RESUMEN
Phenolphthalein (800 and 2400 mg/kg/day by gavage and 2400 mg/kg/day by diet) and bisacodyl (800-500, 4000-2000, and 8000 mg/kg/day by gavage) were administered to 15 male and 15 female and 20 male and 20 female p53(+/-) mice respectively for 26 weeks to investigate the potential carcinogenicity of each compound. Toxicokinetic analyses confirmed systemic exposure. p-Cresidine was administered by gavage (400 mg/kg/day) and served as the positive control agent in each study. Dietary phenolphthalein reduced survival in both sexes and early deaths were attributed to thymic lymphoma. No bisacodyl-related neoplasms were observed. Regardless of route of administration to p53(+/-) mice, phenolphthalein but not bisacodyl was unequivocally genotoxic, causing increased micronuclei in polychromatic erythrocytes. In the Syrian hamster embryo (SHE) cell transformation assay, phenolphthalein caused increases in morphologically transformed colonies, thereby corroborating NTP's earlier reports, showing phenolophthalein has potential carcinogenic activity. Bisacodyl was negative in the SHE assay. Results of these experiments confirm an earlier demonstration that dietary phenolphthalein causes thymic lymphoma in p53(+/-) mice and show that (1) phenolphthalein causes qualitatively identical results in this transgenic model regardless of route of oral administration, (2) phenolphthalein shows evidence of micronucleus induction in p53(+/-) mice for up to 26 weeks, (3) phenolphthalein induced transformations in the in vitro SHE assay, and (4) bisacodyl in p53(+/-) mice induces neither drug-related neoplasm, nor micronuclei in polychromatic erythrocytes, and did not induce transformations in the in vitro SHE assay.
Asunto(s)
Bisacodilo/toxicidad , Catárticos/toxicidad , Micronúcleos con Defecto Cromosómico/inducido químicamente , Fenolftaleína/toxicidad , Neoplasias del Timo/inducido químicamente , Animales , Bisacodilo/sangre , Bisacodilo/farmacocinética , Carcinógenos/farmacocinética , Carcinógenos/toxicidad , Catárticos/farmacocinética , Transformación Celular Neoplásica , Células Cultivadas , Cricetinae , Femenino , Genes p53 , Linfoma/inducido químicamente , Linfoma/patología , Masculino , Mesocricetus/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pruebas de Micronúcleos , Fenolftaleína/sangre , Fenolftaleína/farmacocinética , Timo/efectos de los fármacos , Timo/patología , Neoplasias del Timo/patología , Proteína p53 Supresora de TumorRESUMEN
The Tg.AC transgenic mouse carries a v-Ha-ras transgene. Skin papillomas develop in Tg.AC mice upon repeated dermal application of tumor promoters and carcinogens. The transgene is inserted at a single site on chromosome 11 in a multiple-copy array. Although most of the >or= 40 copies are arranged in a direct-repeat orientation, two copies of the transgene are inserted in a palindromic, inverted-repeat orientation. Deletion of the palindromic transgene promoter sequence is associated strongly with and diagnostic of loss of phenotypic responsiveness to Tg.AC papillomagens, such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Unexpectedly, a loss of palindromic transgene sequence, in the absence of an observable reduction in copy number of the direct-repeat-oriented transgene sequence, is seen in DNA from papillomas when compared to genomic DNA from tail clips or skin samples away from the application site. Transgene-derived transcripts were detectable in all Tg.AC papillomas sampled. The transgene locus was hypomethylated in papillomas but not in samples from tail clips from the same animal or from skin samples away from the application site in responder Tg.AC mice, as shown by loss of resistance to digestion by HpaII. A cell line derived from a Tg.AC squamous cell carcinoma showed complete loss of the palindromic transgene sequence, hypomethylation of the transgene locus, and strong expression of v-Ha-ras mRNA. These data indicate that the palindromic transgene sequence, which appears to be necessary for initial responsiveness to tumorigens, may be susceptible to deletion during rapid cellular proliferation and is not required for transgene expression in later phases of papilloma growth.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes ras , Papiloma/genética , Neoplasias Cutáneas/genética , Animales , Carcinógenos/toxicidad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Ratones , Ratones Transgénicos , Papiloma/inducido químicamente , Regiones Promotoras Genéticas , ARN Mensajero/genética , Eliminación de Secuencia , Neoplasias Cutáneas/inducido químicamente , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
Microarrays are a new technology used to study global gene expression and to decipher biological pathways. In the current study, microarrays were used to examine gene expression patterns associated with cisplatin-mediated nephrotoxicity. Sprague-Dawley rats received either single or seven daily ip doses of cisplatin (0.5 or 1 mg/kg/day) or the inactive isomer transplatin (1 or 3 mg/kg/day). Histopathological evaluation revealed renal proximal tubular necrosis in animals that received cisplatin for 7 days, but no hepatotoxic findings. Microarray analyses were performed using rat specific arrays containing 250 toxicity-related genes. Prominent gene expression changes were observed only in the kidneys of rats that received cisplatin for 7 days. Mechanistically, the gene expression pattern elicited by cisplatin (e.g., Bax upward arrow and SMP-30 downward arrow) suggested the occurrence of apoptosis and the perturbation of intracellular calcium homeostasis. The induction of multidrug resistance genes (MDR1 upward arrow, P-gp upward arrow) and tissue remodeling proteins (clusterin upward arrow, IGFBP-1 upward arrow, and TIMP-1 upward arrow) indicated the development of cisplatin resistance and tissue regeneration. Select gene expression changes were further confirmed by TaqMan analyses. Gene expression changes were not observed in the liver following cisplatin administration. In contrast to these in vivo findings, studies using NRK-52E kidney epithelial cells and clone-9 liver cells suggested that liver cells were more sensitive to cisplatin treatment. The discrepancies between the in vivo and in vitro results suggest that caution should be taken when extrapolating data from in vivo to in vitro systems. Nonetheless, the current study elucidates the biochemical pathways involved in cisplatin toxicity and demonstrates the utility of microarrays in toxicological studies.
Asunto(s)
Cisplatino/toxicidad , Expresión Génica/efectos de los fármacos , Riñón/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Cisplatino/administración & dosificación , Clusterina , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Genes MDR/efectos de los fármacos , Glicoproteínas/metabolismo , Hepatocitos/efectos de los fármacos , Inyecciones Intraperitoneales , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Necrosis Tubular Aguda/inducido químicamente , Necrosis Tubular Aguda/metabolismo , Hígado/efectos de los fármacos , Masculino , Chaperonas Moleculares/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Sulfotransferasas , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Proteína X Asociada a bcl-2RESUMEN
The Tg.AC transgenic mouse carries the v-Ha-ras oncogene under the control of the zeta-globin promoter and is currently being used in a short-term carcinogenesis assay for safety testing of pharmaceuticals. A subset of hemizygous Tg.AC mice was found to be nonresponsive to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, which characteristically induces skin papillomas in these mice with repeated dermal applications. We previously showed that responder and nonresponder hemizygous Tg.AC mice carry about 40 copies of transgene but that the nonresponders had lost a 2-kb BamHI fragment containing the zeta-globin promoter sequence. The present restriction enzyme and S1 nuclease digestion experiments strongly suggested that the 2-kb BamHI fragment resulted from the orientation of two transgenes in an inverted repeat formation. Two subsets of nonresponder Tg.AC mice were identified. Restriction enzyme and S1 nuclease digestion experiments suggested that one nonresponder genotype was produced by a large deletion of one or more near complete copies of transgene sequence and the other genotype was produced by a small deletion near the apex of the "head-to-head" juncture of the inverted repeat. Polymerase chain reaction amplification, cloning, and sequencing results confirmed the palindromic orientation of transgene in Tg.AC mice. Our results indicated that, despite the presence of multiple copies of transgene in a direct repeat orientation, loss of symmetry in the palindromic array of transgene sequence results in the loss of the responder phenotype in Tg.AC mice. Mol. Carcinog. 30:99-110, 2001. Published 2001 Wiley-Liss, Inc.
Asunto(s)
Genes ras/genética , Globinas/genética , Ratones Transgénicos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Transgenes/genética , Animales , Secuencia de Bases , Southern Blotting , ADN/metabolismo , Eliminación de Gen , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fenotipo , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido Nucleico , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismoRESUMEN
Oxymetholone has been identified as a suspected nongenotoxic carcinogen and has recently completed testing in a conventional National Toxicology Program (NTP) 2-yr rodent bioassay program. As a synthetic androgen with a limited historical database in toxicology, oxymetholone is an ideal candidate for prospective examination of the performance of short-term transgenic mouse models in the detection of carcinogenic activity. In the present series of 3 articles, studies are described where oxymetholone was evaluated prior to disclosure of the results of the NTP 2-yr bioassay. The accompanying articles provide evidence showing that oxymetholone is devoid of mutagenic activity yet elicits a positive carcinogenic response in the Tg.AC transgenic mouse model. In the present study, oxymetholone was administered by oral gavage to p53 heterozygous male and female mice for 26 wk at doses of 125, 625, and 1,250 mg/kg/day. The vehicle was 0.5% aqueous methylcellulose. Positive controls consisted of mice treated daily by oral gavage with 200 or 400 mg/kg/day of p-cresidine in corn oil. The oxymetholone-treated females showed significantly increased body weight gain and clitoral enlargement attributable to drug treatment. In addition, significant alterations in kidney, liver, and testis weights were attributable to oxymetholone. However, there were no neoplastic lesions that were attributable to oxymetholone in either sex. p-Cresidine produced unequivocal bladder neoplasms in both sexes at the high dose and in males at the lower dose. The absence of a neoplastic response with oxymetholone is consistent with the selectivity of the p53-/- mouse model for detecting carcinogens that act by genotoxic mechanisms.
Asunto(s)
Anabolizantes/toxicidad , Oximetolona/toxicidad , Animales , Peso Corporal/efectos de los fármacos , Pruebas de Carcinogenicidad , Carcinógenos/toxicidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Genes p53/genética , Heterocigoto , Riñón/efectos de los fármacos , Riñón/patología , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Tamaño de los Órganos/efectos de los fármacos , Papiloma/inducido químicamente , Papiloma/genética , Papiloma/patología , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Análisis de Supervivencia , Testículo/efectos de los fármacos , Testículo/patologíaRESUMEN
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.
Asunto(s)
Genes p53/genética , Polipropilenos/efectos adversos , Sarcoma Experimental/patología , Transductores/efectos adversos , Anabolizantes/toxicidad , Animales , Carcinógenos/toxicidad , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Femenino , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oximetolona/toxicidad , Sarcoma Experimental/etiología , Sarcoma Experimental/genética , Piel/efectos de los fármacos , Piel/patología , Piel/ultraestructura , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Análisis de SupervivenciaRESUMEN
Several rodent models are under examination as possible alternatives to the classical 2-yr carcinogenicity bioassay. The Tg.AC transgenic mouse has been proposed as a shorter term model offering the possibility of detecting nongenotoxic and genotoxic carcinogenic agents. Retrospective studies of chemicals with established carcinogenic potential have revealed a close correlation between classical bioassay results and the production of skin tumors in the Tg.AC mouse model. Oxymetholone is a synthetic testosterone derivative that is a suspected carcinogen but has shown no evidence of genotoxic activity in a comprehensive battery of genetic toxicity assays. It currently is being tested by the National Toxicology Program (NTP) in a 2-yr rat carcinogenicity bioassay. Because of its nongenotoxicity and the ongoing chronic bioassay, oxymetholone was considered an ideal candidate for a prospective evaluation of the predictive validity of the Tg.AC dermal carcinogenicity model. Consequently, a 6-mo dermal study with oxymetholone in the Tg.AC mouse model was initiated and completed prior to disclosure of the NTP rat bioassay results. In this study, male and female hemizygous Tg.AC mice, 7-8 wk old, were housed individually in suspended plastic cages. An area of dorsal skin was shaved to accommodate dermal applications of 200-microl doses of vehicle control (acetone), drug (1.2, 6.0, or 12 mg oxymetholone in dimethylsulfoxide:acetone, 20:80), or positive control (1.25 microg 12-o-tetradecanoyl-phorbol-13-acetate [TPA]) solutions. Mice received oxymetholone or acetone daily or TPA twice weekly for 20 wk followed by a 6-wk recovery period. The acetone control groups exhibited low spontaneous incidences of papillomas, whereas dermal application of oxymetholone produced dose-related increases in the numbers of papilloma-bearing mice and the numbers of papillomas per animal. Females showed a somewhat greater response to the androgen than did the males. TPA caused an unequivocal increase in papillomas, with males exhibiting a greater response than females. The results of this study indicate that this nongerotoxic androgenic compound possesses proliferative properties. The results predict that chronic systemic administration of oxymetholone will most likely be associated with increased incidences of neoplasms.
Asunto(s)
Anabolizantes/toxicidad , Oximetolona/toxicidad , Animales , Pruebas de Carcinogenicidad , Carcinógenos/toxicidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales/inducido químicamente , Papiloma/mortalidad , Papiloma/patología , Factores Sexuales , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Tasa de Supervivencia , Aumento de Peso/efectos de los fármacosRESUMEN
Assessment of the carcinogenic potential of chemical agents continues to rely primarily upon the chronic rodent bioassay, a resource-intensive exercise. Recent advances in transgenic technology offer a potential resource conserving approach to carcinogen detection. Incorporation of oncogenes with known roles in the development of neoplasms into the genomes of laboratory rodents may provide new models with the potential of quickly and accurately separating carcinogenic from noncarcinogenic chemicals. The insertion of the v-Ha-ras oncogene into the genome of FVB/N mice imparts the qualities of genetically initiated skin in the transgenic mouse line designated as Tg.AC. The skin of either hemizygous (animals carrying the transgene on 1 allele) or homozygous (transgene copies on both alleles) Tg.AC mice promptly responds to the application of nongenotoxic carcinogens, such as the classical tumor promoting phorbol esters, with the development of squamous papillomas. Tumor production generally begins after 8-10 applications of 2.5 micrograms/mouse (3 times/wk) of 12-O-tetradecanoylphorbol 13-acetate (TPA). Maximal tumor response is usually in evidence within 20 wk. If this transgenic mouse line is to be useful in the identification of carcinogenic chemicals, experimental protocols must be systematically optimized. Experiments were conducted to compare the relative responsiveness of male and female hemizygous and homozygous Tg.AC mice to the dermal application of TPA and the known human leukemogen, benzene. Results revealed shipment-related variabilities in the relative responsiveness of hemizygous male and female mice to the application of the proliferative agent. Homozygous mice of both sexes were more reliable and uniform in responsiveness to both TPA and benzene. Therefore, our standard protocol for the conduct of bioassays with the Tg.AC mouse line specifies the use of homozygous males and/or females.
Asunto(s)
Benceno/toxicidad , Carcinógenos/toxicidad , Ratones Transgénicos/fisiología , Neoplasias Cutáneas/inducido químicamente , Acetato de Tetradecanoilforbol/toxicidad , Administración Tópica , Animales , Benceno/administración & dosificación , Carcinógenos/administración & dosificación , Femenino , Masculino , Ratones , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol/administración & dosificaciónRESUMEN
2,5-Hexanedione (2,5-HD) exposure in the rat produces irreversible testicular atrophy, a model of human male infertility that can be used for mechanistic and therapeutic studies. Following testicular injury by 2,5-HD, stem cell factor (SCF), a Sertoli cell-derived growth factor that binds the c-kit receptor on spermatogonia, is altered in its expression, changing from predominantly membrane SCF to predominantly soluble SCF. The goals of this study were 2-fold: first, evaluate leuprolide, a GnRH agonist, as a therapy for 2,5-HD-induced testicular atrophy, and second, examine changes in SCF expression during testicular injury and following recovery from injury. Rats exposed to 2,5-HD showed a nearly complete testicular atrophy that could be reversed by leuprolide therapy. Using RT-PCR, preferential expression of membrane SCF was associated with spermatogenesis, whereas soluble SCF expression was associated with atrophy. In conclusion, 2,5-HD exposure altered the form of SCF expressed and disrupted spermatogenesis; leuprolide therapy allowed recovery of spermatogenesis, which correlated with a normalization in growth factor expression in an otherwise irreversibly atrophic testis.
Asunto(s)
Hexanonas/toxicidad , Leuprolida/farmacología , Espermatogénesis/efectos de los fármacos , Factor de Células Madre/biosíntesis , Testículo/efectos de los fármacos , Animales , Atrofia , Masculino , Ratas , Ratas Endogámicas F344 , Testículo/patología , Testículo/fisiopatologíaRESUMEN
To study transgene expression in the adult rat testis in vivo, an adenovirus vector carrying a lacZ transgene with a nuclear localization signal was used as a marker. The adenovirus vector was first tested on rat Sertoli cell-germ cell cocultures in vitro; it efficiently mediated transgene expression in Sertoli cells but not germ cells. This vector was then delivered to the interstitial compartment of adult rat testes by intratesticular injection, resulting in Leydig cells expressing the transgene. Alternatively, delivering the vector to the intratubular compartment by rete testis injection resulted in expression of the transgene by Sertoli cells of the seminiferous epithelium and principal cells of the epididymis. In vivo, each cell type expressed the transgene by 2 days postinfection, and expression persisted for at least 10 days; however, later time points were associated with a loss of transgene expression and focal interstitial inflammation. This study documents the ability of adenovirus to mediate gene transfer to specific testicular cells, providing a powerful tool to study the short-term effects of specific genes on spermatogenesis in vivo.
Asunto(s)
Adenoviridae/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Testículo/metabolismo , beta-Galactosidasa/genética , Animales , Células Cultivadas , Epidídimo/metabolismo , Expresión Génica , Células Intersticiales del Testículo/metabolismo , Masculino , Ratas , Ratas Endogámicas F344 , Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismoRESUMEN
2,5-Hexanedione (2,5-HD) is a Sertoli cell toxicant that causes irreversible testicular atrophy in rats. After toxicant exposure, only Sertoli cells, stem cells, and a few committed type A spermatogonia remain in the seminiferous epithelium. A majority of the stem cell progeny differentiate into type A spermatogonia, but then, rather than continuing to differentiate, undergo apoptosis. We hypothesized that the cause for germ cell apoptosis was, at least in part, a deficiency in the function of stem cell factor (SCF), a paracrine growth factor normally made by Sertoli cells. To test this hypothesis, rats were exposed to 1% 2,5-HD for 5 wk and killed at various times after toxicant exposure. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine that, unlike what was observed in control testes, the majority of SCF was expressed in the soluble form after 2,5-HD injury. In vitro co-culture experiments were used to establish the appropriate dose of SCF to administer in vivo. A continuous intratesticular delivery system was established and used to expose 2,5-HD-treated rats to SCF for 2 wk. Animals were exposed to bromodeoxycytidine (BrdCyd) for 2 days before being killed in order to assess the effect of SCF on germ cell proliferation. SCF caused a statistically significant increase in the number of germ cells positive for bromodeoxyuridine (BrdUrd), indicating that SCF promoted survival and/or stimulated proliferation of the remaining germ cells. We conclude that SCF expression is disrupted after 2,5-HD-induced testicular atrophy and that exogenous administration of SCF promotes recovery of spermatogenesis.
Asunto(s)
Hexanonas/toxicidad , Factor de Células Madre/administración & dosificación , Factor de Células Madre/fisiología , Enfermedades Testiculares/fisiopatología , Testículo/patología , Animales , Apoptosis , Atrofia , Secuencia de Bases , División Celular , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Masculino , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Espermatozoides/patología , Factor de Células Madre/genética , Enfermedades Testiculares/inducido químicamente , Enfermedades Testiculares/patología , Testículo/efectos de los fármacosRESUMEN
2,5-hexanedione (2,5-HD) is a Sertoli cell toxicant which causes germ cell loss and testicular atrophy in the rat. The mechanism of germ cell death over the course of 2,5-HD treatment is not known nor is the reason why residual germ cells do not repopulate the seminiferous epithelium following toxicant withdrawal. In the current study, the role of apoptosis in germ cell loss was studied. Male Fischer rats were treated for up to 5 weeks with 1% 2,5-HD in the drinking water and killed between 0 and 12 weeks after the start of toxicant exposure. Apoptosis was assessed in control and treated animals by (1) DNA fragmentation detected by gel electrophoresis, (2) cellular morphology on plastic sections, and (3) DNA fragmentation in situ by terminal deoxy-nucleotidyl transferase-mediated digoxigenin-UTP nick end label (TUNEL) staining of testis cross sections. All three indices demonstrated a substantial increase in apoptosis which peaked at 5 weeks of 2,5-HD treatment. Morphological analysis determined that apoptosis occurred in germ cells of the seminiferous epithelium. DNA fragmentation determined by gel electrophoresis was barely detectable until 5-6 weeks of toxicant exposure. However, TUNEL staining of testis cross sections indicated that germ cell apoptosis increased after as early as 2 weeks of toxicant exposure, providing a highly sensitive biological marker of toxicant-induced testicular injury. These data also suggested a differential sensitivity of germ cells to toxicant exposure with spermatid apoptosis occurring first at 4-5 weeks of treatment followed by apoptosis of spermatocytes and spermatogonia between 6 and 12 weeks. Together, these data demonstrate that apoptosis is the mechanism of germ cell loss in 2,5-HD-induced testicular injury.
Asunto(s)
Apoptosis/efectos de los fármacos , Hexanonas/toxicidad , Espermatozoides/efectos de los fármacos , Espermatozoides/patología , Testículo/efectos de los fármacos , Testículo/patología , Animales , Atrofia/inducido químicamente , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , ADN/análisis , ADN/efectos de los fármacos , ADN/metabolismo , ADN Nucleotidilexotransferasa , Digoxigenina , Electroforesis en Gel de Agar , Hexanonas/farmacocinética , Masculino , Ratas , Ratas Endogámicas F344 , Coloración y Etiquetado/métodos , Uridina TrifosfatoRESUMEN
Cyclopentadienyl manganese tricarbonyl (CMT) produces acute pulmonary injury following cytochrome P450 mixed-function oxidase (CYP450) activation. The current studies were designed to characterize the role of hepatic and/or pulmonary CMT activation and the subsequent pneumotoxicity of this compound following subcutaneous injection in the male Sprague-Dawley rat. Both pulmonary and hepatic tissues were capable of CYP450-dependent CMT metabolism in vitro. Phenobarbital pretreatment, which induced hepatic but not pulmonary CMT metabolism, protected against CMT-depended pneumotoxicity suggesting escape of an active CMT metabolite from the liver is not responsible for the pneumotoxic response. Animals were also pretreated with either m-xylene or 3-methylindole, each of which reduce CMT metabolism in the lung but not in the liver. These pretreatments also reduced CMT-dependent pulmonary damage. Protection against toxicity by two compounds that inhibit pulmonary but not hepatic CMT metabolism provides strong evidence that CMT-induced pneumotoxicity is due to the activation of CMT within the lungs. Histopathological studies revealed that CMT induced an alveolar injury without apparent damage to the bronchiolar airways. Based on this pattern of injury, studies were performed with freshly isolate alveolar type II (ATII) cells as these cells are thought to contain significant CYP450 activity. However, CMT metabolism was not detectable in ATII cells in vitro. Although CMT was cytotoxic to ATII cells in vitro, this response was not inhibited by metyrapone indicating CYP450 activation was not involved in the in vitro phenomenon. Together these data suggest in situ activation of CMT is necessary for the alveolar toxicity of this compound; however, activation does not occur in ATII cells.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Pulmón/efectos de los fármacos , Intoxicación por Manganeso , Compuestos Organometálicos/toxicidad , Animales , Biotransformación , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Hígado/efectos de los fármacos , Hígado/metabolismo , Pulmón/metabolismo , Pulmón/patología , Masculino , Manganeso/metabolismo , Microsomas/enzimología , Compuestos Organometálicos/metabolismo , Fenobarbital/farmacología , Alveolos Pulmonares/citología , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/patología , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Escatol/farmacologíaRESUMEN
The effect of m-xylene on the rat nasal cytochrome P450 (P450) mixed function oxidase system was analyzed in vitro utilizing microsomes isolated 2, 12, and 24 h following intraperitoneal administration of this solvent in vivo. For comparative purposes, pulmonary and hepatic activities were also measured. Benzyloxyresorufin O-deethylation (BROD) and ethoxyresorufin O-deethylation (EROD), catalytic activities linked with P450 isozymes IIB1 and IA1, respectively, were inhibited in nasal tissue at all times following m-xylene administration. Pulmonary tissue mimicked this m-xylene-dependent inhibition of BROD activity but did not display significant inhibition of EROD activity. In contrast, m-xylene caused a dramatic induction of both BROD and EROD activity in hepatic tissue. The metabolism of a third P450 substrate, cyclopentadienyl manganese tricarbonyl (CMT), was also analyzed. m-Xylene caused significant inhibition of CMT metabolism at all time points in both nasal and pulmonary microsomes but was without effect on hepatic microsomal metabolism of this compound. These data show an inhibitory effect of m-xylene on rat nasal and pulmonary but not hepatic cytochrome P450-dependent metabolism.
Asunto(s)
Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Oxigenasas de Función Mixta/efectos de los fármacos , Mucosa Nasal/enzimología , Xilenos/toxicidad , Animales , Citocromo P-450 CYP1A1 , Citocromo P-450 CYP2B1 , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/enzimología , Pulmón/enzimología , Masculino , Microsomas/enzimología , Oxigenasas de Función Mixta/metabolismo , Compuestos Organometálicos/metabolismo , Oxidorreductasas/metabolismo , RatasRESUMEN
A PB-PK model has been developed for nasal nonreactive vapor uptake in the F344 rat which incorporates nasal enzyme distribution as well as nasal airflow patterns. Nasal tissue is separated into respiratory and olfactory mucosal areas with each area containing mucus, epithelial, and submucosal compartments. Metabolic activities are distributed among the compartments in accordance with published histochemical data, and intercompartmental transfer rate constants are based on molecular diffusivity. Two airflow paths are assumed: a lateral/ventral path which passes over only respiratory mucosa and a dorsal medial path which passes over both respiratory and olfactory mucosa. This model was fit to F344 rat nasal uptake data obtained for five vapors: acetone (an unmetabolized vapor), isoamyl alcohol (alcohol dehydrogenase substrate), ethyl acetate (carboxylesterase substrate), and o-xylene and bromobenzene (mixed function oxidase substrates). These vapors cover a wide span of uptake values ranging from a fractional uptake of less than 0.01 for xylene to 0.80 for isoamyl alcohol. The model accurately predicted the fractional uptake of these vapors, the maximal deviation between actual and predicted values being 0.04. The best fit for the data was obtained assuming: (1) an asymmetric blood flow pattern (with the majority of the blood flow being apportioned to the respiratory mucosa), and (2) only a small fraction of the total airflow (8%) following the dorsal medial pathway and penetrating to the olfactory mucosa. Since only a small fraction of the inspired air passes over the olfactory mucosa, a site with high metabolic activity, an inspiratory flow rate dependence on inspired vapor metabolism results. At low flows, olfactory metabolism is limited by low vapor delivery to that site, an effect that does not occur at high flows. This highlights the potential importance of regional nasal airflow and enzymatic activity patterns in influencing uptake and metabolism of inspired vapors and indicates the need for physiologically relevant models incorporating these factors.
Asunto(s)
Modelos Biológicos , Mucosa Nasal/metabolismo , Farmacocinética , Animales , Ratas , Ratas Endogámicas F344 , Sensibilidad y Especificidad , VolatilizaciónRESUMEN
Inhalation exposure of rodents to high concentrations of acetaldehyde produces lesions in the upper respiratory tract (URT, all regions of the respiratory tract anterior to and including the larynx). Information on the inhalation dosimetric relationships for this vapor are needed for a comprehensive understanding of its inhalation toxicity. Toward this end, uptake of acetaldehyde was measured in the surgically isolated URT of the urethane-anesthetized male F344 rat under unidirectional (50, 100, 200, or 300 ml/min) and cyclic (100 ml/min) flow conditions at inspired concentrations of 1, 10, 100, or 1000 ppm. Under all flow conditions URT deposition efficiency was strongly dependent on inspired concentration. URT deposition efficiency (under cyclic flow) averaged 76, 48, 41, and 26% at 1, 10, 100, and 1000 ppm, respectively. Nasal acetaldehyde dehydrogenase activity averaged 1.2 micrograms/min. Absolute acetaldehyde deposition rates (micrograms/min) at 100 and 1000 ppm exceeded this activity by 5- to 100-fold, suggesting a possible mechanism for the reduced deposition efficiency at high concentrations. URT deposition under unidirectional flow was strongly dependent on the inspiratory flow rate. The effect of flow rate on deposition was reasonably predicted by the mass-transfer model of Aharonson et al. (J. Appl. Physiol. 37, 654-657, 1974). The uptake coefficients determined from the unidirectional flow studies were used to predict uptake under cyclic flow by integration of the model. The predicted cyclic deposition efficiencies differed from the observed efficiencies by 2.3 +/- 4.3% (mean +/- SEM), suggesting this model might provide a reasonable first approximation for acetaldehyde uptake under cyclic breathing conditions.