Purified Rat Liver Cytochrome P-450 Research Articles

A specific interaction between NADPH-cytochrome reductase and phosphatidylserine and phosphatidylinositol.

In the present study the interaction of NADPH-cytochrome reductase with phospholipids was investigated using 31P-NMR, thin-layer chromatography combined with chemical analysis, fluorescence spectroscopy and kinetic studies with purified rat liver cytochrome P450 IIB1. 31P-NMR analysis demonstrates that the composition of the phospholipids that remain associated to NADPH-cytochrome reductase upon its purification is significantly different from the phospholipid composition of the microsomal membrane. Thin-layer chromatography followed by chemical analysis of the phospholipid composition demonstrates that the isolated NADPH-cytochrome reductase was enriched in L-alpha-1,2-diacyl-sn-glycero-3-phosphoserine (acyl2GroPSer) and L-alpha-1,2-diacyl-sn-glycero-3-phosphoinositol (acyl2GroPIns) compared to the microsomal membrane. The observed preference of NADPH-cytochrome reductase for acyl2GroPSer and acyl2GroPIns appeared not to be a result of the procedure for solubilisation and/or purification of the protein. The specific interaction of NADPH-cytochrome reductase with acyl2GroPSer and acyl2GroPIns was further investigated by comparison of the effect of acyl2GroPSer and acyl2GroPIns with that of acyl2GroPCho and acyl2GroPEtn on the 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3- phosphocholine-(DphPamGroPCho)-dependent quenching of the tryptophan fluorescence of purified NADPH-cytochrome reductase. The results demonstrate that the addition of acyl2GroPSer or acyl2GroPIns affects the DphPamGroPCho-dependent quenching of the tryptophan fluorescence in a manner significantly different from the addition of acyl2GroPCho or acyl2GroPEtn. The relatively larger DphPamGroPCho-induced quenching of the tryptophan fluorescence of NADPH-cytochrome reductase in the presence of acyl2GroPSer and acyl2GroPIns must result from a change in the conformation of NADPH-cytochrome reductase induced by the latter two lipids. Finally, the possible consequences of this special interaction of acyl2GroPSer and acyl2GroPIns with NADPH-cytochrome reductase on the kinetic characteristics of the cytochrome P450 system were studied using cytochrome-P450-IIB1-dependent O-dealkylation of pentoxyresorufin as the model reaction. These studies demonstrate that a 1:1 mixture of acyl2GroPCho and acyl2GroPSer results in a significantly higher apparent maximum rate (V) of O-dealkylation than a 1:1 mixture of acyl2GroPCho and acyl2ProPEtn or acyl2GroPCho alone. This increase in the apparent V can be ascribed to an acyl2GroPSer-dependent improvement of the interaction of NADPH-cytochrome reductase with cytochrome P450. This improvement of the interaction of the proteins cannot, however, be exclusively ascribed to the negative charge of acyl2GroPSer, since the other negatively charged phospholipid investigated, namely acyl2GroPIns, resulted in a significant decrease in the apparent V.(ABSTRACT TRUNCATED AT 400 WORDS)

The enzymology of doxorubicin quinone reduction in tumour tissue

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521–526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 ± 4.6 nmol/min/mg; cytosolic activity, 94.3 ± 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b 5 reductase (15.6 ± 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 ± 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol ( ic 50 approx. 50 μM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.

Cytochrome P-450 arachidonic acid epoxygenase. Regulatory control of the renal epoxygenase by dietary salt loading.

The rat kidney microsomal epoxygenase catalyzed the asymmetric epoxidation of arachidonic acid to generate as major products: 8(R),9(S)-, 11(R),12(S)- and 14(S),15(R)-epoxyeicosatrienoic acids with optical purities of 97, 88, and 70%, respectively. Inhibition studies utilizing a panel of polyclonal antibodies to several rat liver cytochrome P-450 isoforms, indicated that the renal epoxygenase(s) belongs to the cytochrome P-450 2C gene family. Dietary salt, administered either as a 2-2.5% (w/v) solution in the drinking water or as a modified solid diet containing 8% NaCl (w/w), resulted in marked and selective increases in the renal microsomal epoxygenase activity (416 and 260% of controls, for the liquid and solid forms of NaCl, respectively) with no significant changes in the microsomal omega/omega-1 oxygenase or in the hepatic arachidonic acid monooxygenase reaction. Immunoblotting studies demonstrated that dietary salt induced marked increases in the concentration of a cytochrome P-450 isoform(s) recognized by polyclonal antibodies raised against human liver cytochrome P-450 2C10 or rat liver cytochrome P-450 2C11. Comparisons of the stereochemical selectivity of the induced and non-induced microsomal epoxygenase(s) with that of purified rat liver cytochrome P-450 2C11 suggest that the salt-induced protein(s) is catalytically and structurally different from liver cytochrome P-450 2C11. The in vivo significance of dietary salt in regulating the activities of the kidney endogenous arachidonic acid epoxygenase was established by the demonstration of a salt-induced 10-20-fold increase in the urinary output of epoxygenase metabolites. These results, in conjunction with published evidence demonstrating the potent biological activities of its metabolites, suggest a role for the epoxygenase in the renal response to dietary salt.

Low kinetic hydrogen isotope effects in the dehydrogenation of 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (nifedipine) by cytochrome P-450 enzymes are consistent with an electron/proton/electron transfer mechanism

Cytochrome P-450 enzymes have been postulated to oxidize amines through a variety of mechanisms. One of the means of distinguishing among potential pathways involves the use of kinetic hydrogen isotope effects: low isotope effects are characteristic of aminium radical mechanisms while high values are consistent with hydrogen atom abstraction, a process documented in alkane hydroxylation. Nifedipine [1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic+ ++ acid dimethyl ester] was prepared with isotopic substitution at the 4-position and utilized in determinations of the deuterium and tritium kinetic isotope effects. The noncompetitive intermolecular deuterium [DV, D(V/K)] and competitive intermolecular tritium [T(V/K)] isotope effects observed in the oxidation of nifedipine with liver microsomes prepared from untreated male rats were less than 2. Similar low deuterium and tritium isotope effects (less than 2) were also found with six other types of rat liver microsomal preparations and with three different human liver microsomal preparations, even though the rates of oxidation (expressed per nanomole of total cytochrome P-450) varied by an order of magnitude and other investigations indicate that several different cytochrome P-450 enzymes can catalyze the reaction. Similar low deuterium and tritium isotope effects were measured with a purified rat liver cytochrome P-450 preparation and horseradish peroxidase. These results are interpreted in terms of an electron/proton/electron transfer mechanism previously postulated for the oxidation of other dihydropyridines by cytochrome P-450 enzymes and model one-electron acceptors and argue against a mechanism involving hydrogen atom abstraction from nifedipine.

Effect of topical application of clotrimazole to rats on epidermal and hepatic monooxygenase activities and cytochrome P-450

Clotrimazole, an N-substituted imidazole, is a widely used topical agent for the treatment of superficial fungal infections. In this study, the effect of application of clotrimazole to the skin of neonatal rats on the induction response of the cytochrome P-450-dependent monooxygenase system in epidermis and liver has been examined. A single topical application of clotrimazole (10 mg/100 g) to rats resulted in a 53% increase in hepatic cytochrome P-450 content. Clotrimazole treatment also resulted in significant induction of epidermal 7-ethoxycoumarin-O-deethylase activity. Hepatic p-nitrophenol hydroxylase, an enzyme, catalyzed principally by the ethanol inducible cytochrome P-450 isozyme, was also significantly induced (58%) by topically applied clotrimazole. This enzyme activity was undetectable in epidermal microsomes. Further characterization of the cytochrome P-450 isozymes induced in liver by clotrimazole treatment was based on monoclonal antibodies (MAbs) raised against purified rat liver cytochrome P-450 isozymes induced by phenobarbital (MAb 2-66-3) and ethanol (MAb 1-98-1). Hepatic microsomes prepared from clotrimazole-treated rats showed significant immunoreactivity on Western blot with both the MAbs whereas no reactivity occurred in epidermal microsomes. Our data indicate that topical application of clotrimazole to rats results in the induction of selected cytochrome P-450 isozyme(s) in liver and epidermis which may have implications for the therapeutic use of this compound.

Cytochrome P-450 isozyme/isozyme functional interactions and NADPH-cytochrome P-450 reductase concentrations as factors in microsomal metabolism of warfarin.

The basis for our previous observations [Kaminsky, L.S., Guengerich, F.P., Dannan, G.A. & Aust, S.D. (1983) Arch. Biochem. Biophys. 225, 398-404] that rates of microsomal metabolism of warfarin were markedly less than the sum of rates of the reconstituted constituent isozymes of cytochrome P-450 has been investigated. Metabolism of warfarin to 4'-, 6-, 7-, 8-, and 10-hydroxywarfarin and dehydrowarfarin by highly purified rat liver cytochrome P-450 (P-450) isozymes reconstituted with NADPH-cytochrome P-450 reductase and by hepatic microsomes from variously pretreated rats was used to probe functional consequences of P-450 isozyme/isozyme interactions and of the effect of microsomal reductase concentrations. Binary mixtures of P-450 isozymes were reconstituted and the regioselectivity and stereoselectivity were used to probe metabolism by each individual isozyme. The isozymes specifically inhibited each other to variable extents and the order of inhibitory potency was: P-450UT-F greater than P-450PB-D greater than or equal to P-450UT-A greater than or equal to P-450BNF/ISF-G greater than P-450PB/PCN-E greater than P-450PB-B greater than or equal to P-450PB-C greater than or equal to P-450BNF-B. The inhibition, possibly a consequence of aggregation, explains the low rate of microsomal metabolism relative to the metabolic potential of the component P-450 isozymes. When purified reductase was added to microsomes it appeared to bind to microsomes at different sites from endogenous reductase and it enhanced warfarin hydroxylase activity only to a minor extent, thus possibly precluding low reductase concentrations from being a major factor in the relatively low rates of microsomal metabolism. Antibody to the reductase differentially inhibited microsomal metabolism of warfarin by the various P-450 isozymes. The results suggest that the reductase and P-450 isozymes may be located differently relative to one another in the various microsomal preparations.

Reactivity of 4',4"-diethylstilbestrol quinone, a metabolic intermediate of diethylstilbestrol.

In a search for the carcinogenic metabolite of diethylstilbestrol, the interactions of 4',4"-diethylstilbestrol quinone with peptides and nucleic acids were investigated. Nonextractable binding of 4',4"-diethylstilbestrol quinone to calf thymus DNA or poly G were observed. However, adduct nucleosides could not be isolated subsequent to enzymatic digestion of nucleic acids. Binding to dGMP or dAMP also occurred, but the initially bound stilbene estrogen could mostly be extracted with 18 extractions using various organic solvents. Non-covalent interactions of 4',4"-diethylstilbestrol quinone with calf thymus DNA were observed spectrally only after exhaustive dialysis of the DNA versus water, but not with native DNA. In chemical reactions of 4',4"-diethylstilbestrol quinone and nucleosides, nucleotides, and amines such as n-pentyl amine, only Z,Z-dienestrol could be identified as reaction product. The quinone did react with mercaptoethanol via Michael addition to the unsaturated carbonyl system to form a stable adduct, 4-(2-hydroxyethylthio)-3,4-di(p-hydroxyphenyl)-2-hexene. It also reacted covalently with sulfur-containing peptides such as reduced glutathione or bovine serum albumin. Partially purified rat liver cytochrome P-450 reductase reduced 4',4"-diethylstilbestrol quinone to E- and Z-diethylstilbestrol. It is proposed that 4',4"-diethylstilbestrol quinone forms unstable adduct intermediates with DNA which decompose with time. Also, covalent binding of 4',4"-diethylstilbestrol quinone to important proteins via thioether linkages may play a role in carcinogenesis.

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene.

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.

Reduction kinetics of purified rat liver cytochrome P-450. Evidence for a sequential reaction mechanism dependent on the hemoprotein spin state

The anaerobic reduction kinetics of purified rat liver ferric cytochrome P-450 from phenobarbital-treated rat liver microsomes, reconstituted with saturating NADPH-cytochrome P-450 reductase, have been investigated and were shown not to be monophasic. From experiments correlating changes in the rate of fast-phase reduction with the spin state of the heme iron existing at preequilibrium, data were obtained consistent with a model for spin-state control of cytochrome P-450 reduction wherein the high-spin form of the hemoprotein is more rapidly reduced than the low-spin form. In addition, the temperature dependence of the reduction process in the presence of the substrate benzphetamine was studied. From the results obtained it is suggested that the endothermic nature of the low- to high-spin transition largely accounts for the apparent activation energy observed for the reduction of high-spin cytochrome P-450 being relatively temperature insensitive when compared to the rate constant for reduction of the membrane-bound form of the hemoprotein.

Regio- and stereoselective metabolism of two C19 steroids by five highly purified and reconstituted rat hepatic cytochrome P-450 isozymes.

High pressure liquid chromatographic systems capable of resolving at least 28 known and potential metabolites of 17 beta-hydroxy-4-androsten-3-one (testosterone) and 4-androstene-3,17-dione (androstenedione) were used to quantitatively assess the metabolism of the two steroids in monooxygenase systems reconstituted with five purified rat liver cytochrome P-450 isozymes. Cytochromes P-450a, -b, -c, -d, and -e catalyzed the oxidation of testosterone at overall rates of 21, 27, 2, 0.7, and 3 nmol/min/nmol of cytochrome P-450, respectively; while the corresponding rates for total androstenedione metabolism were 12, 62, 1.5, 0.3, and 5. Cytochrome P-450a catalyzed the oxidation of testosterone and androstenedione almost exclusively to their respective 7 alpha-hydroxy metabolites. Cytochrome P-450b catalyzed the oxidation of testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone in approximately equal molar ratios. However, this same hemoprotein exhibited a marked stereoselectivity in the metabolism of androstenedione since the molar ratio of 16 alpha- and 16 beta-hydroxyandrostenedione was greater than 1:10. Cytochrome P-450e catalyzed the oxidation of both steroids to the same products as cytochrome P-450b, but at approximately 10% of the rate. Cytochromes P-450c and P-450d catalyzed the oxidation of testosterone and androstenedione regio- and stereospecifically to their respective 6 beta-hydroxy metabolites. These results indicate that certain cytochrome P-450 isozymes show marked positional specificity in the metabolism of both testosterone and androstenedione, and that the rate as well as stereoselectivity of the oxidative reactions can be markedly dependent on subtle differences in the structure of the steroid substrate.

Determination of partition ratios for allylisopropylacetamide during suicidal processing by a phenobarbital-induced cytochrome P-450 isozyme from rat liver.

Allylisopropylacetamide is shown to be a suicide substrate for the phenobarbital-inducible cytochromes P-450. In phenobarbital-induced rat liver microsomes about 70% of the cytochrome P-450-mediated N,N-dimethylaniline N-demethylase activity is sensitive to allylisopropylacetamide inactivation; the residual 30% of the N-demethylase activity is incapable of allylisopropylacetamide turnover and insensitive to allylisopropylacetamide inactivation. The partition number for inactivation of the susceptible population of cytochrome P-450 indicates turnover of 201 molecules of allylisopropylacetamide per molecule of P-450 inactivated. A purified phenobarbital-induced isozyme of cytochrome P-450, when reconstituted with purified rat liver cytochrome P-450 reductase, is also inactivated by allylisopropylacetamide in a suicide fashion with a corrected partition ratio of 184 turnovers of allylisopropylacetamide per inactivation event. This partition number is corrected for the competing O2-dependent autoinactivation of cytochrome P-450 which we have previously shown to occur with the purified isozyme (Loosemore, M., Light, D. R., and W#alsh, C. (1980) J. Biol. Chem. 255, 9017-9020). The 201 product molecules of cytochrome P-450-mediated turnover of allylisopropylacetamide in either the microsomal or purified enzyme system are probably the epoxide, are reactive toward alkylation of cellular nucleophiles, and covalently modify protein and exogenous calf thymus DNA molecules.

Stereoselectivity of rat liver cytochrome P-450 c on formation of benzo[a]pyrene 4,5-oxide

The facial selectivity of purified rat liver cytochrome P-450 c toward epoxidation of benzo[a]pyrene (BP) at its 4,5-position (K-region) was determined by formation, separation and quantitation of diastereomeric trans-addition products of glutathione with enzymatically produced benzo[a]pyrene 4,5-oxide (BP 4,5-oxide). Non-enzymatic trans addition of glutathione to synthetic (+)-(4S,5R)- and (−)-(4R,5S)-BP 4,5-oxides occurs equally at carbons 4 and 5 to give two diastereomeric pairs of positional isomers. The position of the glutathionyl moiety in each isomer was determined by acetylation, oxidation of the thioether to the sulfoxide, cis -elimination of the sulfoxide, and identification of the acetoxybenzo[a]pyrene formed. Correlation of the glutathione conjugates obtained from BP 4,5-oxide derived from cytochrome P-450 c catalyzed oxidation of BP with those obtained from the synthetic enantiomers indicated that ≥97% of the enzymatically formed arene oxide was the (+)-(4S,5R) enantiomer.

Inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion (diethyl p-nitrophenyl phosphorothionate).

The mechanism of the inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion has been investigated with special reference to the covalent binding to the enzyme of the atomic sulfur (S) released upon oxidation of the compound. In addition to the previously reported loss of cytochrome P-450 detectable as its carbon monoxide complex, a loss of heme detectable as pyridine-hemochromagen, and a loss of enzymatic activity toward benzphetamine and ethoxycoumarin are shown. The loss of heme is 80% of the loss of cytochrome P-450, which in turn is less than the loss of benzphetamine demethylase activity, the turnover number of the modified enzyme being two-thirds that of the native based on the amount of cytochrome P-450 detectable as its carbon monoxide complex. Treatment with performic acid or dithiothreitol removes 75% of the 35S covalently bound to the cytochrome P-450 during parathion metabolism. However, the dithiothreitol treatment does not restore cytochrome P-450 detectable as its carbon monoxide complex or enzymatic activity. Likewise, no protection against the loss of heme, cytochrome P-450, or enzymatic activity is afforded by inclusion of 1 mM dithiothreitol during the incubation with parathion, although the amount of irreversibly bound sulfur, i.e. sulfur not dissociable by dithiothreitol, is significantly reduced. In the absence of dithiothreitol, 4 nmol of 35S become bound to the cytochrome P-450 for each nanomole of P-450 no longer detectable as its carbon monoxide complex. Evidence is presented for the covalent binding of sulfur to at least three other amino acids than cysteine. It is suggested that structural changes resulting from the covalent binding of atomic sulfur to cysteine residues are responsible for at least some of the loss of enzymatic activity observed.

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 m m. 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 μ m. 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 μ m) 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.