Abstract: There is ongoing concern regarding the toxicity of nanoparticles with sizes less than 100 nm as compared to larger particles of the same nominal substance. Two commercial ZnO types, one sold as a 8-10 nm powder and the other described as -325 mesh (<44 mum) powder, were evaluated in human colon-derived RKO cells. The powders had a volume-to-surface area ratio equivalent to 40 and 330 nm spheres, respectively. Both materials formed micrometer-sized agglomerates in cell culture media. The nanosized ZnO was more cytotoxic than the micrometer-sized ZnO with LC(50) values of 15 +/- 1 and 29 +/- 4 mug/cm(2), respectively. Transfer of Zn from the solid phase to the cell culture media in the presence of RKO cells was time- and concentration-dependent. However, direct particle-cell contact was required for RKO cell cytotoxicity, and the toxicity of particles was independent of the amount of soluble Zn in the cell culture media. The mechanism of cell death includes the disruption of mitochondrial function. Robust markers of apoptosis, Annexin V staining, loss of mitochondrial potential, and increased generation of superoxide were observed when cells were treated with ZnO particulate matter but not when treated with comparable concentration of a soluble Zn salt. Both ZnO samples induced similar mechanisms of toxicity, but there was a statistically significant increase in potency per unit mass with the smaller particles.
Abstract: The mammalian inner ear forms from a thickened patch of head ectoderm called the otic placode. The placodal ectoderm invaginates to form a cup whose edges cinch together to establish a fluid-filled sac called the otic vesicle or otocyst. The progenitor cells lining the otocyst lumen will give rise to sensory and non-sensory cells of the inner ear. These formative stages of inner ear development are initiated during the first week of postimplantation embryonic development in the mouse. The inaccessibility of the inner ear in utero has hampered efforts to gain insight into the molecular mechanisms regulating essential developmental processes. An experimental embryological method to misexpress genes in the developing mammalian inner ear is presented. Expression plasmid encoding a gene of interest is microinjected through the uterine wall into the lumen of the otocyst and electroporated into otic epithelial progenitor cells. Downstream analysis of the transfected embryonic or postnatal inner ear is then conducted to gain insight into gene function.
Abstract: Sensory hair cells in the mammalian cochlea convert mechanical stimuli into electrical impulses that subserve audition. Loss of hair cells and their innervating neurons is the most frequent cause of hearing impairment. Atonal homologue 1 (encoded by Atoh1, also known as Math1) is a basic helix-loop-helix transcription factor required for hair-cell development, and its misexpression in vitro and in vivo generates hair-cell-like cells. Atoh1-based gene therapy to ameliorate auditory and vestibular dysfunction has been proposed. However, the biophysical properties of putative hair cells induced by Atoh1 misexpression have not been characterized. Here we show that in utero gene transfer of Atoh1 produces functional supernumerary hair cells in the mouse cochlea. The induced hair cells display stereociliary bundles, attract neuronal processes and express the ribbon synapse marker carboxy-terminal binding protein 2 (refs 12,13). Moreover, the hair cells are capable of mechanoelectrical transduction and show basolateral conductances with age-appropriate specializations. Our results demonstrate that manipulation of cell fate by transcription factor misexpression produces functional sensory cells in the postnatal mammalian cochlea. We expect that our in utero gene transfer paradigm will enable the design and validation of gene therapies to ameliorate hearing loss in mouse models of human deafness.
