Bioscience Horizons Advance Access originally published online on April 19, 2009
Bioscience Horizons 2009 2(2):197-204; doi:10.1093/biohorizons/hzp023
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Can ESR be used to assess the levels of oxidative stress in fat-loaded human hepatocytes and hepatic stellate cells?
University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK
* Corresponding author: Institute of Molecular and Cellular Biology, Garstang Building (8.53), University of Leeds, Leeds LS2 9JT, UK. Email: bslfw{at}leeds.ac.uk
Supervisors: Prof. Ian Mason, Dr Geoff Beckett, and Dr Forbes Howie, Section of Clinical Biochemistry, Division of Reproductive and Developmental Sciences, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionFundingReferencesAuthor Biography |
|---|
Non-alcoholic fatty liver disease (NAFLD) is a growing clinical problem, which manifests itself particularly in obese subjects who may have the metabolic syndrome. A two-hit hypothesis for the pathogenesis of the disease has been proposed. The first hit is the development of insulin resistance leading to fat accumulation specifically in the liver. The second hit involves oxidative damage to the liver when intracellular triglyceride is metabolized by beta-oxidation in the mitochondria to produce harmful reactive oxygen species (ROS) and their hydroperoxide by-products. An in vitro model for NAFLD along with a method to detect the levels of oxidative stress would be useful for testing this hypothesis. Such a model would also allow investigation of the ability of antioxidants such as selenium to prevent oxidative damage. This study aimed to develop a method for assessing the levels of oxidative stress in cultured fat-loaded human hepatocytes (C3A cells) and hepatic stellate cells (LX-2 cells) using electron spin resonance with the spin trap 1-hydroxy-2,2,6,6-tetramethyl-4-oxopiperidine (TEMPONE-H). Cells were fat-loaded with either LPON (lactate, pyruvate, octanoate and NH4+) or oleate. Initial experiments showed that the culture media alone generated free radicals but this was minimal when Dulbecco's phosphate-buffered saline was used as the TEMPONE-H carrier. It proved difficult to detect the free radical production by cells cultured in the basal state; however, when marked oxidative stress was induced in the cells by adding tertiary butyl hydroperoxide (t-BuOOH), free radical production by cells could be identified. Pre-treating cells with selenium, to induce the synthesis of selenoenzymes with antioxidant action, protected cells from the harmful effects of t-BuOOH. This supported selenium's role as an antioxidant, which may have the potential to prevent the onset of non-alcoholic steato-hepatitis. The human vascular endothelial cell line EAhy926 also accumulates lipid as triglyceride when pre-treated with oleate but not with LPON. This suggests that the use of LPON rather than oleate may be a more appropriate model of NAFLD.
Key words: NAFLD, ESR, TEMPONE-H, C3A cells, free radicals, oxidative stress
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionFundingReferencesAuthor Biography |
|---|
Non-alcoholic fatty liver disease (NAFLD) is considered as the hepatic manifestation of the metabolic syndrome and presents itself with a wide spectrum of severity. In its most benign form, fat accumulates in the liver as triglycerides (steatosis) while in some patients there is evidence of non-alcoholic steato-hepatitis (NASH), an associated inflammatory disease, which can progress to fibrosis, cirrhosis and ultimately liver failure or cancer.
In 1998, Day and James1 proposed a two-hit hypothesis for the pathogenesis of NAFLD. The first hit leads to hepatic steatosis and the second to hepatocyte injury and inflammation. If prolonged, this inflammatory response will activate hepatic stellate cells resulting in increased collagen production and clinical fibrosis or cirrhosis. Initial metabolic abnormalities that lead to the accumulation of lipid droplets within hepatocytes are not fully understood. The evidence so far indicates that NAFLD is associated with mitochondrial dysfunction.2
NAFLD is common in patients suffering from type II diabetes, hence insulin resistance is often regarded as the first hit, leading to fatty acid mobilization from adipose tissue and their subsequent uptake and storage in the liver. Lipids are a rich energy source for hepatic cells and they can be transported to the mitochondria where they undergo oxidative metabolism. Reactive oxygen species (ROS) are a natural by-product of this metabolism.
In the respiratory chain, oxygen is normally converted to water but it can also undergo incomplete one-electron reduction, producing ROS with unpaired valence shell electrons (e.g. superoxide and hydroxyl radicals). These ROS may react with lipids in the cells producing harmful lipid peroxides (Fig. 1), peroxynitrite, DNA damage and inactivation of essential enzyme and protein systems.3 ROS also play a role in cell signalling and oxidative stress.4
|
The second hit is often regarded as unopposed oxidative stress in the liver, leading to oxidative damage within the cell, inflammation and disordered cytokine production. In healthy liver, endogenous defence mechanisms counter oxidative stress and prevent oxidative damage. These defence mechanisms include enzymes with antioxidant actions, including families of superoxide dismutases (SODs), catalases, glutathione peroxidises (GPXs) and thioredoxin reductases (TRs), which may reduce harmful free radicals and/or peroxides. If ROS production in hepatocytes overcomes the antioxidant capacity of the cell, it is argued that the occurrence of cell damage leads to inflammatory cytokine production and thus NASH.
Excess ROS and inflammatory cytokines may leak from the hepatocytes and activate adjacent hepatic stellate cells. On activation, stellate cells synthesize collagen, which can lead to fibrosis and cirrhosis. It is also possible that NAFLD also leads to increased ROS production within stellate cells themselves, thus promoting collagen synthesis per se. It is yet to be determined whether stellate cells accumulate triglyceride in vivo although they do accumulate vitamin A in the quiescent state.
Selenoenzymes have important antioxidant actions and, in addition, selenium has anti-inflammatory properties and is capable of improving glycaemic control in diabetic mice.5 A model is required to test whether or not selenium, acting via increased expression of selenoenzymes with antioxidant action, such as GPXs and TRs, can prevent oxidative damage in fatty liver and thus the onset of NASH. Such a model could be used to determine whether selenium can diminish ROS production and thus attenuate the activation of stellate cells preventing the development of NASH and cirrhosis.
GPXs and TRs incorporate selenium as selenocysteine, the ‘21st amino acid’, at their active site. They provide protection from the harmful effects of oxidative stress throughout the cell. In humans, maximal expression of selenoenzymes requires a dietary intake of at least 70 µg/day. In the UK, selenium intake is approximately half of this optimal level, leading to sub-optimal expression of selenoenzymes. Up-regulation of selenoenzymes within hepatocytes and stellate cells, using selenium supplementation, may curb oxidative stress, reducing the number of ROS and lipid hydroperoxides, in turn reducing hepatocellular damage and its pro-fibrogenic consequences.
When investigating the development of diseases thought to be mediated by oxidative damage, it is important to be able to measure the ROS produced by the cell. 6 An in vitro model of hepatocytes and hepatic stellate cells under oxidative stress would assist the development of regimens for NAFLD treatment.
At present, two in vitro cell models of fat-loaded cultured hepatocytes have been described. The Liver Unit at Edinburgh's Royal Infirmary have used an immortalized human hepatocyte cell line (C3A) and found that when grown in the presence of LPON (Lactate, Pyruvate, Octanoate and Nitrogen in the form of NH4Cl)7 these cells accumulate fat droplets comprised of triglycerides (personal communication, Dr J. Plevris). Gomez-Lechon et al.8 have used the immortalized human hepatic cell line HepG2 and reported that these cells, when grown in the presence of long-chain fatty acids (oleate or palmitate), also accumulate triglyceride. Such fat-loaded cells were more resistant to oxidative damage induced by tertiary butyl hydroperoxide (t-BuOOH) than were control cells grown in the absence of long-chain fatty acids. It is therefore clearly important to compare the models to determine which one most closely represents fatty liver and increased oxidative stress.
Differences in oxidative stress might be expected between these models since long-chain fatty acids (e.g. oleate) are converted to fatty acyl CoA via a carnitine transport system before entering the mitochondria. Octanoate is a medium-chain fatty acid and can enter the mitochondria independently of the carnitine transport system and undergoes preferential oxidation. Thus it might be expected that the model using octanoate (i.e. the LPON model) may be subject to higher degrees of oxidative stress than the oleate model.9
A human immortalized hepatic stellate cell line (LX-2) is available, which represents partially activated cells that are able to up-regulate the production of procollagen1 mRNA when exposed to the cytokine TGFβ. Preliminary work in our laboratory has suggested that LX-2 cells when exposed to LPON or oleate also accumulate triglyceride in the form of lipid droplets. The effect of this lipid accumulation on oxidative stress in LX-2 cells has not been reported. A method that quantifies oxidative stress in C3A and LX-2 cells would assist research in this area.
It is very difficult to quantify free radical production by cells in vivo due to their low production, instability, efficient antioxidant defence mechanisms and intracellular location of their production.10 To increase the stability of the free radicals, 1-hydroxy-2,2,6,6-tetramethyl-4-oxopiperidine (TEMPONE-H) can be employed as a spin trap. Its sensitivity for the detection of peroxynitrite and superoxide radicals is 10-fold that of more common spin traps such as 5,5-dimethylpyrrolidine-1-oxide and 2,2,4-trimethyl-2H-imidazole-1-oxide.11
Free radicals possess the property of paramagnetic resonance owned to their unpaired electron. As a result of their ability to rotate, unpaired electrons are magnetic. TEMPONE-H on collision with a free radical is oxidized to the more stable TEMPONE, which can be detected by electron spin resonance (ESR)12 (Fig. 2).
|
Past studies have assessed tissues from animals, human biopsies or isolated mitochondria.13 Possible differences between animal and human models of NAFLD may exclude the use of animal models, and significant amounts of human biopsy material are difficult to obtain. We did not have a ready access to large amounts of normal human liver tissue and therefore sought model systems that employed immortalized LX-2 and C3A cells. These in vitro models could potentially be used to develop treatments for NAFLD without the initial need for large numbers of human or animal biopsies.
The objective of this study was to determine whether ESR along with spin trapping could be used to assess the levels of oxidative stress in fat-loaded human hepatocytes and hepatic stellate cells. In addition, it would be useful to measure the differences in oxidative stress between the LPON and oleate models and determine whether selenium could prevent oxidative damage to these cells induced by t-BuOOH. The ability of the vascular endothelial cell line EAhy926 to accumulate triglyceride in the LPON and oleate treatments was also investigated.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionFundingReferencesAuthor Biography |
|---|
Comparing the ESR Intensities for Various Media in the Absence of Cells
The ESR signals generated by various culture media incubated at 37°C were investigated in the absence of cells. For these experiments, 1 ml of distilled water, phosphate-buffered saline (PBS) solution, Dulbecco's phosphate-buffered saline solution (DPBS), foetal calf serum (FCS; 10%) medium, insulin transferrin (IT) medium (final concentrations 10 mg/l bovine insulin and 5.5 mg/l human transferrin), IT medium supplemented with selenium as 40 nM selenite (ITS) was used. The signal generated was assessed 10 min after the addition of 10 µl of TEMPONE-H (100 mM) to the cells. The effect of vortex mixing on free radical production in PBS and DPBS was also assessed as well as varying the concentration of TEMPONE-H.
Assessing Extracellular Free Radicals in the Presence of Pre-treated and Untreated C3A or LX-2 Cells
Cell Culture
Cell model systems comprising the human hepatocyte cell line C3A or the human stellate cell line LX-2 were used. Dr Plevris (University of Edinburgh) kindly provided the C3A cell line and Dr Friedman (Mount Sinai, School of Medicine, New York) the LX-2 cell line. Cells were maintained using the culture conditions described previously by Campbell et al.14 For experiments that involved weakening the endogenous defence mechanisms of the cells, cells were initially cultured in FCS medium but then transferred to IT medium to deplete cells of selenium and thus to also diminish the expression of selenoenzymes with anti-oxidant action. Cells were also exposed to ITS medium to act as a control where selenoenzymes were maximally expressed.
Cell Plating
LX-2 and C3A cells were plated on Day 0 at sub-confluence with 25 000 and 50 000 cells/ml, respectively, into 12-well plates and allowed to grow at 37°C in a CO2 incubator for 5 days until confluent. For studying the effect of cell density on ESR signal, triplicate wells of each 300 000, 150 000, 75 000 and 37 500 cells/ml were plated. Treated (LPON, oleate or t-BuOOH) and untreated cells were analysed by ESR with TEMPONE-H spin trapping as described above.
Cell Treatment
All incubation conditions were performed using triplicate wells. Untreated cells and wells containing culture media in the absence of cells (as controls) were prepared on Day 0. On Day 1, 40 µl of LPON (20 mM lactate, 2 mM pyruvate, 4 mM octanoate, and 4 mM nitrogen in the form of NH4Cl) was added to wells containing C3A or LX-2 cells. In addition, C3A or LX-2 cells were treated with oleate or t-BuOOH at final concentrations of 0.025 and 0.2 mM, respectively, the latter being used to induce oxidative stress in a reproducible manner.5
Preparation of ESR Samples
After 3 days of incubation with the various agents, cells on Day 4 were washed and the control medium or medium containing the various treatments was replaced with either 0.5 ml of PBS or DPBS. Cells and reagents were kept at 37°C throughout. At time 0 min TEMPONE-H was added to the PBS/DPBS media in each of the wells and samples for ESR measurement were then taken at the specified time intervals.
Assessing Intracellular Free Radical Production
TEMPONE-H was added to the culture media covering the cells and incubated overnight. Cells were either lysed by osmotic shock with water or sonicated, and ESR samples were taken from the cell lysates at 10-min intervals up to 90 min. The effect of pre-lysing the cells with cell lysis buffer prior to TEMPONE-H addition was also investigated.
Free radicals trapped by the TEMPONE-H were quantified using a Miniscope MS200 ESR machine. ESR spectra were visualized using Miniscope software (Magnet Tech., Berlin). Settings for the Magnet Tech Miniscope MS200 ESR machine were as follows: BO = 3555 G, sweep = 55 G, sweep time = 30 s, smooth = 0, steps = 4096, number of passes = 1, mod = 1500, MW atten = 7, gain = 1E1 and phase 180.
Cell Harvesting prior to Assay for Protein and Triglyceride
Cells were harvested on ice using 150 µl of cell lysis buffer (0.125 M sodium phosphate, 1 mM EDTA, 0.1% (v/v) TritonX-100, pH 7.4) and sonicated prior to assay of total protein and triglyceride.
Total Protein Assay
The Bradford assay15 adapted for use on the Cobas Fara Centrifugal Analyser (Roche Diagnostics, Welwyn Garden City, UK) was used to measure total protein in the C3A cell lysates using BSA as the standard.
Triglyceride Concentration of EAhy926 Cells
A commercial kit (Alpha Laboratories Ltd, UK) adapted for use on the Cobas Fara Centrifugal Analyser was utilized, according to the manufacturers instructions, to measure total triglyceride in the cell homogenate. All triglyceride measurements were corrected for total protein measured using this method.
Quantifying Lactate Dehydrogenase Activity
The amount of cell damage was quantified by assessing release of lactate dehydrogenase (LDH) activity.16 A commercial kit (Alpha Laboratories Ltd, UK) adapted for use on the Cobas Fara Centrifugal Analyser (Roche Diagnostics Ltd) was utilized, according to the manufacturer's instructions.
Data Analysis
ESR intensity was expressed on an arbitrary scale based on the area under the curve of the first derivative of the first peak in the spectra generated. All data were handled using Microsoft Excel and Graphpad. Data were analyzed using the one-way ANOVA test and the Tukey test.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionFundingReferencesAuthor Biography |
|---|
Effects of Culture Medium and Cell Number on ESR Signal Generation
Initial experiments using C3A cells cultured in FCS were encouraging as they appeared to show significant differences in the ESR signal generation when cells were pre-treated with LPON or t-BuOOH. Cells pre-treated with t-BuOOH as a positive control produced a large ESR signal. This was as predicted as t-BuOOH is known to cause high levels of oxidative stress in cells.5 LPON-treated cells appeared to produce a large ESR signal in comparison to untreated cells. However, subsequent experiments showed that this result was not reproducible and that ESR intensities were modified by the culture media per se even in the absence of cells (Fig. 3). It was also found that the ESR signal generated was independent of cell number (Table 1) in contrast to the findings of Shi et al.17 Thus, it was concluded that in our system the free radical ESR signal being generated arose from the culture medium per se with little or no contribution arising from cells. It was thus essential to choose a culture medium that had minimal signal generation through free radical production in order that any free radical production by the cells could be assessed.
|
|
The more complex media containing FCS or transferrin generated particularly high ESR signals (Fig. 3) probably due to the presence of Fe in the culture medium, which would promote free radical production by the Fenton reaction. These culture media were thus considered to be unsuitable for assessing cellular free radical production with a high degree of sensitivity due to their high background free radical production. ITS medium is supplemented with selenium and generated lower ESR intensities than IT medium (Fig. 3), suggesting that selenium itself can lower the number of free radicals in the absence of cells.
The medium with the lowest ESR signal was deionized water (Fig. 3). Unfortunately, this did not generate a sigmoidal ESR spectrum characteristic of TEMPONE-H oxidation; the signal was low but erratic. Furthermore, water was unsuitable to sustain viable cells in culture and cells quickly became detached from the surface of the culture dish.
DPBS was subsequently used as the medium of choice as it had not only a low background ESR signal (Fig. 3) but also allowed cells to remain attached to the plate and viable (due to the calcium and magnesium ions present in the medium). DPBS also allowed a good sigmoidal ESR signal.
Experiments with Cultured C3A Cells
When free radical generation in C3A cells cultured in various conditions was assessed using DPBS as the TEMPONE-H carrier, no significant differences in the generation of an ESR signal between cell pre-treatments were seen (Fig. 4). However, these cells were cultured in FCS medium that contained some selenium, suggesting that the endogenous defence mechanisms (GPX and TR levels) were optimal and able to cope with any oxidative stress caused by each pre-treatment in which case this result would be expected. The ESR signals produced by cells cultured in FCS medium fall into the same range of ESR signals recorded for DPBS in the absence of cells over the same time course (Fig. 3). This suggests that the ESR signals shown in Fig. 4 were background signals generated by the DPBS alone. Subsequently, in later experiments, cells were cultured in IT medium, which contains no selenium in efforts to weaken the endogenous defence mechanisms and amplify the ESR signal produced by pre-treated cells.
|
Assessing Intracellular Free Radical Levels of C3A Cells
As attempts to determine extracellular free radical levels were unsuccessful, attention was turned towards intracellular free radical levels. Several studies, utilizing TEMPONE-H have assessed intracellular free radical levels in endothelial cells, suggesting that TEMPONE-H is able to enter cells.17
Incubation of pre-treated C3A cells with TEMPONE-H added to FCS culture medium 24 h prior to lysis by osmotic shock with water showed very low and erratic ESR signals (results not shown). This observation may be due to the fact that any TEMPONE-H getting into the cells would have to compete with other endogenous free radical trappers. A similar experiment, whereby TEMPONE-H was added to cells, post-osmotic shock, gave similar erratic results with low ESR signals (results not shown). This may be due to the low background ESR signal being caused by the release of proteins, DNA and other molecules that compete with TEMPONE-H to trap free radicals.
Weakening the Endogenous Defence Mechanisms of C3A Cells
Final attempts to try to identify free radical production by cells involved weakening their endogenous defence mechanisms by culturing them in IT medium rather than FCS to deplete cells of selenium. IT medium does not contain FCS (and therefore no selenium source or other factors that may allow the cell to build up its antioxidant defences) thus theoretically making the cells more susceptible to oxidative damage.18
Using these IT-treated cells, it was found that the ESR intensity forming in DPBS in the absence of cells was greater than when cells were present. This suggested that the cells were scavenging the free radicals generated in the DPBS TEMPONE-H carrier. In contrast, when cells were treated with t-BuOOH to induce oxidative stress, free radical accumulation in the DPBS was higher in the presence of cells compared with when cells were absent. This suggested a net movement of free radicals from t-BuOOH-treated cells to the DPBS, an effect that was dependent on the concentration of t-BuOOH used (Fig. 5).
|
When the same experiment was performed using cells grown in the presence of added selenium (ITS, selenium added as 40 nM selenite), the net movement of free radicals from cells into DPBS appeared to be diminished particularly when t-BUOOH (0.2 mM) was used (Fig. 5). This is possibly because cells grown in ITS have access to a selenium supply that increases their endogenous antioxidant defence mechanisms by inducing selenoenzymes that would diminish free radical production induced by t-BuOOH. This experiment would need to be repeated to be conclusive but it did show promise as a method that could be used for assessing free radical production by cells. If these results were reproducible, the method could thus be applied to LPON- or oleate-treated C3A cells.
Protection by Selenium of Cell Damage Induced by t-BuOOH
To further show that selenium was protecting the cells from oxidative damage, release of LDH from cells was assessed in cells exposed to increasing concentrations of t-BuOOH (Fig. 6). This investigation showed ITS medium protected cells from cell death even at the highest doses of t-BuOOH. The use of FCS medium also prevented cell death in cells treated with t-BuOOH. This protective effect appears to be due to the selenium supply in the FCS and ITS culture media that allows optimal expression of the antioxidant selenoenzymes glutathione peroxidase and thioredoxin reductase.19 These data support a role for selenium as an antioxidant and suggest that it may potentially be used therapeutically to prevent progression of NAFLD to NASH.
|
It would be of value to pre-treat cells with LPON or oleate to determine whether there is any increased free radical production when cells are conditioned in IT medium and whether conditioning with media containing selenium (ITS and FCS) can counteract this production by up-regulation of selenoenzymes.18 To further weaken the endogenous defence mechanisms, a superoxide dismutase inhibitor could also be used, such as diethyldithiocarbamic acid (DETCA) to render the organism more susceptible to oxygen toxicity,20 theoretically increasing oxidative stress.
One study has isolated mitochondria from mouse brain tissue and claimed to have successfully used ESR with TEMPONE-H as a spin trap to quantify oxidative stress in these cells.13 The quantities of mitochondria obtainable from the cultured C3A cell line would be a foreseeable obstacle to such experiments.
Experiments with Cultured LX-2 cells
Results using LX-2 cells failed to show any evidence of extracellular free radical production as with C3A cells. Owing to time restrictions LX-2 cells treated with IT media and t-BuOOH were not studied.
Lipid Accumulation in EAhy926 Cells
The uses of LPON or oleate treated C3A cells have both been suggested as models to mimic fatty liver as in both circumstances the cells accumulate triglyceride.7 Experiments were therefore carried out to determine whether the ability to fat load on exposure to oleate or LPON is confined to hepatocytes. To do this, EAhy926 cells (a human umbilical vein endothelial cell line) were treated with the culture conditions of the oleate and LPON model and the triglyceride content was assessed. These data showed that endothelial cells cannot accumulate triglycerides in the presence of LPON but can with oleate (Fig. 7). This suggests that only liver cells, but not endothelial cells, can produce triglycerides in the presence of medium-chain fatty acids. This is possibly due to a lack of the enzyme system in endothelial cells that allows medium-chain fatty acids to be elongated to synthesize long-chain fatty acids.21 Owing to time restrictions this experiment could not be repeated using an octonoate (LPON)-treated C3A hepatocyte control, which would have further highlighted the lack of triglycerides produced by octonoate (LPON)-treated EAHy926 cells.
|
In conclusion, the methods used in this study were unable to determine the intracellular or extracellular levels of free radicals in cultured human hepatocytes and hepatic stellate cells. However, it seems that weakening the endogenous defence mechanisms of these cells may be the key to the development of a successful method and a step towards an in vitro model for NAFLD that can test the effects of antioxidants such as selenium. This study favoured the use of the LPON model over the oleate model and showed that selenium was able to protect fat-loaded C3A cells from oxidative damage induced by t-BuOOH as has been shown previously in EAhy926 cells.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionFundingReferencesAuthor Biography |
|---|
The University of Edinburgh.
|
Acknowledgements |
|---|
My sincere thanks to Prof. Ian Mason, Dr Geoff Beckett and Dr Forbes Howie for all their help and support throughout my time in the laboratory, Dr J. Allan, Dr M. Miller and the staff at the QMRI in Edinburgh and also to Dr Plevris, Dr Filipi and Dr Freidman for kindly supplying the cell lines and my family, flatmates, friends and the University of Edinburgh for their support.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionFundingReferencesAuthor Biography |
|---|
- Day CP, James O. Steatohepatitis: a tale of two "hits"? Gasteroenterology (1998) 114:842–845.[CrossRef][Web of Science][Medline]
- Pessayre D, Fromenty B. NASH: a mitochondrial disease. J. Hepatol (2005) 42:928–940.[CrossRef][Web of Science][Medline]
- Vickers T, Young IS, McEneny J. Lipoprotein oxidation and atherosclerosis. Biochem Soc Trans (2001) 29(Pt 2):358–362. http://schools-wikipedia.org/images/834/83467.png.htm.[CrossRef][Web of Science][Medline]
- Maulik N. Reactive oxygen species drives myocardial angiogenesis? Antioxid Redox Signal (2006) 8:2161–2168.[CrossRef][Web of Science][Medline]
- Beckett GJ, Arthur JR. Selenium and endocrine systems. J Endocrinol (2005) 184:455–465.
[Abstract/Free Full Text] - Dikalov S, Skatchkov M, Fink B, et al. Quantification of superoxide radicals and peroxynitrite in vascular cells using oxidation of sterically hindered hydroxylamines and electron spin resonance. Nitric Oxide (1997) 1:423–431.[CrossRef][Web of Science][Medline]
- Filippi C, Keatch S, Rangar D, et al. Improvement of C3A cell metabolism for usage in bioartificial liver support systems. J Hepatol (2005) 41:599–605.[Web of Science]
- Gomez-Lechon MJ, Donato MT, Martinez-Romero A, et al. A human hepatocellular in vitro model to investigate steatosis. Chem Biol Interactions (2007) 165:106–116.[CrossRef][Web of Science][Medline]
- Alaoui-Talibi Z, Moravec J. Carnitine transport and free fatty acid oxidation in chronically volume-overloaded rat hearts. Biochim Biophys Acta (1989) 1003:109–14.[Medline]
- Batandier C, Fontaine E, Keriel C, et al. Determination of mitochondrial reactive oxygen species: methodological aspects. J Cell Mol Med (2002) 6:175–187.[Web of Science][Medline]
- Halliwell B, Whiteman M. Measuring reactive oxygen species and oxidative damage in vivo and in cell culture, how should you do it and what do the results mean? Br J Pharmacol (2004) 142:231–255.[CrossRef][Web of Science][Medline]
- Rohn S, Kroh LW. Electron spin resonance – a spectroscopic method for determining the antioxidative activity. Mol Nutr Food Res (2005) 49:898–907.[CrossRef][Web of Science][Medline]
- Paterson AWJ, Curtis JC, Macleod NK, et al. Complex I specific increase in superoxide formation and respiration rate by PrP-null mouse brain mitochondria. J Neurochem (2007) 105:177–191.[CrossRef][Web of Science][Medline]
- Campbell L, Howie F, Arthur JR, et al. Selenium and sulforaphane modify the expression of selenoenzymes in the human endothelial cell line Eahy926 and protect cells from oxidative damage. Nutrition (2007) 23:138–144.[CrossRef][Web of Science][Medline]
- Bradford MM. A rapid and sensitive method for quantification of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem (1976) 72:248–254.[CrossRef][Web of Science][Medline]
- Gay RJ, McComb RB, Bowes GN Jr. Optimum reaction conditions for human lactate dehydrogenase activity. Clin Chem (1968) 14:740–753.[Abstract]
- Shi H, Timmins G, Monske M, et al. Evaluation of spin trapping agents and trapping conditions for detection of cell generated reactive oxygen species. Arch Biochem Biophys (2005) 437:59–68.[CrossRef][Web of Science][Medline]
- Lewin MH, Arthur JR, Riemersima RA, et al. Selenium supplementation acting through the induction of thioredoxin reductase and glutathione peroxidase protects the human endothelial cell line EAhy926 from damage by lipid hydroperoxides. Biochim Biophys Acta (2002) 1593:85–92.[Medline]
- Saito Y, Yoshida Y, Akazawa T, et al. Cell death caused by selenium deficiency and protective effect of antioxidants. J Biol Chem (2003) 278:39428–39434.
[Abstract/Free Full Text] - Kelner MJ, Bagnell R, Hale B, et al. Inactivation of copper–zinc superoxide dismutase by copper chelating agents without glutathione depletion and methemoglobin formation. Free Radic Biol Med (1989) 6:355–360.[CrossRef][Web of Science][Medline]
- Dagher Z, Ruderman N, Tornhein K, et al. Acute regulation of fatty acid oxidation and AMP activated protein kinase in human umbilical vein endothelial cells. Circ Res (2001) 88:1276–1282.
[Abstract/Free Full Text]
|
Author Biography |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionFundingReferencesAuthor Biography |
|---|
-
After studying for four years at the University of Edinburgh, Laura Wetherill graduated with a 2:1 degree with Honours in Biochemistry in June 2008. At present she is studying for an MRC-funded PhD at the University of Leeds, investigating the mechanism by which human papilloma virus (HPV) evades the innate immune system and causes cervical cancer. Laura is also interested in biofluids and biomarkers associated with clinical diseases such as type II diabetes, cardiovascular disease and non-alcoholic fatty liver disease (NAFLD).
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||