Cytochrome P450 enzymes are composed of many isozymes and involved in the biotransformation of both exogenous and endogenous substances. A growing number of studies have found that the P450 enzymes do not always follow the classical Michaelis-Menten kinetics, but show atypical kinetic behavior, which is also the current research hotspot. In this paper, the category and mechanisms of atypical kinetics of the P450 enzyme were reviewed, providing theoretical basis for the research of enzyme kinetics.
Animals ; Benzoflavones ; pharmacology ; Binding Sites ; Cytochrome P-450 CYP3A ; chemistry ; metabolism ; Cytochrome P-450 Enzyme System ; chemistry ; metabolism ; Enzyme Activation ; Humans ; Kinetics ; Protein Binding ; Substrate Specificity

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypic halogenated aromatic hydrocarbon (HAH), is known as one of the most potent toxicants. At least a part of its toxic effects appears to be derived from its ability to induce TNF-alpha production. However, the signaling pathway of TCDD that leads to TNF-alpha expression has not been elucidated. In this study, we investigated the signaling mechanism of TCDD-induced TNF-alpha expression in PMA-differentiated THP-1 macrophages. TCDD induced both mRNA and protein expression of TNF-alpha in a dose- and time-dependent manner. Alpha-Naphthoflavone (NF), an aryl hydrocarbon receptor (AhR) inhibitor, prevented the TCDD-induced expression of TNF-alpha at both mRNA and protein levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, and PD153035, an EGFR inhibitor, also blocked the increase of TNF-alpha expression by TCDD, indicating the role of EGFR in TCDD-induced TNF-alpha expression. On the other hand, PP2, a c-Src specific inhibitor, did not affect TCDD-induced TNF-alpha expression. EGFR phosphorylation was detected as early as 5 min after TCDD treatment. TCDD-induced EGFR activation was AhR-dependent since co-treatment with alpha-NF prevented it. ERK was found to be a downstream effector of EGFR activation in the signaling pathway leading to TNF-alpha production after TCDD stimulation. Activation of ERK was observed from 30 min after TCDD treatment. PD98059, an inhibitor of the MEK-ERK pathway, completely prevented the TNF-alpha mRNA and protein expression induced by TCDD, whereas inhibitors of JNK and p38 MAPK had no effect. PD153035, an EGFR inhibitor, as well as alpha-NF significantly reduced ERK phosphorylation, suggesting that ERK activation by TCDD was mediated by both EGFR and AhR. These results indicate that TNF-alpha production by TCDD in differentiated THP-1 macrophages is AhR-dependent and involves activation of EGFR and ERK, but not c-Src, JNK, nor p38 MAPK. A signaling pathway is proposed where TCDD induces sequential activation of AhR, EGFR and ERK, leading to the increased expression of TNF-alpha.
Animals ; Benzoflavones/pharmacology ; Cell Differentiation ; Cell Line, Tumor ; Enzyme Activation ; Genistein/pharmacology ; Hazardous Substances/*toxicity ; Humans ; MAP Kinase Signaling System/drug effects/physiology ; Macrophages/*metabolism ; Mice ; Phosphorylation ; Pyrimidines/pharmacology ; Quinazolines/pharmacology ; RNA, Messenger/metabolism ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptors, Aryl Hydrocarbon/antagonists & inhibitors ; Signal Transduction ; Tetrachlorodibenzodioxin/*toxicity ; Tumor Necrosis Factor-alpha/*biosynthesis ; src-Family Kinases/antagonists & inhibitors/metabolism

To study the enzyme kinetics of ligustilide metabolism and the effects of selective CYP450 inhibitors on the metabolism of ligustilide in liver microsomes of rat, a LC-MS method was established for quantitative analysis of ligustilide in liver microsomes incubation system with nitrendipine as internal standard. The determination m/z for ligustilide was 173, and for nitrendipine, 315. An optimum incubation system was found and various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of ligustilide. The results showed that enzyme kinetics of ligustilide could be significantly inhibited by ketoconazole, trimethoprim and a-naphthoflavon but scarcely inhibited by omeprazole, 4-methylpyrazole and quinidine. Therefore, CYP3A4, CYP2C9 and CYP1A2 are the major isoenzyme participated in in vitro metabolism of ligustilide.
4-Butyrolactone ; analogs & derivatives ; metabolism ; Animals ; Benzoflavones ; pharmacology ; Cytochrome P-450 CYP1A2 ; Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; Cytochromes ; antagonists & inhibitors ; Ketoconazole ; pharmacology ; Kinetics ; Male ; Microsomes, Liver ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trimethoprim ; pharmacology