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Sensitivity to microcystins: A comparative study in human cell lines with and without multidrug resistance phenotype
Cell Biology International 31 (2007) 1359e1366 www.elsevier.com/locate/cellbi
Sensitivity to microcystins: A comparative study in human cell lines with and without multidrug resistance phenotype Ana Paula de Souza Votto a,b, Viviane Plasse Renon a, Jo~ao Sarkis Yunes c, Vivian Mary Rumjanek d, Ma´rcia Alves Marques Capella e, Vivaldo Moura Neto f, Marta Sampaio de Freitas g, Laura Alicia Geracitano a,b, Jose´ Marı´a Monserrat a,b, Gilma Santos Trindade a,b,* a
Departamento de Cieˆncias Fisiolo´gicas, Fundac¸~ao Universidade Federal do Rio Grande (FURG), Rio Grande 96201-900, Brazil b Programa de Po´s-graduac¸~ao em Cieˆncias Fisiolo´gicas, Fisiologia Animal Comparada, FURG, Rio Grande 96201-900, Brazil c Departamento de Quı´mica, Unidade de Pesquisa em Cianobacte´rias, FURG, Rio Grande 96201-900, Brazil d Laborato´rio de Imunologia Tumoral, Departamento de Bioquı´mica Me´dica, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21941-590, Brazil e Laborato´rio de Fisiologia Renal, Instituto de Biofı´sica Carlos Chagas Filho, UFRJ, Rio de Janeiro 21941-590, Brazil f Instituto de Cieˆncias Biome´dicas, Departamento de Anatomia, UFRJ, Rio de Janeiro 21941-590, Brazil g Centro Biome´dico, Departamento de Farmacologia e Psicobiologia, Universidade do Estado do Rio de Janeiro (UEFRJ), Rio de Janeiro 20551-030, Brazil Received 29 March 2006; revised 5 April 2007; accepted 12 May 2007
Abstract Multidrug resistance (MDR) is an obstacle in cancer treatment. An understanding of how tumoral cells react to oxidants can help us elucidate the cellular mechanism involved in resistance. Microcystins are cyanobacteria hepatotoxins known to generate oxidative stress. The aim of this study was to compare the sensitivity to microcystins of human tumoral cell lines with (Lucena) and without (K562) MDR phenotype. Endpoints analyzed were effective microcystins concentration to 50% of exposed cells (EC50), antioxidant enzyme activity, lipid peroxidation, DNA damage, reactive oxygen species (ROS) concentration, and tubulin content. Lucena were more resistant and showed lower DNA damage than K562 cells (P < 0.05). Although microcystins did not alter catalase activity, a higher mean value was observed in Lucena than in K562 cells. Lucena cells also showed lower ROS concentration and higher tubulin content. The higher metabolism associated with the MDR phenotype should increase ROS concentration and make for an improved antioxidant defense against the toxic effects of microcystins. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: MDR phenotype; Microcystins; Oxidative stress; DNA damage
1. Introduction Multidrug resistance (MDR) is a phenomenon by which tumors that initially respond to determined chemotherapy, acquire resistance to chemically and non-chemically related * Corresponding author. Departamento de Cieˆncias Fisiolo´gicas, Fundac¸~ao Universidade Federal do Rio Grande (FURG), Campus Carreiros, Avenida Italia, km 8, Rio Grande 96201-900, RS, Brazil. Tel.: þ55 53 3233 6855; fax: þ55 53 3233 6850. E-mail address: [email protected] (G.S. Trindade).
drugs. The best understood mechanism of MDR is the one conferred by the membrane P-glycoprotein (Pgp), which acts by pumping several unrelated drugs out from the cells (Gottesman and Pastan, 1993). Despite the resistance process to be multifactorial, MDR phenotype exhibits some main characteristics: (1) resistance to non-related drugs (Kartner and Ling, 1989; Tiirikainen and Krusius, 1991), (2) expression of protein as the P-glycoprotein (Pgp) (Gottesman and Pastan, 1993), (3) extrusion of rhodamine dye (Neyfakh, 1988), and (4) reversion of the resistance induced by agents like trifluoperazine, verapamil and cyclosporin A (Ford and Hait, 1990; Sikic, 1993).
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.05.010
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Rumjanek et al. (1994, 2001) established a leukemic MDR cell line by using the method of Tsuruo et al. (1983), and called K562-Lucena1 (Lucena). It has already been shown that Lucena cells possess the characteristics mentioned above (Maia et al., 1996a,b; Marques-Silva, 1996; Orind et al., 1997). The reactive oxygen species (ROS) are also supposed to be involved in the cytotoxicity of some anticancer agents, and it has been suggested that multidrug-resistant (MDR) cells might also be resistant to oxidative stress (Capella et al., 2001; Trindade et al., 1999, 2000). Microcystins are toxins produced by the cyanobacterium Microcystis aeruginosa, among several other species (Mackintosh et al., 1990). These toxins are cyclic heptapeptides presenting a general chemical structure of D-Ala-X-D-erythrob-methylAsp-Z-Adda-D-Glu-N-methyldehydroAla, where positions X and Z represent variable L-amino acids, which generate a family of different molecular forms of microcystins (Pflugmacher et al., 1998). Microcystin-LR is the most common cyanotoxin with a leucine (L) and an arginine (R) in X and Z positions, respectively (Mackintosh et al., 1990; Dawson, 1998.). Pouria et al. (1998) found that microcystins provoked the death of several dialysis patients after acute hepatic failure, due to cyanobacteria contamination of water used during this treatment. In Southern Brazil, cyanobacteria blooms, dominated by Microcystis aeruginosa, have allegedly given microcystin concentrations as high as 244.8 mg/l in estuarine waters of Patos Lagoon (Yunes et al., 1996; Matthiensen et al., 1999). Microcystins are potent inhibitors of serine/threonine phosphatases PP1 and PP2A, leading to an increase of protein phosphorylation, inducing changes in the control of cell growth and differentiation, resulting in tumor promotion after prolonged exposure to sub-acute doses (Eriksson et al., 1990; Mackintosh et al., 1990; Falconer and Yeung, 1992; Mackintosh and Mackintosh, 1994; Runnegar et al., 1995a; Toivola and Eriksson, 1999). The hepatotoxicity of this toxin is linked to the uptake mechanism in hepatocytes by biliary acid transporters (Runnegar et al., 1995b). According to these authors, the liver is the primary microcystin target because of its specific transport mechanism, which enables the uptake of microcystin into hepatocytes. However, Fischer et al. (2005) recently reported that members of the organic anion transporting polypeptide super family are involved in microcystins uptake, including the human OATP1A2 transporter that is expressed both in liver and brain. So, this finding opens the possibility that targets for microcystins are not only liver cells but also brain cells. In this context, it is important to stress that, although several in vitro studies have been conducted with primary cultures of hepatocytes, some authors have already observed microcystins cytotoxicity in others cell lines; for example, Zhan et al. (2004) reported genotoxicity of these toxins, inducing micronucleus formation as well as gene mutations in human lymphoblastoid TK6. Most of the physiological effects observed during microcystins exposure are probably secondary to the phosphatases proteins inhibition (Mikhailov et al., 2003). Runnegar et al. (1995b) concluded that microcystinstoxicity is a consequence
of irreversible phosphatase inhibition, although they did not exclude the possibility that other mechanisms might be involved. Some authors have considered oxidative stress generation as another possible toxicity mechanism. Ding et al. (1998) reported that extracts from M. aeruginosa were able to induce oxidative cellular damage in hepatocytes of rats, manifested by high levels of lipid peroxides and reactive oxygen species (ROS). Guzman and Solter (1999) verified hepatic oxidative damage after microcystin-LR exposure evidenced by an increase of malonaldehyde levels, a lipid peroxidation byproduct. Microcystins are also able to induce nuclear DNA damage in human hepatoma cell line HepG2, an effect involving ROS generation (Zegura et al., 2003). Pinho et al. (2003) demonstrated that the hepatopancreas of crabs exposed to microcystins exhibited higher GST and CAT activity than that observed in control crabs at the same experimental time, indicating that the antioxidant defense system was activated after exposure to these cyanotoxins. Considering that microcystins may interfere in antioxidant enzyme activities and induce lipid peroxidation, and that MDR cells are resistant to oxidative stress, the aim of this study was to compare the sensitivity of Lucena, a MDR cell line, and of K562, its non MDR parental cell line, to these toxins. 2. Materials and methods 2.1. Cells and culture conditions K562 and Lucena cells were obtained from the Tumoral Immunology Laboratory at the Medical Biochemistry Department of the Rio de Janeiro Federal University, Brazil. Parental K562 cells were grown in RPMI 1640 (Gibco) medium supplemented with sodium bicarbonate (0.2 g/l) (Vetec), L-glutamine (0.3 g/l) (Vetec), Hepes (25 mM) (Acros) and b-mercaptoethanol (5 105 M) (Sigma), with 10% fetal bovine serum (Gibco), 1% of antibiotic (penicillin (100 U/ml) and streptomycin (100 mg/ml) (Gibco) and antimicotic (0.25 mg/ml) (Sigma), in disposable plastic flasks, at 37 C. The MDR Lucena cells were grown under the same conditions above with 60 nM vincristine (VCR) (Sigma) in order to preserve the MDR phenotype.
2.2. Preparation of Microcystis aeruginosa extracts Cells of Microcystis aeruginosa from strain RST 9501 were cultured in BG11 ( 8.82 mM of NaNO3) medium (Rippka et al., 1979) at 25 1 C and employed as toxin source. Characterization of microcystins produced by the strain RST 9501 was done by Matthiensen et al. (1999, 2000). These authors found that methanolic extracts of strain RST 9501 presented one major peak and three minor peaks with UV spectra characteristic of microcystins, with a lmax of 238e239 nm. Interestingly, the retention time of the major peak (15.22 min) was different to that of microcystin-LR. Further analysis confirmed that the most abundant microcystin produced by strain RST 9501 is a [D-Leu1]-microcystin-LR, a variant with a similar potency in terms of phosphatases inhibition respect the common [D-Ala1]-microcystin-LR. The extract was prepared using lyophilized cells of Microcystis aeruginosa and the protocol of Coyle and Lawton (1996). Briefly, the cells were dissolved in absolute methanol (Sigma), sonicated 3 times and centrifuged (10,000 g) at 4 C, during 10 min. Extracts were evaporated at 40 C and then re-dissolved in ultra-pure water. Finally, samples were centrifuged and the supernatant was collected and stored at 20 C. Microcystins content was determined using a commercial enzyme-linked immunoassay (ELISA) with polyclonal antibodies (EnviroLogix Inc., Portland, ME, USA). Different concentrations of microcystin were prepared after appropriate dilutions with saline phosphate buffer (Ca2þ and Mg2þ free) (PBS). Taking into account
A.P.S. Votto et al. / Cell Biology International 31 (2007) 1359e1366 the streaking difference in microcystin sensitivity between the 2 lines (see Section 3), additional experiments were perform to characterize K562 and Lucena lines, in terms of a- and b-tubulins content (see Section 2.7).
2.3. Microcystins exposure The cells were grown for 2 days (K562) or 3 days (Lucena) before the beginning of experimentation (Trindade et al., 1999). For each experiment, cells were centrifuged, washed with PBS and suspended in medium without b-mercaptoethanol at 2 105 cells/ml. The cells were treated in medium with different concentrations of microcystins (0.2, 0.4, 0.8 or 1.6 mg/ml) plus a control group that received the same volume of PBS without toxins. The cells were incubated at 37 C in 24-well culture plates. During the experiments, no VCR was added to Lucena cell cultures.
2.4. Assessment of the sensitivity of the two cell lines to microcystins Cell viability was assessed by trypan blue (Gibco) exclusion after 0, 24 and 48 h exposure. In some instances (24 h exposure to microcystins), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium (MTT; Sigma) assay was also used, according to Trindade et al. (1999). The EC50 (concentration at which viability was reduced 50%) was estimated for the 2 cell lines after 24 h and 48 h of exposure for cell viability assessed by trypan blue exclusion and after 24 h for MTT assay. The concentration (0.2 mg/ml) that presented no significant difference in viability respect to the control (cell line K562) after 24 h of exposure was employed in posterior tests (see Results).
2.5. Effects of microcystins in activity of antioxidants enzymes and oxidative effects For preparation of each sample for enzyme activity and lipid peroxidation (LPO) determinations, one pool of 3 wells of the culture plates of each treatment (control cells and cells treated with 0.2 mg/ml of microcystin during 24 h) were centrifuged at 250 g for 2 min at room temperature to obtain a pellet with 6.0 105 cells/ml. For enzyme activity determinations, the homogenates were prepared in cold (4 C) buffer solution containing 20 mM Tris-Base (Gibco), 1 mM EDTA (Gibco), 1 mM dithiothreitol (Sigma), 500 mM sucrose (Sigma), 150 mM KCl (Gibco), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), pH 7.60. Samples were centrifuged at 9000 g for 30 min at 4 C. The supernatant of each sample was stored at 80 C until enzyme activity was determined. Total protein content in the homogenate was measured using a commercial reagent kit (Doles Reagentes Ltda.), which is based on the Biuret protein assay. Both enzymatic and protein content determinations were performed at least in duplicate. Catalase (CAT) activity was determined by the method of Bleuter (1975). Enzyme activity was expressed in CAT units, where one unit is the amount of enzyme needed to hydrolyze 1 mmol of H2O2 (Merck) per minute, and normalized by being expressed per mg of total proteins present in the homogenate at 30 C and pH 8.00. Glutathione-S-transferase (GST) activity was determined according to Habig et al. (1974), and Habig and Jakoby (1981). Enzyme activity values were expressed in GST units, where one unit is the amount of enzyme necessary to conjugate 1 mmol of 1-chloro-2,4-dinitrobenzene (CDNB; Sigma) per min and expressed per mg of total protein present in the homogenate, at 30 C and pH 7.0. Lipid peroxidation (LPO) was determined according to Hermes-Lima et al. (1995). Samples were homogenized (10% W/V) in 100% cold (4 C) methanol. The homogenate was centrifuged at 1000 g, for 10 min at 4 C. The supernatant was used for LPO determinations (580 nm). Cumene hydroperoxide (CHP; Sigma) was employed as standard. DNA damage was evaluated by the alkaline single cell electrophoresis (comet) assay, performed as described by Singh et al. (1988) and Tice et al. (2000), with some modifications. An aliquot (50 ml) of cell suspension of
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each sample (2 105 cell/ml) was mixed with 30 ml of 1.73% low melting point agarose (BMA) and added to fully frozen slides which had been covered with a layer of 0.65% normal melting point agarose (BMA). Following the layer solidification, cells in slides were lysed [2.5 M NaOH (Sigma), 0.1 M EDTA, 0.01 M Tris (Gibco), 1% sodium sarcosinate (Fisher Scientific), 1% Triton X-100 (Aldrich), and 10% dimethyl sulfoxide (Aldrich), pH 10.0] overnight at 4 C. Subsequently, samples were placed in the electrophoresis solution (300 mM NaOH and 1 mM EDTA, pH 13) for 35 min to allow DNA unwinding. Then, electrophoresis was performed during 25 min at 25 V and 280 mA. Finally, the slides were neutralized with 0.4 M Tris buffer (pH 7.5) (Gibco), stained with 95 ml of ethidium bromide (20 mg/ml) (Sigma) and analyzed using an epifluorescence microscope Zeiss Axioplan (400 magnification). In 50 randomly selected cells in duplicated slides, DNA damage was scored on the basis of undamaged (class 0), cells presenting short migration of DNA (class 1), medium migration (class 2), long migration (class 3), and complete migration (no nucleus remaining, class 4). The final score were made multiplying the category for the nucleus number in this category, resulting score 0 for no damage and 200 for maximum damage.
2.6. Assessment of intracellular ROS formation Suspensions of both cell lines (4.0 105 cells/ml) (control cells and cells treated with 0.2 mg/ml of microcystins during 24 h) were washed twice with PBS and incubated for 30 min at 37 C with the fluorogenic compound, 20 ,70 -dichlorofluorescin diacetate (H2DCF-DA, Molecular Probes) at a final concentration of 40 mM, according to Myhre and Fonnum (2001). H2DCFDA passively diffuses through cellular membranes and, once inside, the acetates are cleaved by intracellular esterases. Thereafter, the non-fluorescent compound H2DCF is oxidized by ROS to the fluorescent compound DCF. Each treatment was performed in triplicate. After the loading with H2DCFDA, the cells were washed twice with PBS and suspended in fresh PBS. Aliquots of 160 ml of each sample (five replicates) were placed onto an ELISA plate and the fluorescence intensity determined during 90 min at 37 C, using a fluorometer (Victor 2, Perkin Elmer), with excitation and emission wavelengths of 485 and 520 nm, respectively. ROS levels were expressed in terms of fluorescence area, after fitting fluorescence data to a second order polynomial and the estimated functions integrated between 0 and 90 min in order to obtain its area.
2.7. Quantification of a- and b-tubulins content on the two cell lines For preparation of each sample for a- and b-tubulins content, a suspension of 1.0e1.5 106 cells/ml was washed 3 times with ice-cold PBS. Whole cell extracts were obtained by suspending cells in lysis buffer [50 mM MES (Sigma), pH 6.4, 1 mM MgCl2 (Sigma), 10 mM EDTA, 1 mM PMSF, 1 mM benzamidine (Sigma) and 1% Triton X-100]. Triton-insoluble and -soluble cytoskeletal fractions were obtained using the lysis buffer followed by centrifugation at 13,000 g for 10 min at 25 C. Supernatants were collected and pellets were suspended in the same buffer. Cytosolic extracts were obtained by suspending cells in modified RIPA buffer [PBS, 0.5% sodium deoxycholate (Aldrich), 1% Nonidet P-40 (Sigma), 0.1% sodium dodecyl sulfate (SDS; Sigma), 10 mg/ml PMSF, 10 mg/ml aprotinin (Sigma), 10 mg/ml leupeptin (Sigma), 1 mM benzamidine], incubating on ice for 30 min before the supernatant was collected by centrifugation at 10,000 g for 10 min. Protein content of all cell extracts were determined by Bradford’s method (Bradford, 1976). Whole extract and cytoskeletal fractions proteins were denatured with Laemmli sample buffer composed of 50 mM Tris buffer (pH 6.8), 1% SDS, 140 mM b-mercaptoethanol, 10% glycerol (Sigma), 0.001% bromophenol blue (Sigma) and by heating in a boiling water bath for 3 min (Laemmli, 1970). Protein samples (30 mg) were subjected to gel electrophoresis and immunoblotting. SDSepolyacrylamide gel electrophoresis (SDSePAGE) was performed on 10% acrylamide (Sigma) according to discontinuous system of Laemmli (1970). Afterwards, the proteins were transferred to nitrocellulose sheets (Hybond-C Extra, Amersham Biosciences) during 90 min electrophoresis (30 mA) in Triseglycine buffer [25 mM TriseHCl (Gibco), pH 7.0, 192 mM glycine (Sigma)] containing 20% methanol (Towbin et al., 1979).
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The nitrocellulose sheets were incubated with TBS [20 mM Tris-HCl, pH 7.5, 500 mM NaCl (Sigma), 0.1% Tween-20 (Gibco)] containing 5% skimmed milk followed by incubation with mouse (monoclonal) anti-a-tubulin antibody (1:1000; Sigma), or mouse (monoclonal) anti-b-tubulin antibody (1:1000; Amersham Biosciences), overnight at 4 C. After extensive washing in TBS, the nitrocellulose sheets were incubated with anti-mouse IgG antibody peroxidase-conjugated (1:5000, Gibco). Immunoreactive proteins were visualized by ECLÔ Western Blotting Kit (Amersham Biosciences) and exposed to Kodak X-Omat films. Autoradiograms were quantified by densitometry on a model GS-690 Imaging densitometer equipped with Molecular Analyst software version 1.4.1 (Bio-Rad Laboratories).
2.8. Statistical analysis In all cases, three independent experiments were done using triplicates in each experiment. Data are expressed as mean standard error and analyzed with ANOVA followed by Tukey’s multiple range test. Significance level was fixed at P < 0.05. Values of EC50 and their respective 95% confidence intervals were estimated according to the American Public Health Association (1976).
3. Results Exposure of K562 cells to microcystins induced a decrease in cell viability, as determined by trypan blue exclusion, in a concentration- and time-dependent manner (Fig. 1a,b). There were no differences in the viability (P > 0.05) between the control and treated cells immediately after exposure to microcystins. After 24 h of microcystins exposure, a significant decrease of cell viability (P < 0.05) was observed with 0.4 mg/ ml (66.62 9.31%), 0.8 mg/ml (39.02 12.72%) and 1.6 mg/ ml (6.65 4.74%), as compared to control cells (96.41 2.32%) (Fig. 1a), in contrast to cells exposed to 0.2 mg/ml microcystin (89.07 7.03%) (P > 0.05). After 48 h, no viable cells were observed at concentrations higher than 0.2 mg/ml (Fig. 1b). In the lowest microcystins concentration (0.2 mg/ ml) cell viability was 52.82 11.46% (Fig. 1b). For the Lucena cell line, no differences (P > 0.05) were observed in cell viability determined by trypan blue exclusion, between the control and treated cells immediately after exposure. No differences (P > 0.05) were also observed between control (87.35 2.18%) and microcystins exposed cells after 24 h, except at the highest concentration (35.55 17.37%) (P < 0.05; see Fig. 1a). After 48 h of exposure, a decrease of cell viability (P < 0.05) was observed at all microcystins concentrations (Fig. 1b). Similar results were obtained when the MTT assay was used after 24 h of microcystins exposure (Fig. 2). Also, in terms of EC50 values, Lucena cell line was more resistant to microcystins than K562 cell line, as demonstrated by the significantly (P < 0.05) higher EC50 values obtained for Lucena cell line after 24 h and 48 h of exposure (Tables 1 and 2). Catalase activity was significantly higher (P < 0.05) in the Lucena cell line respect K562 cell line; however, microcystins exposure for 24 h did not alter this enzyme activity in both cell lines (P > 0.05; see Fig. 3). The capability of LPO induction by microcystins was investigated after 24 h of exposure to 0.2 mg/ml of microcystins. Neither K562 cells (30.21 0.81 and 52.03 2.05 nmol
Fig. 1. Viability (%) of K562 (non-MDR phenotype) and Lucena (MDR phenotype) cells exposed to different concentrations of microcystins during 24 (a) or 48 h (b), according to trypan blue exclusion test. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
Fig. 2. Effect of different concentrations of microcystins on K562 (non-MDR phenotype) and Lucena (MDR phenotype) cells, as measured by MTT assay after 24 h exposure. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
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Table 1 Estimated microcystins concentration (mg/ml) that reduced 50% of cell viability (EC50) in K562 and Lucena cell lines, according to trypan blue exclusion test Exposure time
K562
Lucena
P
24 h 48 h
0.60 (0.53e0.68) 0.21 (0.19e0.23)
1.41 (1.21e1.73) 0.32 (0.26e0.37)
<0.05 <0.05
Values in parentheses represent the 95% confidence interval for each EC50 estimate. The significance of the statistical comparison (P) between EC50 values at each exposure time is also indicated.
CHP/mg pellet for control and exposed group, respectively) nor Lucena cells (78.24 12.33 and 48.72 13.89 nmol CHP/mg pellet for control and exposed group, respectively) presented statistical differences (P > 0.05) in the LPO content. DNA damage score was analyzed after 24 h of exposure to microcystins. Fig. 4 shows that microcystins induced significant DNA damage (P < 0.05) in K562 cells compared to control cells. Significant DNA damage (P < 0.05) was also observed in Lucena cells microcystins exposure as compared to control cells, however DNA damage was much less intense than in the parental K562 cells. No differences (P > 0.05) after 24 h exposure to microcystins in GST activity were observed between the cell lines or treatments (K562 control group: 0.03 0.01 U GST; K562 treated group: 0.05 0.01 U GST; Lucena control group: 0.03 0.01 U GST; Lucena treated group: 0.04 0.003 U GST). The ROS concentration in both cell lines was evaluated after exposure for 24 h to microcystins. Fig. 5 shows that a significantly higher ROS level (P < 0.05) was observed in K562 cells after microcystins exposure, compared to the controls. However, in Lucena cells, no significant difference (P > 0.05) was observed. The quantification of microtubule cytoskeletal proteins demonstrated a markedly enrichment of a-tubulin subunits content in both polymers and monomers (P < 0.05) from Lucena cells as compared to K562 cells (Fig. 6a). However, when b-tubulin subunits content was analyzed, no differences (P > 0.05) were observed between K562 cells and Lucena cells (Fig. 6b).
Fig. 3. Catalase activity (U CAT) in K562 (non-MDR phenotype) and Lucena (MDR phenotype) cells lines exposed during 24 h to 0.2 mg/ml of microcystins. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
whole embryo cultured cells (Frangez et al., 2003), human colon carcinoma CaCo2 cell line (Botha et al., 2004), and human lymphoblastoid TK6 (Zhan et al., 2004). The results of these studies and ours obtained herein lead us to conclude that microcystins are toxic to non-hepatic cells. As mentioned in Section 1, the fact that transporters of organic anion transporting polypeptide super family are also expressed in the brain (Fischer et al., 2005) opens up the possibility of additional targets for microcystins other than liver cells or even other transport mechanisms being available for microcystins uptake. Although no information about OATP family transporters being expressed in Lucena and K562 cell lines is available, the responsiveness to microcystins concentrations as low as 0.2 mg/ml indicates a sensitivity 100 times higher than TK-6 cell lines. This in turn suggests that the entry of microcystins to these two cell lines is not limiting the toxicity these cyanotoxins. Lucena cells were more resistant to microcystins toxicity than their parental non-MDR cell line in our assay of cell
4. Discussion Previous studies have reported the toxic effect of microcystins on immortalized liver cells (Battle et al., 1997), human oral epidermoid carcinoma cells KB (Chong et al., 2000), Table 2 Estimated microcystins concentration (mg/ml) that reduced 50% of cell viability (EC50) in K562 and Lucena cell lines, according to MTT assay Exposure time
K562
Lucena
P
24 h
0.63 (0.56e0.70)
1.31 (1.19e1.46)
<0.05
Values between brackets represent the 95% confidence interval for each EC50 estimate. The significance of the statistical comparison (P) between EC50 values at each exposure time is also indicated.
Fig. 4. Quantification of DNA damage (score) in K562 (non MDR phenotype) and Lucena (MDR phenotype) cells lines exposed during 24 h to 0.2 mg/ml of microcystins. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
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Fig. 5. Quantification of ROS production (fluorescence area) in K562 (non MDR phenotype) and Lucena (MDR phenotype) cells lines exposed during 24 h to 0.2 mg/ml of microcystins. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
Fig. 6. Quantification of (a) a-tubulin and (b) b-tubulin contents (arbitrary units) in K562 (non MDR phenotype) and Lucena (MDR phenotype) cell lines. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
viability, as in the lower EC50 values after 24 h and 48 h of exposure. Also these cells presented lower DNA damage and ROS level. Interestingly, the concentration of microcystins (0.2 mg/ml), which did not interfere with cell viability, was similar to that determined previously in Patos Lagoon after blooms dominated by Microcystis (Matthiensen et al., 1999). Considering that microcystins can generate oxidative stress, the difference in sensitivity in the two cell lines can be related to dissimilar antioxidant defenses. In this context, the higher catalase activity can help explain, at least partially, the resistance of Lucena cells to microcystins exposure. Higher catalase activity in Lucena cells has been described by Trindade et al. (1999) in a study of the resistance of this cell line to UVA radiation and hydrogen peroxide. The fact that the pumping activity through Pgp consumes ATP (Uchiumi et al., 1993) raises the possibility of a higher cellular metabolism to cope with the augmented energetic demands. A higher ATP turnover should increase oxidative phosphorylation, producing more ROS (Reichelt and Schachtschabel, 2001). In fact, it is possible to observe in our results higher ROS levels in control Lucena cells when compared to control K562 cells (Fig. 5). Taking into account that no differences in GST activity were observed between cell lines or treatments, it is concluded that the resistance of Lucena cells to microcystins is not due to the extrusion of GSH-microcystin conjugates by Pgp. However it cannot be discarded the possibility of the Pgp pumps non-conjugated microcystins. Considering the ability of microcystins to induce DNA damage, Lucena cells presented lower damage scores when exposed to the toxin than the parental cell line, but these hepatotoxins exerted a significant effect on both cell lines. In agreement with this, Rao and Bhattacharya (1996) showed that microcystin can cause DNA damage in mouse liver cells. DNA fragmentation has also been reported in baby hamster kidney cell line BHK21 and mouse embryo fibroblasts (Rao et al., 1998). According to Zegura et al. (2003), the capacity of microcystins to cause DNA damage can be related to ROS generation, once it had been demonstrated that endonuclease III (Endo III) was involved. This protein shares in the repair systems associated with oxidative damage, promoting the digestion of oxidized pyrimidine bases. MDR cell line (Lucena) with higher catalase activity also showed a lower DNA damage than its parental cell line (K562), suggesting the involvement of ROS in the toxicity exerted by this cyanotoxin. In fact, previous reports indicate that microcystins exposure augment directly or indirectly H2O2 production (Li et al., 2003) and Zegura et al. (2003) observed lower DNA damage induced by microcystins if a hydroxyl radical scavenger (DMSO) was co-administered with the toxins. In this context, the significant increase in ROS production observed in the K562 cells, in contrast to Lucena cells, when both cell lines were exposed to microcystins, suggest that the MDR cells, at least in part, were more resistant to microcystins due to a higher antioxidant competence. The MDR phenotype of Lucena cells was obtained to select of progressively higher concentrations of VCR, an anti-mitotic drug. VCR binds to tubulin dimers constituted by a- and
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b-tubulin subunits, leading to destabilization of microtubules (Himes et al., 1976; Lee, 1995). Erokhina et al. (1997) showed that the first steps of MDR phenotype acquisition were accompanied by significant alterations of cytoskeleton elements, as well as changes in microtubule distribution. They suggested that these alterations are essential for evolution of MDR phenotype. According to this, our results showed a differential microtubule dynamics between K562 and Lucena cells. Lucena cells presented an a- tubulin subunit overexpression in Lucena cells in polymer and monomer forms. This indicates that the MDR phenomenon could be associated with greater availability of these proteins to repair (monomers) or in giving stability to the cell (polymers), once this MDR cell line is continuously exposed to VCR. Considering the capacity of microcystininduced ROS generation to cause disruption of cytoskeleton organization (Ding et al., 2001), it is possible that the higher availability of a-tubulin monomers in Lucena cells should be another feature that explain the lower sensitivity of this cell line to microcystins toxicity. To sum up, the present study provides further evidence of differential microcystins sensitivity in non-hepatic cells, supporting previous findings that suggested that mechanisms of these cyanotoxins are not limited to phosphatase inhibition but also to oxidative stress generation. The acquisition of an MDR phenotype should be important when cells are confronted to microcystin because of the higher antioxidant competence of these cells and also by the higher a-tubulin content that should aid in the preservation of cytoskeletal structure after microcystins exposure. Acknowledgments This work was supported by FAPERGS (Grants 00/2150.4, 04/0023.1 and 00/1137.0). A.P.S.V. received a graduate fellowship from Brazilian CAPES. J.M.M., J.S.Y., V.M.R., M.A.M.C., V.M.N. and M.S.de F. are research fellows from Brazilian CNPq. L.A.G. is a post-doctoral fellow from CAPES (PRODOC program). We are thankful to Dr. Paulo Abreu for the use of the epifluorescence microscope, to Dr. Euclydes dos Santos Filho and Dr. Adalto Bianchini for revision of the manuscript, and to the personnel from Cyanobacteria Research Unit (FURG) for logistic support. References American Public Health Association. American WaterWorks Association, Water Pollution Control Federation. Standard Methods for the Examination of Water and Wastewaters. Washington, DC: American Public Health Association; 1976. Battle T, Touchard C, Moulsdale HJ, Dowsett B, Stacey GN. New Cell substrates in vitro evaluation of microcystin hepato-cytotoxicity. Toxicol In Vitro 1997;11:557e67. Bleuter E. Red cell metabolism: a manual of biochemical methods. New York: Grune & Stratton; 1975. Botha N, Gehringer MM, Downing TG, Venter M, Shephard EG. The role of microcystin-LR in the induction of apoptosis and oxidative stress in CaCo2 cells. Toxicon 2004;43:85e92.
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Rippka R, Deruelles J, Waterbury JB, Herdman M, Stanier RY. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 1979;111:1e61. Rumjanek VM, Lucena M, Campos MM, Marques-Silva VM, Maia RC. Multidrug resistance in leukemias: the problem and some approaches to its circumvention. Cienc Cult 1994;46:63e9. Rumjanek VM, Trindade GS, Wagner-Souza K, Meletti-de-Oliveira MC, Marques-Santos LF, Maia RC, et al. Multidrug resistance in tumour cells: characterisation of the multidrug resistant cell line K562-Lucena 1. Acad Bras Cienc 2001;73:57e69. Runnegar M, Berndt N, Kong S, Lee EYC, Zhang L. In vivo and in vitro binding of microcystin to protein phosphatases 1 and 2A. Biochem Biophys Res Commun 1995a;216:162e9. Runnegar M, Berndt N, Kaplowitz N. Microcystin uptake and inhibition of protein phosphatases: effects of chemoprotectants and self-inhibition in relation to known hepatic transporters. Toxicol Appl Pharmacol 1995b;134:264e72. Sikic B. Modulation of multidrug resistance: at the threshold. J Clin Oncol 1993;11:1629e35. Singh NP, McCoy M, Tice RR, Schneider E. A simple technique for quantitation of low levels of DNA damage in single cells. Exp Cell Res 1988; 175:184e91. Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, et al. Single cell gel/Comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000;35:206e21. Tiirikainen MI, Krusius T. Multidrug resistance. Annu Med 1991;23:509e20. Toivola DM, Eriksson JE. Toxins affecting cell signaling and alteration of cytoskeletal structure. Toxicol in Vitro 1999;1991(13):521e30. Trindade GS, Capella MAM, Capella LS, Affonso-Mitidier OR, Rumjanek VM. Differences in sensitivity to UVC, UVB and UVA radiation of a multidrug-resistant cell line overexpressing P-glycoprotein. Photochem Photobiol 1999;69:694e9. Trindade GS, Farias SLA, Rumjanek VM, Capella MAM. Methylene blue reverts multidrug resistance: sensitivity of multidrug resistant cells to this dye and its photodynamic action. Cancer Lett 2000;151:161e7. Tsuruo T, Lida H, Ohkochi E, Tsukagoshi S, Sakurai Y. Establishment and properties of a vincristine-resistant human myelogenous leukemia K562. J Cancer Res 1983;74:751e8. Uchiumi T, Kohno K, Tanimura H, Matsuo K, Sato S, Uchida Y, et al. Enhanced expression of the human multidrug resistance 1 gene in response to UV light irradiation. Cell Growth Differ 1993;4:147e57. Yunes JS, Salomo´n PS, Matthiensen A, Beattie KA, Raggett SL, Codd GA. Toxic blooms of cyanobacteria in the Patos Lagoon Estuary, southern Brazil. J Aquat Ecosyst Health 1996;5:223e9. Zegura B, Sedmak B, Filipic M. Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2. Toxicon 2003;41:41e8. Zhan L, Sakamoto H, Sakuraba M, Wu D-S, Zhang L-S, Suzuki T, et al. Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells. Mutat Res 2004;557:1e6.
Sensitivity to microcystins: A comparative study in human cell lines with and without multidrug resistance phenotype Ana Paula de Souza Votto a,b, Viviane Plasse Renon a, Jo~ao Sarkis Yunes c, Vivian Mary Rumjanek d, Ma´rcia Alves Marques Capella e, Vivaldo Moura Neto f, Marta Sampaio de Freitas g, Laura Alicia Geracitano a,b, Jose´ Marı´a Monserrat a,b, Gilma Santos Trindade a,b,* a
Departamento de Cieˆncias Fisiolo´gicas, Fundac¸~ao Universidade Federal do Rio Grande (FURG), Rio Grande 96201-900, Brazil b Programa de Po´s-graduac¸~ao em Cieˆncias Fisiolo´gicas, Fisiologia Animal Comparada, FURG, Rio Grande 96201-900, Brazil c Departamento de Quı´mica, Unidade de Pesquisa em Cianobacte´rias, FURG, Rio Grande 96201-900, Brazil d Laborato´rio de Imunologia Tumoral, Departamento de Bioquı´mica Me´dica, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21941-590, Brazil e Laborato´rio de Fisiologia Renal, Instituto de Biofı´sica Carlos Chagas Filho, UFRJ, Rio de Janeiro 21941-590, Brazil f Instituto de Cieˆncias Biome´dicas, Departamento de Anatomia, UFRJ, Rio de Janeiro 21941-590, Brazil g Centro Biome´dico, Departamento de Farmacologia e Psicobiologia, Universidade do Estado do Rio de Janeiro (UEFRJ), Rio de Janeiro 20551-030, Brazil Received 29 March 2006; revised 5 April 2007; accepted 12 May 2007
Abstract Multidrug resistance (MDR) is an obstacle in cancer treatment. An understanding of how tumoral cells react to oxidants can help us elucidate the cellular mechanism involved in resistance. Microcystins are cyanobacteria hepatotoxins known to generate oxidative stress. The aim of this study was to compare the sensitivity to microcystins of human tumoral cell lines with (Lucena) and without (K562) MDR phenotype. Endpoints analyzed were effective microcystins concentration to 50% of exposed cells (EC50), antioxidant enzyme activity, lipid peroxidation, DNA damage, reactive oxygen species (ROS) concentration, and tubulin content. Lucena were more resistant and showed lower DNA damage than K562 cells (P < 0.05). Although microcystins did not alter catalase activity, a higher mean value was observed in Lucena than in K562 cells. Lucena cells also showed lower ROS concentration and higher tubulin content. The higher metabolism associated with the MDR phenotype should increase ROS concentration and make for an improved antioxidant defense against the toxic effects of microcystins. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: MDR phenotype; Microcystins; Oxidative stress; DNA damage
1. Introduction Multidrug resistance (MDR) is a phenomenon by which tumors that initially respond to determined chemotherapy, acquire resistance to chemically and non-chemically related * Corresponding author. Departamento de Cieˆncias Fisiolo´gicas, Fundac¸~ao Universidade Federal do Rio Grande (FURG), Campus Carreiros, Avenida Italia, km 8, Rio Grande 96201-900, RS, Brazil. Tel.: þ55 53 3233 6855; fax: þ55 53 3233 6850. E-mail address: [email protected] (G.S. Trindade).
drugs. The best understood mechanism of MDR is the one conferred by the membrane P-glycoprotein (Pgp), which acts by pumping several unrelated drugs out from the cells (Gottesman and Pastan, 1993). Despite the resistance process to be multifactorial, MDR phenotype exhibits some main characteristics: (1) resistance to non-related drugs (Kartner and Ling, 1989; Tiirikainen and Krusius, 1991), (2) expression of protein as the P-glycoprotein (Pgp) (Gottesman and Pastan, 1993), (3) extrusion of rhodamine dye (Neyfakh, 1988), and (4) reversion of the resistance induced by agents like trifluoperazine, verapamil and cyclosporin A (Ford and Hait, 1990; Sikic, 1993).
1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.05.010
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Rumjanek et al. (1994, 2001) established a leukemic MDR cell line by using the method of Tsuruo et al. (1983), and called K562-Lucena1 (Lucena). It has already been shown that Lucena cells possess the characteristics mentioned above (Maia et al., 1996a,b; Marques-Silva, 1996; Orind et al., 1997). The reactive oxygen species (ROS) are also supposed to be involved in the cytotoxicity of some anticancer agents, and it has been suggested that multidrug-resistant (MDR) cells might also be resistant to oxidative stress (Capella et al., 2001; Trindade et al., 1999, 2000). Microcystins are toxins produced by the cyanobacterium Microcystis aeruginosa, among several other species (Mackintosh et al., 1990). These toxins are cyclic heptapeptides presenting a general chemical structure of D-Ala-X-D-erythrob-methylAsp-Z-Adda-D-Glu-N-methyldehydroAla, where positions X and Z represent variable L-amino acids, which generate a family of different molecular forms of microcystins (Pflugmacher et al., 1998). Microcystin-LR is the most common cyanotoxin with a leucine (L) and an arginine (R) in X and Z positions, respectively (Mackintosh et al., 1990; Dawson, 1998.). Pouria et al. (1998) found that microcystins provoked the death of several dialysis patients after acute hepatic failure, due to cyanobacteria contamination of water used during this treatment. In Southern Brazil, cyanobacteria blooms, dominated by Microcystis aeruginosa, have allegedly given microcystin concentrations as high as 244.8 mg/l in estuarine waters of Patos Lagoon (Yunes et al., 1996; Matthiensen et al., 1999). Microcystins are potent inhibitors of serine/threonine phosphatases PP1 and PP2A, leading to an increase of protein phosphorylation, inducing changes in the control of cell growth and differentiation, resulting in tumor promotion after prolonged exposure to sub-acute doses (Eriksson et al., 1990; Mackintosh et al., 1990; Falconer and Yeung, 1992; Mackintosh and Mackintosh, 1994; Runnegar et al., 1995a; Toivola and Eriksson, 1999). The hepatotoxicity of this toxin is linked to the uptake mechanism in hepatocytes by biliary acid transporters (Runnegar et al., 1995b). According to these authors, the liver is the primary microcystin target because of its specific transport mechanism, which enables the uptake of microcystin into hepatocytes. However, Fischer et al. (2005) recently reported that members of the organic anion transporting polypeptide super family are involved in microcystins uptake, including the human OATP1A2 transporter that is expressed both in liver and brain. So, this finding opens the possibility that targets for microcystins are not only liver cells but also brain cells. In this context, it is important to stress that, although several in vitro studies have been conducted with primary cultures of hepatocytes, some authors have already observed microcystins cytotoxicity in others cell lines; for example, Zhan et al. (2004) reported genotoxicity of these toxins, inducing micronucleus formation as well as gene mutations in human lymphoblastoid TK6. Most of the physiological effects observed during microcystins exposure are probably secondary to the phosphatases proteins inhibition (Mikhailov et al., 2003). Runnegar et al. (1995b) concluded that microcystinstoxicity is a consequence
of irreversible phosphatase inhibition, although they did not exclude the possibility that other mechanisms might be involved. Some authors have considered oxidative stress generation as another possible toxicity mechanism. Ding et al. (1998) reported that extracts from M. aeruginosa were able to induce oxidative cellular damage in hepatocytes of rats, manifested by high levels of lipid peroxides and reactive oxygen species (ROS). Guzman and Solter (1999) verified hepatic oxidative damage after microcystin-LR exposure evidenced by an increase of malonaldehyde levels, a lipid peroxidation byproduct. Microcystins are also able to induce nuclear DNA damage in human hepatoma cell line HepG2, an effect involving ROS generation (Zegura et al., 2003). Pinho et al. (2003) demonstrated that the hepatopancreas of crabs exposed to microcystins exhibited higher GST and CAT activity than that observed in control crabs at the same experimental time, indicating that the antioxidant defense system was activated after exposure to these cyanotoxins. Considering that microcystins may interfere in antioxidant enzyme activities and induce lipid peroxidation, and that MDR cells are resistant to oxidative stress, the aim of this study was to compare the sensitivity of Lucena, a MDR cell line, and of K562, its non MDR parental cell line, to these toxins. 2. Materials and methods 2.1. Cells and culture conditions K562 and Lucena cells were obtained from the Tumoral Immunology Laboratory at the Medical Biochemistry Department of the Rio de Janeiro Federal University, Brazil. Parental K562 cells were grown in RPMI 1640 (Gibco) medium supplemented with sodium bicarbonate (0.2 g/l) (Vetec), L-glutamine (0.3 g/l) (Vetec), Hepes (25 mM) (Acros) and b-mercaptoethanol (5 105 M) (Sigma), with 10% fetal bovine serum (Gibco), 1% of antibiotic (penicillin (100 U/ml) and streptomycin (100 mg/ml) (Gibco) and antimicotic (0.25 mg/ml) (Sigma), in disposable plastic flasks, at 37 C. The MDR Lucena cells were grown under the same conditions above with 60 nM vincristine (VCR) (Sigma) in order to preserve the MDR phenotype.
2.2. Preparation of Microcystis aeruginosa extracts Cells of Microcystis aeruginosa from strain RST 9501 were cultured in BG11 ( 8.82 mM of NaNO3) medium (Rippka et al., 1979) at 25 1 C and employed as toxin source. Characterization of microcystins produced by the strain RST 9501 was done by Matthiensen et al. (1999, 2000). These authors found that methanolic extracts of strain RST 9501 presented one major peak and three minor peaks with UV spectra characteristic of microcystins, with a lmax of 238e239 nm. Interestingly, the retention time of the major peak (15.22 min) was different to that of microcystin-LR. Further analysis confirmed that the most abundant microcystin produced by strain RST 9501 is a [D-Leu1]-microcystin-LR, a variant with a similar potency in terms of phosphatases inhibition respect the common [D-Ala1]-microcystin-LR. The extract was prepared using lyophilized cells of Microcystis aeruginosa and the protocol of Coyle and Lawton (1996). Briefly, the cells were dissolved in absolute methanol (Sigma), sonicated 3 times and centrifuged (10,000 g) at 4 C, during 10 min. Extracts were evaporated at 40 C and then re-dissolved in ultra-pure water. Finally, samples were centrifuged and the supernatant was collected and stored at 20 C. Microcystins content was determined using a commercial enzyme-linked immunoassay (ELISA) with polyclonal antibodies (EnviroLogix Inc., Portland, ME, USA). Different concentrations of microcystin were prepared after appropriate dilutions with saline phosphate buffer (Ca2þ and Mg2þ free) (PBS). Taking into account
A.P.S. Votto et al. / Cell Biology International 31 (2007) 1359e1366 the streaking difference in microcystin sensitivity between the 2 lines (see Section 3), additional experiments were perform to characterize K562 and Lucena lines, in terms of a- and b-tubulins content (see Section 2.7).
2.3. Microcystins exposure The cells were grown for 2 days (K562) or 3 days (Lucena) before the beginning of experimentation (Trindade et al., 1999). For each experiment, cells were centrifuged, washed with PBS and suspended in medium without b-mercaptoethanol at 2 105 cells/ml. The cells were treated in medium with different concentrations of microcystins (0.2, 0.4, 0.8 or 1.6 mg/ml) plus a control group that received the same volume of PBS without toxins. The cells were incubated at 37 C in 24-well culture plates. During the experiments, no VCR was added to Lucena cell cultures.
2.4. Assessment of the sensitivity of the two cell lines to microcystins Cell viability was assessed by trypan blue (Gibco) exclusion after 0, 24 and 48 h exposure. In some instances (24 h exposure to microcystins), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium (MTT; Sigma) assay was also used, according to Trindade et al. (1999). The EC50 (concentration at which viability was reduced 50%) was estimated for the 2 cell lines after 24 h and 48 h of exposure for cell viability assessed by trypan blue exclusion and after 24 h for MTT assay. The concentration (0.2 mg/ml) that presented no significant difference in viability respect to the control (cell line K562) after 24 h of exposure was employed in posterior tests (see Results).
2.5. Effects of microcystins in activity of antioxidants enzymes and oxidative effects For preparation of each sample for enzyme activity and lipid peroxidation (LPO) determinations, one pool of 3 wells of the culture plates of each treatment (control cells and cells treated with 0.2 mg/ml of microcystin during 24 h) were centrifuged at 250 g for 2 min at room temperature to obtain a pellet with 6.0 105 cells/ml. For enzyme activity determinations, the homogenates were prepared in cold (4 C) buffer solution containing 20 mM Tris-Base (Gibco), 1 mM EDTA (Gibco), 1 mM dithiothreitol (Sigma), 500 mM sucrose (Sigma), 150 mM KCl (Gibco), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), pH 7.60. Samples were centrifuged at 9000 g for 30 min at 4 C. The supernatant of each sample was stored at 80 C until enzyme activity was determined. Total protein content in the homogenate was measured using a commercial reagent kit (Doles Reagentes Ltda.), which is based on the Biuret protein assay. Both enzymatic and protein content determinations were performed at least in duplicate. Catalase (CAT) activity was determined by the method of Bleuter (1975). Enzyme activity was expressed in CAT units, where one unit is the amount of enzyme needed to hydrolyze 1 mmol of H2O2 (Merck) per minute, and normalized by being expressed per mg of total proteins present in the homogenate at 30 C and pH 8.00. Glutathione-S-transferase (GST) activity was determined according to Habig et al. (1974), and Habig and Jakoby (1981). Enzyme activity values were expressed in GST units, where one unit is the amount of enzyme necessary to conjugate 1 mmol of 1-chloro-2,4-dinitrobenzene (CDNB; Sigma) per min and expressed per mg of total protein present in the homogenate, at 30 C and pH 7.0. Lipid peroxidation (LPO) was determined according to Hermes-Lima et al. (1995). Samples were homogenized (10% W/V) in 100% cold (4 C) methanol. The homogenate was centrifuged at 1000 g, for 10 min at 4 C. The supernatant was used for LPO determinations (580 nm). Cumene hydroperoxide (CHP; Sigma) was employed as standard. DNA damage was evaluated by the alkaline single cell electrophoresis (comet) assay, performed as described by Singh et al. (1988) and Tice et al. (2000), with some modifications. An aliquot (50 ml) of cell suspension of
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each sample (2 105 cell/ml) was mixed with 30 ml of 1.73% low melting point agarose (BMA) and added to fully frozen slides which had been covered with a layer of 0.65% normal melting point agarose (BMA). Following the layer solidification, cells in slides were lysed [2.5 M NaOH (Sigma), 0.1 M EDTA, 0.01 M Tris (Gibco), 1% sodium sarcosinate (Fisher Scientific), 1% Triton X-100 (Aldrich), and 10% dimethyl sulfoxide (Aldrich), pH 10.0] overnight at 4 C. Subsequently, samples were placed in the electrophoresis solution (300 mM NaOH and 1 mM EDTA, pH 13) for 35 min to allow DNA unwinding. Then, electrophoresis was performed during 25 min at 25 V and 280 mA. Finally, the slides were neutralized with 0.4 M Tris buffer (pH 7.5) (Gibco), stained with 95 ml of ethidium bromide (20 mg/ml) (Sigma) and analyzed using an epifluorescence microscope Zeiss Axioplan (400 magnification). In 50 randomly selected cells in duplicated slides, DNA damage was scored on the basis of undamaged (class 0), cells presenting short migration of DNA (class 1), medium migration (class 2), long migration (class 3), and complete migration (no nucleus remaining, class 4). The final score were made multiplying the category for the nucleus number in this category, resulting score 0 for no damage and 200 for maximum damage.
2.6. Assessment of intracellular ROS formation Suspensions of both cell lines (4.0 105 cells/ml) (control cells and cells treated with 0.2 mg/ml of microcystins during 24 h) were washed twice with PBS and incubated for 30 min at 37 C with the fluorogenic compound, 20 ,70 -dichlorofluorescin diacetate (H2DCF-DA, Molecular Probes) at a final concentration of 40 mM, according to Myhre and Fonnum (2001). H2DCFDA passively diffuses through cellular membranes and, once inside, the acetates are cleaved by intracellular esterases. Thereafter, the non-fluorescent compound H2DCF is oxidized by ROS to the fluorescent compound DCF. Each treatment was performed in triplicate. After the loading with H2DCFDA, the cells were washed twice with PBS and suspended in fresh PBS. Aliquots of 160 ml of each sample (five replicates) were placed onto an ELISA plate and the fluorescence intensity determined during 90 min at 37 C, using a fluorometer (Victor 2, Perkin Elmer), with excitation and emission wavelengths of 485 and 520 nm, respectively. ROS levels were expressed in terms of fluorescence area, after fitting fluorescence data to a second order polynomial and the estimated functions integrated between 0 and 90 min in order to obtain its area.
2.7. Quantification of a- and b-tubulins content on the two cell lines For preparation of each sample for a- and b-tubulins content, a suspension of 1.0e1.5 106 cells/ml was washed 3 times with ice-cold PBS. Whole cell extracts were obtained by suspending cells in lysis buffer [50 mM MES (Sigma), pH 6.4, 1 mM MgCl2 (Sigma), 10 mM EDTA, 1 mM PMSF, 1 mM benzamidine (Sigma) and 1% Triton X-100]. Triton-insoluble and -soluble cytoskeletal fractions were obtained using the lysis buffer followed by centrifugation at 13,000 g for 10 min at 25 C. Supernatants were collected and pellets were suspended in the same buffer. Cytosolic extracts were obtained by suspending cells in modified RIPA buffer [PBS, 0.5% sodium deoxycholate (Aldrich), 1% Nonidet P-40 (Sigma), 0.1% sodium dodecyl sulfate (SDS; Sigma), 10 mg/ml PMSF, 10 mg/ml aprotinin (Sigma), 10 mg/ml leupeptin (Sigma), 1 mM benzamidine], incubating on ice for 30 min before the supernatant was collected by centrifugation at 10,000 g for 10 min. Protein content of all cell extracts were determined by Bradford’s method (Bradford, 1976). Whole extract and cytoskeletal fractions proteins were denatured with Laemmli sample buffer composed of 50 mM Tris buffer (pH 6.8), 1% SDS, 140 mM b-mercaptoethanol, 10% glycerol (Sigma), 0.001% bromophenol blue (Sigma) and by heating in a boiling water bath for 3 min (Laemmli, 1970). Protein samples (30 mg) were subjected to gel electrophoresis and immunoblotting. SDSepolyacrylamide gel electrophoresis (SDSePAGE) was performed on 10% acrylamide (Sigma) according to discontinuous system of Laemmli (1970). Afterwards, the proteins were transferred to nitrocellulose sheets (Hybond-C Extra, Amersham Biosciences) during 90 min electrophoresis (30 mA) in Triseglycine buffer [25 mM TriseHCl (Gibco), pH 7.0, 192 mM glycine (Sigma)] containing 20% methanol (Towbin et al., 1979).
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The nitrocellulose sheets were incubated with TBS [20 mM Tris-HCl, pH 7.5, 500 mM NaCl (Sigma), 0.1% Tween-20 (Gibco)] containing 5% skimmed milk followed by incubation with mouse (monoclonal) anti-a-tubulin antibody (1:1000; Sigma), or mouse (monoclonal) anti-b-tubulin antibody (1:1000; Amersham Biosciences), overnight at 4 C. After extensive washing in TBS, the nitrocellulose sheets were incubated with anti-mouse IgG antibody peroxidase-conjugated (1:5000, Gibco). Immunoreactive proteins were visualized by ECLÔ Western Blotting Kit (Amersham Biosciences) and exposed to Kodak X-Omat films. Autoradiograms were quantified by densitometry on a model GS-690 Imaging densitometer equipped with Molecular Analyst software version 1.4.1 (Bio-Rad Laboratories).
2.8. Statistical analysis In all cases, three independent experiments were done using triplicates in each experiment. Data are expressed as mean standard error and analyzed with ANOVA followed by Tukey’s multiple range test. Significance level was fixed at P < 0.05. Values of EC50 and their respective 95% confidence intervals were estimated according to the American Public Health Association (1976).
3. Results Exposure of K562 cells to microcystins induced a decrease in cell viability, as determined by trypan blue exclusion, in a concentration- and time-dependent manner (Fig. 1a,b). There were no differences in the viability (P > 0.05) between the control and treated cells immediately after exposure to microcystins. After 24 h of microcystins exposure, a significant decrease of cell viability (P < 0.05) was observed with 0.4 mg/ ml (66.62 9.31%), 0.8 mg/ml (39.02 12.72%) and 1.6 mg/ ml (6.65 4.74%), as compared to control cells (96.41 2.32%) (Fig. 1a), in contrast to cells exposed to 0.2 mg/ml microcystin (89.07 7.03%) (P > 0.05). After 48 h, no viable cells were observed at concentrations higher than 0.2 mg/ml (Fig. 1b). In the lowest microcystins concentration (0.2 mg/ ml) cell viability was 52.82 11.46% (Fig. 1b). For the Lucena cell line, no differences (P > 0.05) were observed in cell viability determined by trypan blue exclusion, between the control and treated cells immediately after exposure. No differences (P > 0.05) were also observed between control (87.35 2.18%) and microcystins exposed cells after 24 h, except at the highest concentration (35.55 17.37%) (P < 0.05; see Fig. 1a). After 48 h of exposure, a decrease of cell viability (P < 0.05) was observed at all microcystins concentrations (Fig. 1b). Similar results were obtained when the MTT assay was used after 24 h of microcystins exposure (Fig. 2). Also, in terms of EC50 values, Lucena cell line was more resistant to microcystins than K562 cell line, as demonstrated by the significantly (P < 0.05) higher EC50 values obtained for Lucena cell line after 24 h and 48 h of exposure (Tables 1 and 2). Catalase activity was significantly higher (P < 0.05) in the Lucena cell line respect K562 cell line; however, microcystins exposure for 24 h did not alter this enzyme activity in both cell lines (P > 0.05; see Fig. 3). The capability of LPO induction by microcystins was investigated after 24 h of exposure to 0.2 mg/ml of microcystins. Neither K562 cells (30.21 0.81 and 52.03 2.05 nmol
Fig. 1. Viability (%) of K562 (non-MDR phenotype) and Lucena (MDR phenotype) cells exposed to different concentrations of microcystins during 24 (a) or 48 h (b), according to trypan blue exclusion test. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
Fig. 2. Effect of different concentrations of microcystins on K562 (non-MDR phenotype) and Lucena (MDR phenotype) cells, as measured by MTT assay after 24 h exposure. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
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Table 1 Estimated microcystins concentration (mg/ml) that reduced 50% of cell viability (EC50) in K562 and Lucena cell lines, according to trypan blue exclusion test Exposure time
K562
Lucena
P
24 h 48 h
0.60 (0.53e0.68) 0.21 (0.19e0.23)
1.41 (1.21e1.73) 0.32 (0.26e0.37)
<0.05 <0.05
Values in parentheses represent the 95% confidence interval for each EC50 estimate. The significance of the statistical comparison (P) between EC50 values at each exposure time is also indicated.
CHP/mg pellet for control and exposed group, respectively) nor Lucena cells (78.24 12.33 and 48.72 13.89 nmol CHP/mg pellet for control and exposed group, respectively) presented statistical differences (P > 0.05) in the LPO content. DNA damage score was analyzed after 24 h of exposure to microcystins. Fig. 4 shows that microcystins induced significant DNA damage (P < 0.05) in K562 cells compared to control cells. Significant DNA damage (P < 0.05) was also observed in Lucena cells microcystins exposure as compared to control cells, however DNA damage was much less intense than in the parental K562 cells. No differences (P > 0.05) after 24 h exposure to microcystins in GST activity were observed between the cell lines or treatments (K562 control group: 0.03 0.01 U GST; K562 treated group: 0.05 0.01 U GST; Lucena control group: 0.03 0.01 U GST; Lucena treated group: 0.04 0.003 U GST). The ROS concentration in both cell lines was evaluated after exposure for 24 h to microcystins. Fig. 5 shows that a significantly higher ROS level (P < 0.05) was observed in K562 cells after microcystins exposure, compared to the controls. However, in Lucena cells, no significant difference (P > 0.05) was observed. The quantification of microtubule cytoskeletal proteins demonstrated a markedly enrichment of a-tubulin subunits content in both polymers and monomers (P < 0.05) from Lucena cells as compared to K562 cells (Fig. 6a). However, when b-tubulin subunits content was analyzed, no differences (P > 0.05) were observed between K562 cells and Lucena cells (Fig. 6b).
Fig. 3. Catalase activity (U CAT) in K562 (non-MDR phenotype) and Lucena (MDR phenotype) cells lines exposed during 24 h to 0.2 mg/ml of microcystins. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
whole embryo cultured cells (Frangez et al., 2003), human colon carcinoma CaCo2 cell line (Botha et al., 2004), and human lymphoblastoid TK6 (Zhan et al., 2004). The results of these studies and ours obtained herein lead us to conclude that microcystins are toxic to non-hepatic cells. As mentioned in Section 1, the fact that transporters of organic anion transporting polypeptide super family are also expressed in the brain (Fischer et al., 2005) opens up the possibility of additional targets for microcystins other than liver cells or even other transport mechanisms being available for microcystins uptake. Although no information about OATP family transporters being expressed in Lucena and K562 cell lines is available, the responsiveness to microcystins concentrations as low as 0.2 mg/ml indicates a sensitivity 100 times higher than TK-6 cell lines. This in turn suggests that the entry of microcystins to these two cell lines is not limiting the toxicity these cyanotoxins. Lucena cells were more resistant to microcystins toxicity than their parental non-MDR cell line in our assay of cell
4. Discussion Previous studies have reported the toxic effect of microcystins on immortalized liver cells (Battle et al., 1997), human oral epidermoid carcinoma cells KB (Chong et al., 2000), Table 2 Estimated microcystins concentration (mg/ml) that reduced 50% of cell viability (EC50) in K562 and Lucena cell lines, according to MTT assay Exposure time
K562
Lucena
P
24 h
0.63 (0.56e0.70)
1.31 (1.19e1.46)
<0.05
Values between brackets represent the 95% confidence interval for each EC50 estimate. The significance of the statistical comparison (P) between EC50 values at each exposure time is also indicated.
Fig. 4. Quantification of DNA damage (score) in K562 (non MDR phenotype) and Lucena (MDR phenotype) cells lines exposed during 24 h to 0.2 mg/ml of microcystins. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
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Fig. 5. Quantification of ROS production (fluorescence area) in K562 (non MDR phenotype) and Lucena (MDR phenotype) cells lines exposed during 24 h to 0.2 mg/ml of microcystins. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
Fig. 6. Quantification of (a) a-tubulin and (b) b-tubulin contents (arbitrary units) in K562 (non MDR phenotype) and Lucena (MDR phenotype) cell lines. Data are expressed as mean 1 standard error. Similar letters indicate absence of significant differences (P > 0.05).
viability, as in the lower EC50 values after 24 h and 48 h of exposure. Also these cells presented lower DNA damage and ROS level. Interestingly, the concentration of microcystins (0.2 mg/ml), which did not interfere with cell viability, was similar to that determined previously in Patos Lagoon after blooms dominated by Microcystis (Matthiensen et al., 1999). Considering that microcystins can generate oxidative stress, the difference in sensitivity in the two cell lines can be related to dissimilar antioxidant defenses. In this context, the higher catalase activity can help explain, at least partially, the resistance of Lucena cells to microcystins exposure. Higher catalase activity in Lucena cells has been described by Trindade et al. (1999) in a study of the resistance of this cell line to UVA radiation and hydrogen peroxide. The fact that the pumping activity through Pgp consumes ATP (Uchiumi et al., 1993) raises the possibility of a higher cellular metabolism to cope with the augmented energetic demands. A higher ATP turnover should increase oxidative phosphorylation, producing more ROS (Reichelt and Schachtschabel, 2001). In fact, it is possible to observe in our results higher ROS levels in control Lucena cells when compared to control K562 cells (Fig. 5). Taking into account that no differences in GST activity were observed between cell lines or treatments, it is concluded that the resistance of Lucena cells to microcystins is not due to the extrusion of GSH-microcystin conjugates by Pgp. However it cannot be discarded the possibility of the Pgp pumps non-conjugated microcystins. Considering the ability of microcystins to induce DNA damage, Lucena cells presented lower damage scores when exposed to the toxin than the parental cell line, but these hepatotoxins exerted a significant effect on both cell lines. In agreement with this, Rao and Bhattacharya (1996) showed that microcystin can cause DNA damage in mouse liver cells. DNA fragmentation has also been reported in baby hamster kidney cell line BHK21 and mouse embryo fibroblasts (Rao et al., 1998). According to Zegura et al. (2003), the capacity of microcystins to cause DNA damage can be related to ROS generation, once it had been demonstrated that endonuclease III (Endo III) was involved. This protein shares in the repair systems associated with oxidative damage, promoting the digestion of oxidized pyrimidine bases. MDR cell line (Lucena) with higher catalase activity also showed a lower DNA damage than its parental cell line (K562), suggesting the involvement of ROS in the toxicity exerted by this cyanotoxin. In fact, previous reports indicate that microcystins exposure augment directly or indirectly H2O2 production (Li et al., 2003) and Zegura et al. (2003) observed lower DNA damage induced by microcystins if a hydroxyl radical scavenger (DMSO) was co-administered with the toxins. In this context, the significant increase in ROS production observed in the K562 cells, in contrast to Lucena cells, when both cell lines were exposed to microcystins, suggest that the MDR cells, at least in part, were more resistant to microcystins due to a higher antioxidant competence. The MDR phenotype of Lucena cells was obtained to select of progressively higher concentrations of VCR, an anti-mitotic drug. VCR binds to tubulin dimers constituted by a- and
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b-tubulin subunits, leading to destabilization of microtubules (Himes et al., 1976; Lee, 1995). Erokhina et al. (1997) showed that the first steps of MDR phenotype acquisition were accompanied by significant alterations of cytoskeleton elements, as well as changes in microtubule distribution. They suggested that these alterations are essential for evolution of MDR phenotype. According to this, our results showed a differential microtubule dynamics between K562 and Lucena cells. Lucena cells presented an a- tubulin subunit overexpression in Lucena cells in polymer and monomer forms. This indicates that the MDR phenomenon could be associated with greater availability of these proteins to repair (monomers) or in giving stability to the cell (polymers), once this MDR cell line is continuously exposed to VCR. Considering the capacity of microcystininduced ROS generation to cause disruption of cytoskeleton organization (Ding et al., 2001), it is possible that the higher availability of a-tubulin monomers in Lucena cells should be another feature that explain the lower sensitivity of this cell line to microcystins toxicity. To sum up, the present study provides further evidence of differential microcystins sensitivity in non-hepatic cells, supporting previous findings that suggested that mechanisms of these cyanotoxins are not limited to phosphatase inhibition but also to oxidative stress generation. The acquisition of an MDR phenotype should be important when cells are confronted to microcystin because of the higher antioxidant competence of these cells and also by the higher a-tubulin content that should aid in the preservation of cytoskeletal structure after microcystins exposure. Acknowledgments This work was supported by FAPERGS (Grants 00/2150.4, 04/0023.1 and 00/1137.0). A.P.S.V. received a graduate fellowship from Brazilian CAPES. J.M.M., J.S.Y., V.M.R., M.A.M.C., V.M.N. and M.S.de F. are research fellows from Brazilian CNPq. L.A.G. is a post-doctoral fellow from CAPES (PRODOC program). We are thankful to Dr. Paulo Abreu for the use of the epifluorescence microscope, to Dr. Euclydes dos Santos Filho and Dr. Adalto Bianchini for revision of the manuscript, and to the personnel from Cyanobacteria Research Unit (FURG) for logistic support. References American Public Health Association. American WaterWorks Association, Water Pollution Control Federation. Standard Methods for the Examination of Water and Wastewaters. Washington, DC: American Public Health Association; 1976. Battle T, Touchard C, Moulsdale HJ, Dowsett B, Stacey GN. New Cell substrates in vitro evaluation of microcystin hepato-cytotoxicity. Toxicol In Vitro 1997;11:557e67. Bleuter E. Red cell metabolism: a manual of biochemical methods. New York: Grune & Stratton; 1975. Botha N, Gehringer MM, Downing TG, Venter M, Shephard EG. The role of microcystin-LR in the induction of apoptosis and oxidative stress in CaCo2 cells. Toxicon 2004;43:85e92.
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