Biocompatible Azide-Alkyne "Click" Reactions for Surface Decoration of Glyco-Engineered Cells.

Bio-orthogonal copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) has been widely used to modify azide- or alkyne-bearing monosaccharides on metabolic glyco-engineered mammalian cells. Here, we present a systematic study to elucidate the design space for the cytotoxic effects of the copper catalyst on NIH 3T3 fibroblasts and on HEK 293-F cells. Monitoring membrane integrity by flow cytometry and RT-PCR analysis with apoptotic and anti-apoptotic markers elucidated the general feasibility of CuAAC, with exposure time of the CuAAC reaction mixture having the major influence on biocompatibility. A high labeling efficiency of HEK 293-F cells with a fluorescent alkyne dye was rapidly achieved by CuAAC in comparison to copper free strain-promoted azide-alkyne cycloaddition (SPAAC). The study details effective and biocompatible conditions for CuAAC-based modification of glyco-engineered cells in comparison to its copper free alternative.

Differential resistance of drinking water bacterial populations to monochloramine disinfection.

The impact of monochloramine disinfection on the complex bacterial community structure in drinking water systems was investigated using culture-dependent and culture-independent methods. Changes in viable bacterial diversity were monitored using culture-independent methods that distinguish between live and dead cells based on membrane integrity, providing a highly conservative measure of viability. Samples were collected from lab-scale and full-scale drinking water filters exposed to monochloramine for a range of contact times. Culture-independent detection of live cells was based on propidium monoazide (PMA) treatment to selectively remove DNA from membrane-compromised cells. Quantitative PCR (qPCR) and pyrosequencing of 16S rRNA genes was used to quantify the DNA of live bacteria and characterize the bacterial communities, respectively. The inactivation rate determined by the culture-independent PMA-qPCR method (1.5-log removal at 664 mg·min/L) was lower than the inactivation rate measured by the culture-based methods (4-log removal at 66 mg·min/L). Moreover, drastic changes in the live bacterial community structure were detected during monochloramine disinfection using PMA-pyrosequencing, while the community structure appeared to remain stable when pyrosequencing was performed on samples that were not subject to PMA treatment. Genera that increased in relative abundance during monochloramine treatment include Legionella, Escherichia, and Geobacter in the lab-scale system and Mycobacterium, Sphingomonas, and Coxiella in the full-scale system. These results demonstrate that bacterial populations in drinking water exhibit differential resistance to monochloramine, and that the disinfection process selects for resistant bacterial populations.

Toxicity ranking of heavy metals with screening method using adult Caenorhabditis elegans and propidium iodide replicates toxicity ranking in rat.

The utility of any model system for toxicity screening depends on the level of correlation between test responses and toxic reactions in humans. Assays in Caenorhabditis elegans can be fast and inexpensive, however few studies have been done comparing toxic responses in this easily cultured nematode with data on mammalian toxicity. Here we report that a screening assay for acute toxicity, using adult C. elegans grown in axenic liquid culture, replicated LD50 toxicity ranking in rat for five metals. This assay utilized the COPAS Biosort and propidium iodide (PI) as a fluorescent indicator of morbidity and mortality after 30-h exposures. We found that chronic toxicity assays of 2-week treatment duration, followed by analysis of PI induced red fluorescence levels, produced less consistent results than the acute assays. However, other chronic toxicity endpoints were compound and concentration specific, including changes in vulval and gonadal morphology, intestinal thickness and integrity, and the presence of retained internal eggs in post-reproductive animals. Some of these endpoints reflect similar findings in mammals, indicating that measurements of morbidity and mortality in conjunction with morphology analyses in C. elegans may have the potential to predict mammalian toxic responses.

Assessment of the genotoxic potential of Hoechst 33342, SYBR-14 and PI using the SOS ChromoTest(™).

Fluorochromes in combination with flow cytometry can be used for laboratory assessment of semen quality in humans and domestic animals. Some studies have reported the potential toxicity of these fluorochromes toward the cells analyzed, but not toward the laboratory technician who operates the analytical instrument. We tested the genotoxic potential of three fluorochromes, SYBR-14, propidium iodide, and Hoechst 33342, using the colorimetric SOS ChromoTest(™). The test revealed no genotoxic potential for any of the three fluorochromes within the dilution ranges investigated. We conclude that occasional direct contact with these fluorescent probes does not necessarily pose a genotoxic hazard.

Impaired mitochondrial bioenergetics determines glutamate-induced delayed calcium deregulation in neurons.

BACKGROUND: Accumulation of glutamate in ischaemic CNS is thought to amplify neuronal death during a stroke. Exposure of neurons to toxic glutamate concentrations causes an initial transient increase in [Ca(2+)](c) followed by a delayed increase commonly termed delayed [Ca(2+)](c) deregulation (DCD). METHODS: We have used fluorescence imaging techniques to explore differences in glutamate-induced DCD in rat hippocampal neurons after different periods of time in culture (days in vitro; DIV). RESULTS: The amplitude of both the initial [Ca(2+)](c) signal and the number of cells showing DCD in response to glutamate increased with the duration of culture. The capacity of mitochondria to accumulate calcium in permeabilised neurons decreased with time in culture, although mitochondrial membrane potential at rest did not change. The rate of ATP consumption, measured as an increase in [Mg(2+)](c) following inhibition of ATP synthesis, was lower in 'young' neurons. The sensitivity of 'young' neurons to glutamate-induced DCD approximated to that of 'old' neurons when mitochondrial function was impaired using either FCCP or oligomycin. Further, following such treatment, cells showed a DCD-like response to increased [Ca(2+)](c) induced by KCl induced depolarisation which was never otherwise seen. GENERAL SIGNIFICANCE: Thus, changes in cellular bioenergetics dictate the onset of DCD in response to glutamate.

Low osmolar contrast medium induces cellular injury and disruption of calcium homeostasis in rat glomerular endothelial cells in vitro.

The mechanisms of contrast medium (CM)-induced renal impairment at cellular level are not fully understood. The purpose of this study was to investigate the toxic effects of non-ionic low osmolar contrast medium (LOCM) on glomerular endothelial cells (GECs). Ioversol, the most representative LOCM used in clinic, was chosen to act on primary cultured rat GECs. When rat GECs were treated with various amount of ioversol, a dose-dependent reduction in cell viability was observed by tetrazolium dye (MTT) assay. After exposure to ioversol at dose of 100 microl/ml for 24h, nuclear condensation, nuclear fragmentation, cell apoptosis and necrosis were found in GECs. The intracellular free Ca(2+) concentrations ([Ca(2+)]i) detected by confocal laser scanning were markedly elevated in GECs treated with ioversol. The [Ca(2+)]i increase, LDH release and expression changes of apoptotic associated proteins in GECs induced by ioversol could be reversed by pretreatment with intracellular Ca(2+) chelator, 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (BAPTA/AM). These results suggest that LOCM can decrease cell viability and cause cell injury of GECs. The elevation of [Ca(2+)]i released from intracellular calcium stores likely contribute to the LOCM-induced cell injury of GECs.

Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay.

Genotoxicity can be assessed by monitoring expression of a GADD45a-GFP reporter in the human lymphoblastoid cell line TK6. A flow cytometric method has been developed to effectively distinguish GFP fluorescence from coloured and fluorescent test samples as well from the S9 liver extracts used to generate metabolites from pro-genotoxins. The method includes the use of propidium iodide exclusion for the determination of cellular viability. This paper describes the method development, the derivation of decision thresholds for the identification of genotoxins using the method, and presents data from a 56-compound validation study of the method. The results illustrate that the method permitted the detection of the majority of pro-genotoxins tested and, importantly, the high specificity of the GADD45a-GFP assay was maintained.

The impact of skin viability on drug metabolism and permeation -- BSA toxicity on primary keratinocytes.

For testing cutaneous absorption of drugs, ingredients of cosmetics and also for risk assessment of industrial compounds predictable in vitro test protocols are under investigation using excised skin or reconstructed human epidermis. Since the metabolizing enzymes expressed by viable skin can influence the absorption behaviour of substances by changing their structure and thereby their physicochemical characteristics, the metabolic capacity should be considered in the design of the test protocols of compounds susceptible to metabolism. Then data, generated using viable reconstructed epidermis may reflect the in vivo situation. Interestingly, bovine serum albumin (BSA) commonly used in receptor media in permeation studies to facilitate solubility of highly lipophilic substances strongly inhibited the metabolism of topically applied prednicarbate in reconstructed epidermis. Here, we show that 5% BSA is toxic to reconstructed epidermis and keratinocytes which was consistent with the earlier findings. While media toxicity (deficiency media) was at least partly the cause of both apoptotic and necrotic processes in keratinocytes, BSA only slightly increased the rate of necrotic cells. Moreover, caspase inhibitors did not reduce BSA toxicity. Yet, the results show that BSA toxicity on keratinocytes has to be carefully considered if this protein is used in permeation studies with reconstructed epidermis.

Effect of Bcl-2 on oxidant-induced cell death and intracellular Ca2+ mobilization.

The mechanism by which Bcl-2 inhibits cell death is unknown. It has been suggested that Bcl-2 functions as an antioxidant. Because Bcl-2 is localized mainly to the membranes of the endoplasmic reticulum (ER) and the mitochondria, which represent the main intracellular storage sites for Ca2+, we hypothesized that Bcl-2 might protect cells against oxidant injury by altering intracellular Ca2+ homeostasis. To test this hypothesis, we examined the effect of oxidant treatment on viability in normal rat kidney (NRK) cells and in NRK cells stably transfected with Bcl-2 in the presence or absence of intracellular Ca2+, and we compared the effect of Bcl-2 expression on oxidant-induced intracellular Ca2+ mobilization and on ER and mitochondrial Ca2+ pools. NRK cells transfected with Bcl-2 (NRK-Bcl-2) were significantly more resistant to H2O2-induced cytotoxicity than control cells. EGTA-AM, an intracellular Ca2+ chelator, as well as the absence of Ca2+ in the medium, reduced H2O2-induced cytotoxicity in both cell lines. Compared with controls, cells overexpressing Bcl-2 showed a delayed rise in intracellular Ca2+ concentration ([Ca2+]i) after H2O2 treatment. After treatment with the Ca2+ ionophore ionomycin, Bcl-2-transfected cells showed a much quicker decrease after the maximal rise than control cells, suggesting stronger intracellular Ca2+ buffering, whereas treatment with thapsigargin, an inhibitor of the ER Ca2+-ATPases, transiently increased [Ca2+]i in control and in Bcl-2-transfected cells. Estimates of mitochondrial Ca2+ stores using an uncoupler of oxidative phosphorylation show that NRK-Bcl-2 cells have a higher capacity for mitochondrial Ca2+ storage than control cells. In conclusion, Bcl-2 may prevent oxidant-induced cell death, in part, by increasing the capacity of mitochondria to store Ca2+.

Selective labeling by propidium iodide injected into the lateral cerebral ventricle of the rat.

Injections of propidium iodide (PI) into the lateral cerebral ventricle of the rat resulted in a bilateral labeling in the septohippocampal nuclei, substantia nigra (SN), ventral tegmental area (VTA), retrorubral nuclei (rr), dorsal and median raphe nuclei, regions within and dorsal to the medial lemniscus of the caudal midbrain, and Purkinje cells in the cerebellum. No labeled neurons were seen in other areas of the brain. The data suggest that PI appears to exhibit selective labeling, and the mechanism underlying the selective labeling is discussed. Combined with Faglu histofluorescence, it was found that all PI-labeled cells in SN-VTA-rr were catecholamine (CA) neurons. After a transection of the medial forebrain bundle immediately before the PI injection, an accumulation of PI was only seen in the distal segments of severed nigrostriatal CA fibers. This provides a strong evidence that PI labeling of CA cells in SN-VTA-rr is due to axonal uptake and retrograde transport.