BENZENE MYELOCLASTOGENICITY AND METABOLISM IN CD-1 MICE: A CORRELATION

Benzene was studied in its target organ of effect, the bone marrow, with the micronucleus test and metaphase chromosomal analysis. Groups of 5 or 10, male and female CD-1 mice were treated with one or two p.o. or i.p. doses of benzene (440 mg/kg) or toluene (430, 860 or 1720 mg/kg) or both, and sacrificed 30 or 54h after the first dose. Benzene-treated animals were pretreated with phenobarbital (PB), 3-methylcholanthrene (3MC), (beta)-naphthoflavone ((beta)NF), SKF-525A, or Aroclor 1254. Toluene showed no clastogenic activity and reduced the clastogenic effect of co-administered benzene. None of the pretreatments protected against benzene clastogenicity. 3MC and (beta)NF greatly promoted benzene myeloclastogenicity. Dose response curves for benzene myeloclastogenicity were much steeper with 3MC induction than without. Micronuclei (MN) were 4-6 times higher by p.o. than i.p. benzene administration. This was not due to bacterial flora since no difference was found between germ-free and conventional males gavaged with benzene. A sensitive high-pressure liquid chromatographic method was developed and used to explore the relation between metabolic profiles of benzene in urine and MN after various pretreatments. Phenol (PH), trans-trans-muconic acid (MA) and hydroquinone (HQ) in the 48h male mouse urine accounted, respectively, for 12.8-22.8, 1.8-4.7 and 1.5-3.7% of the single oral dose of benzene (880, 440 and 220 mg/kg). Catechol (CT) was seen in trace amounts. MA was identified by ultraviolet and infrared spectroscopy and elemental analysis. Urinary metabolites--especially MA, HQ, and phenol glucuronide--correlated well with MN and were dependent on both the dose and the metabolism of benzene. Benzene metabolism was most inducible by cytochrome P-448 enzyme inducers, by p.o. > i.p., in males > females, and inhibited by toluene. Ph, CT or HQ administered p.o., 250, 150 and 250 mg/kg, respectively, or at 150 mg/kg x 2 after 3MC pretreatment, failed to reproduce the potent myeloclastogenicity of benzene. In fact, only HQ was mildly clastogenic. ^

Characterization of the cytochrome P-450 drug metabolism system of LS174T human colon tumor cell line

The human colon tumor cell line, LS174T, has been shown to have four major components of the drug metabolizing system; cytochrome b$\sb5$ reductase, cytochrome b$\sb5$, cytochrome P450 reductase and cytochrome P450, by activity measurements, spectral studies and antibody cross-reactivity. Cytochrome P450IA1 is induced by benzanthracene in these cells as shown by activity with the specific substrate, ethoxyresorufin, cross-reactivity with rabbit antibodies to rat IA1, and by a hybridizing band on a Northern blot to a rat IA1 probe.^ Further, this system has proven responsive to various inducers and conditions of growth. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 $\mu$mol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b$\sb5$ per milligram and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone treatment showed a consistent, but not always significant increase in the NADPH and NADH cyt c reducing activity and benzanthracene treatment an increase in the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5mM) caused a significant decrease in the specific activity of all enzyme contents and activities tested.^ Finally, the cytochrome b$\sb5$ to cytochrome P450, by the coordinate induction of the cytochrome b$\sb5$ pathway by P450 inducers, by the high ratio of NADH to NADPH ethoxycoumarin deethylase activity in uninduced cell microsomes, and by the increase in NADH and NADPH ethoxycoumarin deethylase activity when the microsomes were treated with potassium cyanide, a desaturase inhibitor. ^

Expression, induction and regulation of the cytochrome P450 monooxygenase system in the rat glioma C6 cell line

The cytochrome P450 monooxygenase system consists of NADPH- cytochrome P450 reductase (P450 reductase) and cytochromes P450, which can catalyze the oxidation of a wide variety of endogenous and exogenous compounds, including steroid hormones, fatty acids, drugs, and pollutants. The functions of this system are as diverse as the substrates. P450 reductase transfers reducing equivalents from NADPH to P450, which in turn catalyzes metabolic reactions. This enzyme system has the highest level of activity in the liver. It is also present in other tissues, including brain. The functions of this enzyme system in brain seem to include: neurotransmission, neuroendocrinology, developmental and behavioral modulation, regulation of intracellular levels of cholesterol, and potential neurotoxicity.^ In this study, we have set up the rat glioma C6 cell line as an in vitro model system to examine the expression, induction, and tissue-specific regulation of P450s and P450 reductase. Rat glioma C6 cells were treated with P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). The presence of P450 reductase and of cytochrome P450 1A1, 1A2, 2A1, 2B1/2, 2C7, 2D1-5 and 2E1 was detected by reverse transcription followed by polymerase chain reaction (RT-PCR) and confirmed by restriction digestion. The induction of P450 1A1 and 2B1/2 and P450 reductase was quantified using competitive PCR. Ten- and five-fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected. Western blot analysis of microsomal preparations of glioma C6 cells demonstrated the presence of P450 1A, 2B and P450 reductase at the protein level. ELISAs showed that BA and PB induce P450 1A and 2B proteins 7.3- and 13.5-fold, respectively. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 CO difference spectra with absorption at or near 450 nm. Microsomes prepared from rat glioma C6 cells demonstrated much higher levels of ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-dealkylase (PROD) activity, when treated with BA or PB, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active cytochrome P450 monooxygenase system which can be induced by P450 inducers. The mRNAs of P450 1A1 and 2B1/2 can not bind to the oligo(dT) column efficiently, indicating they have very short poly(A) tails. This finding leads us to study the tissue specific regulation of P450s at post-transcriptional level. The half lives of P450 1A1 and 2B1/2 mRNA in glioma C6 cells are only 1/10 and 1/3 of that in liver. This may partly contribute to the low expression level of P450s in glial cells. The induction of P450s by BA or PB did not change their mRNA half lives, indicating the induction may be due to transcriptional regulation. In summary of this study, we believe the presence of the cytochrome P450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors. ^

Polymorphisms, linkage and mapping of four enzyme loci in the fish genus Xiphophorus (Poeciliidae).

Electrophoretic variants at four additional enzyme loci--two esterases (Est-2, Est-3), retinal lactate dehydrogenase (LDH-1) and mannose phosphate isomerase (MPI)--among three species and four subspecies of fish of the genus Xiphophorus were observed. Electrophoretic patterns in F1 hybrid heterozygotes confirmed the monomeric structures of MPI and the esterase and the tetrametric structure of LDH in these fishes. Variant alleles of all four loci displayed normal Mendelian segregation in backcross and F2 hybrids. Recombination data from backcross hybrids mapped with Haldane's mapping function indicate the four loci to be linked as Est-2--0.43--Est3--0.26--LDH-1--0.19--MPI. Significant interference was detected and apparently concentrated in the Est-3 to MPI region. No significant sex-specific differences in recombination were observed. This group (designated linkage group II) was shown to assort independently from the three loci of linkage group I (adenosine deaminase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) and from glyceraldehyde-3-phosphate dehydrogenase and two isocitrate dehydrogenase loci. Evidence for conservation of the linkage group, at least in part, in other vertebrate species is presented.

Three linked enzyme loci in fishes: implications in the evolution of vertebrate chromosomes.

A three-point linkage group comprised of loci coding for adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phospho-gluconate dehydrogenase (6PGD) is described in fish of the genus Xiphophorus (Poeciliidae). The alleles at loci in this group were shown to assort independently from the alleles at three other loci--isocitrate dehydrogenase 1 and 2, and glyceraldehyde-3-phosphate dehydrogenase 1. Alleles at the latter three loci also assort independently from each other. Data were obtained by observing the segregation of electrophoretically variant alleles in reciprocal backcross hybrids derived from crosses between either X. helleri guentheri or X. h. strigatus and X. maculatus. The linkage component of chi2 was significant (less than 0.01) in all crosses, indicating that the linkage group is conserved in all populations of both species of Xiphophorus examined. While data from X. h. guentheri backcrosses indicate the linkage relationship ADA--6%--G6PDH--24%--6PGD, and ADA--29%--6PGD (30% when corrected for double crossovers), data from backcrosses involving strigatus, while supporting the same gene order, yielded significantly different recombination frequencies. The likelihood of the difference being due to an inversion could not be separated from the possibility of a sex effect on recombination in the present data. The linkage of 6PGD and G6PDH has been shown to exist in species of at least three classes of vertebrates, indicating the possibility of evolutionary conservation of this linkage.

Intracellular phosphorylation of ornithine decarboxylase: Regulation of enzyme activity

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis exists as two major and one minor ionic form in the macrophage cell line, RAW 264. The forms have the same molecular weight, 55,000, but differ in their isoelectric points, 5.2, 5.1, and 4.9-5.0. The hypothesis that phosphorylation accounts for the differences in the two major ionic forms and that phosphorylation is involved in the regulation of enzyme activity was investigated. Metabolic-radiolabeling of cells with $\sp{32}$P-orthophosphate indicated that only one of the major forms of the protein can be explained by phosphorylation: treatment of purified ODC with alkaline phosphatase resulted in the loss of the phosphorylated form of the protein, pl 5.1, with a concomitant increase in the unphosphorylated, pl 5.2, form of the protein. Characterization of the phosphorylation sites showed that serine was the present. Tryptic digests of $\sp{32}$P-labeled ODC, analyzed by either two dimensional tryptic peptide mapping or reverse-phase HPLC, contained only one major radiolabeled peptide.^ The role phosphorylation plays in the regulation of enzyme activity was also investigated. Treatment of purified ODC with alkaline phosphatase resulted in the loss of enzyme activity. A positive linear correlation exists between enzyme activity and the amount of phosphorylated form of the protein present.^ To ascertain if the two major forms of the protein were also found in animal cells, ODC was immunoprecipitated from various rat tissues, fractionated by isoelectric focusing, and detected by immunoblotting. ODC was present in rat tissues in a single major form, which comigrated with the pl 5.1, phosphorylated form of ODC present in RAW 264 cell.^ This study concludes that ODC exists as a phosphorylated form, pl 5.1, and an unphosphorylated form, pl 5.2 in RAW 264 cells. The amount of the phosphorylated form of ODC correlates well with the enzyme activity. ^

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

The angiotensin-converting enzyme insertion/deletion polymorphism and its associations with carotid artery wall thickness and coronary heart disease: The atherosclerosis risk in communities study

Coronary heart disease (CHD) is the leading cause of death in the United States. Recently, renin-angiotensin system (RAS) was found associated with atherosclerosis formation, with angiotensin II inducing vascular smooth muscle cell growth and migration, platelet activation and aggregation, and stimulation of plasminogen activator inhibitor-1. Angiotensin II is converted from angiotensin I by angiotensin I-converting enzyme (ACE) and this enzyme is mainly genetically determined. The ACE gene has been assigned to chromosome 17q23 and an insertion/deletion (I/D)polymorphism has been characterized by the presence/absence of a 287 bp fragment in intron 16 of the gene. The two alleles form three genotypes, namely, DD, ID and II and the DD genotype has been linked to higher plasma ACE levels and cell ACE activity.^ In this study, the association between the ACE I/D polymorphism and carotid artery wall thickness measured by B-mode ultrasound was investigated in a biracial sample, and the association between the gene and incident CHD was investigated in whites and if the gene-CHD association in whites, if any, was due to the gene effect on atherosclerosis. The study participants are from the prospective Atherosclerosis Risk in Communities (ARIC) Study, including adults aged 45 to 65 years. The present dissertation used a matched case-control design for studying the associations of the ACE gene with carotid artery atherosclerosis and an unmatched case-control design for the association of the gene with CHD. A significant recessive effect of the D allele on carotid artery thickness was found in blacks (OR = 3.06, 95% C.I: 1.11-8.47, DD vs. ID and II) adjusting for age, gender, cigarette smoking, LDL-cholesterol and diabetes. No similar associations were found in whites. The ACE I/D polymorphism is significantly associated with coronary heart disease in whites, and while stratifying data by carotid artery wall thickness, the significant associations were only observed in thin-walled subgroups. Assuming a recessive effect of the D allele, odds ratio was 2.84 (95% C.I:1.17-6.90, DD vs. ID and II) and it was 2.30 (95% C.I:1.22-4.35, DD vs. ID vs. II) assuming a codominant effect of the D allele. No significant associations were observed while comparing thick-walled CHD cases with thin-walled controls. Following conclusions could be drawn: (1) The ACE I/D polymorphism is unlikely to confer appreciable increase in the risk of carotid atherosclerosis in US whites, but may increases the risk of carotid atherosclerosis in blacks. (2) ACE I/D polymorphism is a genetic risk factor for incident CHD in US whites and this effect is separate from the chronic process of atherosclerosis development. Finally, the associations observed here are not causal, since the I/D polymorphism is in an intron, where no ACE proteins are encoded. ^

CYTOCHROME P450 MEDIATED REDUCTIVE DEHALOGENATION OF HEXACHLOROBENZENE

The role of the cytochrome (CYT) P-450 mixed-function oxidase (MFO) in the biotransformation of hexachlorobenzene (HCB) was investigated, since in vivo interaction between this enzyme and chemical is very probable. HCB is a type I substrate with (Fe('3+)) CYT P-450 isozymes present in untreated, b-naphthoflavone (BNF) and phenobarbital (PB) induced rat liver microsomes. HCB dependent and saturable type I binding titrations yield spectral dissociation constants (K(,s)) of 180 and 83 uM for the isozymes present in untreated and PB induced microsomes, respectively. Purified CYT P-450b, the major isozyme induced by PB, produces HCB dependent and saturable type I spectra with a K(,s) of 0.38 uM.^ CYT P-450 mediated reductive dehalogenation occurs in microsomes and purified/reconstituted MFO systems and produces pentachlorobenzene (PCB) as the initial and major metabolite under both aerobic and anaerobic conditions. In microsomal reactions secondary metabolism of PCB occurs in the presence of oxygen. Pentachlorophenol (PCP) is produced only in aerobic reactions with PB induced microsomes with a concomitant decrease in PCB production. PCP is not detected in aerobic reactions with BNF induced microsomes, although PCB production is decreased compared to anaerobic conditions. A reaction scheme for the production of phenolic metabolities from PCB is deduced.^ CYT P-450 dependent and NADPH independent modes of PCB production occur with purified/reconstituted MFO systems and are consistent with dehalogenation pathways observed with microsomal experiments. The NADPH independent production of PCB requires native microsomal or purified MFO protein components and may be the result of nucleophilic displacement of a chlorine atom from HCB mediated or coupled with redox active functions (primary, secondary, tertiary and quarternary structures) of the proteins. CYT P-450 dependent production of PCB from HCB is isozyme dependent: CYT P-450c = CYT P-450d > CYT P-450a > CYT 450b. The low apparent specific activity may be due to non-optimal reconstitution conditions (e.g., isozyme choice and requirement of other microsomal elecron transport components) and secondary metabolism of PCB and the phenols derived from PCB. CYT P-450 mediated dehalogenation may be catalyzed through attack, by the iron oxene (postulated intermediate of CYT P-450 monooxygenations), at the chlorines of HCB instead of the aromatic nucleus. (Abstract shortened with permission of author.) ^

IN VITRO METABOLISM OF 6-THIOGUANINE IN RAT SUBCELLULAR FRACTIONS

The metabolism of the antitumor agent 6-thioguanine (TG, NSC-752) by rat liver was studied in vitro. Livers from adult male Sprague-Dawley rats were homogenized and the "liver homogenate" was subjected to differential centrifugation to obtain the "10,000 x g pellet", the "post-mitochondrial fraction", the "cytosol fraction", and the "microsomes". The homogenity of each fraction was estimated by appropriate marker enzyme assays. To delineate the in vitro metabolism of TG by rat liver, 0.2 mM of {8-('14)C}TG was incubated with different subcellular fractions in KCl-Tris-MgCl(,2) buffer, pH 7.4 at 37(DEGREES). The metabolites formed were identified by chromatography, UV spectrometry, as well as mass spectrometry. After a 1 hr incubation, TG was metabolized by the liver homogenate, the 10,000 x g pellet and the post-mitochondrial fraction mainly to 6-thioguanosine (TGR), accompanied by varying lesser amounts of 6-thiouric acid (TUA), allantoin, guanine-6-sulfinic acid (G-SO(,2)H) and an unknown product. In comparison, the cytosal fraction converted TG almost entirely to TGR and TUA in equal amounts. The formation of TGR from TG was limited by the endogenous supply of ribose-1-phosphate. With the microsomal fraction, however, TG was metabolized significantly to G-SO(,2)H and the unknown, accompanied with some TGR. After a 5 hr incubation the metabolism of TG was changed to favor the catabolic route, yielding mostly TUA in the post-mitochondrial and cytosol fractions; but mainly allantoin in the liver homogenate fraction. The kinetic studies of TG metabolism by the subcellar fractions indicated that the formation of TGR served as a depot form of TG. The level of TGR decreased when the catabolism of TG became prominent. The oxidation of TG to GSO(,2)H mediated by the hepatic microsomes represented a new catabolic pathway of TG. This GSO(,2)H, under acidic conditions, readily decomposes to guanine and inorganic sulfate. In the presence of reduced glutathione in Tris buffer, pH 7.8 at 25(DEGREES), GSO(,2)H is adducted to glutathione chemically to form S-(2-amino-purin-6-yl) glutathione and conceivably, inorganic sulfate. Therefore, the formation of GSO(,2)H from TG might have implication in the desulfuration mechanism of TG. On the other hand, the unknown formed from TG by the action of the microsomal enzymes appeared to be a TG conjugate. However, it is neither a glutathione, a glucuronide, nor a ribose conjugate. Additionally, the deamination of TG by guanine deaminase (E.C.3.5.4.3) isolated from rat liver was also investigated. TG is a poorer substrate (Km = 4.8 x 10('-3)M) for guanine deaminase than that of guanine (Km = 4.7 x 10('-6)M) at pH 7.25, optimal pH for TG as a substrate. TG is also a competitive inhibitor of guanine for guanine deaminase, with a ki of 2.2 x 10('-4)M. ^

PREPARATION AND CHARACTERIZATION OF COVALENTLY LINKED ENZYME-ANTIBODY CONJUGATES AND THEIR USE IN MEASURING MINUTE QUANTITIES OF PROTEIN ANTIGENS

The discovery and characterization of oncofetal proteins have led to significant advances in early cancer diagnosis and therapeutic monitoring of patients undergoing cancer chemotherapy. These tumor-associated antigens are presently measured by sensitive, specific immunoassay techniques based on the detection of minute amounts of labeled antigen or antibody incorporated into immune complexes, which must be isolated from free antigen and antibody.^ Since there are several disadvantages with using radioisotopes, the most common immunolabel, one major objective was to prepare covalently coupled enzyme-antibody conjugates and evaluate their use as a practical alternative to radiolabeled immune reagents. An improved technique for the production of enzyme-antibody conjugates was developed that involves oxidizing the carbohydrate moieties on a glycoprotein enzyme, then introducing antibody in the presence of polyethylene glycol (PEG). Covalent enzyme-antibody conjugates involving alkaline phosphatase and amyloglucosidase were produced and characterized.^ In order to increase the sensitivity of detecting the amyloglucosidase-antibody conjugate, an enzyme cycling assay was developed that measures glucose, the product of maltose cleavage by amyloglucosidase, in the picomole range. The increased sensitivity obtained by combined usage of the amyloglucosidase-antibody conjugate and enzyme cycling assay was then compared to that of conventional enzyme immunoassay (EIA).^ For immune complex isolation, polystyrene tubes and protein A-bearing Staphylococcus aureus were evaluated as solid phase matrices, upon which antibodies can be immobilized. A sandwich-type EIA, using antibody-coated S. aureus, was developed that measures human albumin (HSA) in the nanogram range. The assay, using an alkaline phosphatase-anti-HSA conjugate, was applied to the determination of HSA in human urine and evaluated extensively for its clinical applicability.^ Finally, in view of the clinical significance of alpha-fetoprotein (AFP) as an oncofetal antigen and the difficulty with its purification for use as an immunogen and assay standard, a chemical purification protocol was developed that resulted in a high yield of immunochemically pure AFP. ^

NADPH-CYTOCHROME P-450 REDUCTASE: EVIDENCE FOR TWO SEPARATE STRUCTURAL DOMAINS AND ISOLATION AND CHARACTERIZATION OF THE MEMBRANE ASSOCIATED SEGMENT

Homogenous detergent-solubilized NADPH-Cytochrome P-450 reductase was incorporated into microsomes and liposomes. This binding occurred spontaneously at temperatures between 4(DEGREES) and 37(DEGREES) and appeared to involve hydrophobic forces as the binding was not disrupted by 0.5 M sodium chloride. This exogenously-added reductase was active catalytically towards native cytochrome P-450, suggesting an association with the microsomal membrane similar to endogenous reductase. Homogeneous detergent-solubilized reductase was disaggregated by Renex-690 micelles, confirming the presence of a hydrophobic combining region on the enzyme. In contrast to these results, steapsin protease-solubilized reductase was incapable of microsomal attachment and did not interact with Renex-690 micelles. Detergent-solubilized reductase (76,500 daltons) was converted into a form with the electrophoretic mobility of steapsin protease-solubilized reductase (68,000 daltons) and a 12,500 dalton peptide (as determined by polyacrylamide-SDS gel electrophoresis) when the liposomal-incorporated enzyme was incubated with steapsin protease. The 68,000 dalton fragment thus obtained had properties identical with steapsin protease-solubilized reductase, i.e. it was catalytically active towards cytochrome c but inactive towards cytochrome P-450 and did not bind liposomes. The 12,500 dalton fragment remained associated with the liposomes when the digest was fractionated by gel filtration, suggesting that this is the segment of the enzyme which is embedded in the phospholipid bilayer. Thus, detergent-solubilized reductase appears to contain a soluble catalytic domain and a separate and separable membrane-binding domain. This latter domain is required for attaching the enzyme to the membrane and also to facilitate the catalytic interaction between the reductase and its native electron acceptor, cytochrome P-450. The membrane-binding segment of the reductase was isolated by preparative gel electrophoresis in SDS following its generation by proteolytic treatment of liposome-incorporated reductase. The peptide has a molecular weight of 6,400 as determined by gel filtration in 8 M guanidine hydrochloride and has an amino acid composition which is not especially hydrophobic. Following removal of SDS and dialysis out of 6 M urea, the membrane-binding peptide was unable to inhibit the activity of a reconstituted system containing purified reductase and cytochrome P-450. Moreover, when reductase and cytochrome P-450 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptide were used. Thus, the membrane-binding peptide was ineffective as an inhibitor of mixed function oxidase activity, suggesting perhaps that it facilitates catalysis by anchoring the catalytic domain of the reductase proximal to cytochrome P-450 (i.e. in the same mixed micelle) rather than through a specific interaction with cytochrome P-450. ^

DRUG METABOLISM IN LIVER TUMORS: PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME-P450 REDUCTASE AND THE RESOLUTION OF MULTIPLE FORMS OF CYTOCHROME P-450

The hydroxylation of N- and O-methyl drugs and a polycyclic hydrocarbon has been demonstrated in microsomes prepared from two transplantable Morris hepatomas (i.e., 7288C. t.c. and 5123 t.c.(H). The hydroxylation rates of the drug benzphetamine and the polycyclic hydrocarbon benzo {(alpha)} pyrene by tumor microsomes were inducible 2 to 3-fold and 2-fold, respectively by pretreatment of rats with phenobarbital/hydrocortisone. Hepatoma 5123t.c.(h) microsomal hydroxylation activities were more inducible after these pretreatments than hepatoma 7288C.t.c. Two chemotherapeutic drugs (cyclophosphamide and isophosphamide) were shown to be mutagenic after activation by the tumor hemogenate with the TA100 strain of Salmonella typhimurium bacteria. NADPH-cytochrome P-450 was purified from phenobarbital/hydrocortisone treated rat hepatoma 5123t.c.(H) microsomes 353-fold with a specific activity 63.6 nmol of cytochrome c reduced per min per mg of protein. The purified enzyme, has an apparent molecular weight of 79,500 daltons, and contained an equal molar ratio of FMN and FAD, with a total flavin content of 16.4 nmol per mg of protein. The purified enzyme also catalyzed electron transfer to artificial electron acceptors with the K(,m) values of the hepatoma reductase similar to those of purified liver reductase. The K(,m) value of the hepatoma reductase (13 uM) for NADPH was similar to that of purified liver reductase (5.0 uM). In addition the purified hepatoma reductase was immunochemically similar to the liver reductase.^ Hepatoma cytochrome P-450, the hemeprotein component of the hepatoma microsomes of rats pretreated with phenobarbital/hydrocortisone. The resolution of the six forms was achieved by the DE-53 ion-exchange chromatography, and further purified by hydroxyapatite. The six different fractions that contained P-450 activity, had specific contents from 0.47 to 1.75 nmol of cytochrome P-450 per mg of protein, and indicated a 2 to 9-fold purification as compared to the original microsomes. In addition, difference spectra, molecular weights and immunological results suggest there are at least six different forms of cytochrome P-450 in hepatoma 5123 t.c.(H). ^