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Genotoxic effects of sodium arsenite on human cells
Mutation Research, 284 (1992) 215-221
215
© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
MUT 05179
Genotoxic effects of sodium arsenite on h u m a n cells * A.N. Jha
a M. Noditi a, R. Nilsson c and A.T.
N a t a r a j a n a,b
a Department of Radiation Genetics and Chemical Mutagenesis, State Unicersity of Leiden, Netherlands, h J.A. Cohen Institute, lnterunicersity Research Institute for Radiopathology and Radiation Protection, Leiden, Netherlands and " National Swedish Chemicals lnspectorate, Dicision for Scientific Documentation and Research, Solna, Sweden (Received 29 April 1992) (Revision received 16 June 1992) (Accepted 18 June 1992)
Keywords: Sodium arsenite; Chromosomal aberrations; Sister-chromatid exchanges; Micronucleus test
Summary The effects of sodium arsenite (SA) were studied either alone or in combination with X-rays in peripheral blood lymphocytes, and with short-wave ultraviolet (UV) radiation in primary human fibroblast culture systems. It was found that SA (i) inhibited the cell cycle progression of phytohaemagglutinin (PHA)-responsive lymphocytes, (ii) induced chromatid-type aberrations and sister-chromatid exchanges (SCEs) as a function of concentration and (iii) potentiated the X-ray- and UV-induced chromosomal damage. Our results suggest that SA interferes with the DNA repair process, presumably by inhibiting the ligase activity. This accounted for an increase in the DNA replication-dependent processes, chromatid aberrations and SCEs and synergistic enhancement of the X-ray- and UV-induced chromosomal damage. This ability of arsenite may be responsible for its comutagenic properties with different types of mutagens and hence its carcinogenicity.
Epidemiological studies have recognised arsenic (As) compounds or arsenicals as known carcinogens (Hernberg, 1977; IARC, 1980). Arsenic contamination of water in different parts of the world has also caused symptoms of poisoning which is manifested by skin, gastrointestinal and neurological disorders (Cebrian et al., 1983). These have led to growing concern about the
Correspondence: Prof. A.T. Natarajan, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Wassenaarseweg 72, 2333 AL Leiden, Netherlands. * Dedicated to Prof. B.A. Kihlman on the occasion of his 70th birthday.
health hazards due to arsenicals. Studies on animals using various routes of exposure have, however, failed to detect direct carcinogenic effects of arsenicals (Byron et al., 1967). Furthermore, in spite of the fact that most carcinogens are also mutagens, arsenicals have been found to be nonmutagenic in either bacteria or Chinese hamster cells (L6froth and Ames, 1978; Amacher and Pailler, 1980; Rossman et al., 1980). The lack of mutagenic activity of arsenicals therefore emphasised the studies pertaining to its co-mutagenicity with UV, X-rays and alkylating agents. It was reported that inorganic arsenicals inhibit the removal of ultraviolet (UV)-induced thymine dimers from the DNA of human SF 34 cells and potenti-
216 ate the lethal effects of UV in excision-proficient normal and xeroderma pigmentosum (XP) variant cells but not in excision-defective XP group A cells (Okui and Fujiwara, 1986). It was also found that sodium arsenite (SA), which is the probable physiologically active form (Bertolero et al., 1987), inhibits the repair of N-methyl-N-nitrosourea (MNU)-induced lesions by affecting the incorporation of dNMPs into damaged DNA template or by interfering with the ligation step (Li and Rossman, 1989a). The mechanisms by which arsenic compounds induce mutagenic effects are not very clear. It has been found that inorganic trivalent arsenicals can inhibit the activities of thiol-containing enzymes, especially those which contain two sulphydryl groups (Sunderman, 1979; Jenette, 1981). DNA ligases which contain essential sulphydryl groups (S6derhall and Lindahl, 1987) are considered to be suspected targets for arsenite action (Li and Rossman, 1989b). This indicates a possible mechanism of arsenite's co-mutagenic effects. Although numerous studies have indicated co-mutagenic properties of SA, substantial evidence to show its co-clastogenic effects with Xrays and UV especially in human cells is lacking. The determination of potentiating effects of SA on X-ray- and UV-induced genetic damage particularly in the light of its possible inhibitory effect on DNA ligase may help in our understanding of its enhancing effects. We have, therefore, carried out different cytogenetic investigations to test the possible effects of SA, either alone or in combination with X-rays or short-waVe UV radiation, using peripheral blood lymphocytes and primary cultured human fibroblasts. The parameters considered are (i) chromosomal aberrations (CA), (ii) sister-chromatid exchanges (SCEs) and (iii) micronuclei (MN). Materials and methods
Experiments using the lymphocyte culture system Peripheral blood samples from healthy donors were collected and lymphocytes were isolated in Ficoll Histopaque gradients (Boehringer). These lymphocytes were used to analyse the clastogenic and SCE-inducing capability of SA and to verify its potentiating effect on X-ray-induced CA. In
preliminary experiments, we employed a wholeblood culture technique for the experiments but because of possible interaction of red cells with arsenite, the effects were very low. Therefore, isolated and subsequently frozen lymphocytes were used in the present study. First, a pilot experiment was done to analyse the proliferation kinetics of phytohaemagglutinin (PHA)-responsive lymphocytes using the fluorescence plus Giemsa (FPG) staining technique, which allows recognition of cells which have grown in presence of 5-bromodeoxyuridine (BrdU) for one, two or more cycles. The lymphocytes were cultured in complete F-10 medium supplemented with 5 /~M of SA. This concentration has been found to be non-toxic to the cells (Li and Rossman, 1989a). After 24 h of initiation, the cells were washed twice with serum-free medium and allowed to grow for a further 48 and 72 h. The cells were arrested with colcemid, treated with hypotonic solution (0.075 M KCI) and fixed in acetic acid-methanol (1:3). Metaphase spreads were prepared and the slides were processed for FPG staining employing standard protocols. At least 100 metaphase were analysed for the proliferation rate of the cells at each fixation time. Five different concentrations, viz. 1, 5, 10, 20 and 50 /~M, of SA were used to evaluate the induction of CA and SCEs. Lymphocytes were allowed to grow in complete medium for 48 h with different concentrations of SA. The cells were washed twice with serum-free medium and allowed to grow in the presence of BrdU for a further 24 h (for CA) or 48 h (for SCEs). Metaphase spreads were prepared and the slides were processed as described earlier. At least 100 metaphases for CA and 50 for SCEs were analysed at each concentration. To analyse the effects of SA on X-ray-induced CA, lymphocytes were preincubated with 5 p.M of SA in serum-free medium for 2 h after which they were irradiated with 1 and 2 Gy of X-rays. The cells were allowed to remain in the same medium (containing SA) for 30 min at 37°C and then transferred to complete medium for 24 h after which they were washed twice. The cells were reincubated in complete medium containing BrdU and allowed to grow for a final period of 72 h. The cells were fixed and metaphase spreads
217
~
were p r e p a r e d as per standard protocol. At least 200 cells were analysed from two independent experiments to score dicentrics, rings and excess acentrics. One set of slides from these experiments was also processed for F P G staining to analyse the proportion of different division cells at this fixation time.
Experiments using the fibroblast culture system We analysed two cytogenetic parameters, i.e., frequencies of micronuclei (an indicator of induced CA) and SCEs, in human primary fibroblast cells to determine if SA has any modifying effect on the UV-induced genetic damage. Human fibroblasts (VH16 cell line) were allowed to grow to confluence in petri dishes. The cells were irradiated with 7.5 J / m 2 of U V (254 nm) and allowed to grow in conditioned medium in either the presence or the absence of a non-toxic concentration of SA (5/x M) for 24 h. The ceils were then washed, trypsinised and plated in four dishes. They were allowed to grow in the presence of cytochalasin B (for MN) or BrdU (for SCEs) for 48 and 72 h. Cytochalasin B inhibits cytokinesis, thus creating binucleated cells which represent cells which have divided once since the addition of cytochalasin B. The cells were fixed by standard methods and prepared slides were stained for scoring of MN and SCEs. The MN were analysed from both the fixation times but SCEs could be analysed only after 48 h of fixation as after 72 h most of the ceils were in the third or subsequent division stage.
1st
2nd 3rd (+)
3rd (+)
72 hours fixation time 96 hours fixation time Fig. 1. Proportion of lymphocytes at first, second and third cell cycle stages after culturing for 72 or 96 h in a medium containing 5/zM SA.
60
- - * - - Aberrant ceils
~,
Chromatid breaks
../' .- • ""
50
40
~
,.L~"
30
10
0
~
~
/
,// ,
i
,
2
i
,
4
I
,
6
i
,
8
I
10
C o n c e n t r a t i o n (pM)
Fig. 2. Frequencies of chromatid-type aberrations induced by
different concentrations of SA in human peripheral lymphocytes cultured in medium containing different concentration of SA.
25
Results 20
The proportions of different division cells in PHA-responsive lymphocytes at two different fixation times (72 and 96 h) after treatment with 5 /zM of SA for 24 h are presented in Fig. 1. In contrast to untreated cells which showed up to 90% ceils in second or subsequent division, lymphocytes grown in the presence of SA were delayed in cell cycle progression, with 73 and 32% of cells still in first division at fixation times of 72 and 96 h, respectively. A dose-dependent increase in the yield of aberrations in lymphocytes grown in the presence of SA was found and is presented in Fig. 2. Only a few metaphases could
15
10
i
2
,
~
4
,
i
6
,
i
8
,
i
10
C o n c e n t r a t i o n (!JM)
Fig. 3. Frequencies of SCEs induced in human lymphocytes cultured in medium containing different concentrations of SA.
218 100
Donor A
100
Dicentrics [ - ~ J Deletions + Rings
80
Donor B Oit-J=ntrir'~
80
60
60
40
40
20
20
[--I
n=l=tinn~
+
0
0 SA
1 Oy
SA + 1 Gy Treatment
2 Gy
SA + 2 Gy
SA
1 Gy
SA + 1 Gy Treatment
2 Gy
SA + 2 Gy
Fig. 4. Frequencies of dicentrics plus rings and deletions induced by X-rays in human lymphocytes and treated with SA from two different donors (A and B).
100 2nd d i v . ~
3rd div.
80
~
6O
g.
40 20
0 SA
1 Gy
1 Gy +SA Treatment
2 Gy
B
2 Gy -~SA
Fig. 5. Percentages of first and subsequent division lymphocytes at 72 h fixation time at which chromosome aberrations were analysed.
be scored at SA concentrations over 1 0 / z M . The aberrations were predominantly chromatid breaks including a few isochromatid breaks or terminal deletions. Chromatid- or chromosome-type exchanges were not observed in 400 cells scored for different concentrations. The frequencies of SCEs also showed a dose-dependent increase, with an about 5-fold increase at the concentration of 10 /zM (Fig. 3). The effect of SA on X-ray-induced unstable types of CA showed a 2-fold increase in the yield of dicentrics and rings. Excess acentrics also showed a 1.3-2.5-fold increase following SA treatment. X-Ray-induced c h r o m o s o m e aberration data could not be pooled for these two experiments as the donors showed differences for the yield of excess acentrics. The frequencies of
100 m 80
4 8 hour fixation
~
72 hour fixation
60
on o
40
20
0
0 -SA -UV
-SA +UV +SA -UV Treatment
+SA +UV
Fig. 6. Frequencies of micronuclei (MN) in human fibroblasts irradiated with U V and post-incubated with or without SA.
-SA -UV
i!11 11 +SA -UV -SA +UV Treatment
+SA +UV
Fig. 7. Frequencies of SCEs in human fibroblasts irradiated with UV and incubated with or without SA.
219 these unstable types of CA are presented in Figs. 4. The proportion of different division cells as revealed by the FPG staining after various treatments is given in Fig. 5. This further supports the earlier observation that SA treatment delays the cell cycle progression. Figs. 6 and 7 show that both UV and SA are capable of inducing MN and SCEs. SA significantly potentiated the frequency of UV-induced MN by 1.6- and 1.4-fold at the two fixation times respectively. There was, however, no synergistic effect of SA on UV-induced SCEs (Fig. 7). Discussion
Earlier studies have shown that arsenicals inhibit the proliferation of cultured human and bovine lymphocytes under in vitro conditions (Petres et al., 1977; Wen et al., 1981; McCabe et al., 1983). The inhibitory effect of arsenicals on cell cycle progression has been suggested to be primarily due to its affinity for proteins containing sulphydryl groups (L6onard and Lauwerys, 1980). Lymphocyte proliferation kinetics has also been found to be slower in individuals chronically exposed to hydroarsenicism in Mexico. It has been speculated that such a delay in lymphocyte proliferation could represent an impairment of the cellular immune response and may eventually lead to malignancy (Ostrosky-Wegman et al., 1991). In a study with several inorganic metal salts, it was found that arsenic compounds are capable of inducing SCEs and chromatid-type aberrations in human lymphocytes and Syrian hamster embryo ceils. It was further reported that arsenic salts are less efficient in inducing SCEs in human whole blood than in purified lymphocyte cultures (Larramendy et al., 1981). This is supported by the results of the present study where induction of both endpoints showed a concentration-dependent increase. It is conceivable that SA can induce DNA synthesis-dependent effects such as chromatid aberrations and SCEs, if it interferes with the ligation process. Inhibitors of DNA repair process have been found to potentiate the yield of CA induced by various clastogens (Natarajan et ai., 1982; Preston, 1982). X-Rays mainly induce base damages,
single- and double-strand breaks (ssb and dsb) in DNA, the latter being mainly responsible for the production of chromosomal aberrations (Natarajan and Obe, 1984). The final step in the repair of such induced breaks involves a ligation. If this step is inhibited, it will result in an enhanced frequency of CA. The results presented in this study support this expectation as SA was found to increase the frequencies of X-ray-induced dicentrics plus rings and excess acentrics. It has been found that gamma-irradiated CHO and human fibroblast cells either pre- or post-treated with SA show a much longer time for the recovery of altered D N A conformation than untreated samples. It has been suggested that a decrease in DNA ligase activity may be responsible for the delay of DNA repair after irradiation (Huang et al., 1991). The induction of MN by UV radiation and the synergistic effect of arsenite on it may be explained on the basis of an earlier observation that UV-induced DNA lesions on parental strands may produce double-strand breaks through replication (Wang and Smith, 1986). It was found that the rate of excision repair is greatest before the onset of DNA replication (Lipman et al., 1989) and DNA repair-associated ligase activity in cells increases after exposure to UV radiation or alkylating agent (Mezzina and Nocentini, 1978; Sarasin, 1985). It is therefore reasonable to assume that impairment in the excision repair process through DNA ligase activity may account for the enhancing effect of arsenite on UV clastogenicity. However, SA has been found to inhibit the excision of pyrimidine dimers and unscheduled DNA synthesis in normal human fibroblasts and to potentiate UV-induced killings of excision-proficient normal human and XP variant cells, but not that of excision-defective XP group A ceils (Okui and Fujiwara, 1980). Huang et al. (1992) have found an enhanced frequency of chromatid exchanges, which are supposed to result from misjoining of strand breaks, after combined treatment of UV and SA in late G~- and S-phase CHO-K1 cells. These results indicate the possibility of inhibition of steps other than ligation in the excision of pyrimidine dimers for the cytotoxic effects of arsenite. Li and Rossman (1989b) have, however, reported that both consti-
220
tutive and MNU-inducible levels of D N A ligase II are inhibited by SA. In this context, it has also been reported that 3-aminobenzamide, a possible inhibitor of D N A ligase, potentiates the yield of exchanges and deletions induced by restriction enzymes in G~ CHO-K1 cells (Chung et al., 1991). Using alkaline sucrose gradient sedimentation and pulse-chase labelling, it has recently been shown that, unlike aphidicolin and hydroxyurea, SA does not accumulate UV-induced D N A strand breaks. Rather, it strongly inhibits the chain elongation of nascent D N A in CHO-K1 cells. It simply indicates that SA inhibits the rejoining of strand breaks but not the activity of D N A polymerase o~ in UV-irradiated cells (Lee-Chert et al., 1992). However, differences in the processing of UV-induced damage and therefore the target for SA action may exist between CHO and human cells. The lack of synergistic effect on UV-induced SCEs in the present study may be due to the fact that SA was washed off before seeding the cells for division. SCE is an S-dependent process hence absence of SA during this phase could not potentiate the residual UV-induced lesions synergistically. Lee et al. (1985) have also observed an additive effect for the induction of SCEs with a combined treatment of low doses of UV and SA and less than additive values using a combination of higher doses of UV and SA, even though a dose-dependent increase in the frequency of SCEs was obtained after a single treatment with UV or SA in CHO cells. In conclusion, the present study supports the contention that arsenic compounds interfere with the action of ligase involved in rejoining of strand breaks. If SA interferes with ligation, it is conceivable that it by itself can induce D N A synthesis-dependent effects, such as chromatid aberrations and SCEs. In the repair of lesions induced by both X-rays and UV, a ligation step is involved and an inhibition of ligase activity is expected to be responsible for the subsequent biological effects, including chromosomal damage. Acknowledgement The work was supported by the European Economic Community Radiation Protection Programme.
References Amacher, D.E., and S.C. Pailler (1980) Induction of trifluorothymidine-resistant mutants by metal ions in L5178Y/ TK +/- cells, Mutation Res., 78, 279-288. Bertolero, F., G. Pozzi, E. Sabbioni and U. Saffiotti (1987) Cellular uptake and metabolic reduction of pentavalent to trivalent arsenic as determinants of cytotoxicity and morphological transformation, Carcinogenesis, 8, 8113-808. Byron, W.R., J. Bierlower and J.A. Dipaolo (1967) Pathologic changes in rats and dogs from two-year feeding of sodium arsenite and sodium arsenate, Toxicol. Appl. Pharmacol., 10, 132-140. Cebrian, M.E., A. Albores, M. Aguilar and E. Blakely (1983) Chronic arsenic poisoning in the north of Mexico, tlum. Toxicol., 2, 121-133. Chung, H.W., J.W. Phillips, R.A. Winegar, R.J. Preston and W.F. Morgan (1991) Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulse-field gel analysis, Radiation Res., 125, 107-113. Hernberg, S. (1977) Incidence of cancer in population with environmental exposure to metals, in: H.H. Hiatt, J.D. Watson and J.A. Winston (Eds.), Origin of Human Cancer, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 147 157. Huang, H, S.H. Tyan and S.G. Lin (1991) The effect of sodium arsenite on the DNA repair of gamma-irradiated mammalian cells, in: J.D. Chapman, W.C. Dewey and G.F. Whitmore (Eds.), Radiat. Res., Vol. 1, 9th ICRR Congress Abstracts, 359 pp. Huang, H., C.F. Huang, J.S. Huang, T.C. Wang and K.Y. Jan (1992) The transition from late G1 to early S phase is most vulnerable to the co-clastogenic effect of ultraviolet radiation plus arsenite, Int. J. Radiat. Biol., 61, 57-62. 1ARC (1980) Carcinogenesis of arsenic compounds, IARC Monograph on Evaluation of Carcinogenic Risks, Vol. 23, IARC, Lyon, pp. 37-141. Jennette, K.W. (1981) The role of metals in carcinogenesis: Biochemistry and metabolism, Environ. Health Perspect., 40, 233-252. 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 in Syrian hamster cell strains, Environ. Mutagen., 3, 597-6(16. Lee-Chert, S.F., C.T. Yu and K.Y. Jan (1992) Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cell, Mutagenesis, 7, 51-55. Lee, T.-C., R.Y. Huang and K.Y. Jan (1985) Sodium arsenite enhances the cytotoxieity, clastogenicity, and 6thioguanine-resistant mutagenicity of ultraviolet light in Chinese hamster ovary cells, Mutation Res., 148, 83-89. L~onard, A., and R.R. Lauwerys (1980) Carcinogenicity, teratogenicity and mutagenicity of arsenic, Mutation Res., 75, 49-62. Li, J.-H., and T.G. Rossman (1989a) Mechanism of co-mutagenesis of sodium arsenite with N-methyI-N-nitrosourea, Biol. Trace Element Res., 21,373-381.
221 Li, J.-H., and T.G. Rossman (1989b) Inhibition of DNA ligase activity by arsenite: a possible mechanism of its co-mutagenesis, Mol. Toxicol., 2, 1-9. Lipman, J.M., A..Applegate-Stevens, L.A. Soyka and R.W. Hart (1989) Cell-cycle defect of DNA repair in progeria skin fibroblasts, Mutation Res., 219, 273-281. L6froth, G., and B.N. Ames (1978) Mutagenicity of inorganic compounds in Salmonella typhimurium: arsenic, chromium, and selenium, Mutation Res., 53, 65-66. McCabe, M., D. Maguire and M. Nowak (1983) The effects of arsenic compounds on human and bovine lymphocyte mitogenesis in vitro, Environ. Res., 31,323-331. Mezzina, M., and S. Nocentini (1978) DNA ligase activity in UV-irradiated monkey kidney cells, Nucleic Acids Res., 5, 4317-4328. Natarajan, A.T., and G. Obe (1984) Molecular mechanisms involved in the production of chromosomal aberrations. 111. Restriction endonucleases, Chromosoma, 90, 120-127. Natarajan, A.T., 1. Csukas, F. Degrassi, A.A. van Zeeland, F. Palitti, C. Tanzarella, R. de Saliva and M. Fiore (1982) Influence of inhibition of repair enzymes on the induction of chromosomal aberrations by physical and chemical agents, in: A.T. Natarajan, G. Obe and H. Altman (Eds.), Progress in Mutation Research, Vol. 4, Elsevier, Amsterdam, pp. 47-59. Okui, T., and Y. Fujiwara (1986) Inhibition of human excision DNA repair by inorganic arsenic and the co-mutagenic effect in V79 Chinese hamster cells, Mutation Res., 172, 69-76. Ostrosky-Wegman, P., M.E. Gonsebatt, R. Montero, L. Vega,
H. Barba, J. Espinosa, A. Palao, C. Cortinas, G. GarciaVargas, L.M. del Razo and M. Cebrian (1991) Lymphocyte proliferation kinetics and genotoxic findings in a pilot study on individuals chronically exposed to arsenic in Mexico, Mutation Res., 250, 477-482. Petres, J., D. Baron and M. Hagedorn (1977) Effects of arsenic cell metabolism and cell proliferation: cytogenetic and biochemical studies, Environ. Health Perspect., 19, 223-227. Preston, R.J. (1982) The use of inhibitors of DNA repair in the study of the mechanisms of induction of chromosome aberrations, Cytogenet. Cell Genet., 33, 20-26. Rossman, T.G., D. Stone, M. Molina and W. Troll (1980) Absence of arsenite mutagenicity in E. coli and Chinese hamster cells, Environ. Mutagen., 2, 371-379. Sarasin, A. (1985) SOS response in mammalian cells, Cancer Invest., 3, 163-174. S6derhall, S., and T. Lindahl (1987) DNA ligase of eukaryotes, FEBS Lett., 67, 1-8. Sunderman, F.W. (1979) Mechanism of metal carcinogenesis, Biol. Trace Elements Res., 1, 63-86. Wang, T.C.V., and K.C. Smith (1986) Postreplication repair in ultraviolet-irradiated human fibroblasts: formation and repair of DNA double-strand breaks, Carcinogenesis, 7, 389-392. Wen, W.N., L.T. 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.
215
© 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
MUT 05179
Genotoxic effects of sodium arsenite on h u m a n cells * A.N. Jha
a M. Noditi a, R. Nilsson c and A.T.
N a t a r a j a n a,b
a Department of Radiation Genetics and Chemical Mutagenesis, State Unicersity of Leiden, Netherlands, h J.A. Cohen Institute, lnterunicersity Research Institute for Radiopathology and Radiation Protection, Leiden, Netherlands and " National Swedish Chemicals lnspectorate, Dicision for Scientific Documentation and Research, Solna, Sweden (Received 29 April 1992) (Revision received 16 June 1992) (Accepted 18 June 1992)
Keywords: Sodium arsenite; Chromosomal aberrations; Sister-chromatid exchanges; Micronucleus test
Summary The effects of sodium arsenite (SA) were studied either alone or in combination with X-rays in peripheral blood lymphocytes, and with short-wave ultraviolet (UV) radiation in primary human fibroblast culture systems. It was found that SA (i) inhibited the cell cycle progression of phytohaemagglutinin (PHA)-responsive lymphocytes, (ii) induced chromatid-type aberrations and sister-chromatid exchanges (SCEs) as a function of concentration and (iii) potentiated the X-ray- and UV-induced chromosomal damage. Our results suggest that SA interferes with the DNA repair process, presumably by inhibiting the ligase activity. This accounted for an increase in the DNA replication-dependent processes, chromatid aberrations and SCEs and synergistic enhancement of the X-ray- and UV-induced chromosomal damage. This ability of arsenite may be responsible for its comutagenic properties with different types of mutagens and hence its carcinogenicity.
Epidemiological studies have recognised arsenic (As) compounds or arsenicals as known carcinogens (Hernberg, 1977; IARC, 1980). Arsenic contamination of water in different parts of the world has also caused symptoms of poisoning which is manifested by skin, gastrointestinal and neurological disorders (Cebrian et al., 1983). These have led to growing concern about the
Correspondence: Prof. A.T. Natarajan, Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Wassenaarseweg 72, 2333 AL Leiden, Netherlands. * Dedicated to Prof. B.A. Kihlman on the occasion of his 70th birthday.
health hazards due to arsenicals. Studies on animals using various routes of exposure have, however, failed to detect direct carcinogenic effects of arsenicals (Byron et al., 1967). Furthermore, in spite of the fact that most carcinogens are also mutagens, arsenicals have been found to be nonmutagenic in either bacteria or Chinese hamster cells (L6froth and Ames, 1978; Amacher and Pailler, 1980; Rossman et al., 1980). The lack of mutagenic activity of arsenicals therefore emphasised the studies pertaining to its co-mutagenicity with UV, X-rays and alkylating agents. It was reported that inorganic arsenicals inhibit the removal of ultraviolet (UV)-induced thymine dimers from the DNA of human SF 34 cells and potenti-
216 ate the lethal effects of UV in excision-proficient normal and xeroderma pigmentosum (XP) variant cells but not in excision-defective XP group A cells (Okui and Fujiwara, 1986). It was also found that sodium arsenite (SA), which is the probable physiologically active form (Bertolero et al., 1987), inhibits the repair of N-methyl-N-nitrosourea (MNU)-induced lesions by affecting the incorporation of dNMPs into damaged DNA template or by interfering with the ligation step (Li and Rossman, 1989a). The mechanisms by which arsenic compounds induce mutagenic effects are not very clear. It has been found that inorganic trivalent arsenicals can inhibit the activities of thiol-containing enzymes, especially those which contain two sulphydryl groups (Sunderman, 1979; Jenette, 1981). DNA ligases which contain essential sulphydryl groups (S6derhall and Lindahl, 1987) are considered to be suspected targets for arsenite action (Li and Rossman, 1989b). This indicates a possible mechanism of arsenite's co-mutagenic effects. Although numerous studies have indicated co-mutagenic properties of SA, substantial evidence to show its co-clastogenic effects with Xrays and UV especially in human cells is lacking. The determination of potentiating effects of SA on X-ray- and UV-induced genetic damage particularly in the light of its possible inhibitory effect on DNA ligase may help in our understanding of its enhancing effects. We have, therefore, carried out different cytogenetic investigations to test the possible effects of SA, either alone or in combination with X-rays or short-waVe UV radiation, using peripheral blood lymphocytes and primary cultured human fibroblasts. The parameters considered are (i) chromosomal aberrations (CA), (ii) sister-chromatid exchanges (SCEs) and (iii) micronuclei (MN). Materials and methods
Experiments using the lymphocyte culture system Peripheral blood samples from healthy donors were collected and lymphocytes were isolated in Ficoll Histopaque gradients (Boehringer). These lymphocytes were used to analyse the clastogenic and SCE-inducing capability of SA and to verify its potentiating effect on X-ray-induced CA. In
preliminary experiments, we employed a wholeblood culture technique for the experiments but because of possible interaction of red cells with arsenite, the effects were very low. Therefore, isolated and subsequently frozen lymphocytes were used in the present study. First, a pilot experiment was done to analyse the proliferation kinetics of phytohaemagglutinin (PHA)-responsive lymphocytes using the fluorescence plus Giemsa (FPG) staining technique, which allows recognition of cells which have grown in presence of 5-bromodeoxyuridine (BrdU) for one, two or more cycles. The lymphocytes were cultured in complete F-10 medium supplemented with 5 /~M of SA. This concentration has been found to be non-toxic to the cells (Li and Rossman, 1989a). After 24 h of initiation, the cells were washed twice with serum-free medium and allowed to grow for a further 48 and 72 h. The cells were arrested with colcemid, treated with hypotonic solution (0.075 M KCI) and fixed in acetic acid-methanol (1:3). Metaphase spreads were prepared and the slides were processed for FPG staining employing standard protocols. At least 100 metaphase were analysed for the proliferation rate of the cells at each fixation time. Five different concentrations, viz. 1, 5, 10, 20 and 50 /~M, of SA were used to evaluate the induction of CA and SCEs. Lymphocytes were allowed to grow in complete medium for 48 h with different concentrations of SA. The cells were washed twice with serum-free medium and allowed to grow in the presence of BrdU for a further 24 h (for CA) or 48 h (for SCEs). Metaphase spreads were prepared and the slides were processed as described earlier. At least 100 metaphases for CA and 50 for SCEs were analysed at each concentration. To analyse the effects of SA on X-ray-induced CA, lymphocytes were preincubated with 5 p.M of SA in serum-free medium for 2 h after which they were irradiated with 1 and 2 Gy of X-rays. The cells were allowed to remain in the same medium (containing SA) for 30 min at 37°C and then transferred to complete medium for 24 h after which they were washed twice. The cells were reincubated in complete medium containing BrdU and allowed to grow for a final period of 72 h. The cells were fixed and metaphase spreads
217
~
were p r e p a r e d as per standard protocol. At least 200 cells were analysed from two independent experiments to score dicentrics, rings and excess acentrics. One set of slides from these experiments was also processed for F P G staining to analyse the proportion of different division cells at this fixation time.
Experiments using the fibroblast culture system We analysed two cytogenetic parameters, i.e., frequencies of micronuclei (an indicator of induced CA) and SCEs, in human primary fibroblast cells to determine if SA has any modifying effect on the UV-induced genetic damage. Human fibroblasts (VH16 cell line) were allowed to grow to confluence in petri dishes. The cells were irradiated with 7.5 J / m 2 of U V (254 nm) and allowed to grow in conditioned medium in either the presence or the absence of a non-toxic concentration of SA (5/x M) for 24 h. The ceils were then washed, trypsinised and plated in four dishes. They were allowed to grow in the presence of cytochalasin B (for MN) or BrdU (for SCEs) for 48 and 72 h. Cytochalasin B inhibits cytokinesis, thus creating binucleated cells which represent cells which have divided once since the addition of cytochalasin B. The cells were fixed by standard methods and prepared slides were stained for scoring of MN and SCEs. The MN were analysed from both the fixation times but SCEs could be analysed only after 48 h of fixation as after 72 h most of the ceils were in the third or subsequent division stage.
1st
2nd 3rd (+)
3rd (+)
72 hours fixation time 96 hours fixation time Fig. 1. Proportion of lymphocytes at first, second and third cell cycle stages after culturing for 72 or 96 h in a medium containing 5/zM SA.
60
- - * - - Aberrant ceils
~,
Chromatid breaks
../' .- • ""
50
40
~
,.L~"
30
10
0
~
~
/
,// ,
i
,
2
i
,
4
I
,
6
i
,
8
I
10
C o n c e n t r a t i o n (pM)
Fig. 2. Frequencies of chromatid-type aberrations induced by
different concentrations of SA in human peripheral lymphocytes cultured in medium containing different concentration of SA.
25
Results 20
The proportions of different division cells in PHA-responsive lymphocytes at two different fixation times (72 and 96 h) after treatment with 5 /zM of SA for 24 h are presented in Fig. 1. In contrast to untreated cells which showed up to 90% ceils in second or subsequent division, lymphocytes grown in the presence of SA were delayed in cell cycle progression, with 73 and 32% of cells still in first division at fixation times of 72 and 96 h, respectively. A dose-dependent increase in the yield of aberrations in lymphocytes grown in the presence of SA was found and is presented in Fig. 2. Only a few metaphases could
15
10
i
2
,
~
4
,
i
6
,
i
8
,
i
10
C o n c e n t r a t i o n (!JM)
Fig. 3. Frequencies of SCEs induced in human lymphocytes cultured in medium containing different concentrations of SA.
218 100
Donor A
100
Dicentrics [ - ~ J Deletions + Rings
80
Donor B Oit-J=ntrir'~
80
60
60
40
40
20
20
[--I
n=l=tinn~
+
0
0 SA
1 Oy
SA + 1 Gy Treatment
2 Gy
SA + 2 Gy
SA
1 Gy
SA + 1 Gy Treatment
2 Gy
SA + 2 Gy
Fig. 4. Frequencies of dicentrics plus rings and deletions induced by X-rays in human lymphocytes and treated with SA from two different donors (A and B).
100 2nd d i v . ~
3rd div.
80
~
6O
g.
40 20
0 SA
1 Gy
1 Gy +SA Treatment
2 Gy
B
2 Gy -~SA
Fig. 5. Percentages of first and subsequent division lymphocytes at 72 h fixation time at which chromosome aberrations were analysed.
be scored at SA concentrations over 1 0 / z M . The aberrations were predominantly chromatid breaks including a few isochromatid breaks or terminal deletions. Chromatid- or chromosome-type exchanges were not observed in 400 cells scored for different concentrations. The frequencies of SCEs also showed a dose-dependent increase, with an about 5-fold increase at the concentration of 10 /zM (Fig. 3). The effect of SA on X-ray-induced unstable types of CA showed a 2-fold increase in the yield of dicentrics and rings. Excess acentrics also showed a 1.3-2.5-fold increase following SA treatment. X-Ray-induced c h r o m o s o m e aberration data could not be pooled for these two experiments as the donors showed differences for the yield of excess acentrics. The frequencies of
100 m 80
4 8 hour fixation
~
72 hour fixation
60
on o
40
20
0
0 -SA -UV
-SA +UV +SA -UV Treatment
+SA +UV
Fig. 6. Frequencies of micronuclei (MN) in human fibroblasts irradiated with U V and post-incubated with or without SA.
-SA -UV
i!11 11 +SA -UV -SA +UV Treatment
+SA +UV
Fig. 7. Frequencies of SCEs in human fibroblasts irradiated with UV and incubated with or without SA.
219 these unstable types of CA are presented in Figs. 4. The proportion of different division cells as revealed by the FPG staining after various treatments is given in Fig. 5. This further supports the earlier observation that SA treatment delays the cell cycle progression. Figs. 6 and 7 show that both UV and SA are capable of inducing MN and SCEs. SA significantly potentiated the frequency of UV-induced MN by 1.6- and 1.4-fold at the two fixation times respectively. There was, however, no synergistic effect of SA on UV-induced SCEs (Fig. 7). Discussion
Earlier studies have shown that arsenicals inhibit the proliferation of cultured human and bovine lymphocytes under in vitro conditions (Petres et al., 1977; Wen et al., 1981; McCabe et al., 1983). The inhibitory effect of arsenicals on cell cycle progression has been suggested to be primarily due to its affinity for proteins containing sulphydryl groups (L6onard and Lauwerys, 1980). Lymphocyte proliferation kinetics has also been found to be slower in individuals chronically exposed to hydroarsenicism in Mexico. It has been speculated that such a delay in lymphocyte proliferation could represent an impairment of the cellular immune response and may eventually lead to malignancy (Ostrosky-Wegman et al., 1991). In a study with several inorganic metal salts, it was found that arsenic compounds are capable of inducing SCEs and chromatid-type aberrations in human lymphocytes and Syrian hamster embryo ceils. It was further reported that arsenic salts are less efficient in inducing SCEs in human whole blood than in purified lymphocyte cultures (Larramendy et al., 1981). This is supported by the results of the present study where induction of both endpoints showed a concentration-dependent increase. It is conceivable that SA can induce DNA synthesis-dependent effects such as chromatid aberrations and SCEs, if it interferes with the ligation process. Inhibitors of DNA repair process have been found to potentiate the yield of CA induced by various clastogens (Natarajan et ai., 1982; Preston, 1982). X-Rays mainly induce base damages,
single- and double-strand breaks (ssb and dsb) in DNA, the latter being mainly responsible for the production of chromosomal aberrations (Natarajan and Obe, 1984). The final step in the repair of such induced breaks involves a ligation. If this step is inhibited, it will result in an enhanced frequency of CA. The results presented in this study support this expectation as SA was found to increase the frequencies of X-ray-induced dicentrics plus rings and excess acentrics. It has been found that gamma-irradiated CHO and human fibroblast cells either pre- or post-treated with SA show a much longer time for the recovery of altered D N A conformation than untreated samples. It has been suggested that a decrease in DNA ligase activity may be responsible for the delay of DNA repair after irradiation (Huang et al., 1991). The induction of MN by UV radiation and the synergistic effect of arsenite on it may be explained on the basis of an earlier observation that UV-induced DNA lesions on parental strands may produce double-strand breaks through replication (Wang and Smith, 1986). It was found that the rate of excision repair is greatest before the onset of DNA replication (Lipman et al., 1989) and DNA repair-associated ligase activity in cells increases after exposure to UV radiation or alkylating agent (Mezzina and Nocentini, 1978; Sarasin, 1985). It is therefore reasonable to assume that impairment in the excision repair process through DNA ligase activity may account for the enhancing effect of arsenite on UV clastogenicity. However, SA has been found to inhibit the excision of pyrimidine dimers and unscheduled DNA synthesis in normal human fibroblasts and to potentiate UV-induced killings of excision-proficient normal human and XP variant cells, but not that of excision-defective XP group A ceils (Okui and Fujiwara, 1980). Huang et al. (1992) have found an enhanced frequency of chromatid exchanges, which are supposed to result from misjoining of strand breaks, after combined treatment of UV and SA in late G~- and S-phase CHO-K1 cells. These results indicate the possibility of inhibition of steps other than ligation in the excision of pyrimidine dimers for the cytotoxic effects of arsenite. Li and Rossman (1989b) have, however, reported that both consti-
220
tutive and MNU-inducible levels of D N A ligase II are inhibited by SA. In this context, it has also been reported that 3-aminobenzamide, a possible inhibitor of D N A ligase, potentiates the yield of exchanges and deletions induced by restriction enzymes in G~ CHO-K1 cells (Chung et al., 1991). Using alkaline sucrose gradient sedimentation and pulse-chase labelling, it has recently been shown that, unlike aphidicolin and hydroxyurea, SA does not accumulate UV-induced D N A strand breaks. Rather, it strongly inhibits the chain elongation of nascent D N A in CHO-K1 cells. It simply indicates that SA inhibits the rejoining of strand breaks but not the activity of D N A polymerase o~ in UV-irradiated cells (Lee-Chert et al., 1992). However, differences in the processing of UV-induced damage and therefore the target for SA action may exist between CHO and human cells. The lack of synergistic effect on UV-induced SCEs in the present study may be due to the fact that SA was washed off before seeding the cells for division. SCE is an S-dependent process hence absence of SA during this phase could not potentiate the residual UV-induced lesions synergistically. Lee et al. (1985) have also observed an additive effect for the induction of SCEs with a combined treatment of low doses of UV and SA and less than additive values using a combination of higher doses of UV and SA, even though a dose-dependent increase in the frequency of SCEs was obtained after a single treatment with UV or SA in CHO cells. In conclusion, the present study supports the contention that arsenic compounds interfere with the action of ligase involved in rejoining of strand breaks. If SA interferes with ligation, it is conceivable that it by itself can induce D N A synthesis-dependent effects, such as chromatid aberrations and SCEs. In the repair of lesions induced by both X-rays and UV, a ligation step is involved and an inhibition of ligase activity is expected to be responsible for the subsequent biological effects, including chromosomal damage. Acknowledgement The work was supported by the European Economic Community Radiation Protection Programme.
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221 Li, J.-H., and T.G. Rossman (1989b) Inhibition of DNA ligase activity by arsenite: a possible mechanism of its co-mutagenesis, Mol. Toxicol., 2, 1-9. Lipman, J.M., A..Applegate-Stevens, L.A. Soyka and R.W. Hart (1989) Cell-cycle defect of DNA repair in progeria skin fibroblasts, Mutation Res., 219, 273-281. L6froth, G., and B.N. Ames (1978) Mutagenicity of inorganic compounds in Salmonella typhimurium: arsenic, chromium, and selenium, Mutation Res., 53, 65-66. McCabe, M., D. Maguire and M. Nowak (1983) The effects of arsenic compounds on human and bovine lymphocyte mitogenesis in vitro, Environ. Res., 31,323-331. Mezzina, M., and S. Nocentini (1978) DNA ligase activity in UV-irradiated monkey kidney cells, Nucleic Acids Res., 5, 4317-4328. Natarajan, A.T., and G. Obe (1984) Molecular mechanisms involved in the production of chromosomal aberrations. 111. Restriction endonucleases, Chromosoma, 90, 120-127. Natarajan, A.T., 1. Csukas, F. Degrassi, A.A. van Zeeland, F. Palitti, C. Tanzarella, R. de Saliva and M. Fiore (1982) Influence of inhibition of repair enzymes on the induction of chromosomal aberrations by physical and chemical agents, in: A.T. Natarajan, G. Obe and H. Altman (Eds.), Progress in Mutation Research, Vol. 4, Elsevier, Amsterdam, pp. 47-59. Okui, T., and Y. Fujiwara (1986) Inhibition of human excision DNA repair by inorganic arsenic and the co-mutagenic effect in V79 Chinese hamster cells, Mutation Res., 172, 69-76. Ostrosky-Wegman, P., M.E. Gonsebatt, R. Montero, L. Vega,
H. Barba, J. Espinosa, A. Palao, C. Cortinas, G. GarciaVargas, L.M. del Razo and M. Cebrian (1991) Lymphocyte proliferation kinetics and genotoxic findings in a pilot study on individuals chronically exposed to arsenic in Mexico, Mutation Res., 250, 477-482. Petres, J., D. Baron and M. Hagedorn (1977) Effects of arsenic cell metabolism and cell proliferation: cytogenetic and biochemical studies, Environ. Health Perspect., 19, 223-227. Preston, R.J. (1982) The use of inhibitors of DNA repair in the study of the mechanisms of induction of chromosome aberrations, Cytogenet. Cell Genet., 33, 20-26. Rossman, T.G., D. Stone, M. Molina and W. Troll (1980) Absence of arsenite mutagenicity in E. coli and Chinese hamster cells, Environ. Mutagen., 2, 371-379. Sarasin, A. (1985) SOS response in mammalian cells, Cancer Invest., 3, 163-174. S6derhall, S., and T. Lindahl (1987) DNA ligase of eukaryotes, FEBS Lett., 67, 1-8. Sunderman, F.W. (1979) Mechanism of metal carcinogenesis, Biol. Trace Elements Res., 1, 63-86. Wang, T.C.V., and K.C. Smith (1986) Postreplication repair in ultraviolet-irradiated human fibroblasts: formation and repair of DNA double-strand breaks, Carcinogenesis, 7, 389-392. Wen, W.N., L.T. 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.