Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays.

Degradation and toxicity assessment of the nonionic surfactant Triton™ X-45 by the peroxymonosulfate/UV-C process.

The degradation and mineralization of the nonionic surfactant octylphenol ethoxylate (OPEO), commercially known as Triton™ X-45, by the peroxymonosulfate (PMS)/UV-C process were investigated. Three different toxicity tests (Daphnia magna, Vibrio fischeri and Pseudokirchneriella subcapitata) as well as the Yeast Estrogen Screen (YES) bioassay were undertaken to evaluate the potential toxic and estrogenic effects of OPEO and its oxidation products. OPEO removal was very fast and complete after 7 min via PMS/UV-C treatment under the investigated reaction conditions (OPEO = 20 mg L(-1) (47 µM); TOC = 12 mg L(-1); PMS = 2.5 mM; initial reaction pH = 6.5; applied UV-C dose = 21 Wh L(-1)). TOC removal also proceeded rapidly; a gradual decrease was observed resulting in an overall TOC removal of 84%. The toxic responses of PMS/UV-C treated OPEO solutions varied according to the test organism used in the bioassay. Daphnia magna was found to be most sensitive to aqueous OPEO, whereas Pseudokirchneriella subcapitata appeared to be the least sensitive one. Daphnia magna and Vibrio fischeri tests revealed that the inhibitory effect of OPEO decreased significantly during the course of treatment. On the other hand, PMS/UV-C oxidation products exhibited a high toxic effect towards Pseudokirchneriella subcapitata (around 60%). YES test results underlined the need for improving the PMS/UV-C treatment performance to remove the estrogenic activity of OPEO and its oxidation products.

Identification of cornifelin and early growth response-1 gene as novel biomarkers for in vitro eye irritation using a 3D reconstructed human cornea model MCTT HCE™.

Evaluation of the eye irritation is essential in the development of new cosmetic products. Draize rabbit eye irritation test has been widely used in which chemicals are directly applied to rabbit eye, and the symptoms and signs of eyes are scored. However, due to the invasive procedure, it causes substantial pain and discomfort to animals. Recently, we reported in vitro eye irritation test method using a 3D human corneal epithelial model (MCTT HCE™) which is reconstructed from remaining human tissues after a corneal transplantation. This model exhibited an excellent predictive capacity for 25 reference chemicals (sensitivity 100%, specificity 77% and accuracy 88% vs. GHS). To improve the test performance, we explored new biomarkers for the eye irritation through transcriptomic approach. Three surfactants were selected as model eye irritants that include sodium lauryl sulfate, benzalkonium chloride and triton X-100. After test chemicals were treated, we investigated differentially expressed genes through a whole-gene microarray (Affymetrix GeneChip(®) Human Gene 2.0 ST Array, 48,000 probes). As a result, we identified that mRNAs of cornifelin (CNFN), a constituent of the insoluble cornified cell envelope of stratified squamous epithelia, and early growth response-1 (EGR1), a nuclear transcriptional regulator, were significantly up-regulated by all three irritants. Up-regulation of CNFN and EGR1 was further confirmed by Q-RT-PCR, and immunohistochemistry revealed increased level of CNFN in irritant-treated tissues, supporting the relevance of CNFN and EGR1 as new biomarkers for eye irritation.

Screening structure and predicting toxicity of pesticide adjuvants using molecular dynamics simulation and machine learning for minimizing environmental impacts.

Surfactants as synergistic agents are necessary to improve the stability and utilization of pesticides, while their use is often accompanied by unexpected release into the environment. However, there are no efficient strategies available for screening low-toxicity surfactants, and traditional toxicity studies rely on extensive experimentation which are not predictive. Herein, a commonly used agricultural adjuvant Triton X (TX) series was selected to study the function of amphipathic structure to their toxicity in zebrafish. Molecular dynamics (MD) simulations, transcriptomics, metabolomics and machine learning (ML) were used to study the toxic effects and predict the toxicity of various TX. The results showed that TX with a relatively short hydrophilic chain was highly toxic to zebrafish with LC50 of 1.526 mg/L. However, TX with a longer hydrophilic chain was more likely to damage the heart, liver and gonads of zebrafish through the arachidonic acid metabolic network, suggesting that the effect of surfactants on membrane permeability is the key to determine toxic results. Moreover, biomarkers were screened through machine learning, and other hydrophilic chain lengths were predicted to affect zebrafish heart health potentially. Our study provides an advanced adjuvants screening method to improve the bioavailability of pesticides while reducing environmental impacts.

Cell-based dose responses from open-well microchambers.

Cell-based assays play a critical role in discovery of new drugs and facilitating research in cancer, immunology, and stem cells. Conventionally, they are performed in Petri dishes, tubes, or well plates, using milliliters of reagents and thousands of cells to obtain one data point. Here, we are introducing a new platform to realize cell-based assay capable of increased throughput and greater sensitivity with a limited number of cells. We integrated an array of open-well microchambers into a gradient generation system. Consequently, cell-based dose responses were examined with a single device. We measured IC50 values of three cytotoxic chemicals, Triton X-100, H2O2, and cadmium chloride, as model compounds. The present system is highly suitable for the discovery of new drugs and studying the effect of chemicals on cell viability or mortality with limited samples and cells.

Advances in quality control: mouse embryo morphokinetics are sensitive markers of in vitro stress.

STUDY QUESTION: Can time-lapse analysis of cell division timings [morphokinetics (MK)] in mouse embryos detect toxins at concentrations that do not affect blastocyst formation? SUMMARY ANSWER: An MK algorithm enhances assay sensitivity while providing results 24-48 h sooner than the traditional mouse embryo assay (MEA). WHAT IS KNOWN ALREADY: Current quality control testing methodology is sensitive but further improvements are needed to assure optimal culture conditions. MKs of embryo development may detect small variations in culture conditions. STUDY DESIGN: Cross sectional-control versus treatment. Mouse embryo development kinetics of 466 embryos were analyzed according to exposure to various concentrations of toxins and toxic mineral oil. MATERIALS, SETTING, METHODS: Cryopreserved 1-cell embryos from F1 hybrid mice were cultured with cumene hydroperoxide (CH) (0, 2, 4, 6 and 8 µM) and Triton X-100 (TX-100; 0, 0.0008, 0.0012, 0.0016 and 0.002%). Using the Embryoscope, time-lapse images were obtained every 20 min for 120 h in seven focal planes. End-points were timing and pattern of cell division and embryo development. The blastocyst rate (BR) was defined as the percentage of embryos that developed to the expanded blastocyst stage within 96 h. MAIN RESULTS AND THE ROLE OF CHANCE: BR was not affected for embryos cultured in the three lowest concentrations of CH and the four lowest concentrations of TX-100. In contrast, a unique MK model detected all concentrations tested (P < 0.05). The MK model identified toxicity in two lots of toxic mineral oil that did not affect BR (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: A limited number of toxins were used so that the results may not apply to all potential embryo toxins. A larger sample size may also demonstrate other statistically significant developmental kinetic parameters. WIDER IMPLICATIONS OF THE FINDINGS: MKs in mouse embryos are a sensitive and efficient method for quality control testing of in vitro culture conditions. BR, the end-point of traditional quality control assays, did not detect sublethal concentrations of toxins in the culture milieu in our study. This study demonstrates that temporal variation at key developmental stages reflects the quality of the culture environment. An MEA that incorporates MK will provide enhanced sensitivity and faster turn-around times.

A combination of in vitro comet assay and micronucleus test using human lymphoblastoid TK6 cells.

The comet assay has been widely used as a genotoxicity test for detecting primary DNA damage in individual cells. The micronucleus (MN) test is also a well-established assay for detecting clastogenicity and aneugenicity. A combination of the comet assay (COM) and MN test is capable of detecting a variety of genotoxic potentials as an in vitro screening system. Although the in vitro MN test has a robust protocol and Organisation for Economic Co-operation and Development (OECD) test guideline, the in vitro COM does not. To establish a robust protocol for the COM and to compare its sensitivity with that of the MN, we conducted COM and MN concurrently for five genotoxic agents (ethyl methanesulfonate, methyl methanesulfonate, hydrogen peroxide, gamma-rays and mitomycin C) and one non-genotoxic agent (triton X-100), using human lymphoblastoid TK6 cells. Relative cell count (RCC), relative population doubling (RPD), relative increase in cell count (RICC) and relative cell viability determined by trypan blue dye-exclusion assay (TBDE) were employed as cytotoxic measurements. However, the relative cell viability determined by TBDE just after the treatment was not an appropriate parameter of cytotoxicity for the genotoxic agents because it remained constant even at the highest doses, which showed severe cytotoxicity by RCC, RPD and RICC. The results of the COM showed qualitative agreement (positive or negative) with those of the MN except for mitomycin C, which is an interstrand cross-linker. The COM always required higher doses than the MN to detect the genotoxic potential of the genotoxic agents under the test conditions applied here. The doses that induced a comet tail always yielded <50% RICC, and do not accord to the OECD test guideline for MN because of their high cytotoxicity. These results are helpful for interpreting the results of the COM and MN in in vitro genotoxic hazard assessments. Further investigation is required to standardise the COM.

Biodegradation of Triton X-100 and its primary metabolites by a bacterial community isolated from activated sludge.

A set of studies was carried using a continuous flow biodegradation unit in order to isolate a microbial community capable of efficient and complete utilization of octylphenol ethoxylates from activated sludge. Increasing concentrations of Triton X-100 (in the range of 1-1000 mg/l) were applied over a time period of 35 days in order to select microorganisms, which exhibit high tolerance towards this surfactant. The fate of the surfactant and its primary degradation products was assessed by HPLC/MS. It was observed that even small doses of the surfactant contributed to the disruption of the activated sludge, due to adsorption of primary Triton X-100 metabolites (octylphenol and short-chained ethoxylates) on the cells, although the long-chain octylphenol ethoxylates were efficiently degraded during the isolation process. The toxicity assessment of octylphenol as well as octylphenol di- and monoethoxylates towards activated sludge allowed for determination of EC50 values (8 and 55 mg/l, respectively). The identification of the residual microorganisms revealed the presence of Acinetobacter junii, Acinetobacter calcoaceticus, Aeromonas hydrophilia, Alcaligenes spp., Pseudomonas fluorescens and Sphingomonas capsulata. The isolated community exhibited a high resistance towards Triton X-100 and was capable of growth even at 10,000 mg/l, with the highest specific growth rate (0.47 h(-1)) observed at 4000 mg/l. Under aerobic conditions both octylphenol and the short-chained ethoxylates were completely degraded while no toxic effect towards the isolated bacterial community was observed.

Dosing metric in cellular experiments: The mol/cell metric has its limitations.

It has been argued that the mol/cell metric is more universal than concentration of the toxic agent since in many cases the effect of dose expressed as mol/cell is independent of ex-perimental setup. We confirmed it for hemolysis of erythrocytes in phosphate-buffered saline induced by hypochlorite where the amount of femtomoles/cell of hypochlorite needed for 50% hemolysis was independent of erythrocyte concentration. However, in the presence of blood plasma this metric became dependent on cell concentration. Similarly, the effect of 3-bromopyruvic acid (3-BP) on PEO1 cells as a function of mol/cell ratio depended on the volume of the 3-BP containing medium, due to the reaction of 3-BP with components of the medium. Hemolytic amounts of sodium dodecyl sulfate and Triton X-100 expressed as mol/cell decreased with increasing cell concentration while the effect of DMSO on the viability of a constant number of fibroblasts was independent of the volume of DMSO-containing medium. These results demonstrate that the mol/cell metric is still dependent on experimental conditions when the toxic agent interacts with components of the medium or when its physical state is modified by the target cells, and the effect is independent of the mol/per cell ratio for high excess of a cell damaging agent.

Changes in the aquatic ecotoxicological effects of Triton X-100 after UV photodegradation.

Various spray adjuvants including surfactants are widely used in agricultural pesticide formulations, and some of them may remain in soils and waters and impose more adverse effects than active pesticide ingredients on organisms. However, previous studies are more focused on the active pesticide ingredients than the adjuvants. Thus, this study investigates the changes in toxic effects of surfactants during photodegradation, which is one way of naturally degrading contaminants in natural waters. Triton X-100, a water-soluble non-ionic surfactant, was degraded using different types of UV radiation (UVA, UVB, and UVC), and the changes in the toxic effects were determined using bioluminescent bacteria and water flea. The Triton X-100 removals were negligible with UVA within 24 h, while its removal was 81% with UVB and almost complete with UVC. The NMR spectra indicated possible molecule rearrangement after photolysis. On the other hand, the toxic effects based on the mortality of Daphnia magna and the bioluminescence of Aliivibrio fischeri increased (i.e., lower EC50 values) after photodegradation, suggesting the generation of photoproducts that are likely to have higher toxic effects or higher bioavailability. Furthermore, the sensitivities of D. magna and A. fischeri for Triton X-100 and the photodegraded Triton X-100 were different. This study suggests that the changes in the chemical composition of the Triton X-100 containing water with photodegradation can lead to changes in the relative toxic effects on different aquatic organisms. Therefore, not only the management of parent compound (i.e., Triton X-100) but also the photoproducts generated from the parent compound need to be considered when managing water environment subject to photodegradation.

Attenuation of lipid levels in triton induced hyperlipidemia rats through rosuvastatin calcium nanoparticles: Pharmacokinetic and pharmacodynamic studies.

The aim of this research was to study the effect of marketed tablet (Crestor®) powder suspension (MTPS) and nanoparticle formulation of rosuvastatin calcium (RC) on the pharmacokinetic (PK) and pharmacodynamic (PD) parameters in hyperlipidemia rats. The hyperlipidemia is induced by intraperitoneal injection of Triton-X-100 in 0.9 %w/v saline solution. The marketed tablet was dispersed into suspension. The RC loaded nanoparticles (RC-NPs) are prepared by homogenization method. The prepared RC-NP formulation was characterized for size, drug excipient compatibility and crystallization by differential scanning calorimeter (DSC), morphology by SEM, stability at room temperature, in-vitro dissolution and in-situ absorption in rats. Further, the pharmacokinetic and pharmacodynamic studies were conducted in hyperlipidemia rats. The size of the RC-NP formulation was found to be 183.4 ± 4.5 nm and to be nearly spherical by SEM. DSC studies revealed that no interaction and RC converted to amorphous form in RC-NP formulation. RC-NP formulation was physically and chemically stable over two months at room temperature. The drug release was found to be 25.8 ± 2.5 and 89.96 ± 2.8 % in five mins, respectively from MTPS and RC-NP formulations. The Peff of MTPS and NP of RC was 1.8 ± 0.2 × 10-5 and 2.7 ± 0.3 × 10-5 cm/s, respectively. From the PK studies, the enhancement in the oral bioavailability was found to be 2.4-folds when compared to MTPS formulation and statistically significant (p < 0.05). PD study of RC-NP formulation in hyperlipidemic rats exhibited decrease in lipid profile for 24 h, while MTPS exhibited a decrease in lipid profile for 12 h. Therefore, the results conclusively demonstrate the nanoparticles of RC showed significant enhancement in the PK and PD parameters.

Oxytocin mediated cardioprotection is independent of coronary endothelial function in rats.

The cardioprotective effect of oxytocin (OT) has been well established. However, there are no related studies on the role of endothelia in oxytocin-induced cardioprotection. Endothelial dysfunction (ED) model was established by injection of 0.01 % Triton X-100 in the isolated rat heart. Oxytocin pretreatment was conducted at the end of stabilization for 40 min, followed by 30 min global ischemia and 60 min reperfusion to induce I/R injury. Coronary perfusion pressure, hemodynamics and arrhythmia severity scores were measured respectively. High-sensitivity cardiac troponin T (hs-cTnT) was evaluated by enzyme-linked immunosorbent assay. Infarct size was detected by triphenyltetrazolium chloride staining. The morphological changes in coronary endothelium were observed by scanning electron microscopy. Injection of 0.01 % Triton X-100 caused significant reduction of CPP induced by histamine and endothelium removal from scanning electron microscopy, but SNP had no significant effect. Oxytocin pretreatment showed significant recovery in LVDP, ±dp/dtmax, RPP and SI after reperfusion (P <  0.05). Additionally, I/R injury led to a rise of arrhythmia severity score, hs-cTnT and infarct size. No significant differences between ED-OT-I/R and OT-I/R groups were found in arrhythmia severity score, hs-cTnT, and infarct size (P >  0.05). I/R injury exacerbated the decrease in CPP and worsened the migration, deformation, and fracture of coronary endothelium, while oxytocin reversed these injuries. Despite the presence of endothelial damages, oxytocin partially alleviated I/R- and Triton-induced endothelial damages. The cardioprotective effects of oxytocin are independent of endothelial function in alleviating I/R injury and I/R-induced coronary endothelial dysfunction.

Lipidomic analysis of human corneal epithelial cells exposed to ocular irritants highlights the role of phospholipid and sphingolipid metabolisms in detergent toxicity mechanisms.

Detergent chemicals, widely used in household products, in pharmaceutical, medical, cosmetic and industrial fields, have been linked to side effects and involved in several eye diseases. On the ocular surface, detergents can interfere with the corneal epithelium, the most superficial layer of the cornea, representing a line of defence against external aggression. Despite its major role in numerous biological functions, there is still little data regarding disruption of lipid homeostasis induced by ocular irritants. To this purpose, a lipidomic analysis using UPLC-HRMS/MS-ESI ± was performed on human corneal epithelial (HCE) cells incubated with three widely known ocular irritants: benzalkonium chloride (BAK), sodium lauryl sulfate (SLS) and Triton X-100 (TXT). We found that these ocular irritants lead to a profound modification of the HCE cell lipidome. Indeed, the cell content of ceramide species increased widely while plasmalogens containing polyunsaturated fatty acid species, especially docosahexaenoic acids, decreased. Furthermore, these irritants upregulated the activity of phospholipase A2. The present study demonstrates that BAK, SLS and TXT induced disruption of the cell lipid homeostasis, highlighting that lipids mediate inflammatory and cell death processes induced by detergents in the cornea. Lipidomics may thus be regarded as a valuable tool to investigate new markers of corneal damage.

Development of reduced vancomycin susceptibility in methicillin-susceptible Staphylococcus aureus.

BACKGROUND: Most cases of reduced vancomycin susceptibility in Staphylococcus aureus reported in the literature have been in methicillin-resistant strains. We report the development of reduced vancomycin susceptibility in a series of clonally related, methicillin-susceptible S. aureus (MSSA) clinical isolates. This isogenic series permitted us to determine whether the evolution of reduced vancomycin susceptibility in MSSA is similar to that seen in MRSA. METHODS: Differences in vancomycin population analysis profiles; chemical autolysis; vancomycin, oxacillin, and daptomycin minimum inhibitory concentrations; and bactericidal activities were examined. RESULTS: Progressive vancomycin resistance correlated with increasing daptomycin nonsusceptibility. Chemical autolysis and the bactericidal activity of vancomycin, oxacillin, and daptomycin were reduced in the final, vancomycin-intermediate S. aureus isolate, compared with the vancomycin-susceptible MSSA progenitor. CONCLUSIONS: Clinicians should recognize that reduced vancomycin susceptibility can occur in S. aureus irrespective of background methicillin susceptibility and that development of intermediate vancomycin susceptibility in MSSA may result in increased tolerance to several classes of anti-staphylococcal antibiotics.

Differential roles for Bak in Triton X-100- and deoxycholate-induced apoptosis.

We recently reported that Bax activation occurs downstream of caspase activation in Triton X-100 (TX)-induced apoptosis. Here, Bak was found to be activated in TX-induced apoptosis. Although z-VAD-fmk completely suppressed Bax activation, it only partially attenuated TX-induced Bak activation. Moreover, activation of both Bak and Bax was detected in apoptosis induced by deoxycholate, a physiological detergent in bile. z-VAD-fmk completely suppressed deoxycholate-induced Bak as well as Bax activation. Furthermore, Bak siRNA attenuated TX- but not deoxycholate-induced caspase activation. These results suggest that Bak activation may occur upstream of caspase activation in TX- but not deoxycholate-induced apoptosis and that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis at least with regard to the role for Bak.

Potential method to determine irritant potency in vitro - Comparison of two reconstructed epidermal culture models with different barrier competency.

Testing chemicals for their ability to cause skin irritation is required for all ingredients of products that come into contact with the skin. Here, we describe a potential method for determining the irritant potency of a chemical in vitro and apply the method to two different reconstructed epidermis models which exhibit different barrier properties. Two surfactants: sodium dodecyl sulphate, Triton X100 and two non-surfactants: 2-4-di-nitro-chloro-benzene, cinnamaldehyde were applied topically in a dose response for 24h. Biomarkers IL-1alpha, IL-1RA, IL-8 and MTT were assessed and EC(50) values determined. Variation in barrier properties between the epidermal models led to variation in the extent of penetration of surfactants, but not of non-surfactants which in turn influenced the EC(50) value obtained from surfactants. Furthermore, EC(50) values showed that no single biomarker could be classed as the most sensitive biomarker since biomarker sensitivity differed between the different chemicals studied. However, the ranking of the chemicals in order of strong to weak irritant was the same irrespective of the model used and also independent of the biomarker used (Triton X100>DNCB>SDS>CA). This study describes a method which not only distinguishes an irritant from a non-irritant but which may possibly also be used to determine irritant potency.

Qualitative electron microscopic analysis of cultured chick embryonic cardiac and skeletal muscle cells: the cellular effect of coenzyme q10 after exposure to triton x-100.

The numerous protective effects of coenzyme Q10 (CoQ10) evoked the question of whether it might be able to elicit protection to cell membranes after being challenged by the membrane disrupter Triton X-100. Cardiac and skeletal muscle tissue from chick embryos was cultured and exposed to increasing concentrations of CoQ10 and Triton X-100. Scanning electron microscopy was used to study cell morphology. Results suggested the ability of CoQ10 to offer protection to cells challenged by Triton X-100. The authors suggest that CoQ10 may offer protection to muscle cells, by enhancing membrane repair via patch formation by an unknown mechanism that possibly involves Ca(2+)-dependent ion channel activation.

Ecotoxicological assessment of synthetic and biogenic surfactants using freshwater cladoceran species.

Surfactants have been continuously detected within aquatic environments as a consequence of their use on a global scale. Lipopeptides are biosurfactants naturally produced by Bacillus subtilis that have been explored as green alternatives. The assessment of ecotoxicological parameters of synthetic and biogenic surfactants are required for evaluating toxicity values and to verify the eco-friendly behaviour of the biological compounds. This study aimed to conduct toxicity testing for different surfactants - sodium dodecyl sulphate and Triton X-100 - and biosurfactants - surfactin, iturin and fengycin - at different concentrations using Daphnia magna as model organism and Dendrocephalus brasiliensis as alternative test species for monitoring of pollutants in tropical freshwaters. According results, both species showed high sensitivity for the anionic compound SDS concerning the recommended dosage use, exhibiting EC50-48h values of 24.1 and 15.4 mg/L for D. magna and D. brasiliensis, respectively. Although the biological source, surfactin showed the lower safety behaviour among the biogenic surfactants, while iturin and fengycin revealed very low toxicity effects on both organisms. Besides, data exhibited a higher responsiveness of D. brasiliensis for all tested compounds in comparison to D. magna, highlighting the importance of this species for monitoring of pollutants in tropical and subtropical environments.

Microglial and astroglial reaction in the olfactory bulb of mice after Triton X-100 application.

Gliosis including microgliosis and astrogliosis is a response to central nervous system inflammation. The purpose of this study was to evaluate whether olfactory bulbs are influenced by intranasal exposure to the detergent Triton X-100, a non-ionic surfactant. In this experiment, we measured olfactory function in mice based on the time needed to identify hidden pellets. Our results found that more time was needed to find the buried pellets by mice exposed to Triton X-100 compared with mice without Triton X-100 exposure, up to day 7. Histopathological examination revealed inflammatory cells in the olfactory mucosa and olfactory bulbs in mice treated with Triton X-100. Western blot analysis revealed significant downregulation of olfactory marker proteins in the olfactory mucosa and bulbs of mice after intranasal exposure to Triton X-100. In the olfactory bulbs of mice exposed to Triton X-100, microgliosis and astrogliosis were evident using immunohistochemistry. Cathepsin D was also upregulated in Iba-1-positive microglia/macrophages and GFAP-positive astrocytes in the olfactory bulbs of mice exposed to Triton X-100. In mice, Triton X-100 induced olfactory sensory neuron death in the nasal cavity and gliosis in olfactory bulbs with concurrent downregulation of olfactory marker protein expression, resulting in transient olfactory dysfunction.

Real-time viability assay based on 51Cr retention in adherent cells.

The chromium (51Cr) release assay has been widely used for viability measurements, even though it has major disadvantages such as high manual workload and poor time resolution. By the use of LigandTracer 51Cr release viability measurements on adherent cells can be significantly simplified and improved. LigandTracer enables a time-resolved detection of 5SCr in target cells, with the result that the effect of toxic material is updated continuously throughout the experiment. Here we explain the principle behind this novel real-time viability assay and show viability curves for known toxic compounds on A431 and U343MGaCl2:6 cell lines.