• BMB Reports
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• v.37 no.5
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• pp.629-633
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• 2004

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450, and catalyzes the one-electron reduction of many drugs and foreign compounds. Various forms of spectrophotometric titration have been performed to investigate the electron-accepting properties of CPR, particularly, to examine its ability to reduce cytochrome c and ferricyanide. In this study, the reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) by CPR was assessed as a means of monitoring CPR activity. The principle advantage of DPPH is that its reduction can be assayed directly in the reaction medium by a continuous spectrophotometry. Thus, electrons released from NADPH by CPR were transferred to DPPH, and DPPH reduction was then followed spectrophotometrically by measuring $A_{520}$ reduction. Optimal assay concentrations of DPPH, CPR, potassium phosphate buffer, and NADPH were first established. DPPH reduction activity was found to depend upon the strength of the buffer used, which was optimal at 100 mM potassium phosphate and pH 7.6. The extinction coefficient of DPPH was $4.09\;mM^{-1}\;cm^{-1}$. DPPH reduction followed classical Michaelis-Menten kinetics ($K_m\;=\;28\;{\mu}M$, $K_{cat}\;=\;1690\;min^{-1}$). This method uses readily available materials, and has the additional advantages of being rapid and inexpensive.

• BMB Reports
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• v.31 no.4
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• pp.391-398
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• 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.

• YAKHAK HOEJI
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• v.43 no.6
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• pp.834-842
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• 1999

The metabolism and pharmacokinetics of M-l, which is metabolite of pentoxifylline, have been studied in human liver microsomes. Biphasic kinetics was observed from the Eadie-Hofstee plot for the formation of both metabolites of M-l. For the kinetics of pentoxifylline, mean values of $V_{max1}{\;}and{\;}V_{max2}$ were 1,648 and 5,622 nmol/min/mg protein, and the estimated values of $K_{ml}{\;}and{\;}K_{m2}$ were 0.180 and 4.829 mM, respectively. For M-3, mean values of $V_{max1}{\;}and{\;}V_{max2}$ were 0.062 and 0.491 nmol/min/mg protein, and estimated values of $K_{ml}{\;}and{\;}K_{m2}$ were 0.025 and 1.216 mM. The formations of pentoxifylline and M-3 from M-1 were indentified by using several selective inhibitors of cytochrome P450 isoformes at 0.05-5 mM concentration of M-1 in human liver microsomes. For the analysis of low (0.05 mM) concentration of M-1, where the affinity was expected as low, indicated that CYPlA2 and CYP3A4 were major P450 isoforms responsible for pentoxifylline and M-3 formation. CYP3A4 and CYP2A6 appeared to be P450 isoforms responsible for M-3 formation at high (5 mM) concentration of M-1.

• Preventive Nutrition and Food Science
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• v.8 no.2
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• pp.130-136
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• 2003

The protective effects of onion extract (OE), onion powder extracted in ethanol for 2 days. on carbon tetrachloride ($CCl_4$)-induced hepatotoxicities and the possible mechanisms involved in this protection were investigated in mice. Pretreatment with OE prior to the administration of $CCl_4$ significantly reduced the increase in serum alanine and aspartate aminotransferase activities and hepatic lipid peroxidation in a dose-dependent manner. In addition, pretreatment with OE significantly prevented the depletion of reduced glutathione content in the liver of $CCl_4$-intoxicated mice. $CCl_4$-induced hepatotoxicity was also prevented, as indicated by a liver histopathologic findings. The effects of OE on the cytochrome P450 (P450) 2E1, the major isozyme involved in $CCl_4$ biotransformation were investigated. Treatment of mice with OE resulted in a significant decrease in P450 2E1-dependent p-nitrophenol and aniline hydroxylation in a dose-dependent manner. Consistent with these observations, the P450 2E1 expressions were also decreased, as determined by immunoblot analysis. OE also exhibited antioxidant effects in FeCl$_2$-ascorbate induced lipid peroxidation in rat liver homogenates and in superoxide radical scavenging activity. These results show that the protective effects of OE against the $CCl_4$-induced hepatotoxicity may be due to its ability to block bioactivation of $CCl_4$, mainly tty inhibiting the expression and activities of P450 2E1 and by scavenging free radicals.

• Korean Journal of Biological Psychiatry
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• v.7 no.1
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• pp.14-20
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• 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.

• The Korean Journal of Pharmacology
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• v.32 no.3
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• pp.407-416
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• 1996

The reactive intermediates formed during the metabolism of therapeutic agents, toxicants and carcinogens by cytochromes P450 are frequently capable of covalently binding to tissue macromolecules and causing tissue damage. It has been shown that YH439, a congener of malotilate, is effective in suppressing hepatic P450 2E1 expression. The present study was designed to further establish the mechanistic basis of YH439 protection against toxicant by assessing its effects against chemical-mediated potentiated hepatotoxicity. Retinoyl palmitate (Vit-A) pretreatment of rats for 7 days substantially enhanced carbon tetrachloride hepatotoxicity, as supported by an ${\sim}5-fold$ increase in serum alanine aminotransferase (ALT) activity, as compared to $CCl_4$ treatment alone. The elevation of ALT activity due to Vit-A was completely blocked by the treatment of $GdCl_3$ a selective inhibitor of Kupffer cell activity. Concomitant pretreatment of rats with both YH439 and Vit-A resulted in a 94% decrease in Vit-A-potentiated $CCl_4$ hepatotoxicity. YH439 was also effective against propyl sulfide-potentiated $CCl_4-induced$ hepatotoxicity. Whereas propyl sulfide (50 mg/kg, 7d) enhanced $CCl_4-induced$ hepatotoxicity by >5-fold, relative to $CCl_4$ treatment alone, concomitant treatment of animals with both propyl sulfide and YH439 at the doses of 100 and 200 mg/kg prevented propyl sulfide-potentiated $CCl_4$ hepatotoxicity by 35% and 90%, respectively. Allyl sulfide, a suppressant of hepatic P450 2E1 expression, completely blocked the propyl sulfide-enhanced hepatotoxicity, indicating that propyl sulfide potentiation of $CCl_4$ hepatotoxicity was highly associated with the expression of P450 2E1 and that YH439 blocked the propyl sulfide-enhanced hepatotoxicity through modulation of P450 2E1 levels. Propyl sulfide- and $CCl_4-induced$ stimulation of lipid peroxidation was also suppressed by YH439 in a dose-related manner, as supported by decreases in malonedialdehyde production. The role of P450 2E1 induction in the potentiation of $CCl_4$ toxicity and the effects of YH439 were further evaluated using pyridine as a P450 2E1 inducer. Pyridine pretreatment substantially enhanced the $CCl_4$ hepatotoicity by 23-fold, relative to $CCl_4$ alone. YH439, however, failed to reduce the pyridine-potentiated toxicity, suggesting that the other form(s) of cytochroms P450 inducible by pyridine, but not suppressible by YH439 treatment, may play a role in potentiating $CCl_4-induced$ hepatotoxicity. YH439 was capable of blocking cadmium chloride-induced liver toxicity in mice. These results demonstrated that YH439 efficiently blocks Vit-A-enhanced hepatotoxiciy through Kupffer cell inactivation and that the suppression of P450 2E1 expression by YH439 is highly associated with blocking of propyl sulfide-mediated hepatotoxicity.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.6
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• pp.759-767
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• 1997

In the present study, we characterized the non-vascular smooth muscle relaxant effects of a novel benzoyran derivative ,SKP-450 (2-[2'(1',3'-dioxolone)-2-methyl-4- (2'-oxo-1'-pyrrolidinyl) -6-nitro-2H-1- benzopyran) and its metabolite, SKP-310, in comparison with levcromakalim (LCRK). In the rat stomach fundus, the spontaneous motility stimulated by $10^{-6.5}\;M$ bethanechol was completely eliminated not only by $10^{-7}\;M$ SKP-450 but also by $10^{-6}\;M$ LCRK, which were blocked by $10^{-6}\;M$ glibenclamide. The inhibitory effect of SKP-450 $pD_2,\;3.94{\pm}0.66)$ was much less than LCRK $(pD2,\;5.73{\pm}0.38,\;p<0.05)$. In the bethanechol $(10{-6.5 }\;M)-stimulated$ urinary bladder, the tonus was decreased in association with elimination of spontaneous motility by $10^{-7}\;M$ M SKP-450 and $10^{-6}\;M\;LCRK\;(pD2,\;6.77{\pm}0.06)\;(P<0.05)$, which were inhibitable by $10^{-6}\;M$ glibenclamide. The inhibitory effect of SKP-450 $(pD2,\;7.66{\pm}0.05)$ was significantly more potent than that of LCRK $(pD2,\;6.77{\pm}0.06,\;p<0.05)$. In the rat uterus stimulated by $PGF_{2\alpha}\;(10^{-7}\;M)$, both increased tonus and spontaneous motility were eliminated by $10^{-6}\;M$ LCRK with slight depression of the tonus, but not by SKP-450 $(10^{-5}\;M)$. The stimulated trachea of guinea-pig by $10^{-6.5}\;M$ bethanechol was moderately suppressed by SKP-450 $(10^{-6}{sim}10^{-5}\;M)$ but little by SKP-310. In association with the relaxant effects, SKP-450 $(10^{-6}\;M)$ and LCRK $(10^{-5}\;M)$ caused a significant stimulation of the $^{86}Rb$ efflux from rat urinary bladder and stomach fundus, which were antagonized by $10^{-5}\;M$ glibenclamide, whereas the $K^+$ channel openers could not exert a stimulation of the $^{86}Rb$ efflux from rat uterus. In conclusion, it is suggested that SKP-450 exerts potent relaxant effects on the urinary bladder detrusor muscle and duodenum, whereas it shows much less effect on stomach fundus and uterus as contrasted to LCRK.

• Korean Journal of Veterinary Research
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• v.38 no.1
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• pp.17-28
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• 1998

Drug-metabolizing activities of Korean native cattle and swine were investigated from viewpoints of the cytochrome P-450's level, their dependent mixed function oxidase activities, the reactive oxygen species formation and cytosolic enzyme acitivities from each liver homogenates. Level of cytochrome P-450 in the liver microsome of Korean native cattle was $0.28{\pm}0.05nmole/mg$ and that in pigs $0.35{\pm}0.03nmole/mg$. Level of cytochrome $b_5$ of Korean native cattle was $0.24{\pm}0.06nmole/mg$, and that of pigs $0.2{\pm}0.05nmole/mg$, showing no difference between two species. NADPH P-450 reductase were higher in Korean native cattle ($58.3{\pm}5.3nmole/mg/min$) than in pigs ($29.9{\pm}3.8nmole/mg/min$)(p<0.01). The activities of cytochrome P-450 dependent monooxygenases such as ethoxyresorufin O-deethylase (cattle, $96.5{\pm}12.5nmole/mg/min$ ; pigs, $13.6{\pm}2.1nmole/mg/min$), N-benzphetamine N-demethylase (cattle, $5.23{\pm}0.82nmole/mg/min$ ; pigs, $0.76{\pm}0.3nmole/mg/min$) and aniline hydroxylase (cattle, $0.95{\pm}0.1nmole/mg/min$ ; pigs, $0.33{\pm}0.08nmole/mg/min$) were much higher in Korean native cattle than in swine(p<0.01). However, the activity of testosterone $7{\alpha}$-hydroxylase was higher in swine ($90.4{\pm}1.2nmole/mg/min$) than cattle (cattle, $32.8{\pm}1.2nmole/mg/min$). Interestingly, testosterone $16{\alpha}$-hydroxylase, a marker enzyme for P-450 IIA was not detected in both animal species. These results suggest that Korean native cattle and pigs have high contents of P-450 IA1 and P-450 IIIA. Total sulfhydryl compound (cattle, $10.3{\pm}1.1nmole/mg$ ; Pigs, $14.5{\pm}1.8nmole/mg$) and glutathione related enzymes except glutathione reductase (cattle, $38.1{\pm}7.9nmole/mg/min$; swine, $22{\pm}3.6nmole/mg/min$) showed higher levels in swine than in Korean native cattle. Superoxide dismutase (cattle, $7.64{\pm}0.84nmole/mg/min$ ; pigs, $4.47{\pm}0.94nmole/mg/min$) and catalase (cattle, $30.4{\pm}3.7nmole/mg/min$ ; pigs, $17.2{\pm}1.8nmole/mg/min$) were remarkably higher in Korean native cattle than in swine (p<0.05).

• Biomolecules & Therapeutics
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• v.32 no.4
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• pp.474-480
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• 2024

Streptomyces avermitilis genome includes 33 genes encoding monooxygenation-catalyzing cytochrome P450 enzymes. We investigated the structure of CYP107P2 and its interactions with terpenoid compounds. The recombinant CYP107P2 protein was expressed in Escherichia coli and the purified enzyme exhibited a typical P450 spectrum upon CO-binding in its reduced state. Type-I substrate-binding spectral titrations were observed with various terpenoid compounds, including α-pinene, β-pinene, α-terpinyl acetate, and (+)-3-carene. The calculated binding affinities (Kd) ranged from 15.9 to 50.8 µM. The X-ray crystal structure of CYP107P2 was determined at 1.99 Å resolution, with a well-conserved overall P450 folding conformation. The terpenoid compound docking models illustrated that the structural interaction between monoterpenes and CYP107P2, with the distance between heme and terpenes ranging from 3.4 to 5.4 Å, indicates potential substrate binding for P450 enzyme. This study suggests that CYP107P2 is a Streptomyces P450 enzyme capable of catalyzing terpenes as substrates, signifying noteworthy advancements in comprehending a novel P450 enzyme's involvement in terpene reactions.

• Toxicological Research
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• v.26 no.1
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• pp.47-52
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• 2010

The Malassezia fungi are responsible for various human skin disorders including dandruff and seborrheic dermatitis. Of the Malassezia fungi, Malassezia globosa (M. globosa) is one of the most common in human scalp. The completed genome sequence of M. globosa contains four putative cytochrome P450 genes. To determine the roles of Malassezia P450 enzymes in the biosynthesis of ergosterol, we isolated MGL3996 gene from M. globosa chromosomal DNA by PCR. The MGL3996 gene encodes an enzyme of 616 amino acids, which shows strong similarity with known CYP52s of other species. MGL3996 gene was cloned and expressed in Pichia pastoris (P. pastoris) heterologous yeast expression system. Using the yeast microsomes expressing MGL3996 protein, a typical P450 CO-difference spectrum was shown with absorption maximum at 448 nm. SDS-PAGE analysis revealed a protein band of apparent molecular weight 69 kDa and Western blot with anti-histidine tag antibody showed that MGL3996 was successfully expressed in P. pastoris. Cloning and expression of a new P450 gene is an important step to study the P450 monooxygenase system of M. globosa and to understand the role of P450 enzymes in pathophysiology of dandruff.