RESUMEN
BACKGROUND/AIM: We previously reported that the half maximal inhibitory concentration (IC50) values of cisplatin and epirubicin correlated in 13 triple-negative breast cancer (TNBC) cell lines between two-dimensional (2D) and three-dimensional (3D) culture methods. However, the IC50 values of docetaxel (DTX) did not correlate between the two culture methods. We hypothesized that this non-correlation is partly associated with differences in expression of the ß-tubulin isoform, the target molecule of DTX and in morphology depending on the culture method. MATERIALS AND METHODS: We investigated the expression levels of ß-tubulin isoforms by real-time polymerase chain reaction and morphology of spheroid formation in the 13 TNBC cell lines cultured using the 2D and 3D culture methods. RESULTS: Tubulin ß class I (TUBB) expression levels were negatively correlated with the IC50 value of DTX in the 2D culture method (R=-0.360), whereas tubulin ß class IIa (TUBB2a) expression levels were positively correlated in the 3D culture method (R=0.398). There was no significant difference in the expression levels of ß-tubulin isoforms between the 2D and 3D culture methods. The spheroids were classified morphologically into three types: round, mass, and grape-like. However, no clear association was found between DTX sensitivity and morphology. CONCLUSION: The non-correlation of the IC50 values of DTX between the 2D and 3D culture methods does not appear to be due to the changes in ß-tubulin isoforms. Morphology in the 3D culture method may play some role in drug sensitivity.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Tubulina (Proteína) , Línea Celular , Línea Celular Tumoral , Cisplatino , Docetaxel/farmacología , Epirrubicina/farmacología , Humanos , Isoformas de Proteínas , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Tubulina (Proteína)/metabolismoRESUMEN
BACKGROUND AND AIM: Capsular contracture is the most common complication with smooth-type silicone implants. We investigated the preventive effect of an active metabolite of tamoxifen, 4-hydroxytamoxifen (4-OH TAM), on capsular contracture. METHODS: A silicone sheet was implanted into the back of 28 female ICR mice. Mixtures of gel with 0.2% 4-OH TAM and 0.1% 4-OH TAM were administered transdermally once a day for 4 weeks. Saline was administered to the control. After killing the mice, capsular thickness was measured in H&E-stained specimens. Estrogen receptor (ER), α-smooth muscle actin (α-SMA), and transforming growth factor-ß (TGF-ß) expressions were immunohistochemically investigated in the capsules. RESULTS: The capsule was thinner in the 0.2% 4-OH TAM gel group than in the control group (control, 0.1% 4-OH TAM gel, 0.2% 4-OH TAM gel: 52.8 ± 3.4 µm, 54.2 ± 6.8 µm, 46.4 ± 3.3 µm, respectively). ER was found in most fibroblasts of all samples. α-SMA expression in the capsule was significantly lower in the 4-OH TAM gel groups than in the control group (control = 70.0 ± 3.4%, 0.1% 4-OH TAM = 57.0 ± 3.4%, 0.2% 4-OH TAM = 49.4 ± 4.9%). TGF-ß expression was significantly reduced by the 4-OH TAM gel injections dose-dependently (control = 67.3 ± 2.2%, 0.1% 4-OH TAM = 52.4 ± 3.1%, 0.2% 4-OH TAM = 45.1 ± 2.4%). CONCLUSIONS: The transdermal administration of 0.1% and 0.2% 4-OH TAM gels inhibited capsule development. The inhibition of TGF-ß expression is a mechanism by which 4-OH TAM suppresses fibroblast growth, preventing capsular formation.
Asunto(s)
Implantación de Mama , Implantes de Mama , Neoplasias de la Mama , Contractura , Administración Cutánea , Animales , Implantación de Mama/efectos adversos , Implantes de Mama/efectos adversos , Neoplasias de la Mama/complicaciones , Contractura/etiología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos ICR , Geles de Silicona , Tamoxifeno/farmacologíaRESUMEN
Three-dimensional (3D) culture reflects tumor biology complexities compared with two-dimensional (2D) culture. Thus, 3D culture has attracted attention in cell biology studies including drug sensitivity tests. Herein, we investigated differences in anticancer drug sensitivities between 2D and 3D culture systems in triple-negative breast cancer (TNBC) cell lines. Thirteen TNBC cell lines were maintained in 2D and 3D cultures for 3 days before drug exposure. Cell morphology in the 3D culture was examined by phase-contrast microscopy. Sensitivities to epirubicin (EPI), cisplatin (CDDP), and docetaxel (DTX) were investigated by cell viability assay in both cultures and compared. The IC50s of all 3 drugs were significantly higher in the 3D culture than in the 2D culture in most cell lines. Those were correlated between the 2D and 3D cultures in EPI (R = 0.555) and CDDP (R = 0.955), but not in DTX (R = 0.221). Round spheroid-forming cells were more resistant to agents than grape-like types. In conclusion, 3D culture was more resistant to all 3 drugs than 2D culture in most TNBC cell lines. Sensitivity to CDDP was highly correlated between the 2D and 3D cultures, but not to DTX. 2D culture may be acceptable for sensitivity test for DNA-damaging agents.
Asunto(s)
Antineoplásicos/farmacología , Técnicas de Cultivo de Célula , Cisplatino/farmacología , Docetaxel/farmacología , Resistencia a Antineoplásicos , Epirrubicina/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Concentración 50 Inhibidora , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND: Many triple-negative breast cancers (TNBCs) show impaired breast cancer susceptibility gene I (BRCA1) function, called BRCAness. BRCAness tumors may show similar sensitivities to anticancer drugs as tumors with BRCA1 mutations. In this study, we investigated the association of BRCA mutations or BRCAness with drug sensitivities in TNBC. METHODS: BRCAness was evaluated as BRCA1-like scores, using multiplex ligation-dependent probe amplification in 12 TNBC cell lines, including four with mutations. Sensitivities to docetaxel, cisplatin, and epirubicin were compared with BRCA mutations and BRCA1-like scores. Cisplatin sensitivity was examined in BRCA1 knockdown Michigan Cancer Foundation-7 cell lines. RESULTS: Eight and four cell lines had characteristics of BRCAness and non-BRCAness, respectively. The 50% inhibitory concentration of docetaxel was higher in BRCA mutant and BRCAness cell lines than their counterparts. BRCA1-like scores showed a weak positive correlation with docetaxel sensitivity (r = 0.377; P = 0.039). Regarding cisplatin, scores were lower in BRCA mutants and BRCAness tumors than their counterparts. A negative correlation was found between BRCA1-like scores and cisplatin sensitivity (r = -0.407; P = 0.013). No differences were found for epirubicin. BRCA1 gene knockdown increased the cisplatin sensitivity of Michigan Cancer Foundation-7 cells. CONCLUSIONS: BRCA1-like scores were associated with cisplatin sensitivity and docetaxel resistance. BRCA1-like score is hence a promising indicator for estimating drug sensitivities in TNBC.
Asunto(s)
Antineoplásicos/farmacología , Proteína BRCA1/genética , Resistencia a Antineoplásicos/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Proteína BRCA1/análisis , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Cisplatino/farmacología , Cisplatino/uso terapéutico , Docetaxel/farmacología , Docetaxel/uso terapéutico , Femenino , Humanos , Mutación , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
The tumor-promoting effects of oxfendazole (OX), a benzimidazole anthelmintic, were investigated using a medium-term rat hepatocarcinogenesis model. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were given a powdered diet containing 0 or 500 ppm OX for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after OX treatment. The numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in the livers of rats treated with OX, with concomitantly increased cell proliferation, compared with those in the livers of the DEN alone group. Quantitative real-time RT-PCR analysis revealed that OX induced not only mRNA expression of phase I enzymes Cyp1a1, Cyp1a2, but also Nrf2-regulated phase II enzymes such as Gpx2, Nqo1, Yc2, Akr7a3 and Gstm1, presumably due to an adaptive response against OX-induced oxidative stress. Reactive oxygen species production increased in microsomes isolated from the livers of OX-treated rats. Furthermore, OX enhanced oxidative DNA damage (as assessed by 8-hydroxydeoxyguanosine; 8-OHdG) and lipid peroxidation (as assessed by thiobarbituric acid-reactive substances; TBARS). These results suggest that administration of OX at a high dose and for a long term enhances oxidative stress responses, which may contribute to its tumor-promoting potential in rats.
Asunto(s)
Antihelmínticos/farmacología , Bencimidazoles/farmacología , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Inmunohistoquímica , Neoplasias Hepáticas Experimentales/enzimología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Especies Reactivas de Oxígeno/metabolismo , Factores de TiempoRESUMEN
To determine the threshold dose of piperonyl butoxide (PBO) that induces hepatocellular tumor-promoting effects, reactive oxygen species (ROS) generation, and drug-metabolizing enzymes that protect against ROS generation, partial hepatectomized rats were fed diets containing 0, 0.015, 0.03, 0.06, 0.125, 0.25, or 0.5% PBO after an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, Glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in a dose-dependent manner in rats given 0.25% PBO or higher. The formation of microsomal ROS in the liver was significantly increased in 0.25 and 0.5% PBO. Real-time RT-PCR showed that the expression of the CYP1A1, UDPGTr-2, and Mrp3 genes was significantly upregulated in rats given 0.03% PBO or higher. These results suggest that 0.25% is the threshold dose of PBO that induces ROS-mediated hepatocarcinogenesis in rats, although the CYP1A1 gene that is related to ROS generation and the UDPGTr-2 and Mrp3 genes that are involved in protection against ROS were induced in the livers of rats even at a PBO dose of 0.03%.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas Experimentales/inducido químicamente , Sinergistas de Plaguicidas/toxicidad , Butóxido de Piperonilo/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Animales , Peso Corporal/efectos de los fármacos , Carcinógenos/toxicidad , Dietilnitrosamina/toxicidad , Relación Dosis-Respuesta a Droga , Glutatión Transferasa/metabolismo , Hepatectomía , Inmunohistoquímica , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas Experimentales/metabolismo , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Endogámicas F344RESUMEN
We previously found that administration of ascorbic acid (AA) enhances the liver tumor-promoting activity of kojic acid (KA) in mice. To examine the reproducibility of these results in rats and the underlying mechanism of this effect, we employed a two-stage liver carcinogenesis model using male F344 rats. Two weeks after initiation with diethylnitrosamine (DEN), the animals received a diet containing 2% KA and drinking water with or without 5,000 ppm AA for a period of 7 weeks. A DEN-alone group was also established as a control. One week after the commencement of the administration, the animals were subjected to two-thirds partial hepatectomy. At the end of the experiment, the livers were analyzed immunohistochemically, and the mRNA expression level and extent of lipid peroxidation were measured. AA treatment enhanced the KA-induced tumor-promoting activity in terms of the number and area of liver cell foci that were positive for glutathione-S-transferase placental form. AA coadministration increased the number of hepatocytes positive for proliferating cell nuclear antigen and inversely decreased the number of TUNEL-positive cells. However, the increased level of thiobarbituric acid reactive substances resulting from KA treatment was suppressed by coadministration of AA. Gene expression analyses using low-density microarrays and real-time RT-PCR showed that coadministration of AA resulted in upregulation of genes related to cell proliferation and downregulation of those involved in apoptosis and/or cell cycle arrest. These results indicate that the concerted effects of AA on cell proliferation and apoptosis/cell cycle arrest probably through its antioxidant activity are involved in this enhancement.
Asunto(s)
Ácido Ascórbico/toxicidad , Carcinógenos/toxicidad , Hígado/efectos de los fármacos , Pironas/toxicidad , Animales , Apoptosis , Ciclo Celular , Proliferación Celular , Dietilnitrosamina , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Peroxidación de Lípido/efectos de los fármacos , Hígado/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas F344 , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
To examine the possible modifying effect of the extract of Siraitia grosvenori (SGE), a naturally occurring antioxidative agent, on piperonyl butoxide (PBO)-promoted hepatocarcinogenesis, male F344 rats were administered a single intraperitoneal injection of N-diethylnitrosamine (DEN) as an initiator followed by administration of a diet containing 2% PBO for 7 weeks with or without SGE (1,000 ppm) in the drinking water. To enhance cellular proliferation, all animals underwent two-thirds partial hepatectomy 1 week after the commencement of PBO administration. Pretreatment with SGE was also applied to the PBO + SGE group for 2 weeks prior to DEN initiation. Liver immunohistochemistry revealed that although the PBO-mediated increase in the number of glutathione S-transferase placental form (GST-P)-positive foci and proliferating cell nuclear antigen-positive cells remained unaltered with SGE coadministration, the area of the GST-P-positive foci was increased. On the contrary, real-time RT-PCR showed that coadministration of SGE increased hepatic GST and glutathione peroxidase (GSH-Px) antioxidant activities and mRNA expression levels of the phase II enzymes that are known to be transcriptionally up-regulated through the Nrf 2-Keap1-antioxidant responsive element (ARE) as well as the phase III enzymes. Furthermore, measurement of thiobarbituric acid-reactive substances showed a decrease in lipid peroxidation by SGE coadministration. The results suggest that SGE may exert hepatic antioxidant activity by up-regulating the genes under the control of the Nrf 2-Keap1-ARE transcriptional machinery; however, this activity was neither effective nor sufficient for suppression of PBO-promoted early hepatocarcinogenesis.
Asunto(s)
Cucurbitaceae/química , Neoplasias Hepáticas Experimentales/metabolismo , Animales , Antioxidantes/metabolismo , Perfilación de la Expresión Génica , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Butóxido de Piperonilo , Extractos Vegetales/farmacología , Ratas , Ratas Endogámicas F344 , Especies Reactivas de Oxígeno/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
To clarify whether oxidative stress is involved in the development of hepatocellular preneoplastic foci induced by fenofibrate (FF), a peroxisome proliferator-activated receptor alpha agonist, male F344/N rats were fed a diet containing 6,000, 3,000, or 0 ppm of FF for 13 weeks after N-diethylnitrosamine initiation. Two-third partial hepatectomy was performed 1 week after the FF treatment. Histopathologically, the number of hepatocellular altered foci significantly increased in the FF-treated groups with a concomitant increase in the number of hepatocytes positive for anti-Ki-67 antibody, but the number and area of glutathione S-transferase placental form (GST-P)-positive foci decreased in these groups, as compared to those in the controls. Microarray analysis or quantitative real-time reverse transcription-polymerase chine reaction demonstrated the significant up-regulations of Aco and Cyp4a1 (genes related to lipid metabolism); Gpx2, Yc2, Cat, Cyp2b15, and Ugt1a6 (metabolic oxidative stress-related genes); Apex1, Mgmt, Xrcc5, Nbn, and Gadd45a (DNA repair-related genes); and Ccnd1 (cell cycle-related genes) in the FF-treated groups, and the significant down-regulations of Cyp1a2, Gsta2, Gstm2, and Gstm3 (phase I or II metabolism-related genes); Mlh1 and Top1 (DNA repair-related genes); and Cdkn1a, Cdkn1b, Chek2, and Gadd45b (cell cycle/apoptosis-related genes) in these rats. FF-treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl-CoA oxidizing system, and catalase in the liver, but not superoxide dismutase in the liver. In addition, 8-OHdG level in liver DNA, lipofuscin deposition in hepatocytes, and in vitro reactive oxygen species production in microsomes significantly increased due to FF treatment. These results suggest that oxidative stress is involved in the development of FF-induced hepatocellular preneoplastic foci in rats.
Asunto(s)
Fenofibrato/toxicidad , Hipolipemiantes/toxicidad , Neoplasias Hepáticas Experimentales/inducido químicamente , Estrés Oxidativo/fisiología , 8-Hidroxi-2'-Desoxicoguanosina , Alquilantes/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Carcinógenos/toxicidad , ADN/biosíntesis , ADN/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/farmacología , Dietilnitrosamina/toxicidad , Ingestión de Alimentos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Hepatectomía , Antígeno Ki-67/biosíntesis , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas Experimentales/patología , Masculino , Tamaño de los Órganos/efectos de los fármacos , PPAR alfa/agonistas , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/patología , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Ratas , Ratas Endogámicas F344 , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The tumour-promoting effects of beta-naphthoflavone (BNF), a novel aryl hydrocarbon receptor (AhR) agonist, were investigated using a medium-term hepatocarcinogenesis model in rats. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) at a dose of 200mg/kg body weight and were fed a diet containing 0% (basal diet), 0.5% or 1% BNF for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after the BNF treatment. The number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the livers of rats treated with BNF with concomitantly increased cell proliferation compared to those in the livers of the DEN alone group. Global gene expression analysis and subsequent quantitative real-time reverse transcription-polymerase chain reaction revealed that BNF induced not only the 'AhR gene battery'Cyp1a1, Cyp1a2, Cyp1b1, Nqo1, Aldh3a1 and Ugt1a6 but also the transcription factor NF-E2-related factor 2 (Nrf2)-regulated genes such as Gstm1, Gpx2, Akr7a3 and Yc2 (and also Nqo1), presumably due to the adaptive response against BNF-triggered oxidative stress responses. Reactive oxygen species production increased in microsomes isolated from the livers of BNF-treated rats, and this enhancement was suppressed by the P450 inhibitor SKF-525A. Furthermore, BNF enhanced oxidative DNA damage and lipid peroxidation, estimated by the levels of 8-hydroxydeoxyguanosine (8-OHdG) and thiobarbituric acid-reactive substances. These results suggest that the administration of BNF at a high dose and over a long-term enhance oxidative stress responses which may contribute to its hepatocarcinogenic potential in rats.
Asunto(s)
Dietilnitrosamina/toxicidad , Inhibidores Enzimáticos/toxicidad , Hepatectomía , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/patología , Estrés Oxidativo/efectos de los fármacos , Lesiones Precancerosas/inducido químicamente , beta-naftoflavona/toxicidad , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Glutatión Transferasa/metabolismo , Inmunohistoquímica , Hígado/enzimología , Hígado/patología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Lesiones Precancerosas/patología , Ratas , Ratas Endogámicas F344 , Especies Reactivas de Oxígeno/metabolismo , Receptores de Hidrocarburo de Aril/efectos de los fármacos , Receptores de Hidrocarburo de Aril/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
In order to clarify the possible mechanism of hepatocarcinogenesis induced by piperonyl butoxide, we attempted to identify the transcription factor activated by piperonyl butoxide in the male ICR mouse liver. Administration of 0.6% piperonyl butoxide for 24 h elevated the level of liver nuclear proteins that bind to an AP-1 consensus oligonucleotide, and these proteins demonstrated a supershift with the anti-c-Jun antibody. Additionally, immunoblot analysis revealed that piperonyl butoxide induced c-Jun phosphorylation within 8 h of administration, and phosphorylated ATF-2 was detected after 24 h of piperonyl butoxide treatment. Immunohistochemical analysis also demonstrated the presence of phosphorylated ATF-2 in the hepatocyte nuclei of mice fed with 0.6% piperonyl butoxide for 24 h. Furthermore, piperonyl butoxide induced ATF-2 phosphorylation in TLR-3, a mouse immortalized hepatocyte cell line. These results indicated that piperonyl butoxide activated c-Jun and ATF-2 in mouse hepatocytes during the early stage of hepatocarcinogenesis.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Carcinógenos/toxicidad , Hepatocitos/efectos de los fármacos , Butóxido de Piperonilo/toxicidad , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Línea Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Hepatocitos/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos ICR , Fosforilación , Factores de Tiempo , Factor de Transcripción AP-1/metabolismoRESUMEN
To examine the tumor modification activity of kojic acid (KA) by sodium ascorbic acid (AA), 5-week-old male ICR mice were administered intraperitoneally with N-diethylnitrosamine (DEN) as an initiation treatment. Two weeks after the initiation treatment, animals were fed basal diet containing 0 (Group 1: DEN alone) or 3% KA (Group 3: DEN+KA), drinking water containing 5,000 ppm AA (Group 2: DEN+AA) or 3% KA and 5,000 ppm AA (Group 4: DEN+KA+AA) for 6 weeks. One week after the administration of KA and/or AA, all mice were subjected to two-thirds partial hepatectomy. At the end of the experimental period, all surviving mice were sacrificed and removed the liver. The liver weights of the Groups 3 and 4 were significantly increased, and the number of proliferating cell nuclear antigen positive hepatocytes and the gene expressions of Ccnc, Ccnd1, Ercc and Cyp7a1 were significantly increased in the Group 4, as compared to the Group 1. These results of the present study suggest that AA enhances the hepatocellular proliferative activity of KA in mice.
Asunto(s)
Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Neoplasias Hepáticas Experimentales/patología , Pironas/farmacología , Animales , Peso Corporal/efectos de los fármacos , Dietilnitrosamina/metabolismo , Interacciones Farmacológicas , Inmunohistoquímica , Hígado/efectos de los fármacos , Hígado/metabolismo , Neoplasias Hepáticas Experimentales/inducido químicamente , Masculino , Ratones , Ratones Endogámicos ICR , Análisis de Secuencia por Matrices de Oligonucleótidos , Tamaño de los Órganos/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba/efectos de los fármacosRESUMEN
To evaluate the carcinogenicity of troglitazone in rasH2 mice, 7-week-old male and female rasH2 mice were fed a diet containing 0, 3,000 or 6,000 ppm troglitazone for 26 weeks. An increased tendency in the incidence of vascular tumors was observed in females of the 6,000 ppm group. The preliminary analysis using a high-density oligonucleotide microarray on a splenic hemangiosarcoma of a high dose female that could be obtained as a fresh sample showed that several genes related to the ras/MAPK pathway activation, angiogenesis, cell cycle and cell multiplication were up-regulated. In addition, most of the genes up-regulated were confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR). These results may suggest that the carcinogenic susceptibility of rasH2 mice to troglitazone is relatively low and up-regulations of the ras/MAPK pathway and angiogenesis-related genes are probably involved in the production of splenic hemangiosarcomas in rasH2 mice given troglitazone.
Asunto(s)
Cromanos/toxicidad , Expresión Génica/efectos de los fármacos , Genes ras/genética , Predisposición Genética a la Enfermedad , Tiazolidinedionas/toxicidad , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/patología , Administración Oral , Animales , Pruebas de Carcinogenicidad/métodos , Cromanos/administración & dosificación , Relación Dosis-Respuesta a Droga , Femenino , Perfilación de la Expresión Génica , Hemangiosarcoma/genética , Hemangiosarcoma/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/toxicidad , Masculino , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Sexuales , Tiazolidinedionas/administración & dosificación , Factores de Tiempo , Troglitazona , Vacuolas/efectos de los fármacosRESUMEN
To clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by piperonyl butoxide (PBO), male F344 rats were administered an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Two weeks later, the rats were administered a PBO-containing (0, 1, or 2%) diet for 6 weeks and subjected to a two-third partial hepatectomy 1 week later. After sacrificing them on week 8, their livers were histopathologically examined and analyzed for gene expression using a microarray and real-time RT-PCR. Reactive oxygen species (ROS) products were also measured using liver microsomes. Hepatocytes exhibited centrilobular hypertrophy and increased glutathione S-transferase placental form (GST-P) positive foci formation. ROS products increased significantly in liver microsomes. In the microarray analysis, the expressions of genes related to metabolism and oxidative stress - NAD(P)H dehydrogenase, quinone 1 (Nqo1), UDP-glucuronosyltransferase (UDPGTR-2), glutathione peroxidase 2 (Gpx2), glutathione reductase (GRx) - multidrug resistance associated protein 3 (Abcc3), and solute carrier family 7 (cationic amino acid transporter, y+ system) member 5 (Slc7a5) were up-regulated in the PBO group in comparison to the 0% PBO group; this was confirmed by real-time RT-PCR. Additionally, a significant up-regulation of stress response related genes such as CYP1A1 was observed in PBO-treated groups in real-time RT-PCR. HPLC analysis revealed that the level of 8-OHdG in the 2% PBO group was significantly higher than that in the 0% PBO group. This suggests that PBO has the potential to generate ROS via metabolic pathways and induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in rats.
Asunto(s)
Neoplasias Hepáticas Experimentales/metabolismo , Estrés Oxidativo , Sinergistas de Plaguicidas , Butóxido de Piperonilo , Animales , ADN/metabolismo , Desoxiadenosinas/metabolismo , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/patología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The effect of oxfendazole (OX), a benzimidazole anthelmintic, on hepatic gene expression was investigated in the liver of rats as a preliminary study to elucidate the possible mechanism of its non-genotoxic hepatocarcinogenesis. The liver from a male F344/N rat given a diet containing 500 ppm of OX for 3 weeks was examined by global gene expression analysis in comparison with an untreated rat. Microarray analysis revealed that phase I and phase II detoxifying enzymes were up-regulated in an OX-treated rat. In addition to these genes, the expressions of several upregulated genes related to xenobiotic metabolism and oxidative stress [e.g. Cyp1a1; NAD(P)H dehydrogenase, quinone 1 (Nqo1); glutathione peroxidase 2 (Gpx2); glutathione S-transferase Yc2 subunit (Yc2)], were confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). Furthermore, rats were administered 500 or 1,000 ppm of OX for 9 weeks, and the effect of OX on oxidative stress responses was evaluated by real-time RT-PCR along with conventional toxicological assays, including lipid peroxidation (thiobarbituric acid-reactive substance; TBARS). A longer treatment period and/or a higher dose of OX tended to increase the gene expressions of not only phase I (Cyp1a1 and Cyp1a2) but also phase II (Nqo1, Gpx2, Yc2, and Akr7a3) drug metabolizing enzymes. Toxicological parameters, such as TBARS, serum aspartate aminotransferase (AST), and serum alkaline phosphatase (ALP), showed slight but significant increases after treatment with OX for 9 weeks. These results indicate that OX elicits adaptive responses against oxidative stress in the liver and suggest that the imbalance in redox status might be one of the factors triggering the initial step of OX-induced non-genotoxic carcinogenesis in the liver of rats.
Asunto(s)
Antihelmínticos/toxicidad , Bencimidazoles/toxicidad , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hígado/efectos de los fármacos , Fosfatasa Alcalina/sangre , Animales , Aspartato Aminotransferasas/sangre , Perfilación de la Expresión Génica , Hígado/metabolismo , Hígado/patología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas F344 , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Regulación hacia ArribaRESUMEN
Siraitia grosvenori extract has been used as a food additive. As a part of the safety assessment of the extracts, a 13-week repeated dose toxicity study was performed in Wistar Hannover (GALAS) rats. Male and female rats were divided into five groups consisting of eight animals each and given diet containing 0%, 0.04%, 0.2%, 1%, and 5% of S. grosvenori extract for 13 weeks. During the experiment, no deaths were observed in any groups, and there were no remarkable changes in general appearance, body weight, food and water consumption, hematological and serum biochemical parameters, organ weight and histopathological findings between the control and treated groups. On the basis of these data, the no-observed-adverse effect level (NOAEL) of S. grosvenori extract in Wistar Hannover rats was considered to be 5% (2520 mg/kg/day in males and 3200 mg/kg/day in females) or more.
Asunto(s)
Cucurbitaceae/química , Aditivos Alimentarios/toxicidad , Extractos Vegetales/toxicidad , Pruebas de Toxicidad/métodos , Animales , Peso Corporal/efectos de los fármacos , Pruebas de Química Clínica , Dieta , Relación Dosis-Respuesta a Droga , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Femenino , Aditivos Alimentarios/administración & dosificación , Pruebas Hematológicas , Masculino , Nivel sin Efectos Adversos Observados , Tamaño de los Órganos/efectos de los fármacos , Extractos Vegetales/administración & dosificación , Ratas , Ratas WistarRESUMEN
To investigate the relationship between fenofibrate (FF) and oxidative stress, enzymatic, histopathological, and molecular biological analyses were performed in the liver of male F344 rats fed 2 doses of FF (Experiment 1; 0 and 6000 ppm) for 3 weeks and 3 doses (Experiment 2; 0, 3000, and 6000 ppm) for 9 weeks. FF treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl-CoA oxidizing system, and catalase in the liver. However, it decreased those of superoxide dismutase in the liver in both experiments. Increased 8-hydroxy-2'-deoxyguanosine levels in liver DNA and lipofuscin accumulation were observed in the treated rats of Experiment 2. In vitro measurement of reactive oxygen species (ROS) in rat liver microsomes revealed a dose-dependent increase due to FF treatment. Microarray (only Experiment 1) or real-time reverse transcription-polymerase chain reaction analyses revealed that the expression levels of metabolism and DNA repair-related genes such as Aco, Cyp4a1, Cat, Yc2, Gpx2, Apex1, Xrcc5, Mgmt, Mlh1, Gadd45a, and Nbn were increased in FF-treated rats. These results provide evidence of a direct or indirect relationship between oxidative stress and FF treatment. In addition, increases in the expression levels of cell cycle-related genes such as Chek1, Cdc25a, and Ccdn1; increases in the expression levels of cell proliferation-related genes such as Hdgfrp3 and Vegfb; and fluctuations in the expression levels of apoptosis-related genes such as Casp11 and Trp53inp1 were observed in these rats. This suggests that cell proliferation induction, apoptosis suppression, and DNA damage due to oxidative stresses are probably involved in the mechanism of hepatocarcinogenesis due to FF in rats.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Daño del ADN , Fenofibrato/toxicidad , Hígado/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proliferadores de Peroxisomas/toxicidad , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Biotransformación/genética , Catalasa/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica/métodos , Antígeno Ki-67/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/metabolismo , Factores de TiempoRESUMEN
Microarray and RT-PCR analyses were performed for the transgene and Ras-related genes in forestomach squamous cell carcinomas (SCCs) induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rasH2 mice; these results were compared with our previous molecular data of N-ethyl-N-nitrosourea-induced forestomach SCCs and urethane-induced lung adenomas in rasH2 mice. Overexpression of the transgene was detected in the DMBA-induced SCCs, suggesting that the transgene plays an important role in enhanced carcinogenesis in rasH2 mice. In addition, the mouse endogenous ras genes were up-regulated in the DMBA-induced SCCs, and are probably involved in the tumorigenesis of forestomach SCCs. Genes such as osteopontin, Cks1b, Tpm1, Reck, gelsolin, and amphiregulin that were commonly altered in these three different carcinogen-induced tumors may contribute to the development of tumors in rasH2 mice.
Asunto(s)
Carcinoma de Células Escamosas/genética , Perfilación de la Expresión Génica , Genes ras/genética , Neoplasias Gástricas/genética , 9,10-Dimetil-1,2-benzantraceno , Animales , Carcinógenos , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/patología , Análisis por Conglomerados , Humanos , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Neoplasias Gástricas/inducido químicamente , Neoplasias Gástricas/patologíaRESUMEN
Our previous studies showed the possibility that oxidative stress, including oxidative DNA damage, is involved in the mechanism of dicyclanil (DC)-induced hepatocarcinogenesis at the preneoplastic stage in mice. In this study, the expression analyses of genes, including oxidative stress-related genes, were performed on the tissues of hepatocellular tumors in a two-stage liver carcinogenesis model in mice. After partial hepatectomy, male ICR mice were injected with N-diethylnitrosamine (DEN) and given a diet containing 0 or 1500 ppm of DC for 20 weeks. Histopathological examinations revealed that the incidence of hepatocellular tumors (adenomas and carcinomas) significantly increased in the DEN + DC group. Gene expression analysis on the microdissected liver tissues of the mice in the DEN + DC group showed the highest expression levels of oxidative stress-related genes, such as Cyp1a1 and Txnrd1, in the tumor areas. However, no remarkable up-regulation of Ogg1-an oxidative DNA damage repair gene-was observed in the tumor areas, but the expression of Trail-an apoptosis-signaling ligand gene-was significantly down-regulated in the tumor tissues. These results suggest the possibility that the inhibition of apoptosis and a failure in the ability to repair oxidative DNA damage occur in the hepatocellular DC-induced tumors in mice.
Asunto(s)
Carcinoma Hepatocelular/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hepáticas Experimentales/patología , Lesiones Precancerosas/patología , Animales , Apoptosis/genética , Peso Corporal/efectos de los fármacos , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Cocarcinogénesis , Enzimas Reparadoras del ADN/genética , Dietilnitrosamina , Hepatectomía , Hormonas Juveniles/toxicidad , Rayos Láser , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/genética , Masculino , Redes y Vías Metabólicas/genética , Ratones , Ratones Endogámicos ICR , Microdisección/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Estrés Oxidativo/genética , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Piperonyl butoxide (PBO), alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes--cytochrome P450 (Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase (Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.