HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in Cultured Cells and Animal Tissues

Abstract

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2'-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxodGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO 3 , and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples. Video LinkThe video component of this article can be found at

8-OxoG in GC-rich Sp1 binding sites enhances gene transcription during adipose tissue development in juvenile mice

“…Coefficient of variation of 8-oxoG levels in different tissues was less than 32%. These global concentrations of 8-oxoG were comparable to the background range of 8-oxoG levels in pig liver (0.0002% to 0.0441%), HeLa cells (0.0001% to 0.0214%) (Escodd 2002) or commercially available calf thymus DNA (0.032%) (Chepelev et al 2015) . The ratios of total dG to total dC were 1.03, 1.00, and 0.91 in lung, liver, and adipose tissues, respectively, indicating that there was no detection bias in the analysis.…”

Carnosic Acid, a Natural Diterpene, Attenuates Arsenic‐Induced Hepatotoxicity via Reducing Oxidative Stress, MAPK Activation, and Apoptotic Cell Death Pathway

“…The DNA precipitates were heated to 90°C for 10 min in 5% trichloroacetic acid and quantitatively estimated by colorimetric reaction with diphenylamine [ 29 ]. DNA oxidation was evaluated by RP-HPLC analysis and was represented as 7,8-hydroxy-2′-deoxyguaosine/2′-deoxyguaosine (8-OHdG/2-dG) ratio [ 30 , 31 ]. Briefly, DNA was isolated from hepatic tissue by pronase-ethanol method followed by enzymatic digestion as the protocol by Chepelev et al [ 31 ].…”

“…DNA oxidation was evaluated by RP-HPLC analysis and was represented as 7,8-hydroxy-2′-deoxyguaosine/2′-deoxyguaosine (8-OHdG/2-dG) ratio [ 30 , 31 ]. Briefly, DNA was isolated from hepatic tissue by pronase-ethanol method followed by enzymatic digestion as the protocol by Chepelev et al [ 31 ]. 8-OHdG stock solution (35 mmol/l) was prepared in water, while 2-dG stock solution (37 mmol/l) was prepared in 0.5 mol/l NH 4 OH [ 30 ].…”

Transcriptional Profiling of Dibenzo[def,p]chrysene-induced Spleen Atrophy Provides Mechanistic Insights into its Immunotoxicity in MutaMouse

“…Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) analysis. DNA was extracted by the DNAzol method as it produces minimal amounts of artificial DNA oxidation (Mangal et al, 2009), as described elsewhere (Chepelev et al, 2015c). Approximately 15 mg of spleen was used (N=5, 4and 24-h post-exposure samples).…”

“…DNA oxidation in the spleen was quantified using a modified HPLC method that measures an established DNA oxidation marker, dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) (Chepelev et al, 2015c). In contrast to the other genotoxicity endpoints (i.e., DNA adducts, lacZ mutant frequency, and micronucleus formation), 8-oxo-dG levels increased only at the highest dose and only at the 4 h post-exposure time-point (1.9-fold increase vs. control, Fig.…”