Cytochrome P-450 Species Research Articles

Electronic Structure Makes a Difference: Cytochrome P-450 Mediated Hydroxylations of Hydrocarbons as a Two-State Reactivity Paradigm

This paper describes a reactivity paradigm called two-state reactivity (TSR) in C ± H bond activation by metal oxenoid cations (e.g., FeO‡). The paradigm is applied to the hydroxylation of alkanes by the active species of the enzyme cytochrome P-450, and a mechanistic scheme is proposed based on the competition between TSR pathways and single-state-reactivity (SSR) pathways. Generally, the oxide cations of the late transition metals (MO‡) possess the same bonding patterns as the O2 molecule, having a high-spin ground state and an adjacent low-spin excited state. The adjacency of the spin states, together with the poor bonding capability of the high-spin state and the good bonding capability of the low-spin state, leads to a spin crossover along the reaction coordinate and opens a low-energy TSR path for hydroxylation. The competing pathway is SSR, in which the reaction starts, occurs and ends in the same spin state. The TSR/SSR competition is modulated by the probability of spin crossover. Generally, TSR involves concerted pathways that conserve stereochemical information, while SSR results in stepwise mechanisms that scramble this information. The TSR/SSR competition is used to shed some light on recent results which are at odds with the commonly accepted mechanism of P-450 hydroxylation. The fundamental features of the paradigm are outlined and the theoretical and experimental challenges for its articulation are spelled out.

Cytochrome P450 Monooxygenases Metabolizing Herbicides

ABSTRACTCytochrome P450 monooxygenases catalyze oxidative reactions in biosynthesis of secondary metabolites as well as in metabolism of xenobiotics in higher plants. The enzyme system localized on the microsomes of plant cells consists of many cytochrome P450 (P450 or CYP) species and one or a few species of NADPH-cytochrome P450 reductase (P450 reductase). Particularly, P450 species metabolizing herbicides play an important role in r herbicide selectivity and resistance. However, molecular information on plant P450 species metabolizig herbicides was quite limited. It. was found that tobacco cultured S401 cells treated with 2,4-D metabolized chlortoluron to give ring-methyl hydroxylated and N-demethylated metabolites. Then, we cloned P450 cDNAs from the S401 cells treated with 2,4-D by RT-PCR methods. Among four novel P450 cDNA clones, CYP71A11 and CYP81B2 were each expressed in the yeast Saccharomyces cerevisiae and were found to catalyze both ring-methyl hydroxylation and N-demethylation of chlortoluron, and ring-methylhydroxylation, respectively. It was also found that several mammalian P450 species including human P4501A1 metabolized chlortoluron through ring-methyl hydroxylation and N-demethylation, and atrazine through N-deal- kylation. When rat P450IA1 (CYP 1 Al) and its fused enzyme with yeast P450 reductase were each expressed in potato and tobacco plants, respectively, both transgenic plants showed resistance to the herbicides chlortoluron. Expression of a mammalian P450 species with overlapping broad substrate-specificity towards xenobiotics in plants seems to produce transgenic plants not only resistant to herbicides but also enhanced in metabolism of various environmental contaminants.

Effects of cytochrome P450 inhibitors and of steroid hormones on the formation of 7-hydroxylated metabolites of pregnenolone in mouse brain microsomes.

Hydroxylations of pregnenolone (PREG) at the 7 alpha- and 7 beta-positions have been reported in numerous murine tissues and organs and responsible cytochrome P450 (CYP) species await identification. Using thin layer chromatography and gas chromatography-mass spectrometry, we report identification of 7 alpha-hydroxy-PREG and 7 beta-hydroxy-PREG metabolites produced in mouse brain microsome digests and kinetic studies of their production with apparent KM values of 0.5 +/- 0.1 microM and 5.1 +/- 0.6 microM for 7 alpha- and 7 beta-hydroxylation respectively. Investigation of CYP inhibitors and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylations of PREG showed that: (i) different CYP were involved in 7 alpha- and 7 beta-hydroxylation of PREG because solely 7 alpha-hydroxylation was extensively inhibited by metyrapone, alpha-naphthoflavone, ketoconazole and 3 beta-hydroxysteroids, (ii) CYP 1A2, 2D6, 2B1 and 2B11 were not responsible for 7 alpha- and 7 beta-hydroxylation of PREG because respective specific inhibitors furafylline, quinidine and chloramphenicol triggered no inhibition, (iii) CYP 1A1 was responsible for only part of the 7 beta-hydroxylation of PREG because use of alpha-naphthoflavone, which inhibits specifically CYP 1A1, did not suppress entirely 7 beta-hydroxylation, while ketoconazole, metyrapone and antipyrine, which do not inhibit CYP 1A1, decreased part of the 7 beta-hydroxylation, (iv) 7 alpha-hydroxylation of PREG may be shared with other 3 beta-hydroxysteroids such as isoandrosterone and 5-androstene-3 beta,17 beta-diol which were strong inhibitors, but not with dehydroepiandrosterone which was a non-competitive inhibitor as weak as 3-oxosteroids, and (v) 7 beta-hydroxylation of PREG was not markedly changed by other steroids. Taken together, these findings will be of use for identification of the CYP species responsible for 7 alpha- and 7 beta-hydroxylation of PREG and for studies of their activities in brain.

Mechanism for intracellular localization of cytochrome P450.

Different molecular species of cytochrome P450 exist in two intracellular organella, the endoplasmic reticulum and mitochondria in eukaryotic cells. Microsomal P450s possess uncleavable signal sequences whose orientation in the membrane is inverted to the signal peptides of secretory proteins. Their unique function is dependent on the balance between the hydrophobic segment and its amino-terminal charges. Mitochondrial P450s possess extension peptides which target the precursor molecules to mitochondria. The precursors are recognized, kept in an import competent state, and targeted to mitochondria by mitochondrial import stimulation factor (MSF).

Molecular Cloning and Expression of Rabbit Sterol 12α-Hydroxylase

Sterol 12alpha-hydroxylase is an important enzyme in bile acid biosynthesis, responsible for the balance between formation of cholic acid and chenodeoxycholic acid. The enzyme has been purified to apparent homogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, K., and Coon, M. J. (1992) J. Biol. Chem. 267, 21319-21323), and we here describe the cloning and sequencing of a cDNA coding for this enzyme. After tryptic digestion of purified protein in a polyacrylamide gel, eight different peptides were isolated and sequenced. Using oligonucleotides deduced from the amino acid sequences, clones were isolated from a rabbit liver cDNA library. In addition to several overlapping clones, one full-length clone was obtained that coded for a polypeptide of 500 amino acids, corresponding to a molecular mass of 57 kDa. All of the eight peptides and the reported NH2-terminal amino acid sequence were matched against the sequence. The peptide sequence showed a 39% similarity with human prostacyclin synthase (CYP8) and 31% similarity with the rate-limiting enzyme in over-all synthesis of bile acids, the cholesterol 7alpha-hydroxylase (CYP7) of the rabbit. The similarity with most other sterol cytochrome P-450 hydroxylases was less. Thus, this species of cytochrome P-450 should belong to a group of its own, here denoted CYP12. Transfection of COS cells with the coding part of the cDNA resulted in a significant expression of sterol 12alpha-hydroxylase activity toward 7alpha-hydroxy-4-cholesten-3-one. Northern blotting showed that the enzyme was exclusively expressed in the liver. The major mRNA fraction in rabbit liver had a size of approximately 2.9 kilobases, and those found in rat and human liver were about 2.5 and 4.5 kilobases, respectively. Fasting of rats and mice led to a severalfold increase in both enzyme activity and mRNA levels. In contrast, starvation of rabbits had little or no stimulatory effect on enzyme activity and mRNA levels.

Studies of the enzyme complex responsible for pregnenolone and dehydroepiandrosterone 7α-hydroxylation in mouse tissues

7α-Hydroxylation of pregnenolone (PREG) and dehydroepiandrosterone (DHEA) is known to take place in numerous tissues of mouse and rat. The responsible cytochrome P450 species has not yet been identified. Interest in the production of 7α-hydroxylated steroid derivatives results from their ability to increase the immune response in mice. Using crystallizations to constant specific activity and gas chromatography-mass spectrometry, 7α-hydroxy-PREG and 7α-hydroxy-DHEA metabolites produced by microsomes of liver, brain, thymus, and spleen were identified. Study of the 7α-hydroxylating enzyme in these tissues indicated that microsomes contained most of the activity, except for brain, where it was primarily mitochondrial. Production yields of 7α-hydroxy-PREG and 7α-hydroxy-DHEA by microsomes from heart, spleen, thymus, brain, and liver of 7-week-old mice were higher than those of 1-week-old and (except for liver) 41-week-old animals. At the optimal pH (7.4) and in all tested tissues but liver, microsomal 7α-hydroxylation was more extensive for PREG than for DHEA. With brain and thymus microsomes, K M were lower for PREG than for DHEA and decreased when phosphate was used instead of Tris buffer. With brain microsomes, the use of 1 mM EDTA increased 7α-hydroxylating activity. Complete inhibition was obtained with 0.1 mM Zn 2+ or Cu 2+ and with 1 mM Fe 2+ or Fe 3+. 7α-Hydroxylation of PREG was activated only by 0.5 mM Ca 2+ and that of DHEA only by 0.25 mM Mg 2+. Since the production rates of 7α-hydroxy-PREG and 7α-hydroxy-DHEA in tissues may be a key to the triggering of immune defenses, and since both 7α-hydroxylation and immunity decrease with aging, these data will prove to be useful in studies of the enzyme responsible and of the mechanisms that control its activity.

Cytochrome P450 species involved in the metabolism of quinoline.

Quinoline is a hepatocarcinogen in rats and mice and a well-known mutagen in bacteria after incubation with rat liver microsomes. The specific cytochrome P450 enzymes involved in quinoline metabolism in human and rat liver microsomes were determined using cDNA-expressed cytochrome P450s, correlations with specific cytochrome P450-linked monooxygenase activities in human liver microsomes and inhibition by specific inhibitors and antibodies. CYP2A6 is the principal cytochrome P450 involved in the formation of quinoline-1-oxide in human liver microsomes (correlation coefficient r = 0.95), but is formed in only minute quantities in rat liver microsomes. CYP2E1 is the principal cytochrome P450 involved in the formation of 3-hydroxyquinoline (r = 0.93) in human liver microsomes and is involved in the formation in rat liver microsomes. A high correlation coefficient (r = 0.91) between CYP2A6 activity and quinoline-5,6-diol formation in human liver microsomes was observed, but this most likely reflects the involvement of CYP2A6 in the formation of quinoline-5,6-epoxide, from which the quinoline-5,6-diol is formed, as conversion of quinoline-5,6-epoxide to quinoline-5,6-diol on incubation of the epoxide with CYP2A6 could not be demonstrated. A cDNA-expressed human microsomal epoxide hydrolase, however, efficiently converted the epoxide to the diol and the microsomal epoxide inhibitor cyclohexene oxide inhibited quinoline-5,6-diol formation in rat liver microsomes. A preliminary kinetic analysis of quinoline metabolism in human liver microsomes was carried out and Eadie-Hofstee plots indicate that the formation of quinoline-5,6-diol is monophasic, while that of quinoline-1-oxide and 3-hydroxyquinoline is biphasic.

Involvement of CYP2E in 8-hydroxylation of theophylline in mouse hepatic microsomes-difference from its N-demethylations.

We examined which cytochrome P-450 (P-450) species other than CYP1A participates in the oxidative metabolism of theophylline (TP) in mouse hepatic microsomes. Among the three metabolic pathways of TP, only 8-hydroxylation was selectively enhanced by acetone, a potent inducer of CYP2E. We assumed that two P-450 populations with different metabolic ability were involved in this metabolic process, and kinetic analyses revealed that the enhancement was due to the induction of a high-capacity P-450 population. The 8-hydroxylation at a substrate concentration, where most of the total activity was attributed to the catalysis of the high-capacity phase, was markedly impaired by CYP2E inhibitors such as 4-methylpyrazole and aminoacetonitrile, whereas the N-demethylations were little affected by these agents. The activity of TP 8-hydroxylation was significantly correlated with that of p-nitrophenol hydroxylation, a probe for CYP2E, in untreated microsomes. The activities of these oxidative reactions were modified to a similar degree by known enzyme inhibitors with a range of inhibitory potencies and affinity for P-450 isoforms. On the other hand, a relationship between TP N-demethylations and p-nitrophenol hydroxylation was not apparent, but there was a behavioral similarity between the two types of N-demethylations. The results indicated that TP 8-hydroxylation, which accounts for a large portion of TP oxidations, involves CYP2E, and that its N-demethylations are mediated by a common or closely similar P-450 species distinct from CYP2E.

Characterisation of taurochenodeoxycholic acid 6 alpha-hydroxylase from pig liver microsomes.

A fraction of cytochrome P-450 catalysing an efficient 6 alpha-hydroxylation of taurine-conjugated 3 alpha,7 alpha-dihydroxy-5 beta- cholanoic acid (taurochenodeoxycholic acid) was partially purified from pig liver microsomes. The specific content of cytochrome P-450 was 6 nmol/mg protein and the preparation showed two major protein bands upon SDS/PAGE. These two bands were isolated after SDS/PAGE and protein blotting. The protein band with a molecular mass of 53 kDa had an N-terminal amino acid sequence and internal sequences resembling that of the cytochrome P-450 4A subfamily (CYP 4A). Polyclonal antibodies raised against this protein were able to, after SDS/PAGE and immunoblotting, detect the protein in microsomal fractions as well as in the purified cytochrome P-450 fraction. Furthermore, addition of these antibodies to a reconstituted system containing the cytochrome P-450 fraction, inhibited 6 alpha-hydroxylation of taurochenodeoxycholic acid by up to 90%. Experiments with irrelevant antibodies did not show inhibition of 6 alpha-hydroxylation. The purified cytochrome P-450 fraction catalysed in addition omega- and omega-1 hydroxylation of lauric acid and 6 alpha-hydroxylation of 3 alpha-hydroxy-5 beta-cholanoic acid (lithocholic acid). However, these hydroxylase activities were rather low compared to 6 beta-hydroxylation of taurochenodexycholic acid. The enzyme fraction did not show hydroxylase activities towards cholesterol and 5 beta-cholestane-3 alpha,7 alpha-diol. These results indicate that 6 alpha-hydroxylation of taurochenodeoxycholic acid is catalysed by a specific species of cytochrome P-450 that, according to N-terminal amino acid sequence as well as catalytic properties, could be a member of the CYP 4A subfamily.

Multiplicity of cytochrome P-450 species involved in theophylline metabolism in mouse hepatic microsomes.

To ascertain the multiplicity of the cytochrome P-450 (P-450) species participating in the individual metabolic conversion of theophylline by 8-hydroxylation, 3-demethylation and 1-demethylation in mice, kinetics were studied under various conditions using untreated and inducer-treated mouse hepatic microsomes. Eadie-Hofstee plots of 1-demethylation in untreated microsomes exhibited a straight line, whereas those of 8-hydroxylation and 3-demethylation were curved lines. The biphasic kinetics indicated the contribution of two P-450 populations to the respective metabolic pathways; one characterized by high affinity and low capacity, the other by low affinity and high capacity. The high affinity population was efficiently induced by beta-naphthoflavone (beta-NF), and was highly susceptible to inhibition by a specific CYP1A inhibitor. The low affinity population was sensitive to induction by phenobarbital (PB), and was markedly inhibited by preferential inhibitors for PB-inducible P-450 species. The present results indicated that two P-450 populations contributed to the theophylline metabolism in mouse hepatic microsomes, and that the high and low affinity populations corresponded, respectively, to CYP1A, and a PB-inducible P-450 species having a much higher capacity than CYP1A.

Hydrogen peroxide-mediated inactivation of microsomal cytochrome P450 during monooxygenase reactions

Cytochrome P450 can undergo inactivation following monooxygenase reactions in liver microsomes of untreated, phenobarbital and 3-methylcholanthrene-treated rats and rabbits. The acceleration of cytochrome P450 loss in the presence of catalase inhibitors (sodium azide, hydroxylamine) indicates that hydrogen peroxide is involved in hemoprotein degradation. It was revealed that cytochrome P450 is inactivated mainly by H 2O 2 formed through peroxy complex breakdown, whereas H 2O 2 formed via the dismutation of superoxide anions produces a slight inactivating effect. The hydrogen peroxide added outside or formed by a glucose-glucose oxidase system has less of an inactivating effect than H 2O 2 produced within the cytochrome P450 active center. Self-inactivation of cytochrome P450 during oxygenase reactions is highly specific. Other components of the monooxygenase system, such as cytochrome b 5, NADH- and NADPH-specific flavorproteins, undergo no inactivation. The alterations in phospholipid content and in the rate of lipid peroxidation were not observed as well. The inactivation of cytochrome P450 by H 2O 2 is the result of heme loss or destruction without cytochrome P420 formation. Such. a mechanism operates with different substrates and cytochrome P450 species catalyzing the partially coupled monooxygenase reactions.

Effect of Dietary Alpha-Linolenic, Eicosapentaenoic and Docosahexaenoic Acids on Liver Microsomal Mixed-Function Oxidases in Rats

This study was undertaken to compare the effects of three major dietary n-3 fatty acids, i.e. alpha-linolenic acid (C18:3, n-3; LN), eicosapentaenoic acid (C20:5, n-3; EPA) and docosahexaenoic acid (C22:6, n-3; DHA), on liver microsomal mixed-function oxidase activities with and without phenobarbital (PB) induction. Male Sprague-Dawley rats were fed a 5% lipid diet containing either LN, EPA or DHA as major constituent fatty acid (about 40%) of dietary lipids for 15 days. The fatty acid compositions of liver microsomal lipids of each n-3 fatty acid group were characteristically different, particularly in the n-3 fatty acid compositions, but were nearly the same with and without PB treatment. Dietary LN, EPA and DHA did not affect the levels of PB-inducible cytochrome P-450 species nor the activities of aminopyrine N-demethylase and aniline hydroxylase. However, the PB-uninduced constitutive levels of cytochromes P-450 increased in the following order with a slight but significant difference between LN and DHA groups: LN group < EPA group < DHA group. Both the PB-uninduced activities of aminopyrine N-demethylase and aniline hydroxylase changed in a very similar manner to the constitutive levels of cytochromes P-450. These results suggest that the uninduced constitutive level of certain cytochrome P-450 species is modulated differentially by dietary LN, EPA and DHA, whereas dietary LN, EPA and DHA do not affect the PB-inducible levels of cytochrome P-450 species.

Evidence of partial involvement of P-450 2D in mutagenic activation of benzo(a)pyrene in liver S-9 fraction from untreated rats.

In order to clarify which species of cytochrome P-450 is involved in activation of benzo(a)pyrene (BP) in untreated rat liver, strain and sex differences in the ability of rat liver 9000 g supernatant (S-9) to mutagenically activate BP was investigated using Ames test. The numbers of histidine revertants in Ames test after pre-incubation of TA 98 strain of Salmonella typhimurium and BP with liver S-9 from male rats were markedly higher than those obtained using female rats. In addition, a marked strain difference (Wistar > DA) in the ability of liver S-9 from Wistar and DA rats to activate BP was observed. Antibody against cytochrome P-450 2D inhibited up to 50% of the revertant formation by the activation of BP with liver S-9 from male Wistar rats. These results indicate the partial involvement of cytochrome P-450 2D subfamily as well as cytochrome P-450 species specific to male rats in activation of BP to ultimate mutagen in untreated rat liver.

Stereoselectivity in the hydroxylation of propafenone enantiomers in mouse hepatic microsomes.

Stereoselectivity in the oxidative metabolism of propafenone (PPF) enantiomers to 5-hydroxypropafenone was studied with untreated and inducer-treated mouse hepatic microsomes. In the untreated microsomes, Lineweaver-Burk plots of the 5-hydroxylation of R(-)- and S(+) PPF. single lines, and the intrinsic clearance (Vmax/Km) value of R(-)-PPF was 1.3-fold higher than that of S(+)-PPF. When racemic PPF was used as a substrate, the plot was shifted to the upper region, in comparison with that estimated from the sum of the individual 5-hydroxylase activities of each enantiomer, suggesting enantiomer/enantiomer interaction. In phenobarbital-induced microsomes, the Lineweaver-Burk plot for R(-)-PPF was a single line, but that for S(+)-PPF was biphasic. When racemic PPF was used as a substrate, the plot was biphasic and was shifted to the upper region in comparison with that estimated from the sum of the individual 5-hydroxylase activities of each enantiomer. The observed value of intrinsic clearance of the PPF racemate at lower concentrations (Vmax/Km1) was consistent with the estimated value, suggesting no interaction between R(-)- and S(+)-PPF. These findings indicate that most of the 5-hydroxylation of R(-)- and S(+)-PPF is catalyzed by common cytochrome P-450 species, but a part of S(+)-PFF 5-hydroxylation is catalyzed by another phenobarbital inducible cytochrome P-450 species, particularly at lower substrate concentrations.

Sex-related differences in rat liver microsomal enzymes and their induction by doxapram.

The effects of doxapram on the hepatic microsomal mono-oxygenase system of male and female rats were investigated. Male and female rats were administered doxapram (10-120 mg kg-1 day-1, i.p.) for 4 days. In female rats, administration of doxapram (20, 40, 60, 80, 100 and 120 mg kg-1) elevated the parameters in a dose-dependent manner while doxapram (100 and 120 mg kg-1) elevated the levels of cytochrome P450 and hexobarbitone hydroxylase in male rats. Doxapram (40 mg kg-1) caused induction of hepatic drug metabolism typified by an increase of hepatic microsomal cytochrome P450 content and activities of hexobarbitone hydroxylase, benzphetamine N-demethylase and ethylmorphine N-demethylase in female rats, but no change in male rats. These findings were supported by the results of SDS/polyacrylamide-gel electrophoresis. However, 7-ethoxycoumarin O-de-ethylase and arylhydrocarbon hydroxylase activities were significantly increased in male rats. NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities, and cytochrome b5 content were unaffected in rats of both sexes. The sex-dependent cytochrome P450 species may be selectively sensitive to the action of doxapram.

Cytochrome P-450-dependent biotransformation of QA 208-199 in cultured rat conceptuses

It has been shown, using the method of rat post-implantation embryo culture, that the rat conceptus metabolizes the lipoxygenase inhibitor N-hydroxy- N-methyl-7-propoxy-2-naphthalenethan-amine (QA 208-199, QAB) in vitro. The capacity to metabolize QAB and accumulate its main metabolites depends on the developmental stage and length of exposure. In the present study, pregnant Han Wistar dams were given ip injections of either Aroclor 1254 (AC), 3-methylcholanthrene (3-MC) or phenobarbital (PB) before the dissection and culture of 9.5- and 10.5-day-old conceptuses in order to identify possible inducible conceptal cytochrome P-450 species. Conceptuses preinduced in utero were exposed to QAB in vitro for 48 hr, after which metabolites in the culture medium and conceptual tissues were determined with HPLC. Embryotoxic effects were evaluated in the same conceptuses. Preinduction in utero with AC, 3-MC or PB revealed that pretreatment with PB led, after 48 hr of culture, to a statistically significantly increased impairment of embryonic differentiation in 9.5-day-old conceptuses in vitro in comparison with vehicle-induced QAB-exposed conceptuses. Preinduction with PB led to higher levels of QAA, the main metabolite in vivo, in the culture medium. In the conceptal tissues, metabolite levels were significantly lower after preinduction with AC. In vehicle-induced 10.5-day-old embryos QAB caused less embryotoxicity in comparison with 9.5-day-old ones. In all preinduced conceptuses cultured from 10.5 to 12.5 days, yolk-sac vascularization was less affected and fewer embryonic anomalies were found in comparison with conceptuses cultured from 9.5 to 11.5 days. AC and PB pretreatment caused a decreased morphological score without changes in overall growth. Analysis of QAB and its metabolites in culture media and conceptal tissues at 12.5 days showed different levels of metabolites depending on the inducer used. To find further evidence for the involvement of cytochrome P-450 enzymes in the conceptal metabolism of QAB, conceptuses preinduced in utero (3-MC or PB) were exposed to QAB in vitro and gassed during the second half of the culture period with a mixture containing 35% carbon monoxide (CO), an inhibitor of cytochrome P-450 enzymes. CO treatment led to an inhibition of conceptal metabolism of QAB in comparison with that in conceptuses cultured under normal gassing conditions (without CO). These results strongly suggest the involvement of cytochrome P-450-dependent monooxygenases in the conceptal metabolism of QAB in vitro. 10.5-day-old conceptuses, after preexposure in utero to PB, were less susceptible to QAB embryotoxicity in vitro than were 9.5-day-old ones. In addition, the response to preinduction appeared to be age dependent.

Purification and characterization of cytochrome P450 from an isobutene-forming microorganism, Rhodotorula minuta.

A cytochrome P450 was purified from microsomes of Rhodotorula minuta. The optical spectrum of the purified cytochrome was characteristic of a low-spin ferric heme protein. Isovalerate caused a type I spectral change in it. The amino-terminal sequence of the cytochrome was different from those of other known microsomal cytochrome P450s. These results indicate that the cytochrome, which is tentatively named P450rm, is a novel species of cytochrome P450.

Study on the regioselectivity and mechanism of the aromatic hydroxylation of monofluoroanilines

The in vitro and in vivo metabolism of monofluoroanilines was investigated. Special attention was focussed on the regioselectivity of the aromatic hydroxylation by cytochromes P-450 and the mechanism by which this reaction might proceed. The results clearly demonstrate that the in vitro and in vivo regioselectivity of the aromatic hydroxylation by cytochromes P-450 is dependent on the fluoro-substituent pattern of the aromatic aniline-ring. Results from experiments with liver microsomes from differently pretreated rats demonstrate that the observed regioselectivity for the aromatic hydroxylation is not predominantly determined by the active site of the cytochromes P-450. To investigate the underlying reason for the observed regioselectivity, semi-empirical molecular orbital calculations were performed. Outcomes of these calculations show that neither the frontier orbital densities of the LUMO/LUMO + 1 (lowest unoccupied molecular orbital) of the monofluoroanilines nor the spin-densities in their NH • radicals can explain the observed regioselectivities. The frontier orbital densities of the HOMO/HOMO − 1 (highest occupied molecular orbital) of the monofluoroanilines however, qualitatively correlate with the regioselectivity of the aromatic hydroxylation. Based on these results it is concluded that the cytochrome P-450 dependent aromatic hydroxylation of monofluoroanilines does not proceed by hydrogen or electron abstraction from the aniline substrate to give an aniline-NH • radical. The results rather suggest that cytochrome P-450 catalyzed aromatic hydroxylation of monofluoroanilines proceeds by an electrophilic attack of the (FeO) 3+ species of cytochrome P-450 on a specific carbon atom of the aromatic aniline-ring.

Improved sample preparation for the testosterone hydroxylation assay using disposable extraction columns

The preparation of samples for injection into a high-performance liquid chromatography from assay mixtures for the determination of cytochrome P-450-dependent testosterone hydroxylation has been substantially facilitated. By replacing the multiple cumbersome extraction steps of the conventional method with a single column extraction the time for sample preparation was reduced from hours to minutes. The new procedure also yields better recoveries for most of the testosterone metabolites than the original protocol. The use of extraction columns for sample preparation allows the simultaneous treatment of a large number of samples or even the automation of the whole assay procedure. The modified procedure is a straightforward, easy-to-perform method that should greatly facilitate the implementation of the testosterone hydroxylation assay for sharply discriminating between many individual cytochrome P-450 species in routine enzyme diagnostics.

Cytochrome P-450 induction in human lung tumor-derived cell lines. Characterisation and effects of inflammatory mediators.

Cytochrome P-450 species (P-450) comprise a polymorphic multigene family of heme-containing enzymes which are essential to the phase-I metabolism of xenobiotics. Induction of P-450 species by drugs and carcinogens has been extensively studied; endogenous regulation of P-450 also occurs during normal development and disease. The aim of this project was to study the in-vitro induction of P-450 and its modulation by inflammatory mediators in the human lung tumor-derived cell lines NCI H322 and NCI H358. The cell lines expressed detectable levels of 7-ethoxyresorufin O-deethylase which could be induced by benzanthracene. After benzanthracene treatment, a protein tentatively identified as isozyme CYP1A1 was detected by Western-blot analysis and a concommitant increase in CYP1A mRNA expression was observed. Optimal induction was observed at a benzanthracene concentration of 5 micrograms/ml with cells grown in RPMI medium containing 10% fetal calf serum. The effects of endotoxin, dexamethasone and five recombinant DNA-derived cytokines, interleukin-1 beta, tumor necrosis factor, and interferons alpha, beta and gamma, on constitutive and benzanthracene-induced ethoxyresorufin O-deethylase activity were examined in NCI H322 cells. Of all the lymphokines studied, only interferon gamma had any marked effect. Administration of this lymphokine strongly suppressed ethoxyresorufin O-deethylase activity in both control and benzanthracene-treated cells.