RESUMEN
A diet high in linoleic acid (an ω-6 PUFA) increased the formation of miscoding etheno (ε)--DNA adducts in WBC-DNA of women, but not in men (Nair et al., Cancer Epidemiol Biomark Prev 1997;6:597-601). This gender specificity could result from an interaction between ω-6 PUFA intake and estrogen catabolism, via redox-cycling of 4-hydroxyestradiol (4-OH-E(2) ) and subsequent lipid peroxidation (LPO). In this study, we investigated whether in premenopausal women LPO-derived adducts in WBC-DNA are affected by serum concentration of 2- and 4-hydroxyestradiol metabolites and by fatty acid intake. DNA extracted from buffy coat and plasma samples, both blindly coded from healthy women (N = 124, median age 40 year) participating in the EPIC-Heidelberg cohort study were analyzed. Three LPO-derived exocyclic DNA adducts, εdA, εdC and M(1) dG were quantified by immuno-enriched (32) P-postlabelling and estradiol metabolites by ultra-sensitive GC-mass spectrometry. Mean M(1) dG levels in WBC-DNA were distinctly higher than those of εdA and εdC, and all were positively and significantly interrelated. Serum levels of 4-OH-E(2) , but not of 2-OH-E(2) , metabolites were positively related to etheno DNA adduct formation. Positive correlations existed between M(1) dG levels and linoleic acid intake or the ratios of dietary linoleic acid/oleic acid and PUFA/MUFA. Aerobic incubation of 4-OH-E(2) , arachidonic acid and calf thymus DNA yielded two to threefold higher etheno DNA adduct levels when compared with assays containing 2-OH-E(2) instead. In conclusion, this study is the first to compare M(1) dG and etheno-DNA adducts and serum estradiol metabolites in human samples in the same subjects. Our results support a novel mechanistic link between estradiol catabolism, dietary ω-6 fatty acid intake and LPO-induced DNA damage supported by an in vitro model. Similar studies in human breast epithelial tissue and on amplification of DNA-damage in breast cancer patients are in progress.
Asunto(s)
Aductos de ADN/biosíntesis , Grasas de la Dieta/administración & dosificación , Estrógenos/metabolismo , Ácidos Grasos Omega-6/administración & dosificación , Peroxidación de Lípido , Linfocitos/metabolismo , Premenopausia , Adulto , Estudios de Cohortes , Daño del ADN , Estradiol/análogos & derivados , Estradiol/sangre , Estrógenos/sangre , Estrógenos de Catecol , Femenino , Humanos , Ácido Linoleico/administración & dosificación , Ácido Linoleico/metabolismo , Linfocitos/citología , Ácido Oléico/metabolismoRESUMEN
Methylation of cytidine at dCpdG sequences regulates gene expression and is altered in many chronic inflammatory diseases. Inflammation generates lipid peroxidation (LPO) products which can react with deoxycytidine, deoxyadenosine, and deoxyguanosine in DNA to form pro-mutagenic exocyclic etheno-nucleoside residues. Since 5-methyl-2'-deoxycytidine (5mdC) residues exhibit increased nucleophilicity at N3, they should be even better targets for LPO products. We synthesized and characterized 3,N(4)-etheno-5-methyl-2'-deoxycytidine-3'-phosphate and showed that LPO products can indeed form the corresponding etheno-5mdC (ε5mdC) lesion in DNA in vitro. Our newly developed (32)P-postlabeling method was subsequently used to detect ε5mdC lesions in DNA from human white blood cells, lung, and liver at concentrations 4-10 times higher than that observed for etheno adducts on nonmethylated cytidine. Our new detection method can now be used to explore the hypothesis that this DNA lesion perturbs the DNA methylation status.
Asunto(s)
Aductos de ADN , ADN/metabolismo , Desoxicitidina/análogos & derivados , Acetaldehído/análogos & derivados , Acetaldehído/metabolismo , Ácido Araquidónico/metabolismo , Metilación de ADN , Desoxicitidina/metabolismo , Femenino , Genoma , Humanos , Leucocitos/metabolismo , Peroxidación de Lípido , Hígado/metabolismo , Pulmón/metabolismoRESUMEN
Tobacco smoke contains many alkylating agents that can react with DNA to produce O(4)-ethylthymidine (O(4)-etT) and several other types of promutagenic base modifications. Our aims were (i) to confirm results of a pilot study (Godschalk, R., Nair, J., Schooten, F. J., Risch, A., Drings, P., Kayser, K., Dienemann, H. and Bartsch, H. (2002) Comparison of multiple DNA adduct types in tumor adjacent human lung tissue: effect of cigarette smoking. Carcinogenesis, 23, 2081-2086) on the formation of O(4)-etT in smokers' lung; (ii) to explore associations between levels of O(4)-etT and smoking status and (iii) to investigate whether a correlation exists between levels of O(4)-etT and bulky (polycyclic aromatic hydrocarbons-derived) DNA adducts. Archived DNA samples originated from histologically normal peripheral lung tissues of 64 Hungarian lung cancer patients, who underwent lung resection. O(4)-etT was determined by an immunoenriched (32)P-postlabelling-high-performance liquid chromatography method. Levels of bulky DNA adducts were determined by the nuclease P1 adduct-enriched (32)P-postlabelling. O(4)-etT levels ranged from 0.01 to 3.91 adducts/10(8) thymidines. In the combined group of subjects who smoked until surgery or gave up smoking at most 1 year before it, the mean level of O(4)-etT was 1.7-fold (P = 0.015) and of bulky DNA adducts 2.2-fold (P < 0.0001) higher than in long-term ex-smokers (LES) and never-smokers (NS) combined. We found no significant correlation between the individual levels of the two DNA adduct types. No dose-response was detected between O(4)-etT formation and smoking dose. In one-third of LES, O(4)-etT levels were above the 2.0-fold mean level of adducts found in NS, indicating its high persistence. Our results confirm the smoking-related formation of O(4)-etT in human lung DNA that should be explored as biomarker. Its long persistence in target tissue implicates a role of this potentially miscoding lesion in tobacco smoking-associated cancers.
Asunto(s)
Aductos de ADN/metabolismo , Pulmón/metabolismo , Fumar/metabolismo , Timidina/análogos & derivados , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Timidina/química , Timidina/metabolismoRESUMEN
BACKGROUND: Oxidative stress enhances lipid peroxidation (LPO), which both are implicated in the promotion and progression stages of carcinogenesis, in particular under conditions of chronic inflammation and infections. Exocyclic etheno-DNA adducts, which are formed by LPO-products such as 4-hydroxy -2-nonenal, are strongly pro-mutagenic DNA lesions. METHODS: The development of ultra-sensitive detection methods for etheno-adducts in human tissues, white blood cells( WBC) and urine has provided evidence that these adducts are elevated in affected organs of cancer-prone patients, probably acting as a driving force to malignancy. RESULTS: Two recent studies that yielded some new insights into disease causation are briefly reviewed: DNA-damage in WBC of mother-newborn child pairs, and lipid peroxidation derived DNA damage in patients with cancer-prone liver diseases. Our results indicate that biomonitoring of etheno-DNA adducts in humans are promising tools (i) to better understand disease aetiopathogenesis, allowing hazard identification(ii) to monitor disease progression and (iii) to verify the efficacy of chemopreventive and therapeutic interventions. Such clinical trials are warranted.
Asunto(s)
Biomarcadores/análisis , Carcinógenos Ambientales/toxicidad , Neoplasias/epidemiología , Animales , Transformación Celular Neoplásica , Daño del ADN , Progresión de la Enfermedad , Humanos , Leucocitos/fisiología , Peroxidación de Lípido , Hepatopatías/patología , Neoplasias/fisiopatología , Estrés OxidativoRESUMEN
Exocyclic etheno-DNA adducts are formed by the reaction of lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE) with DNA bases to yield 1,N (6)-etheno-2'-deoxyadenosine (εdA), 3,-N (4)-etheno-2'-deoxycytidine (εdC), and etheno-2'-deoxyguanosine. These adducts act as a driving force for many human malignancies and are elevated in the organs of cancer-prone patients suffering from chronic inflammation and infections. Here, we describe the ultrasensitive and specific techniques for the detection of εdA and εdC in tissue and white blood cell (WBC) DNA. This approach is based on -combined immunopurification by monoclonal antibodies and (32)P-postlabeling analysis. The detection limit is about five adducts per 10(10) parent nucleotides, requiring 5-10 µg of DNA. In addition, we describe techniques for immunohistochemical detection of εdA and εdC in tissue biopsies, and the approaches for the -analysis of εdA and εdC excreted in urine. The utility of these detection methods for human studies is based on: (1) high sensitivity and specificity, (2) low amounts of DNA required, (3) capability to detect "background" levels of etheno-DNA adducts in biopsies, WBC, and urine samples of healthy subjects, and (4) reliable monitoring of the disease-related increase of these substances in patients.The described methods are useful in diagnosis and monitoring of chronic degenerative diseases, including cancer, atherosclerosis, and neurodegenerative disorders.
Asunto(s)
Aductos de ADN/orina , Inmunohistoquímica/métodos , Marcaje Isotópico/métodos , Leucocitos/metabolismo , Especificidad de Órganos , Aldehídos , Animales , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , ADN/aislamiento & purificación , Fluorescencia , Humanos , Hidrólisis , Hígado/metabolismo , Hígado/patología , Radioisótopos de Fósforo , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chronic infection by the liver fluke Opisthorchis viverrini is a strong risk factor for cholangiocarcinoma. To clarify the involvement of oxidative stress and lipid peroxidation-derived DNA damage, etheno (epsilon)-DNA adducts (epsilondA, epsilondC) in WBC and plasma alpha-tocopherol were measured in samples collected from O. viverrini-infected Thai patients (n = 50) and healthy noninfected volunteers (n = 20). epsilondA and epsilondC levels were three to five times higher (P < 0.001) in infected patients than in controls; O. viverrini infection also increased two to three times in the plasma inflammatory indicators, 8-isoprostane, malondialdehyde, and nitrate/nitrite. Mean plasma alpha-tocopherol levels were two times lower in patients than in healthy controls (P < 0.001). Two months after a single dose to infected patients of the antiparasitic drug praziquantel, epsilondA and epsilondC levels in WBC were decreased to control level (P < 0.03); plasma 8-isoprostane, malondialdehyde, nitrate/nitrite, and alkaline phosphatase (ALP) were concomitantly lowered. epsilondA and epsilondC levels in WBC were positively correlated with plasma 8-isoprostane, malondialdehyde, and nitrate/nitrite levels and ALP activity, whereas plasma alpha-tocopherol levels showed inverse correlations. We conclude that chronic O.viverrini infection induces an accumulation of lipid peroxidation-derived DNA damage through oxidative/nitrative stress, which is lowered by the plasma alpha-tocopherol and by antiparasitic drug therapy. Etheno adducts in WBC and urine should be explored as a risk marker for opisthorchiasis-related cholangiocarcinoma, and to assess the efficacy of preventive and therapeutic interventions.
Asunto(s)
Antihelmínticos/uso terapéutico , Aductos de ADN/sangre , Peroxidación de Lípido/efectos de los fármacos , Opistorquiasis/sangre , Praziquantel/uso terapéutico , alfa-Tocoferol/sangre , Animales , Neoplasias de los Conductos Biliares/sangre , Neoplasias de los Conductos Biliares/parasitología , Neoplasias de los Conductos Biliares/prevención & control , Conductos Biliares Intrahepáticos/metabolismo , Conductos Biliares Intrahepáticos/parasitología , Colangiocarcinoma/sangre , Colangiocarcinoma/parasitología , Colangiocarcinoma/prevención & control , Aductos de ADN/efectos de los fármacos , Dinoprost/análogos & derivados , Dinoprost/sangre , Femenino , Humanos , Leucocitos/efectos de los fármacos , Masculino , Malondialdehído/sangre , Persona de Mediana Edad , Nitratos/sangre , Nitritos/sangre , Opistorquiasis/tratamiento farmacológico , Opisthorchis , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiologíaRESUMEN
Chronic inflammatory processes induce oxidative and nitrative stress that trigger lipid peroxidation (LPO), whereby DNA-reactive aldehydes such as trans-4-hydroxy-2-nonenal (HNE) are generated. Miscoding etheno-modified DNA adducts including 1,N(6)-etheno-2'-deoxyadenosine (epsilondA) are formed by reaction of HNE with DNA-bases which are excreted in urine, following elimination from tissue DNA. An ultrasensitive and specific immunoprecipitation/HPLC-fluorescence detection method was developed for quantifying epsilondA excreted in urine. Levels in urine of Thai and European liver disease-free subjects were in the range of 3-6 fmol epsilondA/micromol creatinine. Subjects with inflammatory cancer-prone liver diseases caused by viral infection or alcohol abuse excreted massively increased and highly variable epsilondA-levels. Groups of Thai subjects (N=21) with chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (HCC) due to HBV infection had 20, 73 and 39 times higher urinary epsilondA levels, respectively when compared to asymptomatic HBsAg carriers. In over two thirds of European patients (N=38) with HBV-, HCV- and alcohol-related liver disease, urinary epsilondA levels were increased 7-10-fold compared to healthy controls. Based on this pilot study we conclude: (i) high urinary epsilondA-levels, reflecting massive LPO-derived DNA damage in vivo may contribute to the development of HCC; (ii) epsilondA-measurements in urine and target tissues should thus be further explored as a putative risk marker to follow malignant progression of inflammatory liver diseases in affected patients; (iii) etheno adducts may serve as biomarkers to assess the efficacy of (chemo-)preventive and therapeutic interventions.
Asunto(s)
Daño del ADN , Desoxiadenosinas/orina , Hepatitis B/orina , Peroxidación de Lípido , Cirrosis Hepática Alcohólica/orina , Neoplasias Hepáticas/orina , Adulto , Anciano , Consumo de Bebidas Alcohólicas , Aldehídos/orina , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/orina , Estudios de Casos y Controles , Aductos de ADN/orina , Europa (Continente) , Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/metabolismo , Humanos , Cirrosis Hepática Alcohólica/genética , Neoplasias Hepáticas/genética , Masculino , Persona de Mediana Edad , Proyectos PilotoRESUMEN
UNLABELLED: Oxidative stress is thought to play a major role in the pathogenesis of hepatocellular cancer (HCC), a frequent complication of alcoholic liver disease (ALD). However, the underlying mechanisms are poorly understood. In hepatocytes of ALD patients, we recently detected by immunohistochemistry significantly increased levels of carcinogenic etheno-DNA adducts that are formed by the reaction of the major lipid peroxidation product, 4-hydroxynonenal (4-HNE) with nucleobases. In the current study, we show that protein-bound 4-HNE and etheno-DNA adducts both strongly correlate with cytochrome P450 2E1 (CYP2E1) expression in patients with ALD (r = 0.9, P < 0.01). Increased levels of etheno-DNA adducts were also detected in the liver of alcohol-fed lean (Fa/?) and obese (fa/fa) Zucker rats. The number of nuclei in hepatocytes stained positively for etheno-DNA adducts correlated significantly with CYP2E1 expression (r = 0.6, P = 0.03). To further assess the role of CYP2E1 in the formation of etheno-DNA adducts, HepG2 cells stably transfected with human CYP2E1 were exposed to ethanol with or without chlormethiazole (CMZ), a specific CYP2E1 inhibitor. Ethanol increased etheno-DNA adducts in the nuclei of CYP2E1-transfected HepG2 cells in a concentration-dependent and time-dependent manner, but not in vector mock-transfected control cells. CMZ blocked the generation of etheno-DNA adducts by 70%-90% (P < 0.01). CONCLUSION: Our data support the assumption that ethanol-mediated induction of hepatic CYP2E1 leading inter alia to highly miscoding lipid peroxidation-derived DNA lesions may play a central role in hepatocarcinogenesis in patients with ALD.
Asunto(s)
Aldehídos/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Aductos de ADN/metabolismo , Hepatopatías Alcohólicas/metabolismo , Adulto , Anciano , Animales , Biopsia , Línea Celular Tumoral , Etanol/efectos adversos , Femenino , Humanos , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Ratas , Ratas ZuckerRESUMEN
In thalassemia patients, iron overload can stimulate lipid peroxidation (LPO), thereby generating miscoding DNA adducts. Adducted DNA was measured in the lymphocytes of beta-Thal/Hb E patients and healthy controls and in the organs of thalassemic mice. epsilondA, epsilondC and M(1)dG residues were quantified by (32)P-postlabeling-TLC/HPLC. M(1)dG levels in lymphocyte DNA from patients were 4 times as high as in controls, while the increase in epsilondA and epsilondC was not significant. Adducted DNA accumulated in the liver of thalassemic mice having >2.7 mg Fe/g tissue dry weight; DNA adducts and iron were highly correlated. epsilondA was not specifically generated in certain mouse liver cell types as revealed by immunohistochemical staining. We found elevated LPO-induced DNA damage in the liver of thalassemic mouse and in lymphocytes, implicating that massive DNA damage occurs in the liver of thalassemia patients. We conclude that promutagenic LPO-derived DNA lesions are involved in the onset of hepatocellular carcinoma in these patients.
Asunto(s)
Aductos de ADN , Sobrecarga de Hierro/metabolismo , Peroxidación de Lípido , Hepatopatías/metabolismo , Linfocitos/patología , Talasemia beta/metabolismo , Adolescente , Adulto , Animales , Estudios de Casos y Controles , Niño , Daño del ADN , Desoxiadenosinas/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Glutatión Peroxidasa/metabolismo , Humanos , Técnicas para Inmunoenzimas , Sobrecarga de Hierro/genética , Sobrecarga de Hierro/patología , Hepatopatías/genética , Hepatopatías/patología , Linfocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Adulto Joven , Globinas beta/fisiología , Talasemia beta/genética , Talasemia beta/patologíaRESUMEN
OBJECTIVE: Oxidative DNA damage is believed to be involved in tumor formation and may be an important biomarker for malignant transition or relapse. A decrease of such damage has been observed in human and animal studies following dietary intervention and/or changes in lifestyle such as physical exercise at different levels of intensity. The purpose of this study was to carry out a clinical trial comparing the effects of a short-term (2 weeks) exercise program of moderate intensity (0.3-0.4 x maximal exercise capacity) (MI) versus high intensity (0.5-0.6 x maximal exercise capacity) (HI) on individual urinary excretion of 8-oxo-dG before and after completion of the exercise programs. MATERIAL AND METHODS: In this short-term, prospective and randomized trial, 19 patients with colorectal cancer were allocated to the MI group following primary therapy and 29 to the HI group. Urinary 8-oxo-dG excretion concentration was determined by a highly sensitive detection method using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS). Concentrations were determined immediately before and after completion of the exercise programs. RESULTS: Using HPLC-ESI-MS, it was shown that MI exercise significantly reduced urinary 8-oxo-dG excretion levels from 8.47 +/- 1.99 to 5.81 +/- 1.45 (ng/mg creatinine, mean +/- SE, p = 0.02), whereas HI exercise resulted in a non-significant increase from 5.00 +/- 1.31 to 7.11 +/- 1.63 (ng/mg creatinine, p = 0.18). Clinical characteristics (gender, age, body mass index (BMI), diet, chemotherapy/irradiation) were not associated/correlated with urinary 8-oxo-dG levels. CONCLUSIONS: By using HPLC-ESI-MS it was shown that short-term MI exercise after primary therapy in patients with colorectal cancer was associated with lower levels of urinary 8-oxo-dG, suggesting decreased oxidative DNA damage. In contrast, HI exercise tended to increase DNA damage. A prospective trial is now warranted to prove that reduced oxidative DNA damage lowers the risk of relapse of colorectal cancer in treated patients.
Asunto(s)
Carcinoma/terapia , Neoplasias Colorrectales/terapia , Daño del ADN/genética , Desoxiguanosina/análogos & derivados , Terapia por Ejercicio/métodos , Estrés Oxidativo/genética , 8-Hidroxi-2'-Desoxicoguanosina , Biomarcadores de Tumor/orina , Carcinoma/genética , Carcinoma/orina , Cromatografía Líquida de Alta Presión/métodos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/orina , Terapia Combinada/métodos , Desoxiguanosina/orina , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodos , Factores de Tiempo , Resultado del TratamientoRESUMEN
Chronic infection by Opisthorchis viverrini (OV) is a strong risk factor for developing cholangiocarcinoma (CCA). To clarify the involvement of oxidative stress and lipid peroxidation (LPO)-derived DNA damage, the excretion of LPO-derived etheno DNA adducts was measured in urine samples collected from healthy volunteers and OV-infected Thai subjects. 1,N(6)-etheno-2'-deoxyadenosine (epsilondA) and 3,N(4)-etheno-2'-deoxycytidine (epsilondC) levels were quantified by immunoprecipitation/high-performance liquid chromatography/fluorescence detection and (32)P-postlabeling TLC. Excreted etheno adduct levels were related to indicators of inflammatory conditions [malondialdehyde (MDA) and nitrate/nitrite levels in urine and plasma alkaline phosphatase (ALP) activity]. Mean epsilondA and epsilondC levels were 3 to 4 times higher in urine of OV-infected patients; MDA, nitrate/nitrite, and ALP were also increased up to 2-fold. MDA and ALP were positively related to epsilondA excretion. Two months after a single dose of the antiparasitic drug Praziquantel, epsilondA and epsilondC concentrations in urine of OV-infected subjects were decreased; MDA, nitrate/nitrite, and ALP were concomitantly lowered. We conclude that chronic OV infection through oxidative/nitrative stress leads to increased urinary excretion of the etheno-bridged deoxyribonucleosides, reflecting high LPO-derived DNA damage in vivo. These promutagenic DNA etheno adducts in bile duct epithelial cells may increase the risk of OV-infected patients to later develop CCA. Urinary epsilondA and epsilondC levels should be explored (a) as noninvasive risk markers for developing opisthorchiasis-related CCA and (b) as promising biomarkers to assess the efficacy of preventive and therapeutic interventions.
Asunto(s)
Antihelmínticos/farmacología , Aductos de ADN/orina , Daño del ADN/efectos de los fármacos , Desoxiadenosinas/orina , Desoxicitidina/análogos & derivados , Opistorquiasis/orina , Praziquantel/farmacología , Adulto , Animales , Neoplasias de los Conductos Biliares/epidemiología , Neoplasias de los Conductos Biliares/etiología , Neoplasias de los Conductos Biliares/prevención & control , Conductos Biliares Intrahepáticos , Colangiocarcinoma/epidemiología , Colangiocarcinoma/etiología , Colangiocarcinoma/prevención & control , Cromatografía Líquida de Alta Presión , Desoxicitidina/orina , Heces/parasitología , Femenino , Humanos , Inmunoprecipitación , Incidencia , Masculino , Persona de Mediana Edad , Opistorquiasis/complicaciones , Opistorquiasis/epidemiología , Opisthorchis/aislamiento & purificación , Estrés Oxidativo , Factores de Riesgo , Tailandia/epidemiologíaRESUMEN
Thalassemic diseases including homozygous beta-thalassemia and beta-thalassemia/Hb E (beta-Thal/Hb E) are prevalent in Southeast Asia. Iron overload is a common complication in beta-thalassemia patients which induces intracellular oxidative stress and lipid peroxidation (LPO). LPO end products generate miscoding etheno adducts in DNA which after their repair are excreted in urine. We investigated whether urinary levels of 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) can serve as putative cancer risk markers in beta-Thal/Hb E patients. epsilondA and epsilondC levels were assayed in collected urine samples by immunoprecipitation-HPLC-fluorescence and 32P-postlabeling TLC, respectively. Mean epsilondA (fmol/micromol creatinine) levels in urine of beta-Thal/Hb E patients ranged from 4.8 to 120.4 (33.8+/-3.9; n=37) and were 8.7 times higher compared to asymptomatic controls (1.4-13.8; 3.9+/-0.8; n=20). The respective epsilondC levels ranged from 0.15 to 32.5 (5.2+/-1.3; n=37) and were increased some 13 times over controls (0.04-1.2; 0.4+/-0.7; n=20). epsilondC levels were correlated positively with NTBI (r=0.517; P=0.002), whereas epsilondA showed only a trend (r=0.257; P=0.124). We conclude that the strongly increased urinary excretion of etheno adducts indicates elevated LPO-induced DNA damage in internal organs such as the liver. These highly promutagenic lesions may contribute to the increased risk of thalassemia patients to develop hepatocellular carcinoma.
Asunto(s)
Daño del ADN , Desoxiadenosinas/orina , Desoxicitidina/análogos & derivados , Peroxidación de Lípido , Talasemia/orina , Adulto , Biomarcadores de Tumor/orina , Desoxicitidina/orina , Femenino , Humanos , Hígado/metabolismo , MasculinoRESUMEN
Persistent oxidative stress and excess lipid peroxidation (LPO), induced by inflammatory processes, impaired metal storage, and/or dietary imbalance, cause accumulations and massive DNA damage. This massive DNA damage, along with deregulation of cell homeostasis, leads to malignant diseases. Reactive aldehydes produced by LPO, such as 4-hydroxy-2-nonenal, malondialdehyde, acrolein, and crotonaldehyde, react directly with DNA bases or generate bifunctional intermediates which form exocyclic DNA adducts. Modification of DNA bases by these electrophiles, yielding promutagenic exocyclic adducts, is thought to contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. Ultrasensitive detection methods have facilitated studies of the concentrations of promutagenic DNA adducts in human tissues, white blood cells, and urine, where they are excreted as modified nucleosides and bases. Thus, immunoaffinity-(32)P-postlabeling, high-performance liquid chromatography-electrochemical detection, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, immunoslotblot assay, and immunohistochemistry have made it possible to detect background concentrations of adducts arising from endogenous LPO products in vivo and studies of their role in carcinogenesis. These background adduct levels in asymptomatic human tissues occur in the order of 1 adduct/10(8) and in organs affected by cancer-prone inflammatory diseases these can be 1 or 2 orders of magnitude higher. In this review, we critically discuss the accuracy of the available methods and their validation and summarize studies in which measurement of exocyclic adducts suggested new mechanisms of cancer causation, providing potential biomarkers for cancer risk assessment in humans with cancer-prone diseases.
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Aductos de ADN , Daño del ADN , Inflamación/metabolismo , Peroxidación de Lípido/fisiología , Neoplasias/etiología , Adenina/análogos & derivados , Adenina/análisis , Animales , Biomarcadores/análisis , Desoxiadenosinas/análisis , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Humanos , Malondialdehído/químicaRESUMEN
Intake of linoleic acid (LA) increased etheno-DNA adducts induced by lipid peroxidation (LPO) in white blood cells (WBC) of female but not of male volunteers [J. Nair, C.E. Vaca, I. Velic, M. Mutanen, L.M. Valsta, H. Bartsch, High dietary omega-6 polyunsaturated fatty acids drastically increase the formation of etheno-DNA adducts in white blood cells of female subjects, Cancer Epidemiol. Biomarkers Prev. 6 (1997) 597-601]. Etheno-adducts were measured in rats gavaged with LA, oleic acid (OA) and saturated fatty acid rich coconut oil for 30 days. DNA from organs and total WBC was analyzed for 1, N(6)-ethenodeoxyadenosine (varepsilondA) and 3, N(4)-ethenodeoxycytidine (varepsilondC) by immunoaffinity/(32)P-postlabeling. Colon was the most affected target with LA-treatment, where etheno-adducts were significantly elevated in both sexes. In WBC both adducts were elevated only in LA-treated females. Unexpectedly, OA treatment enhanced etheno-adduct levels in prostate 3-9 fold. Our results in rodents confirm the gender-specific increase of etheno-adducts in WBC-DNA, likely due to LPO induced by redox-cycling of 4-hydroxyestradiol. Colon was a target for LPO-derived DNA-adducts in both LA-treated male and female rats, supporting their role in omega-6 PUFA induced colon carcinogenesis.
Asunto(s)
Aductos de ADN/biosíntesis , Ácido Linoleico/toxicidad , Ácido Oléico/toxicidad , Aceites de Plantas/toxicidad , Animales , Bovinos , Aceite de Coco , Colon/efectos de los fármacos , Colon/metabolismo , Neoplasias del Colon/etiología , Aductos de ADN/análisis , Desoxiadenosinas/análisis , Desoxicitidina/análogos & derivados , Desoxicitidina/análisis , Femenino , Humanos , Técnicas In Vitro , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Ácido Linoleico/administración & dosificación , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ácido Oléico/administración & dosificación , Especificidad de Órganos , Aceites de Plantas/administración & dosificación , Ratas , Caracteres SexualesRESUMEN
Increased oxidative stress and subsequent lipid peroxidation (LPO) are thought to be critical events in the formation of atherosclerotic lesions in apolipoprotein E deficient mice (ApoE-KO). LPO derived reactive aldehydes react with DNA to form exocyclic etheno-DNA adducts. These pro-mutagenic DNA lesions are known to be involved in the initiation of carcinogenesis, but their role in the development of atherosclerosis is unknown. In the present study we show that levels of the LPO derived 1,N(6)-ethenodeoxyadenosine (varepsilondA) and 3,N(4)-ethenodeoxycytidine (varepsilondC) were both significantly lower in aorta of 12 weeks old ApoE-KO mice as compared to their wild type controls (1.6+/-0.3 versus 3.2+/-0.8 varepsilondA per 10(8) parent nucleotides, P=0.04 and 4.8+/-0.8 versus 9.2+/-2.1 for varepsilondC, P=0.02). Moreover, levels of both DNA adduct types were inversely related with total plasma cholesterol levels. Consequently, lowest etheno-DNA adduct levels were observed in ApoE-KO mice on a high fat diet. Hypercholesterolemia has previously been associated with increased expression of base excision repair (BER) enzymes, which could explain the lower levels of etheno-DNA adducts in ApoE-KO mice as compared to wild type controls. Indeed, increased staining for the BER-specific DNA repair enzyme apurinic/apyrimidinic endonuclease (Ape1/Ref1) was observed by immunohistochemistry in the endothelium and the first layers of arterial smooth muscle cells of ApoE-KO mice as compared to their wild type counterparts. A high fat diet further increased overall Ape1/Ref1 protein expression in ApoE-KO mice. Although these data suggest no role for increased LPO derived DNA damage in the onset of atherogenesis in ApoE-KO mice, the potentially modulating role of Ape1/Ref1 in the arterial wall deserves further attention.
Asunto(s)
Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Daño del ADN , Peroxidación de Lípido/genética , Animales , Apolipoproteínas E/genética , Aterosclerosis/genética , Aductos de ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Femenino , Inmunohistoquímica , Lípidos/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Estrés Oxidativo/genéticaRESUMEN
Atherosclerosis and cancer are characterized by uncontrolled cell proliferation and share common risk factors, such as cigarette smoking, dietary habits and ageing. Growth of smooth muscle cells (SMCs) in atherosclerotic plaques may result from DNA damage, caused either by exogenous mutagens or by agents endogenously generated due to oxidative stress and lipid peroxidation (LPO). Hydroxy-2-nonenal (HNE), a major LPO product, binds covalently to cellular DNA to form the exocyclic etheno-DNA-base adducts, 1,N(6)-ethenodeoxyadenine (varepsilondA) and 3,N(4)-ethenodeoxycytosine (varepsilondC). By applying an ultrasensitive (32)P-postlabeling-immunoaffinity method, varepsilondA and varepsilondC were quantified in abdominal aorta SMCs from 13 atherosclerotic patients and 3 non-smoking subjects without atherosclerotic lesions. The levels of etheno-adducts ranged for varepsilondA from 2.3 to 39.6/10(8)dA and for varepsilondC from 10.7 to 157.7/10(8)dC, with a high correlation between varepsilondA and varepsilondC (r=0.84, P=0.0001). Etheno-adduct levels were higher in atherosclerotic smokers than in ex-smokers for both varepsilondA (means 15.2 versus 7.3, P=0.06) and varepsilondC (71.9 versus 51.6, not significant). varepsilondC levels were higher in either ex-smokers (P=0.03) or smokers (P=0.07) than in non-smokers. There was a poor correlation between either varepsilondA or varepsilondC and 8-hydroxy-2'-deoxyguanosine, whereas significant positive correlations were detected with the levels of several postlabeled bulky aromatic DNA adducts. In conclusion, two different types of DNA damage may be involved in atherosclerotic plaque formation and progression: (i) bulky aromatic compounds, to which aorta SMCs are chronically exposed in smokers, can either covalently bind to DNA, induce redox-cycling via quinone intermediates and/or activate local chronic inflammatory processes in the arterial wall; ii) this in turn leads to a self perpetuating generation of reactive oxygen species, LPO-products and increasing DNA-damage, as documented by the presence of high levels of miscoding etheno-DNA adducts in human aorta SMCs.
Asunto(s)
Aorta Abdominal/metabolismo , Aterosclerosis/metabolismo , Aductos de ADN/metabolismo , Desoxiadenosinas/metabolismo , Desoxicitidina/análogos & derivados , Peroxidación de Lípido , Anciano , Anciano de 80 o más Años , Animales , Aterosclerosis/etiología , Aductos de ADN/química , Desoxiadenosinas/química , Desoxicitidina/química , Desoxicitidina/metabolismo , Humanos , Peroxidación de Lípido/genética , Masculino , Persona de Mediana Edad , Estructura Molecular , Fumar/efectos adversos , Fumar/metabolismoRESUMEN
BACKGROUND AND AIMS: Chronic inflammation, induced by biological, chemical, and physical factors, was associated with increased risk of human cancer at various sites. Chronic inflammatory processes induce oxidative/nitrosative stress and lipid peroxidation (LPO), thereby generating excess reactive oxygen species (ROS), reactive nitrogen species (RNS), and DNA-reactive aldehydes. Miscoding etheno- and propano-modified DNA bases are generated inter alia by reaction of DNA with these major LPO products. Steady-state levels of LPO-derived (etheno-) DNA adducts in organs affected by persistent inflammatory processes were investigated as potential lead markers for assessing progression of inflammatory cancer-prone diseases. RESULTS: Using ultrasensitive and specific detection methods for the analysis of human tissues, cells, and urine, etheno-DNA adduct levels were found to be significantly elevated in the affected organs of subjects with chronic pancreatitis, ulcerative colitis, and Crohn's disease. Patients with alcohol-related liver diseases showed excess hepatic DNA damage progressively increasing from hepatitis, fatty liver, to liver cirrhosis. Ethenodeoxyadenosine excreted after DNA repair in urine of hepatitis B virus-related chronic hepatitis and liver cirrhosis patients was increased up to 90-fold. Putative mechanisms that may control DNA damage in inflamed tissues including impaired or imbalanced DNA repair pathways are reviewed. CONCLUSION: Persistent oxidative/nitrosative stress and excess LPO are induced by inflammatory processes in a self-perpetuating process and cause progressive accumulation of DNA damage in target organs. Together with deregulation of cell homeostasis, the resulting genetic changes act as driving force in chronic inflammation-associated human disease pathogenesis. Thus steady-state levels of DNA damage caused by ROS, RNS, and LPO end products provide promising molecular signatures for risk prediction and potential targets and biomarkers for preventive measures.
Asunto(s)
Daño del ADN , Inflamación/complicaciones , Peroxidación de Lípido , Neoplasias/etiología , Estrés Oxidativo , Enfermedad Crónica , Aductos de ADN/química , Aductos de ADN/metabolismo , Reparación del ADN , Humanos , Hepatopatías/complicaciones , Hepatopatías/metabolismo , Neoplasias/genética , Factores de RiesgoRESUMEN
Chronic inflammatory processes induce oxidative stress and lipid peroxidation (LPO), hereby generating DNA-reactive aldehydes such as trans-4-hydroxy-2-nonenal (HNE). Etheno-modified DNA bases are inter alia generated by reaction of DNA with HNE. Using an immunoaffinity-(32)P-postlabeling method, the authors have investigated etheno-DNA adduct levels 1,N (6)-ethenodeoxyadenosine (epsilondA) and of 3,N (4)-ethenodeoxycytidine (epsilondC) in the pancreas of chronic pancreatitis patients and in the colon of patients with inflammatory bowel disease. Both epsilondA and epsilondC levels were found to be significantly, 3 and 28 times, respectively, elevated in the inflamed pancreatic tissue. In contrast, only epsilondC was found to be increased in affected colonic mucosa of Crohn's disease (19 times) and of ulcerative colitis patients (4 times) when compared to asymptomatic tissues. In all three cancer-prone diseases, the mean epsilondC-levels in tissues were five- to ninefold higher than those of epsilondA. Differential or impaired DNA repair pathways of these adducts, known to occur by two different glycosylases are implicated. K-ras in pancreatic tumors and K-ras and p53 in colon mucosa in long-standing inflammatory bowel disease are known to be highly mutated. The conclusion is that promutagenic etheno-DNA adducts are generated as a consequence of chronic inflammation, acting as a driving force to malignancy in cancer-prone inflammatory diseases.
Asunto(s)
Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Aductos de ADN/metabolismo , Pancreatitis Crónica/metabolismo , Biomarcadores/análisis , Colon/citología , Colon/metabolismo , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Oxidación-Reducción , Páncreas/citología , Páncreas/metabolismoRESUMEN
The goal of this study was to determine whether short-term fasting changes in urinary biomarkers related to oxidative stress: malondialdehyde (MDA), 8-isoprostaglandin F2alpha (8-isoPGF), 8-hydroxydeoxy-guanosine (8-OHdG) and 1,N6-ethenodeoxyadenosine (epsilondA) among female volunteers participating in the short-term fasting program in South Korea. The study subjects were 52 healthy women (mean age 28, range 15-48 years old) who provided urine samples both before and after the fasting program (average 7.2, range: 3-11 days). Urinary MDA was measured by HPLC-UV and epsilondA levels were measured by immuno-affinity purification followed by HPLC-fluorescence detection. Urinary 8-OHdG and 8-isoPGF concentrations were determined by ELISA. Plasma leptin levels were also measured by radioimmunoassay. Information on demographic characteristics, personal habits (smoking and alcohol consumption) and previous medical history were collected by a self-administered questionnaire. Percent loss of body weight (average 6.3%, 4.28 +/- 0.25 kg) was significantly correlated with fasting duration (r = 0.70, n = 52, P < 0.01). The plasma leptin levels after fasting (5.89 +/- 1.10 ng/ml) were significantly lower than before fasting (6.91 +/- 1.13 ng/ml) (n = 27, P = 0.05). Urinary MDA levels after fasting (0.18 +/- 1.10 mg/g creatinine) were significantly lower than before fasting (0.37 +/- 1.11) (n = 51, P < 0.01). Urinary 8-isoPGF also were significantly reduced after fasting (n = 47, P < 0.01). However, there was no significant difference in 8-OHdG or epsilondA. There was a statistically significant correlation between % change of urinary MDA level with % change of 8-isoPGF level (partial correlation coefficient r = 0.57, n = 46, P = 0.01). The correlations between % change of 8-OHdG and plasma leptin was also significant (partial correlation coefficient r = 0.51, n = 27, P = 0.02). Our results demonstrate that the short-term fasting reduces lipid peroxidation products but does not affect oxidative stress-induced DNA damage.
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Daño del ADN/fisiología , Ayuno/orina , Peroxidación de Lípido/fisiología , Estrés Oxidativo/fisiología , 8-Hidroxi-2'-Desoxicoguanosina , Adolescente , Adulto , Cromatografía Líquida de Alta Presión , Desoxiadenosinas/orina , Desoxiguanosina/análogos & derivados , Desoxiguanosina/orina , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Leptina/sangre , Malondialdehído/orina , Persona de Mediana Edad , Prostaglandinas A/orina , Factores de TiempoRESUMEN
Reactive oxygen species (ROS) and lipid peroxidation (LPO) play a role in aging and degenerative diseases. To correlate oxidative stress and LPO-derived DNA damage, we determined etheno-DNA-adducts in liver and brain from ROS overproducing OXYS rats in comparison with age-matched Wistar rats. Liver DNA samples from 3- and 15-month-old OXYS and Wistar rats were analyzed for 1,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) by immunoaffinity/32P-postlabelling. While epsilondA and epsilondC levels were not different in young rats, adduct levels were significantly higher in old OXYS rats when compared to old Wistar or young OXYS rats. Frozen rat brain sections were analyzed for epsilondA by immunostaining of nuclei. Brains from old OXYS rats accumulated epsilondA more frequently than age-matched Wistar rats. Our results demonstrate increased LPO-induced DNA damage in organs of OXYS rats which correlates with their known shorter life-span and elevated frequency of chronic degenerative diseases.