Effects of Toluene on the Development of the Inner Ear and Lateral Line Sensory System of Zebrafish / 生物医学与环境科学（英文）

Objective@#The aim of this study was to explore the ototoxicity of toluene in the early development of zebrafish embryos/larvae.@*Methods@#Zebrafish were utilized to explore the ototoxicity of toluene. Locomotion analysis, immunofluorescence, and qPCR were used to understand the phenotypes and molecular mechanisms of toluene ototoxicity.@*Results@#The results demonstrated that at 2 mmol/L, toluene induced zebrafish larvae death at 120 hours post fertilization (hpf) at a rate of 25.79% and inhibited the rate of hatching at 72 hpf. Furthermore, toluene exposure inhibited the distance travelled and average swimming velocity of zebrafish larvae while increasing the frequency of movements. As shown by fluorescence staining of hair cells, toluene inhibited the formation of lateral line neuromasts and middle line 1 (Ml @*Conclusion@#This study indicated that toluene may affect the development of both the inner ear and lateral line systems in zebrafish, while the lateral line system may be more sensitive to toluene than the inner ear.

Melatonin has a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/secretion / Melatonina tem efeito estimulador em osteoblastos, pela regulação positiva da expressão/secreção de COL-I e OPN

ABSTRACT Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.

RESUMO A melatonina (MLT) é uma molécula potencial de sinalização na homeostase do metabolismo ósseo e pode ser um importante mediador da formação e estimulação óssea. O objetivo deste estudo in vitro foi avaliar o efeito da MLT na viabilidade, na expressão do mRNA da proteína e mineralização de células préosteoblásticas. As concentrações de MLT 5, 2,5, 1, 0,1 e 0,01 mM foram testadas em células pré-osteoblásticas da linhagem MC3T3 em comparação ao controle (sem MLT), avaliando a proliferação e a viabilidade celular (C50), expressão gênica (rtPCR) e secreção (Elisa) de Colágeno tipo 1 (COL-I) e osteopontina (OPN) às 24, 48 e 72 horas, além da formação de nódulos minerais por meio do teste vermelho de Alizarina fast red após 10 dias de tratamento. MLT a 5 e 2,5 mM provou ser tóxico (C50). Portanto, as concentrações de 0,01, 0,1 e 1 mM foram utilizadas para as análises subsequentes. A expressão do mRNA da OPN aumentou com MLT a 0,1 mM-1mM, seguida pela secreção aumentada de OPN às 24 e 72 horas em comparação aos demais grupos (p<0,05). O mRNA de COL-I e a secreção de COL-I seguiram o mesmo padrão do OPN a 0,1 mM de MLT em 72 horas de tratamento (p<0,05). Em relação à mineralização, todas as doses de MLT (exceto 1mM) causaram aumento (p<0,05) na formação de nódulos minerais em comparação ao controle. A MLT na concentração entre 0,01mM a 1 mM teve um efeito estimulador sobre os osteoblastos, ao regular positivamente a expressão e secreção de COL-I e OPN, além da mineralização, favorecendo a osteogênese.

Endothelin A Receptor Blockade Influences Apoptosis and Cellular Proliferation in the Developing Rat Kidney

Endothelin systems are believed to play important roles in the emergence and maintenance of functions of various organs during perinatal development, including the kidney. The present study was designed to investigate the roles of endothelin systems on physiologic renal growth and development. Newborn rat pups were treated with either Bristol-Myers Squibb-182874 (30 mg/kg/day), a selective endothelin A receptor (ET(A)R) antagonist, or vehicle for 7 days. To identify cellular changes, kidneys were examined for apoptotic cells by terminal deoxynucleotide transferasemediated nick-end labeling stain and proliferating cell nuclear antigen (PCNA) by immunohistochemical (IHC) stain. To clarify the molecular control of these processes, immunoblots and reverse transcriptase-polymerase chain reaction for Clusterin, Bcl-2, Bcl-X(L), Bax, and p53 were performed. ETAR antagonist treatment resulted in reduced kidney weights, decreased PCNA-positive proliferating cells, and increased apoptotic cells. The protein expressions of renal Bcl-X(L) and Bax in the ETAR antagonist-treated group were significantly decreased, whereas the mRNA expressions of these genes were not changed. There were no significant differences in the expressions of Clusterin, Bcl-2, and p53. In conclusion, inhibition of endogenous endothelins impairs renal growth, in which decreased cellular proliferation, increased apoptosis and decreased expressions of renal Bcl-X(L) and Bax are possibly implicated.

Spatiotemporal regulation of fibroblast growth factor signal blocking for endoderm formation in Xenopus laevis

We have previously shown that the inhibition of fibroblast growth factor (FGF) signaling induced endodermal gene expression in the animal cap and caused the expansion of the endodermal mass in Xenopus embryos. However, we still do not know whether or not the alteration of FGF signaling controls embryonic cell fate, or when FGF signal blocking is required for endoderm formation in Xenopus. Here, we show that FGF signal blocking in embryonic cells causes their descendants to move into the endodermal region and to express endodermal genes. It is also interesting that blocking FGF signaling between fertilization and embryonic stage 10.5 promotes endoderm formation, but persistent FGF signaling blocking after stage 10.5 restricts endoderm formation and differentiation.

Increased expression of SNARE proteins and synaptotagmin IV in islets from pregnant rats and in vitro prolactin-treated neonatal islets

During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic â-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion.

Vitamin A mediated limb deformities in the common Indian toad, Bufo melanostictus (Schneider).

Several types of limb deformities were induced by vitamin A in B. melanostictus. These ranged from total suppression of all the limbs (ectromelia) to partial development of either the forelimb or the hindlimb or both (mesomelia) to reduction or absence of digits in either the forelimbs or hindlimbs or both and absence of long bones in either the forelimbs or hindlimbs or both (phocomelia) or duplication of the hindlimbs (polymelia). All the limb abnormalities were induced in the developing limbs of the tail amputated tadpoles of B. melanostictus following vitamin A treatment, which is all the more interesting. The results suggest that vitamin A induces the above mentioned abnormalities by either switching on or over-expressing or disrupting the limb-specific hox genes by yet unknown mechanisms.