Department of Pharmacology and Toxicology and School of Environmental Studies, Queens University, Kingston K7L 3N6, Ontario, Canada
Received September 11, 2002; accepted December 6, 2002
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ABSTRACT |
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Key Words: benzene; reactive oxygen species; oxidative stress; homologous recombination.
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INTRODUCTION |
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ROS include the superoxide radical anion, the hydroperoxyl radical, hydrogen peroxide, and the highly reactive hydroxyl radical and are generated in many physiological processes. While the cell has developed an array of nonenzymatic and enzymatic antioxidative mechanisms to detoxify ROS, oxidative stress can occur with xenobiotic bioactivation, leading to an imbalance between ROS formation and detoxification favoring a net increase in the formation of ROS (Gutteridge and Halliwell, 2000). Molecular targets of ROS-initiated damage include protein, lipids, and DNA, which may initiate carcinogenesis. Benzene metabolites have been shown to initiate oxidative damage in HL60 cells (Kolachana et al., 1993; Shen et al., 1996
) and to cause lipid oxidation in an animal model (Gaido and Wierda, 1987
), supporting a role for ROS in benzene-initiated toxicity.
DNA damage is a critical cellular lesion and is involved in cell death and carcinogenesis (Elliott and Jasin, 2002). ROS-initiated DNA damage includes oxidized bases, abasic sites, DNA-DNA intrastrand adducts, DNA strand breaks, and DNA-protein cross-links (reviewed in Cadet et al., 1999
). Several studies have demonstrated that benzene metabolites can cause DNA damage (Andreoli et al., 1997
; Tsutsui et al., 1997
) including DNA strand breaks (Kawanishi et al., 1989
; Lee and Garner, 1991
; Li and Trush, 1994
; Sze et al., 1996
) and DNA oxidation (Oikawa et al., 2001
). Oxidized DNA can be repaired by base excision repair and nucleotide excision repair (Lindahl and Wood, 1999
). During replication, areas of single-stranded DNA, produced by base excision and nucleotide excision repair can be converted to double-strand breaks, which are then repaired via homologous recombination (Haber, 1999
; Nickoloff and Little, 1997
). Therefore, ROS formation can directly induce DNA double-strand breaks and can also oxidize nucleotides that are subsequently converted to double-strand breaks during DNA replication (Brennan and Schiestl, 1998
; Haber, 1999
).
While homologous recombination is a repair mechanism, like all repair mechanisms it is not error-free, and therefore there is an increased risk of error with increased recombination frequency. Erroneous repair via homologous recombination can produce deleterious changes, such as loss of heterozygosity, gene deletions, or duplications, which can lead to genome instability and carcinogenesis (Moynahan and Jasin, 1997; Ramel et al., 1996
) and may be a potential underlying mechanism for benzene-initiated toxicity. Previously, studies have shown that benzene can initiate increased homologous recombination in both mammalian (Aubrecht et al., 1995
; Helleday et al., 1998
) and yeast cells (Brennan and Schiestl, 1998
). Furthermore, in yeast cells, benzene-initiated homologous recombination can be reduced by the free radical scavenger, N-acetyl cysteine (Brennan and Schiestl, 1998
). In the present study, to test the hypothesis that benzene and its metabolites initiate hyper-recombination in mammalian cells and to investigate the potential role of ROS, a previously characterized Chinese hamster ovary recombination cell line (CHO 36; Deng and Nickoloff, 1994
) was used to determine whether benzene and/or its metabolites initiated homologous recombination and whether the antioxidant catalase could abolish these effects. This study provides evidence supporting the hypothesis that increased homologous recombination is a molecular mechanism mediating the toxicity of benzene metabolites and suggests a role for ROS in this mechanism.
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MATERIALS AND METHODS |
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Cell culture.
The previously characterized neo recombination CHO cell line (CHO 36) was used to study homologous recombination initiated by benzene and its metabolites (Deng and Nickoloff, 1994). These cells were obtained from Jac A. Nickoloff, Department of Molecular Genetics and Microbiology, University of New Mexico, U.S. Briefly, these cells have a single, stably integrated tandem repeat neo recombination substrate, which upon homologous recombination confers resistance to the antibiotic Geneticin® (G418; Fig. 1
). Cells were maintained in
-minimum essential media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in 5% CO2.
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To study the role of ROS in benzene-initiated toxicity, further experiments evaluated whether catalase could reduce homologous recombination initiated by phenol, hydroquinone, catechol, or 1,4-benzoquinione. In these experiments, cells were plated as described above for both plating efficiency and homologous recombination studies. Cells were then exposed to catalase (2000 U/ml) just prior to being exposed to phenol, hydroquinone, catechol, or 1,4-benzoquinione (10 µM). The catalase concentration of 2000 U/ml was chosen because it has previously been demonstrated to protect against benzoquinone-initiated ROS (Shen et al., 1996). Homologous recombination frequency was then determined as described above.
Statistical analysis.
Results were analyzed using a standard, computerized statistical program (GraphPad Prism 3.0). Groups were compared using a one-factor analysis of variance (ANOVA). The minimum level of significance used throughout was p < 0.05.
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RESULTS |
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DISCUSSION |
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The results of this study, demonstrating that benzoquinone is the most potent metabolite in its ability to increase recombination, are similar to those obtained by Sze et al. (1996), who found that benzoquinone, of all the benzene metabolites that they tested, showed the highest potency in inducing DNA strand breaks in CHO cells. As stated by these authors, while CHO cells are not the natural target of benzene toxicity, the levels of bioactivating and detoxifying enzymes in these cells is similar to levels found in the human lymphoblast TK6 cell line (McGregor et al., 1991
). In studies evaluating micronucleus formation in human lymphocytes, benzoquinone was also the most potent metabolite (Yager et al., 1990
). However, studies using Chinese hamster V79 cells demonstrated that the increase in micronucleus formation and sister chromatid exchange was greatest after exposure to hydroquinone versus other metabolites of benzene, including benzoquinone (Glatt et al., 1989
). In contrast, in the present study it appears that hydroquinone is the least potent metabolite in causing homologous recombination. Differences in these studies are most likely due to varying levels of bioactivating and detoxification enzymes between the different cell lines.
It is now recognized that free radicals, including ROS, play a large role in many cellular processes, and that the cell contains mechanisms to balance radical production and radical detoxification. Oxidative stress, however, can occur when this balance somehow becomes disturbed. Increased oxidative stress has been implicated in over 100 diseases, including ischemia/reperfusion injury, cancer, inflammation, degenerative diseases, and aging (Gutteridge and Halliwell, 2002). One mechanism the cell uses to combat increased oxidative stress is the antioxidative enzyme, catalase, which is a heme protein found in the cytoplasm and peroxisomes. Catalase removes hydrogen peroxide from the cell by catalyzing its conversion to water. If not detoxified, hydrogen peroxide can interact with iron to form the highly reactive hydroxyl radical, which can initiate a series of toxic reactions that can irreversibly damage essential macromolecule targets.
The enzymatic bioactivation of benzene leading to the formation of ROS and subsequent increased oxidative stress is thought to play a significant role in benzene-initiated toxicity. Mice treated with benzene, phenol, catechol, or hydroquinone have significantly increased levels of oxidized DNA (Kolachana et al., 1993). Furthermore, bone marrow cells from benzene-treated mice have increased DNA binding activity for the transcription factor activator protein-1 (AP-1), a known target of oxidative stress (Ho and Witz, 1997
). These findings are consistent with increased levels of ROS after benzene exposure. Benzene metabolites have also been shown to increase myeloid cell growth in vitro by the formation of ROS (Wiemels and Smith, 1999
). Results from the present study demonstrate that the antioxidative enzyme catalase can completely block the observed increase in homologous DNA recombination initiated by exposure to phenol, catechol, hydroquinone, or benzoquinone, supporting the hypothesis that ROS can mediate the toxicity observed with exposure to these metabolites. Furthermore, these results are consistent with studies demonstrating that exposure to oxidative carcinogens including benzene leads to increased DNA recombination in the yeast Saccharomyces cerevisiae (Brennan et al., 1994
; Brennan and Schiestl, 1998
), which can be reduced by the presence of the free-radical scavenger N-acetyl cysteine (Brennan and Schiestl, 1998
). The protective effects of catalase observed in this study are consistent with numerous in vitro studies showing a protective effect of catalase against ROS production and ROS-initiated damage (Mann et al., 1997
; Noble et al., 1994
; Shen et al., 1996
).
Previous studies have shown that other toxicants, including carcinogens such as benzo[a]pyrene, 1-nitrosopyrene, and N-acetoxy-2-acetylamino-fluorene, can initiate DNA damage and homologous recombination in mammalian cells (Liskay et al., 1984; Wang et al., 1988
), yeast cells (Kunz and Haynes, 1981
; Schiestl et al., 1989
), and bacteria (Quinto and Radman, 1987
), supporting the hypothesis that genetic recombination is involved in the pathogenesis of cancer potential via the loss of wild-type alleles of critical genes (reviewed in Bishop and Schiestl, 2000
). Furthermore, it has been shown that fibroblasts from patients with ataxia telangiectasia, who have a 100-fold increase in the incidence of cancer, have high levels of spontaneous recombination frequencies (Meyn, 1993
).
Benzene-initiated leukemia has been associated with chromosomal translocations (reviewed in Synder, 2002), which may be mediated via aberrant recombination (Hutt and Kalf, 1996). Recently in studies with transgenic mice, it has been shown that 90% of benzene-induced thymic lymphomas exhibited loss of the functional p53 allele locus; the authors concluded this loss was likely due to aberrant recombination (Boley et al., 2000
). A potential mechanism mediating aberrant recombination is via the inhibition of the sulfhydryl (SH)-dependent endonuclease topoisomerase II (Topo II), which is essential for proper DNA recombination. Topo II exists in two isoforms, Topo II
and Topo IIß. Both isoforms catalyze the cleavage and subsequent religation of both strands of duplex DNA, thereby functioning to relax supercoiled DNA. Hutt and Kalf (1996)
demonstrated that both hydroquinone and benzoquinone could inhibit the activity of Topo II in vitro. Furthermore, Frantz et al. (1996)
also observed similar in vitro results when examining a series of putative benzene metabolites. Recent studies by Eastmond et al.(2001)
, showing that both benzene itself and its metabolites were able to inhibit the activity of Topo II in an isolated in vitro enzyme system, a leukemia cell line derived from human bone marrow, and additionally, in vivo, in the bone marrow of treated mice, further supported a role for this enzyme in benzene-initiated toxicity. However, additional studies investigating the mechanism of Topo II inhibition by benzene metabolites demonstrated that the metabolites did not stabilize the Topo II
-DNA cleavage complex, leading to decreased religation of the nicked DNA strand as previously hypothesized (Baker et al., 2001
). Instead these authors found that both benzoquinone and hydroquinone inhibited Topo II
-DNA binding, suggesting that inhibition of Topo II
is not involved in benzene-initiated recombination. Further studies investigating the potential inhibition of Topo IIß by benzene metabolites would enhance the understanding of the role of this enzyme in benzene-initiated toxicity.
In conclusion, the evidence presented in this paper demonstrates that the benzene metabolites phenol, catechol, hydroquinone, and benzoquinone can initiate increased frequencies of homologous DNA recombination in the CHO 36 recombination cell line. This increased frequency of recombination can be completely blocked by the activity of the antioxidative enzyme catalase, supporting the hypothesis that increased oxidative stress plays a role in benzene-initiated toxicity.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Aubrecht, J., Rugo, R., and Schiestl, R. H. (1995). Carcinogens induce intrachromosomal recombination in human cells. Carcinogenesis 11, 28412846.
Baker, R. K., Kurz, E. U., Pyatt, D. W., Irons, R. D., and Kroll, D. J. (2001). Benzene metabolites antagonize etoposide-stabilized cleavage complexes of DNA topoisomerase IIa. Blood 98, 830833.
Bishop, A. J. R., and Schiestl, R. H. (2000). Homologous recombination as a mechanism for genome rearrangements: Environmental and genetic effects. Hum. Mol. Genet. 9, 24272434.
Boley, S. E., Anderson, E. E., French, J. E., Donehower, L. A., Walker, D. B., and Recio, L. (2000). Loss of p53 in benzene-induced thymic lymphomas in p53 +/ mice: Evidence of chromosomal recombination. Cancer Res. 60, 28312835.
Brennan, R. J., and Schiestl, R. H. (1998). Free radicals generated in yeast by the Salmonella test-negative carcinogens benzene, urethane, thiourea and auramine O. Mutat. Res. 403, 6573.[CrossRef][ISI][Medline]
Brennan, R. J., Swoboda, B. E. P., and Schiestl, R. H. (1994). Oxidative mutagens induce intrachromosomal recombination in yeast. Mutat. Res. 308, 159167.[ISI][Medline]
Cadet, J., Delatour, T., Douki, T., Gasparutto, D., Pouget, J.-P., Ravanat, J.-L., and Sauvaigo, S. (1999). Hydroxyl radicals and DNA base damage. Mutat. Res. 424, 921.[CrossRef][ISI][Medline]
Deng, W. P., and Nickoloff, J. A. (1994). Preferential repair of UV damage in highly transcribed DNA diminishes UV-induced intrachromosomal recombination in mammalian cells. Mol. Cell. Biol. 14, 391399.[Abstract]
Eastmond, D. A., Schuler, M., Frantz, C., Chen, H., Parks, R., Wang, L., and Hasegawa, L. (2001). Characterization and mechanisms of chromosomal alterations induced by benzene in mice and humans. Res. Rep. Health Eff. Inst. 103, 168.[Medline]
Elliott, B., and Jasin, M. (2002). Double-strand breaks and translocations in cancer. Cell. Mol. Life Sci. 59, 373385.[CrossRef][ISI][Medline]
Frantz, C. E., Chen, H., and Eastmond, D. A. (1996). Inhibition of human topoisomerase II in vitro by bioactive benzene metabolites. Environ. Health Perspect. 104(Suppl. 6), 13191323.[ISI][Medline]
Gaido, K. W., and Wierda, D. (1987). Suppression of bone marrow stromal cell function by benzene and hydroquinone is ameliorated by indomethacin. Toxicol. Appl. Pharmacol. 89, 378390.[CrossRef][ISI][Medline]
Glatt, H., Padykula, R., Berchtold, G. A., Ludewig, G., Platt, K. L., Klein, J., and Oesch, F. (1989). Multiple activation pathways of benzene leading to products with varying genotoxic characteristics. Environ. Health Perspect. 82, 8189.[ISI][Medline]
Golding, B. T., and Watson, W. P. (1999). Possible mechanisms of carcinogenesis after exposure to benzene. IARC Scientific Pub. 150, 7588.
Gutteridge, J. M., and Halliwell, B. (2002) Free radicals and antioxidants in the year 2000. A historical look to the furture. Ann. N.Y. Acad. Sci. 899, 136147.
Haber, J. E. (1999). DNA recombination: The replication connection. Trends Biochem. Sci. 24, 271275.[CrossRef][ISI][Medline]
Helleday, T., Arnaudeau, C., and Jenssen, D. (1998). Effects of carcinogenic agents upon different mechanisms for intragenic recombination in mammalian cells. Carcinogenesis 19, 973978.[Abstract]
Ho, T.-Y., and Witz, G. (1997). Increased gene expression in human premyeloid leukemia cells exposed to trans, trans-muconaldehyde, a hematotoxic benzene metabolite. Carcinogenesis 18, 739744.[Abstract]
Hutt, A. M., and Kalf, G. F. (1996). Inhibition of human DNA Topoisomerase II by hydroquinione and p-benzoquinone, reactive metabolites of benzene. Environ. Health Perspect. 104, 12651269.[ISI][Medline]
Kawanishi, S., Inoue, S., and Kawanishi, M. (1989). Human DNA damage induced by 1,2,4-benzenetriol, a benzene metabolite. Cancer Res. 49, 164168.[Abstract]
Kolachana, P., Subrahmanyam, V. V., Meyer, K. B., Zhang, L., and Smith, M. T. (1993). Benzene and its phenolic metabolites produce oxidative DNA damage in HL60 cells in vitro and in the bone marrow in vivo. Cancer Res. 53, 10231026.[Abstract]
Kunz, B. A., and Haynes, R. H. (1981). Phenomenology and genetic control of mitotic recombination in yeast. Annu. Rev. Genet. 15, 5789.[CrossRef][ISI][Medline]
Lee, E. W., and Garner, C. D. (1991). Effects of benzene on DNA strand breaks in vivo versus benzene metabolite-induced DNA strand breaks in vitro in mouse marrow cells. Toxicol. Appl. Pharmacol. 108, 497508.[CrossRef][ISI][Medline]
Li, Y., and Trush, M. A. (1994). Reactive oxygen-dependent DNA damage resulting from the oxidation of phenolic compounds by a copper-redox cycle mechanism. Cancer Res. 54, 1895S1898S.[Medline]
Lindahl, T., and Wood, R. D. (1999). Quality control by DNA repair. Science 286, 18971905.
Liskay, R. M., Stachelek, J. L., and Letsou, A. (1984). Homologous recombination between repeated chromosomal sequences in mouse cells. Cold Spring Harbor Symp. Quant. Biol. 49, 183189.[ISI][Medline]
Mann, H., McCoy, M. T., Subramaniam, J., Van Remmem, H., and Cadet, J. L. (1997). Over expression of superoxide dismutase and catalase in immortalized neural cells: Toxic effects of hydrogen peroxide. Brain Res. 770, 163168.[CrossRef][ISI][Medline]
McGregor, D. B., Edwards, I., Wolf, C. R., Forrester, L. M., and Caspary, W. J. (1991). Endogenous xenobiotic enzyme levels in mammalian cells. Mutat. Res. 261, 2939.[ISI][Medline]
Meyn, M. S. (1993). High spontaneous intrachromosomal recombination rates in ataxia-telangiectasia. Science 260, 13271330.[ISI][Medline]
Moynahan, M. E., and Jasin, M. (1997). Loss of heterozygosity induced by a chromosomal double-strand break. Proc. Natl. Acad. Sci. U.S.A. 94, 89888993.
Nickoloff, J. A., and Little, J. B. (1997). Recombination. In Encyclopedia of Cancer, vol III. Academic Press.
Noble, P. G., Antel, J. P., and Yong, V. W. (1994). Astrocytes and catalase prevent the toxicity of catecholamines to oligodendrocytes. Brain Res. 633, 8390.[CrossRef][ISI][Medline]
Oikawa, S., Hirosawa, I., Hirakawa, K., and Kawanishi, S. (2001). Site specificity and mechanism of oxidative DNA damage induced by carcinogenic catechol. Carcinogenesis 22, 12391245.
Quinto, I., and Radman, M. (1987). Carcinogenic potency in rodents versus genotoxic potency in E. coli: A correlation analysis for bifunctional alkylating agents. Mutat. Res. 181, 235242.[ISI][Medline]
Ramel, C., Cederberg, H., Magnusson, J., Vogel, E., Natarajan, A. T., Mullender, L. H., Nivard, J. M., Parry, J. M., Leyson, A., Comendador, M. A., Sierra, L. M., Ferreiro, J. A., and Consuegra, S. (1996). Somatic recombination, gene amplification, and cancer. Mutat. Res. 353, 85107.[ISI][Medline]
Schiestl, R. H., Gietz, R. D., Mehta, R. D., and Hastings, P. J. (1989). Carcinogens induce intrachromosomal recombination in yeast. Carcinogenesis 10, 14451455.[Abstract]
Shen, Y., Shen, H.-M., Shi, C.-Y., and Ong, C.-N. (1996). Benzene metabolites enhance reactive oxygen species generation in HL60 human leukemia cells. Hum. Exp. Toxicol. 15, 422427.[ISI][Medline]
Snyder, R. (2002). Benzene and leukemia. Crit. Rev. Toxicol. 32, 155210.[ISI][Medline]
Sze, C.-C., Shi, C.-Y., and Ong, C.-N. (1996). Cytotoxicity and DNA strand breaks induced by benzene and its metabolites in Chinese hamster ovary cells. J. Appl. Toxicol. 16, 259264.[CrossRef][ISI][Medline]
Tsutsui, T., Hayashi, N., Maizumi, H., Huff, J., and Barrett, J.C. (1997). Benzene-, catechol-, hydroquinone-, and phenol-induced cell transformation, gene mutations, chromosome aberrations, aneuploidy, sister chromatid exchanges, and unscheduled DNA synthesis in Syrian hamster embryo cells. Mutat. Res. 373, 113123.[ISI][Medline]
Wang, Y. Y., Maher, V. M., Liskay, R. M., and McCormick, J. J. (1988). Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells. Mol. Cell. Biol. 8, 196202.[ISI][Medline]
Wiemels, J., and Smith, M. T. (1999). Enhancement of myeloid cell growth by benzene metabolites via the production of active oxygen species. Free Radical Res. 30, 93103.[ISI][Medline]
Yager, J. W., Eastmond, D. A., Robertson, M. L., Paradisin, W. M., and Smith, M. T. (1990). Characterization of micronuclei induced in human lymphocytes by benzene metabolites. Cancer Res. 50, 393399.[Abstract]