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1.
BMC Genomics ; 11: 9, 2010 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-20053287

RESUMO

BACKGROUND: Non-genotoxic carcinogens are notoriously difficult to identify as they do not damage DNA directly and have diverse modes of action, necessitating long term in vivo studies. The early effects of the classic rodent non-genotoxic hepatocarcinogen phenobarbital have been investigated in the Fisher rat using a combination of metabolomics and transcriptomics, to investige early stage mechanistic changes that are predictive of longer term pathology. RESULTS: Liver and blood plasma were profiled across 14 days, and multivariate statistics used to identify perturbed pathways. Both metabolomics and transcriptomics detected changes in the liver which were dose dependent, even after one day of exposure. Integration of the two datasets associated perturbations with specific pathways. Hepatic glycogen was decreased due to a decrease in synthesis, and plasma triglycerides were decreased due to an increase in fatty acid uptake by the liver. Hepatic succinate was increased and this was associated with increased heme biosynthesis. Glutathione synthesis was also increased, presumably in response to oxidative stress. Liquid Chromatography Mass Spectrometry demonstrated a remodeling of lipid species, possibly resulting from proliferation of the smooth endoplasmic reticulum. CONCLUSIONS: The data fusion of metabolomic and transcriptomic changes proved to be a highly sensitive approach for monitoring early stage changes in altered hepatic metabolism, oxidative stress and cytochrome P450 induction simultaneously. This approach is particularly useful in interpreting changes in metabolites such as succinate which are hubs of metabolism.


Assuntos
Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Metaboloma , Fenobarbital/toxicidade , Animais , Análise por Conglomerados , Sistema Enzimático do Citocromo P-450/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Fígado/metabolismo , Fígado/patologia , Espectroscopia de Ressonância Magnética , Masculino , Análise Multivariada , Estresse Oxidativo , Plasma/efeitos dos fármacos , Plasma/metabolismo , Análise de Componente Principal , Ratos , Ratos Endogâmicos F344
2.
Chem Biol Interact ; 166(1-3): 323-31, 2007 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-16870169

RESUMO

Chloroprene (2-chloro-1,3-butadiene, 1) is oxidised by cytochrome P450 enzymes in mammalian liver microsomes to several metabolites, some of which are reactive towards DNA and are mutagenic. Much less of the metabolite (1-chloroethenyl)oxirane (2a/2b) was formed by human liver microsomes compared with microsomes from Sprague-Dawley rats and B6C3F1 mice. Epoxide (2a/2b) was a substrate for mammalian microsomal epoxide hydrolases, which showed preferential hydrolysis of the (S)-enantiomer (2b). The metabolite 2-chloro-2-ethenyloxirane (3a/3b) was rapidly hydrolysed to 1-hydroxybut-3-en-2-one (4) and in competing processes rearranged to 1-chlorobut-3-en-2-one (5) and 2-chlorobut-3-en-1-al (6). The latter compound isomerised to (Z)-2-chlorobut-2-en-1-al (7). In microsomal preparations from human, rat and mouse liver, compounds 4, 5 and 7 were conjugated by glutathione both in the absence and presence of glutathione transferases. There was no evidence for the formation of a chloroprene diepoxide metabolite in any of the microsomal systems. The major adducts from the reaction of (1-chloroethenyl)oxirane (2a/2b) with calf thymus DNA were identified as N7-(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (20) and N3-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyuridine (23), with the latter being derived by alkylation at N-3 of 2'-deoxycytidine, followed by deamination. Adducts in DNA were identified by comparison with those derived from individual deoxyribonucleosides. The metabolite (Z)-2-chlorobut-2-en-1-al (7) formed principally two adducts with 2'-deoxyadenosine which were identified as a pair of diastereoisomers of 3-(2'-deoxy-beta-d-ribofuranosyl)-7-(1-hydroxyethyl)-3H-imidazo[2,1-i]purine (25). The chlorine atom of chloroprene thus leads to different intoxication and detoxication profiles compared with those for butadiene and isoprene. The results infer that in vivo oxidations of chloroprene catalysed by cytochrome P450 are more important in rodents, whereas hydrolytic processes catalysed by epoxide hydrolases are more pronounced in humans. The reactivity of chloroprene metabolites towards DNA is important for the toxicology of chloroprene, especially when detoxication is incomplete.


Assuntos
Cloropreno/metabolismo , Cloropreno/toxicidade , Animais , Cloropreno/química , DNA/metabolismo , Adutos de DNA/metabolismo , Epóxido Hidrolases/metabolismo , Óxido de Etileno/metabolismo , Glutationa/metabolismo , Humanos , Hidrólise/efeitos dos fármacos , Inativação Metabólica , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Nucleosídeos/química , Nucleosídeos/metabolismo , Oxirredução/efeitos dos fármacos , Ratos
3.
Appl Environ Microbiol ; 69(6): 3650-2, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12788775

RESUMO

4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1 acts by dehydrogenation of its substrate to give a quinone methide, which is then hydrated to an alcohol. It was shown to be active with a range of 4-alkylphenols as substrates. 4-n-propylphenol, 4-n-butylphenol, chavicol, and 4-hydroxydiphenylmethane were hydroxylated on the methylene group next to the benzene ring and produced the corresponding chiral alcohol as the major product. The alcohols 1-(4'-hydroxyphenyl)propanol and 1-(4'-hydroxyphenyl)-2-propen-1-ol, produced by the biotransformation of 4-n-propylphenol and chavicol, respectively, were shown to be R(+) enantiomers. 5-Indanol, 6-hydroxytetralin, 4-isopropylphenol, and cyclohexylphenol, with cyclic or branched alkyl groups, gave the corresponding vinyl compounds as their major products.


Assuntos
Oxigenases de Função Mista/metabolismo , Fenóis/metabolismo , Pseudomonas putida/enzimologia , Cromatografia Gasosa-Espectrometria de Massas , Fenóis/química , Estereoisomerismo , Especificidade por Substrato
4.
Chem Res Toxicol ; 15(12): 1549-60, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12482237

RESUMO

(1-Chloroethenyl)oxirane is a major mutagenic metabolite of chloroprene, an important large-scale petrochemical used in the manufacture of synthetic rubbers. The reactions of (1-chloroethenyl)oxirane with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The adducts from the nucleosides were isolated by reversed-phase HPLC, and characterized by their UV absorbance and (1)H and (13)C NMR spectroscopic and mass spectrometric features. The reaction with 2'-deoxyguanosine gave one major adduct, N7-(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGI), and eight minor adducts which were identified as diastereoisomeric pairs of N1-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyguanosine (dGII, dGIII), N3,N7-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGIV, dGV), N7,N9-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGVI, dGVII), and N1,N7-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGVIII, dGIX). The reaction of 2'-deoxyadenosine with (1-chloroethenyl)oxirane gave two adducts: N1-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (dAI) and N(6)-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (dAII). The adduct dAII was shown to arise via a Dimroth rearrangement of adduct dAI. The HPLC analyses of the reaction mixtures of (1-chloroethenyl)oxirane with 2'-deoxycytidine and thymidine showed the formation of one major product in each reaction. The adduct from 2'-deoxycytidine was identified as N3-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyuridine (dCI) derived by alkylation at N-3 followed by deamination. The adduct from thymidine was identified as N3-(3-chloro-2-hydroxy-3-buten-1-yl)-thymidine (TI). Reaction of (1-chloroethenyl)oxirane with calf thymus DNA gave all of the adducts observed from the individual nucleosides except dGII and dGIII. However, there was selectivity for the formation of dGI and dCI. The adduct levels in DNA were 9,630 (dGI), 240 (dCI), 83 (dAI), 6 (dAII), and 28 (TI) pmol/mg DNA, respectively. The preferred formation of dCI may be relevant to chloroprene mutagenesis.


Assuntos
Adutos de DNA/isolamento & purificação , Adutos de DNA/metabolismo , DNA/metabolismo , Desoxirribonucleosídeos/metabolismo , Óxido de Etileno/análogos & derivados , Óxido de Etileno/metabolismo , Mutagênicos/metabolismo , Alquilação , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , DNA/química , Adutos de DNA/química , Desaminação , Desoxirribonucleosídeos/química , Óxido de Etileno/química , Mutagênicos/química , Mutagênicos/isolamento & purificação , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrofotometria Ultravioleta , Estereoisomerismo
5.
Chem Res Toxicol ; 16(10): 1287-97, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14565770

RESUMO

Chloroprene (2-chloro-1,3-butadiene, 1) is an important industrial chemical, which is carcinogenic in experimental animals and possibly in humans. It is metabolized to the monoepoxides, 2-chloro-2-ethenyloxirane (2a,b) and (1-chloroethenyl)oxirane (3a,b), together with electrophilic chlorinated aldehydes and ketones. This study has investigated the detoxication of these chloroprene metabolites in vitro by glutathione (GSH) and epoxide hydrolase (EH) in liver microsomes from Sprague-Dawley rats, B6C3F1 mice, and humans. In incubations of chloroprene with liver microsomes containing GSH, several GSH conjugates were identified. These were 1-hydroxy-4-(S-glutathionyl)butan-2-one (13), 1,4-bis-(S-glutathionyl)butan-2-one (15), and (Z)-2-(S-glutathionyl)but-2-en-1-al (16). A fourth GSH conjugate was identified as either 2-chloro-3-hydroxy-4-(S-glutathionyl)butene (12a,b) or 1-chloro-4-(S-glutathionyl)-butan-2-one (14), which were indistinguishable by LC/MS. Structural assignments of metabolites were based on chromatographic and spectroscopic comparisons with synthetic standards. There were significant differences between species in the amounts of 3a,b formed in microsomal incubations, the order being mouse > rat > human. Hydrolysis by microsomal EHs showed a distinct selectivity for S-(1-chloroethenyl)oxirane (3b) resulting in an accumulation of the R-enantiomer; the ratio of the amounts between species was 20:4:1 for mouse:rat:human, respectively.


Assuntos
Cloropreno/metabolismo , Cloropreno/toxicidade , Epóxido Hidrolases/metabolismo , Glutationa/metabolismo , Animais , Cloropreno/química , Cromatografia Líquida de Alta Pressão , Compostos de Epóxi/metabolismo , Humanos , Hidrólise , Inativação Metabólica , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Oxirredução , Ratos , Estereoisomerismo
6.
Chem Res Toxicol ; 16(7): 933-44, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12870896

RESUMO

Isoprene (2-methylbuta-1,3-diene) is a large scale petrochemical used principally in the manufacture of synthetic rubbers. It is also produced by plants and trees and is formed endogenously in mammals as a major endogenous hydrocarbon. Mammalian metabolism of isoprene involves cytochrome P450-dependent monooxygenases to give the regioisomeric monoepoxides, prop-2-enyloxirane and 2-ethenyl-2-methyloxirane. The isoprene monoepoxides are further oxidized to the mutagenic diepoxides, 2-methyl-2,2'-bioxiranes. The present studies have investigated the stereochemistry and comparative rates of the metabolic epoxidation in vitro of isoprene to mono- and diepoxides by liver microsomes from rat, mouse, and human in order to identify stereochemical and kinetic differences between species in the formation of these epoxide metabolites, which are key to understanding the toxicology of isoprene. The assignments of stereochemistry were based on comparisons with synthetic standards, the syntheses for which are described. Comparative enzyme kinetic parameters (apparent K(m) and apparent V(max) values) for the in vitro formation of all of the monoepoxide and diepoxide stereoisomers have been obtained. The rates of formation of both mono- and diepoxides were greater in the rodent systems as compared with the human in vitro system. The results provide comparative kinetic data that have potential for modeling and assessing the relevance of the animal carcinogenicity data for man. The possibility of human interindividual variation was also investigated with liver preparations from several individual humans, but significant differences between individuals were not observed in the formation of the monoepoxides from isoprene.


Assuntos
Butadienos/metabolismo , Compostos de Epóxi/metabolismo , Hemiterpenos/metabolismo , Microssomos Hepáticos/metabolismo , Pentanos/metabolismo , Animais , Butadienos/química , Butadienos/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Compostos de Epóxi/análise , Compostos de Epóxi/química , Hemiterpenos/química , Hemiterpenos/toxicidade , Humanos , Técnicas In Vitro , Cinética , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Oxirredução , Pentanos/química , Pentanos/toxicidade , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Estereoisomerismo
7.
Arch Toxicol ; 78(7): 410-7, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15141287

RESUMO

Male F344 rats exposed to bromodichloromethane (BDCM) by gavage at 50 or 100 mg/kg/day for 5 days a week for 28 days excreted large amounts of formic acid in their urine, which was accompanied by a change in urinary pH. Male B6C3F1 mice exposed to BDCM at 25 or 50 mg/kg/day for 5 days a week for 28 days also excreted increased amounts of formic acid in their urine. In rats, formate excretion was dose and time dependant, being markedly elevated after four doses and remaining at that level after 3 weeks of dosing at 100 mg/kg/day BDCM, while at 50 mg/kg/day there was some suggestion of a decline after 3 weeks. In contrast, in mice formate excretion did not start to a major extent until 3 weeks of dosing, with the biggest response at 4 weeks. There was no increase in clinical chemistry markers of liver or kidney injury in either rats or mice following 28-day exposure to BDCM. However, morphological examination of the kidneys showed some mild renal tubule injury in two out of five rats exposed to 100 mg/kg/day BDCM. This was associated with a marked increase in cell proliferation in the renal cortex of all rats exposed to 100 mg/kg/day. No increase in cell proliferation was seen in the renal cortex of rats exposed to BDCM at 50 mg/kg/day, or in mice exposed to 25 or 50 mg/kg/day BDCM for 28 days. Long-term exposure to formic acid is known to cause kidney damage, suggesting that excretion of this acid may be a contributory factor to the increase in cell proliferation and kidney damage seen in the longer-term studies with BDCM.


Assuntos
Formiatos/urina , Túbulos Renais/efeitos dos fármacos , Trialometanos/toxicidade , Poluentes Químicos da Água/toxicidade , Administração Oral , Animais , Peso Corporal/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Creatinina/metabolismo , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Córtex Renal/efeitos dos fármacos , Córtex Renal/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Ratos , Ratos Endogâmicos F344 , Especificidade da Espécie , Fatores de Tempo , Trialometanos/administração & dosagem , Poluentes Químicos da Água/administração & dosagem
8.
Biomarkers ; 8(6): 472-90, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-15195679

RESUMO

N-Phenylanthranilic acid (NPAA) causes renal papillary necrosis (RPN) in the rat following repeated oral dosing. Non-invasive early detection of RPN is difficult, but a number of potential biomarkers have been investigated, including phospholipid and uronic acid excretion. This study used 1H-nuclear magnetic resonance (NMR) spectroscopic analysis of urine to investigate urinary metabolic perturbations occurring in the rat following exposure to NPAA. Male Alderley Park rats received NPAA (300, 500 or 700 mg kg(-1) day(-1) orally) for 7 days, and urine was collected on days 7-8, 14-15, 21-22 and 28-29. In a separate study, urine was collected on days 1-2, 3-4, 5-6 and 7-8 from rats receiving 500 mg kg(-1) day(-1). Samples were analysed by 1H NMR spectroscopy combined with multivariate data analysis and clinical chemistry. Histopathology and clinical chemistry analysis of terminal blood samples was carried out following termination on days 4, 6, 8 and 29 (4 week time course) and days 2, 4, 6 and 8 (8 day study). Urine analysis revealed a marked, though variable, excretion of beta-hydroxybutyrate, acetoacetate and acetone (ketone bodies) seen on days 3-4, 5-6 and 7-8 of the study. It is postulated that the ketonuria might be secondary to an alteration in fatty acid metabolism due to inhibition of prostaglandin synthesis. In addition, an elevation in urinary ascorbate was observed during the first 8 days of the study. Ascorbate is considered to be a biomarker of hepatic response, probably reflecting an increased hepatic activity due to glucuronidation of NPAA.


Assuntos
Necrose Papilar Renal/diagnóstico , Ressonância Magnética Nuclear Biomolecular/métodos , ortoaminobenzoatos/efeitos adversos , Animais , Ácido Ascórbico/urina , Biomarcadores/urina , Peso Corporal , Relação Dose-Resposta a Droga , Corpos Cetônicos/urina , Necrose Papilar Renal/induzido quimicamente , Necrose Papilar Renal/urina , Masculino , Ratos , Ratos Endogâmicos , Fatores de Tempo , Urinálise/métodos
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