RESUMO
Antiestrogen resistance of breast cancer has been related to enhanced growth factor receptor expression and activation. We have previously shown that ectopic expression and subsequent activation of the insulin-like growth factor-1 receptor (IGF1R) or the epidermal growth factor receptor (EGFR) in MCF7 or T47D breast cancer cells results in antiestrogen resistance. In order to identify novel therapeutic targets to prevent this antiestrogen resistance, we performed kinase inhibitor screens with 273 different inhibitors in MCF7 cells overexpressing IGF1R or EGFR. Kinase inhibitors that antagonized antiestrogen resistance but are not directly involved in IGF1R or EGFR signaling were prioritized for further analyses. Various ALK (anaplastic lymphoma receptor tyrosine kinase) inhibitors inhibited cell proliferation in IGF1R expressing cells under normal and antiestrogen resistance conditions by preventing IGF1R activation and subsequent downstream signaling; the ALK inhibitors did not affect EGFR signaling. On the other hand, MEK (mitogen-activated protein kinase kinase)1/2 inhibitors, including PD0325901, selumetinib, trametinib and TAK-733, selectively antagonized IGF1R signaling-mediated antiestrogen resistance but did not affect cell proliferation under normal growth conditions. RNAseq analysis revealed that MEK inhibitors PD0325901 and selumetinib drastically altered cell cycle progression and cell migration networks under IGF1R signaling-mediated antiestrogen resistance. In a group of 219 patients with metastasized ER + breast cancer, strong pMEK staining showed a significant correlation with no clinical benefit of first-line tamoxifen treatment. We propose a critical role for MEK activation in IGF1R signaling-mediated antiestrogen resistance and anticipate that dual-targeted therapy with a MEK inhibitor and antiestrogen could improve treatment outcome.
Assuntos
Neoplasias da Mama , Moduladores de Receptor Estrogênico , Quinase do Linfoma Anaplásico , Benzamidas , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Difenilamina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB , Antagonistas de Estrogênios/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Moduladores de Receptor Estrogênico/uso terapêutico , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Fator de Crescimento Insulin-Like I , Quinases de Proteína Quinase Ativadas por Mitógeno , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptor IGF Tipo 1 , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêuticoRESUMO
We recently developed two biomarker sets for oxidative damage: one for determination of lipid peroxidation (LPO) degradation products; acetaldehyde, propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal, malondialdehyde and acetone, by a gas chromatography-electron capture detection method, and the other for protein oxidation products such as o,o'-dityrosine, by an isotope dilution high performance liquid chromatography-tandem mass spectrometry method. In the present study, we explored the possibility to utilize these biomarkers for determining the oxidative damage in liver mammalian cells in vitro. Two different treatments were chosen for inducing oxidative stress in Chinese Hamster ovary cells: menadione and copper plus hydrogen peroxide (Cu2+/H2O2). Cells were incubated with the model compounds in the presence or absence of vitamin E and C, and cytotoxicity was evaluated by a nuclear-dye method. Results were compared to two fluorescent probes, H2DCF-DA and C11 -BODIPY581/591, which have been used for determining the formation of free radicals in the cells. From ten LPO degradation products, eight were increased significantly following incubation with menadione in cell lysate or incubation media. Menadione-induced oxidative stress was also confirmed by oxidation of fluorescent probes. However, no increased formation of protein oxidation products was observed. Vitamin E and C did not diminish the formation of LPO degradation products that were increased by menadione. Although Cu2+/H2O2 did not induce oxidation of fluorescent probes, it induced formation of six out of ten LPO degradation products. Vitamin E and C did not diminish the formation of LPO degradation products; vitamin C even substantially increased the formation of acetaldehyde and propanal, which is in line with its reported prooxidant action under certain conditions. Vitamin C also caused two-fold increase in Cu2+/H2O2-induced o,o'-dityrosine formation when applied simultaneously. In conclusion, our present results show that the LPO biomarker set can be used for evaluation of oxidant capacity and the toxic potential of various chemicals in an in vitro cell model. These biomarkers might even be more sensitive than measuring protein oxidation products or oxidation of fluorescent probes.
Assuntos
Compostos de Boro , Fluoresceínas , Corantes Fluorescentes , Peroxidação de Lipídeos , Proteínas/metabolismo , Animais , Ácido Ascórbico/farmacologia , Biomarcadores , Células CHO , Sobrevivência Celular , Cricetinae , Malondialdeído/análise , Oxirredução , Tirosina/análogos & derivados , Tirosina/metabolismo , Vitamina E/farmacologiaRESUMO
We have carried out molecular dynamics (MD) simulations and free energy calculations on the alpha-subtype of the human estrogen receptor ligand-binding domain (ERalpha LBD) complexed with a number of known agonists and putative xenoestrogens. Our dynamical simulations of ligand-receptor complexes underscore the highly structured nature of the complex and offer some interesting insights into the structure-activity relationship (SAR) for these ligands. With traditional thermodynamic integration (TI) calculations, we calculate relative binding free energies for three known agonists, in good agreement with experimental values. The sheer number of possible xenoestrogenic compounds makes an approach using traditional free energy calculations unfeasible. Instead, we have made use of a single-step perturbation methodology that allows the calculation of relative free energies for a large number of related polyaromatic hydrocarbons (PAHs) from a single simulation. Our results show good (maximum deviation 3.3 kJ mol(-1)) agreement with experimental data, suggesting the possibility of large-scale xenoestrogen screening in silico to obtain strongly estrogenic compounds for subsequent experimental testing.
Assuntos
Estrogênios/química , Receptores de Estrogênio/química , Xenobióticos/química , Dietilestilbestrol/química , Dietilestilbestrol/metabolismo , Estradiol/química , Estradiol/metabolismo , Congêneres do Estradiol/química , Congêneres do Estradiol/metabolismo , Estrogênios/metabolismo , Genisteína/química , Genisteína/metabolismo , Humanos , Ligantes , Modelos Moleculares , Hidrocarbonetos Policíclicos Aromáticos/química , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Estrogênio/metabolismo , Termodinâmica , Xenobióticos/metabolismoRESUMO
We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for determining the inhibition and activity of CYP2D6 in heterologously expressed systems and human liver microsomes. The high-throughput screening (HTS) assay can be used both in an end-point and real-time configuration, and is easy to use, rapid and sensitive. In addition, end-point measurements by means of flow injection analysis have also successfully been performed. The HTS-assay was validated by performing inhibition experiments for several low- and high-affinity ligands (n=6) of CYP2D6, and comparing the findings to those obtained with the standard O-demethylation assay of dextromethorphan. The results indicate that all compounds tested display competitive inhibition in both the MAMC and dextromethorphan assay, and the K(i) values reveal a very good correlation (R(2)=0.984) between the two assays. To further demonstrate the usefulness of the HTS-assay, IC(50) values of a series of five N-substituted analogs of 3, 4-methylenedioxyamphetamine for CYP2D6 have been determined. The results obtained demonstrate that the current HTS-assay represents a significant improvement over previous assays, with a higher turnover of MAMC and a higher selectivity for CYP2D6.
Assuntos
Cumarínicos/farmacocinética , Citocromo P-450 CYP2D6/metabolismo , Dextrometorfano/farmacocinética , Microssomos Hepáticos/enzimologia , Microssomos/enzimologia , Calibragem , Linhagem Celular , Citocromo P-450 CYP2D6/análise , Humanos , Cinética , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodos , Especificidade por SubstratoRESUMO
A series of six structural analogs of 7-methoxy-4-(aminomethyl)-coumarin (MAMC), a recently developed high-throughput substrate of P450 2D6 (CYP2D6), was synthesized to investigate the influence of N-substitution on the metabolism by cytochrome P450s, as well as on P450 selectivity. The analogs were obtained by introducing alkyl substituents at the amino group of MAMC and by replacing this moiety with a pyridine group. Competition experiments using heterologously expressed CYP2D6 demonstrated that the introduction and elongation of alkyl substituents strongly decreased the IC(50) values toward dextromethorphan O-demethylation. Metabolism studies showed that the regioselectivity of metabolism was unaffected by the varying N substituents, as only O-dealkylation of the analogs and no N-dealkylation was observed. In excellent agreement with the competition experiments, metabolism studies also showed that elongation of the alkyl chain dramatically increased the affinity of the compounds toward CYP2D6, as indicated by an up to 100-fold decrease in K(m) values. The V(max) values displayed a much less pronounced decrease with an increasing N-alkyl chain, resulting in as much as a 30-fold increase in the V(max)/K(m) value. Interestingly, due to the higher fluorescent yield of the N-alkyl metabolites compared with the metabolite of MAMC, O-dealkylation of N-methyl MAMC by CYP2D6 can be measured with a more than 3-fold higher sensitivity. Studies on P450 selectivity showed that only CYP1A2 and CYP2D6 contribute to the O-dealkylation of the N-alkyl analogs in both heterologously expressed P450s and human liver microsomes. In sharp contrast to CYP2D6, N-alkylation of MAMC did not significantly affect the K(m) values of O-dealkylation by CYP1A2, but it did result in higher V(max) values. Finally, CYP1A2 also N-dealkylated the analogs.
Assuntos
Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Alquilação , Ligação Competitiva/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Remoção de Radical Alquila , Humanos , Hidroxilação , Técnicas In Vitro , Isoenzimas/metabolismo , Cinética , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Modelos Biológicos , Conformação Proteica , Proteínas/metabolismoRESUMO
Previous studies have indicated that isolated preneoplastic rat hepatocytes in vitro fail to induce nuclear p53 protein and fail to block replication in response to genotoxic compounds. This suggests that defects in the protection of genomic integrity are part of their premalignant character. In the present study, we have investigated if similar defects occur in vivo. Preneoplastic glutathione-S-transferase (GST) 7-7-positive foci were induced in male Wistar rats by diethylnitrosamine (DEN) initiation and promotion with 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PH). The response to genotoxic damage was studied by X-irradiation. p53 protein was moderately expressed in nuclei in surrounding hepatocytes. This nuclear p53 staining had decreased 2 weeks after 2-AAF treatment. In foci, the protein was detected in the cytoplasm whereas the nuclei were negative. Levels of p21(waf1/cip1) protein were high in nuclei and cytoplasm of surrounding hepatocytes, whereas the expression in foci was low. A low level of Mdm2 in nuclei was observed in surrounding liver, while both Mdm2 and Bcl-2 protein were strongly expressed in the cytoplasm in foci. X-ray exposure further induced nuclear expression of p53, p21(waf1/cip1), and Mdm2 in surrounding hepatocytes, but focal nuclei were still negative. DNA replication was strongly reduced by X-irradiation in surrounding hepatocytes, but only partially reduced in the foci. These results indicate that the p53 pathway of response to genomic stress is impaired in preneoplastic cells in vivo. This may support their clonal expansion and their further malignant transformation because protection against genetic damage is diminished.
Assuntos
Dano ao DNA , Neoplasias Hepáticas/química , Fígado/química , Proteínas Nucleares , Lesões Pré-Cancerosas/química , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas/análise , Proteína Supressora de Tumor p53/análise , Animais , Divisão Celular , Aberrações Cromossômicas , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/análise , DNA/biossíntese , Glutationa Transferase/metabolismo , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Masculino , Lesões Pré-Cancerosas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Ratos , Ratos WistarRESUMO
Clonal expansion of initiated cells is an important process in carcinogenesis. Loss of functional p53 protein in initiated, preneoplastic cells might be involved in this process because such a loss would favour cell growth at the expense of normal cells upon exposure to genotoxic compounds. We have tested the hypothesis that p53 is not expressed in preneoplastic cells in the rat liver. Hepatocytes were isolated from livers of 10-week-old female rats that contained foci of preneoplastic hepatocytes, generated by 6-7 weekly injections of diethylnitrosamine (0.15 mmol/kg body wt intraperitoneally (i.p.)), starting 24 h after birth. The mixture of phenotypically normal and preneoplastic hepatocytes was exposed to X-rays or N-acetoxy-acetylaminofluorene (NAAAF), both causing DNA damage directly. At 24 and 48 h after exposure the cells were fixed and double stained for glutathione-S-transferase 7-7 (GST7-7), to identify preneoplastic cells, and p53. The percentage of p53-positive cells was much lower in GST7-7 positive (GST7-7+) than in GST7-7 negative (GST7-7-) hepatocytes. Exposure of cells to X-rays or NAAAF induced p53 in GST7-7- cells after 24 h, but GST7-7+ hepatocytes failed to do so. These results suggest that preneoplastic cells do not express p53 or have an attenuated p53 response to genotoxic treatments. This was confirmed when the cells were exposed to a proteasome inhibitor, PSI, which inhibits p53 degradation: a 12-fold increase in p53-positive cells was found after 48 h in GST7-7- hepatocytes, but in GST7-7+ hepatocytes no increase was observed. The percentage of GST7-7+ hepatocytes among surviving cells was increased after exposure to NAAAF, suggesting that these are more resistant to NAAAF than GST7-7- cells. This was not observed with PSI. These results indicate that preneoplastic hepatocytes have a lower p53 protein content and are not able to increase p53 upon inhibition of p53 breakdown or upon induction of DNA damage. Therefore, loss of p53 may favour clonal expansion of preneoplastic hepatocytes in the rat after administration of hepatocarcinogens or X-rays.
Assuntos
Neoplasias Hepáticas/metabolismo , Lesões Pré-Cancerosas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetoxiacetilaminofluoreno , Animais , Carcinógenos , Cisteína Endopeptidases , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Feminino , Imuno-Histoquímica , Neoplasias Hepáticas/etiologia , Complexos Multienzimáticos , Lesões Pré-Cancerosas/etiologia , Complexo de Endopeptidases do Proteassoma , Ratos , Ratos Sprague-Dawley , Raios XRESUMO
The urinary excretion of seven aldehydes, acetone, coproporphyrin III and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) as non-invasive biomarkers of oxidative damage was measured in rats treated with diquat or N-nitrosodimethylamine (NDMA), two compounds causing hepatic damage by different mechanisms. Furthermore, the effect of co-administration of the aldehyde dehydrogenase inhibitor, calcium carbimide (CC) on the urinary excretion of the aldehydes was determined. Slight hepatotoxicity was found at the end of the experiment after treatment with NDMA (0.5, 4 and 8 mg/kg at t = 0, 48 and 96 h, respectively) or diquat (6.8 and 13.6 mg/kg at t = 0 and 48 h, respectively). In diquat treated rats slight nephrotoxicity was also found. Urinary excretion of aldehydes, acetone and coproporphyrin III remained largely unchanged in rats treated with NDMA. In the rats treated with diquat, the urinary excretion of several aldehydes was several-fold increased. An increase was also found in the urinary excretion of 8-OH-dG after the second dose of diquat. Treatment of rats with CC did not significantly influence the urinary excretion of aldehydes in control and NDMA rats. However, in rats treated with diquat, CC caused a potentiating effect on the excretion of acetaldehyde, hexanal and malondialdehyde (MDA), indicating that oxidation of aldehydes to carbonylic acids by aldehyde dehydrogenases (ALDHs) might be an important route of metabolism of aldehydes. In conclusion, increased urinary excretion of various aldehydes, acetone, coproporphyrin III and 8-OH-dG was observed after administration of diquat, probably reflecting oxidative damage induced by this compound. No such increases were found after NDMA administration, which is consistent with a different toxicity mechanism for NDMA. Therefore, excretion of aldehydes, acetone, coproporphyrin III and 8-OH-dG might be used as easily accessible urinary biomarkers of free radical damage.
Assuntos
Biomarcadores/urina , Cianamida/toxicidade , Diquat/toxicidade , Inibidores Enzimáticos/toxicidade , Herbicidas/toxicidade , Compostos Nitrosos/toxicidade , 8-Hidroxi-2'-Desoxiguanosina , Acetona/urina , Aldeído Desidrogenase/antagonistas & inibidores , Aldeído Desidrogenase/metabolismo , Aldeídos/urina , Animais , Peso Corporal/efeitos dos fármacos , Coproporfirinas/urina , Cianamida/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Diquat/metabolismo , Combinação de Medicamentos , Interações Medicamentosas , Inibidores Enzimáticos/metabolismo , Radicais Livres/metabolismo , Herbicidas/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Compostos Nitrosos/metabolismo , Ratos , Ratos WistarRESUMO
Free radical damage is an important factor in many pathological and toxicological processes. Despite extensive research efforts in biomarkers in recent years, yielding promising results in experimental animals, there is still a great need for additional research on the applicability of, especially non-invasive, biomarkers of free radical damage in humans. This review gives an overview of the applications in experimental and human situations of four main groups of products resulting from free radical damage, these include: lipid peroxidation products, isoprostanes, DNA-hydroxylation products and protein hydroxylation products.
Assuntos
Radicais Livres/metabolismo , Animais , Antioxidantes/metabolismo , Biomarcadores , DNA/metabolismo , Dano ao DNA , Humanos , Hidroxilação , Peroxidação de Lipídeos , Prostaglandinas/metabolismo , Proteínas/metabolismoRESUMO
The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and its metabolite 2-bromoacrolein (2BA) are very potent bacterial mutagens in Salmonella typhimurium (S. typhimurium) TA 100. In this study, we showed that 2BA and Tris-BP are also mutagenic in S. typhimurium TA 104, which detects mutations at AT base pairs, while TA 100 detects mutations at CG basepairs. We also studied the mutagenicity of 2BA in mammalian cells in vitro and in the rat in vivo. Firstly, 2BA was tested in the human lymphoblastoid cell line TK6. The results showed that there was no increase in mutation frequency at the hprt locus, whereas there was a large decrease in cell survival. Secondly, a shuttle vector system was used to study the induction of mutations by 2BA:DNA adducts. The vector was modified by insertion of a single-stranded oligonucleotide containing on average one 2BA:DNA adduct. No increase in mutation frequency above background was detected after replication of this vector in SV40 transformed normal human fibroblasts. Because the liver is a major site for bioactivation of Tris-BP to 2BA in vivo, we tested the initiating capacity of Tris-BP in the rat liver in a modified Solt & Farber initiation and promotion system. Administration of Tris-BP resulted in a small increase in the number of preneoplastic gamma-glutamyl-transpeptidase positive (GGT+) foci in the liver compared to control animals (only significant in the lowest size class). Modification of the experimental protocol by performing partial hepatectomy 24 h after the administration of Tris-BP, did not increase the number of GGT+ or glutathione S-transferase-P (GST-P+) positive foci above the control level. Taken together, these results indicate that, in spite of a high mutagenicity in S. typhimurium, 2BA and Tris-BP have low or negligible mutagenic effects in mammalian systems. The lack of mutagenic activity may explain why Tris-BP is not a carcinogen in the rat liver.
Assuntos
Acroleína/análogos & derivados , Retardadores de Chama/toxicidade , Testes de Mutagenicidade , Mutagênicos/toxicidade , Organofosfatos/toxicidade , Salmonella typhimurium/genética , Acroleína/metabolismo , Acroleína/toxicidade , Animais , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/efeitos dos fármacos , Retardadores de Chama/metabolismo , Vetores Genéticos , Humanos , Hipoxantina Fosforribosiltransferase/genética , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/patologia , Mutagênicos/metabolismo , Organofosfatos/metabolismo , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/patologia , Salmonella typhimurium/efeitos dos fármacosRESUMO
There is an increasing need for biomarkers of oxidative stress in animals and man. In this study, we have evaluated in the rat the utility of various endogenous products that are excreted in urine as potential noninvasive biomarkers of oxidative stress in the kidney. Renal oxidative damage was induced by daily i.p. injections of ferric nitrilotriacetate (Fe-NTA) for a period of 13 days. The daily dose of Fe-NTA was increased during the experiment from 6 to 40 mg Fe/kg body wt. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), coproporphyrin III (COPRO III), seven aldehydes, and acetone were determined in fractionated urine samples and compared with commonly used urinary and plasma clinical chemical parameters for toxicity. The parameters that showed the earliest increase were acetaldehyde (ACET), propanal (PROPA), and COPRO III. Their increase was significantly earlier than that of classical clinical chemical parameters indicative of renal damage such as urinary concentration of glucose (GLU) and protein (PRT), and N-acetyl-beta-D-glucosaminidase (NAG) activity. The excretion of 8-OHdG was increased only after administration of the highest dose of Fe-NTA. Urinary excretion of acetone, form-aldehyde (FOR), butanal (BUTA), pentanal (PENTA) hexanal (HEXA), and malondialdehyde (MDA) was also increased; however, their increase occurred only slightly before or simultaneously with that of the urinary clinical chemical parameters. In conclusion, 8-OHdG, acetone, FOR, BUTA, PENTA, HEXA, and MDA may possibly serve as biomarkers for oxidative kidney damage. COPRO III, ACET, and PROPA might even be used as biomarkers of production of reactive oxygen species at an early stage.
Assuntos
Biomarcadores/urina , Compostos Férricos/toxicidade , Rim/efeitos dos fármacos , Ácido Nitrilotriacético/análogos & derivados , Estresse Oxidativo , 8-Hidroxi-2'-Desoxiguanosina , Animais , Coproporfirinas/urina , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Rim/patologia , Peróxidos Lipídicos/urina , Masculino , Ácido Nitrilotriacético/toxicidade , Ratos , Ratos WistarRESUMO
Carbon tetrachloride (CCl4) is a model compound for inducing free radical damage in liver. In this study 10 biomarkers in rats treated i.p. with three different single doses of CCl4 (0.25, 0.50, and 1.00 ml/kg body wt) were measured dose and time dependently and compared to evaluate these urinary products as noninvasive biomarkers for radical damage. Eight degradation products of lipid peroxides, namely, formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal, and malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and coproporphyrin III were measured in this study. As general measures of toxicity, several clinical chemical parameters (n = 12) and histopathological damage were determined. A dose-dependent increase in both the clinical parameters and the lipid degradation products was found. Increases in lipid degradation products were statistically significant at doses of 0.5 and 1 ml/kg CCl4. An increase in these products was already found in the first 12 h after exposure. At the lowest dose, 0.25 ml/kg CCl4, acetaldehyde and propanal already showed a statistically significant increase as well. No change in the urinary levels of 8-OH-dG could be found in this study and a decrease in the urinary excretion of coproporphyrin III was found. It is concluded that 8-OH-dG and coproporphyrin III are not useful biomarkers for radical damage induced by CCl4. Lipid degradation products, however, are promising noninvasive biomarkers for in vivo radical damage, although the precise specificity of these biomarkers for damage induced by radicals needs to be further investigated.
Assuntos
Biomarcadores/urina , Tetracloreto de Carbono/toxicidade , Fígado/efeitos dos fármacos , 8-Hidroxi-2'-Desoxiguanosina , Acetona/urina , Alanina Transaminase/sangue , Alanina Transaminase/efeitos dos fármacos , Aldeídos/urina , Animais , Aspartato Aminotransferases/sangue , Coproporfirinas/urina , Desoxiguanosina/análogos & derivados , Desoxiguanosina/urina , Relação Dose-Resposta a Droga , Radicais Livres/toxicidade , Injeções Intraperitoneais , Fígado/patologia , Masculino , Ratos , Ratos WistarRESUMO
P53 protein plays an important role in regulation of the cell cycle. Recently, a role in tumour genesis has also been suggested. The protein is induced after various forms of DNA damage. Immunohistochemical detection of p53 protein showed positive cells in human skin after UV-irradiation, in mouse skin after benzo[a]pyrene treatment and in mouse spleen, thymus and bone after gamma-irradiation. However, no staining was found in mouse and rat liver with traditional immunohistochemical staining methods due to the low amount of p53 present. This seriously hampered studies on the role of p53 in hepatocarcinogenesis. We have developed a more sensitive immunohistochemical method for staining of p53 in paraffin-embedded sections of rat liver using microwave irradiation for antigen retrieval, avidin-biotin complexing and tyramide amplification. A strong, specific fluorescence signal for p53 was found in hepatocytes of rats that had received the hepatocarcinogen N-hydroxy-2-acetylaminofluorene; in control liver no such p53 staining was observed. The fluorescence was located in the nucleus of hepatocytes in zone 1 of the liver. This agrees with the fact that N-hydroxy-2-acetylaminofluorene causes cytotoxicity in this zone.
Assuntos
Genes p53/efeitos dos fármacos , Hidroxiacetilaminofluoreno/toxicidade , Fígado/efeitos dos fármacos , Proteína Supressora de Tumor p53/biossíntese , Animais , Carcinógenos/toxicidade , Corantes Fluorescentes , Humanos , Imuno-Histoquímica/métodos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Parafina , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/análiseRESUMO
In the present study the urinary excretion of seven aldehydes, acetone and coproporphyrin III as non-invasive in vivo biomarkers of free radical damage was measured in rats after treatment with three nephrotoxic compounds: cisplatin, mercuric chloride (HgCl2) and N -acetyl- S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-Nac). A clear difference between the different nephrotoxic compounds was found in the time interval between dosage and maximal toxicity, as measured by clinical chemical parameters in urine. In rats treated with TFE-Nac and HgCl2 this was fast: 12 h and 24 h after treatment, respectively. In the rats treated with cisplatin, however, nephrotoxicity occurred later: 96 h-108 h after treatment. Urinary creatinine excretion was decreased in all treatments. Therefore, the excretion of the proposed biomarkers was expressed as amount excreted per 12 h urine fraction as well as amount excreted per mol creatinine in each 12 h urine fraction. Urinary excretion of coproporphyrin III was decreased in almost all 12 h urine fractions with all treatments, however, when expressed per mol creatinine, increases were found in urine of rats treated with cisplatin and HgCl2. In cisplatin-treated rats an increase was found in the excretion of formaldehyde per 12 h, but acetaldehyde, propanal and MDA levels were decreased. Expressed per mol creatinine, MDA levels were decreased, but other aldehydes were increased. In HgCl2-treated rats urinary aldehyde excretion expressed per mol creatinine was increased. In TFE-Nac treated animals the urinary levels of acetaldehyde per 12 h were increased and per mol creatinine the levels of some aldehydes were only slightly increased. With none of the treatments did the increase in the biomarkers expressed per mol creatinine exceed the decrease in creatinine excretion. Similar time intervals were found between dosage and maximal excretion of biomarkers as for the time intervals between dosage and maximal toxicity. With all treatments significant increases in the excretion of acetone were found both per 12 h and per mol creatinine, probably related to the increased glucose excretion. It was concluded that no convincing evidence for free radical damage was found in the present study with the employed biomarkers.
RESUMO
The genotoxic agent 2-acetylaminofluorene induces, upon metabolic activation, two main types of DNA adducts in animal tissue, i.e., (deoxyguanine-8-yl)-aminofluorene (dG-C8-AF) and N-(deoxyguanine-8-yl)-acetylaminofluorene (dG-C8-AAF). Quantification of the frequency of these adducts usually relies on the use of radioactively labeled 2-acetylaminofluorene. Here, we report the development of a sensitive, non-radioactive method for the quantification of dG-C8-AF and dG-C8-AAF. Essentially, the modified DNA bases are separated by high-performance liquid chromatography (HPLC) and quantified by electrochemical detection. We established that both modified bases guanine-C8-aminofluorene and guanine-C8-acetylaminofluorene are electrochemically active. Subsequently, a procedure was developed to quantify dG-C8-AF and dG-C8-AAF in genomic DNA. Following DNA hydrolysis the adducted bases were extracted by ethyl acetate, separated by HPLC, and detected electrochemically. This procedure has been applied in the analysis of dG-C8-AAF in N-acetoxy-2-acetylaminofluorene-modified calf thymus DNA and in the detection of dG-C8-AAF and dG-C8-AF in liver DNA of mice injected intraperitoneally with 150-450 mg N-hydroxy-2-acetylaminofluorene/kg. The quantification of relatively low dG-C8-AF and dG-C8-AAF adduct levels (i.e., 0.1-1 adduct/10(6) nucleotides) in mouse liver DNA demonstrates the sensitivity of this electrochemical detection procedure. The detection limit of the method is 1 adduct per 10(6) nucleotides for both adducts using 20 micrograms of DNA and 4 adducts per 10(8) nucleotides using 500 micrograms DNA.
Assuntos
2-Acetilaminofluoreno/toxicidade , Adutos de DNA/análise , Adutos de DNA/efeitos dos fármacos , Eletroquímica/métodos , Acetilação , Animais , Bovinos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/estatística & dados numéricos , DNA/química , DNA/efeitos dos fármacos , Adutos de DNA/química , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Eletroquímica/estatística & dados numéricos , Técnicas In Vitro , Masculino , Camundongos , Mutagênicos/toxicidadeRESUMO
One of the major processes that occur as a result of radical-induced oxidative stress is lipid peroxidation (LPO). Degradation of lipid peroxides results in various products, including a variety of carbonyl compounds. In the present study eight different lipid degradation products, i.e., formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal and malondialdehyde were identified and measured simultaneously and quantitatively in rat urine after derivatization with O-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine hydrochloride, extraction with heptane and using gas chromatography-electron-capture detection (GC-ECD). The identity of the respective oximes in urine was confirmed by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS). Simultaneously measured standard curves were linear for all oxime-products and the detection limits were between 39.0 +/- 5.3 (n=9) and 500 +/- 23 (n=9) fmol per microl injected sample. Recoveries of all products from urine or water were 73.0 +/- 5.2% and higher. In urine of CCl4-treated rats an increase in all eight lipid degradation products in urine was found 24 h following exposure. ACON showed the most distinct increase, followed by PROPA, BUTA and MDA. It is concluded that the rapid, selective and sensitive analytical method based on GC-ECD presented here is well suited for routine measurement of eight different lipid degradation products. These products appear to be useful as non-invasive biomarkers for in vivo oxidative stress induced in rats by CCl4.
Assuntos
Acetona/urina , Aldeídos/urina , Tetracloreto de Carbono/toxicidade , Peroxidação de Lipídeos , Estresse Oxidativo , Animais , Biomarcadores/urina , Cromatografia Gasosa , Masculino , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The tumour suppressor gene p53 is expressed in response to DNA-damage; its protein product blocks cells in the G1-phase of the cell cycle. This gives cells additional time to repair their DNA-damage. However, it may trigger apoptosis if damage is too high. Loss of p53 function appears to be an important step in carcinogenesis because 50% of human tumours have lost functional p53. In order to study the role of p53 in experimental hepatocarcinogenesis, we determined the expression of p53 in rat liver in response to various hepatocarcinogenic and hepatotoxic compounds. Administration of hepatocarcinogenic compounds increased p53 protein levels in the liver as detected by immunoprecipitation followed by SDS-PAGE and Western blotting with ECL-detection. The hepatocarcinogens included N-hydroxy-2-acetylaminofluorene, aflatoxin B1, and diethylnitrosamine. Their structural analogues N-hydroxy-4-acetylaminobiphenyl and ethyl methane-sulphonate which are not hepatocarcinogenic, did not induce p53. Also, two hepatotoxic compounds (carbon tetrachloride, D-galactosamine) did not induce p53. Other compounds that induced p53 in the rat liver were 2-aminofluorene (administered by drinking water for two weeks) and tris-(2,3-dibromopropyl)phosphate. Benzo[a]pyrene did not induce p53. N-Hydroxy-2-acetylaminofluorene, aflatoxin B1, and diethylnitrosamine are potent hepatic tumour promoters. At the same time, they induce p53 protein expression and inhibit proliferation of normal hepatocytes. Because this is not observed with non-hepatocarcinogenic analogues, it suggests an involvement of p53 expression in hepatic tumour promotion. A possible mechanism is discussed.
Assuntos
Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Alanina Transaminase/sangue , Animais , Benzo(a)pireno/farmacologia , Carcinógenos/farmacologia , Divisão Celular , Adutos de DNA , Dietilnitrosamina/farmacologia , Metanossulfonato de Etila/farmacologia , Hepatectomia , Hidroxiacetilaminofluoreno/farmacologia , Fígado/citologia , Masculino , Testes de Precipitina , Antígeno Nuclear de Célula em Proliferação , Ratos , Ratos WistarRESUMO
1. 4-Hydroxy-2,3-nonenal (HNE) is an end product of lipid peroxidation (LPO) and a well known cytotoxic aldehyde that exhibits a variety of biological effects. In this study the in vivo disposition and covalent binding of i.p. administered [2-3H]HNE was examined in the rat. 2. It was found that several metabolites of [2-3H]HNE are excreted in urine among which at least four mercapturic acids. 1,4-Dihydroxynonane mercapturic acid (DHN-MA) appeared to be the most abundant mercapturic acid excreted in urine (3.5% of the dose) and the excretion of the other three mercapturic acids amounted to 2% of the dose. 3. Within 48 h following i.p. administration of 5 or 25 mumol/kg bodyweight [2-3H]HNE (specific activity 4 microCi/mumol) about 25% of the radioactivity was excreted in urine, whereas 18% of the radioactivity appeared in the faeces. 4. After 48 h, 7% of the radioactivity was still present in the liver and 0.2% in other organs, but this radioactivity appeared to not to be covalently bound to cellular macromolecules. It was found that only 0.13% of the radioactivity was covalently bound in the liver and even less in other organs.
Assuntos
Aldeídos/farmacocinética , Peroxidação de Lipídeos , Aldeídos/urina , Animais , Fezes , Injeções Intraperitoneais , Masculino , Ratos , Ratos Wistar , Distribuição Tecidual , TrítioRESUMO
The metabolism of tris(2,3-dibromopropyl) phosphate (Tris-BP) was compared with that of completely deuterated Tris-BP (D15-Tris-BP) in an isolated, recirculating rat liver perfusion system in order to determine the relative quantitative importance of two different biotransformation pathways of Tris-BP: (i) cytochrome P450-mediated metabolism and (ii) GSH S-transferase-mediated metabolism. To accomplish this we quantitated the biliary excretion of S-(3-hydroxypropyl)glutathione (GSOH) as a marker metabolite for cytochrome P450-mediated metabolism and that of S-(2,3-dihydroxypropyl) glutathione (GSOHOH) as a marker metabolite for GSH S-transferase-mediated metabolism. Complete deuterium substitution of Tris-BP significantly decreased the formation of GSOH, whereas there was no effect on the formation of GSOHOH. Because our previous studies showed a large decrease in genotoxicity of D15-Tris-BP compared to Tris-BP, the present results support our hypothesis that cytochrome P450-mediated metabolism is responsible for the genotoxic effects of Tris-BP in the rat liver.
Assuntos
Deutério/efeitos adversos , Retardadores de Chama/metabolismo , Fígado/metabolismo , Organofosfatos/metabolismo , Animais , Bile/metabolismo , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Eletroquímica , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Técnicas In Vitro , Marcação por Isótopo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Ratos , Ratos WistarRESUMO
The haloalkane 1,2-dibromo-3-chloropropane (DBCP), an environmental pollutant that was widely used as a soil fumigant, is a carcinogen and a mutagen and displays target-organ toxicity to the testes and the kidneys. Because little is known about effects of stereochemistry on the metabolism and toxicity of halogenated alkyl compounds and because DBCP, which has a chiral center at C-2, may show enantioselectivity in its metabolism and/or toxicities, the optically pure enantiomers of DBCP were tested in vivo in rats for organ toxicity as well as for bacterial mutagenicity. Organ toxicity studies showed that (S)-DBCP was slightly more renal toxic than (R)-DBCP but was not significantly more toxic than the racemate, and that no significant differences were observed in the extents of testicular necrosis and atrophy caused by either enantiomer or the racemate. In contrast, (R)-DBCP was more mutagenic than either (S)-DBCP or the racemate to Salmonella typhimurium (S. typhimurium) strains TA 100 and TA104. However, there was little or no enantioselectivity in glutathione S-transferase (GST)-catalyzed conjugation reactions of glutathione with DBCP based on the lack of selectivity in the rates of disappearance of the enantiomers of DBCP in the presence of glutathione (GSH) and GSTs as monitored by chiral gas chromatography (GC).