Down-regulation of the DNA-repair endonuclease 8-oxo-guanine DNA glycosylase 1 (hOGG1) by sodium dichromate in cultured human A549 lung carcinoma cells
N.J. Hodges1,3 and
J.K. Chipman2
1 Institute of Occupational Health and
2 School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
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Abstract
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Hexavalent chromium is a genotoxic human pulmonary carcinogen that elevates DNA oxidation, apparently through the generation of reactive DNA-damaging intermediates including CrV, CrIV and reactive oxygen species. We tested the hypothesis that elevation of DNA oxidation may also be through inhibition of the expression of the repair glycosylase for 8-oxo deoxyguanine (hOGG1) in cultured A549 human lung epithelial cells. Treatment with sodium dichromate (0100 µM, 16 h) resulted in a concentration-dependent decrease in the levels of OGG1 mRNA as measured by both RTPCR and RNase protection assay. Sodium dichromate at 25 µM and above gave a marked reduction of OGG1 mRNA expression which was not seen at 1 µM and below. No effect on the expression of the apurinic endonuclease hAPE or the house-keeping gene GAPDH was observed at any of the concentrations of sodium dichromate investigated. Treatment of cells with the pro-oxidant H2O2 (0200 µM) for 16 h had no detectable effect on the levels of OGG1 mRNA or protein expression suggesting that the effect of sodium dichromate is not mediated by H2O2. Western blotting demonstrated that sodium dichromate (100 µM; 16 h and >25 µM; 28 h) markedly reduced levels of OGG1 protein in nuclear cell extracts. Additionally, treatment of cells with sodium dichromate (>25 µM, 28 h) resulted in a concentration-dependent decrease in the ability of nuclear extracts to nick a synthetic oligonucleotide containing 8-oxo deoxyguanine (8-oxo dG). We conclude that the elevation of 8-oxo dG levels observed in A549 cells treated with sodium dichromate may be, at least in part, due to a reduced capacity to repair endogenous and hexavalent chromium-induced 8-oxo dG.
Abbreviations: CrVI, hexavalent chromium; ROS, reactive oxygen species; HO-1, haem oxygenase-1; Nf
B, nuclear transcription factor-
B; CBP, cAMP-response element-binding protein-binding protein; 8-oxo dG, 8-oxo 2-deoxyguanine; FaPy, formamidopyrimidine
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Introduction
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Hexavalent chromium (CrVI) is known to induce lung cancer (1) and is genotoxic in a number of in vitro systems causing DNA-strand breaks (2,3), DNADNA cross links (4), DNAprotein cross links (5) and Cr-DNA adducts (6). Although the mechanism of CrVI-genotoxicity remains to be fully elucidated, intracellular reduction of CrVI by cellular antioxidants to reactive intermediates such as CrV (7), CrIV (8) and possibly reactive oxygen species (ROS) including HOo, Oo, O2o and H2O2 (911) is believed to be important. Such reductive processes cause intracellular oxidation a priori. It is hypothesized that the resulting oxidative stress is central to many of the cellular effects of hexavalent chromium, being not only responsible for DNA damage but also for chromium-mediated changes in gene expression, including induction of nuclear transcription factor-
B (NF
B) (8,12), haem oxygenase-1 (HO-1) (13) and activation of p53 (10,11). In addition, oxidative stress has also been implicated in chromium-dependent activation of mitogen-activated protein kinases (14). Cellular targets of ROS are numerous and include lipids, proteins and DNA (1517). Support for a role of DNA oxidation comes from the findings that CrV and possibly CrIV are able to oxidize nucleotides and DNA (18,19). One of the principal lesions produced in DNA following oxidative stress is 8-oxo 2-deoxyguanine (8-oxo dG), which as a result of mis-pairing to adenine during DNA-replication results in the formation of G to T transversions, a commonly observed mutation in the gene of the tumour suppressor p53 in human cancers including lung cancer (2023). CrVI and CrIII have been demonstrated to cause 8-oxo dG adducts in isolated DNA (24,25). Recently, we have demonstrated that culture of human A549 lung epithelial cells with sodium dichromate (10 µM) results in elevated levels of both 8-oxo dG as determined by immunocytochemistry and DNA-strand breaks introduced by formamidopyrimidine (FaPy) DNA-glycosylase (comet assay), known to be involved in the repair of this lesion (2). These findings support the hypothesis that formation of 8-oxo dG may represent an important mechanism by which hexavalent chromium initiates lung cancer.
As a consequence of the mutagenic potential of 8-oxo dG, cells have evolved a complex repair system termed the GO-system (26) whose function is to recognize and repair 8-oxo dG. In eukaryotic cells one of the key components of this system is 8-oxo guanine-DNA glycosylase 1 (OGG1). OGG1 cleaves the glycosidic bond of 8-oxo dG preferentially at 8-oxo dG:C base pairs (27). The phosphodiester bond at the resulting apurinic (AP)-site is cleaved by ß-elimination and the process of base excision repair completed by the sequential action of apurinic endonuclease, DNA polymerase ß and DNA-ligase III. Chemical induction of OGG1 considered to be mediated by ROS has been demonstrated in certain in vivo (28) and in vitro (29) systems but not others (30). In the current study we investigated mRNA and protein expression, and activity of OGG1 in human alveolar epithelial A549 cells exposed to sodium dichromate in vitro to determine whether CrVI is able to modulate expression of hOGG1, either by induction via ROS or by inhibition and consequently contributing to the observations of elevation of 8-oxo dG.
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Materials and methods
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Cell culture
Human lung carcinoma A549 cells (European Cell Culture Collection No. 86012804) were grown to 100% confluency in T25 (Falcon) culture flasks at 37°C in a humidified, 5% CO2 atmosphere using DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Fetal bovine serum was included in the medium for all treatments.
ATP and TUNEL assays
To investigate the possible effects of sodium dichromate (0500 µM, 16 h) on cellular ATP-levels and the frequency of apoptosis, A549 cells were grown to confluency in 12-well plates (Nunc, Naperville, US) and chamber slides (Nunc, Lab-Tek II) respectively. Following treatment, intracellular ATP-levels were measured using an ATP-bioluminescent kit according to the manufacturer's instructions (Sigma, FL-AA, Dorset, UK). The frequency of apoptosis was assessed by the TUNEL method using a commercially available kit (Promega, Southampton, UK).
RNA isolation and RNase protection assay
Total cellular RNA was isolated using a NucleoSpin RNAII kit (Clontech, Basingstoke, UK) according to the manufacturer's instructions. hOGG1 (495 bp), hAPE (419 bp) and GAPDH (304 bp) cDNA were synthesized by reverse transcription (RT)PCR carried out on 0.1 µg of RNA using a Superscript One-step RTPCR kit (InVitrogen, Paisley, UK) according to the manufacturer's instructions. Primers, hOGG1 (forward, 5'-atctgttcctccaacaacaa-3'; and reverse 5'-gccagcataaggtccccacag-3'), hAPE (forward, 5'-ataggcgatgaggatcatga-3': and reverse 5'-caacattcttggatcgagca-3) and GAPDH (forward, 5'-aacgggaagctcactggcatg-3'; and reverse, 5'-tccaccaccctgttgctgtag-3') were synthesized by MWG Biotech UK, cDNA was purified using quikspin columns (Ambion, Texas, US). Biotinylated anti-sense RNA probes were synthesized from purified cDNA using a Lig'n scribe PCR cloning kit (Ambion) followed by in vitro transcription with a maxiscript kit (Ambion, Texas, US) and bio-16-UTP (Sigma) according to the manufacturer's instructions. RNase protection assays were carried out on 10 µg of total RNA using a RPAIII kit (Ambion). Protected fragments were resolved on a 5% acrylamideurea gel and transferred to positively charged nylon membrane (Brightstar Plus, Ambion) and bands detected by chemiluminescence (Brightstar, Ambion). Quantification of OGG1 mRNA relative to GAPDH was carried out by densitometry using the Scion software package. The results of the RNase protection assay were further supported by semi-quantitative RTPCR using 0.1 µg of RNA and a previously published PCR cycle (28). Levels of hOGG1 mRNA were assessed relative to GAPDH albeit without quantification relative to an internal standard.
Preparation of nuclear protein extracts
Cells in lysis buffer (0.6% NP-40, 150 mM NaCl, 10 mM HEPES pH 7.5, 1 mM EDTA) supplemented with protease inhibitors (10 µl/ml mammalian protease inhibitor cocktail, Sigma) were incubated on ice for 20 min and the nuclei pelleted by centrifugation at 4°C (15 min, 3000 g). The nuclear pellet was homogenized in high salt buffer (25% v/v glycerol, 420 mM NaCl, 20 mM HEPES pH 7.5, 0.2 mM EDTA, 1.5 mM MgCl2, 0.5 mM DTT, containing 10 µl/ml mammalian protease inhibitor cocktail, Sigma) and the lysate incubated on ice for 30 min. Following centrifugation (18 000 g, 30 min, 4°C) the supernatant was retained for analysis.
Western blotting
Nuclear extracts (10 µg) were resolved on a 10% SDSpolyacrylamide gel, transferred to PVDF membrane (Amersham Pharmacia Biotech) and blocked overnight at 4°C (TBS0.05% Tween 20, 5% low fat milk). Membranes were incubated with polyclonal rabbit anti-OGG1 (1:500 dilution, Novus Biologicals) in blocking buffer for 1 h at room temperature. Following washing (3 x 10 min, TBS-0.05% Tween 20) membranes were incubated with horseradish peroxidase-conjugated anti-rabbit antibody (1:1000 dilution, Sigma) for 1 h at room temperature and washed (as above). Bands were detected using enhanced chemiluminescence detection (Amersham Pharmacia Biotech).
Endonuclease nicking assay
A single-stranded 24 mer (15 pmol) containing 8-oxo dG at position 10 (Trevigen, Gaithersburg, US) was 5'-end labelled with [
-32P]ATP (NEN) using a DNA 5' End-Labeling System (Promega) according to the manufacturer's instructions and un-incorporated label removed by centrifugation through a G-10 spin column (Sigma). The end-labelled oligomer was annealed to a 1.2-fold excess (18 pmol) of a complementary 24 mer (Trevigen) by heating to 95°C for 10 min followed by gradual cooling to room temperature. Endonuclease nicking assays were performed in 10 mM TrisHCl pH 7.5, 100 mM KCl, 10 mM EDTA containing 1 µg of nuclear protein and 1 pmol of the annealed oligomer at 37°C for 1 h. Reactions were terminated by the addition of loading buffer (95% formamide, 0.5 mM EDTA, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol) and heating to 95°C for 5 min followed by cooling on ice. Samples were resolved on 20% polyacrylamide, 8 M urea gels and bands visualized by exposing to X-ray film (Kodak, Herts, UK).
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Results
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Cytotoxicity
No statistically significant decrease in intracellular ATP levels were observed following treatment with either sodium dichromate (0100 µM) or H2O2 (0200 µM) for 16 h in cultured A549 cells (Figure 1A and 1B
). In contrast, treatment with 500 µM sodium dichromate resulted in a statistically significant (P < 0.01) decrease in ATP levels. There was also no evidence that sodium dichromate (16 h, 0500 µM) induced apoptosis in confluent A549 cells as assessed by the TUNEL assay (<0.01% cells apoptotic). As a positive control, incubation of cells with DNase resulted in >95% labelling index (images not shown). Based upon these results, sodium dichromate (0100 µM) and H2O2 (0200 µM) were chosen to investigate treatment-mediated changes in hOGG1 gene expression.


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Fig. 1. Effect of sodium dichromate (A) and H2O2 (B) on intracellular ATP levels in cultured A549 cells. Cells were treated for 16 h at 37°C. Values are the mean of three separate experiments ± SD (n = 3). **Significantly different from control (P < 0.01, paired t-test).
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Sodium dichromate decreases OGG1 mRNA and protein expression
Initial experiments showed that sodium dichromate (0100 µM, 2 and 4 h) had no detectable effect on the expression of OGG1 at either the mRNA or protein level as determined by RT-PCR and western blotting respectively (data not shown). To determine the effect of longer-term treatment with hexavalent chromium on the expression of OGG1 mRNA, cells were treated with sodium dichromate (0100 µM) for 16 h. Under these experimental conditions sodium dichromate treatment resulted in a concentration-dependent inhibition of OGG1 mRNA expression as detected by RTPCR (Figure 2
) and RNase protection (Figure 3
). Both RTPCR and RNase protection showed a marked reduction in OGG1 mRNA levels (70.0% reduction by densitometry: RNase protection) at 25 µM (Figures 2 and 3
). There was no evidence for an effect at 1 µM and below. The specificity of this effect was demonstrated by the lack of effect on the expression of the apurinic endonuclease hAPE or the house-keeping gene GAPDH at any of the concentrations of sodium dichromate investigated (Figures 2 and 3
). Additionally, treatment of cells with the pro-oxidant H2O2 (0200 µM) for 16 h had no detectable effect on the levels of OGG1 mRNA as measured by RNase protection (Figure 6A
), suggesting that the effect of sodium dichromate on hOGG1 expression is not mediated by the generation of H2O2.

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Fig. 2. Effect of sodium dichromate on OGG1, APE and GAPDH mRNA expression as assessed by RT-PCR. A549 cells were treated for 16 h at 37°C.
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Fig. 3. Effect of sodium dichromate on OGG1 and GAPDH mRNA expression as assessed by the RNase protection assay. A549 cells were treated for 16 h at 37°C.
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Fig. 6. Effect of H2O2 on OGG1 mRNA (A) and nuclear protein (B) expression as determined by RNase protection and western blotting respectively. A549 cells were treated for 16 h at 37°C. OGG1 protein is apparent as a band at 39 kDa (B).
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Sodium dichromate-mediated repression of OGG1 mRNA was also reflected at the protein level as assessed by western blotting. Treatment of cells with sodium dichromate (100 µM) for 16 h and (>25 µM) for 28 h markedly reduced detectable levels of OGG1 protein in nuclear cell extracts (Figures 4 and 5
). There was no evidence for an effect on nuclear levels of OGG1 protein at concentrations of 10 µM and below even after 48 h (Figure 5
). OGG1 protein was not detectable in the cytoplasm of either control or treated cells (Figure 4
) in agreement with previous reports that OGG1 is primarily nuclear in location. Treatment with H2O2 (0200 µM, 16 h) had no detectable effect on the levels of OGG1 protein in nuclear extracts (Figure 6B
).

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Fig. 4. Effect of sodium dichromate on OGG1 protein expression as determined by western blotting. A549 cells were treated for 16 h at 37°C. OGG1 protein is apparent as a band at 39 kDa.
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Fig. 5. Effect of sodium dichromate on OGG1 nuclear protein levels in cultured A549 lung cells as determined by western blotting. Cells were treated for 28 h and 48 h at 37°C. OGG1 protein is apparent as a band at 39 kDa.
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Sodium dichromate decreases DNA-repair capacity of nuclear protein extracts
To ascertain whether the observed decrease in OGG1 mRNA and protein expression represents a functionally significant effect on the capacity of A549 cells to repair 8-oxo dG, we assessed the ability of nuclear cell extracts to nick a synthetic oligonucleotide containing an 8-oxo dG nucleotide. Nuclear extracts from control cells proved to be efficient at this process (Figure 7
). However, pre-treatment of cells with sodium dichromate (0100 µM) for 28 h resulted in a concentration-dependent inhibition in the ability of nuclear extracts to cut the 8-oxo dG containing synthetic oligonucleotide (Figure 7
).

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Fig. 7. Effect of sodium dichromate on the ability of A549 nuclear protein extracts to cut a synthetic oligonucleotide containing a single 8-oxo dG. Cells were treated for 28 h at 37°C and nuclear protein extracts analyzed as described in the Materials and methods section. As a positive control the synthetic oligonucleotide was incubated with formamidopyrimidine (FaPy) DNA glycosylase, an endonuclease with specific activity towards 8-oxo dG. The negative control consisted of oligonucleotide alone.
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Discussion
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ROS and consequently 8-oxo dG are continually produced in cells either by normal cellular metabolism or by exposure to a wide range of physical and chemical agents. 8-oxo dG paired to cytosine in DNA is recognized and repaired by a DNA-glycosylase/endonuclease (27) encoded by the Fpg gene in E.coli and by the structurally unrelated but functionally similar OGG1 in eukaryotic cells. The importance of these proteins has been demonstrated in several genetic studies where E.coli strains containing Fpg mutations and yeast strains containing OGG1 mutations show a spontaneous mutator phenotype characterized by an increased frequency of G to T transversions (3134). The human and mouse forms of this gene (hOGG1 and mOGG1) have been cloned (3540) and further studies have demonstrated a possible role for OGG1 in carcinogenesis. For example, mice lacking functional OGG1 protein accumulate abnormal levels of 8-oxo dG and show elevated spontaneous mutation frequencies in non-proliferative tissue (41). In humans, somatic mutations and loss of heterozygosity in hOGG1 have been associated with lung, renal and possibly headneck tumours (4245), but not ovarian tumours (46). Recently the promoter region of hOGG1 has been cloned (30); analysis revealed two CpG islands, several SP1 binding sites and a putative NRF2 antioxidant response element but no TATA or CAAT boxes. These findings suggest that the hOGG1 gene is essentially a house-keeping gene, whose expression may potentially be modulated by oxidative stress. In support of this hypothesis, several groups have demonstrated that OGG1 is inducible at the mRNA level by ROS in several experimental systems (28,29). However, other studies have failed to observe induction of OGG1 by ROS (30) suggesting that modulation of OGG1 expression by ROS is dependent on the experimental model chosen. In the current study we have demonstrated that culture of A549 human epithelial lung cells with sodium dichromate (>25 µM) results in a concentration-dependent decrease in the expression of OGG1 mRNA and protein as measured by RTPCR and RNase protection assay and western blotting respectively. In addition nuclear extracts from sodium dichromate-treated cells (28 h) show a concentration-dependent decrease in ability to nick a synthetic oligonucleotide containing 8-oxo dG, suggesting that inhibition of OGG1 expression by sodium dichromate is functionally important in this cell line. Inhibition of OGG1 mRNA and protein expression was observed in the absence of changes in hAPE or GAPDH gene expression or loss of membrane integrity, and under conditions in which ATP concentrations were not reduced compared with controls. The effects are therefore not secondary to mitochondrial damage or cytotoxicity. It is known, however, that concentrations of
20 µM and above can affect mitogen-activated signal transduction pathways and clonogenicity in lung epithelial cells (7,14).
Interestingly, treatment of A549 cells with H2O2 (0200 µM, 16 h) had no effect on OGG1 mRNA levels as assessed by RNase protection or nuclear protein levels as assessed by western blotting, suggesting that sodium dichromate-mediated inhibition of OGG1 is not dependent on H2O2. However, the possibility that H2O2 has a transient effect on OGG1 expression cannot be completely excluded. Reduction of hexavalent chromium to CrV by GSH-reductase is known to result in the generation of O and subsequently H2O2 by the action of superoxide dismutase (47). Pentavalent chromium is believed to be among the principal species responsible for chromium-mediated oxidative stress and is capable of oxidizing DNA either directly (18) or possibly via the formation of highly oxidizing CrV-peroxo complexes (19). Furthermore, catalase has been demonstrated to inhibit sodium dichromate-induced DNA damage in human peripheral blood lymphocytes (48). These observations suggest that although H2O2 may be important in the mechanism of sodium dichromate-induced DNA damage, it does not appear to play a role in sodium dichromate-mediated inhibition of hOGG1 gene expression.
The apurinic endonuclease APE has been reported to be induced by ROS-generating systems such as H2O2, HOCl and bleomycin (49) but not alkylating agents. The mechanism of the apparent ROS-specific induction of APE remains to be established, but may be related to the presence of a NF
B consensus sequence (but lack of a functional AP-1 binding site) in the APE promoter. In the current study, sodium dichromate had no effect on the expression of APE as measured by RTPCR at any of the concentrations investigated, suggesting that sodium dichromate may not produce the spectrum of ROS required to induce APE in the A549 cell line. Furthermore, Dubrovskaya and Wetterhahn (13) failed to observe induction of ROS-inducible genes including catalase, Cu-, Zn- and Mn-superoxide dismutase in either LL24 or A549 lung cells treated with a range of concentrations of sodium dichromate (0100 µM). The same study observed induction of HO-1 mRNA in LL24 (>50 µM) but not A549 lung cells. In our system, we observed induction of HO-1 by RNase protection and western blotting in the A549 cell line at high concentrations of sodium dichromate (100 µM) (unpublished data). The mechanism of sodium dichromate-dependent induction of HO-1 remains to be established, but may be related to the presence of the metal response element in the HO-1 promoter rather than as a result of sodium dichromate-mediated oxidative stress.
The observation that hexavalent chromium can negatively regulate gene expression is not unique. For example, Maier et al. (50) demonstrated that sodium dichromate is able to inhibit dioxin-mediated induction of cytochrome P450 1A1 and NAD(P)H quinone oxidoreductase 1, and Shumilla et al. (51) have demonstrated that in A549 cells, sodium dichromate (>20 µM) inhibits NF
B-dependent TNF
induction of IL-8. The same study also demonstrated that this inhibition was not a result of reduced NF
B binding to the IL-8 promoter. Rather it was considered to be as a result of decreased interaction between the transcription factor p65 and CBP, a coactivating molecule that links enhancer-bound transcription factors (e.g. TFIIB and TATA-binding protein) to the basal transcription machinery and is essential for NF
B-enhanced transcriptional activity (52,53). CrIII (the final cellular metabolite of hexavalent chromium) is known to bind to tridentite amino acid residues and proteins (5456); therefore CrIII may decrease the interaction of p65 with CBP by directly altering either CBP of p65 recognition sequences. Alternatively up-regulation of other transcription factors such as c-jun (51) by sodium dichromate may result in competitive or non-competitive inhibition of p65 binding to CBP. Expression of the gene for O6-methyl guanine DNA transferase is also dependent upon CBP binding (57), but it is unknown whether CBP-binding is a requirement for hOGG1 gene expression. In addition, binding of the transcription factor SP1 to its cognate target DNA sequence (GC box, present in the hOGG1 promoter) is inhibited by substitution of a guanine base for 8-oxo dG (58,59). Furthermore, decreases in the expression of genes regulated by SP1 binding and considered to be related to oxidative stress, have been observed in vivo (60).
We conclude that inhibition of hOGG1 gene expression by sodium dichromate contributes to genotoxicity as a result of decreased capacity to repair endogenous and chromate-induced 8-oxo dG. It has been estimated that chromium levels up to 5 µM may be attained in the blood of chrome pigment production workers (1,61) and it is possible that higher concentrations may be achieved locally in lung tissue. Furthermore, substantially higher exposure levels have been used in animal carcinogenicity studies (1). Therefore, this process may also contribute to the mechanism of chromium-mediated lung carcinogenicity. Current work in our laboratory is aimed at understanding the molecular mechanism(s) of this inhibition.
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Notes
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3 To whom correspondence should be addressed Email: N.Hodges{at}bham.ac.uk 
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Received July 25, 2001;
revised October 2, 2001;
accepted October 4, 2001.