Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.     

Covalent binding of polychlorinated biphenyls to proteins by reconstituted monooxygenase system containing cytochrome P-450

Incubation in the presence of NADPH and molecular oxygen of ¹⁴C-labeled polychlorinated biphenyls (PCBs) and two tetrachlorobiphenyl (TCB) isomers with a reconstituted system containing NADPH-cytochrome P-450 reductase and cytochrome P-450, both purified from liver microsomes of phenobarbital(PB)-pretreated rabbits, led to covalent binding of radioactive metabolites of PCBs and TCBs to the protein components of the system. A rabbit liver cytosol fraction added to the system provided more binding sites for the activated metabolites and thus increased the extent of binding markedly. The binding reaction depended absolutely on the reductase, cytochrome P-450 and NADPH, and required dilauroyl phosphatidylcholine and sodium cholate for maximal activity. A further stimulation of the binding was attained by including cytochrome b₅ in the reconstituted system. Four forms of cytochrome P-450, purified from liver microsomes of PB- and 3-methylcholanthrene(MC)-treated rabbits and rats, were used to reconstitute the PCB- and TCB-metabolizing systems, and it was found that PB-inducible forms of the cytochrome from both animals were more active than those inducible by MC in catalyzing the PCB- and TCB-binding reaction. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis indicated that, in the system containing the reductase, cytochrome P-450 and cytochrome b₅, PCB metabolites bound to the reductase and cytochrome P-450, but not to cytochrome b₅. In the presence of the liver cytosol fraction, the binding took place to many cytosolic proteins in addition to the reductase and cytochrome P-450.     

Substrate-binding region of cytochrome P-450scc (P-450 XIA1). Identification and primary structure of the cholesterol binding region in cytochrome P-450scc

Cytochrome P-450_scc (P-450 XIA1) from bovine adrenocortical mitochondria was investigated using a suicide substrate: [¹⁴C]methoxychlor. [¹⁴C]Methoxychlor irreversibly abolished the activity of the side-chain cleavage enzyme for cholesterol (P-450_scc) and the inactivation was prevented in the presence of cholesterol. The binding of [¹⁴C]methoxychlor and cytochrome P-450_scc occurred in a molar ratio of 1:1 and the cholesterol-induced difference spectrum of cytochrome P-450_scc was similar with the methoxychlor-induced difference spectrum. [¹⁴C]Methoxychlor-binding peptides were purified from tryptic-digested cytochrome P-450_scc modified with [¹⁴C]methoxychlor. Determination of the sequence of the amino-acid residues of a [¹⁴C]methoxychlor-binding peptide allowed identification of the peptide comprising the amino-terminal amino-acid residues 8 to 28.     

Microsomal activation of thioacetamide-S-oxide to a metabolite(s) that covalently binds to calf thymus DNA and other polynucleotides

In the presence of NADPH liver microsomes isolated from phenobarbital-pretreated rats catalyze the conversion of [³H]thioacetamide-S-oxide to a reactive intermediate(s) which covalently binds to calf thymus DNA, calf liver RNA, polyguanylic acid (poly(G)) and polyadenylic acid (poly(A)). The highest level of binding of radioactivity was obtained with poly(G), followed by poly(A), RNA and DNA. The incorporation of radioactivity into DNA was linear for 30 min and there was a requirement for NADPH for time-dependent covalent binding to occur. Performing the microsomal incubations in an atmosphere of 80% CO/20% O₂ or adding partially purified anti cytochrome P-450 immune serum to the microsomal incubations inhibited the total metabolism of thioacetamide-S-oxide and had a small, but insignificant, inhibitory effect on binding of radioactivity to calf thymus DNA. Using a reconstituted monooxygenase system containing cytochrome P-450 purified from phenobarbital-treated rats we were unable to detect any metabolism of thioacetamide-S-oxide. Only background levels of radioactivity were incorporated into calf thymus DNA when microsomes isolated from phenobarbital-treated rats were incubated with [³H]thioacetamide in the presence of NADPH. These results suggest that thioacetamide-S-oxide is an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s) which binds to calf thumus DNA.     

N-Hydroxylation of arylamides by the rat and guinea pig: Evidence for substrate specificity and participation of cytochrome P1-450

The hypothesis that N-hydroxylation of arylamides is essential for carcinogenicity was examined in vivo and in vitro with N-2-fluorenylacetamide, a potent carcinogen, and with N-3-fluorenylacetamide, an isomer with marginal carcinogenicity. About 10–20% of 2-[9-¹⁴C]fluorenylacetamide administered intraperitoneally to the rat was excreted in the bile as the N-hydroxy-2-[9-¹⁴C]-derivative, whereas <0.1% of 3-[G-³H]fluorenylacetamide was found as the N-hydroxy metabolite in bile and urine. N-Hydroxylation of the 2- isomer by hepatic microsomes of untreated or 3-methylcholanthrene-treated rats was 40 to 50-fold greater than that of the 3- isomer. The role of cytochromes P-450 and P₁-450 in N-hydroxylation of arylamides by rat liver microsomes was shown by inhibition of the reaction with carbon monoxide and cobaltous chloride. Interaction of the arylamides with cytochrome P₁-450 was also demonstrated by binding spectra obtained on addition on 2- and 3-fluorenylacetamide to hepatic chromosomes of methylcholanthrene-treated rats. There appeared to be no correlation between the magnitude of the spectra and the extent of N-hydroxylation. N-Hydroxylation of the 2- isomer by hepatic microsomes of the guinea pig, a species resistant to the carcinogenecity of this compound, was markedly less than N-hydroxylation by rat liver microsomes, even though, as judged by the appearance of the binding spectra, both 2- and 3- isomers were bound by cytochrome P₁-450 of guinea pig-liver microsomes. The results are in agreement with the view that the microsomal N-hydroxylation of arylamides parallels their carcinogenicity.     

Increased metabolism of retinoic acid after chronic ethanol consumption in rat liver microsomes

In rat liver microsomes, all-trans-[11,12-³H]retinoic acid was found to be metabolized to polar products in the presence of NADPH. One of the metabolites was coeluted with 4-hydroxyretinoic acid on reverse-phase high-pressure liquid chromatography (HPLC). This reaction required oxygen and was inhibited by carbon monoxide as well as aminopyrine, aniline, and ethanol, suggesting the involvement of cytochrome P-450. Isolated rat hepatocytes also metabolized all-trans[³H]retinoic acid to polar compounds, with an elution pattern on HPLC similar to that in microsomal preparations. Microsomal activity was compared in rats pair-fed with diets containing either ethanol or isocaloric carbohydrate for 4–6 weeks. Ethanol-fed rats showed enhanced microsomal retinoic acid metabolism (50%, P < 0.01) accompanied by increased microsomal cytochrome P-450 content (34%, P < 0.005). On the other hand, microsomal β-glucuronidation of retinoic acid in the presence of uridine diphosphoglucuronic acid (UDPGA) was not affected by chronic ethanol feeding. The increased hepatic microsomal cytochrome P-450-dependent metabolism of retinoic acid after chronic ethanol consumption may contribute to the accelerated catabolism of retinoic acid in vivo.     

Partial inhibition of hepatic microsomal aminopyrine N-demethylase by caffeine in partially purified cytochrome P450

Cytochrome P-450 substrate interactions were studied with cytochrome P-450 partially purified from livers of untreated, phenobarbital-treated, benzo[a]pyrene-treated and caffeine-treated rats. Partial inhibition of aminopyrine N-demethylase in presence of in vitro caffeine observed with intact microsomes was further investigated in a reconstituted system composed of partially purified cytochrome c reductase. Caffeine addition (in vitro) to partially purified cytochrome P-450 altered the hexobarbital, aniline and ethylisocyanide induced spectral change, and decreased NADPH oxidation in presence of substrates aminopyrine and acetanilide. NADPH oxidation was found to be increased in presence of aminopyrine and unaltered in presence of acetanilide in reconstituted system having partially purified cytochrome P-450 from caffeine-treated rats. Our studies suggest that caffeine acts as a true modifier of cytochrome P-450 and is possibly responsible for the formation of abortive complexes with aminopyrine.     

Covalent binding of benzo[a]pyrene to cytochrome P-450βNF-B2 and other proteins in reconstituted mixed-function oxidase systems

A reconstituted mixed-function oxidase system, containing the major β-naphthoflavone-induced isozyme of rat liver cytochrome P-450 bound benzo[a]pyrene covalently in the presence of NADPH. NADPH-cytochrome P-450 reductase was required for binding and a maximum rate of adduct formation was obtained at 8 units of reductase per nmol cytochrome P-450. Phosphatidylcholine inhibited this reaction. Benzo[a]pyrene was bound to the cytochrome, but not to the reductase, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Approximately 6 molecules of benzo[a]pyrene bound to each molecule cytochrome P-450 during prolonged incubations. No binding occurred when the β-naphthoflavone-induced isozyme of cytochrome P-450 was replaced by the major isozyme induced by phenobarbital, but both cytochromes incorporated benzo[a]pyrene to approximately the same extent when they were incubated together in the presence of the reductase and NADPH. Metabolically activated benzo[a]pyrene also bound covalently to purified epoxide hydrodrolase, when this enzyme was added to the reconstituted mixed-function oxidase system.     

Reconstitution of the reduced nicotinamide adenine dinucleotide phosphate- and reduced nicotinamide adenine dinucleotide-peroxidase activities from solubilized components of rat liver microsomes.

The liver microsomal enzyme system that catalyzes the oxidation of NADPH by organic hydroperoxides has been solubilized and resolved by the use of detergents into fractions containing NADPH-cytochrome c reductase, cytochrome P-450 (or P-448), and microsomal lipid. Partially purified cytochromes P-450 and P-448, free of the reductase and of cytochrome b₅, were prepared from liver microsomes of rats pretreated with phenobarbital (PB) and 3-methylcholanthrene (3-MC), respectively, and reconstituted separately with the reductase and lipid fractions prepared from PB-treated animals to yield enzymically active preparations functional in cumene hydroperoxide-dependent NADPH oxidation. The reductase, cytochrome P-450 (or P-448), and lipid fractions were all required for maximal catalytic activity. Detergent-purified cytochrome b₅ when added to the complete system did not enhance the reaction rate. However, the partially purified cytochrome P-450 (or P-448) preparation was by itself capable of supporting the NADPH-peroxidase reaction but at a lower rate (25% of the maximal velocity) than the complete system. Other heme compounds such as hematin, methemoglobin, metmyoglobin, and ferricytochrome c could also act as comparable catalysts for the peroxidation of NADPH by cumene hydroperoxide and in these reactions, NADH was able to substitute for NADPH. The microsomal NADH-dependent peroxidase activity was also reconstituted from solubilized components of liver microsomes and was found to require NADH-cytochrome b₅ reductase, cytochrome P-450 (or P-448), lipid, and cytochrome b₅ for maximal catalytic activity. These results lend support to our earlier hypothesis that two distinct electron transport pathways operate in NADPH- and NADH-dependent hydroperoxide decomposition in liver microsomes.     

Microsomal redox systems in brown adipose tissue: high lipid peroxidation,low cholesterol biosynthesis and no detectable cytochrome P-450

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-¹⁴C]acetate and [2-¹⁴C]-mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b ₅ in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.Abbreviations BAT Brown Adipose Tissue - MDA malondialdehyde     

The drug and carcinogen metabolism system of rat colon microsomes

The hydroxylation of N- and O-methyl drugs and polycyclic hydrocarbons has been demonstrated in microsomes prepared from colon mucosal cells. The hydroxylation of the drugs benzphetamine, ethylmorphine, p-nitroanisole, and p-nitrophenetole by colon microsomes is inducible two- to fourfold by pretreatment with phenobarbital/hydrocortisone. Colon microsomal benzo[α]pyrene hydroxylation is inducible 35-fold by pretreatment with β-naphthoflavone. Phenobarbital/hydrocortisone pretreatment also induces a fourfold increase in the specific content of colon microsomal cytochrome P-450, while β-naphthoflavone pretreatment causes a shift in the reduced CO difference spectrum peak to 448 nm and an eightfold increase in the specific content of this cytochrome. SKF 525-A inhibits the hydroxylation of the drug benzphetamine by colon microsomes or liver microsomes by 77% at a concentration of 2.0 mm. 7,8-Benzoflavone, on the other hand, inhibits the hydroxylation of the polycyclic hydrocarbon benzo[α]pyrene by colon microsomes by 76% and by liver microsomes by 44% at a concentration of 10 μm. Carbon monoxide, an inhibitor of oxygen interaction with cytochromes P-450 and P-448, inhibits benzphetamine hydroxylation and benzpyrene hydroxylation by colon microsomes 30 and 51%, respectively, at an oxygen to carbon monoxide ratio of 1:10. The K_m values of colon microsomal cytochrome P-450 reductase for the artificial electron acceptors cytochrome c, dichloroindophenol, and ferricyanide (10–77 μm) are in agreement with those for purified rat liver cytochrome P-450 reductase. These data support the conclusions that hydroxylation of drugs and polycyclic hydrocarbons is catalyzed by colon mucosal microsomes and that the hydroxylation activity is attributable to a cytochrome P-450-dependent drug metabolism system similar to that found in liver microsomes.     

Inactivation of glutathione reductase by 2-chloroethyl nitrosourea-derived isocyanates.

The specific inactivation of yeast glutathione reductase (GSSG-reductase) by 2-chloroethyl isocyanate and cyclohexyl isocyanate derived from their respective 2-chloroethyl nitrosoureas has been demonstrated. Titration of the enzyme with 2-chloroethyl isocyanate or [¹⁴C] labeling with 1-(2-chloroethyl)-3-(1-¹⁴C-cyclohexyl)-1-nitrosourea or 1,3-bis (2-¹⁴C-chloroethyl)-1-nitrosourea resulted in near stoichiometric inactivation and/or covalent labeling of the enzyme. In addition to 1,3-bis (2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea, several other 2-chloroethyl nitrosoureas were capable of inactivation of not only purified GSSG-reductase, but also the activity of this enzyme in cell-free extracts of murine lymphoma L5178Y ascites tumor cells and murine bone marrow.     

Assay and partial purification of epoxide hydrase from rat liver microsomes

Solubilized cytochrome P-450 monooxygenase and epoxide hydrase activities from rat liver microsomes have been separated by column chromatography. The highly active epoxide hydrase fraction is still contaminated with cytochrome P-450, which has very low monooxygenase activity. The highly purified cytochrome P-450 fraction possesses high monooxygenase activity and is essentially devoid of epoxide hydrase activity. Purification factors for the epoxide hydrase through four purification steps are similar with [³H]styrene oxide, [³H]naphthalene oxide, [³H]cyclohexene oxide, and benzene oxide as substrates. Failure of benzene oxide to inhibit hydration of styrene or naphthalene oxide in the most purified preparations in indicative of the presence of at least two hydrases. These purified cytochrome monooxygenase and hydrase preparations represent valuable tools for the study of the intermediacy of arene oxides in drug metabolism. Thus, with naphthalene, only naphthol is formed with the monooxygenase, while both naphthol and the dihydrodiol are formed in the presence of monooxygenase and hydrase. A convenient radiochemical synthesis of [³H]naphthalene 1,2-oxide and assays for the measurement of the hydration of [³H]naphthalene oxide and benzene oxide, based on differential extractions and high-pressure liquid chromatography, respectively, are described.     

1-chloro-2,2,2-trifluoroethyl radical: Formation from halothane by human cytochrome P-450 in reconstituted vesicles and binding to phospholipids

We report the first complete structural characterization of a metabolically-produced halocarbon radical bound to a phospholipid. Human cytochrome P-450 and NADPH cytochrome P-450 reductase were reconstituted into vesicles composed of dioleoylphosphatidylcholine and egg phosphatidylethanolamine. The vesicles were incubated under argon with NADPH and [¹⁴C]-halothane (1-[¹⁴C]-2-bromo-2-chloro-1,1,1-trifluoroethane), the dioleoylphosphatidylcholine fraction was isolated and subjected to transesterification. Separation of the resulting fatty acid methyl esters resulted in one radioactive fraction which gas chromatography-mass spectrometry revealed to be a mixture of 9- and 10-(1-chloro-2,2,2-trifluoroethyl)-stearate methyl ester.     

Spin-trapping studies of hydroxyl radical production involved in lipid peroxidation.

Evidence presented in this report suggests that the hydroxyl radical (OH.), which is generated from liver microsomes is an initiator of NADPH-dependent lipid peroxidation. The conclusions are based on the following observations: 1) hydroxyl radical production in liver microsomes as measured by esr spin-trapping correlates with the extent of NADPH induced microsomal lipid peroxidation as measured by malondialdehyde formation; 2) peroxidative degradation of arachidonic acid in a model OH · generating system, namely, the Fenton reaction takes place readily and is inhibited by thiourea, a potent OH · scavenger, indicating that the hydroxyl radical is capable of initiating lipid peroxidation; 3) trapping of the hydroxyl radical by the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide prevents lipid peroxidation in liver microsomes during NADPH oxidation, and in the model system in the presence of linolenic acid. The possibility that cytochrome P-450 reductase is involved in NADPH-dependent lipid peroxidation is discussed. The optimal pH for the production of the hydroxyl radical in liver microsomes is 7.2. The generation of the hydroxyl radical is correlated with the amount of microsomal protein, possibly NADPH cytochrome P-450 reductase. A critical concentration of EDTA (5 × 10^?5m) is required for maximal production of the hydroxyl radical in microsomal lipid peroxidation during NADPH oxidation. High concentrations of Fe²⁺-EDTA complex equimolar in iron and chelator do not inhibit the production of the hydroxyl radical. The production of the hydroxyl radical in liver microsomes is also promoted by high salt concentrations. Evidence is also presented that OH radical production in microsomes during induced lipid peroxidation occurs primarily via the classic Fenton reaction.     

Characteristcs of microsomal enzyme controls in primary cultures of rat hepatocytes.

Cytochrome P-450, NADPH-cytochrome c reductase, biphenyl hydroxylase, and epoxide hydratase have been compared in intact rat liver and in primary hepatocyte cultures. After 10 days in culture, microsomal NADPH-cytochrome c reductase and epoxide hydratase activities declined to a third of the liver value, while cytochrome P-450 decreased to less than a tenth. Differences in the products of benzo[a]pyrene metabolism and gel electrophoresis of the microsomes indicated a change in the dominant form(s) of cytochrome P-450 in the cultured hepatocytes. Exposure of the cultured cells to phenobarbital for 5 days resulted in a threefold induction in NADPH-cytochrome c reductase and epoxide hydratase activities which was typical of liver induction of these enzymes. Exposure of the cells to 3-methylcholanthrene did not affect these activities. Cytochrome P-450 was induced over two times by phenobarbital and three to four times by 3-methylcholanthrene. The λ_max of the reduced carbon monoxide complex (450.7 nm) and analysis of microsomes by gel electrophoresis showed that the phenobarbital-induced cytochrome P-450 was different from the species induced by 3-methylcholanthrene (reduced carbon monoxide λ_max = 447.9 nm). However, metabolism of benzo[a]pyrene (specific activity and product distribution) was similar in microsomes of control and phenobarbital- and 3-methylcholan-threne-induced hepatocytes and the specific activity per nmole of cytochrome P-450 was higher than in liver microsomes. The activities for 2- and 4-hydroxylation of biphenyl were undetectable in all hepatocyte microsomes even though both activities were induced by 3-methylcholanthrene in the liver. Substrate-induced difference spectra and gel electrophoresis indicated an absence in phenobarbital-induced hepatocytes of most forms of cytochrome P-450 which were present in phenobarbital-induced rat liver microsomes. It is concluded that the control of cytochrome P-450 synthesis in these hepatocytes is considerably different from that found in whole liver, while other microsomal enzymes may be near to normal. Hormonal deficiencies in the culture medium and differential hormonal control of the various microsomal enzymes provide a likely explanation of these effects.     

Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

Human liver cytochrome P-450 was isolated from autopsy samples using cholate extraction and chromatography on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose gels. Purified preparations contained as much as 14 nmol cytochrome P-450 mg^?1 protein, were free of other hemoproteins, and were active in the mixed-function oxidation of d-benzphetamine and 7-ethoxycoumarin when coupled with either rat or human liver NADPH-cytochrome P-450 reductase. Some of the preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; apparent subunit M_rs estimated for several preparations were 53,000 or 55,500. The amino acid composition of one preparation was determined and found to resemble those of rat liver cytochromes P-450, although some variations were noted. Rabbit antibodies raised to phenobarbital-treated rat liver cytochrome P-450 were more effective in inhibiting d-benzphetamine N-demethylase activity in human liver microsomes than were antibodies raised to 3-methylcholanthrene-treated rat liver cytochrome P-450. These antibodies also inhibited benzo(a)pyrene hydroxylation in human liver microsomes, although the inhibition patterns did not follow a general pattern as in the case of benzphetamine demethylase activity. Microsomes prepared from three different human liver samples were more effective in eliciting complement fixation with antibodies raised to phenobarbitalthan to 3-methylcholanthrene-treated rat liver cytochrome P-450. Complement fixation in such systems appears to result from similarity of certain rat and human liver cytochrome P-450 antigenic determinants, as fixation could be inhibited by removal of cytochrome P-450-directed antibodies from the total immunoglobulin population and purified human cytochrome P-450 was more effective (on a protein basis) than liver microsomes in producing fixation. Human liver microsomes prepared from five different individuals all produced ≥ 90% complement fixation, but variations were observed in the fixation curves plotted either versus microsomal protein or versus spectrally detectable microsomal cytochrome P-450.These results indicate that human liver microsomal cytochromes P-450 can be isolated using modifications of techniques developed for laboratory animals and that human and rat liver cytochromes P-450 share certain features of structural, functional, and immunological similarity. The available data suggest the existence of multiple forms of human liver microsomal cytochrome P-450, but possible artifacts associated with the use of autopsy samples suggest caution in advancing such a conclusion.     

Non-β-oxidizable ω-[18F]fluoro long chain fatty acid analogs show cytochrome P-450-mediated defluorination: Implications for the design of PET tracers of myocardial fatty acid utilization

The nature of the in vivo defluorination of non-β-oxidizable no-carrier-added ω-[¹⁸F]fluoro long chain fatty acid (LCFA) analogs was studied with the aim of developing PET tracers of LCFA utilization. Extensive defluorination of 15-[¹⁸F]fluoro-3-thia-pentadecanoic acid (FTPA) in mouse was evidenced by radioactivity uptake by bone. [¹⁸F]Fluoride in the blood was verified analytically. Incubations of FTPA in rat-liver homogenates and subcellular fractions thereof showed a strong defluorination process in microsomes which was O₂- and NADPH-dependent. In contrast, defluorination of FTPA was relatively slow in Langendorff perfused rat heart. High bone uptake in mouse was also observed with 14-[¹⁸F]fluoro-13, 13-dimethyl-3-thia-tetradecanoic acid, where gem-dimethyl substitution precludes direct elimination of H¹⁸F. These data indicate that the defluorination of non-β-oxidizable ω-[¹⁸F]fluoro LCFA analogs is primarily governed by cytochrome P-450-mediated ω-oxidation.Therefore, labeling at the (ω-3) carbon was proposed to provide a more stabile ¹⁸F-label. Defluorination of the (ω-3)-labeled 13 (R,S)-[¹⁸F]fluoro-3-thia-hexadecanoic acid was lower than that of FTPA in mouse and was independent of O₂ and NADPH in vitro. Thus, (ω-3) labeling with ¹⁸F is preferable to ω labeling of non-β-oxidizable LCFA analogs.     

Spin trapping and its application in the study of lipid peroxidation and free radical production with liver microsomes.

Lipid peroxidation in microsomes was studied using a spin-trapping technique. Free radical adducts of phenyltertiarybutylnitrone (PBN) were produced as detected by electron spin resonance during induced lipid peroxidation of microsomes with a system consisting of NADPH, Fe²⁺, and pyrophosphate. The adducts were identified as intermediates of the substrates added to the microsomal system and not OH · or HO₂ radicals. The production of the adduct parallels the NADPH-dependent formation of malondialdehyde (MDA). Analyses of the electron spin resonance hyperfine splitting constants allowed in some instances identification of the adducts. Purified preparations of cytochrome P-450 mimic the results of the microsomes. The carcinogens dimethyl and diethylnitrosoamine were metabolized in this system yelding reactive free radicals and free NO, suggesting an alternate mechanism for the activity of these compounds as ultimate carcinogens.     

Incorporation of radioactive- -aminolevulinic acid into microsomal cytochrome P 450 : selective breakdown of the hemoprotein by allylisopropylacetamide and carbon tetrachloride

Administration of allylisopropylacetamide (AIA) or CCl₄ to rats previously treated with phenobarbital leads to a rapid decrease in cytochrome P₄₅₀ within 1 hr. The amount of cytochrome b₅ and NADPH cytochrome c reductase in liver microsomes remains unchanged following AIA treatment. In contrast, CCl₄ administration causes a decrease in total microsomal protein thus leading to a net loss in cytochrome b₅ and NADPH cytochrome c reductase. By using ³H-δ-aminolevulinic acid to label microsomal cytochrome P₄₅₀ heme, the effect of AIA and CCl₄ on this cytochrome was shown to be caused by destruction of preexisting CO-binding pigment and not from inhibition of synthesis. In addition, the breakdown products of cytochrome P₄₅₀ heme accumulate in the liver after AIA or CCl₄ treatment.