, Genetika, 13, 1089-1092. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Martell, A.E., and R.M. Smith (1977) Critical Stability Constants, Vol. 3, Plenum, New York. McCann, J. (1978) Mutagenesis, Carcinogenesis, and the Salmonella test, in: J. Becky and P.C. Sherod (Eds.), In Vitro Toxicity Testing, Franklin Press, Philadelphia, pp. 280-295.
Natarajan, A.T., A.D. Tates, P.P.W. van Buul, M. Meijers and N. de Vogel (1976) Cytogenetic effects of mutagens/carcinogens after activation in a microsomal system in vitro, I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in the presence of rat liver microsomes, Mutation Res., 37, 83-90. Nishioka, H. (1975) Mutagenic activities of metal compounds in bacteria, Mutation Res., 31, 185-189. Parisi, A.F., and B.L. Vallee (1970) Isolation of a zinc 2-macroglobulin from human serum, Biochemistry, 9, 2421-2426, Rubin, H. (1975) Nonspecific nature of the stimulus to DNA synthesis in cultures of chick embryo cells, Proc. Natl. Acad. Sci. (U.S.A.), 72, 1676-1680. Sandstead, H.W., and G.W. Evans (1984) Zinc, in: Nutrition Review, Present Knowledge in Nutrition, 5th edn., Nutrition Foundation Inc., Washington, DC. Skare, J.A., T.K. Wong, B.L.B. Evans and D.B. Cody (1986) DNA-repair studies with sodium fluoride: comparative evaluation using density gradient ultracentrifugation and autoradiography, Mutation Res., 172, 77-87. Thompson, E.D., W.J. Coppinger, R. Valencia and J. lavicoli (1984) Mutagenicity testing of diethylene glycol monobutyl ether, Environ. Health Perspect., 57, 105-112. Underwood, E.J. (1977) Trace Elements in Human and Animal Nutrition, Academic Press, New York, pp. 196-242. Williams, G.M. (1978) Further improvements in the hepatocyte primary culture DNA repair test for carcinogens: detection of carcinogenic biphenyl derivatives, Cancer Lett., 4, 69-75. Williams, G.M., E. Bermudez and D. Scaramuzzino (1977) Rat hepatocyte primary cell cultures, III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium, In vitro, 13, 809-817.
267
MUTGEN 01419
Genotoxicity of zinc in 4 short-term mutagenicity assays E.D. Thompson,
J.A. M c D e r m o t t ,
T.B. Zerkle, J.A. Skare, B.L.B. Evans and D.B. Cody
The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, OH 45239-8707 (U.S.A.)
(Received 3 February 1987) (Revisio~a received 8 December 1988) (Accepted 5 January 1989)
Keywords: Zinc, genotoxicity; Short-term mutagenicity assays, zinc; Zinc-2,4-pentanedione; UDS; Rat hepatocytes; L5178Y mouse lymphoma
Summary The genotoxicity of zinc was examined in 4 short-term mutagenicity assays. Zinc acetate produced dose-related positive responses in the L5178Y mouse lymphoma assay and an in vitro cytogenetic assay with Chinese hamster ovary cells, but was negative in the Salmonella mutation assay and did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. Zinc-2,4-pentanedione produced frameshift mutations in Salmonella tester strains TA1538 and TA98, but did not induce unscheduled DNA synthesis in primary cultures of rat hepatocytes. The effect of ligand binding of zinc in the in vitro test systems is discussed.
Zinc is an essential metal which functions as a cofactor for more than 70 enzymes (Sandstead and Evans, 1984). In addition to the cofactor role, zinc is involved in stabilizing D N A by binding to the phosphate portions (Eichhorn and Shin, 1968) and also functions in the regulation of D N A synthesis and cell division in mammalian cells (Rubin, 1975). The mutagenicity of zinc has recently been reviewed by Hansen and Stern (1984). In vitro genotoxicity test results for zinc are mixed.
CAS numbers: Zinc acetate, 557-34-6; 2,4-Pentanedione, 12354-6; Zinc 2,4-pentanedione, 14024-63-6. Correspondence: Dr. E.D. Thompson, The Procter and Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, OH 45239-8707 (U.S.A.).
Kalinina et al. (1977) reported ZnC12 to be a frameshift mutagen in Salmonella, whereas Gocke et al. (1981) reported ZnSO 4 to be negative in the Salmonella assay. ZnC12 has been shown to be inactive in the rec assay with Bacillus subtilis (Kanematsu et al., 1980; Nishioka, 1975). In mammalian cells, ZnC12 has been shown to be a weak clastogen in human lymphocytes (Deknudt and Deminatti, 1978), but did not induce mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells (Amacher and Paillet, 1980). Zinc salts do not appear to be genotoxic when fed to whole animals. ZnSO 4 was negative in a Drosophila sex-linked recessive lethal assay and a bone-marrow micronucleus assay in mice (Gocke et al., 1981). Deknudt and Gerber (1979) reported that zinc chloride induced chromosome aberrations in bone-marrow cells of calcium-deficient mice, but not mice fed a standard diet.
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
268
Materials and methods
Zinc acetate was purchased from Fisher Scientific, Springfield, NJ. Zinc 2,4-pentanedionate was purchased from Alfa Products, Danvers, MA. 2,4-pentanedione was purchased from Aldrich, Milwaukee, WI. All other chemicals were purchased from commercial sources.
Mutagenicity assays
Results
This paper describes the genotoxicity of zinc acetate in 4 short-term mutagenicity assays. A rationale for the conflicting genotoxicity data for zinc is proposed.
Salmonella / mammafian microsome assay. The S a l m o n e l l a / m a m m a l i a n microsome plate-incorporation assay was performed with strains TA1535, TA100, TA1537, TA1538 and TA98 as described by Maron and Ames (1983). Toxicity was estimated by measuring the extent of reduction of the bacterial lawn in the overlay agar with strain TA100. L 5 1 7 8 Y T K + / - mouse lymphoma assay. L5178Y mouse l y m p h o m a cells were received from D. Clive, Burroughs, Welcome Co., Research Triangle, NC. Zinc acetate was tested according to the procedures of Clive and Spector (1975) and Clive et al., 1979). In vitro cvtogenetic C H O assay. The methods used to measure the induction of chromosome aberrations in vitro were a modification of those described by Natarajan et al. (1976). The method has been described in detail by T h o m p s o n et al. (1984).
The results of the S a l m o n e l l a / m a m m a l i a n microsome plate-incorporation assay for zinc acetate were uniformly negative. Zinc acetate was neither toxic (determined by reduction of bacterial lawn) nor mutagenic to any of the 5 strains tested over a dose range of 5 0 - 7 2 0 0 / ~ g / p l a t e (data not shown). However, when zinc was complexed with an organic ligand, 2,4-pentanedione, mutagenic results were obtained with strains TA1538 and TA98, and toxicity was obtained with both strains at 400 /~g/plate (an approx. 75% reduction in bacterial lawn). The results for TA1538 are shown in Fig. 1. As shown in Fig. 1, the addition of $9 diminished the response in a dose-dependent manner. The response obtained with strain TA98 was dose-related, but relatively weak (approx. 4-fold over background at 400/~g/ml). 2,4-Pentanedione was neither toxic nor mutagenic to any of the 5 tester strains at 800 /ag/plate, indicating the mutagenic response and toxicity was due to zinc. 200o)
Unscheduled D N A synthesis in rat hepatocytes. The unscheduled D N A synthesis assay was conducted by a modified procedure originally described by Williams et al. (1977) and Williams (1978). The modifications were recently described in detail by Skare et al. (1986). Materials Aroclor-induced rat liver $9 from SpragueDawley rats was purchased from Microbiological Associates, Bethesda, M D 20816. Aroclor 1254-induced rat liver $9 was prepared in 0.15 M KC1 for the Salmonella and cytogenetic assays. A 2 : 1 mixture of Aroclor 1242:1254 was used to induce rat livers for the mouse lymphoma assay. This $9 was prepared in 0.25 M sucrose.
1130¢r
I
J
1oo
20o
I
400 Dose ~g/plate)
l
800
Fig. 1. Mutagenic response of strain TA1538 to zinc 2,4-pentanedione and the effects of Aroclor-induced rat liver $9 on that response. Zinc 2,4-pentanedione and 2,4-pentanedione were dissolved in DMSO. o, zinc 2,4-pentanedione without $9. X, zinc 2,4-pentanedione with 1% $9 mix. zx, zinc 2,4-pentanedione with 4% $9 mix. D, zinc 2,4-pentanedione with 10% $9 mix. ©, 2,4-pentanedione.
269 TABLE 1 RESULTS OF THE L5178Y TK +/ MOUSE LYMPHOMA ASSAY ON ZINC ACETATE With $9 activation
Without $9 activation Concentration (/~g/ml)
Colonies per TFT plate
Colonies per VC plate
H2Ocontrol
61_+ 1 a
146_+ 9
0.8
-
H2Ocontrol
H 20 control
68_+ 7
127_+15
1.1
-
H 20 control
59_+ 1 61_+ 6 62_+ 6 59-+ 1 59_+ 1 60+ 3 54-+14 117-+14 194-+12
138_+ 5 113_+ 3 152_+ 5 151-+11 133-+ 5 152_+ 5 144_+14 127_+ 5 92-+ 2
0.9 1.1 0.8 0.8 0.9 0.8 0.8 1.8 4.2
79 61 113 119 82 130 82 60 31
1.3 1.8 2.4 3.2 4.2 5.6 7.5 10 13
Pos. control EMS (0.5 ~l/ml) 251_+19 (1.0~l/ml) 142_+ 7
80_+ 3 17_+ 2
Mutation frequency per 104 surviving cells
6.3 16.7
% Concentration total (/zg/plate) growth
35 4
Colonies per TFT plate
Colonies per VC plate
Mutation % frequency total per 104 growth surviving cells
62_+ 4
143+ 8
0.9
-
38_+ 1
145_+11
0.5
-
4.2 5.6 7.5 10 13 18 24 32 42
63+ 4 92_+ 7 87_+ 7 78+ 3 94_+ 4 108+ 8 194_+ 7 275-+12 230_+ 2
162___ 5 169_+ 6 151+ 6 132+21 159+10 130_+14 138_+10 135_+ 8 62-+ 5
0.8 1.1 1.2 1.2 1.2 1.7 2.8 4.1 7.4
111 112 79 74 79 49 33 19 8
Pos. control DMBA (5.0 ~g/ml) (7.5 ~g/ml)
145_+ 1 194_+ 6
132-+ 4 2.2 125_+13 3.1
64 57
a Standard deviation. The results of the T K +/- m o u s e l y m p h o m a assay with zinc acetate are shown i n T a b l e 2. D o s e - d e p e n d e n t positive responses were o b t a i n e d in the presence a n d absence of the $9 m e t a b o l i c activation system with a d o u b l i n g of the m u t a t i o n frequency occurring at 10 / ~ g / m l for b o t h portions of the assay. The results of the C H O cell in vitro cytogenetic assay with zinc acetate are s h o w n in T a b l e 2. D o s e - d e p e n d e n t positive responses were o b t a i n e d in the presence a n d absence of the $9 activation system, although the $9 reduced b o t h the clastogenic response a n d the toxicity. Both zinc acetate a n d zinc 2 , 4 - p e n t a n e d i o n e were tested for i n d u c t i o n of U D S in p r i m a r y cultures of rat hepatocytes. N e i t h e r form of zinc i n d u c e d U D S or p r o d u c e d toxicity i n these cells over a range of 1 0 - 1 0 0 0 / ~ g / m l (data n o t shown).
Discussion As p o i n t e d out i n the i n t r o d u c t i o n , the results of short-term m u t a g e n i c i t y assays for zinc are
mixed. T h e reason for the conflicting results m a y be the form of zinc tested a n d the possible interactions b e t w e e n m e d i u m c o m p o u n d s a n d the zinc forms. F o r instance, w h e n i n o r g a n i c zinc (ZnC12 or Z n S O 4) is a d d e d to growth m e d i u m , the zinc is c o m p l e t e l y dissociated a n d n u m e r o u s c o n s t i t u e n t s in the m e d i u m (especially serum proteins) are c a p a b l e of b i n d i n g the free zinc (Parisi a n d Vallee, 1970). Thus, i n o r g a n i c zinc m a y be b o u n d to m e d i u m c o m p o n e n t s , a n d be u n a v a i l a b l e for t r a n s p o r t into the cell. O n the other h a n d , when zinc is tested as a n organic complex, there exists a c o m p e t i t i o n b e t w e e n that complex a n d m e d i u m c o n s t i t u e n t s for the zinc. If the organic c o m p o u n d b i n d s zinc with a higher affinity t h a n the m e d i u m c o m p o n e n t s , the zinc m a y be t r a n s p o r t e d as the organic complex. S u b s e q u e n t m e t a b o l i s m within the cell w o u l d then release the zinc. Thus, the presence or absence of a m u t a g e n i c response in a s h o r t - t e r m assay w o u l d d e p e n d o n the type of m e d i u m , the type of cell, a n d the b i n d i n g c o n s t a n t of the m e t a l complex. T h e b u l k of the in vitro m u t a g e n i c i t y d a t a appears to be c o n s i s t e n t with this proposal, as discussed below.
270 TABLE 2 RESULTS OF IN VITRO C Y T O G E N E T I C ASSAY W I T H CHO CELLS ON ZINC ACETATE Dose
Metabolic
Relative
Aberration types
(g g / m l )
activation
cloning efficiency
Chromatid b
Chromatid
gaps
break
Neg. Control (water)
-
100
25 34 45
-
Pos. control ( T E M - 0.5 g g / m l )
-
Neg. control (water)
+
45 60 80
+ + +
Pos. control (cyclophosphamide 35 g g / m l )
+
Fragments
Exchanges
Rings
> 10 Aberrations
% Polyploidy
Aber rations c per cell
2
0
0
0
0
0
2
0
100 36 53
2 1 3
0 5 10
0 1 5
0 15 22
0 0 0
0 0 5
4 4 4
0 0.42 1.74
2
4
23
26
42
1
8
4
3.44
0
0
0
0
0
0
0
0
73 76 58
3 2 1
7 17 7
0 2 0
10 21 5
0 0 0
1 3 5
4 6 0
0.54 1.40 1.24
9
9
28
17
45
0
4
4
2.60
(58)
a
100 (33) a
The cloning efficiency for water is arbitrarily set at 100. The number in parentheses is the number of clones formed when 200 cells (was determined by hemacytomer counts) were plated. The relative cloning efficiency of each dose equals the number of viable clones for that dose divided by the viable clones in the water control, times 100. b Counted, but not used in the aberrations per cell calculation. c Aberrations per cell is the total number of aberrations (excluding gaps) divided by the number of cells scored (50 for all doses). Cells with > 10 aberrations are counted as 10 aberrations. a
4 types of zinc have been tested in the Salmonella assay, 2 inorganic and 2 organic forms. Kalinina et al. (1977) and Gocke et al. (1981) tested ZnC12 and ZnSO4 respectively. The negative response by Gocke et al. is consistent with inorganic zinc binding to agar constituents. McCann (1978) has also demonstrated that inorganic metals are not transported through procaryotic cell walls. The positive response reported by Kalinina et al. appears to be inconsistent with the data presented here as well as those of Gocke et al. (1981). However, a close examination of the methods used by Kalininia et al. (1977) indicate their data are probably consistent with those reported here. We employed a standard plate-incorporation assay for the Salmonella studies in which the inorganic salts and loosely bound forms of zinc are negative. Kalinina et al. performed a suspension-type assay in which they suspended
the cells in a phosphate buffer which contained 0.2% sodium citrate, then added ZnC12 and incubated the mixture for 30 min. Under these conditions, the citrate will rapidly exchange Z n 2+ for N a +. Thus, Kalinina et al. were most likely testing a mixture of zinc citrate and ZnC1 z- Citrate has a log binding constant for zinc of 4 - 5 (Martell and Smith, 1977) which is close to that of the zinc 2,4-pentanedione complex (log binding constant of 5.06). The negative response with zinc acetate in the Salmonella assay suggests that binding of zinc by acetate (log of binding constant = 1.57, Martell and Smith, 1977) is either not sufficiently strong to compete with the agar medium or that zinc acetate does not cross the bacterial cell wall. The zinc 2,4-pentanedione complex (log of binding constant = 5.06) appears to be sufficiently stable to retain the zinc in the agar medium and is transported across the bacterial cell wall. The fact
271
that the mutagenic responses obtained by Kalinina et al. (1977) and those reported here agree, i.e. zinc-induced frameshift type mutations, supports the contention that the mutagenicity of zinc 2,4pentanedione is due to zinc. The disparity with the mouse lymphoma results reported by Amacher and Paillet (1980) and those reported here is also consistent with testing different forms of zinc. The ZnC12 tested by Amacher and Paillet will dissociate and likely be bound to serum proteins, whereas a significant portion of the zinc acetate may be available for transport. Deknudt and Deminatti (1978) reported ZnC12 to be "a weak clastogen in cultured human lymphocytes, whereas Gasiorek and Bauchinger (1981) reported zinc acetate to be negative in cultured human lymphocytes. However, Gasiorek and Bauchinger treated freshly isolated lymphocytes for 3 h, then induced replication. Thus the cells were in G o during treatment. Since cytogenetic events occur most readily in dividing cells, it is not surprising that a negative response was obtained. It is also possible that the 3 h exposure induced metallothionein which could bind zinc before cell replication occurred (Enger et al., 1983). Our data show zinc acetate to be a potent clastogen in CHO cells at concentrations equivalent to those tested by Deknudt and Deminatti (1978) and Gasiorck and Bauchinger (1981). The differences in the responses reported here and those reported by Deknudt and Deminatti may be differences in transport of the two forms of zinc tested. The differences in our results and those of Gasiorek and Bauchinger (1981) are most likely due to the cell-cycle stage during treatment, although metallothionein levels cannot be ruled out since CHO cells normally have very low levels of this protein (Enger et al., 1986). The results of the UDS assay in primary cultures of rat hepatocytes suggest that these cell are not susceptible to D N A damage by zinc. Whether this is due to lack of transport of the organic ligands by the hepatocytes or unique metabolic capabilities such as constitutive production of the metal detoxifying enzyme metallothionein (Enger et al., 1986) is not known. In vivo genotoxicity studies with zinc are generally negative (Gocke et al., 1981). A single report of clastogenicity in bone marrow of calcium-deft-
cient mice has appeared (Deknudt and Gerber, 1979), but the exposure conditions were so exaggerated the data are nearly uninterpretable e.g. the mice were maintained for 30 days on a diet which resulted in average body weights of 30 g in the controls and 12 g in the treated group. The same authors reported no effects by zinc on mice fed a standard diet. Thus, zinc fed to animals under realistic conditions is not likely to be genotoxic because of the homeostatic controls of zinc absorption (Underwood, 1977) and the fact that zinc is almost entirely bound to serum proteins or metalloproteins in the cells (Parisi and Vallee, 1970). The results presented here indicate zinc is an effective mutagen and clastogen when presented to a susceptible cell population in an appropriate form. While mutagenic responses were obtained in cultured mammalian fibroblasts at levels lower than may be found in many tissues in vivo, homeostatic controls of absorption and protein binding preclude the likelihood of zinc being genotoxic in vivo under standard feeding conditions.
Acknowledgements We thank Drs. R.L. Anderson and R.A. LeBoeuf for critical review of this manuscript. We are indebted to Ms. L.R. Schroer for assistance in preparation of the manuscript.
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