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Effect of trivalent and pentavalent arsenic in causing chromosome alterations in cultured Chinese hamster ovary (CHO) cells
Toxicology Letters
Effect of trivalent and pentavalent arsenic in causing chromosome alterations in cultured Chinese hamster ovary (CHO) cells T.S. Kochhar*, W. Howard, S. Hoffman, L. Brammer-Carleton Division of hfathematics
and Sciences, Kentucky
State University, Franfort, KY 40601, USA
Pkceived 22 May 1995; revision received 25 August 1995;accepted 28 August 1995
Abstract Sodium salts of trivalent and pentavalent arsenic were tested for their effect in inducing chromosome aberrations and sister-chromatid exchange (SCE) in cultured Chinese hamster ovary (CHO) cells. It was discovered that arsenite (As 3) produced excessive endoreduplication of the chromosomes at higher levels. No endoreduplication was observed with arsenate (As 5) treatment. These agents also elevated the frequencies of SCE, but less so compared to aberrations. The results obtained indicate that arsenic may be carcinogenic in animal system. Keywords: Arsenicals; Carcinogenic; Chromosome aberrations; Sister-chromatid exchange
1. lntroduetion
It has been known for a long time that several metal compounds enhance carcinogenesis in humans. Arsenic medication was suggested to cause skin cancer more than a century ago [ 11. Since then numerous published reports and epidemiological studies concerning medical and occupational exposure, as well as exposure through drinking water, have strengthened evidence that arsenic can be a human carcinogen [2,3]. Considering the situation with this m.etal and other environmental agents, there is a pressing need for identification of * Corresponding author, Tel.: 502 227 6070; Fax: 502 227 6403.
early biological markers that may reveal genetic
damage. This would help toward early diagnosis and/or prevention of cancer and genetic disorders in humans. One of the more accepted procedures is to investigate in vitro or in vivo chromosome changes in cells of the higher organisms. These assays are rapid, sensitive and relatively inexpensive. Cytogenetic investigations conducted on the influence of arsenicals have, so far, yielded conflicting results. Increased frequencies of chromosome aberrations have been reported in humans exposed to the metal for medical or occupational reasons [4-61. Nonetheless, negative results have also been noticed with this regard [7]. In vitro studies of Jacobson-Kram and Montalbano [8] showed that
0378-4274196/$15.00 0 1996 Ekvier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)03536-T
38
T.S. Kochhar et al. / Toxicology Letters 84 (19%)
arsenic was clastogenic and induced sisterchromatid exchange (SCE) in mammalian cells. Also, Burgdorf et al. [7] and Wen et al. [9] showed enhanced SCE in human lymphocytes due to arsenic exposure while Nordenson et al. [5] failed to see any such increase. Comparing the effect of trivalent and pentavalent arsenic, Nakamura and Sayoto [lo] showed that chromosome breaking activity in cultured leukocytes was significantly higher for trivalent compounds than pentavalents. These workers further demonstrated that trivalent arsenic was considerably more toxic in colony forming capacity of human tibroblasts compared to pentavalent salts. Working on similar lines, Anderson [l l] noted that exposure of human lymphocytes to arsenite (As 3) for 48 and 72 h substantially increased SCE frequencies, whereas in P3880, cells, As 3 exposure had a marginal effect. Other As compounds (cacodylic acid and arsenate; As 5) did not increase SCE. As noted here, the cytogenetic studies dealing with effect of arsenicals are still controversial and inconclusive. Therefore, it is necessary that further studies be continued to evaluate the influence of this important metal on mammalian chromosomes. In the present paper, a comparative effect of trivalent and pentavalent arsenic on their abilities to induce chromosome aberrations and SCE is reported. 2. Materials and methods Exponentially growing Chinese hamster ovary (CHO) cells were cultured in 75-cm2 Coming plastic flasks in 10 ml McCoy’s 5A medium supplemented with 10% fetal bovine serum and antibiotics consisting of penicillin-G (100 U/ml) and streptomycin (100 &ml). The cells were incubated in an atmosphere of 5% CO2 in air at 37°C. Approximately 1.5 x 10V6cells were seeded in each flask 24 h prior to exposure to test chemicals. The CHO cells were obtained from Environmental Health Research and Testing (Lexington, KY) and were regularly thawed from liquid nitrogen storage and maintained by subculturing twice a week. NaAsOz and Na2HAs02 were purchased from Sigma Chemical Company (St. Louis, MO).
37-42
For assessing the chromosome aberrations, the cultures were exposed to 10m7,10e6, lo-’ and 10m4 M of the arsenicals suspended in the medium for 12 h. The controls were maintained under identical conditions. The procedure for chromosome preparation and scoring for aberrations has been described in previous reports [ 12,131. The number and types of aberrations were scored by examining 100 well-spread metaphases for each treatment and the controls. The endoreduplication was scored by the presence of characteristic diplochromosomes (2 pairs of homologous chromosomes) in metaphase plates. This condition was first described by Levan and Hauschka [14] to name type of endomitotic chromosome replication without mitotic division and resulting in cells with diplochromosomes. The experiment for chromosome aberrations was repeated 3 times. Since SCE is comparatively a sensitive assay, the cultures were treated with lo-*, lo-‘, 10m6and 10m5levels of the test compounds. At the same time treatments were made, the culture flasks including the controls were wrapped in foil and injected with 10 PM 5-bromo-2-deoxyuridine (BrdU) under OC Kodak safe light and incubated for another 24 h. The cell harvest for SCE was carried out by mitotic shake-off method. About 2-3 h before harvest, 0.1 &ml colcemid was added to each flask. The flasks were gently shaken and contents were poured into labeled centrifuge tubes and spun at 550 rev./min for 10 min. The supematant was discarded and the cells (pellet) were suspended in 5 ml of 0.075 M KC1 and allowed to stand for 10 min. After spinning at 550 rev./mm for 5 min, the supematant was discarded again and the cells resuspended in 1 ml freshly prepared fixative consisting of distilled methanol:glacial acetic acid (3: 1) followed by centrifugation for 10 min. The fixation procedure was repeated twice. The harvest of SCE cultures was carried out under safe light up to the second fixation to avoid photoactivation of BrdU. The cells were finally suspended in 0.5 ml fixative and concentrated suspension dropped onto chilled, wet slides at 45” angle and air dried overnight. The SCE slides were stained with Hoechst dye, essentially according to the method of Perry and Wolff [ 151with black light modification of Goto et
T.S. Kochhar et al. / Toxicology Letters 84 (19%) 37-42
al. [ 161.The SCE were scored under oil immersion at 1000 x magnification. For each of 2 slides/treatment 25 well-spread metaphase plates were examined and average SCE calculated. Only spreads of 21 f 2 chromosomes were scored. The mean SC&e11 was assessed by dividing total SCE by 25. Finally, standard error of the mean of observed SCE and controls was calculated. 3. Results and discussion The observations on the frequencies of chromosome aberrations produced by trivalent and pentavalent arsenic are described in Table 1. It was noticed that As 3 treatment substantially increased chromosome aberrations compared to the controls. The increase a.ppeared to be dose dependent. NaAsOz ( 10m5 M) caused the most profound effect by revealing excessive number of metaphase plates with endoreduplication of chromosomes. The increase in endloreduplication was statistically significant at this level and as well at lob6 M. The percentage of cells with aberrations also showed a significant increase over the controls at these 2 levels of NaAsOz. Otlher aberrations observed were the chromosomes with more than one centromere and the chromatid type of aberrations - mainly the breaks. NaAsOz (10m4 M) was toxic and, therefore, the chromosomes could not be observed at this level. The arsenicals tested also produced elevated SCE frequency (Ta.ble 2). It was noticed that the increase was significant at all levels of the test compounds. The enhancement of SCE was more than 3-fold with both compounds (in lop6 and 10s5 M) compared to the controls and did not appear to be dose dependent. Arsenic is one of the few environmental chemicals which has ample evidence for carcinogenicity in humans but inadequate evidence in animals. Since chromosome alterations and cell transformation assays in vitro have been linked to neoplastic development, it was worthwhile to study the effect of As using animal cells. The results obtained in the present study on CHO cells showed that both trivalent and pentavalent arsenic enhanced the rates of chromosome aberrations and SCE. Similar arsenic compounds induced a
39
high frequency of methotrexate-resistant mouse 3T6 cells, that were shown to have amplified copies of dihydrofolate reductase (DHFR) gene [ 171.Also, Lee, et al. [ 181had previously reported that sodium arsenite was effective in transformation of Syrian hamster embryo cells at l-10 PM level. Interestingly, the induction of DHFR gene of Syrian amplification, the transformation hamster embryo cells and the chromosome damage observed in the present study occurred around similar levels of sodium arsenite. These studies raise a possibility that arsenic could be carcinogenic by itself in animal system. However, some earlier studies have indicated that arsenic promotes carcinogenesis by cogenotoxic effect in combination with other agents [3,19-211. The mechanism by which arsenic induces changes in genetic material of the cell is unclear. As far as direct DNA damage measured by gene mutation or unscheduled DNA synthesis, the evidence is negligible or limited [22,23]. However, the chromosome damage (aberrations and SCE) in higher organisms is not doubted. Arsenicals are known to affect a variety of enzymes with sulfiydry1 groups as well as other interactions [24], this indicates that the target for arsenic-caused transformation is not DNA but proteins, possibly ones involved in DNA replication. The result of this supposed interaction may be DNA damage. An excessive amount of chromosomal endoreduplication by As 3 treatment seen in the present study may have a role in transformation of cultured Syrian hamster embryo cells observed by others [18,25]. It is possible that arsenic-induced transformation is a result of numerical chromosome alterations produced through endoreduplication. Oshimura et al. [26] have observed that asbestos and other mineral dusts produce tetraploidy, possibly by inhibition of cytokinesis. On the other hand, Lee et al. [18] suggest that tetraploidy correlates with toxicity, while aneuploidy and chromosome aberrations correlate with transformation. Furthermore, early passage asbestos-transformed cells have primarily nonrandom chromosome changes such as trisomy of chromosome 11. Thus, arsenic may be acting by inducing segmental aneuploidy or chromosome rearrangement. A recent study by Gurr et al. [27]
4.3 5.3 5.3 6.3 8.0
NalHAs04 10-a 10-7 10-s 10-s 10-4
0.6 1.5 2.0. 2.3**
2.3 2.7 2.3 2.3 2.7
f 1.2 l 1.2 f 0.6 zt 0.6 f 0.6
4.0 l o* 2.0 l 1.0 4.0 * o* 4.0 l I.02 -
2.3 * 0.6
More than 1 centromere
1.2 1.2 0 1.0
0 0.3 + 0.6* 0.7 f 1.2* 0.3 f 0.6* 0.7 l 1.2*
1.7 f 2.3 f 2.0 f 2.0 f -
2.6 f 1.5
Gaps
Aberrations/100 metaphases
1.0 1.0 2.0 0
0 0.3 ztz0.6 0 0.7 zt 0.6 0
1.0 f 1.0 l 2.0 f 2.0 l
0.7 ztz0.6
Rings
f zt t f
1.3 * 1.7 * 3.0 f 3.0 f 4.0 *
3.0 3.3 4.3 5.0 -
1.5 0.6 0 0 o*
1.0 0.6* 0.6* 1.0*
1.7 f 0.6
Chromatid breaks
0 0 0 0 0.3 zt 0.6
0 0 0 3.7 * 1.5; -
0
Chromatid exchanges
0 0 0 0 0
0 2.0 l 1.0 5.1 zt 0.6* 22.0 zt 4.6** -
0
Endoreduplication
All values represent means f S.D. of 3 independent experiments. ‘Included fragmentation and the stickiness of chromosomes. lP < 0.05; l*P < 0.001. Significant difference from controls by planned contrast following randomized complete block ANOVA.
* 1.0 l 1.5 zt 0.6 za 1.2 f l.O*
7.1 f 7.1 l 11.0 -f 36.1 f -
5.3 f 2.1
Ceils with aberrations W
NaAsOz 10-s 10-7 10-6 10-s 10-4
Control
(Ml
Treatment and concentration
Table 1 Frequencies of chromosome aberrations produced at metaphase by various levels of arsenic compounds in cultured CHO cells
0.3 0 0.1 0.7 2.0
2.0 2.7 3.0 3.3 -
0
f 1.2 f 0.6 * 0’
f 0.6
f 2.0* f 0.6* h o+ ZIZ0.6;
Others’
T.S. Kochhar et al. /Toxicology
Table 2 Frequencies of SCE produced by sodium arsenide arsenate in cultured CHO cells Treatment
Control NaAsO*
Na,HAsO,
Concentration (M)
SCEKell
l
SE”
10-s 10-7 10-s 10-s
4.0 zt 0.28 10.94 * 0.37* Il.58 f 0.53* 12.50 f 0.42* 14.08 f 0.51.
10-s 10-7 10-6 10-5
11.38 f 12.88 f 12.36 * 12.84 zt
‘Means f S.E. *Significant at P < 0.05 by Student’s
0.49* 0.54* 0.44* 0.46*
and Sodium Range
2.0- 7.0 8.0-14.5 8.0-15.5 9.0-17.0 10.0-18.5 7.5-16.5 8.5-18.5 9.0-16.5 9.0-18.0
r-test.
has shown that treatment of CHO-Kl cells with As 3 during G2 phase induced cell cycle delay and poor condensation of chromosomes resulting in doubling of chromosome number in second mitosis. It has been further shown that endoreduplication frequently leads to formation of hyperploidy during the progression of tumors [28,29]. At the present moment little is known about the biochemical mechanism responsible for endoreduplication of chromosomes. Experimental evidence has shown that it may happen in cell by interference with progression of G2 phase [30]. This view is further supported by recent findings of Huang et al. (311 which show that arsenic significantly delayed mitotic division and inhibited the assembly of mitotic spindle in human Iibroblasts. This inhibition of spindle fiber assembly may interfere with progression of Gz phase. The same study revealed that sodium arsenite caused inhibition of protein phosphatase activity. These investigators strongly suspect that the induction of endoreduplication by As 3 may be brought about by its inhibitory effect on the activity of protein phosphotase which is required for sister chromatid separation. From the present study and that of others [IO,11,321, it is clearly evident that the trivalent species of arsenic (as As 3) are much more powerful inducers of chromosome changes than pen-
Letters 84 (19%)
37-k
41
tavalent arsenic in mammalian cells. As to why As 3 is more effective, Bertolero et al. 133) have shown that As 3 was absorbed and retained much more efficiently by mouse embryo BALB/3T3 cells than by As 5. The more rapid cellular uptake of As 3 than As 5 is attributed to the fact that As 3 is an uncharged form at physiological pH [34] and therefore can readily pass through the cell membrane. Uptake of trivalent arsenic had further been shown to be oxygen independent in rat tissue slices 1351,which would indicate no energy requirement for cellular uptake. It has been also shown that As 5 is reduced to As 3 [36]. In this regard, Bertolero et al. [33] showed that As 3-exposed BALB/3T3 cells retained only trivalent arsenic, whereas As 5exposed cells converted most of cytosoloic arsenic to the trivalent form. Beside this enzyme-catalyzed transformation, As 5 reduction can partly be influenced by non-enzyme components like glutathione [35]. It should, however, be noted that one has to be cautious in interpreting results obtained with BALB/3T3 or CHO cells as these cell lines are not comparable to normal cells as they may be aneuploid and have unstable karyotype. Acknowledgements
This investigation supported was NIIUNIGMS Grant # GM 0523 1.
by
References 111Hutchinson, J. (1888) Arsenic-keratosis and arseniccancer. Trans. Pathol. Sot. (London) 39, 352-363.
121IARC (1980) Carcinogenesis of arsenic and arsenic compounds. IARC monograph on evaluation of carcinogenic risk to humans. IARC Suppl. 23, 37-141. I31 Ostrosky-Wegman, P., Gonsebatt, M.E., Montero, R., Vega, L., Barba, H., Espinosa, J., Palao, A., Cortinas, C., Garcia-Vargas, G., del Razo, L.M. and Cebriin, M. (1991) Lymphocyte proliferation and genotoxic findings in a pilot study on individuals chronically exposed to arsenic in Mexico. Mutat. Res. 250, 477-482. 141 Nordenson, I., Beckman, G., Beckman, L. and Nordstriim, S. (1978) Occupational and environmental risks in and around a smelter in northern Sweden. II. Chromosomal aberrations in workers exposed to arsenic. Heredities 88, 47-50. [51 Nordenson, I., Salmonsson, S.. Brun, E. and Beckman, G. (1979) Chromosome aberrations in psoriatic patients treated with arsenic. Hum. Genet. 48, l-8.
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[6] Petres, J., Baron, D. and Hagedom, M. (1977) Effect of
arsenic cell metabolism and cell proliferation: cytogenetic and biochemical studies. Environ. Health Perspect. 19, 223-227. [7] Burgdorf, W., Kurvink, K. and Cervenka, J. (1977) Elevated sisterthromatid exchange rate in lymphocytes of subjects treated with arsenic. Hum. Genet. 36, 69-72. [S] Jacobson-Kram, D. and Montalbano, D. (1985) The reproductive effects assessment group’s report of mutagenecity of inorganic arsenic. Environ. Mutagen. 7, 707-804. 191 Wen, W.N., Lieu, T.L., Chang, H.J., Wuu, SW., Yau, M.L. and Jan, K.Y. (1981) Baseline and sodium arseniteinduced sister chromatid exchanges in cultured lymphocytes from patients with Blackfoot disease and healthy persons. Hum. Genet. 59, 201-203. [IO] Nakamura, K. and Sayoto, Y. (1981) Comparative studies of chromosomal aberrations induced by trivalent and pentavalent arsenic. Mutat. Res. 88, 73-80. 1111 Anderson, 0. (1983) Effect of coal combustion products and metal compounds on sister-chromatid exchange (SCE) in a macrophage like cell line. Environ. Health Perspect. 47, 239-253. [12] Kochhar, T.S. (1982) Effect of polycychc hydrocarbons in the induction of chromosomal aberrations in absence of an exogenous metabolic activation system in cultured hamster cells. Experientia 38, 845-846. [ 13) Kochhar, T.S. (1985) Inducibility of chromosome aberrations by steroid hormones in cultured Chinese hamster ovary cells. Toxicol. L&t. 29, 201-206. 1141 Levan, A. and Hauschka, T.S. (1953) Endomitotic reduplication mechanisms in ascites tumors of mouse. J. Natl. Cancer Inst. 14, l-43. [I51 Perry, P. and Wolff, S. (1974) New Giemsa method for differential staining of sister chromatids. Nature (London) 261, 156. [16] Goto, K., Maeda, S., Kano, Y. and Sugiyama, T. (1978) Factors involved in differential Giemsa-staining of sister chromatids. Chromosoma 66, 351-359. [17] Lee, T.C., Tanaka, N., Lamb, P.W., Gilmer, T.M. and Barrett, J.C. (1989) Induction of gene amplification by arsenic. Science 241, 79-81. [18] Lee, T.C., Oshimura, M. and Barrett, J.C. (1985) Comparison of arsenic-induced cell transformations, cytotoxicity, mutation and cytogenetic effects in Syrian hamster embryo cells in culture. Carcinogenesis 6, 1421-1426. 1191 Rossman, T.G. (1981) Enhancement of UV-mutagenesis by low concentrations of arsenite in E. colt Mutat. Res. 91, 207-211. [20] Lee, T.C., Haung, R.Y. and Jan, K.Y. (1985) Sodium arsenite enhances the cytotoxicity, clastogenicity and 6thioguinine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cell. Mutat. Res. 148, 83-89. 1211 Lee,T.C., Wang-Wuu, S., Haung,R.Y., Lee, K.C.C. and Jan, K.Y. (1986) Differential effects of pre and post treatment of sodium arsenite on the genotoxicity of methyl methanesulfonate in Chinese hamster ovary cells. Cancer Res. 46, 1854-1857.
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[22] IARC (1982) Monograph on the evaluation of carcinogenic risks of chemicals to humans. IARC Suppl. 14, 50-51. [23] Waters, M.D., Garrett, N.E., Covone-de Serres, C.M., Howard, B.E. and Stack, H.F. (1984) Genetic toxicology of some known or suspected human carcinogens. In: F.J. de Serres, (Ed.), Chemical Mutagens, Vol. 8, Plenum Press, N.Y., pp. 261-341. [24] Squibb, KS. and Fowler, B.A. (1983) The toxicity of arsenic and its compounds. In: B.A. Fowler (Ed.), Biological and Environmental Effects of Arsenic, Elsevier, Amsterdam, pp. 233-269. [25] DiPaolo, J.A. and Castro, B.C. (1979) Quantitative studies of in vitro morphological transformation of Syrian hamster cells by inorganic metal salts. Cancer Res. 39, 1008-1013. [26] Oshimura, M., Hesterberg, T.W., Tsutsui, T. and Barrett, J.C. (1984) Correlation of asbestos-induced cytogenetic effects with cell transformation of Syrian hamster embryo cells in culture. Cancer Res. 44, 5017-5022. [27] Gurr, J.R., Lin, Y.C., Ho, I.C., Jan, K.Y. and Lee+T.C. (1993) Induction of chromatid breaks and tetraploidy in Chinese hamster ovary cells by treatment with sodium arsenite during the G2 phase. Mutat. Res. 319, 135-142. [28] Muleris, M., Delattre, O., Olschwang, S., Dutrillaux, A.M., Remvikos, Y., Salmon, R.J., Thomas, G. and Dutrillaux, B. (1990) Cytogenetic and molecular ap proaches of polyploidization in colorectal adenocarcinemas. Cancer Genet. Cytogenet. 44, 107- 118. [29] Dutrillaux, B., Gerbault-Seureau, M., Remvikos, Y., Zafrani, B. and Prieur, M. (1991) Breast cancer genetic evolution I: Data from cytogenetics and DNA content. Breast Cancer Res. Treat. 19, 245-255. [30] Matsumoto, K. and Ohta, K. (1992) Sensitive period for the induction of endoreduplication by rotenone in cultured Chinese hamster cells. Chromosoma 102, 60-65. [31] Huang, R.N., Ho, I.C., Yih, L.H. and Lee, T.C. (1995) Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphotase activity in human tibroblasts. Environ. Mol. Mutagenesis 25, 188-196. [32] Paton, G.R. and Allison, A.C. (1972) Chromosome damage in human cell cultures induced by metal salts. Mutat. Res. 16, 332-336. [33] Bertolero, F., Pozxi, G., Sabbioni, E. and Saffiotti, U. (1987) Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformations. Carcinogenesis 8, 803-808. [34] Lerman, S.A., Clarkson, T.W. and Gerson, R.J. (1983) Arsenic uptake and metabolism by liver cells is dependent on arsenic oxidation state. Chem.-Biol. Interact. 45, 401-406. [35] Georis, B., Cardenas, A., Buchet, J.P. and Lauwerys, R. (1990) Inorganic arsenic methylation by rat tissue slices. Toxicology 63, 73-84. [36] Vahter, M. and Envall, J. (1983) In vivo reduction of arsenate in mice and rabbits. Environ. Res. 32, 14-21.
Effect of trivalent and pentavalent arsenic in causing chromosome alterations in cultured Chinese hamster ovary (CHO) cells T.S. Kochhar*, W. Howard, S. Hoffman, L. Brammer-Carleton Division of hfathematics
and Sciences, Kentucky
State University, Franfort, KY 40601, USA
Pkceived 22 May 1995; revision received 25 August 1995;accepted 28 August 1995
Abstract Sodium salts of trivalent and pentavalent arsenic were tested for their effect in inducing chromosome aberrations and sister-chromatid exchange (SCE) in cultured Chinese hamster ovary (CHO) cells. It was discovered that arsenite (As 3) produced excessive endoreduplication of the chromosomes at higher levels. No endoreduplication was observed with arsenate (As 5) treatment. These agents also elevated the frequencies of SCE, but less so compared to aberrations. The results obtained indicate that arsenic may be carcinogenic in animal system. Keywords: Arsenicals; Carcinogenic; Chromosome aberrations; Sister-chromatid exchange
1. lntroduetion
It has been known for a long time that several metal compounds enhance carcinogenesis in humans. Arsenic medication was suggested to cause skin cancer more than a century ago [ 11. Since then numerous published reports and epidemiological studies concerning medical and occupational exposure, as well as exposure through drinking water, have strengthened evidence that arsenic can be a human carcinogen [2,3]. Considering the situation with this m.etal and other environmental agents, there is a pressing need for identification of * Corresponding author, Tel.: 502 227 6070; Fax: 502 227 6403.
early biological markers that may reveal genetic
damage. This would help toward early diagnosis and/or prevention of cancer and genetic disorders in humans. One of the more accepted procedures is to investigate in vitro or in vivo chromosome changes in cells of the higher organisms. These assays are rapid, sensitive and relatively inexpensive. Cytogenetic investigations conducted on the influence of arsenicals have, so far, yielded conflicting results. Increased frequencies of chromosome aberrations have been reported in humans exposed to the metal for medical or occupational reasons [4-61. Nonetheless, negative results have also been noticed with this regard [7]. In vitro studies of Jacobson-Kram and Montalbano [8] showed that
0378-4274196/$15.00 0 1996 Ekvier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)03536-T
38
T.S. Kochhar et al. / Toxicology Letters 84 (19%)
arsenic was clastogenic and induced sisterchromatid exchange (SCE) in mammalian cells. Also, Burgdorf et al. [7] and Wen et al. [9] showed enhanced SCE in human lymphocytes due to arsenic exposure while Nordenson et al. [5] failed to see any such increase. Comparing the effect of trivalent and pentavalent arsenic, Nakamura and Sayoto [lo] showed that chromosome breaking activity in cultured leukocytes was significantly higher for trivalent compounds than pentavalents. These workers further demonstrated that trivalent arsenic was considerably more toxic in colony forming capacity of human tibroblasts compared to pentavalent salts. Working on similar lines, Anderson [l l] noted that exposure of human lymphocytes to arsenite (As 3) for 48 and 72 h substantially increased SCE frequencies, whereas in P3880, cells, As 3 exposure had a marginal effect. Other As compounds (cacodylic acid and arsenate; As 5) did not increase SCE. As noted here, the cytogenetic studies dealing with effect of arsenicals are still controversial and inconclusive. Therefore, it is necessary that further studies be continued to evaluate the influence of this important metal on mammalian chromosomes. In the present paper, a comparative effect of trivalent and pentavalent arsenic on their abilities to induce chromosome aberrations and SCE is reported. 2. Materials and methods Exponentially growing Chinese hamster ovary (CHO) cells were cultured in 75-cm2 Coming plastic flasks in 10 ml McCoy’s 5A medium supplemented with 10% fetal bovine serum and antibiotics consisting of penicillin-G (100 U/ml) and streptomycin (100 &ml). The cells were incubated in an atmosphere of 5% CO2 in air at 37°C. Approximately 1.5 x 10V6cells were seeded in each flask 24 h prior to exposure to test chemicals. The CHO cells were obtained from Environmental Health Research and Testing (Lexington, KY) and were regularly thawed from liquid nitrogen storage and maintained by subculturing twice a week. NaAsOz and Na2HAs02 were purchased from Sigma Chemical Company (St. Louis, MO).
37-42
For assessing the chromosome aberrations, the cultures were exposed to 10m7,10e6, lo-’ and 10m4 M of the arsenicals suspended in the medium for 12 h. The controls were maintained under identical conditions. The procedure for chromosome preparation and scoring for aberrations has been described in previous reports [ 12,131. The number and types of aberrations were scored by examining 100 well-spread metaphases for each treatment and the controls. The endoreduplication was scored by the presence of characteristic diplochromosomes (2 pairs of homologous chromosomes) in metaphase plates. This condition was first described by Levan and Hauschka [14] to name type of endomitotic chromosome replication without mitotic division and resulting in cells with diplochromosomes. The experiment for chromosome aberrations was repeated 3 times. Since SCE is comparatively a sensitive assay, the cultures were treated with lo-*, lo-‘, 10m6and 10m5levels of the test compounds. At the same time treatments were made, the culture flasks including the controls were wrapped in foil and injected with 10 PM 5-bromo-2-deoxyuridine (BrdU) under OC Kodak safe light and incubated for another 24 h. The cell harvest for SCE was carried out by mitotic shake-off method. About 2-3 h before harvest, 0.1 &ml colcemid was added to each flask. The flasks were gently shaken and contents were poured into labeled centrifuge tubes and spun at 550 rev./min for 10 min. The supematant was discarded and the cells (pellet) were suspended in 5 ml of 0.075 M KC1 and allowed to stand for 10 min. After spinning at 550 rev./mm for 5 min, the supematant was discarded again and the cells resuspended in 1 ml freshly prepared fixative consisting of distilled methanol:glacial acetic acid (3: 1) followed by centrifugation for 10 min. The fixation procedure was repeated twice. The harvest of SCE cultures was carried out under safe light up to the second fixation to avoid photoactivation of BrdU. The cells were finally suspended in 0.5 ml fixative and concentrated suspension dropped onto chilled, wet slides at 45” angle and air dried overnight. The SCE slides were stained with Hoechst dye, essentially according to the method of Perry and Wolff [ 151with black light modification of Goto et
T.S. Kochhar et al. / Toxicology Letters 84 (19%) 37-42
al. [ 161.The SCE were scored under oil immersion at 1000 x magnification. For each of 2 slides/treatment 25 well-spread metaphase plates were examined and average SCE calculated. Only spreads of 21 f 2 chromosomes were scored. The mean SC&e11 was assessed by dividing total SCE by 25. Finally, standard error of the mean of observed SCE and controls was calculated. 3. Results and discussion The observations on the frequencies of chromosome aberrations produced by trivalent and pentavalent arsenic are described in Table 1. It was noticed that As 3 treatment substantially increased chromosome aberrations compared to the controls. The increase a.ppeared to be dose dependent. NaAsOz ( 10m5 M) caused the most profound effect by revealing excessive number of metaphase plates with endoreduplication of chromosomes. The increase in endloreduplication was statistically significant at this level and as well at lob6 M. The percentage of cells with aberrations also showed a significant increase over the controls at these 2 levels of NaAsOz. Otlher aberrations observed were the chromosomes with more than one centromere and the chromatid type of aberrations - mainly the breaks. NaAsOz (10m4 M) was toxic and, therefore, the chromosomes could not be observed at this level. The arsenicals tested also produced elevated SCE frequency (Ta.ble 2). It was noticed that the increase was significant at all levels of the test compounds. The enhancement of SCE was more than 3-fold with both compounds (in lop6 and 10s5 M) compared to the controls and did not appear to be dose dependent. Arsenic is one of the few environmental chemicals which has ample evidence for carcinogenicity in humans but inadequate evidence in animals. Since chromosome alterations and cell transformation assays in vitro have been linked to neoplastic development, it was worthwhile to study the effect of As using animal cells. The results obtained in the present study on CHO cells showed that both trivalent and pentavalent arsenic enhanced the rates of chromosome aberrations and SCE. Similar arsenic compounds induced a
39
high frequency of methotrexate-resistant mouse 3T6 cells, that were shown to have amplified copies of dihydrofolate reductase (DHFR) gene [ 171.Also, Lee, et al. [ 181had previously reported that sodium arsenite was effective in transformation of Syrian hamster embryo cells at l-10 PM level. Interestingly, the induction of DHFR gene of Syrian amplification, the transformation hamster embryo cells and the chromosome damage observed in the present study occurred around similar levels of sodium arsenite. These studies raise a possibility that arsenic could be carcinogenic by itself in animal system. However, some earlier studies have indicated that arsenic promotes carcinogenesis by cogenotoxic effect in combination with other agents [3,19-211. The mechanism by which arsenic induces changes in genetic material of the cell is unclear. As far as direct DNA damage measured by gene mutation or unscheduled DNA synthesis, the evidence is negligible or limited [22,23]. However, the chromosome damage (aberrations and SCE) in higher organisms is not doubted. Arsenicals are known to affect a variety of enzymes with sulfiydry1 groups as well as other interactions [24], this indicates that the target for arsenic-caused transformation is not DNA but proteins, possibly ones involved in DNA replication. The result of this supposed interaction may be DNA damage. An excessive amount of chromosomal endoreduplication by As 3 treatment seen in the present study may have a role in transformation of cultured Syrian hamster embryo cells observed by others [18,25]. It is possible that arsenic-induced transformation is a result of numerical chromosome alterations produced through endoreduplication. Oshimura et al. [26] have observed that asbestos and other mineral dusts produce tetraploidy, possibly by inhibition of cytokinesis. On the other hand, Lee et al. [18] suggest that tetraploidy correlates with toxicity, while aneuploidy and chromosome aberrations correlate with transformation. Furthermore, early passage asbestos-transformed cells have primarily nonrandom chromosome changes such as trisomy of chromosome 11. Thus, arsenic may be acting by inducing segmental aneuploidy or chromosome rearrangement. A recent study by Gurr et al. [27]
4.3 5.3 5.3 6.3 8.0
NalHAs04 10-a 10-7 10-s 10-s 10-4
0.6 1.5 2.0. 2.3**
2.3 2.7 2.3 2.3 2.7
f 1.2 l 1.2 f 0.6 zt 0.6 f 0.6
4.0 l o* 2.0 l 1.0 4.0 * o* 4.0 l I.02 -
2.3 * 0.6
More than 1 centromere
1.2 1.2 0 1.0
0 0.3 + 0.6* 0.7 f 1.2* 0.3 f 0.6* 0.7 l 1.2*
1.7 f 2.3 f 2.0 f 2.0 f -
2.6 f 1.5
Gaps
Aberrations/100 metaphases
1.0 1.0 2.0 0
0 0.3 ztz0.6 0 0.7 zt 0.6 0
1.0 f 1.0 l 2.0 f 2.0 l
0.7 ztz0.6
Rings
f zt t f
1.3 * 1.7 * 3.0 f 3.0 f 4.0 *
3.0 3.3 4.3 5.0 -
1.5 0.6 0 0 o*
1.0 0.6* 0.6* 1.0*
1.7 f 0.6
Chromatid breaks
0 0 0 0 0.3 zt 0.6
0 0 0 3.7 * 1.5; -
0
Chromatid exchanges
0 0 0 0 0
0 2.0 l 1.0 5.1 zt 0.6* 22.0 zt 4.6** -
0
Endoreduplication
All values represent means f S.D. of 3 independent experiments. ‘Included fragmentation and the stickiness of chromosomes. lP < 0.05; l*P < 0.001. Significant difference from controls by planned contrast following randomized complete block ANOVA.
* 1.0 l 1.5 zt 0.6 za 1.2 f l.O*
7.1 f 7.1 l 11.0 -f 36.1 f -
5.3 f 2.1
Ceils with aberrations W
NaAsOz 10-s 10-7 10-6 10-s 10-4
Control
(Ml
Treatment and concentration
Table 1 Frequencies of chromosome aberrations produced at metaphase by various levels of arsenic compounds in cultured CHO cells
0.3 0 0.1 0.7 2.0
2.0 2.7 3.0 3.3 -
0
f 1.2 f 0.6 * 0’
f 0.6
f 2.0* f 0.6* h o+ ZIZ0.6;
Others’
T.S. Kochhar et al. /Toxicology
Table 2 Frequencies of SCE produced by sodium arsenide arsenate in cultured CHO cells Treatment
Control NaAsO*
Na,HAsO,
Concentration (M)
SCEKell
l
SE”
10-s 10-7 10-s 10-s
4.0 zt 0.28 10.94 * 0.37* Il.58 f 0.53* 12.50 f 0.42* 14.08 f 0.51.
10-s 10-7 10-6 10-5
11.38 f 12.88 f 12.36 * 12.84 zt
‘Means f S.E. *Significant at P < 0.05 by Student’s
0.49* 0.54* 0.44* 0.46*
and Sodium Range
2.0- 7.0 8.0-14.5 8.0-15.5 9.0-17.0 10.0-18.5 7.5-16.5 8.5-18.5 9.0-16.5 9.0-18.0
r-test.
has shown that treatment of CHO-Kl cells with As 3 during G2 phase induced cell cycle delay and poor condensation of chromosomes resulting in doubling of chromosome number in second mitosis. It has been further shown that endoreduplication frequently leads to formation of hyperploidy during the progression of tumors [28,29]. At the present moment little is known about the biochemical mechanism responsible for endoreduplication of chromosomes. Experimental evidence has shown that it may happen in cell by interference with progression of G2 phase [30]. This view is further supported by recent findings of Huang et al. (311 which show that arsenic significantly delayed mitotic division and inhibited the assembly of mitotic spindle in human Iibroblasts. This inhibition of spindle fiber assembly may interfere with progression of Gz phase. The same study revealed that sodium arsenite caused inhibition of protein phosphatase activity. These investigators strongly suspect that the induction of endoreduplication by As 3 may be brought about by its inhibitory effect on the activity of protein phosphotase which is required for sister chromatid separation. From the present study and that of others [IO,11,321, it is clearly evident that the trivalent species of arsenic (as As 3) are much more powerful inducers of chromosome changes than pen-
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tavalent arsenic in mammalian cells. As to why As 3 is more effective, Bertolero et al. 133) have shown that As 3 was absorbed and retained much more efficiently by mouse embryo BALB/3T3 cells than by As 5. The more rapid cellular uptake of As 3 than As 5 is attributed to the fact that As 3 is an uncharged form at physiological pH [34] and therefore can readily pass through the cell membrane. Uptake of trivalent arsenic had further been shown to be oxygen independent in rat tissue slices 1351,which would indicate no energy requirement for cellular uptake. It has been also shown that As 5 is reduced to As 3 [36]. In this regard, Bertolero et al. [33] showed that As 3-exposed BALB/3T3 cells retained only trivalent arsenic, whereas As 5exposed cells converted most of cytosoloic arsenic to the trivalent form. Beside this enzyme-catalyzed transformation, As 5 reduction can partly be influenced by non-enzyme components like glutathione [35]. It should, however, be noted that one has to be cautious in interpreting results obtained with BALB/3T3 or CHO cells as these cell lines are not comparable to normal cells as they may be aneuploid and have unstable karyotype. Acknowledgements
This investigation supported was NIIUNIGMS Grant # GM 0523 1.
by
References 111Hutchinson, J. (1888) Arsenic-keratosis and arseniccancer. Trans. Pathol. Sot. (London) 39, 352-363.
121IARC (1980) Carcinogenesis of arsenic and arsenic compounds. IARC monograph on evaluation of carcinogenic risk to humans. IARC Suppl. 23, 37-141. I31 Ostrosky-Wegman, P., Gonsebatt, M.E., Montero, R., Vega, L., Barba, H., Espinosa, J., Palao, A., Cortinas, C., Garcia-Vargas, G., del Razo, L.M. and Cebriin, M. (1991) Lymphocyte proliferation and genotoxic findings in a pilot study on individuals chronically exposed to arsenic in Mexico. Mutat. Res. 250, 477-482. 141 Nordenson, I., Beckman, G., Beckman, L. and Nordstriim, S. (1978) Occupational and environmental risks in and around a smelter in northern Sweden. II. Chromosomal aberrations in workers exposed to arsenic. Heredities 88, 47-50. [51 Nordenson, I., Salmonsson, S.. Brun, E. and Beckman, G. (1979) Chromosome aberrations in psoriatic patients treated with arsenic. Hum. Genet. 48, l-8.
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[22] IARC (1982) Monograph on the evaluation of carcinogenic risks of chemicals to humans. IARC Suppl. 14, 50-51. [23] Waters, M.D., Garrett, N.E., Covone-de Serres, C.M., Howard, B.E. and Stack, H.F. (1984) Genetic toxicology of some known or suspected human carcinogens. In: F.J. de Serres, (Ed.), Chemical Mutagens, Vol. 8, Plenum Press, N.Y., pp. 261-341. [24] Squibb, KS. and Fowler, B.A. (1983) The toxicity of arsenic and its compounds. In: B.A. Fowler (Ed.), Biological and Environmental Effects of Arsenic, Elsevier, Amsterdam, pp. 233-269. [25] DiPaolo, J.A. and Castro, B.C. (1979) Quantitative studies of in vitro morphological transformation of Syrian hamster cells by inorganic metal salts. Cancer Res. 39, 1008-1013. [26] Oshimura, M., Hesterberg, T.W., Tsutsui, T. and Barrett, J.C. (1984) Correlation of asbestos-induced cytogenetic effects with cell transformation of Syrian hamster embryo cells in culture. Cancer Res. 44, 5017-5022. [27] Gurr, J.R., Lin, Y.C., Ho, I.C., Jan, K.Y. and Lee+T.C. (1993) Induction of chromatid breaks and tetraploidy in Chinese hamster ovary cells by treatment with sodium arsenite during the G2 phase. Mutat. Res. 319, 135-142. [28] Muleris, M., Delattre, O., Olschwang, S., Dutrillaux, A.M., Remvikos, Y., Salmon, R.J., Thomas, G. and Dutrillaux, B. (1990) Cytogenetic and molecular ap proaches of polyploidization in colorectal adenocarcinemas. Cancer Genet. Cytogenet. 44, 107- 118. [29] Dutrillaux, B., Gerbault-Seureau, M., Remvikos, Y., Zafrani, B. and Prieur, M. (1991) Breast cancer genetic evolution I: Data from cytogenetics and DNA content. Breast Cancer Res. Treat. 19, 245-255. [30] Matsumoto, K. and Ohta, K. (1992) Sensitive period for the induction of endoreduplication by rotenone in cultured Chinese hamster cells. Chromosoma 102, 60-65. [31] Huang, R.N., Ho, I.C., Yih, L.H. and Lee, T.C. (1995) Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphotase activity in human tibroblasts. Environ. Mol. Mutagenesis 25, 188-196. [32] Paton, G.R. and Allison, A.C. (1972) Chromosome damage in human cell cultures induced by metal salts. Mutat. Res. 16, 332-336. [33] Bertolero, F., Pozxi, G., Sabbioni, E. and Saffiotti, U. (1987) Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformations. Carcinogenesis 8, 803-808. [34] Lerman, S.A., Clarkson, T.W. and Gerson, R.J. (1983) Arsenic uptake and metabolism by liver cells is dependent on arsenic oxidation state. Chem.-Biol. Interact. 45, 401-406. [35] Georis, B., Cardenas, A., Buchet, J.P. and Lauwerys, R. (1990) Inorganic arsenic methylation by rat tissue slices. Toxicology 63, 73-84. [36] Vahter, M. and Envall, J. (1983) In vivo reduction of arsenate in mice and rabbits. Environ. Res. 32, 14-21.