RESUMEN

Chemical functionalization broadens carbon nanotube (CNT) applications, conferring new functions, but at the same time potentially altering toxicity. Although considerable experimental data related to CNT toxicity, at the molecular and cellular levels, have been reported, there is very limited information available for the corresponding mechanism involved (e.g. cell apoptosis and genotoxicity). The threshold dose for safe medical application in relation to both pristine and functionalized carbon nanotubes remains ambiguous. In this study, we evaluated the in vitro cytotoxicity of pristine and functionalized (OH, COOH) multi-walled carbon nanotubes (MWCNTs) for cell viability, oxidant detection, apoptosis and DNA mutations, to determine the non-toxic dose and influence of functional group in a human lung-cancer cell line exposed to 1-1000µg/ml MWCNTs for 24, 48 and 72h. The findings suggest that pristine MWCNTs induced more cell death than functionalized MWCNTs while functionalized MWCNTs are more genotoxic compared to their pristine form. The level of both dose and dispersion in the matrix used should be taken into consideration before applying further clinical applications of MWCNTs.

Asunto(s)

Nanotubos de Carbono/toxicidad , Células A549 , Apoptosis/efectos de los fármacos , Ácidos Carboxílicos/química , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Humanos , Hidróxidos/química , L-Lactato Deshidrogenasa/metabolismo , Nanotubos de Carbono/química , Necrosis/inducido químicamente

RESUMEN

G-quadruplex structures, formed from guanine rich sequences, have previously been shown to be involved in various physiological processes including cancer-related gene expression. Furthermore, G-quadruplexes have been found in several oncogene promoter regions, and have been shown to play a role in the regulation of gene expression. The mutagenic properties of oxidative stress on DNA have been widely studied, as has the association with carcinogenesis. Guanine is the most susceptible nucleotide to oxidation, and as such, G-rich sequences that form G-quadruplexes can be viewed as potential "hot-spots" for DNA oxidation. We propose that oxidation may destabilise the G-quadruplex structure, leading to its unfolding into the duplex structure, affecting gene expression. This would imply a possible mechanism by which oxidation may impact on oncogene expression. This work investigates the effect of oxidation on two biologically relevant G-quadruplex structures through 500 ns molecular dynamics simulations on those found in the promoter regions of the c-Kit and c-Myc oncogenes. The results show oxidation having a detrimental effect on stability of the structure, substantially destabilising the c-Kit quadruplex, and with a more attenuated effect on the c-Myc quadruplex. Results are suggestive of a novel route for oxidation-mediated oncogenesis and may have wider implications for genome stability.

Asunto(s)

Biología Computacional , G-Cuádruplex , Simulación de Dinámica Molecular , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-myc/genética , 8-Hidroxi-2'-Desoxicoguanosina , ADN/química , ADN/genética , ADN/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Regulación de la Expresión Génica , Oxidación-Reducción

RESUMEN

Intracellular vitamin C acts to protect cells against oxidative stress by intercepting reactive oxygen species (ROS) and minimising DNA damage. However, rapid increases in intracellular vitamin C may induce ROS with subsequent DNA damage priming DNA repair processes. Herein, we examine the potential of vitamin C and the derivative ascorbate-2-phosphate (2-AP) to induce a nucleotide excision repair (NER) response to DNA damage in a model of peripheral blood mononuclear cells. Exposure of cells to elevated levels of vitamin C induced ROS activity, resulting in increased levels of deoxycytidine glyoxal (gdC) and 8-oxo-2'-deoxyguanosine (8-oxodG) adducts in DNA; a stress response was also induced by 2-AP, but was delayed in comparison to vitamin C. Evidence of gdC repair was also apparent. Measurement of cyclobutane thymine-thymine dimers (T < >T) in DNA and culture supernatant were included as a positive marker for NER activity; this was evidenced by a reduction in DNA and increases in culture supernatant levels of T < >T for vitamin C-treated cells. Genomics analysis fully supported these findings confirming that 2-AP, in particular, induced genes associated with stress response, cell cycle arrest, DNA repair and apoptosis, and additionally provided evidence for the involvement of vitamin C in the mobilisation of intracellular catalytic Fe.

Asunto(s)

Ácido Ascórbico/farmacología , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Vitaminas/farmacología , 8-Hidroxi-2'-Desoxicoguanosina , Apoptosis/efectos de los fármacos , Ácido Ascórbico/análogos & derivados , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , ADN/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Marcadores Genéticos , Genómica , Glioxal/metabolismo , Humanos , Hierro/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Modelos Biológicos , Dímeros de Pirimidina/metabolismo

RESUMEN

Polyunsaturated fats have been linked to occurrences of sporadic colon cancer. One possible cause may be degradation of polyunsaturated fats during cooking, resulting in multiple reactive carbonyl species (RCS) that can damage nuclear DNA and proteins, particularly in rapidly dividing colon crypt cells. This study describes a novel antiserum against RCS-modified DNA, with apparent order of reactivity to DNA modified with 4-hydroxy-trans-2-nonenal > glyoxal > acrolein > crotonaldehyde > malondialdehyde; some reactivity was also observed against conjugated Schiff base-type structures. Anti-(RCS-DNA) antiserum was successfully utilised to demonstrate formation of RCS-DNA in a human colon cell model, exposed to RCS insult derived from endogenous and exogenous lipid peroxidation sources. Further utilisation of the antiserum for immunohistochemical analysis confirmed RCS-modified DNA in crypt areas of 'normal' colon tissue. These results fully support a potential role for dietary lipid peroxidation products in the development of sporadic colon cancer.

Asunto(s)

Colon/metabolismo , Neoplasias del Colon/metabolismo , ADN/metabolismo , Grasas Insaturadas/metabolismo , Animales , Bovinos , Línea Celular Tumoral , Neoplasias del Colon/patología , ADN de Neoplasias/metabolismo , Epitelio/patología , Humanos , Inmunoglobulina G/química , Peroxidación de Lípido , Conejos , Bases de Schiff/metabolismo , Albúmina Sérica/metabolismo

RESUMEN

Global gene expression profiles of livers from mice, fed diets differing in alpha-tocopherol content, were compared using DNA microarray technology. Three hundred and eighty nine genes were found to significantly differ in their expression level by a factor of 2 or higher between the high and the low alpha-tocopherol group. Functional clustering using the EASE software identified 121 genes involved in transport processes. Twenty-one thereof were involved in (synaptic) vesicular trafficking. Up-regulation of syntaxin 1C (Stx1c), vesicle-associated membrane protein 1 (Vamp1), N-ethylmaleimide-sensitive factor (Nsf) and syntaxin binding protein 1 (Stxbp1, Munc18-1) was verified by real time PCR. At a functional level, alpha-tocopherol increased the secretory response in RBL and PC12 cells. Although here detected in liver, the alpha-tocopherol-responsive pathways are also relevant to neurotransmission. A role of alpha-tocopherol in the vesicular transport might not only affect its own absorption and transport but also explain the neural dysfunctions observed in severe alpha-tocopherol deficiency.

Asunto(s)

Antioxidantes/administración & dosificación , Dieta , Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Vesículas Transportadoras/genética , alfa-Tocoferol/administración & dosificación , Animales , Antioxidantes/análisis , Transporte Biológico/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , alfa-Tocoferol/análisis

RESUMEN

Oxidation of PUFAs in the diet has the potential to be genotoxic and hence carcinogenic. Such carcinogenic processes originate within stem cells of the colon. These cells appear to be predisposed to the carcinogenic process. In colon cells (CRL-1807) exposed to chemical reactions simulating exogenous and endogenous peroxidation reactions, we have observed that undifferentiated cells could mount an effective recombinational repair/TCR response to an endogenous peroxidative DNA damage insult, but not to an external exogenous peroxidative insult as one would encounter from a dietary source. This may suggest that defects in such specific DNA repair may play a role in tumour development in undifferentiated colonocytes exposed to a diet-derived lipid peroxides.

Asunto(s)

Amidinas/toxicidad , Colon/fisiología , Neoplasias del Colon/genética , Reparación del ADN/efectos de los fármacos , Perfilación de la Expresión Génica , Peróxidos/toxicidad , Ciclo Celular/efectos de los fármacos , Colon/citología , Colon/efectos de los fármacos , Neoplasias del Colon/inducido químicamente , Daño del ADN/efectos de los fármacos , Dieta , Genes BRCA1/efectos de los fármacos , Calor , Humanos

RESUMEN

The product of oxidative damage to DNA, 8-hydroxy-2'-deoxyguanosine (8-OHdG), when detected in urine, is considered to be a global, noninvasive biomarker of in vivo oxidative DNA damage. In this paper we describe a novel approach to confirm the presence of oligonucleotides containing 8-OHdG in human urine. Fractions of urine were prepared by gel-filtration chromatography, and the presence of oligonucleotides was confirmed by ELISA using a monoclonal anti-(single-stranded DNA) antibody. Pools of urine fractions were subsequently prepared according to ELISA reactivity, each containing oligonucleotides with a known range of base numbers. The level of 8-OHdG in each pool was subsequently determined using a commercial ELISA kit. Results confirmed that oligonucleotides containing 8-OHdG are present in urine and, most significantly, oligomers of <30-55 bases were found to be associated with 8-OHdG. This finding strongly supports the involvement of nucleotide excision repair (NER) in the removal of 8-OHdG from the cell. The novel approach adopted in this study was validated using cell culture supernatant obtained from an in vitro model comprising CCRF cells exposed to vitamin C; this model has previously been shown to stimulate removal of 8-OHdG from the cell by an NER-dependent process.

Asunto(s)

Desoxiguanosina/análogos & derivados , Oligonucleótidos/orina , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Desoxiguanosina/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Liofilización , Humanos , Masculino , Oligonucleótidos/química

RESUMEN

To investigate the hypothesis that the micronutrient ascorbic acid can modulate the functional genome, T cells (CCRF-HSB2) were treated with ascorbic acid (up to 150 microM) for up to 24 h. Protein expression changes were assessed by two-dimensional electrophoresis. Forty-one protein spots which showed greater than two-fold expression changes were subject to identification by matrix-assisted laser desorption ionisation time of flight MS. The confirmed protein identifications were clustered into five groups; proteins were associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of phosphatidylinositol transfer protein (promotes intracellular signalling) within 5 min of ascorbic acid treatment was confirmed by Western blotting. Together, these observations suggest that ascorbic acid modulates the T cell proteome in a time- and dose-dependent manner and identify molecular targets for study following antioxidant supplementation in vivo.

Asunto(s)

Ácido Ascórbico/farmacología , Proteoma/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Linfocitos T/química , Western Blotting/métodos , Línea Celular Transformada , Dermatoglifia del ADN , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Humanos , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteoma/genética , Proteoma/metabolismo , Transducción de Señal/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de Tiempo

RESUMEN

Vascular monocyte retention in the subintima is pivotal to the development of cardiovascular disease and is facilitated by up-regulation of adhesion molecules on monocytes/endothelial cells during oxidative stress. Epidemiological studies have shown that cardiovascular disease risk is inversely proportional to plasma levels of the dietary micronutrients, vitamin C and vitamin E (alpha-tocopherol). We have tested the hypothesis that alpha-tocopherol supplementation may alter endothelial/monocyte function and interaction in subjects with normal ascorbate levels (> 50 microM), as ascorbate has been shown to regenerate tocopherol from its oxidised tocopheroxyl radical form in vitro. Healthy male subjects received alpha-tocopherol supplements (400 IU RRR-alpha-tocopherol/day for 6 weeks) in a placebo-controlled, double-blind intervention study. There were no significant differences in monocyte CD11b expression, monocyte adhesion to endothelial cells, plasma C-reactive protein or sICAM-1 concentrations post-supplementation. There was no evidence for nuclear translocation of NF-kappaB in isolated resting monocytes, nor any effect of alpha-tocopherol supplementation. However, post-supplementation, sVCAM-1 levels were decreased in all subjects and sE-selectin levels were increased in the vitamin C-replete group only; a weak positive correlation was observed between sE-selectin and alpha-tocopherol concentration. In conclusion, alpha-tocopherol supplementation had little effect on cardiovascular disease risk factors in healthy subjects and the effects of tocopherol were not consistently affected by plasma vitamin C concentration.

Asunto(s)

Proteína C-Reactiva/metabolismo , Monocitos/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/sangre , alfa-Tocoferol/farmacología , Adulto , Antioxidantes/administración & dosificación , Antioxidantes/farmacología , Transporte Biológico/efectos de los fármacos , Antígeno CD11b/metabolismo , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Suplementos Dietéticos , Método Doble Ciego , Ensayo de Cambio de Movilidad Electroforética , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Monocitos/citología , Monocitos/metabolismo , FN-kappa B/metabolismo , Solubilidad , alfa-Tocoferol/administración & dosificación , alfa-Tocoferol/sangre

RESUMEN

Vitamin C (or ascorbic acid) is regarded as the most important water-soluble antioxidant in human plasma and mammalian cells which have mechanisms to recycle and accumulate it against a concentration gradient, suggesting that the vitamin might also have important intracellular functions. In this review we summarize evidence from human trials that have attempted an association between vitamin C supplementation and an effect on biomarkers of oxidative DNA damage. Most studies reviewed herein showed either a vitamin C-mediated reduction in oxidative DNA damage or a null effect, whereas only a few studies showed an increase in specific base lesions. We also address the possible beneficial effects of vitamin C supplementation for the prevention of cancer and cardiovascular disease. Finally, we discuss the contribution of cell culture studies to our understanding of the mode of action of vitamin C and we review recent evidence that vitamin C is able to modulate gene expression and cellular function, with a particular interest in cell differentiation.

Asunto(s)

Antioxidantes/metabolismo , Antioxidantes/farmacología , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacología , Daño del ADN , Suplementos Dietéticos , Expresión Génica , Humanos , Oxidantes/metabolismo , Oxidantes/farmacología , Estrés Oxidativo , Transducción de Señal/fisiología

RESUMEN

The repair of oxidatively damaged DNA is integral to the maintenance of genomic stability, and hence prevention of a wide variety of pathological conditions, such as aging, cancer and cardiovascular disease. The ability to non-invasively assess DNA repair may provide information regarding repair pathways, variability in repair capacity, and susceptibility to disease. The development of assays to measure urinary DNA lesions offered this potential, although it rapidly became clear that possible contribution from diet and cell turnover may influence urinary lesion levels. Whilst early studies attempted to address these issues, up until now, much of the data appears conflicting. However, recent work from our laboratories, in which human volunteers were fed highly oxidatively modified 15N-labelled DNA demonstrates that diet does not appear to contribute to urinary levels of 8-hydroxyguanine and 7,8-dihydro-8-oxo-2'-deoxyguanosine. Furthermore, we propose that a number of literature reports form an argument against a contribution from cell death. Indeed we, and others, have presented evidence, which strongly suggests the involvement of cell death to be minimal. Taken together, these data would appear to rule out various confounding factors, leaving DNA repair pathways as the principal source of urinary purine, if not DNA, lesions enabling such measurements to be used as indicators of repair.

Asunto(s)

Daño del ADN , Reparación del ADN , ADN/orina , Desoxiguanosina/análogos & derivados , Guanina/análogos & derivados , Guanina/orina , Estrés Oxidativo , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Muerte Celular , Desoxiguanosina/orina , Dieta , Humanos , Modelos Biológicos , Ratas

RESUMEN

Familial amyotrophic lateral sclerosis (FALS) is caused, in 20% of cases, by mutations in the Cu/Zn superoxide dismutase gene (SOD1). Although motor neuron injury occurs through a toxic gain of function, the precise mechanism(s) remains unclear. Using an established NSC34 cellular model for SOD1-associated FALS, we investigated the effects of mutant SOD1 specifically in cells modelling the vulnerable cell population, the motor neurons, without contamination from non-neuronal cells present in CNS. Using gene expression profiling, 268 transcripts were differentially expressed in the presence of mutant human G93A SOD1. Of these, 197 were decreased, demonstrating that the presence of mutant SOD1 leads to a marked degree of transcriptional repression. Amongst these were a group of antioxidant response element (ARE) genes encoding phase II detoxifying enzymes and antioxidant response proteins (so-called 'programmed cell life' genes), the expression of which is regulated by the transcription factor NRF2. We provide evidence that dysregulation of Nrf2 and the ARE, coupled with reduced pentose phosphate pathway activity and decreased generation of NADPH, represent significant and hitherto unrecognized components of the toxic gain of function of mutant SOD1. Other genes of interest significantly altered in the presence of mutant SOD1 include several previously implicated in neurodegeneration, as well as genes involved in protein degradation, the immune response, cell death/survival and the heat shock response. Preliminary studies on isolated motor neurons from SOD1-associated motor neuron disease cases suggest key genes are also differently expressed in the human disease.

Asunto(s)

Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Neuronas Motoras/metabolismo , Mutación , Superóxido Dismutasa/genética , Transactivadores/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Antioxidantes , Apoptosis/genética , Línea Celular , Supervivencia Celular/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neuronas Motoras/patología , Factor 2 Relacionado con NF-E2 , Degeneración Nerviosa/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Elementos de Respuesta , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1

RESUMEN

Colorectal cancer (CRC) is responsible for the second highest associated mortality in Western Europe and the United States. Approximately 95% of CRC is sporadic and believed to involve environmental agents and chronic inflammation as causal elements. Several recent studies have suggested a link with diet, in particular, red meat, dietary fats, and low consumption of vegetables. Lipid peroxidation and arachidonic acid metabolism have specifically been implicated in genotoxicity, tumor initiation, and promotion. We have examined the global gene expression profiles (Affymetrix; HU133A) of differentiated vs. undifferentiated colonocytes (CRL-1807), with and without vitamin E supplementation, while undergoing a lipid peroxidative stress. Malondialdehyde and hydroxynonenal, generated by heating a mixture of linoleic and linolenic acid, caused DNA adduct formation identified by immunofluoresence. We also observed a decreased ability for vitamin E to upregulate detoxifying enzymes against free-radical peroxidation, with the exception of mitochondrial superoxide dismutase in undifferentiated cells. However, there was an increased ability in undifferentiated, rather than in differentiated, colonic cells to detect DNA damage, initiate cytostasis, and then effect subsequent DNA repair and apoptosis, in the presence of vitamin E. The expression profile implies less genotoxic stress is experienced in vitamin E-supplemented colonocytes, particularly undifferentiated cells, and points to a mechanism by which dietary supplementation may prevent genotoxic damage and subsequent carcinogenic events in the colon, by both antioxidant and non-antioxidant-related mechanisms.

Asunto(s)

Neoplasias del Colon/etiología , Neoplasias del Colon/genética , Dieta , Expresión Génica/efectos de los fármacos , Vitamina E/farmacología , Anticarcinógenos/farmacología , Antioxidantes/farmacología , Ácido Araquidónico/metabolismo , Aductos de ADN/análisis , Suplementos Dietéticos , Perfilación de la Expresión Génica , Humanos , Peroxidación de Lípido , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/efectos de los fármacos , ARN Mensajero/análisis , Células Tumorales Cultivadas

RESUMEN

The application of an antiserum to ultraviolet radiation (UVR)-damaged DNA is presented. A novel experimental system was employed to ascertain the limits of detection for this antiserum. Using a DNA standard containing a known amount of dimer, the limits of detection were found to be 0.9 fmol of dimer. This was compared to a limit of 20-50 fmol dimer using gas chromatography-mass spectrometry (GC-MS). Induction of thymine dimers in DNA following UVR exposure, as assessed using this antiserum in an enzyme-linked immunosorbent assay (ELISA), was compared with GC-MS measurements. The ELISA method successfully demonstrated the induction of lesions in DNA irradiated either with UVC or UVB, although despite high sensitivity, no discernible binding was seen to UVA-irradiated DNA. The antiserum was also shown to be applicable to immunocytochemistry, localising damage in the nuclei of UVR exposed keratinocytes in culture. The ability of the antiserum to detect DNA damage in skin biopsies of individuals exposed to sub-erythemal doses of UVR was also demonstrated. Moreover, the subsequent removal of this damage, as evidenced by a reduction in antiserum staining, was noted in sections of biopsies taken in the hours following irradiation.

Asunto(s)

Daño del ADN , Reparación del ADN/efectos de la radiación , Inmunoquímica/métodos , Rayos Ultravioleta/efectos adversos , Animales , Antígenos/análisis , Bovinos , Línea Celular , ADN/química , ADN/inmunología , ADN/efectos de la radiación , Ensayo de Inmunoadsorción Enzimática/métodos , Cromatografía de Gases y Espectrometría de Masas , Humanos , Técnicas In Vitro , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Microscopía Confocal , Dímeros de Pirimidina/análisis , Dímeros de Pirimidina/inmunología , Dímeros de Pirimidina/efectos de la radiación , Piel/metabolismo , Piel/efectos de la radiación

RESUMEN

The involvement of oxidatively modified low density lipoprotein (LDL) in the development of CHD is widely described. We have produced two antibodies, recognizing the lipid oxidation product malondialdehyde (MDA) on whole LDL or ApoB-100. The antibodies were utilized in the development of an ELISA for quantitation of MDA-LDL in human plasma. Intra- and inter-assay coefficients of variation (% CV) were measured as 4.8 and 7.7%, respectively, and sensitivity of the assay as 0.04 micro g/ml MDA-LDL. Recovery of standard MDA-LDL from native LDL was 102%, indicating the ELISA to be specific with no interference from other biomolecules. Further validation of the ELISA was carried out against two established methods for measurement of lipid peroxidation products, MDA by HPLC and F(2)-isoprostanes by GC-MS. Results indicated that MDA-LDL is formed at a later stage of oxidation than either MDA or F(2)-isoprostanes. In vivo analysis demonstrated that the ELISA was able to determine steady-state concentrations of plasma MDA-LDL (an end marker of lipid peroxidation). A reference range of 34.3 +/- 8.8 micro g/ml MDA-LDL was established for healthy individuals. Further, the ELISA was used to show significantly increased plasma MDA-LDL levels in subjects with confirmed ischemic heart disease, and could therefore possibly be of benefit as a diagnostic tool for assessing CHD risk.

Asunto(s)

Ensayo de Inmunoadsorción Enzimática/métodos , Lipoproteínas LDL/sangre , Malondialdehído/sangre , Isquemia Miocárdica/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apolipoproteínas B/sangre , Apolipoproteínas B/inmunología , Estudios de Casos y Controles , Cobre/farmacología , F2-Isoprostanos/metabolismo , Femenino , Cromatografía de Gases y Espectrometría de Masas , Humanos , Inmunoglobulinas/inmunología , Peroxidación de Lípido/fisiología , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Isquemia Miocárdica/patología , Oxidación-Reducción , Conejos , Factores de Riesgo , Tiobarbitúricos/metabolismo

Asunto(s)

Reparación del ADN , Desoxiguanosina/análogos & derivados , Homeostasis , Oxidación-Reducción , 8-Hidroxi-2'-Desoxicoguanosina , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Daño del ADN , Desoxiguanosina/sangre , Desoxiguanosina/metabolismo , Desoxiguanosina/orina , Regulación de la Expresión Génica , Glutatión/fisiología , Estrés Oxidativo , Factor de Transcripción AP-1/metabolismo

RESUMEN

Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Although more than 20 base lesions have been identified, only a fraction of these have received appreciable study, most notably 8-oxo-2'deoxyguanosine. This lesion has been the focus of intense research interest and been ascribed much importance, largely to the detriment of other lesions. The present work reviews the basis for the biological significance of oxidative DNA damage, drawing attention to the multiplicity of proteins with repair activities along with a number of poorly considered effects of damage. Given the plethora of (often contradictory) reports describing pathological conditions in which levels of oxidative DNA damage have been measured, this review critically addresses the extent to which the in vitro significance of such damage has relevance for the pathogenesis of disease. It is suggested that some shortcomings associated with biomarkers, along with gaps in our knowledge, may be responsible for the failure to produce consistent and definitive results when applied to understanding the role of DNA damage in disease, highlighting the need for further studies.

Asunto(s)

Daño del ADN , Predisposición Genética a la Enfermedad , Mutación , Estrés Oxidativo , Aductos de ADN/química , Aductos de ADN/metabolismo , Reparación del ADN , Humanos , Neoplasias/genética , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

The induction and repair of DNA damage has been shown to occur heterogeneously throughout the mammalian genome. As a consequence, analysis of these parameters at a global genome level may not reflect important gene-level events. Few techniques have been established to explore quantitatively gene-specific DNA damage and repair. Most of these are polymerase chain reaction (PCR)-based assays and are relatively insensitive, relying on decreased PCR amplification arising from damage in template DNA. We have developed a quantitative assay that combines specific immunocapture of damaged DNA by an antiserum specific for thymine dimers (IgG479), with PCR amplification of a 149 bp fragment of the human H-ras proto-oncogene. Quantification of DNA damage was based upon proportionality between the amount of the PCR product and the initial amount of damage. Detection of thymine dimers was possible with nanogram amounts of genomic DNA and increased in a linear, dose-responsive manner. Using this assay, gene-level induction of thymine dimers was shown to be directly proportional to levels induced in the global genome of ultraviolet radiation (UVR)-exposed, extracted DNA as measured by gas chromatography-mass spectrometry (GC-MS). This result suggests that global damage assessments do indeed reflect gene-level events although we predict that this relationship may not be maintained when applied to a cellular system. These findings demonstrate the suitability of this approach to the detection of UVR-induced DNA damage at the level of individual genes.

Asunto(s)

Daño del ADN , Daño del ADN/inmunología , Inmunoglobulina G/inmunología , Reacción en Cadena de la Polimerasa/métodos , Dímeros de Pirimidina/análisis , Rayos Ultravioleta/efectos adversos , Especificidad de Anticuerpos , Daño del ADN/genética , Ensayo de Inmunoadsorción Enzimática , Genes ras/genética , Genes ras/inmunología , Genes ras/efectos de la radiación , Humanos , Poli T/inmunología , Poli T/efectos de la radiación , Proto-Oncogenes Mas , Dímeros de Pirimidina/genética

RESUMEN

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p > 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC(50) values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2'-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Aductos de ADN/metabolismo , Daño del ADN , Desoxicitidina/metabolismo , Glioxal/metabolismo , Animales , Anticuerpos Monoclonales/química , Sitios de Unión de Anticuerpos , Línea Celular Transformada , ADN/química , ADN/efectos de los fármacos , ADN/metabolismo , Aductos de ADN/análisis , Aductos de ADN/inmunología , Desoxicitidina/análogos & derivados , Desoxicitidina/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Cromatografía de Gases y Espectrometría de Masas , Glioxal/inmunología , Glioxal/toxicidad , Queratinocitos , Ratones , Ratones Endogámicos BALB C , Estrés Oxidativo

RESUMEN

Epidemiological studies strongly suggest associations between chronic exposure to endogenous oestrogens and the development of breast and gynaecological tumours. Two mechanisms by which 17 beta-oestradiol (E2) may enhance tumorigenesis are: (i) enhancement of cell proliferation and (ii) the production of reactive, genotoxic metabolites. Here we suggest an additional mechanism, inhibition of DNA repair. The removal of UV-induced thymine dimers from human keratinocytes, reflective of nucleotide excision repair, was significantly attenuated by treatment of cells with E2. In contrast, treatment with 17 alpha-oestradiol had no effect. Mechanisms are proposed for this effect of E2, which may contribute to its carcinogenic potential.

Asunto(s)

Reparación del ADN/efectos de los fármacos , Estradiol/farmacología , Queratinocitos/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , ADN/química , ADN/metabolismo , ADN/efectos de la radiación , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Dímeros de Pirimidina/análisis , Dímeros de Pirimidina/metabolismo , Estereoisomerismo , Rayos Ultravioleta