Analysis of 7,8-Dihydro-8-oxo-2′-deoxyguanosine in Cellular DNA during Oxidative Stress

Abstract

Analysis of cellular 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 ± 0.4 8-oxo-dGuo/107 dGuo (mean ± standard deviation) or 5.5 ± 1.0 8-oxo-dGuo/108 nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose−response for 8-oxo-dGuo formation (r2 = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.

Aryl Hydrocarbon Receptor Facilitates DNA Strand Breaks and 8-Oxo-2′-deoxyguanosine Formation by the Aldo-Keto Reductase Product Benzo[a]pyrene-7,8-dione

“…Detection of DNA Strand Breaks by Comet Assay-DNA strand breaks in cells were measured using a hOGG1-coupled comet assay as previously published (25,34). The cells (1 ϫ 10 5 cells) were mixed with 85 l of 0.7% low melting point agarose.…”

Citotoxicidade do corante dinitrofenilazo CI DB291: biotransformação, estresse oxidativo e alteração epigenética em células HepG2

“…O dano nas bases do DNA gerado oxidativamente não é resultante de um agente químico específico que apresenta capacidade de modificar o DNA, mas é o resultado da interação do DNA com ROS e RNS (MANGAL et al, 2009;BANOUB e LIMBACH, 2010;CADET et al, 2010CADET et al, , 2011ANDREOLI et al, 2011). LOUREIRO et al, 2002;HALLIWELL e GUTERIDGE, 2007).…”

“…Inúmeros estudos correlacionam um aumento no nível de 8-oxodG com a ocorrência de dano gerado oxidativamente em múltiplos compartimentos do organismo (RAVANAT et al, 2002;WUA et al, 2004;HAGHDOOST et al, 2005;COOKE et al, 2008;CADET et al, 2008;MANGAL et al, 2009;CADET et al, 2010CADET et al, , 2011ANDREOLI et al, 2011;BROHEM et al, 2012). Não obstante, altos níveis de 8-oxodG também são correlacionados com tabagismo e doenças, como o diabetes e a artrite reumatóide (HAJIZADEH, et al, 2003;SIMONE et al, 2008;ROSSNER et al, 2009;BROEDBAEK et al, 2011) Devido a sua elevada incidência no DNA genômico, a 8-oxodG assume um papel fundamental nos estudos de caracterização de dano ao DNA.…”

“…A magnitude dos níveis basais detectados em DNA de células humanas normais é de 5-50 lesões a cada 10 7 desoxiguanosina não modificada (RAVANAT et al, 2002;KNERR et al, 2006;HANZALOVA et al, 2010). Infelizmente, é extremamente fácil induzir a formação artefatual de 8-oxodG no DNA celular durante as etapas de extração e hidrólise (RAVANAT et al, 2002;ESCODD, 2002;GEDIK et al, 2004;MANGAL et al, 2009) Adutos de DNA exocíclicos surgem a partir da reação de DNA com produtos da peroxidação lipídica, tais como malonaldeído, 4-hidroxi-2(E)-nonenal, 4-oxo-2(E)-nonenal, 2,4-decadienal, acroleína e crotonaldeído (NAIR et al, 2007;MEDEIROS, 2009;GARCIA et al, 2010). A adição direta de aldeídos α,β-insaturados ao DNA acaba gerando adutos com um anel de seis membros resultante da ligação de três átomos de carbono às bases (propano).…”

“…SUZUKI et al, 1997;FACOMPRÉ et al, 2000;IACOMINO et al, 2006 Estudos cinéticos do ciclo celular demonstraram que o bloqueio da progressão do ciclo na fase G2/M é um requisito para a deflagração da apoptose (LIU et al, 2005). Considerando que a homeostase mitocondrial desempenha um papel importante nessa regulação, podemos dizer que os efeitos do corante DB291 sobre as mitocôndrias estão relacionados com a parada no ciclo celular, conforme o observado por outros autores em diferentes tipos de linhagens celulares (LI et al, 1999;FACOMPRÉ et al, 2000;LIU et al, 2005 (BIANCHINI et al, 2001;CADET et al, 2008) e esforços têm sido feitos nos últimos anos para minimizar sua formação artefatual durante o preparo do DNA para análise (RAVANAT et al, 2002;ESCODD, 2002;GEDIK et al, 2004;MANGAL et al, 2009 GEHRING et al, 2009;NEIHRS, 2009;HU et al, 2012), o que sugere a existência de uma correlação entre níveis de lesões em DNA e a perda de 5-metil-dC do genoma (HU et al, 2012).…”