The Importance of Measuring Oxidative and Nitrosative Stress Damage in DNA/RNA

Oxidative and nitrosative stress damage have a significant impact on the etiology and progression of a multitude of human pathologies, including cardiovascular, autoimmune, and oncological conditions. By way of reactive oxygen species (ROS) and reactive nitrogen species (RNS), respectively, oxidative and nitrosative stress can result in cellular damage, enzyme inactivation, and epigenetic impairment of DNA. A better understanding of stress damage—disease relationships can therefore be beneficial for diagnostic and therapeutic purposes. As a consequence, several biomarkers of oxidative and nitrosative stress have been identified, including nucleic acid modifications like 8-hydroxyguanosine (8-OhG) and 8-nitroguanine (8-NG).

8-OhG is a modified nucleoside found in cellular RNA. Formed by the oxidation of guanosine, this modification is generated by hydroxyl radical, singlet oxygen, and one-electron oxidant ROS. 8-OhG present in mRNA can induce base mispairing during gene expression, leading to aberrant translation. Furthermore, 8-OhG-modified transcripts in the chromatin modifying enzymes pathway can alter chromatin organization and regulation. This mutagenic potential for abnormal protein production, unsurprisingly, contributes to the role of 8-OhG in various diseases. Indeed, elevated 8-OhG levels have been associated with cancer, neurodegenerative disorders, coronary heart disease, diabetes, and age-related conditions, and are closely correlated with exposure to harmful environmental factors such as ionizing radiation, industrial chemicals, air pollution, and cigarette smoking.

8-NG is a modified nucleobase found in both RNA and DNA, typically formed through a reaction between guanine and RNS. During DNA replication, this modified guanine may mispair with adenine instead of cytosine, leading to a G:C to T:A transversion mutation that can disrupt the normal functioning of genes and regulatory elements, potentially contributing to the development of inflammation, cancer, and other diseases. Additionally, 8-NG’s induction of DNA lesions such as single-strand breaks can indirectly influence epigenetic regulation by modulating chromatin structure and gene expression patterns near damaged sites.

While urine and serum assays mainly reflect the balance between oxidative or nitrosative stress damage and the repair rate of the whole body, direct quantification of 8-OhG or 8-NG content in different cells and tissues under normal and disease states would allow site-specific damage of DNA/RNA to be identified. Such a method would provide more useful information about oxidative/nitrosative stress damage-disease relationships to benefit disease diagnostics and therapeutics. Chromatography-based techniques, such as HPLC-ECD and LC-MS, are time-consuming, expensive, and have low throughput. Also, currently used competitive ELISA methods are less accurate and cannot directly use intact DNA or RNA isolated from cells or tissues.

To address these issues, EpigenTek has developed the EpiQuik 8-OhG RNA Damage Quantification Direct Kit and the EpiQuik Nitrosative (8-Nitroguanine) DNA/RNA Damage Quantification Kit, which use a unique procedure to directly quantify 8-OhG or 8-NG in cells/tissues. These kits offer the following advantages:

• Simple, reliable, and consistent ELISA with easy-to-follow steps and a rapid protocol time of just 3 hours.
• High sensitivity, with a detection limit as low as 2 pg of 8-OhG or 8-NG.
• High specificity, without cross-reactivity to G or its modified derivatives within the indicated concentration range of the sample DNA/RNA.
• Direct detection of 8-OhG or 8-NG using fully intact DNA/RNA isolated from cells or tissues, without the need for additional sample processing like digestion or hydrolysis.
• Universal positive and negative controls are included, which are suitable for quantifying 8-OhG or 8-NG from any species.
• Strip-well microplate format makes the assay flexible for either manual or high throughput analysis.

References

1. Dai DP, Gan W, Hayakawa H, Zhu JL, Zhang XQ, Hu GX, Xu T, Jiang ZL, Zhang LQ, Hu XD, Nie B, Zhou Y, Li J, Zhou XY, Li J, Zhang TM, He Q, Liu DG, Chen HB, Yang N, Zuo PP, Zhang ZX, Yang HM, Wang Y, Wilson SH, Zeng YX, Wang JY, Sekiguchi M, Cai JP. Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells. Proc Natl Acad Sci U S A. 2018 Apr 17;115(16):4218-4222. doi: 10.1073/pnas.1718363115. Epub 2018 Apr 2. PMID: 29610342; PMCID: PMC5910836.
2. Gonzalez-Rivera JC, Sherman MW, Wang DS, Chuvalo-Abraham JCL, Hildebrandt Ruiz L, Contreras LM. RNA oxidation in chromatin modification and DNA-damage response following exposure to formaldehyde. Sci Rep. 2020 Oct 6;10(1):16545. doi: 10.1038/s41598-020-73376-7. PMID: 33024153; PMCID: PMC7538935.
3. Ohshima H, Sawa T, Akaike T. 8-nitroguanine, a product of nitrative DNA damage caused by reactive nitrogen species: formation, occurrence, and implications in inflammation and carcinogenesis. Antioxid Redox Signal. 2006 May-Jun;8(5-6):1033-45. doi: 10.1089/ars.2006.8.1033. PMID: 16771693.