Is glutathione an important neuroprotective effector molecule against amyloid beta toxicity?

Cellular thiols are critical moieties in signal transduction, regulation of gene expression, and ultimately are determinants of specific protein activity. Whilst protein bound thiols are the critical effector molecules, low molecular weight thiols, such as glutathione, play a central role in cytoprotection through (1) direct consumption of oxidants, (2) regeneration of protein thiols and (3) export of glutathione containing mixed disulphides. The brain is particularly vulnerable to oxidative stress, as it consumes 20% of oxygen load, contains high concentrations of polyunsaturated fatty acids and iron in certain regions, and expresses low concentrations of enzymic antioxidants. There is substantial evidence for a role for oxidative stress in neurodegenerative disease, where excitotoxic, redox cycling and mitochondrial dysfunction have been postulated to contribute to the enhanced oxidative load. Others have suggested that loss of important trophic factors may underlie neurodegeneration. However, the two are not mutually exclusive; using cell based model systems, low molecular weight antioxidants have been shown to play an important neuroprotective role in vitro, where neurotrophic factors have been suggested to modulate glutathione levels. Glutathione levels are regulated by substrate availability, synthetic enzyme and metabolic enzyme activity, and by the presence of other antioxidants, which according to the redox potential, consume or regenerate GSH from its oxidised partner. Therefore we have investigated the hypothesis that amyloid beta neurotoxicity is mediated by reactive oxygen species, where trophic factor cytoprotection against oxidative stress is achieved through regulation of glutathione levels. Using PC12 cells as a model system, amyloid beta 25-35 caused a shift in DCF fluorescence after four hours in culture. This fluorescence shift was attenuated by both desferioxamine and NGF. After four hours, cellular glutathione levels were depleted by as much as 75%, however, 24 hours following oxidant exposure, glutathione concentration was restored to twice the concentration seen in controls. NGF prevented both the loss of viability seen after 24 hours amyloid beta treatment and also protected glutathione levels. NGF decreased the total cellular glutathione concentration but did not affect expression of GCS. In conclusion, loss of glutathione precedes cell death in PC12 cells. However, at sublethal doses the surviving fraction respond to oxidative stress by increasing glutathione levels, where this is achieved, at least in part, at the gene level through upregulation of GCS. Whilst NGF does protect against oxidative toxicity, this is not achieved through upregulation of GCS or glutathione.

Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source

Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m2 resulted in increased tissue transglutaminase activity when measured either in cell homogenates, "in situ" by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the epsilon(gamma-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m2 resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m2 resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.

The significance of hand wash compliance on the transfer of dermal lipids in contact lens wear

Aim: The aim of this study was to assess the impact of hand washing regimes on lipid transference to contact lenses. The presence of lipids on contact lenses can affect visual acuity and enhance spoilation. Additionally, they may even mediate and foster microbial transfer and serve as a marker of potential dermal contamination. Methods and materials: A social hand wash and the Royal College of Nursing (RCN) hand wash were investigated. A 'no-wash regime' was used as control. The transfer of lipids from the hand was assessed by Thin Layer Chromatography (TLC). Lipid transference to the contact lenses was studied through fluorescence spectroscopy (FS). Results: Iodine staining, for presence of lipids, on TLC plates indicated the 'no-wash regime' score averaged at 3.4 ± 0.8, the social wash averaged at 2.2 ± 0.9 and the RCN averaged at 1.2 ± 0.3 on a scale of 1-4. The FS of lipids on contact lenses for 'no washing' presented an average of 28.47 ± 10.54 fluorescence units (FU), the social wash presented an average of 13.52 ± 11.12. FU and the RCN wash presented a much lower average 6.47 ± 4.26. FU. Conclusions: This work demonstrates how the method used for washing the hands can affect the concentration of lipids, and the transfer of these lipids onto contact lenses. A regime of hand washing for contact lens users should be standardised to help reduce potentially transferable species present on the hands. © 2011 British Contact Lens Association.

The role of tissue transglutaminase in 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in differentiated human SH-SY5Y neuroblastoma cells

Tissue transglutaminase (TG2) can induce post-translational modification of proteins, resulting in protein cross-linking or incorporation of polyamines into substrates, and can also function as a signal transducing G protein. The role of TG2 in the formation of insoluble cross-links has led to its implication in some neurodegenerative conditions. Exposure of pre-differentiated SH-SY5Y cells to the Parkinsonian neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) resulted in significant dose-dependent reductions in TG2 protein levels, measured by probing Western blots with a TG2-specific antibody. Transglutaminase (TG) transamidating activity, on the other hand, monitored by incorporation of a polyamine pseudo-substrate into cellular proteins, was increased. Inhibitors of TG (putrescine) and TG2 (R283) exacerbated MPP+ toxicity, suggesting that activation of TG2 may promote a survival response in this toxicity paradigm.

The cellular response to transglutaminase-cross-linked collagen

Collagen, type I, is a highly abundant natural protein material which has been cross-linked by a variety of methods including chemical agents, physical heating and UV irradiation with the aim of enhancing its physical characteristics such as mechanical strength, thermal stability, resistance to proteolytic breakdown, thus increasing its overall biocompatibility. However, in view of the toxicity of residual cross-linking agents, or impracticability at large scales, it would be more useful if the collagen could be cross-linked by a milder, efficient and more practical means by using enzymes as biological catalysts. We demonstrate that on treating native collagen type I (from bovine skin) with both tissue transglutaminase (TG2; tTG) and microbial transglutaminase (mTG; Streptoverticillium mobaraense) leads to an enhancement in cell attachment, spreading and proliferation of human osteoblasts (HOB) and human foreskin dermal fibroblasts (HFDF) when compared to culture on native collagen. The transglutaminase-treated collagen substrates also showed a greater resistance to cell-mediated endogenous protease degradation than the native collagen. In addition, the HOB cells were shown to differentiate at a faster rate than on native collagen when assessed by measurement of alkaline phosphatase activity and osteopontin expression. © 2005 Elsevier Ltd. All rights reserved.

Towards development of a dermal rudiment for enhanced wound healing response

Enhancement of collagen's physical characteristics has been traditionally approached using various physico-chemical methods frequently compromising cell viability. Microbial transglutaminase (mTGase), a transamidating enzyme obtained from Streptomyces mobaraensis, was used in the cross-linking of collagen-based scaffolds. The introduction of these covalent bonds has previously indicated increased proteolytic and mechanical stability and the promotion of cell colonisation. The hypothesis behind this research is that an enzymatically stabilised collagen scaffold will provide a dermal precursor with enhanced wound healing properties. Freeze-dried scaffolds, with and without the loading of a site-directed mammalian transglutaminase inhibitor to modulate matrix deposition, were applied to full thickness wounds surgically performed on rats’ dorsum and explanted at three different time points (3, 7 and 21 days). Wound healing parameters such as wound closure, epithelialisation, angiogenesis, inflammatory and fibroblastic cellular infiltration and scarring were analysed and quantified using stereological methods. The introduction of this enzymatic cross-linking agent stimulated neovascularisation and epithelialisation resisting wound contraction. Hence, these characteristics make this scaffold a potential candidate to be considered as a dermal precursor.

Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems

Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC(50) values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.

Hydrogels for dermal applications

This thesis is concerned with the development of hydrogels that adhere to skin and can be used for topical or trans dermal release of active compounds for therapeutic or cosmetic use. The suitability of a range of monomers and initiator systems for the production of skin adhesive hydro gels by photopolymerisation was explored and an approximate order of monomer reactivity in aqueous solution was determined. Most notably, the increased reactivity of N-vinyl pyrrolidone within an aqueous system, as compared to its low rate of polymerisation in organic solvents, was observed. The efficacy of a series of photoinitiator systems for the preparation of sheet hydrogels was investigated. Supplementary redox and thermal initiators were also examined. The most successful initiator system was found to be Irgacure 184, which is commonly used in commercial moving web production systems that employ photopolymerisation. The influence of ionic and non-ionic monomers, crosslinking systems, water and glycerol on the adhesive and dynamic mechanical behaviour of partially hydrated hydrogel systems was examined. The aim was to manipulate hydrogel behaviour to modify topical and transdermal delivery capability and investigated the possibility of using monomer combinations that would influence the release characteristics of gels by modifying their hydrophobic and ionic nature. The copolymerisation of neutral monomers (N-vinyl pyrrolidone, N,N-dimethyl acrylamide and N-acryloyl morpholine) with ionic monomers (2-acrylamido-2-methylpropane sulphonic acid; sodium salt, and the potassium salt of 3-sulphopropyl acrylate) formed the basis of the study. Release from fully and partially hydrated hydrogels was studied, using model compounds and a non-steroidal anti-inflammatory drug, Ibuprofen. Release followed a common 3-stage kinetic profile that includes an initial burst phase, a secondary phase of approximate first order release and a final stage of infinitesimally slow release such that the compound is effectively retained within the hydrogel. Use of partition coefficients, the pKa of the active and a knowledge of charge-based and polar interactions of polymer and drug were complementary in interpreting experimental results. In summary, drug ionisation, hydrogel composition and external release medium characteristics interact to influence release behaviour. The information generated provides the basis for the optimal design of hydrogels for specific dermal release applications and some understanding of the limitations of these systems for controlled release applications.

Toxicity as a factor influencing the distribution of gammorus pulex in some Midland rivers

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

The development of functional in vitro toxicity tests

In vitro toxicity tests which detect evidence of the formation of reactive metabolites have previously relied upon cell death as a toxicity end point. Therefore these tests determine cytotoxicity in terms of quantitative changes in specified cell functions. In the studies involving the CaC0-2 cell model, there was no significant change in the transport of [3H] L-proline by the cell after eo-incubation with either dapsone or cyclophosphamide (50µM) and rat liver microsomal metabolite generating system. The pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation groups. Studies involving the L6 cell model showed that there was no significant effect in the cell signalling pathway producing the second messenger cAMP, after incubation with dapsone or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. There was also no significant affect on the vasopressin stimulated production of the second messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although there were some morphological changes observed with the cells at the highest concentration of dapsone hydroxylamine (100µM). With the test involving the NG115-401 L-C3 cell model, there was no significant changes in DNA synthesis in terms of [3H] thymidine incorporation, after eo-incubation with either phenytoin or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. In the one compartment erythrocyte studies, there were significant decreases in glutathione with cyclophosphamide (50µM) (0.44 ± 0.04 mM), sulphamethoxazole (50µM) (0.43 ± 0.08mM) and carbamazepine (50µM) (0.47 ± 0.034 mM), when eoincubated with the rat microsomal system, compared to the control (0.52 ± 0.07mM). There was no significant depletion in glutathione when the erythrocytes were eoincubated with phenytoin and the rat microsomal system. In the two compartment erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with cyclophosphamide (50µM) (0.953 ± 0110mM) when co-incubated the rat microsomal system, compared to the control (1.124 ± 0.032mM). Differences were considered statistically significant for p<0.05, using the Student's two tailed 't' test with Bonferroni's correction. There was no significant depletion of glutathione with phenytoin, carbamazepine and sulphamethoxazole when co-incubated with the rat microsomalsystem, compared to the control.

Investigation of aspects of reactive gliosis in human astrocytoma cell lines:application for toxicity assessment

The process of astrogliosis, or reactive gliosis, is a typical response of astrocytes to a wide range of physical and chemical injuries. The up-regulation of the astrocyte specific glial fibrillary acidic protein (GFAP) is a hallmark of reactive gliosis and is widely used as a marker to identify the response. In order to develop a reliable, sensitive and high throughput astrocyte toxicity assay that is more relevant to the human response than existing animal cell based models, the U251-MG, U373-MG and CCF-STTG 1 human astrocytoma cell lines were investigated for their ability to exhibit reactive-like changes following exposure to ethanol, chloroquine diphosphate, trimethyltin chloride and acrylamide. Cytotoxicity analysis showed that the astrocytic cells were generally more resistant to the cytotoxic effects of the agents than the SH-SY5Y neuroblastoma cells. Retinoic acid induced differentiation of the SH-SY5Y line was also seen to confer some degree of resistance to toxicant exposure, particularly in the case of ethanol. Using a cell based ELISA for GFAP together with concurrent assays for metabolic activity and cell number, each of the three cell lines responded to toxicant exposure by an increase in GFAP immunoreactivity (GFAP-IR), or by increased metabolic activity. Ethanol, chloroquine diphosphate, trimethyltin chloride and bacterial lipopolysaccharide all induced either GFAP or MTT increases depending upon the cell line, dose and exposure time. Preliminary investigations of additional aspects of astrocytic injury indicated that IL-6, but not TNF-α. or nitric oxide, is released following exposure to each of the compounds, with the exception of acrylamide. It is clear that these human astrocytoma cell lines are capable of responding to toxicant exposure in a manner typical of reactive gliosis and are therefore a valuable cellular model in the assessment of in vitro neurotoxicity.