Cytochrome P-450 ligands: Metyrapone revisited

doi:10.1016/0003-9861(85)90562-4

Archives of Biochemistry and Biophysics

Volume 241, Issue 2, September 1985, Pages 397-402

https://doi.org/10.1016/0003-9861(85)90562-4 Get rights and content

Abstract

Expressing metyrapone interactions with ferrous cytochrome P-450 as ligand saturation by cytochrome, rather than the more conventional cytochrome saturation by ligand, an extinction coefficient of 68.5 ± 1.8 mm⁻¹ cm⁻¹ for the metyrapone complex of dithionite-reduced rat hepatic microsomal cytochrome P-450 was derived. Utilizing this new extinction coefficient, the increased cytochrome P-450 present after phenobarbital induction was almost exclusively that which is able to both bind to metyrapone and form a metabolic-intermediate complex from norbenzphetamine. However, it was not the only subpopulation present in microsomes that was able to bind metyrapone, nor the only one capable of forming a metabolic intermediate complex from norbenzphetamine. Thus, neither technique alone can be used to quantitate the “phenobarbital-induced form” of cytochrome P-450.

References (18)

A.G. Hildebrandt et al.
Biochem. Biophys. Res. Commun
(1969)
H.G. Jonen et al.
Biochem. Pharmacol
(1974)
K.M. Ivanetich et al.
Biochem. Biophys. Res. Commun
(1982)
J. Werringloer et al.
Arch. Biochem. Biophys
(1975)
J.A. Peterson et al.
Arch. Biochem. Biophys
(1971)
V. Luu-The et al.
Biochem. Biophys. Res. Commun
(1980)
M.R. Franklin et al.
Arch. Biochem. Biophys
(1971)
O.H. Lowry et al.
J. Biol. Chem
(1951)
F. Mitani et al.
FEBS Lett
(1982)

There are more references available in the full text version of this article.

Cited by (14)

Comparative expression of liver cytochrome P450-dependent monooxygenases in the horse and in other agricultural and laboratory species
2003, Veterinary Journal
The apoprotein expression and the catalytic activities of cytochrome P450s involved in the biotransformation of xenobiotics were investigated in horse liver microsomes and compared with those of food producing (cattle, pigs, broiler chicks, and rabbits) and laboratory species (rats). Western blot analysis revealed the presence of proteins immunorelated to rat CYP 1A, CYP 2B, CYP 2E, and CYP 3A subfamilies in hepatic microsomes from horses and from any other examined species. With the exception of the N-demethylation of N-nitrosodimethylamine in broiler chicks, all the recorded interspecies differences were quantitative in nature. Equine preparations proved the most active in the biotransformation of the CYP 1A substrates ethoxy- and methoxyresorufin and the least active in the metabolism of aminopyrine and ethoxycoumarin. On a comparative basis, large differences were observed in the rate of the in vitro metabolism of model substrates between “minor” (rabbits, horses) and “major” food producing species. Taken in due consideration the limitations of the in vitro approach, results from this study reinforce the conclusion that studies on drug efficacy and residue depletion should be performed in each target species.
Induction of rat hepatic glucocorticoid-inducible cytochrome P450 3A by metyrapone
1994, Journal of Steroid Biochemistry and Molecular Biology
The bipyridyl compound metyrapone is a potent inhibitor of cytochromes P450, a gene superfamily of haemoproteins involved in the metabolism of many xenobiotics as well as endogenous compounds such as steroid hormones. Administration of metyrapone to male rats induces the expression of the cytochrome P450 sub-family 3A (CYP3A). In order to determine whether metyrapone was causing the induction of CYP3A by blocking endogenous glucocorticoid metabolism, CYP3A levels were examined in rat hepatocytes cultured in serum-free medium supplemented with hydrocortisone 21-hemisuccinate plus or minus metyrapone. Western blotting indicated that metyrapone alone induces CYP3A and that hydrocortisone 21-hemisuccinate is ineffective. However, hydrocortisone 21-hemisuccinate enhanced the levels of CYP3A induced by metyrapone. In contrast, glucocorticoid-inducible tyrosine aminotransferase (TAT) activity was unaffected by metyrapone but metyrapone enhanced the levels induced by hydrocortisone 21-hemisuccinate. An examination of the metabolism of hydrocortisone by rat hepatocytes in vitro indicated that metyrapone perturbed the catabolism of hydrocortisone under conditions which give rise to an enhancement of hydrocortisone 21-hemisuccinate and hydrocortisone-dependent TAT induction. However, evidence is presented to suggest that such a perturbation of hydrocortisone metabolism could not account for the glucocorticoid potency amplifying property of metyrapone. Thus the induction of CYP3A and the enhancement of glucocorticoid-mediated TAT induction appears not to be associated with any perturbation in glucocorticoid metabolism but with some other as yet undefined mechanism(s).
Formation of ligand and metabolite complexes as a means for selective quantitation of cytochrome P450 isozymes
1993, Biochemical Pharmacology
The suitability of triacetyloleandomycin (TAO) metabolite complex formation and metyrapone binding to reduced cytochrome P450 as a means for selective isozyme quantitation has been studied. Although isozymes of both subfamilies bind metyrapone in the reduced state, selective quantitation of 2B isozymes through the metyrapone complex is possible after complex formation of P450 3A with a TAO metabolite. Thus, consecutive application of both reactions allows the spectroscopic quantitation of P450 3A and 2B isozymes. Complete conversion of P450 3A into the complex, a precondition for P450 3A quantitation, requires NADH in addition to NADPH. A precise collective quantitation of 3A + 2B isozymes as metyrapone complexes alone is not possible because the corresponding complexes possess different molar extinction coefficients, i.e 71.5 and 52 mM⁻¹ cm⁻¹ at 446–490 nm, respectively. The formation of the TAO complex appears to be quite specific, since it correlates well with 3A-specific enzymatic activities, i.e. TAO N-demethylation and formation of 2β-hydroxy-, 15β-hydroxy- and 6-dehydrotestosterone. P450 3A levels in liver microsomes of male rats either untreated or treated with TAO, dexamethasone (DEX), phenobarbital or hexachlorobenzene amount to 13%, 78%, 66%, 24% and 11% of total P450, respectively. Good correlation between these values and P450 3A-specific enzymatic activities is obtained. By the spectroscopic method, P450 2B isozymes could not be detected in microsomes of untreated rats. With TAO, DEX and hexachlorobenzene the microsomal 2B level is elevated to about 20% of total P450, i.e. to 0.8, 0.4 and 0.4 nmol P450/mg protein, respectively. 2B levels of about 60% of total P450 (0.75 nmol P450/mg protein) are obtained by phenobarbital treatment. Immunoblotting with anti-P450 2B shows that the ratio of expressed 2B1 and 2B2 differs depending on the type of inducer. DEX predominantly leads to induction of 2B2, which may explain the low pentoxyresorufin O-depentylase activity in these microsomes.
Identification of the hepatic cytochrome P-450 isozymes induced and decreased by picloram
1988, Biochemical Pharmacology
Microsomes from male rats treated with plcloram (100 mg/kg/day) for 7 days showed a 48% decrease in 16α-hydroxylase activity when incubated with (4-¹⁴C) androstenedione. These data are consistent with the assertion that picloram decreases the titer of hepatic male specific cytochrome P 450h. Several lines of evidence suggested that picloram is an inducer of hepatic cytochrome P-450d in male rats. First, SDS polyacrylamide gel electrophoresis revealed an intensified hepatic microsomal polypeptide (MW 54,000) following picloram pretreatment. This polypeptide co-migrated with protein bands which were correspondingly intensified after pretreatment with known inducers of cytochrome P-450d (3-methylcholanthrene and isosafrole). Second, no increase in the binding of metyrapone to plcloram treated microsomes was noted compared with controls, suggesting no increase in phenobarbitalindudble forms of cytochrome P-450. Third, hepatic microsomes from plcloram treated rats activated 2-amino-3-methylimidazo [4,5-f] quinoline (a cytochrome P-450d mediated catalysis) causing a 5-fold increase in the number of induced Salmonella typhimurium TA98' revertant colonies formed compared with control microsomes. Fourth, the binding of n-octylamine to hepatic microsomes from plcloramtreated rats showed, like microsomes from 3-methylcholanthrene-treated rats, an increase in the proportion of high-spin cytochrome P-450 present. Cytochrome P-450d is known to be a high spin haemoprotein.
High magnitude hepatic cytochrome P-450 induction by an N-substituted imidazole antimycotic, clotrimazole
1987, Biochemical Pharmacology
A 4-fold induction of hepatic microsomal cytochrome P-450 following 3 days of treatment of rats with clotrimazole (75mg/kg), a potent monooxygenase inhibitor, greatly exceeded that evident from similar phenobarbital and dexamethasone treatment. The clotrimazole-induced microsomes exhibited a pattern of monooxygenase activities similar to that seen in microsomes from both phenobarbital- and dexamethasone-treated animals. Precautions were necessary to determine both monooxygenase activities and the full amount of cytochrome P-450 present in microsomes because of interference by residual clotrimazole in the microsomes.
Carbon tetrachloride and trichloroethylene toxicities to rat hepatocytes in primary monolayer culture: Its relationship to the level of cytochrome P-450
1986, Toxicology Letters
The effects of carbon tetrachloride (CCl₄) and trichloroethylene (TCE) on the synthesis and the secretion of triacylglycerols (TGs) in primary cultured rat hepatocytes were investigated in relation to the level of cellular cytochrome P-450 (P-450). Pretreatment of rats with phenobarbital (PB) and plating the hepatocytes in the presence of metyrapone attained a marked preservation of P-450 during the preparation of monolayer. CC1₄ was able to cause the accumulation of cellular TG in the hepatocytes when the content of P-450 was retained at the level equivalent to that in the liver in vivo.

View all citing articles on Scopus

View full text

Cytochrome P-450 ligands: Metyrapone revisited

Abstract

Biochem. Biophys. Res. Commun

Biochem. Pharmacol

Biochem. Biophys. Res. Commun

Arch. Biochem. Biophys

Arch. Biochem. Biophys

Biochem. Biophys. Res. Commun

Arch. Biochem. Biophys

J. Biol. Chem

FEBS Lett