RESUMEN
Ralfinamide is an α-aminoamide derivative with ion channel blocking properties, acting both peripherally and centrally through different molecular targets important in pain control. Absorption, blood and plasma time courses, and urinary and faecal excretion of total radioactivity were assessed in 6 male healthy volunteers administered a single oral dose of 320 mg ¹4C-(S)-ralfinamide. Pharmacokinetics of the parent drug were investigated over 120 h, urinary and plasma metabolites up to 192 h post-dose. ¹4C-(S)-ralfinamide was rapidly and completely absorbed. Ralfinamide and the dealkylated ralfinamide metabolite (NW-1716) represented the majority of plasma radioactivity. Plasma elimination of the parent compound occurred mono-exponentially (half-life approx. 15 h). ¹4C-radioactivity was eliminated in a bi-phasic manner (terminal half-life of 60 and 24 h for plasma and whole blood, respectively). Plasma-concentrations of unchanged ralfinamide were significantly lower than radioactivity concentrations, indicating metabolism of the parent compound. At 192 h post-dose the total balance of radioactivity was almost complete (95%). The main route of excretion was via the kidneys (94% of the dose). Major metabolites identified in urine and plasma were the N-dealkylated acid of ralfinamide and deaminated ralfinamide acid (NW-1799). Other metabolites, in particular the product of glucuronide conjugation N-dealkylated-ß-glucuronide, were identified.
Asunto(s)
Fluorobencenos/farmacocinética , Bloqueadores de los Canales de Sodio/farmacocinética , Administración Oral , Adulto , Semivida , Humanos , Riñón/metabolismo , Masculino , Factores de Tiempo , Adulto JovenAsunto(s)
Neoplasias Colorrectales/etiología , Aductos de ADN/química , ADN de Neoplasias/química , Desoxiguanosina/metabolismo , Malondialdehído/metabolismo , Neoplasias Colorrectales/genética , Daño del ADN , Grasas de la Dieta , Femenino , Humanos , Mucosa Intestinal/química , Peroxidación de Lípido , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
Cyclooxygenases (COX) catalyse the oxygenation of arachidonic acid to prostaglandin (PG) endoperoxides. Activity of one of the COX isoforms, COX-2, results in production of prostaglandin E(2) (PGE(2)) via the endoperoxide PGH(2). COX-2 has been implicated in the pathogenesis of colorectal cancer. Malondialdehyde (MDA) is a mutagen produced by spontaneous and enzymatic breakdown of PGH(2). MDA reacts with DNA to form adducts, predominantly the pyrimidopurinone adduct of deoxyguanosine (M(1)G). Here the hypothesis was tested that COX-2 activity in human colon cells results in formation of MDA and generation of M(1)G adducts. M(1)G was detected in basal cultures of human non-malignant colon epithelial (HCEC) and malignant SW48, SW480, HT29 and HCA-7 colon cells, at levels from 77 to 148 adducts/10(8) nucleotides. Only HCA-7 and HT29 cells expressed COX-2 protein. Levels of M(1)G correlated significantly (r = 0.98, P < 0.001) with those of intracellular MDA determined colorimetrically in the four malignant cell types, but neither parameter correlated with expression of COX-2 or PG biosynthesis. Induction of COX-2 expression by phorbol 12-myristate 13-acetate in HCEC cells increased PGE(2) production 20-fold and MDA concentration 3-fold. Selective inhibition of COX-2 activity in HCA-7 cells by NS-398 significantly inhibited PGE(2) production, but altered neither MDA nor M(1)G levels. Malondialdehyde treatment of HCEC cells resulted in a doubling of M(1)G levels. These results show for the first time in human colon cells that COX-2 activity is associated with formation of the endogenous mutagen, MDA. Moreover, they demonstrate the correlation between MDA concentration and M(1)G adduct levels in malignant cells.
Asunto(s)
Colon/metabolismo , Aductos de ADN/biosíntesis , Desoxiguanosina/metabolismo , Isoenzimas/metabolismo , Malondialdehído/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Neoplasias del Colon/metabolismo , Ciclooxigenasa 2 , ADN/metabolismo , Dinoprostona/biosíntesis , Dinoprostona/metabolismo , Humanos , Proteínas de la Membrana , Células Tumorales CultivadasRESUMEN
Helicobacter hepaticus infection is associated with chronic hepatitis and the development of liver tumours in mice. The underlying mechanism of this liver carcinogenesis is not clear but the oxidative stress associated with H. hepaticus infection may result in induction of lipid peroxidation and the generation of malondialdehyde. Malondialdehyde can react with deoxyguanosine in DNA resulting in the formation of the cyclic pyrimidopurinone N-1,N(2) malondialdehyde-deoxyguanosine (M1dG) adduct. This adduct has the potential to cause mutations that may ultimately lead to liver carcinogenesis. The objective of this study was to determine the control and infection-related levels of M1dG in the liver DNA of mice over time, using an immunoslot-blot procedure. The level of M1dG in control A/J mouse livers at 3, 6, 9 and 12 months averaged 37.5, 36.6, 24.8 and 30.1 adducts per 10(8) nucleotides, respectively. Higher levels of M1dG were detected in the liver DNA of H. hepaticus infected A/JCr mice, with levels averaging 40.7, 47.0, 42.5 and 52.5 adducts per 10(8) nucleotides at 3, 6, 9 and 12 months, respectively. There was a significant age dependent increase in the level of M1dG in the caudate and median lobes of the A/JCr mice relative to control mice. A lobe specific distribution of the M1dG adduct in both infected and control mice was noted, with the left lobe showing the lowest level of the adduct compared with the right and median lobes at all time points. In a separate series of mice experimentally infected with H. hepaticus, levels of 8-hydroxy-deoxyguanosine were significantly greater in the median compared with the left lobe at 12 weeks after treatment. In conclusion, these results suggest that M1dG occurs as a result of oxidative stress associated with H. hepaticus infection of mice, and may contribute to liver carcinogenesis in this model.
Asunto(s)
Aductos de ADN/metabolismo , ADN/química , Infecciones por Helicobacter/metabolismo , Hígado/química , Malondialdehído/química , Animales , Cromatografía Líquida de Alta Presión , Infecciones por Helicobacter/microbiología , Masculino , RatonesRESUMEN
Curcumin prevents colon cancer in rodent models. It inhibits lipid peroxidation and cyclooxygenase-2 (COX-2) expression and induces glutathione S-transferase (GST) enzymes. We tested the hypothesis that 14 days of dietary curcumin (2%) affects biomarkers relevant to cancer chemoprevention in the rat. Levels of inducible COX-2, as reflected by prostaglandin E(2) production by blood leukocytes, were measured ex vivo. Total GST activity and adducts of malondialdehyde with DNA (M(1)G), which reflect endogenous lipid peroxidation, were measured in colon mucosa, liver, and blood leukocytes. Curcumin and its metabolites were analyzed by high-performance liquid chromatography in plasma, and its pharmacokinetics were compared following a diet containing 2% curcumin versus intragastric (i.g.) administration of curcumin suspended in an amphiphilic solvent. The curcumin diet did not alter any of the markers in the blood but increased hepatic GST by 16% and decreased colon M(1)G levels by 36% when compared with controls. Administration of carbon tetrachloride during the treatment period increased colon M(1)G levels, and this increase was prevented by dietary curcumin. Dietary curcumin yielded low drug levels in the plasma, between 0 and 12 nM, whereas tissue concentrations of curcumin in liver and colon mucosa were 0.1--0.9 nmol/g and 0.2--1.8 micromol/g, respectively. In comparison with dietary administration, suspended curcumin given i.g. resulted in more curcumin in the plasma but much less in the colon mucosa. The results show that curcumin mixed with the diet achieves drug levels in the colon and liver sufficient to explain the pharmacological activities observed and suggest that this mode of administration may be preferable for the chemoprevention of colon cancer.
Asunto(s)
Antineoplásicos/farmacocinética , Curcumina/farmacocinética , Aductos de ADN/metabolismo , Mucosa Gástrica/metabolismo , Glutatión Transferasa/metabolismo , Hígado/metabolismo , Animales , Antineoplásicos/uso terapéutico , Neoplasias del Colon/prevención & control , Curcumina/uso terapéutico , Aductos de ADN/efectos de los fármacos , Dieta , Femenino , Mucosa Gástrica/efectos de los fármacos , Glutatión Transferasa/efectos de los fármacos , Hígado/efectos de los fármacos , Malondialdehído/metabolismo , Ratas , Ratas Endogámicas F344RESUMEN
Helicobacter pylori infection is associated with elevated gastric mucosal concentrations of the lipid peroxidation product malondialdehyde and reduced gastric juice vitamin C concentrations. Malondialdehyde can react with DNA bases to form the mutagenic adduct malondialdehyde-deoxyguanosine (M(1)-dG). We aimed to determine gastric mucosal levels of M(1)-dG in relation to H. pylori infection and malondialdehyde and vitamin C concentrations. Patients (n = 124) attending for endoscopy were studied. Levels of antral mucosal M(1)-dG were determined using a sensitive immunoslot-blot technique; antral mucosal malondialdehyde was determined by thiobarbituric acid extraction, and gastric juice and antral mucosal ascorbic acid and total vitamin C were determined by high-performance liquid chromatography. Sixty-four H. pylori-positive patients received eradication therapy, and endoscopy was repeated at 6 and 12 months. Levels of M(1)-dG did not differ between subjects with H. pylori gastritis (n = 85) and those with normal mucosa without H. pylori infection (n = 39; 56.6 versus 60.1 adducts/10(8) bases) and were unaffected by age or smoking habits. Malondialdehyde levels were higher (123.7 versus 82.5 pmol/g; P < 0.001), gastric juice ascorbic acid was lower (5.7 versus 15.0 micromol/ml; P < 0.001), and antral mucosal ascorbic acid was unchanged (48.0 versus 42.7 micromol/g) in H. pylori gastritis compared with normal mucosa. Multiple regression analysis revealed that M(1)-dG increased significantly with increasing levels of malondialdehyde, antral ascorbic acid, and total antral vitamin C. M(1)-dG levels were unchanged 6 months (63.3 versus 87.0 adducts/10(8) bases; P = 0.24; n = 38) and 12 months (66.7 versus 77.5 adducts/10(8) bases; P = 0.8; n = 13) after successful eradication of H. pylori. M(1)-dG thus is detectable in gastric mucosa, but is not affected directly by H. pylori.
Asunto(s)
Ácido Ascórbico/farmacología , Desoxiguanosina/análisis , Mucosa Gástrica/química , Infecciones por Helicobacter/complicaciones , Adulto , Anciano , Cromatografía Líquida de Alta Presión , Desoxiguanosina/análogos & derivados , Endoscopía , Femenino , Jugo Gástrico/química , Mucosa Gástrica/microbiología , Infecciones por Helicobacter/tratamiento farmacológico , Humanos , Inmunoensayo , Peroxidación de Lípido , Masculino , Persona de Mediana EdadRESUMEN
As part of a large programme on food risk assessment, we have become Interested in the endogenous production of genotoxic agents from dietary precursors. Malondialdehyde (MDA), a product of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in bacterial and mammalian systems. MDA reacts with DNA, and the major adduct (M1-dG) has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to large numbers of individuals or a large variety of samples. Often, only a few micrograms of DNA from human tissues are available for analysis, and a very sensitive assay is needed to detect background levels of M1-dG in very small amounts of DNA. In this paper, we describe the development of an immunoslot-blot (ISB) assay for the measurement of M1-dG in 1 microgram of DNA. The limit of detection of the assay is about 5 adducts per 10(8) bases. The advantages of ISB over other assays for DNA adduct detection, such as the possibility of analysing 1 microgram DNA per sample and the fact that it is less time-consuming and laborious, mean that it can be more easily used for routine analysis of large numbers of samples in biomonitoring. A series of human samples was analysed, and levels of 0.3-6.43 M1-dG per 10(7) normal bases were detected in 42 gastric biopsy samples and 0.7-16.65 M1-dG per 10(7) normal bases in 28 samples of leukocyte DNA. In an initial study in five human volunteers on standardized diets, the levels of M1-dG in leukocyte DNA changed in relation to meat, vegetable and tea intake.
Asunto(s)
Aductos de ADN/análisis , ADN/análisis , Desoxiguanosina/análogos & derivados , Immunoblotting/métodos , Biomarcadores , Cromatografía Líquida de Alta Presión , Aductos de ADN/sangre , Desoxiguanosina/análisis , Desoxiguanosina/sangre , Dieta , Mucosa Gástrica/metabolismo , Humanos , Leucocitos/metabolismo , Modelos Químicos , Sensibilidad y EspecificidadRESUMEN
Malondialdehyde (MDA) is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic and the major adduct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M1-dG), has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to a large number of individuals or variety of samples. Often, only a few microg of DNA from human tissues are available for analysis and a very sensitive assay is needed in order to detect background levels of M1-dG in very small amounts of DNA. In this paper, the development of an immunoslot blot (ISB) assay for the measurement of MI-dG in 1 microg of DNA is described. The limit of detection of the assay is 2.5 adducts per 10(8) bases. A series of human samples were analysed and levels of 5.6-9.5 (n = 8) and 3.1-64.3 (n = 42) of M1-dG per 10(8) normal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those obtained using an HPLC/32P-post-labelling (HPLC/PPL) method previously developed and indicated a high correlation between M1-dG levels measured by the two assays. The advantages of ISB over other assays including HPLC/PPL, such as the possibility of analysing 1 microg DNA/sample and the fact that it is less time-consuming and laborious, means that it can be more easily used for routine analysis of a large number of samples in biomonitoring studies.
Asunto(s)
Aductos de ADN/análisis , Daño del ADN , ADN/efectos de los fármacos , Malondialdehído/toxicidad , Animales , Cromatografía Líquida de Alta Presión , ADN/metabolismo , Humanos , Immunoblotting , Malondialdehído/metabolismo , RatonesRESUMEN
A coordinated study was carried out on the development, evaluation and application of biomonitoring procedures for populations exposed to environmental genotoxic pollutants. The procedures used involved both direct measurement of DNA or protein damage (adducts) and assessment of second biological effects (mutation and cytogenetic damage). Adduct detection at the level of DNA or protein (haemoglobin) was carried out by 32P-postlabelling, immunochemical, HPLC or mass spectrometric methods. Urinary excretion products resulting from DNA damage were also estimated (immunochemical assay, mass spectrometry). The measurement of adducts was focused on those from genotoxicants that result from petrochemical combustion or processing, e.g. low-molecular-weight alkylating agents, PAHs and compounds that cause oxidative DNA damage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei, chromosome aberrations and sister chromatid exchanges) and mutation frequency was estimated at a number of loci including the hprt gene and genes involving in cancer development. Blood and urine samples from individuals exposed to urban pollution were collected. Populations exposed through occupational or medical sources to larger amounts of some of the genotoxic compounds present in the environmental samples were used as positive controls for the environmentally exposed population. Samples from rural areas were used as negative controls. The project has led to new, more sensitive and more selective approaches for detecting carcinogen-induced damage to DNA and proteins, and subsequent biological effects. These methods were validated with the occupational exposures, which showed evidence of DNA and/or protein and/or chromosome damage in workers in a coke oven plant, garage workers exposed to diesel exhaust and workers exposed to ethylene oxide in a sterilization plant. Dose reponse and adduct repair were studied for methylated adducts in patients treated with methylating cytostatic drugs. The biomonitoring methods have also demonstrated their potential for detecting environmental exposure to genotoxic compounds in nine groups of non-smoking individuals, 32P-postlabelling of DNA adducts being shown to have the greatest sensitivity.
Asunto(s)
Carcinógenos Ambientales/toxicidad , Monitoreo del Ambiente/métodos , Antineoplásicos Alquilantes/toxicidad , Proteínas Sanguíneas/efectos de los fármacos , Estudios de Casos y Controles , Aductos de ADN/sangre , Daño del ADN , Exposición a Riesgos Ambientales , Epiclorhidrina/toxicidad , Óxido de Etileno/toxicidad , Humanos , Cloruro de Metileno/toxicidad , Mutágenos/toxicidad , Óxidos de Nitrógeno/toxicidad , Exposición Profesional , Petróleo/toxicidad , Hidrocarburos Policíclicos Aromáticos/toxicidad , Estireno , Estirenos/toxicidadRESUMEN
To date only a few studies have been undertaken on DNA adducts formed by epoxybutene (EB) and diepoxybutane (DEB), the two active metabolites of 1,3-butadiene. Our interests have focused on further investigating DNA alkylation by the two epoxides, especially in relation to the development of a method for human biomonitoring. Here, following the reaction of deoxyadenosine monophosphate and poly(dA-dT)(dA-dT) with DEB and subsequent HPLC, we have identified an adenine adduct. MS analyses indicate the structure of an adenine adducted by DEB at the N6 position. A HPLC/32P-postlabelling method was developed for its measurement in DNA samples and the adduct was detected in calf thymus DNA and DNA from Chinese hamster ovary cells exposed to DEB. The 100% labelling efficiency during postlabelling, the amount of the adduct and its elution before the normal nucleotides during HPLC suggest it could be a suitable indicator of BUT exposure.
Asunto(s)
Adenina/análisis , Adenina/metabolismo , Butadienos/metabolismo , Butadienos/toxicidad , Aductos de ADN/análisis , Aductos de ADN/metabolismo , Daño del ADN , ADN/análisis , Contaminantes Ambientales/metabolismo , Contaminantes Ambientales/toxicidad , Compuestos Epoxi/análisis , Compuestos Epoxi/metabolismo , Adamantano/análogos & derivados , Adamantano/metabolismo , Animales , Células CHO , Bovinos , Cromatografía Líquida de Alta Presión , Cricetinae , ADN/efectos de los fármacos , ADN/metabolismo , Aductos de ADN/aislamiento & purificación , Compuestos Epoxi/toxicidad , Marcaje Isotópico/métodos , Radioisótopos de Fósforo , Poli dA-dT/metabolismo , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
1,2-Epoxy-3-butene and 1,2:3,4-diepoxybutane, the two oxidative metabolites of 1,3-butadiene, are considered to be involved in some of the carcinogenicity of the parent compound. Diepoxybutane is a bifunctional alkylating agent and reacts with DNA to form monoadducts and cross-links. We investigated DNA alkylation after exposure to diepoxybutane in order to develop a method for human biomonitoring. After reacting dAMP and then poly(dA-dT) (dA-dT) with diepoxybutane, we identified a major adenine adduct. Preliminary mass spectrometry indicated an adenine adducted by diepoxybutane at the N6 position. A high-performance liquid chromatography/32P-postlabelling method was developed, and the adduct was detected in calf thymus DNA and in DNA from Chinese hamster cells after exposure to diepoxybutane. The labelling efficiency, the amount of the adduct and its stability suggest that it could be a suitable indicator of exposure to butadiene.
Asunto(s)
Adenina/metabolismo , Butadienos/análisis , Exposición a Riesgos Ambientales , Compuestos Epoxi/metabolismo , Mutágenos/análisis , Adenosina Monofosfato/metabolismo , Alquilación , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Cricetinae , Cricetulus , ADN/metabolismo , Desoxiadenosinas/metabolismo , Femenino , OvarioRESUMEN
We have developed an HPLC-32P-postlabelling procedure to detect DNA adducts formed by epoxybutene and diepoxybutane. The method exploits the interaction of the two epoxides with deoxynucleotides and polydeoxynucleotides to optimize the HPLC enrichment of adducted nucleotides before 32P-postlabelling. Using this approach, a number of guanine adducts were identified after the exposure of dGMP, poly(dG-dC) or calf thymus DNA to epoxybutene and diepoxybutane, and a major adenine adduct was identified in poly(dA-dT) and calf thymus DNA exposed to diepoxybutane.