Mutagenicity of reactive oxygen and nitrogen species as detected by co-culture of activated inflammatory leukocytes and AS52 cells

Ha Won Kim1, Akira Murakami1, Marshall V. Williams2 and Hajime Ohigashi1,3

1 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and
2 Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, 2078 Graves Hall, 333 W. 10th Avenue, Columbus, OH 43210, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Activated inflammatory leukocytes generate a variety of reactive oxygen and nitrogen species (RONS) that may have roles in mutagenesis and carcinogenesis. The purpose of the present study was to explore the relationship between inflammatory leukocyte activation and mutagenesis using co-culture systems. We investigated the mutagenic potentials of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated differentiated HL-60 (human promyelocytic leukemia cells), and RAW 264.7 cells (murine macrophages) stimulated with lipopolysaccharide (LPS) and interferon (IFN)-{gamma} by co-culturing each cell line with AS52 cells, a transgenic Chinese hamster ovary cell line. HL-60 cells rapidly generated superoxide (O2) 15 min to 1 h (peak at 30 min) following TPA stimulation. RAW 264.7 cells stimulated with LPS and IFN-{gamma} produced O2, nitric oxide (NO) and peroxynitrite (ONOO) continuously for 5–25 h. There was a 2.0-fold increase in the mutation frequency of the gpt gene in AS52 cells co-cultured with TPA stimulated HL-60 cells, when compared with non-treated cells. Importantly, this increase in mutation frequency was significantly suppressed by antioxidants, such as superoxide dismutase (SOD) and diphenylene iodonium (DPI), an NADPH oxidase inhibitor (inhibition rates: IRs = 18.2 and 35.1%, respectively). Similarly, co-culture of AS52 cells with LPS/IFN-{gamma}-stimulated RAW 264.7 cells also increased the mutation frequency of the gpt gene by 2.6-fold, and this increase in mutation frequency was suppressed by SOD, DPI and N5-(1-iminoethyl)-L-ornithine dihydrochloride (L-NIO), an specific iNOS inhibitor (IRs = 58.3, 70.8 and 70.8%, respectively). In co-culture experiments, activated HL-60 and RAW 264.7 cells increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in AS52 cells when compared with non-treated controls (1.7- and 1.6-fold, respectively). Treatment of AS52 cells with hydrogen peroxide (H2O2, 100 µM), ONOO (100 µM) and SIN-1 (100 µM), a ONOO generator, also increased the mutation frequency of the gpt gene (4.6-, 5.4- and 2.8-fold, respectively). Taken together, these results support the hypothesis that RONS, derived from activated inflammatory leukocytes, are mutagenic in the biological systems, and that RONS generation inhibitors are potentially anti-mutagenic, and thus may be useful in cancer preventive strategies.

Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; DPI, diphenylene iodonium; EDRF, endothelium-derived relaxing factor; EGCG, (–)epigallo-catechin gallate; FBS, fetal bovine serum, gpt, xanthine-guanine phospho-ribosyltransferase; HBSS, Hank’s balanced salt solution; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; H2O2, hydrogen peroxide; IFN, interferon; LPS, lipopolysaccaride; MPA, mycophenolic acid; L-NIO, N5-(1-iminoethyl)-L-ornithine, dihydrochloride; NO, nitric oxide; NOS, nitric oxide synthase; oOH; hydroxyl radical; 8-OHdG, 8-hydroxy-2'-deoxyguanosine, ONOO, peroxynitrite; PMN, polymorphonuclear leukocyte; ROS, reactive oxygen species; SIN-1, 3-(4-morpholinyl) sydnonimine, hydrochloride; SOD, superoxide dismutase; TG, thioguanine; TPA, 12-O-tetradecanoylphorbol-13-acetate; XOD, xanthine oxidase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In respiring cells, a small amount of the consumed oxygen is reduced, yielding a variety of highly reactive chemical entities. These are collectively called reactive oxygen and nitrogen species (RONS), including nitric oxide (NO), superoxide (O2), hydroxyl radical (•OH), hydrogen peroxide (H2O2) and peroxynitrite (ONOO). RONS are recognized as important signal transduction mediators that regulate gene expression, cell differentiation, immune activation and apoptosis (1). However, RONS are potentially toxic and excess production of RONS is known to play causative roles in the onset of a variety of diseases and aging (24). Numerous previous studies have shown that RONS cause lipid peroxidation, oxidation of amino acid residues, formation of protein–protein cross links, oxidation of polypeptide backbones resulting in protein fragmentation, DNA damage and DNA strand breaks (57).

While numerous studies have demonstrated the mutagenicity of RONS (812), they were performed using in vitro systems, in which target cells or isolated DNA were exposed to chemically generated RONS. Under such circumstances, the permeation rate, half-life and dose of RONS that are important in vivo are not taken into account. Thus, these in vitro systems may not reflect conditions in vivo. Therefore, a model system that can be employed to demonstrate the mutagenic potential of biologically generated RONS needs to be established. In this regard, a co-culture experiment using RONS generating cells, instead of chemically synthesized RONS, may compensate for these insufficient in vitro conditions and thus possibly mimic biological environment in vivo.

Activated inflammatory leukocytes are a major source of RONS generation with O2 and NO being produced initially in most RONS producing pathways (13,14). 12-O-Tetradecanoylphorbol-13-acetate (TPA) activates dimethylsulfoxide (DMSO)-differentiated HL-60 cells to induce the NADPH oxidase activity, leading to O2 generation. Conversely, bacterial lipopolysaccharide (LPS) or interferon (IFN)-{gamma} induces the production of NO from the inducible nitric oxide synthase (iNOS) gene in macrophages, including RAW 264.7. NO reacts with O2 to form ONOO, which is highly toxic when compared with O2 or NO (15). ONOO modifies amino acid residue (e.g. 3-nitrotyrosine) (16) and induces DNA damage (e.g. 8-nitroguanine) (17).

Various mammalian cell lines have been used to study the mechanisms of mutagenesis. For this study, we chose to use the Chinese hamster ovary (CHO) cell line AS52. AS52 cells are transgenic, lacking the normal X-linked mammalian hypoxanthine guanine phosphoribosyltransferase (hprt) gene, but containing a single functional copy of the Escherichia coli xanthine guanine phosphoribosyltransferase (gpt) gene stably integrated into the CHO genome (1820). As the gpt gene is not essential for growth or survival of AS52 cells, it is possible to isolate mutants containing a variety of mutations, including: point, interchromosomal deletion, mitotic recombinations, gene–chromosomal conversions and multilocus deletions. We, as well as others, have demonstrated that AS52 cells can be used to determine molecular and mechanistic features associated with mutagenesis in mammalian cells (21,22).

In the present study, we co-cultured either TPA-stimulated, differentiated HL-60 cells or LPS/IFN-{gamma}-stimulated RAW 264.7 cells with AS52 cells to evaluate the mutagenic potential of RONS produced by the activated inflammatory leukocytes. Furthermore, by employing specific agents that either inhibited RONS-generating enzyme activity or scavenged specific RONS, we were able to determine the RONS responsible for inducing mutations in the gpt gene of AS52 cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell cultures
Human promyelocytic leukemia HL-60 cells, obtained from American Type Culture Collection (ATCC), were maintained in RPMI 1640 medium containing L-glutamine supplemented with 10% fetal bovine serum (FBS). The murine macrophage RAW 264.7 cells, obtained from ATCC were cultivated in DMEM medium containing L-glutamine supplemented with 10% FBS. AS52 cells, kindly donated by Dr Kenneth Tindall, National Institute for Environmental Health Science, Research Triangle Park, NC, were maintained in Ham’s F-12 medium containing L-glutamine supplemented with 5% FBS, and MPA additives (10 µg/ml MPA, 25 µg/ml adenine, 50 µM thymidine, 250 µg/ml xanthine and 3 µM aminopterin). These cell lines were maintained at 37°C in a humidified 5% CO2 atmosphere.

Chemicals
RPMI 1640, DMEM and IFN-{gamma} were purchased from Gibco BRL (Grand Island, NY). Cytochrome c and diphenylene iodonium (DPI) were obtained from Sigma Chemical (St Louis, MO). LPS (E.coli serotype 0127, B8) was purchased from Difco Labs (Detroit, MI), and Ham’s F-12 from Nihonseiyaku (Tokyo, Japan). N5-(1-iminoethyl)-L-ornithine, dihydrochloride (L-NIO), 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1) and ONOO solution were purchased from Dojindo Labs (Kumamoto, Japan). All other chemicals were purchased from Wako Pure Chemicals (Osaka, Japan).

Determination of O2 level
The O2 level following TPA treatment of HL-60 cells was determined, with some modification, as described previously (23). HL-60 cells were pre-incubated with 1.25% DMSO at 37°C in a 5% CO2 incubator for 6 days, differentiating them into granulocytes. Differentiated HL-60 cells or RAW 264.7 cells were suspended in RPMI 1640 or DMEM medium at a density of 1x106 cells/ml, and incubated at 37°C for 15 min. After stimulating differentiating HL-60 cells with TPA (100 nM) or RAW 264.7 cells with the combination of LPS (100 ng/ml) and IFN-{gamma} (100 U/ml), each cell suspension was incubated for 15 min to 24 h. Cytochrome c solution (1 mg/ml) was added to the cell suspension 15 min before measurement. The reaction mixture was centrifuged at 4000 g for 1 min, and absorption at 550 nm was measured. The level of O2 production was calculated from the formula: O2 (nmol/ml) = 47.7xA550nm.

Determination of NO level
RAW 264.7 cells were suspended in DMEM medium at a density of 2x105 cells/ml, and then treated with LPS (100 ng/ml) and IFN-{gamma} (100 U/ml). HL-60 cells were differentiated as described above, and suspended at a density of 2x105 cells/ml before addition of TPA (100 nM). Cells were treated for 1–24 h, and NO synthesis was determined using the Griess reaction. Briefly, 0.5 ml of the cell medium supernatant was added to a solution (0.5 ml) of the Griess reagent (1% sulfanilamide, 0.1% naphythyl ethylene diaminedihydrochloride in 5% H2PO4), and absorption at 543 nm was monitored. The amount of NO production was indirectly measured by the level of nitrite (NO2), one of the NO metabolites in media. NO2 was calculated from the formula: NO2 (µM) = (A543 nm – 0.042)/0.091. The concentration of NO in cell culture media without cells was measured as a background control and subtracted.

Determination of ONOO level
The formation of peroxynitrite was measured by the peroxynitrite-dependent oxidation of dihydrorhodamine 123 to rhodamine 123, based on the method described previously by Ischiropoulos et al. (24). Briefly, RAW 264.7 cells were LPS/IFN-{gamma} treated for 1–24 h. The medium was replaced with PBS containing 5 µM dihydrorhodamine 123, and 1 h later the amount of rhodamine formed was determined. The fluorescence of rhodamine 123 was measured using VersaFluor fluorometer (Bio-Rad, Hercules, CA) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm (slit widths, 2.5 and 3.0 nm, respectively), and the peroxynitrite concentrations were calculated using standard curves obtained with authentic peroxynitrite.

AS52 mutation assay
AS52 mutation assay was performed as reported previously (2022). Briefly, on day –1, AS52 cells were pre-incubated in Ham’s F-12 medium lacking MPA additives at a density of 106 cells/100 mm dish. On day 0, cells were washed with Hank’s Balanced Salt Solution (HBSS) three times, and treated with the test agents or cell cultures for specified duration. After treatment, the cells were washed with HBSS three times, and incubated with Ham’s F-12 medium without MPA additives. On days +3 and +6, the cells were subcultured at a density of 106. On day +9, cells were cultured in 100 mm dishes at a density of 2x105 cells/plate in Ham’s F-12 medium containing 5% FBS and 10 µM TG. Cells were incubated for another 10 days and examined for the development of TG resistant (TGr) clones. Cells were fixed with a solution containing methanol, acetic acid and water (50:7:43), and then stained with a 1% crystal violet solution. Only those colonies containing at least 50 cells/colony were counted. Mutation frequency was expressed as mutants/106 clonable cells.

Cytotoxicity was determined on day +4 and plating efficiency on day +7. Briefly, AS 52 cells were plated at a density of 200 cells/60 mm dish in Ham’s F-12 medium lacking MPA additives, and incubated at 37°C for 7 days (three to five dishes). Only those clones containing at least 50 cells/colony were counted.

AS52 mutation assay co-cultured with HL-60 cells
AS52 cells were plated at a density of 5x105 cells/100 mm dish in Ham’s F-12 medium containing 5% FBS lacking MPA additives 12 h prior to co-culturing with differentiated HL-60 cells. On day 0, differentiated HL-60 cells were co-cultured with AS52 cells at a ratio of 1:1 in the presence or absence of inhibitors and then, treated with TPA (100 nM). After incubating at 37°C for 1 h, HL-60 cells were removed by decantation. AS52 cells were washed three times with HBSS, and then subcultured for cytotoxicity and mutagenesis studies. Procedures from day +3 were the same as described above.

AS52 mutation assay co-cultured with RAW 264.7 cells
AS52 and RAW 264.7 cells were plated together in Ham’s F-12 medium lacking MPA additives at a ratio of 1:1 (5x105, respectively), and then incubated at 37°C for 12 h. On day 0, the co-cultured cells were treated with LPS (100 ng/ml) and IFN-{gamma} (100 U/ml). After incubating at 37°C for 24 h, cells were washed three times with HBSS, and then subcultured for cytotoxicity and mutagenesis studies. Procedures from day +3 were the same as described above. No colonies formed from RAW 264.7 cells treated with 6-TG (10 µM) for 10 days.

8-Hydroxy-2'-deoxyguanosine (8-OHdG) measurement
AS52 cells (106) were plated on a 100 mm dish for 12 h. After treatment with different agents, the cellular DNA was isolated using DNeasy tissue kit (QIAGEN, Germany). Ten micrograms of DNA was converted to single-stranded DNA by incubation with 180 U Exonuclease III (Takara Biotech., Japan) at 37°C for 1 h. The DNA was heated at 95°C for 5 min and rapidly chilled on ice, and was digested to nucleosides by incubation with 0.6 U nuclease P1 (Takara) at 37°C for 1 h and followed by treatment with 0.6 U E.coli alkaline phosphatase (Takara) for another 1 h. The reaction mixture was centrifuged (6000 g for 1 min) and the supernatant was used for the 8-OHdG assay. The amount of 8-OHdG was measured according to the protocol of the competitive ELISA kit (8-OHdG check, Japan Institute for the Control of Aging, Shizuoka, Japan).

Statistical analysis and inhibition rate (IR)
Each experiment was performed at least three times, and the data are shown as mean ± standard deviation (mean ± SD). The statistical significance of differences between groups in each assay was assessed by a Student’s t-test (two sided) that assumed unequal variance. The IR in each assay was calculated by the following equation: IR (%) = {1 – [(test sample data) – (negative control data)][(positive control data) – (negative control data)]–1}x100.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Measurement of O2, NO and ONOO production in TPA-stimulated, differentiated HL-60 and LPS/IFN-{gamma}-stimulated RAW 264.7 cells
We determined the concentration of O2 and NO in the media from TPA-stimulated differentiated HL-60 cells cultured alone or co-cultured with AS52 cells. TPA treatment of HL-60 cells resulted in the rapid generation of O2 with a maximum concentration of 32.7 µM being formed by 1 h. This was followed by a time dependent decay over 24 h (Figure 1AGo). TPA treatment of HL-60 cells did not result in the induction of NO synthesis (Figure 1AGo). Co-culturing of TPA-stimulated HL-60 with AS52 cells did not alter the production of O2 and NO when compared with HL-60 cells cultured alone (Figure 1BGo compared with A).



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Fig. 1. Time course of superoxide and nitric oxide production in TPA-stimulated differentiated HL-60 cells and in LPS/IFN-{gamma}-stimulated RAW 264.7 cells. (A) HL-60 cells cultured alone, (B) HL-60 co-cultured with AS52 cells, (C) RAW 264.7 cells cultured alone, (D) RAW 264.7 cells co-cultured with AS52 cells. Open circle ({circ}) indicates O2 generation and closed circle (•) indicates NO generation. (E) Time course of peroxynitrite producition in LPS/IFN-{gamma}-stimulated RAW 264.7 cells. Open square ({square} indicates RAW 264.7 cultured alone, closed square ({blacksquare}) indicates RAW 264.7 cells co-cultured with AS52 cells. HL-60 cells (106 cells/ml) were differentiated by incubation in RPMI 1640 medium containing 1.25% DMSO for 6 days. The differentiated HL-60 cells or RAW 264.7 cells (106 cells/ml) were co-cultured with AS52 cells at a ratio of 1:1. For the determination of O2 production, the cells were incubated with 100 nM TPA at 37°C for 1–25 h. Cytochrome c (1 mg/ml) was added to the medium 15 min before measurement. The extracelluar O2 was determined using a cytochrome c reduction method. For the determination of NO, the cells were incubated with LPS (100 ng/ml), IFN-{gamma} (100 U/ml) and L-arginine (2 mM) at 37°C for 1–25 h. The amount of NO2 produced was determined using the Griess method. For the determination of ONOO, LPS/IFN-{gamma}-treated RAW 264.7 cells were incubated for 1–24 h. The medium was replaced with PBS containing 5 µM dihydrorhodamine 123, 60 min before measurement. The fluorescence of rhodamine 123 was determined at an excitation wavelength of 490 nm and an emission wavelength of 520 nm (slit widths, 2.5 and 3.0 nm, respectively).

 
We also determined the concentration of O2 and NO in the media from LPS/IFN-{gamma}-treated RAW 264.7 cells cultured alone or co-cultured with AS52 cells. There was a time-dependent increase in NO production that began 5 h following LPS/IFN-{gamma} stimulation of RAW 264.7 cells (Figure 1CGo). There was also a linear time-dependent production of O2 in the LPS/IFN-{gamma}-stimulated RAW 264.7 cells. It is notable that the time course for O2 generation in RAW 264.7 cells was different from that observed in TPA-treated HL-60 cells (Figure 1A and CGo). Co-culturing of LPS/IFN-{gamma}-stimulated RAW 264.7 cells with AS52 cells did not alter the production of O2 and NO when compared with LPS/IFN-{gamma}-stimulated RAW 264.7 cells cultured alone (Figure 1DGo compared with C).

The concentration of ONOO, a product formed from the interaction of O2 with NO, in the media of LPS/IFN-{gamma}-stimulated RAW 264.7 cultured alone and co-cultured with AS52 cells was determined by quantifying the ONOO-dependent oxidation of dihydrorhodamine 123 to rhodamine 123. As hydrogen peroxide, which is also produced by activated macrophages, can oxidize dihydrorhodamine 123 to rhodamine 123 (32), we estimated the contribution of hydrogen peroxide to the formation of rhodamine 123. In the present study, we incubated control and LPS/IFN-{gamma}-stimulated cells with PBS and without dihydrorhodamine 123 for 60 min, followed by a 60 min incubation at room temperature to decompose peroxynitrite in the buffer, followed by an additional 60 min incubation with 5 µM dihydrorhodamin 123. Under these conditions, peroxynitrite decomposes, while hydrogen peroxide remains stable (32). ONOO formation (1.9–3.5 µM) was observed in LPS/IFN-{gamma}-stimulated RAW 264.7 cultured alone and co-cultured with AS52 cells (Figure 1EGo), whereas no H2O2 formation was detectable (data not shown). There were no detectable amounts of O2, NO and ONOO in non-stimulated cells (data not shown).

Mutagenicity of H2 O2, ONOO and SIN-1 in AS52 cells
To determine whether RONS induced mutations in AS52 cells, we examined the mutagenic potential of H2O2 and ONOO in AS52 cells. The data, which are shown in Table IGo, demonstrate that AS52 cells treated with H2O2 (100 µM) or ONOO (100 µM) for 1 h exhibited an increased frequency of TGr mutants (4.6- and 5.4-fold, respectively) when compared with the spontaneous mutation frequency obtained with non-treated cells. Treatment of AS52 cells with SIN-1, an ONOO generator, at the concentrations of 50 and 100 µM for 1 h significantly increased the mutation frequency of the gpt gene in treated cells when compared with non-treated control (Table IIGo).


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Table I. Mutagenesis of AS52 cells following treatments with hydrogen peroxide and peroxynitrite
 

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Table II. Mutagenesis of AS52 cells following treatment with SIN-1
 
Mutagenicity of TPA-stimulated, differentiated HL-60 cells as detected by co-culture with AS52 cells
To investigate the relationship between activated inflammatory leukocytes and mutagenesis, we co-cultured TPA-stimulated, differentiated HL-60 cells with AS52 cells, and conducted several experiments to determine whether RONS production from the activated HL-60 cells was responsible for the gpt gene mutations. When AS52 cells were co-cultured with HL-60 cells in the presence of TPA (100 nM), a significant increase in the frequency of TGr mutants was observed when compared to the mutation frequency determined for AS52 cells co-cultured with non-TPA-treated HL-60 cells or to AS52 cultured alone (2.0- and 1.7-fold, respectively) (Table IIIGo). To confirm that the increased mutation frequency was observed in AS52 cells co-cultured with TPA-treated HL-60 cells was due to O2 generation from TPA-stimulated HL-60 cells, SOD (100 U/ml, a superoxide scavenger), DPI (1 mM, a NADPH oxidase inhibitor), or allopurinol (100 µM, a XOD inhibitor), were added to the culture media. Treatments of TPA-stimulated co-cultured cells with SOD or DPI, inhibiting O2 generation in TPA-stimulated HL-60 mono-culture (83.6 and 92.1%, respectively) led to a significant reduction in the number of TGr mutants isolated (IR = 18.2 and 35.1%, respectively). It is of interest to note that allopurinol, a less potent O2 generation suppressant, was inactive for inhibiting mutagenesis.


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Table III. Mutagenesis of AS52 cells following co-culturing with TPA-stimulated HL-60 cells
 
Mutagenicity of LPS/IFN-{gamma}-stimulated RAW 264.7 cells as detected by co-culture with AS52 cells
We also examined the mutagenicity of NO generated from LPS/IFN-{gamma}-stimulated RAW 264.7 cells. When AS52 cells were co-cultured with RAW 264.7 cells in the presence of LPS (100 ng/ml) and IFN-{gamma} (100 U/ml), a significant increase in the mutation frequency was observed when compared with that observed in AS52 cells co-cultured with RAW 264.7 cells which were not stimulated with LPS/IFN-{gamma} or with AS52 cultured alone (2.6- and 2.5-fold, respectively) (Table IVGo). Treatment of LPS/IFN-{gamma}-stimulated RAW 264.7 cells co-cultured with AS52 cells with DPI (1 mM), SOD (100 U/ml), L-NIO (100 µM, a specific iNOS inhibitor) significantly reduced the number of TGr mutants (IR = 70.8, 36.1 and 70.8%, respectively). However, allopurinol (100 µM) did not inhibit the enhanced mutation frequency observed in the co-cultured cells. Inhibition of O2 and NO generation in RAW 264.7 cell mono-cultured by these antioxidants showed linear correlation with mutation frequency of AS52 cells.


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Table IV. Mutagenesis of AS52 cells following co-culturing with LPS/IFN-{gamma}-stimulated RAW 264.7 cell
 
Measurement of 8-OHdG in AS52 cells
The concentrations of 8-OHdG in the DNA of AS52 cells treated with H2O2, ONOO and SIN-1, as well as in those co-cultured with stimulated HL-60 or RAW 264.7 cells are shown in Figure 2Go. Treatment of AS52 cells with H2O2 (100 µM), ONOO (100 µM) and SIN-1 (100 µM) for 1 h significantly increased the levels of 8-OHdG in the DNA when compared with that observed in the DNA of non-treated cells (1.7-, 1.8- and 2.3-fold, respectively). There was no increase in the level of 8-OHdG in the DNA of AS52 cells co-cultured with non-activated HL-60 or RAW 264.7 cells. Conversely, there was a significant increase in the levels of 8-OHdG in the DNA of AS52 cells co-cultured with either TPA-stimulated HL-60 cells or LPS/IFN-{gamma}-stimulated RAW 264.7 (1.7- and 1.6-fold, respectively).



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Fig. 2. 8-OHdG levels in the DNA of AS52 cells DNA following treatment of cells with RONS or RONS generator, and after co-culturing with HL-60 cells or RAW 264.7 cells. Cells were treated with H2O2, ONOO, SIN-1 (100 µM, respectively) for 1 h, or co-cultured with TPA-stimulated HL-60 cells for 1 h or LPS/IFN-{gamma}-stimulated RAW 264.7 cells for 24 h. The 8-OHdG levels were measured by the competitive ELISA kit as described in Materials and methods. *P < 0.01 versus blank. **P < 0.01 versus AS52/HL-60. ***P < 0.05 versus AS52/RAW 264.7.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been proposed that RONS generated from activated inflammatory leukocytes may be involved in mutagenic processes, and thus, play a pivotal role in mutagenesis and carcinogenesis. Several studies using co-culture model system have demonstrated that RONS generated from cells induced mutations. For example, co-incubation of rat lung epithelial cells with {alpha}-quartz or carbon black-treated bronchoalveolar lavage (BAL) cells increased the mutation frequency of the hprt gene and this increase in mutation frequency was inhibited by the addition of catalase (25), suggesting that RONS are responsible for the increased mutation. Knaapen et al. (26) demonstrated that activated human polymorphonuclear leukocytes (PMN) induced DNA damage in co-cultured alveolar epithelial cells. Similarly, Ariza et al. (27) showed that the co-culture of AS52 cells with peripheral blood lymphocytes (PBLs) from SENCAR mice treated with either TPA or 7, 12-dimethylbenz[a]anthracene (DMBA) and TPA, resulted in a 7–160-fold increase in the mutation frequency of the gpt gene in AS52 cells when compared with that induced by PBLs isolated from mice treated with either acetone or DMBA. The increased mutation frequency was markedly inhibited by the antioxidant (–)epigallocatechin gallate (EGCG). Recently, Watanabe et al. (28) reported that hepatocytes co-cultured with activated macrophages exhibited increased levels of NO release and increased levels of single-strand breaks in DNA, and these alterations were attenuated by NO inhibitors.

In the present study, we demonstrated that the treatment of AS52 cells with H2O2, ONOO and SIN-1 resulted in an increase in the mutation frequency of the gpt gene when compared with the non-treated controls. This is the first demonstration that ONOO and SIN-1 are mutagenic in AS52 cells and confirms a previous study demonstrating the mutagenic potential of H2O2 [29]. The gpt mutation frequency of AS52 cells treated with 100 µM H2O2 was 93 TGr mutants/106 clonable cells which is similar to the mutation frequency of 115 mutants/106 clonable cells reported for treatment of cells with 150 µM H2O2.

Stimulation of HL-60 cells with TPA resulted in the rapid production of O2 rapidly whereas there was no significant generation of NO. Co-culturing of HL-60 and AS52 cells followed by TPA stimulation resulted in the production of O2 that exhibited a similar time course as to what was observed with HL-60 cells cultured alone, suggesting that O2 generation in this co-culture was derived from the stimulated HL-60 cells. On the other hand, LPS/IFN-{gamma}-stimulated RAW 264.7 cells produced significant levels of NO, O2 and ONOO. Previous studies have reported that NO and O2 are generated from iNOS, and interact with each other to form the potent oxidant ONOO in L-arginine (L-Arg) depleted RAW 264.7 macrophages (3032). Based on the above reports, O2 and ONOO in the present study are probably generated from iNOS in RAW 264.7 cells, because L-Arg in the media is consumed time-dependently after stimulation. In this experiment, Griess method detecting NO2 level was utilized for assuming NO levels. Although the NO2 levels were not always equal to the NO levels under all experimental conditions, the Griess assay is nevertheless a convenient and simple method for estimating NO levels.

Co-culturing AS52 cells with TPA-stimulated HL-60 cells resulted in a significant increase in the frequency of gpt gene mutation. Treatment of the co-cultured cells with SOD or a NADPH oxidase inhibitor, DPI, but not allopurinol, markedly decreased the mutation frequency, demonstrating that O2 and/or occurring downstream RONS, e.g. H2O2, oOH, generated from NADPH oxidase, induced mutagenesis of AS52 cells. Co-culturing of LPS/IFN-{gamma}-stimulated RAW 264.7 cells with AS52 cells also increased the frequency of TGr AS52 mutants. SOD, iNOS inhibitors, DPI and L-NIO, strongly suppressed the mutation frequency, indicating that NO, O2, and/or its oxidative metabolites generated from LPS/IFN-{gamma}-stimulated RAW 264.7 cells induced mutagenesis of AS52 cells. There are three possible means for the reduction of mutation frequency by iNOS inhibitors. (i) Inhibition of downstream RONS production by suppressing O2 generation; (ii) inhibition of nitrosoamine formation which causes DNA alkylation (33) by suppressing NO production; and (iii) inhibition of ONOO production by suppressing both NO and O2 generation. Additionally, XOD, the other major source of O2 generation, probably does not contribute to the mutagenesis of AS52 cells under these conditions, because allopurinol did not suppress the mutation frequency. In addition, while a previous study reported that allopurinol also possesses •OH scavenging activity (34), we are not able to determine the involvement of oOH in gpt gene mutation on account of the limitation of a method for •OH measurement. Furthermore, although we confirmed that activated RAW 264.7 cells generated ONOO time-dependently, it still remains unknown whether ONOO was involved in gpt gene mutation in this experiment. Additional experiments will be required to elucidate the ONOO involvement in gpt gene mutation in the future.

8-OHdG is recognized as a useful marker for the estimation of DNA damage produced by RONS (35) and previous studies also reported that hepatocytes co-cultured with activated macrophages increased 8-OHdG/dG ratio (28). Oxidation at the C8 position of guanine one of the most common types of DNA damage and it is also a major mutagenic lesion, producing predominately G-T transversion mutation. In this study, co-culturing of TPA-stimulated HL-60 cells or LPS/IFN-{gamma}-stimulated RAW 264.7 cells with AS52 cells increased the 8-OHdG levels in the DNA of AS52 cells when compared with AS52 cells cultured alone. It is not surprising that the levels of 8-OHdG do not correlate with the mutation frequency, but with the oxidative damage levels of whole cellular DNA, because other types of DNA base modifications (e.g. 8-hydroxyadenine, 2-hydroxyadenine) may occur by RONS-producing cells. In any case, the present results strongly support the hypothesis that excessive RONS production from activated inflammatory leukocytes induces DNA mutation in mammalian cells. There are few known studies showing particular DNA mutation patterns of cells under oxidative co-culture. The sequencing of mutant gpt genes is now being investigated in our laboratory.

We have isolated and identified some active constituents, e.g. 1’-acetoxychavicol acetate (36,37), curcumin (38), cardamonin (39), glycerol glycolipids (40), pheophorbides (41), auraptene (42) and nobiletin (43); which suppress the production of O2 and NO from TPA-stimulated differentiated HL-60 cells and LPS/IFN-{gamma}-stimulated RAW 264.7 cells. It is important to note that most of these compounds prevented experimental carcinogenesis (37,4143), possibly through suppression of RONS generation. Thus, agents that suppress the production of RONS from activated leukocytes may be useful in cancer prevention.

In conclusion, while we were not able to identify precisely which RONS were responsible for mutagenesis in AS52 cells, the results of the present study strongly support the hypothesis that RONS generated from activated inflammatory leukocytes are responsible for inducing mutations in mammalian cells, and RONS generation inhibitors are potentially anti-mutagenic.


    Notes
 
3 To whom correspondence should be addressed Email: ohigashi{at}kais.kyoto-u.ac.jp Back


    Acknowledgments
 
This study was performed with Special Coordination Funds from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government and supported by a Grant-in-Aid for Cancer Research from Ministry of Health, Labor and Welfare for Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 5, 2002; revised October 18, 2002; accepted October 29, 2002.