RESUMO
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.
Assuntos
Ácido Ascórbico/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Vitaminas/farmacologia , 8-Hidroxi-2'-Desoxiguanosina , Apoptose/efeitos dos fármacos , Ácido Ascórbico/análogos & derivados , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Marcadores Genéticos , Genômica , Glioxal/metabolismo , Humanos , Ferro/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Modelos Biológicos , Dímeros de Pirimidina/metabolismoRESUMO
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.
Assuntos
Desoxiguanosina/análogos & derivados , Oligonucleotídeos/urina , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Desoxiguanosina/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Liofilização , Humanos , Masculino , Oligonucleotídeos/químicaRESUMO
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.
Assuntos
Antioxidantes/administração & dosagem , Dieta , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Vesículas Transportadoras/genética , alfa-Tocoferol/administração & dosagem , Animais , Antioxidantes/análise , Transporte Biológico/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , alfa-Tocoferol/análiseRESUMO
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.
Assuntos
Nanotubos de Carbono/toxicidade , Células A549 , Apoptose/efeitos dos fármacos , Ácidos Carboxílicos/química , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Humanos , Hidróxidos/química , L-Lactato Desidrogenase/metabolismo , Nanotubos de Carbono/química , Necrose/induzido quimicamenteRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Neurônios Motores/metabolismo , Mutação , Superóxido Dismutase/genética , Transativadores/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Antioxidantes , Apoptose/genética , Linhagem Celular , Sobrevivência Celular/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neurônios Motores/patologia , Fator 2 Relacionado a NF-E2 , Degeneração Neural/genética , Análise de Sequência com Séries de Oligonucleotídeos , Elementos de Resposta , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1RESUMO
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.
Assuntos
Dano ao DNA , Predisposição Genética para Doença , Mutação , Estresse Oxidativo , Adutos de DNA/química , Adutos de DNA/metabolismo , Reparo do DNA , Humanos , Neoplasias/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Dano ao DNA , Suplementos Nutricionais , Expressão Gênica , Humanos , Oxidantes/metabolismo , Oxidantes/farmacologia , Estresse Oxidativo , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Dano ao DNA , Reparo do DNA , DNA/urina , Desoxiguanosina/análogos & derivados , Guanina/análogos & derivados , Guanina/urina , Estresse Oxidativo , 8-Hidroxi-2'-Desoxiguanosina , Animais , Morte Celular , Desoxiguanosina/urina , Dieta , Humanos , Modelos Biológicos , RatosRESUMO
Oxidative DNA damage has been implicated to be important in the pathogenesis of many diseases, including cancer and heart disease. The assessment of damage in various biological matrices, such as DNA, serum, and urine, is vital to understanding this role and subsequently devising intervention strategies. Despite the numerous techniques to measure oxidative DNA damage products in urine, it remains unclear what these measurements truly represent. Sources of urinary lesions may include the diet, cell death, and, of most interest, DNA repair. Were it possible to exclude the two former contributions, a noninvasive assay for DNA repair would be invaluable in the study of DNA damage and disease. This review highlights that, although progress has been made, significant work remains. Diet, cell death, and repair need continued examination to further elucidate the kinetics of lesion formation and clearance in vivo. Studies from our laboratory and others are making appreciable progress towards the interpretation of urinary lesion measurements along with the development of urinary assays to evaluate DNA repair. Upon establishment of these details, urinary oxidative DNA damage measurements may become more than a reflection of generalized oxidative stress.
Assuntos
Dano ao DNA , DNA/metabolismo , DNA/urina , Estresse Oxidativo , Urinálise , Dieta , Substâncias Perigosas , HumanosRESUMO
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.
Assuntos
Reparo do DNA/efeitos dos fármacos , Estradiol/farmacologia , Queratinócitos/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , DNA/química , DNA/metabolismo , DNA/efeitos da radiação , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Dímeros de Pirimidina/análise , Dímeros de Pirimidina/metabolismo , Estereoisomerismo , Raios UltravioletaRESUMO
DNA is susceptible to damage by reactive oxygen species (ROS). ROS are produced during normal and pathophysiological processes in addition to ionizing radiation, environmental mutagens, and carcinogens. 8-oxo-2'-deoxyguanosine (8-oxodG) is probably one of the most abundant DNA lesion formed during oxidative stress. This potentially mutagenic lesion causes G --> T transversions and is therefore an important candidate lesion for repair, particularly in mammalian cells. Several pathways exist for the removal, or repair, of this lesion from mammalian DNA. The most established is via the base excision repair enzyme, human 8-oxoguanine glycosylase (hOgg1), which acts in combination with the human apurinic endonuclease (hApe). The latter is known to respond to regulation by redox reactions and may act in combination with hOgg1. We discuss evidence in this review article concerning alternative pathways in humans, such as nucleotide excision repair (NER), which could possibly remove the 8-oxodG lesion. We also propose that redox-active components of the diet, such as vitamin C, may promote such repair, affecting NER specifically.
Assuntos
Reparo do DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , 8-Hidroxi-2'-Desoxiguanosina , Biomarcadores/urina , Humanos , Oxirredução , Espécies Reativas de OxigênioRESUMO
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.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Lipoproteínas LDL/sangue , Malondialdeído/sangue , Isquemia Miocárdica/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apolipoproteínas B/sangue , Apolipoproteínas B/imunologia , Estudos de Casos e Controles , Cobre/farmacologia , F2-Isoprostanos/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Imunoglobulinas/imunologia , Peroxidação de Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Isquemia Miocárdica/patologia , Oxirredução , Coelhos , Fatores de Risco , Tiobarbitúricos/metabolismoRESUMO
Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p =.001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p =.0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected.
Assuntos
Ácido Ascórbico/farmacologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Desoxicitidina/metabolismo , Glioxal/metabolismo , Adutos de DNA/metabolismo , Desoxicitidina/sangue , Suplementos Nutricionais , Ensaio de Imunoadsorção Enzimática , Feminino , Glioxal/sangue , Humanos , Masculino , Malondialdeído/sangue , Dímeros de Pirimidina/sangue , Dímeros de Pirimidina/urina , Fatores de TempoRESUMO
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.
Assuntos
Dano ao DNA , Dano ao DNA/imunologia , Imunoglobulina G/imunologia , Reação em Cadeia da Polimerase/métodos , Dímeros de Pirimidina/análise , Raios Ultravioleta/efeitos adversos , Especificidade de Anticorpos , Dano ao DNA/genética , Ensaio de Imunoadsorção Enzimática , Genes ras/genética , Genes ras/imunologia , Genes ras/efeitos da radiação , Humanos , Poli T/imunologia , Poli T/efeitos da radiação , Proto-Oncogene Mas , Dímeros de Pirimidina/genéticaRESUMO
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.
Assuntos
Dano ao DNA , Reparo do DNA/efeitos da radiação , Imunoquímica/métodos , Raios Ultravioleta/efeitos adversos , Animais , Antígenos/análise , Bovinos , Linhagem Celular , DNA/química , DNA/imunologia , DNA/efeitos da radiação , Ensaio de Imunoadsorção Enzimática/métodos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Técnicas In Vitro , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Microscopia Confocal , Dímeros de Pirimidina/análise , Dímeros de Pirimidina/imunologia , Dímeros de Pirimidina/efeitos da radiação , Pele/metabolismo , Pele/efeitos da radiaçãoRESUMO
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.
Assuntos
Neoplasias do Colo/etiologia , Neoplasias do Colo/genética , Dieta , Expressão Gênica/efeitos dos fármacos , Vitamina E/farmacologia , Anticarcinógenos/farmacologia , Antioxidantes/farmacologia , Ácido Araquidônico/metabolismo , Adutos de DNA/análise , Suplementos Nutricionais , Perfilação da Expressão Gênica , Humanos , Peroxidação de Lipídeos , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
Due to various confounding factors, namely dietary contribution and cell death, measurement of urinary 8-oxo-2'-deoxyguanosine (8-oxodG) has long been considered to be no more than a marker of generalised oxidative stress. Indeed, the action of no single enzyme has been reported to excise 8-oxodG from DNA. However, analysis of recent research has suggested that these confounders may be circumvented, which, combined from work from the authors' laboratory, indicates that urinary 8-oxodG has the potential to become a most important marker of oxidative damage to, and repair of, DNA.
Assuntos
Biomarcadores/análise , Reparo do DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Doenças Urológicas/urina , 8-Hidroxi-2'-Desoxiguanosina , Animais , Cromatografia Líquida de Alta Pressão , Dano ao DNA , Humanos , Estresse Oxidativo , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Biologia Computacional , Quadruplex G , Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-myc/genética , 8-Hidroxi-2'-Desoxiguanosina , DNA/química , DNA/genética , DNA/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Regulação da Expressão Gênica , OxirreduçãoAssuntos
Reparo do DNA , Desoxiguanosina/análogos & derivados , Homeostase , Oxirredução , 8-Hidroxi-2'-Desoxiguanosina , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Dano ao DNA , Desoxiguanosina/sangue , Desoxiguanosina/metabolismo , Desoxiguanosina/urina , Regulação da Expressão Gênica , Glutationa/fisiologia , Estresse Oxidativo , Fator de Transcrição AP-1/metabolismoRESUMO
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.