Reduced constitutive 8-oxoguanine-DNA glycosylase expression and impaired induction following oxidative DNA damage in the tuberin deficient Eker rat

Samy L. Habib1, Minh N. Phan1, Sonal K. Patel1, Donghui Li2, Terrence J. Monks1 and Serrine S. Lau1,3

1 Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712-1074 and
2 Department of Gastrointestinal Medical Oncology, The University of Texas M.D.Anderson Cancer Center, Houston, TX 77030, USA


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The Tsc-2 tumor suppressor gene encodes the protein tuberin, a multi-functional protein with sequence homology to the GTPase activating protein (GAP) for Rap1. Mutations in the Tsc-2 gene are associated with the development of renal tumors. The Eker rat (Tsc-2EK/+) bears a mutation in one allele of the Tsc-2 gene, which predisposes these animals to renal cancer. Treatment of wild-type (Tsc-2+/+) and mutant (Tsc-2EK/+) Eker rats with 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ; 7.5 µmol/kg. i.v.), a potent redox active and nephrotoxic metabolite of hydroquinone increases the incidence of renal tumors only in animals carrying the mutant Tsc-2EK/+ allele. We now show that the constitutive expression of 8-oxoguanine-DNA glycosylase (OGG1) in Tsc-2EK/+ rats is three-fold lower than in wild-type Tsc-2+/+ rats. Moreover, treatment of wild-type and mutant Eker rats with TGHQ greatly increases 8-oxo-deoxyguanosine (8-oxo-dG) levels within the outer stripe of the outer medulla. Tsc-2EK/+ rats, with lower constitutive renal OGG1 expression, experience substantially higher levels of 8-oxo-dG than do wild type Tsc-2+/+ rats. Interestingly, whereas OGG1 expression was rapidly (4 h) induced in Tsc-2+/+ rats following exposure to TGHQ, it was significantly reduced in Tsc-2EK/+ rats. The combination of the higher constitutive expression of OGG1 in Tsc-2+/+ rats, and its rapid induction in response to TGHQ treatment, coupled to the initial decrease in OGG1 expression in Tsc-2EK/+ rats, results in Tsc-2EK/+ OGG1 protein levels just 5% of those seen in Tsc-2+/+ rats 8 h after treatment. Coincidentally, 8-oxo-dG levels in Tsc-2+/+ rats 8 h after treatment with TGHQ are just 5% of those that occur in Tsc-2EK/+ rats. The results indicate that the Tsc-2 gene influences constitutive OGG1 expression and the ability of OGG1 to respond to an oxidative stress, consistent with the proposal that Tsc-2 is an acute-phase response gene. In keeping with this view, acute TGHQ-induced cytotoxicity was greater in Tsc-2EK/+ rats than in Tsc-2+/+ rats. The mechanism(s) coupling tuberin expression to the regulation of OGG1 are not known and are under investigation.

Abbreviations: FITC, fluorescein isothiocynates; {gamma}-GT, {gamma}-glutamyl transpeptidase; GST, glutathione-S-transferase; LOH, loss of heterozygosity; OGG1, 8-oxoguanine-DNA glycosylase; OSOM, outer stripe of the outer medulla; 8-oxo-dG, 8-oxo-deoxyguanine; PI, propidium iodide; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; ROS, reactive oxygen species; TBS, tris-buffered saline; TBS-T, tris-buffered saline containing 0.1% Tween 20; TGHQ, 2,3,5-tris-(glutathion-S-yl)hydroquinone; Tsc-2, tuberous sclerosis-2; Tsc-2EK/+, mutant Eker carrier rats; Tsc-2+/+, wild-type Eker non-carrier rats.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
The Tsc-2 tumor suppressor gene encodes the protein tuberin (1). Tuberin appears to be a multi-functional protein; it was first identified as having sequence homology to a portion of the catalytic GTPase activating protein (GAP) for Rap1 (1). Rap 1 is a member of the Ras superfamily of small GTP-binding proteins, which can couple mitogenic signaling from the cell surface to the nucleus (24) via the b-Raf/MEK/MAPK cascade. Rap1GAP is a negative regulator of Rap1 activity (5), and tuberin/Rap1 may participate in the regulation of the G0G1/S phase transition by regulating the activity of the cyclin-dependent kinases (CDKs) Cdk2 or Cdk4 (6). Interestingly, tuberin coimmunoprecipitates with CDK1, cyclin B and cyclin A (7) suggesting that its cell cycle regulatory effects are more complex than first thought. Rap1 is also required for the cAMP-mediated inhibition of Ras-dependent extracellular-regulated kinase (ERK) activation (8). Transfection of Tsc-2 cDNA into tuberin-negative renal epithelial cells suppresses ERK activity (9) and cyclin D1 is over-expressed in the same tuberin-negative renal cells (10). In addition, tuberin modulates steroid hormone receptor-mediated transcription, suggesting that tuberin behaves as a nuclear receptor coactivator (11). Tuberin also colocalizes with Rap1 in the Golgi (12), and exhibits GAP activity toward Rab5 (13), another small GTPase involved in vesicular trafficking, implicating a role for tuberin in endocytosis. Finally, tuberin also associates with harmartin, the product of the Tsc-1 gene, to form a stable complex (14). This interaction is regulated by the reversible phosphorylation of tuberin on serine and tyrosine residues (15). Disease causing mutations that can alter the phosphorylation status of tuberin, hinder its ability to interact with harmartin (15).

Mutations in the Tsc-2 gene are associated with the development of renal tumors (1618). The Eker rat (Tsc-2EK/+) is a derivative of the Long–Evans strain, bearing a mutation in one allele of the Tsc-2 gene, which predisposes these animals to renal cancer (1620). A germline insertion of an endogenous retrovirus in Tsc-2 gene is responsible for the predisposing Eker mutation (21,22). Treatment of wild-type (Tsc-2+/+) and mutant (Tsc-2EK/+) Eker rats with 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ), a potent nephrotoxic metabolite of hydroquinone (23), induced preneoplastic lesions, including toxic tubular dysplasias, and increased the incidence of renal tumors only in animals carrying the mutant Tsc-2EK/+ allele (24). Loss of heterozygosity (LOH) at the Tsc-2 locus occurred in the tumors and the toxic tubular dysplasias, consistent with TGHQ-induced loss of tumor suppressor function of the Tsc-2 gene (24). Moreover, TGHQ increased cell proliferation in the kidney of both Tsc-2EK/+ and Tsc-2+/+ rats, indicating that cell proliferation per se was not sufficient for tumor development (9). Interestingly, tuberin expression was initially induced following acute renal injury, suggesting that the Tsc-2 gene may function as an acute-phase response gene, limiting the proliferative response after injury (9).

TGHQ-induced mutations are consistent with the generation of reactive oxygen species (ROS) (25), and TGHQ is capable of transforming primary rat kidney epithelial cells derived from Eker Tsc-2EK+ rats (26). Moreover, the cytotoxicity of TGHQ in renal proximal tubule epithelial cells is dependent upon the generation of ROS (27). ROS-induced mutations may activate oncogenes, or inactivate tumor suppressor genes, altering the control of cell growth (28) and several ROS-induced base modifications are promutagenic (2933). 8-Oxo-deoxyguanine (8-oxo-dG) is a quantitatively major form of oxidative DNA damage (34,35), inducing mainly G to T and A to C substitutions (36). 8-Oxo-dG in DNA is repaired primarily via the DNA base excision repair pathway. The gene coding for the DNA repair enzyme that recognizes and excises 8-oxo-dG is 8-oxoG-DNA glycosylase (OGG1) (37). Mice lacking functional OGG1 protein accumulate abnormal levels of 8-oxo-dG in their genomes and display a moderately elevated spontaneous mutation rate in nonproliferative tissues (38). LOH at the OGG1 allele, located on chromosome 3p25, was found in 85% of 99 human kidney clear cell carcinoma samples (39). Cytogenetic abnormalities and LOH at human chromosome 3p occur at high frequency in sporadic forms of renal cell carcinoma. (39). In the present study we therefore investigated whether differences exist in the generation and clearance of 8-oxo-dG in TGHQ-treated Tsc-2EK/+ and Tsc-2+/+ Eker rats, and whether such potential differences might contribute to the susceptibility of Tsc-2EK/+ rats to TGHQ-induced renal tumors.


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Chemicals
GSH, nuclease P1, 8-oxo-dG, and Salmon sperm DNA were purchased from Sigma Chemical Co. (St Louis, MO). RNase T1, RNase 1A and proteinase K were obtained from Boehringer-Mannheim (Indianapolis, IN). Phenol:chloroform:isoamyl alcohol (25:24:1) and buffer saturated phenol were obtained from Fisher Scientific (Pittsburgh, PA). Horseradish peroxidase conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Primary 8-oxo-dG monoclonal antibody and secondary goat anti-mouse IgG conjugated with fluorescein isothiocyanate were purchased from Pharmingen (San Diego, CA). TSATM Fluorescence systems kits were purchased from NEN Life Science Products (Boston, MA). Propidium iodide (PI) and normal horse serum were purchased from Vector Laboratories, (Burlingame, CA). Phase Lock GelTM Eppendorf tubes were purchased from Eppendorf Scientific (Westbury, NY). TGHQ was synthesized according to previously established methodology (40) and used at >98% purity, as determined by high performance liquid chromatography.

Animals and dosing
Two-month-old Eker male rats (wild-type, Tsc-2+/+ and mutant Tsc-2EK/+) were provided from a breeding colony maintained in house at the University of Texas MD Anderson Cancer Center, Smithville, TX. The animals were allowed food and water ad libitum prior to and during the experiments. Animals were treated with a single dose of TGHQ (7.5 mmol/kg in phosphate buffered saline (PBS), pH 7.4, i.v.). Control rats were administered PBS only. Animals were euthanized at 0, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h following TGHQ treatment. Kidneys were removed and dissected longitudinally. Half of the kidney was preserved in 10% formalin in PBS, 0.01 M, pH 7.4. The outer stripe of the outer medulla (OSOM) regions of the kidney were excised from the remaining kidney sections and frozen immediately in liquid nitrogen.

Histological assessment of toxicity
The half of the kidney preserved in formalin was embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin for histological examination by light microscopy.

Biochemical assessment of nephrotoxicity
Rats were individually housed in metabolism cages equipped for the collection of urine. Urine was collected from each rat over dry ice and ice, in the dark at 4, 8, 12, 24, 48 and 72 h following treatment with TGHQ. Urinary excretion of {gamma}-glutamyl transpeptidase activity ({gamma}-GT) was used as a marker of brush border membrane damage and determined as described in Sigma Technical Bulletin 545, using {gamma}-glutamyl-p-nitroanilide as a substrate. One unit of {gamma}-GT activity is defined as 1 µmol of p-nitroanilide formed per min at 37°C. Urinary excretion of glutathione-S-transferase (GST) was measured as an indicator of the loss of cell membrane integrity, and determined using dinitrochlorobenzene and glutathione (GSH) as substrate and co-substrate respectively (41). One unit of GST activity is defined as the formation of 1 µmol of dinitrochlorobenzene GSH conjugate per min at pH 6.5 and 25°C.

8-Oxo-dG assay using HPLC-EC
DNA was isolated from frozen samples of kidney tissue by the phenol extraction procedure with modifications (42). Approximately 20–30 mg of frozen kidney tissue was homogenized in 500 µl of 10 mM EDTA (pH 8.0). The homogenate was incubated with 15 µl RNase 1A (20 mg/ml in 10 mM Tris–HCl, pH 8.0) and 15 µl RNase T1 (3330 U/ml) in an Eppendorf tube for 1 h at 37°C. Subsequently, proteins were digested by adding 34 µl 20% SDS, 35 µl 1 M Tris (pH 7.5), and 6 µl proteinase K (50 mg/ml in 50 mM Tris containing 5 mM calcium acetate, pH 8.0), vortexing, and incubating for 1 h at 37°C. The incubation mixture was then transferred to an Eppendorf tube containing ‘Heavy’ Phase Lock GelTM. Tris–HCl saturated phenol (750 µl) was added and mixed by vortexing, then centrifuged. The aqueous phase was transferred to an Eppendorf tube and DNA was extracted with 300 µl of phenol/chloroform/isoamyl alcohol (25:24:1, v/v) and washed with 300 µl chloroform/isoamyl alcohol (24:1, v/v). DNA was precipitated with 0.14 M sodium acetate and 50% (v/v) isopropanol, washed once with 100 µl of 70% ethanol, and redissolved in 250 µl 1.0 mM EDTA. The RNase, proteinase K, and extraction steps were repeated once for greater purity. The DNA was collected by centrifugation, washed twice with 70% ethanol, and resuspended in 150 µl 1.0 mM EDTA, pH 8.0. DNA purity and concentration was determined spectrophotometrically. An OD of 1 at 260 nm corresponded to ~50 µg/ml DNA. Only samples with A260/230 above 2.20, indicating a high degree of DNA purity, were used.

Detection of dG and 8-oxo-dG was performed as follows: a mixture of 10 µg of DNA in 66 µl 2 mM sodium acetate buffer (pH 4.5) 10 µl 0.1 mM zinc chloride, and 4 µl nuclease P1 (0.5 µg/µl) were incubated for 2 h at 37°C in an Eppendorf tube. DNA digests were further incubated with 6 µl 80 mM Tris base and 4 µl alkaline phosphatase (0.1 unit/µl) for 1 h at 37°C. Aliquots (90 µl) of DNA hydrolysates were injected onto a Partisil 5 µm ODS-3 reverse-phase analytical column (25 cm x 4.6 mm inner diameter; Whatman, Clifton, NJ) maintained at 25°C. The mobile phase contained 4 mM citric acid, 8 mM ammonium acetate, 20 mg EDTA/l, and 5% methanol at pH 4.0. The flow rate was 1 ml/min. The eluate was monitored with a UV photodiode array (Shimadzy SPD M10A) and electrochemical (EC) detectors (ESA CoulArray). The latter system contained four coulometric detectors set at +50, +200, +290, and +380 mV. 8-Oxo-dG was eluted with a retention time of 15.0 min and was quantified by analysis of EC channel with +290 mV. Deoxycytidine, thymidine, deoxyguanosine and deoxyadenosine, which usually elute with retention times of 5.6, 9.9, 11.6, and 20.6 min, respectively, were quantified by ultraviolet absorption at 254 nm. Authentic standards of 8-oxo-dG and dG were analyzed along with every batch of the samples. Salmon sperm DNA (5–50 µg) was used as a positive control for DNA digestion reactions. Standard curves for dG and 8-oxo-dG were prepared and quantitation achieved by linear regression analyses. Data are expressed as 8-oxo-dG/105 x dG in 90 µl of DNA hydrolysate.

Immunohistochemical detection of 8-oxo-dG
A double fluorescent labeling method was used as previously described (43) with minor modifications. Kidney sections (5 µm) were deparaffinized with xylene and hydrated with serial ethanol and then boiled in a microwave oven for 3 min for antigen retrieval, followed by RNase and pepsin digestion. After washing with PBS, non-specific binding sites were blocked with normal horse serum. The primary monoclonal antibody against 8-oxo-dG (Pharmingen, San Diego, CA) was applied at a dilution of 1:1000, and tissue sections were then incubated at 4°C overnight. After washing with PBS, the sections were reacted with a goat anti-mouse IgG (1:200) conjugated with fluorescein isothiocyanates (FITC) at 37°C for 30 min. The slides were reacted with Vectashield Mounting Medium with propidium iodide (PI) (Vector Laboratories, Burlingame, CA). In this assay, DNA was labeled with PI, and 8-oxo-dG was identified by the primary monoclonal antibody and FITC-conjugated secondary antibody. FITC green signals for 8-oxo-dG were detected using a filter with excitation range 450–490 nm, and PI red signals to nuclear DNA were detected using a filter with excitation at 535 nm. To demonstrate staining specificity, control sections were stained without primary antibody.

Western blot analysis
The expression of OGG1 was evaluated within the OSOM of rat kidney. The OSOM was homogenized with lysis buffer [1X PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS containing phenylmethylsulfonyl fluoride, aprotinin, sodium orthovanadate and protease inhibitor tablet [(completeTM Mini, Boehringer-Mannheim) containing antipain dihydrochloride (50 mg/ml), bestatin (40 mg/ml), chymostatin (60 mg/ml), E-64 (10 mg/ml), leupeptin (0.5 mg/ml), pepstatin (0.7 mg/ml), phosphoramidon (300 mg/ml), pefabloc SC (1 mg/ml), EDTA disodium salt (0.5 mg/ml), and aprotinin (2 mg/ml)]. The tissue was centrifuged at 14 000 xg for 30 min at 4°C and protein concentrations were determined with the Bradford assay (44) using bovine serum albumin as a standard. Protein (50 or 100 µg) was subjected to 8% SDS–polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes at a constant voltage of 1000 V for 1 h. The PVDF membranes were blocked in 5% nonfat dried milk in TBS-0.1% tween buffer [25 mM Tris–HCl, 0.2 mM NaCl; 0.1% Tween 20 (v/v) pH 7.6] (TBS-T) overnight. The membrane was washed 2X with TBS- and then incubated with 1:1000 anti-OGG1 antibody, generously provided by Dr S.Mitra (45). After extensive washing with TBS-T buffer, anti-rabbit immunoglobulin conjugated with horseradish peroxidase was added at a 1:3000 dilution and incubated for 1 h at room temperature. An enhanced chemiluminescence kit (Amersham, NJ) was used for western blot to identify OGG1 expression. Expression of OGG1 was quantified by densitometry and normalized against ß-actin expression.

Statistics
All data expressed as mean ± SE. Multigroup studies were analyzed by ANOVA followed by Student Dunnett’s (Exp.vs.Control) test using 1 trial analysis.


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Comparative nephrotoxicity of TGHQ in Tsc-2EK/+ and Tsc-2+/+ rats
TGHQ-induced nephrotoxicity in Tsc-2EK/+ and Tsc-2+/+ rats was monitored by measuring the urinary excretion of the brush border membrane protein, {gamma}-GT, and the cytosolic protein, GST. Increases in urinary {gamma}-GT activity tended to precede elevations in urinary GST activity (Figure 1Go), consistent with the view that shedding of the brush border membrane into the tubular lumen precedes cell death. Excretion of {gamma}-GT reached maximum levels at 8 h in Tsc-2+/+ (16-fold) and Tsc-2EK/+ (19-fold) rats, but whereas levels progressively decreased thereafter in Tsc-2+/+ rats, high levels of {gamma}-GT excretion were sustained for at least 48 h in Tsc-2EK/+ rats. Total urinary {gamma}-GT excretion over the initial 48 h was ~8-fold higher in Tsc-2EK/+ rats compared with Tsc-2+/+ rats. Excretion of GST reached maximum levels (~4-fold) 12–24 h after TGHQ administration in Tsc-2+/+ rats, and at 24 h (15-fold) in Tsc-2EK/+ rats (Figure 1Go). The biochemical indices of nephrotoxicity suggest that animals carrying the mutant Tsc-2 allele are more susceptible to acute toxicity following administration of TGHQ, than are the wild-type animals.



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Fig. 1. Tsc-2EK/+ rats are more susceptible than Tsc-2+/+ rats to TGHQ-induced acute renal injury. Renal injury was determined by measuring the urinary excretion of {gamma}-GT (A) and GST (B) in Tsc-2+/+ and Tsc-2EK/+ rats following TGHQ administration (7.5 µmol/kg, i.v.). Values represent the mean ± SE (n = 4) and are expressed as a percentage of the corresponding PBS vehicle control. *Significantly different from control at P < 0.01. Control values for {gamma}-GT and GST were 6.3 ± 1.4 and 0.36 ± 0.06 units/time in Tsc-2+/+ rats and 1.34 ± 0.57 and 0.06 ± 0.01 units/time in Tsc-2EK/+ respectively.

 
Immunohistochemical fluorescence analysis of 8-oxo-dG formation in Tsc-2+/+ and Tsc-2EK/+ rats
The toxicity of TGHQ to renal epithelial cell cultures is dependent upon the generation of ROS (27), and 8-oxo-dG formation has been detected in vitro with DNA treated with TGHQ (46). We therefore developed an immunohistochemical assay to determine whether the location of 8-oxo-dG formation within the kidney corresponded to the sites of toxicity, and whether differences in 8-oxo-dG formation existed between Tsc-2+/+ and Tsc-2EK/+ rats. Staining of 8-oxo-dG confirmed the formation and localization of 8-oxo-dG in TGHQ-treated rats to proximal tubular epithelial cells within the S3 segment of the OSOM, the site of cell necrosis (Figure 2BGo). Consistent with the biochemical data (see below), immunohistochemical fluorescence staining for 8-oxo-dG was more intense in Tsc-2EK/+ rat kidneys than in kidneys from Tsc-2+/+ rats at 8 h (Figure 2CGo), a time at which no overt tubular epithelial cell necrosis is evident (Figure 2DGo). Moreover, 8-oxo-dG-positive cells were seen earlier, and persisted for a longer period of time in the Tsc-2EK/+ rats (Figure 3Go), than in Tsc-2+/+ rats (Figure 4Go) again consistent with the biochemical indices of toxicity (see below). Maximum numbers of 8-oxo-dG-positive cells were observed by 8 h in Tsc-2EK/+ rats and by 24 h in Tsc-2+/+ rats.



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Fig. 2. 8-Oxo-dG formation colocalizes with the region of TGHQ-induced renal injury. 8-Oxo-dG and tissue injury were visualized by IHC and H&E staining respectively in kidney sections from Tsc-2+/+ and Tsc-2EK/+ rats 8 h following TGHQ (7.5 µmol/kg, i.v.) administration. (A and B) represent 8-oxo-dG labeled with FITC-conjugated antibody in kidney sections obtained from untreated and TGHQ-treated rats respectively, C: cortex. O : OSOM, (bar = 500 µm). (C) represents overlay of 8-oxo-dG and DNA staining with PI (bar = 50 µm). (D) represents hematoxylin and eosin staining within the OSOM (bar = 50 µm).

 


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Fig. 3. Time course of 8-oxo-dG formation in Tsc-2EK/+ Eker rats following TGHQ treatment (7.5 µmol/kg, i.v.). (A) represents 8-oxo-dG labeled with FITC-conjugated antibody. (B) represents DNA labeled with PI. (C) represents overlay of 8-oxo-dG and DNA staining (bar = 100 µm).

 


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Fig. 4. Time course of 8-oxo-dG formation in Tsc-2+/+ Eker rats following TGHQ treatment (7.5 µmol/kg, i.v.). (A) represents 8-oxo-dG labeled with FITC-conjugated antibody. (B) represents DNA labeled with PI. (C) represents overlay of 8-oxo-dG and DNA staining (bar = 100 µm).

 
Relationship between renal OGG1 expression and 8-oxo-dG formation in Tsc-2+/+ and Tsc-2EK/+ rats
8-Oxo-dG formation was significantly higher within the OSOM than in the renal cortex of all animals treated with TGHQ (Figure 5Go). Significant increases in 8-oxo-dG were detected as early as 4 h in both Tsc-2EK/+ and Tsc-2+/+ rats (Figure 5Go). In Tsc-2EK/+ rats, levels of 8-oxo-dG in OSOM reached a maximum (47-fold) 8 h following TGHQ administration (Figure 5AGo), and corresponded with the maximum suppression of OGG1 protein expression (20% of untreated controls, see below). In contrast, 8-oxo-dG levels were substantially lower in Tsc-2+/+ rats at all time points examined (Figure 5Go) consistent with constitutively higher levels of OGG1 protein in these animals compared with the Tsc-2EK/+ rats (Figure 6AGo). Moreover, OGG1 protein levels were rapidly (4 h) induced in Tsc-2+/+ rats following exposure to TGHQ, and these elevated levels were sustained throughout the duration of the experiment (72 h) (Figure 6BGo). The combination of the higher constitutive expression of OGG1 in Tsc-2+/+ rats (Figure 6AGo), and its rapid induction in response to TGHQ treatment (Figure 6BGo), coupled to the initial decrease in OGG1 expression in Tsc-2EK/+ rats, results in Tsc-2EK/+ OGG1 protein levels just 5% of those seen in Tsc-2+/+ rats 8 h after treatment (Figure 6CGo). Coincidentally, 8-oxo-dG levels in Tsc-2+/+ rats 8 h after treatment with TGHQ are just 5% of those that occur in Tsc-2EK/+ rats. Despite the sustained (4–72 h) induction of OGG1 expression in Tsc-2+/+ rats, 8-oxo-dG levels in these animals continues to increase and reach a maximum at 24 h (Figure 5AGo). It is likely that despite the efficient induction of OGG1 in Tsc-2+/+ rats, the repair process remains saturated at the earlier time points, requiring several hours to overcome the initial insult. The fact that renal proximal tubular epithelial cells in these animals succumb to necrotic cell death is consistent with this view. Nonetheless, the efficient induction of OGG1 in Tsc-2+/+ rats appears to limit the effects of TGHQ treatment in these animals compared with the more extensive cellular damage observed in Tsc-2EK/+ rats (Figure 1Go).



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Fig. 5. 8-Oxo-dG formation is greater in the Tsc-2EK/+ rats than in the Tsc-2+/+ rats following administration of TGHQ (7.5 µmol/kg, i.v.). Values for 8-oxo-dG/105 x dG represent the mean ± SE, and are expressed as a percentage of the corresponding control; (A) OSOM and (B) cortex. *Significantly different from controls at P < 0.01. Control values were 0.38 ± 0.26 and 0.93 ± 0.4 in OSOM, and 1.4 ± 0.37 and 0.82 ± 0.14 in the cortex of Tsc-2EK/+ and Tsc-2+/+ rats, respectively.

 


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Fig. 6. Constitutive OGG1 expression is lower in Tsc-2EK/+ rats and its induction is attenuated following administration of TGHQ (7.5 µmol/kg, i.v.). (A) Constitutive OGG1 expression in Tsc-2+/+ and Tsc-2EK/+ rats determined by western analysis. (B) Kinetics of OGG1 expression in Tsc-2+/+ and Tsc-2EK/+ rats following TGHQ treatment. (C) Comparative expression of OGG1 in Tsc-2+/+ and Tsc-2EK/+ rats relative to a control value of 1 for constitutive expression in the Tsc-2EK/+. Values represent the mean ± SE (n = 4). *Significantly different from control at P < 0.01.

 

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Constitutive renal OGG1 expression in rats carrying a germ line mutation in the tuberous sclerosis tumor suppressor gene (Tsc-2EK/+) is three-fold lower than in wild type Tsc-2+/+ rats (Figure 6AGo). The basis for the suppression of renal OGG1 in Tsc-2EK/+ rats is not known. However, the decrease in OGG1 expression in Tsc-2EK/+ rats has important functional consequences, compromising the ability of these animals to respond to oxidative stress. The nephrotoxicity of TGHQ is dependent upon the generation of ROS (27). Therefore, factors that influence the ability of cells and tissues to respond to ROS would be expected to have a major impact on the response to TGHQ. Consistent with this view, Tsc-2EK/+ rats, with lower constitutive renal OGG1 expression, experience substantially higher levels of 8-oxo-dG than do wild type Tsc-2+/+ rats (Figure 5Go). Moreover, the poor ability of Tsc-2EK/+ rats to cope with the TGHQ-induced oxidative stress is exacerbated by their inability to rapidly upregulate this enzyme. Indeed, for at least 12 h after challenge with TGHQ, OGG1 expression is suppressed by as much as 80% in Tsc-2EK/+ rats (Figure 6Go). Other pro-oxidants, such as nitric oxide and peroxynitrite, inhibit OGG1 activity without affecting protein expression (47). Moreover, sodium dichromate decreases OGG1 mRNA and protein levels in human A549 lung carcinoma cells, an affect that appears independent of its ability to generate hydrogen peroxide (48). Thus addition of hydrogen peroxide to A549 cells had no effect on OGG1 mRNA levels. The basis for the lack of induction of OGG1 in Tsc-2EK/+ rats is not known. In humans and rats, the Tsc-2 (16p13.3 [49] and 10q [18] respectively) and OGG1 (3p26.5 [50] and 4 [51] respectively) genes are located on different chromosomes. Clearly however, the presence of a germline insertion in one allele of the Tsc-2 gene somehow influences the regulation of the OGG1 gene. To our knowledge this is the first evidence linking the Tsc-2 and OGG1 genes. A single treatment of primary renal epithelial cells derived from Tsc-2EK/+ mutant or Tsc-2+/+ wild-type rats with TGHQ for 4 h induced cell transformation only in mutant Tsc-2EK/+ cells (26). Transformation was associated with loss of tuberin expression, suggesting that a single exposure to TGHQ has the potential to induce LOH at the Tsc-2 locus. Such a genetic event may subsequently influence the regulation of OGG1 expression. Indeed, tuberin, the product of the Tsc-2 gene, behaves as a transcriptional coregulator, binding and selectively modulating members of the steroid receptor superfamily of genes, including the retinoid x receptor (RXR), the peroxisome proliferator receptor (PPAR), the vitamin D receptor (VDR), and the glucocorticoid receptor (11). Tuberin translocation to the nucleus and modulation of steroid receptor-mediated transcription may be regulated by phosphorylation (52). It is possible that the lower levels of tuberin expression in Tsc-2EK/+ rat kidney (9) contribute to the lower constitutive expression of OGG1 in these animals.

In contrast to Tsc-2EK/+ rats, OGG1 is rapidly induced in Tsc-2+/+ rats (Figure 6Go), and this seems to effectively limit increases in 8-oxo-dG levels in these animals (Figure 5Go). OGG1 is also rapidly upregulated in mouse brain following ischemia–reperfusion injury (53). As noted, despite the efficient and prolonged induction of OGG1 in Tsc-2+/+ rats, 8-oxo-dG levels continue to rise, and reach a maximum at 24 h after administration of TGHQ. This suggests that the repair process is saturated in Tsc-2+/+ rats despite the upregulation of OGG1. Consistent with this view, OGG1 protein alone may not be rate limiting for the repair of 8-oxo-dG, at least under normal growth conditions (54). The repair of much larger quantities of 8-oxo-dG in the Tsc-2EK/+ rats, despite the somewhat attenuated induction of OGG1, may then be due to the coordinated induction of additional DNA repair proteins triggered by the extensive oxidative stress in these animals. Interestingly, mutations in the OGG1 gene occur in human lung and kidney tumors (55,56). Thus, the combination of chemical-induced loss of tuberin in TGHQ-treated Tsc-2EK/+ rats coupled to the subsequent deregulated expression of OGG1 may together selectively predispose these animals to TGHQ-induced renal tumors (24).

An intriguing aspect of the present work is the time course of 8-oxo-dG formation in both groups of rats. TGHQ is rapidly eliminated from the circulation following administration to rats, being undetectable 3 h after dosing (24). 8-oxo-dG levels are maximal 8 h after TGHQ administration in Tsc-2EK/+ rats, and at 24 h in Tsc-2+/+ rats (Figure 5Go). The source of the ROS required to catalyze the generation of 8-oxo-dG at these later time-points is unclear. Although TGHQ is rapidly cleared from the circulation, a major fraction of the dose reaching the kidney is sequestered there, a much smaller fraction of metabolites being excreted in the urine (57). In addition to being redox active, oxidation of TGHQ and its metabolites gives rise to electrophilic intermediates capable of alkylating tissue macromolecules (58). Thus, following infusion of 10 µmol TGHQ directly into the kidney, a significant fraction (35.6%, greater at lower doses) becomes covalently bound to protein (57) and the nucleus seems to be a preferred target of reactive quinone-thioether metabolites (58). It is thus likely that metabolites of TGHQ bind to nuclear proteins, where they continue to redox cycle and generate ROS. Following depletion of nuclear antioxidant defenses, nuclear generated ROS would then cause extensive oxidative DNA damage. The more extensive oxidative DNA damage in Tsc-2EK/+ rats being reflected in more extensive tissue necrosis compared to Tsc-2+/+ rats. Thus, although both groups of animals initially shed brush border membrane into the tubular lumen (Figure 1Go), the ability of Tsc-2+/+ rats to contain the oxidative DNA damage results in less cell death (cytosolic GST excretion), and less overt tissue damage.

In summary, constitutive renal OGG1 expression in rats carrying a germ line mutation in the tuberous sclerosis tumor suppressor gene (Tsc-2EK/+) is lower than in wild type Tsc-2+/+ rats. Moreover, the ability of OGG1 to respond to oxidative DNA damage is impaired in Tsc-2+/+ rats, leading to excessive levels of 8-oxo-dG. The inability to efficiently repair this DNA damage results in more extensive cell death and tissue necrosis in Tsc-2EK/+ rats compared to the wild type Tsc-2+/+ rats. The deregulation of OGG1 in Tsc-2EK/+ rats may be coupled to the ability of tuberin, the Tsc-2EK/+ gene product, to behave as a transcriptional coactivator.


    Notes
 
3 To whom correspondence should be addressed Email: slau{at}mail.utexas.edu Back


    Acknowledgments
 
The authors would like to acknowledge Dr S.Mitra at the University of Texas M.D.Anderson and Sealy Center for Molecular Science, Galveston, TX, for providing the OGG1 antibody. This work was supported in part by an award from the National Institutes of Health (GM39338 to S.S.L.) and Center Grant ES07784.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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