RESUMEN

Ultraviolet (UV) radiation is one of the most genotoxic, universal agents present in the environment. UVB (280-315 nm) radiation directly damages DNA, producing cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs). These photolesions interfere with essential cellular processes by blocking transcription and replication polymerases, and may induce skin inflammation, hyperplasia and cell death eventually contributing to skin aging, effects mediated mainly by keratinocytes. Additionally, these lesions may also induce mutations and thereby cause skin cancer. Photolesions are repaired by the Nucleotide Excision Repair (NER) pathway, responsible for repairing bulky DNA lesions. Both types of photolesions can also be repaired by distinct (CPD- or 6-4PP-) photolyases, enzymes that specifically repair their respective photolesion by directly splitting each dimer through a light-dependent process termed photoreactivation. However, as photolyases are absent in placental mammals, these organisms depend solely on NER for the repair of DNA UV lesions. However, the individual contribution of each UV dimer in the skin effects, as well as the role of keratinocytes has remained elusive. In this study, we show that in NER-deficient mice, the transgenic expression and photorepair of CPD-photolyase in basal keratinocytes completely inhibited UVB-induced epidermal thickness and cell proliferation. On the other hand, photorepair by 6-4PP-photolyase in keratinocytes reduced but did not abrogate these UV-induced effects. The photolyase mediated removal of either CPDs or 6-4PPs from basal keratinocytes in the skin also reduced UVB-induced apoptosis, ICAM-1 expression, and myeloperoxidase activation. These findings indicate that, in NER-deficient rodents, both types of photolesions have causal roles in UVB-induced epidermal cell proliferation, hyperplasia, cell death and inflammation. Furthermore, these findings also support the notion that basal keratinocytes, instead of other skin cells, are the major cellular mediators of these UVB-induced effects.

Asunto(s)

Desoxirribodipirimidina Fotoliasa , Animales , ADN , Reparación del ADN , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Femenino , Hiperplasia , Inflamación , Queratinocitos/metabolismo , Mamíferos/genética , Ratones , Placenta/metabolismo , Embarazo

RESUMEN

Aldehydes are abundantly present in tobacco smoke and in urban air pollution and are endogenously generated as products of the lipid peroxidation process. These molecules can react with DNA bases forming mutagenic exocyclic adducts, which have been used as biomarkers of aldehyde exposure and as potential tools for the study of inflammation, metal storage diseases, neurodegenerative disorders, and cancer. High-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) provides a highly precise, specific and ultrasensitive method for the detection of exocyclic DNA adducts. Here we present and describe a validated micro-HPLC-Electro Spray Ionization (ESI)-MS/MS method for the quantification of 1,N2-propanodGuo, an adduct produced following the reaction between 2'-deoxyguanosine and acetaldehyde or crotonaldehyde.

Asunto(s)

Aductos de ADN/metabolismo , Daño del ADN , Pulmón/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Animales , Cromatografía Líquida de Alta Presión , Desoxiguanosina/metabolismo , Ratas

RESUMEN

Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell reactivation (HCR) assays showed that XP-D/CS and TTD cells have severely impaired repair capacity of oxidised lesions in plasmid DNA, and alkaline comet assays demonstrated the induction of significantly higher amounts of DNA strand breaks after treatment with photoactivated MB in these cells compared to wild-type cells. All XPD-mutated cells presented strong S/G2 arrest and persistent γ-H2AX staining after photoactivated MB treatment. Taken together, these results indicate that XPD participates in the repair of lesions induced by the redox process, and that XPD mutations lead to differences in the response to oxidatively induced damage.

Asunto(s)

Mutación , Estrés Oxidativo , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Biomarcadores , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Ensayo Cometa , Daño del ADN , Reparación del ADN , Relación Dosis-Respuesta en la Radiación , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Mutación/efectos de los fármacos , Mutación/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Rayos Ultravioleta

RESUMEN

Extensive mineral extractivism in the Brazilian Iron Quadrangle (IQ) region has destroyed large areas of land, decimating plant species, and their associated microbiota. Very little is known about the microbiota of the region; hence, cultivable bacteria associated with plants of its soils were investigated for their biotechnological potential. Samples were collected from nine plant species and six soils, and 65 cultivable bacterial isolates were obtained. These represent predominantly gram-positive bacilli (70%) capable of producing amylases (55%), proteases (63%), cellulases (47%), indole acetic acid (IAA) (46%), siderophores (26%), and to solubilize phosphate (9%). In addition, 65% of these were resistant to ampicillin, 100% were sensitive to tetracycline, and 97% were tolerant to high arsenic concentrations. Three isolates were studied further: the isolate FOB3 (Rosenbergiella sp.) produced high concentrations of IAA in vitro in the absence of tryptophan - shown by the significant improvement in plant germination and growth rate where the isolate was present. For isolates C25 (Acinetobacter sp.) and FG3 (Serratia sp.), plasmids were purified and inserted into Escherichia coli cells where they modified the physiological profile of the transformed strains. The E. coli::pFG3B strain showed the highest capacity for biofilm production, as well as an increase in the replication rate, arsenic tolerance and catalase activity. Moreover, this strain increased DNA integrity in the presence of arsenic, compared to the wild-type strain. These results help to explain the importance of bacteria in maintaining plant survival in ferruginous, rocky soils, acting as plant growth promoters, and to highlight the biotechnological potential of these bacteria. IMPORTANCE  The Iron Quadrangle region is responsible for ∼60% of all Brazilian iron production and, at the same time, is responsible for housing a wide diversity of landscapes, and consequently, a series of endemic plant species and dozens of rare species - all of which have been poorly studied. Studies exploring the microbiota associated with these plant species are limited and in the face of the continuous pressure of extractive action, some species along with their microbiota are being decimated. To understand the potential of this microbiota, we discovered that cultivable bacterial isolates obtained from plants in the ferruginous rocky soil of the Iron Quadrangle region have diverse biotechnological potential, revealing a genetic ancestry still unknown.

RESUMEN

Air pollution is a major environmental risk for human health. Acetaldehyde is present in tobacco smoke and vehicle exhaust. In this study, we show that [13C2]-acetaldehyde induces DNA modification with the formation of isotopically labeled 1, N2-propano-2'-deoxyguanosine adducts in the brain and lungs of rats exposed to concentrations of acetaldehyde found in the atmosphere of megacities. The adduct, with the addition of two molecules of isotopically labeled acetaldehyde [13C4]-1, N2-propano-dGuo, was detected in the lung and brain tissues of exposed rats by micro-HPLC/MS/MS. Structural confirmation of the products was unequivocally performed by nano-LC/ESI+-HRMS3 analyses. DNA modifications induced by acetaldehyde have been regarded as a key factor in the mechanism of mutagenesis and may be involved in the cancer risks associated with air pollution.

Asunto(s)

Acetaldehído/toxicidad , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Aductos de ADN/biosíntesis , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Acetaldehído/administración & dosificación , Acetaldehído/química , Animales , Isótopos de Carbono , Aductos de ADN/química , Aductos de ADN/aislamiento & purificación , Masculino , Estructura Molecular , Ratas , Ratas Wistar

RESUMEN

The occurrence of biochemical alterations that last for a long period of time in diabetic individuals even after adequate handling of glycemia is an intriguing phenomenon named metabolic memory. In this study, we show that a kidney pathway is gradually altered during the course of diabetes and remains persistently changed after late glycemic control in streptozotocin-induced diabetic rats. This pathway comprises an early decline of uric acid clearance and pAMPK expression followed by fumarate accumulation, increased TGF-ß expression, reduced PGC-1α expression, and downregulation of methylation and hydroxymethylation of mitochondrial DNA. The sustained decrease of uric acid clearance in treated diabetes may support the prolonged kidney biochemical alterations observed after tight glycemic control, and this regulation is likely mediated by the sustained decrease of AMPK activity and the induction of inflammation. This manuscript proposes the first consideration of the possible role of hyperuricemia and the underlying biochemical changes as part of metabolic memory in diabetic nephropathy development after glycemic control.

Asunto(s)

Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Riñón/metabolismo , Riñón/patología , Adenilato Quinasa/metabolismo , Animales , Glucemia/metabolismo , ADN Mitocondrial/metabolismo , Diabetes Mellitus Experimental/fisiopatología , Ayuno/sangre , Fumaratos/metabolismo , Hiperglucemia/sangre , Hiperglucemia/fisiopatología , Riñón/fisiopatología , Masculino , Malondialdehído/metabolismo , Mitocondrias/metabolismo , Modelos Biológicos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Fosforilación , Ratas Wistar , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Air pollution is a major risk for human health. Acetaldehyde is an environmental pollutant present in tobacco smoke, vehicle exhaust and several food products. Formation of DNA adducts has been regarded as a critical factor in the mechanisms of acetaldehyde mutagenicity and carcinogenesis. Acetaldehyde reacts with 2'-deoxyguanosine in DNA to primarily form N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dGuo). The subsequent reaction of N(2)-ethylidene-dGuo with another molecule of acetaldehyde gives rise to 1,N(2)-propano-2´-deoxyguanosine (1,N(2)-propanodGuo). In this study, on-line reverse-phase high-performance liquid chromatography (HPLC) separation with tandem mass spectrometry detection was utilized for the accurate quantification of 1,N(2)-propanodGuo and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-edGuo) in tissues of rats exposed to 12 ppb, 33 ppb and 96 ppb acetaldehyde in atmospheric air for 50 days. A significant increase in the levels of 1,N(2)-propanodGuo was observed in lung tissues of rats exposed to 12 ppb (7.8/10(8) dGuo); 33 ppb (8.9/10(8) dGuo) and 96 ppb (11.6/10(8) dGuo) compared to controls (4.2/10(8) dGuo). For comparative purposes, the levels of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-edGuo), which is produced from a,b-unsaturated aldehydes formed during the lipid peroxidation process were also measured. Elevated levels of 1,N(2)-edGuo were observed only in lung tissues of animals exposed to 96 ppb acetaldehyde. 1,N(2)-propanodGuo also differed quantitatively in liver but not in brain. The monitoring of 1,N(2)-propanodGuo levels in tissues provides important information on acetaldehyde genotoxicity and may contribute to the elucidation of the mechanisms associated with acetaldehyde exposure and cancer risk. Supported byFAPESP:2011/10048-5, CAPES, INCT Redoxoma:573530/2008-4,NAP Redoxoma: 2011.1.9352.1.8, CEPID Redoxoma:2013/07937-8.

RESUMEN

Acetaldehyde and crotonaldehyde are genotoxic aldehydes present in tobacco smoke and vehicle exhaust. The reaction of these aldehydes with 2'-deoxyguanosine in DNA produces α-methyl-γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo). Online HPLC-tandem mass spectrometry was utilized to accurately quantify 1,N(2)-propanodGuo in human urinary samples from 47 residents of São Paulo City (SP) and 35 residents of the rural municipality of São João da Boa Vista (SJBV) in the state of São Paulo. Significantly higher 1,N(2)-propanodGuo levels were found in the samples from SP donors than in samples from SJBV donors. Our results provide the first evidence that elevated levels of 1,N(2)-propanodGuo in urinary samples may be correlated with urban air pollution.

Asunto(s)

Contaminantes Atmosféricos/química , Aductos de ADN/orina , ADN/química , Desoxiguanosina/análogos & derivados , Acetaldehído/química , Adolescente , Adulto , Contaminantes Atmosféricos/toxicidad , Contaminantes Atmosféricos/orina , Aldehídos/química , Cromatografía Líquida de Alta Presión , ADN/efectos de los fármacos , Aductos de ADN/aislamiento & purificación , Desoxiguanosina/aislamiento & purificación , Desoxiguanosina/orina , Humanos , Masculino , Persona de Mediana Edad , Fumar , Extracción en Fase Sólida , Espectrometría de Masa por Ionización de Electrospray , Emisiones de Vehículos/toxicidad , Adulto Joven

RESUMEN

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance.

Asunto(s)

Restricción Calórica/métodos , Dieta Reductora/métodos , Grasa Intraabdominal/metabolismo , Músculo Esquelético/metabolismo , Obesidad/dietoterapia , Receptor de Insulina/metabolismo , Adiposidad , Animales , Western Blotting , Peso Corporal , Glucosa/metabolismo , Intolerancia a la Glucosa/metabolismo , Humanos , Insulina/metabolismo , Proteínas Sustrato del Receptor de Insulina/análisis , Proteínas Sustrato del Receptor de Insulina/biosíntesis , Masculino , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/biosíntesis , Nitrocompuestos , Obesidad/metabolismo , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/antagonistas & inhibidores

RESUMEN

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) and 1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2'-deoxyguanosine and ca. 500 amol of adducts in 100 microg of hydrolyzed DNA in the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2'-deoxyguanosine and 1,N(2)-propano-2'-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilondGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilondGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/10(8) dGuo; brain, 2.96 +/- 1.43 1,N(2)-epsilondGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanodGuo/10(8) dGuo; and lung, 0.87 +/- 0.34 1,N(2)-epsilondGuo/10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental animals with pathological conditions and after air pollution exposure.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Aductos de ADN/análisis , ADN/química , Desoxiadenosinas/análisis , Desoxiguanosina/análogos & derivados , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Bovinos , Línea Celular , Desoxiguanosina/análisis , Humanos , Masculino , Ratas , Ratas Wistar , Espectrometría de Masas en Tándem

RESUMEN

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93)-->Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.

Asunto(s)

Esclerosis Amiotrófica Lateral/metabolismo , Apoptosis , Cromatina/metabolismo , Daño del ADN , Neuroblastoma/metabolismo , Superóxido Dismutasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Esclerosis Amiotrófica Lateral/patología , Núcleo Celular/enzimología , Núcleo Celular/patología , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Humanos , Técnicas para Inmunoenzimas , Peroxidación de Lípido , Superóxido Dismutasa-1 , Células Tumorales Cultivadas

RESUMEN

Lipid peroxidation produces a large number of reactive aldehydes as secondary products. We have previously shown that the reaction of cytochrome c with trans,trans-2,4-decadienal (DDE), an aldehyde generated as a product of lipid peroxidation in cell membranes, results in the formation of adducts. Mass spectrometry analysis indicated that His-33, Lys-39, Lys-72 and Lys-100 in cytochrome c were modified by DDE. In the present work, we investigated the effect of DDE on isolated rat liver mitochondria. DDE (162 microM) treatment increases the rate of mitochondrial oxygen consumption. Extensive mitochondrial swelling upon treatment with DDE (900 nM-162 microM) was observed by light scattering and transmission electron microscopy experiments. DDE-induced loss of inner mitochondrial membrane potentials, monitored by safranin O fluorescence, was also observed. Furthermore, DDE-treated mitochondria showed an increase in lipid peroxidation, as monitored by MDA formation. These results suggest that reactive aldehydes promote mitochondrial dysfunction.

Asunto(s)

Diclorodifenil Dicloroetileno/administración & dosificación , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/fisiología , Estrés Oxidativo/efectos de la radiación , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratas , Ratas Sprague-Dawley

RESUMEN

A ruthenium oxide hexacyanoferrate (RuOHCF) modified electrode was developed. Hydrodynamic voltammetry was employed to demonstrate the remarkable electrocatalytic activity toward the oxidation of 2'-deoxyguanosine. The RuOHCF modified electrode was used as amperometric detector for 2'-deoxyguanosine determination in a FIA apparatus. The influence of various experimental conditions was explored for optimum analytical performance, and at these experimental conditions, the method exhibited a linear response range to 2'-deoxyguanosine extending from 3.8 to 252 micromol L(-1) with detection limit of 94 nmol L(-1). Applications in DNA samples were examined, and the results for determination of 2'-deoxyguanosine were in good agreement with those obtained by HPLC analysis. Studies on the kinetics of the in vitro consumption of 2'-deoxyguanosine by acetaldehyde were also performed.

Asunto(s)

Daño del ADN , ADN/química , Desoxiguanosina/análisis , Ferrocianuros/química , Análisis de Inyección de Flujo/métodos , Potenciometría/métodos , Compuestos de Rutenio/química , Catálisis , Electrodos , Análisis de Inyección de Flujo/instrumentación , Potenciometría/instrumentación

RESUMEN

Epidemiological studies testing the effect of beta-carotene in humans have found a relative risk for lung cancer in smokers supplemented with beta-carotene. We investigated the reactions of retinal and beta-apo-8'-carotenal, two beta-carotene oxidation products, with 2'-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N(2)-etheno-2'-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with beta-carotene or beta-carotene oxidation products, significantly increased levels of 1,N(2)-etheno-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of beta-carotene oxidation products.

Asunto(s)

Antioxidantes/metabolismo , Aductos de ADN/análisis , Daño del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , beta Caroteno/metabolismo , Animales , Carotenoides/metabolismo , Bovinos , Cromatografía Líquida de Alta Presión , ADN/análisis , Desoxiguanosina/aislamiento & purificación , Espectrometría de Masas , Estructura Molecular , Oxidación-Reducción , Retinaldehído/metabolismo , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

Modification of cellular DNA upon exposure to reactive oxygen and nitrogen species is the likely initial event involved in the induction of the mutagenic and lethal effects of various oxidative stress agents. Evidence has been accumulated for the significant implication of singlet oxygen (1O(2)), generated as the result of UVA activation of endogenous photosensitizers as porphyrins and flavins. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) has been shown to be the exclusive product of the reaction of 1O(2) with the guanine moiety of cellular DNA, in contrast to the hydroxyl radical, which reacts almost indifferently with all the nucleobases and the sugar moiety of DNA. Furthermore 8-oxodGuo is also produced by other oxidants and can be used as an ubiquitous biomarker of DNA oxidation but can not be a specific marker of any particular species. The role of DNA etheno adducts in mutagenic and carcinogenic processes triggered by known occupational and environmental carcinogens has also been studied. Much interest in etheno adducts resulted from the detection of increased levels of 1,N(6)-etheno-2'-deoxyadenosine and 3,N(4)-etheno-2'-deoxycytidine in DNA from human, rat and mouse tissues under pathophysiological conditions associated with oxidative stress. A method involving on-line HPLC with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysates. This method provides the first evidence of the occurrence of 1,N(2)-epsilondGuo as a basal endogenous lesion and may be utilized to better assess the biological consequences of etheno DNA damage under normal and pathological conditions. This work addresses the importance of isotope labeling associated with mass spectrometry technique for biomolecule damage studies.

Asunto(s)

Alquilantes/toxicidad , Carcinógenos/toxicidad , Daño del ADN/genética , Neoplasias/genética , Especies Reactivas de Oxígeno/toxicidad , Aductos de ADN , Humanos , Peroxidación de Lípido , Mutágenos/toxicidad , Neoplasias/inducido químicamente , Estrés Oxidativo , Oxígeno Singlete/toxicidad

RESUMEN

A method involving on-line reversed-phase high-performance liquid chromatography with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysate. Using the newly developed technique, basal levels of 1,N(2)-etheno-2'-deoxyguanosine were determined in commercial calf thymus DNA (1.70 +/- 0.09 adducts/10(7) dGuo), in cultured mammalian cells (CV1-P) DNA (4.5 +/- 0.4 adducts/10(7) dGuo), and in untreated female rat liver DNA (5.22 +/- 1.37 adduct/10(7) dGuo). The mutagenicity of 1,N(2)-etheno-2'-deoxyguanosine had already been demonstrated by in vitro and in vivo systems. The method described here provides the first evidence of the occurrence of 1,N(2)-etheno-2'-deoxyguanosine as a basal endogenous lesion and may be usefully employed to assess the biological consequences of etheno DNA damage under normal and pathological conditions.

Asunto(s)

Aductos de ADN/análisis , Daño del ADN , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Animales , Cromatografía Liquida/métodos , Femenino , Ratas , Ratas Wistar , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/métodos