Homologous Recombination Initiated by Benzene Metabolites: A Potential Role of Oxidative Stress

Louise M. Winn1

Department of Pharmacology and Toxicology and School of Environmental Studies, Queen’s University, Kingston K7L 3N6, Ontario, Canada

Received September 11, 2002; accepted December 6, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzene is a ubiquitous pollutant and known human leukemogen. Benzene can be enzymatically bioactivated to reactive intermediates that can lead to increased formation of reactive oxygen species (ROS). ROS formation can directly induce DNA double-strand breaks, and also oxidize nucleotides that are subsequently converted to double-strand breaks during DNA replication that can be repaired through homologous recombination, which is not error-free. Therefore increased DNA double-strand-break levels may induce hyper-recombination, which can lead to deleterious genetic changes. To test the hypothesis that benzene and its metabolites can initiate hyper-recombination and to investigate the potential role of ROS, a Chinese hamster ovary (CHO) cell line containing a neo direct repeat recombination substrate (CHO 3–6), was used to determine whether benzene or its metabolites phenol, hydroquinone, catechol, or benzoquinone initiated increased homologous recombination and whether this increase could be diminished by the coincubation of cells with the antioxidative enzyme catalase. Results demonstrated that cells exposed to benzene (1, 10, 30, or 100 µM) for 24 h did not exhibit increased homologous recombination. Increased recombination occurred with exposure to phenol (1.8-, 2.6-, or 2.9-fold), catechol (1.9-, 2-, 5-, or 3.2-fold), or benzoquinone (2.7-, 5.5-, or 6.9-fold) at 1, 10, and 30-µM concentrations, respectively, and with exposure to hydroquinone at 10 and 30 µM concentrations (1.5–1.9-fold; p < 0.05). Studies investigating the effects of catalase demonstrated that increased homologous recombination due to exposure to phenol, hydroquinone, catechol, or benzoquinone (10 µM) could be completely abolished by the addition of catalase. These data support the hypothesis that increased homologous recombination mediates benzene-initiated toxicity and supports a role for oxidative stress in this mechanism.

Key Words: benzene; reactive oxygen species; oxidative stress; homologous recombination.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzene is a ubiquitous environmental chemical that is used mainly as a precursor in the synthesis of numerous products including drugs, dyes, insecticides, and plastics (Golding and Watson, 1999Go). It is found in unleaded gasoline, cigarette smoke, and industrial emissions (Golding and Watson, 1999Go). Based on epidemiological studies, human exposure to benzene causes bone marrow depression, acute myelogenous leukemia, acute lymphocytic leukemia, myelotoxicity, non-Hodgkin’s lymphoma, lung cancer, and nasopharyngeal cancer (Golding and Watson, 1999Go; reviewed in Snyder, 2002Go). In animals, benzene exposure leads to leukemia and neoplasms in the Zymbal gland, nasal cavity, liver, and mammary tissue (reviewed in Golding and Watson, 1999Go). Whereas the mechanism(s) by which benzene causes leukemia still remain unknown, it is generally accepted that, in order for benzene to exert its toxicity, it first must be metabolized in the body by cytochromes P450 (primarily CYP2E1) to phenol, catechol, and hydroquinone (Snyder, 2002Go). These metabolites can accumulate in bone marrow, where they can be further bioactivated by myleoperoxidases and other heme-protein peroxidases, to reactive semiquinones and quinones, which can further lead to the formation of reactive oxygen species (ROS).

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., 1993Go; Shen et al., 1996Go) and to cause lipid oxidation in an animal model (Gaido and Wierda, 1987Go), 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, 2002Go). 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., 1999Go). Several studies have demonstrated that benzene metabolites can cause DNA damage (Andreoli et al., 1997Go; Tsutsui et al., 1997Go) including DNA strand breaks (Kawanishi et al., 1989Go; Lee and Garner, 1991Go; Li and Trush, 1994Go; Sze et al., 1996Go) and DNA oxidation (Oikawa et al., 2001Go). Oxidized DNA can be repaired by base excision repair and nucleotide excision repair (Lindahl and Wood, 1999Go). 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, 1999Go; Nickoloff and Little, 1997Go). 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, 1998Go; Haber, 1999Go).

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, 1997Go; Ramel et al., 1996Go) 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., 1995Go; Helleday et al., 1998Go) and yeast cells (Brennan and Schiestl, 1998Go). Furthermore, in yeast cells, benzene-initiated homologous recombination can be reduced by the free radical scavenger, N-acetyl cysteine (Brennan and Schiestl, 1998Go). 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 3–6; Deng and Nickoloff, 1994Go) 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Benzene, phenol, 1,4-benzoquinone, hydroquinone, catechol, catalase, and crystal violet were all obtained from Sigma Chemical Co. (St. Louis, MO). {alpha}-Minimum essential media, fetal bovine serum, penicillin/streptomycin, and Geneticin® (G418) were from Gibco Life Technologies (Burlington, ON, Canada). All other reagents were of analytical grade.

Cell culture.
The previously characterized neo recombination CHO cell line (CHO 3–6) was used to study homologous recombination initiated by benzene and its metabolites (Deng and Nickoloff, 1994Go). 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. 1Go). Cells were maintained in {alpha}-minimum essential media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in 5% CO2.



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FIG. 1. The CHO 3–6-recombination substrate. Structure of the tandem repeat neo recombination substrate in CHO 3–6 cells. The first neo is regulated by the MMTV promoter but is inactivated by the HindIII site. While the second neo has wild-type coding capacity, it lacks a promoter and is therefore also inactive. Homologous recombination via gene conversion or deletion events leads to the loss of the HindIII site and confers resistance to the antibiotic G418 (modified from Deng and Nickoloff, 1994Go).

 
Recombination studies.
Homologous recombination frequency was determined by plating CHO 3–6 cells at a density of 1 x 106 per 10-cm culture dish. For the determination of plating efficiency (cell survival), cells were plated at a density of 300 per 10-cm dish. After 5 h, cells were treated with benzene, phenol, hydroquinone, catechol, or benzoquinone (1-, 10-, 30-, or 100-µM concentrations) or the vehicle control (media) for 24 h. After drug exposure, cells used in recombination studies were washed with phosphate-buffered saline (PBS) and grown in fresh media containing G418 (500 µg/ml). Cells used for plating efficiency studies were treated the same way except these cells were not exposed to G418. Cells were grown for either 1 week (plating efficiency studies) or two weeks (recombination studies) and then stained with crystal violet (1% in methanol). Homologous recombination frequency was determined by counting the number of G418-resistant colonies versus the total number of surviving cells plated.

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., 1996Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzene- and metabolite-initiated cell death.
Cell survival in the different treatment groups was determined and used to calculate the recombination frequency. Exposure to benzene and phenol for 24 h at concentrations as high as 100 µM did not lead to increased cell death in the CHO 3–6 cells when compared to vehicle controls (Fig. 2Go). Hydroquinone caused a significant increase in cell death at 30 and 100 µM, while catechol increased cell death at concentrations of 10 µM and higher, and benzoquinone increased cell death at 100 µM (p < 0.05; Fig. 2Go).



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FIG. 2. Benzene and metabolite-initiated cell death. Percentage of CHO 3–6 cell survival after a 24-h exposure to benzene, phenol, hydroquinone, catechol, or benzoquinone (0-, 1-, 10-, 30-, or 100-µM). Cell survival was determined by calculating the number of cells forming colonies after one week, divided by the number of cells plated; n = 4 for each group. *Difference from the 0 µM treatment (media alone) for each metabolite tested (p < 0.05).

 
Benzene- and metabolite-initiated homologous recombination.
Due to the high level of cell death in CHO 3–6 cells exposed to benzene metabolites at 100 µM concentrations, homologous recombination data at this concentration was not determined except for benzene itself. CHO 3–6 cells exposed to benzene for 24 h did not demonstrate increased homologous recombination frequency compared to vehicle control (Fig. 3Go), even at the 100 µM concentration (data not shown). However exposure of CHO 3–6 cells to benzene metabolites did lead to increased homologous recombination (Fig. 3Go). Exposure to hydroquinone at 10 and 30 µM increased homologous recombination by 1.5- and 1.9-fold respectively (p < 0.05; Fig. 3Go). Increased recombination occurred with exposure to phenol (1.8-, 2.6-, and 2.9-fold), catechol (1.9-, 25-, and 3.2-fold), or benzoquinone (2.7-, 5.5- and 6.9-fold) at 1, 10, and 30 µM concentrations, respectively, when compared to vehicle controls (p < 0.05; Fig. 3Go). There was variability in the recombination frequency amongst the different control groups for each experiment. CHO 3–6 cells have a spontaneous level of recombination frequency, and given that the cells used to conduct the different treatment groups were not necessarily from the same passage, this might potentially explain the variability. Nevertheless, cells from the same passage were used for groups within the same treatment regimen, and thus, the results within these experiments should not be affected by this variability.



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FIG. 3. Benzene and metabolite-initiated homologous recombination. Frequency of homologous DNA recombination in CHO 3–6 cells treated with benzene, phenol, hydroquinone, catechol, or benzoquinone (0-, 1-, 10-, 30-, or 100-µM) for 24 h. Homologous recombination frequency was determined by counting the number of G418-resistant colonies compared with the total number of surviving cells plated; n = 10 for each group. *Difference from the 0 µM treatment (media alone) for each metabolite tested (p < 0.05).

 
Effects of catalase on metabolite-initiated cell death and homologous recombination.
In studies investigating the effects of the antioxidative enzyme catalase on metabolite-mediated homologous recombination, both hydroquinone and benzoquinone at a concentration of 10 µM, significantly enhanced cell death in CHO 3–6 recombination cells, while exposure to 10 µM phenol or catechol did not significantly decrease cell survival in these cells (p < 0.05; Fig. 4AGo). Increased cell death due to hydroquinone or benzoquinone exposure was completely reduced by coincubation with catalase in these studies (p < 0.05; Fig. 4AGo). At 10-µM strength, increased recombination occurred with exposure to either phenol (5.9 fold), hydroquinone (4.7 fold), catechol (6.2 fold), or benzoquinone (7.1 fold) when compared to vehicle controls (p < 0.05; Fig. 4BGo). The increased frequency of homologous recombination in these cells, due to exposure to these metabolites, was completely reduced to control levels by the addition of catalase (Fig. 4BGo).



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FIG. 4. Effects of catalase on metabolite-initiated cell death and homologous recombination: (A) The effects of catalase (2000 units/ml) on the percentage of CHO 3–6 cell survival after a 24-h exposure to phenol (PHEN), hydroquinone (HQ), catechol (CATE), or benzoquinone (BQ; 10 µM). Open bars, data from cells treated with drug alone; filled bars, data from cells coincubated with catalase and drug. For each group n = 4. *Difference from the 0 µM treatment (CON; p < 0.05). (B) The effects of catalase (2000 U/ml) on the frequency of DNA recombination after a 24-h exposure to phenol (PHEN), hydroquinone (HQ), catechol (CATE), or benzoquinone (BQ; 10 µM). Open bars, data from cells treated with drug alone; filled bars, data from cells coincubated with catalase and drug. For each group n = 10. *Difference from the 0 µM treatment (CON; p < 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzene is an industrial and environmental contaminant that continues to be a significant medical concern. In humans, acute myelogenous leukemia is the major health risk associated with exposure to low levels of benzene (reviewed in Snyder, 2002Go). However, while benzene is a known human leukemogen, the mechanism of its carcinogenicity still remains to be elucidated. Hematotoxicity caused by benzene exposure is thought to occur via the metabolites phenol, catechol, and hydroquinone that can be further metabolized to reactive intermediates including benzoquinone. The results of this study demonstrate that phenol, hydroquinone, catechol, and benzoquinone, all metabolites of benzene, caused a dose-dependent increase in the frequency of homologous DNA recombination in the CHO 3–6 recombination cell line, but that benzene itself did not. Previous studies have shown that exposure to benzene can increase DNA recombination in other mammalian recombination cell lines but at concentrations that were 3-fold higher than those used in this experiment (Aubrecht et al., 1995Go; Helleday et al., 1998Go). Therefore it is possible that, at higher concentrations, benzene would have initiated higher frequencies of recombination in CHO 3–6 cells.

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)Go, 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., 1991Go). In studies evaluating micronucleus formation in human lymphocytes, benzoquinone was also the most potent metabolite (Yager et al., 1990Go). 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., 1989Go). 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, 2002Go). 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., 1993Go). 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, 1997Go). 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, 1999Go). 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., 1994Go; Brennan and Schiestl, 1998Go), which can be reduced by the presence of the free-radical scavenger N-acetyl cysteine (Brennan and Schiestl, 1998Go). 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., 1997Go; Noble et al., 1994Go; Shen et al., 1996Go).

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., 1984Go; Wang et al., 1988Go), yeast cells (Kunz and Haynes, 1981Go; Schiestl et al., 1989Go), and bacteria (Quinto and Radman, 1987Go), 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, 2000Go). 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, 1993Go).

Benzene-initiated leukemia has been associated with chromosomal translocations (reviewed in Synder, 2002), which may be mediated via aberrant recombination (Hutt and Kalf, 1996Go). 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., 2000Go). 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{alpha} 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)Go demonstrated that both hydroquinone and benzoquinone could inhibit the activity of Topo II in vitro. Furthermore, Frantz et al. (1996)Go also observed similar in vitro results when examining a series of putative benzene metabolites. Recent studies by Eastmond et al.(2001)Go, 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{alpha}-DNA cleavage complex, leading to decreased religation of the nicked DNA strand as previously hypothesized (Baker et al., 2001Go). Instead these authors found that both benzoquinone and hydroquinone inhibited Topo II{alpha}-DNA binding, suggesting that inhibition of Topo II{alpha} 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 3–6 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.


    ACKNOWLEDGMENTS
 
These studies were supported through grants from the Institute of Human Development, Child and Youth Health of the Canadian Institute of Health Research and the Queen’s University Research Advisory Committee.


    NOTES
 
1 For correspondence via fax: (613) 533-6412. Email: winn1{at}biology.queensu.ca Back


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 DISCUSSION
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