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Cancer Epidemiology Biomarkers & Prevention Vol. 14, 497-505, February 2005
© 2005 American Association for Cancer Research

Interindividual Variability in Response to Sodium Dichromate–Induced Oxidative DNA Damage: Role of the Ser326Cys Polymorphism in the DNA-Repair Protein of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine DNA Glycosylase 1

Amanda J. Lee, Nikolas J. Hodges and James K. Chipman

School of Biosciences, The University of Birmingham, Birmingham, United Kingdom

Requests for reprints: Nikolas J. Hodges, School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 121-4145422; Fax: 121-4145925. E-mail: N.Hodges{at}bham.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the genotoxic mechanism(s) of hexavalent chromium (CrVI) carcinogenicity remain to be fully elucidated, intracellular reduction of CrVI and concomitant generation of reactive intermediates including reactive oxygen species and subsequent oxidative damage to DNA is believed to contribute to the process of carcinogenesis. In the current study, substantial interindividual variation (7.19-25.84% and 8.79-34.72% tail DNA as assessed by conventional and FPG-modified comet assay, respectively) in levels of DNA strand breaks after in vitro treatment of WBC with sodium dichromate (100 µmol/L, 1 hour) was shown within a group of healthy adult volunteers (n = 72) as assessed by both comet and formamidopyrimidine glycosylase–modified comet assays. No statistically significant correlation between glutathione S-transferases M1 or T1, NADPH quinone oxidoreductase 1 (codon 187) and X-ray repair cross complementation factor 1 (codon 194) genotypes and individual levels of DNA damage were observed. However, individuals homozygous for the Cys326 8-oxo 7,8-dihydro-2'-deoxyguanosine glycosylase 1 (OGG1) polymorphism had a statistically significant elevation of formamidopyrimidine glycosylase–dependent oxidative DNA damage after treatment with sodium dichromate when compared with either Ser326/Ser326 or Ser326/Cys326 individuals (P = 0.008 and P = 0.003, respectively). In contrast, no effect of OGG1 genotype on background levels of oxidative DNA damage was observed. When individuals were divided on the basis of OGG1 genotype, Cys326/Cys326 individuals had a statistically significant (P < 0.05, one-way ANOVA followed by Tukey test) higher ratio of oxidative DNA damage to plasma antioxidant capacity than either Ser326/Ser326 or Ser326/Cys326 individuals. The results of this study suggest that the Cys326/Cys326 OGG1 genotype may represent a phenotype that is deficient in the repair of 8-oxo-7,8-dihydro-2'-deoxyguanosine, but only under conditions of cellular oxidative stress. We hypothesize that this may be due to oxidation of the Cys326 residue. In conclusion, the homozygous Cys326 genotype may represent a biomarker of individual susceptibility of lung cancer risk in individuals that are occupationally exposed to CrVI.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epidemiologic studies, supported by animal and in vitro data, suggest that hexavalent chromium (CrVI) compounds are carcinogenic, and the IARC has classified CrVI as a group I carcinogen (1). CrVI compounds are used industrially as catalysts, corrosion inhibitors, and as alloying metal for making stainless steel and various superalloys. Subsequently, CrVI is present in various welding fumes (2-5). In the workplace, exposure to chromium can be oral and dermal; however, the respiratory tract is the major site of exposure and, consequently, there is an increased risk of cancers of the respiratory system in exposed individuals (2).

CrVI compounds including sodium dichromate are genotoxic and cause DNA strand breaks (6, 7), chromium-DNA adducts (8), DNA-DNA (9), DNA-protein cross-links (10), and oxidative base damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG; ref. 6) in vitro. Although the mechanism(s) involved remains to be fully elucidated (2), intracellular reduction of CrVI by cellular reductants such as reduced glutathione (GSH) and ascorbic acid and the subsequent generation of partially reduced chromium species such as CrV and CrIV are believed to be important (11). In addition, reduction of CrVI may also result in the concomitant generation of reactive oxygen species including hydroxyl radicals (·OH), singlet oxygen (O), superoxide (O2·) and hydrogen peroxide (12-15). In addition to reductive activation of CrVI, the antioxidant nature of GSH may also play a protective role in detoxification of reactive oxygen species and partially reduced chromium species. Interestingly, recent studies have indicated that human lung cells are able to internalize particulate chromium, resulting in high local intracellular concentrations of CrVI whose effects seem to be mediated by reactive oxygen species (16, 17).

The alkaline version of the single cell gel electrophoresis assay or comet assay represents a sensitive technique for the detection of ssDNA breaks in cells exposed to genotoxic carcinogens. Collins et al. (18) developed the use of formamidopyrimidine glycosylase (FPG) in the comet assay for the specific detection of oxidized purines, predominantly 8-oxo-dG. Because of its sensitivity, many studies have used the comet assay to investigate CrVI-induced DNA damage in vitro (e.g., 6, 19-24).

Biomarkers of susceptibility are indicators of an inherent or acquired limitation of an organism's ability to respond to the challenge of exposure to a specific xenobiotic substance (25). Examples include interindividual differences in metabolism (activation and detoxification) of carcinogenic chemicals, DNA repair, and the functions of proto-oncogenes or tumor suppressor genes, as well as interindividual differences in nutritional, hormonal, and immunologic factors (26).

The intracellular reduction of sodium dichromate is extremely complex and involves multiple pathways including metabolism by enzymes for which functional polymorphisms in human populations are known to exist (e.g., NQO1 and cytochrome P450s 27-29). A major defense system against the damaging effects of oxidative stress are the glutathione S-transferases (GST), which catalyze the nucleophilic addition of glutathione (GSH) to electrophilic acceptors including toxic products generated from tissue damage (e.g.,glutathionealkenes, epoxides, hydroperoxides, and aldehydes) produced as a result of lipid peroxidation (30).

A wide range of low molecular weight cellular antioxidants (e.g., GSH, ascorbic acid, melatonin, and vitamins D and E) also plays a role in the reduction and metabolic activation of CrVI (31, 32). It is likely that intracellular levels of these molecules will be influenced by both genetic factors and environmental factors including diet, alcohol consumption, and smoking habit. In addition, several components of the base excision repair pathway, including the DNA glycosylase 8-oxo-7,8-dihydro-2'-deoxyguanosine glycosylase 1 (OGG1) and X-ray repair cross complementation 1 (XRCC1), are also polymorphic in the human population. Base excision repair is the principal pathway for repair of oxidative DNA damage including 8-oxo-dG in eukaryotic cells. Therefore, functional polymorphisms in components of the cellular base excision repair machinery may also be expected to influence levels of sodium dichromate–induced oxidative DNA damage.

Because of the above factors, it is likely that there will be a considerable heterogeneity in individual response to sodium dichromate–induced DNA damage. The aim of the current study, therefore, is to utilize the conventional and FPG-modified comet assay to study interindividual differences in DNA damage in human WBC after treatment in vitro with sodium dichromate. The relationship between differences in life-style factors, plasma antioxidant capacity, and genetic polymorphisms in detoxification (GSTM1, GSTT1, NQO1) and DNA repair (OGG1, XRCC1) genes and levels of sodium dichromate–induced DNA damage were investigated in an attempt to identify potential biomarkers of susceptibility to sodium dichromate–induced DNA damage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All chemicals were obtained from (Sigma-Aldrich, Poole, Dorset, United Kingdom) and were the highest purity available unless stated otherwise.

Sample Collection
Ethical approval was granted from South Birmingham, United Kingdom, Local Research Ethics Committee before commencement of the study. Subjects for study were healthy adults ages 17 to 62 years (n = 72). After obtaining their informed consent, blood samples (2 mL) were obtained by venipuncture and collected in Vacutainer tubes (Haemograd, Becton Dickinson, United Kingdom) containing either EDTA or sodium heparin anticoagulant. All samples were stored on ice until required and were processed within 3 hours of sample collection. Volunteers completed a detailed questionnaire at the time of sampling in order to gather information on diet, smoking habit, age, and alcohol consumption.

Comet Assay
The comet assay was done as described by Singh et al. (33) with the following alterations. Whole blood (10 µL; EDTA anticoagulant) was suspended in 1 mL HBSS containing 100 µmol/L sodium dichromate and incubated for 1 hour at 37°C. Although cells respond to lower concentrations of sodium dichromate (34) this concentration was used to provide sufficient sensitivity to discriminate between individual responses. After treatment, cells were pelleted by centrifugation (200 x g, 5 minutes). Cell pellets were resuspended in 0.5% low-melting-point agarose (110 µL) and transferred to a glass microscope slide (Surgipath, Peterborough, United Kingdom) that had been precoated with 0.5% normal-melting-point agarose. The slides were coverslipped and placed on ice for a further 10 minutes. Coverslips were removed and the slides lysed for 1 hour at 4°C in lysis buffer (2.5 mol/L NaCl, 100 mmol/L Na2EDTA, 10 mmol/L Tris base, 1% sodium N-lauryl sarcosinate, 10% DMSO, and 1% Triton X-100). Slides were then transferred to a horizontal electrophoresis tank (Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom) containing electrophoresis buffer (300 mmol/L NaOH, 1 mmol/L EDTA, pH 13). After exactly 20 minutes to allow DNA unwinding, they were subjected to electrophoresis for 20 minutes using an electrophoresis power supply (Pharmacia LKB, Cambridge, United Kingdom). After electrophoresis, slides were neutralized by flooding with three changes of neutralization buffer (0.4 mol/L Tris, pH 7.5) for 5 minutes each wash. The slides were then stained with 50 µL ethidium bromide solution (20 µg/mL).

FPG-Modified Comet Assay
The modified comet assay was carried out as described by Collins et al. (18). Briefly, The comet assay was carried out as described above, with the exception that after lysis slides were washed 3 x 5 minutes with FPG buffer (40 mmol/L HEPES, 0.1 mol/L KCl, 0.5 mmol/L EDTA, 0.2 mg/mL bovine serum albumin, pH 8). After this time, the slides were incubated with 1 unit of FPG enzyme (Trevigen, Gaithersburg, MD) in 50 µL FPG buffer at 37°C for 1 hour. After this time, the slides were incubated with 1 unit FPG enzyme (Trevigen) in 50 mL FPG buffer at 37°C for 1 hour as optimized by Collins et al. (18). One unit FPG is defined as the amount of enzyme required to cleave 1 pmol 32P-labeled oligonucleotide probe containing 8-oxo-dG within an oligonucleotide duplex in 1 hour at 37°C. To minimize potential variation in FPG activity, the same batch number (3632LO, stored at –80°C in aliquots) was used in all experiments. Control slides were incubated with 50 µL FPG buffer only. DNA unwinding and electrophoresis were then completed as described above.

Slide Visualization and Analysis
Slides were examined at 320x magnification (32x/0.40 dry objective) using a fluorescence microscope (Zeiss, Welwyn Garden City, Hertforshire, United Kingdom), fitted with a 515- to 560-nm excitation filter and a barrier filter of 590 nm. A video camera (Kinetic Pulnix TM-765) received the images, which were analyzed with a personal computer–based image analysis system Komet 3.0 Europe (Kinetic Imaging Ltd., Liverpool, United Kingdom). One hundred randomly selected nuclei were analyzed per slide using the equipment described. We note that our sample numbers are 8 to 30 per group compared with the n = 3 to 5 commonly used in standard testing experiments and our replicates are within the range reported by Duez et al. (35). Measurements of percent tail DNA were determined to assess the extent of DNA damage. This parameter is the preferred measurement as it is linearly related to the DNA break frequency over a wide range of levels of damage (36). The unmodified version of the comet assay was carried out in parallel so levels of FPG-dependent oxidative damage (exceeding background levels of DNA strand breaks and labile site) could be determined.

Genotyping Analyses
Genomic DNA was isolated from whole blood (~1 mL) using a QIAamp blood spin column midi kit (Qiagen, Crawley, United Kingdom), according to the manufacturer's instructions. PCR genotyping reactions were carried out as previously reported to detect deletions in GSTM1 and GSTT1 (37), with the exception that GST and albumin (internal control) primers sequences were as detailed previously (38). In addition, previously reported RFLP-PCR methods were used to detect polymorphisms in NQO1 at codon 609 (39), OGG1 at codon 326 (40), and XRCC1 at codon 194 (41).

Plasma Preparation and Plasma Antioxidant Test
Whole blood (500 µL; heparin anticoagulant) was centrifuged for 15 minutes at 1500 x g at room temperature. Plasma was stored at –70°C until analysis. Plasma antioxidant capacity was determined by measuring the ability of samples to inhibit superoxide-dependent pholasin fluorescence using a commercially available bioluminescence ABEL kit (Knight Scientific, Plymouth, United Kingdom) according to the manufacturer's instructions. Briefly, bioluminescence was measured using a plate reader luminometer (Tecan Instruments, Reading, Berkshire, United Kingdom) and antioxidant capacity of each individual plasma sample was expressed as the percentage reduction in fluorescence emission compared with a negative control containing no plasma.

Statistical Analyses
All data were analyzed using SPSS version 10 statistical package (SPSS, Working, Surrey, United Kingdom). Differences between both mean and median tail DNA before and after treatment were calculated using a one-tailed paired Student's t test as recommended by Duez et al. (35). In comparison of tail DNA values between individuals, the comet values were log transformed (mean values only) before analysis. If there were two genotypes in the group (GSTM1 and GSTT1) a one-way ANOVA test was used to analyze differences between the mean tail DNA with or without treatment. For NQO1, OGG1 and XRCC1 (three genotypes per group) the one-way ANOVA was accompanied by a post hoc Scheffe test. All correlations between comet tail DNA and plasma antioxidant capacity were determined by Pearson's correlation coefficient test (2-tailed).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We have previously shown that sodium dichromate induces detectable DNA strand breaks at concentrations as low as 100 nmol/L (34), which is relevant to potential human exposure. However, we emphasize that a higher concentration of 100 µmol/L for 1-hour exposure was most appropriate for the current study because of the need to optimize sensitivity in the search for interindividual differences. We were careful to ensure that this concentration did not cause cytotoxicity as assessed by ATP content and frequency of apoptosis over the period investigated (6, 34).

Characteristics of the Study Population
The mean age of volunteers was 34.8 ± 11.3 (SD, n = 72) years. Of the 72 subjects studied, 40 (55.6%) were female and 32 (44.4%) were male. Information gathered from the self-administered questionnaires revealed that 5.6% of the population were vegetarian, 23.6% were smokers, and 86.1% were Caucasian (Table 1). A general linear univariate ANOVA model was constructed to identify potential confounding factors. Age, ethnicity, gender, alcohol consumption (in the previous week), cigarettes smoked per day on average, vegetable and fruit consumption (average number of servings consumed per day), and meat and fish consumption (average number of servings consumed per week) were analyzed as potential confounding factors. In the current study, none of these parameters was found to have a statistically significant effect on levels of DNA damage or plasma antioxidant status either before or after treatment (data not shown). Therefore, these factors were excluded from further analyses.


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  		Table 1. General characteristics of the study population (mean age, 34.8 ± 11.3 years)

 
Comet Assay Results
We determined the coefficient of variation of the comet assay between different experiments using a cell line (mouse embryonic fibroblasts European Cell Culture Collection No. 99072801) with a range of concentrations of sodium dichromate. These values ranged from 14.1% to 26.1% without FPG and from 14.2% to 26.3% with FPG (n = 8 experiments in duplicate). Furthermore, we have shown previously that intraindividual variability of DNA strand breaks in human WBC over an 8- to 12-month period was of a relatively small coefficient of variation (24%) either with or without treatment of cells with sodium dichromate (34). A survey of previous studies indicates that these values compare favorably. Mean tail DNA was calculated for the population using WBC (with and without FPG incubation) without treatment (control) and after treatment with 100 µmol/L sodium dichromate for 1 hour at 37°C (Fig. 1). This concentration of sodium dichromate does not reduce intracellular levels of ATP or induce apoptosis within the period of incubation as assessed by the terminal deoxynucleotidyl transferase–mediated nick end labeling assay (data not shown) indicating that DNA strand breaks observed were not secondary to toxicity.



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  		Figure 1. Interindividual range, mean + SE of comet tail DNA (%) with and without FPG incubation in human WBC from individuals before and after treatment with 100 µmol/L sodium dichromate for 1 hour at 37°C. ****, P < 0.0005 (significantly different). Both median values and log-transformed mean values were analyzed by one-tailed paired t test.

 
The mean background level of DNA strand breaks in untreated WBC was 6.54 ± 0.16%. Background levels of DNA damage increased after FPG treatment to 13.45 ± 0.52%, indicating the presence of endogenous oxidative damage. After treatment with sodium dichromate there was a 1.6-fold increase in DNA strand breaks (mean, 10.14 ± 0.32%). A similar 1.5-fold increase was observed in levels of FPG-sensitive sites after sodium dichromate treatment (mean, 20.01 ± 0.71%). Analysis using a one-tailed paired Student's t test (both using mean and median values) showed a statistically significant increase in tail DNA after treatment with sodium dichromate using both the conventional comet assay (P < 0.0005) and the FPG-modified comet assay (P < 0.0005). In addition, treatment with FPG significantly increased tail DNA levels in both control nuclei (P < 0.0005) and nuclei from CrVI-treated cells (P < 0.0005).

Interindividual ranges of DNA strand breaks in controls measured by the conventional and FPG-modified comet assay were 4.06% to 11.97% and 6.02% to 18.48%, respectively. After dichromate treatment, the range of DNA strand breaks was 7.19% to 25.84% in the conventional comet assay and 8.79% to 34.72% in the FPG-modified comet assay. These data indicate substantial interindividual variation in levels of both background and dichromate-mediated DNA strand breaks and oxidative DNA damage (Fig. 1). The mean level of FPG-dependent oxidative damage (FPG-dependent oxidative damage was calculated by subtracting the percent tail DNA value obtained by the conventional comet assay from the percent tail DNA value obtained after inclusion of FPG) was 6.91 ± 0.57%, and this was significantly increased (P < 0.001, one-tailed paired t test) to 9.87 ± 0.66% after treatment with 100 µmol/L sodium dichromate for 1 hour at 37°C.

DNA Strand Breaks in Relation to Genotype
There was no statistically significant effect of any of the genotypes investigated on levels of frank DNA strand breaks as assessed by the conventional comet assay (data not shown). In addition, GSTM1, GSTT1, NQO1 (codon 187) and XRCC1 (codon 194) polymorphic genotypes had no statistically significant effect on levels of oxidative DNA damage as assessed by the FPG-modified comet assay in either controls or after treatment with sodium dichromate (summarized in Table 2).


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  		Table 2. Mean levels of control and dichromate-induced FPG-dependent oxidative DNA damage in WBC and relationship to GST, NQO1, and XRCC genotype (n = 72)

 
Individuals were analyzed for the OGG1 Cys326 polymorphism by PCR-RFLP (Fig. 2). No significant difference in control levels of tail DNA between Ser326/Ser326 and Ser326/Cys326 individuals (13.70 ± 4.49% versus 11.98 ± 3.22%, P = 0.369) or the Ser326/Ser326 and Cys326/Cys326 individuals (13.70 ± 4.49% versus 15.24 ± 5.64, P = 0.655) was detected as assessed by the FPG-modified comet assay. Furthermore, the difference between the mean tail DNA values in control (untreated) cells between the Ser326/Cys326 and Cys326/Cys326 individuals was also not statistically significant (P = 0.219). In contrast, after dichromate treatment, a role of codon 326 polymorphism in OGG1 was apparent. There was a statistically significant difference in mean tail DNA levels between Cys326/Cys326 homozygotes and both wild-type (26.56 ± 5.19% versus 19.61 ± 6.10%, P = 0.008) and Ser326/Cys326 individuals (26.56 ± 5.19% versus 18.12 ± 4.40, P = 0.003) as measured by the FPG-modified comet assay (Fig. 3). Wild-type and heterozygous individuals did not differ significantly (P = 0.639; Fig. 3).



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  		Figure 2. Representative agarose gel showing PCR-RFLP genotyping of codon 326 of OGG1 as described previously (39). As a positive control the identity of individuals of known genotype were confirmed by sequencing (data not shown).

 


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  		Figure 3. Mean levels + SE of DNA strand breaks as analyzed by both conventional and FPG-modified comet assay in WBC before (Control) and after treatment with sodium dichromate (100 µmol/L, 1 hour, 37°C). Individuals are grouped according to OGG1 codon 326 genotype. **, significantly different at the 1% level (one-way ANOVA and post hoc Scheffe test).

 
Interestingly, the same profile was observed when the data were analyzed as FPG-dependent oxidative DNA damage (Fig. 4). No significant difference in control levels of tail DNA between Ser326/Ser326 and Ser326/Cys326 individuals (6.99 ± 4.38% versus 5.71 ± 3.13%, P = 0.560) or the Ser326/Ser326 and Cys326/Cys326 individuals (6.99 ± 4.38% versus 9.09 ± 5.60%, P = 0.442 was detected. Furthermore, the difference between the mean control tail DNA levels between the Ser326/Cys326 and Cys326/Cys326 individuals was also not statistically significant (P = 0.183; Fig. 4). In contrast, after dichromate treatment, a role of codon 326 polymorphism in OGG1 was again apparent. There was a statistically significant difference in mean tail DNA levels between Cys326/Cys326 homozygotes and both wild-type (15.24 ± 6.59% versus 9.54 ± 5.48%, P = 0.023) and Ser326/Cys326 individuals (15.24 ± 6.59% versus 8.30 ± 3.88, P = 0.011) as measured by the FPG-dependent comet assay (Fig. 4). Wild-type and heterozygous individuals did not differ significantly (P = 0.700; Fig. 4).



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  		Figure 4. Mean levels + SE of FPG-dependent DNA strand breaks as analyzed by comet assay in WBC before (Control) and after treatment with sodium dichromate (100 µmol/L, 1 hour, 37°C). Individuals are grouped according to OGG1 codon 326 genotype. *, significantly different at the 5% level (one-way ANOVA and post hoc Scheffe test). FPG-dependent oxidative damage was calculated by subtracting the tail DNA percent (TD %) value obtained by the conventional comet assay from the tail DNA percent value obtained after inclusion of FPG.

 
Correlation between DNA Strand Breaks and Plasma Antioxidant Capacity
To investigate possible relationships between antioxidant status and dichromate-induced DNA strand breaks, plasma antioxidant capacity was compared against levels of DNA strand breaks (measured using the FPG-modified comet assay). The mean plasma antioxidant capacity of the study population was 69.92 ± 10.50%. Age, ethnicity, gender, alcohol consumption (in the previous week), cigarettes smoked per day on average, vegetable and fruit consumption (average number of servings consumed per day), and meat and fish consumption (average number of servings consumed per week) were all analyzed as potential confounding factors. None of these parameters was found to have a statistically significant effect on plasma antioxidant status in the relatively small numbers of subjects in this study (data not shown). However, a statistically significant positive correlation between levels of FPG-dependent oxidative DNA damage and antioxidant capacity significant (r = 0.253, P = 0.037) was observed. When individuals were divided based on OGG1 genotype, wild-type and heterozygous individuals continued to display a positive trend (r = 0.201 and 0.403, respectively). In contrast, Cys326 homozygote individuals displayed a negative trend (r = –0.349; Fig. 5A). When the data were analyzed as a ratio of oxidative DNA damage to plasma antioxidant capacity, Cys326/Cys326 individuals had a statistically significant (P < 0.05, one-way ANOVA followed by Tukey test) higher tail DNA percent/antioxidant capacity ratio than either heterozygous or wild-type individuals (Fig. 5B). There was no statistically significant difference (P > 0.05) between the Ser326/Ser326 and Ser326/Cys326 individuals (Fig. 5B).



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  		Figure 5. Correlation between plasma antioxidant capacity (%) and levels of dichromate-induced oxidative DNA damage. A. FPG-dependent oxidative DNA damage related to OGG1 (codon 326) genotype (n = 68). Colored symbols, different OGG1 genotypes. B. Columns, mean of ratio (tail DNA % / antioxidant capacity) grouped according to OGG1 genotype; bars, SE. Difference between the means calculated using one-way ANOVA and Tukey test. *, P < 0.05 (significantly different).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study we observed 2.9- and 3.6-fold ranges in control levels of DNA strand breaks in WBC from the study population (n = 72), as measured by the conventional and FPG-modified comet assays, respectively. Previous studies have observed similar variable levels of DNA strand break in cells from human populations in the absence of any chemical insult (42-44). We have shown previously that intraindividual variability of DNA strand breaks over an 8- to 12-month period was of a relatively small coefficient of variation (24%) either with or without treatment of cells with sodium dichromate (34). After treatment with sodium dichromate (100 µmol/L; 1 hour) 3.1- and 3.9-fold ranges in levels of DNA strand breaks were observed as assessed by the conventional and FPG-modified comet assay, respectively. Possible causes of this variation include age, gender, diet, exercise, infection, sunlight, and smoking [reviewed by Møller et al. (45)] as well as genetic factors. Analysis using a general linear univariate ANOVA model showed that age, gender, ethnicity, alcohol consumption, vitamin intake, fruit, vegetable, meat and fish consumption, and smoking habit had no statistically significant effect on the measured DNA damage in either control or dichromate-induced DNA damage. In agreement with our findings, previous studies have also observed no significant association between these factors and levels of DNA damage (46-54).

Other potential sources of variability in response to dichromate treatment include interindividual differences in the intracellular reduction of chromium and the capacity to detoxify reactive intermediates. For example, large interindividual differences in the reduction of CrVI in plasma (55, 56) and blood (57) from human subjects and differences in chromium uptake in lymphoblastic cell lines derived from three different individuals (58) have been reported. In this study, potential differences in chromium uptake due to variability in reductive capacity of plasma will not contribute to the variability because in our experiments extracellular conditions were uniform. Variability could also be related to differences in DNA repair capacity. In support of this, Collins et al. (59) and Janssen et al. (60) have reported large interindividual differences in repair of 8-oxo-dG by human lymphocytes, although the basis of this variability has not been determined.

Genetic polymorphisms may affect the level of expression, the structure, or the catalytic activity of metabolic or DNA repair enzymes (61), thereby potentially influencing individual susceptibility to sodium dichromate–induced DNA damage. Six common and potentially relevant genetic polymorphisms were considered in this study and related to levels of background and sodium dichromate–induced levels of DNA strand breaks and oxidative DNA damage. GSTM1 and GSTT1 genotype had no significant effect on levels of either background or dichromate-induced DNA suggesting that neither GSTM1 nor GSTT1 play a rate-limiting role in metabolic detoxification or activation of sodium dichromate. Alternatively compensatory mechanisms may exist to overcome GST deficiency, and this study cannot completely exclude a role for GSTM1 and T1 in the metabolic activation of CrVI. The polymorphism in NQO1 at codon 187 results in a protein with only 2% of the enzymic activity of the wild-type protein (39). However, lack of correlation of this polymorphism with sodium dichromate–induced strand breaks suggests that NQO1 does not play a major role in the metabolic reduction of CrVI to species capable of causing either DNA strand breaks or oxidative DNA damage or in the metabolic detoxification of sodium dichromate. This finding is in agreement with a study in rat hepatocytes that showed inhibition of NQO1 had little effect on the cytotoxicity of CrVI (62).

The gene products XRCC1 and OGG1 are involved in base excision repair of oxidative DNA damage. In this study, the polymorphism in XRCC1 at codon 194 had no significant effect on levels of DNA strand breaks or oxidative DNA damage either before or after dichromate treatment. OGG1 has been shown to be a glycosylase/AP lyase that cleaves the glycosidic bond of 8-oxo-dG preferentially at 8-oxo-dG:C base pairs (63). The importance of OGG1 has been shown in a number of studies. For example, yeast strains containing OGG1 mutations show a spontaneous mutator phenotype, characterized by an increased frequency of G to T transversions (64). Mice lacking functional OGG1 protein accumulate abnormal levels of 8-oxo-dG and have elevated spontaneous mutation frequencies in liver and kidney cells (65-68). In addition, Collins et al. (59) observed a lack of repair activity on a DNA substrate containing 8-oxo-dG by a protein extract from homozygous OGG1 knockout mouse embryonic fibroblasts in a modified version of the comet assay. In contrast, mammalian cells overexpressing OGG1 repair 8-oxo-dG more rapidly after toxic insult with oxidants (69).

The human OGG1 gene is located on chromosome 3p25-p26 and allelic deletions of this region occur frequently in a variety of human cancers (70) and this has been associated with elevated 8-oxo-dG levels in lung tumors (40). Analysis of the OGG1 gene in normal and tumor tissues has revealed somatic mutations and polymorphisms, such as the polymorphism at codon 326 that results in a Ser->Cys amino acid substitution in the protein (71, 72). The latter has been associated with an increased risk of orolaryngeal (73), lung (74), and esophageal (75) cancers. There is evidence that the Cys326 polymorphism has a functional effect. For example, OGG1 Cys326 protein was shown to have approximately seven times lower activity in a complementation assay of an E. coli mutant defective in the repair of 8-oxo-dG (76). In addition, the catalytic efficiency of excision for 8-oxo-dG from -irradiated DNA by OGG1-Ser326 protein is twice that of OGG1 Cys326 (77). Very recently Chen et al. have showed that the Cys326/Cys326 OGG1 genotype represents a repair deficient phenotype compared with wild-type (78). In contrast, another study has indicated no difference in enzymic activity between the Ser326 and Cys326 variants of OGG1 in human peripheral blood lymphocytes (60).

Our study, as outlined below, suggests that deficiency of the Cys326 variant is evident only under conditions of oxidative stress. We found no statistically significant difference in the mean levels of background DNA damage between wild-type individuals and individuals with either one or two copies of the Cys326 allele, as measured by the conventional or FPG-modified comet assay, was observed. Moreover, after dichromate treatment (100 µmol/L; 1 hour), there was no significant difference in mean levels of DNA strand breaks between the three groups as assessed by the conventional comet assay. In contrast, individuals homozygous for the Cys326 allele had statistically significantly higher levels of DNA strand breaks after FPG incubation than either wild-type (P = 0.008) or heterozygous (P = 0.003) individuals. Furthermore, the same trend was also observed when the data were analyzed as levels of FPG-dependent oxidative damage (P = 0.02 and P = 0.01, respectively). Although this association has been shown to be statistically significant, we recognize that a relatively small number (n = 8) of Cys326/Cys326 OGG1 genotype individuals have been studied and these individuals all had an antioxidant capacity above 70%. We recognize that further analyses in a larger population and a wider range of antioxidant capacities are required for confirmation of our findings. In support of our observations a similar correlation between 8-oxo-dG in leukocytes and OGG1 genotype has been made by Tarng et al. (79) in a group of patients undergoing hemodialysis.

These data suggest a deficiency in the repair of sodium dichromate–induced but not background oxidative DNA damage in Cys326/Cys326 individuals. No such deficiency was evident in heterozygotes compared with wild-type individuals. This suggests that a single copy of the Ser326 allele may represent sufficient functional reserve to repair the nature and extent of dichromate-induced oxidative damage in a manner indistinguishable from wild-type individuals. We hypothesize that the Cys326 amino acid in variant OGG1 is susceptible to thiol modification under conditions of oxidative stress so as to reduce the functional capacity of the enzyme. This would not contradict the observations of Janssen et al. (60) who reported no difference in the enzymic activities of purified OGG1 proteins containing either Ser326 or Cys326, but did not investigate the effect of oxidative stress. Interestingly, in a similar way, a recent study (80) has reported that activity of the DNA repair protein XPA is modulated by redox modification to a zinc finger motif present in its active site. In addition, reduced repair capacity of variant OGG1 protein only under conditions of oxidative stress would explain why no difference between background levels of oxidative DNA damage was observed between the three groups. Interestingly, a reduction in activity of FPG (the functional homologue of OGG1 in Escherichia coli) has already been shown as a result of oxidation of cysteine residues in the zinc finger region (81). In addition, OGG1 has been shown to be regulated by posttranslational phosphorylation of an unidentified serine residue apparently by protein kinase C resulting in translocation of OGG1 from the chromatin and nuclear matrix to the condensed chromatin during mitosis (82). Ser326 is not thought to be a constituent of a potential protein kinase C consensus sequence in OGG1. It is possible, however, that phosphorylation of Ser326 by another kinase may also posttranslationally regulate OGG1. Substitution of Ser326 with a cysteine residue may therefore potentially alter OGG1 activity and/or cellular location either by thiol oxidation or by avoidance of phosphorylation.

As with levels of DNA damage, large interindividual variations in levels of plasma antioxidant capacity (mean, 69.2 ± 10.5%; range, 27.8-91.6%) were observed. The relationship between plasma antioxidant capacity and WBC intracellular redox status is complex. There is evidence of a positive correlation between intracellular and plasma (extracellular) levels of glutathione, ascorbate, and vitamin E (83-85). Furthermore, intracellular levels of both glutathione and ascorbate are negatively correlated with 8-oxo-dG levels in lymphocytes (86). In addition, plasma levels of antioxidants including ascorbate, ß-carotenoids, lycopenes, and vitamin E are also negatively correlated with levels of DNA oxidation in human lymphocytes (87-90). Therefore, plasma antioxidant capacity is likely to be an indicator of WBC intracellular redox status and may be related to the ability to activate sodium dichromate within the cell by the reductive antioxidants GSH and ascorbate. In this study we observed a statistically significant (P = 0.037) positive correlation between sodium dichromate–induced FPG-dependent oxidative DNA damage and plasma antioxidant capacity. When the population was analyzed on the basis of OGG1 genotype, Ser326/Ser326 and Ser326/Cys326 individuals continued to display a positive correlation (r = 0.201 and 0.403, respectively). In contrast, for Cys326/Cys326 individuals a negative correlation (r = –0.349) was observed. Further analysis revealed that Cys326/Cys326 individuals had a statistically significant (P < 0.05, one-way ANOVA followed by Tukey test) higher tail DNA percent/antioxidant capacity ratio than either heterozygous or wild-type individuals. These data suggest that Cys326/Cys326 individuals have a functionally different relationship between plasma antioxidant capacity and sodium dichromate–induced oxidative DNA damage than either heterozygous or wild-type individuals. Despite the relatively low power of this study for the Cys326/Cys326 genotype, the finding of a statistically significant difference between these and Ser326/Ser326 individuals emphasizes the biological importance of this polymorphism in the context of responsiveness to chromium-mediated oxidative DNA damage.

Although the basis of this difference remains to be determined, we hypothesize that in wild-type and heterozygous individuals the major determinant of DNA damage by dichromate is reductive activation by GSH (as shown in Fig. 6), which correlates with plasma antioxidant activity. In contrast, although GSH still plays a role in sodium dichromate activation in Cys326/Cys326 individuals, impaired DNA repair capacity of variant OGG1 protein under conditions of oxidative stress is proposed to become rate limiting through thiol oxidation and enzyme inactivation. Therefore, Cys326/Cys326 individuals with a relatively low antioxidant (and GSH) capacity are expected to have higher levels of dichromate-induced DNA oxidation despite less activation because of lower cellular reductants. We propose that the reason why the codon 326 Cys residue in OGG1 impairs function is due to higher chances of thiol modification of this residue under conditions of high oxidative stress. As antioxidant levels increase, thiol modification becomes less prevalent and the Cys residue no longer impairs OGG1 function and therefore no difference is found between these two proteins in their ability to repair DNA damage (see Fig. 6).



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  		Figure 6. Proposed relationship between OGG1 genotype, plasma antioxidant capacity, and levels of dichromate-induced oxidative DNA damage. See text for explanation and discussion.

 
In summary, the results of this study suggest that the Cys326/Cys326 OGG1 genotype is responsible for a substantial proportion of the interindividual variation in response to dichromate-induced oxidative DNA damage. We hypothesize that this is a result of reduced repair activity of variant OGG1 protein (conferred by the Cys326 residue) under conditions of cellular oxidative stress and that the Cys326/Cys326 OGG1 genotype represents a phenotype that is deficient in the repair of sodium dichromate–induced oxidative DNA damage. Consequently, OGG1 genotype is a candidate biomarker of susceptibility for CrVI-induced lung cancer in occupationally exposed individuals.


   Acknowledgments
 
We thank Jonathan D. Pritchard for his assistance in carrying out preliminary experiments to optimize the conditions for the comet assay before the commencement of the main population study.


   Footnotes
 
Grant support: COLT Foundation and ICI.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: A.J. Lee is currently in Biological and Biomedical Sciences, The University of Durham Science Laboratories, Durham, DH1 3LE, United Kingdom.

Received 4/20/04; revised 9/16/04; accepted 9/30/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. IARC. Chromium, nickel and welding. IARC monographs on the evaluation of carcinogenic risks to humans. Vol. 49. Lyon: IARC; 1990.
  2. Cohen MD, Kargacin B, Klein CB, Costa M. Mechanism of chromium carcinogenicity and toxicity. Crit Rev Toxicol 1993;23:255–81.[Medline]
  3. Waterhouse JAH. Cancer among chromium platers. Br J Cancer 1975;32:262.[Medline]
  4. Alderson MR, Rattan NS, Bidstrup L. Health of workmen in the chromate-producing industry in Britain. Br J Ind Med 1981;38:117–24.[Medline]
  5. Werfel U, Langen V, Eickhoff I, et al. Elevated DNA single-strand breakage frequencies in lymphocytes of welders exposed to chromium and nickel. Carcinogenesis 1998;19:413–8.[Abstract/Free Full Text]
  6. Hodges NJ, Ádám B, Lee AJ, Cross HJ, Chipman JK. Induction of DNA-strand breaks in human peripheral blood lymphocytes and A549 lung cells by sodium dichromate: association with 8-oxo 2 deoxy guanosine formation and inter-individual variation. Mutagenesis 2001;16:467–74.[Abstract/Free Full Text]
  7. Gao M, Binks SP, Chipman JK, Levy LS, Braithwaite RA, Brown SS. Induction of DNA strand breaks in peripheral lymphocytes by soluble chromium compounds. Hum Exp Toxicol 1992;11:77–82.[Medline]
  8. Xu J, Manning FC, Patierno S. Preferential formation and repair of chromium-induced DNA adducts and DNA-protein cross-links in nuclear matrix DNA. Carcinogenesis 1994;15:1443–50.[Abstract/Free Full Text]
  9. Bridgewater LC, Manning FCR, Patierno SR. Base-specific arrest of in vitro DNA replication by carcinogenic chromium: relationship to DNA interstrand cross linking. Carcinogenesis 1994;15:2421–7.[Abstract/Free Full Text]
  10. Cupo DY, Wetterhahn KE. Binding of chromium to chromatin and DNA from liver and kidney of rats. Cancer Res 1985;45:1146–51.[Abstract/Free Full Text]
  11. Snow ET. A possible role for chromium (III) in genotoxicity. Environ Health Perspect 1991;92:75–81.[Medline]
  12. Aiyer J, Borges KM, Floyd RA, Wetterhahn KE. Role of chromium(V), glutathionyl and hydroxyl radical intermediates in chromium(VI)-induced DNA damage. Toxicol Environ Chem 1989;22:135–48.
  13. Shi X, Mao Y, Knapton AD, et al. Reaction of Cr(VI) with ascorbate and hydrogen peroxide generates hydroxyl radicals and causes DNA damage: role of a Cr(VI)-mediated Fenton-like reaction. Carcinogenesis 1994;15:2475–8.[Abstract/Free Full Text]
  14. Ye J, Wang S, Leonard SS, et al. Role of reactive oxygen species and p53 in chromium(VI)-induced apoptosis. J Biol Chem 1999;274:34974–80.[Abstract/Free Full Text]
  15. Wang S, Leonard SS, Ye J, Ding M, Shi, X. The role of hydroxyl radical as a messenger in CrVI-induced p53 activation. Am J Physiol Cell Physiol 2000;273:C868–83.
  16. Singh J, Pritchard DE, Carlisle DL, et al. Internalisation of carcinogenic lead chromate particles by cultured normal lung epithelial cells: formation of intracellular lead-inclusion bodies and induction of apoptosis. Toxicol Appl Pharmacol 1999;161:240–8.[CrossRef][Medline]
  17. Leonard SS, Roberts JR, Antonini JM, Castranova V, Shi X. PbCrO4 mediates cellular responses via reactive oxygen species. Mol Cell Biochem 2004;225:171–9.
  18. Collins AR, Duthie SJ, Dobson VL. Direct enzymic detection of endogenous base damage in human lymphocyte DNA. Carcinogenesis 1993;14:1733–5.[Abstract/Free Full Text]
  19. Blasiak J, Kowalik JA. Comparison of the in vitro genotoxicity of tri- and hexavalent chromium. Mutat Res 2000;469:135–45.[Medline]
  20. Trzeciak A, Kowalik J, Malecka-Panas E, Drzewoski J, Wojewódzka M, Ko TI. Genotoxicity of chromium in human gastric mucosa cells and peripheral blood lymphocytes evaluated by the single cell gel electrophoresis (comet) assay. Med Sci Monit 2000;6:24–9.[Medline]
  21. Blasiak J, Trzeciak A, Malecka-Panas E, et al. DNA damage and repair in human lymphocytes and gastric mucosa cells exposed to chromium and curcumin. Teratog Carcinog Mutagen 1999;19:19–31.[CrossRef][Medline]
  22. Merk O, Reiser K, Speit G. Analysis of chromate-induced DNA-protein crosslinks with the comet assay. Mutat Res 2000;471:71–80.[Medline]
  23. Liu K, Husler J, Ye J, et al. On the mechanism of Cr(VI)-induced carcinogenesis: dose dependence of uptake and cellular response. Mol Cell Biochem 2001;222:221–9.[CrossRef][Medline]
  24. Ueno S, Kashimoto T, Susa N, et al. Detection of dichromate (VI)-induced DNA strand breaks and formation of paramagnetic chromium in multiple mouse organs. Toxicol App Pharmacol 2001;170:56–62.[CrossRef][Medline]
  25. Amaral-Mendes JJ, Pluygers E. Use of biochemical and molecular biomarkers for cancer risk assessment in humans. In: Cogliano VI, Luebeck EG, Zapponi GA, editors. Perspectives on biologically based cancer risk assessment. NATO series on challenges of modern society, vol. 25. New York: Kluwer Academic/Plenum Publishers; 1999. p. 81–182.
  26. Perera FP, Weinstein IB. Molecular epidemiology: recent advances and future directions. Carcinogenesis 2000;21:517–24.[Abstract/Free Full Text]
  27. Standeven AM, Wetterhahn KE. Is there a role for reactive oxygen species in the mechanism of chromium(VI) carcinogenesis? Chem Res Toxicol 1991;4:616–25.[CrossRef][Medline]
  28. Pourahmad J, O'Brian PJ. Biological reactive intermediates that mediate chromium (VI) toxicity. Adv Exp Med Biol 2001;500:203–7.[Medline]
  29. Garcia JD, Jennette KW. Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate. J Inorg Biochem 1981;14:281–95.[Medline]
  30. Pickett CB. Glutathione S-transferases: gene structure, regulation, and biological function. Annu Rev Biochem 1989;8:743–64.
  31. De Flora S, Serra D, Camoirano A, Zanacchi P. Metabolic reduction of chromium, as related to its carcinogenic properties. Biol Trace Elem Res 1989;21:179–87.[Medline]
  32. Shi X, Chiu A, Chen CT, Halliwell B, Castranova V, Vallyathan V. Reduction of chromium(VI) and its relationship to carcinogenesis. J Toxicol Environ Health B Crit Rev 1999;2:87–104.[CrossRef][Medline]
  33. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184–91.[CrossRef][Medline]
  34. Lee AJ, Hodges NJ, Chipman JK. The modified comet assay as a biomarker of sodium dichromate-induced oxidative DNA damage: optimisation and reproducibility. Biomarkers. Biomarkers 2004;9:103–15.[CrossRef][Medline]
  35. Duez P, Dehon G, Kumps A, Dubois J. Statistics of the comet assay: a key to discriminate between genotoxic effects. Mutagenesis 2003;2:159–66.
  36. Hartmann A, Agurell E, Beevers C, et al. Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis 2003;18:45–51.[Abstract/Free Full Text]
  37. Arand M, Muhlbauer R, Hengstler J, Jager E, Fuchs J, Winkler L. Multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms. Anal Biochem 1996;236:184–6.[CrossRef][Medline]
  38. Nair UJ, Nair J, Mathew B, Bartsch H. Glutathione S-transferase M1 and T1 null genotypes as risk factors for oral leukoplakia in ethnic Indian betel quid/tobacco chewers. Carcinogenesis 1999;20:743–8.[Abstract/Free Full Text]
  39. Traver RD, Siegel D, Beall HD, et al. Characterization of a polymorphism in NAD(P)H: quinone oxidoreductase (DT-diaphorase). Br J Cancer 1997;75:69–75.[Medline]
  40. Hardie LJ, Briggs JA, Davidson LA, et al. The effect of OGG1 and glutathione peroxidase I genotypes and 3p chromosomal loss on 8-hydroxydeoxyguanosine levels in lung cancer. Carcinogenesis 2000;21:167–72.[Abstract/Free Full Text]
  41. Lunn RM, Langlois RG, Gsieh LL, Thompson CL, Bell DA. XRCC1 polymorphisms: effects on aflatoxin B1-DNA adducts and glycophorin A variant frequency. Cancer Res 1999;59:2557–61.[Abstract/Free Full Text]
  42. Betti C, Davini T, Giannessi L, Loprieno N, Barale R. Microgel electrophoresis assay (comet test) and SCE analysis in humans. Mutat Res 1994;307:323–33.[Medline]
  43. Collins AR, Dusinská M, Franklin M, et al. Comet assay in human biomonitoring studies: reliability, validation and applications. Environ Mol Mutagen 1997;30:139–46.[CrossRef][Medline]
  44. Dhawan A, Mathur N, Seth PK. The effect of smoking and eating habits on DNA damage in Indian population as measured in the comet assay. Mutat Res 2001;474:121–8.[Medline]
  45. Møller P, Knudsen LE, Loft S, Wallin H. The comet assay as a rapid test in biomonitoring occupational exposure to DNA-damaging agents and effect of confounding factors. Cancer Epidemiol Biomarkers Prev 2000;9:1005–15.[Abstract/Free Full Text]
  46. Singh NP, Danner DB, Tice RR, Brant L, Schneider EL. DNA damage and repair with age in individual human lymphocytes. Mutat Res 1990;237:123–30.[CrossRef][Medline]
  47. Betti C, Davini T, Giannessi L, Loprieno N, Barale R. Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects. Mutat Res 1995;343:201–7.[CrossRef][Medline]
  48. Frenzilli G, Betti C, Davini T, et al. Evaluation of DNA damage in leukocytes of ex-smokers by single cell gel electrophoresis. Mutat Res 1997;375:117–23.[Medline]
  49. Møller P, Knudsen LE, Frentz G, Dybdahl M, Wallin H, Nexø BA. Seasonal variation of DNA and repair in patients with non-melanoma skin cancer and referents with and without skin cancer. Mutat Res 1998;407:25–34.[Medline]
  50. Strám RJ. Effect of glutathione S-transferase M1 polymorphisms on biomarkers of exposure and effect. Environ Health Perspect 1998;106:231–9.[Medline]
  51. Wojewódzka M, Kruszewski M, Iwanenko T, Collins AR, Szumiel I. Application of the comet assay for monitoring DNA damage in workers exposed to chronic low-dose irradiation. Mutat Res 1998;416:21–35.[Medline]
  52. Van Zeeland AA, de Groot AJL, Hall J, Donato F. 8-Hydroxyydeoxyguanosine in DNA from leukocytes of healthy adults: relationship with cigarette smoking, environmental tobacco smoke, alcohol and coffee consumption. Mutat Res 1999;439:249–57.[Medline]
  53. Lodovici M, Casalini C, Cariaggi R, Michelucci L, Dolara P. Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes. Free Radic Biol Med 2000;28:13–7.[CrossRef][Medline]
  54. Giovannelli L, Saieva C, Masala G, et al. Nutritional and lifestyle determinants of DNA oxidative damage: a study in a Mediterranean population. Carcinogenesis 2002;23:1483–9.[Abstract/Free Full Text]
  55. Harzdorf C, Lewalter J. Analytical methodology for biological monitoring of chromium. Regul Toxicol Pharmacol 1997;26:S86–93.[Medline]
  56. Miksche LW, Lewalter J. Health surveillance and biological effect monitoring for chromium-exposed workers. Regul Toxicol Pharmacol 1997;26:S94–9.[Medline]
  57. Corbett GE, Dodge DG, O'Flaherty E, et al. In vitro reduction kinetics of hexavalent chromium in human blood. Environ Res 1998;78:7–11.[Medline]
  58. Zhang Q, Kluz T, Salnikow K, Costa M. Comparison of the cytotoxicity, cellular uptake, and DNA-protein crosslinks induced by potassium chromate in lymphoblast cell lines derived from three different individuals. Biol Trace Elem Res 2002;86:11–22.[Medline]
  59. Collins AR, Dusinská M, Horvá thová E, Munro E, Savio M, Stetina R. Inter-individual differences in repair of base oxidation, measured in vitro with the comet assay. Mutagenesis 2001;16:297–01.[Abstract/Free Full Text]
  60. Janssen K, Sclink K, Götte W, Hippler B, Kaina B, Oesch F. DNA repair activity of 8-oxoguanine DNA glycosylase 1 (OGG1) in human lymphocytes is not dependent on genetic polymorphism Ser326/Cys326. Mutat Res 2001;486:207–16.[Medline]
  61. Miller MC, Mohrenweiser HW, Bell DA. Genetic variability in susceptibility and response to toxicants. Toxicol Lett 2001;120:269–80.[CrossRef][Medline]
  62. Gunaratnam M, Grant MH. The role of glutathione reductase in the cytotoxicity of chromium (VI) in isolated rat hepatocytes. Chem Biol Int 2001;134:191–2.[CrossRef][Medline]
  63. Hazra TK, Izumi T, Maidt L, Floy RA, Mitra S. The presence of two distinct 8-oxoguanine repair enzymes in human cells: their potential complementary roles in preventing mutation. Nucleic Acids Res 1998;26:5116–22.[Abstract/Free Full Text]
  64. Thomas D, Scot AD, Barbet R, Padula M, Boiteux S. Inactivation of OGG1 increases the incidence of GC-TA transversions in Saccharomyces cerevisiae: evidence for endogenous oxidative damage to DNA in eukaryotic cells. Mol Gen Genet 1997;254:171–8.[CrossRef][Medline]
  65. Klungland A, Rosewell I, Hollenbach S, et al. Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage. Proc Nat Acad Sci U S A 1999;96:113300–5.
  66. Osterod M, Hollenbach S, Hengstler JG, Barnes DE, Lindahl T, Epe B. Age-related and tissue-specific accumulation of oxidative DNA base damage in 7,8-dihydro-8-oxoguanine-DNA glycosylase (OGG1) deficient mice. Carcinogenesis 2001;22:1459–63.[Abstract/Free Full Text]
  67. Arai T, Kelly VP, Minowa O, Noda T, Nishimura S. High accumulation of oxidative DNA damage, 8-hydroxyguanine, in Mmh/OGG1 deficient mice by chronic oxidative stress. Carcinogenesis 2002;23:2005–10.[Abstract/Free Full Text]
  68. Epe B. Role of endogenous oxidative DNA damage in carcinogenesis: what can we learn from repair-deficient mice. Biol Chem 2002;383:467–75.[CrossRef][Medline]
  69. Hollenbach S, Dhénaut A, Eckert I, Radicella JP, Epe B. Overexpression of OGG1 in mammalian cells: effects on induced and spontaneous oxidative DNA damage and mutagenesis. Carcinogenesis 1999;20:1863–8.[Abstract/Free Full Text]
  70. Shinmura K, Yokota J. The OGG1 gene encodes a repair enzyme for oxidatively damaged DNA and is involved in human carcinogenesis. Antioxid Redox Signal 2001;3:597–609.[CrossRef][Medline]
  71. Boiteux S, Radicella JP. Base excision repair of 8-hydroxyguanine protects DNA from endogenous oxidative stress. Biochimie 1999;81:59–67.[Medline]
  72. Boiteux S, Radicella JP. The human OGG1 gene: structure, functions, and its implication in the process of carcinogenesis. Arch Biochem Biophys 2000;377:1–8.[CrossRef][Medline]
  73. Elahi A, Zheng Z, Park J, Eyring K, MaCaffrey T, Lazarus, P. The human OGG1 DNA repair enzyme and its association with orolaryngeal cancer risk. Carcinogenesis 2002;23:1229–34.[Abstract/Free Full Text]
  74. Sugimura H, Kohno T, Wakai K, et al. OGG1 Ser326Cys polymorphism and lung cancer susceptibility. Cancer Epidemiol Biomarkers Prev 1999;8:669–74.[Abstract/Free Full Text]
  75. Xing DY, Tan W, Song N, Lin DX. Ser326Cys polymorphism in OGG1 gene and risk of esophageal cancer in a Chinese population. Int J Cancer 2001;95:140–3.[CrossRef][Medline]
  76. Kohno T, Shinmura K, Tosaka M, et al. Genetic polymorphisms and alternative splicing of the OGG1 gene, that is involved in the repair of 8-hydroxyguanine in damaged DNA. Oncogene 1998;16:3219–25.[CrossRef][Medline]
  77. Dherin C, Radicella JP, Dizdaroglu M, Boiteux S. Excision of oxidatively damaged DNA bases by the human {alpha}-OGG1 protein and the polymorphic {alpha}-OGG1 (Ser326/Cys326) protein which is frequently found in human populations. Nucleic Acids Res 1999;27:4001–7.[Abstract/Free Full Text]
  78. Chen SK, Hsieh WA, Tsai MT, et al. Age-associated decrease of oxidative repair enyzmes human 8-oxoguanine DNA glycosylases (OGG1), in human aging. J Radiat Res 2003;44:31–5.
  79. Tarng DC, Tsai TJ, Chen WT, Liu TY, Wei YH. Effect of human OGG1 1245C->G gene polymorphism on 8-hydroxy-2'-deoxyguanosine levels of leukocyte DNA among patients undergoing chronic hemodialysis. J Am Soc Nephrol 2001;12:2338–47.[Abstract/Free Full Text]
  80. Blessing H, Kraus S, Heindl P, Bal W, Hartwig A. Interaction of selenium compounds with zinc finger proteins involved in DNA repair. Eur J Biochem 2004;15:3190–9.
  81. Hartwig A, Asmuss M, Ehleben I, et al. Interference by toxic metal ions with DNA repair processes and cell cycle control: molecular mechanisms. Environ Health Perspect 2002;110:797–9.
  82. Dantzer F, Luna L, Bjørås M, Seeberg E. Human OGG1 undergoes serine phosphorylation and associates with the nuclear matrix and mitotic chromatin in vivo. Nucleic Acids Res 2002;30:2349–57.[Abstract/Free Full Text]
  83. Evans RM, Currie L, Campbell A. The distribution of ascorbic acid between various cellular components of blood, in normal individuals and its relation to plasma concentration. Br J Nutr 1982;47:473–82.[CrossRef][Medline]
  84. Lenton KJ, Sane AT, Therriault H, Cantin AM, Payette H, Wagner JR. Vitamin C augments lymphocyte glutathione in subjects with ascorbate deficiency. Am J Clin Nutr 2003;77:189–95.[Abstract/Free Full Text]
  85. Lehmann J, Rao DD, Canary JJ, Judd JT. Vitamin E and relationship among tocopherols in human plasma, platelets, lymphocytes and red blood cells. Am J Clin Nutr 1998;47:470–4.
  86. Lenton KJ, Therriault H, Fulop T, Payette H, Wagner JR. Glutathione and ascorbate are negatively correlated with oxidative DNA damage in human lymphocytes. Carcinogenesis 1999;20:607–13.[Abstract/Free Full Text]
  87. Fillion L, Collins AR, Southon S. ß-Carotene enhances the recovery of lymphocytes from oxidative DNA damage. Acta Biochim Pol 1998;45:183–90.[Medline]
  88. Collins AR, Olmedilla B, Southon S, Granado F, Duthie SJ. Serum carotenoids and oxidative DNA damage in human lymphocytes. Carcinogenesis 1998;19:2159–62.[Abstract/Free Full Text]
  89. Duthie SJ, Ma A, Ross MA, Collins AR. Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res 1996;56:1291–5.[Abstract/Free Full Text]
  90. Perez DD, Strobel P, Foncea R, et al. Wine, diet, antioxidant defenses and DNA damage. Ann N Y Acad Sci 2002;957:136–45.[Medline]



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P. Rossner Jr., M. B. Terry, M. D. Gammon, F. F. Zhang, S. L. Teitelbaum, S. M. Eng, S. K. Sagiv, M. M. Gaudet, A. I. Neugut, and R. M. Santella
OGG1 Polymorphisms and Breast Cancer Risk.
Cancer Epidemiol. Biomarkers Prev., April 1, 2006; 15(4): 811 - 815.
[Abstract] [Full Text] [PDF]

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