RESUMEN
Bifunctional acid urease can not only remove urea from rice wine, but also effectively remove ethyl carbamate (EC), thereby reducing harmful substances in rice wine. Acid urease from Providencia rettgeri JN-B815 was expressed in Escherichia coli BL21(DE3) as an inclusion body. Subsequently, acid urease was refolded gradually using dilution-ultrafiltration and specific activity of 12.3 U mg-1 was obtained. The acid urease thus obtained was immobilized on graphene oxide/chitosan beads. The recovery of immobilized urease was 77% for the graphene oxide/chitosan composite when coupled for 8 h with 5% glutaraldehyde. Steady-state kinetic analysis showed that the optimum temperature for urease activity was 37°C. Time-dependent thermal inactivation studies showed that the stability of the immobilized urease improved at 37°C and the t1/2 (half-life) of decay in urease activity was 12 h, whereas it was 43 days for soluble and chitosan/graphene oxide immobilized urease at 4°C. A significant change in Km occurred during the immobilization; the Km for urea and EC in the free state were 9.14 mM and 386 mM, respectively, whereas they were 5.28 mM and 180 mM, respectively, in the immobilized state. The values of Vmax for immobilized EC and urea were 1.08 µmol min-1 and 1.16 µmol min-1, respectively, whereas they were 0.782 µmol min-1 and 0.931 µmol min-1, respectively, for soluble EC and urea. Furthermore, 90% of the original activity of graphene oxide/chitosan-urease beads were retained after the beads were used 10 times, indicating excellent reusability.
Asunto(s)
Quitosano/química , Enzimas Inmovilizadas , Grafito/química , Pliegue de Proteína , Ureasa , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Microesferas , Temperatura , Urea/farmacocinética , Ureasa/química , Ureasa/metabolismo , Uretano/farmacocinética , VinoRESUMEN
Ethyl carbamate (EC), a chemical substance widely present in fermented food products and alcoholic beverages, has been classified as a Group 2A carcinogen by the International Agency for Research on Cancer (IARC). New evidence indicates that long-term exposure to EC may cause neurological disorders. Formation of EC in food and its metabolism have therefore been studied extensively and analytical methods for EC in various food matrices have been established. Due to the potential threat of EC to human health, mitigation strategies for EC in food products by physical, chemical, enzymatic, and genetic engineering methods have been developed. Natural products are suggested to provide protection against EC-induced toxicity through the modulation of oxidative stress. This review summarizes knowledge on the formation and metabolism of EC, detection of EC in food products, toxic effects of EC on various organs, and mitigation strategies including prevention of EC-induced tumorigenesis and genotoxicity by natural products.
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Contaminación de Alimentos , Uretano/análisis , Uretano/toxicidad , Bebidas Alcohólicas/análisis , Carcinógenos/análisis , Alimentos Fermentados , Contaminación de Alimentos/análisis , Humanos , Neoplasias/inducido químicamente , Neoplasias/prevención & control , Uretano/farmacocinéticaRESUMEN
Urethane is often used as a sole anaesthetic agent for non-recovery studies in laboratory animals. However, the use of urethane is controversial, in part, because the electroencephalogram after urethane administration is similar to the electroencephalogram recorded from unanaesthetized animals. Here, we assessed the minimum alveolar concentration (MAC)-sparing effects of urethane by measuring the effect of two doses of urethane on the MAC of isoflurane in male Sprague Dawley rats. Isoflurane MAC was measured before and after intravenous administration of urethane at 1.0 g/kg (Group G1, n = 6) and 1.5 g/kg (Group G1.5, n = 6), or an equal volume of 0.9% saline (Group Gs, n = 6). Baseline isoflurane MAC was not statistically different between groups (isoflurane concentration: 1.47 ± 0.08%, 1.40 ± 0.19% and 1.42 ± 0.12% for G1, G1.5 and Gs, respectively). Intravenous injection of saline did not alter isoflurane MAC (post-saline MAC: 1.43 ± 0.11%). After urethane administration, isoflurane MAC decreased in a dose-dependent manner (new MAC G1: 0.19 ± 0.06%; G1.5: 0.03 ± 0.01%; P < 0.05). The isoflurane MAC after 1.5 g/kg urethane was not significantly different from room air isoflurane concentrations (0.01 ± 0.01%), demonstrating a 100% MAC reduction at this dose. In conclusion, high-dose urethane (1.5 g/kg intravenously) was suitable as a sole anaesthetic agent to prevent gross purposeful movement during the conditions of the study, whereas low-dose urethane (1.0 g/kg intravenously) was not.
Asunto(s)
Anestésicos por Inhalación/farmacocinética , Anestésicos Intravenosos/farmacocinética , Isoflurano/farmacocinética , Alveolos Pulmonares/metabolismo , Uretano/farmacocinética , Animales , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Hemodinámica/efectos de los fármacos , Hemodinámica/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
INTRODUCTION: The aim of the work was to establish the impact of urethane-chloralose anaesthesia on pharmacokinetic-pharmacodynamic (PK-PD) properties of carvedilol in control rats and L-NAME hypertensive animals. METHODS: Male Wistar Rats were randomly divided into: control (n=12) with tap water to drink and L-NAME rats (n=12) with L-NAME solution (40 mg/kg/day) to drink for 2 weeks. Effects of carvedilol (1 mg kg(-1), i.v.) on blood pressure and heart rate were recorded during 3 h in conscious and urethane (500 mg kg(-1), i.p.) - chloralose (50 mg kg(-1), i.p.) anaesthetized rats. Carvedilol plasma pharmacokinetics was studied by means of traditional blood sampling. PK-PD modeling of carvedilol was made by means of an effect compartment model. RESULTS: Neither urethane-chloralose nor L-NAME modified estimation of pharmacokinetic parameters of carvedilol. Although urethane-chloralose did not modify potency of carvedilol comparing with awake animals in control and hypertensive group, maximal negative chronotropic response was significantly greater in anaesthetized L-NAME rats in comparison to awake animals. Conversely, anaesthesia did not modify maximal chronotropic response to carvedilol in control rats. Whilst no differences were found in the estimated potency of carvedilol hypotensive response comparing control and L-NAME rats in both awake and anaesthetized conditions, maximal hypotensive effect of carvedilol was significantly greater in anaesthetized control and L-NAME animals in comparison to conscious rats. L-NAME rats showed a greater maximal hypotensive response comparing to control group. DISCUSSION: Urethane-chloralose anaesthesia is an acceptable experimental condition for the evaluation of PK-PD properties of carvedilol, considering that it does not affect the potency of carvedilol for its chronotropic and hypotensive effect. Conclusions obtained from urethane-chloralose anaesthetized animals, regarding the impact of l-NAME treatment on PK-PD properties of carvedilol, did not differ from those obtained from conscious animals. Anaesthesia did not modify pharmacokinetic behaviour of carvedilol in both normotensive and L-NAME hypertensive rats.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacocinética , Anestesia , Carbazoles/farmacocinética , Cloralosa/farmacocinética , Propanolaminas/farmacocinética , Uretano/farmacocinética , Administración Oral , Antagonistas Adrenérgicos beta/administración & dosificación , Antagonistas Adrenérgicos beta/sangre , Algoritmos , Animales , Área Bajo la Curva , Presión Sanguínea/efectos de los fármacos , Carbazoles/administración & dosificación , Carbazoles/sangre , Carvedilol , Cloralosa/administración & dosificación , Interpretación Estadística de Datos , Modelos Animales de Enfermedad , Esquema de Medicación , Sinergismo Farmacológico , Semivida , Frecuencia Cardíaca/efectos de los fármacos , Hipertensión/inducido químicamente , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Masculino , Modelos Biológicos , NG-Nitroarginina Metil Éster/efectos adversos , NG-Nitroarginina Metil Éster/química , Propanolaminas/administración & dosificación , Propanolaminas/sangre , Ratas , Ratas Wistar , Soluciones/química , Uretano/administración & dosificaciónRESUMEN
Urethane is an established animal carcinogen and has been classified as "reasonably anticipated to be a human carcinogen." Until recently, urethane metabolism via esterase was considered the main metabolic pathway of this chemical. However, recent studies in this laboratory showed that CYP2E1, and not esterase, is the primary enzyme responsible for urethane oxidation. Subsequent studies demonstrated significant inhibition of urethane-induced genotoxicity and cell proliferation in Cyp2e1-/- compared to Cyp2e1+/+ mice. Using Cyp2e1-/- mice, current studies were undertaken to assess the relationships between urethane metabolism and carcinogenicity. Urethane was administered via gavage at 1, 10, or 100 mg/kg/day, 5 days/week, for 6 weeks. Animals were kept without chemical administration for 7 months after which they were euthanized, and urethane carcinogenicity was assessed. Microscopic examination showed a significant reduction in the incidences of liver hemangiomas and hemangiosarcomas in Cyp2e1-/- compared to Cyp2e+/+ mice. Lung nodules increased in a dose-dependent manner and were less prevalent in Cyp2e1-/- compared to Cyp2e+/+ mice. Microscopic alterations included bronchoalveolar adenomas, and in one Cyp2e1+/+ mouse treated with 100 mg/kg urethane, a bronchoalveolar carcinoma was diagnosed. Significant reduction in the incidence of adenomas and the number of adenomas/lung were observed in Cyp2e1-/- compared to Cyp2e1+/+ mice. In the Harderian gland, the incidences of hyperplasia and adenomas were significantly lower in Cyp2e1-/- compared to Cyp2e+/+ mice at the 10 mg/kg dose, with no significant differences observed at the high or low doses. In conclusion, this work demonstrated a significant reduction of urethane-induced carcinogenicity in Cyp2e1-/- compared to Cyp2e1+/+ mice and proved that CYP2E1-mediated oxidation plays an essential role in urethane-induced carcinogenicity.
Asunto(s)
Citocromo P-450 CYP2E1/metabolismo , Neoplasias del Ojo/inducido químicamente , Glándula de Harder/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Pulmonares/inducido químicamente , Uretano/análogos & derivados , Animales , Citocromo P-450 CYP2E1/genética , Neoplasias del Ojo/enzimología , Neoplasias del Ojo/patología , Glándula de Harder/enzimología , Inactivación Metabólica , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Noqueados , Uretano/farmacocinética , Uretano/toxicidadAsunto(s)
Seguridad de Productos para el Consumidor/normas , Contaminación de Alimentos , Alimentos/normas , Acrilamida/análisis , Acrilamida/farmacocinética , Acrilamida/toxicidad , Animales , Cadmio/administración & dosificación , Cadmio/análisis , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Humanos , Bifenilos Polibrominados/administración & dosificación , Bifenilos Polibrominados/análisis , Bifenilos Polibrominados/farmacocinética , Bifenilos Polibrominados/toxicidad , Hidrocarburos Policíclicos Aromáticos/administración & dosificación , Hidrocarburos Policíclicos Aromáticos/análisis , Hidrocarburos Policíclicos Aromáticos/farmacocinética , Hidrocarburos Policíclicos Aromáticos/toxicidad , Estaño/administración & dosificación , Estaño/farmacocinética , Estaño/toxicidad , Naciones Unidas , Uretano/análisis , Uretano/farmacocinética , Uretano/toxicidad , Organización Mundial de la SaludRESUMEN
Urethane is a fermentation by-product and a potent animal carcinogen. Human exposure to urethane occurs through consumption of alcoholic beverages and fermented foods. Recently, CYP2E1 was identified as the primary enzyme responsible for the metabolism of [(14)C]carbonyl-labeled urethane. Subsequently, attenuation of urethane-induced cell proliferation and genotoxicity in CYP2E1-/- mice was reported. The present work compares the metabolism of single versus multiple exposures of CYP2E1-/- and CYP2E1+/+ mice to (14)C-ethyl-labeled urethane. Urethane was administered as a single 10 or 100 mg/kg gavage dose or at 100 mg/kg/day for 5 consecutive days. CYP2E1+/+ mice administered single or multiple doses exhaled 78 to 88% of dose as (14)CO(2)/day. CYP2E1-/- mice eliminated 30 to 38% of a single dose as (14)CO(2) in 24 h and plateaued after day 3 at approximately 52% of dose/day. The concentrations of urethane-derived radioactivity in plasma and tissues were dose-dependent, increased as a function of the number of doses administered, and were significantly higher in CYP2E1-/- versus CYP2E1+/+ mice. Whereas urethane was the main chemical found in the plasma and tissues of CYP2E1-/- mice, it was not detectable in CYP2E1+/+ mice. In conclusion, multiple dosing led to considerable bioaccumulation of urethane in mice of both genotypes; however, greater retention occurred in CYP2E1-/- versus CYP2E1+/+ mice. Furthermore, greater bioaccumulation of (14)C-ethyl-labeled than [(14)C]carbonyl-labeled urethane was observed in mice. Comparison of the metabolism of ethyl-versus carbonyl-labeled urethane was necessary for tracing the source of CO(2) and led us to propose for the first time that C-hydroxylation is a likely pathway of urethane metabolism.
Asunto(s)
Citocromo P-450 CYP2E1/metabolismo , Uretano/metabolismo , Animales , Dióxido de Carbono/metabolismo , Radioisótopos de Carbono , Carcinógenos/toxicidad , Citocromo P-450 CYP2E1/deficiencia , Citocromo P-450 CYP2E1/genética , Dieta , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Noqueados , Mutágenos/toxicidad , Distribución Tisular , Uretano/farmacocinética , Uretano/toxicidadRESUMEN
In an attempt to achieve post-inhalation modulation of drug release rate, Bhavane et al. have recently proposed a microparticle agglomerate of nano-sized liposomal particles, with the agglomeration process consisting of chemical cross-linkages that are capable of cleavage [Bhavane et al. J. Cont. Rel 93 (2003) 15-28.]. There, the in vitro modulation of release from agglomerated liposomes encapsulating the antibiotic ciprofloxacin was demonstrated. However, the cleaving agents used in the previous studies are not acceptable for in vivo use. In the present work therefore, a new generation of in vivo compatible agglomerated liposomes has been developed. The release rate of encapsulated compounds from these carriers can be modulated by the addition of mild thiolytic cleaving agents such as cysteine. Specifically, an amino terminated PEG conjugate has been successfully synthesized, similar to the conjugate proposed by Zalipsky [Bioconjugate Chemistry, 10 (5) (1999) 703-707.]. This conjugate contains a dithiobenzyl urethane linkage between the lipid and the PEG, cleavable by the addition of cysteine. The amines at the distal ends of the PEG are used to cross-link the liposomes into agglomerates by the addition of a suitable cross-linking agent reactive towards amines. The cross-linkages were cleaved by cysteine at the DTB sites, resulting in changes in the size distribution of the agglomerates, as well as changes in the release rate of the encapsulated drug.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Liposomas/administración & dosificación , Liposomas/síntesis química , Pulmón/metabolismo , Administración por Inhalación , Liposomas/farmacocinética , Pulmón/efectos de los fármacos , Polietilenglicoles/administración & dosificación , Polietilenglicoles/farmacocinética , Uretano/administración & dosificación , Uretano/análogos & derivados , Uretano/farmacocinéticaRESUMEN
Phenytoin is an anticonvulsant drug known to interact with many other drugs. Previous data suggest that chronic administration of phenytoin delays urethane-induced loss of righting reflex (LRR) and this phenomenon being potentiated by concomitant administration of ascorbic acid (ASC). Therefore, we examined how phenytoin at two different doses combined or not with ASC (fixed dose) interact with both the latency to, and the duration for urethane-induced LRR in experimental rats. The results showed that lower dose of phenytoin (60 mg/kg rat b.i.d.) has significantly shortened the duration of LRR while higher dose of the drug (120 mg/kg b.i.d.) has delayed the latency to LRR (cut-off period of 15 min). Furthermore, addition of ASC to any of the two doses of phenytoin gave results similar to that observed for latency to and duration of LRR when phenytoin was given alone at the higher dose (120 mg/kg b.i.d.). Our data suggest a resistant effect of chronic administration of phenytoin on latency to and/or duration of loss righting reflex induced by urethane in experimental animals. A dose-response relationship of phenytoin in this regard is expected and needs further investigations. The resistant effect of phenytoin on righting reflex has been augmented when ASC was chronically given in combination. This result supports a possible interaction between the two drugs and needs further investigations at both experimental and clinical levels.
Asunto(s)
Ácido Ascórbico/farmacocinética , Quimioterapia Combinada , Fenitoína/farmacocinética , Administración Oral , Anestésicos Intravenosos , Animales , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/farmacocinética , Ácido Ascórbico/administración & dosificación , Esquema de Medicación , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Inyecciones Intraperitoneales , Intubación Gastrointestinal , Masculino , Fenitoína/administración & dosificación , Equilibrio Postural/efectos de los fármacos , Equilibrio Postural/fisiología , Ratas , Factores de Tiempo , Uretano/administración & dosificación , Uretano/metabolismo , Uretano/farmacocinéticaRESUMEN
Urethane ([carbonyl-(14)C]ethyl carbamate) is a fermentation by-product in alcoholic beverages and foods and is classified as reasonably anticipated to be a human carcinogen. Early studies indicated that while CYP2E1 is involved, esterases are the primary enzymes responsible for urethane metabolism. Using CYP2E1-null (KO) mice, current studies were undertaken to elucidate CYP2E1's contribution to urethane metabolism. [Carbonyl-(14)C]urethane was administered by gavage to male CYP2E1-null and wild-type mice at 10 or 100 mg/kg and its metabolism and disposition were investigated. CO(2) was confirmed as the main metabolite of urethane. Significant inhibition of urethane metabolism to CO(2) occurred in CYP2E1-null versus wild-type mice. Pharmacokinetic modeling of (14)CO(2) exhalation data revealed that CYP2E1 is responsible for approximately 96% of urethane metabolism to CO(2) in wild-type mice. The contributions of other enzymes to urethane metabolism merely account for the remaining 4%. The half-life of urethane in wild-type and CYP2E1-null mice was estimated at 0.8 and 22 h, respectively. Additionally, the concentration of urethane-derived radioactivity in blood and tissues was dose-dependent and significantly higher in CYP2E1-null mice. High-performance liquid chromatography analysis showed only urethane in the plasma and liver extracts of CYP2E1-null mice. Because the lack of CYP2E1 did not completely inhibit urethane metabolism, the disposition of 10 mg/kg urethane was compared in mice pretreated with the P450 inhibitor, 1-aminobenzotriazole or the esterase inhibitor, paraoxon. Unlike paraoxon, 1-aminobenzotriazole resulted in significant inhibition of urethane metabolism to CO(2) in both genotypes. In conclusion, this work demonstrated that CYP2E1, not esterase, is the principal enzyme responsible for urethane metabolism.
Asunto(s)
Citocromo P-450 CYP2E1/metabolismo , Uretano/metabolismo , Animales , Dióxido de Carbono/metabolismo , Cromatografía Líquida de Alta Presión , Simulación por Computador , Citocromo P-450 CYP2E1/genética , Heces/química , Semivida , Hígado/enzimología , Masculino , Ratones , Ratones Noqueados , Modelos Biológicos , Paraoxon/metabolismo , Distribución Tisular , Triazoles/metabolismo , Uretano/farmacocinética , Uretano/orinaAsunto(s)
Anestésicos Intravenosos/farmacología , Receptores de Neurotransmisores/efectos de los fármacos , Uretano/farmacología , Anestésicos Intravenosos/farmacocinética , Animales , Activación del Canal Iónico/efectos de los fármacos , Receptores de Neurotransmisores/metabolismo , Uretano/farmacocinéticaRESUMEN
A urethane-based analogue containing an azo aromatic linkage in the backbone was synthesized for use in colon-specific delivery systems by reacting toluene-2,6-diisocyanate with a mixture of an aromatic azo diol, (bis-4-hydroxyphenyl)-4,4'-diazobiphenyl, poly(ethylene glycol) (Mn = 4000; number-average molecular weight) and 1,2-propanediol (propylene glycol). The resultant compounds (UR-1 and UR-2) were characterized by IR spectroscopy, 1H NMR spectroscopy, DSC studies, X-ray diffraction studies and molecular weight determination by gel permeation chromatography. The compounds exhibited low molecular weight, lacked film-forming properties and crystallinity in the structure. An in-vitro bacterial degradation test to demonstrate the susceptibility of azo bond to bacterial enzymes was performed using media inoculated with lactobacillus culture. The results indicated degradation of films by azoreductase. In-vitro permeation of 5-aminosalicylic acid was studied in control and lactobacilli-treated films. The permeability of the lactobacilli treated films was significantly increased suggesting the potential of these compounds for application in colonic targeting.
Asunto(s)
Antineoplásicos/síntesis química , Compuestos Azo/síntesis química , Colon/efectos de los fármacos , Lactobacillus/efectos de los fármacos , Uretano/análogos & derivados , Antineoplásicos/farmacocinética , Compuestos Azo/farmacocinética , Biodegradación Ambiental , Biopelículas , Técnicas de Cultivo de Célula , Técnicas de Química Analítica , Sistemas de Liberación de Medicamentos , Humanos , Lactobacillus/fisiología , Permeabilidad , Uretano/síntesis química , Uretano/farmacocinéticaRESUMEN
Inbred strains of mice exhibit differing susceptibilities to formation of lung tumors induced by procarcinogens including ethyl carbamate (EC) and vinyl carbamate (VC). Strain A/J mice are susceptible, whereas C57BL/6 mice are resistant to lung tumor development. In this study, we tested the hypothesis that differential susceptibilities of A/J, CD-1, and C57BL/6 mice to lung tumor development are associated, in part, with their capacities for VC bioactivation and with the extents of DNA adduct formation. Previous studies have shown that the P450 isozyme CYP2E1 and microsomal carboxylesterases are involved in activation and detoxication of VC, respectively. Bioactivation capacity, as estimated by ratios of p-nitrophenol hydroxylase, a CYP2E1 catalytic marker, to carboxylesterase activities, was greater in control A/J (1.32 +/- 0.18 x 10(-6)) and CD-1 (1.25 +/- 0.29 x 10(-6)) mice than in control C57BL/6 (0.69 +/- 0.12 x 10(-6)) mice. The ratios were reduced in all three strains of mice treated with VC. Covalent binding of [(14)C-carbonyl]-VC to lung proteins was time- and dose-dependent, and was significantly higher in A/J and CD-1 mice than in C57BL/6 mice. Experiments using (32)P-postlabeling/thin-layer chromatography showed formation of the DNA adducts 1,N:(6)-ethenodeoxyadenosine and 3,N:(4)-ethenodeoxycytidine in lungs of mice treated with VC. The DNA adducts were detected at 30 min after treatment, peaked at 60 min, and declined thereafter. Levels of 1,N:(6)-ethenodeoxyadenosine and 3,N:(4)-ethenodeoxycytidine were about 70% higher in A/J and CD-1 mice than in C57BL/6 mice. These results indicated that formation of VC metabolites in these murine strains is linked to their bioactivation capacities, and suggested that this attribute may confer differing susceptibilities to lung tumor development.
Asunto(s)
Aductos de ADN/biosíntesis , Uretano/análogos & derivados , Uretano/farmacocinética , Animales , Autorradiografía , Carboxilesterasa , Hidrolasas de Éster Carboxílico/metabolismo , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Citocromo P-450 CYP2E1/metabolismo , ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Pulmón/efectos de los fármacos , Pulmón/enzimología , Ratones , Ratones Endogámicos C57BL , Microsomas/efectos de los fármacos , Microsomas/enzimología , Radioisótopos de Fósforo , Especificidad de la EspecieRESUMEN
This paper considers the utility of a stochastic model of carcinogenesis proposed by Yakovlev and Polig [Math. Biosci. 132 (1996) 1-33] in the analysis of experimental data on multiple tumors induced by chemical carcinogens. The model provides a good description of published data on multiple tumors developing in the lungs of mice in response to different schedules of urethane. The distribution of pulmonary tumor counts appears to be negative binomial for each period of time after exposure to urethane. Our results suggest that the rate of administration of urethane has little effect both on the mean number of initiated cells per unit dose and on the rate of formation of lesions responsible for cell death. As our estimates show, more than 80% of initiated cells die in the course of tumor promotion. The model is robust to variations in the rate of urethane excretion given a fixed total dose of the carcinogen. Some prospects for further development of the model to allow for expansion of promoted cell clones are discussed.
Asunto(s)
Carcinógenos/efectos adversos , Neoplasias Pulmonares/inducido químicamente , Modelos Biológicos , Neoplasias Primarias Múltiples/inducido químicamente , Uretano/efectos adversos , Animales , Distribución Binomial , Carcinógenos/administración & dosificación , Carcinógenos/farmacocinética , Muerte Celular/fisiología , Intervalos de Confianza , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Funciones de Verosimilitud , Masculino , Ratones , Procesos Estocásticos , Factores de Tiempo , Uretano/administración & dosificación , Uretano/farmacocinéticaRESUMEN
Urethane, a byproduct of fermentation found in alcoholic beverages, is carcinogenic in rodents and is classified by the International Agency for Research on Cancer as a possible human carcinogen. The United States Food and Drug Administration nominated urethane for study because of the widespread exposure of humans through the consumption of fermented foods and beverages and because of a lack of adequate dose-response data about the carcinogenicity of urethane with and without the coadministration of ethanol. Comparative studies of urethane in drinking water and in 5% ethanol were conducted to investigate possible effects of ethanol on urethane toxicity. Toxicokinetic studies of urethane in drinking water and in 5% ethanol and genetic toxicity studies of urethane in vivo and in vitro were also conducted. Groups of 10 male and 10 female F344/N rats and B6C3F1 mice, 6 weeks of age, received 0, 110, 330, 1,100, 3,300, or 10,000 ppm urethane in drinking water or in 5% ethanol for 13 weeks. Toxicokinetic evaluations were performed for urethane in the plasma of male mice after 13 weeks of administration in drinking water or 5% ethanol. The mutagenicity of urethane in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, and TA1537 with and without S9 was tested at doses up to 16,666 micrograms/plate; urethane was also tested for induction of sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells and sex-linked recessive lethal mutations and chromosomal reciprocal translocations in Drosophila melanogaster. The frequency of micronucleated erythrocytes induced in peripheral blood and bone marrow cells of mice by urethane in drinking water and in 5% ethanol was also evaluated. In rats that received urethane in drinking water, seven males and four females administered 10,000 ppm and one female administered 3,300 ppm died before the end of the study; body weight gains were reduced at these concentrations. Two males and all females given 10,000 ppm urethane in 5% ethanol died during the study, and the body weight gains of males and females that received 3,300 ppm were lower than those of the controls. Relative right kidney, liver, and lung weights of males and females and relative right testis weights of males administered 1,100 ppm or greater were generally higher than those of the controls in each study. Leukopenia and lymphopenia were observed in rats receiving urethane in either drinking water or ethanol and occurred in males receiving 330 ppm or greater and females receiving 110 ppm or greater. Other differences in hematology and clinical chemistry variables were not considered to be biologically significant. Lymphoid depletion of the spleen, lymph nodes, and thymus was observed in male and female rats receiving 1,100, 3,300, or 10,000 ppm urethane in drinking water. Cellular depletion of the bone marrow occurred in males and females in the 10,000 ppm groups. Hepatocellular fatty changes and clear cell foci of alteration were noted in the liver of males and females that received 3,300 or 10,000 ppm. The incidences of nephropathy were significantly increased in female rats that received 1,100 ppm or greater; the severity of this lesion in exposed males and females was greater than that in the controls. Females that received 330 ppm or greater had higher incidences of cardiomyopathy than the controls; the severity of this lesion was greater in males in the 10,000 ppm group and females in the 3,300 and 10,000 ppm groups than in the controls. In rats that received urethane in 5% ethanol, lymphoid depletion occurred in males and females in the 3,300 and 10,000 ppm groups. Cellular depletion of the bone marrow was observed in males and females in the 10,000 ppm groups. Only males in the 10,000 ppm group had hepatocellular fatty change (8/10) and clear cell foci (1/10); the incidence and severity of nephropathy in males and females and cardiomyopathy in males were similar to those in rats administered urethane in drinking water; however, no cardiomyopathy was observed in females receiving urethane in ethanol. The estrous cycle length of females receiving urethane in ethanol appeared to be longer than that of females receiving urethane in drinking water. Because cycle length was longer in the 10,000 ppm groups than in the controls in both the drinking water and ethanol vehicle studies, this difference may represent an exacerbation of the toxicity of urethane. A longer estrous cycle may be a sign of reproductive impairment and correlates with a decrease in female fecundity. All mice administered 10,000 ppm urethane in either vehicle died. All mice that received 3,300 ppm urethane in drinking water died, while only one male and four females receiving 3,300 ppm urethane in 5% ethanol died. Body weight gains of males and females in all 1,100 ppm groups were less than those of the respective controls, but the weight gains of mice receiving 1,100 ppm urethane in 5% ethanol were greater than those of mice receiving urethane in drinking water. The mean body weights of the lower exposure groups were similar to those of the respective controls, and there were no other differences between the body weights of mice receiving urethane in drinking water and those receiving urethane in 5% ethanol. Fluid consumption, and therefore total urethane intake, appeared lower in mice receiving the 5% ethanol vehicle than in those receiving the water vehicle. The relative right kidney, liver, and lung weights of males and females administered urethane in drinking water or ethanol were generally greater than those of the controls. Clearance of urethane from the plasma of male mice was complete within 2 hours after urethane was administered in water, but urethane was not cleared 12 hours after administration in 5% ethanol. At the end of 13 weeks of urethane administration, the plasma urethane elimination half-life was 0.8 hours; the kinetics were similar for concentrations of 110, 330, and 1,100 ppm urethane in water and in ethanol. However, at each exposure level, the plasma urethane concentration was four times greater for urethane administered in 5% ethanol than for urethane administered in drinking water, indicating a possible inhibition of urethane metabolism by ethanol. Kinetic measurements for elimination by female mice could not be obtained from the data collected. In mice administered urethane in drinking water, lung inflammation occurred in males and females that received 1,100 ppm or greater. Alveolar epithelial hyperplasia occurred in the lungs of males in the 330 and 1,100 ppm groups and females in the 1,100 ppm group; one male mouse in the 330 ppm group had an alveolar/bronchiolar adenoma (see the following summary table). Mice receiving urethane in 5% ethanol had lower incidences and severity of lung inflammation but generally greater incidences and severity of alveolar epithelial hyperplasia than mice receiving the same concentrations of urethane in drinking water. Alveolar/bronchiolar adenomas occurred in four males and one female administered urethane in ethanol. [table: see text] Nephropathy was observed in males and females that received urethane in either vehicle, and the lesions in female mice were more severe than those in male mice; ethanol did not appear to increase the incidence or severity of nephropathy. Cardiomyopathy occurred in males and females that received 1,100 or 3,300 ppm urethane in drinking water and in females that received 3,300 ppm urethane in ethanol. Lymphoid depletion occurred in mice that received 3,300 or 10,000 ppm urethane; 5% ethanol did not appear to enhance these effects. However, urethane in 5% ethanol induced ovarian atrophy; the incidence of this lesion was lower in females receiving urethane in drinking water. A concentration of 1,100 ppm urethane in either drinking water or ethanol effectively stopped estrous cycling. Urethane is clearly genotoxic in vitro and in vivo. In vitro, urethane induced mutations in Salmonella typhimurium strain TA1535 in the presence of liver S9 enzymes. Sister chromatid exchanges were induced in cultured Chinese hamster ovary (CHO) cells with and without S9. However, no induction of chromosomal aberrations was observed in CHO cells treated with urethane, with or without S9. In vivo, urethane induced sex-linked recessive lethal mutations and reciprocal translocations in germ cells of adult male Drosophila melanogaster fed urethane. Significantly increased frequencies of micronucleated erythrocytes were observed in peripheral blood obtained from male and female mice after 45 days of exposure and in bone marrow and peripheral blood obtained after 13 weeks of exposure to urethane in drinking water. There appeared to be no significant difference in the magnitude of the response in the peripheral blood micronucleus test between mice administered urethane in drinking water and mice administered urethane in 5% ethanol. In summary, concentrations of 1,100 ppm urethane or greater in drinking water caused lymphoid and bone marrow cell depletion and hepatocellular lesions and increased the severity of nephropathy and cardiomyopathy in male and female rats. The lethal effects of 10,000 ppm urethane were slightly exacerbated by 5% ethanol in female rats. Urethane administered in drinking water induced lung inflammation, alveolar and bronchiolar hyperplasia, alveolar/bronchiolar adenomas, nephropathy, cardiomyopathy, lymphoid and bone marrow cell depletion, seminiferous tubule degeneration, and ovarian atrophy and follicular degeneration in mice. In female mice, 5% ethanol appeared to exacerbate ovarian atrophy. Mice administered urethane in 5% ethanol consumed less fluid, and therefore less urethane, than mice receiving urethane in drinking water. Coadministration of urethane and ethanol inhibited the clearance of urethane from plasma. (ABSTRACT TRUNCATED)
Asunto(s)
Uretano/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Recuento de Células Sanguíneas , Peso Corporal/efectos de los fármacos , Células CHO , Cricetinae , Ciclo Estral/efectos de los fármacos , Femenino , Masculino , Ratones , Ratones Endogámicos , Pruebas de Mutagenicidad , Ratas , Ratas Endogámicas F344 , Caracteres Sexuales , Motilidad Espermática/efectos de los fármacos , Uretano/farmacocinética , Enfermedades Vaginales/inducido químicamente , Enfermedades Vaginales/patología , Contaminantes Químicos del Agua/farmacocinética , Abastecimiento de Agua/análisisRESUMEN
Vinyl carbamate epoxide (VCO) is believed to be the metabolite of ethyl carbamate (EC) ultimately responsible for its carcinogenic effects. This study investigates the role of glutathione (GSH) in protection against VCO-mediated adduct formation, and the involvement of glutathione S-transferases (GSTs) in detoxification of VCO. Formation of 1,N6-ethenoadenosine from VCO and adenosine in vitro was employed as a measure of VCO toxicity. GSH inhibited formation of ethenoadenosine in a concentration-dependent manner at concentrations ranging from 1 to 8 mM. This effect was significantly enhanced by addition of rat liver GST. Mouse liver cytosol was also found to inhibit formation of ethenoadenosine in a concentration-dependent manner, and the inhibition was relieved by addition of S-octylglutathione, a competitive inhibitor of GST. Pretreatment of mice with 1% dietary (2(3)-tert-butyl-4-hydroxyanisole (BHA) caused parallel increases in cytosolic GST activity and cytosolic enhancement of detoxification of VCO by GSH. Furthermore, BHA increased hepatic steady-state concentrations of GSH greater than twofold. The effect of BHA on detoxification of EC in vivo was examined using formation of 2-oxoethylvaline (OEV) adducts of hemoglobin as a biomarker. Pretreatment with BHA decreased overall formation of OEV adducts 23%. The major conclusions of this study are (1) VCO can be detoxified by spontaneous conjugation with GSH, (2) conjugation of VCO with GST can be catalyzed by GST(s), (3) pretreatment with BHA protects against binding of active EC metabolites in vitro and in vivo, and (4) the protective effect of BHA against EC is mediated by increases in GST activity and GSH concentration.
Asunto(s)
Carcinógenos/farmacocinética , Glutatión Transferasa/farmacocinética , Uretano/análogos & derivados , Uretano/farmacocinética , Animales , Hidroxianisol Butilado/farmacología , Catálisis , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Glutatión/farmacología , Glutatión Transferasa/farmacología , Hemoglobinas/efectos de los fármacos , Hemoglobinas/metabolismo , Inactivación Metabólica , Hígado/química , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos A , Uretano/toxicidadRESUMEN
Ethyl carbamate (EC) is bioactivated by CYP2E1 through vinyl carbamate to its epoxide, a reactive electrophile. This carcinogen reacts with macromolecules, including hemoglobin (Hb). This report defines a method to examine levels of N-(2-oxoethyl) adduct on the N-terminal valine of Hb after EC treatment at carcinogenic doses. Concentrations were determined 24 hr following an oral dose of EC (1 mg/g body wt) to strains A/J and C57BL/6 mice. Globin samples were isolated by precipitation in acidified acetone, washed, dried, and stored frozen at -20 degrees C until analyzed. Weighed aliquots were treated with sodium borohydride to reduce the aldehyde of the 2-oxoethyl group to the N-(2-hydroxyethyl) adduct. The adduct valine was cleaved using phenylisothiocyanate to form a substituted phenylthiohydantoin derivative of N-(2-hydroxyethyl)valine in a modified Edman degradation. After reaction with N,O-bis(trimethylsilyl)trifluoroacetamide, the resultant product, 1-(2'-trimethylsilyloxy)ethyl-5-isopropyl-3-phenyl-2-thiohydantoin , was quantified by GC/MS with selected ion monitoring of the molecular ion using synthetic N-(3-hydroxypropyl)valine as an internal standard. No adducts were detected without NaBH4 reduction. Strain A/J mice treated with EC (1 mg/g, N = 10) yielded mean +/- standard deviation (SD) adduct level values of 13.3 +/- 1.03 nmol/g globin; saline-treated A/J controls (N = 7) gave background levels of 4.43 +/- 0.69 nmol/g globin. Strain C57BL/6 mice treated with EC (1 mg/g, N = 6) exhibited mean +/- SD values of 12.0 +/- 1.92 nmol/g globin, while control mice of this strain (N = 4) had adduct levels of 7.23 +/- 1.19 nmol/g globin. These results are consistent with findings of others that bioactivation of EC produces N-(2-oxoethyl)valine hemoglobin adducts. Although the difference between mouse strains in mean total adduct levels following EC treatment was not significant, the differences evident in comparisons within strains due to treatment, between strains in endogenous background levels, and between strains in estimates of mean increases in adduct concentrations resulting from EC treatment were highly significant (p < 0.01). This assay provides a biomarker system for assessment of production from EC of the electrophilic metabolites which are believed to be genotoxic following metabolic activation in vivo.
Asunto(s)
Carcinógenos/toxicidad , Hemoglobinas/efectos de los fármacos , Hemoglobinas/metabolismo , Uretano/toxicidad , Animales , Biotransformación , Calibración , Carcinógenos/farmacocinética , Cromatografía de Gases y Espectrometría de Masas , Hidrólisis , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Oxidación-Reducción , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/análisis , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Uretano/farmacocinética , Valina/análisis , Valina/metabolismoAsunto(s)
Médula Ósea/efectos de los fármacos , Mutágenos , Uretano/análogos & derivados , Uretano/metabolismo , Animales , Biotransformación , Células de la Médula Ósea , Carcinógenos/metabolismo , Carcinógenos/farmacocinética , Ratones , Pruebas de Micronúcleos , Uretano/farmacocinética , Uretano/toxicidadRESUMEN
Ethyl carbamate is an animal carcinogen when administered in large doses; it is naturally present in minute concentrations in fermented foods and beverages. Previous studies from this laboratory have demonstrated that ethanol, in vivo, inhibits the metabolism of ethyl carbamate in mice, but the enzyme system has not been identified. In an effort to further characterize the enzyme system responsible, the metabolic products of ethanol metabolism were studied to determine whether ethanol or either of its metabolites is inhibitory. Acetaldehyde (400 mg/kg) is a potent inhibitor of ethyl carbamate metabolism for about 2 hr in vitro, but sodium acetate is not. Paraldehyde (250 mg/kg) has a slower onset and longer duration of inhibition, suggesting that its conversion to acetaldehyde produces the inhibitory molecule. Disulfiram (200 mg/kg) has a prolonged inhibitory effect; this effect is enhanced and extended when the disulfiram is combined with acetaldehyde (400 mg/kg). D-Penicillamine, given in a regimen of 1.2 g/kg 0.5 hr before and 0.6 g/kg 1.5 and 3.5 hr after ethyl carbamate, is not inhibitory; however, it abolishes the inhibitory effect of acetaldehyde, presumably from sequestration of acetaldehyde. These studies demonstrate that acetaldehyde is an inhibitor of the metabolism of ethyl carbamate and suggest that acetaldehyde is one, and perhaps the only, molecule responsible for the inhibition seen when ethanol is administered to mice. In vitro incubation studies determined that ethyl carbamate was not metabolized by human plasma.