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Tin-protoporphyrin potentiates arsenite-induced DNA strand breaks, chromatid breaks and kinetochore-negative micronuclei in human fibroblasts
Mutation Research 452 Ž2000. 41–50 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres
Tin-protoporphyrin potentiates arsenite-induced DNA strand breaks, chromatid breaks and kinetochore-negative micronuclei in human fibroblasts I-Ching Ho, Ling-Huei Yih, Cheng-Yuan Kao, Te-Chang Lee ) Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan Received 4 November 1999; received in revised form 15 February 2000; accepted 17 March 2000
Abstract Numerous reports have shown that oxidative stress is involved in arsenite-induced genetic damage. Arsenite is also a potent inducer of heme oxygenase ŽHO.-1. To understand whether HO-1 could function as a cellular antioxidant and protect cells from arsenite injury, the effects of tin-protoporphyrin ŽSnPP., a competitive inhibitor of HO-1, on arsenite-induced genetic damage were examined in human skin fibroblasts ŽHFW.. In the present study, we found that SnPP at 100 mM significantly potentiated arsenite-induced cytotoxicity, DNA strand breaks Žassayed by alkaline single cell gel electrophoresisŽSCGE.., and chromatid breaks. Although arsenite alone mainly induced kinetochore-plus micronuclei ŽKq-MN., SnPP only synergistically enhanced kinetochore-negative micronuclei ŽKy-MN.. The increase in Ky-MN by SnPP cotreatment was consistent with the increase in DNA strand breaks and chromatid breaks caused by SnPP. However, at higher arsenite doses, Kq-MN was significantly reduced by SnPP. Pretreatment of HFW cells with hemin, an inducer of HO-1, significantly attenuated the cytotoxicity of arsenite. Therefore, the present results suggest that HO-1 induction by arsenite plays certain roles in protecting cells from arsenite-induced injury. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Arsenite; Heme oxygenase; Tin-protoporphyrin; DNA strand breaks; Single cell gel electrophoresis assay; Chromatid breaks; Micronuclei; Kinetochore
1. Introduction Evidence shows that arsenite induces oxidative stress and damage in mammalian cells w1–4x. Antioxidants, such as N-acetyl cysteine, glutathione ŽGSH., catalase, and squalene reduce arsenite-induced chro-
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Corresponding author. Tel.: q886-2-26523055; fax: q886-227829142. E-mail address: [email protected] ŽT.-C. Lee..
mosome aberrations, sister chromatid exchanges, and micronuclei ŽMN., and protect cells from arsenite insults w3,5–7x. In our previous study, we demonstrated that exposure of human fibroblasts ŽHFW. to arsenite increased the formation of fluorescent dichlorofluorescein by oxidation of its nonfluorescent form and modulated the cellular antioxidant defense activities in HFW, such as increasing GSH, superoxide dismutase, heme oxygenase ŽHO.-1, and ferritin, but decreasing catalase w8x. Arsenite-enhanced dichlorofluorescein fluorescence could be
0027-5107r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 0 0 . 0 0 0 3 5 - X
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abolished by an antioxidant, butylated hydroxytoluene w8x. These results indicate that arsenite-induced oxidative damage plays certain roles in arsenite genotoxicity. HO is the rate-limiting enzyme in the oxidative degradation of heme to bile pigments and carbon monoxide. Since the heme molecule is the prosthetic moiety of hemeproteins involved in cell respiration, energy generation, oxidative biotransformation, and the growth differentiation process, HO apparently performs vital roles in maintaining cellular homeostasis w9,10x. Two isoforms of HO have been identified: HO-1 and HO-2 w9x. HO-2 is the noninducible form found primarily in the central nervous system, whereas HO-1, an inducible isoform of HO, is not only induced by its substrate, heme molecules, but is also remarkably induced by a variety of stress-associated agents, including tumor-promoting phorbol esters, UV irradiation, hyperthermia, and heavy metals such as arsenite and cadmium w11–14x. Currently, the induction of HO-1 is generally thought to be a landmark of oxidative stress w10,11,15x. This point of view is supported by numerous studies that showed anti-oxidative activities of HO-1 in a variety of in vitro and in vivo systems w16–19x. However, several studies have shown that HO-1 expression did not protect cells against oxidative injury w20–22x. In HO-1 knock-out mice, genetic evidence clearly demonstrated that HO-1 was required for iron recycling w23x. Since iron is a potent oxidant, iron liberation accelerates the generation of reactive oxygen radicals and damages cellular macromolecules. The detection of HO-1 expression in atherosclerotic lesions also implied the pathogenicity of HO-1 w24x. Thus, the functional significance of HO-1 induction following oxidative stress is still not fully understood. Arsenite is a well-known HO-1 inducer. However, evidence showing the protective role of HO-1 against arsenite-induced genetic damage remains inadequate. To explore this issue, we treated HFW with a combination with arsenite and tin-protophorphyrin ŽSnPP., a potent inhibitor of HO-1 w25x, and examined whether SnPP could potentiate arsenite-induced genetic damage such as DNA strand breaks, chromosome aberrations, and MN. In this report, we found that SnPP could potentiate arsenite-induced DNA strand breaks, chromatid
breaks, and MN in HFW cells, indicating that HO-1 induction is not only a response to arsenite-induced oxidative stress but that HO-1 induction also participates in decreasing arsenite-induced genetic injury.
2. Materials and methods 2.1. Chemicals and cell culture Sodium arsenite ŽNaAsO 2 ., a trivalent arsenic compound obtained from E. Merck ŽDarmstadt, Germany., was used throughout the experiments. Media and chemicals used for cell culture were purchased from GIBCO ŽGrand Island, NY.. Fetal bovine serum ŽFBS. was obtained from HyClone Laboratories ŽLogan, UT.. Tin-protoporphyrin ŽSnPP. was purchased from Porphyrin Products ŽLogan, UT.. HFW, derived from the foreskin of a newborn Chinese infant, were cultured in Dulbecco’s modified Eagle medium Žhigh glucose, 430-2800EG. supplemented with 10% FBS, and antibiotics Ž100 unitsrml penicillin, 100 mgrml streptomycin. w26x. KB cells, derived from a human oral epidermal carcinoma, were obtained from American Type Culture Collection ŽRockville, MD. and grown in RPMI 1640 medium supplemented with 10% FBS and antibiotics Ž100 unitsrml penicillin, 100 mgrml streptomycin. w27x. 2.2. Assay of cell surÕiÕal Colony forming assay was used to determine the cytotoxicity of arsenite to HFW and KB cells. Cells were plated at a density of 3–5 = 10 5 cells per 60-mm dish. After 24-h incubation, cells were treated with drugs Žarsenite, SnPP, or arsenite plus SnPP. for 24 h, and then trypsinized and replated at 200– 1000 cells per 100-mm dish in triplicate. HFW cells were incubated for 14 days with one medium change, while KB cells were incubated for 10 days without medium change. Colonies were stained with a 1% crystal violet solution and counted under a dissecting microscope w7x. The colony forming efficiency of control HFW and KB cells were 53 " 12% and 64 " 3%, respectively.
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2.3. Western blotting analysis of HO The expression of HO-1 was analyzed by Western blotting technique w28x. Logarithmically growing cells were treated with drugs as described above. At the end of drug treatment, the cells were scraped from culture dishes with a rubber policeman, and prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously w26x. An aliquot of 10 mg cellular proteins was electrophoretically separated on a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane by a semi-dry electrotransfer system ŽATTO, Japan.. The membranes were immunoblotted with antibodies aganist rabbit HO-1 ŽStressGen Biotechnologies, Victoria, B.C., Canada. or the carboxy terminus of human actin ŽSanta Cruz Biotechnology, Santa Cruz, CA., and then visualized by an enhanced chemilluminescent technique according to the procedure provided by the manufacturer ŽPierce Chemical, Rockford, IL.. 2.4. Assay of DNA strand breaks The single cell gel electrophoresis ŽSCGE. assay or ‘Comet assay’ was used to monitor DNA strand breaks. Immediately after drug treatment, an aliquot of 1 = 10 5 HFW cells was subjected to alkaline SCGE according to the method described by Lynn et al. w29x. The slides were then stained with Sybr green ŽMolecular Probes, Eugene, OR.. Under a fluorescence microscope, DNA tail or comet tail examination was performed by the method described by Miyamae et al. w30x with slight modification. Accordingly, DNA comet tails were classified into four types: type I, without significant tail; type II, with short tail Žtail length less than head diameter.; type III, with long tail Žtail length greater than head diameter.; type IV, with small head. For each treatment, at least 500 cells were examined under a fluorescent microscope with naked eyes. 2.5. Assay of chromosome aberrations Logarithmically, growing HFW cells were treated with arsenite, SnPP, or arsenite plus SnPP for 24 h. Colcemid at the concentration of 0.05 mgrml was added 3 h prior to the end of treatment. The cells
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were subsequently trypsinized, treated with hypotonic solution, fixed, and prepared for chromosome analysis as previously described w26x. Chromosome aberrations were identified by following the criteria described by Buckton and Evans w31x. For each treatment, 50 metaphases were examined for the frequency of various types of chromosome aberrations. 2.6. Assay of MN The method of cytokinesis-block MN assay w32x with slight modification w7x was adopted to analyze the effect of SnPP on arsenite-induced MN. In brief, subsequent to drug treatment Žas described above., HFW cells were further incubated with 1 mgrml cytochalasin B, a cytokinesis inhibitor, for 24 h. Afterwards, cells were trypsinized and an aliquot of cell suspension was cytospun onto a clean slide. The slides were fixed with methanol at y208C for 10 min, air-dried, and stored at y208C in the dark. Immunostaining with CREST anti-kinetochore antibody ŽAntibody, Davis, CA. and fluroescein-conjugated secondary antibody ŽSigma, St. Louis, MO. was performed as previously described w7x. Meanwhile, chromosomes were counterstained with 0.1 mgrml 4,6-diamino-2-phenyl-indole. The frequency of MN with kinetochore ŽKq-MN. or without kinetochore ŽKy-MN. was scored from at least 500 binucleated cells under a fluorescence microscope.
3. Results 3.1. Enhancement of arsenite cytotoxicity by SnPP to HFW According to clonogenic assay, treatment with NaAsO 2 for 24 h resulted in a dose-dependent killing effect to HFW cells ŽFig. 1A.. Western blotting analysis indicated that a significant amount of HO-1 was induced and accumulated in HFW cells treated with arsenite at concentrations above 1.25 mM ŽFig. 1B.. To understand whether the induction of HO-1 played a protective role on arsenite cytotoxicity, HFW cells were treated in combination with 1.25 mM arsenite and various concentrations of SnPP. As
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with a combination of 5 mM arsenite and various concentrations of SnPP ŽFig. 2B.. When HFW cells were pretreated with hemin, a HO-1 inducer, HO-1 accumulation was clearly demonstrated in HFW cells by Western blotting technique ŽFig. 3A.. When hemin-pretreated HFW cells were subsequently challenged with arsenite, arsenite’s cytotoxicity was significantly reduced Ž p - 0.01, according to two-way ANOVA analysis; Fig. 3B.. 3.2. Enhancement of arsenite-induced DNA strand breaks by SnPP Fig. 1. Cytotoxicity and HO-1 induction by sodium arsenite in HFW. ŽA. HFW cells were treated with various concentrations of sodium arsenite for 24 h, and then replated to assess the survival rate by colony-forming assay as described in Section 2. Bars, SD of three independent experiments. ŽB. After a 24-h treatment with sodium arsenite, the cellular proteins were electrophoretically separated, immunoblotted with antibody against to HO-1 or actin, and then visualized by an enhanced chemilluminescent technique as described in Section 2.
shown in Fig. 2A, SnPP alone at the concentrations up to 100 mM did not show a significant killing effect to HFW cells, while SnPP dose-dependently enhanced the cytotoxicity of arsenite at 1.25 mM Ž p - 0.01, according to two-way ANOVA analysis.. Similar findings were observed in KB cells treated
Fig. 2. Enhancement of sodium arsenite cytotoxicity by SnPP to HFW and KB cells. HFW ŽA. or KB ŽB. cells were cotreated with various concentrations of SnPP and 1.25 or 5 mM sodium arsenite, respectively, for 24 h. At the end of treatment, the survival rate was determined by colony forming assay as described in Section 2. Bars, SD of three to four independent experiments. `, SnPP alone; v, arsenite plus SnPP.
SCGE assay is a sensitive technique to detect DNA strand breaks. The tail size reflects the levels of DNA strand breaks. Accordingly, we classified the DNA comet tails observed in our experiments into four types as illustrated in Fig. 4A. As reported by Lynn et al. w29,33x, SCGE assay was able to demonstrate the induction of DNA strand breaks by arsenite in Chinese hamster ovary and bovine aortic endothelial cells, respectively. Similarly, in the present study, arsenite treatment caused DNA strand breaks in HFW cells, i.e. type II and III DNA comet
Fig. 3. Induction of HO-1 and protection of HFW cells from the killing effects of sodium arsenite by hemin pretreatment. HFW cells were pretreated with 50 mM hemin for 24 h. ŽA. HO-1 expression was examined by Western blotting analysis as described in Section 2. Actin served as an internal control. ŽB. The hemin-pretreated HFW cells were further treated with various concentrations of sodium arsenite for 24 h, and then the survival rate was determined by colony forming assay described in Section 2. Bars, SD of three independent experiments. `, without hemin pretreatment; v, with hemin pretreatment.
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Fig. 4. Enhancement of sodium arsenite-induced DNA strand breaks by SnPP in HFW cells. HFW cells were cotreated with 100 mM SnPP and various concentrations of sodium arsenite for 24 h. DNA strand breaks were analyzed by Comet assay as described in Section 2. ŽA. The classification of comets by shape. ŽB. The incidence of each Comet type. Bars, SD of three to four independent experiments. p-values are the statistical comparison by the Mann–Whitney rank sum test between the treatments with and without SnPP.
tails were significantly increased by arsenite treatment ŽFig. 4B.. If comet tail types I–IV were assigned numerical values of 0–3, respectively, the averaged numerical values for 0, 1.25, 2.5 and 5 mM arsenite treatment were calculated to be 0.105 " 0.030, 0.386 " 0.060, 0.583 " 0.064, 0.909 " 0.024, respectively. These results indicated that arsenite dose-dependently induced DNA strand breaks in HFW cells Ž p - 0.001, according to ANOVA analysis.. The mean levels of comet tails in cells treated with 0–5 mM arsenite plus 100 mM SnPP were 0.152 " 0.029, 0.799 " 0.020, 1.006 " 0.081, 1.164 " 0.030, respectively. By the Mann–Whitney rank sum test, SnPP alone at 100 mM did not induce DNA strand breaks, but significantly potentiated arsenite-induced DNA strand breaks at all three doses of arsenite used Ž p values shown in Fig. 4B.. To exclude the formation of comet tails was originated from arsenite-induced dead or apoptotic cells, we have examined the cell status using dye exclusion
and DNA fluorescent dye staining techniques. Immediately after drug treatment, the trypan blue-stained cells were less than - 4% and no apoptotic cell could be found. Therefore, the comet tails observed were unlikely attributed to the dead or apoptotic cells. 3.3. Enhancement of arsenite-induced chromosome aberrations by SnPP Consistent with our previous study w26x, chromatid breaks were the major type of chromosome aberrations induced by arsenite in HFW cells. Arsenite at the doses of 2.5 mM and above induced a significant number of chromatid breaks ŽFig. 5.. However, arsenite at the concentration of 1.25 showed no significant induction of chromatid breaks Ž p s 0.114 according to Mann–Whitney rank sum test., indicating that a threshold might be required
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Fig. 5. Enhancement of sodium arsenite-induced chromatid breaks by SnPP in HFW cells. HFW cells were cotreated with 100 mM SnPP and various concentrations of sodium arsenite for 24 h. Chromosome analysis was performed as described in Section 2. Bars, SD of four independent experiments. `, arsenite alone; v, arsenite plus SnPP.
for chromatid break induction in HFW cells treated with arsenite. In contrast, cotreatment of HFW cells with various concentrations of arsenite and 100 mM SnPP resulted in a linear increase in chromatid breaks Ž p - 0.001, according to ANOVA analysis; Fig. 5.. A strong interaction between arsenite and SnPP was also observed Ž p - 0.001 according to two-way ANOVA analysis., suggesting that chromatid breaks induced by arsenite were significantly potentiated by SnPP in HFW cells. At the time of sampling for chromosome aberration analysis, the cell number was slightly increased to approximately 114% of
untreated cultures at the dose of 1.25 mM but decreased to 96% and 72% at 2.5 and 5 mM, respectively. In the cotreated cultures Žvarious oncentrations of arsenite plus 100 mM SnPP., the cell numbers were further decreased 10% in average as compared to the cultures treated with arsenite alone. Thus, under our experimental conditions, arsenite at the doses used did not seriously suppress the cell growth and hence distorted the analysis of chromosome aberrations.
3.4. Enhancement of arsenite-induced MN by SnPP The cytokinesis-block MN technique was used to confirm the potentiation effects of SnPP on arseniteinduced chromosomal damage. In our previous study, we demonstrated that treatment of HFW cells with arsenite at the same dose range used in this study mainly induced Kq-MN MN Ž70% of total MN. w7x. To determine whether SnPP cotreatment could alter the types of MN induced by arsenite, immunostaining with CREST anti-kinetochore antibody was performed to differentiate Kq-MN and Ky-MN. Consistent with our previous study results w7x, arsenite at the dose range and protocol used mainly induced Kq-MN in HFW cells ŽTable 1.. Arsenite at 5 mM also induced a significant amount of Ky-MN. SnPP synergistically enhanced the total number of
Table 1 Effects of SnPP on arsenite-induced MN a Treatment ŽmM. Sodium arsenite
SnPP
No. of binucleated cells per 1000 cells
MNr1000 binucleated cells Total Kq
Ky
0 1.25 2.5 5 0 1.25 2.5 5
0 0 0 0 100 100 100 100
401 453 347 320 365 364 361 315
9 "5 35 " 5 62 " 9 67 " 13 15 " 5 66 " 8 Ž41. ) 68 " 14 Ž68. 85 " 13 Ž73.
6 "4 9 "2 10 " 6 21 " 8 8 "2 49 " 9 Ž11. ) ) ) 53 " 10 Ž12. ) ) ) 70 " 15 Ž23. ) ) )
3 "2 25 " 4 52 " 7 46 " 6 7 "4 17 " 2 Ž29. 14 " 7 Ž56. ) ) ) 15 " 9 Ž50. ) ) )
Numbers in parentheses are the expected values calculated as the summation of the values of MN induced by single treatment with arsenite and SnPP minus the value of no treatment w60x. Asterisks indicate significant difference between the observed and expected values. a Data are averages" SD of three independent experiments. ) p - 0.05 according to x 2 analysis. ))) p - 0.001 according to x 2 analysis.
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MN production by arsenite only at the dose of 1.25 mM Ž p - 0.05 according to x 2 analysis, Table 1.. However, only Ky-MN was enhanced by SnPP. Arsenite at higher doses, such as 2.5 or 5 mM, induced a large number of MN; SnPP cotreatment did not further increase the frequency of total MN but resulted in a significant increase in Ky-MN Ž3–4.4-fold to the expected values. and a significant decrease in Kq-MN ŽTable 1., i.e. Ky-MN instead of Kq-MN became the major type of MN in HFW treated with arsenite plus SnPP. Although there was no evidence showing the interconversion Kq-MN to Ky-MN and Kq-MN, the present study still suggested that HO-1 played a role in preventing from the formation of Ky-MN that was mainly derived from chromosome fragments. According to the binucleate indexes ŽTable 1., our experimental conditions did not severely suppress the cell growth.
4. Discussion Inorganic arsenic is a well documented human carcinogen w34x. In the present study, the induction of chromatid breaks and MN by arsenite in HFW cells is consistent with our previous studies w7,26x. Using Comet assay, induction of DNA strand breaks by inorganic arsenite has been shown in human lymphocytes and bovine aortic endothelial cells w29,35,36x. Our results further confirm that inorganic arsenite causes DNA strand breaks in HFW cells. Furthermore, the present results also show that arsenite-induced DNA strand breaks, chromatid breaks, and MN are significantly potentiated by cotreatment with SnPP. Since SnPP is a competitive inhibitor of HO-1, these results suggest that HO-1 plays certain roles in reducing arsenite-induced genetic injury. Since our unpublished data showed that SnPP had no effect on the uptake of arsenite, we still could not rule out the possibility that the potentiation effects of SnPP are due to its unknown functions. Although how inorganic arsenite induces genetic injury is not fully elucidated, oxidative damage is likely involved in arsenite-induced DNA strand breaks, chromatid breaks, MN, and even in apoptosis in a variety of cell systems w4,7,29,37x. Several different mechanisms have been reported to generate oxidative stress by arsenite. Arsenite-induced DNA
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strand breaks and MN in Chinese hamster ovary cells were reported to be mediated through the generation of nitric oxide w33,37x. Reactive oxygen intermediates were shown to be involved in the generation of DNA strand breaks by dimethylarsenic acid w38x. In HFW cells, we previously demonstrated that reactive oxygen radicals were enhanced by arsenite in HFW cells w8x. In contrast to Chinese hamster ovary cells, arsenite treatment apparently did not enhance nitric oxide production in HFW cells, since we could not detect the production of nitrite Žan indicator for estimation of nitric oxide production. in cultural medium of HFW cells treated with arsenite Ždata not shown.. Therefore, the protective roles of HO-1 from arsenite-induced genetic injury may be attributed to HO-1’s antioxidant activity as has been shown in a variety of in vitro and in vivo systems w16–19x. Although inhibition of HO-1 apparently potentiated the genetic injury caused by arsenite, the levels of HO-1 expression remained positively associated with the increase of cytotoxicity, DNA strand breaks, chromatid breaks, and MN in arsenite-treated HFW cells. Pre-induction of HO-1 by hemin only partially protected HFW cells from the cytotoxic effects of arsenite. These results indicate that arsenite’s toxic effects are pleiotropic. The toxic effects of arsenite have generally been considered to result from its strong interaction with sulfhydryl groups, particularly vicinyl–sulfhydryl groups, of functional molecules, such as cytoskeleton molecules and ubiquitin conjugation enzymes w39–42x. Arsenite treatment may also alter cellular functions by changing phosphorylation profiles of cellular proteins w43,44x. In addition, several other mechanisms of arsenite genotoxicity and carcinogenicity, such as modulation of the DNA methylation status w45,46x and inhibition of DNA repair enzymes w33,47–49x, have also been well noticed in the literature. Therefore, the protective function of HO-1 is only partial and oxidative stress is only one of the mechanisms involved in arsenite genotoxicity. By using MN assay and chromosome specific probes, arsenite has been shown to have both aneugenic and clastogenic properties in lymphocytes and exfoliated epithelial cells w50–53x. Although the mechanisms by which arsenite exerts its cytogenetic effects remain unclear, we have previously demon-
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strated that the aneugenic or clastogenic manifestation of arsenite was associated with different treatment protocols w7x, e.g. arsenite at a low dose range Ž1.25–10 mM for 24 h. mainly induced Kq-MN, whereas at a high dose range Ž5–80 mM for 4 h. mainly induced Ky-MN. Consistent with this previous study, the protocol used in the present study for arsenite treatment mainly resulted in Kq-MN. Although our previous results suggested that oxidative stress was involved in the generation of Kq-MN and Ky-MN in arsenite treated HFW cells w7x, we found that SnPP changes the patterns of MN types induced by arsenite, i.e. with Kq-MN decreases and Ky-MN increases. Different mechanisms are involved in the generation of these two types of MN w54x. In general, Kq-MN derived from whole chromosomes is mainly produced by interference with mitosis, while Ky-MN derived from chromosome fragments is mainly formed from DNA breaks. The increase of Ky-MN by SnPP cotreatment may be due to the increase of DNA and chromatid breaks. However, the reason why SnPP cotreatment reduces the formation of Kq-MN is not clear. We suspect that HO-1 induction interferes with the homeostasis of cellular hemeproteins and hence influences chromosome segregation. Further investigation is required for clearer understanding of the vital roles of HO induction. The curve of chromatid break induction by arsenite alone ŽFig. 5. showed a slightly upward concave, indicating that a threshold might be required for chromatid break induction in HFW cells treated with arsenite. As reviewed by Rudel et al. w55x, sublinear dose–response relationships for arsenic-induced chromosome aberrations were repeatedly established in a variety of cell systems. However, no significant threshold was observed in arsenite-induced cytotoxicity, MN, and DNA strand breaks in HFW cells. In fact, the shapes of dose–response to arsenite were usually dependent on cell types used w1x, methods for genetic damage assay w55x, and the protocols of drug treatment, such as the levels of doses and the length of drug exposure w7x. Although oxidative stress has been recently considered to play certain roles in arsenic-induced genetic damages, how oxidative stress leads to various endpoints of genetic damages, such as DNA strand breaks, MN, sister chromatid exchanges, and chromosome aberrations, is not clear.
Furthermore, the dose–response relationships for arsenic-induced genetic damages are seriously affected by the cellular antioxidant molecules. Further investigation is essentially required for establishment of an appropriate dose–response model for assessing the human health risks of arsenic exposure. In conclusion, arsenite-induced DNA strand breaks, chromatid breaks, and Ky-MN were significantly potentiated by SnPP, suggesting that HO-1 plays antioxidant roles to prevent cells from arsenite-induced oxidative stress. Our results also confirm the involvement of oxidative stress in arsenite-induced genetic injury. Over generation of oxygen free radicals is considered to play a role in the development of several chronic diseases such as cancer, aging, inflammatory disorders, etc. w56–58x. Oxidative stress was demonstrated to induce the expression of numerous genes involved in the restoration of cellular homeostasis w59x. Since HO-1 is one of important antioxidant defense enzyme and performs vital roles in maintaining cellular homeostasis w9,10,18,19x, the physiological functions of HO-1 induction in arsenite exposed cells warrant our further investigation.
Acknowledgements We thank Mr. Douglas J. Platt for carefully reading this progress report. This work was supported by grants from Academia Sinica and from the National Science Council ŽNSC 87-2314-B001-009 and NSC 88-2314-B001-003., Republic of China.
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w50x D.A. Eastmond, J.D. Tucker, Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody, Environ. Mol. Mutagen. 13 Ž1989. 34–43. w51x L. Vega, M.E. Gonsebatt, P. Ostrosky-Wegman, Aneugenic effect of sodium arsenite on human lymphocytes in vitro: an individual susceptibility effect detected, Mutat. Res. 334 Ž1995. 365–373. w52x L.E. Moore, M.L. Warner, A.H. Smith, D. Kalman, M.T. Smith, Use of the fluorescent micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells, Environ. Mol. Mutagen. 27 Ž1996. 176–184. w53x M.E. Gonsebatt, L. Vega, A.M. Salazar, R. Montero, P. Guzman, A. ´ J. Blas, L.M. Del Razo, G. Garcıa-Vargas, ´ Albores, M.E. Cebrıan, M. Kelsh, P. Ostrosky-Wegman, ´ Cytogenetic effects in human exposure to arsenic, Mutat. Res. 386 Ž1997. 219–228. w54x H. Stopper, S.O. Muller, Micronuclei as a biolgoical end¨ point for genotoxicity: a review, Toxicol. in Vitro 11 Ž1997. 661–667. w55x R. Rudel, T.M. Slayton, B.D. Beck, Implications of arsenic genotoxicity for dose response of carcinogenic effects, Regul. Toxicol. Pharmacol. 23 Ž1996. 87–105. w56x B.N. Ames, L.S. Gold, Endogenous mutagens and the causes of aging and cancer, Mutat. Res. 250 Ž1991. 3–16. w57x D. Dreher, A.F. Junod, Role of oxygen free radicals in cancer development, Eur. J. Cancer 32A Ž1996. 30–38. w58x H. Wiseman, B. Halliwell, Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer, Biochem. J. 313 Ž1996. 17–29. w59x T.P. Dalton, H.G. Shertzer, A. Puga, Regulation of gene expression by reactive oxygen, Annu. Rev. Pharmacol. Toxicol. 39 Ž1999. 67–101. w60x W.F. Morgan, J.E. Cleaver, 3-Aminobenzamide synergistically increase sister-chromatid exchanges in cells exposed to methyl methanesulfonate but not to ultraviolet light, Mutat. Res. 104 Ž1982. 361–366.
Tin-protoporphyrin potentiates arsenite-induced DNA strand breaks, chromatid breaks and kinetochore-negative micronuclei in human fibroblasts I-Ching Ho, Ling-Huei Yih, Cheng-Yuan Kao, Te-Chang Lee ) Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan Received 4 November 1999; received in revised form 15 February 2000; accepted 17 March 2000
Abstract Numerous reports have shown that oxidative stress is involved in arsenite-induced genetic damage. Arsenite is also a potent inducer of heme oxygenase ŽHO.-1. To understand whether HO-1 could function as a cellular antioxidant and protect cells from arsenite injury, the effects of tin-protoporphyrin ŽSnPP., a competitive inhibitor of HO-1, on arsenite-induced genetic damage were examined in human skin fibroblasts ŽHFW.. In the present study, we found that SnPP at 100 mM significantly potentiated arsenite-induced cytotoxicity, DNA strand breaks Žassayed by alkaline single cell gel electrophoresisŽSCGE.., and chromatid breaks. Although arsenite alone mainly induced kinetochore-plus micronuclei ŽKq-MN., SnPP only synergistically enhanced kinetochore-negative micronuclei ŽKy-MN.. The increase in Ky-MN by SnPP cotreatment was consistent with the increase in DNA strand breaks and chromatid breaks caused by SnPP. However, at higher arsenite doses, Kq-MN was significantly reduced by SnPP. Pretreatment of HFW cells with hemin, an inducer of HO-1, significantly attenuated the cytotoxicity of arsenite. Therefore, the present results suggest that HO-1 induction by arsenite plays certain roles in protecting cells from arsenite-induced injury. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Arsenite; Heme oxygenase; Tin-protoporphyrin; DNA strand breaks; Single cell gel electrophoresis assay; Chromatid breaks; Micronuclei; Kinetochore
1. Introduction Evidence shows that arsenite induces oxidative stress and damage in mammalian cells w1–4x. Antioxidants, such as N-acetyl cysteine, glutathione ŽGSH., catalase, and squalene reduce arsenite-induced chro-
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Corresponding author. Tel.: q886-2-26523055; fax: q886-227829142. E-mail address: [email protected] ŽT.-C. Lee..
mosome aberrations, sister chromatid exchanges, and micronuclei ŽMN., and protect cells from arsenite insults w3,5–7x. In our previous study, we demonstrated that exposure of human fibroblasts ŽHFW. to arsenite increased the formation of fluorescent dichlorofluorescein by oxidation of its nonfluorescent form and modulated the cellular antioxidant defense activities in HFW, such as increasing GSH, superoxide dismutase, heme oxygenase ŽHO.-1, and ferritin, but decreasing catalase w8x. Arsenite-enhanced dichlorofluorescein fluorescence could be
0027-5107r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 0 0 . 0 0 0 3 5 - X
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I.-C. Ho et al.r Mutation Research 452 (2000) 41–50
abolished by an antioxidant, butylated hydroxytoluene w8x. These results indicate that arsenite-induced oxidative damage plays certain roles in arsenite genotoxicity. HO is the rate-limiting enzyme in the oxidative degradation of heme to bile pigments and carbon monoxide. Since the heme molecule is the prosthetic moiety of hemeproteins involved in cell respiration, energy generation, oxidative biotransformation, and the growth differentiation process, HO apparently performs vital roles in maintaining cellular homeostasis w9,10x. Two isoforms of HO have been identified: HO-1 and HO-2 w9x. HO-2 is the noninducible form found primarily in the central nervous system, whereas HO-1, an inducible isoform of HO, is not only induced by its substrate, heme molecules, but is also remarkably induced by a variety of stress-associated agents, including tumor-promoting phorbol esters, UV irradiation, hyperthermia, and heavy metals such as arsenite and cadmium w11–14x. Currently, the induction of HO-1 is generally thought to be a landmark of oxidative stress w10,11,15x. This point of view is supported by numerous studies that showed anti-oxidative activities of HO-1 in a variety of in vitro and in vivo systems w16–19x. However, several studies have shown that HO-1 expression did not protect cells against oxidative injury w20–22x. In HO-1 knock-out mice, genetic evidence clearly demonstrated that HO-1 was required for iron recycling w23x. Since iron is a potent oxidant, iron liberation accelerates the generation of reactive oxygen radicals and damages cellular macromolecules. The detection of HO-1 expression in atherosclerotic lesions also implied the pathogenicity of HO-1 w24x. Thus, the functional significance of HO-1 induction following oxidative stress is still not fully understood. Arsenite is a well-known HO-1 inducer. However, evidence showing the protective role of HO-1 against arsenite-induced genetic damage remains inadequate. To explore this issue, we treated HFW with a combination with arsenite and tin-protophorphyrin ŽSnPP., a potent inhibitor of HO-1 w25x, and examined whether SnPP could potentiate arsenite-induced genetic damage such as DNA strand breaks, chromosome aberrations, and MN. In this report, we found that SnPP could potentiate arsenite-induced DNA strand breaks, chromatid
breaks, and MN in HFW cells, indicating that HO-1 induction is not only a response to arsenite-induced oxidative stress but that HO-1 induction also participates in decreasing arsenite-induced genetic injury.
2. Materials and methods 2.1. Chemicals and cell culture Sodium arsenite ŽNaAsO 2 ., a trivalent arsenic compound obtained from E. Merck ŽDarmstadt, Germany., was used throughout the experiments. Media and chemicals used for cell culture were purchased from GIBCO ŽGrand Island, NY.. Fetal bovine serum ŽFBS. was obtained from HyClone Laboratories ŽLogan, UT.. Tin-protoporphyrin ŽSnPP. was purchased from Porphyrin Products ŽLogan, UT.. HFW, derived from the foreskin of a newborn Chinese infant, were cultured in Dulbecco’s modified Eagle medium Žhigh glucose, 430-2800EG. supplemented with 10% FBS, and antibiotics Ž100 unitsrml penicillin, 100 mgrml streptomycin. w26x. KB cells, derived from a human oral epidermal carcinoma, were obtained from American Type Culture Collection ŽRockville, MD. and grown in RPMI 1640 medium supplemented with 10% FBS and antibiotics Ž100 unitsrml penicillin, 100 mgrml streptomycin. w27x. 2.2. Assay of cell surÕiÕal Colony forming assay was used to determine the cytotoxicity of arsenite to HFW and KB cells. Cells were plated at a density of 3–5 = 10 5 cells per 60-mm dish. After 24-h incubation, cells were treated with drugs Žarsenite, SnPP, or arsenite plus SnPP. for 24 h, and then trypsinized and replated at 200– 1000 cells per 100-mm dish in triplicate. HFW cells were incubated for 14 days with one medium change, while KB cells were incubated for 10 days without medium change. Colonies were stained with a 1% crystal violet solution and counted under a dissecting microscope w7x. The colony forming efficiency of control HFW and KB cells were 53 " 12% and 64 " 3%, respectively.
I.-C. Ho et al.r Mutation Research 452 (2000) 41–50
2.3. Western blotting analysis of HO The expression of HO-1 was analyzed by Western blotting technique w28x. Logarithmically growing cells were treated with drugs as described above. At the end of drug treatment, the cells were scraped from culture dishes with a rubber policeman, and prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described previously w26x. An aliquot of 10 mg cellular proteins was electrophoretically separated on a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane by a semi-dry electrotransfer system ŽATTO, Japan.. The membranes were immunoblotted with antibodies aganist rabbit HO-1 ŽStressGen Biotechnologies, Victoria, B.C., Canada. or the carboxy terminus of human actin ŽSanta Cruz Biotechnology, Santa Cruz, CA., and then visualized by an enhanced chemilluminescent technique according to the procedure provided by the manufacturer ŽPierce Chemical, Rockford, IL.. 2.4. Assay of DNA strand breaks The single cell gel electrophoresis ŽSCGE. assay or ‘Comet assay’ was used to monitor DNA strand breaks. Immediately after drug treatment, an aliquot of 1 = 10 5 HFW cells was subjected to alkaline SCGE according to the method described by Lynn et al. w29x. The slides were then stained with Sybr green ŽMolecular Probes, Eugene, OR.. Under a fluorescence microscope, DNA tail or comet tail examination was performed by the method described by Miyamae et al. w30x with slight modification. Accordingly, DNA comet tails were classified into four types: type I, without significant tail; type II, with short tail Žtail length less than head diameter.; type III, with long tail Žtail length greater than head diameter.; type IV, with small head. For each treatment, at least 500 cells were examined under a fluorescent microscope with naked eyes. 2.5. Assay of chromosome aberrations Logarithmically, growing HFW cells were treated with arsenite, SnPP, or arsenite plus SnPP for 24 h. Colcemid at the concentration of 0.05 mgrml was added 3 h prior to the end of treatment. The cells
43
were subsequently trypsinized, treated with hypotonic solution, fixed, and prepared for chromosome analysis as previously described w26x. Chromosome aberrations were identified by following the criteria described by Buckton and Evans w31x. For each treatment, 50 metaphases were examined for the frequency of various types of chromosome aberrations. 2.6. Assay of MN The method of cytokinesis-block MN assay w32x with slight modification w7x was adopted to analyze the effect of SnPP on arsenite-induced MN. In brief, subsequent to drug treatment Žas described above., HFW cells were further incubated with 1 mgrml cytochalasin B, a cytokinesis inhibitor, for 24 h. Afterwards, cells were trypsinized and an aliquot of cell suspension was cytospun onto a clean slide. The slides were fixed with methanol at y208C for 10 min, air-dried, and stored at y208C in the dark. Immunostaining with CREST anti-kinetochore antibody ŽAntibody, Davis, CA. and fluroescein-conjugated secondary antibody ŽSigma, St. Louis, MO. was performed as previously described w7x. Meanwhile, chromosomes were counterstained with 0.1 mgrml 4,6-diamino-2-phenyl-indole. The frequency of MN with kinetochore ŽKq-MN. or without kinetochore ŽKy-MN. was scored from at least 500 binucleated cells under a fluorescence microscope.
3. Results 3.1. Enhancement of arsenite cytotoxicity by SnPP to HFW According to clonogenic assay, treatment with NaAsO 2 for 24 h resulted in a dose-dependent killing effect to HFW cells ŽFig. 1A.. Western blotting analysis indicated that a significant amount of HO-1 was induced and accumulated in HFW cells treated with arsenite at concentrations above 1.25 mM ŽFig. 1B.. To understand whether the induction of HO-1 played a protective role on arsenite cytotoxicity, HFW cells were treated in combination with 1.25 mM arsenite and various concentrations of SnPP. As
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with a combination of 5 mM arsenite and various concentrations of SnPP ŽFig. 2B.. When HFW cells were pretreated with hemin, a HO-1 inducer, HO-1 accumulation was clearly demonstrated in HFW cells by Western blotting technique ŽFig. 3A.. When hemin-pretreated HFW cells were subsequently challenged with arsenite, arsenite’s cytotoxicity was significantly reduced Ž p - 0.01, according to two-way ANOVA analysis; Fig. 3B.. 3.2. Enhancement of arsenite-induced DNA strand breaks by SnPP Fig. 1. Cytotoxicity and HO-1 induction by sodium arsenite in HFW. ŽA. HFW cells were treated with various concentrations of sodium arsenite for 24 h, and then replated to assess the survival rate by colony-forming assay as described in Section 2. Bars, SD of three independent experiments. ŽB. After a 24-h treatment with sodium arsenite, the cellular proteins were electrophoretically separated, immunoblotted with antibody against to HO-1 or actin, and then visualized by an enhanced chemilluminescent technique as described in Section 2.
shown in Fig. 2A, SnPP alone at the concentrations up to 100 mM did not show a significant killing effect to HFW cells, while SnPP dose-dependently enhanced the cytotoxicity of arsenite at 1.25 mM Ž p - 0.01, according to two-way ANOVA analysis.. Similar findings were observed in KB cells treated
Fig. 2. Enhancement of sodium arsenite cytotoxicity by SnPP to HFW and KB cells. HFW ŽA. or KB ŽB. cells were cotreated with various concentrations of SnPP and 1.25 or 5 mM sodium arsenite, respectively, for 24 h. At the end of treatment, the survival rate was determined by colony forming assay as described in Section 2. Bars, SD of three to four independent experiments. `, SnPP alone; v, arsenite plus SnPP.
SCGE assay is a sensitive technique to detect DNA strand breaks. The tail size reflects the levels of DNA strand breaks. Accordingly, we classified the DNA comet tails observed in our experiments into four types as illustrated in Fig. 4A. As reported by Lynn et al. w29,33x, SCGE assay was able to demonstrate the induction of DNA strand breaks by arsenite in Chinese hamster ovary and bovine aortic endothelial cells, respectively. Similarly, in the present study, arsenite treatment caused DNA strand breaks in HFW cells, i.e. type II and III DNA comet
Fig. 3. Induction of HO-1 and protection of HFW cells from the killing effects of sodium arsenite by hemin pretreatment. HFW cells were pretreated with 50 mM hemin for 24 h. ŽA. HO-1 expression was examined by Western blotting analysis as described in Section 2. Actin served as an internal control. ŽB. The hemin-pretreated HFW cells were further treated with various concentrations of sodium arsenite for 24 h, and then the survival rate was determined by colony forming assay described in Section 2. Bars, SD of three independent experiments. `, without hemin pretreatment; v, with hemin pretreatment.
I.-C. Ho et al.r Mutation Research 452 (2000) 41–50
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Fig. 4. Enhancement of sodium arsenite-induced DNA strand breaks by SnPP in HFW cells. HFW cells were cotreated with 100 mM SnPP and various concentrations of sodium arsenite for 24 h. DNA strand breaks were analyzed by Comet assay as described in Section 2. ŽA. The classification of comets by shape. ŽB. The incidence of each Comet type. Bars, SD of three to four independent experiments. p-values are the statistical comparison by the Mann–Whitney rank sum test between the treatments with and without SnPP.
tails were significantly increased by arsenite treatment ŽFig. 4B.. If comet tail types I–IV were assigned numerical values of 0–3, respectively, the averaged numerical values for 0, 1.25, 2.5 and 5 mM arsenite treatment were calculated to be 0.105 " 0.030, 0.386 " 0.060, 0.583 " 0.064, 0.909 " 0.024, respectively. These results indicated that arsenite dose-dependently induced DNA strand breaks in HFW cells Ž p - 0.001, according to ANOVA analysis.. The mean levels of comet tails in cells treated with 0–5 mM arsenite plus 100 mM SnPP were 0.152 " 0.029, 0.799 " 0.020, 1.006 " 0.081, 1.164 " 0.030, respectively. By the Mann–Whitney rank sum test, SnPP alone at 100 mM did not induce DNA strand breaks, but significantly potentiated arsenite-induced DNA strand breaks at all three doses of arsenite used Ž p values shown in Fig. 4B.. To exclude the formation of comet tails was originated from arsenite-induced dead or apoptotic cells, we have examined the cell status using dye exclusion
and DNA fluorescent dye staining techniques. Immediately after drug treatment, the trypan blue-stained cells were less than - 4% and no apoptotic cell could be found. Therefore, the comet tails observed were unlikely attributed to the dead or apoptotic cells. 3.3. Enhancement of arsenite-induced chromosome aberrations by SnPP Consistent with our previous study w26x, chromatid breaks were the major type of chromosome aberrations induced by arsenite in HFW cells. Arsenite at the doses of 2.5 mM and above induced a significant number of chromatid breaks ŽFig. 5.. However, arsenite at the concentration of 1.25 showed no significant induction of chromatid breaks Ž p s 0.114 according to Mann–Whitney rank sum test., indicating that a threshold might be required
I.-C. Ho et al.r Mutation Research 452 (2000) 41–50
46
Fig. 5. Enhancement of sodium arsenite-induced chromatid breaks by SnPP in HFW cells. HFW cells were cotreated with 100 mM SnPP and various concentrations of sodium arsenite for 24 h. Chromosome analysis was performed as described in Section 2. Bars, SD of four independent experiments. `, arsenite alone; v, arsenite plus SnPP.
for chromatid break induction in HFW cells treated with arsenite. In contrast, cotreatment of HFW cells with various concentrations of arsenite and 100 mM SnPP resulted in a linear increase in chromatid breaks Ž p - 0.001, according to ANOVA analysis; Fig. 5.. A strong interaction between arsenite and SnPP was also observed Ž p - 0.001 according to two-way ANOVA analysis., suggesting that chromatid breaks induced by arsenite were significantly potentiated by SnPP in HFW cells. At the time of sampling for chromosome aberration analysis, the cell number was slightly increased to approximately 114% of
untreated cultures at the dose of 1.25 mM but decreased to 96% and 72% at 2.5 and 5 mM, respectively. In the cotreated cultures Žvarious oncentrations of arsenite plus 100 mM SnPP., the cell numbers were further decreased 10% in average as compared to the cultures treated with arsenite alone. Thus, under our experimental conditions, arsenite at the doses used did not seriously suppress the cell growth and hence distorted the analysis of chromosome aberrations.
3.4. Enhancement of arsenite-induced MN by SnPP The cytokinesis-block MN technique was used to confirm the potentiation effects of SnPP on arseniteinduced chromosomal damage. In our previous study, we demonstrated that treatment of HFW cells with arsenite at the same dose range used in this study mainly induced Kq-MN MN Ž70% of total MN. w7x. To determine whether SnPP cotreatment could alter the types of MN induced by arsenite, immunostaining with CREST anti-kinetochore antibody was performed to differentiate Kq-MN and Ky-MN. Consistent with our previous study results w7x, arsenite at the dose range and protocol used mainly induced Kq-MN in HFW cells ŽTable 1.. Arsenite at 5 mM also induced a significant amount of Ky-MN. SnPP synergistically enhanced the total number of
Table 1 Effects of SnPP on arsenite-induced MN a Treatment ŽmM. Sodium arsenite
SnPP
No. of binucleated cells per 1000 cells
MNr1000 binucleated cells Total Kq
Ky
0 1.25 2.5 5 0 1.25 2.5 5
0 0 0 0 100 100 100 100
401 453 347 320 365 364 361 315
9 "5 35 " 5 62 " 9 67 " 13 15 " 5 66 " 8 Ž41. ) 68 " 14 Ž68. 85 " 13 Ž73.
6 "4 9 "2 10 " 6 21 " 8 8 "2 49 " 9 Ž11. ) ) ) 53 " 10 Ž12. ) ) ) 70 " 15 Ž23. ) ) )
3 "2 25 " 4 52 " 7 46 " 6 7 "4 17 " 2 Ž29. 14 " 7 Ž56. ) ) ) 15 " 9 Ž50. ) ) )
Numbers in parentheses are the expected values calculated as the summation of the values of MN induced by single treatment with arsenite and SnPP minus the value of no treatment w60x. Asterisks indicate significant difference between the observed and expected values. a Data are averages" SD of three independent experiments. ) p - 0.05 according to x 2 analysis. ))) p - 0.001 according to x 2 analysis.
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MN production by arsenite only at the dose of 1.25 mM Ž p - 0.05 according to x 2 analysis, Table 1.. However, only Ky-MN was enhanced by SnPP. Arsenite at higher doses, such as 2.5 or 5 mM, induced a large number of MN; SnPP cotreatment did not further increase the frequency of total MN but resulted in a significant increase in Ky-MN Ž3–4.4-fold to the expected values. and a significant decrease in Kq-MN ŽTable 1., i.e. Ky-MN instead of Kq-MN became the major type of MN in HFW treated with arsenite plus SnPP. Although there was no evidence showing the interconversion Kq-MN to Ky-MN and Kq-MN, the present study still suggested that HO-1 played a role in preventing from the formation of Ky-MN that was mainly derived from chromosome fragments. According to the binucleate indexes ŽTable 1., our experimental conditions did not severely suppress the cell growth.
4. Discussion Inorganic arsenic is a well documented human carcinogen w34x. In the present study, the induction of chromatid breaks and MN by arsenite in HFW cells is consistent with our previous studies w7,26x. Using Comet assay, induction of DNA strand breaks by inorganic arsenite has been shown in human lymphocytes and bovine aortic endothelial cells w29,35,36x. Our results further confirm that inorganic arsenite causes DNA strand breaks in HFW cells. Furthermore, the present results also show that arsenite-induced DNA strand breaks, chromatid breaks, and MN are significantly potentiated by cotreatment with SnPP. Since SnPP is a competitive inhibitor of HO-1, these results suggest that HO-1 plays certain roles in reducing arsenite-induced genetic injury. Since our unpublished data showed that SnPP had no effect on the uptake of arsenite, we still could not rule out the possibility that the potentiation effects of SnPP are due to its unknown functions. Although how inorganic arsenite induces genetic injury is not fully elucidated, oxidative damage is likely involved in arsenite-induced DNA strand breaks, chromatid breaks, MN, and even in apoptosis in a variety of cell systems w4,7,29,37x. Several different mechanisms have been reported to generate oxidative stress by arsenite. Arsenite-induced DNA
47
strand breaks and MN in Chinese hamster ovary cells were reported to be mediated through the generation of nitric oxide w33,37x. Reactive oxygen intermediates were shown to be involved in the generation of DNA strand breaks by dimethylarsenic acid w38x. In HFW cells, we previously demonstrated that reactive oxygen radicals were enhanced by arsenite in HFW cells w8x. In contrast to Chinese hamster ovary cells, arsenite treatment apparently did not enhance nitric oxide production in HFW cells, since we could not detect the production of nitrite Žan indicator for estimation of nitric oxide production. in cultural medium of HFW cells treated with arsenite Ždata not shown.. Therefore, the protective roles of HO-1 from arsenite-induced genetic injury may be attributed to HO-1’s antioxidant activity as has been shown in a variety of in vitro and in vivo systems w16–19x. Although inhibition of HO-1 apparently potentiated the genetic injury caused by arsenite, the levels of HO-1 expression remained positively associated with the increase of cytotoxicity, DNA strand breaks, chromatid breaks, and MN in arsenite-treated HFW cells. Pre-induction of HO-1 by hemin only partially protected HFW cells from the cytotoxic effects of arsenite. These results indicate that arsenite’s toxic effects are pleiotropic. The toxic effects of arsenite have generally been considered to result from its strong interaction with sulfhydryl groups, particularly vicinyl–sulfhydryl groups, of functional molecules, such as cytoskeleton molecules and ubiquitin conjugation enzymes w39–42x. Arsenite treatment may also alter cellular functions by changing phosphorylation profiles of cellular proteins w43,44x. In addition, several other mechanisms of arsenite genotoxicity and carcinogenicity, such as modulation of the DNA methylation status w45,46x and inhibition of DNA repair enzymes w33,47–49x, have also been well noticed in the literature. Therefore, the protective function of HO-1 is only partial and oxidative stress is only one of the mechanisms involved in arsenite genotoxicity. By using MN assay and chromosome specific probes, arsenite has been shown to have both aneugenic and clastogenic properties in lymphocytes and exfoliated epithelial cells w50–53x. Although the mechanisms by which arsenite exerts its cytogenetic effects remain unclear, we have previously demon-
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strated that the aneugenic or clastogenic manifestation of arsenite was associated with different treatment protocols w7x, e.g. arsenite at a low dose range Ž1.25–10 mM for 24 h. mainly induced Kq-MN, whereas at a high dose range Ž5–80 mM for 4 h. mainly induced Ky-MN. Consistent with this previous study, the protocol used in the present study for arsenite treatment mainly resulted in Kq-MN. Although our previous results suggested that oxidative stress was involved in the generation of Kq-MN and Ky-MN in arsenite treated HFW cells w7x, we found that SnPP changes the patterns of MN types induced by arsenite, i.e. with Kq-MN decreases and Ky-MN increases. Different mechanisms are involved in the generation of these two types of MN w54x. In general, Kq-MN derived from whole chromosomes is mainly produced by interference with mitosis, while Ky-MN derived from chromosome fragments is mainly formed from DNA breaks. The increase of Ky-MN by SnPP cotreatment may be due to the increase of DNA and chromatid breaks. However, the reason why SnPP cotreatment reduces the formation of Kq-MN is not clear. We suspect that HO-1 induction interferes with the homeostasis of cellular hemeproteins and hence influences chromosome segregation. Further investigation is required for clearer understanding of the vital roles of HO induction. The curve of chromatid break induction by arsenite alone ŽFig. 5. showed a slightly upward concave, indicating that a threshold might be required for chromatid break induction in HFW cells treated with arsenite. As reviewed by Rudel et al. w55x, sublinear dose–response relationships for arsenic-induced chromosome aberrations were repeatedly established in a variety of cell systems. However, no significant threshold was observed in arsenite-induced cytotoxicity, MN, and DNA strand breaks in HFW cells. In fact, the shapes of dose–response to arsenite were usually dependent on cell types used w1x, methods for genetic damage assay w55x, and the protocols of drug treatment, such as the levels of doses and the length of drug exposure w7x. Although oxidative stress has been recently considered to play certain roles in arsenic-induced genetic damages, how oxidative stress leads to various endpoints of genetic damages, such as DNA strand breaks, MN, sister chromatid exchanges, and chromosome aberrations, is not clear.
Furthermore, the dose–response relationships for arsenic-induced genetic damages are seriously affected by the cellular antioxidant molecules. Further investigation is essentially required for establishment of an appropriate dose–response model for assessing the human health risks of arsenic exposure. In conclusion, arsenite-induced DNA strand breaks, chromatid breaks, and Ky-MN were significantly potentiated by SnPP, suggesting that HO-1 plays antioxidant roles to prevent cells from arsenite-induced oxidative stress. Our results also confirm the involvement of oxidative stress in arsenite-induced genetic injury. Over generation of oxygen free radicals is considered to play a role in the development of several chronic diseases such as cancer, aging, inflammatory disorders, etc. w56–58x. Oxidative stress was demonstrated to induce the expression of numerous genes involved in the restoration of cellular homeostasis w59x. Since HO-1 is one of important antioxidant defense enzyme and performs vital roles in maintaining cellular homeostasis w9,10,18,19x, the physiological functions of HO-1 induction in arsenite exposed cells warrant our further investigation.
Acknowledgements We thank Mr. Douglas J. Platt for carefully reading this progress report. This work was supported by grants from Academia Sinica and from the National Science Council ŽNSC 87-2314-B001-009 and NSC 88-2314-B001-003., Republic of China.
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