Potential role for 8-oxoguanine DNA glycosylase ... - The FASEB Journal

©2004 FASEB

The FASEB Journal express article 10.1096/fj.04-2278fje. Published online December 1, 2004.

Potential role for 8-oxoguanine DNA glycosylase in regulating inflammation Jon G. Mabley,*,† Pál Pacher,†,‡ Amitabha Deb,† Rebecca Wallace,† Rhoderick H. Elder,§ and Csaba Szabó†,║ *School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, United Kingdom; †Inotek Pharmaceuticals Corp., Beverly, Massachussetts, USA. ‡National Institutes of Health, NIAAA, Laboratory of Physiologic Studies, Rockville, Maryland, USA; §CR-UK Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom; ║Institute of Human Physiology and Clinical Experimental Research, Semmelweis University of Medicine, Budapest, Hungary Corresponding author: Jon G Mabley, DPhil., School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, UK. E-mail: [email protected] ABSTRACT OGG-1 DNA glycosylase (OGG-1) is an enzyme involved in DNA repair. It excises 7,8dihydro-8-oxoguanine, which is formed by oxidative damage of guanine. We have investigated the role of OGG-1 in inflammation using three models of inflammation: endotoxic shock, diabetes, and contact hypersensitivity. We found that OGG-1−/− mice are resistant to endotoxin (lipopolysaccharide, LPS)-induced organ dysfunction, neutrophil infiltration and oxidative stress, when compared with the response seen in wild-type controls (OGG+/+). Furthermore, the deletion of the OGG-1 gene was associated with decreased serum cytokine and chemokine levels and prolonged survival after LPS treatment. Type I diabetes was induced by multiple low-dose streptozotocin treatment. OGG-1−/− mice were found to have significantly lower blood glucose levels and incidence of diabetes as compared with OGG-1+/+ mice. Biochemical analysis of the pancreas showed that OGG-1−/− mice had greater insulin content, indicative of a greater β-cell mass coupled with lower levels of the chemokine MIP-1α and Th1 cytokines IL-12 and TNF-α. Levels of protective Th2 cytokines, IL-4 and IL-10 were significantly higher in the pancreata of OGG-1−/− mice as compared with the levels measured in wild-type mice. In the contact hypersensitivity induced by oxazolone, the OGG-1−/− mice showed reduced neutrophil accumulation, chemokine, and Th1 and Th2 cytokine levels in the ear tissue. The current studies unveil a role for OGG-1 in the regulation of inflammation. Key words: OGG-1 • diabetes • shock • hypersensitivity • DNA repair

Page 1 of 18 (page number not for citation purposes)

I

ncreased levels of reactive nitrogen and oxygen species are generated during the inflammatory response. These reactive species have been implicated in the etiology of a wide variety of diseases (1) including endotoxic and septic shock (2–4), diabetes (4), contact hypersensitivity (4), arthritis (3, 5) and colitis (3, 5). These reactive species are genotoxic and induce a wide variety of DNA lesions, including oxidized bases and DNA strand breaks (6, 7). In response to DNA damage, various nuclear enzymes are activated such as poly (ADP-ribose) polymerase (PARP), alkylpurine-DNA-N-glycosylase (APNG), and 8-oxoG-DNA glycosylase (OGG-1). PARP, in addition to orchestrating cellular responses after DNA injury, has been shown to play important roles as an inflammatory/immune system modulator in a variety of disease states (8). A major site of attack of DNA by oxidizing species is at the C-8 position of guanine to form 7,8dihydro-8-oxoguanine (8-oxoG). This 8-oxoG is strongly mutagenic as it can mispair with adenine residues instead of the more usual cysteine residues, leading to an increased frequency of G.C to T.A transversion mutations (9–11). To prevent this event, two distinct repair enzymes are present in mammalian cells to excise 8-oxoG from DNA: termed OGG-1 and OGG-2. OGG1 is an 8-oxoG-DNA glycosylase/abasic lysase, which removes the base lesion from DNA via a glycolytic mechanism and concomitantly cleaves the DNA at the resulting abasic site via βelimination (12–17). OGG-1 is primarily responsible for removing the 8-oxoG from in situ DNA, where it has resulted in an 8-oxoG:C base pairing; in contrast, OGG-2 also removes 8oxoG from DNA but is antigenically distinct from OGG-1 and removes predominantly 8-oxoG from 8-oxoG:A pairs that are formed by misincorporation of 8-oxoG into nascent DNA (18). OGG-1 gene knockout mice have been generated recently (19) and found to exhibit moderately increased spontaneous mutations, but no malignancies or any pathological changes (19). These results suggest an alternative pathway by which 8-oxoG can be removed from or prevented from entering the genome, two other enzymes MutY homologue (MYH) and MutT homologue 1 (MTH1) are two such enzymes. MYH removes adenine, which has mispaired opposite the 8oxoG, allowing it to be replaced by a C so giving OGG-1 a second chance at removing the 8oxoG, MTH1 acts much earlier to prevent the 8-oxoG being incorporated into the DNA by breaking down 8-oxo-dGTP to 8-oxo-dGMP, which can then be degraded for excretion (18). The aim of the present study was to determine whether OGG-1 plays a role in the regulation of the inflammatory response. To this end, we selected three different models of inflammation; endotoxic shock (a model of acute, severe systemic inflammation), diabetes (a model of autoimmunity with a strong proinflammatory component), and contact hypersensitivity (a model of allergy). The current data demonstrate that OGG-1 gene disruption is markedly protective in these distinct models of inflammation. MATERIALS AND METHODS Animal models All animal experiments were carried out in accordance with the guidelines published by the NIH in Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985).


Generation of transgenic mice Breeding pairs of OGG-1+/− were obtained from [NTH1,OGG1]+/− mice (20) on a mixed 129/C57Bl/6J background; the original OGG-1−/− deletion was that described by Klungland et al. (19). Breeding pairs of OGG-1+/− mice were bred under normal conditions and their offspring genotyped as described (19, 21, 22). Briefly, DNA was prepared from a tail snip taken when mice reach 3 weeks of age. PCR was preformed using 4 primers; Primer 1 mOGG-1 Exon 5 sense (5′-GTG GCT GAC TGC ATC TGC TT-3′), Primer 2 mOGG-1 Exon 6 anti-sense (5′GCA TAA GGT CCC CAC AGA TTC-3′), Primer 3 mOGG-1 Exon 3 sense (5′-GCT TCC CAA ACC TCC ATG C-3′), and Primer 4 Neo antisense (5′-GCC GAA TAG CCT CTC CAC CCA AGC-3′). For wild-type mice (OGG-1+/+), Primer 1 and Primer 2 give a 0.4 kbar band, whereas for gene-disrupted mice (OGG−/−), Primer 3 and Primer 4 give a 1.0 kbar band (Fig. 1). Induction of endotoxic shock Male OGG-1+/+ or OGG-1−/− mice (6–8 weeks of age) were used in studies investigating lipopolysaccharide (LPS) induced cytokine production, organ damage, neutrophil infiltration, oxidative stress, and survival. For cytokine production, mice were subjected to LPS (1 mg/kg ip) for 90 min before the blood was removed and serum taken for analysis (23). For organ damage, neutrophil infiltration and oxidative stress mice were challenged with LPS (80 mg/kg ip) for 12 h, the mice were killed and serum removed and stored at –80°C for organ function assays. The lungs, heart, liver, kidney, and gut were removed for analysis of neutrophil infiltration (MPO levels) and oxidative stress (MDA levels) (24). For survival studies, mice were subjected to LPS (55 mg/kg ip) and survival followed over the following 72 h (23). Induction of diabetes Male OGG-1+/+ or OGG-1−/− were treated with streptozotocin (40 mg/kg dissolved in citrate buffer, pH 4.5) or vehicle (citrate buffer) intraperitoneally for five consecutive days (25). Blood glucose was measured on days 1, 7, 14, and 21 from blood obtained from the tail vein. Hyperglycemia was defined as a nonfasting blood glucose level ≥200 mg/dl. Cumulative incidence of diabetes was calculated as a percentage of hyperglycemic mice at each time point. Biopsies of pancreas were removed on day 21 for biochemical and cytokine assays (26). Induction of contact hypersensitivity Female OGG-1+/+ and OGG-1−/− mice were sensitized on the shaved abdominal wall by the administration of 100 µL 2% oxazolone solubilized in acetone: olive oil 4:1 or 100 µl vehicle (27). After 7 days, all animals were challenged by applying 20 µl 0.5% oxazolone on each ear. After 24 h, mice were killed by CO2, and ears were immediately removed and frozen for biochemical analysis.


Biochemical determinations Cytokine levels Serum was diluted 1:5 before being assayed for cytokines using specific ELISA kits (28). Tissue samples were removed and snap frozen in liquid nitrogen; the sample was then homogenized in 700 µl of a TRIS-HCl buffer containing protease inhibitors (26). Samples were centrifuged for 30 min, and the supernatant frozen at –80°C until assay. Cytokine levels were determined from undiluted samples using specific ELISA kits. Myeloperoxidase (MPO) activity Tissue samples were homogenized (50 mg/ml) in 0.5% hexadecyltrimethylammonium bromide in 10 mM 3-N-morpholinopropanesulfonic acid (MOPS) and centrifuged at 15,000 g for 40 min (5). The suspension was then sonicated 3 times for 30 s. An aliquot of supernatant (20 µl) was mixed with a solution of 1.6 mM tetra-methyl-benzidine and 1 mM hydrogen peroxide. Activity was measured spectrophotometrically as the change in absorbance at 650 nm at 37°C, using a Spectramax microplate reader (Molecular Devices, Sunnyvale, CA). Results are expressed as milliunits of MPO activity per milligram protein, which were determined with the Bradford assay (29). Malondialdehyde assay (MDA) Malondialdehyde formation was used to quantify the lipid peroxidation in the tissue samples and measured as thiobarbituric acid-reactive material (5). Tissues were homogenized (100 mg/ml) in 1.15% KCl buffer. 200 µl of the homogenates were then added to a reaction mixture consisting of 1.5 ml 0.8% thiobarbituric acid, 200 µl 8.1% sodium dodecyl sulfate, 1.5 ml 20% acetic acid (pH 3.5), and 600 µl distilled H2O. The mixture was then heated at 90°C for 45 min (30, 31). After cooling to room temperature, the samples were cleared by centrifugation (10,000 g, 10 min), and their absorbance measured at 532 nm, using 1,1,3,3-tetramethoxypropane as an external standard. The level of lipid peroxides was expressed as nmol MDA /mg protein (Bradford assay) (29). Organ function Liver and kidney function was assessed by serum levels of alanine aminotransferase (ALT), creatinine, and blood urea nitrogen (BUN) determined using a VetScan chemistry analyzer using the comprehensive diagnostic profile rotors (24). Pancreatic insulin content Insulin contents in pancreata of the mice were determined from a pancreas biopsy, which was homogenized in acidified ethanol (75% ethanol, 1.5% 12mol/l HCl, and 23.5% H2O), then incubated for 72 h at 4°C and centrifuged (32). Care was taken to remove biopsies from the same location of the pancreas (body) to avoid differences between the regions of the pancreas in regard to insulin content. The insulin content of the supernatant was determined using an ELISA kit (Alpco). Pancreatic insulin content was expressed as nanograms of insulin per milligram protein, which was determined by the Bradford assay (29).


Statistical analysis The results are presented as means ± SEM; statistical analysis was preformed using either oneway ANOVA followed by Student-Newman-Keuls multiple comparisons post hoc analysis, Fisher’s exact test, Mann-Whitney test, or Kaplan-Meier survival analysis as appropriate, with a P value of