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PubMed1983

Regulation of monooxygenation during development of chick embryo liver: role of cytochrome P-450 isozymes and NADPH.

The endoplasmic reticulum of the liver cell, supplied with molecular oxygen and NADPH, can monooxygenate a variety of lipid soluble compounds including steroids and many drugs. The essential enzymatic steps are carried out by cytochrome P-450 and NADPH cytochrome P-450 reductase. There are several molecular forms (isozymes) of cytochrome P-450 in liver, and it has been shown that their relative concentrations change during development and in response to phenobarbital. Because substrate specificities of the isozymes differ to some extent, changes in the isozyme pattern should alter the pattern of substrate utilization if the isozymes are controlling the rates of monooxygenation. We found that the rates of testosterone hydroxylation and the oxidative N-demethylations of benzphetamine, codeine, and ethylmorphine were equal in endoplasmic reticulum isolated from chick embryo liver on day 11 of egg incubation, and this equality was unaffected by age or phenobarbital. Thus, monooxygenation rates do not appear to be controlled by the isozymes of cytochrome P-450. A factor that might affect the pattern of steroid and drug utilization by endoplasmic reticulum may be the availability of reducing equivalents. Lowering the concentration of NADPH to one half-optimum values in our reaction mixtures (0.5 to 0.25 mM) diminished the monooxygenation rate of benzphetamine by 8%, of testosterone by 40%, and of codeine and ethylmorphine by 60%. These differential effects were independent of age and phenobarbital. Thus, the pattern of substrate utilization by the monooxygenase system in vivo is probably altered by changes in the tissue concentration of NADPH.

Animals↗
PubMed2001

Evidence supporting the interaction of CYP2B4 and CYP1A2 in microsomal preparations.

Recent studies have demonstrated that the catalytic behavior of one cytochrome P450 (P450) enzyme can be influenced by the presence of a second P450. This effect has been observed using reconstituted systems containing reductase, CYP2B4, and CYP1A2, primarily at subsaturating reductase. Addition of 1A2 caused a 75% inhibition of CYP2B4-dependent 7-pentoxyresorufin-O-dealkylation (PROD). Conversely, CYP2B4-dependent benzphetamine (bzp) demethylation did not exhibit this response after CYP1A2 addition. Addition of CYP2B4 to a reconstituted system containing reductase and CYP1A2 caused synergism of CYP1A2-dependent 7-ethoxyresorufin-O-dealkylation (EROD). This behavior was consistent with the formation of heteromeric CYP1A2-CYP2B4 complexes with altered catalytic properties. Although such responses have been documented in reconstituted systems, they have not been demonstrated in microsomal preparations. The goal of the present study was to determine whether such interactions were observed in rabbit liver microsomes. In an effort to detect such changes, we took advantage of the differential effect of CYP1A2 on CYP2B4-selective PROD and bzp metabolism. Rabbits were treated with phenobarbital (PB), beta-naphthoflavone (betaNF), and both PB + betaNF-conditions that enrich microsomes with CYP2B4, CYP1A2, or both enzymes, respectively. Benzphetamine demethylation activity was equivalently elevated in both the PB and the PB + betaNF groups, consistent with the induction of CYP2B4 in both groups. In contrast, PROD activity in the PB + betaNF group was less than 25% of that found in the PB-treated rabbits. These results demonstrate that the interactions observed in reconstituted systems are not an artifact of reconstitution but are observed under the more natural conditions of the microsomal membrane.

Animals↗
PubMedDate not supplied

Effects of conditions for reconstitution with cytochrome b5 on the formation of products in cytochrome P-450-catalyzed reactions.

Evidence is presented that the method of reconstitution of the cytochrome P-450-containing liver microsomal enzyme system with cytochrome b5 (b5), including the order of addition of the components, the concentration of the b5, and the length of incubation prior to initiation of the reaction by NADPH, governs the steady state catalytic activity obtained. For example, the addition to cytochrome P-450 isozyme 2, NADPH-cytochrome P-450 reductase, and phosphatidylcholine of concentrated b5 (0.4 microM) results in extensive inhibition of benzphetamine demethylation and NADPH oxidation, whereas the addition of dilute b5 (0.02 microM) to the other components results in extensive stimulation of the demethylation reaction. The inhibition is partly relieved by prolonged incubation. The effects of pH and buffer concentration were determined, and the optimal molar ratio of b5 to cytochrome P-450 isozyme 2 was shown to be about 2.0 for stimulation of benzphetamine demethylation, dimethylaniline demethylation, and cyclohexanol oxidation to cyclohexanone. Cytochrome P-450 isozyme 4-catalyzed aminopyrine demethylation and aniline p-hydroxylation are not stimulated by b5, as predicted from a model based on stopped flow kinetic measurements [Pompon and Coon: J. Biol. Chem. 259, 15377 (1984)]. End-point stoichiometry measurements were carried out with cytochrome P-450 isozyme 2 in the absence of b5 or in the presence of b5 under optimal conditions. The results indicate that when b5 is reconstituted with the cytochrome P-450 isozyme 2 enzyme system under optimal conditions, substrate monooxygenation is enhanced, NADPH oxidation is unaffected, and hydrogen peroxide formation is decreased.

Aminopyrine↗
PubMed1986

N-alkylaminobenzotriazoles as isozyme-selective suicide inhibitors of rabbit pulmonary microsomal cytochrome P-450.

To produce potent, isozyme-selective suicide inhibitors of cytochrome P-450 (P-450), a series of N-alkylated 1-aminobenzotriazole (ABT) derivatives was synthesized; these included the N-methyl, N-butyl (BuBT), N-benzyl (BBT), and N-alpha-methylbenzyl (alpha MB) analogues of ABT. The suicide inhibitors showing the greatest potency and isozyme selectivity were BBT and alpha MB, compounds which included molecular features for P-450 inactivation (the ABT moiety) and similarity to benzphetamine. ABT and its N-alkylated derivatives were tested as suicide inhibitors in rabbit lung microsomes, whose P-450 monooxygenase system has been well characterized in both untreated and beta-naphthoflavone- or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated animals. ABT (10 mM) destroyed up to 99% of the total P-450 content of lung microsomes of untreated rabbits. At equimolar concentrations (10 microM), ABT was less effective than the N-alkylated compounds for the inhibition of P-450 isozyme 2-catalyzed benzphetamine N-demethylation (BND); in fact, BuBT, BBT, and alpha MB completely inhibited BND activity at this concentration and destroyed less than 40% of total pulmonary P-450. However, these compounds also inactivated 69-85% of isozyme 6-catalyzed 7-ethoxyresorufin O-deethylation. The most potent and isozyme-selective suicide inhibitor prepared was alpha MB: at 1 microM this compound inhibited approximately 80% of isozyme 2-catalyzed and 20% of isozyme 6-catalyzed monooxygenase activity but spared P-450 isozyme 5; at 2.5 microM it caused a near-complete loss (96 +/- 2%) of BND activity. The partition ratio of alpha MB, i.e., the molar ratio of inhibitor present to that of the P-450 destroyed, was 11 +/- 2, further demonstrating the potency of this compound. Experiments with BBT- and sodium phenobarbital-treated rats showed that the mechanism for suicidal inactivation of P-450 by this N-alkylated compound was by benzyne release, the same mechanism demonstrated earlier for the parent compound ABT.

Alkylation↗
PubMed1985

On the mechanism of the inactivation of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 by chloramphenicol.

The mechanism of the inactivation of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 (P-450 PB-B2) by chloramphenicol has been investigated. Preparations of the enzyme from animals treated in vivo with chloramphenicol (CAP PB-B2) have been isolated, and their catalytic, spectral, and physical properties have been compared with those of the native PB-B2. The CAP PB-B2 exhibited: 1) a 60-70% loss in the rate of NADPH-supported monooxygenase activity with the substrates benzphetamine, 7-ethoxycoumarin, and p-nitroanisole; 2) a 60% decrease in the extent of enzymatic P-450 reduction catalyzed by NADPH-cytochrome P-450 reductase under both aerobic and anaerobic conditions; 3) a 60% decrease in the steady-state level of the ferrous dioxygen complex in the presence of substrates; 4) a 60% decrease in the magnitude of the type I spectral change induced by benzphetamine; and 5) a shift in the wavelength maximum for the chemically reduced ferrous-carbonyl complex from 450 to 451.5 nm. On the other hand, the ability of the CAP PB-B2 to catalyze the iodosobenzene-supported metabolism of 7-ethoxycoumarin and p-nitroanisole was unaltered. The results are consistent with a scheme whereby the binding of metabolites of chloramphenicol to amino acid residues in the PB-B2 close to the heme moiety blocks electron transport from NADPH-cytochrome P-450 reductase, thereby leading to a loss of monooxygenase activity.

7-Alkoxycoumarin O-Dealkylase↗
PubMedDate not supplied

[Activity of the liver monooxygenase system after induction with phenobarbital in the early neonatal period].

Activity of enzymes participating iH metabolism of xenobiotics was studied after their induction by means of phenobarbital in rats of Wistar strain within first weeks of life. After administration of phenobarbital into the neonatal rats content of cytochrome P-450 and activity of NADPH-cytochrome c reductase were increased in liver microsomes. The rates of N-demethylation of benzphetamine and benz(alpha)pyrene hydroxylation were also increased after administration of the inducing agent. Immunochemical studies with antibodies towards some forms of cytochrome P-450 showed that enzymatic forms of cytochrome P-450 PB were induced; this enzymatic form is predominant in adult animals treated with phenobarbital. When the metabolism of benzphetamine and benz(alpha)pyrene was inhibited by antibodies, several forms of cytochrome P-450, involved in these metabolic reactions were found in neonatal rats, whereas in the adult animals only one form of the enzyme exhibited activity towards these substrates.

Animals↗
PubMed1985

[Stoichiometry of microsomal oxidation reactions. Distribution of redox-equivalents in monooxygenase and oxidase reactions catalyzed by cytochrome P-450].

The stoichiometry of NADPH oxidation in rabbit liver microsomes was studied. It was shown that in uncoupled reactions cytochrome P-450, besides O2- generation catalyzes direct two- and four-electron reduction of O2 to produce H2O2 and water, respectively. With an increase in pH and ionic strength, the amount of O2 reduced via an one-electron route increases at the expense of the two-electron reaction. In parallel, with a rise in pH the steady-state concentration of the oxy-complex of cytochrome P-450 increases, while the synergism of NADPH and NADH action in the H2O2 formation reaction is replaced by competition. The four-electron reduction is markedly accelerated and becomes the main pathway of O2 reduction in the presence of a pseudo-substrate--perfluorohexane. Treatment of rabbit with phenobarbital, which induces the cytochrome P-450 isozyme specific to benzphetamine results in a 2-fold increase in the degree of coupling of NADPH and benzphetamine oxidation. The experimental results suggest that the ratio of reactions of one- and two-electron reduction of O2 is controlled by the ratio of rates of one- and two-electron reduction of cytochrome P-450. In the presence of pseudo-substrates cytochrome P-450 acts predominantly as a four-electron oxidase; one of possible reasons for the uncoupling of microsomal monooxygenase reactions is the multiplicity of cytochrome P-450 isozymes.

Aniline Hydroxylase↗
PubMed1979

Kinetics of reduction of purified liver microsomal cytochrome P-450 in the reconstituted enzyme system studied by stopped flow spectrophotometry.

Stopped flow studies were undertaken to examine the kinetics of reduction of 5,6-benzoflavone-inducible P-450 LM4 by NADPH in the presence of NADPH-cytochrome P-450 reductase and phospholipid under anaerobic CO at 25 degrees C. The reaction exhibited biphasic kinetics irrespective of NADPH concentration or of the molar ratio of reductase to P-450 LM4. The apparent first order rate constants for the fast and slow phases were determined to be 0.9 to 1.0 and 0.25 s-1, respectively. With the reductase and P-450 LM4 present in equimolar amounts, the total amount of P-450 LM4 reduced increased linearly with NADPH concentration; the titration gave a stoichiometry of 2 mol of NADPH per mol of reductase-cytochrome complex. The NADPH concentration had no appreciable effect on the magnitude of the first order rate constants for the fast and slow phases. The kinetics obtained in the presence of benzphetamine were essentially indistinguishable from those seen in the absence of this substrate, while the amount of P-450 LM4 reduced in the fast phase, but not the rate constant for this phase, decreased when phospholipid was omitted from the reaction mixture. Nearly maximal rates of NADPH oxidation by P-450 LM2 OR LM4 were obtained with a molar ratio of reductase to P-450 LM of 1.0. Benzphetamine enhanced the oxidation of NADPH by P-450 LM2 but had no effect on the activity of P-450 LM4. Rates of NADPH oxidation in the presence of P-450 LM2 and LM4 decreased by 80 and 40%, respectively, when phospholipid was omitted from the reconstituted enzyme system. These studies provide evidence for the formation of a catalytically functional 1:1 complex between the reductase and P-450 LM4, and indicate that P-450 LM2 and LM4 differ in their dependence on phospholipid.

Animals↗
PubMed1980

The pulmonary clara cell as a target for toxic chemicals requiring metabolic activation; studies with carbon tetrachloride.

Oral administration of carbon tetrachloride to rats or mice caused striking decreases in rat lung microsomal cytochrome P-450 and benzphetamine demethylase activity and in the enzyme-mediated covalent binding of 4-ipomeanol in preparations of rat and mouse lung microsomes, mouse lung slices and isolated whole-mouse lungs. Although it is not yet known whether cytochrome P-450 and benzphetamine demethylase activities are present in substantial amounts in more than one lung cell type in mice or rats, previous studies have indicated that cytochrome P-450 enzymes located in pulmonary bronchiolar Clara cells of these mediate the covalent binding of 4-ipomeanol to lung macromolecules. Histologic examinations of lungs of animals given doses of CCl4, orally or by inhalation, revealed striking morphologic changes in Clara cells, including severe dilation of endoplasmic reticulum and occasional cellular necrosis. Because of cytochrome P-450 enzymes are capable of mediating the formation of highly reactive and potentially toxic-free radicals from CCl4, the present results support the view that pulmonary Clara cells are susceptible to CCl4-induced injury due to their capacity to metabolically activate the chemical.

Animals↗
PubMed1981

Neonatal imprinting and hepatic cytochrome P-450. II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450.

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16 alpha-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7 alpha- and 6 beta-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16 alpha- and 7 alpha-hydroxylase activities in a concentration-dependent manner, without impairing 6 beta-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16 alpha-) and nonimprintable (7 alpha-) testosterone hydroxylase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Expression of cytochrome P450 3A5 in Escherichia coli: effects of 5' modification, purification, spectral characterization, reconstitution conditions, and catalytic activities.

Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5'-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601) to the 3A5 cDNA. Two constructs were made, differing by their identity with the modified 3A4 N-terminal sequence (E. M. J. Gillam, T. Baba, B-R. Kim, S. Ohmori, and F. P. Guengerich, (1993) Arch. Biochem. Biophys. 305, 123-131). The first modified sequence (3A5#1) was identical to recombinant P450 3A4 up to codon 15, the 3A5 sequence being introduced thereafter. In the other (3A5#2), the successful 3A4 N-terminal nucleotide sequence was attached to codon 30. The yield was greater than fourfold higher in the first construct [up to 260 nmol (liter culture)-1]. The recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed previously for P450 3A4, with an overall yield of approximately 40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6 beta-hydroxylation, aflatoxin 3 alpha-hydroxylation and 8,9-epoxidation, ethylmorphine N-demethylation, erythromycin N-demethylation, and d-benzphetamine N-demethylation. The reconstitution of nifedipine oxidation, testosterone 6 beta-hydroxylation, and the aflatoxin oxidation activities showed dependence upon the presence of cytochrome b5, divalent cations, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-demethylations of ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly dependent upon modifications of reconstitution systems [corrected].

Amino Acid Sequence↗
PubMed1996

Substrate influence on interaction between cytochrome P450 and cytochrome b5 in microsomes.

We have been able to demonstrate that cytochrome b4 interacts closely with cytochrome P450 in the microsomal membrane, and that substrates can serve to order the interaction: Using the water-soluble carbodiimide EDC we could crosslink cytochrome b5 and CYP2B4 in microsomes from phenobarbital-treated rabbits and cytochrome b5 and CYP1A2 in microsomes from beta-naphthoflavone-treated animals. The substrate benzphetamine increased the specific interaction between cytochrome b5 and CYP2B4, decreasing the formation of higher molecular weight oligomeric complexes with cytochrome b5. In contrast, no ordering of the interactions were obtained on addition of 7-ethoxycoumarin, a substrate of CYP1A2, or of benzphetamine to microsomes of beta-naphthoflavone-treated animals in the presence of EDC. Of interest, although evidence could be shown for complementary charge-pairing between cytochrome b5 and a number of other microsomal proteins in the membranes, and while the extent of CYP1A2 and CYP2B4 interaction with cytochrome b5 each exceeded 30% in the presence of substrate, no significant complexation of the P450s was obtained with any other microsomal proteins.

Animals↗
PubMed2000

Mechanistic studies of cytochrome P450 2B1 inactivation by xanthates.

Xanthates have previously been shown to inactivate the phenobarbital-inducible rat cytochrome P450 2B1 as well as its human homologue P450 2B6. The inactivation was mechanism-based and the loss in enzymatic activity was due to covalent binding of a reactive xanthate intermediate to the P450 2B1 apoprotein. In this report, we investigated various mechanistic events to elucidate the individual step(s) in the P450 catalytic cycle that are compromised due to the inactivation by xanthates. Different xanthates displayed typical type I binding spectra and the spectral binding constants were in the low-millimolar range. A dramatic loss in 7-ethoxy-4-(trifluoromethyl)coumarin activity was observed when P450 2B1 was incubated with five different xanthates in the presence of NADPH. With the exception of the C14 xanthate, virtually no loss of absorbance at 418 or 450 nm in the reduced-CO complex was observed. Long-chain xanthates were able to affect the rate of the first electron transfer in the P450 catalytic cycle by stabilizing the heme in its low-spin state. n-Octyl xanthate (C8) metabolism led to very little observable oxy-ferro intermediate complex formation. The alternate oxidant tert-butyl hydroperoxide was able to support the inactivation reaction of C8 in the absence of reductase or NADPH. The rates of reduction of native, C8-exposed, and C8-inactivated P450 2B1 were measured. The C8-inactivated P450 had a 62% lower rate of reduction in the absence or presence of benzphetamine compared to the native enzyme. Product formation of the three enzyme preparations was quantified with benzphetamine as the substrate. The C8-inactivated P450 2B1 exhibited a much lower rate of NADPH consumption and formation of formaldehyde. However, the ratio of H2O2 to formaldehyde production increased from 1:1 for the native enzyme to 2.8:1 for the inactivated P450. Together these observations indicate that the covalent modification of P450 2B1 by a reactive intermediate of xanthates reduces the rate of the first electron transfer by the reductase and also leads to uncoupling of electron transfer from product formation by diverting a greater proportion of the electrons to H2O2 formation.

Animals↗
PubMed1987

Interactions of the histamine H2-receptor antagonist etintidine with rat liver cytochrome P-450: a comparison with cimetidine.

The two imidazole histamine H2-receptor antagonists etintidine and cimetidine interact with the rat liver microsomal cytochrome P-450. From type II spectral changes follows that the affinity of rat liver microsomal preparations for etintidine is about 5 times as high as for cimetidine when comparing both high and low affinity binding sites. After pretreatment with phenobarbital etintidine inhibited benzphetamine N-demethylation competitively (app. Ki: 4.0 mmol/l). Cimetidine inhibited benzphetamine N-demethylation in the same range. After pretreatment with phenobarbital both drugs inhibited the oxidation of benzo(a)pyrene for which etintidine showed a higher inhibitory potency than cimetidine. However, this oxidation could not be inhibited when microsomes of 5,6-benzoflavone pretreated rats were used. After pretreatment with 5,6-benzoflavone only etintidine but not cimetidine inhibited the O-deethylation of ethoxyresorufin competitively (app. Ki: 0.2 mmol/l). Etintidine and cimetidine were metabolized by rat liver microsomes to their corresponding sulphoxides. In conclusion, etintidine may cause mainly the same drug interactions as cimetidine but seems to be a more potent inhibitor.

Animals↗
PubMed1996

Interactions of 8-anilino-1-naphthalenesulfonic acid (ANS) and cytochrome P450 2B1: role of ANS as an effector as well as a reporter group.

Interactions of cytochrome P450 2B1 were probed using 8-anilino-1-naphthalenesulfonic acid (ANS), a well known and frequently used reporter group for hydrophobic interactions. Titration of cytochrome P450 2B1 with ANS revealed 6.6 +/- 0.2 ANS binding sites per molecule of P450 2B1 with a Kd value of 42 +/- 2 microM. In our evaluation of the consequences of the binding of ANS to cytochrome P450 2B1, we found that the binding of ANS to P450 2B1 increased benzphetamine demethylation activity by 1.5-fold, indicating a role for ANS as an effector in addition to its role as a reporter group. Kinetic analysis of the effects of ANS on P450 2B1-dependent demethylation activity revealed that ANS increased both the Vmax and K(m) of the benzphetamine demethylation reaction. ANS stimulation of activity appears not to be due to the replacement or augmentation of the role of lipid since studies of binding and catalytic activities in the presence and absence of added lipid gave the same array of effects. These results demonstrate that ANS can bind to cytochrome P450 2B1 as would be expected of a reporter group probe but show in addition that this probe also acts as an effector molecule stimulating catalytic activity. Thus, results of ANS studies should be viewed with caution since the molecule may play more than one role in its reaction with a protein.

Anilino Naphthalenesulfonates↗
PubMed1989

Distribution and induction of cytochrome P-450 and two cytochrome P-450-dependent monooxygenase activities in rat liver parenchymal cell subpopulations separated by centrifugal elutriation.

Liver parenchymal cells from the periportal and centrilobular zones differ in their morphological, biochemical and functional characteristics. In an effort to obtain fractions enriched in either periportal or centrilobular cells, isolated rat liver parenchymal cells were separated into five subpopulations by centrifugal elutriation. The mean diameters of the cells present in fractions I-V were 19.6, 21.1, 21.8, 22.7 and 23.5 micron, respectively. The content of cytochrome P-450 as well as benzphetamine N-demethylase and 7-ethoxyresorufin O-deethylase activities were higher in the larger parenchymal cells than in the smaller ones. After administration of phenobarbital the content of cytochrome P-450 was approximately two-fold greater in the cells present in fractions 3-5, when compared to the same subpopulations isolated from untreated rats; the activity of benzphetamine N-demethylase was enhanced to a similar extent in all five fractions. 3-Methylcholanthrene treatment resulted in a significant increase of cytochrome P-450 content and 7-ethoxyresorufin O-deethylase activity in all five fractions: both parameters were slightly higher in fractions 4 and 5 than in fractions 1 and 2. In conclusion, the elutriated liver parenchymal cells seem to preserve the biochemical heterogeneity observed in the intact liver; the potential enrichment of periportal and centrilobular cells in the different fractions by centrifugal elutriation is discussed.

Alanine Transaminase↗
PubMed1991

Effects of acetone administration on cytochrome P-450-dependent monooxygenases in hamster liver, kidney, and lung.

The effects of acetone on liver, kidney, and lung monooxygenases were studied using hamsters administered 8% acetone in drinking water. Binding of aniline to liver microsomes induced a type II difference spectrum, and the spectral binding was enhanced in hamsters pretreated with acetone. Administration of acetone caused significant increases of cytochrome P-450 and cytochrome b5 contents in liver microsomes. The increases of the hemeproteins were associated with induction of monooxygenase activities toward test substrates, aniline, N-nitrosodimethylamine, benzphetamine, benzo(a)pyrene, and 7-ethoxycoumarin. In the kidneys, acetone administration increased microsomal contents of the hemeprotein and monooxygenase activities toward aniline. N-nitrosodimethylamine, and 7-ethoxycoumarin, but not benzphetamine or benzo(a)pyrene. In the lungs, acetone pretreatment increased aniline hydroxylase activity without affecting the levels of N-nitrosodimethylamine demethylase, cytochromes P-450 and b5. In marked contrast to the inductive effects in the liver, acetone administration markedly decreased lung microsomal benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities. Gel electrophoresis of liver and kidney microsomes from control and acetone-treated hamsters revealed that acetone treatment enhanced the intensity of a protein band(s) in the cytochrome P-450 molecular weight region. Immunoblotting of the microsomal proteins showed that the protein band induced by acetone in hamster liver, kidney and lung was cross-reactive with antibody raised against ethanol-inducible human liver cytochrome P-450. These results demonstrate that acetone has the ability to uniformly induce a specific form of cytochrome P-450, designated as IIE1, and to cause differential changes of monooxygenase activities in the hamster tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetone↗
PubMed1983

Comparative kinetic studies of the dealkylation reaction and steroid hydroxylations in various oxygen and carbon monoxide:oxygen atmospheres.

The time-course kinetics of the cytochrome P-450-catalyzed dealkylations of the exogenous compounds benzphetamine, ethylmorphine, codeine, and 7-ethoxycoumarin were compared to the hydroxylation of the endogenous compound testosterone. Using liver microsomes from phenobarbital-induced rats, the time course of the demethylations of ethylmorphine, codeine, and especially benzphetamine was characterized by a fast initial phase of enzymatic activity and then a steady decline in the rate throughout the remainder of the reaction. In contrast, under the same experimental conditions, both the dealkylation of 7-ethoxycoumarin and the hydroxylation of testosterone showed no initial fast phase of activity and a constant rate of product formation for most of the remainder of the time course. The difference also held for the carbon monoxide inhibition studies in which the degree inhibition of the demethylation reactions by a variety of CO:O2 mixtures was time dependent, in contrast to the constant, time-independent degree of CO inhibition of the other two reactions. The kinetics of the demethylation reactions could not be explained by enzyme destruction, back reaction, or product adduct formation and were further confirmed by measurements of the rate of O2 utilization and NADPH oxidation. The complexity of the demethylation reaction should be taken into consideration in any detailed studies of the monooxygenation reaction system.

Animals↗