Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202
Received February 3, 2000; accepted April 11, 2000
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ABSTRACT |
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Key Words: oxidative stress; OH8dG; SHE cell; morphological transformation; carcinogenicity; singlet oxygen.
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INTRODUCTION |
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Among the molecular targets of ROS, DNA is particularly important in neoplasia formation and progression (Ames et al., 1995). To date, over 20 specific oxidized modifications of DNA resulting from ROS have been identified (Dizdaroglu, 1992
). Of these adducts, 8-hydroxy-2'-deoxyguanosine (OH8dG) is the most predominant DNA modification, therefore, formation of OH8dG has been used as a biomarker of oxidative DNA damage. OH8dG has also been shown to be mutagenic (Kamiya et al., 1992
; Moriya et al., 1991
; Shibutani et al., 1991
; Wood et al., 1990
). In an in vitro study, Shibutani et al. showed that either dC or dA could be incorporated opposite OH8dG (Shibutani et al., 1991
). Similar findings were seen in prokaryotic and eukaryotic cells using OH8dG-containing vectors (Wood et al., 1990
; Moriya et al., 1991
; Kamiya et al., 1992
). In addition, ROS can react with dGTP in the nucleotide pool to form OH8dG (Akiyama et al., 1989
). During DNA replication, OH8dG has been postulated to be incorporated into DNA from the oxidized nucleotide pool (opposite dC or dA on the template strand) by DNA polymerases, resulting in A:T to C:G transversions during the next round of scheduled DNA replication (Cheng et al., 1992
; Maki and Sekiguchi, 1992
). The mutagenicity observed following OH8dG formation suggests that oxidative DNA damage produced by agents causing oxidative stress contributes to their carcinogenicity. This oxidative DNA modification may be involved in the tumor initiation and/or progression stages of the cancer process by mutating or modifying the expression of tumor-suppressor genes or oncogenes. OH8dG has also been reported to interfere with the normal function of DNA methyltransferase (Turk et al., 1995
). Using synthetic oligonucleotides containing OH8dG at specific positions, Turk and colleagues found that OH8dG diminished the ability of DNA methyltransferase to methylate a target cytosine when OH8dG was one or two bases 3' from the cytosine on the same strand (Turk et al., 1995
). DNA methylation status regulates gene expression. A hypomethylated gene thus has a higher probability of being expressed compared to a hypermethylated gene (Razin and Cedar, 1991
; Vorce and Goodman, 1989
). It is believed that hypomethylation may be a nongenotoxic mechanism facilitating aberrant gene expression in the carcinogenesis process (Counts and Goodman, 1994
; Goodman and Counts, 1993
). The effect of OH8dG on methyltransferase suggests that this DNA modification may be involved in carcinogenesis through nongenotoxic mechanisms.
The above studies collectively support an involvement of OH8dG formation in carcinogenesis through either genotoxic or nongenotoxic mechanisms. Thus, OH8dG formation appears to be a causative or contributing factor to the carcinogenesis process. Indeed, previous studies have revealed an association between OH8dG formation and cancer development (Floyd, 1990). However, there is no direct experimental evidence to show whether this association reflects a causative relationship between OH8dG formation and carcinogenesis. The purpose of the present study was to examine the effect of OH8dG formation/incorporation in Syrian hamster embryo (SHE) cells on cellular transformation. An advantage of the SHE-cell transformation model is that these cells exhibit multistage neoplastic morphological and molecular transformation similar to that observed in vivo (Isfort and LeBoeuf, 1995
). Morphological transformation appears to be an initial stage in SHE-cell carcinogenesis. Morphological transformation of SHE cells demonstrates a high concordance rate (85%) with rodent bioassay results (LeBoeuf et al., 1996
) and therefore provides a useful model for detecting carcinogens and studying the mechanisms of carcinogenesis.
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MATERIALS AND METHODS |
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Cell culture and treatment.
SHE cells were isolated from 13-day-old embryos of Syrian Golden hamsters, as previously described (Kerchaert et al., 1996), and cryopreserved. For OH8dG analysis, SHE cells were plated at a density of 2.5 x 106/T75 culture flask in 15 ml of SHE complete medium (LeBoeuf's modified Dulbecco's modified Eagle's medium containing 200 mM L-glutamine and 20% fetal bovine serum). The cells (80% confluency) were treated with 0, 1.25, 2.5, 5, 10, and 20 µM of methylene blue in the dark for 1 h; 50% of the cultures were then exposed to fluorescent light at a distance of 11 cm for 1 h. The remaining half of the cultures were maintained in the dark for 1 h as a control. After treatment, the cells were washed twice with calcium-free, magnesium-free Hank's balanced salt solution (CMF-HBSS), and collected by trypsinization.
Analysis of OH8dG.
OH8dG was measured as described previously (Shigenaga et al., 1994). Briefly, DNA was isolated using a sodium iodide chaotropic isolation method (Wang et al., 1994
). Isolated DNA was dissolved in Tris-HCl buffer (10 mM, pH 7.0) containing 2 mM butylated hydroxytoluene (BHT) and 0.1 mM desferal. DNA (100200 µg) was digested with 10 units nuclease P1 (37°C, 30 min) followed by 14 units of alkaline phosphatase (37°C, 60 min). After centrifugation (10,000 x g, 10 min, 4°C), the supernatant was removed for analysis. OH8dG was detected by HPLC with an electrochemical detector (ESA, Inc., Chelmsford, MA) set at E1: 100 mV, 1 µA and E2: 400 mV, 5 µA. 2'-Deoxyguanosine (2'-dG) was detected using a Waters Photodiode Array Detector set at 260 nm (Waters Inc., Milford, MA). The amount of OH8dG and 2'-dG was quantified from standard curves and expressed as the ratio of OH8dG to 2'-dG. Each experiment was performed in duplicate.
SHE-cell transformation assay.
SHE-cell transformation was conducted as described previously (Kerckaert et al., 1996). Briefly, in this assay, a nondividing feeder-cell layer of SHE cells is initially prepared. To this feeder-cell layer, SHE target cells are subsequently seeded onto the feeder layer. The feeder cells were prepared by reconstituting a vial of frozen cells at 37°C and cultured in a T150 culture flask containing 30 ml of SHE complete medium at 37°C and 10% CO2. The cells were incubated for 72 h to achieve 7090% confluency. The cells were then rinsed twice with CMF-HBSS, and detached with 5 ml of 0.05% trypsin-EDTA. After detachment, the cells were suspended in 30 ml of culture medium in a T150 flask, and exposed to X-ray (~ 5000 rad) so the feeder cells were still viable but no longer capable of replication. The feeder cells were plated at a density of 2 x 104/ml in 60-mm culture dishes (2 ml/dish) and incubated for 24 h. Target cells (freshly reconstituted SHE cells) were cultured in a T25 flask containing 5 ml complete medium. After 24 h, target cells (the cells to be used for clonal growth) were plated onto the nondividing feeder cells at a density of 85 cells/dish in 2 ml complete medium. After 24-h incubation, cells were treated as described above with methylene blue. After treatment with methylene blue, cell cultures were refed and allowed to grow for 7 days. Colonies were then fixed with methanol, stained with Giemsa, and scored for morphological transformation. Each transformation experiment was performed in duplicate as defined previously (Kerckaert et al., 1996
).
In vitro treatment of DNA and transfection.
DNA was isolated from SHE cells as described above. Isolated DNA was dissolved in TE buffer (10 mM TrisHCl, pH 8.0, 1 mM EDTA). DNA (500 µg) was treated with 20 µM Mbyte plus light for 5, 15, and 30 min. After treatment, DNA was ethanol-precipitated and washed twice with 70% ethanol. The resultant pellet was resuspended in sterile TE buffer. OH8dG concentration was then measured as described above. The SHE cells to be transfected were cultured in T25 tissue culture flasks at a density of 2.5 x 106/flask in 6 ml complete medium. The DNA containing methylene blue-induced OH8dG or DNA from untreated SHE cells was transfected into recipient SHE cells using a cationic liposome-mediated transfection kit (TfxTM-50, Promega, Madison, WI). DNA (7.5 µg) and TfxTM-50 reagent (11.25 µl) (final charge ratio 2:1) was vortexed, adjusted to 2 ml with serum-free medium, and incubated at room temperature for 15 min. Recipient SHE cells were washed once with PBS. Two milliliters of the TfxTM-50 reagent/DNA mixture was added to the SHE cells, and incubated for 2 h. Transfection was confirmed using a plasmid containing a ß-galactosidase reporter gene. At the end of this 2-h incubation, medium was changed and replaced with complete medium, and incubated for 24 h at 37°C. After incubation, the SHE cells were detached with 0.005% trypsin-EDTA and plated onto the feeder cells for the SHE-cell transformation assay. This experiment was repeated twice.
In a separate study, isolated DNA was treated with 20 µM methylene blue plus light for 5, 15, 30, and 60 min. After treatment, DNA was ethanol-precipitated and resuspended in sterile TE buffer. DNA (100 µg) from each group was digested with EcoR I at 37°C for 2 h. Samples were then separated by electrophoresis using low-melting-point agarose gel (0.8% containing 500 µg/l ethidium bromide in TAE buffer at 100 mV). DNA fragments (564 bp to 2322 bp) were excised, purified (QIAquick gel extraction kit, Qiagen, Valencia, CA), and used to transfect SHE cells as described above. The morphological transformation of the transfected SHE cells was determined. The remaining DNA fragments were excised, purified and used for the measurement of OH8dG level as described above. These studies were performed in duplicate.
Statistics.
Data were analyzed using ANOVA followed by Duncan's test for OH8dG studies, Fisher exact test for morphological transformation studies, and regression analysis for correlative analysis.
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RESULTS |
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DISCUSSION |
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In addition to inducing the formation of OH8dG, photoactivated methylene blue also induced a dose-dependent increase in morphological transformation in SHE cells (Table 1). In the absence of photoactivation, methylene blue had no significant effect on either OH8dG formation or morphological transformation in SHE cells (Fig. 2
and Table 1
). Regression analysis revealed a significant log linear relationship between morphological transformation and OH8dG formation (p < 0.01, Fig. 2
). The observed log linear relationship between OH8dG and morphological transformation may be due to the DNA repair mechanisms in the SHE cells. Three OH8dG repair enzymes have been identified and their genes (MutM, MutT, and MutY) cloned in E. coli and in mammalian cells (Rosenquist et al., 1997
; Sakumi et al., 1993
; Slupska et al., 1996
; Tchou and Grollman, 1993
). These three enzymes apparently function at different stages of OH8dG formation and incorporation to reduce the potential adverse effects of OH8dG. The expression of these repair enzymes may be upregulated following oxidative stress (Bases et al., 1992
; Wani et al., 1998
) and may explain the presence of a log linear relationship instead of a simple linear relationship between the formation of OH8dG and morphological transformation. After a threshold amount of OH8dG is produced, these repair enzymes may be upregulated by the insult, and thus repair OH8dG more efficiently. Therefore, the increase of morphological transformation plateaued. The correlation between OH8dG and morphological transformation may not be restricted to a causative relationship, because photoactivated methylene blue has been shown to produce other oxidative effects, including lipid peroxidation and protein damage (Girotti et al., 1979
; Kamat and Devasagayam, 1996
). Recently, Kress et al. reported that photoactivated methylene blue stimulated rat primary cutaneous afferents via impeding sodium inactivation and blocking potassium currents (Kress et al., 1997
). Furthermore, methylene blue was found to inhibit the stimulation of soluble guanylyl cyclase by NO and other nitrovasodilators in cell-free systems (Miki et al., 1977
; Murad et al., 1978
). Photoactivated methylene blue blocked smooth muscle relaxation induced by nitrovasodilators or acetylcholine (Gruetter et al., 1981
; Holzmann, 1982
). In order to address and exclude these other cellular actions of photoactivated methylene in the transformation process, studies were performed to examine the effect of transfection of DNA containing different levels of OH8dG in SHE cells on cellular transformation. Transfected DNA has been shown to integrate into the genome of the recipient cells by homologous recombination (Smithies et al., 1985
). Therefore, transfection of DNA containing OH8dG will incorporate OH8dG into the genomic DNA. Transfection of SHE cells with DNA containing different concentrations of OH8dG induced morphological transformation (Table 2
). Thus, it appears that the formation and incorporation of OH8dG, and not necessarily the other cellular effects of photoactivated methylene blue, were responsible for the observed morphological transformation. Regression analysis also revealed a log linear relationship (p < 0.01, Fig. 3
) between these two end points. In an additional study, DNA fragments of a similar size (564 pb2322 pb) that contained different concentrations of OH8dG were transfected in SHE cells. Morphological transformation of these transfected SHE cells was also dependent on the relative amount of OH8dG in the transfected DNA (Table 3
). However, the association between the amount of OH8dG and the frequency of morphological transformation produced a different pattern from that seen in the earlier transfection study (Fig. 3
). The relationship between OH8dG and morphological transformation was sigmoidal rather than log linear (Fig. 4
). The reason why the relationship is different is unclear. One possibility is that DNA fragment length may have an effect on transfection efficiency or homologue recombination, and thus subsequently affect the final concentration and position of OH8dG in genomic DNA.
DNA modifications induced by photosensitizers including methylene blue are mediated through two competitive mechanisms: type I and type II photosensitization reactions (Foote, 1991). The type I reaction involves direct electron or hydrogen transfer from the substrate (DNA) to the excited, triplet-state photosensitizer, resulting in two radicals (substrate and photosensitizer radicals). The type II reaction involves the energy transfer from the excited photosensitizer to ground-state oxygen to generate singlet oxygen (Foote, 1991
). The formation of OH8dG by photoactivated methylene blue has been shown to be mediated by the type II reaction (Epe et al., 1989
; Epe et al., 1990
; Floyd et al, 1989
). ß-Carotene is a potent singlet oxygen scavenger with a quenching constant of 14 x1 09 M1s1 (Di Mascio et al., 1989
). Results from the present study showed that ß-carotene inhibited the morphological transformation induced by photoactivated methylene blue (Table 4
), suggesting that type II reaction products are responsible for the morphological transformation induced by photoactivated methylene blue. The type I reaction is apparently not involved in the above processes either, because type I reaction products are not produced or are produced at a level not high enough to result in a measurable effect on morphological transformation in SHE cells. Therefore, the potential effect of type I reaction products produced by photoactivated methylene blue on morphological transformation can be excluded.
Besides OH8dG, 2,6-diamino-4-hydroxy-5formamidopyrimidine (FapyGua) is reported as the only detectable type II reaction product in photoactivated methylene bluetreated double-strand DNA or cells (Ravanat and Cadet, 1995). However, the level of FapyGua produced has been reported to be less than 5% of the level of OH8dG (Boiteux et al., 1992
; Ravanat and Cadet, 1995
). Although OH8dG is mutagenic, the FapyGua lesion blocks the progression of DNA polymerase, and therefore is more likely to be lethal rather than mutagenic (Boiteux and Laval, 1983; O'Connor et al., 1988
). Due to its minor amount and lethal properties, FapyGua formation is not likely to contribute to the morphological transformation induced by photoactivated methylene blue in SHE cells. Therefore, OH8dG (the type II reaction product) appears to be responsible for the induction of morphological transformation in SHE cells. The formation of OH8dG by photoactivated methylene blue is also blocked by the cotreatment of SHE cells with ß-carotene. It provides further evidence that OH8dG is the factor involved in methylene blue plus lightinduced morphological transformation in SHE cells.
The cytotoxicity of photoactivated methylene blue does not appear to correlate with OH8dG formation. No cytotoxicity was observed in SHE cells transfected with high levels of OH8dG. In addition, blockage of type II reaction products by ß-carotene did not reduce the cytotoxic effects of photoactivated methylene blue measured by relative plating efficiency (Table 4). ROS have been shown to induce apoptosis and necrosis (Kim et al., 1999
; Payne et al., 1995
). Noodt et al. (1998) reported that a methylene blue derivative induced apoptosis in the presence of light (Noodt et al., 1998
). Additionally, Rose Bengal, another photosensitive dye that generates singlet oxygen, has been reported to induce apoptosis in cultured cells in the presence of light (Manev et al., 1995
). Reactive oxygen generation can produce necrosis via direct cellular effects or indirectly through stimulation of inflammatory cells (Kim et al., 1999
). Obviously, in the present study only the former would apply. Therefore, the cytotoxicity of photoactivated methylene blue observed in the present studies may be due to either apoptotic and/or necrotic mechanisms.
Collectively, the results of the present study show that OH8dG formation and incorporation into SHE-cell DNA is an important factor in the induction of morphological transformation in SHE cells by photoactivated methylene blue. Future studies will focus on the cellular and molecular mechanisms by which OH8dG induces morphological transformation in SHE cells.
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NOTES |
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