Classical Cytochrome P450 Research Articles

Cytochrome P450 1B1, a new keystone in gene-environment interactions related to human head and neck cancer?

Alcohol consumption and tobacco smoking are major causes of head and neck cancers, and regional differences point to the importance of research into gene-environment interactions. Much interest has been focused on polymorphisms of CYP1A1 and of GSTM1 and GSTT1, but a number of studies have not demonstrated significant effects. This has mostly been ascribed to small sample sizes. In general, the impact of polymorphisms of metabolic enzymes appears inconsistent, with some reports of weak-to-moderate associations, and with others of no elevation of risks. The classical cytochrome P450 isoenzyme considered for metabolic activation of polycyclic aromatic hydrocarbons (PAH) is CYP1A1. A new member of the CYP1 family, CYP1B1, was cloned in 1994, currently representing the only member of the CYP1B subfamily. A number of single nucleotide polymorphisms of the CYP1B1 gene have been reported. The amino acid substitutions Val432Leu ( CYP1B1*3) and Asn453Ser ( CYP1B1*4), located in the heme binding domain of CYP1B1, appear as likely candidates to be linked with biological effects. CYP1B1 activates a wide range of PAH, aromatic and heterocyclic amines. Very recently, the CYP1B1 codon 432 polymorphism ( CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squamous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has pointed to interactive effects. This provides new molecular evidence that tobacco smoke-specific compounds relevant to head and neck carcinogenesis are metabolically activated through CYP1B1 and is consistent with a major pathogenetic relevance of PAH as ingredients of tobacco smoke.

Induction by xenobiotics of phase I and phase II enzyme activities in the human keratinocyte cell line NCTC 2544

This study analyses the expression and induction of several drug-metabolising enzyme activities involved in either phase I or phase II biotransformations in NCTC 2544 human keratinocytes. The phase I activities 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depenthylase (PROD) were easily detectable in basal conditions. During incubations lasting up to 144 h in the presence of the classical cytochrome P450 inducers β-naphthoflavone (BNF), 3-methylcholanthrene (MC) and phenobarbital (PB), a considerable and significant increase in all the three activities was observed. PROD activity was induced up to 4.5-fold after 96 h in the presence of PB. The MC-induced ECOD and EROD activities were also dose-dependently inhibited by α-naphothflavone, which was given to the cells during the incubation with CYP 1A1 inducers. Also the PB-induced PROD activity was decreased by the simultaneous addition of the CYP 2B inhibitor metyrapone. Both cytochrome P450 inhibitors were used at non-cytotoxic concentrations. The phase II enzymes glutathione S-transferase, aldehyde dehydrogenase and quinone reductase were all highly expressed and inducible by MC. The exposure (24 h) of the cells to four hair dyes used in cosmetic formulations resulted in a marked increase in ECOD activity. All data give sustained evidence for the suitability of NCTC 2544 cell line to skin toxicology studies.

The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism.

Organisms encounter a wide range of foreign compounds--or 'xenobiotics'--with potentially harmful consequences. The cytochrome P450 (CYP) enzymes metabolize xenobiotics and thus are a primary defence against these compounds. Increased expression of specific CYP genes in response to particular xenobiotics is a central component of this defence, although such induction can also increase production of toxic metabolites. Here we show that the nuclear receptor CAR mediates the response evoked by a class of xenobiotics known as the 'phenobarbital-like inducers'. The strong activation of Cyp2b10 gene expression by phenobarbital, or by the more potent TCPOBOP, is absent in mice lacking the CAR gene. These animals also show decreased metabolism of the classic CYP substrate zoxazolamine and a complete loss of the liver hypertrophic and hyperplastic responses to these inducers. Cocaine causes acute hepatotoxicity in wild-type mice previously exposed to phenobarbital-like inducers and this toxicity is also absent in the CAR-deficient animals. Thus, loss of CAR function alters sensitivity to toxins, increasing or decreasing it depending on the compound. Modulation of CAR activity in humans may significantly affect metabolism of drugs and other xenobiotics.

The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells.

The polarized processes of cell elongation play a crucial role in morphogenesis of higher plants. We reported previously that the rotundifolia3 (rot3) mutant of Arabidopsis has a defect in the polar elongation of leaf cells. In the present study, we isolated two additional alleles with mutations in the ROT3 gene. The ROT3 gene was cloned by a T-DNA-tagging method and isolation of the gene was confirmed by a molecular analysis of three rot3 mutant alleles obtained from different mutagenesis. The ROT3 gene encodes a cytochrome P-450 (CYP90C1) with domains homologous to regions of steroid hydroxylases of animals and plants. Expression of the ROT3 gene was detected in all major plant organs. Especially, higher expression was detected in the tissues that had high activity of cell division. We confirmed that the ROT3 gene controls polar elongation specifically in leaf cells by an analysis of three rot3 mutants obtained from different mutagenesis experiments. Our results imply that the ROT3 protein is a member of a new class of cytochrome P-450 encoding putative steroid hydroxylases, which is required for the regulated polar elongation of cells in leaves of Arabidopsis.

Effects of cytochrome P450 inducers on tamoxifen genotoxicity in female mice in vivo

We recently reported that administration of the antiestrogen tamoxifen (TAM) gives rise to two groups of DNA adducts in female mouse liver in vivo, as measured by 32P-postlabeling, and provided evidence that 4-hydroxytamoxifen and α-hydroxytamoxifen are proximate carcinogenic metabolites leading to group I and group II adducts, respectively (Randerath et ed., Carcinogenesis 15: 2087-2094, 1994). Because cytochrome P450 (CYP) enzymes play an important role in TAM metabolism, in this investigation we tested the hypothesis that induction of liver CYP enzymes may affect TAM metabolism profoundly, resulting in increased or decreased TAM-DNA adduct formation in vivo. To this end, we treated female ICR mice with TAM either alone or in combination with one of several classic CYP inducers, i.e. phenobarbital (PB), ß-naphthoflavone (BNF), and pregnenolone-16α-carbonitrile (PCN), and determined the levels of 32P-postlabeled TAM-DNA adducts and the activities of several CYP-dependent enzymes. Each of the inducers greatly diminished levels of group II, but did not affect group I adducts. TAM elicited induction of benzphetamine N-demethylase activity in liver, while activities of other enzymes were not affected. TAM, when given in combination with BNF, elicited a synergistic induction of ethoxyresorufin O-deethylase (EROD) (CYP1A1) and methoxyresorufin O-demethylase (MROD) (CYP1A2) activities. Likewise, PCN given along with TAM caused synergistic induction of EROD and ethylmorphine N-demethylase activities. There was no synergism between PB and TAM, however. Overall, the results further support the existence of two pathways of TAM metabolism to DNA-reactive electrophiles and strongly suggest that the classic CYP inducers tested enhance detoxication of TAM to non-genotoxic metabolites.

Characterization of Inducible Nitric-oxide Synthase by Cytochrome P-450 Substrates and Inhibitors: INHIBITION BY CHLORZOXAZONE

Nitric-oxide synthases (NOS, EC 1.14.13.39) are heme-containing enzymes that catalyze the formation of nitric oxide from L-Arg. General cytochrome P-450 inhibitors and cytochrome P-450 isoform-selective substrates and inhibitors were used to characterize the activity of recombinant human inducible NOS (iNOS). Classical cytochrome P-450 ligands such as the mechanism-based inactivator 1-aminobenzotriazole did not inhibit iNOS. Of a panel of 30 human cytochrome P-450 isoform-selective substrates and inhibitors, only chlorzoxazone, a cytochrome P-450 2E1 (CYP2E1) substrate, showed any significant inhibition of iNOS activity. Chlorzoxazone was not a substrate for iNOS but was a potent competitive inhibitor with respect to L-Arg with Ki = 3.3+/-0.7 microM. The binding of chlorzoxazone to iNOS and human and rat liver microsomal cytochrome P-450 induced a high spin, type I spectra, which was reversed by imidazole. Although the binding of chlorzoxazone to iNOS and its inhibition of iNOS activity suggest some similarity between iNOS and CYP2E1 activity, other CYP2E1 substrates and inhibitors including zoxazolamine were not inhibitors of iNOS. Overall, iNOS activity is distinctly different from the major cytochrome P-450 enzymes in human liver microsomes.

Developmental regulation of flavin-containing monooxygenase (FMO) isoforms 1 and 2 in pregnant rabbit

Mammalian flavin-containing monooxygenase (FMO, EC 1.14.13.8) metabolizes a vast number of structurally diverse xenobiotics containing a soft-nucleophile, typically a nitrogen or sulfur. FMO is not inducible by the classical cytochrome P450 (CYP) inducers, such as phenobarbital, polycyclic aromatic hydrocarbons, ethanol or macrolide antibiotics. Evidence does exist from a number of laboratories, however, for developmental and hormonal regulation of FMO. Our laboratory has confirmed previous observations of enhanced FMO activity during mid- and late-gestation in maternal rabbit lung and have demonstrated that this response is due to increased protein and catalytic activity associated with FMO2. The time course of expression of FMO2 during mid- and late-gestation correlates to plasma peaks of progesterone or Cortisol. FMO2 also peaks at parturition in maternal kidney, coincident with plasma cortisol levels. FMO2 is induced by s.c. administration of either progesterone or dexamethasone in lung, or by dexamethasone in kidney. Correlation of plasma progesterone or cortisol levels during gestation and postpartum support a role for progesterone, but not cortisol in regulation of FMO2 in maternal rabbit lung. The levels of FMO1 also appear to be increased during mid- and late-gestation in liver. FMO1 in liver may also be regulated during gestation by progesterone or glucocorticoids as administration of these steroids enhanced FMO1 mRNA levels 4-fold.

The proximal pathway of metabolism of the chlorinated signal molecule differentiation-inducing factor-1 (DIF-1) in the cellular slime mould Dictyostelium

Stalk cell differentiation during development of the slime mould Dictyostelium is induced by a chlorinated alkyl phenone called differentiation-inducing factor-1 (DIF-1). Inactivation of DIF-1 is likely to be a key element in the DIF-1 signalling system, and we have shown previously that this is accomplished by a dedicated metabolic pathway involving up to 12 unidentified metabolites. We report here the structure of the first four metabolites produced from DIF-1, as deduced by m.s., n.m.r. and chemical synthesis. The structures of these compounds show that the first step in metabolism is a dechlorination of the phenolic ring, producing DIF metabolite 1 (DM1). DM1 is identical with the previously known minor DIF activity, DIF-3. DIF-3 is then metabolized by three successive oxidations of its aliphatic side chain: a hydroxylation at omega-2 to produce DM2, oxidation of the hydroxy group to a ketone group to produce DM3 and a further hydroxylation at omega-1 to produce DM4, a hydroxyketone of DIF-3. We have investigated the enzymology of DIF-1 metabolism. It is already known that the first step, to produce DIF-3, is catalysed by a novel dechlorinase. The enzyme activity responsible for the first side-chain oxidation (DIF-3 hydroxylase) was detected by incubating [3H]DIF-3 with cell-free extracts and resolving the reaction products by t.l.c. DIF-3 hydroxylase has many of the properties of a cytochrome P-450. It is membrane-bound and uses NADPH as co-substrate. It is also inhibited by CO, the classic cytochrome P-450 inhibitor, and by several other cytochrome P-450 inhibitors, as well as by diphenyliodonium chloride, an inhibitor of cytochrome P-450 reductase. DIF-3 hydroxylase is highly specific for DIF-3: other closely related compounds do not compete for the activity at 100-fold molar excess, with the exception of the DIF-3 analogue lacking the chlorine atom. The Km for DIF-3 of 47 nM is consistent with this enzyme being responsible for DIF-3 metabolism in vivo. The two further oxidations necessary to produce DM4 are also performed in vitro by similar enzyme activities. One of the inhibitors of DIF-3 hydroxylase, ancymidol (IC50 67 nM) is likely to be particularly suitable for probing the function of DIF metabolism during development.

Characterization of Rat Cytochrome P450 Isozymes Involved in the Covalent Binding of Cyclosporine A to Microsomal Proteins

Cyclosporin A (CsA) is an immunosuppressant drug which is extensively metabolized by the hepatic microsomal monooxygenases. Among other toxicities, CsA is nephrotoxic and hepatotoxic. In the present study, the NADPH-dependent cytochrome P450-supported metabolism of CsA to reactive metabolite(s) capable of covalently binding to proteins was studied. The covalent binding was inhibitable in vitro with classical cytochrome P450 inhibitors. The covalent binding of CsA metabolite(s) was induced six- to eightfold in liver microsomes from rats of both sexes treated with dexamethasone, suggesting that a P450 3A-related protein was involved in the covalent binding of CsA metabolite(s). However, the isozyme responsible was not P450 3A1 or 3A2, since inhibitory monoclonal antibodies to these isozymes did not inhibit the covalent binding. The binding was, however, inhibitable in vitro with cytochrome P450 3A substrates and inhibitors such as erythromycin and triacetyloleandomycin. Greater amounts of CsA covalent binding occurred in liver microsomes from adult uninduced female rats than males or immature rats of either sex. Therefore, a female-specific isozyme of P450 present in adult female rat liver microsomes, which may or may not be identical to a dexamethasone-inducible isozyme, is also involved in the metabolism of CsA to form covalent binding metabolites. The covalent binding of CsA was 50% inhibited by glutathione. However, mannitol and superoxide dismutase did not affect the binding. This suggested that at least some of the metabolites of CsA involved in covalent binding were electrophilic in nature; however, hydroxyl radicals and superoxide anion radicals were not involved.

Heme-heme oxygenase complex. Structure of the catalytic site and its implication for oxygen activation.

Heme oxygenase, a central monooxygenase enzyme of the heme catabolism and the associated generation of carbon monoxide, forms a 1:1 stoichiometric complex with iron protoporphyrin IX, which is a prosthetic active center and at the same time the substrate of the enzyme. By using EPR, resonance Raman, and optical absorption spectroscopic techniques, we have determined the axial ligand coordination of the enzyme-heme complex. The ferric heme iron in the heme-enzyme complex at neutral pH is six-coordinate high spin, while at alkaline pH (pKa 7.6), the complex becomes low spin. Spectra of ferrous forms of the complex indicate that histidine serves as the iron proximal axial ligand and that the residue is in its neutral imidazole rather than its imidazolate protonation state. Thus, the active site of the heme-heme oxygenase complex has a myoglobin-like structure rather than an active site similar to the large cytochrome P-450 class of monooxygenases. As a consequence, the activated form of the heme-heme oxygenase complex, a peroxo intermediate, is different from that of the cytochrome P-450 monooxygenases, in which the activated form is an oxo intermediate. The overall catalytic mechanism is probably more closely related to that of other monooxygenases with myoglobin-like active sites, such as secondary amine monooxygenase.

Inhibition by SKF-525A of the aldehyde oxidase-mediated metabolism of the experimental antitumour agent acridine carboxamide

Oxidation of the experimental anti-tumour agent N-[(2'-dimethylamino) ethyl]acridine-4-carboxamide (AC; NSC 601316; acridine carboxamide) to the 9(10 H)acridone, followed by ring hydroxylation and glucuronidation, appears to be the main pathway of detoxication of AC in the rat and mouse. The acridone formation has been further characterized in vitro using an enzyme-enriched fraction where activity per milligram protein is increased approximately 10-fold compared with the cytosolic fraction. Inhibition by amsacrine [4'-(9-acridinylamino)methanesulphon- m-anisidide; NSC 249992] and menadione (50% inhibition at 6.4 and 1.8 μM, respectively) but not allopurinol (to 30μM) indicates that the activity is due to aldehyde oxidase, without the involvement of xanthine oxidase. Interestingly, acridone formation in both the cytosolic and enzyme-enriched fractions is highly sensitive to the classical cytochrome P450 inhibitor SKF-525A [proadifen hydrochloride; 2'-(diethylamino)ethyl 2,2-diphenylpentenoate] (50% inhibition at 9.2 and 1.9 μM, respectively). Further analysis indicates mixed non-competitive type inhibition by SKF-525A ( K is , 0.3 μM; K ii , 4.9 μM). Little or no inhibition was seen with cimetidine, metyrapone or methimazole. No NADPH-dependent acridone formation was observed with the microsomal fraction. These data indicate that acridone formation previously observed in isolated rat hepatocytes and in vivo is most likely due to aldehyde oxidase rather than cytochrome P450.

Interaction of cimetidine with cytochrome P450 and effect on mixed-function oxidase activities of liver microsomes.

The histamine H2-receptor antagonist drug, cimetidine (CM), was investigated to determine its effect on the metabolism of 'model' alkoxyphenoxazone substrates ethoxyresorufin (ER) and pentoxyresorufin (PR). The investigation was carried out under different conditions in rat liver microsomes from rodents pretreated with various classical cytochrome P450 inducers. CM exerted a dual effect on uninduced and PB-induced liver microsomal ethoxyresorufin-O-de-ethylase (EROD) activities; it was initially stimulatory but became inhibitory. However, when 3-MC-induced preparations were used. CM only exerted an inhibitory effect on EROD activity over the whole concentration range (0.01 microM-20 mM). Pre-incubating PB-induced and uninduced liver microsomes with NADPH before the addition of ER and CM decreased the stimulatory effect of CM and increased the inhibitory effect in the concentration range (5-20 mM). With 3-MC-induced preparations, the inhibition of EROD was only marginally potentiated. Overall conclusions for the diphasic effects of CM on biological activities were due to CM binding with differing affinities to different P450s. Subsequent decreases and increases in stimulation and inhibition, respectively, on pre-incubation with NADPH were thought to be due to an increased affinity of CM for reduced cytochrome P450.

Evidence for tangeretin O-demethylation by rat and human liver microsomes.

1. Tangeretin, a polymethoxylated flavone, was studied as a substrate for cytochrome P450-catalysed demethylation reactions by rat and human liver microsomes. Evidence has been presented for the production of formaldehyde in the presence of tangeretin and NAD(P)H. Kinetic studies showed a Km value for tangeretin of about 18 microM in both species. 2. The reaction was inhibited by CO, piperonyl butoxide, 7,8-benzoflavone, propyl gallate, aminobenzothiazole and metyrapone. 3. Rats pretreated with classical cytochrome P450 inducers (Aroclor 1254, 3-methylcholanthrene, phenobarbital, dexamethasone and ciprofibrate) or with flavonoids (flavone, flavanone, quercetin and tangeretin) resulted in increased microsomal demethylation of tangeretin after 3-methylcholanthrene and flavone only. Tangeretin did not enhance its own metabolism. 4. Tangeretin interacted with the oxidized form of cytochrome P450 to produce a reverse type I spectrum. 5. Results indicate that tangeretin is metabolized in liver microsomes by an O-demethylation reaction involving cytochrome P450.

DNA binding and mutagenicity of aflatoxin B1 catalyzed by isolated rabbit lung cells.

The abilities of different rabbit lung cell types to bioactivate aflatoxin B1 (AFB1) to a DNA-binding and mutagenic metabolite have been examined. Microsomes were prepared from centrifugal elutriation-enriched preparations of isolated rabbit lung cell types. The activation of [3H]AFB1 (5.0 or 200 microM), measured indirectly as covalent binding to calf thymus DNA, was concentrated in microsomes from the non-ciliated bronchiolar epithelial (Clara) cell-rich fractions (13-22 times the activity of whole lung microsomes). Microsomes from type II cell-rich fractions had minimal activity. Significant correlations were detected between the rates of microsomal DNA binding and the percentages of Clara cells in the fractions. Prior treatment of rabbits with the cytochrome P450 class 1A inducer beta-naphthoflavone had no significant effect on the microsomal activation of AFB1. In other experiments, intact, enriched isolated rabbit lung cells were incubated with AFB1 (0-1.5 microM) in a modification of the Ames mutagenicity assay, using Salmonella typhimurium strain TA100. The ability to activate AFB1 to mutagenic metabolite(s) in this system was localized in Clara cell-rich fractions, with no significant activity being detected in other fractions. The results of these studies indicate that the biotransformation of AFB1 to DNA-binding and mutagenic metabolite(s) in rabbit lung is heterogeneous, and that the Clara cell is specifically implicated in this ability.

Microsomal lauric acid hydroxylase activities after treatment of rats with three classical cytochrome P450 inducers and peroxisome proliferating compounds

In order to investigate a proposed relationship between induction of hepatic microsomal lauric acid hydroxylase activity and peroxisome proliferation in the liver, male Wistar rats were treated with peroxisome proliferating compounds, and the lauric acid hydroxylase activity, the immunochemical detectable levels of cytochrome P450 4A1 and the activities of peroxisomal enzymes were determined. In addition, the levels of cytochrome P450 4A1 and lauric acid hydroxylase activities were studied after treatment of rats with three cytochrome P450 inducers. After treatment with aroclor-1254, phenobarbital or 3-methylcholanthrene total cytochrome P450 was 1.7–2.7 times induced. However, no induction of lauric acid ω-hydroxylase activities or P450 4A1 levels were found. After treatment of rats with di(2-ethylhexyl)phthalate (DEHP) a dose-dependent induction of lauric acid ω-hydroxylase activities, levels of cytochrome P450 4A1 and peroxisomal fatty acid β-oxidation was found. Even at a dose-level of 100 mg DEPH/kg body weight per day a significant induction of these activities was observed. the main metabolites of DEHP, mono(2-ethylhexyl)phthalate and 2-ethyl-1-hexanol, also caused an induction of levels of P450 4A1, lauric acid ω-hydroxylase activities and the activity of peroxisomal palmitoyl-CoA oxidase. 2-Ethyl-1-hexanoic acid did not influence lauric acid ω-hydroxylase activities, but did induce levels of P450 4A1 and palmitoyl-CoA oxidase activities. Three other compounds (perfluoro-octanoic acid, valproate and nafenopin) induced both lauric acid ω-hydroxylase activity and peroxisomal palmitoyl-CoA oxidase activity. the plasticizer, di(2-ethylhexyl)adipate, did not induce leveis of P450 4A1, lauric acid ω-hydroxylase activities or palmitoyl-CoA oxidase activities. With the compounds tested a close association between the induction of lauric acid ω-hydroxylase activities and peroxisomal palmitoyl-CoA oxidase activity was found. These data support the theory that peroxisome proliferating compounds do induce lauric acid ω-hydroxylase activities and that there might be a mechanistic inter-relationship between peroxisome proliferation and induction of lauric acid ω-hydroxylase activities.

Role of heme in phenobarbital induction of cytochromes P450 and 5-aminolevulinate synthase in cultured rat hepatocytes maintained on an extracellular matrix

When hepatocytes are cultured on matrigel, a reconstituted basement membrane matrix, mRNAs for cytochrome P450 class IIB1/2 and class III genes can be induced by treatment with phenobarbital. We took advantage of this new system to critically evaluate the role of heme as a regulator of these cytochromes P450 and of 5-aminolevulinate synthase (ALA-S), the rate-limiting enzyme in heme biosynthesis. Phenobarbital treatment of rat cultures increased the total amount of cytochrome P450, activities catalyzed by IIB1/2 (benzyloxy- and pentoxyresorufin O-dealkylases) and ALA-S activity, and ALA-S mRNA. Treatments with phenobarbital combined with succinyl acetone, an inhibitor of heme biosynthesis at the step of 5-aminolevulinate dehydrase, blocked the induction of the proteins for cytochrome P450IIB1/2 and cytochrome P450IIIAI, as indicated by spectral, immunological, and enzymatic assays. However, at the same time, succinyl acetone cotreatment failed to inhibit the induction of the mRNAs for cytochrome P450IIB1/2 and cytochrome P450IIIA. Lack of effect on the cytochrome P450 mRNAs was selective inasmuch as treatment with phenobarbital combined with succinyl acetone synergistically increased both ALA-S activity and ALA-S mRNA, presumably by blocking formation of heme, the feedback repressor of ALA-S. Indeed, the increase in ALA-S mRNA caused by the combined treatment was abolished by adding heme itself to the cultures. In contrast to earlier concepts, we conclude that in the intact hepatocyte, phenobarbital-induced cytochrome P450 induction is independent of changes in heme synthesis.

Cytochrome P450 activity and distribution in the human colon mucosa.

Enzymatic activity associated with the mixed-function oxidase system was determined in microsomes prepared from the mucosal cells extracted from normal human colons. A high activity toward nitrogen oxidation reactions was observed. 1,2-Dimethylhydrazine, a colon-specific carcinogen, was metabolized at a higher rate in vitro by human colon microsomes as compared with the rat, and exhibited a km ten-fold lower, 1.03 mmol/l versus 9.68 mmol/l, respectively. This activity was inhibited by classic cytochrome P450 inhibitors; 70% inhibition was achieved using 70 mmol/l metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone), 20 mmol/l; SKF-525A (diethylaminoethyl-2,-2-diphenylvalerate HCl), or 350 mumol/l n-octylamine. These data suggest the presence of a stable, active mixed-function oxidase system in the human colon mucosa which has a preferential activity toward nitrogenous compounds and provides a mechanism for the activation of carcinogens. Its distribution in the colon appears to parallel the reported incidence of human colonic carcinomas.

Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria (insecta, orthoptera)

It is most generally accepted that the last three enzymatic reactions in the biosynthetic pathway of ecdysone are, in this order, the hydroxylations at positions C-25, C-22 and C-2. Using high specific activity tritiated ecdysone precursors (2,22,25-trideoxyecdysone, 2,22-dideoxyecdysone and 2-deoxyecdysone) we have characterized the hydroxylases involved in these reactions, in the major biosynthetic tissue of ecdysone, i.e. the prothoracic glands. We show that C-2 hydroxylase is a mitochondrial oxygenase which differs from conventional cytochrome P-450-dependent monooxygenases by its relative insensitivity to CO. In contrast, C-22 and C-25 hydroxylases appear as classical cytochrome P-450 monooxygenases; C-22 hydroxylase is a mitochondrial enzyme whereas our data point to a microsomal localization of the C-25 hydroxylase.

Effects of chemical and enzymic probes on microsomal covalent binding of bromobenzene and derivatives. Evidence for quinones as reactive metabolites.

1. The chemical reactivity of bromobenzene metabolite(s) responsible for its protein covalent binding was investigated by determining the effects of many chemical and enzymic probes on the metabolism and covalent binding of [3,5-3H]bromobenzene with rat liver microsomes in vitro. 2. Classical cytochrome P-450 enzyme inhibitors decreased both metabolism and binding in parallel, whereas scavenging agents for reactive oxygen species and free radicals exhibited little or no effect. Sulphur nucleophiles were extremely efficient in decreasing binding with little or no effect on metabolism. Reducing agents such as ascorbate and diaphorase decreased binding slightly more than metabolism. 3. UDP-Glucuronic acid inhibited neither metabolism nor binding, but all three mono-bromophenols decreased binding more than metabolism. Trichloropropene oxide was unique in decreasing metabolism more than binding. 4. The effects of ascorbate, glutathione, bisulphite and butylated hydroxytoluene (BHT) on metabolism and binding of five ortho-substituted bromobenzene derivatives (o-BrC6H4X; X = OCH3, CH3, Br, CF3, and CN) were similar to their effects on the metabolism and binding of bromobenzene. 5. Collectively these results support a major role for quinones as the reactive metabolites responsible for the majority of the protein covalent binding of bromobenzene and its ortho-substituted derivatives in microsomal systems in vitro.

Rat liver subcellular fractions catalyze aerobic binding of 1-nitro[14C]pyrene to DNA.

The recently characterized environmental mutagen and potential carcinogen 1-nitropyrene (NP) is known to bind DNA in Salmonella typhimurium, and also in anaerobic incubations catalyzed by purified xanthine oxidase. In this study we show that rat liver S9 supernatant, microsomal and cytosolic subcellular fractions are also able to catalyze the binding of 1-nitropyrene labeled with 14C to calf thymus DNA in vitro. In incubations conducted under air, S9 and microsomes from Charles River CD rats were the most active fractions, and NADPH was required for maximum activity (25-100 pmole NP bound/mg DNA/mg protein in 1 hr). S9 and microsomes had about one-fourth the activity under nitrogen, although less of this activity was NADPH-dependent. Binding in cytosolic incubations was generally low (1 to 5 pmole NP/mg DNA/mg protein in 1 hr), was somewhat enhanced under N2, and was more extensive in the absence of NADPH. Treatment of rats (Harlan Sprague-Dawley) with the inducing agents phenobarbital (PB), Aroclor 1254 (A), or 3-methylcholanthrene (3-MC) enhanced NADPH-dependent binding in aerobic S9 (2- to 5-fold) and microsomal (10- to 20-fold) incubations. The effects of induction regimen on binding assays conducted under N2 were more equivocal: 3-MC produced a 2-fold increase in binding in both S9 and microsomes, while the other two agents decreased binding from 50 to 75%. These results indicate that classic cytochrome P-450 inducers were able to stimulate activation of NP, but that this activation is not mediated solely by cytochrome P-450.