OBJECTIVE: To explore the inhibitive effects of 1,3,8-trihydroxy-5-methoxyxanthone (TMX) on cytochrome P450s (CYP450s) in human liver microsomes. METHODS: Probe drugs were incubated with and without adding TMX to determine the changes of enzyme activities. The concentration ratio of metabolites to probe drugs was used to present enzyme activities. Concentrations of the probe drugs and their metabolites in the incubated mixture were detected by high performance liquid chromatography. RESULTS: The variations (mean, 95%CI) of the activities of CYP1A2, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 were 2.95 x 10(-3) (2.03 x 10(-3), 3.88 x 10(-3)), 3.14 x 10(-2) (1.87 x 10(-2), 4.42 x 10(-2)), 2.27 x 10(-3) (-1.4 x 10(-2),1.81 x 10(-2)), 7.72 x 10(-2) (-0.83 x 10(-2), 0.2374), and -0.2548 (-2.9802, 2.4707), respectively. The activities of CYP1A2 and CYP2C9 were significantly reduced in the present of TMX. CONCLUSION TMX (10 micromol/L) has significant inhibitive effect on the activities of CYP1A2 and CYP2C9, but no significant inhibitive effect on the activities of CYP2C19, CYP2E1 and CYP3A4.
Cytochrome P-450 Enzyme System ; metabolism ; Humans ; Microsomes, Liver ; drug effects ; enzymology ; Xanthones ; pharmacology

Cytochrome P450 3A4 (CYP3A4), is the dominant human liver hemoprotein enzyme localized in the endoplasmic reticulum (ER), and is responsible for the metabolism of more than 50% of clinically relevant drugs. While we were studying CYP3A4 expression and activity in human liver, we found that anti-CYP3A4 antibody cross-reacted with a lower band in liver cytoplasmic fraction. We assessed the activities of CYP3A4 and its truncated form in the microsomal and cytoplasmic fraction, respectively. In the cytoplasmic fraction, truncated CYP3A4 showed catalytic activity when reconstituted with NADPH-cytochrome P-450 reductase and cytochrome b5. In order to determine which site was deleted in the truncated form in vitro, we transfected cells with N-terminal tagged or C-terminal tagged human CYP3A4 cDNA. The truncated CYP3A4 is the N-terminal deleted form and was present in the soluble cytoplasmic fraction. Our result shows, for the first time, that N-terminal truncated, catalytically active CYP3A4 is present principally in the cytoplasm of human liver cells.
Blotting, Western ; Catalysis ; Cell Line ; Cytochrome P-450 CYP3A/chemistry/*metabolism ; Cytoplasm/*enzymology ; Humans ; Microsomes, Liver/*enzymology

Methylation catalyzed by methyltransferases is a major metabolic pathway for an inactivation of some catecholamines, niacinamide as well as aliphatic sulfhydryl drugs and toxic hydrogen sulfides. To investigate the effects of obstructive jaundice in an animal model, common bile duct ligation (CBDL) was performed in the rat and enzyme activities of S-adenosyl-L-methionine-dependent arylamine N-methyltransferase and thiol methyltransferase were examined in liver cell fractions and serum for a period of 42 d after CBDL. Both mitochondrial and microsomal arylamine N-methyltransferase showed significant increases in their activities between the 1st through the 7th day (P < or = 0.05 to 0.001), and between the 1st through the 28th day (P X or = 0.01 to 0.001) post-ligation, although the cytosolic arylamine N-methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. The mitochondrial as well as microsomal thiol methyltransferase showed significant increases in their activities between the 1st through the 28th day (P < or = 0.05 to 0.01 and P < or = 0.01 to 0.001, respectively) post-ligation, although the cytosolic thiol methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. Arylamine N-methyltransferase and thiol methyltransferase in the serum from cholestatic rats also showed significant increases in their activities between the 1st through 28th day (P < or = 0.01 to 0.001), and between the 0.5th through the 42nd day (P < or = 0.05 to 0.001) post-ligation compared to the sham-operated control, respectively. Enzyme kinetic parameters (Km and Vmax) of hepatic membrane-bound arylamine N-methyltransferase and thiol methyltransferase were analyzed with the preparation from the 7th day post-ligation, using tryptamine or 4-chlorothiophenol as substrates and S-Adenosyl-L-[methyl-3H]methionine as co-substrate. The results indicate that although the Km values were about the same as the sham-operated control, the Vmax values of both enzymes increased significantly (P < or = 0.01 and 0.001, respectively). These results suggest that the biosynthesis of arylamine N-methyltransferase and thiol methyltransferase have been induced in response to obstructive jaundice.
Animal ; Bile Ducts/surgery ; Cholestasis/*enzymology ; Ligation ; Liver/*enzymology ; Methyltransferases/blood/*metabolism ; Microsomes, Liver/enzymology ; Mitochondria, Liver/enzymology ; Rats ; Rats, Sprague-Dawley ; Time Factors

5'-nucelotidase and glucose-6-phosphatase are liver plasma and microsomal membranes markers and their respective activities were determined. In the liver homogenate, the activities of 5'-nucleotidase were 0.58 +/- 0.08 and 0.29 +/- 0.07 micromols/mg protein/10min in the control and 3'-methyl-4-dimethyl aminoazobenzene (3'-Me DAB) groups respectively. The enzyme activities m the partially purified plasma membranes were 2.15 +/- 0.25 and 1.31 +/- 0.23 micromols/mg protein/10min in the control and 3'-Me DAB groups respectively. The glucose-6-phosphatase activities in the homogenates of the control and 3'-Me DAB groups were 0.23 +/- 0.10, and 0.45 +/- 0.25 micromols/mg protein/10min, and in the microsomal fraction, 1.14 +/- 0.32, and 0.63 +/- 0.11 micromols/mg protein/10min, respectively, The concentrations of glucose carrier in the plasma membranes from the control and 3'-Me DAB group were 25, and 35 pmols/mg membrane protein, respectively, and the Ka values for cytochalsin B in each group were 5.20 X 109. and 5.14 X 109ml/mol, respectively. However in the microsomal fraction, no significant differences of glucose carrier were found between the control and 3'-Me DAB groups from the DEAE Sephadex A-50 ion exchange chromatography, fractions I and ll were obtained. Electrophoretic analysis of fraction I revealed a major protein band with a molecular weight of 45,000 and minor bands with MWs of 50,000, 55,000 and 15,000. Following AcA 34 gel filtration, a major protein band with a MW of 45,000 was obtained. From these results, it can be concluded that the glucose carrier protein was increased on plasma membrane of hepatoma induced by 3'-Me DAB, and the carrier protein showed similar molecular weight to other glucose carrier found in the RBC, muscle cells and adipocyte.
Animal ; Cell Membrane/enzymology ; Cell Membrane/metabolism ; Liver Neoplasms, Experimental/metabolism* ; Male ; Methyldimethylaminoazobenzene* ; Microsomes, Liver/enzymology ; Microsomes, Liver/metabolism ; Monosaccharide Transport Proteins/isolation & purification ; Monosaccharide Transport Proteins/metabolism* ; Rats ; Rats, Inbred Strains ; p-Dimethylaminoazobenzene*/analogs & derivatives

OBJECTIVEQingkailing injection (QKLI), Jinnaduo injection (JNDI), Shuxuetong injection (SXTI), Shenmai injection (SMI) and Kangai injection (KAI) are widely used in China. To predict the herb-drug interactions in clinical application, they were evaluated for their in vitro inhibition effect on CYP3A in rat liver microsomes.

METHODThe rat liver microsomes were incubated with different doses of 5 kinds of traditional Chinese medicine injections (TCMIs) in the present of testosterone, a specific substrate of CYP3A. 6beta-hydroxytestosterone, the metabolite of testosterone, was monitored by HPLC to compare the inhibition effect of 5 TCMIs on CYP3A in rat liver microsomes. Ketoconazole was used as a positive control.

RESULT10% QKLI reduced the formation of 6beta-hydroxytestosterone by approximately 93.0%, which is more significant than other four TCMIs. The half maximal inhibitory concentration (IC50) and the enzyme-inhibitor constant K(i) were 1.0% and 0.7% respectively.

CONCLUSIONQKLI showed much stronger inhibition activity against CYP3A, comparing to other 4 TCMIs. The results revealed that QKLI may be involved in herb-drug interactions by inhibition of CYP3A.

Animals ; Cytochrome P-450 CYP3A Inhibitors ; Injections ; Male ; Medicine, Chinese Traditional ; Microsomes, Liver ; drug effects ; enzymology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the stereoselectivity of R-(+) and S-(-)-propranolol glucuronidation and metabolic interaction between R(+)- and S-(-)-propranolol.

METHODSA RP-HPLC analytical method was developed for determination of R-(+)-and S-(-)-propranolol glucuronide (PG) incubated with rat hepatic microsome induced with phenobarbital (PB). The method was applied to investigate the stereoselectivity metabolism of racemic propranolol glucuronidation in vitro.

RESULTIn control and PB group, the concentration of R-(+)-PG produced at different substrates was higher than that of S-(-)-PG. Compared with the control, the V(max) and Cl(int) for R(+)-and S-(-)-propranolol increased significantly the K(m) for R(+)-propranolol was elevated, while that for S-(-) propranolol was decreased.

CONCLUSIONThere is a stereoselectivity in glucuronidation of propranolol in rat hepatic microsome induced with PB and R-(+)-propranolol is preferred. Metabolic interaction between R-(+)-and S-(-)-propranolol exists with a concentration-dependent mode.

Animals ; Enzyme Induction ; Microsomes, Liver ; enzymology ; Phenobarbital ; pharmacology ; Propranolol ; analogs & derivatives ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stereoisomerism

The present study is aimed to investigate the in vitro metabolic interconversion between baicalin (BG) and baicalein (B) in rat liver, kidney, intestine and bladder. BG and B were separately incubated with rat hepatic, renal, and intestinal microsomes, as well as bladder homogenates, for 30 min. The metabolites were identified and quantified by HPLC and metabolic kinetic parameters were obtained by fitting the data to the Michaelis-Menten equation. In hepatic microsomes, renal microsomes and bladder homogenates, but not in intestinal microsomes, BG was transformed into B, the hydrolysis metabolite of BG, with K(m) values being (44.65 +/- 6.01), (92.73 +/- 11.41), (74.60 +/- 3.68) micromol x L(-1), respectively, and V(max) values being (12.32 +/- 0.56), (3.30 +/- 0.18), (5.93 +/- 0.12) micromol x min(-1) x g(-1) (protein), respectively. In incubations with hepatic, renal, and intestinal microsomes and bladder homogenates, B was also transformed into BG, the glucuronidation metabolite of B, with K(m) values being (67.46 +/- 10.49), (226.7 +/- 71.59), (177.3 +/- 35.85), and (18.33 +/- 2.53) micromol x L(-1), respectively, and V(max) values being (14.74 +/- 0.97), (5.91 +/- 1.03), (38.14 +/- 3.60), and (1.22 +/- 0.05) micromol x min(-1) x g(-1) (protein), respectively. The results showed that the activity of UDP-glucuronosyltranferase (UGT) in intestinal microsomes was the highest among the four organs, and the activities of UGT were higher than that of glucuronidase (GUS) in hepatic, renal and intestinal microsomes, but the activity of GUS was higher than that of UGT in bladder homogenates.
Animals ; Anti-Infective Agents ; pharmacokinetics ; Antioxidants ; pharmacokinetics ; Biotransformation ; Flavanones ; pharmacokinetics ; Flavonoids ; pharmacokinetics ; Glucuronidase ; metabolism ; Glucuronosyltransferase ; metabolism ; Hydrolysis ; Intestines ; enzymology ; metabolism ; Kidney ; enzymology ; metabolism ; Liver ; enzymology ; metabolism ; Male ; Microsomes ; enzymology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Urinary Bladder ; enzymology ; metabolism

To study the effect of Siwu decoction (SWD) compound and its combined administration on hepatic P450 enzymatic activity and mRNA expression in rats. Rats were orally administered with SWD and water decoction combined with other medicines for two weeks, and then sacrificed. Their livers were perfused with normal saline to prepare liver micrisomes. Mixed probe and liver microsome in vitro incubation method were adopted to detect the effect of SWD on hepatic cytochrome P450. The real-time quantitative polymerase chain reaction (Q-PCR) was used to detect the effect of SWD on the expression of hepatic cytochrome P450. Compared with the control group, the SWD compound group showed higher CYP1A2 enzymatic activity (P < 0.05); Rehmanniae-paeoniae, angelicae-paeoniae, angelicae-rhizome, paeoniae-rhizome groups had lower CYP1A2 and CYP2C19 enzymatic activities (P < 0.05); And the compound group, the single component group and the combination group showed lower CYP2B6 enzymatic activities (P < 0.05). The compound could up-regulated the mRNA expression of CYP2B1 (P < 0.05); And the four single components could down-regulated the mRNA expression of CYP2B1 (P < 0.05). SWD compound had the effect in inducing CYP1A2 enzymatic activity. The rehmanniae-paeoniae group and the angelicae-paeoniae group had identical enzymatic activity with the control group, but significant down-regulation in CYP1A2 enzymatic activity after being combined with paeoniae. The compound and its combined administration showed the inhibitory effect on CYP2B6 enzymatic activity, particularly being combined with angelicae. The compound showed identical effect with the four single components in terms of CYP1A2 mRNA expression and enzymatic activity.
Animals ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Liver ; drug effects ; enzymology ; Microsomes, Liver ; drug effects ; enzymology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar

To investigate the effect of Jiawei Foshou San and its various combined administration on hepatic P450 enzyme activity and hepatocyte morphology in rats. Rats were orally administered with drugs for four weeks and then sacrificed to prepare liver microsomes. The liver microsomes were incubated with the cocktail method; The metabolites were determined with the rapid liquid chromatography with tandem mass spectrometry (LC-MS/MS) to investigate the hepatocyte P450 enzyme activity. In addition, the hepatic pathological changes were observed by using the hematoxylin and eosin (HE) staining. Compared with the control group, the enzyme activity of CYP1A2 and CYP3A4 in the Jiawei Foshou san group showed a significant rise (P < 0.05); the enzyme activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in the ferulic acid + ligustrazine group and the ligustrazine + tetrahydropalmatine group showed a significant rise (P < 0.05) ; the enzyme activity of CYP1A2, CYP2D6 and CYP2E1 in the ligustrazine group showed a significant rise (P < 0.05); the enzyme activity of CYP3A4 in the ferulic acid group showed a significant reduction (P < 0.05). After the administration with various drugs, the hepatocyte morphologies in the ferulic acid group and the ligustrazine group were normal. The pathological changes were observed in the tetrahydropalmatine group, such as unclear boundary of hepatic lobules, disordered hepatic cell arrangement, blurred edge, anisokaryosis and infiltration of inflammatory cells. The ferulic acid + tetrahydropalmatine group, the ligustrazine + tetrahydropalmatine group and the Jiawei Foshou San group also showed inflammatory infiltration, but with less pathological changes, particularly the Jiawei Foshou San group. The study result shows that Jiawei Foshou San can induce the enzyme activity of CYP1A2 and CYP3A4, and ligustrazine may be the effective substance for inducing CYP1A2. Its combination with ferulic acid and ligustrazine can significantly reduce the liver toxicity of tetrahydropalmatine.
Animals ; Cytochrome P-450 Enzyme System ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Hepatocytes ; drug effects ; enzymology ; Microsomes, Liver ; drug effects ; enzymology ; Rats ; Rats, Sprague-Dawley

To illustrate the compability rule of Jinlingizi powder, by investigating the effects of Jinlingzi Powder with different compatibility on the enzymatic activity of cytochrome P1 A2 (CYP1A2) from rat liver microsome. The different compability of Jinlingizi powder is designed, based on the orthogonal array L9 (3(4)). In vitro test, rat liver microsomes incubation system is applied to detect the 50% inhibitory concentraton of Jinlingzi powder with different compatibility to cytochrome P1A2 (CYP1A2) enzyme. In vivo experiments, rats is treated orally with the different compability of Jinlingizi powder for 5 days, then be injected with probe drug phenacetin. The biosample from liver tissue is obtained by microdialysis probe, then analysisd by HPLC. The concentration-time data are modulated by software WinNonlin. IC50 data show no significant inhibitory activty to cytochrome P1 A2. Acetaminophen and phenacetin PK parameters indicate that the different compability of Jinlingizi powder can modulate the CYP 1A2 mediated metabolism, which is associate with the compatibility of Jinlingzi powder.
Animals ; Cytochrome P-450 CYP1A2 Inhibitors ; Drugs, Chinese Herbal ; pharmacology ; Male ; Medicine, Chinese Traditional ; Microsomes, Liver ; drug effects ; enzymology ; Powders ; Rats ; Rats, Wistar