Hepatoprotective effect of 7-Hydroxycoumarin against Methyl glyoxal toxicity via activation of Nrf2.

Methyl glyoxal (MG), a major precursor of advanced glycation end-products, has been identified as significant in the progression of several diseases including aging, diabetes and neurodegenerative diseases as well as causing hepatic damages. 7-hydroxycoumarin (7-HC), a natural-occurring derivative of coumarin from fruits and plants, has been reported to exert antioxidant and free radical-scavenging properties, protecting cells from aldehydes and oxidants. In this study, the ability of 7-HC to protect human HepG2 cells against MG-induced toxicity and oxidative stress was investigated. Results show that 7-HC pretreatment significantly attenuates MG-induced cytotoxicity, apoptotic changes and ROS accumulation and that this protection is shown to be associated with the induction of the nuclear factor erythroid 2-related factor 2 (NRF2) and its downstream detoxifying enzymes. In response to 7-HC, NRF2 protein translocates from cytosol to the nuclei. In addition, depletion of NRF2 by siRNA significantly reduces the protective effect of 7-HC against MG, suggesting that NRF2 plays an important role in the protective function of 7-HC. These findings highlight the potential for the interventional activation of the NRF2 induction via the non-toxic natural phytochemical 7-HC as a novel therapeutic approach towards the detoxification of MG, with the aim of halting the progression of diseases in which MG has been implicated.

Observation of coherent delocalized phonon-like modes in DNA under physiological conditions.

Underdamped terahertz-frequency delocalized phonon-like modes have long been suggested to play a role in the biological function of DNA. Such phonon modes involve the collective motion of many atoms and are prerequisite to understanding the molecular nature of macroscopic conformational changes and related biochemical phenomena. Initial predictions were based on simple theoretical models of DNA. However, such models do not take into account strong interactions with the surrounding water, which is likely to cause phonon modes to be heavily damped and localized. Here we apply state-of-the-art femtosecond optical Kerr effect spectroscopy, which is currently the only technique capable of taking low-frequency (GHz to THz) vibrational spectra in solution. We are able to demonstrate that phonon modes involving the hydrogen bond network between the strands exist in DNA at physiologically relevant conditions. In addition, the dynamics of the solvating water molecules is slowed down by about a factor of 20 compared with the bulk.

Umbelliferone and esculetin protect against N-nitrosodiethylamine-induced hepatotoxicity in rats.

N-nitrosodiethylamine (NDEA), a nitrosamine compound, is known to cause liver damage through the generation of reactive oxygen species (ROS), resulting in oxidative damage to macromolecules such as DNA, and the consequent development of cancer. The present study examines the protective effects of two antioxidant coumarin compounds umbelliferone (Umb) and esculetin (Esc) against NDEA-induced hepatotoxicity when administered in the diet to male Wistar rats. The results show that treatment with Umb (0.5% w/w) and Esc (0.5% w/w) in the diet for 7 days significantly attenuates NDEA-induced liver damage, lowering serum alanine transaminase (ALT) levels, decreasing hepatic lipid peroxidation, and restoring total glutathione levels. To investigate the mechanism for the observed protective effect, the levels of the key protective enzymes NAD(P)H: quinone oxidoreductase 1 (NQO1), heme oxygenase (HO1), and glutathione S-transferase Pi (GSTP1) were measured by Western blotting following Umb and Esc administration. The results showed that Umb and Esc administration significantly increased the expression of NQO1 by 3.6- and 2.7-fold, HO1 by 2.7- and 3.2-fold, and GSTP1 by 2.8- and 3.2-fold, respectively. In conclusion, Umb and Esc are capable of protecting liver from NDEA-induced hepatotoxicity, and this is associated with the induction of protective enzymes.

Heme oxygenase-1 attenuates cadmium-induced mitochondrial-caspase 3- dependent apoptosis in human hepatoma cell line.

BACKGROUND: Cadmium (Cd) is a well known environmental and industrial toxicant causing damaging effects in numerous organs. In this study, we examined the role of heme oxygenase-1 (HO-1) in modulating the Cd-induced apoptosis in human hepatoma (HepG2) cells after 24 h exposure. METHODS: HepG2 cells were exposed to 5 and 10 µM Cd as CdCl2 for 24 h while other sets of cells were pre-treated with either 10 µM Cobalt protoporphyrin (CoPPIX) or 10 µM Tin protoporphyrin (SnPPIX) for 24 h, or 50 µM Z-DEVD-FMK for 1 h before exposure to 5 and 10 µM CdCl2 for 24 h. Expressions of caspase 3, cytosolic cytochrome c, mitochondrial Bax and anti-apoptotic BCL-xl proteins were assessed by western blot. Intracellular reactive oxygen species (ROS) production was determined using the dihydrofluorescein diacetate (H2DFA) method. Cell viability was assessed by MTT assay, while a flow cytometry method was used to assess the level of apoptosis in the cell populations. RESULTS: Our results show that there were a significant increase in the expression of cytosolic cytochrome c, mitochondrial Bax protein, and caspase 3 at 5 and 10 µM compared to the control, but these increases were attenuated by the presence of CoPPIX. The presence of SnPPIX significantly enhanced Cd-induced caspase 3 activities. CoPPIX significantly decreased the level of ROS production by 24.6 and 22.2 % in 5 and 10 µM CdCl2, respectively, but SnPPIX caused a significant increase in ROS production in the presence of CdCl2. HepG2 cell viability was also significantly impaired by 13.89 and 32.53 % in the presence of 5 and 10 µM CdCl2, respectively, but the presence of CoPPIX and Z-DEVD-FMK significantly enhanced cell survival, while SnPPIX enhanced Cd-impaired cell viability. The presence of CoPPIX and Z-DEVD-FMK also significantly decreased the population of apoptotic and necrotic cells compared with Cd. CONCLUSION: In summary, the present study showed that HO-1 attenuates the Cd-induced caspase 3 dependent pathway of apoptosis in HepG2 cells, probably by modulating Cd-induced oxidative stress.

Nrf2-mediated adaptive response to methyl glyoxal in HepG2 cells involves the induction of AKR7A2.

Methyl glyoxal (MG), a highly reactive dicarbonyl metabolite, causes a range of changes within the cell. It forms adducts with DNA and protein and contributes to the progression of several diseases as well as causing hepatic damage. In this study, we have used human hepatoma (HepG2) cells as a model to investigate the induction of protective enzymes in response to MG exposure. We have shown that treating HepG2 cells with sub-lethal concentrations of MG increases the level of NADPH:quinone oxidoreductase (NQO1) mRNA by 4.5-fold, AKR1C3 mRNA by 14-fold and AKR7A2 mRNA by 4-fold. Levels of AKR7A2 protein are increased by 2.1- and 1.8-fold following 9h and 24h exposure of cells to 50 µM MG. The role of AKR7A2 in protecting HepG2 cells against MG toxicity was further investigated using specific siRNAs against AKR7A2 and Nrf2. Knockdown of AKR7A2 in HepG2 shows that AKR7A2 is responsible for up to 50% of the protection against MG toxicity in HepG2 cells. We have also shown that MG was able to induce the translocation of the transcription factor Nrf2 to the nucleus. HepG2 cells in which Nrf2 had been knocked down exhibited decreased NQO1 and AKR7A2 mRNA levels compared to control cells. In conclusion, these findings indicate that protective enzymes are significantly up-regulated in response to low concentrations of MG in HepG2 cells and that AKR7A2 contributes to protection against MG-induced toxicity. Nrf2 is critical in mediating MG induced expression of protective genes.

Metabolism of gamma hydroxybutyrate in human hepatoma HepG2 cells by the aldo-keto reductase AKR1A1.

Gamma hydroxybutyrate (GHB) is a recreational and date-rape drug, for which the detection following ingestion is hampered by rapid metabolism and its endogenous presence. GHB catabolism occurs mainly by its oxidation to succinic semialdehyde (SSA), which converts to succinate and enters the tricarboxylic acid cycle. A high Km aldehyde reductase has previously been reported to catalyse the NADP-dependent oxidation of GHB at high concentrations. It is assumed that this enzyme is identical to the aldo-keto reductase AKR1A1, but its role in GHB oxidation has not been fully evaluated. In this study, the extent of AKR1A1 in GHB metabolism has been determined in HepG2 cells using RNA-interference technology. The gene encoding AKR1A1 was targeted by siRNA. Results demonstrate a successful knock-down of the AKR1A1 gene with 92% reduction in total mRNA and 93% reduction in protein expression. Demolishing AKR1A1 expression in HepG2 cells leads to significant 82% decrease in NADP-dependent GHB-dehydrogenase activity at high concentration (10mM) of GHB. Moreover, when exposing the cells to 50 µM of GHB for 24h, and measuring intracellular and extracellular GHB levels by GC/MS, a significant two-fold increase was observed on GHB intracellular level in silenced cells. In contrast, measuring SSA-reductase activity in silenced cells indicated that AKR1A1 is not involved in endogenous GHB production. These findings describe a pathway for GHB metabolism in the liver which should be useful in GHB exposure cases, and will enable a better understanding of the enzymes participating in its metabolism at natural and overexposed levels.

Terahertz underdamped vibrational motion governs protein-ligand binding in solution.

Low-frequency collective vibrational modes in proteins have been proposed as being responsible for efficiently directing biochemical reactions and biological energy transport. However, evidence of the existence of delocalized vibrational modes is scarce and proof of their involvement in biological function absent. Here we apply extremely sensitive femtosecond optical Kerr-effect spectroscopy to study the depolarized Raman spectra of lysozyme and its complex with the inhibitor triacetylchitotriose in solution. Underdamped delocalized vibrational modes in the terahertz frequency domain are identified and shown to blue-shift and strengthen upon inhibitor binding. This demonstrates that the ligand-binding coordinate in proteins is underdamped and not simply solvent-controlled as previously assumed. The presence of such underdamped delocalized modes in proteins may have significant implications for the understanding of the efficiency of ligand binding and protein-molecule interactions, and has wider implications for biochemical reactivity and biological function.

Aldo-keto reductase 7A5 (AKR7A5) attenuates oxidative stress and reactive aldehyde toxicity in V79-4 cells.

Aldo-keto reductase (AKR) enzymes are critical in the detoxification of endogenous and exogenous aldehydes. In previous studies, we have shown that AKR7A5 enzyme is catalytically active towards aldehydes arising from lipid peroxidation (LPO) and that it can significantly protect against 4-hydroxynonenal-induced apoptosis, suggesting a protective role against the consequences of oxidative stress. The aim of this study was to elucidate the cytoprotective effect of AKR7A5 against oxidative stress using a transgenic mammalian cell line expressing AKR7A5. Results show that expression of AKR7A5 in V79-4 cells provides significant protection against the cytotoxicity of H2O2 and menadione, with its expression altering the IC50 of H2O2 from 1.1 to 2.3 mM and the IC50 of menadione from 8.6 to 9.6 µM, thus providing direct evidence for its anti-oxidant activity. Cells expressing AKR7A5 were also found to be more resistant to several LPO-derived aldehydes--trans-2-nonenal, hexanal and methylglyoxal. In addition the ability of AKR7A5 to enable the cells to cope with ROS accumulation and glutathione depletion was assessed. V79-4 cells overexpressing AKR7A5 were able to lower cellular ROS levels following treatment with H2O2 and menadione. AKR7A5 was also able to maintain cellular glutathione homeostasis in the presence of H2O2 and menadione. These findings indicate the importance of AKR7A5 in protecting cells from the damaging effects of oxidative stress, and that this cytoprotective function is carried out through multiple pathways.

4-Hydroxynonenal induces an increase in expression of Receptor for Activating C Kinase 1 (RACK1) in Chinese hamster V79-4 lung cells.

4-Hydroxy-trans-2-nonenal (HNE) is a cytotoxic α,ß-unsaturated aldehyde implicated in the pathology of several diseases that have an oxidative stress mechanism, including atherosclerosis, diabetes, alcohol-induced liver disease, and neurodegenerative disorders. As the most toxic aldehydic product of lipid peroxidation, HNE is known to exert a range of biological effects in a concentration-dependent manner. In this study, the effect of HNE on the levels of proteins in V79-4 Chinese hamster lung cells was investigated using two-dimensional electrophoresis and mass spectrometry. The results revealed that the expression of 23 proteins was increased by at least 2-fold and the expression of 19 proteins was decreased by at least 2-fold after exposure to 10 µM HNE for 24 h. Decreased proteins included the metabolic enzyme phosphoglycerate kinase 1 (PGK1), levels of which were decreased by 47%. Levels of the apoptotic indicator Lamin C were decreased by 33%. In contrast, levels of the scaffolding protein Receptor for Activating C Kinase 1 (RACK1) were increased by 2-fold after treatment with 10 µM HNE for 24h, and this was confirmed using quantitative PCR of reverse-transcribed mRNA and Western blots. The role of RACK1 in mediating the induction of apoptosis in response to 10 µM HNE was confirmed using RACK1-specific siRNA. The results from this study provide new information on the mechanism of adaptive stress response to HNE and also identify potential new biomarkers of exposure to HNE.

The diversity of microbial aldo/keto reductases from Escherichia coli K12.

The genome of Escherichia coli K12 contains 9 open reading frames encoding aldo/keto reductases (AKRs) that are differentially regulated and sequence diverse. A significant amount of data is available for the E. coli AKRs through the availability of gene knockouts and gene expression studies, which adds to the biochemical and kinetic data. This together with the availability of crystal structures for nearly half of the E. coli AKRs and homologues of several others provides an opportunity to look at the diversity of these representative bacterial AKRs. Based around the common AKR fold of (ß/α)8 barrel with two additional α-helices, the E. coli AKRs have a loop structure that is more diverse than their mammalian counterparts, creating a variety of active site architectures. Nearly half of the AKRs are expected to be monomeric, but there are examples of dimeric, trimeric and octameric enzymes, as well as diversity in specificity for NAD as well as NADP as a cofactor. However in functional assignments and characterisation of enzyme activities there is a paucity of data when compared to the mammalian AKR enzymes.

Aldo-keto reductases mediate constitutive and inducible protection against aldehyde toxicity in human neuroblastoma SH-SY5Y cells.

Reactive aldehydes including methyl glyoxal, acrolein and 4-hydroxy-2-nonenal (4-HNE) have been implicated in the progression of neurodegenerative diseases. The reduction of aldehydes to alcohols by the aldo-keto reductase (AKR) family of enzymes may represent an important detoxication route within neuronal cells. In this study, the ability of AKR enzymes to protect human neuroblastoma SH-SY5Y cells against reactive aldehydes was assessed. Using gene-specific RNA interference (RNAi), we report that AKR7A2 makes a significant contribution to the reduction of methyl glyoxal in SH-SY5Y cells, with its knockdown altering the IC(50) from 410 to 25.8µM, and that AKR1C3 contributes to 4-HNE reduction, with its knockdown lowering the IC(50) from 1.25 to 0.58µM. In addition, we have shown that pretreatment of cells with sub-lethal concentrations of 4-HNE or methyl glyoxal leads to a significant increase in IC(50) when cells are exposed to higher concentrations of the toxic aldehyde. The IC(50) for methyl glyoxal increased from 410µM to 1.9mM, and the IC(50) for 4-HNE increased from 120 to 690nM. To investigate this protection, we show that pretreatment of cells with the AKR inhibitor sorbinil lead to decreased resistance to aldehydes. We show that AKR1C can be induced 8-fold in SH-SY5Y cells by treatment with sub-lethal concentrations of methyl glyoxal, and 5-fold by 4-HNE treatment. AKR1B is not induced by methyl glyoxal but is induced 10-fold by 4-HNE treatment. Furthermore, we have shown that this adaptive response can also be induced using the chemoprotective agent tert-butyl hydroquinone (t-BHQ), and that this also evokes an increase in the expression and activity of AKR1B and AKR1C. These findings highlight the potential for the interventional upregulation of AKR via non-toxic derivatives or natural compounds as a novel therapeutic approach towards the detoxication of aldehydes, with the aim of halting the progression of aldehyde-dependent neurodegenerative diseases.

Neuroprotective effects of umbelliferone and esculetin in a mouse model of Parkinson's disease.

The production of reactive oxygen species and mitochondrial dysfunction in the brain are both associated with the progression of several neurodegenerative diseases, including Parkinson's disease. These characteristics are also observed when rodents are exposed to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a compound that causes nigrostriatal dopaminergic neurotoxicity and that has been used previously for assessing the effectiveness of neuroprotective agents. In this study, the neuroprotective effects of two coumarins, umbelliferone and esculetin, against MPTP-induced neurotoxicity were examined in C57BL/6J mice. The results show that dietary administration of umbelliferone and esculetin significantly attenuated MPTP-induced neurotoxicity in the substantia nigra pars compacta but not striatum, as measured by tyrosine hydroxylase staining. Both coumarins also prevented an MPTP-induced increase in nitrosative stress as measured by 3-nitrotyrosine immunoreactivity and also maintained glutathione levels in MPTP-exposed mice as well as in cell lines exposed to the metabolite 1-methyl-4-phenylpyridinium. Umbelliferone and esculetin also prevented MPTP-dependent caspase 3 activation, an indicator of apoptosis, but did not inhibit monoamine oxidase activity. This is the first time that the neuroprotective capabilities of these coumarins have been demonstrated, and the results indicate that umbelliferone and esculetin can protect against MPTP-induced neurotoxicity in the mouse. These compounds can cross the blood-brain barrier, so their effectiveness indicates that they have the potential to protect in neurodegenerative disease such as Parkinson's disease.

Inducible protection of human astrocytoma 1321N1 cells against hydrogen peroxide and aldehyde toxicity by 7-hydroxycoumarin is associated with the upregulation of aldo-keto reductases.

Reactive oxygen species (ROS) and consequent aldehydic lipid peroxidation products have been identified as significant in the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's. Understanding and enhancing endogenous cellular protection against oxidants and aldehydes is therefore of interest in developing strategies to combat these diseases. In this study the role of the aldo-keto reductases AKR7A2 and AKR1C3 in protecting human astrocytoma 1321N1 cells against oxidant and aldehyde toxicity was investigated using siRNA gene silencing. Results show that both enzymes are responsible for part of the intrinsic protection against aldehydes and oxidants. Treating cells with sub-lethal concentrations of oxidant or aldehyde stress or with the natural coumarin 7-hydroxycoumarin (umbelliferone) revealed that endogenous resistance to aldehydes and oxidants can be induced significantly. The basis of the inducible protection by 7-hydroxycoumarin was shown to be associated with induction of the aldo-keto reductases AKR7A2 (1.5-fold) and AKR1C (3-fold), and this inducible protection was sufficient to overcome siRNA silencing of AKR1C3. These results indicate the importance of AKR family members in the detoxication of aldehydes, and also show that the natural phytochemical 7-hydroxycoumarin is a potential therapeutic candidate for neurodegenerative diseases.

Phospholipase C mediates cadmium-dependent apoptosis in HEK 293 cells.

Cadmium is a heavy metal that is known to cause toxicity to cells and, at low concentrations, can initiate apoptosis. This study was undertaken with the aim of defining the role of phospholipase C (PLC) in mediating cadmium-induced apoptosis in human embryonic kidney (HEK 293) cells. We have shown that intracellular Ca(2+) levels increased significantly in HEK 293 cells after 24-hr exposure to Cd. The activity of the calcium-dependent protease calpain rose by four times. The PLC-specific inhibitor, U73122, prevented the Cd-dependent increase in Ca(2+) levels and also abolished Cd-dependent calpain and caspase 3 activation as well as Cd-dependent mitochondrial Bax accumulation. Inhibition of PLC also leads to an increased cell viability following exposure to Cd. Taken together, the results show that the PLC pathway is involved in mediating Cd-induced apoptosis in HEK 293 cells.

Human aldo-keto reductase AKR7A2 protects against the cytotoxicity and mutagenicity of reactive aldehydes and lowers intracellular reactive oxygen species in hamster V79-4 cells.

Aldo-keto reductase (AKR) enzymes are critical for the detoxication of endogenous and exogenous aldehydes. Previous studies have shown that the AKR7A2 enzyme is catalytically active toward aldehydes arising from lipid peroxidation, suggesting a potential role against the consequences of oxidative stress, and representing an important detoxication route in mammalian cells. The aim of this study was to determine the ability of AKR7A2 to protect cells against aldehyde cytotoxicity and genotoxicity and elucidate its potential role in providing resistance to oxidative stress. A transgenic mammalian cell model was developed in which AKR7A2 was overexpressed in V79-4 cells and used to evaluate the ability of AKR7A2 to provide resistance against toxic aldehydes. Results show that AKR7A2 provides increased resistance to the cytotoxicity of 4-hydroxynonenal (HNE) and modest resistance to the cytotoxicity of trans, trans-muconaldehyde (MUC) and methyglyoxal, but provided no protection against crotonaldehyde and acrolein. Cells expressing AKR7A2 were also found to be less susceptible to DNA damage, showing a decrease in mutation rate cause by 4-HNE compared to control cells. Furthermore, the role of the AKR7A2 enzyme on the cellular capability to cope with oxidative stress was assessed. V79 cells expressing AKR7A2 were more resistant to the redox-cycler menadione and were able to lower menadione-induced ROS levels in both a time and dose dependent manner. In addition, AKR7A2 was able to maintain intracellular GSH levels in the presence of menadione. Together these findings indicate that AKR7A2 is involved in cellular detoxication pathways and may play a defensive role against oxidative stress in vivo.

The chemopreventive effects of aged garlic extract against cadmium-induced toxicity.

Garlic has been reported in many previous studies as a potent chemopreventive agent. The protective effect of garlic has been ascribed to the presence of organosulphur compounds (OSC). In this study, the efficacy of aged garlic extract (AGE) compared to diallyl disulfide (DADS) in protecting against toxicity induced by cadmium (Cd) in 1321N1 and HEK293 cells was investigated. The involvement of the transcription factor Nrf2 in this protection was also examined. The results show that AGE significantly prevented loss of cell viability in Cd-treated 1321N1 and HEK293 cells. In comparison DADS had no significant effect in protecting HEK293 cells but did protect 1321N1 cells. AGE significantly reduced Cd-induced TBARS production and LDH leakage in the two cell lines, and AGE and DADS both increased GSH levels in Cd-treated cell lines. Pre-treatment of cells with AGE or DADS increased expression of the protective enzyme NAD(P)H:quinone oxidoreductase (NQO1), and this was associated with the accumulation of the transcription factor Nrf2. These results show that AGE and DADS have beneficial effects against Cd-induced toxicity, and this protection appears to be mediated via induction of cytoprotective enzymes in an Nrf2-dependent manner. This indicates the potential for using AGE as a chemoprevention strategy for Cd toxicity.

Nrf2-mediated adaptive response to cadmium-induced toxicity involves protein kinase C delta in human 1321N1 astrocytoma cells.

Cadmium (Cd) is a toxic heavy metal, and exposure to Cd causes a range of changes within the cell. At high concentrations, Cd causes damage to cells via a range of mechanisms. At low concentrations, Cd can stimulate expression of genes that are part of an adaptive response. In this study, we have used the astrocytoma cell line 1321N1 as a model to investigate the induction of protective enzymes in response to Cd. We have shown that expression of NAD(P)H:quinone oxidoreductase and haem oxygenase enzymes are induced as the protein level by -fold and -fold, and in response to 5 and 10 µM Cd. Levels of NQO1 and HO1 mRNA are also increased by -fold and -fold following 24h exposure to 5 and 10 µM cadmium. An increase in the nuclear accumulation of the transcription factor Nrf2 was also observed following Cd treatment. Through the use of the protein kinase C inhibitor bisindolylmaleimide (VIII) acetate we have demonstrated the involvement PKC in the Nrf2-mediated response of 1321N1 cells to 5-10 µM Cd. We have also shown through the used of 10 µM rottlerin that PKCδ is the isoform responsible for mediating this response.

A miniaturised integrated biophotonic point-of-care genotyping system.

This paper reports the development of a novel genotyping device specifically designed for point-of-care applications. As the results of the human genome project are applied to clinical practice there is an increasing requirement for simple to operate high-speed, potentially low-cost genotyping devices for use in the clinic. The aim of such devices is not to specifically detect a full gene sequence but to monitor the presence of specific Single Nucleotide Polymorphisms (SNPs). The instrument is designed to fulfil this specific clinical requirement. Using a FRET-based assay the instrument completes a full PCR process and then performs a melting point test to determine the exact SNPs present in the sample. Results are presented in which the instrument produces results within 18 min based upon saliva samples provided by the patient. The paper also reports successful results both with purified DNA samples and saliva-based samples which were taken from subjects after experiments deliberately aimed at confusing the instrument.

The role of aldehyde reductase AKR1A1 in the metabolism of γ-hydroxybutyrate in 1321N1 human astrocytoma cells.

The role of the aldehyde reductase AKR1A1 in the biosynthesis of gamma-hydroxybutyrate (GHB) has been investigated in cell lines using a specific double stranded siRNA designed to knock down expression of the enzyme. This enzyme, along with the aldo-keto reductase AKR7A2, has been proposed previously to be one of the major succinic semialdehyde reductases in brain. The AKR1A1 siRNA was introduced into the human astrocytoma cell line (1321N1) and AKR1A1 expression was monitored using quantitative reverse-transcriptase PCR and Western blots. Results show an 88% reduction in mRNA levels and a 94% reduction in AKR1A1 protein expression 72 h after transfection with the siRNA. Aldehyde reductase activity was examined in silenced cells by following the aldehyde-dependent conversion of NADPH to NADP at 340 nm. This revealed a 30% decrease in pNBA reductase activity in cell extracts after AKR1A1 silencing. Succinic semialdehyde reductase activity was significantly lower in silenced cells when measured using high concentrations (1mM) of succinic semialdehyde, but not with low concentrations (10 µM). The effect of silencing on intracellular and extracellular GHB levels was measured using gas chromatography-mass spectrometry. Results show that AKR1A1 has little effect on the production of GHB, indicating that in this cell line alternative enzymes such as the AKR7A2 are likely to play a more significant role in GHB biosynthesis.

Esculetin-induced protection of human hepatoma HepG2 cells against hydrogen peroxide is associated with the Nrf2-dependent induction of the NAD(P)H: Quinone oxidoreductase 1 gene.

Esculetin (6,7-dihydroxy coumarin), is a potent antioxidant that is present in several plant species. The aim of this study was to investigate the mechanism of protection of esculetin in human hepatoma HepG2 cells against reactive oxygen species (ROS) induced by hydrogen peroxide. Cell viability, cell integrity, intracellular glutathione levels, generation of reactive oxygen species and expression of antioxidant enzymes were used as markers to measure cellular oxidative stress and response to ROS. The protective effect of esculetin was compared to a well-characterized chemoprotective compound quercetin. Pre-treatment of HepG2 cells with sub-lethal (10-25 µM) esculetin for 8h prevented cell death and maintained cell integrity following exposure to 0.9 mM hydrogen peroxide. An increase in the generation of ROS following hydrogen peroxide treatment was significantly attenuated by 8h pre-treatment with esculetin. In addition, esculetin ameliorated the decrease in intracellular glutathione caused by hydrogen peroxide exposure. Moreover, treatment with 25 µM esculetin for 8h increased the expression of NAD(P)H: quinone oxidoreductase (NQO1) at both protein and mRNA levels significantly, by 12-fold and 15-fold, respectively. Esculetin treatment also increased nuclear accumulation of Nrf2 by 8-fold indicating that increased NQO1 expression is Nrf2-mediated. These results indicate that esculetin protects human hepatoma HepG2 cells from hydrogen peroxide induced oxidative injury and that this protection is provided through the induction of protective enzymes as part of an adaptive response mediated by Nrf2 nuclear accumulation.