Mutagenesis vol. 19 no. 3 pp. 195-201,
May 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Profiles of gene expression changes in L5178Y mouse lymphoma cells treated with methyl methanesulfonate and sodium chloride
1Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland, MI 48674, USA and 2Battelle Toxicology, 505 King Avenue, Columbus, OH, USA
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Treatment of cells with genotoxic chemicals is expected to set into motion a series of events including gene expression changes to cope with the damage. We have investigated gene expression changes in L5178Y TK+/– mouse lymphoma cells in culture following treatment with methyl methanesulfonate (MMS), a direct acting genotoxin, and sodium chloride (NaCl), which induces mutations in these cells through indirect mechanisms at high concentrations. The mouse lymphoma cells were treated for 4 or 24 h and the cells were harvested for RNA isolation at the end of the treatment. Analysis of the transcriptome was performed using Clontech Mouse 1.2K cDNA microarrays (1185 genes) and hybridized using 32P-labeled cDNA. The microwell methodology was used to quantify the mutagenic response. Of the genes examined, MMS altered the expression (1.5-fold or more) of only five (four at 4 h and one after 24 h treatment). NaCl altered two genes after 4 h treatment, but after 24 h it altered 19 genes (13 down- and six up-regulated). Both compounds altered the expression of several genes associated with apoptosis and NaCl altered genes involved in DNA damage/response and GTP-related proteins. This, along with other data, indicates that the widely used L5178Y TK+/–mouse lymphoma cells in culture are relatively recalcitrant in terms of modulating gene expression to deal with genotoxic insult.
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Introduction |
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The advent of microarray technology opened a potentially new opportunity for genome-wide analysis to examine the effects of chemicals at the molecular level. It is becoming an integral part of drug discovery research. The use of microarrays has also become a part of toxicology (toxicogenomics) as a means of studying mechanisms of action and has opened up the prospect of becoming a useful tool for predicting toxicity. For toxico-genomics to be a predictor of toxicity it is assumed that most, if not all, toxicants alter gene regulation (Farr and Dunn, 1999
). This altered regulation may be part of the toxic response, a secondary response or an adaptation to the original insult (protective). However, to predict possible toxic effects, it is imperative that a set of genes must be identified that would allow for accurate classification of the compounds of interest. It has been shown that a small, carefully selected set of genes can predict an unknown chemical’s classification better than a large set of genes, even if other genes are altered by the chemical (Thomas et al., 2001; Hamadeh et al., 2002). To identify these predictive genes, classic toxicants must be analyzed to determine the most appropriate genes that will represent each class of chemical or toxic end-point. This information could then be maintained in a database where new transcription profile patterns can be compared to determine which class of compounds they represent.
This study examined the gene expression changes induced by two chemicals which damage DNA through distinctly different mechanisms of action. Methyl methanesulfonate (MMS) is an alkylating agent which shows high SN2 reactivity causing N-alkylation of purines (N7-methylguanine and N3-methyladenine). These N-alkylations are rapidly repaired and have a minimal potential for mutagenicity. Therefore, it is believed that the mutagenicity of MMS is through abasic sites by ß-elimination of the N-glycosylic bond or by a DNA glycosylase repair enzyme (Glaab et al., 1998). Sodium chloride (NaCl), although not commonly recognized as a DNA-damaging agent, has been shown to cause chromosomal aberrations and double-strand breaks in many different types of cells (Galloway et al., 1987; Uchida et al., 1987; Kalweit et al., 1990; Nowak, 1990; Kültz and Chkravarty, 2001). There are several possible mechanisms by which NaCl, or the resultant hypertonicity, could cause double-strand breaks. First, cell shrinkage may cause changes in DNA rigidity and bending through increased ionic strength, macromolecular crowding and physical distortion of the nuclear matrix. This can enhance the possibility of breakage of DNA. Second, double-strand breaks could be formed by free radicals. It has been shown that hyperosmolarity can increase the activity of free radical scavenging enzymes (Shukla et al., 1993). Finally, chromatin compactness and DNA accessibility changes may disturb the balance between repair and damage. This could also enhance access to certain regions of DNA by nucleases or free radicals (Kültz and Chkravarty, 2001).
The L5178Y/TK+/–-3.7.2C mouse lymphoma cell line was chosen for these experiments due to its use in regulatory genetic toxicology testing for the detection of forward mutations. The L5178Y cell line has a single functional copy of the thymidine kinase (Tk) gene. Therefore, when DNA damage occurs to the functional Tk allele, a total loss of TK activity results. This loss of TK activity (Tk–/– cells) can then be determined by selecting with the toxic thymidine analog trifluorothymidine (Clive and Spector, 1975). The L5178Y cells also have point mutations in both p53 alleles, one of which encodes a stop codon (Storer et al., 1997; Clark et al., 1998). Because p53 is an important protein in the DNA damage response in the cell, the L5178Y cell line is inadequate in its response to DNA damage, which is arguably important for enhanced assay sensitivity for the detection of genotoxic compounds.
In this study, we examined the alteration in genomic expression caused by the DNA-damaging agents MMS and NaCl in L5178Y/TK+/–-3.7.2C cells utilizing microarrays containing 1185 genes. The goal of this study was to find both unique and commonly altered genes between two different types of DNA-damaging agents in this mouse lymphoma cell line for possible use in defining modes of genotoxic action.
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Cell culture
L5178Y/TK+/–-3.7.2C clonal mouse lymphoma cells (originally obtained from D.Clive of Burroughs Wellcome Laboratory) were cultured in Fischer’s medium (F10P; Quality Biological, Gaithersburg, MD) supplemented with 2 mM L-glutamine (Gibco, Grand Island, NY), 0.22 mg/ml sodium pyruvate (Sigma Chemical Co., St Louis, MO), 1 mg/ml Plurionic F68® (Gibco), 10% (v/v) heat-inactivated (56°C, 30 min) horse serum (Hyclone Laboratories, Logan, UT) and antibiotics (20 U/ml penicillin G and 20 µg/ml streptomycin sulfate; Gibco). Cells, in capped tubes gassed with air containing 5% CO2, were maintained at 37°C in a shaker incubator. The conditioned medium was F10P from which cells had been removed; this medium was used during treatments. F0P is F10P without the 10% horse serum. Experimental conditions and analysis were performed using the standard protocol for the mouse lymphoma assay.
Two concentrations of MMS (CAS no. 66-27-3) (Sigma) and sodium chloride (CAS no. 76-471-45) (Fisher Scientific, Pittsburgh, PA) were used for these experiments (MMS, 0.1 and 10 µg/ml; NaCl, 500 and 5000 µg/ml) based on previous experiments run in our laboratory. The two concentrations selected for analyses were chosen to yield relatively little and high degrees of both cytotoxicity and mutant frequency. Since one of the objectives of this study was to evaluate gene expression changes under conditions of excessive cytotoxicity by an indirect acting agent, NaCl concentrations exceeding the generally recommended upper limit of 10 mM were employed. Cell treatment medium was a 1:1 mixture of conditioned medium and fresh medium (F0P for 4 h or F10P for 24 h treatment). After treatment, cells were washed with F10P and resuspended in RNALaterTM (Ambion, Austin, TX) for future RNA isolation. Cells placed in RNALaterTM were stored overnight at 4°C and then transferred to –20°C until RNA isolation. Two independent experiments (Experiments 1 and 2) were performed with both MMS and NaCl.
Evaluation of toxicity and mutant frequency
After treatment, cells were grown in F10P medium in a roller drum at 37°C in capped tubes gassed with air at 5% CO2. Following an additional 24 h incubation, cultures were counted and those with a density of >4 x 105 cells/ml were diluted to a concentration of 3 x 105 cells/ml with F10P. After another 24 h, the cells were plated in 96-well microtiter plates at 1.6 and 2000 cells/well for cell viability (cloning efficiency) and mutant selection, respectively. The F10P medium was used to determine cloning efficiency and F10P containing trifluorothymidine (TFT) (Sigma) (3 µg/ml for experiment 1 and 1 µg/ml for experiment 2), which selects cells that have mutated the single functional Tk gene, was used for mutant selection. The cells were grown for an additional 10–12 days before determining the number of wells with and without colonies. Standard procedures for the microwell methodology were used to calculate cloning efficiency and mutant frequency (Clements, 2000).
RNA isolation and microarray screening
Total RNA was isolated using an AtlasTM Pure Total RNA Labeling System (Clontech, Palo Alto, CA), which is based on a modification of the guanidinium thiocyanate/phenol/chloroform method of Chomczynski and Sacchi (1987). RNA isolation was carried out as described by the manufacturer. In brief, cells were resuspended in denaturing solution to break the cells open and the cellular debris was removed by centrifugation. Then the supernatant was extracted twice with phenol/chloroform and the RNA was precipitated and resuspended. Next, RNA samples were treated with DNase I, followed by phenol/chloroform and then chloroform extraction. The DNase-treated RNA was precipitated, quantitated and aliquoted. RNA samples were run on a denaturing agarose gel to check RNA integrity and the absence of DNA contamination. Poly(A)+ RNA was isolated by incubating total RNA with biotinylated oligo(dT) and allowing the poly(A)+ RNA to hybridize. Magnetic beads coated with streptavidin were then added and allowed to interact with the biotin on the oligo(dT). Finally, the magnetic bead–biotinylated oligo(dT)–poly(A)+ RNA complex was separated from the rest of the RNA using a magnet and then washed. The probes were made by incubating the poly(A)+ RNA with specific primers, against the genes spotted on the array, and making 32P-labeled cDNA using reverse transcriptase. AtlasTM Mouse 1.2K (Clontech) arrays were pre-hybridized for 30 min then the [32P]cDNA probe was denatured, added to the prehybridization mixture and allowed to hybridize overnight. The arrays were washed, exposed to a phosphorimaging plate and scanned using a Molecular Dynamic (Sunnyvale, CA) phosphorimager. In each experiment, RNA samples from each treatment where labeled and hybridized on two separate occasions (Experiments 1A, 1B, 2A and 2B) to a microarray membrane, except for Experiment 1, in which the 4 h treatment RNA was run only once. In each experiment the membrane arrays were from the same manufacturer lot number.
Microarray analysis
Each array was aligned with the appropriate template using AtlasTM Imager (Clontech) and the open area around the gene panels was used as background. These raw data were then analyzed using GeneSpringTM (Silicon Genetics, Redwood City, CA) software. For a gene to be considered expressed, the phosphorimage value had to be of an intensity >20 (
2 times background) on one of the blots (control, low or high dose) after background subtraction. These genes were then analyzed to determine if they had altered gene expression. A two step normalization was used: (i) each array was normalized to the median of all measurements for that array (to correct for differences in exposure/specific activity) and (ii) each gene was normalized to the solvent control array. To be considered induced or repressed on an array, a gene must have had a normalized value of >1.5 or <0.67, respectively, on one of the two treated arrays. For a gene to have been considered altered due to treatment, it must have been induced or repressed on all four microarray runs (two experiments, each RNA analyzed twice), except at 4 h, when only three sets of arrays were run. In the figures these normalized fold change values were converted to log2 for graphing purposes.
Real-time PCR analysis
In order to corroborate the relative changes in gene expression induced by MMS or NaCl exposure, some of the genes identified by microarray analysis were selected for verification by real-time quantitative reverse transcription PCR (RT–PCR). The gene sequences were obtained from the GenBank database and used to design mouse-specific PCR primer probes (Table I) using Primer ExpressTM Version 2.0 primer design software (PE Applied Biosystems, Foster City, CA). cDNA was synthesized with random hexamer primers and total RNA using a Reverse Transcription kit (PE Applied Biosystems) according to the manufacturer’s instruction. Preliminary experiments were done with each primer pair to determine the overall quality and specificity of the primer design. RT–PCR was performed using 10 ng of the cDNA on a 5700 Prism machine using a SYBR Green PCR Reagent kit (PE Applied Biosystems) according to the manufacturer’s instructions for quantitation of relative gene expression. All samples were run in triplicate. Production of a single PCR product was confirmed by dissociation curve analysis of the product.
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Results |
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Doses for MMS and NaCl were chosen based on previous work that was performed in our laboratory (data not shown). Table II shows the relative total growth (RTG) and mutant frequency for the 4 and 24 h treatments from two separate experiments in which RNA was isolated. MMS at 10 µg/ml induced increases in mutant frequency in both experiments. Sodium chloride at 5000 µg/ml induced an elevation in mutant frequency that is considered to represent a positive response following both 4 (one experiment) and 24 h (two experiments) treatments. The lower concentration of MMS and NaCl employed induced very little to no cytotoxicity or mutagenicity.
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Of the 1185 genes present on the array, NaCl altered the expression of 21 genes and MMS altered the expression of only five (Figure 1). Between these two chemicals, only one gene was in common, i.e. P-selectin glycoprotein ligand 1 precursor (Psgl1), which was up-regulated at 4 h with MMS and 24 h with NaCl. The vast majority of gene expression changes with NaCl were observed in cultures treated for 24 h. The minimal number of gene expression changes in MMS-treated cultures at similar levels of excessive cytotoxicity as in NaCl following 24 h treatment suggests different mechanisms of cytotoxic action. Interestingly, none of the genes altered by MMS are known to be involved in DNA repair. However, NaCl altered the expression of six genes involved in DNA damage/repair and cell cycle control, including p53, which is non-functional in this cell line. Both MMS and NaCl altered the expression of genes that have been associated with apoptosis. Genes altered by MMS were all up-regulated, while the majority of genes altered by NaCl were down-regulated. Granzyme A and G-protein-coupled receptor 27 were the most highly altered genes overall after 24 h treatment with NaCl.
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GDP dissociation inhibitor ß), Gnb2 (guanine nucleotide-binding protein ß), Gapdh (glyceraldehyde 3-phosphate dehydrogenase), Stra13 (stimulated by retinoic acid 13).Real-time quantitative RT–PCR was performed on several genes using the RNA from Experiment 1 for all treatments and times to confirm the results seen with microarray (Table III). Two genes that were called altered by treatment using microarray were considered unaltered by RT–PCR. Psgl1 showed 1.6-fold induction with low dose MMS after 4 h treatment by microarray but RT–PCR showed only a 1.1-fold change. However, RT–PCR after 24 h high dose MMS treatment showed a 4.3-fold increase in Psgl1 compared to 1.6-fold for microarrays in Experiment 1. Similarly, Gpcr27 showed induction by both low and high dose NaCl after 24 h treatment with microarray but only the high dose effect was confirmed by RT–PCR.
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Discussion |
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The microarray results obtained in this study with a mouse lymphoma cell line comparing the alkylating agent MMS and NaCl show a distinct difference in their gene expression profiles. Only one gene, Psgl1, had altered expression in common between these two chemicals in microarray analysis, but after RT–PCR analysis Psgl1 was not shown to be altered by MMS. It is interesting to note that MMS did not affect the expression of any genes related to DNA damage response/repair or cell cycle control. On the other hand, NaCl affected genes involved in both DNA damage response/repair and cell cycle control: Rad21 (Birkenbihl and Subramani, 1992
), myb-related protein 2 (Oh and Reddy, 1999), p53 (Balint and Vousden, 2001), Cdc46 (Kimura et al., 1996), Gapdh (Meyer-Siegler et al., 1992) and Tgfß1 (Ewan et al., 2002). Though L5178Y cells have two non-functional copies of p53, which is a key protein in the control of apoptosis, they can undergo delayed apoptosis (Endlich et al., 2000). It was found that both MMS and NaCl altered the regulation of genes that have been associated with apoptosis. For MMS, the three genes were Bax membrane isoform 
(Oltvai et al., 1993), cytoplasmid dynein light chain 1 (Dick et al., 1996) and thromboxane A2 receptor (Ushikubi et al., 1993). For NaCl, Rad21 (Chen et al., 2002), p53 (Balint and Vousden, 2001), granzyme A (Beresford et al., 1999), Gapdh (Ishitani and Chuang, 1996) and interleukin-2 receptor 
subunit (Il2rg) (Akbar et al., 1996) expression were altered. Interestingly, a high induction level of granzyme A was also seen when L5178Y cells were treated with the DNA-damaging agents hydrogen peroxide and bleomycin (Seidel et al., 2003). This induction of granzyme A may be related to the generation of free radicals, since both hydrogen peroxide (Termini, 2000) and bleomycin (Dedon and Goldberg, 1992) produce free radicals. One possible mechanism for NaCl inducing DNA damage is through the generation of free radicals (Shukla et al., 1993). Though there are no data on granzyme A being regulated in response to free radicals, it is known that granzyme A cleaves/inactivates Ape1 (Fan et al., 2003). Normally, Ape1 translocates to the nucleus in response to an increase in reactive oxygen species, where it is involved in DNA repair and also reducing oxidized immediate-early transcription factors (Xanthoudakis and Curran, 1992; Hirota et al., 1997), including p53 (Gaiddon et al., 1999). It is believed that granzyme A cleaves/inactivates Ape1 to help facilitate apoptosis by preventing cellular repair/recovery through Ape1 (Fan et al., 2003). Though several of the MMS/NaCl affected genes have been associated with DNA damage response/repair, cycle control and/or apoptosis, many were not altered as anticipated (induction/repression) in response to DNA damage. This may likely be due to the fact that most of these genes have other functions besides being part of the DNA damage response and the changes observed could be for these other functions, especially those due to NaCl treatment, as the change in osmolarity has an enormous effect on the cell.
Another group of genes altered by NaCl treatment has been associated with GTP. Transducin ß-2 subunit (guanine nucleotide-binding protein) contributes to the functional specificity of G proteins (Gao et al., 1987). Little is known about G protein-coupled receptor 27 except that, based on its sequence, it is a member of the G protein-coupled receptors (O’Dowd et al., 1998). Rho GDP dissociation inhibitor ß (Gdiß) decreases the rate of GDP dissociation from rho proteins, which is important in regulating the activity of rho proteins since they are only active in the GTP-bound state (Guillemot et al., 1996). Finally, Rab24 is a member of the Rab GTPase family, the members of which are involved in protein trafficking (Takai et al., 2001). These GTP-connected proteins are most likely not involved in the response to DNA damage caused by NaCl but more likely an effect of hypertonicity such as cell shrinkage.
Confirmation of the microarray results using real-time PCR gave relatively good concordance across all times and treatments in general (Table III). Two microarray results were not confirmed as being altered, Gpcr27 by NaCl at 24 h after low dose treatment and Psgl1 by MMS at 4 h after low dose treatment. The most likely reason that Gpcr27 was not confirmed is the fact that the overall values from the microarray were around the cut-off value set to consider a gene being expressed. A small change to a low end expression value could result in a large fold change, therefore, experimental variability has a significant impact on these values (Rockett et al., 2002). The lack of confirmation for Psgl1 could be due to variability of the microarray (Ting Lee et al., 2000) since the value of experiment 1 was just above our cut-off of 1.5-fold for considering a gene as altered. Therefore, random experimental variability could lead to a false negative or false positive call in such cases. The values for Psgl1 in experiment 2 showed considerable variability, with one replicate showing a 2-fold induction and the other no change (Figure 1B). These observations support the need to confirm microarray results, especially for those gene expression changes that are identified as marginal. Another trend seen is that large fold changes are underestimated by microarrays compared with RT–PCR (e.g. high dose NaCl after 24 h for granzyme A and Gpcr27). However, this is a known artifact of using cDNA arrays, which tend to compress values compared with the true expression (Yuen et al., 2002).
It is known that high concentrations of NaCl activate several MAPKs in many different cell types (Kültz et al., 1998; Ying and Sanders, 2002). In turn, MAPKs produce a cascade of other events in the cell. Several genes identified with altered regulation after 24 h NaCl treatment have been connected to MAPK activation. Tgfß1 is regulated by AP-1, which is activated by MAPKs (Hamaguchi et al., 2000). MAPK also increases the production of interleukin 2 (IL2) (Matthews and O’Neill, 1999), which is not present on the array used, however, an increase in IL2RG is seen. After IL2 binds and transduces its signal through IL2RG, along with the other IL2 receptor subunits (which are not present on the array used), the receptor is internalized and transported to lysosomes (Hemar et al., 1995). Therefore, more IL2RG needs to be produced to replace that lost by internalization. Internalization of the IL2 receptor (containing IL2RG) is controlled by two proteins, called RhoA and Rac1, which are small GTP-binding proteins that are inactive when GDP is bound (Lamaze et al., 2001). RhoA and Rac1 are controlled by GDIs and it has been shown that a GDI can totally prevent IL2R uptake (Lamaze et al., 2001). In the present study, after 24 h NaCl treatment, Gdiß, a GDI, was down-regulated. Therefore, it is possible that Gdiß repression by NaCl allows RhoA and Rac1 to be active subsequently, allowing internalization of the IL2 receptor. Another gene that is down-regulated after NaCl treatment is Rab24, which is involved in vesicle trafficking. Not much is known about RAB24, but it plays a role in the trafficking of late endosomes to lysosomes, which is where IL2R is degraded (Takai et al., 2001).
Salt (NaCl) is generally thought of as relatively benign. However, this study (Table II) and others have shown that high levels of salt can cause DNA damage and genotoxicity in vitro and in vivo (Galloway et al., 1987; Uchida et al., 1987; Kalweit et al., 1990; Nowak, 1990; Moore and Brock, 1988; Kültz and Chkravarty, 2001; Balakrishnan et al., 2002). Significantly, hyperosmolality per se does not appear to be responsible for the DNA damage. Hyperosmolarity caused by NaCl, but not urea, has been shown to cause double-strand breaks, which are consistent with NaCl causing an increase in cancer (Kültz and Chkravarty, 2001; Tsugane et al., 2004). It is also interesting to note that the same study showed that NaCl, but not urea, activated MAPKs (Kültz and Chkravarty, 2001). Further, it has been shown that the sodium salts of ascorbate, glutamate, aspartate, citrate, erthorbate, bicarbonate and, to a lesser extent, chloride, can generate urothelial tumorigenesis in rat bladders (Cohen et al., 1995). Both epidemiology and animal data suggest that high salt diets are a risk factor for stomach cancer (Kono and Hirohata, 1996; MacGregor, 1997; Tsugane et al., 2004).
The lack of transcriptome changes in L5178Y mouse lymphoma cells in response to exposure to MMS could indicate that these cells may be relatively refractory in reacting to genotoxic insult induced by alkylating agents. Alternatively, it is possible that the transcriptional machinery of the cell was adversely impacted through the macromolecular alkylation induced by MMS. MMS altered the regulation of only four genes after 4 h treatment and only a single gene after 24 h treatment. Assuming that the genes on our array are a good representation of the whole mouse genome this would mean that 122 and 30 genes would be altered after 4 and 24 h treatment, respectively, in the entire genome of these cells. In comparison, yeast cells exposed for 1 h to 1 mg/ml MMS altered 401 genes (325 up- and 76 down-regulated) (Jelinsky and Samson, 1999). Extrapolation of the present study data to the whole mouse transcriptome would suggest that expression of only 0.4% of the genes would be altered in these cells, compared with 6.5% for yeast. There is a high homology in structure, function and response of genes involved in DNA damage/repair between yeast and mouse cells (Herrlich et al., 1997; Eckardt-Schupp and Klaus, 1999) and of the genes altered in the yeast treated with MMS, many were involved in stress response/detoxification, DNA synthesis/repair and cell cycle control (Jelinsky and Samson, 1999). The same trend is also seen with NaCl treatment of yeast. Yeast treated with 58.4 mg/ml NaCl for 90 min altered the regulation of 354 genes, which is 5.8% of the yeast genes (Yale and Bohnert, 2001), compared with 1.6% of the mouse genes if the present results from 24 h treatment are extrapolated to the whole mouse genome. Similar to MMS-treated yeast, many of these genes are involved in the stress response, detoxification and a few with DNA repair. It is speculated that at least part of this difference is due to the fact that L5178Y cells have no functional p53, which also makes them more sensitive to DNA damage since the normal repair process will not function properly (Storer et al., 1997; Clark et al., 1998).
In a previous paper (Seidel et al., 2003), we used L5178Y cells to analyze the effects of two other mutagens, bleomycin and H2O2, which cause damage through the generation of free radicals. Several points can be made by comparing the genes that were altered by the four chemicals we have studied thus far in this test system. First, L5178Y cells are relatively non-responsive when treated with DNA-damaging compounds. Of the genes that do respond, all the genotoxins altered genes associated with apoptosis, but only NaCl altered genes associated with DNA damage/response. This is interesting since the other three chemicals are well-known mutagens where NaCl is not normally thought of as being genotoxic. This diminished responsiveness of these cells to modulate the expression of DNA damage/response genes may in part explain the higher incidence of positive responses when chemicals are screened for mutagenicity in this test system. There are also several genes that were altered in common by several of these chemicals. The most significant one is granzyme A, which NaCl, bleomycin and H2O2 strongly induced at the 24 h time point. This could potentially be a marker for free radical-based DNA-damaging agents since bleomycin (Dedon and Goldberg, 1992) and H2O2 (Termini, 2000) are known to act through this mechanism and it has been hypothesized as the mechanism for NaCl (Shukla et al., 1993).
In conclusion, the results presented here, along with our earlier studies (Seidel et al., 2003), indicate that mouse lymphoma cells may be relatively non-responsive in modulating gene expression when coping with the stress induced by mutagens. These results also demonstrate that gene expression data could be used to discern differing modes of genotoxic action.
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Acknowledgements |
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This work was performed as part of the Genotoxicity Working Group of the International Life Sciences Institute (ILSI) Technical Committee on Application of Genomics to Mechanism-Based Risk Assessment.
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3To whom correspondence should be addressed. Tel: +1 989 638 6155; Fax: +1 989 638 9305; Email: sseidel2{at}dow.com
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