ABSTRACT
Avarol, a sesquiterpenoid hydroquinone, its quinone avarone, both main secondary metabolites from the marine sponge Dysidea avara and nine of their natural and synthetic derivatives were tested for ability to interact selectively with rat liver microsomal phenobarbital (PB)- or 3-methylcholanthrene (3-MC)-induced cytochrome (cyt.) P-450 isoenzymatic forms. Ethoxy- and pentoxyresorufin, aminopyrine and ethoxycoumarin were the specific substrates used for assaying cyt. P-450-dependent mono-oxygenase activities. All compounds were more effective in inhibiting the enzymatic activities measured in microsomes from PB-induced rat liver than those measured in microsomes from 3-MC-treated rats. Avarol and avarone, which were the most active inhibitors, act as mixed-type inhibitors of pentoxyresorufin-O-dealkylase activity. Mono- and diacetyl-derivatives of avarol, being deacetylated by rat liver microsomes, were almost as effective as the parent compound. Conversely, avarone derivatives, where one or two hydrogen atoms of the quinone ring have been substituted, were less effective inhibitors than the parent compound. The selective inhibition of PB-inducible cyt.P-450IIB by avarol and avarone was confirmed by their ability to inhibit the mutagenic activation of cyclophosphamide, as opposed to that of benzo[a]pyrene, which is activated mainly by the 3-MC-inducible cyt.P-450IA.
Subject(s)
Antineoplastic Agents/toxicity , Microsomes, Liver/drug effects , Sesquiterpenes/toxicity , 7-Alkoxycoumarin O-Dealkylase/antagonists & inhibitors , 7-Alkoxycoumarin O-Dealkylase/metabolism , Animals , Cyclohexenes , Cytochrome P-450 CYP1A1 , Cytochrome P-450 CYP2B1 , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Drug Interactions , Male , Methylcholanthrene/toxicity , Microsomes, Liver/enzymology , Mutagenicity Tests , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/metabolism , Phenobarbital/toxicity , Rats , Rats, Inbred Strains , Sesquiterpenes/pharmacologyABSTRACT
We measured the response of HepG2 cells to the classic cytochrome (cyt.) P-450 inducers 3-methylcholanthrene (3-MC) and phenobarbital (PB), by evaluating oxidative and/or reductive metabolism of the nitroarenes, 1-NP and 1,6-dinitropyrene (1,6-DNP), in control and induced cells. In HepG2 cells, 3-MC induces ring-hydroxylation of 1-NP, whereas PB stimulates its nitroreduction. PB induces NADPH-cyt. c reductase, but does not affect other cytosolic and microsomal enzymes which contribute to 1-NP nitroreduction in these cells. However, PB-inducible nitroreductase activity seems to be associated primarily with cyt. P-450 isoenzymatic form(s), as indicated by the requirement for NADPH and the response to specific inhibitors such as alpha-naphthoflavone and CO.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Methylcholanthrene/pharmacology , Phenobarbital/pharmacology , Pyrenes/metabolism , Carcinoma, Hepatocellular/enzymology , Enzyme Induction , Humans , Liver Neoplasms/enzymology , Mutagens/metabolism , Subcellular Fractions/enzymology , Tumor Cells, CulturedABSTRACT
Nitrofluoranthenes (NFs) are mutagenic and carcinogenic environmental pollutants found in incomplete combustion products and urban air particulate. We have studied both oxidative and reductive metabolism in vitro of different NF isomers mediated by subcellular rat liver fractions. Under aerobic conditions only ring hydroxylation of NFs by rat liver microsomes occurred and the isomeric position of the nitro group affected both the amount and the type of phenolic metabolites formed. Liver microsomes from 3-methylcholanthrene-induced rats were most effective in giving ring hydroxylated 7- and 8-nitrofluoranthene, whereas liver microsomes from phenobarbital-pretreated rats were the most active in metabolizing 1- and 3-nitrofluoranthene. Under anaerobic conditions, only reduction of NFs mediated by both cytosolic and microsomal rat liver enzymes occurred. Cofactor requirements and inhibition experiments indicated that the reductase activity in rat liver cytosolic fractions could be ascribed to DT-diaphorase, aldehyde oxidase and/or other unknown enzymes. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride and n-octylamine, and slightly by cytochrome c; flavin mononucleotide greatly enhanced this activity. 3-Nitrofluoranthene microsomal nitroreductase activity was increased by phenobarbital rat pretreatment and this increment correlated well with the content of cytochrome P450. These results indicate a participation of cytochrome P450 in the reductive metabolism of NFs by rat liver microsomes.
Subject(s)
Carcinogens/metabolism , Fluorenes/metabolism , Microsomes, Liver/enzymology , Mutagens/metabolism , Animals , Cytochrome P-450 Enzyme System/physiology , In Vitro Techniques , Male , Oxidation-Reduction , RatsABSTRACT
The effect of 1-aminopyrene (1-AP) and N-acetylaminopyrene (1-NAAP) on rat hepatic microsomal monooxygenase system was investigated. Both drugs increased the total content of cytochrome P-450 (cyt. P-450). The substrate specificity and the electrophoretic pattern of 1-AP and 1-NAAP induced cytochrome(s) were compared with those of the major forms of cyt. P-450 induced by 3-methylcholanthrene (3-MC) and phenobarbital (PB). The results suggest that the form of cyt. P-450 induced by 1-AP and 1-NAAP resembles that one induced by 3-MC. Furthermore the abilities of liver microsomes from control or differently induced rats to ring hydroxylate and to activate 1-nitropyrene (1-NP) metabolites to species mutagenic for bacteria were compared. It was observed that: (1) 1-NAAP is a good substrate for microsome-mediated ring hydroxylation, whereas 1-AP is oxygenated only at a low extent; (2) 3-MC, 1-AP and 1-NAAP-stimulated microsomes are more active than control or PB-ones to ring hydroxylate 1-NAAP. As phenolic derivatives of 1-NAAP show high mutagenic activity, these results indicate that 1-AP and 1-NAAP induce toxification pathways of 1-NP in similar way, even if in less extent, as compared to 3-MC.