Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

Toxicological implications of the mixed-function oxidase catalyzed metabolism of carbon disulfide.

The results of these studies have indicated that the decrease in the activity of the hepatic mixed-function oxidase enzyme system and the concentration of cytochrome P-450 seen on incubation of carbon disulfide (CS2) with rat liver microsomes in the presence of NADPH is the result of the binding of the sulfur atom released in the mixed-function oxidase catalyzed metabolism of CS2 to carbonyl sulfide (COS). Moreover, it appears that COS is further metabolized by the mixed-function oxidase enzyme system to CO2 and that, analogous to the metabolism of CS2 to COS, the sulfur atom released in this reaction also binds to the microsomes and inhibits benzphetamine metabolism and decreases the concentration of cytochrome P-450 detectable as its carbon monoxide complex. The results of these studies also suggest that the decrease in the concentration of cytochrome P-450 and the liver damage seen on in vivo administration of CS2 to phenobarbital pretreated rats, is due to the mixed-function oxidase catalyzed release and binding of the sulfur atoms of CS2. The decrease in the concentration of cytochrome P-450 seen on incubation of CS2 with rat liver microsomes in the presence of NADPH does not appear to be the result of destruction of the heme group or its dissociation from the apoenzyme since the total amount of protoheme is unchanged in microsomes which have been incubated with CS2 and NADPH as compared to those not incubated with these compounds.     

Cytochrome P-450-mediated oxidation of 2-hydroxyestrogens to reactive intermediates

2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17α-ethynylestradiol, oxidation products of naturally occurring estrogens and synthetic estrogens in some oral contraceptives were found to be converted by rat liver microsomes to reactive metabolites that become irreversibly bound to microsomal protein. The irreversible binding required microsomes, oxygen and NADPH. The NADPH could be replaced by a xanthine-xanthine oxidase system which is known to generate superoxide anions. The irreversible binding was substantially inhibited by superoxide dismutase, 30% in those incubations containing NADPH and 98% in those incubations containing the xanthine-xanthine oxidase system. Further studies with 2-hydroxyestradiol showed that microsomal cytochrome P-450 was rate limiting in the NADPH-dependent irreversible binding, because the binding was inhibited 62% by an antibody against NADPH-cytochrome $\text{c}$ reductase and 70% in an atmosphere of CO:O₂ (9:1) when compared to an atmosphere of N₂:O₂ (9:1). Phenobarbital, a known inducer of cytochrome P-450, had no effect on the irreversible binding of 2-hydroxyestradiol, whereas another inducer of P-450, pregnenolone-16α-carbonitrile, markedly increased the irreversible binding. In contrast, cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450, decreased both P-450 and the irreversible binding. These results are consistent with a mechanism for irreversible binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

Evidence that covalent binding of metabolically activated phenol to microsomal proteins is caused by oxidised products of hydroquinone and catechol

The metabolic activation of [14C]phenol resulting in covalent binding to proteins has been studied in rat liver microsomes. The covalent binding was dependent on microsomal enzymes and NADPH and showed saturation kinetics for phenol with a Km-value of 0.04 mM. The metabolites hydroquinone and catechol were formed at rates which were 10 or 0.7 times that of the binding rate of metabolically activated phenol. The effects of cytochrome P-450 inhibitors and cytochrome P-450 inducers on the metabolism and binding of phenol to microsomal proteins, suggest that cytochrome P-450 isoenzyme(s) other than P-450 PB-B or P-450 beta NF-B catalyses the metabolic activation of phenol. Furthermore, reconstituted mixed-function oxidase systems containing cytochrome P-450 PB-B and P-450 beta NF-B were (on basis of cytochrome P-450 content) 6 and 11 times less active in catalysing the formation of hydroquinone than microsomes. The isolated metabolites hydroquinone and catechol bound more extensively to microsomal proteins than phenol and the binding of these was not stimulated by NADPH. The binding occurring during the metabolism of phenol could be predicted by the rates of formation of hydroquinone and catechol and the rates by which the isolated metabolites were bound to proteins.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Metabolism of prostaglandin A1 by hepatic microsomal monooxygenase P-450 system in the guinea pig and rat.

This study demonstrates the metabolic transformation of prostaglandin A₁ (PGA₁) by guinea pig and rat liver microsomes. The transformation, which required NADPH and oxygen, yielded polar (presumably hydroxylated) products. Incubations with guinea pig liver microsomes yielded one zone of product on tlc, whereas rat liver microsomes produced two discernable metabolic zones. It was demonstrated that PGA₁ metabolism in the guinea pig and the rat was inhibited by the addition of SKF-525A, metyrapone, carbon monoxide and cytochrome $\text{C}$; nicotinamide (10 mM) inhibited only the guinea pig system. These findings indicate that the enzymatic activity responsible for PGA₁ metabolism is composed of a typical cytochrome P-450 monooxygenase system.     

Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique.

We have developed a specific radioimmunoassay to quantify NADPH: cytochrome P-450 reductase. The assay is based on the use of 125I-labelled NADPH: cytochrome P-450 reductase as the radiolabelled antigen and can detect quantities of this protein in amounts as low as 30 pg. The results of the radioimmunoassay demonstrates that the 2.7-fold increase in enzyme activity in rat liver microsomal membranes after phenobarbital treatment is due to increased amounts of the protein. beta-Naphthoflavone treatment, however, did not alter the activity or the quantity of this enzyme in microsomes. The quantification of NADPH: cytochrome P-450 reductase in the microsomes isolated from control and phenobarbital- and beta-naphthoflavone-treated animals permits the calculation of the ratio of this protein to that of total cytochromes P-450. A molar ratio of 15:1 (cytochromes P-450/NADPH: cytochrome P-450 reductase) was calculated for control and phenobarbital-treated animals. This ratio increased to 21:1 after beta-naphthoflavone treatment. Thus the molar ratio of these proteins in liver microsomes can vary with exposure of the animals to particular xenobiotics.     

Transformation of an arachidonic acid hydroperoxide into epoxyhydroxy and trihydroxy fatty acids by liver microsomal cytochrome P-450

In the absence of NADPH, the addition of an arachidonic acid hydroperoxide, 15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid, to liver microsomes, prepared from phenobarbital-treated rats, resulted in the formation of two major metabolites and several minor products, some of which have been purified by reverse-phase high-performance liquid chromatography. We propose the structures of the two major products to be 13-hydroxy-14,15-epoxyeicosa-5,8,11-trienoic acid and 11,14,15-trihydroxyeicosa-5,8,12-trienoic acid based on spectral characteristics and mass spectral analysis of derivatives of the compounds. A potential heterolytic cleavage product, 15-hydroxyeicosa-5,8,11,13-tetraenoic acid, was not a product of the reaction. Ferric cytochrome P-450 catalyzed the formation of these products as shown by the inability of boiled microsomes to support the reaction, the inhibition of epoxyhydroxy and trihydroxy fatty acid formation by imidazole derivatives which bind tightly to the ferric heme iron of cytochrome P-450, and the inability of carbon monoxide (which binds to ferrous P-450) and free iron chelators (EDTA and diethylenetriaminepentaacetic acid) to inhibit product formation. These results show that liver microsomal cytochrome P-450, in addition to its role in the NADPH-dependent metabolism of arachidonic acid, can utilize a hydroperoxide to produce an interesting series of potentially important arachidonic acid metabolites.     

Stereoselective hydroxylation at the aliphatic carbons of 7,8- and 9,10-dihydrobenzo[a]pyrenes by rat liver microsomes

Optically active 7-hydroxy-7,8-dihydrobenzo[a]pyrene and 8-hydroxy-7,8-dihydrobenzo[a]pyrene were identified as two of the major metabolites formed by incubation of 7,8-dihydrobenzo[a]pyrene with rat liver microsomes. Optically active 9-hydroxy-9,10-dihydrobenzo[a]pyrene and 10-hydroxy-9,10-dihydrobenzo[a]pyrene were similarly identified as two of the minor metabolites of 9,10-dihydrobenzo[a]pyrene. The formation of these metabolites was abolished either by prior treatment of liver microsomes with carbon monoxide or the absence of NADPH, but was not inhibited by an epoxide hydrolase inhibitor. The results indicate that the aliphatic carbons of dihydro polycyclic aromatic hydrocarbons may undergo stereoselective hydroxylation reactions catalyzed by the cytochrome P-450 system of rat liver microsomes.     

Induction of specific cytochrome P-450 isozymes by methylenedioxyphenyl compounds and antagonism by 3-methylcholanthrene

Two methylenedioxyphenyl compounds, isosafrole (5-propenyl-1,3-benzodioxole) and an analog, 5-t-butyl-1,3-benzodioxole (BD), differ markedly as inducers of cytochrome P-450 isozymes in rat liver microsomes. Isosafrole is a mixed-type inducer, inducing P-450b, P-450c, and P-450d. In contrast, BD is a phenobarbital-type inducer, increasing P-450b, but producing little or no increase in P-450c or P-450d. Similarly, isosafrole increases the amount of translatable mRNA for P-450b, c and d, while BD induces only the mRNA for P-450b. Dimethylation of the methylene bridge carbon of BD to give 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBD) blocks the formation of NADPH-reduced Type III metabolite-P-450 complexes in vitro, and diminishes but does not abolish the ability of the compound to induce P-450b. Western blots of microsomes from isosafrole and BD-treated rat livers confirm that in contrast to isosafrole, BD does not induce P-450d or P-450c. However, the antibody to P-450d recognizes two new polypeptides (approximately 50K Mr) from sodium dodecyl sulfate-polyacrylamide gels of liver microsomes from BD-treated rats. These polypeptides are not observed in control, isosafrole, 3-methylcholanthrene (3-MC), or DBD-treated rats. They are intensified by coadministration of 3-MC with BD and may represent either modified isozyme-metabolite adducts or degradation products of P-450d. However, the polypeptides could not be generated in vitro by addition of BD to 3-MC-induced microsomes with NADPH under conditions which produced spectral metabolite complexes, or in a reconstituted system with P-450d. The two methylenedioxyphenyl compounds do not form stable metabolite complexes with the same P-450 isozymes. BD formed distinct spectral metabolite complexes in vitro with both P-450b and P-450c but not with P-450d in a reconstituted system. In contrast, isosafrole forms metabolite complexes with all three isozymes. Coadministration of 3-MC with BD blocked induction of P-450b by 80% and produced a similar repression of its translatable mRNA. This finding indicates that 3-MC type inducers not only induce certain cytochrome P-450 isozymes, but also repress synthesis of other isozymes.     

Studies of cytochrome P-450 in testis microsomes

Studies on the role of cytochrome P-450 in mouse, rat, and chick testis microsomes showed that this CO-binding hemoprotein is involved in the activity of the 17α-hydroxylase. A 70–80% inhibition by CO of the 17α-hydroxylase activity was detected in rat and chick testis microsomes. In the mouse testis, the level of the enzyme activity is ten times greater than that of the rat. This partly explains why an acceleration of NADPH oxidation by progesterone can be observed in mouse but not in rat testis microsomes. In rat testis microsomes, type I binding spectra of cytochrome P-450 was observed with pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, and testosterone. The apparent K_s values for progesterone and 17-hydroxyprogesterone were 0.50 and 1.00 μm, respectively.When NADPH is used to measure cytochrome P-450 levels in rat testis microsomes, CO formation resulting from a stimulation in lipid peroxidation by phosphate or Fe²⁺ was sufficient to bind with 50% of the total amount of cytochrome P-450. Substitution of phosphate by Tris reduced the amount of lipid peroxidation to minimal levels. On a comparable basis, no CO formation was observed in avian testis microsomes.An increase in the testicular levels of cytochrome P-450 resulted upon the administration of HCG and cyclic-AMP to 1-day-old chicks. The lack of stimulation of the cytochrome P-450 levels by progesterone and pregnenolone suggest that the hormonal stimulation of the P-450 levels is not due to substrate induction.     

Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney.

Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.     

Suicidal inactivation of microsomal cytochrome P-450 by halothane under hypoxic conditions

Under anaerobic conditions the addition of halothane to NADPH-reduced liver microsomes from phenobarbital-pretreated male rats resulted in a pronounced inactivation of microsomal cytochrome P-450, presumably produced by covalent binding of reactive halothane metabolites such as the CF₃CHCl-radical. Compared with microsomes from phenobarbital-pretreated rats, the loss of active cytochrome P-450 was markedly decreased in microsomes from both 3-methylcholanthrene-pretreated and untreated rats. Increasing the O₂-partial pressure decreased the amount of cytochrome P-450 inactivated by halothane metabolites. At an O₂-partial pressure of approximately 40 mm Hg the inactivation was virtually eliminated.     

Leukotriene B4 metabolism by hepatic cytochrome P-450

Leukotriene B4 (LTB) was found to be metabolized by suspensions of rat liver microsomes in the presence of NADPH and oxygen. The rate of LTB metabolism was also measured in reconstituted systems of both micelles and phospholipid vesicles containing cytochrome P-450-LM2, NADPH cytochrome P-450 reductase, and cytochrome b5. A 1 microM concentration of LTB was metabolized by rat hepatic microsomes at a rate of 4 pmol LTB/min/nmole P-450, and by vesicle and micelle reconstituted systems at 3 pmole/min/nmole P-450-LM2. At this rate a 10 g rat liver exposed to 1 microM LTB can metabolize 30 micrograms per hour. In that the leukotrienes are pharmacologically active at nanomolar concentrations, hepatic metabolism may be an important pathway of leukotriene inactivation.     

12alpha-hydroxylation of 7alpha-hydroxy-4-cholesten-3-one by a reconstituted system from rat liver microsomes

A preparation of partially purified cytochrome P-450 from rat liver microsomes was found to catalyze 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one in the presence of NADPH and phosphatidyl choline. The reaction was stimulated two- to four-fold by addition of a preparation of cytochrome P-450 reductase. The reaction was inhibited by carbon monoxide to a considerably less extent than other hydroxylations catalyzed by the reconstituted system. In the presence of optimal concentrations of cytochrome P-450 reductase, cytochrome P-450 prepared from livers of starved rats catalyzed the 12α-hydroxylation more efficiently than cytochrome P-450 prepared from livers of normal rats or rats treated with phenobarbital.     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

Evidence for the involvement of cytochrome P-450 in tiaramide N-oxide reduction

Tiaramide N-oxide, a major metabolite of tiaramide, is reduced anaerobically to tiaramide by rat liver microsomes. The reaction requires NADPH and is inhibited by oxygen and carbon monoxide. Both phenobarbital and 3-methylcholanthrene treatments induced the reductase activity with increasing cytochrome P-450 content. Tiaramide N-oxide produced a pronounced spectral change with reduced cytochrome P-450 and the difference spectrum showed a peak of absorbance at 442 nm.These findings provide evidence in support of an essential role for cytochrome P-450 in the process of the N-oxide reduction.     

Formation of epoxyeicosatrienoic acids from arachidonic acid by cultured rat aortic smooth muscle cell microsomes.

The vasodilatory effect of epoxyeicosatrienoic acids (EpETrE), especially 5(6)-EpETrE, has been reported recently and a role of P-450-dependent arachidonic acid monooxygenase metabolites was suggested in vasoregulation. Accordingly, the presence of P-450-dependent arachidonic acid monooxygenase was investigated in rat aortic smooth muscle cells. Incubation of the microsomes of rat cultured aortic smooth muscle cells with 14C-arachidonic acid in the presence of 1 mM NADPH resulted in the formation of oxygenated metabolites. The metabolites were separated and purified by reverse phase and straight phase high performance liquid chromatography and identified by gas chromatography-mass spectrometry. Identified metabolites were 5(6)-EpETrE, 5,6-dihydroxyeicosatrienoic acid (DiHETrE), and 14,15-DiHETrE. The formation of these metabolites was totally dependent on the presence of NADPH, and inhibitors of cytochrome P-450-dependent enzymes, SKF-525A and metyrapone, reduced the formation of these metabolites. This is the first report that cytochrome P-450-dependent arachidonic acid metabolites, especially 5(6)-EpETrE and 14(15)-EpETrE, can be produced in the microsomes of vascular smooth muscle cells of rats.     

Selective induction of cytochrome b5 and NADH cytochrome b5 reductase by propylthiouracil

Both the cytochrome b₅ level and NADH cytochrome b₅ reductase activity in rat liver microsomes were increased 2-fold by repeated i.p. administration of 1.5 mmol/kg propylthiouracil (PTU) for 2 weeks, but neither the cytochrome P-450 level nor NADPH cytochrome P-450 reductase activity were affected by the treatment. Liver microsomes from PTU-treated rats showed a significant decrease in aminopyrine N-demethylation, but not in benzphetamine N-demethylation, aniline hydroxylation or 7-ethoxycoumarin O-deethylation. A single administration of the compound had no effect on any components of the system. $\text{In vitro}$, drug hydroxylation activities were not affected by PTU up to 1.0 mM. From the above evidence, repeated administration of PTU selectively induced cytochrome b₅ and NADH cytochrome b₅ reductase in rat liver microsomes.