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Anticlastogenic and antigenotoxic effects of selenomethionine on doxorubicin-induced damage in vitro in human lymphocytes
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Food and Chemical Toxicology 46 (2008) 671–677 www.elsevier.com/locate/foodchemtox
Anticlastogenic and antigenotoxic effects of selenomethionine on doxorubicin-induced damage in vitro in human lymphocytes Raquel Alves dos Santos a
a,*
, Catarina Satie Takahashi
a,b
Department of Genetics, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeira˜o Preto, SP, Brazil b Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeira˜o Preto, University of Sa˜o Paulo, Av. Bandeirantes, 3900, 14040-901 Ribeira˜o Preto, SP, Brazil Received 12 March 2007; accepted 12 September 2007
Abstract The use of antioxidants during chemotherapy has been shown to reduce or prevent the undesirable effects experienced by healthy cells. Micronutrient selenium is well known for its antioxidant properties; however, selenium exhibits a bimodal nature in that both its beneficial and toxic properties lie within a limited and narrow dose range. The present study investigated the possible protective effects of selenomethionine (SM) on the cytotoxicity, genotoxicity and clastogenicity of the chemotherapic doxorubicin (DXR), a key chemotherapic used in cancer treatment. Human peripheral lymphocytes were treated in vitro with varying concentrations of SM (0.25 lM, 0.5 lM, 1.0 lM and 2.0 lM), tested in combination with DXR (0.15 lg/mL). SM alone was not cytotoxic and when combined with DXR treatment, reduced the DNA damage index significantly, the frequency of chromosomal aberrations, the number of aberrant metaphases and the frequency of apoptotic cells. The mechanism of chemoprotection of SM may be related to its antioxidant properties as well as its ability to interfere with DNA repair pathways. Therefore this study showed that SM is effective in reducing the genetic damage induced by the antitumoral agent DXR. 2007 Elsevier Ltd. All rights reserved. Keywords: Selenium; Selenomethionine; Doxorubicin; Comet assay; Chromosomal aberrations
1. Introduction A large number of dietary components, such as vitamin C and E, lycopene and selenium are known for their antioxidant properties. Selenium is an essential micronutrient for animals, humans and microorganisms. A direct relationship between selenium intake and cancer risk in humans has been reported, indicating that selenium defi-
Abbreviations: CAs, chromosomal aberrations; CT, chromatid breaks; DC, dicentric; DI, DNA damage index; DXR, doxorubicin; IC, chromosome breaks; MI, mitotic index; QR, quadriradial; R, ring; SM, selenomethionine; TR, triradial. * Corresponding author. Tel.: +55 016 3602 3082; fax: +55 016 3602 3761. E-mail address: [email protected] (R.A.d. Santos). 0278-6915/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.09.090
ciency enhances the probability of developing cancer (Postovsky et al., 2003; Li et al., 2004). However selenium, like some other trace elements, is bimodal in nature whereby its beneficial properties occur in a limited range of daily intake below which it cannot perform its essential functions, and above which it is toxic (Alaejos et al., 2000). As a result of these properties, selenium can be included in the class of ‘‘Janus compounds’’, having two ‘faces’ on the same head. It is well established that selenium participates in the process of detoxification by forming part of glutathione peroxidase, a cellular enzyme that maintains appropriately low levels of hydrogen peroxide within a cellular environment (Tapiero et al., 2003). Despite its antioxidant properties and requirement for human and animal nutrition, the appropriate form of selenium for supplementation continues to be debated. There exists a wide variety of selenium forms including sodium
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selenite, sodium selenate, selenomethionine, selenocysteine and selenocystine. The L-isomer of selenomethionine (SM) is the most important natural food form of selenium found in seafood, grain products, meat and abundantly in Brazilian nuts (Alaejos et al., 2000; Schrauzer, 2000). Some chemotherapeutic approaches have proposed the use of antioxidants to minimize cytotoxicity and the damage induced in normal tissues by antitumor agents that produce free radicals (Antunes and Takahashi, 1999). Although chemotherapy plays a major role in treating various cancers, especially in controlling advanced stages of malignancies in clinical settings, most cytotoxic chemotherapeutic agents used in the treatment of a wide range of cancers are not selective touching, neoplastic and health cells by its clastogenic effects (Ferguson and Pearson, 1996). These events may have important consequences in cancer chemotherapy since mutations may lead to drug resistance, limiting further therapy. Moreover, germ cell mutations may be transmitted to future generations and mutations may result in the development of secondary tumors from cells that were originally benign (Baguley and Ferguson, 1998). The chemotherapeutic doxorubicin (DXR), an anthracycline antibiotic commonly used to treat a wide variety of cancers (Quiles et al., 2002), produces an increase in DNA strand breakage, in the percentage of abnormal frequencies of chromosomal damage in the FISH and conventional chromosomal aberration assays and also increases micronucleus formation in human lymphocytes in vitro (Anderson et al., 1997; Dhawan et al., 2003), related to its activity when generating reactive oxygen species and inhibiting topoisomerase II complex (Gewitz, 1999). Therefore, the present study was undertaken to investigate the possible antigenotoxic and anticlastogenic effects of selenomethionine (SM) in DNA damage induced by DXR in human lymphocytes in vitro. Damage was detected by assessing the mitotic index, chromosomal aberrations, comet assays and apoptotic cells. 2. Materials and methods 2.1. Cells and medium Human peripheral blood was collected using heparinized vials from six healthy donors aged 26–30 years. Lymphocytes were isolated with plasma and cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 20% fetal calf serum (Cultilab, Campinas, SP, Brazil), penicillin (5 lg/mL), streptomycin (10 lg/mL) and 2% phytohemagglutinin (Life Technologies, Grand Island, NY). Cells were cultured at 37 C in culture flasks containing 5 ml of complete medium. The National Research Ethics Committee (process no. 8709/2001) approved the protocol of the experiments and written consent was obtained from each blood donor prior to joining the study.
2.2. Chemicals L-Selenomethionine (CAS# 1464-42-2) was purchased from Acros Organics (New Jersey, USA) and DXR (CAS# 23214-92-8) (Laborato´rios Eurofarma, Sa˜o Paulo, Brazil) was donated from the Chemotherapy
Center, Faculty of Medicine, USP, Ribeira˜o Preto. Both SM and DXR were dissolved in sterile distilled water prior to use.
2.3. Culture treatments The concentrations of SM (0.25 lM, 0.5 lM, 1.0 lM and 2.0 lM) tested together with DXR were established in preliminary experiments; concentrations of SM above 2 lM were cytotoxic and reduced significantly mitotic index and cell viability (data not shown). The concentration of DXR (0.15 lg/mL) was similarly defined in preliminary experiments and besides the mitotic index, it was considered the number of chromosomal aberrations. In combined treatments (SM plus DXR) the values of mitotic index was similar to the negative control. The toxicity of the combined treatment is a critical factor because cytotoxic and cytostatic effects can mimic antimutagenicity because they interfere with the appearance of the mutant cells (Zeiger, 2007). Three different types of SM treatment were assessed on DXR-induced damage cells. The cultures were treated with DXR 24 h after initiation of incubation and treatment with SM was performed either 2 h before, simultaneously, or 2 h after DXR treatment. After each respective treatment, DXR and SM remained in the cultures until harvesting. Cells were harvested after 24 h of DXR treatment and then assessed for cell viability, apoptosis (apoptotic assay) and DNA damage of individual cells (comet assay), and after 26 h, for the analysis mitotic index and chromosomal aberrations, as DXR induces a delay in the cell cycle.
2.4. Comet assay An aliquot of 300 lL from each culture was taken after 48 hours of incubation to test for cell viability by trypan blue exclusion and for the alkaline version of the Comet assay as described by Singh et al. (1988). Briefly, 300 lL of the cell suspension was centrifuged for 5 minutes (500 rpm) in a refrigerated microcentrifuge (Eppendorff). The resulting pellet was homogenized with 80 lL of a low melting point agarose (0.5%), spread onto microscope slides pre-coated with a normal melting point agarose (1.5%), and covered with a coverslip. After 5 min at 4 C, the coverslip was removed and the slides were immersed in cold lysis solution (2.4 M NaCl; 100 mM EDTA; 10 mM Tris, 10% DMSO and 1% TritonX, pH 10) for 24 h. After lysis, the slides were placed in an electrophoresis chamber and covered with electrophoresis buffer (300 mM NaOH per 1 mM EDTA, pH > 13), for a remaining 20 min to allow for unwinding of DNA. The electrophoresis proceeded for 20 min (25 V and 300 mA). Afterwards, the slides were submerged for 15 min in a neutralization buffer (0.4 M Tris–HCl, pH 7.5), dried at room temperature and fixed in 100% ethanol for 5 min. Slide staining was performed immediately before analyzing using ethidium bromide (20 lg/mL). Slides were prepared in duplicate and 100 cells were screened per sample (50 cells from each slide) in a fluorescent microscope (ZEISS, Germany) equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm using a 40· objective. The nucleus was classified visually according to the migration of the fragments in: class 0 (no damage); class 1 (little damage with a short tail length smaller than the diameter of the nucleus); class 2 (medium damage with a tail length one or two times the diameter of the nucleus); 3 (significant damage with a tail length between two and a half to three times the diameter of the nucleus); class 4 (significant damage with a long tail of damage greater than three times the diameter of the nucleus).
2.5. Metaphases preparation To analyze chromosomal aberrations (CAs), a metaphase preparation was performed after 50 h of cell culture. Ninety minutes before harvesting, 12.5 lL colchicine (0.016%, Sigma) was added to each culture. The conventional cell harvest procedure was followed according to Moorhead et al. (1960). The cells were treated with hypotonic KCl solution (0.075 M) for 10 min, fixed with methanol:acetic acid (3:1), air-dried and stained with Giemsa: So¨rensen buffer (1:30) for 5 min. All slides were coded. The same scorer analyzed the slides in a blind test and a total of 100 cells per
R.A.d. Santos, C.S. Takahashi / Food and Chemical Toxicology 46 (2008) 671–677 treatment per individual were scored for the presence of CAs classified according to Savage (1976) and 2000 cells per treatment per individual were scored for MI.
2.6. Morphological characterization of normal, apoptotic and necrotic cells The frequencies of normal, apoptotic and necrotic cells were determined only where SM was added 2 h before DXR treatment, using 2 lL of staining solution [fluorescein diacetate dissolved in dimethyl-sulfoxide (15 lg/mL), propidium iodide (5 lg/mL) and Hoechtst 33342 (2 lg/mL) all from Sigma] mixed with 100 lL of cell suspension. Five hundred cells were analyzed per treatment, for three independent experiments, using an epi-fluorescence microscope (Carl Zeiss, Germany). A blue and intact nucleus was considered normal, a blue and fragmented nucleus was considered apoptotic and a red nucleus was considered necrotic.
2.7. Statistical analysis Six independent experiments were analyzed and statistical analysis was performed using the Kruskal–Wallis One-way Method of Variance followed by Student–Newman–Keuls considering a confidence interval of 95%.
3. Results Table 1 shows the frequencies of cells with the Comet and DNA damage index (DI) in human lymphocytes treated with different concentrations of SM and with DXR (0.15 lg/mL). DXR alone and SM alone (0.5 lM, 1.0 lM and 2.0 lM) increased the DI, the frequencies of chromosomal aberrations, abnormal metaphases and the frequen-
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cies of apoptotic cells (Tables 1–4). However, the combined treatment of DXR and SM led to a statistically significant reduction in DI and in the frequencies of comet positive cells relative to the DXR treatment alone. The reduction was between 96% and 97.5% and was neither dose-dependent nor different for all treatments. Tables 2 and 3 show the anticlastogenic effect of different treatments with SM. There was a significant reduction in the number of cells with aberrations and in the total number of chromosomal aberrations however, this reduction was similar in all tested concentrations of SM. Table 2 also shows the mean of mitotic index (MI) obtained in each treatment of SM combined with DXR, with variations from 3.2 to 4.7. The number of chromosomal aberrations in cells was significantly reduced by SM treatments (Table 3) when compared to the positive control. The types of chromosomal aberrations detected are also depicted in Table 3. Chromatid and chromosome gaps were excluded from the analysis. When calculating the total number of chromatid type breaks (chromatid breaks, triradial and quadriradial) or isochromatid breaks (chromosome breaks, dicentric and rings), it was observed that SM reduced both types similarly when compared to the DXR treatment alone. Table 4 demonstrates that SM treatment significantly reduced the number of apoptotic cells induced by DXR and, consequently, increased the number of viable cells.
Table 1 Antigenotoxic effect of SM in human lymphocytes treated in vitro with DXR Treatment
Cells with Comet (%)
Distribution of Comet classes 0 (%)
1 (%)
2 (%)
3 (%)
4 (%)
DNA damage index X ± SE
Reduction (%)
Negative control DXR 0.25 lM-SM 0.5 lM-SM 1.0 lM-SM 2.0 lM-SM
29.3 88.1 48.3 56.5 60.5 67.8
70.7 11.8 51.7 43.5 39.5 32.1
25.1 32.1 34.5 40.4 40.8 45.4
3.5 38.7 11.3 14.1 15.5 19.9
0.7 11.7 2.1 1.5 3.8 2.5
0 5.7 0.4 0.5 0.4 0.1
0.34 ± 0.03 1.67a ± 0.09 0.64a ± 0.09 0.75a ± 0.09 0.84a ± 0.07 0.93b ± 0.06
– – – – – –
SM 2 h before DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
73.8 68.3 60.8 60.6
31.4 31.7 39.1 39.4
45.4 45 44.5 41.1
20.2 20.8 14 16.7
2.2 2.4 1.7 2.7
0.8 0.1 0.7 0.1
0.96b ± 0.04 0.94b ± 0.12 0.80b ± 0.05 0.79b ± 0.04
96.2 96.3 97.1 97.2
Simultaneous treatment 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXaR 2.0 lM-SM + DXR
59.6 54 58.1 61
40 46 41.8 39
40.7 35.5 39.7 41
16.4 16.8 16.4 16.5
1.8 1.7 2 3
0.7 0 0.1 0.5
0.82b ± 0.06 0.74b ± 0.07 0.74b ± 0.05 0.85b ± 0.09
97 97.5 97.5 96.8
SM 2 h after DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
62.3 62.1 64.3 62.1
37.7 37.8 35.7 37.8
39.4 44 44.4 43.4
20.8 16.9 16.7 15.5
1.8 0.8 2.6 2.8
0.3 0.5 0.6 0.5
0.88b ± 0.02 0.82b ± 0.03 0.88b ± 0.07 0.84b ± 0.06
96.6 97 96.6 96.9
Six hundred nuclei were analyzed in each treatment (100 nuclei/treatment/experiment). DXR-doxorubicin at 0.15 lg/mL concentration, SM-selenomethionine. a p < 0.05 different from the negative control. b p < 0.01 different from the positive control (DXR).
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Table 2 Mean and Standard Error (X ± SE) of Mitotic Index (MI) and abnormal metaphases observed in human lymphocytes submitted to different treatments with SM and/or DXR in vitro Treatment
MI X ± SE
Abnormal metaphases
Control DXR 0.25 lM-SM 0.5 lM-SM 1.0 lM-SM 2.0 lM-SM + DXR
5.4 ± 0.2 2.7a ± 1.8 4.4 ± 0.3 4.4 ± 0.2 3.9 ± 0.2 4.1 ± 0.3
9 102 14 23 25 26
1.5 ± 0.6 17a ± 1.4 2.3 ± 0.8 3.8a ± 2.0 4.1a ± 2.3 4.3a ± 1.8
SM 2 h before DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
3.8 ± 0.2 4.1 ± 0.3 4.7b ± 0.5 3.8 ± 0.3
31 30 21 42
5.1a,b ± 1.5 5.0a,b ± 0.3 3.5a,b ± 1.2 7.0a,b ± 1.3
76.7 77.4 87 64.5
Simultaneous treatment 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
3.2 ± 0.4 3.7 ± 0.4 3.4 ± 0.3 3.4 ± 0.5
34 36 30 30
5.6a,b ± 0.6 6.0a,b ± 0.6 5.0a,b ± 1.4 5.0a,b ± 1.2
73.5 70.9 77.4 77.4
SM 2 h after DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
4.2 ± 0.2 4.1 ± 0.4 3.8 ± 0.3 3.9 ± 0.3
32 37 44 38
5.3a,b ± 2.0 6.1a,b ± 1.1 7.3a,b ± 0.6 6.3a,b ± 1.1
75.4 70.3 62.5 69
Total
X ± SE
Reduction (%) – – – – – –
Six hundred metaphases were analyzed in each treatment (100 metaphases/treatment/experiment). DXR-doxorubicin at 0.15 lg/mL concentration, SM-selenomethionine. a p < 0.01 different from the negative control. b p < 0.01 different from the positive control (DXR).
4. Discussion The trace element selenium is an essential micronutrient of fundamental importance to human health as it is a component of selenoproteins with important enzymatic functions. SM is a non-redoxing selenium compound that does not generate superoxide and is less toxic than inorganic selenium forms (Stewart et al., 1999). The present study investigated the antigenotoxic and anticlastogenic effects of SM against genetic damages induced by DXR, a potent genotoxic anticancer drug that generates undesirable effects in healthy cells. It was observed that SM treatment alone did not reduce MI significantly. The cytotoxic and antiproliferative effects of SM have been extensively investigated; it exhibits no cytotoxicity in BALB/c MK-2 cells exposed to concentrations up to 250 lg/mL (Stewart et al., 1999) or in normal human keratinocytes (Shen et al., 2001). The experiments herein employed DXR as the DNAdamaging agent, a recognized topoisomerase II poison that generates reactive oxygen species (ROS). Treatments with DXR were performed 24 h after the beginning of the cell cultures, when most of the stimulated lymphocytes are in the middle of the S phase. It is noteworthy that SM treatment alone (0.5, 1.0 and 2.0 lM) increased the frequencies of chromosomal aberrations, abnormal metaphases and
apoptotic cells. Despite these significant genotoxic effects, treatments with SM in all tested concentrations were effective in reducing genotoxic and clastogenic effects of DXR. A similar ‘dual behavior’ was also observed with vitamin C which exhibited both pro-oxidative and anti-oxidative activity by decreasing cell death, membrane damage and lipid peroxidation subsequent to oxygen exposure; this dual role is probably due to its opposing action on two types of oxidative stress (Shi et al., 2005). However, the lower concentration of SM (0.25 lM) did not show a clastogenic effect and reduced the frequencies of chromosomal aberrations significantly. Roussyn et al. (1996) demonstrated that SM at 0.1 mM was able to suppress in 75% of single-strand break formation. Our results showed that SM had the ability to reduce the frequencies of chromosomal aberrations and aberrant metaphases. This can be explained by a cell cycle perturbation by selective cell killing, reducing the frequency of mitosis available for the chromosomal aberration record. Although SM treatment increased the number of apoptotic cells, it was not sufficient to reduce the number of viable cells and the MI index was not reduced. Furthermore, when combined with DXR treatment, SM prevented cell death induced by this chemotherapeutic agent and increased the mitotic index, thereby reducing the cytotoxicity of DXR. Moreover, it is important to consider that the comet assay is performed only with viable cells and our results showed that SM significantly reduced the DNA damage index. This is due to both the reduction in the number of cells with comets as well as the reduction in the size of comet tails. Whereas most of the comets observed in the DXR treatment belonged to classes 2, 3 and 4, the SM treated cultures exhibited predominantly class 1 comets. It is interesting to observe that the protective effects of SM demonstrated here did not occur in a dose-dependent manner and the ability of SM to reduce the DXR-induced damage equally in the lowest and in the highest concentrations tested here, can be explained by its antioxidant properties. In vitro supplementation with inorganic selenium or various forms of organoselenium compounds has been shown to inhibit both chemically and physically-induced oxidative damage (El-Bayoumy, 2001). Furthermore, protection against oxidative damage was proposed because SM induces glutathione peroxidase (GPx) and thioredoxin reductase which are seleno-enzymes capable of scavenging free radicals directly and may also play a role in protection against DNA damage (Ganther, 1999). Bordoni et al. (2003) demonstrated that SM at 10–50 nM increased the GPx activity but, 0.5 lM was the most effective concentration. They found that even with concentrations at 1 lM and 10 lM, the activity of GPx was the same. Thus, the plateau of GPx activity could explain the lack of doseresponse observed in the present results. In the present study, data from the chromosomal aberration assay also showed the ability of SM to reduce chromatid and isochromatid breaks induced by DXR, which are more drastic lesions than those detected by the comet
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Table 3 Number and type of chromosomal aberrations in human lymphocytes submitted to different treatments with SM and/or DXR in vitro Treatment
Number of chromosome aberrations
Reduction (%)
Type of break ct
tr
qr
Totalc
ic
r
dic
Totald
Totalc+d
CA (X ± SE)
Chromatid
Isochromatid
Control DXR 0.25 lM-SM 0.5 lM-SM 1.0 lM-SM 2.0 lM-SM SM4
6 76 11 19 21 21
0 3 0 0 0 1
0 1 0 0 0 0
6 80 11 19 21 22
3 26 3 6 6 5
0 0 0 0 0 0
0 5 4 1 6 5
3 31 4 7 6 5
9 111a 15 26a 27a 27a
1.5 ± 0.6 18.5a ± 1.8 2.5 ± 0.8 4.3a ± 2.0 4.5a ± 2.3 4.5a ± 1.5
– – – – – –
– – – – – –
SM 2 h before DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
25 23 11 33
0 0 0 0
0 0 0 0
25 23 11 33
8 9 10 12
0 0 0 1
1 1 0 1
9 10 10 14
34 33 21 47
5.6a,b ± 1.2 5.5a,b ± 0.3 3.5a,b ± 1.8 7.8a,b ± 1.3
74.3 77 93.2 63.5
78.5 75 75 60.7
Simultaneous treatment 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
30 29 20 23
0 1 1 0
0 0 0 0
30 30 21 23
5 10 11 11
0 0 0 0
1 0 1 0
6 10 12 11
36 40 33 34
6a,b ± 1.4 6.6a,b ± 0.8 5.5a,b ± 1.1 5.6a,b ± 1.4
67.5 67.5 81.0 77
89.2 75 67.8 71.4
SM 2 h after DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
23 29 41 29
1 0 0 0
0 0 0 0
24 29 41 29
9 12 9 11
0 0 0 0
1 0 0 0
10 12 0 11
34 41 50 40
5.6a,b ± 1.7 6.8a,b ± 1.8 8.3a,b ± 0.6 6.6a,b ± 1.2
75.6 68.9 52.7 68.9
75 67.8 78.5 71.4
Six hundred metaphases cells were analyzed in each treatment (100 metaphases/treatment/experiment). DXR-doxorubicin at 0.15 lg/m concentration; SM-selenomethionine; CA-chromosomal aberrations. a p < 0.05 different from the negative control. b p < 0.01 different from the positive control (DXR). c Chromatid breaks (i.e. chromatid breaks – ct; triradial – tr; quadriradial – qr). d Isochromatid breaks (i.e. chromosome breaks – ic; dicentric – dic; ring – r).
Table 4 Percentage of apoptotic cells observed in human lymphocytes submitted to different treatments with SM and/or DXR Treatment
Cells scored
Viable cells (%)
Apoptotic cells (%)
Necrotic cells (%)
Control DXR 0.25 lM 0.5 lM-SM 1.0 lM-SM 2.0 lM-SM 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
1500 1500 1500 1500 1500 1500 1500 1500 1500 1500
96.9 67 93.8 93.4 91.6 92.6 88.8 89.7 87.6 86.4
2 29.6a 5.2a 5.8a 6.9a 6.3a 10.4b 11b 10.8b 12.2b
1.1 3.4 1 0.8 1.5 1.1 0.8 1.3 1.6 1.4
Five hundred cells were scored in each one of the three repetitions. DXR-doxorubicin at 0.15 lg/m concentration; SM-selenomethionine. a p < 0.05 different from the negative control. b p < 0.01 different from the positive control (DXR).
assay. In a previous study using an in vivo model, we demonstrated that SM and sodium selenite prevented the clastogenic effects of DXR reducing the frequencies of micronucleated erythrocytes and the extent of DNA damage detected by comet assay. In the same study, even after DXR treatment, selenium compounds maintained the thiobarbituric acid reactive substances, increased the hepatic
concentration of glutathione and allowed for the maintenance of hepatic concentrations of vitamin E (Santos et al., 2007). Selenium also prevented partially the DXRinduced oxidative damage in cardiac tissue of rats supplemented with selenium enriched diet (Danesi et al., 2006). The role of selenium modulating the antioxidant defense could render loss of effectiveness during chemotherapy, however, recently, other mechanisms related to SM antioxidant properties have been proposed to explain the chemoprotective effects of this selenium compound. Seo et al. (2002a) demonstrated that SM concentration is a determinant of basal p53 activity and that protection from UV-induced DNA damage by SM is p53-dependent in a repair pathway where this protein regulates Gadd45a and p48XPE, the gene products of which participate in the nucleotide excision repair pathway (NER). The regulation of p53 in this study was dependent on Ref-1, a substrate for reduction by the thioredoxin reductase system (Brash and Havre, 2002). It was also demonstrated that SM induced DNA repair in human fibroblasts by enhancing DNA repair protein complexes (Seo et al., 2002b). Although the detailed study of Seo et al. (2002b) did not detect cell cycle arrest and/or apoptosis with SM treatment, Kennedy et al. (2004) showed that supplementation with SM allowed the checkpoint genes ATR and CHK2 to be
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expressed at higher levels with or without radiation exposure. Thus, the role of selenium modulating p53 activity is of fundamental importance in non p53-mutant cells (Seo et al., 2002a). Fischer et al. (2007) demonstrated that SM did not induce expression of key NER recognition factors or alter the rate or overall level of DNA repair in p53 null cells or other tumor cell lines tested. The conclusion of this study was that selenium selectively protects genetically normal cell from DNA-damaging chemotherapeutics, while simultaneously offering no detectable protection to cells either completely lacking p53 or only mutant p53. Therefore, in nontarget tissues, an increase in the basal levels of NER damage recognition factors following selenium supplementation promotes an increase in the basal rate of NER, which can better tolerate additional damage from chemotherapy. A preclinical work has shown that nude mice bearing human tumor xenografts that received daily supplementation with SM before and during chemotherapy also better tolerated increasing doses of irinotecan with no evidence of antagonistic effects in antitumor activity (Cao et al., 2004). Regarding the timing of the supplementation with SM the present results showed that pre, pos or simultaneous treatment reduced equally the genotoxic effects induced by DXR in all tested concentrations; similar results were found by Antunes and Takahashi (1999) that treated lymphocytes cultures with vitamin C, a potent antioxidant, before, simultaneously or after DXR treatment. The action of a protective agent by a single mechanism may be the exception rather the rule, and therefore SM may have a protective effect based on different mechanisms (Gebhart, 1992) however, as mentioned above, SM act in the antioxidant defense and modulate the response of DNA repair factors and p53 expression. In the present work, only the lowest concentration of SM (0.25 lM) was not clastogenic and able to reduce the damage induced by DXR treatment. The other tested concentrations of SM exhibited some genotoxic effects however, also reduced the DNA damage induced by DXR. The literature underlines the observation that selenium, in low concentrations, may have anticarcinogenic effects whereas in high concentrations, it can be genotoxic and carcinogenic (Bronzetti et al., 2001). Thus, it is of fundamental importance to determine the optimal concentration of SM that provides protection against genetic damage with the least toxicity. In conclusion, our results demonstrated that SM treatment reduced DNA damage and cytotoxicity induced by DXR in human lymphocytes, and exhibited chemoprotective effect under the conditions used in the present study.
Acknowledgements We would like to thank Alexandre Souto Martinez for statistical support. This investigation was supported with
grants by CAPES (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior), CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico) and FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo). References Alaejos, M.S., Romero, F.J.D., Romero, C.D., 2000. Selenium and cancer: some nutritional aspects. Nutrition 16, 376–383. Anderson, D., Yu, T.W., Browne, M.A., 1997. The use of the same image analysis system to detect genetic damage in human lymphocytes treated with doxorubicin in the comet and fluorescent in situ hybridization (FISH) assays. Mutat. Res. 390, 69–77. Antunes, L.M.G., Takahashi, C.S., 1999. Protection and induction of chromosomal damage by vitamin C in human lymphocytes cultures. Terat. Carcinog. Mutag. 19, 53–59. Baguley, B.C., Ferguson, L.R., 1998. Mutagenic properties of topoisomerase-targeted drugs. Biochim. Biophys. Acta 1400, 213–222. Bordoni, A., Biagi, P.L., Angeloni, C., Leoncini, E., Muccinelli, I., Hrelia, S., 2003. Selenium supplementation can protect cultured rat cardiomyocytes from hypoxia/reoxygenation damage. J. Agric. Food Chem. 51, 1736–1740. Brash, D.E., Havre, P.A., 2002. New careers for antioxidants. PNAS 99, 13969–13971. Bronzetti, G., Cini, M., Andreoli, E., Caltavuturo, L., Panuzio, M., Croce, C.D., 2001. Protective effects of vitamins and selenium compounds in yeast. Mutat. Res. 496, 105–115. Cao, S., Durrani, F.A., Rustum, Y.M., 2004. Selective modulation of the therapeutic efficacy of anticancer drugs by selenium containing compounds against human xenografts. Clin. Cancer Res. 10, 2561– 2569. Danesi, F., Malaguti, M., Di Nunzio, M., Maranesi, M., Biagi, P.L., Bordoni, A., 2006. Counteraction of adriamycin-induced oxidative damage in rat heart by selenium dietary supplementation. J. Agric. Food Chem. 54, 1203–1208. Dhawan, A., Kayani, M.A., Parry, J.M., Parry, E., Anderson, D., 2003. Aneugenic and clastogenic effects of doxorubicin in human lymphocytes. Mutagenesis 18, 487–490. El-Bayoumy, K., 2001. The protective role of selenium on genetic damage and cancer. Mutat. Res. 475, 123–139. Ferguson, L.R., Pearson, A.E., 1996. The clinical use of mutagenic anticancer drugs. Mutat. Res. 355, 1–12. Fischer, J.L., Mihelc, E.M., Pollok, K.E., Smith, M.L., 2007. Chemotherapeutic selectivity conferred by selenium: a role for p53-dependent DNA repair. Mol. Cancer Ther. 6, 355–361. Ganther, H.E., 1999. Selenium metabolism, selenoproteins and mechanisms of cancer prevention: complexities with thioredoxin reductase. Carcinogenesis 20, 1657–1666. Gebhart, E., 1992. Anticlastogenicity in cultured mammalian cells. Mutat. Res. 267, 211–220. Gewitz, D.A., 1999. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem. Pharmacol. 57, 727. Kennedy, A.R., Ware, J.H., Guan, J., Donahue, J., Blaglow, J.E., Zhou, Z., Stewart, J., Vazquez, M., Wan, X.S., 2004. Selenomethionine protects against adverse biological effects induced by space radiation. Free Rad. Biol. Med. 36, 259–266. Li, H., Stampfer, M.J., Giovannucci, E.L., Morris, J.S., Willett, W.C., Gaziano, J.M., Ma, J., 2004. A prospective study of plasma selenium levels and prostate cancer risk. J. Natl. Cancer Inst. 96, 696–703. Moorhead, O.S., Nowell, P.C., Mellman, W.J., Battips, D.M., Hungerford, D.A., 1960. Chromosome preparations of leucocytes cultured from human peripheral blood. Exp. Cell Res. 20, 613–616. Postovsky, S., Arush, M.W., Diamond, E., Elhasid, R., Shoshani, R., Shamir, R., 2003. The prevalence of low selenium levels in newly
R.A.d. Santos, C.S. Takahashi / Food and Chemical Toxicology 46 (2008) 671–677 diagnosed pediatric cancer patients. Pediat. Hematol. Oncol. 20, 273– 280. Quiles, J.L., Huertas, J.R., Battino, M., Mataix, J., Ramı´rez-Tortosa, M.C., 2002. Antioxidant nutrients and adriamycin toxicity. Toxicology 180, 79–95. Roussyn, I., Karlis, B., Masumoto, H., Sies, H., 1996. Seleniumcontaining compounds protect DNA from single-strand breaks by peroxinitrite. Arch. Biochem. Biophys. 330, 216–218. Santos, R.A., Jorda˜o Jr, A.A., Vannucchi, H., Takahashi, C.S., 2007. Protection of doxorubicin-induced DNA damage by sodium selenite and selenomethionine in Wistar rats. Nutr. Res. 27, 343–348. Savage, J.R.K., 1976. Classification and relationships of induced chromosomal structural changes. J. Med. Genet. 13, 103–122. Schrauzer, G.N., 2000. Selenomethionine: a review of its nutritional significance, metabolism and toxicity. J. Nutr. 130, 1653–1656. Seo, Y.R., Kelley, M.R., Smith, M.L., 2002a. Selenomethionine regulation of p53 by a ref1-dependent redox mechanism. PNAS 99, 14548–14553. Seo, Y.R., Sweeney, C., Smith, M.L., 2002b. Selenomethionine induction of DNA repair response in human fibroblasts. Oncogene 21, 3663– 3669.
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Shen, C., Song, W., Pence, B.C., 2001. Interactions of selenium compounds with other antioxidants in DNA damage apoptosis in human normal keratinocytes. Cancer Epidemiol. Biomark. Prev. 10, 385–390. Shi, M., Xu, B., Azakami, K., Morikawa, T., Watanabe, K.O., Marimoto, K., Komatsu, M., Aoyama, K., Takeuchi, T., 2005. Dual role of vitamin C in an oxygen-sensitive system: Discrepancy between DNA damage and cell death,. Free Radic. Res. 39, 213–320. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. A simple technique for quantitation of low DNA levels of DNA damage in individual cells. Exp. Cell Res. 175, 184–191. Stewart, M.S., Spallholz, J.E., Neldner, K.H., Pence, B.C., 1999. Selenium compounds have disparate abilities to impose oxidative stress and induce apoptosis. Free Radic. Biol. Med. 26, 42–48. Tapiero, H., Townsend, D.M., Tew, K.D., 2003. The antioxidant role of selenium and seleno-compounds. Biomed. Pharmacother. 57, 134–144. Zeiger, E., 2007. What is needed for an acceptable antimutagenicity manuscript? Mutat. Res. 626, 1–3.
Food and Chemical Toxicology 46 (2008) 671–677 www.elsevier.com/locate/foodchemtox
Anticlastogenic and antigenotoxic effects of selenomethionine on doxorubicin-induced damage in vitro in human lymphocytes Raquel Alves dos Santos a
a,*
, Catarina Satie Takahashi
a,b
Department of Genetics, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeira˜o Preto, SP, Brazil b Department of Biology, Faculty of Philosophy, Sciences and Letters of Ribeira˜o Preto, University of Sa˜o Paulo, Av. Bandeirantes, 3900, 14040-901 Ribeira˜o Preto, SP, Brazil Received 12 March 2007; accepted 12 September 2007
Abstract The use of antioxidants during chemotherapy has been shown to reduce or prevent the undesirable effects experienced by healthy cells. Micronutrient selenium is well known for its antioxidant properties; however, selenium exhibits a bimodal nature in that both its beneficial and toxic properties lie within a limited and narrow dose range. The present study investigated the possible protective effects of selenomethionine (SM) on the cytotoxicity, genotoxicity and clastogenicity of the chemotherapic doxorubicin (DXR), a key chemotherapic used in cancer treatment. Human peripheral lymphocytes were treated in vitro with varying concentrations of SM (0.25 lM, 0.5 lM, 1.0 lM and 2.0 lM), tested in combination with DXR (0.15 lg/mL). SM alone was not cytotoxic and when combined with DXR treatment, reduced the DNA damage index significantly, the frequency of chromosomal aberrations, the number of aberrant metaphases and the frequency of apoptotic cells. The mechanism of chemoprotection of SM may be related to its antioxidant properties as well as its ability to interfere with DNA repair pathways. Therefore this study showed that SM is effective in reducing the genetic damage induced by the antitumoral agent DXR. 2007 Elsevier Ltd. All rights reserved. Keywords: Selenium; Selenomethionine; Doxorubicin; Comet assay; Chromosomal aberrations
1. Introduction A large number of dietary components, such as vitamin C and E, lycopene and selenium are known for their antioxidant properties. Selenium is an essential micronutrient for animals, humans and microorganisms. A direct relationship between selenium intake and cancer risk in humans has been reported, indicating that selenium defi-
Abbreviations: CAs, chromosomal aberrations; CT, chromatid breaks; DC, dicentric; DI, DNA damage index; DXR, doxorubicin; IC, chromosome breaks; MI, mitotic index; QR, quadriradial; R, ring; SM, selenomethionine; TR, triradial. * Corresponding author. Tel.: +55 016 3602 3082; fax: +55 016 3602 3761. E-mail address: [email protected] (R.A.d. Santos). 0278-6915/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.09.090
ciency enhances the probability of developing cancer (Postovsky et al., 2003; Li et al., 2004). However selenium, like some other trace elements, is bimodal in nature whereby its beneficial properties occur in a limited range of daily intake below which it cannot perform its essential functions, and above which it is toxic (Alaejos et al., 2000). As a result of these properties, selenium can be included in the class of ‘‘Janus compounds’’, having two ‘faces’ on the same head. It is well established that selenium participates in the process of detoxification by forming part of glutathione peroxidase, a cellular enzyme that maintains appropriately low levels of hydrogen peroxide within a cellular environment (Tapiero et al., 2003). Despite its antioxidant properties and requirement for human and animal nutrition, the appropriate form of selenium for supplementation continues to be debated. There exists a wide variety of selenium forms including sodium
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selenite, sodium selenate, selenomethionine, selenocysteine and selenocystine. The L-isomer of selenomethionine (SM) is the most important natural food form of selenium found in seafood, grain products, meat and abundantly in Brazilian nuts (Alaejos et al., 2000; Schrauzer, 2000). Some chemotherapeutic approaches have proposed the use of antioxidants to minimize cytotoxicity and the damage induced in normal tissues by antitumor agents that produce free radicals (Antunes and Takahashi, 1999). Although chemotherapy plays a major role in treating various cancers, especially in controlling advanced stages of malignancies in clinical settings, most cytotoxic chemotherapeutic agents used in the treatment of a wide range of cancers are not selective touching, neoplastic and health cells by its clastogenic effects (Ferguson and Pearson, 1996). These events may have important consequences in cancer chemotherapy since mutations may lead to drug resistance, limiting further therapy. Moreover, germ cell mutations may be transmitted to future generations and mutations may result in the development of secondary tumors from cells that were originally benign (Baguley and Ferguson, 1998). The chemotherapeutic doxorubicin (DXR), an anthracycline antibiotic commonly used to treat a wide variety of cancers (Quiles et al., 2002), produces an increase in DNA strand breakage, in the percentage of abnormal frequencies of chromosomal damage in the FISH and conventional chromosomal aberration assays and also increases micronucleus formation in human lymphocytes in vitro (Anderson et al., 1997; Dhawan et al., 2003), related to its activity when generating reactive oxygen species and inhibiting topoisomerase II complex (Gewitz, 1999). Therefore, the present study was undertaken to investigate the possible antigenotoxic and anticlastogenic effects of selenomethionine (SM) in DNA damage induced by DXR in human lymphocytes in vitro. Damage was detected by assessing the mitotic index, chromosomal aberrations, comet assays and apoptotic cells. 2. Materials and methods 2.1. Cells and medium Human peripheral blood was collected using heparinized vials from six healthy donors aged 26–30 years. Lymphocytes were isolated with plasma and cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 20% fetal calf serum (Cultilab, Campinas, SP, Brazil), penicillin (5 lg/mL), streptomycin (10 lg/mL) and 2% phytohemagglutinin (Life Technologies, Grand Island, NY). Cells were cultured at 37 C in culture flasks containing 5 ml of complete medium. The National Research Ethics Committee (process no. 8709/2001) approved the protocol of the experiments and written consent was obtained from each blood donor prior to joining the study.
2.2. Chemicals L-Selenomethionine (CAS# 1464-42-2) was purchased from Acros Organics (New Jersey, USA) and DXR (CAS# 23214-92-8) (Laborato´rios Eurofarma, Sa˜o Paulo, Brazil) was donated from the Chemotherapy
Center, Faculty of Medicine, USP, Ribeira˜o Preto. Both SM and DXR were dissolved in sterile distilled water prior to use.
2.3. Culture treatments The concentrations of SM (0.25 lM, 0.5 lM, 1.0 lM and 2.0 lM) tested together with DXR were established in preliminary experiments; concentrations of SM above 2 lM were cytotoxic and reduced significantly mitotic index and cell viability (data not shown). The concentration of DXR (0.15 lg/mL) was similarly defined in preliminary experiments and besides the mitotic index, it was considered the number of chromosomal aberrations. In combined treatments (SM plus DXR) the values of mitotic index was similar to the negative control. The toxicity of the combined treatment is a critical factor because cytotoxic and cytostatic effects can mimic antimutagenicity because they interfere with the appearance of the mutant cells (Zeiger, 2007). Three different types of SM treatment were assessed on DXR-induced damage cells. The cultures were treated with DXR 24 h after initiation of incubation and treatment with SM was performed either 2 h before, simultaneously, or 2 h after DXR treatment. After each respective treatment, DXR and SM remained in the cultures until harvesting. Cells were harvested after 24 h of DXR treatment and then assessed for cell viability, apoptosis (apoptotic assay) and DNA damage of individual cells (comet assay), and after 26 h, for the analysis mitotic index and chromosomal aberrations, as DXR induces a delay in the cell cycle.
2.4. Comet assay An aliquot of 300 lL from each culture was taken after 48 hours of incubation to test for cell viability by trypan blue exclusion and for the alkaline version of the Comet assay as described by Singh et al. (1988). Briefly, 300 lL of the cell suspension was centrifuged for 5 minutes (500 rpm) in a refrigerated microcentrifuge (Eppendorff). The resulting pellet was homogenized with 80 lL of a low melting point agarose (0.5%), spread onto microscope slides pre-coated with a normal melting point agarose (1.5%), and covered with a coverslip. After 5 min at 4 C, the coverslip was removed and the slides were immersed in cold lysis solution (2.4 M NaCl; 100 mM EDTA; 10 mM Tris, 10% DMSO and 1% TritonX, pH 10) for 24 h. After lysis, the slides were placed in an electrophoresis chamber and covered with electrophoresis buffer (300 mM NaOH per 1 mM EDTA, pH > 13), for a remaining 20 min to allow for unwinding of DNA. The electrophoresis proceeded for 20 min (25 V and 300 mA). Afterwards, the slides were submerged for 15 min in a neutralization buffer (0.4 M Tris–HCl, pH 7.5), dried at room temperature and fixed in 100% ethanol for 5 min. Slide staining was performed immediately before analyzing using ethidium bromide (20 lg/mL). Slides were prepared in duplicate and 100 cells were screened per sample (50 cells from each slide) in a fluorescent microscope (ZEISS, Germany) equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm using a 40· objective. The nucleus was classified visually according to the migration of the fragments in: class 0 (no damage); class 1 (little damage with a short tail length smaller than the diameter of the nucleus); class 2 (medium damage with a tail length one or two times the diameter of the nucleus); 3 (significant damage with a tail length between two and a half to three times the diameter of the nucleus); class 4 (significant damage with a long tail of damage greater than three times the diameter of the nucleus).
2.5. Metaphases preparation To analyze chromosomal aberrations (CAs), a metaphase preparation was performed after 50 h of cell culture. Ninety minutes before harvesting, 12.5 lL colchicine (0.016%, Sigma) was added to each culture. The conventional cell harvest procedure was followed according to Moorhead et al. (1960). The cells were treated with hypotonic KCl solution (0.075 M) for 10 min, fixed with methanol:acetic acid (3:1), air-dried and stained with Giemsa: So¨rensen buffer (1:30) for 5 min. All slides were coded. The same scorer analyzed the slides in a blind test and a total of 100 cells per
R.A.d. Santos, C.S. Takahashi / Food and Chemical Toxicology 46 (2008) 671–677 treatment per individual were scored for the presence of CAs classified according to Savage (1976) and 2000 cells per treatment per individual were scored for MI.
2.6. Morphological characterization of normal, apoptotic and necrotic cells The frequencies of normal, apoptotic and necrotic cells were determined only where SM was added 2 h before DXR treatment, using 2 lL of staining solution [fluorescein diacetate dissolved in dimethyl-sulfoxide (15 lg/mL), propidium iodide (5 lg/mL) and Hoechtst 33342 (2 lg/mL) all from Sigma] mixed with 100 lL of cell suspension. Five hundred cells were analyzed per treatment, for three independent experiments, using an epi-fluorescence microscope (Carl Zeiss, Germany). A blue and intact nucleus was considered normal, a blue and fragmented nucleus was considered apoptotic and a red nucleus was considered necrotic.
2.7. Statistical analysis Six independent experiments were analyzed and statistical analysis was performed using the Kruskal–Wallis One-way Method of Variance followed by Student–Newman–Keuls considering a confidence interval of 95%.
3. Results Table 1 shows the frequencies of cells with the Comet and DNA damage index (DI) in human lymphocytes treated with different concentrations of SM and with DXR (0.15 lg/mL). DXR alone and SM alone (0.5 lM, 1.0 lM and 2.0 lM) increased the DI, the frequencies of chromosomal aberrations, abnormal metaphases and the frequen-
673
cies of apoptotic cells (Tables 1–4). However, the combined treatment of DXR and SM led to a statistically significant reduction in DI and in the frequencies of comet positive cells relative to the DXR treatment alone. The reduction was between 96% and 97.5% and was neither dose-dependent nor different for all treatments. Tables 2 and 3 show the anticlastogenic effect of different treatments with SM. There was a significant reduction in the number of cells with aberrations and in the total number of chromosomal aberrations however, this reduction was similar in all tested concentrations of SM. Table 2 also shows the mean of mitotic index (MI) obtained in each treatment of SM combined with DXR, with variations from 3.2 to 4.7. The number of chromosomal aberrations in cells was significantly reduced by SM treatments (Table 3) when compared to the positive control. The types of chromosomal aberrations detected are also depicted in Table 3. Chromatid and chromosome gaps were excluded from the analysis. When calculating the total number of chromatid type breaks (chromatid breaks, triradial and quadriradial) or isochromatid breaks (chromosome breaks, dicentric and rings), it was observed that SM reduced both types similarly when compared to the DXR treatment alone. Table 4 demonstrates that SM treatment significantly reduced the number of apoptotic cells induced by DXR and, consequently, increased the number of viable cells.
Table 1 Antigenotoxic effect of SM in human lymphocytes treated in vitro with DXR Treatment
Cells with Comet (%)
Distribution of Comet classes 0 (%)
1 (%)
2 (%)
3 (%)
4 (%)
DNA damage index X ± SE
Reduction (%)
Negative control DXR 0.25 lM-SM 0.5 lM-SM 1.0 lM-SM 2.0 lM-SM
29.3 88.1 48.3 56.5 60.5 67.8
70.7 11.8 51.7 43.5 39.5 32.1
25.1 32.1 34.5 40.4 40.8 45.4
3.5 38.7 11.3 14.1 15.5 19.9
0.7 11.7 2.1 1.5 3.8 2.5
0 5.7 0.4 0.5 0.4 0.1
0.34 ± 0.03 1.67a ± 0.09 0.64a ± 0.09 0.75a ± 0.09 0.84a ± 0.07 0.93b ± 0.06
– – – – – –
SM 2 h before DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
73.8 68.3 60.8 60.6
31.4 31.7 39.1 39.4
45.4 45 44.5 41.1
20.2 20.8 14 16.7
2.2 2.4 1.7 2.7
0.8 0.1 0.7 0.1
0.96b ± 0.04 0.94b ± 0.12 0.80b ± 0.05 0.79b ± 0.04
96.2 96.3 97.1 97.2
Simultaneous treatment 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXaR 2.0 lM-SM + DXR
59.6 54 58.1 61
40 46 41.8 39
40.7 35.5 39.7 41
16.4 16.8 16.4 16.5
1.8 1.7 2 3
0.7 0 0.1 0.5
0.82b ± 0.06 0.74b ± 0.07 0.74b ± 0.05 0.85b ± 0.09
97 97.5 97.5 96.8
SM 2 h after DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
62.3 62.1 64.3 62.1
37.7 37.8 35.7 37.8
39.4 44 44.4 43.4
20.8 16.9 16.7 15.5
1.8 0.8 2.6 2.8
0.3 0.5 0.6 0.5
0.88b ± 0.02 0.82b ± 0.03 0.88b ± 0.07 0.84b ± 0.06
96.6 97 96.6 96.9
Six hundred nuclei were analyzed in each treatment (100 nuclei/treatment/experiment). DXR-doxorubicin at 0.15 lg/mL concentration, SM-selenomethionine. a p < 0.05 different from the negative control. b p < 0.01 different from the positive control (DXR).
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Table 2 Mean and Standard Error (X ± SE) of Mitotic Index (MI) and abnormal metaphases observed in human lymphocytes submitted to different treatments with SM and/or DXR in vitro Treatment
MI X ± SE
Abnormal metaphases
Control DXR 0.25 lM-SM 0.5 lM-SM 1.0 lM-SM 2.0 lM-SM + DXR
5.4 ± 0.2 2.7a ± 1.8 4.4 ± 0.3 4.4 ± 0.2 3.9 ± 0.2 4.1 ± 0.3
9 102 14 23 25 26
1.5 ± 0.6 17a ± 1.4 2.3 ± 0.8 3.8a ± 2.0 4.1a ± 2.3 4.3a ± 1.8
SM 2 h before DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
3.8 ± 0.2 4.1 ± 0.3 4.7b ± 0.5 3.8 ± 0.3
31 30 21 42
5.1a,b ± 1.5 5.0a,b ± 0.3 3.5a,b ± 1.2 7.0a,b ± 1.3
76.7 77.4 87 64.5
Simultaneous treatment 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
3.2 ± 0.4 3.7 ± 0.4 3.4 ± 0.3 3.4 ± 0.5
34 36 30 30
5.6a,b ± 0.6 6.0a,b ± 0.6 5.0a,b ± 1.4 5.0a,b ± 1.2
73.5 70.9 77.4 77.4
SM 2 h after DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
4.2 ± 0.2 4.1 ± 0.4 3.8 ± 0.3 3.9 ± 0.3
32 37 44 38
5.3a,b ± 2.0 6.1a,b ± 1.1 7.3a,b ± 0.6 6.3a,b ± 1.1
75.4 70.3 62.5 69
Total
X ± SE
Reduction (%) – – – – – –
Six hundred metaphases were analyzed in each treatment (100 metaphases/treatment/experiment). DXR-doxorubicin at 0.15 lg/mL concentration, SM-selenomethionine. a p < 0.01 different from the negative control. b p < 0.01 different from the positive control (DXR).
4. Discussion The trace element selenium is an essential micronutrient of fundamental importance to human health as it is a component of selenoproteins with important enzymatic functions. SM is a non-redoxing selenium compound that does not generate superoxide and is less toxic than inorganic selenium forms (Stewart et al., 1999). The present study investigated the antigenotoxic and anticlastogenic effects of SM against genetic damages induced by DXR, a potent genotoxic anticancer drug that generates undesirable effects in healthy cells. It was observed that SM treatment alone did not reduce MI significantly. The cytotoxic and antiproliferative effects of SM have been extensively investigated; it exhibits no cytotoxicity in BALB/c MK-2 cells exposed to concentrations up to 250 lg/mL (Stewart et al., 1999) or in normal human keratinocytes (Shen et al., 2001). The experiments herein employed DXR as the DNAdamaging agent, a recognized topoisomerase II poison that generates reactive oxygen species (ROS). Treatments with DXR were performed 24 h after the beginning of the cell cultures, when most of the stimulated lymphocytes are in the middle of the S phase. It is noteworthy that SM treatment alone (0.5, 1.0 and 2.0 lM) increased the frequencies of chromosomal aberrations, abnormal metaphases and
apoptotic cells. Despite these significant genotoxic effects, treatments with SM in all tested concentrations were effective in reducing genotoxic and clastogenic effects of DXR. A similar ‘dual behavior’ was also observed with vitamin C which exhibited both pro-oxidative and anti-oxidative activity by decreasing cell death, membrane damage and lipid peroxidation subsequent to oxygen exposure; this dual role is probably due to its opposing action on two types of oxidative stress (Shi et al., 2005). However, the lower concentration of SM (0.25 lM) did not show a clastogenic effect and reduced the frequencies of chromosomal aberrations significantly. Roussyn et al. (1996) demonstrated that SM at 0.1 mM was able to suppress in 75% of single-strand break formation. Our results showed that SM had the ability to reduce the frequencies of chromosomal aberrations and aberrant metaphases. This can be explained by a cell cycle perturbation by selective cell killing, reducing the frequency of mitosis available for the chromosomal aberration record. Although SM treatment increased the number of apoptotic cells, it was not sufficient to reduce the number of viable cells and the MI index was not reduced. Furthermore, when combined with DXR treatment, SM prevented cell death induced by this chemotherapeutic agent and increased the mitotic index, thereby reducing the cytotoxicity of DXR. Moreover, it is important to consider that the comet assay is performed only with viable cells and our results showed that SM significantly reduced the DNA damage index. This is due to both the reduction in the number of cells with comets as well as the reduction in the size of comet tails. Whereas most of the comets observed in the DXR treatment belonged to classes 2, 3 and 4, the SM treated cultures exhibited predominantly class 1 comets. It is interesting to observe that the protective effects of SM demonstrated here did not occur in a dose-dependent manner and the ability of SM to reduce the DXR-induced damage equally in the lowest and in the highest concentrations tested here, can be explained by its antioxidant properties. In vitro supplementation with inorganic selenium or various forms of organoselenium compounds has been shown to inhibit both chemically and physically-induced oxidative damage (El-Bayoumy, 2001). Furthermore, protection against oxidative damage was proposed because SM induces glutathione peroxidase (GPx) and thioredoxin reductase which are seleno-enzymes capable of scavenging free radicals directly and may also play a role in protection against DNA damage (Ganther, 1999). Bordoni et al. (2003) demonstrated that SM at 10–50 nM increased the GPx activity but, 0.5 lM was the most effective concentration. They found that even with concentrations at 1 lM and 10 lM, the activity of GPx was the same. Thus, the plateau of GPx activity could explain the lack of doseresponse observed in the present results. In the present study, data from the chromosomal aberration assay also showed the ability of SM to reduce chromatid and isochromatid breaks induced by DXR, which are more drastic lesions than those detected by the comet
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Table 3 Number and type of chromosomal aberrations in human lymphocytes submitted to different treatments with SM and/or DXR in vitro Treatment
Number of chromosome aberrations
Reduction (%)
Type of break ct
tr
qr
Totalc
ic
r
dic
Totald
Totalc+d
CA (X ± SE)
Chromatid
Isochromatid
Control DXR 0.25 lM-SM 0.5 lM-SM 1.0 lM-SM 2.0 lM-SM SM4
6 76 11 19 21 21
0 3 0 0 0 1
0 1 0 0 0 0
6 80 11 19 21 22
3 26 3 6 6 5
0 0 0 0 0 0
0 5 4 1 6 5
3 31 4 7 6 5
9 111a 15 26a 27a 27a
1.5 ± 0.6 18.5a ± 1.8 2.5 ± 0.8 4.3a ± 2.0 4.5a ± 2.3 4.5a ± 1.5
– – – – – –
– – – – – –
SM 2 h before DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
25 23 11 33
0 0 0 0
0 0 0 0
25 23 11 33
8 9 10 12
0 0 0 1
1 1 0 1
9 10 10 14
34 33 21 47
5.6a,b ± 1.2 5.5a,b ± 0.3 3.5a,b ± 1.8 7.8a,b ± 1.3
74.3 77 93.2 63.5
78.5 75 75 60.7
Simultaneous treatment 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
30 29 20 23
0 1 1 0
0 0 0 0
30 30 21 23
5 10 11 11
0 0 0 0
1 0 1 0
6 10 12 11
36 40 33 34
6a,b ± 1.4 6.6a,b ± 0.8 5.5a,b ± 1.1 5.6a,b ± 1.4
67.5 67.5 81.0 77
89.2 75 67.8 71.4
SM 2 h after DXR 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
23 29 41 29
1 0 0 0
0 0 0 0
24 29 41 29
9 12 9 11
0 0 0 0
1 0 0 0
10 12 0 11
34 41 50 40
5.6a,b ± 1.7 6.8a,b ± 1.8 8.3a,b ± 0.6 6.6a,b ± 1.2
75.6 68.9 52.7 68.9
75 67.8 78.5 71.4
Six hundred metaphases cells were analyzed in each treatment (100 metaphases/treatment/experiment). DXR-doxorubicin at 0.15 lg/m concentration; SM-selenomethionine; CA-chromosomal aberrations. a p < 0.05 different from the negative control. b p < 0.01 different from the positive control (DXR). c Chromatid breaks (i.e. chromatid breaks – ct; triradial – tr; quadriradial – qr). d Isochromatid breaks (i.e. chromosome breaks – ic; dicentric – dic; ring – r).
Table 4 Percentage of apoptotic cells observed in human lymphocytes submitted to different treatments with SM and/or DXR Treatment
Cells scored
Viable cells (%)
Apoptotic cells (%)
Necrotic cells (%)
Control DXR 0.25 lM 0.5 lM-SM 1.0 lM-SM 2.0 lM-SM 0.25 lM-SM + DXR 0.5 lM-SM + DXR 1.0 lM-SM + DXR 2.0 lM-SM + DXR
1500 1500 1500 1500 1500 1500 1500 1500 1500 1500
96.9 67 93.8 93.4 91.6 92.6 88.8 89.7 87.6 86.4
2 29.6a 5.2a 5.8a 6.9a 6.3a 10.4b 11b 10.8b 12.2b
1.1 3.4 1 0.8 1.5 1.1 0.8 1.3 1.6 1.4
Five hundred cells were scored in each one of the three repetitions. DXR-doxorubicin at 0.15 lg/m concentration; SM-selenomethionine. a p < 0.05 different from the negative control. b p < 0.01 different from the positive control (DXR).
assay. In a previous study using an in vivo model, we demonstrated that SM and sodium selenite prevented the clastogenic effects of DXR reducing the frequencies of micronucleated erythrocytes and the extent of DNA damage detected by comet assay. In the same study, even after DXR treatment, selenium compounds maintained the thiobarbituric acid reactive substances, increased the hepatic
concentration of glutathione and allowed for the maintenance of hepatic concentrations of vitamin E (Santos et al., 2007). Selenium also prevented partially the DXRinduced oxidative damage in cardiac tissue of rats supplemented with selenium enriched diet (Danesi et al., 2006). The role of selenium modulating the antioxidant defense could render loss of effectiveness during chemotherapy, however, recently, other mechanisms related to SM antioxidant properties have been proposed to explain the chemoprotective effects of this selenium compound. Seo et al. (2002a) demonstrated that SM concentration is a determinant of basal p53 activity and that protection from UV-induced DNA damage by SM is p53-dependent in a repair pathway where this protein regulates Gadd45a and p48XPE, the gene products of which participate in the nucleotide excision repair pathway (NER). The regulation of p53 in this study was dependent on Ref-1, a substrate for reduction by the thioredoxin reductase system (Brash and Havre, 2002). It was also demonstrated that SM induced DNA repair in human fibroblasts by enhancing DNA repair protein complexes (Seo et al., 2002b). Although the detailed study of Seo et al. (2002b) did not detect cell cycle arrest and/or apoptosis with SM treatment, Kennedy et al. (2004) showed that supplementation with SM allowed the checkpoint genes ATR and CHK2 to be
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expressed at higher levels with or without radiation exposure. Thus, the role of selenium modulating p53 activity is of fundamental importance in non p53-mutant cells (Seo et al., 2002a). Fischer et al. (2007) demonstrated that SM did not induce expression of key NER recognition factors or alter the rate or overall level of DNA repair in p53 null cells or other tumor cell lines tested. The conclusion of this study was that selenium selectively protects genetically normal cell from DNA-damaging chemotherapeutics, while simultaneously offering no detectable protection to cells either completely lacking p53 or only mutant p53. Therefore, in nontarget tissues, an increase in the basal levels of NER damage recognition factors following selenium supplementation promotes an increase in the basal rate of NER, which can better tolerate additional damage from chemotherapy. A preclinical work has shown that nude mice bearing human tumor xenografts that received daily supplementation with SM before and during chemotherapy also better tolerated increasing doses of irinotecan with no evidence of antagonistic effects in antitumor activity (Cao et al., 2004). Regarding the timing of the supplementation with SM the present results showed that pre, pos or simultaneous treatment reduced equally the genotoxic effects induced by DXR in all tested concentrations; similar results were found by Antunes and Takahashi (1999) that treated lymphocytes cultures with vitamin C, a potent antioxidant, before, simultaneously or after DXR treatment. The action of a protective agent by a single mechanism may be the exception rather the rule, and therefore SM may have a protective effect based on different mechanisms (Gebhart, 1992) however, as mentioned above, SM act in the antioxidant defense and modulate the response of DNA repair factors and p53 expression. In the present work, only the lowest concentration of SM (0.25 lM) was not clastogenic and able to reduce the damage induced by DXR treatment. The other tested concentrations of SM exhibited some genotoxic effects however, also reduced the DNA damage induced by DXR. The literature underlines the observation that selenium, in low concentrations, may have anticarcinogenic effects whereas in high concentrations, it can be genotoxic and carcinogenic (Bronzetti et al., 2001). Thus, it is of fundamental importance to determine the optimal concentration of SM that provides protection against genetic damage with the least toxicity. In conclusion, our results demonstrated that SM treatment reduced DNA damage and cytotoxicity induced by DXR in human lymphocytes, and exhibited chemoprotective effect under the conditions used in the present study.
Acknowledgements We would like to thank Alexandre Souto Martinez for statistical support. This investigation was supported with
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