• Toxicological Research
• /
• 제11권2호
• /
• pp.329-335
• /
• 1995

Incubation of aflatoxin $B_1$ $(AFB_1)$ with microsomes isolated from human liver number 110 yielded two metabolite peaks which were aflatoxin $Q_1$ $(AFQ_1)$ and $(AFB_1)$-exo-8, 9-epoxide (exo-epoxide) in high performance liquid chromatography. Production ratio of $AFQ_1$ to exo-epoxide was 2.43$\pm $0.04. Metabolism of $(AFB_1)$ to $(AFQ_1)$ and exo-epoxide was inhibited by troleandomycin in a same degree although troleandomycin was not activated as a mechanism-based inhibitor. The inhibitory effect was dependent upon either the incubation time with $(AFB_1)$ or the preincubation time before the addition of $(AFB_1)$. Incubation of troleandomycin and NADPH by the microsomes resulted in the formation of a cytochrome P 450 (P450)-metabollc intermediate (MI) complex and the level was approximately 80% of total P450 3A4 in the microsomes. This figure was similar to that of the inhibitory effect of troleandomycin on $AFB_1$ metabolism. Glutathione which was reported that it prevented the formation of MI complex in rat liver microsomes did not inhibit the formation of MI complex in human liver microsomes. These results suggested that the inhibitory effect of troleandomycin on $AFB_1$ metabolism is due to the formation of MI complex with P450 3A4. And the reaction mechanism of troleandomycin by human liver microsomes might be dlfferent from that one by rat liver microsomes.

• Toxicological Research
• /
• 제30권1호
• /
• pp.33-38
• /
• 2014

The human cytochrome P450 2J2 catalyzes an epoxygenase reaction to oxidize various fatty acids including arachidonic acid. In this study, three recombinant enzyme constructs of P450 2J2 were heterologously expressed in Escherichia coli and their P450 proteins were successfully purified using a $Ni^{2+}$-NTA affinity column. Deletion of 34 amino acid residues in N-terminus of P450 2J2 enzyme (2J2-D) produced the soluble enzyme located in the cytosolic fraction. The enzymatic analysis of this truncated protein indicated the typical spectral characteristics and functional properties of P450 2J2 enzyme. P450 2J2-D enzymes from soluble fraction catalyzed the oxidation reaction of terfenadine to the hydroxylated product. However, P450 2J2-D enzymes from membrane fraction did not support the P450 oxidation reaction although it displayed the characteristic CO-binding spectrum of P450. Our finding of these features in the N-terminal modified P450 2J2 enzyme could help understand the biological functions and the metabolic roles of P450 2J2 enzyme and make the crystallographic analysis of the P450 2J2 structure feasible for future studies.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1995년도 춘계학술대회
• /
• pp.128-128
• /
• 1995

To investigate the mechanism of the regulation of cytochrome P450IA1 gene expression, ethoxyresorufin deethylase(EROD) and benzo(a)pyrene hydroxylase in B6 mouse liver, in isolated perfused rat liver system. and in B6 mouse hepatocyte Hepa-I cells were examined. In C57BL/6N mouse, 3-methylcholan- throne( 3MC ) treatment have resulted in the stimulation of EROD activity based on fluorometry by 2.79 fold comparirng with that of control. Measurement of mRNA of cytochrome P450 was carried out by either nothern blot or dot blot analysis. Findings are similar to that of studies with enzymes. Furhtermore, when RTPCR method was applied to detect mRNA in Hepa I cell and liver tissues the results were more clear. Cytochrome P450IA1 upstream DNA containing CAT construct was transfected into Hepa-1 cells. After transfection of CAT construct, 3MC and flavonoids, such as, chrysin, hesperetin, kaempferol, morin, myricetin and aminoyrine were treated. 48 Hours after treatments, cells were harvested and assayed for CAT mRNA by RTPCR. 3MC treatment to hepa I cells transfected with trout P450IA1-CAT construct increased CAT mRNA by 2.81 fold when it was compared with that of control. This increase CAT mRNA was decreased by concomitantly treated flavonoids and aminopyrine. The level of CAT protein was 29.2-58.0% of 3MC stimulated CAT protein. Results of this study suggested that RTPCR seems to be a very good method to study regulation of gene expression in liver tissue or Hepa cells.

• 환경위생공학
• /
• 제10권3호
• /
• pp.16-28
• /
• 1995

Influences of capsaicin on the activities of cytochrome P45O of liver cell were studied in rats. Rats were provided food and water ad libitum and capsaicin and methylcellulose were gavaged for 6 days. Body weight gain and liver weight/body weight ratio, microsomal protein content and serum HDL- cholesterol content, the activity of cytochrome P450 and erythromycin demethylase, the activities of ethoxyresorufin and pentoxyresorufin O- dealkylase were determined. Capsaicin increased body weight gain but showed no significant changes on liver weight as compared with control group. Capsaicin increased the microsomal protein significantly but decreased the serum HDL- cholesterol. Capsaicin decreased the microsomal cytochrome P4SO significantly and did not show any influences on erythromycin demethylase ( cytochrome P45O III A ). Capsaicin increased the activity of pentoxyresorufin O- dealkylase ( cytochrome P45O II B) and decreased the activity of ethoxyresorufin O-dealkylase ( cytochrome P45O I A). It might be concluded that capsaicin reduced the microsomal cytochrome P45O and induced the CYP III and inhibited the CYP I A. It also might be concluded that capsaicin had no influence on CYP III A and decreased serum HDL- cholesterol. In these results capsaicin can not be used as an anti- atherosclerotic agent by increasing the CYP III A and HDL- cholesterol but it is considered that the more precise study on these theme is necessary.

• Biomolecules & Therapeutics
• /
• 제27권6호
• /
• pp.577-583
• /
• 2019

Human cytochrome P450 2C9 is a highly polymorphic enzyme that is required for drug and xenobiotic metabolism. Here, we studied eleven P450 2C9 genetic variants-including three novel variants F69S, L310V, and Q324X-that were clinically identified in Korean patients. P450 2C9 variant enzymes were expressed in Escherichia coli and their bicistronic membrane fractions were prepared The CO-binding spectra were obtained for nine enzyme variants, indicating P450 holoenzymes, but not for the M02 (L90P) variant. The M11 (Q324X) variant could not be expressed due to an early nonsense mutation. LC-MS/MS analysis was performed to measure the catalytic activities of the P450 2C9 variants, using diclofenac as a substrate. Steady-state kinetic analysis revealed that the catalytic efficiency of all nine P450 2C9 variants was lower than that of the wild type P450 2C9 enzyme. The M05 (R150L) and M06 (P279T) variants showed high $k_{cat}$ values; however, their $K_m$ values were also high. As the M01 (F69S), M03 (R124Q), M04 (R125H), M08 (I359L), M09 (I359T), and M10 (A477T) variants exhibited higher $K_m$ and lower $k_{cat}$ values than that of the wild type enzyme, their catalytic efficiency decreased by approximately 50-fold compared to the wild type enzyme. Furthermore, the novel variant M07 (L310V) showed lower $k_{cat}$ and $K_m$ values than the wild type enzyme, which resulted in its decreased (80%) catalytic efficiency. The X-ray crystal structure of P450 2C9 revealed the presence of mutations in the residues surrounding the substrate-binding cavity. Functional characterization of these genetic variants can help understand the pharmacogenetic outcomes.

• 생명과학회지
• /
• 제11권5호
• /
• pp.405-414
• /
• 2001

흰점박이꽃무지(Protaetia brevitarsis)는 간 질환의 치료를 위해 민간요법 뿐만 아니라 한방에서는 이용하는 전통약물로 알려져 있다. 본 연구는 사염화탄소와 에탄올로 간 손산을 유도한 흰쥐에서 흰점박이꽃무지 추출물(PBE)의 영향을 조사하였다. 사염화탄소는 이물질대사효소(cytochrome P_450, cytochrome b$_{5}$, cytochrome b$_{5}$ reductase), TBARS, 혈청 AST와 ALT, 간 무게비에서 유의한 변화가 확인되는 간독성이 유발하였으며, 에탄올은 혈청 AST와 TBARS의 변화는 유도하지 않았으나 이물질대사효소, ALT, 체중 당 무게비가 유의한 변화를 나타내었다. PBE로 전 처리한 실험군은 이물질대사효소(cytochrome P_450, cytochrome b$_{5}$, cytochrome b$_{5}$, reductase), 혈청 TBARS, AST와 ALT, 간무게비에서 어떠한 유의적인 변화도 관찰되지 않았다. 이러한결과는 적어도 이 실험조건에서는 PBE가 사염호탄소 혹은 에탄올로 유도한 간 독성에 대하여 보호 또는 회복효과가 없다고 판단된다.

• 생물정신의학
• /
• 제7권1호
• /
• pp.14-20
• /
• 2000

Recently atypical antipsychotics have been used as first line agent in the treatment of schizophrenia, and also played a significant role in the treatment of many kinds of psychiatric disorders. The pharmacokinetic and pharmacodynamic properties of these newer antipsychotics are well known through preclinical and early clinical trials. However, it is important to note the limitations of the results due to its relatively short experience. Clozapine is eliminated principally by the hepatic P450 1A2 and 3A4 cytochrome enzymes. 1A2 inducers such as carbamazepine and smoking can reduce its half-life, while 1A2 inhibitors such as SSRIs, especially fluvoxamine can increase its duration of action. Carbamazepine should be avoided in a patient on clozapine because of carbamazepine's potential effects on bone marrow. Benzodiazepines tend to increase the chances of sedation, delirium and respiratory depression. Risperidone is metabolized to 9-hydroxyriperidone by the hepatic P450 2D6 cytochrome enzymes. Fluoxetine and paroxetine, 2D6 inhibitors interfere with metabolism, but 9-hydroxyrisperidone has similar biological activity as parental drug, so it has little affect on the outcome. Olanzapine shows minimal capacity to inhibit cytochrome P450 isoenzymes and shows minimal chance of drug interaction. It is eliminated principally by the hepatic P450 1A2 and 2D6 cytochrome enzymes.

• BMB Reports
• /
• 제31권4호
• /
• pp.391-398
• /
• 1998

In vitro effects of acetone, methanol, and dimethylsulfoxide (DMSO) on liver microsomal cytochrome P450 (P450) content, and P450-dependent arylhydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) activities were studied in rats. Acetone at 1% (v/v) enhanced the content ofP450, assayed spectrally in 3-methylcholanethrene (MC)- and ${\beta}-naphthoflavone$ (BNF)-inducible microsomes by 18 and 7%, respectively. Methanol, up to 5% (v/v) applied, also showed enhancement effects on P450 content in liver microsomes from rats treated with phenobarbital (PB), MC, and BNF, as well as uninduced microsomes with similar but low strength. DMSO, however, did not show such enhancing effects at the ranges of the concentrations applied. AHH and ECOD activities in MC-inducible microsomes were also enhanced by acetone at 1%, which was in proportion to the increase in P450 content by the same concentration. However, the P450 content, and AHH and ECOD activities, were decreased by increasing the concentration of acetone. Methanol at the same concentration with acetone also enhanced ECOD activity but not AHH activity in MCinducible microsomes. The enhancing effect of acetone on the enzymes was negligible when the microsomes were pretreated with a specific monoclonal antibody of MC-inducible isozyme. The difference in the effects of these solvents on P450 system might be due to their different properties that cause the P450 active site to be exposed in milieu.

• 한국독성학회:학술대회논문집
• /
• 한국독성학회 2001년도 International Symposium on Signal transduction in Toxicology
• /
• pp.143-143
• /
• 2001

Human cytochrome P450 (CYP or P450) 3A4 (CYP3A4) is the most abundant among P450s in human liver. We previously reported that the expression of CYP3A4 in membranes prepared from E. coli coexpressed the bicistronic construct of CYP3A4 and NADPH-P450 reductase with cytochrome b$_{*}$ (b5) was showed 20-60% higher than that in membranes from E. coli expressed only the bicistronic construct with culturing longer times (48-72h).(omitted)

• Toxicological Research
• /
• 제7권1호
• /
• pp.1-12
• /
• 1991

The phenotyping of cytochrome P-450 in hepatic mivrosomes induced by phenytoin in the rats was carried out by using several monoclonal antibodies (MAbs) against specific P-450 isozymes. Phenytoin (180 mg/kg) was administered intrapritoneally for three consecutive days to the male Sprague-Dawley rats(100-120g). Solid phase radio-immunoassay showed higher binding affinity of MAb PB 2-66-3 and PCN 2-13-1 to the microsomes from phenytoin treated rats than those to from untreated rats, which was comparable to the level in phenobarbital induced rat hepatic microsomes.