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.

Enzymes involved in the metabolism of gamma-hydroxybutyrate in SH-SY5Y cells: identification of an iron-dependent alcohol dehydrogenase ADHFe1.

The metabolism of the endogenous metabolite gamma-hydroxybutyrate (GHB) has been studied in a human neuroblastoma cell line SH-SY5Y as a model for examining neuronal metabolism. We show that GHB can be synthesized and released from these cells, indicating that pathways for GHB synthesis and secretion are present. Activities for the major enzymes that are involved in GHB metabolism are reported, and transcripts for AKR1A1, AKR7A2, ALDH5A1 and GABA-T can be detected by RT-PCR. We also demonstrate the presence of the ADHFe1 transcript, a gene that has been reported to encode a hydroxyacid-oxoacid transhydrogenase (HOT). We show that the ADHFe1 gene is related to bacterial GHB dehydrogenases and has a conserved NAD-binding site. The potential for using the SH-SY5Y cell line for investigating GHB catabolism is discussed.

Synthesis and catabolism of gamma-hydroxybutyrate in SH-SY5Y human neuroblastoma cells: role of the aldo-keto reductase AKR7A2.

gamma-Hydroxybutyrate (GHB) is an endogenous metabolite synthesized in the brain. There is strong evidence to suggest that GHB has an important role as a neurotransmitter or neuromodulator. The human aldo-keto reductase AKR7A2 has been proposed previously to catalyze the NADPH-dependent reduction of succinic semialdehyde (SSA) to GHB in human brain. In this study we have used RNA interference to evaluate the role of AKR7A2 in GHB biosynthesis in human neuroblastoma SH-SY5Y cells. Quantitative reverse transcription-PCR analysis and immunoblotting revealed that short interfering RNA molecules directed against AKR7A2 led to a significant reduction in both AKR7A2 transcript and protein levels 72 h post-transfection. We have shown that reduced expression of AKR7A2 results in a 90% decrease in SSA reductase activity of cell extracts. Furthermore, we have shown using gas chromatography-mass spectrometry that a decrease in the level of AKR7A2 was paralleled with a significant reduction in intracellular GHB concentration. This provides conclusive evidence that AKR7A2 is the major SSA reductase in these cells. In contrast, short interfering RNA-dependent reduction in AKR7A2 levels had no effect on the GHB dehydrogenase activity of the extracts, and inhibitor studies suggest that another enzyme characteristic of an NAD-dependent alcohol dehydrogenase may be responsible for catalyzing this reverse reaction. Together these findings delineate pathways for GHB metabolism in the brain and will enable a better understanding of the relationship between GHB biosynthesis and catabolism in disease states and in drug overdose.

Mouse aldo-keto reductase AKR7A5 protects V79 cells against 4-hydroxynonenal-induced apoptosis.

We have developed transgenic Chinese hamster V79 cell lines in order to examine the potential for a mouse aldo-keto reductase, AKR7A5, to protect against the toxicity of 4-hydroxynonenal (4-HNE) and related toxic aldehydes. Stable expression of mouse AKR7A5 in V79 cells conferred four-fold increased resistance to 4-HNE cytotoxicity using the MTT assay compared to empty vector-transfected V79 cells. Cells expressing AKR7A5 showed a decrease in mutation rate compared to control cells in the presence of 4-HNE as measured by HGPRT mutagenicity assay. Furthermore, the cells expressing AKR7A5 showed decreased 4-HNE-induced caspase-3 activity in both a time and dose-dependent manner compared to control cells. These results show that in V79 cells 4-HNE mediates apoptosis via caspase-3 activation and that the AKR7A5 enzyme is able to metabolize 4-HNE in cells, thereby attenuating 4-HNE-induced apoptosis. AKR7A isozymes may therefore be important in protecting against toxic aldehydes derived from lipid peroxidation in vivo.

Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4.

The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a Vmax of 2141+/-500 nmol/min/mg and a Km of 11+/-4 microM. This enzyme was inhibited by pyrazole with a KI of 3.1+/-0.57 microM. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a Vmax of 115 nmol/min/mg and a Km of 15+/-2 microM and was not inhibited by pyrazole.

Substrate specificity of mouse aldo-keto reductase AKR7A5.

We have determined the substrate specificity of a mouse aldo-keto reductase (AKR) AKR7A5, an enzyme that is similar to rat aflatoxin aldehyde reductase (AKR7A1) and to human brain succinic semialdehyde reductase (AKR7A2). Previously, we have shown that the mouse enzyme is present in a range of tissues including liver, kidney, testis and brain, and is able to reduce several carbonyl compounds, including succinic semialdehyde, 2-carboxybenzaldehyde, 4-nitrobenzaldehyde and 9,10-phenanthrenequinone [FEBS Lett. 523 (2002) 213]. It has been suggested that it may represent the mouse equivalent of human succinic semialdehyde reductase which is responsible for the biosynthesis of gamma-hydroxybutyrate. In this study, we show that the enzyme is also able to reduce other aromatic aldehydes such as 4-chloro-3-nitrobenzaldehyde, and 3-nitrobenzaldehyde, and has particular high specific activity towards dicarbonyls such as acenapthenequinone, 2,3-bornanedione (camphorquinone), and phenylglyoxal. It has low specific activity towards ketones, and alpha,beta-unsaturated carbonyls such as acrolein and 4-hydroxynonal. The enzyme is inhibited by several compounds including quercitin, ethacrynic acid, indomethacin and sodium valproate. Developing selective inhibitors may lead to a means of modifying the activity of the enzyme in vivo.

Characterisation of a novel mouse liver aldo-keto reductase AKR7A5.

We have characterised a novel aldo-keto reductase (AKR7A5) from mouse liver that is 78% identical to rat aflatoxin dialdehyde reductase AKR7A1 and 89% identical to human succinic semialdehyde (SSA) reductase AKR7A2. AKR7A5 can reduce 2-carboxybenzaldehyde (2-CBA) and SSA as well as a range of aldehyde and diketone substrates. Western blots show that it is expressed in liver, kidney, testis and brain, and at lower levels in skeletal muscle, spleen heart and lung. The protein is not inducible in the liver by dietary ethoxyquin. Immunodepletion of AKR7A5 from liver extracts shows that it is one of the major liver 2-CBA reductases but that it is not the main SSA reductase in this tissue.