The protective role of antioxidants and the aldo-keto reductase AKR7A2 against toxic aldehydes in cell lines

Abstract

Aldehydes and ketones are widely distributed in nature and can be very reactive, causing damage to DNA, proteins and lipids within the cell. A number of antioxidant molecules are able to react with reactive molecules to minimize their damaging effects. The human body also has a series of enzyme systems for protecting itself against reactive aldehydes and ketones including alcohol dehydrogenases (ADH), aldehyde reductases (AKR) and aldehyde dehydrogenases (ALDH). Some antioxidants are also able to increase the expression of these protective enzymes thereby leading to enhanced protection. In this thesis, intrinsic and extrinsic mechanisms of protection against the toxicity of aldehydes were investigated. In the first part of this thesis, the toxicity of acetaldehyde to HepG2 human hepatoma and 1321N1 astrocytoma cells was investigated as a model for liver and brain cells. Acetaldehyde is a toxic metabolite of ethanol, and is thought to be a major determinant in alcohol toxicity. Acetaldehyde did not cause significant toxicity to 1321N1 or HepG2 at concentrations below 1 mM, but was toxic to 1321N1 cells at higher concentrations. However, acetaldehyde was able to cause an increase in activated caspase-ca3 and DNA fragmentation at concentrations between 5 and 25 µM in HepG2 cells, indicating an induction of apoptosis. Three antioxidants (vanillin, gallic acid and resveratrol) were investigated for their capacity to protect cells against the toxicity of reactive aldehydes. Methylglyoxal and acrolein are products of the peroxidation of lipids and arise during exposure of cells to oxidants. The antioxidants vanillin and gallic acid are found in whisky in the non-volatile fraction and resveratrol is found in red wine. The antioxidants were not able to protect the cells against the aldehydes at the concentrations tested, but did increase cell viability. It appears that vanillin can inhibit ADH as well as ALDH activity by binding to the enzymes without altering their expression levels. To investigate whether the AKR enzymes are involved in protection against aldehyde toxicity, human AKR7A2 and the mouse AKR7A5 were purified and characterized with different substrates including muconaldehyde, crotonaldehyde, acetaldehyde and methylglyoxal. Both AKR7A2 and AKR7A5 enzymes showed a high Km and low turnover number for all three of the aldehydes tested, indicating relatively low specificity. To better understand the role of AKR7A2, the gene encoding the enzyme was transiently transfected into 1321N1 and HepG2 cell lines and exposed to crotonaldehyde, muconaldehyde and methylglyoxal. However, AKR7A2 was not able to protect cells against aldehyde toxicity, and, in some cases, its overexpression increased toxicity. Only when crotonaldehyde was tested at a relatively high concentration the transfected cells did show increased viability compared to the control, suggesting that the enzyme AKR7A2 could be involved in crotonaldehyde detoxification. Transfected HepG2 cells showed an increase in viability compared to the non-transfected control cells, at almost all the tested concentrations. Taken together these results show that AKR7A2 is able to protect HepG2 liver cells against the aldehydes, but is not able to protect 1321N1 brain cells.