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1.
1. Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) both inhibit the O-deethylation of 7-ethoxyresorufin (ER) in liver microsomes of benzo(a)pyrene-induced (BP) mice but do not inhibit the O-deethylase activity in liver microsomes of BP-induced rats.

2. Anti-P3-450 and anti-P-450d-c inhibit BP hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this reaction at all in BP-induced rat liver microsomes.

3. Isolated cytochrome P3-450 in a reconstituted monooxygenase system metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, does not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min per nmol cytochrome.

4. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes corresponding to cytochrome P1-450, 55.0 kDa, and cytochrome P3-450, 54.5 kDa. There appeared a single band (cytochrome P3-450) in interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c.  相似文献   

2.
Metabolism of alpha-naphthoflavone (ANF) is increased markedly in rat liver microsomes by 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), two inducers of cytochromes P-450c and P-450d (P-450c and P-450d). Although several indirect lines of evidence in the literature suggest that ANF is metabolized by P-450c, Vyas et al. [J. Biol. Chem. 258:5649-5659 (1983)] reported that ANF metabolism by 3-MC-induced rat liver microsomes was only partially inhibited by antibodies against P-450c. Our laboratory has previously reported clastogenic effects of metabolites of ANF, and in the present study we reexamined the role of P-450c in ANF metabolism by both uninduced and TCDD-induced rat liver microsomes, using monospecific polyclonal antibodies to P-450c and P-450d. ANF metabolism was inhibited to different extents in TCDD-induced microsomes by different preparations of anti-P-450c. One lot of anti-P-450c produced only 50% inhibition of ANF metabolism in TCDD-induced microsomes, whereas another lot of anti-P-450c inhibited ANF metabolism by 80%. Anti-P-450d had no effect on ANF metabolism. Neither anti-P-450c nor anti-P-450d inhibited ANF metabolism in uninduced rat liver microsomes. In a reconstituted enzyme system, purified P-450c metabolized ANF 47 and 510 times more rapidly than P-450d and P-450b, respectively. Metabolites resulting from oxidation at 7,8- or 5,6-positions (7,8-dihydro-7,8-dihydroxy-ANF, 5,6-dihydro-5,6-dihydroxy-ANF, 5,6-oxide-ANF, and 6-hydroxy-ANF) were formed by all preparations of microsomes. An unknown toxic ANF metabolite was formed only with a reconstituted P-450c system and with 3-MC- or TCDD-induced microsomes. Our results indicate that P-450c is responsible for the majority of the metabolism of ANF in TCDD-induced microsomes, whereas other constitutive isozymes are responsible for the metabolism seen in uninduced liver microsomes. The variable inhibition of ANF metabolism with different lots of anti-P-450c probably reflects the differences in the proportion of antibodies to different epitopes important in the binding or metabolism of this substrate.  相似文献   

3.
The hepatic cytochrome P-450 responsible for metabolism of the structurally related macrolides FK506 and rapamycin in humans was identified using in vitro studies. FK506 and rapamycin metabolism was significantly correlated with nifedipine oxidation in human liver microsomes of eight different individuals. Immunoinhibition with anti-P450 3A4 abolished almost all FK506 and rapamycin metabolite formation. Inactivation of P450 3A4 by incubation of human liver microsomes with triacetyl oleandomycin (50 microM) or gestodene (10 microM) inhibited metabolism of FK506 and rapamycin. In liver microsomes from dexamethasone-treated rats FK506 and rapamycin metabolism was increased compared to liver microsomes from uninduced, phenobarbital-, or 3-methylcholanthrene-induced rats. FK506 and rapamycin were metabolized by reconstituted recombinant human liver P450 3A4. It is concluded that in human and rat liver FK506 and rapamycin are metabolized primarily by cytochrome P-450 3A4.  相似文献   

4.
Rats display a marked sex difference in the oxidation of the pyrrolizidine alkaloid senecionine, especially with respect to N-oxidation. This sex difference was largely eliminated following treatment with dexamethasone. These observations suggested the potential involvement of the male-specific cytochrome P-450 UT-A and the P-450 PCN-E in the metabolism of this pyrrolizidine alkaloid. Reconstituted rat P-450 UT-A exhibited a high rate of N-oxidation (15 nmol min-1 nmol P-450-1) which is almost 3-fold higher than the turnover number observed with male rat liver microsomes. In contrast, rat P-450 UT-A displayed a much lower activity toward necine pyrrole [+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine, DHP) formation (1.0 nmol min-1 nmol P-450-1). The N-oxygenation and pyrrole formation activities displayed by rat cytochromes P-450 PB-B and P-450 BNF-B toward senecionine were low, with rates less than 1 nmol min-1 nmol P-450-1. Rabbit antibody to rat P-450 UT-A inhibited the senecionine-N-oxidation activity of untreated male rat liver microsomes by 60%, with lesser inhibition of DHP production. Rabbit antibody to human P-450NF (the human homologue to rat P-450 PCN-E) was a potent inhibitor of DHP production by untreated male rat liver microsomes. With microsomes from dexamethasone-pretreated rats, anti-P-450NF inhibited DHP and N-oxide production in parallel. We conclude that the large sex difference in senecionine N-oxidation probably is the result of the specificity of P-450 isozymes UT-A and PCN-E.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
P-450-male is a male specific form of cytochrome P-450 in rat liver microsomes. Cytochrome P-450 crossreactive with anti-P-450-male antibodies was purified to an electrophoretical homogeneity from liver microsomes of male beagle dogs. The specific content of the purified cytochrome P-450 (P-450-D1) was 16.9 nmol/mg protein. The apparent monomeric molecular weight of P-450-D1 was 48,000, which was smaller than P-450-male (51,000). P-450-D1 showed similarities in spectral properties, N-terminal amino acid sequence, and catalytic activities with some limited exceptions: P-450-D1 did not catalyze 2 alpha-hydroxylation of testosterone and progesterone and catalyzed 21-hydroxylation of progesterone. Based on these results, we propose that P-450-D1 is a form of cytochrome P-450 in the same gene subfamily as P-450-male.  相似文献   

6.
Theophylline is metabolized in the liver by one or more cytochrome P-450 enzymes. To assess the amounts and types of these human cytochromes P-450, we incubated theophylline with microsomes prepared from 22 different human livers in the presence of NADPH, and measured simultaneous rates of 1- and 3-N-demethylations to 3-methylxanthine (3-MX) and 1-methylxanthine (1-MX), respectively; and 8-hydroxylation to 1,3-dimethyluric acid (1,3-DMU). Under optimal conditions, 3-MX, 1-MX, and 1,3-DMU formation proceeded with mean Km values of 2.05, 1.93, and 5.34 mM and Vmax values of 2.28, 2.48, and 23.4 pmol/mg/min, respectively. Formation of 3-MX and 1-MX correlated best with amounts of the immunoreactive protein HLd (P-450IA2) (p less than 0.05), whereas formation of 1,3-DMU correlated with the microsomal content of HLp (P-450IIIA3) and HLj (P-450IIE1). In immunoinhibition experiments, incubations conducted with a polyclonal anti-rat P-450c/d antibody, the formation of all the three theophylline metabolites (p less than 0.05) was significantly inhibited. However, addition of isoform-specific anti-rat-P-450d antibodies to the microsomal mixture significantly inhibited 1-N-demethylation, selectively, with little (if any) inhibition of 3-N-demethylation or 8-hydroxylation. Nonspecific cytochrome P-450 inhibition was ruled out by showing that erythromycin N-demethylation, an activity catalyzed by HLp, was unaffected by either anti-P-450c/d (P-450IA1/IA2) or anti-P-450d. Anti-rat-P-450p antibodies failed to block formation of theophylline metabolism, but did inhibit erythromycin N-demethylase.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

7.
A multitude of xenobiotics have been demonstrated to co-induce either cytochromes P-450c and P-450d or cytochromes P-450b and P-450e in rat hepatic microsomes. Recently, the compounds 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) and 3-methoxy-4-aminoazobenzene (3-MeO-AAB) have been suggested as selective inducers of cytochrome P-450b (Eur. J. Biochem. 151:67 (1985)) and P-450d (Biochem. Biophys. Res. Commun. 133:1072 (1985)), respectively. Since the identification of inducers with such unique characteristics would have implications with regard to the mechanism of induction of all four isozymes, we have examined the induction of cytochromes P-450b and P-450e by HCB and cytochromes P-450c and P-450d by 3-MeO-AAB in liver microsomes from adult male rats. Immunoblot analysis with monoclonal antibodies directed against cytochromes P-450b and P-450e indicate that HCB induces both isozymic species at the three dosage levels examined (10, 90, and 180 mg/kg). Similarly, 3-MeO-AAB does not appear to represent a unique inducer. Immunoblots of hepatic microsomes from animals treated with three different dosage regimens of 3-MeO-AAB demonstrate that, even at the lowest dosage level (50 mg/kg), both cytochromes P-450c and P-450d are induced. Moreover, immunoinhibition of 7-ethoxyresorufin O-deethylase (EROD) activity by monospecific antibody against either cytochrome P-450c or P-450d confirms this result. 3-MeO-AAB increases this enzyme activity 10-fold; approximately one-third of this induced activity is inhibited with monospecific anti-P-450c, while two-thirds is inhibited with monospecific anti-P-450d. This study also demonstrates that hepatic EROD activity is not an accurate estimate of cytochrome P-450c content since the majority of this enzyme activity in control and 3-MeO-AAB-treated rats is inhibited with monospecific anti-P-450d but not with monospecific anti-P-450c.  相似文献   

8.
The monoclonal antibody MAb 1-7-1, which specifically binds to cytochromes P-450IA1 and P-450IA2 in 3-methylcholanthrene-induced rat liver microsomes, was used to identify a cytochrome P-450IA1 homologue in human lung microsomes. Although MAb 1-7-1 had similar affinity constants for human and rat microsomes, the amount bound to human lung microsomes was severalfold lower than that bound to microsomes from untreated rat or rabbit lung and much lower than the amount bound to 3-methylcholanthrene-induced rat lung or liver microsomes. The amount bound to untreated baboon lung microsomes was similar to that bound to human lung microsomes. Three cytochrome P-450IA1-catalyzed activities, 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin, O-deethylase, and aryl hydrocarbon hydroxylase, were measurable in human lung microsomes, but the cytochrome P-450IA2-dependent activity acetanilide 4-hydroxylase was not. MAb 1-7-1 inhibited, and its binding correlated strongly with, 7-ethoxyresorufin O-deethylase activity (r = 0.92, p less than 0.01) in human lung microsomes. 7-Ethoxyresorufin O-deethylase activities in human lung were similar to those measured in untreated baboon lung but considerably lower than those present in untreated rabbit lung, untreated or 3-methylcholanthrene-induced rat lung and liver, or human liver. We conclude that MAb 1-7-1 recognizes a cytochrome P-450IA1 homologue in human lung and that no cytochrome P-450IA2 homologue is detected. Cytochrome P-450IA1 is expressed in human lung at relatively low levels, similar to those observed in untreated primate (baboon) lung. The majority of the 19 human lung samples examined do not exhibit a permanent polycyclic aromatic hydrocarbon-induced state with respect to this isozyme.  相似文献   

9.
Four isozymes, designated P-450m50a, P-450m50b, P-450m51a, and P-450m51b, have been isolated from liver microsomes of untreated male CD-1 mice. Electrophoretograms revealed that the molecular weights of P-450m51a and P-450m51b were about 51,000, whereas those of P-450m50a and P-450m50b were about 49,700 and 49,500, respectively. The present study is concerned mainly with the elucidation of the characteristics of P-450m50b and P-450m51a, since they appear to be major forms of cytochrome P-450 in mouse liver. End group analyses have revealed that these forms differ not only from P1-450, isolated previously from mouse liver, but also from any other known isozymes of cytochrome P-450. Naphthalene was metabolized by both P-450m50b and P-450m51a, but the turnover number of P-450m50b was 2-4 times greater than that of P-450m51a. Moreover, P-450m50b formed predominantly (1R,2S)-naphthalene 1,2-oxide, as determined by the formation of trans-1(R)-hydroxy-2(R)-glutathionyl-1,2-dihydronaphthalene, whereas P-450m51a formed both (1R,2S)- and (1S,2R)-naphthalene 1,2-oxide in about equal amounts. Although cytochrome P-450c, isolated from rat liver, is also known to convert naphthalene predominantly to (1R,2S)-naphthalene 1,-2-oxide, an antibody prepared against cytochrome P-450c failed to inhibit the formation of the glutathione conjugate when the 1R,2S-oxide was formed by either pure P-450m50b or by microsomes from mouse lung. Thus, P-450m50b probably is not orthologous to cytochrome P-450c.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

10.
Acute cannabidiol treatment of mice inactivated hepatic microsomal cytochrome P-450IIIA (P-450IIIA) and markedly inhibited in vitro cannabinoid metabolism. Antibodies raised against purified P-450IIIA inhibited the microsomal formation of quantitatively minor cannabinoid metabolites but had no effect on the major metabolites. Cannabinoid hydroxylation to the major metabolites was used as a functional probe to isolate and purify a P-450 (termed P-450THC) from hepatic microsomes of untreated mice. The purified protein had an apparent molecular weight of 47,000 and a specific content of 15.4 nmol/mg and exhibited an absorbance maximum at 452 nm for the reduced carbon monoxide complex. NH2-terminal sequence analysis of the first 16 residues of P-450THC suggests that it is a member of the P-450IIC subfamily, because its sequence is 85 and 69% identical to published sequences of rat hepatic P-450IIC7 and P-450IIC6, respectively. P-450THC exhibited high activity for cannabinoid hydroxylation and specifically produced 6 alpha- and 7-hydroxy-delta 1-tetrahydrocannabinol, as well as 6 alpha-, 7-, and 4"-hydroxycannabidiol. Unlike anti-P-450IIIA antibody, antibody raised against purified P-450THC markedly inhibited the microsomal formation of all major cannabinoid metabolites. Similar immunoinhibition studies also revealed the existence of orthologs of mouse P-450THC and P-450IIIA in human liver microsomes. Thus, cannabidiol treatment of mice resulted in the inactivation of at least two constitutive P-450 isozymes, which together account for the majority of the detected cannabinoid metabolites.  相似文献   

11.
A form of cytochrome P-450, P-450-D3, cross reactive with antibodies to rat P-450d was purified from liver microsomes of polychlorinated biphenyl (PCB)-treated female Beagle dogs to an electrophoretic homogeneity. Judging from the result of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of P-450-D3 was estimated to be 54,000. The oxidized form of P-450-D3 showed a peak at 416 nm indicating that the cytochrome is mostly in a low spin state. The carbon monoxide bound reduced form of P-450-D3 showed a peak at 448 nm. In a reconstituted system, P-450-D3 catalyzed drug oxidations including benzphetamine and aminopyrine N-demethylations, 7-ethoxycoumarin and p-propoxyaniline O-dealkylations, and aniline and benzo(a)pyrene hydroxylations. The rate of aniline hydroxylation catalyzed by P-450-D3 was similar to that catalyzed by P-450c which is a low spin form of cytochrome P-450 purified from liver microsomes of PCB-treated rats, whereas the catalytic activities of P-450-D3 for 7-ethoxycoumarin O-deethylation and benzo(a)pyrene hydroxylation were considerably lower than those of P-450c. The amino terminal portion of P-450-D3 was found to be highly similar to those of P-450d, human P3-450 and P3-450 when four amino acid deletions were tentatively inserted between fifth and sixth amino acids from the N-terminal, but not that of P-450c which is a low spin form of cytochrome P-448 purified from rat liver microsomes. These results indicate that Beagle dogs possess a low spin form of cytochrome P-450 with spectral properties similar to P-450c but with catalytic and structural properties similar to P-450d.  相似文献   

12.
1. The effect of various cytochrome P-450 inducers, namely acetone, phenobarbital (PB) and 3-methylcholanthrene (MC), on the pharmacokinetics of styrene metabolism was studied. 2. Styrene metabolism in vivo was studied measuring phenylglyoxylic acid (PGA), the enantiomers of mandelic acid (MA), and total thioethers excreted in the urine during a 24 h period of airborne exposure to styrene at 500 cm3/m3 (2100 mg/m3). In acetone-pretreated rats, PGA and MA and thioether formation were elevated 30-50%. The R/S ratio of MA enantiomers was about two in all styrene-exposed groups except PB-pretreated rats, which showed a ratio of four. 3. Styrene metabolism in liver microsomes measured in vitro was increased by styrene 140%, acetone plus styrene by 190%, methylcholanthrene plus styrene by 180% and phenobarbital plus styrene by 250%. 4. N-Nitrosodimethylamine demethylation (NDMAD) and 7-pentoxyresorufin dealkylation (PROD) in liver microsomes were enhanced 100-150% by styrene inhalation. The metabolism of 7-ethoxyresorufin was not significantly enhanced. 5. Monoclonal antibodies to P-450 IA1, IA2, IIB1 and IIE1 were utilized to identify cytochrome P-450s by Western blot analysis. These studies showed clearly that styrene inhalation induced principally cytochrome P450IE1, whereas styrene given by gavage at a high narcotic dosage induced both P450IIE1 (NDMAD, 60%) and P450IIB (PROD, 3000%). 6. Our conclusions are that styrene metabolism in vivo in both autoinduced and induced by other foreign compounds, that cytochrome P450IIE1 induction has a major impact on styrene metabolism and that P450IIB1 induction yields an altered MA metabolite enantiomer ratio.  相似文献   

13.
Phenacetin is metabolized primarily by O-deethylation to paracetamol (POD activity), a reaction catalysed by cytochrome P450. The high affinity component of POD activity is inducible in rat liver by treatment of the animals with polycyclic aromatic hydrocarbons. Following treatment with hydrocarbons such as 3-methylcholanthrene (MC) and isosafrole (ISF) both cytochromes P450c (P450IA1) and P450d (P450IA2) are also induced in rat liver. Studies with the reconstituted enzymes have shown that both forms of P450 catalyse phenacetin O-deethylation at rates that exceeded that of the high affinity component of activity of hepatic microsomal preparations from 3-methylcholanthrene-treated rats (at 4 microM phenacetin: P450c, 440 +/- 40 pmol/nmol/min; P450d, 1030 +/- 10 pmol/nmol/min; microsomal fraction, 163 pmol/mg/min). Specific inhibitory antibodies (both monoclonal and monospecific polyclonal) were used to define the specificity of microsomal POD activity. These studies have shown that hepatic high affinity POD activity is exclusively catalysed by cytochrome P450d in both untreated rats and in rats pretreated with MC.  相似文献   

14.
Chloroperoxidase (CPO) exhibits many physicochemical and catalytic properties similar to those of the bacterial and microsomal cytochromes P-450. Therefore, the possible similarities between the antigenic determinants of CPO and rat liver microsomal cytochrome P-450b were investigated. Polyclonal antibodies against CPO and rat liver cytochrome P-450b were raised in rabbits and used to investigate the antigenic cross-reactivity between CPO and P-450b. Although anti-CPO antibodies were capable of inhibiting the ethyl hydroperoxide-supported N,N-dimethylaniline (DMA) demethylation activity of CPO by more than 80%, they were unable to inhibit the NADPH-supported demethylation of DMA by cytochrome P-450b in the reconstituted system. The ethyl hydroperoxide-supported demethylation of DMA by CPO was not affected by the addition of anti-P-450b antibodies which inhibited cytochrome P-450 activity greater than 90%. In order to probe for the possible existence of common antigenic determinants which were not involved in catalytic activity, the cross-reactivities were investigated using enzyme-linked immunosorbent assays. There was no cross-reactivity between anti-CPO and cytochrome P-450b, or anti-P-450b and CPO using enzyme-linked immunosorbent assays. When control, phenobarbital-, isosafrole-, and beta-naphthoflavone-induced rat and rabbit liver microsomes and CPO were analyzed by Western blotting and developed with anti-P-450 antibodies, only the phenobarbital- and isosafrole-induced microsomes showed a positive reaction in the P-450 region. When anti-CPO antibodies were used on Western blots of the same series of proteins, a positive reaction was observed only with CPO.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

15.
Eighteen methylenedioxyphenyl (MDP) compounds, including some commonly inhaled by people, were tested for the ability to inhibit rabbit nasal microsomal cytochrome P-450-dependent hexamethylphosphoramide (HMPA) N-demethylase. For comparison, liver microsomes were also used. Nasal cytochrome P-450 from rabbits metabolized MDP compounds to form cytochrome P-450-metabolite (P-450-MI) complexes as indicated by difference spectra in the Soret region. Several of the MDP compounds were potent inhibitors of nasal P-450-dependent N-demethylase. If inhibition of nasal P-450 also occurs in vivo after inhibiting MDP compounds are inhaled, the metabolism of concurrently or subsequently inhaled compounds may be altered.  相似文献   

16.
1. The effect of various cytochrome P-450 inducers, namely acetone, phenobarbital (PB) and 3-methylcholanthrene (MC), on the pharmacokinetics of styrene metabolism was studied.

2. Styrene metabolism in vivo was studied measuring phenylglyoxylic acid (PGA), the enantiomers of mandelic acid (MA), and total thioethers excreted in the urine during a 24 h period of airborne exposure to styrene at 500 cm3/m3 (2100 mg/m3). In acetone-pretreated rats, PGA and MA and thioether formation were elevated 30-50%. The R/S ratio of MA enantiomers was about two in all styrene-exposed groups except PB-pretreated rats, which showed a ratio of four.

3. Styrene metabolism in liver microsomes measured in vitro was increased by styrene 140%, acetone plus styrene by 190%, methylcholanthrene plus styrene by 180% and phenobarbital plus styrene by 250%.

4. N-Nitrosodimethylamine demethylation (NDMAD) and 7-pentoxyresorufin dealkylaticn (PROD) in liver microsomes were enhanced 100-150% by styrene inhalation. The metabolism of 7-ethoxyresorufin was not significantly enhanced.

5. Monoclonal antibodies to P-450 IA1, IA2, IIB1 and IIE1 were utilized to identify cytochrcme P-450s by Western blot analysis. These studies showed clearly that styrene inhalation induced principally cytochrome P450IE1, whereas styrene given by gavage at a high narcotic dosage induced both P450HE1 (NDMAD, 60%) and P450IIB (PROD, 3000%).

6. Our conclusions are that styrene metabolism in vivo is both autoinduced and induced by other foreign compounds, that cytochrome P450IIE1 induction has a major impact on styrene metabolism and that P450IIB1 induction yields an altered MA metabolite enantiomer ratio.  相似文献   

17.
Beraprost sodium (BPS), a chemically stable and orally active prostacyclin analogue used for the treatment of chronic occlusive disease and primary pulmonary hypertension, was investigated in terms of its drug-drug interaction mediated by cytochrome P450. In a metabolic enzyme characterization study using P450-expressing insect cell microsomes, beraprost (BP) was slightly metabolized in the presence of CYP2C8, but not metabolized by the other P450 isoforms (CYP1A1, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11) at a concentration of 20 microM. These results suggest that none of the P450 isoforms is a major metabolic enzyme of BP. In a P450 induction study using human hepatocytes, BP did not induce any P450 isoform (CYP1A2, CYP2C9, CYP2C19, and CYP3A4) at concentrations of 1-100 microM. Furthermore, in a P450 inhibition study using human liver microsomes, BP did not inhibit any P450 isoform (CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) at concentrations of 0.05-1 microM. Therefore it is concluded that BP is not involved in drug-drug interaction mediated by P450 isoforms.  相似文献   

18.
Somatic cell hybrids were made between mouse myeloma cells and spleen cells derived from BALB/c female mice immunized with purified phenobarbital-induced rat liver cytochrome P-450 (PB-P-450). Hybridomas were selected in HAT medium, and the monoclonal antibodies (MAbs) produced were screened for binding to the PB-P-450 by radioimmunoassay, for immunoprecipitation of the PB-P-450, and for inhibition of PB-P-450-catalyzed enzyme activity. In two experiments, MAbs of the IgM and IgG1 were produced that bound and, in certain cases, precipitated PB-P-450. None of these MAbs, however, inhibited the PB-P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity. In two other experiments, MAbs to PB-P-450 were produced that bound, precipitated and, in several cases, strongly or completely inhibited the AHH and 7-ethoxycoumarin deethylase (ECD) activities of PB-P-450. These MAbs showed no activity toward the purified 3-methylcholanthrene-induced cytochrome P-450 (MC-P-450), β-naphthoflavone-induced cytochrome P-450 (BNF-P-450) or pregnenolone 16-α-carbonitrile-induced cytochrome P-450 (PCN-P-450) in respect to RIA determined binding, immunoprecipitation, or inhibition of AHH activity. One of the monoclonal antibodies, MAb 2-66-3, inhibited the AHH activity of liver microsomes from PB-treated rats by 43% but did not inhibit the AHH activity of liver microsomes from control, BNF-, or MC-treated rats. The MAb 2-66-3 also inhibited ECD in microsomes from PB-treated rats by 22%. The MAb 2-66-3 showed high cross-reactivity for binding, immunoprecipitation and inhibition of enzyme activity of PB-induced cytochrome P-450 from rabbit liver (PB-P-450LM2). Two other MAbs, 4-7-1 and 4-29-5, completely inhibited the AHH of the purified PB-P-450. MAbs to different cytochromes P-450 will be of extraordinary usefulness for a variety of studies including phenotyping of individuals, species, and tissues and for the genetic analysis of P-450s as well as for the direct assay, purification, and structure determination of various cytochromes P-450.  相似文献   

19.
The purpose of this study was to quantify the oxidative metabolism of dehydroepiandrosterone (3beta-hydroxy-androst-5-ene-17-one; DHEA) by liver microsomal fractions from various species and identify the cytochrome P450 (P450) enzymes responsible for production of individual hydroxylated DHEA metabolites. A gas chromatography-mass spectrometry method was developed for identification and quantification of DHEA metabolites. 7alpha-Hydroxy-DHEA was the major oxidative metabolite formed by rat (4.6 nmol/min/mg), hamster (7.4 nmol/min/mg), and pig (0.70 nmol/min/mg) liver microsomal fractions. 16alpha-Hydroxy-DHEA was the next most prevalent metabolite formed by rat (2.6 nmol/min/mg), hamster (0.26 nmol/min/mg), and pig (0.16 nmol/min/mg). Several unidentified metabolites were formed by hamster liver microsomes, and androstenedione was produced only by pig microsomes. Liver microsomal fractions from one human demonstrated that DHEA was oxidatively metabolized at a total rate of 7.8 nmol/min/mg, forming 7alpha-hydroxy-DHEA, 16alpha-hydroxy-DHEA, and a previously unidentified hydroxylated metabolite, 7beta-hydroxy-DHEA. Other human microsomal fractions exhibited much lower rates of metabolism, but with similar metabolite profiles. Recombinant P450s were used to identify the cytochrome P450s responsible for DHEA metabolism in the rat and human. CYP3A4 and CYP3A5 were the cytochromes P450 responsible for production of 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, and 16alpha-hydroxy-DHEA in adult liver microsomes, whereas the fetal/neonatal form CYP3A7 produced 16alpha-hydroxy and 7beta-hydroxy-DHEA. CYP3A23 uniquely formed 7alpha-hydroxy-DHEA, whereas other P450s, CYP2B1, CYP2C11, and CYP2D1, were responsible for 16alpha-hydroxy-DHEA metabolite production in rat liver microsomal fractions. These results indicate that the stereo- and regioselectivity of hydroxylation by different P450s account for the diverse DHEA metabolites formed among various species.  相似文献   

20.
The in vitro metabolism of cyclosporin A (CsA) was investigated by rabbit liver microsomes in order to identify the form(s) of cytochrome P-450 responsible for its biotransformation. Metabolites including monohydroxy-, N-demethylated, dihydroxy- and dihydroxy-N-demethylated derivatives were detected and quantified by HPLC from incubates of liver microsomes, CsA, and NADPH. Kinetic data indicated that monohydroxy- and N-demethylated derivatives were first generated and then served as substrates for production of dihydroxylated derivatives. Liver microsomes from phenobarbital-, beta-naphthoflavone-, triacetyloleandomycin-, erythromycin-, or rifampicin-treated and untreated rabbits were investigated, but only microsomes from animals treated with macrolide antibiotics (specific inducers of form P-450 3c) exhibited a type I binding spectrum upon CsA addition (Ks = 1.5 +/- 0.5 microM) and extensively metabolized the drug to all groups of derivatives (Km = 5.0 +/- 0.5 microM, Vmax = 1.0 +/- 0.2 nmol/mg/min). A linear correlation existed between CsA oxidase activity and P-450 3c specific content. Antibodies to P-450 3c strongly inhibited CsA oxidase activity of microsomes from macrolide antibiotic-induced animals, whereas antibodies to other forms, including P-450 2, 3b, 4, and 6, did not. When highly purified forms of P-450, including P-450 2, 3b, 3c, and 4, were assayed in a reconstituted system, only P-450 3c exhibited type I binding spectrum upon CsA addition (Ks = 1.4 +/- 0.5 microM) and extensively metabolized the drug to all derivatives. We conclude that the macrolide antibiotic-inducible form P-450 3c (or P-450 3c related from(s)) is responsible for the major part of CsA metabolism by rabbit liver microsomes.  相似文献   

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