ABSTRACT
Docosahexaenoic acid (DHA) has been shown to have neuroprotective effects in Parkinson's disease, but the underlying mechanism has not been fully elucidated. DHA is metabolized to DHA epoxides (EDPs) and hydroxides by cytochrome P450s (P450s), and EDPs are further hydroxylated to the corresponding diols, dihydroxydocosapentaenoic acids (DHDPs) by soluble epoxide hydrolase (sEH). In the present study, we investigated the roles of these DHA metabolites in the beneficial effects of DHA supplementation on a rotenone-induced rat model of Parkinson's disease. Metabolite analysis by LC-MS revealed that CYP2A1, 2C11, 2C13, 2C23, and 2E1 contributed to the formation of EDPs, and these P450s and sEH were expressed in the rat brain. We found that DHA supplementation in rats improved the motor dysfunction induced by rotenone. In addition, DHA reversed the decrease in tyrosine hydroxylase and the increase in lipid peroxidation generated by rotenone in the striatum. DHA supplementation also induced mRNA expression of antioxidant genes, such as sod1 and catalase, and Nrf2 protein expression in the striatum. However, these effects of DHA supplementation were eliminated by cosupplementation with the sEH inhibitor TPPU. Supplementation with DHA increased the amount of 19,20-DHDP in the rat brain, while the amount of EDPs was not significantly increased. In addition, TPPU suppressed the increase in DHDPs and increased EDPs in the brain. In PC12 cells, 19,20-DHDP increased the mRNA levels of sod1 and catalase along with Nrf2 induction. This study suggests that DHA metabolites-DHDPs generated by P450s and sEH-have an important role in improving rotenone-induced Parkinson's disease.
Subject(s)
Docosahexaenoic Acids/administration & dosage , Fatty Acids, Unsaturated/metabolism , Neuroprotective Agents/administration & dosage , Parkinson Disease, Secondary/drug therapy , Animals , Brain/drug effects , Brain/metabolism , Brain/pathology , Catalase/metabolism , Cytochrome P-450 Enzyme System/metabolism , Disease Models, Animal , Docosahexaenoic Acids/metabolism , Epoxide Hydrolases/antagonists & inhibitors , Epoxide Hydrolases/metabolism , Humans , Male , NF-E2-Related Factor 2/metabolism , Neuroprotective Agents/metabolism , Oxidation-Reduction/drug effects , Parkinson Disease, Secondary/chemically induced , Parkinson Disease, Secondary/pathology , Rats , Rotenone/toxicity , Superoxide Dismutase-1/metabolismABSTRACT
P450 (cytochrome P450) enzymes catalyse the mono-oxygenation of a wide range of compounds such as steroids, fatty acids, vitamins and drugs. In the present paper we demonstrate a system for bioconverting diverse compounds [flavanone, DHEA (dehydroepiandrosterone) and 7-ethoxycoumarin] using P450 species expressed in Escherichia coli. First, we expressed four P450 species: rabbit CYP2B (P450 family 2, subfamily B), fruitfly (Drosophila) CYP317A, rat CYP3A23 and mouse CYP2J5. Next, we added substrates directly to the incubation medium. The resulting metabolites were extracted and analysed by HPLC and spectrofluorimetry. The first substrate, 7-ethoxycoumarin, was de-ethylated by CYP2B; CYP2J5 and CYP3A23 showed weak activity, and CYP317A had no activity for 7-ethoxycoumarin. We next used flavanone, a flavonoid, as a substrate for these four P450 species and other P450 species expressed previously. As a result, CYP2B, CYP2C43 and CYP2C29 catalysed flavanone 2-hydroxylation. CYP2A5 catalysed 2- and 4-hydroxylations. Finally, to produce diverse modified compounds, variants of CYP2A5 with point mutations were incubated with a steroid (DHEA) and an antioxidant (flavanone) in vivo. HPLC analysis indicated that two P450 species produced a 7-beta-hydroxy-DHEA and two P450 species produced a 2-alpha-hydroxy-DHEA. Four P450 species catalysed flavanone 2- and 4-hydroxylations. These results indicate that bioconversion by P450 is a useful technique to modify small molecules (steroids, coumarin and flavanone) and produce new, diverse hydroxylated compounds, which could be used for high-throughput screening for drug discovery.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Dehydroepiandrosterone/metabolism , Escherichia coli/genetics , Flavanones/metabolism , Animals , Base Sequence , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme System/genetics , DNA Primers , DNA, Complementary , Genetic Vectors , Humans , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
In higher plants, long-chain fatty acid hydroperoxides are intermediates in the synthesis of a diverse group of bioactive compounds. We used the reverse trascriptase-polymerase chain reaction to isolate a gene responsible for the oxidization of fatty acids from Petunia hybrida. A P450 cDNA not isolated earlier, CYP92B1, contained an open reading frame predicted to encode a polypeptide consisting of 510 amino acid residues. The transcript of the cyp92B1 gene was expressed at a high level in the early stage of flower development. CYP92B1 cDNA was expressed in a yeast, Saccharomyces cerevisiae, and recombinant yeast microsomes containing CYP92B1, a hemoprotein, metabolized lauric acid, linoleic acid, and linolenic acid.