RESUMEN
Nicotine metabolism is exceedingly sensitive to perturbation by numerous host factors. To reduce the large variations and discrepancies in the literature pertaining to nicotine metabolism, investigators in future studies need to recognize and better control these host factors. Recent advances in the understanding of nicotine metabolism have suggested new approaches to elucidating underlying mechanisms of certain toxic effects associated with cigarette smoking.
Asunto(s)
Envejecimiento/metabolismo , Nicotina/farmacocinética , Fumar , Animales , Cimetidina/farmacología , Proteínas en la Dieta/farmacología , Interacciones Farmacológicas , Etanol/farmacología , Femenino , Variación Genética , Humanos , Nicotina/administración & dosificación , Nicotina/metabolismo , Embarazo/metabolismo , Especificidad de la EspecieRESUMEN
Benzene, an important industrial solvent, is also present in unleaded gasoline and cigarette smoke. The hematotoxic effects of benzene in humans are well documented and include aplastic anemia, pancytopenia, and acute myelogenous leukemia. However, the risks of leukemia at low exposure concentrations have not been established. A combination of metabolites (hydroquinone and phenol, for example) may be necessary to duplicate the hematotoxic effect of benzene, perhaps due in part to the synergistic effect of phenol on myeloperoxidase-mediated oxidation of hydroquinone to the reactive metabolite benzoquinone. Because benzene and its hydroxylated metabolites (phenol, hydroquinone, and catechol) are substrates for the same cytochrome P450 enzymes, competitive interactions among the metabolites are possible. In vivo data on metabolite formation by mice exposed to various benzene concentrations are consistent with competitive inhibition of phenol oxidation by benzene. In vitro studies of the metabolic oxidation of benzene, phenol, and hydroquinone are consistent with the mechanism of competitive interaction among the metabolites. The dosimetry of benzene and its metabolites in the target tissue, bone marrow, depends on the balance of activation processes such as enzymatic oxidation and deactivation processes such as conjugation and excretion. Phenol, the primary benzene metabolite, can undergo both oxidation and conjugation. Thus the potential exists for competition among various enzymes for phenol. Zonal localization of phase I and phase II enzymes in various regions of the liver acinus also impacts this competition. Biologically based dosimetry models that incorporate the important determinants of benzene flux, including interactions with other chemicals, will enable prediction of target tissue doses of benzene and metabolites at low exposure concentrations relevant for humans.
Asunto(s)
Benceno/metabolismo , Benceno/toxicidad , Modelos Biológicos , Animales , Médula Ósea/efectos de los fármacos , Citocromo P-450 CYP2E1/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Fenoles/metabolismo , Fenoles/toxicidadRESUMEN
1,3-Butadiene (BD) is oxidized by cytochrome P450 to reactive metabolites, including 1,2-epoxy-3-butene (BMO) and 1,2:3,4-diepoxybutane (BDE), which are thought to be responsible for BD genotoxicity and carcinogenicity. Alveolar-bronchiolar neoplasms were observed in mice but not rats following chronic exposure to BD. The site-specific carcinogenicity of BD in mice may result from metabolic activation in pulmonary tissue. We have incubated bronchioles isolated from both male B6C3F1 mice and male Sprague-Dawley rats with 34 microM BD (final concentration in the aqueous reaction medium) to assess species differences in pulmonary metabolism of BD and to enhance our understanding of species- and site-dependent BD carcinogenicity. Bronchioles from both mice and rats formed BMO, although mouse tissue produced 2-fold more than rat tissue. These preliminary results suggest that pulmonary activation of BD may play a role in the carcinogenicity of BD following inhalation exposure; however, other factors in addition to metabolic differences, probably contribute to the observed differences in susceptibility to BD toxicity.
Asunto(s)
Butadienos/metabolismo , Carcinógenos/metabolismo , Pulmón/metabolismo , Animales , Compuestos Epoxi/metabolismo , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Benzene (BZ) requires oxidative metabolism catalyzed by cytochrome P-450 2E1 (CYP 2E1) to exert its hematotoxic and genotoxic effects. We previously reported that male mice have a two-fold higher maximum rate of BZ oxidation compared with female mice; this correlates with the greater sensitivity of males to the genotoxic effects of BZ as measured by micronuclei induction and sister chromatid exchanges. The aim of this study was to quantitate levels of BZ metabolites in urine and tissues, and to determine whether the higher maximum rate of BZ oxidation in male mice would be reflected in higher levels of hydroxylated BZ metabolites in tissues and water-soluble metabolites in urine. Male and female B6C3F, mice were exposed to 100 or 600 ppm 14C-BZ by nose-only inhalation for 6 h. An additional group of male mice was pretreated with 1% acetone in drinking water for 8 d prior to exposure to 600 ppm BZ; this group was used to evaluate the effect of induction of CYP 2E1 on urine and tissue levels of BZ and its hydroxylated metabolites. BZ, phenol (PHE), and hydroquinone (HQ) were quantified in blood, liver, and bone marrow during exposure and postexposure, and water-soluble metabolites were analyzed in urine in the 48 h after exposure. Male mice exhibited a higher flux of BZ metabolism through the HQ pathway compared with females after exposure to either 100 ppm BZ (32.0 2.03 vs. 19.8 2.7%) or 600 ppm BZ (14.7 1.42 vs. 7.94 + 0.76%). Acetone pretreatment to induce CYP 2E1 resulted in a significant increase in both the percent and mass of urinary HQ glucuronide and muconic acid in male mice exposed to 600 ppm BZ. This increase was paralleled by three- to fourfold higher steady-state concentrations of PHE and HQ in blood and bone marrow of acetone-pretreated mice compared with untreated mice. These results indicate that the higher maximum rate of BZ metabolism in male mice is paralleled by a greater proportion of the total flux of BZ through the pathway for HQ formation, suggesting that the metabolites formed along this pathway may be responsible for the genotoxicity observed following BZ exposure.
Asunto(s)
Acetona/farmacología , Benceno/metabolismo , Solventes/metabolismo , Solventes/farmacología , Administración por Inhalación , Animales , Benceno/administración & dosificación , Benceno/análisis , Médula Ósea/metabolismo , Radioisótopos de Carbono , Citocromo P-450 CYP2E1/biosíntesis , Inducción Enzimática/efectos de los fármacos , Femenino , Hidroquinonas/sangre , Hidroquinonas/orina , Hidroxilación/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Cavidad Nasal , Fenol/sangre , Fenol/orina , Factores SexualesRESUMEN
A new HPLC assay was adapted for radiometric detection of nicotine metabolites in rat bile. Two glucuronides were identified as the principal biliary metabolites of nicotine. In addition to nicotine glucuronide and 3-hydroxycotinine glucuronide, cotinine was also detected in bile after administration to rats of a single subcutaneous dose of (-)-S-nicotine (0.2 or 1.0 mg/kg) that contained a tracer dose of rac-[pyrrolidine-2'-14C]nicotine (20 microCi). Biliary metabolites accounted for only 3% of the [14C]nicotine dose, but phenobarbital pretreatment (100 mg/kg ip for 3 days) increased the amount of [14C]nicotine-derived radioactivity recovered in bile to 8% and also accelerated rates of biliary excretion of all three nicotine metabolites. Dose-dependency of nicotine metabolism occurred: less nicotine glucuronide was excreted at the low dose than at the high dose.
Asunto(s)
Cotinina/análogos & derivados , Cotinina/farmacocinética , Nicotina/metabolismo , Nicotina/farmacología , Animales , Bilis/química , Bilis/metabolismo , Sistema Biliar/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Glucuronatos/farmacocinética , Glucuronatos/farmacología , Masculino , Nicotina/farmacocinética , Fenobarbital/farmacología , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
1,3-Butadiene is carcinogenic in B6C3F1 mice and Sprague-Dawley rats, and has been classified as a probable human carcinogen. The genetic basis for butadiene carcinogenicity is likely mediated by its metabolite, 1,2:3,4-diepoxybutane (BDE). Oxidation of butadiene to 1,2-epoxy-3-butene (BMO) and further activation to BDE is catalysed by cytochrome P450 (CYP) isozymes. The production of BMO from butadiene is mediated by CYP2E1 and, at high butadiene concentrations, by CYP2A6. The purpose of the present study was to identify which human CYP isozymes have the ability to oxidize BMO to BDE, and to determine the extent to which this reaction occurs in B6C3F1 mouse, Sprague-Dawley rat, and human liver microsomes. Of the human cDNA-expressed CYP isozymes tested, only CYP2E1 formed detectable concentrations of BDE at 80 microM BMO. CYP2E1 and CYP3A4 were active at 5.0 mM BMO. Interindividual and interspecies variation in the initial rate of oxidation of 80 microM BMO to BDE was determined using 10 samples of human liver microsomes and single pooled samples from rats and mice. Those experiments revealed a 60-fold variation in activity among 10 human liver samples (range: 0.005-0.324 nmol/mg protein/min). Rates of BMO oxidation for mouse and rat liver microsomes were 0.473 and 0.166 nmol/mg protein/min, respectively. Apparent kinetic constants for the oxidation of BMO to BDE by four human microsomal preparations, and pooled samples from mice and rats were estimated from detailed investigations of BMO oxidation at various BMO substrate concentrations. Apparent Km for the human liver samples ranged from 0.304-0.880 mM, and Vmax values ranged from 0.38 to 1.2 nmol/mg protein/min. The apparent values of Km and Vmax for mouse liver microsomes were 0.141 +/- 0.007 mM (mean +/- SE) and 1.303 +/- 0.141 nmol/mg protein/min, respectively. For rat liver microsomes, apparent Km and Vmax were 0.145 +/- 0.036 mM and 0.408 +/- 0.031 nmol/mg protein/min, respectively. Measured rates of BDE formation correlated well with CYP2E1 protein concentrations in the human microsome samples. These results implicate human CYP2E1 as a hepatic isoform responsible for the oxidation of BMO to BDE at low concentrations of BMO. Moreover, our in vitro results reveal that microsomes prepared from human, rat and mouse liver possess the ability to form BDE from BMO. Previous in vitro results suggest that following exposure to butadiene more BMO would probably be present in mice than in rats or humans. Thus, in mice more BMO would be available for activation to BDE.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Carcinógenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Compuestos Epoxi/metabolismo , Microsomas Hepáticos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Mutágenos/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Biotransformación , Citocromo P-450 CYP2E1 , Citocromo P-450 CYP3A , ADN Complementario , Humanos , Cinética , Masculino , Ratones , Ratones Endogámicos , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismoRESUMEN
In addition to industrial sources, benzene is present in the environment as a component of cigarette smoke and automobile emissions. Toxicity of benzene most likely results from oxidative metabolism of benzene to reactive products. However, susceptibility to these toxic effects may be related to a balance between activation (phase I) and detoxication (phase II) reactions. In the present study, we have estimated kinetic parameters of the two major detoxication reactions for benzene metabolites--phenol sulfation and hydroquinone glucuronidation--in liver subcellular fractions from 10 humans, and single samples from mice and rats. The extent of oxidative metabolism of benzene by these liver samples has been reported previously. Here, initial rates of phenol sulfation varied 3-fold (range 0.309-0.919 nmol/mg protein/min) among human samples. Measured rates were faster in rats (1.195 nmol/mg protein/min) than in mice (0.458 nmol/mg protein/min). Initial rates of hydroquinone glucuronidation by human samples also varied 3-fold (range 0.101-0.281 nmol/mg protein/min). Hydroquinone glucuronidation was more rapid by mouse microsomes (0.218 nmol/mg protein/min) than by rat microsomes (0.077 nmol/mg protein/min). To integrate interindividual differences in various enzyme activities, a physiological compartmental model was developed that incorporates rates of both conjugation reactions and oxidation reactions. Model equations were solved for steady-state concentrations of phenol and hydroquinone attained in human, mouse and rat blood during continuous exposure to benzene (0.01 microM in blood). Among the 10 human subjects, steady-state concentrations of phenol varied 6-fold (range 0.38-2.17 nM) and steady-state concentrations of hydroquinone varied 5-fold (range 6.66-31.44 nM). Predicted steady-state concentrations of phenol were higher in mice compared with rats (2.28 and 0.83 nM respectively). Likewise, higher steady-state concentrations of hydroquinone were predicted in mice than in rats (42.44 and 17.99 nM respectively). Predicted steady-state concentrations of phenol and hydroquinone in mice were higher than predictions for the 10 human subjects, whereas predicted concentrations for rats fell among the human values. As such, our results underscore the importance of considering the balance between activation and detoxication reactions in the elimination of toxicants. Model simulations suggest that both phase I and phase II pathways influence the relative risk from exposure to benzene.
Asunto(s)
Benceno/farmacocinética , Benceno/toxicidad , Hígado/efectos de los fármacos , Hígado/metabolismo , Adolescente , Adulto , Animales , Benceno/metabolismo , Biotransformación , Compartimentos de Líquidos Corporales , Preescolar , Femenino , Glucuronatos/metabolismo , Humanos , Hidroquinonas/metabolismo , Hidroquinonas/farmacocinética , Inactivación Metabólica , Individualidad , Cinética , Masculino , Ratones , Ratones Endogámicos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Persona de Mediana Edad , Mutágenos/metabolismo , Mutágenos/farmacocinética , Fenol , Fenoles/metabolismo , Fenoles/farmacocinética , Ratas , Ratas Sprague-DawleyRESUMEN
Four metabolites of nicotine, including two glucuronides, have been separated by high-performance liquid chromatography. This separation was applied to identification of biliary metabolites of radiolabeled nicotine by radiometric detection. S-(-)-Nicotine N-glucuronide was synthesized and used as a standard in method development.
Asunto(s)
Bilis/química , Cromatografía Líquida de Alta Presión/métodos , Glucuronatos/síntesis química , Nicotina/análogos & derivados , Nicotina/análisis , Animales , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Nicotina/síntesis química , Nicotina/metabolismo , Radiometría , Ratas , Ratas Sprague-DawleyRESUMEN
Low levels of benzene from sources including cigarette smoke and automobile emissions are ubiquitous in the environment. Since the toxicity of benzene probably results from oxidative metabolites, an understanding of the profile of biotransformation of low levels of benzene is critical in making a valid risk assessment. To that end, we have investigated metabolism of a low concentration of [14C]benzene (3.4 microM) by microsomes from human, mouse and rat liver. The extent of phase I benzene metabolism by microsomal preparations from 10 human liver samples and single microsomal preparations from both mice and rats was then related to measured activities of cytochrome P450 (CYP) 2E1. Measured CYP 2E1 activities, as determined by hydroxylation of p-nitrophenol, varied 13-fold (0.253-3.266 nmol/min/mg) for human samples. The fraction of benzene metabolized in 16 min ranged from 10% to 59%. Also at 16 min, significant amounts of oxidative metabolites were formed. Phenol was the main metabolite formed by all but two human microsomal preparations. In those samples, both of which had high CYP 2E1 activity, hydroquinone was the major metabolite formed. Both hydroquinone and catechol formation showed a direct correlation with CYP 2E1 activity over the range of activities present. A simulation model was developed based on a mechanism of competitive inhibition between benzene and its oxidized metabolites, and was fit to time-course data for three human liver preparations. Model calculations for initial rates of benzene metabolism ranging from 0.344 to 4.442 nmol/mg/min are directly proportional to measured CYP 2E1 activities. The model predicted the dependence of benzene metabolism on the measured CYP 2E1 activity in human liver samples, as well as in mouse and rat liver samples. These results suggest that differences in measured hepatic CYP 2E1 activity may be a major factor contributing to both interindividual and interspecies variations in hepatic metabolism of benzene. Validation of this system in vivo should lead to more accurate assessment of the risk of benzene's toxicity following low-level exposure.
Asunto(s)
Benceno/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Oxidorreductasas N-Desmetilantes/metabolismo , Adolescente , Adulto , Animales , Preescolar , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2E1 , Humanos , Individualidad , Cinética , Masculino , Cómputos Matemáticos , Ratones , Ratones Endogámicos , Persona de Mediana Edad , Modelos Biológicos , Oxidación-Reducción , Ratas , Ratas Endogámicas F344RESUMEN
Transgenic CYP2E1 knockout mice (cyp2e1-/-) were used to investigate the involvement of CYP2E1 in the in vivo metabolism of benzene and in the development of benzene-induced toxicity. After benzene exposure, absence of CYP2E1 protein was confirmed by Western blot analysis of mouse liver samples. For the metabolism studies, male cyp2e1-/- and wild-type control mice were exposed to 200 ppm benzene, along with a radiolabeled tracer dose of [14C]benzene (1.0 Ci/mol) by nose-only inhalation for 6 hr. Total urinary radioactivity and all radiolabeled individual metabolites were reduced in urine of cyp2e1-/- mice compared to wild-type controls during the 48-hr period after benzene exposure. In addition, a significantly greater percentage of total urinary radioactivity could be accounted for as phenylsulfate conjugates in cyp2e1-/- mice compared to wild-type mice, indicating the importance of CYP2E1 in oxidation of phenol following benzene exposure in normal mice. For the toxicity studies, male cyp2e1-/-, wild-type, and B6C3F1 mice were exposed by whole-body inhalation to 0 ppm (control) or 200 ppm benzene, 6 hr/day for 5 days. On Day 5, blood, bone marrow, thymus, and spleen were removed for evaluation of micronuclei frequencies and tissue cellularities. No benzene-induced cytotoxicity or genotoxicity was observed in cyp2e1-/- mice. In contrast, benzene exposure resulted in severe genotoxicity and cytotoxicity in both wild-type and B6C3F1 mice. These studies conclusively demonstrate that CYP2E1 is the major determinant of in vivo benzene metabolism and benzene-induced myelotoxicity in mice.