A typical reaction catalyzed by P450s is the hydroxylation of an unfunctionalized alkyl group or epoxidation of an unsaturated carbon-carbon bond28, 29
. However, examples are also known in which P450s catalyze the oxidation of aldehydes to acids with the incorporation of molecular oxygen into the products. Several critical P450-catalyzed reactions have been shown to involve oxidation of aldehydes or hemiacetals, e.g. the aromatase and sterol 14α-demethylase reactions. Human liver microsomes possess the ability to oxidize aromatic aldehydes such as 11-oxo-Δ8
to carboxylic acids via P450 systems. Model substrates, e.g. 9-AA and 4-biphenylaldehyde, have been used to measure the aldehyde oxidation activity of several P450s21, 22
. These fluorigenic substrates provide simple methods to measure the acid products formed from the P450-dependent oxidation of aromatic aldehydes.
The metabolism of lipid peroxidation-derived metabolites, e.g. 4-HNE, has been shown to involve oxidation, reduction, and GSH conjugation in vitro
and in vivo4, 10, 18, 33
. The reduction reactions have been suggested to principally involve aldose reductase or aldo-keto reductases27
. During a study with mouse liver microsomes and the different P450s that oxidize 9-AA and 4-HNE, we found that some P450s are able to reduce these compounds to the corresponding alcohols. We developed a method for the screening of P450 reduction enzyme activity involved in aldehyde reduction by using a fluorescence assay for measurement of reduction of 9-AA to 9-A-MeOH. Differential extraction with ethyl acetate allowed separation of the carboxylic acids and the alcohols in the two different organic phases (acidic and alkaline, respectively). Human recombinant human P450s 2B6, 3A4, and 2J2, murine P450 2c29, and mouse liver microsomes were able to reduce 9-AA to 9-AMeOH. The order of 4-HNE reduction by the individual P450s analyzed was (human) P450 2B6
3A4 > 1A2 > 2J2 > (mouse) P450 2C292c29. The observation of reduction of 4-HNE with the different P450 enzymes in mouse liver microsomes was further confirmed by using P450-selective inhibitors, e.g. miconazole (general P450 inhibitor), troleandomycin (P450 Subfamily 3A-selective inhibitor), phenytoin (P450 2c29-selective inhibitor), and α-naphthoflavone (CYP1A- selective inhibitor)
Reductive catalysis by P450s is not a common reaction, except for substrates like azo, nitro, and quinone containing compounds where the reduction reaction is not easily observed in ambient air and often requires low oxygen tension to measure the reactions. These reduced products are apparently not stable as 1-electron- or 2-electron-reduced intermediates in the presence of molecular oxygen34
. Azo dyes are widely used in cosmetics, food, textiles, and drugs and several (e.g., sulfonazo III and aramanth) are reduced by rat hepatic microsomes35
. The hepatocarcinogen N
-dimethylamino-azobenzene is reduced by rat liver microsomes in an oxygen- and CO-insensitive manner36
has described these oxygen- and CO-insensitive reactions, that the reduction of α,β -unsaturated aldehydes resembles. The bioreductive activation of 2,3,5,6-tetramethy-1,4-benzoquinone is catalyzed by (rat) P450 2B138
P450s require NADPH and oxygen for oxidative metabolism. Therefore in the absence of oxygen, P450-dependent monooxygenation reactions are inhibited. In addition to the use of chemical inhibitors, we were able to demonstrate that the lack of oxygen inhibited the P450-dependent oxidation of both 9-AA and 4-HNE but not the reduction of either compound. By using argon and CO to inhibit oxidative metabolism, we demonstrated the role of P450s in lipid aldehyde reductive metabolism is not altered in the presence of oxygen, suggesting a stable form of the reduced hemoprotein which does not rely upon formation of the perferryl-oxygen intermediate serve as the reducing species of the enzyme39
A proposed pathway for 4-HNE metabolism by P450s is shown in . During the metabolism of substrate, P450s exist in different oxidation states. These oxidation states can influence the product generated by the P450. The oxidation state of the P450 that generates HNA is presumed to be the perferryl (Fe-O3+
) or possibly ferric peroxide (Fe-O2−1
), requiring oxygen and electrons from NADPH: P450 oxidoreductase. Hydrogen is abstracted from 4-HNE forming a carbonyl carbon radical (or possibly the carbon radical of the hydrated gem-diol, e.g. see Guengerich et al
that can then react with the iron-bound hydroxyl radical generating 4-hydroxy-2-nonenoic acid. The P450-dependent reduction reactions are apparently catalyzed by the ferrous (Fe (II)) P450, with the electrons being transferred from NADPH to the P450 by NADPH-P450 reductase41
. It has been noted that the P450 Fe(II) state can occur during conditions of low oxygen tension in certain tissues39
allowing for fascile reduction of compounds like azo dyes, unsaturated aldehydes, etc. This and the Fe(II)-CO P450 form may be the oxidation states of the P450 that generates DHN or 9-A-MeOH, suggesting that the site where substrate binds may not be affected by formation of the ferrous-CO complex.
Scheme for oxidative and reductive transformation of α,β-unsaturated aldehydes, 4-hydroxy-2-nonenal, by P450s.
In conclusion, our results demonstrate that several P450s are efficient catalysts in both the oxidative and reductive transformation of lipid-derived aldehydes to carboxylic acids and alcohols and adds a new facet to the biological activity of these metabolites. Our studies also suggest that P450-mediated metabolism operates in parallel with other metabolic transformations of aldehydes; hence, the P450s could serve as reserve or compensatory mechanisms when other high capacity pathways of aldehyde elimination are compromised due to disease or toxicity. For example, during myocardial infarction, the activity of aldehyde dehydrogenase is inhibited due to the lack of NAD+. In addition, P450s expressed in the liver—e.g. 2B6, 3A4, 2J2—may play major roles in 4-HNE reduction. The role of P450 in vascular metabolism is unclear, in that the aldo-keto reductases and aldehyde dehydrogenases are expressed at relatively high levels relative to the P450s. Finally, because other unsaturated aldehydes, —e.g., acrolein, trans-2-hexenal, and crotonaldehyde—are also food constituents or environmental pollutants, P450s may be significant regulators of toxicity due to their possible roles in oxidation and reduction of xenobiotic aldehydes as well.