Effects of the antimycobacterial compound 2-phenoxy-1-phenylethanone on rat hepatocytes and formation of metabolites

Context: Neolignans are usually dimers formed by oxidative coupling of allyl and propenyl phenols, and the neolignan analogue, 2-phenoxy-1-phenylethanone (LS-2) is a promising antimycobacterial compound showing very weak cytotoxicity in mammalian cells and lack of acute toxicity in murine models. Objectives: To investigate the mechanism of action of LS-2 in rat hepatocytes by evaluating the activity levels of enzymes related to oxidation status and drug-metabolizing activity. Materials and methods: Hepatocytes were treated with LS-2 from 0.05 up to 1 mM, for 24 and 48 h, and reduced glutathione (GSH), lipid peroxidation and cytochrome P450 enzyme (CYP450) activity were assayed. A homologous series of phenoxazone ethers were used as substrates to measure the enzymatic profile. The biotransformation of LS-2 was studied in hepatocytes by gas chromatography-mass spectrometry (GC-MS) for detection and analysis of possible metabolites. Results: Hepatocytes treated with LS-2 up to 1 mM for 24 or 48 h did not induce the formation of GSH and lipid peroxidation. O-Dealkylation activities of the isoenzymes CYP4501A1, CYP4501A2, CYP4502B1 and CYP4502B2 were also not detected in the hepatocytes treated with LS-2 for 24 or 48 h. Discussion and conclusion: The results indicate that LS-2 or its two detected metabolites, 2-phenoxy-1-phenylethanol and 2,4-(2-hydroxy-2-phenylethoxy) phenol, are not cytotoxic to rat hepatocytes. These compounds maintain a balance between the production of pro-oxidant agents and their respective antioxidant systems. The data show that enzymes related to oxidation status and drug-metabolizing activities are not involved in the mechanism of action of LS-2.

Purification, characterization and structural determination of chitinases produced by Moniliophthora perniciosa

The enzyme chitinase from Moniliophthora perniciosa the causative agent of the witches' broom disease in Theobroma cacao, was partially purified with ammonium sulfate and filtration by Sephacryl S-200 using sodium phosphate as an extraction buffer. Response surface methodology (RSM) was used to determine the optimum pH and temperature conditions. Four different isoenzymes were obtained: ChitMp I, ChitMp II, ChitMp III and ChitMp IV. ChitMp I had an optimum temperature at 44-73ºC and an optimum pH at 7.0-8.4. ChitMp II had an optimum temperature at 45-73ºC and an optimum pH at 7.0-8.4. ChitMp III had an optimum temperature at 54-67ºC and an optimum pH at 7.3-8.8. ChitMp IV had an optimum temperature at 60ºC and an optimum pH at 7.0. For the computational biology, the primary sequence was determined in silico from the database of the Genome/Proteome Project of M. perniciosa, yielding a sequence with 564 bp and 188 amino acids that was used for the three-dimensional design in a comparative modeling methodology. The generated models were submitted to validation using Procheck 3.0 and ANOLEA. The model proposed for the chitinase was subjected to a dynamic analysis over a 1 ns interval, resulting in a model with 91.7% of the residues occupying favorable places on the Ramachandran plot and an RMS of 2.68.