The role of 8-oxoguanine DNA glycosylase-1 in inflammation

Abstract

Many, if not all, environmental pollutants/chemicals and infectious agents increase intracellular levels of reactive oxygen species (ROS) at the site of exposure. ROS not only function as intracellular signaling entities, but also induce damage to cellular molecules including DNA. Among the several dozen ROS-induced DNA base lesions generated in the genome, 8-oxo-7,8-dihydroguanine (8-oxoG) is one of the most abundant because of guanine's lowest redox potential among DNA bases. In mammalian cells, 8-oxoG is repaired by the 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated DNA base excision repair pathway (OGG1-BER). Accumulation of 8-oxoG in DNA has traditionally been associated with mutagenesis, as well as various human diseases and aging processes, while the free 8-oxoG base in body fluids is one of the best biomarkers of ongoing pathophysiological processes. In this review, we discuss the biological significance of the 8-oxoG base and particularly the role of OGG1-BER in the activation of small GTPases and changes in gene expression, including those that regulate pro-inflammatory chemokines/cytokines and cause inflammation.

Figure 1

Graphical illustration of 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated genome damage repair. OGG1 is a DNA glycosylase/AP lyase that first hydrolyses the N-glycosyl bond releasing 8-oxoG as a free base and subsequently cleaves the sugar-phosphate backbone directly, generating an apurinic/apyrimidinic (AP)-site(s). Apurinic/apyrimidinic endonuclease 1 (APE1) processes the single-strand gap by removing the 3'-phospho-α,β-unsaturated aldehyde residue to form a 3'-OH end. DNA polymerase β inserts guanine, and the nicks are ligated by ligase II/XRCC1. AP site, apurinic/apyrimidinic site; APE1, apurinic/apyrimidinic endonuclease1; DNA Polβ, DNA polymerase β; XRCC1, X-ray cross-complementing protein 1.

Figure 2

Activation of rat sarcoma–small guanosine triphosphatases (RAS–GTPases) in lungs by 8-oxo-7,8-dihydroguanine (8-oxoG) challenge. (A) Challenge of airways with 8-oxoG, but not 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) or 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) (50 ng per kg) increased the GTP-bound levels of RAS (RAS–GTP) levels. Mice (albino, laboratory-bred strain c; Balb/c) were challenged intranasally (i.n.) for 45 min, and lung extracts then prepared for active RAS pull-down assays, as we previously described [81]. Upper panel: RAS–GTP levels in lung extracts of 3–4 individual mice. RAS–GTP was captured by a RAS pull-down assay from 160 μg of lung extract and immunoblotted using Pan-RAS antibody (Ab). Lower panel: Abundance of total RAS in 20 μg per lane of lung extracts, as shown by pan-RAS Ab. 8-oxoG, 8-oxo-7,8-dihydroguanine; FapyG, 2,6-diamino-4-hydroxy-5-formamido-pyrimi-dine; 8-oxodG, 7,8-dihydro-8-oxo-2'-deoxy-guanosine; (B) Time course of RAS–GTPase activation after 8-oxoG challenge of lungs. Individual mice were challenged with 8-oxoG (50 ng mg per kg, dose is determined in preliminary studies) i.n. and GTP-bound RAS was determined by ELISA pull-down assays [105]. RAS–GTP was captured from 160 μg of lung extract. The percentages of increase were calculated using MS Excel. The abundance of total RAS was determined in 20 μg of lung extract by ELISA pull-down assays. Animal experiments were performed according to the NIH Guide for the Care and Use of Experimental Animals and approved by the UTMB IACUC (no. A0807044).

Figure 3

Expression of inflammatory mediators induced by 8-oxoG base challenge. Eight-week-old female Balb/c mice were used for these studies. Mice (n = 5–6) were challenged i.n. under mild anesthesia with 60 µL of pH (7.4) balanced saline solution containing 50 ng per kg 8-oxoG, or only saline [68]. After 0, 30, 60 and 120 min of exposure, lungs were removed, and RNAs were isolated. After reverse transcription, mRNA levels of selected cytokines were determined using individual primer pairs. qRT-PCR was performed in an ABI7000 Sequencer, and expression levels were calculated by the ΔΔC_t method [106,107]. Animal experiments were performed as described in the legend to Figure 2.

Figure 4

Changes in pro-inflammatory gene expression induced by 8-oxoG, the product of OGG1-BER. Mice were challenged i.n. with 8-oxoG base (50 ng per kg), and RNA was extracted. Total RNA (pooled from five mice) was reverse-transcribed into cDNA and analyzed using a Mouse Inflammatory Cytokines & Receptors PCR Array (Cat # PAMM-011A, SABiosciences, Valencia, CA, USA). Data were analyzed using RT² profiler PCR data analysis template version 2.0. Heat maps were created and unsupervised hierarchical clusters were constructed using GENE-E online software (Broad Institute; Cambridge, MA, USA). Genes were clustered using Spearman’s rank correlation coefficient [108]. The colors in the heat-map show the minimum and maximum values (±3). Animal experiments were performed as described in the legend to Figure 2.

Figure 5

Signaling pathways induced by exposing the lungs to 8-oxoG base. Analysis by PANTHER software included genes that were upregulated in Cluster A and B (Figure 4). The percentages of genes for each category were calculated as described previously [108].

Figure 6

The OGG1–BER product 8-oxoG induces neutrophil recruitment to the lungs. Mice (n = 5–6) were challenged i.n. with 60 µL of pH-balanced 8-oxoG solution (pH: 7.4; 50 ng per kg). TNF-α (20 ng per lung) and 8-oxodG (50 ng per kg) and solvent (saline) were used as controls. Bronchoalveolar lavage fluid was collected after 16 h of challenge, cells on cytospin slides were stained with Wright-Giemsa, and the number of neutrophils was counted, as we previously described [68]. All animal experiments were performed as described in Figure 2. *** p < 0.001. TNF-α, tumor necrosis factor–α; 8-oxoG, 8-oxo-7,8-dihydroguanine; 8-oxodG, 7,8-dihydro-8-oxo-2'-deoxyguanosine.

Figure 7

A proposed model for lung inflammation by repair of oxidatively damaged DNA via OGG1–BER. When environmental exposures occur or during an inflammatory process, Reactive oxygen species (ROS) are generated by cellular oxido-reductases and mitochondrial dysfunction in the airway epithelium. Due to guanine’s lowest oxidation potential, the most frequent base damage in DNA is 8-oxoG, which needs to be repaired to prevent mutations. 8-OxoG base is released by OGG1–BER (Figure 1) and transported to the cytoplasm, where it is bound by OGG1, and the resulting complex (OGG1·8-oxoG) functions as a guanine nucleotide exchange factor and mediates GDP→GTP exchange. Activated small GTPase(s) signal expression of pro-inflammatory genes, which induce an innate immune response. Recruited inflammatory cells induced ROS, DNA damage and repair that may lead to a vicious cycle of OGG1·8-oxoG→RAS–GTP→inflammatory signaling, maintaining chronic inflammation.