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Squalene inhibits sodium arsenite-induced sister chromatid exchanges and micronuclei in Chinese hamster ovary-K1 cells
Genetic Toxicology
ELSEVIER
Mutation Research 368 (1996) 165-169
Squalene inhibits sodium arsenite-induced sister chromatid exchanges and micronuclei in Chinese hamster ovary-K1 cells Shyh-Rong Fan, I-Ching Ho, Fenny Lai-Fun Yeoh, Chi-Jen Lin, Te-Chang Lee * Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, ROC
Received 13 September 1995; revised 27 December 1995; accepted 6 January 1996
Abstract
Arsenic, widely distributed throughout our environment, is a well-established human carcinogen. We report here that squalene, a natural fish oil, is a potential agent in the reduction of sodium arsenite-induced sister chromatid exchange (SCE) and micronuclei in Chinese hamster ovary (CHO-K1) cells. Squalene dose-dependently inhibited sodium arsenite-induced SCE. At the highest concentration (160/zM), squalene reduced the SCE frequency from 8.85 to 6.47 SCEs per cell which is very close to the background level (5.82 SCEs per cell). Sodium arsenite dose-dependently induces micronuclei in CHO-K1 cells, and squalene at 80 /xM significantly inhibits arsenite-induced micronuclei. However, squalene did not eliminate the killing effects of arsenite on the cells and only slightly decreased intracellular accumulation of arsenic. Keywords: Sodium arsenite; Squalene; Sister chromatid exchange; Micronucleus
1. Introduction
Arsenic is a universally distributed toxic element (Ishinishi et al., 1986). Human exposure to arsenic is strongly associated with increased risk of lung, skin and liver cancer (Tseng, 1977; Chen et al., 1985), but little evidence exists for the carcinogenicity of arsenic in animals (IARC, 1980; Leonard and Lauwerys, 1980). Arsenic by itself is inactive in inducing mutations at two genetic loci (Na+,K+-ATPase and hypoxanthine phosphoribosyl transferase) in mammalian cells (Rossman et al., 1980; Lee et al., 1985a,b). However, arsenic compounds have been found to induce chromosomal aberrations and sister
* Corresponding author. Tel: + 886 (2) 7899014; Fax: +886 (2) 7825573; E-mail: [email protected].
chromatid exchanges (SCE) in cultured human and rodent cells (Zanzoni and Jung, 1980; Larramendy et al., 1981; Lee et al., 1985a,b) and in lymphocytes of arsenic-exposed humans (Nordenson et al., 1978; Wen et al., 1981; Lerda, 1994). Arsenite is also a potent inducer of micronuclei in Chinese hamster ovary (CHO) cells (Gurr et al., 1993; Wang and Huang, 1994). Arsenic exposure often occurs via inhalation or ingestion still in human populations (Guha Mazumder et al., 1988; D~az-Barriga et al., 1993; Garc~a-Vargas et al., 1994). However, the search for agents preventing arsenic's genotoxicity has been limited. Recently, numerous reports have shown that certain genotoxic effects of arsenic may be mediated by oxygen radicals (Blair et al., 1990; Wang and Huang, 1994; Lee and Ho, 1995). Furthermore, arsenic-in-
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S.-R. Fan et aL /Mutation Research 368 (1996) 165 169
duced SCEs were significantly reduced by oxygen radical scavenging enzymes superoxide dismutase and catalase in cultured human lymphocytes (Nordenson and Beckman, 1991). Furthermore, arsenic-induced micronuclei are remarkably enhanced in catalase-deficient XRS-5 cells (Wang and Huang, 1994). Thus, antioxidants may function to reduce arsenic genotoxicity. Squalene, a 30-carbon isoprenoid and structurally similar to /3-carotene, is found in large quantities in shark liver oil. Although squalene can be converted into peroxides by ultraviolet irradiation (Picardo et al., 1991; Yeo and Shibamoto, 1992), it has been reported to inhibit the tumor-promoting activity of 12-O-tetradecanoylphorbol- 13-acetate (Murakoshi et al., 1992) and to protect the mice against the effects of ionizing radiation (Storm et al., 1993). In a recent report, squalene has been demonstrated to be an efficient quencher of singlet oxygen (Kohno et al., 1995). Since squalene is a popular Asian folk remedy, a potential protective effect of squalene on sodium arsenite-induced SCE was therefore investigated in CHO-K1 cells.
2. Materials and methods CHO-KI cells were cultured in McCoy's 5A medium supplemented with 0.22% sodium bicarbonate, 100 U / m l penicillin, 100 /zg/ml streptomycin, 0.03% L-glutamine, and 10% fetal calf serum. The cultures were maintained in a humidified atmosphere with 5% CO 2. Squalene (purity 98-99%) was obtained from Sigma (St. Louis, MO), and sodium arsenite from Merck (Darmstadt, German). For drug treatment, 1 X 10 6 cells were plated in a 100-mm dish 1 day prior to experimental manipulation. The cells were treated in combination with sodium arsenite (0-40 /xM) and squalene (0-160 ~M) for 6 h. Incubations were carried out under light-free conditions to avoid photo-oxidation of squalene. At the end of treatment, the cultures were washed twice with phosphate-buffered saline, and refed with fresh medium. For SCE analysis, 20 /zM 5-bromodeoxyuridine was inoculated in medium and the cultures were incubated for another 24 h in dark. Colcemid (0.2 /xg/ml) was added 2 h prior to harvesting mitotic
cells. Metaphase preparation and SCE analysis were performed as described previously (Lee et al., 1985a). Randomly selected 30 second division metaphase cells were scored for SCE frequency in each treatment. For micronuclei analysis, the method of Wang and Huang (1994) was adopted. In brief, subsequent to drug treatment, the cultures were fed with 1 /xg/ml cytochalasin B for an additional 24 h. The dishes were then treated with 0.5% KC1 for 5 min, fixed with Carnoy's solution (20:1, methanol:acetic acid), and stained with a 5% Giemsa solution. In each dish, the number of micronuclei per 1000 binucleated cells was scored microscopically. The effect of squalene on cytotoxicity of sodium arsenite was determined by a colony-forming assay. Briefly, 3 × l0 s cells were plated in a 60-mm dish and incubated overnight. The cells were treated with sodium arsenite and squalene as described above. Following treatment, the colony-forming ability was determined by replating 200 cells per dish in triplicate as described previously (Lee et al., 1985a). The effect of squalene on arsenite accumulation was analyzed by atomic absorption spectrophotometry. Cells were plated and treated with sodium arsenite and squalene using the same protocol as above. These cells were washed 5 times with phosphate-buffered saline containing 1 mM EDTA, and harvested by trypsinization. An aliquot of cells (4 X 10 6 cells) was digested by nitric acid and the total arsenic content was measured by an atomic absorption spectrophotometer (Hitachi Z-8000, Tokyo, Japan) as described elsewhere (Wang and Lee, 1993).
3. Results and discussion Sodium arsenite is not a potent SCE inducer, but it does induce SCE in a variety of mammalian cells in a dose-dependent manner (Zanzoni and Jung, 1980; Larramendy et al., 1981; Lee et al., 1985a,b). In order to clearly demonstrate the preventive effect of squalene on SCE induction by arsenite, we treated CHO-K1 cells with 20 /xM sodium arsenite for 6 h. As compared to control culture, sodium arsenite treatment significantly increased SCE frequency by 50% (Fig. 1). While squalene by itself did not induce SCE in CHO-K1 cells, it significantly and dose-dependently inhibited the frequency of SCE induced by
S. -R. Fan et a L / Mutation Research 368 (1996) 165-169 10
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Squalene(pM) Fig. 1. Inhibition of sodium arsenite-induced sister chromatid exchanges by squalene. CHO-KI cells were treated in combination with either 0 (O) or 20 (Q) /zM sodium arsenite and various concentrations of squalene (0-160 /xM) for 6 h. The cultures were then fed in fresh medium in the presence of 20 ~M bromodeoxyuridine for 24 h. The mitotic cells were harvested and SCE was analyzed. Bars, SD of 5 independent experiments. Asterisks indicate significant difference between cultures with and without squalene treatment (* * p < 0.01, according to the Student t-test).
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sodium arsenite and squalene, the effect of squalene on intracellular arsenite content was determined by atomic absorption spectrophotometry. As shown in Fig. 3A, squalene only slightly affected the intracellular content of arsenite. At the highest concentration, squalene decreased the intracellular arsenite content by 25%. However, squalene apparently did not affect the initial uptake rate (Fig. 3B). The decrease of intracellular arsenite was observed only in cells co-treated with sodium arsenite and squalene for 6 h (Fig. 3B). Thus, the inhibition of arsenite-induced SCE by squalene could not be explained simply by the reduction of arsenite uptake. Furthermore, squalene at the concentration range used in this study did not show cytotoxicity in CHO-K1 cells, and did not protect CHO-K1 cells from sodium arsenite-induced cytotoxicity (Fig. 4). These results suggest that the mechanism of SCE induction by arsenite differed from its killing effect. Although sodium arsenite is a well-known inducer of SCE, chromosomal aberrations, and micronuclei
80
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sodium arsenite (Fig. 1). Sodium arsenite-induced SCE (8.85 SCEs/cell) was almost entirely abolished by the highest concentration of squalene, i.e. 6.47 SCEs/cell vs. 5.82 SCEs/cell in control cultures. Recently, micronuclei are generally thought to arise from chromosome fragments or whole chromosomes that are not incorporated into daughter nuclei. Thus, micronuclei assay was adopted to confirm the palliative effect of squalene on arsenite genotoxicity. As shown by linear regression in Fig. 2, the treatment of CHO-K1 cells with sodium arsenite (0, 5, 10, 20, and 40 tzM for 6 h) resulted in a significant dose-dependent increase in micronuclei ( p < 0.001). However, sodium arsenite-induced micronuclei were significantly inhibited by co-treatment with 80 /xM squalene. By regression analysis, the difference between these two groups (with or without squalene co-treatment) was statistically significant, p < 0.01 (Fig. 2). To confirm whether arsenite accumulation could be inhibited by co-treatment of CHO-K1 cells with
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Sodium arsenite(gM) Fig. 2. Inhibition of sodium arsenite-induced micronuclei by squalene. CHO-K1 cells were treated in combination with either 0 ( O ) or 80 ( O ) /~M squalene and various concentrations of sodium arsenite (0-40 /zM) for 6 h. The cultures were then fed in fresh medium in the presence of 1 /xg/ml cytochalasin B for 24 h. The number of micronuclei per 1000 binucleated cells was determined. Bars, SD of 3 independent experiments. Asterisks indicate significant difference between cultures with and without squalene treatment (* p < 0 . 0 5 , *~ p < 0 . 0 1 , * ~* p < 0 . 0 0 1 , according to the Student t-test).
S.-R. Fan et a l . / Mutation Research 368 (1996) 165-169
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Fig. 3. Effect of squalene on intracellular accumulation of sodium arsenite. A: CHO-KI cells were treated in combination with 20 /xM sodium arsenite and various concentrations of squalene for 6 h. B: the cells were co-treated with 20 /xM sodium arsenite and 0 ( O ) or 160 ( O ) /~M squalene for 0, 0.5, 1, 3, and 6 h. The intracellular arsenite contents were determined by atomic absorption spectrometry. Bars, SD of 3 independent experiments. Asterisks indicate significant difference between control and squalenetreated cultures ( , 2 P < 0.01. * * * p < 0.001, according to the Student t-test).
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Squalene(pM) Fig. 4. Effect of squalene on cytotoxicity of sodium arsenite. CHO-KI cells were treated in combination with either 0 (O) or 20 (Q) /zM sodium arsenite and various concentrations of squalene (0-160 /zM) for 6 h. The survival rates were assessed by a colony-formingassay. Bars, SD of 4 independent experiments.
induction (Zanzoni and Jung, 1980; Larramendy et al., 1981; Lee et al., 1985a,b; Wang and Huang, 1994), the mechanisms still remain elusive. However, sodium arsenite has been shown to inhibit DNA repair enzymes, such as DNA ligases (Li and Rossman, 1989). Interference with DNA repair may lead to an increase in these cytogenetic alterations (Natarajan et al., 1981; Shiraishi et al., 1983). Whether squalene can directly prevent the interaction of arsenite with repair enzymes remains to be determined. Recently, numerous reports have shown that oxygen radicals can be produced during arsenic metabolism (Blair et al., 1990; Lee and Ho, 1995). Furthermore, the oxygen radical scavenging enzymes catalase a n d / o r superoxide dismutase have been shown to effectively reduce the frequency of arsenite-induced SCEs in human peripheral lymphocytes (Nordenson and Beckman, 1991) and micronuclei in CHO-K1 cells and XRS-5 X-ray sensitive cells (Wang and Huang, 1994). These results indicate that certain genotoxic effects of arsenite, including SCEs and micronuclei generation, may be mediated by oxygen radicals. Since squalene could serve as an antioxidant, its inhibitory effects on arsenite-induced SCEs and micronuclei may be due to its antioxidant activity (Storm et al., 1993; Kohno et al., 1995). The squalene capsules derived from shark liver oil are readily available in food stores and drug stores and are taken as health food throughout many Asia countries. Squalene is also widely used in cosmetics as a skin-care agent. Furthermore, squalene has been shown to inhibit the tumor-promoting activity of 12-O-tetradecanoylphobol-13-acetate in mouse skin carcinogenesis (Murakoshi et al., 1992). Therefore, the potential of squalene as a preventive agent against arsenite genotoxicity is an interesting question which warrants further investigation.
Acknowledgements The authors thank Dr. Cyprian V. Weaver for careful review of the manuscript. This work was supported by Academia Sinica and Grants NSC 842621-B-001-003z and NSC85-2621-B-001-002z from the National Science Council, Republic of China.
S.-R. Fan et al./Mutation Research 368 (1996) 165-169
References Blair, P.C., M.B. Thompson, B. Bechtold, R.E. Wilson, M.P. Moorman and B.A. Fowler (1990) Evidence for oxidative damage to red blood cells in mice induced by arsine gas, Toxicology, 63. 25-34. Chen. C.J., Y.C. Chuang, T.M. Lin and H.Y. Wu (1985) Malignant neoplasms among residents of a blackfoot disease-endemic area in Taiwan: high-arsenic artesian well water and cancer, Cancer Res., 45, 5895-5899. Dfaz-Barriga, F., M.A. Santos, J.J. Mej~a, L. Baters, L. Yfifiez, L. Carrizales, E. Vera, L.M. del Razo and M.E. Cebrifin (1993) Arsenic and cadmium exposure in children living near a smelter complex in San Luis Potos[, Mexico, Environ. Res., 62, 242-250. Garcfa-Vargas, G.G., L.M. Del Razo, M.E. Cebrifin, A. Albores, P. Ostrosky-Wegman, R. Montero, M.E. Gonsebatt, C.K. Lim and F. De Matteis (1994) Altered urinary porphyrin excretion in a human population chronically exposed to arsenic in Mexico, Hum. Exp. Toxicol., 13, 839-847. Guha Mazumder, D.N., A.K. Chakraborty, A. Ghose, J.D. Gupta, D.P. Chakraborty, S.B. Dey and N. Chattopadhyay (1988) Chronic arsenic toxicity from drinking tubewell water in rural West Bengal, Bull. WHO, 66. 499-506. Gurr, J.R.. Y.C. Lin, I.C. Ho, K.Y. Jan and T.C. Lee (1993) Induction of chromatid breaks and tetraploidy in Chinese hamster ovary cells by treatment with sodium arsenite during the G2 phase, Mutation Res., 319, 135-142. IARC (1980) Carcinogenesis of arsenic and arsenic compounds, IARC Monographs on Evaluation of Carcinogenic Risk to Humans, 23, 37-141. Ishinishi, N., K. Tsuchiya, M. Vahter and B.A. Fowler (1986) Arsenic, in: L. Friberg, G.F. Nordberg and V. Vouk (Eds.), Handbook on the Toxicology of Metals, Elsevier Science Publishers B.V., Amsterdam, pp. 43-83. Kohno, Y., Y. Egawa, S. Itoh, S. Nagaoka and M. Takahashi (1995) Kinetic study of quenching reaction of singlet oxygen and scavenging reaction of free radical by squalene in nbutanol, Biochim. Biophys. Acta, 1256, 52-56. Larramendy, M.L., N.C. Popescu and J.A. DiPaolo (1981) Induction by inorganic metal salts of sister chromatid exchanges and chromosome aberrations in human and Syrian hamster cell strains, Environ. Mutagen., 3, 597-605. Lee. T.C. and I.C. Ho (1995) Modulation of cellular antioxidant defense activities by sodium arsenite in human fibroblasts, Arch. Toxicol.. 69, 498-504. Lee, T.C., R.Y. Huang and K.Y. Jan (1985a) Sodium arsenite enhances the cytotoxicity, clastogenicity, and 6-thioguanineresistant mutagenicity of ultraviolet light in Chinese hamster ovary cells, Mutation Res., 148, 83-89. Lee, T.C., M. Oshimura and J.C. Barrett (1985b) Comparison of arsenic-induced cell translbrmation, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture, Carcinogenesis, 6, 1421- 1426. Leonard, A. and R.R. Lauwerys (1980) Carcinogenicity, teratogenicity and mutagenicity of arsenic, Mutation Rex., 75, 49-62.
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Lerda. D. (1994) Sister chromatid exchange (SCE) among individuals chronically exposed to arsenic in drinking water, Mutation Res.. 312, 111 - 120. Li, J.-H. and T.G. Rossman (1989) Inhibition of DNA ligase activity by arsenite: a possible mechanism of its comutagenesis, Mol. Toxicol., 2, 1-9. Murakoshi, M., H. Nishino, H. Tokuda, A. Iwashima, J. Okuzumi, H. Kitano and R. lwasaki (1992) Inhibition by squalene of the tumor-promoting activity of 12-O-tetradecanolyphobol-13acetate in mouse skin carcinogenesis, Int. J. Cancer, 52, 950-952. Natarajan, A.T., I. Csukas and A.A. van Zeeland (1981) Contribution of incorporated 5-bromodeoxyuridine in DNA to the frequency of sister-chromatid exchanges by inhibitors of poly(ADP-ribose)polymerase, Mutation Res., 84, 125-131. Nordenson, I., G. Beckman, L. Beckman and S. NordstriSm (1978) Occupational and environmental risks in and around a smelter in northern Sweden, Hereditas, 88, 47-50. Nordenson, I. and L. Beckman (1991) Is the genotoxic effect of arsenic mediated by oxygen free radical?, Hum. Heredity, 41, 71-73. Picardo, M., C. Zompetta, C. De Luca, M. Cirone, A. Faggioni, M. Nazzaro-Porro, S. Passi and G. Prota (1991) Role of skin surface lipids in UV-induced epidermal cell changes, Arch. Dermatol. Res., 283, 191-197. Rossman, T.G., D. Stone, M. Molina and W. Troll (1980) Absence of arsenite mutagenicity in E. coli and Chinese hamster ovary cells, Environ. Mutagen., 2, 371-379. Shiraishi, Y., Y. Tanaka, M. Kato, M. Miawa and T. Sugimura (1983) Effect of poly(ADP-ribose)polymerase inhibitors on the frequencies of sister-chromatid exchanges in Bloom syndrome cells, Mutation Res., 122, 223-228. Storm, H.M., S.Y. Oh, B.F. Kimler and S. Norton (1993) Radioprotection of mice by dietary squalene, Lipids, 28, 555-559. Tseng, W.-P. (1977) Effects and dose-response relationships of skin cancer and blackfoot disease with arsenic, Environ. Health Perspect., 19, 109-119. Wang, H.F. and T.C. Lee (1993) Glutathione S-transferase 3~ facilitates the excretion of arsenic from arsenic-resistant Chinese hamster ovary cells, Biochem. Biophys. Res. Commun., 192, 1093-1099. Wang, T.S. and H. Huang (1994) Active oxygen species are involved in the induction of micronuclei by arsenite in XRS-5 cells, Mutagenesis, 9, 253-257. Wen. W.-N., T.-L. Lieu, H.-J. Chang, S.W. Wuu, M.-L. Yau and K.Y. Jan (1981) Baseline and sodium arsenite-induced sister chromatid exchanges in cultured lymphocytes from patients with blackfoot disease and healthy persons, Hum. Genet., 59, 201-203. Yeo, H.C.H. and T. Shibamoto (1992) Formation of formaldehyde and malonaldehyde by photooxidation of squalene, Lipids, 27, 50-53. Zanzoni, F. and E.G. Jung (1980) Arsenic elevates the sister chromatid exchange (SCE) rate in human lymphocytes in vitro, Arch. Dermatol. Res., 267, 91-95.
ELSEVIER
Mutation Research 368 (1996) 165-169
Squalene inhibits sodium arsenite-induced sister chromatid exchanges and micronuclei in Chinese hamster ovary-K1 cells Shyh-Rong Fan, I-Ching Ho, Fenny Lai-Fun Yeoh, Chi-Jen Lin, Te-Chang Lee * Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, ROC
Received 13 September 1995; revised 27 December 1995; accepted 6 January 1996
Abstract
Arsenic, widely distributed throughout our environment, is a well-established human carcinogen. We report here that squalene, a natural fish oil, is a potential agent in the reduction of sodium arsenite-induced sister chromatid exchange (SCE) and micronuclei in Chinese hamster ovary (CHO-K1) cells. Squalene dose-dependently inhibited sodium arsenite-induced SCE. At the highest concentration (160/zM), squalene reduced the SCE frequency from 8.85 to 6.47 SCEs per cell which is very close to the background level (5.82 SCEs per cell). Sodium arsenite dose-dependently induces micronuclei in CHO-K1 cells, and squalene at 80 /xM significantly inhibits arsenite-induced micronuclei. However, squalene did not eliminate the killing effects of arsenite on the cells and only slightly decreased intracellular accumulation of arsenic. Keywords: Sodium arsenite; Squalene; Sister chromatid exchange; Micronucleus
1. Introduction
Arsenic is a universally distributed toxic element (Ishinishi et al., 1986). Human exposure to arsenic is strongly associated with increased risk of lung, skin and liver cancer (Tseng, 1977; Chen et al., 1985), but little evidence exists for the carcinogenicity of arsenic in animals (IARC, 1980; Leonard and Lauwerys, 1980). Arsenic by itself is inactive in inducing mutations at two genetic loci (Na+,K+-ATPase and hypoxanthine phosphoribosyl transferase) in mammalian cells (Rossman et al., 1980; Lee et al., 1985a,b). However, arsenic compounds have been found to induce chromosomal aberrations and sister
* Corresponding author. Tel: + 886 (2) 7899014; Fax: +886 (2) 7825573; E-mail: [email protected].
chromatid exchanges (SCE) in cultured human and rodent cells (Zanzoni and Jung, 1980; Larramendy et al., 1981; Lee et al., 1985a,b) and in lymphocytes of arsenic-exposed humans (Nordenson et al., 1978; Wen et al., 1981; Lerda, 1994). Arsenite is also a potent inducer of micronuclei in Chinese hamster ovary (CHO) cells (Gurr et al., 1993; Wang and Huang, 1994). Arsenic exposure often occurs via inhalation or ingestion still in human populations (Guha Mazumder et al., 1988; D~az-Barriga et al., 1993; Garc~a-Vargas et al., 1994). However, the search for agents preventing arsenic's genotoxicity has been limited. Recently, numerous reports have shown that certain genotoxic effects of arsenic may be mediated by oxygen radicals (Blair et al., 1990; Wang and Huang, 1994; Lee and Ho, 1995). Furthermore, arsenic-in-
0165-1218/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PH S0165- 1218(96)00008-0
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duced SCEs were significantly reduced by oxygen radical scavenging enzymes superoxide dismutase and catalase in cultured human lymphocytes (Nordenson and Beckman, 1991). Furthermore, arsenic-induced micronuclei are remarkably enhanced in catalase-deficient XRS-5 cells (Wang and Huang, 1994). Thus, antioxidants may function to reduce arsenic genotoxicity. Squalene, a 30-carbon isoprenoid and structurally similar to /3-carotene, is found in large quantities in shark liver oil. Although squalene can be converted into peroxides by ultraviolet irradiation (Picardo et al., 1991; Yeo and Shibamoto, 1992), it has been reported to inhibit the tumor-promoting activity of 12-O-tetradecanoylphorbol- 13-acetate (Murakoshi et al., 1992) and to protect the mice against the effects of ionizing radiation (Storm et al., 1993). In a recent report, squalene has been demonstrated to be an efficient quencher of singlet oxygen (Kohno et al., 1995). Since squalene is a popular Asian folk remedy, a potential protective effect of squalene on sodium arsenite-induced SCE was therefore investigated in CHO-K1 cells.
2. Materials and methods CHO-KI cells were cultured in McCoy's 5A medium supplemented with 0.22% sodium bicarbonate, 100 U / m l penicillin, 100 /zg/ml streptomycin, 0.03% L-glutamine, and 10% fetal calf serum. The cultures were maintained in a humidified atmosphere with 5% CO 2. Squalene (purity 98-99%) was obtained from Sigma (St. Louis, MO), and sodium arsenite from Merck (Darmstadt, German). For drug treatment, 1 X 10 6 cells were plated in a 100-mm dish 1 day prior to experimental manipulation. The cells were treated in combination with sodium arsenite (0-40 /xM) and squalene (0-160 ~M) for 6 h. Incubations were carried out under light-free conditions to avoid photo-oxidation of squalene. At the end of treatment, the cultures were washed twice with phosphate-buffered saline, and refed with fresh medium. For SCE analysis, 20 /zM 5-bromodeoxyuridine was inoculated in medium and the cultures were incubated for another 24 h in dark. Colcemid (0.2 /xg/ml) was added 2 h prior to harvesting mitotic
cells. Metaphase preparation and SCE analysis were performed as described previously (Lee et al., 1985a). Randomly selected 30 second division metaphase cells were scored for SCE frequency in each treatment. For micronuclei analysis, the method of Wang and Huang (1994) was adopted. In brief, subsequent to drug treatment, the cultures were fed with 1 /xg/ml cytochalasin B for an additional 24 h. The dishes were then treated with 0.5% KC1 for 5 min, fixed with Carnoy's solution (20:1, methanol:acetic acid), and stained with a 5% Giemsa solution. In each dish, the number of micronuclei per 1000 binucleated cells was scored microscopically. The effect of squalene on cytotoxicity of sodium arsenite was determined by a colony-forming assay. Briefly, 3 × l0 s cells were plated in a 60-mm dish and incubated overnight. The cells were treated with sodium arsenite and squalene as described above. Following treatment, the colony-forming ability was determined by replating 200 cells per dish in triplicate as described previously (Lee et al., 1985a). The effect of squalene on arsenite accumulation was analyzed by atomic absorption spectrophotometry. Cells were plated and treated with sodium arsenite and squalene using the same protocol as above. These cells were washed 5 times with phosphate-buffered saline containing 1 mM EDTA, and harvested by trypsinization. An aliquot of cells (4 X 10 6 cells) was digested by nitric acid and the total arsenic content was measured by an atomic absorption spectrophotometer (Hitachi Z-8000, Tokyo, Japan) as described elsewhere (Wang and Lee, 1993).
3. Results and discussion Sodium arsenite is not a potent SCE inducer, but it does induce SCE in a variety of mammalian cells in a dose-dependent manner (Zanzoni and Jung, 1980; Larramendy et al., 1981; Lee et al., 1985a,b). In order to clearly demonstrate the preventive effect of squalene on SCE induction by arsenite, we treated CHO-K1 cells with 20 /xM sodium arsenite for 6 h. As compared to control culture, sodium arsenite treatment significantly increased SCE frequency by 50% (Fig. 1). While squalene by itself did not induce SCE in CHO-K1 cells, it significantly and dose-dependently inhibited the frequency of SCE induced by
S. -R. Fan et a L / Mutation Research 368 (1996) 165-169 10
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Squalene(pM) Fig. 1. Inhibition of sodium arsenite-induced sister chromatid exchanges by squalene. CHO-KI cells were treated in combination with either 0 (O) or 20 (Q) /zM sodium arsenite and various concentrations of squalene (0-160 /xM) for 6 h. The cultures were then fed in fresh medium in the presence of 20 ~M bromodeoxyuridine for 24 h. The mitotic cells were harvested and SCE was analyzed. Bars, SD of 5 independent experiments. Asterisks indicate significant difference between cultures with and without squalene treatment (* * p < 0.01, according to the Student t-test).
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sodium arsenite and squalene, the effect of squalene on intracellular arsenite content was determined by atomic absorption spectrophotometry. As shown in Fig. 3A, squalene only slightly affected the intracellular content of arsenite. At the highest concentration, squalene decreased the intracellular arsenite content by 25%. However, squalene apparently did not affect the initial uptake rate (Fig. 3B). The decrease of intracellular arsenite was observed only in cells co-treated with sodium arsenite and squalene for 6 h (Fig. 3B). Thus, the inhibition of arsenite-induced SCE by squalene could not be explained simply by the reduction of arsenite uptake. Furthermore, squalene at the concentration range used in this study did not show cytotoxicity in CHO-K1 cells, and did not protect CHO-K1 cells from sodium arsenite-induced cytotoxicity (Fig. 4). These results suggest that the mechanism of SCE induction by arsenite differed from its killing effect. Although sodium arsenite is a well-known inducer of SCE, chromosomal aberrations, and micronuclei
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sodium arsenite (Fig. 1). Sodium arsenite-induced SCE (8.85 SCEs/cell) was almost entirely abolished by the highest concentration of squalene, i.e. 6.47 SCEs/cell vs. 5.82 SCEs/cell in control cultures. Recently, micronuclei are generally thought to arise from chromosome fragments or whole chromosomes that are not incorporated into daughter nuclei. Thus, micronuclei assay was adopted to confirm the palliative effect of squalene on arsenite genotoxicity. As shown by linear regression in Fig. 2, the treatment of CHO-K1 cells with sodium arsenite (0, 5, 10, 20, and 40 tzM for 6 h) resulted in a significant dose-dependent increase in micronuclei ( p < 0.001). However, sodium arsenite-induced micronuclei were significantly inhibited by co-treatment with 80 /xM squalene. By regression analysis, the difference between these two groups (with or without squalene co-treatment) was statistically significant, p < 0.01 (Fig. 2). To confirm whether arsenite accumulation could be inhibited by co-treatment of CHO-K1 cells with
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Sodium arsenite(gM) Fig. 2. Inhibition of sodium arsenite-induced micronuclei by squalene. CHO-K1 cells were treated in combination with either 0 ( O ) or 80 ( O ) /~M squalene and various concentrations of sodium arsenite (0-40 /zM) for 6 h. The cultures were then fed in fresh medium in the presence of 1 /xg/ml cytochalasin B for 24 h. The number of micronuclei per 1000 binucleated cells was determined. Bars, SD of 3 independent experiments. Asterisks indicate significant difference between cultures with and without squalene treatment (* p < 0 . 0 5 , *~ p < 0 . 0 1 , * ~* p < 0 . 0 0 1 , according to the Student t-test).
S.-R. Fan et a l . / Mutation Research 368 (1996) 165-169
168
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Fig. 3. Effect of squalene on intracellular accumulation of sodium arsenite. A: CHO-KI cells were treated in combination with 20 /xM sodium arsenite and various concentrations of squalene for 6 h. B: the cells were co-treated with 20 /xM sodium arsenite and 0 ( O ) or 160 ( O ) /~M squalene for 0, 0.5, 1, 3, and 6 h. The intracellular arsenite contents were determined by atomic absorption spectrometry. Bars, SD of 3 independent experiments. Asterisks indicate significant difference between control and squalenetreated cultures ( , 2 P < 0.01. * * * p < 0.001, according to the Student t-test).
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Squalene(pM) Fig. 4. Effect of squalene on cytotoxicity of sodium arsenite. CHO-KI cells were treated in combination with either 0 (O) or 20 (Q) /zM sodium arsenite and various concentrations of squalene (0-160 /zM) for 6 h. The survival rates were assessed by a colony-formingassay. Bars, SD of 4 independent experiments.
induction (Zanzoni and Jung, 1980; Larramendy et al., 1981; Lee et al., 1985a,b; Wang and Huang, 1994), the mechanisms still remain elusive. However, sodium arsenite has been shown to inhibit DNA repair enzymes, such as DNA ligases (Li and Rossman, 1989). Interference with DNA repair may lead to an increase in these cytogenetic alterations (Natarajan et al., 1981; Shiraishi et al., 1983). Whether squalene can directly prevent the interaction of arsenite with repair enzymes remains to be determined. Recently, numerous reports have shown that oxygen radicals can be produced during arsenic metabolism (Blair et al., 1990; Lee and Ho, 1995). Furthermore, the oxygen radical scavenging enzymes catalase a n d / o r superoxide dismutase have been shown to effectively reduce the frequency of arsenite-induced SCEs in human peripheral lymphocytes (Nordenson and Beckman, 1991) and micronuclei in CHO-K1 cells and XRS-5 X-ray sensitive cells (Wang and Huang, 1994). These results indicate that certain genotoxic effects of arsenite, including SCEs and micronuclei generation, may be mediated by oxygen radicals. Since squalene could serve as an antioxidant, its inhibitory effects on arsenite-induced SCEs and micronuclei may be due to its antioxidant activity (Storm et al., 1993; Kohno et al., 1995). The squalene capsules derived from shark liver oil are readily available in food stores and drug stores and are taken as health food throughout many Asia countries. Squalene is also widely used in cosmetics as a skin-care agent. Furthermore, squalene has been shown to inhibit the tumor-promoting activity of 12-O-tetradecanoylphobol-13-acetate in mouse skin carcinogenesis (Murakoshi et al., 1992). Therefore, the potential of squalene as a preventive agent against arsenite genotoxicity is an interesting question which warrants further investigation.
Acknowledgements The authors thank Dr. Cyprian V. Weaver for careful review of the manuscript. This work was supported by Academia Sinica and Grants NSC 842621-B-001-003z and NSC85-2621-B-001-002z from the National Science Council, Republic of China.
S.-R. Fan et al./Mutation Research 368 (1996) 165-169
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