Mutagenesis, Vol. 15, No. 4, 311-316,
July 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
The role of glutathione in DNA damage by potassium bromate in vitro
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
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Abstract |
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We have investigated the role of reduced glutathione (GSH) in the genetic toxicity of the rodent renal carcinogen potassium bromate (KBrO3). A statistically significant increase in the concentration of 8-oxodeoxyguanosine (8-oxodG) relative to deoxyguanosine was measured following incubation of calf thymus DNA with KBrO3 and GSH or N-acetylcysteine (NACys). This was dependent on these thiols and was associated with the loss of GSH and production of oxidized glutathione. A short-lived (<6 min) intermediate was apparent which did not react with the spin trap dimethylpyrroline N-oxide. DNA oxidation was not evident when potassium chlorate (KClO3) or potassium iodate (KIO3) were used instead of KBrO3, though GSH depletion also occurred with KIO3, but not with KClO3. Other reductants and thiols in combination with KBrO3 did not cause a significant increase in DNA oxidation. DNA strand breakage was also induced by KBrO3 in human white blood cells (5 mM) and rat kidney epithelial cells (NRK-52E, 1.5 mM). This was associated with an apparent small depletion of thiols in NRK-52E cells at 15 min and with an elevation of 8-oxodG at a delayed time of 24 h. Depletion of intra-cellular GSH by diethylmaleate in human lymphocytes decreased the amount of strand breakage induced by KBrO3. Extracellular GSH, however, protected against DNA strand breakage by KBrO3, possibly due to the inability of the reactive product to enter the cell. In contrast, membrane-permeant NACys enhanced KBrO3-induced DNA strand breakage in these cells. DNA damage by KBrO3 is therefore largely dependent on access to intracellular GSH.
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Introduction |
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Potassium bromate (KBrO3) has been used as a food additive in the treatment of flour and as a constituent of the neutralizer in cold-wave hair solutions (Norris, 1965
; FAO/WHO, 1979). It is also formed as a by-product of reactions such as the disinfection of water by ozonation (Fielding and Hutchinson, 1993). The provisional guideline limit value set by the WHO for KBrO3 in drinking water is 25 µg/l, although a concentration limit of 10 µg/l has been set in the USA and is indicated in the 1998 EC Drinking Water Directive. These limits are based on the practical quantitation limit. KBrO3 has been found to be mutagenic in bacterial mutation assays (Ishidate et al., 1984; Kurokawa et al., 1990), in chromosome aberration tests (Ishidate and Yoshikawa, 1980) and in micronucleus assays (Hayashi et al., 1988). It also caused renal cell tumours, mesotheliomas of the peritoneum and follicular cell tumours of the thyroid in F344 rats (Kurokawa et al., 1986, 1990). This was also observed by DeAngelo et al. (1998), who also found renal cell tumours in male B6C3F1 mice (although the treatment was not dose related). The carcinogenicity of KBrO3 in rodents is believed to be due to its ability to oxidize DNA. This was suggested by Kasai et al. (1987) and Sai et al. (1991, 1992), who observed increased levels of 8-oxodeoxyguanosine (8-oxodG) relative to deoxyguanosine (dG) in the kidney DNA of male treated rats. The oxidation of DNA was found to be inhibited by various antioxidants, in particular by reduced glutathione (GSH) (Sai et al., 1992). These results suggest a protective role of GSH against KBrO3-induced DNA oxidation in vivo. In contrast, an in vitro study by Ballmaier and Epe (1995) suggests that GSH can activate KBrO3 to a reactive species that is capable of oxidizing DNA. It was found that KBrO3 (only in the presence of GSH) produced DNA strand breaks and modifications sensitive to formamidopyrimidine-DNA glycosylase protein (Fpg protein; a repair enzyme recognizing particularly 8-oxodG). This involved an apparent short-lived intermediate that was revealed not to share the characteristics of the hydroxyl radical, based on a comparison of DNA damage profiles.
The mechanism whereby KBrO3 can oxidize DNA is therefore not clear, particularly regarding a direct or indirect interaction. GSH therefore has a potential activation and protection role in KBrO3-mediated DNA oxidation. We have now further tested the role of GSH (and other thiols and reductants) in DNA oxidation and strand breakage by KBrO3 in vitro using isolated calf thymus DNA, human white blood cells and cultured rat kidney epithelial cells (NRK-52E). We have also tested if DNA damage can occur from other halates.
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Materials and methods |
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Chemicals
All chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (Poole, UK). The standard sample of 8-oxodG was synthesized by the Udenfriend hydroxylation system (Kasai and Nishimura, 1984
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DNA incubations
Calf thymus DNA [0.5 mg/ml in Ca2+-free phosphate-buffered saline, pH 7.4 (PBS)] was incubated in a total volume of 1 ml at 37°C. Hydrogen peroxide with iron(II) sulphate (FeSO4) (both 250 µM) was used as a positive control. DNA was precipitated with 4 ml ice-cold ethanol plus 100 µl 0.3 M sodium acetate and stored at –20°C overnight. The DNA precipitate was spun at 2000 g for 10 min and the ethanol decanted. Finally, the DNA was resuspended in 100 µl SSC buffer (5 mM sodium citrate, 20 mM sodium chloride, pH 6.5).
DNA extraction from cultured cells
Rat kidney epithelial cells (NRK-52E, obtained from the European Collection of Cell Cultures, Centre for Applied Microbiology and Research, Salisbury, UK) were cultured in Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum and 1% non-essential amino acids at 37°C in 5% CO2. DNA was extracted using a Puregene DNA isolation kit (Flowgen plc, Lichfield, UK) and throughout the extraction procedure the samples were incubated in the presence of 1% dimethylsulphoxide and gassed with nitrogen as practically possible to avoid artifactual DNA oxidation. The medium from cell culture flasks (75 cm2) containing ~1x107 rat kidney epithelial cells was removed and the cells washed with Ca2+-free PBS. Cell lysis solution (3 ml) was added to the cells and incubated at 37°C for 15 min, after which the lysed cells were transferred to a 4.5 ml polypropylene tube. RNase solution (4 mg/ml, 15 µl) was added to the lysate, the tube inverted ~25 times and incubated for a further 15–60 min at 37°C. The samples were cooled on ice for 5 min and, after the addition of 1 ml protein precipitation solution, were vortexed vigorously for 20 s prior to incubation on ice for a further 5 min. The samples were spun at 2000 g for 10 min at 4°C and the supernatant transferred to a 15 ml centrifuge tube containing 2 ml sodium iodide solution (13.5 M NaI, 20 mM Na2EDTA, 40 mM Tris–HCl, pH 8.0). The tube was inverted several times and the DNA precipitated by the addition of 4.5 ml ethanol and inverted ~50 times until the DNA had formed a visible clump. After centrifuging at 2000 g for 3 min, the supernatant was discarded and the DNA washed with 4.5 ml 70% ethanol. The DNA was centrifuged at 2000 g for 1 min, the supernatant discarded carefully and the pellet air dried for 10 min. The DNA was resuspended in 100 µl SSC buffer and then transferred to a clean Eppendorf tube.
DNA hydrolysis and analysis of 8-oxodG by HPLC
The method used for hydrolysis of DNA was based on that of Adachi et al. (1995) and throughout the hydrolysis procedure the samples were incubated in the presence of 1% dimethylsulphoxide and gassed with nitrogen. The DNA, in SSC buffer, was heated to 95°C for 4 min and, after cooling on ice, 95 µl 20 mM sodium acetate buffer, pH 4.8, containing 0.1 mM ZnCl2 was added followed by 5 U nuclease P1 and this was then incubated for 30 min at 45°C. After this, 90 µl 50 mM Tris–HCl, pH 7.4, and 3 U alkaline phosphatase (Type VII-S) were added and incubation continued for 1 h at 37°C. Samples were centrifuged at 2000 g for 10 min and the supernatants stored at –70°C until required.
The method for reverse phase HPLC of the DNA hydrosylates was modified from the procedure of Floyd et al. (1986). The column was a silica C18 Ultra Techsphere (HPLC Technology, Macclesfield, UK) and was used with a flow rate of 1.0 ml/min with an RP-18 5 µm guard column. The mobile phase consisted of 12.5 mM citric acid, 25 mM sodium acetate, 30 mM sodium hydroxide, 10 mM acetic acid and 1 mM EDTA, pH 5.1, with 5% methanol (v/v). Deoxyguanosine was detected by UV absorption (254 nm) and 8-oxodG using a model LC-4B electrochemical detector attached to a temperature controller (at 37°C). A glassy carbon electrode (potential +0.6 V) was used. Authentic standards for HPLC were used for quantitation as previously described (Faux et al., 1992).
GSH and oxidized glutathione (GSSG) analyses
The method was based on that of Hissin and Hilf (1976). For both analyses, conditions of incubation were identical to those used for studying the effects on calf thymus DNA, except that the latter was omitted. Levels of intracellular thiols in rat kidney epithelial cells (NRK-52E) were measured as in the GSH procedure, after lysing the cells by repetitive freeze–thawing in Ca2+-free PBS
GSH. The final assay mixture (2.0 ml) contained 100 µl of the incubates, 1.8 ml phosphate–EDTA buffer, pH 8.0, and 100 µl 1 mg/ml o-phthalaldehyde (OPT) in methanol. This was left for 15 min at room temperature prior to reading the fluorescence at 420 nm with excitation at 350 nm and quantification from a calibration curve constructed using GSH (0–20 µg/ml).
GSSG. To 100 µl of the incubates was added 5 µl 100 mM dithiothreitol, 10% (v/v) Triton X-100. The samples were vortexed and allowed to stand at room temperature for 30 min. The volume was then made up to 1.9 ml with phosphate–EDTA buffer, pH 8.0, followed by incubation with 100 µl OPT for 15 min and the fluorescence read as described above. The GSH value calculated as above was then subtracted from the total glutathione thus obtained, resulting in a value for GSSG.
Electron spin resonance spectroscopy
Spectra were acquired at 298K using a Bruker ESP 300 continuous wave X-band spectrometer, operating in the TE104 mode (Bruker Analytische Messtechnik GmbH, Karlsruhe, Germany). The modulation frequency was 100 kHz and modulation amplitude was 4 G. The spin trap agent 5,5'-dimethyl-l-pyrroline N-oxide (DMPO) was used for this study.
Alkaline single cell gel electrophoresis (Comet) assay
Human white blood cells (in 5 µl of blood), freshly obtained, were incubated in a total volume of 1 ml Ca2+-free PBS at 37°C for 15 min. Rat kidney epithelial cells were cultured in 25 cm2 tissue culture flasks at 37°C (5% CO2) and then removed by trypsinization (0.05% trypsin, 0.02% EDTA). The method of Singh et al. (1988) was then followed, in which the cells were precipitated by centrifugation (2000 r.p.m., 5 min) and the pellet carefully mixed with 100 µl of 0.5% low melting point agarose (LMPA) in PBS (rat kidney epithelial cells after centrifugation were suspended in 100 µl PBS and 10 µl of the cell suspension added to the LMPA). Cells were kept under subdued yellow light throughout. The cells were then applied on top of 130 µl of 0.5% normal melting point agarose in PBS, which had already been allowed to solidify on a fully frosted microscope slide. A third layer of LMPA was added on top of the cells and, following solidification, the slides were lowered into fresh lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% lauroyl sarcosinate, 10% dimethylsulphoxide, 1% Triton X-100, pH 10.0) for 1 h at 4°C in the dark. The DNA was allowed to unwind by placing the slides in 75 mM NaOH and 1 mM EDTA and, following electrophoresis at 25 V, 300 mA for 20 min, the slides were washed with 0.4 M Tris, pH 7.5, drained and then stained with ethidium bromide (20 µg/ml) before analysis under a fluorescence microscope (Zeiss Axiovert inverted fluorescence microscope, x200 magnification, with a 515–560 nm excitation filter and a 590 nm barrier filter). Fifty cells per slide were automatically analysed using the Komet Image Analysis System (Kinetic Imaging Limited, Liverpool, UK). The mean per cent of the DNA in the comet tails was calculated and also the per cent tail DNA grouped and classified as a modification of that used by Anderson et al. (1994). Cell viability was determined by measurement of the leakage of lactate dehydrogenase (Moldeus et al., 1978).
Statistical analyses
Analyses were made by a two-sample t-test, both with and without log transformation of data from Comet analyses. ANOVA was also used for thiol changes.
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Results |
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KBrO3 alone did not increase the levels of 8-oxodG in calf thymus DNA at concentrations up to 1500 µM (Table I
). However, there was a dose-dependent increase in 8-oxodG in the presence of both KBrO3 (0–1500 µM) and GSH (500 µM), which was highly significant (P < 0.01) at 1500 µM KBrO3 [0.008 ± 0.001 and 0.091 ± 0.019% ratio of 8-oxodG/dG (mean ± SD, n = 4) at 0 and 1500 µM KBrO3, respectively]. The positive control (250 µM H2O2 with 250 µM FeSO4) gave a value of 0.076 ± 0.004% (mean ± SD, n = 2). When GSH was substituted by N-acetylcysteine (NACys) (500 µM), a similar concentration-dependent profile was observed (Table I). The use of other reducing agents (including other thiols) in combination with KBrO3 (Table I) did not significantly elevate the levels of 8-oxodG, although there was a significant increase in the presence of FeSO4 at 1500 µM KBrO3. When KBrO3 and NACys were reacted for various times prior to addition of DNA, oxidation of the latter did not occur if preincubation exceeded 6 min. There was, however, no evidence for a free radical species able to be trapped by DMPO from high concentrations of KBrO3 and GSH, compared with reactive oxygen produced by FeSO4 and H2O2 at the same concentrations used to oxidize DNA (Figure 1).
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Substitution of KBrO3 with KClO3 or KIO3, with and without GSH, at the optimal concentration of 1500 µM (Table II
) did not elevate the levels of 8-oxodG. In a complementary study, the levels of GSH were found to be depleted by a 15 min incubation with KBrO3 or KIO3, but not with KClO3 (Table III), in accord with their relative oxidative potential. The subsequent formation of GSSG was detected on incubation of KBrO3 at 1.5 mM with GSH. However, at a higher concentration of KBrO3 (15 mM), both GSH and GSSG were lowered.
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DNA strand breakage measured by the Comet assay showed an increase in the per cent tail DNA in human white blood cells treated with KBrO3 (5 mM) for 15 min compared with the control (Figure 2
). This effect was reduced by pretreatment with 2 mM diethylmaleate (DEM), a glutathione depletor (Plummer et al., 1981), for 15 min prior to KBrO3 administration and was significantly different (P < 0.002) compared with treatment with KBrO3 alone. Co-addition of KBrO3 with extracellular GSH (5 mM) showed a marked reduction in DNA strand breakage (P < 0.005 compared with KBrO3 alone), whereas treatment with KBrO3 and NACys (5 mM) greatly enhanced the per cent tail DNA (P < 0.005 compared with KBrO3 alone).
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P < 0.005, A study of the time course of DNA damage in rat kidney epithelial cells (Figure 3
) revealed that DNA strand breakage by KBrO3 (1.5 mM) peaked at 15 min, with a decrease thereon up to 4 h. However, at 24 h DNA strand breakage was again enhanced, in contrast to H2O2, which gave a greater response at 15 min than at 24 h. There was a small but not significant elevation in 8-oxodG (0.0034 ± 0.0007% ratio of 8-oxodG/dG; mean ± SD, n = 5) compared with the control (0.0028 ± 0.0003%; n = 4) after 15 min incubation of rat kidney epithelial cells (NRK-52E) with KBrO3 (1.5mM). However, a statistically significant elevation of 8-oxodG (0.0102 ± 0.0005%; n = 5) was observed after 24 h treatment with KBrO3 (Figure 4). The DNA strand breakage seen at 15 min was associated with an apparent small lowering of intracellular thiols (Figure 5). The persistence of DNA strand breakage after a 4 h treatment of rat kidney cells and subsequent cell washing was also investigated (data not shown). The level of DNA strand breaks was found to return to control levels between 4 and 24 h post-exposure and cell wash. The use of H2O2 as a positive control gave a similar profile. KBrO3 was not cytotoxic at 1.5 mM as measured by lactate dehydrogenase leakage at all time points investigated (Figure 6). In preliminary studies (data not shown) we also found no evidence for elevation of apoptosis based on Hoechst 33258 nuclear staining and microscopy.
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Discussion |
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Studies herein aimed to expand upon initial observations (Ballmaier and Epe, 1995
; Chipman et al., 1998), which suggested that GSH plays a role in DNA damage by KBrO3 in vitro. KBrO3 was found to produce 8-oxodG in isolated calf thymus DNA in the presence of either GSH or NACys. This suggests the formation of similar reactive species, which are equipotent, derived from both thiols. The lifetime of the DNA-reactive species was found to be <6 min, in agreement with that estimated by Ballmaier and Epe (1995). Other reductants (including thiols) in combination with KBrO3 were not effective in causing DNA oxidation, although an increase was observed at high concentrations of KBrO3 in the presence of FeSO4, suggesting the possibility of a Fenton-type reaction under these conditions. This, however, needs to be clarified. The apparent importance of a thiol interaction with KBrO3 has also been suggested to play a role in the activity of KBrO3 as a flour `improver' in bread making (Dupuis, 1997). However, a specificity of GSH and NACys thiols was suggested in relation to DNA damage by the observation that the thiols dithiothreitol and ß-mercaptoethanol were not effective in catalysing 8-oxodG formation. The coincident formation of GSSG suggests that GSH is oxidized in formation of the reactive product but, at high concentrations of KBrO3, GSSG is also degraded. The identity of the reactive species is not yet known. Lafleur and Retèl (1993) have reviewed data which indicates that although thiols are effective antioxidants, they also possess potential genotoxic consequences, such as through the formation of thiyl peroxy radicals and other reactants from interaction with singlet oxygen. GSH appears also to form superoxide radical (O2·–) in the presence of iron salt (Rowley and Halliwell, 1982). However, the relationship between these reactions and the synergistic effect of GSH and KBrO3 is not known. Although Ballmaier and Epe (1995) suggest various potential free radical products, we found no evidence for a radical species using the trapping agent DMPO. Using repair endonucleases, Ballmaier and Epe (1995) found that the DNA damage profile produced by KBrO3 in the presence of GSH was not characteristic of hydroxyl, superoxide or thiyl radicals. It was assumed that bromine radicals (Br·) or oxides (BrO· and BrO2·) were likely to be the species responsible for the cellular and cell-free DNA damage observed. Mutagenicity tests (Kurokawa et al., 1990) using Salmonella typhimurium TA102 and TA104 (strains sensitive to oxygen radicals; Levin et al., 1982) suggested that KBrO3 was only mutagenic in the presence of metabolic activation (S9). We now propose that the S9 fraction provides a source of thiols which react with KBrO3.
No effect on 8-oxodG was observed with KClO3 or KIO3 in the presence or absence of GSH. GSH was depleted by KBrO3 and KIO3, but not by KClO3. Therefore the damage to DNA appears to be more specific for KBrO3 and GSH and is not a feature of other halates. Since we have found all three halates to produce DNA strand breaks in NRK-52E cells (Parsons and Chipman, 1999a), there may be an additional, alternative mechanism of DNA damage in cells.
KBrO3 induced DNA strand breakage in both human white blood cells and a rat kidney epithelial cell line (NRK-52E). Strand breakage in these cells by KBrO3 was induced within 15 min, indicating a rapid response. The damage then declined up to 4 h, although a further elevation of DNA strand breakage occurred at 24 h. This was associated with an increase in 8-oxodG at this time point (but not at 15 min). We have also noted the formation of lipid peroxides at 24 h (Parsons and Chipman, 1999b) and are currently investigating the possibility that the delayed DNA damage may be mediated by lipid peroxidation products. A role for GSH in the production of strand breaks was also supported by the observation that depletion of intracellular GSH with DEM in human white blood cells reduced the amount of strand breakage. In contrast, the addition of extracellular GSH with KBrO3 also reduced the amount of strand breakage. This protective influence of extracellular GSH may relate to restricted access of the reactants to the cell. The fact that, in contrast, addition of NACys resulted in an increase in DNA strand breakage suggests that either KBrO3 reacts intracellularly with this membrane-permeable thiol (Traber et al., 1992) or that reactants formed extracellularly by NACys have access to intracellular DNA.
There is, therefore, a predicted dual role for GSH in the genotoxicity of KBrO3. Our results in mammalian cells and in isolated DNA suggest an activating role, with a protective effect of extracellular GSH. There is also an apparent protective effect in vivo, as evidenced by enhanced toxicity with GSH depletion (Sai et al., 1992). Thus, activation of KBrO3 by GSH may occur in vivo largely at a site distant from the kidney, which is a major target organ. A further anomaly is that significant increases in 8-oxodG in vivo were only observed 24 h after oral (Kasai et al., 1987) or i.p. (Sai et al., 1991; Cho et al., 1993) administration. This is not consistent with a rapid interaction with thiols and the production of a short-lived intermediate. Since there is also a coincident formation of lipid peroxides at high doses of KBrO3 (Sai et al., 1991, 1992; Chipman et al., 1998), a potential secondary mechanism of DNA oxidation by lipid peroxides (Hruszkewycz and Bertold, 1988) is therefore a possibility in vivo. Previously, we observed no increase in 8-oxodG in kidney tissue up to 4 h following infusion with KBrO3 at 5 mM and, in the present study, an increase in 8-oxodG in cells treated with KBrO3 occurred at 24 h but not at 15 min. 8-Oxodeoxyguanosine was reported to be elevated in V79 Chinese hamster cells within 1 h, but only at 100 mM KBrO3, which was also reported to be cytotoxic (Speit et al., 1999). The concentrations able to produce strand breaks in our study (1.5 mM) and in the study by Speit (1 mM) are, in contrast, not cytotoxic in either cell line. In this context, it is interesting that chlorate also produced strand breakage in kidney cells but did not produce 8-oxodG in calf thymus DNA when incubated with GSH. Taken together, these findings suggest a lack of correlation between strand breakage by halates and 8-oxodG formation.
Although 8-oxodG formation in rat kidney in vivo is associated with high bolus doses of KBrO3 and lipid peroxidation, it has also been detected in male rats after 7 days of inclusion of KBrO3 in the drinking water at 500 p.p.m. (Umemura et al., 1998). We have also measured an increase in 8-oxodG in kidney following exposure at this concentration in the absence of lipid peroxidation (J.E.Davies, J.L.Parsons and J.K.Chipman, unpublished results). Umemura et al. (1998) showed that the elevation in 8-oxodG was concurrent with kidney cell proliferation and proposed that the elevation was a consequence of this proliferative activity. This was based on the study of Adachi et al. (1994), which gave evidence for increased susceptibility to oxidative DNA damage in regenerating liver. It may also be that repair of 8-oxodG is impaired in dividing cells. It can therefore be proposed that the elevation of 8-oxodG by KBrO3 will occur at dose levels able to cause kidney cell proliferation (e.g. 500 p.p.m. in the drinking water) or with high bolus doses able to cause lipid peroxidation. The mechanism responsible for 8-oxodG elevation may differ for each of these two conditions. It is still not known if 8-oxodG plays a causative role in KBrO3-induced kidney tumours. However, if it does, then a non-linear dose–response relationship is likely based on the discussion above.
DNA damage in vivo and in vitro through lipid peroxides is currently being investigated further by observing etheno- and malondialdehyde-adduct formation to determine the relative importance of these products versus a direct thiol-mediated intermediate in DNA damage. Resolution of the mechanism of DNA damage in vivo is important in allowing a better understanding of the dose–response relationship in KBrO3 carcinogenesis and the target organ specificity.
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Acknowledgments |
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This work was partially supported by the UKWIR and we thank W.Matthews, R.Mitchell, and J.Fawell of WRc (Medmenham, UK) for useful discussions. We also gratefully acknowledge the help of T.L.Green and P.A.Anderson from the School of Chemistry, The University of Birmingham, in the ESR study.
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Notes |
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1 To whom correspondence should be addressed. Tel: +44 121 414 5422; Fax: +44 121 414 3982; Email: j.k.chipman{at}bham.ac.uk
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References |
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Received on September 16, 1999; accepted on February 25, 2000.
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