- Email: [email protected]
Suppression of arsenic-induced chromosome mutagenicity by antimony
Mutation Research 412 Ž1998. 213–218
Suppression of arsenic-induced chromosome mutagenicity by antimony T. Gebel
)
Medical Institute of General Hygiene and EnÕironmental Health, Windausweg 2, 37073 Goettingen, Germany Received 20 May 1997; revised 15 October 1997; accepted 15 October 1997
Abstract Arsenic and antimony are two semimetals sharing some chemical as well as toxicological properties. Both elements are clastogenic but not point mutagenic in their trivalent state of valency. Environmental exposure to arsenic was proven to be associated with increased rates of various types of cancers. Antimony is suspected to be carcinogenic to humans. Arsenic and antimony can be found as environmental co-contaminants resulting in co-exposure to man. However, in most regions where arsenic was found in elevated environmental amounts, it was not investigated whether an additional exposure to antimony was predominating. In this study, the chromosome mutagenicity induced by arsenicŽIII. was significantly suppressed by antimonyŽIII. in the micronucleus test with V79 cells. The results demonstrate the necessity to identify putative environmental co-contaminations of antimony in the regions contaminated with arsenic and to determine the impact of antimony co-exposure on arsenic genotoxicity and carcinogenicity in man in vivo. q 1998 Elsevier Science B.V. Keywords: Arsenic; Antimony; Combined genotoxicity; V79 cell
1. Introduction Arsenic is a widely distributed environmental contaminant and known to be a human carcinogen w1x. Elevated human exposures to this element, mostly caused by the intake of contaminated tap water, were shown to be associated with increased incidences of various cancers. In comparison, antimony, an element similar to arsenic in some chemical and toxicological properties, is less widely distributed in the environment. Antimony is suspected to be a carcino) Corresponding author. Tel.: q49 551 394973; Fax: q49 551 394957; E-mail: [email protected]
gen, which was shown to induce lung tumours in female rats w2x. However, it is not clear whether antimony is carcinogenic to man w3x. Like arsenic, antimony is chromosome but not point mutagenic only in the trivalent state of valency w4,5x. Concerning arsenic, clastogenicity but not aneugenicity seems to be the primary mode of genotoxic action w6x. Arsenic and antimony can be found as environmental co-contaminants, resulting in co-exposure to man and animals w7–9x. However, in most regions where arsenic was found in elevated environmental amounts, it has not been investigated whether an additional co-exposure to antimony was present. Putative consequences of such co-exposure on environ-
1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 8 1 - 2
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T. Gebel r Mutation Research 412 (1998) 213–218
mental health are unclear up to now. In the present study, experiments were carried out to investigate the combined mode of arsenic and antimony action in mammalian cells. The micronucleus test is a valid tool for the detection of arsenic and antimony genotoxicity and can be taken as an early marker for the identification of putative carcinogenic hazards. Thus, the trivalent compounds As 2 O 3 and SbCl 3 were used in single and in combined application in the micronucleus test with V79 Chinese hamster cells to obtain information on the mechanism of combined genotoxicity of arsenic and antimony. Furthermore, combined cytotoxicity was tested in the neutral red assay.
2.2. Cellular intake To determine the cell-associated amounts of arsenic and antimony 24 h incubations with 5 mM arsenic andror 5 mM antimony were performed. Thereafter, the cells were washed three times with PBS, counted and decomposed in a closed system using a microwave apparatus ŽMLS mega 1200, MLS Leutkirch, Germany. with 500 ml suspension containing 10 6 cells added to 0.5 ml 65% nitric acid and 0.5 ml 30% hydrogen peroxide. Arsenic and antimony were determined by the graphite furnace technique with an atomic absorption spectrometer Perkin Elmer SIMAA 6000 with Zeeman background compensation.
2. Materials and methods
2.3. In Õitro micronucleus test
2.1. Cytotoxicity test
Cells were seeded at a number of 2 = 10 5 per plastic dish ŽB6 cm. and cultured in 4 ml medium ŽDMEM, Biochrom Berlin, Germany. supplemented with 10% calf serum and penicillin Ž100 IUrml.rstreptomycin Ž100 mgrml.. SbCl 3 and As 2 O 3 were both obtained in pure grade from Merck ŽDarmstadt, Germany.. SbCl 3 was dissolved in dimethylsulfoxide ŽDMSO. and added to the culture in volumes of 25 ml. In this case, pure DMSO was used as negative control. As 2 O 3 was dissolved in distilled water. In the cytokinesis-block variant of the micronucleus test, 4 h after substance application cytochalasin-B was given to the cultures in order to yield a final concentration of 3 mgrml. 20 h later the cells were harvested. After a 5-min treatment with hypotonic solution Ž0.07 M KClr0.15 M NaCl; 1 q 9., the cells were fixed in methanol and acetic acid Ž9 q 1. for at least 90 min at 48C and sedimented by 155 = g for 10 min. The step of fixation was repeated once with an incubation time of 30 min. Microscopic slides were prepared in duplicate by dropping and stained with 5% Giemsa solution ŽpH 6.8. for 12–15 min. Mitomycin C ŽMMC; Sigma, Deisenhofen, Germany. was included in all experiments as a positive control. Micronuclei, as defined by criteria summarized by w10x, were scored in all or, in the cytokinesis-block variant, in 1000 binucleate cells with two nuclei of approximately equal size. The nuclear division index ŽNDI. was calculated as
V79 cells were grown in a humidified atmosphere with 5% CO 2 in DMEM medium ŽBiochrom Berlin, Germany. supplemented with 10% calf serum and penicillin Ž100 IUrml.rstreptomycin Ž100 mgrml. at 378C. Each well of a 96-well tissue-culture microtitre plate was inoculated with 0.1 medium containing 10 4 cells. The plates were incubated for 24 h. Thereafter, the medium was replaced by medium containing one of the test substances. As 2 O 3 was dissolved in distilled water. SbCl 3 was dissolved in DMSO, pure DMSO Ž12 wells. was used as negative control. Each concentration was tested in 6 wells per plate. After an incubation of another 24 h, the neutral red ŽNR. assay was performed: the medium was removed from the microtitre plates, and 0.1 ml DMEM supplemented with 20 mM Hepes containing 50 mg NRrml was added. After a further incubation of 3 h, the cells were washed with 100 mM phosphate-buffered saline. Then 0.1 ml of a solution of 1% Žvrv. acetic acidr50% Žvrv. ethanol was added to each well to extract the dye. After 10 min on a microtitre plate shaker, a spectrophotometrical analysis at 540 nm with a microplate reader was performed. Non-specific staining of non-viable cells in the negative controls was less than 5% compared to that of the positive controls. Each test was done in duplicate and repeated two or three times.
T. Gebel r Mutation Research 412 (1998) 213–218
215
Fig. 1. Single and combined cytotoxicity of AsŽIII. and SbŽIII. in V79 cells.
given in w6x. The number of micronuclei was analysed statistically with Fisher’s Exact test.
3. Results and discussion The viability of V79 cells was 100% at doses of SbCl 3 up to 10 mM ŽFig. 1.. 50% survival was found with approximately 75 mM SbCl 3 and 12.5 mM As 2 O 3 Ži.e. 25 mM As., respectively. Combined cytotoxicity was tested using 10 and 50 mM SbCl 3 added to As 2 O 3 in doses ranging from 0.5 to 12.5 mM. 50% survival was found with 10 and 50 mM SbCl 3 added at AsŽIII. concentrations of roughly 15
and 10 mM, respectively. Assuming a threefold higher cytotoxicity of AsŽIII. in comparison to SbŽIII., combined cytotoxicity seemed best described by effect addition of AsŽIII. and SbŽIII.. Arsenic and antimony accumulated cell association in comparison to the culture medium 7-fold and 10-fold after 24-h incubations, respectively ŽTable 1.. This suggests that antimony is taken up into cells equally well as is arsenic w11–13x. However, in the combined incubations, the contents of AsŽIII. and SbŽIII. were lower than after the single element applications. This effect was significant only for SbŽIII. Ž p - 0.05, t-test. and may have been caused by competition of binding to intracellular sulfhydryl
Table 1 Cellular association of AsŽIII. and SbŽIII. in V79 cells incubated for 24 h Arsenic Žtotal. mM 5 mM SbCl 3 2.5 mM As 2 O 3 2.5 mM As 2 O 3 q 5 mM SbCl 3
Antimony Žtotal. mM 54.2 " 6.6 Ž n s 4.
33.2 " 11.3 Ž n s 4. 24.6 " 10.7 Ž n s 4.
27.5 " 6.8 Ž n s 4. a
The intracellular concentrations of arsenic and antimony were calculated by the help of data from w26x, who determined the volume of a V79 cell with 1.55 = 10y1 2 l. a p - 0.05 in Student’s t-test lower than SbŽIII. intracellularly in comparison to 5 mM SbCl 3 in single application.
216
T. Gebel r Mutation Research 412 (1998) 213–218
moieties. The amounts of arsenic and antimony intracellularly free might not have been affected, because combined cytotoxicity could be described by effect addition. It does not seem likely that combined genotoxicity was suppressed because of competitive inhibition of cellular uptake of AsŽIII. and SbŽIII., because both elements were found cell-associated in accumulation. Furthermore, AsŽIII. and SbŽIII. are likely to be uncharged under physiological conditions and may enter the cells via diffusion Žreviewed in w14x.. In the V79 micronucleus test, trivalent arsenic proved to be a far more potent chromosome mutagen than trivalent antimony ŽFig. 2.. Significantly elevated frequencies of micronuclei were obtained first with As 2 O 3 first at a dose of 0.25 mM Ž p - 0.05, Fisher’s Exact test.. A significantly increased chromosome mutagenicity was found with SbCl 3 first at a dose of 10 mM. In the experimental co-incubation setup, 0.5 mM As 2 O 3 was added to the same antimony dose regime which had been applied in the single substance setup. The resulting genotoxicity was found lying significantly below the effect obtained by simple addition of the effects of AsŽIII.
and SbŽIII. obtained after single substance application Ž p - 0.05, Fisher’s Exact test.. This effect was also found by using 0.1 mM As 2 O 3 Ždata not shown.. The cytokinesis-block variant of the micronucleus test using cytochalasin B as cytokinesis inhibitor but not nuclear division inhibitor offers several advantages in comparison to the classical form of the micronucleus test. Cellular division, i.e. cell viability, can be proved during the experiment. Micronuclei can be counted in cells having undergone exactly one nuclear division during test substance application. Thus, the lesions generated by test substance application can be detected more specifically. A significantly elevated rate of genetic damage was observed in the cytokinesis-block micronucleus test with As 2 O 3 first at a dose of 1 mM, and with SbCl 3 first at 10 mM, respectively. Effects of cytotoxicity indicated by a decrease in the nuclear division index were observed at 5 mM As 2 O 3 and 50 mM SbCl 3 , respectively. A significantly lower than additive coclastogenicity of AsŽIII. and SbŽIII. was found in the cytokinesis-block variant of the micronucleus test, too ŽTable 2.. This effect was also observed by using 0.1 mM As 2 O 3 Ždata not shown.. Confirming these
Fig. 2. Induction of micronuclei in V79 cells by arsenic and antimony. The combined effect was statistically lower than adding the effects of the single substance dose regimes Ž p - 0.05, Fisher’s exact test..
T. Gebel r Mutation Research 412 (1998) 213–218 Table 2 Induction of micronuclei in V79 cells by arsenic and antimony Žcytokinesis-block variant. Compound As 2 O 3 ŽIII.
MMC SbCl 3 ŽIII.
MMC
Dose ŽmM. MN 0 0.05 0.25 0.5 1 2.5 5 0.5 0 0.5 1 5 10 25 50 0.5
SbCl 3 q2 mM AsŽIII.
MMC
0 1 5 10 25 0.5
SD NDI n
6.5 8.5 9.0 10.0 14.5 a 19.5 b 29.5 b 41.5
0.7 2.1 1.4 1.4 0.7 0.7 2.1 0.7
2.10 2.03 2.00 1.95 1.92 1.85 1.39 1.86
2 2 2 2 2 2 2 2
9.5 10.5 11.0 10.5 12.0 17.5 a 21.0 a 45.5
2.1 2.1 0.0 0.7 0.0 0.7 0.0 2.1
2.02 2.12 2.09 2.02 2.00 2.00 1.84 1.86
2 2 2 2 2 2 2 2
0.8 0.8 1.3 1.3 1.0 4.4
2.00 1.91 1.94 1.93 1.90 1.89
4 4 4 4 4 4
det
calc
11.0 9.5 a 12.5 a 15.8 a 19.5 a 40.5
19.0 18.5 20.0 25.5
det, determined effect; calc, calculated effect Žsimple addition.; MMC, mitomycin C; MN, mean number of micronuclei in 2000 or 4000 binucleate cells counted in total of two or four independent experiments, respectively; n, number of experiments; NDI, nuclear division index calculated as described in Section 2; SD, standard deviation. a p- 0.05; b p- 0.001, statistically significant in Fisher’s Exact test vs. control or vs. calculated in the combination experiments, respectively.
observations, a competitive inhibition of arsenic and antimony combined genotoxicity had been observed previously in the sister chromatid exchange test with human peripheral lymphocytes w9x. Knowledge is scarce on the mechanism of antimony genotoxicity. In contrast, there is evidence that AsŽIII. acts genotoxic through inhibition of enzymes involved in DNA repair w12,15,16x. Antimony, like arsenic, seems to have an affinity to sulfhydryl groups w17,18x. Thus, suppression of combined genotoxicity observed in the present study might possibly be caused by competition at such moieties.
217
Clastogenicity can be considered as a cellular indicator for the early detection of carcinogenic hazards. In the case of elevated internal exposures, both arsenic and antimony were shown to induce chromosomal damage in vivo w19–21x. Environmental contamination with arsenic associated with increased incidences of various types of neoplasia still is a problem in several Asian and Southern American countries w22–25x. However, in these regions, a possible environmental co-contamination with antimony has not been investigated in most cases. Because of the results presented in this study, it seems necessary to identify co-contaminations of antimony in these regions, and to determine the impact of antimony co-exposure on arsenic genotoxicity and carcinogenicity in man in vivo. Acknowledgements The excellent technical assistance of Petra Birkenkamp and Kerstin Kleinschmidt is greatly acknowledged. References w1x IARC, Carcinogenesis of Arsenic and Arsenic Compounds, IARC Monographs, Lyon, 1980, pp. 37–141. w2x D.H. Groth, L.E. Stettler, J.R. Burg, W.M. Busey, G.C. Grant, L. Wong, Carcinogenic effects of antimony trioxide and antimony ore concentrate in rats, J. Toxicol. Environ. Health 18 Ž4. Ž1986. 607–626. w3x R.D. Jones, Survey of antimony workers: mortality 1961– 1992, Occup. Environ. Med. 51 Ž11. Ž1994. 772–776. w4x K. Kuroda, G. Endo, A. Okamoto, Y.S. Yoo, S. Horiguchi, Genotoxicity of beryllium, gallium and antimony in shortterm assays, Mutation Res. 264 Ž4. Ž1991. 163–170. w5x T.G. Rossman, D. Stone, M. Molina, W. Troll, Absence of arsenite mutagenicity in E. coli and Chinese hamster cells, Environ. Mutagen. 2 Ž3. Ž1981. 371–379. w6x D.A. Eastmond, J.D. Tucker, Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an anti-kinetochore antibody, Environ. Mol. Mutagen. 13 Ž1989. 34–43. w7x X. Li, I. Thornton, Arsenic, antimony and bismuth in soil and pasture herbage in some old metalliferous mining areas in England, Environ. Geochem. Health 15 Ž1993. 135–144. w8x T. Gebel, S. Kevekordes, J. Schaefer, H. von Platen, H. Dunkelberg, Assessment of a possible genotoxic environmental risk in sheep bred on grounds with strongly elevated contents of mercury, arsenic and antimony, Mutation Res. 368 Ž3–4. Ž1996. 267–274.
218
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w9x T. Gebel, S. Christensen, H. Dunkelberg, Comparative and environmental genotoxicity of antimony and arsenic, Anticancer Res. 17 Ž4A. Ž1997. 2603–2607. w10x M. Fenech, The cytokinesis-block technique: a detailed description of the method and its application to genotoxicity studies in human populations, Mutation Res. 285 Ž1993. 35–44. w11x F. Bertolero, G. Pozzi, E. Sabbioni, U. Saffiotti, Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation, Carcinogenesis 8 Ž6. Ž1987. 803–808. w12x A. Hartwig, U.D. Groeblinghoff, D. Beyersmann, A.T. Natarajan, R. Filon, L.H. Mullenders, Interaction of arsenicŽIII. with nucleotide excision repair in UV-irradiated human fibroblasts, Carcinogenesis 18 Ž2. Ž1997. 399–405. w13x S.A. Lerman, T.W. Clarkson, R.J. Gerson, Arsenic uptake and metabolism by liver cells is dependent on arsenic oxidation state, Chem.-Biol. Interact. 45 Ž3. Ž1983. 401–406. w14x T. Gebel, Arsenic and antimony: comparative approach on mechanistic toxicology, Chem.-Biol. Interact., Ž1998. in press. w15x J.W. Yager, J.K. Wiencke, Inhibition of polyŽADP-ribose. polymerase by arsenite, Mutation Res. 386 Ž1997. 345–351. w16x S.F. Lee Chen, C.T. Yu, K.Y. Jan, Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cells, Mutagenesis 7 Ž1. Ž1992. 51–55. w17x R. Bailly, R. Lauwerys, J.P. Buchet, P. Mahieu, J. Konings, Experimental and human studies on antimony metabolism: their relevance for the biological monitoring of workers exposed to inorganic antimony, Br. J. Ind. Med. 48 Ž2. Ž1991. 93–97. w18x M.A. Basinger, M.M. Jones, Structural requirements for
w19x
w20x
w21x
w22x
w23x w24x
w25x
w26x
chelate antidotal efficacy in acute antimonyŽIII. intoxication, Res. Commun. Chem. Pathol. Pharmacol. 32 Ž2. Ž1981. 355–363. D. Lerda, Sister-chromatid exchange ŽSCE. among individuals chronically exposed to arsenic in drinking water, Mutation Res. 312 Ž2. Ž1994. 111–120. L.E. Moore, M.L. Warner, A.H. Smith, D. Kalman, M.T. Smith, Use of the fluorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells, Environ. Mol. Mutagen. 27 Ž3. Ž1996. 176–184. N. Hashem, R. Shawki, Cultured peripheral lymphocytes: one biologic indicator of potential drug hazard, Afr. J. Med. Med. Sci. 5 Ž2. Ž1976. 155–163. M.N. Bates, A.H. Smith, C. Hopenhayn Rich, Arsenic ingestion and internal cancers: a review, Am. J. Epidemiol. 135 Ž5. Ž1992. 462–476. C.J. Chen, T.L. Kuo, M.M. Wu, Arsenic and cancers, Lancet 1 Ž8582. Ž1988. 414–415, wletterx. D. Das, A. Chatterjee, B.K. Mandal, G. Samanta, D. Chakraborti, B. Chanda, Arsenic in ground water in six districts of West bengal, India: the biggest arsenic calamity in the world. Part 2. Arsenic concentration in drinking water, hair, nails, urine, skin-scale and liver tissue Žbiopsy. of the affected people, Analyst 120 Ž3. Ž1995. 917–924. W.P. Tseng, Effects and dose-response relationships of skin cancer and blackfoot disease with arsenic, Environ. Health Perspect. 19 Ž1977. 109–119. U. Sehlmeyer, S. Hechtenberg, H. Klyszcz, D. Beyersmann, Accumulation of chromium in Chinese hamster V79-cells and nuclei, Arch. Toxicol. 64 Ž1990. 506–508.
Suppression of arsenic-induced chromosome mutagenicity by antimony T. Gebel
)
Medical Institute of General Hygiene and EnÕironmental Health, Windausweg 2, 37073 Goettingen, Germany Received 20 May 1997; revised 15 October 1997; accepted 15 October 1997
Abstract Arsenic and antimony are two semimetals sharing some chemical as well as toxicological properties. Both elements are clastogenic but not point mutagenic in their trivalent state of valency. Environmental exposure to arsenic was proven to be associated with increased rates of various types of cancers. Antimony is suspected to be carcinogenic to humans. Arsenic and antimony can be found as environmental co-contaminants resulting in co-exposure to man. However, in most regions where arsenic was found in elevated environmental amounts, it was not investigated whether an additional exposure to antimony was predominating. In this study, the chromosome mutagenicity induced by arsenicŽIII. was significantly suppressed by antimonyŽIII. in the micronucleus test with V79 cells. The results demonstrate the necessity to identify putative environmental co-contaminations of antimony in the regions contaminated with arsenic and to determine the impact of antimony co-exposure on arsenic genotoxicity and carcinogenicity in man in vivo. q 1998 Elsevier Science B.V. Keywords: Arsenic; Antimony; Combined genotoxicity; V79 cell
1. Introduction Arsenic is a widely distributed environmental contaminant and known to be a human carcinogen w1x. Elevated human exposures to this element, mostly caused by the intake of contaminated tap water, were shown to be associated with increased incidences of various cancers. In comparison, antimony, an element similar to arsenic in some chemical and toxicological properties, is less widely distributed in the environment. Antimony is suspected to be a carcino) Corresponding author. Tel.: q49 551 394973; Fax: q49 551 394957; E-mail: [email protected]
gen, which was shown to induce lung tumours in female rats w2x. However, it is not clear whether antimony is carcinogenic to man w3x. Like arsenic, antimony is chromosome but not point mutagenic only in the trivalent state of valency w4,5x. Concerning arsenic, clastogenicity but not aneugenicity seems to be the primary mode of genotoxic action w6x. Arsenic and antimony can be found as environmental co-contaminants, resulting in co-exposure to man and animals w7–9x. However, in most regions where arsenic was found in elevated environmental amounts, it has not been investigated whether an additional co-exposure to antimony was present. Putative consequences of such co-exposure on environ-
1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 8 1 - 2
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T. Gebel r Mutation Research 412 (1998) 213–218
mental health are unclear up to now. In the present study, experiments were carried out to investigate the combined mode of arsenic and antimony action in mammalian cells. The micronucleus test is a valid tool for the detection of arsenic and antimony genotoxicity and can be taken as an early marker for the identification of putative carcinogenic hazards. Thus, the trivalent compounds As 2 O 3 and SbCl 3 were used in single and in combined application in the micronucleus test with V79 Chinese hamster cells to obtain information on the mechanism of combined genotoxicity of arsenic and antimony. Furthermore, combined cytotoxicity was tested in the neutral red assay.
2.2. Cellular intake To determine the cell-associated amounts of arsenic and antimony 24 h incubations with 5 mM arsenic andror 5 mM antimony were performed. Thereafter, the cells were washed three times with PBS, counted and decomposed in a closed system using a microwave apparatus ŽMLS mega 1200, MLS Leutkirch, Germany. with 500 ml suspension containing 10 6 cells added to 0.5 ml 65% nitric acid and 0.5 ml 30% hydrogen peroxide. Arsenic and antimony were determined by the graphite furnace technique with an atomic absorption spectrometer Perkin Elmer SIMAA 6000 with Zeeman background compensation.
2. Materials and methods
2.3. In Õitro micronucleus test
2.1. Cytotoxicity test
Cells were seeded at a number of 2 = 10 5 per plastic dish ŽB6 cm. and cultured in 4 ml medium ŽDMEM, Biochrom Berlin, Germany. supplemented with 10% calf serum and penicillin Ž100 IUrml.rstreptomycin Ž100 mgrml.. SbCl 3 and As 2 O 3 were both obtained in pure grade from Merck ŽDarmstadt, Germany.. SbCl 3 was dissolved in dimethylsulfoxide ŽDMSO. and added to the culture in volumes of 25 ml. In this case, pure DMSO was used as negative control. As 2 O 3 was dissolved in distilled water. In the cytokinesis-block variant of the micronucleus test, 4 h after substance application cytochalasin-B was given to the cultures in order to yield a final concentration of 3 mgrml. 20 h later the cells were harvested. After a 5-min treatment with hypotonic solution Ž0.07 M KClr0.15 M NaCl; 1 q 9., the cells were fixed in methanol and acetic acid Ž9 q 1. for at least 90 min at 48C and sedimented by 155 = g for 10 min. The step of fixation was repeated once with an incubation time of 30 min. Microscopic slides were prepared in duplicate by dropping and stained with 5% Giemsa solution ŽpH 6.8. for 12–15 min. Mitomycin C ŽMMC; Sigma, Deisenhofen, Germany. was included in all experiments as a positive control. Micronuclei, as defined by criteria summarized by w10x, were scored in all or, in the cytokinesis-block variant, in 1000 binucleate cells with two nuclei of approximately equal size. The nuclear division index ŽNDI. was calculated as
V79 cells were grown in a humidified atmosphere with 5% CO 2 in DMEM medium ŽBiochrom Berlin, Germany. supplemented with 10% calf serum and penicillin Ž100 IUrml.rstreptomycin Ž100 mgrml. at 378C. Each well of a 96-well tissue-culture microtitre plate was inoculated with 0.1 medium containing 10 4 cells. The plates were incubated for 24 h. Thereafter, the medium was replaced by medium containing one of the test substances. As 2 O 3 was dissolved in distilled water. SbCl 3 was dissolved in DMSO, pure DMSO Ž12 wells. was used as negative control. Each concentration was tested in 6 wells per plate. After an incubation of another 24 h, the neutral red ŽNR. assay was performed: the medium was removed from the microtitre plates, and 0.1 ml DMEM supplemented with 20 mM Hepes containing 50 mg NRrml was added. After a further incubation of 3 h, the cells were washed with 100 mM phosphate-buffered saline. Then 0.1 ml of a solution of 1% Žvrv. acetic acidr50% Žvrv. ethanol was added to each well to extract the dye. After 10 min on a microtitre plate shaker, a spectrophotometrical analysis at 540 nm with a microplate reader was performed. Non-specific staining of non-viable cells in the negative controls was less than 5% compared to that of the positive controls. Each test was done in duplicate and repeated two or three times.
T. Gebel r Mutation Research 412 (1998) 213–218
215
Fig. 1. Single and combined cytotoxicity of AsŽIII. and SbŽIII. in V79 cells.
given in w6x. The number of micronuclei was analysed statistically with Fisher’s Exact test.
3. Results and discussion The viability of V79 cells was 100% at doses of SbCl 3 up to 10 mM ŽFig. 1.. 50% survival was found with approximately 75 mM SbCl 3 and 12.5 mM As 2 O 3 Ži.e. 25 mM As., respectively. Combined cytotoxicity was tested using 10 and 50 mM SbCl 3 added to As 2 O 3 in doses ranging from 0.5 to 12.5 mM. 50% survival was found with 10 and 50 mM SbCl 3 added at AsŽIII. concentrations of roughly 15
and 10 mM, respectively. Assuming a threefold higher cytotoxicity of AsŽIII. in comparison to SbŽIII., combined cytotoxicity seemed best described by effect addition of AsŽIII. and SbŽIII.. Arsenic and antimony accumulated cell association in comparison to the culture medium 7-fold and 10-fold after 24-h incubations, respectively ŽTable 1.. This suggests that antimony is taken up into cells equally well as is arsenic w11–13x. However, in the combined incubations, the contents of AsŽIII. and SbŽIII. were lower than after the single element applications. This effect was significant only for SbŽIII. Ž p - 0.05, t-test. and may have been caused by competition of binding to intracellular sulfhydryl
Table 1 Cellular association of AsŽIII. and SbŽIII. in V79 cells incubated for 24 h Arsenic Žtotal. mM 5 mM SbCl 3 2.5 mM As 2 O 3 2.5 mM As 2 O 3 q 5 mM SbCl 3
Antimony Žtotal. mM 54.2 " 6.6 Ž n s 4.
33.2 " 11.3 Ž n s 4. 24.6 " 10.7 Ž n s 4.
27.5 " 6.8 Ž n s 4. a
The intracellular concentrations of arsenic and antimony were calculated by the help of data from w26x, who determined the volume of a V79 cell with 1.55 = 10y1 2 l. a p - 0.05 in Student’s t-test lower than SbŽIII. intracellularly in comparison to 5 mM SbCl 3 in single application.
216
T. Gebel r Mutation Research 412 (1998) 213–218
moieties. The amounts of arsenic and antimony intracellularly free might not have been affected, because combined cytotoxicity could be described by effect addition. It does not seem likely that combined genotoxicity was suppressed because of competitive inhibition of cellular uptake of AsŽIII. and SbŽIII., because both elements were found cell-associated in accumulation. Furthermore, AsŽIII. and SbŽIII. are likely to be uncharged under physiological conditions and may enter the cells via diffusion Žreviewed in w14x.. In the V79 micronucleus test, trivalent arsenic proved to be a far more potent chromosome mutagen than trivalent antimony ŽFig. 2.. Significantly elevated frequencies of micronuclei were obtained first with As 2 O 3 first at a dose of 0.25 mM Ž p - 0.05, Fisher’s Exact test.. A significantly increased chromosome mutagenicity was found with SbCl 3 first at a dose of 10 mM. In the experimental co-incubation setup, 0.5 mM As 2 O 3 was added to the same antimony dose regime which had been applied in the single substance setup. The resulting genotoxicity was found lying significantly below the effect obtained by simple addition of the effects of AsŽIII.
and SbŽIII. obtained after single substance application Ž p - 0.05, Fisher’s Exact test.. This effect was also found by using 0.1 mM As 2 O 3 Ždata not shown.. The cytokinesis-block variant of the micronucleus test using cytochalasin B as cytokinesis inhibitor but not nuclear division inhibitor offers several advantages in comparison to the classical form of the micronucleus test. Cellular division, i.e. cell viability, can be proved during the experiment. Micronuclei can be counted in cells having undergone exactly one nuclear division during test substance application. Thus, the lesions generated by test substance application can be detected more specifically. A significantly elevated rate of genetic damage was observed in the cytokinesis-block micronucleus test with As 2 O 3 first at a dose of 1 mM, and with SbCl 3 first at 10 mM, respectively. Effects of cytotoxicity indicated by a decrease in the nuclear division index were observed at 5 mM As 2 O 3 and 50 mM SbCl 3 , respectively. A significantly lower than additive coclastogenicity of AsŽIII. and SbŽIII. was found in the cytokinesis-block variant of the micronucleus test, too ŽTable 2.. This effect was also observed by using 0.1 mM As 2 O 3 Ždata not shown.. Confirming these
Fig. 2. Induction of micronuclei in V79 cells by arsenic and antimony. The combined effect was statistically lower than adding the effects of the single substance dose regimes Ž p - 0.05, Fisher’s exact test..
T. Gebel r Mutation Research 412 (1998) 213–218 Table 2 Induction of micronuclei in V79 cells by arsenic and antimony Žcytokinesis-block variant. Compound As 2 O 3 ŽIII.
MMC SbCl 3 ŽIII.
MMC
Dose ŽmM. MN 0 0.05 0.25 0.5 1 2.5 5 0.5 0 0.5 1 5 10 25 50 0.5
SbCl 3 q2 mM AsŽIII.
MMC
0 1 5 10 25 0.5
SD NDI n
6.5 8.5 9.0 10.0 14.5 a 19.5 b 29.5 b 41.5
0.7 2.1 1.4 1.4 0.7 0.7 2.1 0.7
2.10 2.03 2.00 1.95 1.92 1.85 1.39 1.86
2 2 2 2 2 2 2 2
9.5 10.5 11.0 10.5 12.0 17.5 a 21.0 a 45.5
2.1 2.1 0.0 0.7 0.0 0.7 0.0 2.1
2.02 2.12 2.09 2.02 2.00 2.00 1.84 1.86
2 2 2 2 2 2 2 2
0.8 0.8 1.3 1.3 1.0 4.4
2.00 1.91 1.94 1.93 1.90 1.89
4 4 4 4 4 4
det
calc
11.0 9.5 a 12.5 a 15.8 a 19.5 a 40.5
19.0 18.5 20.0 25.5
det, determined effect; calc, calculated effect Žsimple addition.; MMC, mitomycin C; MN, mean number of micronuclei in 2000 or 4000 binucleate cells counted in total of two or four independent experiments, respectively; n, number of experiments; NDI, nuclear division index calculated as described in Section 2; SD, standard deviation. a p- 0.05; b p- 0.001, statistically significant in Fisher’s Exact test vs. control or vs. calculated in the combination experiments, respectively.
observations, a competitive inhibition of arsenic and antimony combined genotoxicity had been observed previously in the sister chromatid exchange test with human peripheral lymphocytes w9x. Knowledge is scarce on the mechanism of antimony genotoxicity. In contrast, there is evidence that AsŽIII. acts genotoxic through inhibition of enzymes involved in DNA repair w12,15,16x. Antimony, like arsenic, seems to have an affinity to sulfhydryl groups w17,18x. Thus, suppression of combined genotoxicity observed in the present study might possibly be caused by competition at such moieties.
217
Clastogenicity can be considered as a cellular indicator for the early detection of carcinogenic hazards. In the case of elevated internal exposures, both arsenic and antimony were shown to induce chromosomal damage in vivo w19–21x. Environmental contamination with arsenic associated with increased incidences of various types of neoplasia still is a problem in several Asian and Southern American countries w22–25x. However, in these regions, a possible environmental co-contamination with antimony has not been investigated in most cases. Because of the results presented in this study, it seems necessary to identify co-contaminations of antimony in these regions, and to determine the impact of antimony co-exposure on arsenic genotoxicity and carcinogenicity in man in vivo. Acknowledgements The excellent technical assistance of Petra Birkenkamp and Kerstin Kleinschmidt is greatly acknowledged. References w1x IARC, Carcinogenesis of Arsenic and Arsenic Compounds, IARC Monographs, Lyon, 1980, pp. 37–141. w2x D.H. Groth, L.E. Stettler, J.R. Burg, W.M. Busey, G.C. Grant, L. Wong, Carcinogenic effects of antimony trioxide and antimony ore concentrate in rats, J. Toxicol. Environ. Health 18 Ž4. Ž1986. 607–626. w3x R.D. Jones, Survey of antimony workers: mortality 1961– 1992, Occup. Environ. Med. 51 Ž11. Ž1994. 772–776. w4x K. Kuroda, G. Endo, A. Okamoto, Y.S. Yoo, S. Horiguchi, Genotoxicity of beryllium, gallium and antimony in shortterm assays, Mutation Res. 264 Ž4. Ž1991. 163–170. w5x T.G. Rossman, D. Stone, M. Molina, W. Troll, Absence of arsenite mutagenicity in E. coli and Chinese hamster cells, Environ. Mutagen. 2 Ž3. Ž1981. 371–379. w6x D.A. Eastmond, J.D. Tucker, Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an anti-kinetochore antibody, Environ. Mol. Mutagen. 13 Ž1989. 34–43. w7x X. Li, I. Thornton, Arsenic, antimony and bismuth in soil and pasture herbage in some old metalliferous mining areas in England, Environ. Geochem. Health 15 Ž1993. 135–144. w8x T. Gebel, S. Kevekordes, J. Schaefer, H. von Platen, H. Dunkelberg, Assessment of a possible genotoxic environmental risk in sheep bred on grounds with strongly elevated contents of mercury, arsenic and antimony, Mutation Res. 368 Ž3–4. Ž1996. 267–274.
218
T. Gebel r Mutation Research 412 (1998) 213–218
w9x T. Gebel, S. Christensen, H. Dunkelberg, Comparative and environmental genotoxicity of antimony and arsenic, Anticancer Res. 17 Ž4A. Ž1997. 2603–2607. w10x M. Fenech, The cytokinesis-block technique: a detailed description of the method and its application to genotoxicity studies in human populations, Mutation Res. 285 Ž1993. 35–44. w11x F. Bertolero, G. Pozzi, E. Sabbioni, U. Saffiotti, Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation, Carcinogenesis 8 Ž6. Ž1987. 803–808. w12x A. Hartwig, U.D. Groeblinghoff, D. Beyersmann, A.T. Natarajan, R. Filon, L.H. Mullenders, Interaction of arsenicŽIII. with nucleotide excision repair in UV-irradiated human fibroblasts, Carcinogenesis 18 Ž2. Ž1997. 399–405. w13x S.A. Lerman, T.W. Clarkson, R.J. Gerson, Arsenic uptake and metabolism by liver cells is dependent on arsenic oxidation state, Chem.-Biol. Interact. 45 Ž3. Ž1983. 401–406. w14x T. Gebel, Arsenic and antimony: comparative approach on mechanistic toxicology, Chem.-Biol. Interact., Ž1998. in press. w15x J.W. Yager, J.K. Wiencke, Inhibition of polyŽADP-ribose. polymerase by arsenite, Mutation Res. 386 Ž1997. 345–351. w16x S.F. Lee Chen, C.T. Yu, K.Y. Jan, Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cells, Mutagenesis 7 Ž1. Ž1992. 51–55. w17x R. Bailly, R. Lauwerys, J.P. Buchet, P. Mahieu, J. Konings, Experimental and human studies on antimony metabolism: their relevance for the biological monitoring of workers exposed to inorganic antimony, Br. J. Ind. Med. 48 Ž2. Ž1991. 93–97. w18x M.A. Basinger, M.M. Jones, Structural requirements for
w19x
w20x
w21x
w22x
w23x w24x
w25x
w26x
chelate antidotal efficacy in acute antimonyŽIII. intoxication, Res. Commun. Chem. Pathol. Pharmacol. 32 Ž2. Ž1981. 355–363. D. Lerda, Sister-chromatid exchange ŽSCE. among individuals chronically exposed to arsenic in drinking water, Mutation Res. 312 Ž2. Ž1994. 111–120. L.E. Moore, M.L. Warner, A.H. Smith, D. Kalman, M.T. Smith, Use of the fluorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells, Environ. Mol. Mutagen. 27 Ž3. Ž1996. 176–184. N. Hashem, R. Shawki, Cultured peripheral lymphocytes: one biologic indicator of potential drug hazard, Afr. J. Med. Med. Sci. 5 Ž2. Ž1976. 155–163. M.N. Bates, A.H. Smith, C. Hopenhayn Rich, Arsenic ingestion and internal cancers: a review, Am. J. Epidemiol. 135 Ž5. Ž1992. 462–476. C.J. Chen, T.L. Kuo, M.M. Wu, Arsenic and cancers, Lancet 1 Ž8582. Ž1988. 414–415, wletterx. D. Das, A. Chatterjee, B.K. Mandal, G. Samanta, D. Chakraborti, B. Chanda, Arsenic in ground water in six districts of West bengal, India: the biggest arsenic calamity in the world. Part 2. Arsenic concentration in drinking water, hair, nails, urine, skin-scale and liver tissue Žbiopsy. of the affected people, Analyst 120 Ž3. Ž1995. 917–924. W.P. Tseng, Effects and dose-response relationships of skin cancer and blackfoot disease with arsenic, Environ. Health Perspect. 19 Ž1977. 109–119. U. Sehlmeyer, S. Hechtenberg, H. Klyszcz, D. Beyersmann, Accumulation of chromium in Chinese hamster V79-cells and nuclei, Arch. Toxicol. 64 Ž1990. 506–508.