RESUMO
trans-Cinnamaldehyde is a widely used natural ingredient that is added to foods and cosmetics as a flavoring and fragrance agent. Male and female F344/N rats and B6C3F(1) mice were exposed to microencapsulated trans-cinnamaldehyde in the feed for three months or two years. All studies included untreated and vehicle control groups. In the three-month studies, rats and mice were given diets containing 4100, 8200, 16,500, or 33,000 ppm trans-cinnamaldehyde. In rats, feed consumption was reduced in all exposed groups. In mice, feed consumption was reduced in the highest dose groups. Body weights of all treated males were less than controls. Body weights were reduced in female rats exposed to 16,500 or 33,000 ppm and female mice exposed to 8200 ppm or greater. All rats survived to the end of the study but some male mice in the highest dose groups died due to inanition from unpalatability of the dosed feed. The incidence of squamous epithelial hyperplasia of the forestomach was significantly increased in rats exposed to 8200 ppm or greater and female mice exposed to 33,000 ppm. In mice, the incidence of olfactory epithelial degeneration of the nasal cavity was significantly increased in males and females exposed to 16,500 ppm and females exposed to 33,000 ppm. In the two-year studies, rats and mice were exposed to 1000, 2100, or 4100 ppm trans-cinnamaldehyde. Body weights were reduced in mice exposed to 2100 ppm and in rats and mice exposed to 4100 ppm. In rats, hippuric acid excretion was dose proportional indicating that absorption, metabolism, and excretion were not saturated. No neoplasms were attributed to trans-cinnamaldehyde in rats or mice. Squamous cell papillomas and carcinomas of the forestomach were observed in male and female mice but the incidences were within the NTP historical control range and were not considered to be related to trans-cinnamaldehyde exposure.
Assuntos
Acroleína/análogos & derivados , Acroleína/toxicidade , Carcinógenos/toxicidade , Aromatizantes/toxicidade , Estômago/efeitos dos fármacos , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Testes de Carcinogenicidade , Relação Dose-Resposta a Droga , Composição de Medicamentos , Feminino , Estudos Longitudinais , Masculino , Camundongos , Camundongos Endogâmicos , Distribuição Aleatória , Ratos , Ratos Endogâmicos F344 , Estômago/patologia , Análise de SobrevidaRESUMO
During the Persian Gulf War, soldiers were injured with depleted uranium (DU) fragments. To assess the potential health risks associated with chronic exposure to DU, Sprague Dawley rats were surgically implanted with DU pellets at 3 dose levels (low, medium and high). Biologically inert tantalum (Ta) pellets were used as controls. At 1 day and 6, 12, and 18 months after implantation, the rats were euthanized and tissue samples collected. Using kinetic phosphorimetry, uranium levels were measured. As early as 1 day after pellet implantation and at all subsequent sample times, the greatest concentrations of uranium were in the kidney and tibia. At all time points, uranium concentrations in kidney and bone (tibia and skull) were significantly greater in the high-dose rats than in the Ta-control group. By 18 months post-implantation, the uranium concentration in kidney and bone of low-dose animals was significantly different from that in the Ta controls. Significant concentrations of uranium were excreted in the urine throughout the 18 months of the study (224 +/- 32 ng U/ml urine in low-dose rats and 1010 +/- 87 ng U/ml urine in high-dose rats at 12 months). Many other tissues (muscle, spleen, liver, heart, lung, brain, lymph nodes, and testicles) contained significant concentrations of uranium in the implanted animals. From these results, we conclude that kidney and bone are the primary reservoirs for uranium redistributed from intramuscularly embedded fragments. The accumulations in brain, lymph nodes, and testicles suggest the potential for unanticipated physiological consequences of exposure to uranium through this route.
Assuntos
Osso e Ossos/metabolismo , Rim/metabolismo , Tantálio/metabolismo , Urânio/farmacocinética , Animais , Encéfalo/metabolismo , Ratos , Ratos Sprague-Dawley , Medição de Risco , Comprimidos , Fatores de Tempo , Distribuição Tecidual , Urânio/sangue , Urânio/urinaRESUMO
During the 1991 Persian Gulf War several US military personnel were wounded by shrapnel fragments consisting of depleted uranium. These fragments were treated as conventional shrapnel and were not surgically removed to spare excessive tissue damage. Uranium bioassays conducted over a year after the initial uranium injury indicated a significant increase in urine uranium levels above natural background levels. The potential mutagenic effects of depleted uranium are unknown. To assess the potential mutagenic effects of long-term exposure to internalized depleted uranium, Sprague-Dawley rats were implanted with depleted uranium and their urine and serum were evaluated for mutagenic potential at various times after pellet implantation using the Ames Salmonella reversion assay. Tantalum, an inert metal widely used in prosthetic devices was used for comparison. Enhancement of mutagenic activity in Salmonella typhimurium strain TA98 and the Ames II mixed strains (TA7001-7006) was observed in urine samples from animals implanted with depleted uranium pellets. In contrast, urine samples from animals implanted with tantalum did not show a significant enhancement of mutagenic activity in these strains. In depleted uranium-implanted animals, urine mutagenicity increased in a dose- and time-dependent manner demonstrating a strong positive correlation with urine uranium levels (r = 0.995, P < 0.001). There was no mutagenic enhancement of any bacterial strain detected in the sera of animals implanted with either depleted uranium or tantalum pellets. The results suggest that uranium content in the urine is correlated with urine mutagenicity and that urinary mutagenicity might be used as a biomarker to detect exposure to internalized uranium.
Assuntos
Testes de Mutagenicidade , Tantálio/sangue , Tantálio/urina , Urânio/sangue , Urânio/urina , Animais , Relação Dose-Resposta a Droga , Masculino , Mutagênicos/toxicidade , Próteses e Implantes/efeitos adversos , Ratos , Ratos Sprague-Dawley , Salmonella/efeitos dos fármacos , Salmonella/genética , Tantálio/toxicidade , Fatores de Tempo , Urânio/toxicidadeRESUMO
A quantitative method was developed for determination of alpha2u-globulin in urine and kidney samples collected from male rats using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS). Samples prepared from urine and kidney homogenates using size exclusion filters were subject to reversed-phase liquid chromatography and the effluent passed into an electrospray ionization source. Quantitative analysis using external standard calibration was based upon selected ion monitoring of protonated molecular ions by the mass spectrometer. Linear calibration curves were developed over the range of approximately 4. 6-370 microg of alpha2u-globulin/microL for spiked urine standards and over the range of approximately 4.6-550 microg of alpha2u-globulin/microL for spiked kidney standards. The precision (relative standard deviation) for repeated injection (using urine samples) and intra-assay precision (using both urine and kidney samples) were within +/-10.4% and +/-13.2%, respectively. Using spiked urine standards, inter-assay precision, intra-assay accuracy, and inter-assay accuracy were within +/-20%, +/-20%, and +/-15%, respectively. Using spiked kidney standards, intra-assay accuracy was within +/-15%. The limits of detection (LOD) for the determination of alpha2u-globulin in urine and kidney samples were approximately 0.41 pg/nL (1.0 fmol injected) and 25 pg/nL ( approximately 13 fmol injected), respectively. The limits of quantitation (LOQ) for determination of alpha2u-globulin in urine and kidney samples were calculated as 1.4 pg/nL (3.7 fmol injected) and 83 pg/nL (45 fmol injected), respectively. Applicability of the LC-ESI/MS method was demonstrated by determination of alpha2u-globulin in both urine and kidney samples collected from male Fischer 344/N rats dosed intravenously with cis-Decalin at concentrations of 0, 2.5, 5.0, 10, and 20 mg/kg. A dose-dependent relationship was found between the amount of cis-Decalin administered and alpha2u-globulin accumulation in kidney samples, whereas no significant change in the urinary levels of alpha2u-globulin occurred. These observations are consistent with excessive accumulation of alpha2u-globulin occurring in protein droplets in renal proximal tubule epithelial cells as a result of decreased catabolic activity due to formation of ligand-protein complexs with Decalin and its metabolite(s). This report demonstrates that LC-ESI/MS may be routinely applied for quantitative analysis of alpha2u-globulin in rat urine and kidney samples to address alpha2u-globulin accumulation and its role in the development of nephrotoxicity associated with chemical exposures.
Assuntos
alfa-Globulinas/análise , alfa-Globulinas/urina , Cromatografia Líquida/métodos , Rim/química , Espectrometria de Massas/métodos , Animais , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
Exposure to ionizing radiation leads to formation of covalent crosslinks between DNA and proteins. The nature, extent and site of the modifications are not well understood due to the difficulty in assessing free radical-induced damage in biopolymers. Electrospray ionization mass spectrometry (ESI-MS) permits direct analyses of intact oligopeptides, permitting characterization of the radiation-induced DNA-protein covalently crosslinked constituents. Our first application of this methodology to free radical-induced damage was in a model system where angiotensin, a small 10-amino acid peptide, is irradiated at various doses in the presence of excess thymine. The relative yield of crosslinks, which ranged from 0.1 to 15%, was linearly related to radiation dose for doses from 0.1 to 100 Gy. Detection of thymine-tyrosine moieties in this model system was possible at doses as low as 0.1 Gy with a signal-to-noise ratio of 4 to 1. ESI-MS revealed that the site of crosslink was located exclusively on the tyrosine residue as expected.
Assuntos
Angiotensina II/química , DNA/química , Proteínas/química , Timina/efeitos da radiação , Tirosina/efeitos da radiação , Sequência de Aminoácidos , Angiotensina II/efeitos da radiação , Reagentes de Ligações Cruzadas , DNA/efeitos da radiação , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Oligopeptídeos/química , Proteínas/efeitos da radiação , Timina/química , Tirosina/químicaRESUMO
Analysis of trace levels of DNA damage as a biomarker of human exposure to radiation levels represents a significant challenge. Capillary isotachophoresis (CITP) is well suited for this application due to its high separation efficiency, large sample loading capacity, and relative ease of interfacing with highly specific and sensitive mass spectrometric detectors. This study describes the use of reversed anionic CITP coupled on-line with electrospray ionization-mass spectrometry (ESI-MS) for the identification and characterization of small quantities of radiation induced damage to deoxynucleotide monophosphates. CITP with ESI-tandem MS also provides structural information on the nature of the damage in addition to the molecular weight. This approach has the attraction of allowing the direct on-line determination of damaged nucleotides, as well as the free bases and nucleosides, and avoids the need for sample hydrolysis and/or derivatization.
Assuntos
Dano ao DNA , DNA/efeitos da radiação , Eletroforese Capilar/métodos , Monitoramento de Radiação/métodos , Ribonucleotídeos/efeitos da radiação , Espectrometria de Massa de Íon Secundário/métodos , Biomarcadores , DNA/análise , Eletroforese Capilar/instrumentação , Exposição Ambiental , Humanos , Ribonucleotídeos/análise , Espectrometria de Massa de Íon Secundário/instrumentaçãoRESUMO
Capillary zone electrophoresis (CZE) and reversed anionic capillary isotachophoresis (CITP) conditions have been developed for the separation of mixtures comprised of monophosphate nucleosides, pyridine and flavin dinucleotides, and monophosphate dinucleosides. Results for the on-line coupling of CZE and CITP with electrospray ionization mass spectrometry (MS) are presented. CITP-tandem MS is utilized to provide both molecular weight and structural information of monophosphate dinucleotides. The fragmentation pattern of dinucleotides in the low collision energy range is described. The resulting mass spectra are readily interpreted in terms of dinucleotide structures. These results demonstrate the new capability for applications for the study of DNA and RNA.
Assuntos
Eletroforese/métodos , Espectrometria de Massas/métodos , Nucleotídeos/isolamento & purificação , Ação Capilar , DNA/química , Fosfatos de Dinucleosídeos/isolamento & purificação , Flavina-Adenina Dinucleotídeo/isolamento & purificação , Estrutura Molecular , Peso Molecular , NAD/isolamento & purificação , RNA/químicaRESUMO
Formation of intramolecular cross links by addition of C(5') deoxyribose radicals to the C(8)-N(7) double bond of an attached adenine base was analyzed by ab initio quantum-chemical methods. Conformational preferences that influence the stereospecificity of the reaction were investigated. A good correlation was found between the ratio of experimental yields of R and S stereoisomers of 8,5'-cyclodeoxyadenosine and the relative energy of conformations of the C(5') radical that are precursors to these isomers. Molecular mechanics based on the AMBER force field was used to model the effect of 8,5'-cyclodeoxyadenosine on the conformation of the DNA dodecamer d(CGCGAATTCGCG)2 with the lesion at the A6 position. The R and S stereoisomers of the intrastrand cross link cause comparable levels of DNA distortion with the major conformational changes occurring in backbone torsional angles at the site of the lesion.
Assuntos
DNA/química , Desoxiadenosinas/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Reagentes de Ligações Cruzadas , Desoxirribose , Radicais Livres , Dados de Sequência Molecular , Estereoisomerismo , TermodinâmicaRESUMO
Ultrasound can damage macromolecules by the mechanical (shearing) and sonochemical (free radical generating) action of ultrasonic cavitation. Attributing macromolecular damage to either direct mechanical stress or to indirect mechanisms involving free radicals or other sonochemicals is a challenging problem. DNA damage induced by ultrasound was evaluated by measuring the formation of purine and pyrimidine products using combined gas chromatography-mass spectrometry with selected ion monitoring. Samples of DNA were prepared in 10 mmol dm-3 phosphate buffered saline (pH 7.4) and saturated with a mixture of argon:oxygen (3:1). Continuous 2.17 MHz ultrasound exposures at 0.82 mPa spatial peak negative pressure amplitude were performed in a 60 rpm rotating tube exposure system. Hydrogen peroxide yields were measured after each exposure to quantify the cavitation activity and ranged up to 350 mumol dm-3 for 1-h exposures. Purine and pyrimidine products identified were those typically observed following exposure of DNA to hydroxyl radical-generating systems, such as ionizing radiation, hypoxanthine/xanthine oxidase, or hydrogen peroxide in the presence of transition metal ions. The yields of these products were directly correlated with cavitation activity as measured by residual hydrogen peroxide concentrations. The yields of DNA products increased in the following order: thymine glycol approximately cytosine glycol > 8-oxoAde > FAPyAde approximately 5-HMU approximately 5,6-diOHCyt > FAPyGua. Unexpectedly, 8-oxoguanine did not exhibit a dose-dependent increase above background levels, and this observation is inconsistent with processes involving metal ion-dependent formation of hydroxyl radicals from hydrogen peroxide. In addition, the product yields were far too large to result from the residual hydrogen peroxide. Thus, ultrasonic cavitation appears to have a mode of action distinct from either ionizing radiation or formation of hydroxyl radicals via Fenton-like reaction with transition metals.
Assuntos
Dano ao DNA , Ultrassom , Adenina/análogos & derivados , Adenina/química , Animais , Bovinos , Citosina/análogos & derivados , Citosina/química , Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Peróxido de Hidrogênio/análise , Radical Hidroxila/química , Purinas/análise , Purinas/química , Pirimidinas/análise , Pirimidinas/química , Timina/análogos & derivados , Timina/químicaRESUMO
Radiation-induced electron migration along DNA is a mechanism by which randomly produced stochastic energy deposition events can lead to non-random types of damage along DNA manifested distal to the sites of the initial energy deposition. Radiation-induced electron migration in nucleic acids has been examined using oligonucleotides containing 5-bromouracil (5-BrU). Interaction of 5-BrU with solvated electrons results in release of bromide ions and formation or uracil-5-yl radicals. Monitoring either bromide ion release or uracil formation provides an opportunity to study electron migration processes in model nucleic acid systems. Using this approach we have discovered that electron migration along oligonucleotides is significantly influenced by the base sequence and strandedness. Migration along 7 base pairs in oligonucleotides containing guanine bases was observed for oligonucleotides irradiated in solution, which compares with mean migration distances of 6-10 bp for Escherichia coli DNA irradiated in solution and 5.5 bp for E. coli DNA irradiated in cells. Evidence also suggests that electron migration can occur preferentially in the 5' to 3' direction along a double-stranded oligonucleotide containing a region of purine bases adjacent to the 5-BrU moiety. Our continued efforts will provide information regarding the contribution of electron transfer along DNA to formation of locally multiply damaged sites created in DNA by exposure to ionizing radiation.
Assuntos
Dano ao DNA , DNA/efeitos da radiação , Sequência de Bases , Bromouracila/efeitos da radiação , DNA/química , ElétronsRESUMO
Electron migration in irradiated solutions of DNA was investigated using 5-bromouracil synthetically incorporated into oligonucleotides of defined base composition as a molecular indicator of electron interactions. Solvated electrons interact quantitatively with 5-bromouracil, leading to a highly reactive 5-yl radical which can abstract an adjacent hydrogen atom to yield uracil. Yields of uracil, or loss of 5-bromouracil, from irradiated oligonucleotide samples were measured using gas chromatography-mass spectrometric analysis of their trimethylsilylated acid hydrolysates. To examine the effects of base composition and DNA conformation on electron migration, a set of oligonucleotides containing 5-bromouracil at selected positions with three base (guanine, cytosine, thymine or adenine) spacers (e.g. [BrU(GGG)3]3) were irradiated in their single- or double-stranded form following annealing with appropriate complementary sequences. Differences in uracil yields suggested that electron migration occurred to different extents in oligonucleotides containing different base sequences. In irradiated single-stranded oligonucleotides, the yield of uracil decreased in the order A > T > > C approximately G. However, in irradiated double-stranded oligonucleotides, the yield of uracil decreased in the order G > C approximately T > A. These differences were attributed to proton-transfer reactions facilitated by base pairing in double-stranded oligonucleotides. The distance over which the electron would migrate was then determined using a series of oligonucleotides containing 5-bromouracil at selected positions with guanine spacers (i.e. [BrU(G)n]3 (n = 3, 5, 7, 9). Oligonucleotides were irradiated in their double-stranded form following annealing with the appropriate complementary sequences. Analysis of the loss of 5-bromouracil revealed that electron migration occurred efficiently over c. 3-4 guanine bases assuming that migration could occur as efficiently in either direction along the DNA molecule. These data can be compared with studies reporting more extensive migration for electrons generated by direct ionization of DNA.
Assuntos
DNA/química , DNA/efeitos da radiação , Elétrons , Raios gama , Oligonucleotídeos/química , Oligonucleotídeos/efeitos da radiação , Sequência de Bases , Bromouracila/química , Cromatografia Gasosa-Espectrometria de Massas , Dados de Sequência Molecular , Soluções/química , Uracila/química , Água/químicaRESUMO
Solvated electrons generated in aqueous solution after exposure to ionizing radiation can be scavenged by DNA and then transferred along the DNA molecule. This mechanism of charge transfer provides an opportunity for radiation damage to be targeted to certain regions in the DNA molecule and is a mechanism by which single-strand breaks contribute to locally multiply damaged sites to enhance cell lethality. Experiments were performed in which different amounts of 5-bromouracil (5-BrU) were substituted for thymine in Escherichia coli DNA. The amount of bromide released was assayed after quantitative reaction of radiation-induced solvated electrons with 5-BrU in DNA samples irradiated in solution and irradiated in the cellular environment. By varying the amount of 5-BrU incorporated in the DNA, the average distance between 5-BrU molecules was systematically changed and, because the number of 5-BrU/electron reactions was monitored by the amount of bromine released, the maximum average electron migration distance along the 5-BrU DNA could be estimated. Using this approach, the maximum average electron migration distance in aqueous solutions of 5-BrU DNA was about 6.5 to 10 base distances in nonhybrid 5-BrU DNA (assuming only intrastrand migration). Similar methods revealed charge migration in 5-BrU DNA incorporated into E. coli, and the maximum average migration distance was about 5 to 6 base distances (assuming only intrastrand migration). Only 11-16% of the electrons produced during radiolysis were scavenged by 5-BrU DNA in aqueous solution, and only 1% resulted in the release of bromide from 5-BrU-DNA inside E. coli.
Assuntos
Bromouracila/química , DNA Bacteriano/efeitos da radiação , Elétrons , Escherichia coli/genética , Escherichia coli/efeitos da radiação , SoluçõesRESUMO
Gas chromatography-mass spectrometry with selected-ion monitoring was used to measure the yields of radiation-induced base products in aqueous solutions of native or heat-denatured DNA irradiated in the dose range 20-100 Gy. These DNA solutions were saturated with nitrous oxide, nitrogen, air or 20% oxygen in nitrous oxide during irradiation. The products measured were as follows: 5,6-dihydrothymine; 5-hydroxy-5,6-dihydrothymine; 5,6-dihydrothymine (thymine glycol); 5-hydroxy-5,6-dihydrocytosine; 5,6-dihydroxy-5,6- dihydrocytosine (cytosine glycol); 4,6-diamino-5-formamidopyrimidine; 7,8-dihydro-8-oxoadenine (8-hydroxyadenine); 2,6-diamino-4-hydroxy-5- formamidopyrimidine; and 7,8-dihydro-8-oxoguanine (8-hydroxyguanine). In oxygenated solutions, 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine and 5-hydroxy-5,6-dihydrocytosine were not formed. The yields of all products, other than 5,6-dihydrothymine, were greater in irradiated DNA samples from N2O-saturated solutions than from N2-saturated solutions. In N2-saturated solutions the yield of 8-hydroxyadenine was low and 8-hydroxyguanine was undetectable. Yields of pyrimidine products in heat-denatured DNA were greater than those in native DNA using all types of gases. However, the effects of DNA conformation on the yields of purine products were dependent on the type of gas used to saturate the irradiated DNA solutions. Yields of formamidopyrimidines were generally lower in solutions of DNA irradiated in the native than in the heat-denatured conformation. In air-saturated solutions of DNA, yields of 8-hydroxypurines were not influenced greatly by DNA conformation. In DNA solutions saturated with N2O/O2, 8-hydroxypurine formation was more favourable in the heat-denatured conformation than in the native conformation. On the other hand, in deoxygenated solutions, formation of 8-hydroxypurines was favoured in the native conformation. Data indicate that DNA conformation and the type of gas used to saturate the irradiated solutions have a profound influence on yields of base products in DNA.
Assuntos
DNA/efeitos da radiação , Conformação de Ácido Nucleico , Purinas , Pirimidinonas , Ar , Nitrogênio , Óxido Nitroso , Oxigênio , Soluções , ÁguaRESUMO
Aqueous solutions of calf thymus deoxyribonucleic acid (DNA) were exposed to hydrogen peroxide in the presence of air. Base products formed in DNA were identified and quantitated following acid hydrolysis and trimethylsilylation using gas chromatography-mass spectrometry. The yields of these products were dependent upon the hydrogen peroxide concentration, and increased in the following order: 8-hydroxyadenine, cytosine glycol, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine, thymine glycol, and 4,6-diamino-5-formamidopyrimidine. Previous studies have shown that these compounds are typically formed in DNA in aqueous solution by hydroxyl radicals generated by ionizing radiation. Hydrogen peroxide is thought to participate in a Fenton-like reaction with transition metals, which are readily bound to DNA in trace quantities, resulting in the production of hydroxyl radicals close to the DNA. This proposed mechanism was examined by exposing DNA to hydrogen peroxide either in the presence of a hydroxyl radical scavenger or following pretreatment of DNA with metal-ion chelators. The results indicate that trace quantities of transition metal ions can react readily with hydrogen peroxide to produce radical species. The production of radical species was monitored by determining the altered bases that resulted from the reaction between radicals and DNA. The yields of the base products were reduced by 40 to 60% with 10 mmol dm-3 of dimethyl sulfoxide. A 100-fold increase in the concentration of dimethyl sulfoxide did not result in a further reduction in hydrogen peroxide-induced base damage. DNA which was freed from bound metal ions by pretreatment with metal ion chelators followed by exhaustive dialysis was found to be an ineffective substrate for hydrogen peroxide. The yields of base products measured in this DNA were at background levels. These results support the role of metal ions bound to DNA in the site-specific formation of highly reactive radical species, most likely hydroxyl radicals, in hydrogen peroxide-induced damage to the bases in DNA.
Assuntos
Dano ao DNA , DNA/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Sequência de Bases , Quelantes/farmacologia , Dimetil Sulfóxido/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Nucleotídeos de Purina , Nucleotídeos de PirimidinaRESUMO
Leukocyte-induced DNA damage may partially account for the known association between chronic inflammation and malignancy. Since elucidation of the chemical nature of leukocyte-induced DNA damage may enhance our understanding of the mechanisms underlying leukocyte-induced DNA damage and the carcinogenesis associated with inflammation, the present study was undertaken to characterize the chemical modifications that occur in DNA exposed to stimulated human neutrophils. Calf thymus DNA was exposed to phorbol myristate acetate (PMA)-stimulated neutrophils in the presence or absence of exogenously added iron ions. DNA samples were subsequently hydrolyzed, derivatized and analyzed by gas chromatography-mass spectrometry with selected-ion monitoring. A variety of base modifications including cytosine glycol, thymine glycol, 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine were identified. The yield of these various base products was increased by the addition of iron ions. Specifically, in the presence of physiologic quantities of iron ions, approximately 7 of every 1,000 DNA bases were modified. Addition of the superoxide anion scavenger, superoxide dismutase, the hydrogen peroxide scavenger, catalase, the hydroxyl scavenger, dimethylsulfoxide, or the iron chelator, deferoxamine, to DNA mixtures containing PMA, neutrophils, and iron ions, greatly decreased the yield of the damaged DNA base products. Our results indicate that stimulated human neutrophils can damage each of the four bases in DNA. It is likely that hydroxyl radical, generated via an iron catalyzed Haber-Weiss reaction, mediates neutrophil-induced DNA base damage, since: (a) the chemical structure of neutrophil-induced DNA base damage is consistent with a hydroxyl radical-mediated mechanism, (b) hydroxyl radical generated via ionizing radiation in aqueous solution produces DNA base modifications that are identical to neutrophil-induced DNA base modifications, (c) iron ions increase neutrophil-induced DNA base damage, and (d) iron chelators or scavengers of superoxide anion, hydrogen peroxide or hydroxyl radical decrease neutrophil-induced DNA base damage.
Assuntos
Dano ao DNA , Neutrófilos/fisiologia , Catalase/farmacologia , Fenômenos Químicos , Química , DNA/efeitos dos fármacos , DNA/metabolismo , Desferroxamina/farmacologia , Dimetil Sulfóxido/farmacologia , Radicais Livres , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hidróxidos/farmacologia , Radical Hidroxila , Ferro/farmacologia , Estrutura Molecular , Superóxido Dismutase/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Gas chromatography-mass spectrometry with selected-ion monitoring was used to study radiation-induced damage to DNA. Quantitative analysis of modified purine and pyrimidine bases resulting from exposure to ionizing radiation using this technique is dependent upon the selection of appropriate internal standards and calibration of the mass spectrometer for its response to known quantities of the internal standards and the products of interest. The compounds 6-azathymine and 8-azaadenine were found to be suitable internal standards for quantitative measurement of base damage in DNA. For the purpose of calibration of the mass spectrometer. relative molar response factors for intense characteristic ions were determined for the trimethylsilyl derivatives of 5-hydroxyuracil, thymine glycol, and 5,6-dihydrothymine using 6-azathymine, and for the trimethylsilyl derivatives of 4,6-diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyguanine using 8-azaadenine. Accurate measurement of the yield of radiation-induced modifications to the DNA bases is also dependent upon two chemical steps in which the purines and pyrimidines are released from the sugar-phosphate backbone and then derivatized to make them volatile for gas chromatography. The completeness of these reactions, in addition to assessing the stability of the modified DNA bases in acid and their trimethylsilylated derivatives over the time necessary to complete the experimental analysis was also examined. Application of this methodology to the measurement of radiation-induced base modification in heat-denatured, nitrous oxidesaturated aqueous solutions of DNA is presented.
Assuntos
Dano ao DNA , DNA/efeitos da radiação , Nucleotídeos de Purina/efeitos da radiação , Nucleotídeos de Pirimidina/efeitos da radiação , Cromatografia Gasosa-Espectrometria de MassasRESUMO
Hydroxyl radicals are known to produce DNA-protein crosslinks in chromatin in vivo and in vitro. Here we investigated DNA-protein crosslinks formed between aliphatic amino acids and thymine in calf thymus nucleohistone exposed predominantly to hydroxyl radicals in gamma-irradiated N2O-saturated aqueous solution. Aliphatic amino acids are the predominant types of amino acids in the core histones of calf thymus, and thus are likely to form crosslinks with DNA. For identification of the crosslinks we first investigated hydroxyl radical-induced crosslinking of thymine to aliphatic amino acids in model systems, i.e. an aqueous mixture of thymine and a single amino acid. Samples were analyzed for possible thymine-amino acid crosslinks by gas chromatography-mass spectrometry. Using this approach the structure of the crosslinks was elucidated, and information on their gas chromatographic and mass spectral properties was obtained. Gas chromatography-mass spectrometry with selected-ion monitoring (GC-MS/SIM) was then used to identify DNA-protein crosslinks in acidic hydrolysates of calf thymus nucleohistone exposed to ionizing radiation in buffered aqueous solution. DNA-protein crosslinks involving thymine and the amino acids Gly, Ala, Val, Leu, Ile and Thr were identified. In some cases several isomers of the same crosslink were observed. The yield of the crosslinks was measured by GC-MS/SIM and was found to be a linear function of radiation dose in the range 49 to 436 Gy. The mechanism for the formation of these DNA-protein crosslinks is thought to involve hydrogen atom abstraction by hydroxyl radicals from the aliphatic amino acid followed by addition of the amino acid radical to the carbon(6)-position of thymine and subsequent oxidation of the adduct radical.
Assuntos
DNA/metabolismo , Histonas/metabolismo , Hidróxidos , Aminoácidos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Radical HidroxilaRESUMO
The formation of (R)- and (S)-8,5'-cycloadenosine 5'-monophosphate (8,5'-cycloAMP), 8-hydroxyadenosine 5'-monophosphate (8-hydroxyAMP), and radiolytic adenine release from irradiated solutions of adenosine 5'-monophosphate (5'-AMP) was measured as a function of increasing liquid-phase oxygen concentration. Three classes of specific molecular damage were identified on the basis of the oxygen dependence for product formation. Major changes in product yield occurred near the range of oxygen concentrations associated with the radiobiological oxygen effect. In addition to these data, systematic increases in the concentration of hydrogen peroxide at the time of irradiation resulted in an increase in the yield of 8-hydroxyAMP and a component of radiolytic adenine release in nitrogen-saturated solutions of 5'-AMP. However, no changes in the yield of the 8,5'-cyclonucleotides were observed under these conditions.