RESUMO
Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.
Assuntos
Argininossuccinato Sintase/antagonistas & inibidores , Ácidos Carboxílicos/farmacologia , Inibidores Enzimáticos/farmacologia , Fumonisinas , Animais , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Cinética , Fígado/enzimologia , Masculino , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/antagonistas & inibidoresRESUMO
HL-60 cells in culture were exposed for 2 h to a sinusoidal 0.1 or 1 mT (1 or 10 Gauss) magnetic field at 60 Hz and pulse labeled after exposure with radioactive isotopes by incubation by using either [(35)S]methionine, [(3)H]leucine, or [(33)P]phosphate. The radioactive labels were incorporated into cellular proteins through synthesis or phosphorylation. Proteins were extracted from electrostatically sorted nuclei, and the heat shock/stress proteins (sp) were analyzed for synthesis and phosphorylation by two-dimensional polyacrylamide gel electrophoresis. In the control cultures (no exposure to the magnetic field), sp 72c (cognate form) was faintly observed. A 0.1 mT exposure did not show sp metabolism to be different from that of the controls; however, after a 1 mT exposure of the HL-60 cells, sp 70i (inducible form) was synthesized ([(35)S]methionine incorporation). Sp 90 was not synthesized at either field level, but was phosphorylated ([(33)P]phosphate incorporation) in the 1 mT exposure. Sp 27 (isoforms a and b) was induced after a 1 mT exposure as reflected by labeling with [(3)H]leucine. These sps were not detected after a 0.1 mT exposure. After a 1 mT exposure and labeling with [(33)P], sp 27 isoforms b and c were phosphorylated whereas isoform 'a' was not observed. Sps 70i, 72c, and 90 were identified by commercial sp antibodies. Likewise, polypeptides a, b, and c were verified as sp 27 isoforms by Western blotting. Statistical evaluation of sp areas and densities, determined from fluorographs by Western-blot analysis, revealed a significant increase in sps 90 and 27a after a 1 mT magnetic field exposure. The 1 mT magnetic field interacts at the cellular level to induce a variety of sp species. Bioelectromagnetics 20:347-357, 1999. Published 1999 Wiley-Liss, Inc.
Assuntos
Campos Eletromagnéticos/efeitos adversos , Proteínas de Choque Térmico/biossíntese , Eletroforese em Gel Bidimensional , Células HL-60 , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/isolamento & purificação , Proteínas de Choque Térmico HSP72 , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico HSP90/isolamento & purificação , Proteínas de Choque Térmico/isolamento & purificação , Humanos , Imuno-Histoquímica , Chaperonas Moleculares , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/isolamento & purificaçãoRESUMO
The use of unfiltered quartz-halogen lamps exposes human skin to radiation that spans much of the ultraviolet (UV) spectrum. Reports indicate that exposure to quartz-halogen lamps is erythemogenic, mutagenic, and carcinogenic. To compare the carcinogenic potential of quartz-halogen lamps with that of other UV sources, we determined the dose dependence for cytotoxicity and neoplastic transformation in neonatal human fibroblasts exposed in vitro to: a 15 W germicidal lamp (primarily 254 nm radiation), a 15 W Cool White fluorescent lamp, and an unfiltered 20 W quartz-halogen lamp. Fluence-survival relationships were multiphasic with linear dose response below about 40% survival, and all three sources produced fluence-dependent transformation as indicated by induction of anchorage-independent growth. Maximum transformation frequencies were observed at fluences of 5-8 J/m2 for the germicidal lamp, 6.3 kJ/m2 for the fluorescent lamp, and 300 J/m2 for the quartz-halogen lamp. These data confirm the carcinogenic potential of the quartz-halogen lamp.
Assuntos
Transformação Celular Neoplásica/efeitos da radiação , Fibroblastos/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Morte Celular/efeitos da radiação , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Transformação Celular Neoplásica/patologia , Células Cultivadas , Relação Dose-Resposta à Radiação , Desenho de Equipamento , Eritema/etiologia , Fibroblastos/patologia , Humanos , Recém-Nascido , Mutagênese/efeitos da radiação , Neoplasias Induzidas por Radiação/etiologia , Neoplasias Induzidas por Radiação/patologiaRESUMO
Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are environmental contaminants and potential human airway carcinogens. Agents of this class show a wide range of potencies for toxicity, mutagenicity and carcinogenicity that are associated with the structure of the PAH and the position of the nitro group. In order to evaluate the effect of nitro substitution on in vitro biological activity, the cytotoxicity and transformation potential of two parent PAHs, pyrene and chrysene, and a series of nitro derivatives were examined in the rat tracheal epithelial (RTE) cell system. The nitro derivatives, but not pyrene or chrysene, produced dose-dependent decreases in the colony forming efficiency of the RTE cells. The most cytotoxic agents were 1,6-dinitropyrene and 6-nitrochrysene with ED(50)s of 1.6 mum and 5.9 mum, respectively, followed by 4-nitropyrene and 1-nitropyrene with ED(50)s of 26.3 mum and 44.5 mum, respectively. These compounds were evaluated for transformation potential at three treatment levels that spanned the cytotoxic range, and the assays were scored for morphologically transformed preneoplastic colonies. The control or spontaneous transformation frequency in this series of experiments was 1.79 +/- 0.47 (x 10(-4)). 6-Nitrochrysene and 1,6-dinitropyrene were the only compounds that produced transformation frequencies (12.17 x 10(-4) and 9.68 x 10(-4), respectively) that were statistically different from control. The maximum transformation frequencies of the compounds were compared with published data for liver tumorigenicity in the newborn mouse assay. The orders for tumorigenicity and transformation were the same (1,6-dinitropyrene > 4-nitropyrene > 1-nitropyrene approximately - pyrene and 6-nitrochrysene > chrysene), and the relative potencies of the compounds were similar in the two assays. These results suggest that RTE cells are capable of metabolizing nitro-PAHs to reactive products, and that, within this limited class of compounds, in vitro transformation data in the RTE cell system may be correlated with tumorigenicity in animal studies.
RESUMO
Using a recently developed enzyme-linked immunosorbent assay specific for 3-(cystein-S-yl)acetaminophen adducts we have quantitated the formation of these specific adducts in liver and serum protein of B6C3F1 male mice dosed with acetaminophen. Administration of acetaminophen at doses of 50, 100, 200, 300, 400 and 500 mg/kg to mice resulted in evidence of hepatotoxicity (increase in serum levels of alanine aminotransferase and aspartate aminotransferase) at 4 hr in the 300, 400 and 500 mg/kg treatment groups only. The formation of 3-(cystein-S-yl)acetaminophen adducts in liver protein was not observed in the groups receiving 50, 100 and 200 mg/kg doses, but was observed in the groups receiving doses above 300 mg/kg of acetaminophen. Greater levels of adduct formation were observed at the higher doses. 3-(Cystein-S-yl)acetaminophen protein adducts were also observed in serum of mice receiving hepatotoxic doses of acetaminophen. After a 400 mg/kg dose of acetaminophen, 3-(cystein-S-yl)acetaminophen adducts in the liver protein reached peak levels 2 hr after dosing. By 12 hr the levels decreased to approximately 10% of the peak level. In contrast, 3-(cystein-S-yl)acetaminophen adducts in serum protein were delayed, reaching a sustained peak 6 to 12 hr after dosing. The dose-response correlation between the appearance of serum aminotransferases and 3-(cystein-S-yl)acetaminophen adducts in serum protein and the temporal correlation between the decrease in 3-(cystein-S-yl)acetaminophen adducts in liver protein and the appearance of adducts in serum protein are consistent with a hepatic origin of the adducts detected in serum protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Acetaminofen/metabolismo , Proteínas Sanguíneas/metabolismo , Cisteína/metabolismo , Fígado/metabolismo , Acetaminofen/imunologia , Acetaminofen/toxicidade , Animais , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Fígado/efeitos dos fármacos , Masculino , Camundongos , Ligação Proteica , Fatores de TempoRESUMO
Male and female Fisher 344 rats (12 per group) were dosed by gavage with either 2 or 10 mg (based on the free amine) pyrilamine maleate containing about 12 and 6 muCi 14C-pyrilamine maleate, respectively, to determine excretion of the activity as a function of dose and sex with time. Urine and feces were collected at timed intervals through 144 h. Most of the dose (about 70%) was eliminated within 48 h through the urine and feces, but only about 80% of the total dose was recovered during the experiment. Less than 1% of the total dose remained in the rats at the end of the test period. In an additional experiment to determine the location of the remainder of the dose (about 20%), male rats were dosed with 2 mg pyrilamine maleate containing 14C-pyrilamine maleate. After 144 h, exhaustive washing of the cages resulted in recovery of approximately 20% of the dose, thus identifying its location. There were no significant sex or dose related differences observed in the total amount of 14C that was eliminated through the urine or feces and recovered. Urine and feces are the major routes of elimination of pyrilamine maleate in the Fischer 344 rat. The urinary route of elimination was more predominant than the fecal route in both sexes at either dose.
Assuntos
Aminopiridinas/metabolismo , Pirilamina/metabolismo , Animais , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Fezes/análise , Feminino , Masculino , Pirilamina/urina , Ratos , Ratos Endogâmicos F344RESUMO
Adult bullfrogs were collected along the Rockey Branch Creek in Arkansas. This area is known to be contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). The levels of 2,3,7,8-TCDD found in livers from these bullfrogs ranged from 0.64 to 48 ppb. These results suggest the utility of bullfrogs as a biological marker for 2,3,7,8-TCDD in the environment.
Assuntos
Dioxinas/análise , Monitoramento Ambiental/métodos , Dibenzodioxinas Policloradas/análise , Animais , Rana catesbeianaRESUMO
Experiments were conducted with male and female rats (12 per group) dosed by gavage with 2 or 20 mg (based on the free amine) doxylamine succinate containing about 10 microCi 14C-doxylamine succinate to determine distribution and excretion of the activity as a function of dose and sex with time. Urine and feces were collected at intervals up to 72 hr. Most of the dose (approximately equal to 70%) was eliminated in the first 24 hr after dosing and 95 to 100% of the dose was recovered during the 72-hr course of the experiments with both sexes and dose levels. Less than 1% of the total dose remained in the rats at the end of the test period. The urinary route of elimination was more predominant than the fecal route in both sexes given the 20-mg dose. The fecal route predominates in low-dose males whereas there is no significant difference between urinary and fecal routes of elimination in low-dose females. Preliminary characterization of urinary metabolite form using extraction techniques shows 99% of the metabolites to be in the polar conjugated form.
Assuntos
Doxilamina/metabolismo , Piridinas/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Doxilamina/análogos & derivados , Doxilamina/urina , Fezes/análise , Feminino , Masculino , Ratos , Ratos Endogâmicos F344 , Distribuição TecidualRESUMO
Absorption, metabolism and tissue distribution studies were conducted in the rat with 14C-biphenyl ring-labeled Direct Blue 15, a 3,3'-dimethoxybenzidine (DiMxBzd) based azo dye; Direct Red 2, based on 3,3'-dimethylbenzidine (DiMeBzd) and corresponding benzidine congener amines. Single oral doses of the 14C-labeled dyes (12 mg/kg, 62 microCi/kg) and molar equivalent doses of the respective amines were administered and urine and fecal samples collected at intervals up to 192 hours. Urine specimens were analyzed for 14C content and further characterized by EC/GC for free amines, acetylated metabolites, and conjugates. Feces were assayed for 14C content and for unchanged dosed dyes or amines by HPLC. A comparison of the metabolism of Direct Blue 15 with its base DiMxBzd, indicated that the base was more extensively metabolized and that most of the 14C in various extracts was identified as known metabolites. The metabolism of Direct Red 2 compared with its base, DiMeBzd, indicated that the base was more extensively metabolized, yet only a small percentage of the 14C in extracts was identified as known metabolites. Most of the 14C present in the urine could not be extracted with benzene nor chloroform, indicating high polarity. Distribution studies conducted with both dyes showed that liver, kidney, and lung accumulated and retained higher levels of 14C than other tissues (at 72 hrs). Peak levels of 14C, which occurred 8-12 hours after dosing, were significantly higher with Direct Red 2 than Direct Blue 15. Tissue distribution data (72 hr) for rats dosed with the free amines compared with the dyes showed a generally lower but similar distribution pattern.
Assuntos
Compostos Azo/metabolismo , Corantes/metabolismo , Naftalenossulfonatos/metabolismo , Absorção , Animais , Radioisótopos de Carbono , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Masculino , Ratos , Ratos Endogâmicos F344 , Distribuição TecidualRESUMO
This study demonstrated the utility of negative ion atmospheric pressure ionization mass spectrometry for the routine quantitation of 2,4,5-trichlorophenoxyacetic acid and its glycine and taurine amide metabolites in mouse blood, urine and feces samples. The quantitation of 2,4,5-trichlorophenoxyacetic acid in blood follows a short cleanup procedure and used 2,4,5-trichlorophenoxy-13C6-acetic acid as the stable label isotope diluent. A more extensive cleanup procedure utilizing high-pressure liquid chromatography was required for the determination of 2,4,5-trichlorophenoxyacetic acid and its two metabolites in urine and feces. The glycine amide metabolite was quantitated by the 13C stable isotope diluent method. The taurine amide relied on an initial separation step followed by using a second 2,4,5-trichlorophenoxy-13C6-acetic acid spike fot its isotope diluent. Alkaline hydrolysis of the metabolites, followed by methylation, allowed the methyl ester of 2,4,5-trichlorophenoxyacetic acid to be solely used as the analyte in the negative ion atmospheric pressure ionization mass spectrometry quantitation step.