RESUMO
The present study aimed to investigate the potentiality of certain biocontrol agents, namely Bacillus subtilis, B. pumilus, B. megaterium, Pseudomonas fluorescens, Serratia marcescens, Trichoderma album, T. harzianum and T. viride, as well as the synthetic fungicide difenoconazole to control celery powdery mildew caused by Erysiphe heraclei DC, in vitro (against conidia germination and germ tube length of E. heraclei) and in vivo (against disease severity and AUDPC). In vitro, it was found that the antifungal activity of the tested biocontrol agents significantly reduced the germination percentage of the conidia and germ tube length of the pathogen. The reduction in conidia germination ranged between 88.2% and 59.6% as a result of the treatment with B. subtilis and T. album, respectively compared with 97.1% by the synthetic fungicide difenoconazole. Moreover, the fungicide achieved the highest reduction in germ tube length (92.5%) followed by B. megaterium (82.0%), while T. album was the least effective (62.8%). Spraying celery plants with the tested biocontrol agents in the greenhouse significantly reduced powdery mildew severity, as well as the area under the disease progress curve (AUDPC), after 7, 14, 21 and 28 days of application. In this regard, B. subtilis was the most efficient followed by B. pumilus, S. marcescens and B. megaterium, with 80.1, 74.4, 73.2 and 70.5% reductions in disease severity, respectively. In AUDPC, reductions of those microorganisms were 285.3, 380.9, 396.7 and 431.8, respectively, compared to 1539.1 in the control treatment. On the other hand, the fungicide difenoconazole achieved maximum efficacy in reducing disease severity (84.7%) and lowest AUDPC (219.3) compared to the other treatments. In the field, all the applied biocontrol agents showed high efficiency in suppressing powdery mildew on celery plants, with a significant improvement in growth and yield characteristics. In addition, they caused an increase in the concentration of leaf pigments, and the activities of defense-related enzymes such as peroxidase (PO) and polyphenol oxidase (PPO) and total phenol content (TPC). In conclusion, the results showed the possibility of using tested biocontrol agents as eco-friendly alternatives to protect celery plants against powdery mildew.
RESUMO
Tuberculosis remains a major public health hazard worldwide, with neonates and young infants potentially more susceptible to infection than adults. BCG, the only vaccine currently available, provides some protection against tuberculous meningitis in children but variable efficacy in adults, and is not safe to use in immune compromised individuals. A safe and effective vaccine that could be given early in life, and that could also potentiate subsequent booster immunization, would represent a significant advance. To test this proposition, we have generated gene-based vaccine vectors expressing Ag85B from Mycobacterium tuberculosis (Mtb) and designed experiments to test their immunogenicity and protective efficacy particularly when given in heterologous prime-boost combination, with the initial DNA vaccine component given soon after birth. Intradermal delivery of DNA vaccines elicited Th1-based immune responses against Ag85B in neonatal mice but did not protect them from subsequent aerosol challenge with virulent Mtb H37Rv. Recombinant adenovirus vectors encoding Ag85B, given via the intranasal route at six weeks of age, generated moderate immune responses and were poorly protective. However, neonatal DNA priming following by mucosal boosting with recombinant adenovirus generated strong immune responses, as evidenced by strong Ag85B-specific CD4+ and CD8+ T cell responses, both in the lung-associated lymph nodes and the spleen, by the quality of these responding cells (assessed by their capacity to secrete multiple antimicrobial factors), and by improved protection, as indicated by reduced bacterial burden in the lungs following pulmonary TB challenge. These results suggest that neonatal immunization with gene-based vaccines may create a favorable immunological environment that potentiates the pulmonary mucosal boosting effects of a subsequent heterologous vector vaccine encoding the same antigen. Our data indicate that immunization early in life with mycobacterial antigens in an appropriate vaccine setting can prime for protective immunity against Mtb.
Assuntos
Aciltransferases/imunologia , Adenoviridae/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Portadores de Fármacos , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Vacinas de DNA/imunologia , Aciltransferases/genética , Administração através da Mucosa , Animais , Antígenos de Bactérias/genética , Carga Bacteriana , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Humanos , Pulmão/microbiologia , Masculino , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Resultado do Tratamento , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Flagellin has been tested as a protein-based vaccine adjuvant, with the majority of studies focused on antibody responses. Here, we evaluated the adjuvant activity of flagellin for both cellular and humoral immune responses in BALB/c mice in the setting of gene-based immunization, and have made several novel observations. DNA vaccines and adenovirus (Ad) vectors were engineered to encode mycobacterial protein Ag85B, with or without flagellin of Salmonella typhimurium (FliC). DNA-encoded flagellin given IM enhanced splenic CD4+ and CD8+ T cell responses to co-expressed vaccine antigen, including memory responses. Boosting either IM or intranasally with Ad vectors expressing Ag85B without flagellin led to durable enhancement of Ag85B-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with significantly improved protection against challenge with pathogenic aerosolized M. tuberculosis. However, inclusion of flagellin in both DNA prime and Ad booster vaccines induced localized pulmonary inflammation and transient weight loss, with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in levels of mucosal CD4+ and CD8+ T cell responses following IM DNA/IN Ad mucosal prime-boosting, although antibody responses were not diminished. These findings indicate that flagellin has differential and route-dependent adjuvant activity when included as a component of systemic or mucosally-delivered gene-based prime-boost immunization. Clear adjuvant activity for both T and B cell responses was observed when flagellin was included in the DNA priming vaccine, but side effects occurred when given in an Ad boosting vector, particularly via the pulmonary route.
Assuntos
Adjuvantes Imunológicos , Flagelina/genética , Flagelina/imunologia , Vacinas de DNA/imunologia , Aciltransferases/genética , Aciltransferases/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Citocinas/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Imunização , Imunização Secundária , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Tuberculose/prevenção & controle , Vacinas de DNA/administração & dosagemRESUMO
BACKGROUND: An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. METHODS: We used ovalbumin (OVA)-based mouse models of asthma and primary CD4(+) T cells. C57BL/6J WT or PARP-1(-/-) mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. RESULTS: Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-γ. These traits were associated with a decrease in splenic CD4(+) T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1(-/-) mice. Adoptive transfer of Th2-skewed OT-II-WT CD4(+) T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-γ, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1(-/-)mice suggesting a role for PARP-1 in CD4(+) T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4(+) T cells while causing a moderate elevation in t-bet and ifn-γ expression in Th1-skewed CD4(+) T cells. CONCLUSIONS: Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials.
Assuntos
Asma/tratamento farmacológico , Asma/imunologia , Linfócitos T CD4-Positivos/imunologia , Técnicas de Inativação de Genes , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Transferência Adotiva , Animais , Antígenos CD/metabolismo , Asma/complicações , Hiper-Reatividade Brônquica/complicações , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Eosinofilia/complicações , Eosinofilia/tratamento farmacológico , Eosinofilia/imunologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Humanos , Imunoglobulina E/biossíntese , Camundongos Endogâmicos C57BL , Muco/metabolismo , Ovalbumina/imunologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Baço/imunologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/metabolismoRESUMO
Stem cells are identified as a novel cell therapy for regenerative medicine because of their ability to differentiate into many functional cell types. We have shown earlier a new model of hepatotoxicity in mice by administering (1500 mg/kg) epigallocatechin-3-gallate (EGCG) intragastric (IG) for 5 days after a single intraperitoneal dose (6 mg/kg) of lipopolysaccharide (LPS). In this study, we aimed to study the effect of intrahepatic (IH) injection of mouse embryonic stem cells (MESCs) on the hepatotoxicity induced by EGCG/LPS in mice. Mice were administered EGCG/LPS and rested for 3 days. MESCs were obtained from American Type Culture Collection and cultured in vitro for 4 days. Stem cells were injected IH. Seven days later, a single dose of LPS (6 mg/kg) followed by daily doses of IG administration of EGCG were re-administered for 5 days. At the end of the experiment, blood samples were collected for analysis of biochemical parameters associated with liver. Results showed that the group of mice that were administered MESCs prior to EGCG/LPS showed lower levels of alanine amino transferase, alkaline phosphatase, and bilirubin, higher albumin/globulin ratio, and less remarkable histopathological lesions. Also, that group of mice showed less expression of oxidative stress biomarkers (oxidized low-density lipoprotein Ox.LDL and chemokine CXCL16), less expression of nuclear protein receptors (retinoic acid receptor and retinoid X receptor), and less expression of inflammatory biomarkers (tumor necrosis factor α and transforming growth factor ß1) compared with other groups of mice that were not given MESCs. In conclusion, MESCs can ameliorate EGCG/LPS-induced hepatotoxicity in mice.
Assuntos
Catequina/análogos & derivados , Doença Hepática Induzida por Substâncias e Drogas/terapia , Células-Tronco Embrionárias , Lipopolissacarídeos , Transplante de Células-Tronco , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Amilases/sangue , Animais , Aspartato Aminotransferases/sangue , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Quimiocina CXCL16 , Quimiocina CXCL6/metabolismo , Lipoproteínas LDL/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
To investigate the ability of topotecan, a topoisomerase I-targeting anticancer drug, to induce dominant lethal mutations in male mouse germ cells, males were treated with single doses of 3, 6 and 12 mg/kg topotecan. Each male was mated at 4-day intervals to virgin females for a total of nine 4-day mating intervals. The two highest doses of topotecan are shown to be mutagenic in post-meiotic cells. The greatest effect occurred in those cells which were in the early-spermatid stage at the time of exposure. Mice treated with 12 mg/kg topotecan showed an additional peak of dominant lethal induction in mature sperm during the first 4-day matings after treatment. The mutagenic effects were directly correlated with free radicals accumulation as an obvious increase in the generation reactive oxygen species and 8-hydroxydeoxyguanosine was noted in animals treated with 6 and 12 mg/kg topotecan. Treatment of male mice with N-acetylcysteine, a free radical scavenger, significantly protected mice from topotecan-induce dominant lethality. Moreover, N-acetylcysteine had no antagonizing effect on topotecan-induce topoisomerase-I inhibition. Our study provides evidence that topotecan is a germ cell mutagen and its effect is more pronounced during the post-meiotic stages through a mechanism that may involves increases in DNA oxidative stress.
Assuntos
Mutagênicos/toxicidade , Espermatozoides/efeitos dos fármacos , Inibidores da Topoisomerase I/toxicidade , Topotecan/toxicidade , 8-Hidroxi-2'-Desoxiguanosina , Acetilcisteína/farmacologia , Animais , Antineoplásicos/toxicidade , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Relação Dose-Resposta a Droga , Feminino , Sequestradores de Radicais Livres/farmacologia , Masculino , Camundongos , Testes de Mutagenicidade , Gravidez , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: To circumvent the paucity of the primary adenovirus (Ad5) receptor and the non-specific Ad5 tropism in the context of uterine leiomyoma cells, Ad5 modification strategies would be beneficial. METHODS: We screened several modified adenoviruses to identify the most efficient and selective virus toward human leiomyoma cells to be used as candidate for delivering therapeutic genes. We propagated: wild-type Ad5-luc, fiber-modified viruses: ad5 RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc, as well as transcriptional targeted viruses: ad5 survivin-luc, Ad5-heparanase-luc, Ad5-MSLN-CRAD-luc and Ad5-SLPI-luc, on 293 cells and purified them by double CsCL density centrifugation. Then we transfected primary cultures of human leiomyoma cells derived from fibroids of four different patients, telomerase-immortalized human leiomyoma cell line (huLM), telomerase-immortalized normal human myometrial cell line (HM9) and immortalized normal human liver cells (THLE3) with the viruses at 5, 10 and 50 plaque-forming units (PFU)/cell. After 48 h, luciferase activities were measured and normalized to the total cellular protein content. RESULTS: Ad5-RGD-luc and Ad5-CAV2-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc at 5, 10 and 50 pfu/cell showed significantly higher expression levels of luciferase activity in both primary and immortalized human leiomyoma cells when compared with Ad5-Luc. Additionally, these modified viruses demonstrated selectivity toward leiomyoma cells, compared with myometrial cells and exhibited lower liver cell transduction, compared with Ad5-luc, at the same dose levels. CONCLUSIONS: Ad5-CAV2-luc, Ad5-RGD-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc are promising delivery vehicles in the context of leiomyoma gene therapy.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Leiomioma/terapia , Leiomioma/virologia , Feminino , Humanos , Fígado/citologia , Mesotelina , Miométrio/citologia , Miométrio/virologia , Receptores Virais/genéticaRESUMO
The objective of the current study was to investigate the ability of orthovanadate to induce aneuploidy in mouse sperm and micronuclei in mouse bone marrow cells at the same dose levels. The BrdU-incorporation assay was performed to test if the chemical treatment altered the duration of the meiotic divisions. It was found that orthovanadate (25mg/kg bw) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated by intraperitoneal (i.p.) injection with 5, 15 or 25mg/kg bw orthovanadate and sperm were sampled from the Caudae epididymes 22 days later. Fluorescence in situ hybridization (FISH) was performed with DNA-probes for chromosomes 8, X or Y. Significant increases in the frequencies of total hyperhaploid sperm (p<0.01) were found with 15 and 25mg/kg bw orthovanadate, indicating induced non-disjunction during male meiosis. The dose-response was described best by a linear equation. Orthovanadate did not significantly increase the frequencies of diploid sperm at any of the three doses tested, indicating that no complete meiotic arrest occurred. Orthovanadate was investigated also by the micronucleus test at i.p. doses of 1, 5, 15 or 25mg/kg bw, followed by bone marrow sampling 24h after treatment. None of the orthovanadate doses caused a significant increase in the rates of micronuclei (MN). Since the results show that orthovanadate induced non-disjunction during male meiosis without an accompanying induction of MN in bone marrow erythrocytes under the present experimental conditions and doses, it is concluded that male germ cells (meiosis) are more sensitive to the aneugenic effects of orthovanadate than somatic cells (mitosis). However, induction of micronuclei was reported in the literature with orthovanadate, vanadylsulfate and ammonium metavanadate, which contradicts the notion that vanadium compounds might be unique germ cell aneugens.
Assuntos
Aneugênicos/toxicidade , Aneuploidia , Eritrócitos/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Espermatozoides/efeitos dos fármacos , Vanadatos/toxicidade , Animais , Células da Medula Óssea/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hibridização in Situ Fluorescente , Masculino , Camundongos , Testes para MicronúcleosRESUMO
The therapeutic value of doxorubicin (DOX) as anticancer antibiotic is limited by its cardiotoxicity. The implication of natural phenolic acids in the prevention of many pathologic diseases has been reported. Herein, the ability of p-coumaric (PC) acid, a member of phenolic acids, to protect rat's heart against DOX-induced oxidative stress was investigated. Three main groups of albino rats were used; DOX, PC, and PC plus DOX-receiving animals. Corresponding control animals were also used. DOX was administered i.p. in a single dose of 15mgkg(-1). PC alone, in a dose of 100mgkg(-1), was orally administered for five consecutive days. In PC/DOX group, rats received PC 5 days prior to DOX. DOX-induced high serum levels of lactic dehydrogenase (LDH) and creatine phosphokinase (CPK), were reduced significantly by PC administration, compared to DOX-receiving rats. Pretreatment with PC ameliorated the cardiac content of glutathione (GSH), and superoxide dismutase (SOD) & catalase (CAT) activities, compared to DOX-receiving rats. On the other hand, accumulation of cardiac content of MDA significantly decreased following PC pretreatment, compared to DOX-treated rats. The data presented here indicate that PC protects rats hearts against DOX-induced oxidative stress in the heart. It may be worthy to consider the usefulness of PC as adjuvant therapy in cancer management.
Assuntos
Antibacterianos/farmacologia , Ácidos Cumáricos/farmacologia , Doxorrubicina/farmacologia , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Creatina Quinase/metabolismo , Glutationa/metabolismo , L-Lactato Desidrogenase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Oxirredução , Propionatos , Proteínas/metabolismo , Ratos , Superóxido Dismutase/metabolismoRESUMO
The topoisomerase II (topo II) inhibitors etoposide (VP-16) and merbarone (MER) were investigated with the in vivo micronucleus test (MN test) combined with fluorescence in situ hybridization (FISH) using the mouse minor satellite DNA probe to discriminate MN of clastogenic and aneugenic origin. All experiments were performed with male (102/ElxC3H/El) F1 mice bred in the mouse colony of the GSF Research Center. The sample size per experimental group was five animals and 2,000 polychromatic erythrocytes (PCE) were scored per animal from coded slides in the conventional MN test. A separate set of coded slides was used for the FISH analysis. All treatments consisted of single intraperitoneal injections. Colchicine (COL, 3 mg/kg) and mitomycin (MMC, 1 mg/kg) were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. A dose of 1 mg/kg VP-16 induced 3.44% MNPCE (compared to the concurrent solvent control of 0.37%, P < 0.001) and of these 39.9% (1.4% MNPCE) showed one or more fluorescent signals. MER (7.5-60 mg/kg) increased the MNPCE frequencies in a dose-dependent manner, with 15 mg/kg being the lowest positive dose. At the highest dose of 60 mg/kg of MER, a total of 4.26% MNPCE were found (compared to 0.31% in the concurrent solvent control, P < 0.001) and of these 46.2% (2.0% MNPCE) contained one or more fluorescent signals. The data demonstrate that VP-16 and MER induced both clastogenic and aneugenic events despite their different modes of topo II inhibition.
Assuntos
Aneugênicos/toxicidade , Medula Óssea/efeitos dos fármacos , Cromossomos/genética , Inibidores Enzimáticos/toxicidade , Etoposídeo/toxicidade , Mutagênicos/toxicidade , Tiobarbitúricos/toxicidade , Aneuploidia , Animais , Antibióticos Antineoplásicos/toxicidade , Colchicina/toxicidade , Sondas de DNA , DNA Satélite , Eritrócitos/efeitos dos fármacos , Supressores da Gota/toxicidade , Hibridização in Situ Fluorescente , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C3H , Testes para Micronúcleos , Mitomicina/toxicidade , Inibidores da Topoisomerase IIRESUMO
Relato de caso de paciente de 16 anos de idade, com diagnóstico de infecçäo cérvico vaginal por Papilomavírus Humano (HPV), sugerido inicialmente através de citologia oncótica pela presença evidente de coilócitos, critério patognomônico para HPV e posteriormente comprovado por Captura Híbrida a presença de HPV de alto potencial oncogênico. Realizou-se também colposcopia, onde se observaram lesöes em colo e vulva características do mesmo agente, contudo o estudo histopatológico demonstrou alteraçöes displásicas, sem entretanto caracterizar presença de qualquer agente específico
Assuntos
Humanos , Feminino , Adolescente , Colposcopia , Papillomaviridae , Infecções por Parvoviridae , Esfregaço Vaginal , Colo do Útero , Técnicas Citológicas , Técnicas Histológicas , VulvaRESUMO
The ability of two topoisomerase II (topo II) inhibitors, etoposide (VP-16) and merbarone (MER), to induce meiotic delay and aneuploidy in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting male mice with 5-bromo-2'-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. At 20-24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). It was found that VP-16 (50mg/kg) treatment induced a meiotic delay of about 24h. A significant reduction of BrdU-labelled sperm was observed at 22 days compared to the controls (VP-16 group: 14.20%; controls: 41.10%, P<0.001). At 23 and 24 days, there were no significant differences between the VP-16 and the control groups. MER (80 mg/kg) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated with 12.5, 25 and 50mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 days after VP-16 treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of total hyperhaploid sperm were found after treatment with 25, 50mg/kg VP-16 (0.074 and 0.122% versus 0.052%) and after treatment with 60 mg/kg MER (0.098% versus 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found after treatment of mice with all three doses of VP-16 (0.024, 0.032 and 0.056% versus 0.004 and 0.00%, respectively) and with 30 and 60 mg/kg MER (0.022 and 0.05% versus 0.004 and 0.002%, respectively). All dose responses could be expressed by linear equations. The results indicate that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors.
Assuntos
Aneuploidia , Antineoplásicos Fitogênicos/farmacologia , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Espermatócitos/efeitos dos fármacos , Tiobarbitúricos/farmacologia , Inibidores da Topoisomerase II , Animais , Bromodesoxiuridina , Aberrações Cromossômicas , Relação Dose-Resposta a Droga , Epididimo/efeitos dos fármacos , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Masculino , Meiose/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C3H , Microscopia de FluorescênciaRESUMO
We previously cloned recA-homolog genes from a basidiomycete, Coprinus cinereus, and obtained the recombinant proteins (Nara et al., Mol. Gen. Genet. 262, 781-789, 1999, see Ref. 1; Nara and Sakaguchi, Biochem. Biophys. Res. Commun. 275, 97-102, 2000, see Ref. 2). The primary purpose of the present study was to characterize the biochemical properties of the recombinant LIM15/DMC1 (CoLIM15) and RAD51 (CoRAD51) proteins. We purified the recombinant proteins, and their molecular masses were 37 and 35 kDa, respectively. Both enzymes showed DNA-dependent ATPase activity and ATP-dependent strand exchange reaction in vitro. CoRad51 was a five- to sixfold stronger DNA-dependent ATPase and showed greater dependency on single-stranded DNA than CoLim15. In meiosis, both enzymes were highly accumulated in the meiotic tissue at leptotene and zygotene stages at which the homologous chromosomes pair, but disappeared just before the pachytene stage at which they recombine. From these and the previously reported results, we discuss here the relationships between the enzymes and meiosis.
Assuntos
Adenosina Trifosfatases/metabolismo , Basidiomycota/enzimologia , Proteínas de Ciclo Celular , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Western Blotting , DNA de Cadeia Simples/metabolismo , Técnicas In Vitro , Rad51 Recombinase , Proteínas Recombinantes/metabolismoRESUMO
Understanding the mechanisms involved with genetic susceptibility to environmental disease is of major interest to the scientific community. We have conducted an in vitro study to elucidate the involvement of polymorphic metabolizing genes on the genotoxicity of benzo[a]pyrene (BP). Blood samples from 38 donors were treated with BP and the induction of sister chromatid exchanges (SCE) and chromosome aberrations (CA) were evaluated. The latter is based on the tandem-probe fluorescence in situ hybridization (FISH) assay. The data indicate that the induction of genotoxicity was clearly determined by the inherited variant genotypes for glutathione-S-transferase (GSTM1) and microsomal epoxide hydrolase (EH). In a comparison of the two biomarkers, the CA biomarker shows a more definite association with the genotypes than does SCE. For example, the presence of the GSTM1 null genotype (GSTM1 0/0) is responsible for the highest level and significant induction of CA, irrespective of the presence of other genotypes in the different donors. This effect is further enhanced significantly by the presence of the excessive activation EH gene allele (EH4*) and decreased by the reduced activation EH gene allele (EH3*). Overall, the modulation of genotoxicity by the susceptibility genotypes provides support of their potential involvement in environmental cancer. Furthermore, the data indicate that the variant enzymes function independently by contributing their metabolic capability toward the expression of biologic activities. Therefore, studies like this one can be used to resolve the complexity of genetic susceptibility to environmental disease in human.
Assuntos
Alelos , Benzo(a)pireno/toxicidade , Biotransformação/genética , Linfócitos/efeitos dos fármacos , Adulto , Benzo(a)pireno/metabolismo , Biomarcadores/sangue , Aberrações Cromossômicas , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Predisposição Genética para Doença , Genótipo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Hibridização in Situ Fluorescente , Linfócitos/metabolismo , Linfócitos/patologia , Pessoa de Meia-Idade , Testes de Mutagenicidade , Polimorfismo Genético , Valor Preditivo dos Testes , Troca de Cromátide Irmã/efeitos dos fármacos , Troca de Cromátide Irmã/genéticaRESUMO
BACKGROUND: Postsynaptic density (PSD)-95 interacts with and mediates clustering of the N-methyl-D-aspartate-receptors (NMDA-R). PSD-95 also interacts with the hDLG-associated protein DAP, which is also called Synapse-associated protein 90-associated protein (SAPAP), and Guanylate kinase-associated protein (GKAP). RESULTS: DAP interacted directly with the dynein light chain (DLC) family of proteins. DLC was contained in the NMDA-R-PSD-95-DAP-neuronal nitric oxide synthase (nNOS) complex. Furthermore, DAP interacted with nNOS and recruited it into the Triton X-100-insoluble fraction of transfected cells. CONCLUSION: DAP interacts directly with DLC and nNOS, and links these proteins to the NMDA-R-PSD-95 complex.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila , Proteínas do Tecido Nervoso/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Especificidade de Anticorpos , Sítios de Ligação/genética , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Linhagem Celular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Proteína 4 Homóloga a Disks-Large , Dineínas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Substâncias Macromoleculares , Proteínas de Membrana , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Óxido Nítrico Sintase Tipo I , Octoxinol , Testes de Precipitina , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Associadas SAP90-PSD95 , Técnicas do Sistema de Duplo-HíbridoRESUMO
AIMS AND BACKGROUND: Nausea and vomiting occur in the majority of patients receiving cisplatin (CDDP) chemotherapy. Ondansetron, a new 5-HT3 receptor antagonist, has been used effectively to control CDDP-induced nausea and vomiting. This study examined the potential of ondansetron to interfere with CDDP antitumor activity and toxicity in Ehrlich ascites carcinoma (EAC). METHODS: The influence of ondansetron on CDDP cytotoxicity was evaluated using EAC cells in culture. In addition, the influence of ondansetron pretreatment on CDDP-induced antitumor activity and host tissue toxicity was studied in EAC-bearing mice. RESULTS: Ondansetron (0.25 microM) enhanced CDDP (0-32 microM) cytotoxicity against EAC cells in vitro. In EAC-bearing mice ondansetron (0.2 mg/kg,ip) administered 1 h before CDDP (7 mg/kg, ip) did not modify the antitumor activity of CDDP. CDDP (7 mg/kg, ip) single treatment induced significant increases in blood urea nitrogen (2-fold) and serum creatinine (2.5-fold) and significant decreases in hematocrit (25%) and white blood cell count (39%) compared to saline treatment. Mice receiving ondansetron 1 h before CDDP showed no significant enhancement of CDDP-induced nephrotoxicity or myelosuppression compared to those pretreated with saline receiving the same dose of CDDP. CONCLUSIONS: This study suggests that the use of ondansetron to control CDDP-induced nausea and vomiting does not affect CDDP antitumor efficacy.
Assuntos
Antieméticos/uso terapêutico , Antineoplásicos/farmacologia , Carcinoma de Ehrlich/tratamento farmacológico , Cisplatino/farmacologia , Náusea/prevenção & controle , Ondansetron/uso terapêutico , Vômito/prevenção & controle , Análise de Variância , Animais , Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Camundongos , Náusea/induzido quimicamente , Células Tumorais Cultivadas , Vômito/induzido quimicamenteRESUMO
Polymorphisms in genes of xenobiotic-metabolizing enzymes are largely responsible for interindividual differences in ability to activate and detoxify genotoxic agents and therefore may influence individual susceptibility to environmental cancer. The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), requires metabolic activation by cytochrome P450 (CYP) enzymes to generate DNA-reactive intermediates that induce mutations and cancer. In the current study, we investigated the role of the polymorphic CYP2E1 and CYP2D6 genes in the genotoxicity of NNK using the tandem-probe fluorescence in-situ hybridization (FISH) chromosome aberration assay as a marker. Our results, using whole blood cultures from 39 volunteers, indicated that NNK (0.12, 0.24 or 0.72 mM) induced a concentration-dependent increase in the frequency of chromosome aberration. The potential role of CYP2E1 and CYP2D6 in NNK-induced genetic damage in cultured human lymphocytes was characterized using specific CYP inhibitors. Treatment of blood cultures with 25 microM diethyldithiocarbamate (DDC), a specific CYP2E1 inhibitor, or 0.5 microM quinidine, a specific CYP2D6 inhibitor, simultaneously with NNK, significantly decreased NNK-induced chromosome aberration. We also studied the role of CYP2E1 and CYP2D6 allelic variants on NNK-induced chromosome aberration. Our results indicate that NNK induced a significantly higher level of chromosome aberration in cells with the CYP2E1 WT/*5B genotype compared to cells with the CYP2E1 WT/WT. In contrast, no difference in NNK-induced chromosome aberration was observed between cells with the CYP2D6 extensive metabolizers compared to cells with the CYP2D6 poor metabolizer genotypes. These results underscore the important role of polymorphic metabolizing genes in influencing the genotoxic responses to environmental mutagens and provide support to the reported findings linking CYP2E1 polymorphism to smoking-related lung cancer.
Assuntos
Carcinógenos/toxicidade , Aberrações Cromossômicas , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2E1/genética , Nitrosaminas/toxicidade , Adulto , Alelos , Testes de Carcinogenicidade , Carcinógenos/metabolismo , Células Cultivadas , Inibidores do Citocromo P-450 CYP2D6 , Inibidores do Citocromo P-450 CYP2E1 , Predisposição Genética para Doença , Variação Genética , Humanos , Hibridização in Situ Fluorescente , Linfócitos/efeitos dos fármacos , Neoplasias/etiologia , Nitrosaminas/metabolismo , Fumar/efeitos adversosRESUMO
Cremophor EL (CR), the paclitaxel vehicle, has previously been reported to alter the pharmacokinetics and/or pharmacodynamics of some anticancer drugs including paclitaxel. Several experimental and clinical studies suggested that cisplatin (CDDP) in combination with paclitaxel results in less hematological toxicity than anticipated. To reveal the role of CR in this important pharmacological interaction, we evaluated the interaction of CR with CDDP in vitro and in vivo using experimental Ehrlich ascites carcinoma (EAC) tumor. CR (1 microg/ml) significantly enhanced the in vitro cytotoxicity of CDDP in cultured EAC cells. This enhancement was not associated with a parallel increase in CDDP cellular uptake. In tumor-bearing mice, CR (2.5 ml/kg, i.v.) given in combination with CDDP (7 mg/kg, i.v.) did not significantly change CDDP pharmacokinetics, antitumor activity or nephrotoxicity. On the other hand, CDDP-induced hematological toxicity was significantly reduced by CR. This protective effect was related to CR-induced inhibition of cellular CDDP accumulation in bone marrow. This study presents evidence that CR may play an important role in the pharmacological interaction between CDDP and paclitaxel. The present data may suggest formulation of CDDP with CR for systemic treatment. Further studies are yet necessary to establish the clinical value of CR as a modifier for CDDP therapeutic index.
Assuntos
Antineoplásicos/toxicidade , Carcinoma de Ehrlich/tratamento farmacológico , Cisplatino/toxicidade , Glicerol/análogos & derivados , Tensoativos/farmacologia , Análise de Variância , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Química Farmacêutica , Cisplatino/farmacocinética , Cisplatino/uso terapêutico , Interações Medicamentosas , Feminino , Glicerol/farmacologia , Camundongos , Veículos Farmacêuticos/farmacologia , Distribuição Tecidual , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Nucleotide excision repair (NER), the most versatile and ubiquitous mechanism for DNA repair, operates to remove many types of DNA base lesions. We have studied the role of p53 function in modulating the repair of DNA damage following UV irradiation in normal and p53-compromised human mammary epithelial cells (HMEC). The effect of UV-induced DNA damage on cellular cytotoxicity and apoptosis was determined in conjunction with global, gene- and strand-specific repair. Cytotoxicity studies, using clonogenic survival and MTT assays, showed that HPV-16 E6-expressing HMEC were more UV sensitive than p53-WT cell lines. High apoptotic index obtained with p53-compromised cells was in conformity to both the low clonogenic survival and the low cellular viability. No discernible differences in the formation of initial UV-induced cyclobutane pyrimidine dimers (CPD) were observed in the cell lines of varying p53 functional status. However, the extent and the rate of damage removal from genome overall were highest for p53-WT cells. Further examination of strand-specific repair in the p53 gene revealed that the removal of CPD in the non-transcribed strand (NTS) was slower in p53-compromised cells compared to the normal p53-WT cell lines. These results suggest that loss of p53 function, in the absence of other genetic alterations, decreased both overall amount of CPD repaired and their removal rate from the genome. Additionally, normal function of p53 is required for the repair of the NTS, but not of the transcribed strand (TS) in genomic DNA in human epithelial cells. Thus, failure of quantitative removal of CPD by global genomic repair (GGR), due to loss of p53 function, causes the enhanced UV sensitivity and increased damage-induced apoptosis via a p53-independent pathway. Nevertheless, recovery of cells from UV damage requires normal p53 function and efficient GGR.