Effects of Hyperoxia in Bovine Bronchial Epithelial Tissue - Dissertation Example

?Running Head: Effects of Hyperoxia Effects of Hyperoxia in Bovine Bronchial Epithelial Tissue Name Date of Submission Effects of Hyperoxia in Bovine Bronchial Epithelial Tissue Introduction Hyperoxia can be simply explained as excess oxygen. The condition takes place when normal partial pressure of the gas is exceeded. Excess of oxygen in body tissues is caused by exposure to the gas at pressure higher than normal atmospheric pressure. Hyperoxia can lead to oxygen toxicity, which can cause serious health hazards and even death. Important Use of Hyperoxia in Intensive Care Unit Hyperoxic inspired gas is essential for patients with hypoxic respiratory failure which can be caused by oxygen deficient conditions like acute infection, neuromuscular impairment, etc. (Altemeler and Sinclair, 2007) In the context of critical care medicine, hyperoxia can be beneficial in implementing certain critical care strategies like early goal directed therapy (Calzia et al, 2010). Moreover, oxygen pressure field theory suggests that hyperoxia just before deep hypoxic circulatory arrest takes advantage of increased oxygen solubility and reduced oxygen consumption to load tissues with excess oxygen, which can effectively manage acid-base states during acute hypothermia entailed in circulatory arrest (Pearl et al, 2000) However, studies also testify that hyperoxia adversely affects cilial abundance and cause ciliary disorientation which can lead to dangerous conditions like ciliary dyskinesia (MacNaughton et al, 2007; Kay et al, 2002; Rutman et al, 1993). Also, hyperoxia may impede the pathways of cell signalling (Lee and Choi, 2003) Side Effects of Reactive Oxygen Species (ROS) on Epithelial Tissue Reactive Oxygen Species (ROS) are oxygen containing molecules which are highly reactive. The unpaired valance shell electrons in ROS are responsible for their high reactivity. ROS are often regarded as a key factor behind cardiovascular diseases, ischemic injury, programmed cell death, etc. They can also cause damage to DNA, lipid peroxidation and critical oxidative stress. (Thannickal, 2003; Fuhrman et al 1997) ROS would cause oxidative stress on the epithelial tissue by increasing the levels of total glutathione. Since glutathione is an anti-oxidant, increased levels of ROS will increase its concentration as well. In the case of glutathione depletion, increase of ROS levels is unbridled which would lead to early activation of apoptic signalling. In vivo studies involving human B lymphoma cell line testify such possibilities (Armstrong et al, 2002). Moreover, it has also been testified that pulmonary macrophages stimulate cell proliferation of bovine bronchial epithelial cells in vitro. The process involves mediation in airway epithelial repair, which can probably be explained by a proactive role of glutathione against ROS (Takizawa et al, 1990). Another side effect of ROS is lipid peroxidation which has been studied in details through epithelial cell behaviour in vivo in rats with chronic parenchymal iron overload (Bacon et al, 1983). Hepatic and brain epithelial lipid peroxidation by ROS obtained from certain pesticides have been widely testified by both in vivo and in vitro studies in rats and humans (Bagchi et al, 1995). Besides, Fuhrman and his associates conducted in vitro and ex vivo studies in humans to testify the high extent of low-density-lipoprotein oxidation by ROS through measurement of thiobarbituric acid reactive substances (TBARS) and lipid peroxides in epithelial cells (Fuhrman et al, 1997). Proteins modification is another major side effect of excess ROS generation that has been studied in vivo. The in vivo study conducted in this context further testified that oxidative protein damage could affect the activities of the DNA repair enzymes in the epithelial cells as well (Wiseman and Halliwell, 1996). Further, in vitro studies have established that generation of ROS target the function of redox-sensitive proteins that act as part of a large sub-membranous signalling complex, thus hampering cellular signalling mechanism (Thannickal, 2003). In regards of DNA damage, in vivo studies establish that non-melanoma skin tumours involving epithelial cells could be produced through misreplication of the 8-OHdG lesions induced by ROS in humans (Wiseman and Halliwell, 1996). In vitro studies also show that the frequency of mutation is greatly increased by exposure of DNA to ROS, possibly by mechanisms entailing rearrangements of the nascent strand-template (Wiseman and Halliwell, 1996). Further, in vitro production of ROS by certain pesticides has been found to be damaging the DNA strands of female Sprague-Dawley rats inside an experimental environment (Bagchi et al, 1995). Methods Utilised in the Context of the Present Research TBARS Assay: It is a reliable method to assess free radical production and resultant oxidative damage. For example, effectiveness of the method to detect oxidative stress has been testified in the context of aquatic toxicology (Oakes and Van Der Kraak, 2003) Comet Assay: This method helps in detecting DNA damage and repair (Collins, 2004). Total Glutathione Assay: It is a reliable colorimetric method to determine glutathione concentrations and evaluate oxidative stress (Dojindo Molecular Technologies Inc, 2009; Owens and Belcher, 1965) OxyBlot Kit: This method is an effective immunoblot technique that can be used to determine the levels of protein carbonyl formation as an indicator of protein denaturation induced by oxidative stress. (Singhal et al, 2002) SDS Page: It is a widely used method in molecular biology, genetics, forensics, etc. to separate the proteins in accordance with their respective electrophoretic mobility (Schagger and von Jagow, 1987). Western Blot: Western blotting powered by Enhanced Chemiluminescence (ECL) has been demonstrated as a fast and sensitive method of protein detection (General Electric Company, 2011). For instance, this technique has been effectively used to examine the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets (Towbin, Staehelin and Gordon, 1979). Aims and Objectives This research is aimed at evaluating and analysing the effects of hyperoxia in bovine bronchial epithelial tissue. The effects are analysed on the basis of the following: 1. Study utilising TBARS assay to determine the relative extent of lipid peroxidation (thiobarbituric acid reactive substances) in the homogenised bovine bronchus tissue cultured in hyperoxic conditions as compared to normoxic conditions. 2. Study utilising Comet assay to determine the extent of DNA damage through comet tail formation in the hyperoxic groups as compared to the normoxic ones. 3. Measurement of total glutathione through total glutathione assay in the hyperoxic and normoxic groups to find out relative oxidative stress. 4. Evaluation of protein oxidation by means of OxyBlot and Western Blotting in the hyperoxic and normoxic tissue samples. This research will provide ample background information for proceeding towards advanced in vivo and in vitro studies on hyperoxia involving humans. Results Results of TBARS Assay TBARS assay has been used to investigate the biochemical dynamics of low-density-lipoprotein (LDL) oxidation. Extent of LDL oxidation has been determined by measuring the formation of conjugated dienes, thiobarbituric acid reactive substances (TBARS) and lipid peroxides (Fuhrman et al, 1997). TBARS assay is thus an established and important method to measure the effect of oxidation on tissue and cells. In the present research, a modified protocol from AMDCC was used. Tissues were chopped into small pieces and homogenised using Potter homogeniser. Samples were then properly centrifuged and incubated. Six samples were used in triplicate and the absorbance was measured at 532 nm using ultraviolet spectrophotometer (SpectraMax 340 PC, Molecular Devices Corporation). The results were converted to a TBARS concentration by calibration against the standard curve were 1,1,3,3 tetramethoxypropane stock was used to prepare serial standard dilution of 50 µM standard with distal water. Figure – 1 (Source: The University of Plymouth) In Figure – 1, TBARS standard curve is shown where serial dilution of 50 µM 1,1,3,3 tetramethoxypropane with water was due. TBARS was measured in the homogenised tissues to determine the lipid peroxidation under the oxidative stress. The T-test analysed results from three animals (six samples per animal, in triplicate). TBARS was significantly in hyperoxia group after 6 days of exposure compared to normoxia group. Statistical analysis shows mean ± SE = 24.21 ± 2.8 in 21 % (normoxia group) and mean ± SE = 35.50 ± 2.1 in 95 % (hyperoxia group) where P < 0.05. Figure – 2 (Source: The University of Plymouth) Figure – 2 shows lipid peroxidation (thiobarbituric acid reactive substance) in extracts of homogenised bovine bronchus tissue cultured under normoxic and hyperoxic conditions. Data are means ± S.E., n = 3. Results of Comet Assay The Comet assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology and fundamental research in DNA damage and repair (Collins, 2004). Comet assay is also called single-cell gel electrophoresis (SCGE) which is used to detect DNA damage in individual cell, where the cells migrate in agarose after subjected to electrophoresis. Cells with DNA damage appear with comet tail (the tail length depend on the damage level) while the undamaged DNA cells appear with a sphere shape. During the present research, slides with embedded cells were carefully and methodologically prepared. The slides were visualised using fluorescence microscopy (Leica DMR). Komet 5.0 image analysis software (Kinetic Imaging) was used to score the individual comet tails. In this way, alkaline SCGE assay (or Comet assay) was used to determine the quantitative DNA oxidative damage. Three animals were used (500 cells per animal for each treatment were analysed, 5 slides were used for each treatment with 100 cells per slide). A significant increase (P = 0.048) in DNA strand breaks was observed in the group treated with 95% oxygen concentration for 4 days compared with 21 % oxygen concentration exposed group. Finally, DNA damage was significantly higher in hyperoxia after 6 days of exposure compared to the control group. Statistical analysis shows mean ± SE = 11.08 ± 1.5 in normoxia and mean ± SE = 18.70 ± 2.22 in hyperoxia where P Read More