Cytochromes P450 catalyze the reduction of α,β-unsaturated aldehydes

Abstract

The metabolism of α,β-unsaturated aldehydes, e.g., 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently, we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O(2), and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 and rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice a diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of the reduction of α,β-unsaturated aldehydes in the liver.

Figure 1

HPLC profile of 4-HNE products formed by recombinant P450 3A4. The metabolic assay was performed in triplicate with 2 mL of reaction mixture containing E. coli-expressed recombinant P450 3A4 (50 nM) and 50 µM [³H]-4-HNE at 37 °C for 20 min in a shaking water bath. The reaction was terminated by flash freezing in liquid nitrogen. The reaction mixture was thawed upon adding 1% CF₃CO₂H (w/v), and after centrifugation to remove particulate materials, the supernatant was injected into a C₁₈ column and measured with a radiometric detector. DHN eluted at t_R 56 min and HNA eluted at t_R 58 min. A. Reaction terminated at 0 minutes, B. Reaction terminated at 20 minutes.

Figure 2

Mass spectrum of DHN generated from P450 3A4-dependent metabolism of [³H]-4-HNE. After metabolism, the samples were analyzed by HPLC and fractions coeluting with a DHN standard at t_R 56 min were collected and subjected to mass spectral analysis. A. Authentic DHN, B. DHN produced by P450 3A4.

Figure 3

Use of specific inhibitors of ALDH (cyanamide) and P450 (miconazole and troleandomycin) on 4-HNE metabolism in primary hepatocytes (Sprague-Dawley rats). Primary rat hepatocytes were prepared as described in Methods with a viability of >95% and 4-HNE was immediately added to the cell culture media. After 20 min, the reactions were quenched by adding 1% CF₃CO₂H (w/v) and frozen at −80 °C. The products were measured by HPLC and radioactivity determined using an in-line radiomatic detector. The effects of 0.5 mM of cyanamide (ALDH), miconazole (P450), and troleandomycin (P450 3A subfamily) on formation of oxidative and reductive metabolites of 4-HNE was determined. Four preparations of rat hepatocytes were used to obtain statistical significance: *p ≤ 05; ** p ≤ 0.001.

Figure 4

Relative enzyme 9-AA oxidative and reductive metabolism of liver microsomal fractions from mice fed normal AIN76A diet or diet containing 0.5% BHA (w/w) for 7 days. Microsomal protein was isolated and the enzyme assays performed as described in Methods. Control liver microsomal fraction from untreated mice fed AIN76A diet (open bar) and liver microsomal fractions from mice fed AIN76A diet containing 0.5% BHA (hatched bar).

Figure 5

Effects of P450 inhibitors on 9-AA transformation to oxidized and reduced products. Liver microsomal fractions from mice fed 0.5% BHA (w/w)in AIN76A diet were treated with either 0.5 mM miconazole, troleandomycin, phenytoin, or α-naphthoflavone. The control activity was 1.05 ± 0.1 nmol 9-A-MeOH produced per minute per mg microsomal protein.

Figure 6

Effect of anaerobiosis or carbon monoxide on the oxidative and reductive metabolism of 9-AA. The reaction mixtures were purged with Ar or CO prior to initiating the reaction. Oxygen-depletion systems were included to achieve anaerobiosis as described in Methods. The control activity was 1.05 ± 0.1 nmol 9-A-MeOH produced per minute per mg microsomal protein.

Figure 7

Scheme for oxidative and reductive transformation of α,β-unsaturated aldehydes, 4-hydroxy-2-nonenal, by P450s.