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J Am Soc Nephrol 12:2338-2347, 2001
© 2001 American Society of Nephrology

Effect of Human OGG1 1245C->G Gene Polymorphism on 8-Hydroxy-2'-Deoxyguanosine Levels of Leukocyte DNA among Patients Undergoing Chronic Hemodialysis

DER-CHERNG TARNG*,{dagger},§, TZUNG-JIUN TSAI{dagger}, WEI-TING CHEN{dagger}, TSUNG-YUN LIU|| and YAU-HUEI WEI*,{ddagger}

* Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan.
{dagger} Faculty of Medicine, National Yang-Ming University, Taipei, Taiwan.
{ddagger} Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.
§ Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.
|| Department of Education and Research, Taipei Veterans General Hospital, Taipei, Taiwan.

Correspondence to Dr. Yau-Huei Wei, Department of Biochemistry and Center for Cellular and Molecular Biology, National Yang-Ming University, No. 155, Section 2, Li-Nung Street, Taipei 112, Taiwan. Phone: +886-2-2826-7118; Fax: +886-2-2826-1132; E-mail: joeman{at}mailsrv.ym.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and MethodsStudy...
 Results
 Discussion
 References
 
Abstract. The effects of the human OGG1 gene (hOGG1) 1245C->G polymorphism on the 8-hydroxy-2'-deoxyguanosine (8-OHdG) contents of peripheral leukocyte DNA were investigated among chronic hemodialysis patients. First, the hOGG1 1245C->G transversion was assessed, by using a PCR-restriction fragment length polymorphism method, among 210 hemodialysis patients and 156 healthy individuals. Second, the 8-OHdG contents in leukocyte DNA were measured, by using an HPLC-electrochemical detection method, for 112 hemodialysis patients and 112 age-, gender-, and genotype-matched healthy control subjects. The three genotypes (as dummy variables) and age, gender, dialysis duration, dialyzer membrane type, blood antioxidant levels, and iron parameters were used as independent variables and the natural logarithm of the leukocyte 8-OHdG concentration was used as a dependent variable in a forward, stepwise, multiple-regression model. The results demonstrated that the allelic frequency of hOGG1 1245G was 64.1% among 210 hemodialysis patients and 62.2% in the whole control population. The genotypic frequencies (CC/CG/GG ratio, 10%/51.9%/38.1%) for the hemodialysis patients did not differ significantly from those (16.7%/42.3%/41.0%) for the control subjects (P > 0.05, {chi}2 test). The mean leukocyte 8-OHdG levels for the patients were significantly higher than those for the healthy control subjects (P < 0.001). Leukocyte 8-OHdG levels were further increased among patients with the 1245GG genotype, compared with patients with the 1245CG or CC genotype (P < 0.001, ANOVA), but levels were similar among healthy control subjects irrespective of the hOGG1 gene polymorphism. It was also observed that patients who underwent dialysis with cellulose membranes exhibited significantly higher leukocyte 8-OHdG levels than did patients who underwent dialysis with polymethylmethacrylate, polysulfone, or vitamin E-bonded membranes (P < 0.001, ANOVA). The multivariate regression analysis revealed that hOGG1 1245C->G polymorphism and dialysis membrane type were the two independent predictors of 8-OHdG contents in leukocyte DNA from hemodialysis patients. This study demonstrated that the extent of oxidative DNA damage among chronic hemodialysis patients not only is influenced by overproduction of reactive oxygen species resulting from leukocyte contacts with complement-activating membranes and by impaired antioxidant defense mechanisms but also is genetically determined.


   Introduction
Top
 Abstract
 Introduction
Materials and MethodsStudy...
 Results
 Discussion
 References
 
Reactive oxygen species (ROS) and peroxides are involved in the pathogenesis of many human diseases, including atherosclerosis and inflammatory diseases, and are thought to participate in the aging process and carcinogenesis (1,2,3). Oxidative damage occurs in a cell when the production of ROS exceeds the antioxidant defense capacity. A growing body of evidence indicates that ROS, which are generated by oxidative metabolism in activated phagocytes during hemodialysis, cause damage to cellular constituents, such as membrane lipids (4,5), proteins (6,7), and DNA (8,9). Membrane lipids and proteins are protected against oxidation by low-molecular weight antioxidants, as well as antioxidant enzymes, whereas DNA is not as well protected against ROS. Therefore, a marker of oxidative DNA damage would be useful for estimation of the extent of oxidative stress among hemodialysis subjects. Among the many types of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the most abundant oxidative products of cellular DNA (2). 8-OHdG is capable of reflecting extremely low levels of oxidative damage, because it can be detected, using HPLC-electrochemical detection methods, in the femtomole range. We have identified peripheral leukocytes of hemodialysis patients as being suitable for the monitoring of 8-OHdG levels in cellular DNA, because they are not only the source but also the target of endogenous ROS (8,9). In our previous work, leukocyte 8-OHdG levels were demonstrated to be highest in chronic hemodialysis patients, followed by non-dialysis-treated patients with advanced renal failure and by healthy control subjects. Levels were further increased among patients who underwent dialysis with cellulose membranes, compared with those treated with synthetic membranes (8) or vitamin E-bonded membranes (9). This finding indicates that uremia can be an important contributor to leukocyte DNA oxidation, which is amplified by extracorporeal hemodialysis, especially with complement-activating membranes. Therefore, leukocyte 8-OHdG content is a surrogate biomarker of oxidant-induced DNA damage among patients undergoing chronic hemodialysis.

Because of the abundance and mutagenicity of 8-OHdG, a number of defense mechanisms have evolved in various organisms to minimize its accumulation within the genome. Primary defense mechanisms include antioxidants and free radical-scavenging enzymes (9,10). Once formed, 8-OHdG lesions are subject to DNA repair, primarily through the base excision repair pathway (11). A key component of this pathway is a specific DNA glycosylase/apurinic lyase, which catalyzes the release of 8-OHdG and the cleavage of DNA at the apurinic site (12). Inactivation of this 8-OHdG glycosylase in Escherichia coli and yeast generates a mutator phenotype characterized by GC->TA transversions (13,14). The human homologue of this gene, hOGG1, has recently been identified and characterized (15,16,17). Like its yeast homologue, the gene product constitutes an 8-OHdG DNA glycosylase/apurinic lyase, which exhibits greatest specificity and activity for 8-OHdG-dC and is completely inactive against 8-OHdG-dA (15,17).

Genetic background has been demonstrated to be involved in the control of damaged DNA repair. A C->G polymorphism at position 1245 in exon 7 of the hOGG1 gene is associated with the substitution of cysteine for serine at codon 326 (18). It was recently reported that the DNA repair activity of the mutant hOGG1-Cys326 protein is lower than that of the wild-type hOGG1-Ser326 protein, on the basis of an E. coli complementation assay (18). This genetic polymorphism is frequently observed in the Japanese population, in both healthy individuals and patients with lung cancer (18). The same polymorphism is also observed, at a similar frequency, among European patients with head and neck tumors or kidney tumors (19). However, the genotypic frequency of this polymorphism in the Chinese population is unknown. Furthermore, to the best of our knowledge, the biologic significance of the hOGG1 1245C->G polymorphism for patients undergoing hemodialysis has not yet been elucidated. The polymorphism of the hOGG1 gene is worth investigation, inasmuch as a population with decreased enzyme activity of the hOGG1 protein would be at risk of accumulating 8-OHdG in nuclear DNA because of incomplete repair of oxidatively damaged DNA. In view of the lower activity of the hOGG1-Cys326 protein, compared with the hOGG1-Ser326 protein, in repair of 8-OHdG, we examined the effects of the hOGG1 1245G allele on the 8-OHdG contents of peripheral leukocyte DNA from patients undergoing chronic hemodialysis. In addition, we demonstrated the effects of complement-activating membranes, defective antioxidant defense, and iron excess on the generation of leukocyte 8-OHdG among hemodialysis patients (8,9), and the roles of these factors in determining leukocyte 8-OHdG contents deserve reassessment. We used four dialysis membranes [cellulose, polymethylmethacrylate (PMMA), polysulfone (PS), and vitamin E-bonded membranes], blood antioxidant levels, and iron parameters (serum ferritin concentrations, serum iron concentrations, and transferrin saturation) as covariables in assessments of the relationship between the hOGG1 1245C->G genotype and leukocyte 8-OHdG contents, in a forward, stepwise, multiple-regression model.


   Materials and Methods Study Population
Top
 Abstract
 Introduction
 Materials and MethodsStudy...
Results
 Discussion
 References
 
For determination of the allelic frequency of the 1245C->G polymorphism in the hOGG1 gene, 210 patients who were undergoing chronic hemodialysis treatment at three dialysis units of the affiliated hospitals of National Yang-Ming University, between July 1999 and June 2000, were investigated. All patients were >20 yr of age and had been maintained on hemodialysis protocols for >3 mo before the study. The mean age of the patients (112 men and 98 women) was 61 ± 15 yr. The primary diagnoses associated with end-stage renal disease were diabetic nephropathy (n = 46), glomerulonephritis (n = 39), interstitial nephritis (n = 26), nephrosclerosis (n = 24), polycystic kidney disease (n = 18), miscellaneous nephropathies (n = 21), and shrunken kidney resulting from unknown causes (n = 36). All patients underwent standard bicarbonate dialysis sessions. Hemodialysis was performed three times each week, for 12 to 12.5 h/wk, using single-use dialyzers with a membrane surface area of 1.6 to 1.7 m2. The mean duration of hemodialysis treatment was 46 ± 43 mo. hOGG1 1245C->G genotyping was also performed for 156 healthy individuals (84 men and 72 women; mean age, 59 ± 16 yr) with normal renal function, as defined on the basis of creatinine clearance values of >100 ml/min. These subjects were recruited from among hospital staff members, university students, or volunteers receiving health check-ups. The protocol was approved by the Committee on Human Research of Taipei Veterans General Hospital. Informed consent was obtained from each of the study subjects.

For determination of whether the 8-OHdG contents of leukocyte DNA were influenced by the 1245C->G polymorphism in the hOGG1 gene, the hOGG1 genotype, leukocyte 8-OHdG contents, blood antioxidant levels, and iron status were analyzed for hemodialysis patients. Patients in this case-control study had no malignancies, no inflammatory or infectious diseases, no habit of tobacco smoking, no supplementation with vitamin C or E, and no use of medications such as oral or intravenous iron supplements, angiotensin-converting enzyme inhibitors, or anti-inflammatory drugs for 3 mo before enrollment. The study group consisted of 122 patients (57 men and 65 women; mean age, 60 ± 14 yr), who had undergone dialysis with the same type of hemodialysis membrane for >3 mo. The patients were treated with single-use dialyzers equipped with one of four membranes, i.e., cellulose (Terumo, Tokyo, Japan) for 44 patients, PS (Fresenius, Borkenberg, Germany) for 21 patients, PMMA (Toray, Tokyo, Japan) for 31 patients, and high-flux, vitamin E-modified, multilayer cellulose (Terumo) for 26 patients. Dialysis machines were sterilized daily, and water treatment circuits and tanks were sterilized weekly. Microorganism colony counts in the water used to prepare dialysis fluid did not exceed 200 colonies/ml (20). Endotoxin levels in dialysates, as assessed weekly using the amebocyte lysate test (Chromogenix, Charleston, SC), were <0.01 EU/ml. One hundred twenty-two age-, gender-, and hOGG1 genotype-matched subjects (57 men and 65 women; mean age, 61 ± 14 yr) from among the 156 healthy individuals served as control subjects.

PCR and Restriction Fragment Length Polymorphism Analyses of the hOGG1 Gene
To study the C->G transition at nucleotide 1245 of the hOGG1 gene, cellular DNA isolated from 1 ml of peripheral blood from the patients and healthy individuals was analyzed by PCR. An aliquot of 100 ng of genomic DNA was added to a 50-µl PCR mixture containing 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 0.1% Triton X-100, 2 mM MgCl2, 0.2 mM levels of each dNTP, 100 pmol of each primer, and 1 U of Taq DNA polymerase (Biotools; B & M Labs, Madrid, Spain). PCR was performed using the primers described by Kohno et al. (18), i.e., 5'-AGGGGAAGGTGCTTGGGGGAA-3' as the forward primer and 5'-ACTGTCACTAGTCTCACCAG-3' as the reverse primer. The thermoprofile consisted of 30 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 40 s, preceded by an initial denaturation step at 94°C for 2 min and followed by a terminal extension at 72°C for 5 min. Identification of the 1245C->G transversion was performed by using restriction fragment length polymorphism analyses. In brief, 10 µl of the 200-bp PCR product was subjected to Fnu4HI digestion (2.5 U of enzyme in a 15-µl digest). The presence of a C->G transversion creates a Fnu4HI recognition site, which leads to digestion of the 200-bp PCR product into two fragments of 100 bp. Heterozygous subjects exhibit two fragments (200 and 100 bp), and a homozygous C->G transversion results in the appearance of a single fragment of 100 bp. Fnu4HI digests of PCR amplification products were observed by electrophoresis on 3% agarose gels, followed by ethidium bromide staining.

Measurements of 8-OHdG Contents in Leukocyte DNA
Venous blood samples were drawn from fasting healthy individuals or from fasting hemodialysis patients at the start of a dialysis session, before heparin administration. Blood (10 ml) was withdrawn into an ethylenediaminetetraacetate (EDTA)-containing Vacutainer tube (Becton Dickinson, Franklin Lakes, NJ) and centrifuged in the same tube at 1300 x g at 4°C for 15 min. The buffy coat fraction was collected and transferred to a 20-ml centrifuge tube on ice. Hypotonic saline solution was added to lyse residual red blood cells. Leukocytes were collected by centrifugation at 500 x g for 5 min and were frozen at -80°C until determination of the 8-OHdG content in the DNA. Total leukocyte DNA was extracted by using the pronase/ethanol method (21), with some modifications. Briefly, nuclear fractions were obtained by centrifugation at 1000 x g for 10 min, after gentle homogenization of leukocytes in 10 ml of 5 mM Tris-HCl buffer (pH 7.6) containing 1% Triton X-100, 320 mM sucrose, and 10 mM MgCl2. The nuclear fraction was vigorously resuspended in 700 µl of SSC (5 mM sodium citrate, 20 mM sodium chloride, pH 6.5). After addition of 200 µl of pronase E (20 mg/ml in SSC), 800 µl of sarcosyl (1.5% in 20 mM EDTA, 20 mM Tris-HCl, pH 8.5), and 100 µl of 5% butylated hydroxytoluene in methanol, the mixture was incubated for 6 h at 45°C. After incubation and after the addition of 800 µl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) and 200 µl of 7.5 M ammonium acetate, cooled ethanol (-20°C) was carefully added up to 70%, with mixing. Precipitated DNA was stored overnight in 95% ethanol containing 0.01% butylated hydroxytoluene. The amount of 8-OHdG was measured by using a HPLC system equipped with an electrochemical detector (Bioanalytical Systems, West Lafayette, IN), as described previously (8,9,22). Deoxyguanosine (dG) (Sigma Chemical Co., St. Louis, MO) and 8-OHdG (Cayman, Ann Arbor, MI) were used as standards. The 8-OHdG levels are expressed as the number of 8-OHdG molecules/106 dG molecules. Intra-assay and interassay coefficients of variance (CV) for the assays were determined by repeated analyses (n = 10) of low (normal) and high (uremic) sample pools. Intra-assay CV ranged from 4 to 10%, and interassay CV ranged from 6 to 12% for leukocyte 8-OHdG contents, with the lower number referring to the CV for the high standard and the higher number referring to the CV for the low standard.

Laboratory Measurements
Immediately after sampling, whole blood (0.5 ml) was deproteinized with an equal volume of 20% TCA, for determination of the reduced glutathione (GSH) level. GSH was quantified as described by Beutler et al. (23). For GSH derivatization, 0.5 ml of whole blood was treated with an equal volume of 12% perchloric acid containing 40 mM N-ethylmaleimide and 2 mM bathophenanthroline disulfonic acid. Oxidized glutathione (GSSG) levels in the derivatized glutathione samples were determined by using a HPLC system similar to that developed by Asensi et al. (24). Serum iron concentrations were determined by using commercial kits, with an autoanalyzer (Hitachi 736-60; Naka, Japan). Total iron-binding capacities were measured by using the TIBC Microtest (Daiichi, Tokyo, Japan), and serum ferritin concentrations were determined by using a RIA (Incstar, Stillwater, MN). Transferrin saturation was calculated as the serum iron concentration/total iron-binding capacity x 100. Plasma ascorbate levels were measured by using a method described by Kyaw (25). Plasma concentrations of {alpha}-tocopherol were determined by using the procedure described by Catignani and Bieri (26), with some modifications. A 50-µl aliquot of internal standard (52.5 mg/L {alpha}-tocopherol acetate in ethanol) and 100 µl of plasma were vortex-mixed for 1 min. For lipid extraction, 200 µl of HPLC-grade hexane was added. After thorough mixing for 1 min, the mixture was separated by centrifugation at 800 x g for 2 min. The hexane layer was withdrawn and evaporated by flushing with nitrogen gas. The residue was redissolved in 50 µl of filtered HPLC-grade methanol. A 20-µl aliquot of each sample was then injected onto a µBondapak C18 column (3.9 x 300 mm; Kanto Chemical Co., Tokyo, Japan) and eluted with a mobile phase of 100% HPLC-grade methanol, at a flow rate of 1.2 ml/min, at room temperature. The detector wavelength was 290 nm, and the concentration of {alpha}-tocopherol was calculated from a calibration curve constructed by using internal standards. Total blood lipid levels were also measured, for adjustment of the {alpha}-tocopherol values. All assays were performed with duplicate samples.

Statistical Analyses
Comparisons of the genotypes and allelic frequencies at nucleotide position 1245 of the hOGG1 gene for hemodialysis patients and healthy individuals were performed by using the {chi}2 test. Potential differences in age, gender, dialysis duration, primary kidney disease, recombinant erythropoietin dose, blood antioxidant levels (ascorbate concentration, {alpha}-tocopherol concentration adjusted for total lipid level, and whole-blood GSH and GSSG concentrations), and iron metabolism indices (serum ferritin concentration, iron concentration, and transferrin saturation) among the three patient groups, according to the hOGG1 1245C->G genotype, were assessed by using ANOVA. Separate comparisons of 8-OHdG contents in leukocyte DNA among the subjects with hOGG1 1245CC, CG, and GG genotypes were conducted by using ANOVA followed by post hoc analyses (Scheffé's test). Comparisons of data for hemodialysis patients and normal control subjects were performed by using the t test or Mann-Whitney U test. Descriptive statistical values included mean ± SD values for continuous data and percentages for categorical data. Because 8-OHdG contents in leukocyte DNA and serum ferritin concentrations were positively skewed, natural logarithmic transformation was used to normalize the distributions for ANOVA, univariate analyses, and multivariate analyses. The relationships between leukocyte 8-OHdG contents and the potentially explanatory variables were analyzed by using Pearson correlation analyses. Forward, stepwise, multiple-regression analysis was performed using the 8-OHdG contents of leukocyte DNA as the dependent variable. The independent effect of each explanatory variable on the dependent variable was assessed with two indicators of the hOGG1 1245C->G genotype and dialyzer membrane type for hemodialysis patients. The 1245CC genotype, a reference category, was equal to 0, corresponding to the two dummy variables (1245CG and 1245GG genotypes), and the cellulose membrane was equal to 0, corresponding to the three dummy variables (PMMA, PS, and vitamin E-bonded membranes). Those dummy variables were forced into the regression equation before testing of other variables and could not be removed. An explanatory variable was considered to have an independent effect on leukocyte 8-OHdG levels if it led to a statistically significant change in r2 values. For the patient and control groups, multivariate regression analyses were also performed to assess the independent effect of the hOGG1 1245C->G genotype, with the covariables of age, gender, group (hemodialysis patients versus control subjects), blood antioxidant levels, and iron indices, on leukocyte 8-OHdG contents (dependent variable). Statistical analyses were performed by using Statistical Package of Social Science computer software (SPSS version 8.0; SPSS Inc., Chicago, IL). A P value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and MethodsStudy...
 Results
Discussion
 References
 
hOGG1 Gene Polymorphism
The allelic frequencies of hOGG1 1245G were 64.1% for 210 hemodialysis patients and 62.2% for healthy individuals (Table 1). In both groups, the distribution of the polymorphic alleles was consistent with a Hardy-Weinberg equilibrium. The distribution of the three genotypes, namely CC, CG, and GG, and the frequencies of each allele were not significantly different between the two groups (P > 0.05, {chi}2 test). Furthermore, the hOGG1 genotypic frequencies (CC/CG/GG ratio, 13.9%/44.3%/41.8%) for the 122 hemodialysis patients for whom 8-OHdG levels of leukocyte DNA had been analyzed did not vary significantly from those (10%/51.9%/38.1%) for the whole study population of 210 chronic hemodialysis patients (P > 0.05, {chi}2 test). The genotypic distribution of this polymorphism according to the use of dialysis membranes is indicated in Table 2. Similarly, there were no differences in the genotypic frequencies with respect to the dialysis membrane type for our patients. This finding indicates that there was no selection pressure for hOGG1 alleles among the hemodialysis patients.


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  		Table 1. Genotypes and allelic frequencies of the hOGG1 1245C->G polymorphism among chronic hemodialysis patients and healthy individuals
 

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  		Table 2. 8-OHdG content/106 dG for leukocyte DNA from healthy control subjects, total hemodialysis patients, and hemodialysis patients treated with four different dialysis membranes, stratified according to the hOGG1 1245C->G polymorphisma
 

Effects of hOGG1 1245C->G Gene Polymorphism on Leukocyte 8-OHdG Levels
Compared with the age-, gender-, and genotype-matched healthy control subjects, the 122 hemodialysis patients exhibited impaired antioxidant defense and increased iron storage and transferrin saturation (plasma ascorbate concentrations, 13.2 ± 7.2 versus 17.2 ± 4.3 mg/L; {alpha}-tocopherol concentrations adjusted for blood lipid levels, 4.1 ± 1.7 versus 4.9 ± 1.3 mg/g; whole-blood GSH concentrations, 713 ± 194 versus 1038 ± 208 µM; whole-blood GSSG concentrations, 98 ± 37 versus 79 ± 15 µM; ferritin concentrations, 704 ± 607 versus 80 ± 43 µg/L; serum iron concentrations, 100 ± 55 versus 77 ± 31 µg/dl; transferrin saturation, 40 ± 17 versus 30 ± 11%; P < 0.001). Twelve (10%), 54 (44%), 26 (21%), and 30 (25%) patients exhibited serum ferritin values of <100 µg/L, 101 to 500 µg/L, 501 to 800 µg/L, and >800 µg/L, respectively. High ferritin levels (>500 µg/L) were attributed to the effects of previous intravenous administration of large amounts of iron (n = 39), multiple previous blood transfusions (n = 12), and hereditary hemochromatosis (n = 5). Furthermore, we observed that the three groups of patients with different hOGG1 genotypes were similar with respect to age distribution, gender distribution, percentage of patients with diabetes mellitus, dialysis duration, dose of recombinant human erythropoietin administered, composition of the leukocyte fraction, blood antioxidant levels, and iron status (P > 0.05) (Table 3).


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  		Table 3. Clinical characteristics, recombinant erythropoietin doses, blood antioxidant levels, and iron metabolism indices for the three hemodialysis patient groups, according to the hOGG1 1245C->G polymorphisma
 

The mean leukocyte 8-OHdG levels were 20 ± 11.2/106 dG for hemodialysis patients versus 7.8 ± 2.6/106 dG for control subjects (P < 0.001). The mean 8-OHdG levels were 24.6/106 dG for patients with the 1245GG genotype versus 8.7/106 dG for control subjects (P < 0.001), 17.9/106 dG for patients with the 1245CG genotype versus 7.4/106 dG for control subjects (P < 0.001), and 13.2/106 dG for patients with the 1245CC genotype versus 6.8/106 dG for control subjects (P < 0.05) (Table 2). Post hoc analyses revealed that the leukocyte 8-OHdG contents for 1245GG patients were significantly greater than those for patients with the 1245CG (P < 0.05) and 1245CC (P < 0.05) genotypes. The 8-OHdG levels of leukocyte DNA were slightly but not significantly different among the healthy subjects with different hOGG1 gene polymorphisms.

Hemodialysis patients were further stratified into four subgroups on the basis of the type of dialysis membrane used, i.e., cellulose, PMMA, PS, or vitamin E-bonded cellulose membranes (Table 2). The mean leukocyte 8-OHdG level for the patients who underwent dialysis with cellulose membranes was significantly higher than that for the patients who underwent dialysis with PMMA, PS, or vitamin E-bonded membranes (P < 0.05). Post hoc analyses demonstrated that there was a significant difference in leukocyte 8-OHdG contents between patients with the 1245GG genotype and patients with the 1245CG (P < 0.05) or 1245CC (P < 0.05) genotype treated using cellulose membranes. Although there was a trend toward increased 8-OHdG levels in the presence of the hOGG1 1245G allele, this result was not statistically significant for the patients treated using PMMA, PS, or vitamin E-bonded membranes (P > 0.05, ANOVA). We further observed that only the patient with the 1245GG genotype who was treated using cellulose membranes exhibited higher leukocyte 8-OHdG levels than did the patients who were treated using the other three membrane types (P < 0.05). A trend toward increased 8-OHdG levels in the cellulose membrane group was not statistically significant for patients with the 1245CG genotype or patients with the 1245CC genotype (P > 0.05, ANOVA).

Predictors of Leukocyte 8-OHdG Levels
Univariate analyses revealed that the 8-OHdG contents in leukocyte DNA from hemodialysis patients were significantly correlated with the hOGG1 genotype (1245GG, yes/no term) and the type of dialysis membrane (cellulose, yes/no term) (P < 0.001) (Table 4). Forward, stepwise, multiple-regression analysis confirmed the distinct influence of the hOGG1 gene polymorphism and the dialysis membrane type on leukocyte 8-OHdG levels of hemodialysis patients, with simultaneous adjustment for clinically significant variables (r = 0.616, P < 0.001) (Table 5). Compared with the 1245CC genotype, the 1245GG genotype resulted in an estimated increase in leukocyte 8-OHdG levels of 81% and the 1245CG genotype resulted in an increase in leukocyte 8-OHdG levels of as much as 37%. The PMMA, PS, and vitamin E-bonded membranes were observed to be associated with decreases in leukocyte 8-OHdG levels of 34, 21, and 32%, respectively, compared with the cellulose membranes.


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  		Table 4. Univariate analysis of the relationships between 8-OHdG contents in leukocyte DNA and potentially explanatory variables
 

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  		Table 5. Stepwise, multiple-regression analysis of major determinants of 8-OHdG levels of leukocyte DNA from hemodialysis patients (n = 122) and analysis of data for hemodialysis patients and control subjects (n = 244)a
 

In overall analyses of the patients and control subjects, the 8-OHdG contents in leukocyte DNA were strongly correlated with the hOGG1 genotype (1245GG, yes/no term), the group factor (hemodialysis, yes/no term), plasma {alpha}-tocopherol concentrations adjusted for blood lipid levels, whole-blood GSH and GSSG concentrations, serum iron levels, and percent saturation of transferrin (P < 0.001) (Table 4). In multivariate regression analysis of leukocyte 8-OHdG levels, the variables of hOGG1 gene polymorphism and hemodialysis treatment entered into the model. This model accounts for approximately 45% of the variation in 8-OHdG contents in leukocyte DNA for all subjects (r = 0.672, P < 0.001) (Table 5). This indicates again that the hOGG1 gene polymorphism is a strong independent predictor of leukocyte 8-OHdG levels. For further elucidation of the pathogenetic relationship between hemodialysis and leukocyte DNA damage, variables of hemodialysis treatment were not included in the stepwise, multiple-regression analysis. In addition to the hOGG1 genotype, the following variables were observed to be independent predictors of leukocyte 8-OHdG levels, after adjustment for the other variables (r = 0.724, P < 0.001): serum iron levels, plasma {alpha}-tocopherol concentrations adjusted for blood lipid levels, whole-blood GSH concentrations, iron saturation, and ferritin concentrations (Table 5). These findings confirmed the results of our previous work; oxidative damage to leukocyte DNA becomes greater with higher serum iron concentrations and lower blood lipid-adjusted {alpha}-tocopherol and GSH concentrations.


   Discussion
Top
 Abstract
 Introduction
 Materials and MethodsStudy...
 Results
 Discussion
References
 
This study demonstrates that the distribution of the hOGG1 1245C->G polymorphism among hemodialysis patients is almost identical to that in the control population. The genotypic frequency of this polymorphism also exhibits the same behavior within the four subgroups of patients treated using different dialysis membranes (Table 2). Because there was no significant difference in the duration of previous dialysis among the three genotype groups, the hOGG1 polymorphism seems not to be related to hemodialysis treatment among our patients. Moreover, the allelic frequencies of hOGG1 1245G are higher in the Chinese population, among both healthy individuals (62.2%) and hemodialysis patients (64.1%), than in the Japanese (40.5 to 43.3%), European (40%), and Caucasian (24 to 26.5%) populations (18,19,27), suggesting ethnic variations.

The most compelling observation of this study is that the extent of the increase in 8-OHdG levels in leukocyte DNA from hemodialysis patients not only is affected by complement-activating membranes and impaired antioxidant defenses (8,9) but also is genetically determined. We demonstrated that leukocyte 8-OHdG levels were significantly increased among hemodialysis patients with the hOGG1 1245GG genotype, compared with subjects with the 1245CG or 1245CC genotype. The observed difference cannot be accounted for by differences in age, gender, dialysis duration, blood antioxidant levels, or serum ferritin or iron concentrations (Table 3). Moreover, stepwise multivariate regression analysis validated the independent effect of the hOGG1 1245C->G gene polymorphism on 8-OHdG contents in peripheral leukocyte DNA. Recently, the 1245C->G transversion in hOGG1, which changes a serine into a cysteine residue at amino acid position 326, was demonstrated to be associated with reduced enzymatic activity of 8-OHdG DNA repair (18). Dherin et al. (28) demonstrated that these two enzymes, when fused to the glutathione S-transferase protein, were functional and excised 8-OHdG from damaged DNA. However, the Kcat/Km values for excision of 8-OHdG from damaged DNA differed significantly (by twofold), with the wild-type hOGG1-Ser326 protein being more active than the mutant hOGG1-Cys326 protein. Furthermore, tumors that had lost one allele of the hOGG1 gene were observed to contain twice as much 8-OHdG in their DNA (29). Our results corroborate the findings of these studies (18,28,29), in which the impairment of mutant alleles of the hOGG1 gene was demonstrated. These observations suggest a pivotal role for hOGG1 proteins in maintaining the stability of the genetic system.

Oxidative damage is a consequence of excessive oxidative stress, insufficient antioxidant protection, or both. The levels of 8-OHdG measured in leukocytes at any time represent the integration of a number of parameters, including ROS production, cellular redox status, and antioxidant defense mechanisms, as well as DNA repair systems. The effectiveness of DNA repair may be subject to modulation by gene polymorphism and gene dosage effects. Leukocyte 8-OHdG levels are modestly but not significantly different among healthy subjects, irrespective of the type of hOGG1 proteins expressed. Kohno et al. (18) also reported that mean 8-OHdG levels were similar in peripheral leukocytes expressing either hOGG1-Ser326 or hOGG1-Cys326 protein. The use of 8-OHdG as a dosimeter for oxidative stress is the subject of debate, in light of the susceptibility to artifactual oxidation during DNA isolation and hydrolysis, as well as in the analytical stages of HPLC with electrochemical detection (30,31,32). However, we used rigorous procedures to avoid the use of phenol and other organic solvents in DNA isolation, to dry the DNA samples under a stream of nitrogen, not to expose the samples to the air or other oxygen-containing gases, and to use an appropriate amount of DNA (>20 µg) for analysis (30,31). Low sample-to-sample variance lends additional credibility to our measurements of 8-OHdG contents in leukocyte DNA. Moreover, the leukocyte 8-OHdG contents measured for our healthy subjects were lower than those observed by other investigators (14,33). Therefore, this study indicates that the intracellular activity of hOGG1 proteins may be high enough to maintain 8-OHdG contents at a steady level in nuclear DNA of healthy subjects, under conditions without severe oxidative stress and impaired antioxidant defenses.

In contrast, because of the imbalance between ROS production and antioxidant defense mechanisms, hemodialysis imposes an additional oxidative stress on patients with end-stage renal failure. Blood-membrane interactions during hemodialysis trigger circulating neutrophils to produce large amounts of ROS, including superoxide anion and hydrogen peroxide. In this situation, with accompanying dialytic loss of low-molecular weight antioxidants, the ROS-scavenging system of blood is overwhelmed, as indicated by decreased plasma levels of vitamin C, vitamin E, and GSH among patients undergoing hemodialysis, compared with healthy control subjects (4, 34,35,36). Moreover, iron stores are increased, mainly because of the intravenous administration of large amounts of iron to reduce recombinant erythropoietin doses. Iron is a transition metal, and its ionic forms are prone to participate in one-electron transfer reactions. This capacity also enables iron to generate free radicals. Under conditions of increased hydrogen peroxide production by activated phagocytes in the presence of Fe2+, very reactive hydroxyl radicals may be formed via the Fenton reaction. This can potentiate oxidative damage to DNA. The production of ROS is further increased during dialysis with cellulose membranes, compared with biocompatible membranes such as PMMA, PS, and vitamin E-bonded membranes (9, 37,38,39). Therefore, this study indicates significant increases in 8-OHdG contents of leukocyte DNA in hemodialysis patients, augmented by complement-activating cellulose membranes. In addition to oxidant-induced DNA damage, DNA repair ability has been reported to be impaired in non-dialysis-treated chronic renal failure (40) and is suppressed during long-term hemodialysis (41). Therefore, increased oxidation, with decreased repair activity, enhances oxidative DNA damage. This is supported by our findings that patients with the hOGG1 1245GG genotype who were undergoing hemodialysis with cellulose membranes exhibited the highest leukocyte 8-OHdG levels, compared with patients treated using other dialysis membranes and those with other hOGG1 genotypes (Table 2).

8-OHdG has been demonstrated to be a mutagen (2,42). An accumulation of 8-OHdG in DNA could increase the risk of DNA mutations and cancer development. Respiratory tract cancers are well known to be related to cigarette smoking and asbestosis. Investigators proposed that tobacco smoking and asbestos exposure could induce 8-OHdG formation in peripheral leukocyte DNA (43,44). Intriguingly, the prevalence of malignancies among chronic hemodialysis patients is higher than that in the general population (45). Among the pathogenetic factors, increased DNA damage is of importance for malignant transformation and cancer formation (46). However, to date, there has been no convincing evidence to demonstrate a cause-effect relationship between oxidative DNA damage and malignancies among hemodialysis patients. Further studies are needed to elucidate the pathologic significance of increased leukocyte 8-OHdG levels among these patients, with respect to cancer development. Increased 8-OHdG accumulation in DNA represents an integration of ROS production, antioxidant defense mechanisms, and DNA repair systems. Among these factors, genetic elements (e.g., hOGG1 1245C->G polymorphism) are permanent and irreversible, whereas other contributors (e.g., ROS overproduction associated with complement-activating membranes and decreased plasma levels of vitamins C and E) are reversible and remediable for hemodialysis patients. Because leukocyte 8-OHdG is a hallmark of oxidative stress among hemodialysis patients, it is important to attenuate ROS-induced DNA damage, to ameliorate its effects and to prevent complications that are closely related to the oxidative damage elicited by ROS (1,2,3). Therefore, for patients with hOGG1 1245GG and 1245CG genotypes, the following treatment options may be considered: dialysis with more biocompatible and bioactive membranes (8,9), supplementation with ascorbate and vitamin E (33,47), and avoidance of excess iron attributable to intravenous iron therapy (47).


   Acknowledgments
 
This study was supported by grants from the National Science Council (Grants NSC 89-2320-B010-134, NSC 89-2320-B010-146, and NSC 89-2316-B010-013) and Taipei Veterans General Hospital (Grant VGH 90-440-11). We are extremely grateful to Drs. Nien-Yung Hsiao and Hung-Hsiang Liou and to Bao-Ju Liao for kind help in the collection of samples at their hemodialysis facilities. We are also deeply indebted to P. C. Lee for expert secretarial assistance.


   References
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 Abstract
 Introduction
 Materials and MethodsStudy...
 Results
 Discussion
 References
 

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Received for publication January 23, 2001. Accepted for publication May 9, 2001.




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