RESUMEN
Moli1901 is a 19 residue polycyclic peptide antibiotic which increases chloride transport and water mobilization in airway epithelium. These properties suggest that it may be a useful treatment for cystic fibrosis (CF). In this study, we used accelerator mass spectrometry (AMS) to quantify Moli1901 following administration of only 0.045 microCi of 14C-Moli1901 per dog. Limits of quantitation of AMS were 0.03 (urine) to 0.3 (feces) ng equiv. Moli1901/g. Administration of 14C-Moli1901 by intratracheal instillation (approximately 100 microg) into the left cranial lobe of the lung of beagle dogs resulted in retention of 64% of the dose in the left cranial lobe for up to 28 days. Whole blood and plasma concentrations of 14C were <5 ng/ml at all times after the dose. Concentrations of 14C in whole blood and plasma declined over the first day after the dose and rose thereafter, with the rise in plasma concentrations lagging behind those in whole blood. During the first 3 days after the dose, plasma accounted for the majority of 14C in whole blood, but after that time, plasma accounted for only 25-30% of the 14C in whole blood. Tissue (left and right caudal lung lobe, liver, kidney, spleen, brain) and bile concentrations were low, always less than 0.25% the concentrations found in the left cranial lung lobe. Approximately 13% of the dose was eliminated in urine and feces in 28 days, with fecal elimination accounting for about 10% of the dose. The data presented here are consistent with that obtained in other species. Moli1901 is slowly absorbed and excreted from the lung, and it does not accumulate in other tissues. Moli1901 is currently in the clinic and has proven to be safe in single dose studies in human volunteers and cystic fibrosis patients by the inhalation route. No information on the disposition of the compound in humans is available. This study in dogs demonstrates the feasibility of obtaining that information using 14C-Moli1901 and AMS.
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Espectrometría de Masas/métodos , Aceleradores de Partículas , Péptidos Cíclicos/farmacocinética , Péptidos Cíclicos/orina , Animales , Radioisótopos de Carbono , Fibrosis Quística/tratamiento farmacológico , Perros , Intubación Intratraqueal , Pulmón/metabolismo , Masculino , Espectrometría de Masas/instrumentaciónRESUMEN
We tested the hypothesis that the small intestine is capable of the first-pass, reductive metabolism of xenobiotics. A simplified version of the isolated vascularly perfused rat small intestine was developed to test this hypothesis with 1,3-dinitrobenzene (1,3-DNB) as a model xenobiotic. Both 3-nitroaniline (3-NA) and 3-nitroacetanilide (3-NAA) were formed and absorbed following intralumenal doses of 1,3-DNB (1.8 or 4.2 mumol) to isolated vascularly perfused rat small intestine. Dose, fasting, or antibiotic pretreatment had no effect on the absorption and metabolism of 1,3-DNB in this model system. The failure of antibiotic pretreatment to alter the metabolism of 1,3-DNA indicated that 1,3-DNB metabolism was mammalian rather than microfloral in origin. All data from experiments initiated with lumenal 1,3-DNB were fit to a pharmacokinetic model (model A). ANOVA analysis revealed that dose, fasting, or antibiotic pretreatment had no statistically significant effect on the model-dependent parameters. 3-NA (1.5 mumol) was administered to the lumen of isolated vascularly perfused rat small intestine to evaluate model A predictions for the absorption and metabolism of this metabolite. All data from experiments initiated with 3-NA were fit to a pharmacokinetic model (model B). Comparison of corresponding model-dependent pharmacokinetic parameters (i.e. those parameters which describe the same processes in models A and B) revealed quantitative differences. Evidence for significant quantitative differences in the pharmacokinetics or metabolism of formed versus preformed 3-NA in rat small intestine may require better definition of the rate constants used to describe tissue and lumenal processes or identification and incorporation of the remaining unidentified metabolites into the models.
Asunto(s)
Dinitrobencenos/metabolismo , Dinitrobencenos/farmacocinética , Absorción Intestinal , Intestino Delgado/metabolismo , Acetanilidas/metabolismo , Compuestos de Anilina/metabolismo , Animales , Radioisótopos de Carbono , Intestino Delgado/irrigación sanguínea , Masculino , Cómputos Matemáticos , Perfusión , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los ResultadosRESUMEN
Remifentanil is a mu opioid receptor agonist, structurally related to fentanyl, being developed for use in anesthesia. Remifentanil was designed to be cleared rapidly by ester hydrolysis. To determine the pharmacokinetics of remifentanil in conscious beagle dogs, venous blood was collected at various times during and after the end of a 25 min intravenous infusion of the compound (0.36 or 36.0 micrograms [free base]/kg/min). In a separate set of studies designed to measure tissue clearance of remifentanil, catheters were implanted in various blood vessels of anesthetized beagle dogs to sample blood entering and leaving selected tissues. Approximately 40 min (steady state achieved) after initiation of remifentanil infusions at the same rates as described above, and while infusions were still in progress, blood was collected from the series of catheters. In a third set of experiments, blood obtained from either untreated dogs or from a dog that had been anesthetized, was incubated with remifentanil (either 10 or 1000 ng (free base)/ml). The observed half-life was used to provide an estimate of in vivo blood metabolic clearance of the compound. Extracts of the blood from all experiments were analyzed by either an HPLC or mass spectrometry assay for remifentanil concentration. There were no differences in systemic clearance (approximately 45 ml/kg/min), volume of distribution at steady state, mean residence time, dose-normalized normalized concentration at steady state, or dose-normalized AUC between the doses administered to conscious dogs; the t1/2 alpha was 3-5 min. In the anesthetized animals, muscle and intestine had the highest tissue clearance rates, but liver, kidneys, and blood each accounted for 1% or less of systemic clearance. The results indicate that, within the range of doses studied, the pharmacokinetics of remifentanil during infusion to steady state in dogs were not dependent on dose, and that the liver contributed very minimally to the overall clearance of the compound.
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Analgésicos Opioides/farmacocinética , Hígado/metabolismo , Piperidinas/farmacocinética , Analgésicos Opioides/administración & dosificación , Analgésicos Opioides/sangre , Animales , Perros , Semivida , Inyecciones Intravenosas , Masculino , Piperidinas/administración & dosificación , Piperidinas/sangre , RemifentaniloRESUMEN
The small intestine can metabolize a variety of substances and can play a role in the presystemic clearance of ingested compounds. Relatively little is known about the ability of small intestine to catalyze the presystemic reductive metabolism of xenobiotics. 1,3-Dinitrobenzene (1,3-DNB), which is known to undergo reductive biotransformation in an intact, oxygenated isolated perfused intestinal preparation, was used as a model substrate for reductive enzymes of the small intestine of the rat. Subcellular fractions from duodenal, jejunal, and ileal regions of rat small intestinal mucosa were used to characterize the enzyme source(s) of those reductive reactions of 1,3-DNB that are relevant in the oxygenated intestinal tissue. 1,3-DNB was reduced to 3-nitroaniline (3-NA) by cytosol from duodenum and jejunum. The rate of reduction was 2 times faster when incubations contained duodenal rather than jejunal cytosol. Jejunal cytosol-catalyzed reduction of 1,3-DNB was supported by hypoxanthine, NADPH, or NADH. Duodenal microsomes catalyzed the reduction of 1,3-DNB to 3-NA in the presence of supplemental NADPH or NADH; however, the reaction was very slow. Jejunal microsomes, ileal microsomes, and ileal cytosol failed to catalyze the reduction of 1,3-DNB. Studies with chemical inhibitors suggested possible roles for DT diaphorase, glutathione reductase, or xanthine oxidase in the jejunal cytosol-catalyzed reaction. Purified, commercially available xanthine oxidase (from buttermilk) catalyzed the reduction of 1,3-DNB to 3-NA when supplemented with NADH or hypoxanthine.
Asunto(s)
Dinitrobencenos/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Citosol/efectos de los fármacos , Citosol/enzimología , Glutatión Reductasa/metabolismo , Técnicas In Vitro , Mucosa Intestinal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Masculino , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Espectrofotometría Ultravioleta , Xantina Oxidasa/metabolismoRESUMEN
Substituted metalloporphyrins, in addition to their use as pharmacological agents, are used to investigate metabolic pathways by inhibiting cytochrome P-450. We have examined the specificity of this approach with cobalt mesoporphyrin (CoMP). In vivo, CoMP (50 mumol/kg, s.c.) decreased rat hepatic microsomal cytochrome P-450, NADPH cytochrome P-450 reductase, benzphetamine N-demethylase (BZPH) activity, and thyroid hormones by > 50%, all of which returned to control levels after 45 days; testosterone levels were also reduced at this dose. The half-life of CoMP was 18 days, which is consistent with this sustained effect. At 10 mumol/kg of CoMP, the reductase activity was decreased, but cytochrome P-450 was unchanged. An effect of residual CoMP on the reductase was ruled out as the CoMP content of tissue fractions was not high enough to inhibit directly the reductase activity (even after 50 mumol CoMP/kg). However, immunoblots indicated a lower level of immunoreactive reductase protein following treatment. After 8 weekly doses of 1 mumol CoMP/kg, BZPH activity was 39% less than control but neither P-450 content nor reductase activity was significantly changed. The P-450 content and reductase activity in rabbits were much less affected by CoMP, perhaps due to differences in the disposition of CoMP. Thyroidectomy decreased reductase activity in rats to an extent that was seen with CoMP at 50 mumol/kg; CoMP treatment of thyroidectomised rats did not further decrease reductase activity. Supplementation with thyroid hormone blocked the CoMP-related decrease. The flavin-containing monooxygenase was decreased by CoMP and by castration, and the decrease was not blocked by the thyroid hormone supplement. Thus in the rat, the CoMP-related decreases in thyroid hormone and testosterone decrease flavoproteins that support or mediate monooxygenase activities. This is contrary to the reported specificity of this class of compound as inhibitors of cytochrome P-450.
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Flavoproteínas/efectos de los fármacos , Mesoporfirinas/farmacología , Testosterona/metabolismo , Hormonas Tiroideas/metabolismo , Animales , Radioisótopos de Cobalto , Inhibidores Enzimáticos del Citocromo P-450 , Esquema de Medicación , Immunoblotting , Masculino , Mesoporfirinas/administración & dosificación , Mesoporfirinas/farmacocinética , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/efectos de los fármacos , Conejos , Ratas , Ratas Endogámicas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
The pharmacokinetics of methanol and formate were characterized in male Fischer-344 rats and rhesus monkeys exposed to methanol vapor concentrations between 50 and 2000 ppm for 6 hr. End-of-exposure blood methanol concentrations were not directly proportional to the atmospheric concentration. The methanol exposures did not cause an elevation in blood formate concentrations. After an intravenous dose of [14C]methanol in rats, metabolism, exhalation, and renal excretion contributed 96.6, 2.6, and 0.8%, respectively, to the elimination of blood methanol concentrations. These values and the calculated renal methanol extraction efficiency (0.007) are nearly identical to those for humans after low doses of methanol. A physiologically based pharmacokinetic model was developed to simulate the in vivo data. In order to simulate the observed blood methanol concentrations in the inhalation studies in rats, a double pathway for methanol metabolism to formaldehyde was used. One path used rodent catalase Km and Vmax values and the other used a smaller Km and Vmax to simulate an enzyme with a higher affinity and lower capacity. The lack of proportionality observed in end-of-exposure blood methanol concentrations may be due to saturation of an enzyme with higher affinity and lower capacity than catalase. The physiologically based pharmacokinetic model was modified to simulate the monkey data and was scaled-up for humans. In order to simulate the monkey blood methanol concentrations, the use of rodent catalase parameters for methanol metabolism was required. This finding suggests that primates and rodents may be similar in the initial step of methanol metabolism after low methanol doses. Previously published human urinary methanol excretion data was successfully simulated by the model. The models were used to predict the atmospheric methanol concentration range over which the laboratory species exhibit quantitative similarities with humans. Below 1200 ppm, all three species exhibit similar end-of-exposure blood methanol concentrations and a linear relationship between atmospheric and blood methanol concentrations. At higher atmospheric concentrations, external and internal methanol concentrations increase desparately, suggesting that delivered dose rather than exposure concentration should be used in interpreting data from high-dose studies.
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Metanol/farmacocinética , Modelos Biológicos , Administración por Inhalación , Animales , Relación Dosis-Respuesta a Droga , Humanos , Inyecciones Intravenosas , Macaca mulatta , Masculino , Ratas , Ratas Endogámicas F344RESUMEN
Acrylonitrile (ACN) has been shown to cause tumors of the brain, stomach and Zymbal's gland in rats in several bioassays, but it has not been tested in other species. The carcinogenic risk of humans exposed to ACN is unclear. ACN is metabolized in the liver to 2-cyanoethylene oxide (CEO), which is believed to be the proximate or ultimate carcinogenic species. Therefore, the kinetics of CEO formation were studied with liver and lung microsomes from mice and humans using a GC-MS assay for CEO, and the data were compared with previously obtained kinetic parameters for rat microsomal enzymes. The rate of CEO formation by human liver microsomes was comparable to that of rat liver microsomes, but less than that of mouse liver microsomes. Liver microsomes produced more CEO than lung microsomes with all three species. CEO formation by microsomes from mice was approximately 4 times greater than that by microsomes from rats or humans, suggesting that mice would have higher CEO concentrations in blood than rats after ACN exposure. However, after oral administration of ACN, the concentration of CEO in mouse blood was one-third that in rat blood at all doses and time points examined. These results show that CEO circulates via the blood, providing exposure to distant sites. The blood concentrations of CEO do not appear to correlate with rates of microsomal CEO formation. This suggests that species differences in the detoxication of CEO may play an important role in determining circulating CEO concentrations and distant organ exposure.
Asunto(s)
Acrilonitrilo/metabolismo , Compuestos Epoxi/sangre , Microsomas/metabolismo , Animales , Carcinógenos/metabolismo , Relación Dosis-Respuesta a Droga , Óxido de Etileno/análogos & derivados , Óxido de Etileno/sangre , Óxido de Etileno/metabolismo , Humanos , Cinética , Pulmón/metabolismo , Pulmón/ultraestructura , Masculino , Ratones , Ratones Endogámicos , Microsomas Hepáticos/metabolismo , Oxidación-Reducción , Ratas , Ratas Endogámicas F344 , Especificidad de la EspecieRESUMEN
The metabolism of radiolabeled monochloronitrobenzene isomers was compared in isolated hepatocytes and hepatic subcellular fractions from male Fischer-344 rats. 2-Chloronitrobenezene was converted by isolated hepatocytes to 2-chloroaniline, 2-chloroaniline-N-glucuronide, and S-(2-nitrophenyl)glutathione in approximately equal quantities (13-19% of the added substrate in 90 min). The major metabolite formed from 3-chloronitrobenzene by isolated hepatocytes was 3-chloroaniline (31% of the added substrate in 90 min). Smaller amounts of 3-chloroaniline-N-glucuronide and 3-chloroacetanilide were formed (7 and 17% of the added 3-chloronitrobenzene, respectively, in 90 min). 4-Chloronitrobenzene was metabolized to 4-chloroacetanilide, 4-chloroaniline, and S-(4-nitrophenyl)glutathione in approximately equal amounts (10-15% of the added substrate in 90 min). Studies with hepatic microsomes showed that reduction of the chloronitrobenzenes to chloroanilines was inhibited by SKF 525-A, metyrapone, and carbon monoxide, suggesting that cytochrome P-450 played a role in the reaction. Thus, the major difference in the in vitro hepatic metabolism of the three isomers of chloronitrobenzene is the failure of 3-chloronitrobenzene to be converted to a glutathione conjugate.
Asunto(s)
Hígado/metabolismo , Nitrobencenos/metabolismo , Animales , Biotransformación , Células Cultivadas , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Hígado/citología , Masculino , Microsomas Hepáticos/metabolismo , Ratas , Ratas Endogámicas F344 , Fracciones Subcelulares/metabolismoRESUMEN
The metabolism of acrylonitrile to the epoxide, 2-cyanoethylene oxide (ANO) was examined in rat liver microsomes, lung microsomes, and isolated enriched lung cell preparations. GC/high resolution MS was used to quantitate ANO in microsomal and cellular extracts by monitoring the fragment ion C2H3N (m/z 41.0265). The limit of detection was 0.05 pmol of ANO/0.5 microliter of standard solution, microsomal extract, or cellular extract injected onto the column, and the linear range of analysis was 0.05 to 12.5 pmol of ANO. Kinetic parameters of Vmax, V/K, and Km were calculated for microsomal ANO formation. Liver microsomes were quantitatively more active than lung microsomes on a mg of protein basis. The Vmax (pmol of ANO formed/min/mg of protein) was 666.61 for liver and 45.07 for lung microsomes. The V/K (pmol of ANO/min/mg of protein/microM) was 12.83 for liver and 0.02 for lung microsomes. The apparent Km was 51.93 microM and 1853.83 microM for liver and lung microsomes, respectively. When calculated as nmol of ANO formed/min/nmol of microsomal P-450, the Vmax for lung was equivalent to the Vmax for liver. ANO formation in the rat lung was cell specific. The rates of metabolism in the Clara cell-enriched fraction, the alveolar type II cell-enriched fraction, and the cell suspension were 2.55, 0.38, and 0.67 pmol of ANO formed/min/mg of protein, respectively. No metabolism was observed in the endothelial (small) cell-enriched fraction or in the alveolar macrophages. The results suggest that the lung contributes to the metabolism and disposition of inhaled acrylonitrile.
Asunto(s)
Acrilonitrilo/metabolismo , Pulmón/metabolismo , Microsomas Hepáticos/metabolismo , Microsomas/metabolismo , Nitrilos/metabolismo , Animales , Sistema Enzimático del Citocromo P-450/metabolismo , Óxido de Etileno/metabolismo , Pulmón/efectos de los fármacos , Masculino , Microsomas/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , RatasRESUMEN
Previous results have suggested that key intermediates in the activation of 2-nitrotoluene and 2,6-dinitrotoluene are 2-aminobenzyl alcohol and 2-amino-6-nitrobenzyl alcohol, respectively. In order to determine the metabolic pathway(s) involved in the activation steps, calf thymus DNA and [14C]-2-aminobenzyl alcohol or [14C]-2-amino-6-nitrobenzyl alcohol were incubated with male Fischer-344 rat hepatic cytosol and PAPS, microsomes and NADPH, or microsomes and cytosol with PAPS, NADPH, and acetyl coenzyme A. DNA was isolated and analyzed for radiolabel bound covalently. Analysis of the incubations containing [14C]-2-aminobenzyl alcohol revealed radiolabel bound covalently to DNA, as well as one major metabolite labile in both sulfatase and acid. The appearance of each required the presence of PAPS and cytosol and was inhibited by the sulfotransferase inhibitor 2,6-dichloro-4-nitrophenol. Neither NADPH nor acetyl coenzyme A played a role in the generation of detectable 14C bound to nucleic acids. 2-Amino-6-nitrobenzyl alcohol was converted to metabolites capable of binding to calf thymus DNA when incubated with cytosol and PAPS or with microsomes and NADPH. However, when cytosol and microsomes were incubated together, activation of 2-amino-6-nitrobenzyl alcohol appeared to require only PAPS, suggesting a minor role for NADPH-dependent enzymes in its activation. The results suggest that the metabolite of 2-nitrotoluene responsible for binding covalently to DNA is 2-aminobenzyl sulfate. There may be more than one pathway involved in the formation of metabolite(s) of 2,6-dinitrotoluene that bind covalently to DNA.
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Acetiltransferasas , Alcoholes Bencílicos/metabolismo , Dinitrobencenos/metabolismo , Tolueno/análogos & derivados , Acetilcoenzima A/metabolismo , Aciltransferasas/metabolismo , Animales , Alcoholes Bencílicos/farmacocinética , Biotransformación , Núcleo Celular/metabolismo , Cromatografía Líquida de Alta Presión , Citosol/metabolismo , Hidrólisis , Técnicas In Vitro , Masculino , Microsomas Hepáticos/metabolismo , NADP/metabolismo , Fosfoadenosina Fosfosulfato/metabolismo , Ratas , Ratas Endogámicas F344 , Tolueno/metabolismoRESUMEN
The metabolism of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) was studied in murine lymphocytes. This carcinogen has previously been shown to be immunosuppressive to lymphocytes regardless of their ability to be induced via the Ah locus and receptor. Experiments were designed to quantify the generation of metabolites of DMBA by lymphocytes incubated with [14C]DMBA and to ascertain whether radioactivity was covalently bound to cellular macromolecules in DMBA-exposed lymphocytes. No significant metabolism of DMBA was detected in culture supernatants, except when cultures were incubated in the presence of Arochlor-induced rat liver 9000 x g supernatants (S9). Covalent binding of 14C to cellular macromolecules was enhanced approximately eightfold in the presence of S9. Inhibition of monooxygenase activity by alpha-naphthoflavone did not modulate the immunosuppressive character of DMBA. Furthermore, addition of S9 did not amplify or ablate DMBA-mediated suppression of lymphocyte proliferation to the mitogen concanavalin A (Con A). Selected metabolites of DMBA were evaluated for immunosuppressive effects in cultures stimulated with mitogens and cellular alloantigens. 7-Hydroxymethyl-12-methylbenz[a]anthracene (OHMe) and 5,6-dihydro-5,6-dihydroxybenz[a]anthracene (Diol) were found to cause only slightly greater suppression of lymphocyte responses than DMBA. Thus, it appears that metabolites of DMBA were not responsible for the immunosuppression observed in lymphocyte cultures and that lymphocytes were not equipped to metabolize any significant amount of DMBA. These data lend support to the hypothesis that parent compound alone is responsible for the immunosuppressive effects observed in murine lymphocyte culture.
Asunto(s)
9,10-Dimetil-1,2-benzantraceno/metabolismo , Linfocitos/metabolismo , 9,10-Dimetil-1,2-benzantraceno/farmacología , Animales , Benzoflavonas/farmacología , Células Cultivadas , Cromatografía Líquida de Alta Presión , Concanavalina A/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Femenino , Terapia de Inmunosupresión , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microsomas/enzimología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Bazo/citologíaRESUMEN
Diminished intercellular communication has been associated with heightened sensitivity of cultured cells to morphological transformation and enhancement of transformation by tumor promoters. Microinjection of Lucifer yellow dye was employed to evaluate intercellular communication between transformable C3H/10T1/2 murine fibroblasts under a variety of culture conditions. Intercellular communication assayed by dye transfer from injected cells to surrounding cells in contact occurred in logarithmically growing cultures, declined to very low levels as confluence was attained, and then resumed upon the formation of mature confluent monolayers. Dye-transfer networks of 50 or more cells resulted from injection of single monolayer cells. Freeze-fracture electron microscopy confirmed the presence of gap junction structures in confluent cultures. Treatment with the initiating agent N-methyl-N'-nitro-N-nitrosoguanidine and/or the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit intercellular communication between C3H/10T1/2 cells during 6-week transformation experiments. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate produced a transient inhibition of dye-coupling upon first introduction to cultures and prolonged the period of diminished dye-coupling at the attainment of confluence, but did not inhibit subsequent interactions between monolayer cells. A simple relationship thus could not be established between levels of dye-coupling within monolayers and focus formation events. Curiously, although the cells of foci in early phases of development did not exhibit dye-transfer capacity, dye-coupling was observed in mass cultures of most transformed cell lines cloned from foci. Co-cultivation of communication-competent transformed cells with nontransformed cells to produce reconstructed foci generally resulted in a cessation of dye-transfer by transformed cells. An often reversible loss of communication competence thus accompanies the growth of transformed C3H/10T1/2 cells as foci and may constitute an adaptive response which facilitates focus growth in the presence of intercellular communication between monolayer cells.
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Carcinógenos/farmacología , Comunicación Celular/efectos de los fármacos , Transformación Celular Neoplásica/fisiopatología , Animales , Línea Celular , Isoquinolinas , Metilcolantreno/farmacología , Metilnitronitrosoguanidina , Ratones , Dibenzodioxinas Policloradas/farmacología , Acetato de Tetradecanoilforbol/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Cellular GSH may influence the metabolism of the rodent brain and forestomach carcinogen acrylonitrile (ACN) and its subsequent binding to tissue macromolecules. To investigate the role of GSH in ACN metabolism and binding to macromolecules, we studied the effect of GSH depletion on the irreversible association of radiolabel with tissue macromolecules in male F-344 rats given a 4 mg/kg dose of [2,3-14C]ACN by inhalation. A combined phorone/buthionine sulfoximine treatment (300 mg/kg and 2 mmol/kg, respectively) was given 30 minutes prior to ACN exposure to deplete GSH. The uptake of ACN vapor by control rats was biphasic and characterized by a rapid phase lasting about 60 min and by a slower phase from 60 min to the end of exposure. The rate of uptake for both phases was linearly related to the initial concentration of ACN in the chamber. GSH depletion caused an increase in the rate of ACN uptake in both phases. It also caused a decrease in total radioactivity recovered in brain, stomach, liver, kidney, and blood and a concomitant decrease in the ACN-derived nondialyzable radioactivity in these organs. In control rats, accumulation of radiolabel was greatest in brain RNA, but no radioactivity was detected in DNA of any organ examined. In GSH-depleted rats, the radiolabel concentration was higher in brain RNA than in the liver or stomach RNA, but was also 50% lower than that observed in brain RNA of control rats. Urinary excretion of thiocyanate (SCN-), a metabolite derived from the epoxide pathway of ACN metabolism, was doubled in GSH-depleted rats. These results suggest that GSH might be involved in the distribution of ACN-derived reactive species and, therefore, might play a role in the binding of ACN-derived species to tissue macromolecules and nucleic acids.
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Acrilonitrilo/farmacocinética , Glutatión/metabolismo , Nitrilos/farmacocinética , Animales , ADN/farmacología , Sustancias Macromoleculares , Masculino , ARN/farmacología , Ratas , Ratas Endogámicas F344 , Tiocianatos/orina , Distribución Tisular , VolatilizaciónRESUMEN
Binding of acrylonitrile and its reactive metabolites to tissue macromolecules, especially nucleic acids, may be responsible for its carcinogenicity in rats. Both acrylonitrile and its primary metabolite, 2-cyanoethylene oxide, also react with glutathione. To better understand the role of glutathione in the manifestation of acrylonitrile toxicity, the irreversible binding to tissue macromolecules was assessed in control and glutathione-depleted F-344 rats treated with a 4 mg/kg dose (po) of [2,3-14C]acrylonitrile. Glutathione was depleted in rat tissues by the administration of a combined intraperitoneal phorone/buthionine sulfoximine treatment (300 mg/kg and 2 mmol/kg, respectively) given 30 min prior to acrylonitrile administration. The amount of total radioactivity recovered from brain, stomach (target organs), liver, kidney, lung, and blood (nontarget organs) was similar between control and glutathione-depleted rats. However, stomach, lung, blood, and liver showed an increase in total radioactivity content after glutathione depletion by phorone/buthionine sulfoximine treatment. Glutathione depletion also caused an increase in acrylonitrile-derived non-dialysable radioactivity (MW greater than 3500 Da) in liver, lung, kidney, stomach, blood, and brain macromolecules between 6 and 24 hr after the dose. There was no organ-specific accumulation of radiolabel in RNA in control rats. However, an increase in the radiolabel associated with nucleic acids in the target organs but not in the nontarget organs was measured in glutathione-depleted rats. Urinary excretion of thiocyanate, a metabolite derived from the epoxide pathway, was also increased by 300% in glutathione-depleted rats. These results suggest that glutathione might play a role in the extent of 2-cyanoethylene oxide formation and in the distribution of the radiolabel among tissues.
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Acrilonitrilo/metabolismo , Glutatión/metabolismo , Nitrilos/metabolismo , Acrilonitrilo/administración & dosificación , Administración Oral , Animales , Sustancias Macromoleculares , Masculino , Ratas , Ratas Endogámicas F344 , Tiocianatos/orinaRESUMEN
The effects of pretreatment with inhibitors of drug metabolism on the biotransformation of cyproheptadine (CPH) and the ability of the drug to deplete pancreatic insulin were investigated. CPH (45 mg/kg p.o.) or water was given once daily for 2 days to rats. SKF-525A (beta-diethylaminoethyl-2,2-diphenylpentanoate) (40 mg/kg i.p.) or water was administered 0.5 hr before the CPH treatment, and the animals were sacrificed 6 hr after the second dose of CPH. The pancreatic insulin concentration was determined, and the levels of CPH and its metabolites in pancreas, liver and lung were measured. It was found that SKF-525A pretreatment significantly protected rats from the insulin loss induced by CPH; SKF-525A alone had no effect on pancreatic insulin. The tissues from animals pretreated with SKF-525A, when compared with those of the control, had 10-fold higher levels of CPH, significantly lower concentrations of the metabolite desmethylcyproheptadine-10,11-epoxide (DMCPH-epoxide) and no change in the levels of desmethylcyproheptadine (DMCPH). The in vivo N-demethylation of N-[methyl-14C]CPH, as measured by the excretion rate of 14CO2, also was inhibited by SKF-525A pretreatment. Similar experiments were performed with another inhibitor of drug metabolism, 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP, 50 mg/kg i.p.); and the same results were obtained. In other experiments using DMCPH as the insulin-depleting compound, SKF-525A or DDEP pretreatment also afforded significant protection against chemical-induced insulin loss.(ABSTRACT TRUNCATED AT 250 WORDS)
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Ciproheptadina/metabolismo , Insulina/metabolismo , Páncreas/efectos de los fármacos , Piridinas/farmacología , Pirimetamina/análogos & derivados , Animales , Ciproheptadina/análogos & derivados , Ciproheptadina/farmacología , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Páncreas/metabolismo , Pirimetamina/farmacología , Ratas , Ratas EndogámicasRESUMEN
All three dinitrobenzene (DNB) isomers cause methemoglobinemia, but only 1,3-DNB produces testicular toxicity in rats. In order to determine whether major differences exist in the routes of DNB metabolism, male Fischer-344 rats were given an oral dose (0.15 mmol/kg) of 14C-labeled 1,2-, 1,3-, or 1,4-DNB, and excreta were collected over 48 hr. Elimination of radiolabel was rapid; 85%, 60%, and 75% of the 1,2-, 1,3-, and 1,4-DNB dose was recovered in 24 hr, respectively. Urine was the primary route of excretion, accounting for 82% of the total dose of 1,2-DNB and 75% of the dose of 1,4-DNB after 48 hr. Radiolabel from 1,3-DNB was excreted to a slightly lesser extent in the urine (63% of the dose). A greater portion of radiolabel was excreted in the feces than with the other isomers (18% of total dose, compared to 8% and 9% with 1,2-DNB and 1,4-DNB, respectively). The major urinary metabolites of 1,2-DNB were S-(2-nitrophenyl)-N-acetylcysteine (42% of the dose), 2-nitroaniline-N-glucuronide (4%), 4-amino-3-nitrophenylsulfate (17%), 2-amino-3-nitrophenylsulfate (1.5%), and 2-(N-hydroxylamino)nitrobenzene (1-2%). The major urinary metabolites of 1,3-DNB were 3-aminoacetanilide (22%), 4-acetamidophenylsulfate (6%), 1,3-diacetamidobenzene (7%), and 3-nitroaniline-N-glucuronide (4%). The major metabolites of 1,4-DNB were 2-amino-5-nitrophenylsulfate (35%), S-(4-nitrophenyl)-N-acetylcysteine (13%), and 1,4-diacetamidobenzene (7%). These results suggest that the DNB isomers are primarily metabolized by nitro group reduction and conjugation with glutathione. The testicular toxicant 1,3-DNB was apparently metabolized exclusively by reduction.
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Dinitrobencenos/metabolismo , Nitrobencenos/metabolismo , Animales , Masculino , Ratas , Ratas Endogámicas F344RESUMEN
The metabolism of dinitrobenzene (DNB) isomers in Fischer-344 rat, rhesus monkey and human erythrocytes was investigated. Erythrocytes from all species metabolized o-DNB and p-DNB to S-(nitrophenyl)glutathione conjugates although there were species differences in the rate and extent of conjugate formation. No metabolites of m-DNB were detected in the erythrocytes of any species. The rank order of the ability of the DNB isomers to produce methemoglobin in vitro varied from species to species, but p-DNB was always the most effective isomer. The data suggest that although the erythrocyte can conjugate DNB isomers with glutathione, this pathway offers no substantial protection from methemoglobinemia induced by dinitrobenzenes.
Asunto(s)
Dinitrobencenos/metabolismo , Eritrocitos/metabolismo , Nitrobencenos/metabolismo , Adulto , Animales , Dinitrobencenos/toxicidad , Eritrocitos/efectos de los fármacos , Glutatión/biosíntesis , Humanos , Macaca mulatta , Masculino , Metahemoglobina/biosíntesis , Ratas , Ratas Endogámicas F344 , Especificidad de la EspecieRESUMEN
There appear to be two major pathways for the metabolism of nitrobenzene and substituted nitrobenzenes. The first of these is reduction of the nitro group to yield aniline or substituted anilines. For nitrobenzene and perhaps for pentachloronitrobenzene, reduction of the nitro group to the amine is accomplished by bacteria of the gastrointestinal tract. Addition of a second nitro group results in easier reduction of one of the nitro groups on dinitrobenzenes, since they can be reduced under aerobic conditions by hepatic and erythrocyte enzymes. Bacterial reduction of the dinitrobenzenes is probably not quantitatively important in vivo. The second pathway is replacement of a nitro group by glutathione. The relative importance of this pathway compared to nitro group reduction depends upon the compound. It has not been demonstrated to occur for nitrobenzene. It is the major route of metabolism for 1,2-dinitrobenzene but is not an important route for 1,3- or 1,4-dinitrobenzene in hepatocytes. Tetrachloronitrobenzene isomers in which the nitro group is flanked by chlorines and pentachloronitrobenzene undergo nitro group replacement, but 2,3,4,5-tetrachloronitrobenzene does not. The most important pathway for the metabolism of mononitrotoluenes is methyl group oxidation. All quantitatively important metabolites are apparently formed from the nitrobenzyl alcohols. The mono- and dinitrotoluenes are not significantly reduced to isolatable metabolites by mammalian enzymes in vivo; intestinal microflora reduce these compounds after biliary excretion of the nitrobenzyl glucuronides. The little available evidence suggests that this is not the case for trinitrotoluenes. Urinary metabolites retain the methyl group and bear one or two amino groups. This suggests either that mammalian enzymes are capable of reducing the nitro groups of trinitrotoluenes in vivo or that intestinal microflora gain access to unmodified trinitrotoluene. The nitropolycyclic aromatic hydrocarbons are apparently metabolized by both nitro reduction and ring oxidation. There is good evidence, at least for 1-nitropyrene and 6-nitrobenzo[a]pyrene, that nitro reduction occurs in intestinal microflora. the complexities of foreign compound metabolism are well illustrated by the biotransformation of the nitroaromatic compounds. Positional isomers are preferentially metabolized by markedly different pathways. Substitution to different degrees or with different functional groups greatly affects the types of metabolites formed. Yet these compounds also offer opportunities for understanding the mechanisms of foreign compound metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Nitrocompuestos/metabolismo , Animales , HumanosRESUMEN
Chloroperoxidase, horseradish peroxidase, hemoglobin, myoglobin, lactoperoxidase, and microperoxidase catalyzed the ethyl hydroperoxide-dependent oxidation of N-methylcarbazole to N-(hydroxymethyl)carbazole and N-formylcarbazole as major products. Mass spectral analysis of the N-(hydroxymethyl)carbazole formed during the peroxidase-catalyzed N-demethylation of N-methylcarbazole in 18O-enriched medium indicated partial incorporation (7.5-25.9%) of solvent water oxygen into the carbinolamine intermediate in all systems investigated, suggesting that the peroxidase active site is partially accessible to solvent water during N-demethylation. In contrast, solvent water oxygen was not incorporated into the N-formylcarbazole formed during the peroxidase-catalyzed oxidation of N-methylcarbazole. N-(Hydroxymethyl)carbazole was not further metabolized by the peroxidases in the presence of ethyl hydroperoxide, indicating that it is not an intermediate in N-formylcarbazole formation. The horseradish peroxidase-catalyzed formation of N-formylcarbazole was decreased by 77% when the hydroperoxide-supported reactions were carried out in a nitrogen atmosphere, while the formation of N-(hydroxymethyl)carbazole was decreased by 46%. When the horseradish peroxidase-catalyzed reactions were carried out in a 18O2 atmosphere, 18O incorporation into N-(hydroxymethyl)carbazole was 64.4% of the total oxygen, while 81.8% of the oxygen incorporated into N-formylcarbazole came from 18O2. These results suggest that there are two different mechanisms for the formation of N-(hydroxymethyl)carbazole, both involving the initial oxidation of N-methylcarbazole to a neutral carbon-centered radical. The radical can be further oxidized in the enzyme active site to an iminium cation, which reacts with water derived from either the oxidant or the medium to form the carbinolamine. Alternatively, the substrate radical can react with molecular oxygen to form a hydroperoxy radical, which decomposes to form the carboxaldehyde and carbinolamine.
Asunto(s)
Carbazoles/metabolismo , Peroxidasas/farmacología , Remoción de Radical Alquila , Radicales Libres , Espectrometría de Masas , Oxidación-Reducción , Isótopos de OxígenoRESUMEN
Pectin-induced changes in microflora have been shown to elevate the covalent binding of 2,6-dinitrotoluene (2,6-DNT)-related materials to total rat hepatic macromolecules. Therefore, the effect of diets varying in pectin content on the induction of foci and hepatic tumors induced by 2,6-DNT was studied in male F344 rats. 2,6-DNT (3.0-3.5 and 0.6-0.7 mg/kg/day) was incorporated into NIH-07 (NIH), an open formula cereal-based diet high in pectin content, AIN-76A (AIN), a purified pectin-free diet, or AIN-76A supplemented with 5% pectin (AP). Hepatic foci were scored after histochemical staining for gamma-glutamyl transpeptidase (GGT), canalicular adenosine triphosphatase or glucose-6-phosphatase following administration of test diets for 3, 6 and 12 months. The number of foci per cm3 of liver increased in a dose- and time-department manner following incorporation of 2,6-DNT into test diets with NIH greater than AP greater than AIN. In the NIH diet, 2,6-DNT did not alter the phenotypic distribution of foci. Animals fed control or 2,6-DNT-containing AIN and AP diets had few or no GGT foci throughout the study. Hepatocellular carcinomas and neoplastic nodules were observed only in rats fed NIH containing 2,6-DNT. The concentrations of 2,6-DNT-related material covalently bound to hepatic macromolecules after a single oral dose of radiolabeled 2,6-DNT given after 12 months on the diets increased in control rats and in rats receiving low dose 2,6-DNT in the diet with AIN less than AP less than NIH. These studies show that the carcinogenicity of 2,6-DNT differs depending on whether rats are fed an NIH or AIN (+/- pectin) diet. The results suggest that diet-induced alterations in the covalent binding of 2,6-DNT are not the sole factor in determining the carcinogenic response to 2,6-DNT. Furthermore, unidentified contaminants in cereal-based diets may influence foci and tumor production in rat liver during carcinogen treatment.