RESUMEN
Corneal neo-vascularization (CNV) is a highly prevalent medical condition which impairs visual acuity. The role of specific proteins in modulating CNV has been extensively reported, although no studies have described the entire human proteome in CNV corneas. In this paper, we performed a proteomic analysis of vascularized vs healthy corneal stroma, in a CNV mouse model and in CNV-affected patients, with a specific focus on extracellular matrix (ECM) proteins. We identified and quantified 2315 murine proteins, 691 human proteins and validated 5 proteins which are differentially expressed in vascularized samples and conserved in mice and humans: tenascin-C and fibronectin-1 were upregulated, while decorin, lumican and collagen-VI were downregulated in CNV samples. Interestingly, among CNV patients, those affected with Acanthamoeba keratitis showed the highest levels of fibronectin-1 and tenascin-C, suggesting a specific role of these two proteins in Acanthamoeba driven corneal CNV. On a broader picture, our findings support the hypothesis that the corneal stroma in CNV samples is disorganized and less compact. We are confident that the dissection of the human corneal proteome may shed new light on the complex pathophysiology of human CNV, and finally lead to improved treatments.
Asunto(s)
Neovascularización de la Córnea/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Mapas de Interacción de Proteínas , Adulto , Anciano , Animales , Neovascularización de la Córnea/patología , Matriz Extracelular/patología , Proteínas de la Matriz Extracelular/análisis , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , ProteómicaRESUMEN
Chronic dialysis treatment is characterized by a series of complex interdependent objective problems, such as the dialysis experience, the individual way to assess it, and some "protective" factors such as social and family support. Progresses in dialysis research show that dialysis patients have important alternatives to passively accept their condition: thanks to adequate psychological and relational aid, they can reach rather advanced adaptation levels, which allow them to modify both their behaviour and way of life, to keep a satisfactory compliance, and to improve their quality of life (QoL). In this adaptation process, both family and social support play an important role, although controversy still exists on it. The results of our study confirm the complexity of this role and show that either haemodialysis or peritoneal dialysis patients' adaptation process and QoL may be directly related to the extent of family member's ("caregiver") support. It is of particular interest the fact that patients, especially those undergoing haemodialysis, provided with a caregiver's assistance but who choose to "act by themselves", do have better adaptation levels and QoL than those who rely only on their caregiver. This fact reassesses the widely accepted point of view that continuous caregiver's support is always a positive and necessary factor in order to improve both the adaptation and the QoL of dialysis patients.
Asunto(s)
Adaptación Psicológica , Familia , Calidad de Vida , Diálisis Renal , Apoyo Social , HumanosRESUMEN
Several N-nitroso compounds, present in foods and beverages or formed in the stomach from their precursors, act as alkylating agents. By using a highly reliable technique (high-resolution gas chromatography-mass spectrometry with negative-ion chemical ionization and selected ion recording), we measured a series of specific O6-alkylguanines in snap-frozen paired stomach tissue samples (tumor and noninvolved mucosa) obtained at surgery from 24 gastric cancer patients identified in Florence, Italy. Samples of noninvolved mucosa had higher levels of total O6-alkylguanines and more frequently detectable levels (54%) than tumor samples (29.2%). O6-propylguanine and O6-methylguanine were the single adducts most frequently detected in noninvolved mucosa and tumor tissue, respectively. Tumor samples showed higher levels of total O6-alkylguanines in female patients (p = 0.03) and among those with a diffuse histological type (p = 0.06) or seronegative for Helicobacter pylori CagA antibodies (p = 0.06). Mean dietary nitrate intake was significantly higher in patients with detectable levels of adducts in tumor samples (p = 0.03). Estimated intakes of dimethylamine and N-nitrosodimethylamine correlated with total levels of O6-alkylguanines in noninvolved gastric mucosa. These findings, although based on a small series of cases, support a role for N-nitroso compounds from dietary sources in the etiology of gastric cancer.
Asunto(s)
Aductos de ADN/análisis , Mucosa Gástrica/química , Guanina/análisis , Compuestos Nitrosos/análisis , Neoplasias Gástricas/química , Anciano , Anticuerpos Antibacterianos/sangre , Dieta , Dimetilaminas/análisis , Dimetilnitrosamina/análisis , Femenino , Análisis de los Alimentos , Cromatografía de Gases y Espectrometría de Masas , Jugo Gástrico/química , Mucosa Gástrica/patología , Helicobacter pylori/inmunología , Helicobacter pylori/patogenicidad , Humanos , Masculino , Persona de Mediana Edad , Compuestos Nitrosos/efectos adversos , Neoplasias Gástricas/etiología , Encuestas y CuestionariosRESUMEN
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine formed in meat and fish during cooking and can be used as a model compound for this class of chemicals possibly involved in human carcinogenesis. Knowing the exposure to heterocyclic amines is important for establishing their role in human diseases. Serum albumin (SA) and globin (Gb) adducts were first tested as biomarkers of exposure to PhIP in male Fischer 344 rats given oral doses of 0.1, 0.5, 1 and 10 mg/kg. Blood samples were collected 24 hr after treatment and PhIP released from SA and Gb after acidic hydrolysis was analyzed by gas chromatography-mass spectrometry or liquid chromatography-tandem mass spectrometry. PhIP-SA and Gb adducts increased linearly with the dose. Studies on 35 volunteers with different dietary habits exhibited that diet was a major determinant in the formation of both adducts. PhIP-SA adducts were significantly higher in meat consumers than in vegetarians (6.7 +/- 1.6 and 0.7 +/- 0.3 fmol/mg SA; respectively, mean +/- SE; p = 0.04, Mann-Whitney U test). The Gb adduct pattern was quantitatively lower but paralleled SA (3 +/- 0.8 in meat consumers and 0.3 +/- 0.1 in vegetarians). PhIP-SA adducts were no different in smokers and in non-smokers. The results show for the first time that PhIP-blood protein adducts are present in humans not given the synthetic compound. Both biomarkers appear to be suitable for assessing dietary exposure and internal PhIP dose and may be promising tools for studying the role of heterocyclic amines in the etiology of colon cancer and other diseases.
Asunto(s)
Carcinógenos/metabolismo , Dieta , Hemoglobinas/metabolismo , Imidazoles/sangre , Albúmina Sérica/metabolismo , Animales , Biomarcadores/sangre , Cromatografía Liquida , Dieta Vegetariana , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hidrólisis , Masculino , Espectrometría de Masas , Carne , Unión Proteica , Ratas , Ratas Endogámicas F344 , Sensibilidad y EspecificidadRESUMEN
The 'Mediterranean diet', a diet rich in cereals, fruit and vegetables, has been associated with lowering the risk of a variety of cancers of the digestive tract and the bladder. In a previous study, we showed that the high phenolic content these dietary components produce in the urine could be associated with higher antimutagenic properties of the urine and lower arylamine-DNA adducts in exfoliated bladder cells. We have conducted a case-control study on 162 bladder cancer patients and 104 hospital controls. Total aromatic DNA adducts were measured in white blood cells (WBC) of all subjects by (32)P-post-labelling. Genetically based metabolic polymorphisms were analysed by PCR-RFLP (NAT2, GSTM1, GSTT1, GSTP1, COMT and NQO1). All subjects were interviewed about their tobacco use, dietary habits and other risk factors. The odds ratio (OR) for the risk of bladder cancer according to the presence/absence of WBC DNA adducts (detection limit 0.1 RALx10(8)) was 3.7 [95% confidence interval (CI) 2.2-6.3] and a dose-response relationship with levels of adducts was apparent. The association between case/control status and the presence of WBC DNA adducts was significantly stronger in the subjects who consumed fewer portions of fruit or vegetables per day (OR 7.80, 95% CI 3.0-20.30 for 0-1 portions of vegetables) than in the heavy consumers (OR 4.98 for consumers of 2 portions daily, OR 1.97 for consumers of > or =3 portions; similar but lower estimates were found for the intake of fruit). No association was noticed between tobacco smoking and WBC DNA adducts. Only NAT-2, among the several genotypes considered, was associated in a statistically significant way with the risk of bladder cancer (OR 1.72, 95% CI 1.03-2.87) and with the levels of WBC DNA adducts. Our report suggests that fruit and vegetables could protect against bladder cancer by inhibiting the formation of DNA adducts.
Asunto(s)
Aductos de ADN/sangre , ADN de Neoplasias/química , Dieta , Conducta Alimentaria , Frutas , Leucocitos/química , Neoplasias de la Vejiga Urinaria/prevención & control , Verduras , Estudios de Casos y Controles , ADN de Neoplasias/aislamiento & purificación , Humanos , Italia/epidemiología , Persona de Mediana Edad , Riesgo , Fumar/epidemiología , Neoplasias de la Vejiga Urinaria/sangre , Neoplasias de la Vejiga Urinaria/epidemiologíaRESUMEN
The excretion of nitrate, nitrite, apparent total N-nitroso compounds and volatile nitrosamines was measured in 24 hr urine from 61 Egyptians, divided into 4 groups: controls, Schistosoma haematobium-infected patients and bladder cancer patients with and without a history of schistosomal infection. Urinary nitrate in S. haematobium-infected patients was significantly higher than in the other 3 groups. Nitrite was below the detection limit of the method (
Asunto(s)
Nitratos/orina , Nitritos/orina , Compuestos Nitrosos/orina , Esquistosomiasis Urinaria/complicaciones , Esquistosomiasis Urinaria/orina , Neoplasias de la Vejiga Urinaria/complicaciones , Neoplasias de la Vejiga Urinaria/orina , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/diagnóstico por imagen , Egipto , Humanos , Masculino , Persona de Mediana Edad , Monitoreo Fisiológico , Valores de Referencia , UltrasonografíaRESUMEN
Genotoxic chemicals are known to react with DNA either directly or after metabolic activation to form adducts, a step thought to be relevant with respect to chemical carcinogenesis. Evaluation of cancer risk due to exposure to chemicals requires information about the internal dose which depends on individual variation in rates of metabolic activation and detoxification. The presence and the amount of specific DNA adducts provide a good indication of chemical exposure and genetic damage resulting from exposure to carcinogens and account for some of the factors affecting individual susceptibility to cancer. Analysis of DNA adducts requires that the sensitivity of the methods be sufficiently high to allow the detection of about 1 adduct/109 normal nucleotides. Most suitable methods are based on 32P-postlabelling, immunoassays or physico-chemical techniques such as HPLC coupled to synchronous fluorescence spectroscopy or gas chromatography-mass spectrometry. These methods have been used to assess human exposure to a variety of chemical carcinogens including polycyclic aromatic hydrocarbons, aromatic amines, heterocyclic aromatic amines or aflatoxins. In some instances, the use of DNA-adducts has given accurate estimates of risk.
Asunto(s)
Carcinógenos/análisis , Carcinógenos/metabolismo , Aductos de ADN/análisis , Aductos de ADN/metabolismo , Neoplasias , Humanos , Inmunoensayo , Medición de Riesgo , Sensibilidad y Especificidad , Análisis EspectralRESUMEN
Epidemiologic studies have suggested that smokers of air-cured tobacco (rich in arylamines) are at higher risk of bladder cancer than smokers of flue-cured tobacco. The risk has been shown to be modulated by the N-acetyltransferase genotype. We analyzed the biopsies of 45 patients with bladder cancer. p53 mutations were sought by direct sequencing, and 4-aminobiphenyl-DNA adducts were measured by negative ion gas chromatography-mass spectrometry. 4-Aminobiphenyl-DNA adducts were higher in smokers of air-cured tobacco and in current smokers, but no relationship with the number of cigarettes smoked was found. Adducts were higher in more advanced histologic grades of tumors. No pattern was evident for p53 mutations. Seven of 9 mutations occurred in grade 3 tumors. No association was found between 4-ABP adducts and GSTM1 or NAT2 genetic polymorphisms.
Asunto(s)
Compuestos de Aminobifenilo/metabolismo , Aductos de ADN , Genes p53 , Fumar , Neoplasias de la Vejiga Urinaria/genética , Adulto , Anciano , Arilamina N-Acetiltransferasa/fisiología , Glutatión Transferasa/fisiología , Humanos , Masculino , Persona de Mediana Edad , Mutación , Plantas Tóxicas , NicotianaRESUMEN
Benzo[a]pyrene diol epoxide adducts with hemoglobin (Hb) were measured to detect human exposure to environmental benzo[a]pyrene from traffic exhaust. Benzo[a]pyrene tetrahydrotetrols (BPTs) released from Hb after acid hydrolysis were quantitated by gas chromatography-mass spectrometry after immunoaffinity chromatography. Fifty three newspaper vendors were enrolled. The median adduct concentration was 0.3 fmol BPTs/mg Hb in high density traffic-exposed vendors and < or = 0.1 fmol BPTs/mg Hb in those exposed to low density traffic; the difference was not significant (P = 0.09). Among non-smokers, adducts were detectable in 60% of high exposure subjects (median 0.3 fmol BPTs/mg Hb) and in 28% of those with low exposure (median < or = 0.1 fmol/mg Hb). This difference was significant (P = 0.02). In low exposure smokers the median of adducts was 0.26 fmol BPTs/mg Hb, while in low exposure non-smokers it was < or = 0.1 fmol BPTs/mg Hb (P = 0.08, not significant). Adduct concentration was no different for low and high density traffic-exposed smokers (P = 0.82). The data indicate a significant difference in adduct concentration related to traffic exhaust exposure among non-smokers.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/análisis , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/metabolismo , Exposición a Riesgos Ambientales , Hemoglobinas/efectos de los fármacos , Hemoglobinas/metabolismo , Exposición Profesional , Emisiones de Vehículos , Adulto , Animales , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Femenino , Cromatografía de Gases y Espectrometría de Masas , Hemoglobinas/análisis , Humanos , Masculino , Ratones , Ratones Endogámicos , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Fumar/efectos adversos , Espectrofotometría UltravioletaRESUMEN
A sensitive and specific method has been developed for the simultaneous analysis of different O6-alkylguanines. The cross-reactivity of two different antibodies raised against O6-methylguanosine and O6-butylguanosine for a series of O6-alkylguanines was exploited for the immunoaffinity purification of biological samples before quantitative analysis by gas chromatography/mass spectrometry. The method can be applied to the detection of O6-alkylguanines in DNA and appears to be useful for studying chemical carcinogen mechanisms in animals and possibly for the detection of human exposure to alkylating agents.
Asunto(s)
Guanina/análogos & derivados , Guanina/aislamiento & purificación , Alquilación , Animales , Anticuerpos , Cromatografía de Gases y Espectrometría de Masas/métodos , Guanosina/análogos & derivados , Guanosina/inmunología , Guanosina/aislamiento & purificación , Masculino , Conejos , Sensibilidad y EspecificidadRESUMEN
N-Nitrosobutyl(4-hydroxybutyl)amine (BBN) is a selective bladder carcinogen in rats. Its organ specificity may depend on several factors, including metabolic activation, DNA alkylation and repair within the target organ. Metabolic activation of BBN, which is asymmetrical, may result in butylating and 4-hydroxybutylating species. To test this view, BBN was administered as a single oral dose of 20 or 120 mg/rat or six doses of 20 mg/rat over 2 weeks. The animals given the single 120 mg dose were killed 3, 6 and 24 h after treatment. Rats given 20 mg or 6 x 20 mg BBN were killed 24 h after the last dose. DNA from liver and urothelial cells was hydrolyzed and analyzed for O6-butylguanine (O6-BuG) and O6-(4-hydroxybutyl)guanine [O6-(4-OH-Bu)G] as their pentafluorobenzyl-trimethylsilyl derivatives by high-resolution gas chromatography--negative ion chemical ionization mass spectrometry with selective ion recording after immunoaffinity extraction. Polyclonal antibodies raised against O6-(4-hydroxybutyl)-guanosine [O6-(4-OH-Bu)GR] were coupled to CNBr-activated Sepharose 4B. This was mixed with a gel coupled to antibodies raised against O6-BuG, already available in the laboratory, and the mixed gel was used for the one-step sample clean-up, enrichment and extraction of O6-(4-OH-Bu)G and O6-BuG from hydrolyzed DNA. O6-BuG in urothelial DNA of rats given a single dose of 120 mg BBN increased from 0.44 +/- 0.12 mumol/mol guanine (mean +/- SE) 3 h after treatment, to 17.9 +/- 7.23 mumol/mol guanine at 24 h. O6-(4-OH-Bu)G in the same tissue was 7.7 +/- 3.19 mumol/mol guanine 3 h after treatment and 12.2 +/- 7.01 mumol/mol guanine at 24 h. O6-BuG and O6-(4-OH-Bu)G were always lower in the liver than in urothelial cells. Twenty-four hours after a single dose of 20 mg BBN, urothelial O6-BuG was 5.41 +/- 1.73 mumol/mol guanine and did not accumulate after six doses of 20 mg/rat BBN, since it was 2.59 +/- 1.23 mumol/mol guanine 24 h after the last dose. O6-BuG in liver DNA was detectable after the single dose of 20 mg, but not after 6 x 20 mg/rat BBN. O6-(4-OH-Bu)G was not detected in either the bladder or the liver after 20 mg or after the six doses of BBN.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Aciclovir/análogos & derivados , Butilhidroxibutilnitrosamina/metabolismo , Butilhidroxibutilnitrosamina/toxicidad , Daño del ADN , ADN/efectos de los fármacos , ADN/metabolismo , Guanina/análogos & derivados , Hígado/efectos de los fármacos , Hígado/metabolismo , Neoplasias de la Vejiga Urinaria/inducido químicamente , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismo , Aciclovir/análisis , Aciclovir/metabolismo , Animales , Anticuerpos , Especificidad de Anticuerpos , ADN/análisis , Ensayo de Inmunoadsorción Enzimática , Guanina/análisis , Guanina/metabolismo , Hígado/química , Masculino , Espectrometría de Masas , Conejos , Ratas , Ratas Endogámicas , Vejiga Urinaria/anatomía & histologíaRESUMEN
O6-Butylguanine was detected in the urine of rats given the butylating agent N-nitroso-N-butylurea. O6-Butylguanine contents in the 24-h rat urine samples after i.p. doses of 50, 100, and 200 mg/kg N-nitroso-N-butylurea were 1.03 +/- 0.41 (SE), 8.30 +/- 1.70, and 59.53 +/- 6.52 pmol, respectively. This suggests that O6-butylguanine formation in nucleic acids might be repaired in vivo, possibly by base excision, besides other mechanisms. After i.v. doses of 0.1 and 1 mg/kg of O6-butylguanine to rats urinary excretion did not exceed 2% of the administered dose, suggesting that the amount of O6-butylguanine effectively released by base excision might be much larger than that detected in the urine after N-nitroso-N-butylurea. Inhibition of the enzyme O6-alkyl-DNA transferase by N-nitrosodimethylamine increased urinary O6-butylguanine resulting from exposure to N-nitroso-N-butylurea (100 mg/kg i.p.) up to four times, thus confirming an alternative DNA repair mechanism.
Asunto(s)
Reparación del ADN , ADN/metabolismo , Guanina/análogos & derivados , Metiltransferasas/metabolismo , Compuestos de Nitrosourea/metabolismo , Animales , Guanina/orina , Masculino , Metiltransferasas/antagonistas & inhibidores , N-Metilaspartato/farmacología , Compuestos de Nitrosourea/administración & dosificación , O(6)-Metilguanina-ADN Metiltransferasa , RatasRESUMEN
In order to establish the importance of the target organ in the activation of bladder carcinogens, we compared rat liver and urothelial cell alpha-hydroxylation activities using as substrates N-nitrosobutyl(4-hydroxybutyl)amine and its metabolite N-nitrosobutyl(3-carboxypropyl)amine, two potent urinary bladder carcinogens in animals. Previous studies have shown that the production of molecular nitrogen can serve as an indicator of nitrosamine alpha-hydroxylation. The use of doubly 15N-labelled nitrosamines and the gas chromatography-mass spectrometric detection of 15N2 formed gives a measurement of the extent of this metabolic step. Various amounts of 15N-labelled substrates were incubated for 60 min at 37 degrees C with rat liver S9 preparations or urothelial cell homogenates in the presence of a NADPH generating system. Both enzyme sources metabolized 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine through the alpha-hydroxylation pathway. Using hepatic S9 fractions, 15N2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine increased from 1.69 +/- 0.02 nmol/h per mg protein (mean +/- S.E.) to 5.78 +/- 0.5 with substrate concentrations ranging between 0.55 and 5.55 mM. 15N2 produced by urothelial cell homogenates was about 40-50% that of the liver S9. 15N-labelled N-nitrosobutyl(3-carboxypropyl)amine was also metabolized through the alpha-hydroxylation pathway both by hepatic S9 and urothelial cell homogenates, though to a lesser extent. 15N2 production was about 10-times less than from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine, but again urothelial cell 15N2 production was about 40-50% that of the liver. Treatment with phenobarbital resulted in a 2.7-fold increase in the 15N2 produced from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9. No effect was observed with urothelial cell homogenates. Acetone treatment had no effect on 15N2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9, but raised 15N2 production by urothelial cell homogenates 1.8 times. Although the liver has a greater capacity than the bladder for activating the 15N-labelled nitrosamines studied, the target organ can metabolize bladder carcinogens, thus increasing the possibility of a local toxic effect. Moreover, the distribution of P-450 isozymes might be different in the bladder and this could affect the metabolism of nitrosamines reportedly formed in the human bladder in some pathological conditions.
Asunto(s)
Butilhidroxibutilnitrosamina/metabolismo , Carcinógenos/metabolismo , Hígado/metabolismo , Nitrosaminas/metabolismo , Vejiga Urinaria/metabolismo , Acetona/farmacología , Animales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Hidroxilación , Hígado/citología , Hígado/efectos de los fármacos , Extractos Hepáticos/metabolismo , Masculino , Nitrógeno/química , Fenobarbital/farmacología , Ratas , Vejiga Urinaria/efectos de los fármacosRESUMEN
Determination of molecular nitrogen formed as a consequence of nitrosamine alpha-hydroxylation provides a useful means for studying the extent of activation of these compounds in target and nontarget organs and tissues. alpha-Hydroxylation in rat liver and urothelial cells was compared using as substrate doubly 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine (15N-NBHBA), a potent bladder carcinogen in rodents. Both enzyme sources metabolized 15N-NBHBA through the alpha-hydroxylation pathway. 15N2 production was dependent on the amount of substrate incubated. Vmax values for 15N2 production by urothelial cells and by liver postmitochondrial supernatant were 4.47 and 3.21 nmol/mg protein per h, respectively.
Asunto(s)
Carcinógenos/metabolismo , Hígado/metabolismo , Nitrosaminas/metabolismo , Vejiga Urinaria/metabolismo , Animales , Biotransformación , Reparación del ADN , Hidroxilación , Técnicas In Vitro , Masculino , Especificidad de Órganos , Ratas , Ratas Endogámicas , Neoplasias de la Vejiga Urinaria/inducido químicamenteRESUMEN
A sensitive, specific, and rapid method for quantitating the minor adduct O6-butylguanine (O6BuG) in hydrolyzed DNA has been developed by combining immunoaffinity chromatography and high resolution gas chromatography-negative ion chemical ionization-mass spectrometry. Polyclonal antibodies raised against O6BuG were coupled to CNBr-activated Sepharose 4B and used for sample clean-up and extraction of the specific O6-alkylguanine. After addition of O6BuG and its deuterium labeled analogue (O6BuG-D7), used as internal standard, hydrolyzed DNA was applied on the immunoaffinity column and washed with water, and the immunoadsorbed butylated guanines were eluted with acetone/water cetome/water (95/5) before gas chromatographic derivatization. O6BuG and O6BuG-D7 were analyzed and quantitated by high resolution gas chromatography-negative ion chemical ionization-mass spectrometry as their pentafluorobenzyl-trimethylsilyl derivatives. Immunoaffinity column capacity and O6BuG recovery from this column were 1.53 nmol O6BuG/column and 62 +/- 5%, respectively. The method was applied to evaluate O6BuG levels in DNA butylated in vitro with 10 mM N-nitroso-Nr-butylurea or isolated from rats given an i.p. dose of 185 mg/kg N-nitroso-N-butylurea or N-nitrosodibutylamine. In the first case the level of modifications present in calf thymus DNA was 104 mumol O6BuG/mol guanine, and in the second case O6BuG in liver DNA was about 6 times higher after N-nitroso-N-butylurea (2.11 mumol O6BuG/mol guanine) than after N-nitrosodibutylamine (0.34 mumol O6BuG/mol guanine) treatment. These results indicate that O6BuG formed in vivo can be isolated and quantitated by this method, which may also be useful for studying DNA damage and repair mechanisms.
Asunto(s)
ADN/análisis , Guanina/análogos & derivados , Animales , Anticuerpos , Formación de Anticuerpos , Calibración , Carcinógenos/metabolismo , Cromatografía de Afinidad/métodos , ADN/metabolismo , Deuterio , Ensayo de Inmunoadsorción Enzimática , Cromatografía de Gases y Espectrometría de Masas/métodos , Guanina/aislamiento & purificación , Guanina/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Hemocianinas/metabolismo , Hígado/química , Masculino , Nitrosaminas/metabolismo , Compuestos de Nitrosourea/metabolismo , Ratas , Albúmina Sérica Bovina/metabolismoRESUMEN
The most widely accepted metabolic pathway leading to the formation of reactive intermediates from nitrosamines involves enzymatic hydroxylation at the carbon atom alpha to the nitroso moiety. All subsequent steps are non-enzymatic reactions and the final result is the stoichiometric formation of a cationic product and molecular nitrogen. Thus the amount of molecular nitrogen evolved can be used as an indicator of alpha-hydroxylation. The use of doubly 15N-labelled nitrosamines and the detection of 15N2 by MS makes it simpler to measure the extent of alpha-hydroxylation. We have studied the alpha-oxidation of doubly 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine (BBN) and its metabolite N-nitrosobutyl(3-carboxypropyl)amine (BCPN), two potent urinary bladder carcinogens in animals, within the target organ. Various amounts of 15N-labelled BBN ranging from 0.1 to 5 mumol were incubated at 37 degrees C for 4 h in the isolated rat bladder and the formation of 15N2 was measured by GC-MS. 15N2 production was linear up to 1 mumol and represented approximately 0.1% of the substrate incubated. Time-course experiments showed that 15N2 production was linear over a 6 h incubation period, ranging from 2.16 +/- 0.05 to 4.55 +/- 0.33 nmol/mg urothelial cell protein. 15N-labelled BCPN (1-5 mumol) was also incubated within the rat isolated bladder. 15N2 production from BCPN was approximately 10 times less than that from BBN. The results indicate that, though to a lower extent, the target organ activates 15N-labelled BBN and BCPN through the alpha-hydroxylation pathway.
Asunto(s)
Butilhidroxibutilnitrosamina/metabolismo , Carcinógenos/metabolismo , Nitrosaminas/metabolismo , Vejiga Urinaria/metabolismo , Animales , Biotransformación , Técnicas In Vitro , Masculino , Oxidación-Reducción , RatasRESUMEN
N-Nitrosodibutylamine and its omega-hydroxylated metabolite N-nitrosobutyl(4-hydroxybutyl)amine (NB4HBA) induce tumors in the urine bladder of different animal species through their common urinary metabolite N-nitrosobutyl(3-carboxypropyl)amine (NB3CPA), resulting from the oxidation of the alcoholic group of NB4HBA to a carboxylic group. NB4HBA disappearance from blood, the formation of its main metabolites, NB3CPA and NB4HBA-glucuronide (NB4HBA-G), and their urinary excretion, were investigated in rats after an i.v. dose of 1 mg/kg (5.7 mumol/kg). NB3CPA and NB4HBA-G formation was readily detectable 2 min after treatment and levels were still measurable at 120 and 30 min, respectively. The parent compound disappeared from blood 90 min after injection. The NB4HBA blood concentration-time profile was adequately described by a one-compartmental linear model. NB4HBA half-life was 8 min, total body clearance and renal clearance were 86.1 and 0.22 ml/min/kg, respectively. The 0-96-h urinary excretion of NB4HBA was 0.3% of the administered dose. NB3CPA half-life was 15 min; NB3CPA and NB4HBA-G urinary excretion were 36 and 11.7%, respectively, urinary excretion of known compounds accounting for less than 50%. After i.v. injection of NB3CPA equimolar to the NB4HBA dose, only 50% of unchanged compound was recovered in the urine and after NB4HBA-G, 41% of the administered dose was excreted unchanged, NB3CPA accounting for 10%. Thus NB3CPA and NB4HBA-G might undergo further biotransformation, suggesting that NB3CPA may not be the ultimate carcinogen responsible for urinary bladder tumor induction.
Asunto(s)
Butilhidroxibutilnitrosamina/farmacocinética , Nitrosaminas/farmacocinética , Animales , Biotransformación , Butilhidroxibutilnitrosamina/sangre , Butilhidroxibutilnitrosamina/metabolismo , Butilhidroxibutilnitrosamina/orina , Tasa de Depuración Metabólica , RatasRESUMEN
The isolated rat urinary bladder was used to study this organ's capacity to metabolize chemical carcinogens. In our experimental conditions, the urinary bladder carcinogen N-nitrosobutyl(4-hydroxybutyl)amine was oxidized to N-nitrosobutyl(3-carboxypropyl)amine. A time-dependent increase was observed in the amount of N-nitrosobutyl(3-carboxypropyl)amine formed and simultaneous disappearance of N-nitrosobutyl(4-hydroxybutyl)amine added, indicating that the bladder can metabolize N-nitrosobutyl(4-hydroxybutyl)amine to the metabolite considered responsible for tumor induction in the urinary bladder of laboratory animals. At 15, 30, 60, and 120 min the percentages of N-nitrosobutyl(3-carboxypropyl)amine formed were 11, 22, 36, and 64%, respectively, and 62, 48, 37, and 26% of N-nitrosobutyl(4-hydroxybutyl)amine remained unchanged. When N-nitrosodibutylamine was introduced into the isolated urinary bladder and incubated for 120 min, its oxidized metabolites N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine were formed, amounting to, respectively, 0.13 and 0.06% of the substrate added. The glucuronide of N-nitrosobutyl(4-hydroxybutyl)amine was incubated in the isolated rat urinary bladder both as a buffer and as a urine solution in order to detect cellular and urinary beta-glucuronidase activity. In both systems N-nitrosobutyl(4-hydroxybutyl)amine released was about 1% at 4 h and this percentage did not increase at 6 h. N-Nitrosobutyl(3-carboxypropyl)amine was detectable at 2 h and reached 0.2% of the substrate incubated at 6 h. The results indicate that the urinary bladder may play a role in activating bladder carcinogens.
Asunto(s)
Butilhidroxibutilnitrosamina/metabolismo , Carcinógenos/metabolismo , Nitrosaminas/metabolismo , Vejiga Urinaria/metabolismo , Animales , Biotransformación , Glucuronatos/metabolismo , Masculino , Ratas , Factores de Tiempo , Neoplasias de la Vejiga Urinaria/inducido químicamenteRESUMEN
The capacity of the isolated rat urinary bladder to metabolize chemical carcinogens was studied. Under our experimental conditions, the bladder carcinogen N-nitrosobutyl-(4-hydroxybutyl)amine (NBHBA) was oxidized to N-nitrosobutyl(3-carboxypropyl)amine (NBCPA). A time-dependent increase in the amount of NBCPA formed and a simultaneous disappearance of NBHBA indicated that the bladder can metabolize NBHBA to the metabolite considered to be responsible for tumour induction in the urinary bladder of laboratory animals. After 15, 30, 60 and 120 min, the percentages of NBCPA formed were 10%, 21%, 35% and 61%, respectively, and 59%, 49%, 36% and 25% of NBHBA remained unchanged. When N-nitrosodi-n-butylamine (NDBA) was introduced into the isolated urinary bladder and incubated for 120 min, its oxidized metabolites NBHBA and NBCPA were formed, in amounts of 0.13% and 0.06% of the substrate added.