RESUMO
In the context of understanding lameness and injury from slipping, our objective was to characterize hoof impact and slide of 5 cows walking on 6 flooring surfaces commonly used in Ontario dairy farms: diamond-grooved concrete (DC), sanded epoxy-covered concrete (EC), grooved rubber mat (GR), high-profile rubber mat (HR), low-profile rubber mat (LR), and turf grass (TG; Kentucky bluegrass/fescue mix). Surface hardness was measured on each surface using a Clegg Impact Soil Tester. Five trained lactating Holstein cows were each walked over all 6 surfaces sequentially in a randomized order. Walking speeds were determined from 60-fps videos. A 3-axis accelerometer attached to the lateral claw of each hindfoot captured continuous horizontal (aH), vertical (aV), lateral (aTLat), and medial (aTMed) accelerations at 2,500 Hz during each trial, from which peak values were identified. Data from 45°-rosette strain gauges glued to the dorsal surface of both medial and lateral hooves allowed for the calculation of principal strains (ε1 and ε2). From continuous data, several data points were extracted from 3 to 6 stances/trial: peak values of aH, aV, and aT for the impact phase of the stance; midstance values of ε1 and ε2 as proxies for force on the foot; magnitudes of normal (i.e., consistent and repeatable) sliding on the surface during the support phase; and 3 timing events to capture the cadence of the motion. All aH and aV signals were inspected onscreen to identify irregularities between the end of impact and beginning of breakover that indicated hoof slipping, which was observed on all surfaces. The effects on all measured variables of surface, cow, speed, and hoof (and all significant higher-order factors) were assessed by ANOVA in SAS 9.4 (SAS Institute Inc.), after verifying data normality. Values of aHmax, indicating grip on the surface from highest to lowest, ranked the surfaces in this order: LR, DC, HR, GR, EC, and TG. Ranking on aVmax, indicating most to least cushioning of the hoof on impact, ranked the surfaces in this order: DC, HR, GR, EC, LR, and TG. Differences in ranking among these and other significant impact variables indicate that future studies of lameness on different surfaces need to include all significant variables identified here. We detected no surface and strain interactions in either the ε1 or ε2 strain, indicating that the surfaces do not affect the overall loads on the foot at midstance. Additionally, lateral and medial hooves may have different roles in a stance. The results highlight the capacity to evaluate flooring types with this technology, and the study provides a tool for future work to examine the role of flooring types in the causation of lameness.
Assuntos
Doenças dos Bovinos , Casco e Garras , Feminino , Bovinos , Animais , Fazendas , Lactação , Borracha , Coxeadura Animal , Ontário , Abrigo para AnimaisRESUMO
Genetically altered mouse models (GAMM) for human cancers have been critical to the investigation and characterization of oncogene and tumor suppressor gene expression and function and the associated cancer phenotype. Similarly, several of the mouse models with defined genetic alterations have shown promise for identification of potential human carcinogens and investigation of mechanisms of carcinogen-gene interactions and tumorigenesis. In particular, both the B6.129N5-Trp53 mouse, heterozygous for a p53 null allele, and the CB6F1-RasH2 mouse, hemizygous for the human H-ras transgene, have been extensively investigated. Using 26-week exposure protocols at or approaching the maximum tolerated dose, the summary results to date indicate the potential for GAMM to identify and, possibly, classify chemicals of potential risk to humans using short-term carcinogenicity experiments. This IWGT session focused on: (1) the development of recommendations for genetic/molecular characterization required in animals, tissues, and tumors before and after treatment for identification of presumptive human carcinogens based on the current state of knowledge, (2) identification of data gaps in our current state of knowledge, and (3) development of recommendations for research strategies for further development of our knowledge base of these particular models. By optimization of protocols and identification of significant outcomes and responses to chemical exposure in appropriate short-term mechanism-based genetically altered rodent models, strategies for prevention and intervention may be developed and employed to the benefit of public health.
Assuntos
Bioensaio/normas , Genes Supressores de Tumor , Proteína Supressora de Tumor p53/genética , Proteínas ras/genética , Animais , Testes de Carcinogenicidade/normas , Modelos Animais de Doenças , Camundongos , Proteína Supressora de Tumor p53/deficiência , Proteínas ras/metabolismoRESUMO
Immunization of transgenic mouse models of Alzheimer disease using amyloid-beta peptide (Abeta) reduces both the Alzheimer disease-like neuropathology and the spatial memory impairments of these mice. However, a therapeutic trial of immunization with Abeta42 in humans was discontinued because a few patients developed significant meningo-encephalitic cellular inflammatory reactions. Here we show that beneficial effects in mice arise from antibodies selectively directed against residues 4-10 of Abeta42, and that these antibodies inhibit both Abeta fibrillogenesis and cytotoxicity without eliciting an inflammatory response. These findings provide the basis for improved immunization antigens as well as attempts to design small-molecule mimics as alternative therapies.
Assuntos
Vacinas contra Alzheimer/uso terapêutico , Peptídeos beta-Amiloides/antagonistas & inibidores , Anticorpos/uso terapêutico , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Animais , Anticorpos/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Transgênicos , Dados de Sequência MolecularRESUMO
The p53 tumor suppressor gene has been shown to be critical in preventing cancer in humans and mice. We have generated and extensively characterized p53-deficient mice lacking one (p53+/-) or both (p53-/-) p53 alleles. The p53-deficient mice are much more susceptible to an array of different tumor types than their wild-type (p53+/+) littermates. The enhanced tumor susceptibility of the p53+/- mice has made them one of several transgenic mouse models that are being considered as substitutes for standard 2-year rodent carcinogenicity assays. In order to fully exploit this model, it will be important to understand some of the basic biological and molecular mechanisms that underlie its enhanced tumor susceptibility. With this in mind, we have explored the fate of the remaining wild-type p53 allele in spontaneously arising p53+/- tumors and have shown that over half of these tumors retain an intact, functional wild-type p53 allele. This suggests that p53 is haploinsufficient for tumor suppression and that mere reduction in p53 dosage is sufficient to promote cancer formation. To support the idea that p53 is indeed a haploinsufficient tumor suppressor, we show here that normal p53+/- cells exhibit reduced parameters of growth control and stress response compared to their p53+/- counterparts. We hypothesize that the reduced p53 dosage in the p53+/- cells provides an environment more conducive to the development of further oncogenic lesions and the initiation of a tumor. Finally, we have assessed p53 loss of heterozygosity (LOH) in carcinogen-induced p53+/- tumors and have found that some agents induce tumors that almost invariably exhibit p53 LOH, whereas other agents induce tumors that often retain the wild-type p53 allele. Our preliminary data suggest that LOH is dependent on both the mechanism of genotoxicity of the agent utilized and the tissue type targeted.
Assuntos
Testes de Carcinogenicidade/métodos , Carcinógenos/toxicidade , Genes p53 , Mutagênicos/toxicidade , Neoplasias Experimentais/induzido quimicamente , Alternativas aos Testes com Animais , Animais , Dosagem de Genes , Predisposição Genética para Doença , Genótipo , Perda de Heterozigosidade , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neoplasias Experimentais/genéticaRESUMO
The performance of the p53-/- transgenic (knockout) mouse model was evaluated through review of the data from 31 short-term carcinogenicity studies with 21 compounds tested as part of the International Life Sciences Institute's (ILSI) Alternatives to Carcinogenicity Testing (ACT) project, together with data from other studies which used comparable protocols. As expected based on the hypothesis for the model, a significant number (12/16 or 75%) of the genotoxic human and/or rodent carcinogens tested were positive and the positive control, p-cresidine, gave reproducible responses across laboratories (18/19 studies positive in bladder). An immunosuppressive human carcinogen, cyclosporin A, was positive for lymphomas but produced a similar response in wild type mice. Two hormones that are human tumorigens, diethylstilbestrol and 17beta-estradiol, gave positive and equivocal results, respectively, in the pituitary with p53-deficient mice showing a greater incidence of proliferative lesions than wild type. None of the 22 nongenotoxic rodent carcinogens that have been tested produced a positive response but 2 compounds in this category, chloroform and diethylhexylphthalate, were judged equivocal based on effects in liver and kidney respectively. Four genotoxic noncarcinogens and 6 nongenotoxic, noncarcinogens were also negative. In total (excluding compounds with equivocal results), 42 of 48 compounds or 88% gave results that were concordant with expectations. The technical lessons learned from the ILSI ACT-sponsored testing in the p53+/- model are discussed.
Assuntos
Testes de Carcinogenicidade/métodos , Carcinógenos/toxicidade , Modelos Animais de Doenças , Genes p53 , Mutagênicos/toxicidade , Neoplasias Experimentais/induzido quimicamente , Alternativas aos Testes com Animais , Animais , Relação Dose-Resposta a Droga , Feminino , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neoplasias Experimentais/genética , Reprodutibilidade dos TestesRESUMO
Symptoms of nasal, pharyngeal and ocular discomfort have been reported among workers in the wood surface-coating industry. Symptoms were reported more often by workers using ultraviolet radiation-curable acrylate coatings (UV coatings), which contain potential chemical sensitizers, than by those using acid-curing coatings. Furthermore, increased levels of eosinophil cationic protein (ECP) and albumin, but not tryptase, in nasal lavage from workers exposed to UV coatings have been observed. To further examine whether air contaminants present in the UV-coating industry are causing the observed increase in symptoms, the inflammatory process in the nasal mucosa of workers exposed to UV coatings was investigated. Clinical and biochemical endpoints were selected to distinguish between specific and non-specific hypersensitivity and to test the hypothesis that the symptoms were consistent with Type IV hypersensitivity. The nasal lavage and nasal biopsy were performed under local anesthetic at the workplace during working hours after a minimum of 2 h of work in both the exposed and control groups. Albumin and ECP, and the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8), were used as inflammatory markers. A multi-probe ribonuclease protection assay was used to attempt to detect cytokine variation in human nasal biopsies. The cytokine genes analyzed were TNF-alpha, GM-CSF, interferon-gamma, IL-2, IL-4 and IL-5. L32 and GAPDH were used as control genes for mRNA expression levels. Mucosal inflammation symptoms correlated with increased levels of albumin, but not with increased levels of ECP, secreted proinflammatory cytokines or cytokine gene mRNA expression. We conclude that the symptoms are non-specific and do not correlate with occupational exposure to UV coatings under the conditions of this investigation.
Assuntos
Indústria Química , Citocinas/genética , Expressão Gênica/genética , Cavidade Nasal/patologia , Exposição Ocupacional , Madeira , Albuminas/metabolismo , Biópsia , Citocinas/imunologia , Citocinas/metabolismo , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-8/genética , Interleucina-8/imunologia , Interleucina-8/metabolismo , Masculino , Líquido da Lavagem Nasal/imunologia , Doenças Profissionais/diagnóstico , Exposição Ocupacional/efeitos adversos , Doenças dos Seios Paranasais/diagnóstico , Doenças dos Seios Paranasais/etiologia , Doenças Faríngeas/diagnóstico , Doenças Faríngeas/etiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Epidemiologic studies support the protective role of dietary antioxidants in preventing cancer. However, emerging evidence from clinical trials and laboratory data suggest that in some cases individual antioxidant supplements may actually exacerbate carcinogenesis. Our goal was to explore these paradoxical activities in a rodent model that possesses genotypic characteristics of human cancers. We selected the p53 haploinsufficient Tg.AC (v-Ha-ras) mouse as a model, because it contains an activated, carcinogeninducible ras oncogene and an inactivated p53 tumor suppressor gene, which are frequent genetic alterations in human cancers. These mice develop chemically induced benign and malignant skin tumors rapidly which can easily be quantified. Mice were fed basal diets with or without 3% N-acetyl-L-cysteine (NAC), a well-recognized antioxidant, prior to, during and after topical application of the carcinogen benzo[a]pyrene (64 microg/mouse) applied twice per week for 7 weeks. Tumor incidence exceeded 90% for both groups, and NAC did not reduce tumor latency. Mice fed NAC displayed a 43% reduction (P < 0.05) in tumor multiplicity and delayed the appearance of lesions (P < 0.05). Dietary NAC also significantly (P < 0.05) improved group survival by 5 weeks. Total tumor yields were reduced in both dietary groups but malignant spindle cell tumors (SCT) increased by 25% in NAC-fed mice. The v-Ha-ras oncogene and p53 protein products were clearly co-expressed in both benign and malignant lesions from both dietary groups. In summary, dietary supplementation with NAC was chemopreventive, but the marginal increase in SCT suggests a paradoxical effect.
Assuntos
Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Genes p53/genética , Genes ras/genética , Neoplasias Cutâneas/prevenção & controle , Animais , Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/prevenção & controle , Cocarcinogênese , Cruzamentos Genéticos , Suplementos Nutricionais , Feminino , Sequestradores de Radicais Livres/farmacologia , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Haplótipos , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transgenes , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
Traditionally, the use of rodent models in assessing the carcinogenic potential of chemicals has been expensive and lengthy, and the relevance of the carcinogenic effect to humans is often not fully understood. Today, however, with the rapid advances in molecular biology, genetically altered mice containing genes relevant to humans (e.g. oncogenes, tumor suppressor genes) and reporter genes (e.g. lacI) provide powerful tools for examining specific chemical-gene interactions thereby allowing a better understanding of the mechanisms of carcinogenesis in a shorter period of time. This paper will cover an overview of ongoing validation efforts, followed by examples of studies using several genetically engineered models including the p53def mouse model and the Big Blue transgenic mouse model. Specifically, examples where transgenic models were integrated into the testing program based on specific hypotheses dealing with genetic alterations in cancer genes and reporter genes will be discussed. The examples will highlight possible ways genetically altered mice may be integrated into a comprehensive research and testing strategy and thereby provide an improved estimation of human health risks.
Assuntos
Testes de Carcinogenicidade , Carcinógenos/toxicidade , Proteínas de Escherichia coli , Genes p53/fisiologia , Modelos Animais , Animais , Proteínas de Bactérias/genética , Benzeno/toxicidade , Benzo(a)pireno/toxicidade , Butadienos/toxicidade , Repressores Lac , Camundongos , Camundongos Transgênicos , Oxazepam/toxicidade , Fenolftaleína/toxicidade , Proteínas Repressoras/genética , Uretana/toxicidadeRESUMO
C57BL/6 p53 (+/-) N5 mice heterozygous for a null p53 allele were given phenolphthalein to learn more about mechanisms of carcinogenesis and to evaluate the p53-deficient mouse as a tool for identifying potential human carcinogens. DNA samples isolated from 10 phenolphthalein-induced thymic lymphomas were analyzed for loss of heterozygosity (LOH) at the Trp53 locus and simple sequence length polymorphic (SSLP) loci. The initial screening revealed remarkable results from only chromosome 11. Allelotyping at approximately five centiMorgan intervals, we found SSLP heterozygosity for C57BL/6 and 129Sv over much of chromosome 11. In the tumors, treatment-related LOH was apparent on chromosome 11 at each of the 28 informative loci examined. The strain-specific polymorphism lost from individual tumors allowed us to deduce the distribution of alleles along the length of the maternal and paternal chromosomes 11. The allelic patterns indicate that mitotic homologous recombination occurred during embryogenesis if breeding protocols were carried out as described. The mitotic recombination observed may be attributable to p53 haploinsufficiency for normal suppression of mitotic recombination.
Assuntos
Perda de Heterozigosidade , Linfoma/induzido quimicamente , Linfoma/genética , Fenolftaleína/toxicidade , Neoplasias do Timo/induzido quimicamente , Neoplasias do Timo/genética , Animais , Primers do DNA/química , DNA de Neoplasias/análise , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo GenéticoRESUMO
In this study, we determined the induction and time-dependent accumulation of micronuclei in the peripheral blood of transgenic C57BL/6 p53+/- mice (p53+/- mice), FVB/N Tg.AC v-Ha-ras mice (Tg.AC mice) and their isogenic parental strains, FVB/N and C57BL/6 following inhalation exposure to benzene. Our objective was to determine the impact of p53 heterozygosity in p53+/- mice and the v-Ha-ras transgene in Tg.AC mice on micronuclei induction following exposure to inhaled benzene. A flow cytometric technique that distinguishes micronucleated red blood cells (MN-RBC) from micronucleated reticulocytes (MN-RET) was used. Mice were exposed to 0, 100 or 200 p.p.m. benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 p.p.m.x hours) to benezene: 100 p.p.m. for 6 h/day, 5 days/week, Monday to Friday (M-F); 100 p.p.m. for 10 h/day, 3 days/week, Monday, Wednesday, Friday (MWF); and 200 p.p.m. for 5 h/day, 3 days/week MWF. Significant elevations of MN-RBC and MN-RET were observed from 1 week exposure in all of the benzene-exposed groups that increased in a time-dependent manner for up to 13 weeks exposure. Fewer MN-RBC and MN-RET were induced in the 200 p.p.m. benzene exposure group than in mice exposed to 100 p.p.m. The reduction in the frequency of MN-RBC in the 200 p.p.m.x5 h benzene exposure group is probably due to metabolic saturation resulting in a lower bone marrow dose (concentration x time) than in the 100 p.p.m. exposure groups. No differences were observed in the frequency of MN-RBC or MN-RET in Tg.AC compared with the FVB/N isogenic controls. At certain time points the frequency of micronuclei was less in the heterozygous p53+/- mice than determined in the wild-type C57BL/6 isogenic parental strain. These results indicate that the heterozygous state in p53+/- mice, but not the v-Ha-ras transgene in Tg.AC mice can influence the induction of micronuclei by benzene.
Assuntos
Benzeno/farmacologia , Genes p53 , Genes ras , Exposição por Inalação , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Micronúcleos com Defeito Cromossômico/metabolismo , Animais , Genes p53/efeitos dos fármacos , Genes ras/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Micronúcleos com Defeito Cromossômico/genética , Testes para Micronúcleos , Reticulócitos/efeitos dos fármacos , Fatores de TempoRESUMO
Mice heterozygous for a null p53 allele were administered three well-characterized carcinogens to learn more about mechanisms of carcinogenesis and to evaluate the p53-deficient mouse as a tool for identifying potential human carcinogens. Benzene-induced sarcomas, p-cresidine-induced bladder carcinomas and phenolphthalein-induced thymic lymphomas were allelotyped at the Trp53 locus and chromosome 11 simple sequence length polymorphic (SSLP) loci. Loss of Trp53 and loss of one copy of chromosome 11 occurred in each of 10 lymphomas examined and each of the eight sarcomas examined. Loss of Trp53 and loss of heterozygosity (LOH) at SSLP loci were sporadic in the bladder carcinomas. However, LOH was detected at two or more SSLP loci in six of the eight bladder tumors examined. Loss of one complete copy of chromosome 11 was implicated in three of the bladder tumors where LOH occurred at seven or more widely dispersed SSLP loci. Loss of one copy of chromosome 11 likely occurred through a p53-mediated selection process since Trp53 is located on mouse chromosome 11 and only one copy harbored a functional gene. The data suggest that loss occurred through a mechanism common among the three tumor types. Allelotype patterns of the maternal chromosome 11 were inconsistent with those expected from a nullizygous C57BL/6-Trp53 (N4) x inbred C57BL/6 cross which was reported for production of the mice under investigation. However, comparison with individual control tissues still allowed deduction of maternal chromosome loss. If the breeding protocols were carried out as described, the unexpected allelotype patterns observed in histologically normal tissues might be due to mitotic homologous recombination during embryogenesis.
Assuntos
Carcinógenos/toxicidade , Genes p53/genética , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Proteína Supressora de Tumor p53/deficiência , Alelos , Compostos de Anilina/toxicidade , Animais , Benzeno/toxicidade , Testes de Carcinogenicidade , Cromossomos/genética , Cruzamentos Genéticos , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Feminino , Heterozigoto , Perda de Heterozigosidade , Linfoma/induzido quimicamente , Linfoma/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenolftaleína/toxicidade , Polimorfismo Genético , Sarcoma Experimental/induzido quimicamente , Sarcoma Experimental/genética , Neoplasias do Timo/induzido quimicamente , Neoplasias do Timo/genética , Proteína Supressora de Tumor p53/genética , Neoplasias da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/genéticaRESUMO
Mutagenic carcinogens rapidly induced tumors in the p53 haploinsufficient mouse. Heterozygous p53-deficient (+/-) mice were exposed to different mutagenic carcinogens to determine whether p53 loss of heterozygosity (LOH) was carcinogen-and tissue-dependent. For 26 weeks, C57BL/6 (N4) [corrected] p53-deficient (+/-) male or female mice were exposed to p-cresidine, benzene or phenolphthalein. Tumors were examined first for loss of the wild-type p53 allele. p-cresidine induced p53 LOH in three of 13 bladder tumors, whereas hepatocellular tumors showed p53 LOH in carcinomas (2/2), but not in adenomas (0/3). Benzene induced p53 LOH in 13 of 16 tumors examined. Finally, phenolphthalein induced p53 LOH in all tumors analyzed (21/21). Analysis of the p-cresidine-induced bladder tumors by cold single-strand conformation polymorphism (SSCP) analysis of exon 4-9 amplicons failed to demonstrate polymorphisms associated with mutations in tumors that retained the p53 wild-type allele. p-cresidine induced a dose-related increase in lacI mutations in bladder DNA. In summary, these data demonstrate that loss of the wild-type allele occurred frequently in thymic lymphomas and sarcomas, but less frequently in carcinomas of the urinary bladder. In the bladder carcinomas other mechanisms may be operational. These might include (i) other mechanisms of p53 inactivation, (ii) inactivating mutations occurring outside exons 4-9 or (iii) p53 haploinsufficiency creating a condition that favors other critical genetic events which drive bladder carcinogenesis, as evidenced by the significant decrease in tumor latency. Understanding the mechanisms of p53 LOH and chemical carcinogenesis in this genetically altered model could lead to better models for prospective identification and understanding of potential human carcinogens and the role of the p53 tumor suppressor gene in different pathways of chemical carcinogenesis.
Assuntos
Carcinógenos/toxicidade , Proteínas de Escherichia coli , Genes p53/efeitos dos fármacos , Perda de Heterozigosidade/efeitos dos fármacos , Neoplasias Experimentais/genética , Proteína Supressora de Tumor p53/deficiência , Alelos , Compostos de Anilina/toxicidade , Animais , Proteínas de Bactérias/genética , Benzeno/toxicidade , Feminino , Genes p53/genética , Repressores Lac , Linfoma/induzido quimicamente , Linfoma/genética , Linfoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese/efeitos dos fármacos , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Fenolftaleína/toxicidade , Polimorfismo Conformacional de Fita Simples , Proteínas Repressoras/genética , Sarcoma Experimental/induzido quimicamente , Sarcoma Experimental/genética , Sarcoma Experimental/patologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Neoplasias da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaAssuntos
Benzeno/toxicidade , Carcinógenos Ambientais/toxicidade , Leucemia Experimental/induzido quimicamente , Leucemia Experimental/genética , Camundongos Transgênicos , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Especificidade de ÓrgãosRESUMO
The purpose of this study was to examine the role of chromosomal recombination in mediating p53 loss in benzene-induced thymic lymphomas in C57BL/6-Trp53 haploinsufficient (N5) mice (p53+/- mice). We characterized loss of heterozygosity (LOH) on chromosome 11 using seven microsatellite markers in 27 benzene-induced and 6 spontaneous thymic lymphomas. Eleven patterns of LOH were found between the induced and spontaneous tumors, with only one pattern being in common between the tumor groups. Nearly 90% (24 of 27) of benzene-induced tumors exhibited loss of the functional p53 allele locus, and 83% (20 of 24) of these tumors retained two copies of the disrupted p53 allele. The results indicate that benzene induces a high frequency of LOH on chromosome 11 in p53+/- mice, likely mediated by aberrant chromosomal recombination.
Assuntos
Cromossomos , Deleção de Genes , Genes p53/genética , Linfoma/induzido quimicamente , Linfoma/genética , Recombinação Genética , Neoplasias do Timo/induzido quimicamente , Neoplasias do Timo/genética , Alelos , Animais , Benzeno , Southern Blotting , Dosagem de Genes , Genótipo , Perda de Heterozigosidade , Camundongos , Camundongos Transgênicos , Repetições de Microssatélites , Modelos Genéticos , Reação em Cadeia da PolimeraseRESUMO
The haplo-insufficient p53 knockout (p53+/-) and zetaglobin v-Ha-ras (Tg.AC) transgenic mouse models were compared to the conventional two rodent species carcinogen bioassay by prospectively testing nine chemicals. Seven of the chemicals classified as carcinogens in the conventional bioassay induced tumors in the liver or kidneys of B6C3F1 mice, and one (pentachlorophenol) also induced tumors in other tissues. Only three chemicals, furfuryl alcohol, pyridine, and pentachlorophenol, induced tumors in rats. The tumorigenic effect of pyridine was seen in F344 rats but not in Wistar strain rats. None of the chemicals induced tumors in the p53+/- transgenic mice, which is consistent with the absence of genotoxicity of these chemicals. Only two of the seven nongenotoxic carcinogens were positive in the Tg.AC model (lauric acid diethanolamine and pentachlorophenol). These results show that these transgenic models do not respond to many chemicals that show strain- or species-specific responses in conventional bioassays.
Assuntos
Carcinógenos/toxicidade , Genes ras , Neoplasias Renais/induzido quimicamente , Neoplasias Hepáticas Experimentais/induzido quimicamente , Proteína Supressora de Tumor p53/genética , Administração Oral , Administração Tópica , Animais , Testes de Carcinogenicidade , Modelos Animais de Doenças , Feminino , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Proteína Supressora de Tumor p53/deficiênciaRESUMO
Recent epidemiological evidence suggests that antioxidants may enhance carcinogenesis by promoting cellular proliferation and/or impeding programmed cell death. We examined the effect of N-acetyl-l-cysteine (NAC) on mitogenesis and apoptosis in splenocytes from p53 haploinsufficient Tg.AC (v-Ha-ras) mice. This model contains genetic lesions found frequently in human cancer and is predisposed to develop carcinogen-induced cancer. Splenocytes were incubated with NAC alone or with the B- and T-cell-specific mitogens Concanavalin A (Con A) and E. coli lipopolysaccharide (LPS), respectively. Mitogenesis increased 17-fold in mitogen-stimulated cultures and 10-fold in cultures incubated with NAC alone. Co-incubation with both NAC (1000 microg/mL) and mitogen increased mitogenesis by 33-fold without changing apoptosis rates. Strikingly, incubation with NAC and LPS attenuated LPS-induced apoptosis. Mitogen alone did not affect GSH levels but NAC-induced increases were significantly depleted by co-incubation with mitogen. Furthermore, NAC increased the number of CD45R+ B cells, but decreased CD3+ T cells showing enhanced survival of B cells under these conditions. These results demonstrate concurrent reduced apoptosis and increased mitogenesis in B lymphocytes that may favor clonal selection of preneoplastic cells.
Assuntos
Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Genes p53/genética , Genes ras/genética , Ativação Linfocitária/efeitos dos fármacos , Mitógenos/farmacologia , Mitose/efeitos dos fármacos , Proteína Supressora de Tumor p53/deficiência , Animais , Apoptose/genética , Apoptose/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Concanavalina A/farmacologia , Feminino , Glutationa/metabolismo , Haplótipos/genética , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Transgênicos , Mitose/imunologia , Proteína Oncogênica p21(ras)/genética , Oxirredução/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The potential for occupational exposure to the esters of acrylic acid (acrylates) is considerable, and, thus, requires a greater understanding of the their toxicity. Confluent (70-90%) cultures of normal human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF). or bronchial epithelium (NHBE) were exposed to the monofunctional ethyl acrylate (EA), the multifunctional tripropylene glycol diacrylate (TPGDA), or TPGDA monomer in a radiation curable lacquer (Lacquer A) at equimolar dosages in order to determine human in vitro cytotoxicity. Viability of the cells after 2-24-h exposure to the representative monofunctional or multifunctional acrylate or solvent control was used to calculate an index of acute cytotoxicity (50% inhibitory dose; ID50) and to determine the shape of the dose-response curves. TPGDA, Lacquer A, and EA were equally cytotoxic (ID50 is approximately equal to 0.1 micromol/cm2) to NHEK at equimolar doses. TPGDA or Lacquer A were more cytotoxic (is approximately equal to 100X) to NHDF or NHBE than EA. Sequential exposure of UV(A) and TPGDA to NHEK indicate the potential for a synergistic cytotoxic response. These findings are consistent with observed decreases in free sulfhydryl groups (e.g., glutathione or cysteine) that parallel the dose-response-related decreases in viability. logether, these data suggest possible differences in toxicity between the monofunctional EA and multifunctional TPGDA to NHEK, NHDF. or NHBE, possibly due to the difference in the number of functional acrylate groups and/or physicochemical differences (e.g., vapor pressure) between the acrylates investigated.
Assuntos
Acrilatos/efeitos adversos , Carcinógenos/efeitos adversos , Pulmão/efeitos dos fármacos , Propilenoglicóis/efeitos adversos , Pele/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Pulmão/citologia , Pele/citologiaRESUMO
Heterozygous p53+/- transgenic mice are being studied for utility as a short-term alternative model to the 2-yr rodent carcinogenicity bioassay. During a 26-wk study to assess the potential carcinogenicity of oxymetholone using p-cresidine as a positive control, glass/polypropylene microchips (radio transponder identification devices) were subcutaneously implanted into male and female p53+/- mice. During week 15, the first palpable mass was clinically observed at an implant site. This rapidly growing mass virtually quadrupled in size by week 25. Microscopic examination of all implant sites revealed that 18 of 177 animals had a subcutaneous histologically malignant sarcoma. The neoplasms were characterized as undifferentiated sarcomas unrelated to drug treatment, as indicated by the relatively even distribution among dose groups, including controls. An unusual preneoplastic mesenchymal change characterized by the term "mesenchymal dysplasia" was present in most groups and was considered to be a prodromal change to sarcoma development. The tumors were observed to arise from dysplastic mesenchymal tissue that developed within the tissue capsule surrounding the transponder. The preneoplastic changes, including mesenchymal dysplasia, appeared to arise at the transponder's plastic anchoring barb and then progressed as a neoplasm to eventually surround the entire microchip. Capsule membrane endothelialization, inflammation, mesenchymal basophilia and dysplasia, and sarcoma were considered unequivocal preneoplastic/neoplastic responses to the transponder and were not related to treatment with either oxymetholone or p-cresidine.
Assuntos
Genes p53/genética , Polipropilenos/efeitos adversos , Sarcoma Experimental/patologia , Transdutores/efeitos adversos , Anabolizantes/toxicidade , Animais , Carcinógenos/toxicidade , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Feminino , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oximetolona/toxicidade , Sarcoma Experimental/etiologia , Sarcoma Experimental/genética , Pele/efeitos dos fármacos , Pele/patologia , Pele/ultraestrutura , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Análise de SobrevidaRESUMO
Transgenic rodent models have emerged as potentially useful tools in the assessment of drug and chemical safety. The transgenic Tg.AC mouse carries an inducible v-Ha-ras oncogene that imparts the characteristic of genetically initiated skin to these animals. The induction of epidermal papillomas in the area of topically applied chemical agents, for duration of not more than 26 weeks, acts as a reporter phenotype that defines the activity of the test article. We describe here the activity of six chemicals that have been previously characterized for activity in the standard 2-year bioassay conducted by the National Toxicology Program (NTP). Homozygous female Tg.AC mice were treated with benzene (BZ), benzethonium chloride (BZTC), o-benzyl-p-chlorophenol (BCP), 2-chloroethanol (2-CE), lauric acid diethanolamine (LADA) and triethanolamine (TEA). BZ and LADA induced skin papillomas in a dose-dependent manner, while BCP induced papillomas only at the highest dose. BZTC, 2-CE, and TEA exhibited no activity. The correspondence of chemical activity in Tg.AC mice with that in the 2-year bioassay was high. A comparison of responsiveness to BZ and LADA was made between hemizygous and homozygous female Tg.AC mice. Both genotypes appear to be equally sensitive to maximum doses of active compounds. The results reported here indicate that the Tg.AC transgenic mouse model can discriminate between carcinogens and noncarcinogens and that both mutagenic and nonmutagenic chemicals can be detected. These studies provide support for the adjunctive use of the Tg.AC transgenic mouse skin tumor model in drug and chemical safety assessment and for the prediction of the carcinogenic potential of chemicals.