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Thiabendazole (TBZ), an antihelminthic and antifungal agent, is associated with a host of adverse effects including nephrotoxicity, hepatotoxicity, and teratogenicity. Bioactivation of the primary metabolite of TBZ, 5-hydroxythiabendazole, has been proposed to yield a reactive intermediate. Here we show that this reactive intermediate can be catalyzed by myeloperoxidase (MPO), a neutrophil-bourne peroxidase. Using a cell viability endpoint, we examined the toxicity of TBZ, 5OH-TBZ, and MPO-generated metabolites in cell-based models including primary rat proximal tubule epithelial cells, NRK-52E rat proximal tubule cells, and H9C2 rat myocardial cells. Timecourse experiments with MPO showed complete turnover of 5OH-TBZ within 15 min and a dramatic leftward shift in dose–response curves after 12 h. After a 24 h exposure in vitro , the LC 50 of this reactive intermediate was 23.3 ± 0.2 μM reduced from greater than 200 μM from 5OH-TBZ alone, an approximately 10-fold decrease. LC 50 values were equal in all cell types used. Comparison of lactate dehydrogenase leakage and caspase 3/7 activity revealed that cell death caused by the reactive intermediate is primarily associated with necrosis rather than apoptosis. This toxicity can be completely rescued via incubation with rutin, an inhibitor of MPO. These results suggest that MPO-mediated biotransformation of 5OH-TBZ yields a reactive intermediate which may play a role in TBZ-induced toxicity.

Preservatives are used in ocular medications to prevent microbial contamination. The use of benzalkonium chloride (BAC), the most widely used preservative in ocular medications, has been scrutinized with a number of studies indicating its toxicity to monolayer cultures of corneal and conjunctival epithelial cells. The purpose of this study was to evaluate and compare the toxicity of BAC and other preservatives and common components of ocular formulations on monolayer and stratified air-lifted cultures of Chang conjunctival cells. Air-lifting Chang cells grown on transwell filters increased stratification as assessed by transepithelial electrical resistance and histology. Unlike monolayer cultures in which ocular medications containing BAC caused near complete loss of cell viability, stratified, air-lifted cultures were not affected by the presence of BAC in ocular medications with up to 30-min exposures. Stratification shifted the dose-response curve to the right for benzalkonium chloride, thimerosal, chlorhexidine digluconate, potassium sorbate and EDTA. These results demonstrate that stratification significantly affects cell viability of Chang conjunctival cells in response to preservatives and additives of ophthalmic preparations.

Big brain(bib) is a neurogenic gene that when mutated causes defects in cell fate determination duringDrosophilaneurogenesis through an unknown mechanism. The protein Big Brain (BIB) has sequence identity with the major intrinsic protein family that includes the water- and ion-conducting aquaporin channels. We show here that BIB expressed heterologously inXenopusoocytes provides a voltage-insensitive, nonselective cation channel function with permeability to K+> Na+≫ tetraethylammonium. The conductance, activated in response to endogenous signaling pathways in BIB-expressing oocytes, is decreased after treatment with 20 μminsulin and is enhanced with 10 μmlavendustin A, a tyrosine kinase inhibitor. Western blot analysis confirms that BIB is tyrosine-phosphorylated. Both tyrosine phosphorylation and the potentiating effect of lavendustin A are removed by partial deletion of the C terminus (amino acids 317–700). Current activation is not observed in control oocytes or in oocytes expressing a nonfunctional mutant (BIB E71N) that appears to be expressed on the plasma membrane by confocal microscopy and Western blotting. These results indicate that BIB can participate in tyrosine kinase-regulated transmembrane signaling and may suggest a role for membrane depolarization in the neurogenic function of BIB in early development.

Drosophila Big Brain (BIB) is a transmembrane protein encoded by the neurogenic gene big brain (bib), which is important for early development of the fly nervous system. BIB expressed in Xenopus oocytes is a monovalent cation channel modulated by tyrosine kinase signaling. Results here demonstrate that the BIB conductance shows voltage- and dose-dependent block by extracellular divalent cations Ca(2+) and Ba(2+) but not by Mg(2+) in wild-type channels. Site-directed mutagenesis of negatively charged glutamate (Glu(274)) and aspartate (Asp(253)) residues had no effect on divalent cation block. However, mutation of a conserved glutamate at position 71 (Glu(71)) in the first transmembrane domain (M1) altered channel properties. Mutation of Glu(71) to Asp introduced a new sensitivity to block by extracellular Mg(2+); substitutions with asparagine or glutamine decreased whole-cell conductance; and substitution with lysine compromised plasma membrane expression. Block by divalent cations is important in other ion channels for voltage-dependent function, enhanced signal resolution, and feedback regulation. Our data show that the wild-type BIB conductance is attenuated by external Ca(2+), suggesting that endogenous divalent cation block might be relevant for enhancing signal resolution or voltage dependence for the native signaling process in neuronal cell fate determination.