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1.
Cell Biol Toxicol ; 24(1): 87-99, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17549590

RESUMO

In a number of adverse drug reactions leading to hepatotoxicity drug metabolism is thought to be involved by generation of reactive metabolites from nontoxic drugs. In this study, an in vitro assay was developed for measurement of the impact of metabolic activation of compound on the cytotoxicity toward a human hepatic cell line. HepG2 cells were treated for 6 h with compound in the presence or absence of rat liver S9-mix, and the viability was measured using the MTT test. The cytotoxicity of cyclophosphamide was substantially increased by S9-mix in the presence of NADPH. Three NADPH sources were tested: NADPH (1 mmol/L) or NADPH regenerating system with either NADP(+)/glucose 6-phosphate (G6P) or NADP(+)/isocitrate. All three NADPH sources increased the cytotoxicity of cyclophosphamide to a similar extent. Eight test compounds known to cause hepatotoxicity were tested. For these, only the cytotoxicity of diclofenac was increased by S9 enzymes when an NADPH regenerating system was used. The increased toxicity was NADPH dependent. Reactive drug metabolites of diclofenac, formed by NADPH-dependent metabolism, were identified by LC-MS. Furthermore, an increase in toxicity, not related to enzymatic activity but to G6P, was observed for diclofenac and minocycline. Tacrine and amodiaquine displayed decreased toxicity with S9-mix, and carbamazepine, phenytoin, bromfenac and troglitazone were nontoxic at all tested concentrations, with or without S9-mix. The results show that this method, with measurement of the cytotoxicity of a compound in the presence of an extracellular metabolizing system, may be useful in the study of cytotoxicity of drug metabolites.


Assuntos
Bioensaio/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fígado/metabolismo , Animais , Biotransformação/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Ciclofosfamida/metabolismo , Ciclofosfamida/toxicidade , Diclofenaco/química , Diclofenaco/metabolismo , Diclofenaco/toxicidade , Glucose-6-Fosfato/metabolismo , Humanos , Fígado/efeitos dos fármacos , Minociclina/metabolismo , Minociclina/toxicidade , NADP/metabolismo , Ratos
2.
Int J Biochem Cell Biol ; 32(10): 1055-68, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11091138

RESUMO

N(1)-(n-octanesulfonyl)spermine (N(1)OSSpm) is a potent calmodulin antagonist. In the present work, its toxicity to DHD/K12/TRb and CaCo-2 cells, two colon carcinoma-derived cell lines, was studied with the aim to identify those properties of the cells, which determine their sensitivity to N(1)OSSpm and related structures. Exposure of the cells to MDL 72527, a compound considered to be a selective inactivator of polyamine oxidase (PAO) increased the cytotoxicity of N(1)OSSpm to both cell lines. In contrast, toxicity of trifluoperazine, a calmodulin antagonist with a polyamine-unrelated structure, was not enhanced by MDL 72527. Combined exposure of cells to 2-(difluoromethyl)ornithine (DFMO) (a selective inactivator of ornithine decarboxylase), MDL 72527 and N(1)OSSpm produced a synergistic cytotoxic effect. Neither the intrinsic PAO activity of the cells (as determined with N(1), N(12)-diacetylspermine as substrate), nor their ability to accumulate the drug was a determinant of the cytotoxic effect of N(1)OSSpm. These data suggest that MDL 72527 has a target unrelated to PAO, which is responsible for the enhancement of N(1)OSSpm (and spermine) toxicity. Identification of this target may be of use if the therapeutic potentials of MDL 72527 are to be exploited.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Putrescina/análogos & derivados , Espermina/análogos & derivados , Sulfonamidas/toxicidade , Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Células CACO-2 , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sinergismo Farmacológico , Eflornitina/farmacologia , Guanidinas/farmacologia , Humanos , Estrutura Molecular , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Poliaminas/análise , Poliaminas/metabolismo , Putrescina/química , Putrescina/farmacologia , Putrescina/uso terapêutico , Ratos , Espermina/toxicidade , Trifluoperazina/toxicidade , Células Tumorais Cultivadas , Poliamina Oxidase
3.
Cancer Lett ; 158(1): 85-91, 2000 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-10940513

RESUMO

Resveratrol, a natural polyphenolic phytoalexine present in grapes and wines, has been reported to exert a variety of important pharmacological effects. We investigated the effects of resveratrol on the growth and polyamine metabolism of CaCo-2 human colon cancer cells. Treatment of the CaCo-2 cells with 25 microM resveratrol caused a 70% growth inhibition. The cells accumulated at the S/G2 phase transition of the cell cycle. No signs of cytotoxicity or apoptosis were detected. Resveratrol caused a significant decrease of ornithine decarboxylase (ODC) activity, a key enzyme of polyamine biosynthesis, which is enhanced in cancer growth. ODC inhibition resulted in the reduction of the intracellular putrescine content, indicating that polyamines might represent one of several targets involved in the anti-proliferative effects of resveratrol.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Rosales/química , Estilbenos/farmacologia , Vinho/análise , Células CACO-2 , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Poliaminas/metabolismo , Resveratrol
4.
Talanta ; 48(1): 127-34, 1999 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18967451

RESUMO

Since the induction of putrescine synthesis by ornithine decarboxylase (ODC) is observed in many pathological and physiological processes, a useful and simple method to assay this enzyme activity should be an interesting tool to quantify the biological importance of its induction. An enzymatic method to assay ODC is reported here. This method is based on the reaction between putrescine and soya diamine oxidase. The reaction releases H(2)O(2), which is measured by a colorimetric method. The validation of this method showed good accuracy (98+/-5% of recovery). High precision and reproducibility were obtained. A linearity with a correlation coefficient of 0.999 in the range of 2.5-25 nmol was obtained. This method is also rugged and specific. The application of the assay of ODC activity showed that it is useful as a rapid and simple tool for assaying ODC activity in vitro. Comparison with the HPLC determination of ODC activity shows strong correlation along with the high accuracy of the two methods.

5.
Eur J Pharmacol ; 342(2-3): R1-R2, 1998 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-9548411

RESUMO

Ifenprodil (NMDA receptor antagonist) was tested as an inhibitor of ornithine decarboxylase. It was found that ifenprodil inhibited ornithine decarboxylase activity with the same potency as alpha-difluoromethylornithine, a major inhibitor of ornithine decarboxylase. This result suggests that ifenprodil could target either the polyamine site on the NMDA receptor complex or/and polyamine biosynthesis.


Assuntos
Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Inibidores da Ornitina Descarboxilase , Piperidinas/farmacologia , Poliaminas Biogênicas/metabolismo , Eflornitina/farmacologia , Escherichia coli/enzimologia , Putrescina/análogos & derivados , Putrescina/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
6.
Pharmazie ; 53(1): 15-9, 1998 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9476255

RESUMO

In order to regulate simultaneously the biosynthesis and the transport of natural polyamines, the synthesis of a series of N-methylated analogues of N,N1-Bis(benzyl)-alkanediamines (propanediamine and butanediamine) was achieved and the cytotoxicity of these compounds on the P388D1 cell line was determined. Experiments were conducted in a growth culture medium 20 microM of 2-mercaptoethanol or 0.1 mM of aminoguanidine. Their cytotoxic effects were compared to those obtained under the same conditions with natural polyamines known as toxic compounds at high concentrations. The IC50 of each compound was found very similar for all experimental conditions (IC50 approximately 150 microM) at the opposite of spermidine and spermine which were less toxic (IC50 > 500 microM) when cells were grown in the presence of aminoguanidine (a specific inhibitor of fetal calf serum's PAO). The DL-difluoromethylornithine (DFMO) and MDL 72527DA, two well known inhibitors of ornithine decarboxylase (ODC) and Polyamine Oxidase (PAO) respectively, had no toxicity on the P388D1 cells compared to our compounds. Our most toxic compound was N1,N4-Bis(benzyl)-N1,N4-bis(methyl)-1,4-butanediamine (6) with an IC50 of 127 +/- 3 microM (in culture medium alone). The synthesis of the beta-aminothioether derivative of N-benzylputrescine (11) and the beta-aminothiol derivative of N-benzylspermidine (13) were also related. The Compound 11 was tested against the P388D1 cells, and did not show any cytotoxic effect. The N-methyl derivatives should give the advantage to be used at low concentrations than those used to test the DFMO.


Assuntos
Antineoplásicos/síntese química , Poliaminas/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/toxicidade , Leucemia P388/tratamento farmacológico , Espectroscopia de Ressonância Magnética , Camundongos , Poliaminas/química , Poliaminas/toxicidade
7.
Cell Biol Toxicol ; 14(6): 419-28, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9879934

RESUMO

The effects of a series of bisbenzyldiamine analogs have been tested on P388D1 cell line in vitro. Their effects on cell growth, polyamine oxidase (PAO) activity and intracellular polyamine content were determined. The cytotoxicity tests were performed in culture medium supplemented with 100 micromol/L aminoguanidine (I), 100 micromol/L aminoguanidine and 100 micromol/L N,N'-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527) (II), and finally 100 micromol/L aminoguanidine and 200 micromol/L D,L-difluoromethylornithine (DFMO) (III). The IC50 values under conditions I and III were similar, suggesting that inhibition of ornithine decarboxylase by DFMO did not affect the biological effect of our derivatives. Spermine and spermidine remained nontoxic in conditions I and III. However in the condition II, the toxicity of all tested compounds (excepted spermidine) was increased, suggesting that the inhibition of cellular PAO increased their toxicity. The enzymatic test of PAO showed that at high doses inhibition of this enzyme by putrescine analogs occurred, while the N-methylated propanediamine derivative increased the enzyme activity; however, these results do not correlate with cytotoxicity tests. When these derivatives were incubated for 48 h with the cells, all of them increased the cell content in putrescine (approximately 160%) and spermine (approximately 145%) and decreased the spermidine content (approximately 75%) without any modification of the total amount of polyamine. The correlation between the cytotoxic results and the intracellular polyamine determination shows that the increase in spermine content along with the inhibition of retroconverting PAO enzyme increases the toxic effect of tested compounds (including spermine), suggesting that spermine toxicity is more important in the absence of intracellular oxidation processes.


Assuntos
Leucemia P388/metabolismo , Poliaminas/metabolismo , Propilaminas/farmacologia , Putrescina/análogos & derivados , Animais , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Leucemia P388/enzimologia , Leucemia P388/patologia , Camundongos , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Propilaminas/química , Putrescina/farmacologia , Células Tumorais Cultivadas , Poliamina Oxidase
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