Involvement Of Cytochrome P-450 System Research Articles

Involvement of CYP450 system in hepatoprotective activity of Malaysian Agricultural Research and Development Institute (MARDI)-produced virgin coconut oils

The present study aims to determine the role of cytochrome P450 (CYP450) enzyme system in hepatoprotective activity of virgin coconut oils produced by Malaysian Agricultural Research and Development Institute (MARDI). Paracetamol (PCM)-induced hepatotoxic rat was used as a model. Liver injury induced by 3 g/kg PCM increased the liver weight and liver enzymes (e.g. alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphate (ALP)) and decreased cell viability indicating liver damage. Histological observation also confirms liver damage indicated by the presence of inflammations and necrosis. Pre-treatment with VCOA or VCOB reversed the significantly (P < 0.05) reversed PCM toxic effect. Groups pre-treated withvirgin coconut oil (VCOs) followed by inhibitor or inducer of CYP450 demonstrated significant (P < 0.0.5) increase in liver weight, liver enzymes levels and decrease in cell viability, which are, however, significantly (P < 0.05) less remarkable as compared to group treated with PCM alone. In conclusion, VCO possessed hepatoprotective effect, which is believed to be mediated via a non-CYP450 system and might be associated partly with the antioxidant potential of the oil. Further studies are warranted to determine the actual mechanisms of hepatoprotection involved. Key words: Malaysian Agricultural Research and Development Institute (MARDI), virgin coconut oil, liver toxicity, paracetamol, cytochrome P450, hepatoprotection.

Stereoselective hydroxylation of 4-methyl-2-cyclohexenone in rats: its relevance to R-(+)-pulegone-mediated hepatotoxicity

R-(+)-Pulegone, a monoterpene ketone, is a potent hepatotoxin. One of the major metabolites of pulegone has been shown to be p-cresol, a glutathione depletor and a known toxin. Allylic hydroxylation of 4-methyl-2-cyclohexenone results in the formation of p-cresol. The present study documents for the first time the involvement of cytochrome P-450 system and the stereochemical preference in this hydroxylation reaction. Incubation of PB-induced rat liver microsomes as well as reconstituted PB-induced cytochrome P-450 system with ±4-methyl-2-cyclohexenone in the presence of NADPH and O 2 resulted in the formation of 4-hydroxy-4-methyl-2-cyclohexenone and p-cresol. From the assay mixture, the unreacted substrate, viz., 4-methyl-2-cyclohexenone was isolated and purified and its optical rotation was found to be 2.2 (in CHCl 3). The observed enantiomeric excess in the recovered substrate was further confirmed by circular dichroism (CD) studies. The CD spectrum has a peak at 292 nm and a trough at 270 nm. The enantiomeric excess in the recovered substrate indicates that the hydroxylation at C-4 position is stereoselective. The significance of these results with respect to pulegone-mediated hepatotoxicity is discussed.

Studies on the microsomal 11α-hydroxylation of progesterone inAspergillus ochraceus: characterization of the hydroxylase system

From the induced vegetative cell cultures ofA. ochraceus, a procedure for the preparation of cell-free extract with high 11α-hydroxylase activity was developed. To obtain optimal hydroxylase activity,edta (10mM), glycerol (10%) anddtt (5mM) were required in the grinding medium. Although the optimum pH for the grinding medium was 8·3, the hydroxylase has a pH optimum of 7·7. Microsomes (2mg) isolated from the crude cell-free extract, hydroxylated progesterone in high yields (85–90% in 30 min) in the presence ofnadph and O2. The apparentK m for NADPH and progesterone were 0·052 mM and 0·625 mM respectively. The involvement of cytochrome P-450 system in the hydroxylation reaction was established by various inhibition studies. The hydroxylase activity was inhibited by metyrapone, carbon monoxide,skf-525A andp-cmb. The presence of high levels ofnadph-cytochromec reductase in the microsomal fraction and the strong inhibition of the hydroxylase system by cytochromec indicated that the reductase could be one of the components of the hydroxylase system. Progesterone has the ability to induce the 11α-hydroxylase system significantly, whereas deoxycorticosterone and phenobarbital failed to bring about the induction. However, deoxycorticosterone acted as a good substrate for the 11α-hydroxylase system. The membrane-bound hydroxylase was solubilized using various ionic and non-ionic detergents. Solubilized membrane fraction contained considerable levels of cytochrome P-450 andnadph-cytochromec reductase, besides hydroxylase activity.

Studies on the microsomal 11α-hydroxylation of progesterone in Aspergillus ochraceus: isolation, characterization, and solubilization of the hydroxylase system

A suitable procedure for the preparation of cell-free extract with high 11α-hydroxylase activity from the induced vegetative cell cultures of Aspergillus ochraceus was developed. The presence of EDTA (10 mM), glycerol (10%), and dithiothreitol (5 mM), in the grinding medium was found to be necessary for optimal hydroxylase activity. Although the pH optimum of the hydroxylase was found to be 7.7, the ideal pH for preparing the cell-free extract from mycelium was 8.3. Microsomes (2.0 mg) isolated from the cell-free extract hydroxylated progresterone in very high yields (85–90% in 30 min) in the presence of NADPH and O2. Cytosolic involvement in the hydroxylation reaction was not noticed. The apparent Km for NADPH and progesterone were 0.052 and 0.625 mM, respectively. The hydroxylase activity was inhibited by metyrapone, carbon monoxide, SKF-525A, and p-chloromercuribenzoate indicating the involvement of cytochrome P-450 system in the reaction. Inhibition of the hydroxylase activity by cytochrome c and the presence of significant levels of NADPH–cytochrome c reductase in the microsomal fraction suggested that the reductase could be one of the components of the hydroxylase system. The membrane-bound hydroxylase was solubilized using sodium cholate. Solubilized membrane fraction contained considerable levels of cytochrome P-450 and NADPH–cytochrome c reductase, besides hydroxylase activity.

Microsomal 11α-hydroxylation of progesterone in [formula omitted]: Part I: Characterization of the hydroxylase system

Microsomes (105,000xg sediment) prepared from induced cells of A.ochraceus was found to hydroxylate progesterone to 11α-hydroxyprogesterone (11α-OHP) in high yields (85–90% in 30 min.) in the presence of NADPH and O 2. The pH optimum for the hydroxylase was found to be 7.7. However, for the isolation of active microsomes grinding of the mycelium should be carried out at pH 8.3. Metyrapone, carbon monoxide, SKF-525A, p-CMB and N-methyl maleimide inhibited the hydroxylase activity indicating the involvement of cytochrome P-450 system. The inhibition of the hydroxylase by cytochrome c and the presence of high levels of NADPH-cytochrome c reductase in induced microsomes suggest that the reductase could be one of the components in the hydroxylase system.