RESUMEN
BACKGROUND: Organophosphate pesticides affect mammalian brain development through mechanisms separable from the inhibition of acetylcholinesterase (AChE) enzymatic activity and resultant cholinergic hyperstimulation. In the brain, AChE has two catalytically similar splice variants with distinct functions in development and repair. The rare, read-through isoform, AChE-R, is preferentially induced by injury and appears to promote repair and protect against neurodegeneration. Overexpression of the more abundant, synaptic isoform, AChE-S, enhances neurotoxicity. OBJECTIVES: We exposed differentiating PC12 cells, a model for developing neurons, to 30 microM chlorpyrifos (CPF) or diazinon (DZN), or CPF oxon, the active metabolite that irreversibly inhibits AChE enzymatic activity, in order to determine whether they differentially induce the formation of AChE-S as a mechanistic predictor of developmental neurotoxicity. We then administered CPF or DZN to neonatal rats on postnatal days 1-4 using daily doses spanning the threshold for AChE inhibition (0-20%); we then evaluated AChE gene expression in forebrain and brainstem on post-natal day 5. RESULTS: In PC12 cells, after 48 hr of exposure, CPF, CPF oxon, and DZN enhanced gene expression for AChE-R by about 20%, whereas CPF and DZN, but not CPF oxon, increased AChE-S expression by 20-40%. Thus, despite the fact that CPF oxon is a much more potent AChE inhibitor, it is the native compound (CPF) that induces expression of the neurotoxic AChE-S isoform. For in vivo exposures, 1 mg/kg CPF had little or no effect, but 0.5 or 2 mg/kg DZN induced both AChE-R and AChE-S, with a greater effect in males. CONCLUSIONS: Our results indicate that nonenzymatic functions of AChE variants may participate in and be predictive of the relative developmental neurotoxicity of organophosphates, and that the various organophosphates differ in the degree to which they activate this mechanism.
Asunto(s)
Acetilcolinesterasa/metabolismo , Cloropirifos/análogos & derivados , Cloropirifos/toxicidad , Diazinón/toxicidad , Acetilcolinesterasa/genética , Animales , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/enzimología , Inhibidores de la Colinesterasa/toxicidad , Femenino , Insecticidas/toxicidad , Masculino , Células PC12 , Prosencéfalo/efectos de los fármacos , Prosencéfalo/enzimología , Isoformas de Proteínas , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The organophosphate insecticide chlorpyrifos (CPF) adversely affects mammalian brain development through multiple mechanisms. To determine if CPF directly affects neuronal cell replication and phenotypic fate, and to identify the vulnerable stages of differentiation, we exposed PC12 cells, a model for mammalian neurodevelopment, to CPF concentrations spanning the threshold for cholinesterase inhibition (5-50 microM) and conducted evaluations during mitosis and in early and mid-differentiation. In undifferentiated cells, exposure to 5 microM CPF for 1-3 days reduced DNA synthesis significantly without eliciting cytotoxicity. At the same time, CPF increased the expression of tyrosine hydroxylase (TH), the enzymatic marker for the catecholamine phenotype, without affecting choline acetyltransferase (ChAT), the corresponding marker for the cholinergic phenotype. Upon exposure to nerve growth factor (NGF), PC12 cells developed neuritic projections in association with vastly increased TH and ChAT expression accompanying differentiation into the two phenotypes. CPF exposure begun at the start of differentiation significantly reduced ChAT but not TH activity. In contrast, when CPF was added in mid-differentiation (4 days of NGF pretreatment), ChAT was unaffected and TH was increased slightly. Thus, CPF exerts stage-specific effects, reducing DNA synthesis in the undifferentiated state, impairing development of the cholinergic phenotype at the start of differentiation, and promoting expression of the catecholaminergic phenotype both in undifferentiated and differentiated cells. CPF administration in vivo produces deficits in the number of neurons and cholinergic function, and because we were able to reproduce these effects in vitro, our results suggest that CPF directly influences the phenotypic fate of neuronal precursors.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Cloropirifos/toxicidad , Plaguicidas/toxicidad , Animales , Colina O-Acetiltransferasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Nervioso/administración & dosificación , Neuronas/efectos de los fármacos , Células PC12 , Fisostigmina/farmacología , Ratas , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The use of dexamethasone (DEX) to prevent respiratory distress in preterm infants is suspected to produce neurobehavioral deficits. We used PC12 cells to model the effects of DEX on different stages of neuronal development, utilizing exposures from 24 h up to 11 days and concentrations from 0.01 to 10 microM, simulating subtherapeutic, therapeutic, and high-dose regimens. In undifferentiated cells, even at the lowest concentration, DEX inhibited DNA synthesis and produced a progressive deficit in the number of cells as evaluated by DNA content, whereas cell growth (evaluated by the total protein to DNA ratio) and cell viability (Trypan blue exclusion) were promoted. When cell differentiation was initiated with nerve growth factor, the simultaneous inclusion of DEX still produced a progressive deficit in cell numbers and promoted cell growth and viability while retarding the development of neuritic projections as monitored by the membrane/total protein ratio. Again, even 0.01 microM DEX was effective. We next assessed effects at mid-differentiation by introducing nerve growth factor for 4 days followed by coexposure to DEX. Although effects on cell number, growth, and neurite extension were still detectable, the outcomes were generally less notable. DEX also shifted the fate of PC12 cells away from the cholinergic phenotype and toward the adrenergic phenotype, with the maximum effect achieved at the outset of differentiation. Our results indicate that DEX directly disrupts neuronal cell replication, differentiation, and phenotype at concentrations below those required for the therapy of preterm infants, providing a mechanistic link between glucocorticoid use and neurodevelopmental sequelae.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dexametasona/toxicidad , Trastornos Mentales , Animales , Recuento de Células , Diferenciación Celular/fisiología , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Trastornos Mentales/inducido químicamente , Trastornos Mentales/patología , Células PC12 , Ratas , Factores de TiempoRESUMEN
Selenium is biologically active through the functions of selenoproteins that contain the amino acid selenocysteine. This amino acid is translated in response to in-frame UGA codons in mRNAs that include a SECIS element in its 3' untranslated region, and this process requires a unique tRNA, referred to as tRNA([Ser]Sec). The translation of UGA as selenocysteine, rather than its use as a termination signal, is a candidate restriction point for the regulation of selenoprotein synthesis by selenium. A specialized reporter construct was used that permits the evaluation of SECIS-directed UGA translation to examine mechanisms of the regulation of selenoprotein translation. Using SECIS elements from five different selenoprotein mRNAs, UGA translation was quantified in response to selenium supplementation and alterations in tRNA([Ser]Sec) levels and isoform distributions. Although each of the evaluated SECIS elements exhibited differences in their baseline activities, each was stimulated to a similar extent by increased selenium or tRNA([Ser]Sec) levels and was inhibited by diminished levels of the methylated isoform of tRNA([Ser]Sec) achieved using a dominant-negative acting mutant tRNA([Ser]Sec). tRNA([Ser]Sec) was found to be limiting for UGA translation under conditions of high selenoprotein mRNA in both a transient reporter assay and in cells with elevated GPx-1 mRNA. This and data indicating increased amounts of the methylated isoform of tRNA([Ser]Sec) during selenoprotein translation indicate that it is this isoform that is translationally active and that selenium-induced tRNA methylation is a mechanism of regulation of the synthesis of selenoproteins.
Asunto(s)
Proteínas/genética , Aminoacil-ARN de Transferencia/genética , Animales , Secuencia de Bases , Células CHO , Codón/genética , Cricetinae , Biosíntesis de Proteínas/genética , SelenoproteínasRESUMEN
Dietary intake of selenium has been implicated in a wide range of health issues, including aging, heart disease and cancer. Selenium deficiency, which can reduce selenoprotein levels, has been associated with several striated muscle pathologies. To investigate the role of selenoproteins in skeletal muscle biology, we used a transgenic mouse (referred to as i6A-) that has reduced levels of selenoproteins due to the introduction and expression of a dominantly acting mutant form of selenocysteine transfer RNA (tRNA[Ser]Sec). As a consequence, each organ contains reduced levels of most selenoproteins, yet these mice are normal with regard to fertility, overall health, behavior and blood chemistries. In the present study, although skeletal muscles from i6A- mice were phenotypically indistinguishable from those of wild-type mice, plantaris muscles were approximately 50% heavier after synergist ablation, a model of exercise overload. Like muscle in wild-type mice, the enhanced growth in the i6A- mice was completely blocked by inhibition of the mammalian target of rapamycin (mTOR) pathway. Muscles of transgenic mice exhibited increased site-specific phosphorylation on both Akt and p70 ribosomal S6 kinase (p70S6k) (P < 0.05) before ablation, perhaps accounting for the enhanced response to synergist ablation. Thus, a single genetic alteration resulted in enhanced skeletal muscle adaptation after exercise, and this is likely through subtle changes in the resting phosphorylation state of growth-related kinases.
Asunto(s)
Músculo Esquelético/crecimiento & desarrollo , Esfuerzo Físico , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Proteínas/fisiología , Selenio/deficiencia , Animales , Ratones , Ratones Transgénicos , Músculo Esquelético/química , Mutación , Tamaño de los Órganos , Fosforilación , Proteínas/análisis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , ARN de Transferencia Aminoácido-Específico/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Selenio/fisiología , Selenoproteínas , Transducción de SeñalRESUMEN
Selenocysteine transfer RNA (tRNA([Ser]Sec)) is a central molecule in the production of selenium-containing proteins, and may play a role in the regulation of their biosynthesis. Selenium concentration influences both the levels of tRNA([Ser]Sec) and the relative abundance of two isoforms. To study the mechanism by which selenium affects tRNA([Ser]Sec) levels, Chinese hamster ovary (CHO) cells were treated with the transcription inhibitor, actinomycin D, and tRNA([Ser]Sec) levels were determined by Northern blotting, primer extension and reverse-phase column chromatography. Turnover of tRNA([Ser]Sec) in CHO cells was faster than the total tRNA population. Supplementation of the culture media with selenium reduced turnover of tRNA([Ser]Sec), but did not influence turnover of a randomly selected serine tRNA. Inhibition of transcription with actinomycin D resulted in a relative increase in the abundance of the isoform containing methylcarboxymethyl-5'-uridine-2'-O-methylribose in the wobble position of the anticodon. Primer extension studies, which permitted the independent evaluation of the tRNA([Ser]Sec) arising from the introduced mouse gene and that derived from the host CHO gene, indicated an accelerated decline in tRNA([Ser]Sec) derived from both the transfected and the native gene. These results provide additional insight into the levels of regulation that control the translation of selenium containing proteins in mammalian cells.