Reactions Catalyzed by the Cytochrome P-450 System

Wislocki, Peter G.; Miwa, Gerald T.; Lu, Anthony Y. H.

doi:10.1016/b978-0-12-380001-5.50013-x

Cited by 86 publications

(53 citation statements)

References 135 publications

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“…The types of reactions performed by S. griseus include aromatic, cyclic, and aliphatic hydroxylations, 0-, S-, and N-oxidations, C-C fission, epoxidation, and 0-and N-dealkylations. These transformations, which occur with compounds such as alkaloids (17,27), coumarins (25), rotenoids (28), chromenes (30), and other complex xenobiotics (7,26), parallel those performed by mammalian cytochromes P-450 (34,36). However, the nature of the enzymatic system(s) employed by S. griseus for such diverse reactions has remained unresolved (26).…”

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confidence: 99%

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

1989

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A soybean flour-induced, soluble cytochrome P-450 (P-450,,.Y) was purified 130-fold to homogeneity from Streptomyces gyiseus. Native cytochrome P-450,,. is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450S exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450O had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin;NADP+ oxidoreductase, purified cytochrome P-450O, catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450SOYA.Streptomyces griseus (ATCC 13273) has the remarkable ability to catalyze the stereo-and regiospecific oxidation of a diverse array of xenobiotics. The types of reactions performed by S. griseus include aromatic, cyclic, and aliphatic hydroxylations, 0-, S-, and N-oxidations, C-C fission, epoxidation, and 0-and N-dealkylations. These transformations, which occur with compounds such as alkaloids (17,27), coumarins (25), rotenoids (28), chromenes (30), and other complex xenobiotics (7,26), parallel those performed by mammalian cytochromes P-450 (34, 36). However, the nature of the enzymatic system(s) employed by S. griseus for such diverse reactions has remained unresolved (26). The biotransformations catalyzed by S. griseus occur primarily following growth of the organism on a complex medium enriched with soybean flour (26). Recently, Sariaslani and Kunz (29) demonstrated that soybean flour and one of its isoflavonoid constituents, genistein, induce the synthesis of cytochrome P-450 in S. griseus and inferred a role for this enzyme in the biotransformation reactions performed by this organism. Here we report the purification and characterization of a soybean flour-induced cytochrome P-450 and demonstrate the transformation of two xenobiotic substrates, precocene II and 7-ethoxycoumarin, by purified preparations of this enzyme.( was determined by the method of Omura and Sato (23). Heme was analyzed as its reduced pyridine hemochrome derivative (10). High-pressure liquid chromatography (HPLC) was performed on a system consisting of an LKB 2152 controller, LKI3 2150 pump, and a Hewlett-Packard 1040A diode array detector.Cytochrome P-450.,y purification protocol. All procedures except steps 4 and 5 were carried out at 4°C. Glycerol (20% [vol/vol]) was included in all buffers following the salt precipitation step to prevent degradation of the P-450 to its inactive P-420 species. Protease inhibitors used in steps 1 to 3 to inhibit a wide range of protease activities were pepstatin (an acid protease inhibitor), leupeptin (a broad-range protease inhibitor), and phenylmethylsulfonyl fluoride (PMSF; a serine protease inhibitor). Fractions were stored between purification steps at -80°C with no discernable loss o...

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confidence: 99%

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

1989

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“…Compounds which produce type I binding spectra are almost invariably substrates for cytochrome P-450 (27). Thus, 7V-hydroxynorcocaine meets at least three of the four criteria commonly used to establish the involvement of cytochrome P-450 in the oxidation of a substrate (28). The reaction of 7V-hydroxynorcocaine with cytochrome P-450 to produce norcocaine nitroxide is not, then, a mixed function oxidation reaction, but rather a form of "substrate activation" by cytochrome P-450.…”

Section: Animal Models and The Role Ofoxidative Enzymes Inmentioning

confidence: 99%

Involvement of nitroxide radicals in cocaine-induced hepatotoxicity

Rauckman¹,

Kloss²,

Rosen³

1982

Can. J. Chem.

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Cocaine administration can produce hepatotoxicity in non-induced mice of at least one strain, DBA/2Ha and hepatotoxicity in induced mice of several strains. Metabolic studies and the administration of metabolites indicate that the minor metabolic pathway, cocaine -» norcocaine -» /V-hydroxynorcocaine -» norcocaine nitroxide, is responsible for the observed cocaine-induced hepatotoxicity. In vitro experiments show that cytochrome P-450 can oxidize /V-hydroxynorcocaine to norcocaine nitroxide. Norcocaine nitroxide is unreactive towards cellular proteins or glutathione but does react directly with reduced pyridine nucleotides and is rapidly reduced enzymatically by the microsomal flavoproteins, NADPH-cytochrome P-450 reductase and FAD-containing monooxygenase. These reactions constitute a futile cycle in which NADPH is consumed and superoxide and hydrogen peroxide are generated. We postulate that the destruction of hydrogen peroxide by glutathione peroxidase results in the accumulation of excess oxidized glutathione which is actively excreted by the cell, since insufficient NADPH is available for glutathione reductase to maintain the GSH/GSSG ratio at an acceptable level. As reduced glutathione levels diminish, the cell can no longer protect itself against toxic lipid hydroperoxides which accumulate as a result of stimulation of lipid peroxidation (caused by the one electron cycling reaction, /V-hydroxynorcocaine to norcocaine nitroxide cycle). Finally, as glutathione is depleted below a certain level, the cell loses the ability to maintain the GSH/GSSG ratio in a range consistent with homeostasis resulting in loss of cellular function. Ultimately, necrosis results. This mechanism is consistent with all the information available concerning cocaine-induced hepatotoxicity. L'administration de cocaine peut produire de l'hépatotoxicité chez les souris non-induites de la souche DBA/ZHA ainsi que de l'hépatotoxicité chez plusieurs souches de souris induites. Des études métaboliques et l'administration des metabolites indiquent que la séquence métabolique mineure: cocaine -» norcocaine -» /V-hydroxynorcocaine -» nitroxyde de cocaine est responsable de rhépatotoxicité induite par la cocaine que Ton a observée. Des expériences in vitro montrent que le cytochrome P-450 peut oxyder la M-hydroxynorcocaine en nitroxyde de cocaine. Le nitroxyde de cocaine n'a aucun effet sur les protéines cellulaires ou sur le glutathion mais il réagit directement avec la pyridine réduite des nucléotides et elle est rapidement réduite enzymatiquement par les flavoprotéines microsomiques: la réductase du NADPH-cytochrome P-450 et la mono-oxygénase contenant le ADF. Ces réactions constituent un cycle non efficace au cours duquel le NADPH est consommé et où un superoxyde et le peroxyde d'hydrogène se forment. Nous pensons que la destruction du peroxyde d'hydrogène par la peroxydase du glutathion provient de l'accumulation d'un excès de glutathion oxydé qui est activement sécrété par la cellule, puisqu'une insuffisance de NADPH est nécessaire à la p...

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“…:A5O enzymes, is one such enzyme (16). Phenobarbital, t h o u 3 not as potent an inducer of ECOD as 3-methylcholanthrene, has been shown to increase ECOD activity (16). In addition, other enzyme activities such as ethylmorphine N-demethylase and aniline hydroxylase, which are not related to ECOD, are also induced by phenobarbital (12,13).…”

Section: Biocheniical Paraiiietersmentioning

confidence: 99%

“…ECOD, a member of the resorufin family ofJ? :A5O enzymes, is one such enzyme (16). Phenobarbital, t h o u 3 not as potent an inducer of ECOD as 3-methylcholanthrene, has been shown to increase ECOD activity (16).…”

Section: Biocheniical Paraiiietersmentioning

confidence: 99%

“…A decrease in cytosolic volume percent was observed with both phenobarbital and SK&F 86002 at day 3 and accompanied by a concomitant increase in the Vv of SER. This increase in Vv of SER is-a common hepatic response to P-450 inducers (16) and was quantitated by Staubli and coworkers in rats administered 100 mg/kg/day phenobarbital for 2 or 5 days (1 l). They demonstrated a 2-fold increase in SER after 2 days and nearly a 3-fold increase after 5 days.…”

Section: Morphologymentioning

confidence: 99%

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Comparative Biochemical and Morphometric Changes Associated with Induction of the Hepatic Mixed Function Oxidase System in the Rat

Howard¹,

Newton²,

Qualls

et al. 1991

Toxicol Pathol

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ABSTRA(=T This study characterized the induction of the rat hepatic cytochrome P-450-dependent mixed function oxidase system by SK&F 86002 [6-(4'-~uorophenyl)-5-(4'-pyridyl)-2,3-dihydroimidazo-(2,l-b)thiazole], an inhibitor of both the cyclooxygenase and 5-lipoxygenase pathways of arachidonic acid metabolism. The induction characteristics of SK&F 86002 were compared to those of the classical inducer, phenobarbital, and morphological features of both SK&F 86002 and phenobarbital induced hepatocellular hypertrophy were quantitated.Rats were administered either SK&F 86002 (6, 18, or 60 mg/kg/day, PO) or phenobarbital (8, 24, 80 mg/ kg/day, ip) for 3 or 14 consecutive days. Liver to body weight ratio, total hepatic microsomal protein and cytochrome P-450 content, ethoxycoumarin-0-deethylase (ECOD) and leukotriene B,(LTB,) w-and w-1 hydroxylase were measured. Ultrastmctural morphometry of the liver from control, and high dose SK&F 86002 (60 mg/kg/day) and phenobarbital (80 mg/kg/day) treated rats was completed.On day 3, phenobarbital increased liver to body weight ratio but only at the 80 mg/kg/day dosage; microsomal protein content was unchanged. ECOD activity increased in a dose-dependent fashion. LTB, wand w-1 hydroxylase activities were unaffected. Administration of SK&F 86002 for 3 days increased the liver to body weight ratio at both the 18 and 60 mg/kg/day dosage; microsomal protein content was unchanged. ECOD activity was significantly increased by the 60 mg/kg/day dosage. LTB, w-, but not w-1 hydroxylase activity was increased by both the 18 and 60 mg/kg/day dosages of SK&F 86002.On day 14, phenobarbital increased the liver to body weight ratio and microsomal protein content but again only at the 80 mg/kg/day dosage. Cytochrome P-450 content was increased by all dosages, A dosedependent increase in ECOD activity was observed but there was no change in LTB, w-or 0-1 hydroxylase activity on day 14.Administration of SK&F 86002 (60 mg/kg/day) for 14 days increased the liver to body weight ratio. Microsomal protein content was unaffected but cytochrome P-450 content and ECOD activity were increased at the 60 mg/kg/day dosage. Similar to the day 3 observation, LTB, w-hydroxylase activity was increased by the 18 and 60 mg/kg/day dosages of SK&F 86002; LTB, w-1 hydroxylase was unchanged.Quantitative evaluation of hepatocyte ultrastructural characteristics indicated that the volume percent (Vv) of smooth endoplasmic reticulum (SER) was increased.hut;fQat cytosol and mitochondrial Vv significantly decreased after 3 days of phenobarbital treatment. Three days-of treatment'with SK&F 86002 also increased the Vv of SER; Vv of cytosol was decreased but the mitochondria1 Vv was unchanged from control values.SER Vv was increased after 14 days of phenobarbital treatment but cytosol Vv was not significantly affected. Similar to the 3 day results, mitochondrial Vv decreased in phenobarbital treated rats. SK&F 86002 treatment for 14 days increased the SER Vv and decreased cytosol Vv. Mitochondria1 Vv was unaffected.These studies...

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Reactions Catalyzed by the Cytochrome P-450 System

Cited by 86 publications

References 135 publications

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

Involvement of nitroxide radicals in cocaine-induced hepatotoxicity

Comparative Biochemical and Morphometric Changes Associated with Induction of the Hepatic Mixed Function Oxidase System in the Rat

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