Ethynylestradiol-mediated induction of hepatic CYP3A9 in female rats: implication for cyclosporine metabolism.

Repeated treatment of female rats with the synthetic estrogen ethynylestradiol (EE(2)) increases the formation of the cyclosporine A (CyA) metabolites AM1c and AM9 by 3-fold, whereas the formation of AM1 and AM4N is not significantly enhanced. The formation of all four CyA metabolites was inhibited by greater than 80% by the CYP3A-selective substrate midazolam or polyclonal anti-rat CYP3A IgGs in liver microsomes of untreated and EE(2)-induced rats. In contrast, anti-rat CYP2C6 IgGs had little effect, indicating the involvement of a CYP3A but not 2C6 in this EE(2)-stimulated CyA metabolism.

Expression and inducibility of cytochrome P450 3A9 (CYP3A9) and other members of the CYP3A subfamily in rat liver.

Quantitative reverse-transcriptase polymerase chain reaction was used to determine the content of mRNA derived from four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in rat liver. CYP3A2 and CYP3A9 gene expression was age- and sex-dependent, whereas CYP3A18 and CYP3A23 mRNA were observed before and after puberty at fairly constant levels that were about 20% higher in males than in females. CYP3A9 mRNA was detected only in adult rats, with a nearly twofold higher expression in females.

In vivo induction of cytochrome P450 CYP3A expression in rat leukocytes using various inducers.

Although the induction of cytochromes P450 3A (CYP3A) is relatively well characterized in liver, its inducibility in an easily available tissue such as the peripheral leukocytes is not known. The purpose of this study was, therefore, to determine if CYP3A is inducible in vivo in peripheral leukocytes. Microsomes from rat leukocytes and liver were examined for CYP3A protein expression using Western blotting with a rabbit polyclonal antibody against rat CYP3A.

Liver microsomal levels of cytochrome P450IA1 as biomarker for exposure and bioavailability of soil-bound polycyclic aromatic hydrocarbons.

The bioavailability of soil-bound polycyclic aromatic hydrocarbons (PAHs) for mammalian species was studied with rats fed with a diet containing contaminated soil preparations. The extent of cytochrome P450IA1 (CYP1A1) induction in the liver correlated with the amount of 5- and 6-ring PAHs in the soil samples but not with the total PAH content. Other cytochromes P450 were much less affected by the soil-contaminants.

A novel CYP3 gene from female rats.

A cDNA library constructed from adult female Sprague Dawley rat liver was screened with polyclonal anti CYP3A-IgG. One of the positive clones, cUT, was found to contain the complete coding sequence of a new gene more similar to hamster gene CYP3A10 (cDNA: 85%, deduced amino acid sequence: 79%) than to the known rat CYP3A genes (cDNA: 75-76%, amino acid sequences: 66-69%). The deduced sequence of the first 28 amino acids is identical to the N-terminus of the testosterone 6 beta-hydroxylase, 6 beta-2 (Nagata, K.

Possible existence of a CYP3A protein in liver microsomes from female rats.

CYP3A proteins are P450 monooxygenases involved in the metabolism of steroids, retinoic acid and several important drugs. In rats, the number of CYP3A genes and proteins, and therefore important aspects of their inducibility, developmental regulation and sex specificity are not known for certain. Using triacetyloleandomycin-metabolite complex formation, testosterone hydroxylase assays and immunoblots from peptide maps, we obtained results suggesting that in liver microsomes from mature rats, at least three, possibly four CYP3A proteins are expressed: one is present in females, another is male-specific, and one or two additional CYP3A proteins are inducible by phenobarbital, steroids, and triacetyloleandomycin.

Beef heart mitochondrial F1-ATPase: inhibition by azidoadenyl-5'-yl imidodiphosphates and cooperative binding of substrate.

Two ATP analogs, 2- and 8-azidoadenyl-5'-yl imidodiphosphate, were synthesized, purified and utilized as inhibitors of soluble beef heart mitochondrial F1-ATPase under non-photolytical conditions. In the range of 5 microM to 3 mM ATP, the initial rates of ATP hydrolysis in the presence and absence of the inhibiting ATP analogs can be adequately described by two pairs of Km and Vmax values (3 microM, 8.5 mumol ATP/min per mg; 255 microM, 42.

Formation of ligand and metabolite complexes as a means for selective quantitation of cytochrome P450 isozymes.

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.

Coupling factor 1 from Escherichia coli lacking subunits delta and epsilon: preparation and specific binding to depleted membranes, mediated by subunits delta or epsilon.

A procedure for the preparation of coupling factor 1 (F1) from Escherichia coli lacking subunits delta and epsilon is described. Using chloroform and dimethyl sulfoxide, we can isolate F1 containing only subunits alpha, beta, and gamma [F1(alpha beta gamma)] directly from membrane vesicles in 10-mg quantities. Pure and active subunits delta and epsilon were prepared from five-subunit F1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

High performance liquid chromatography purification and amino terminal sequence analysis of the subunits of bovine heart mitochondrial F1-ATPase.

All five subunits of bovine heart mitochondrial F1-ATPase have been isolated by reverse-phase HPLC and NH2-terminal sequences determined by gas phase Edman degradations. Bovine gamma exhibits 16 identities in the first 30 residues compared with the NH2-terminus of gamma from E.coli F1.

Bovine heart mitochondrial F6: HPLC purification, NH2-terminal sequence and the possible structural relatedness to other components of ATPase complexes.

The mitochondrial factor F6 has been purified by reverse-phase HPLC and the molecular weight (8500), amino acid composition and about 25% of the amino acid sequence determined. In the NH2-terminal sequence of the first 18 amino acids (NKELDPVQKLFVDKIREY), six identities with the NH2-terminal sequence of the oligomycin-sensitivity conferring protein (OSCP) are apparent, as well as less striking similarities with the OSCP related subunit delta of E. coli F1.

ATP-dependent spectral response of oxonol VI in an ATP-Pi exchange complex.

Energy transduction in an ATPase complex (complex V) has been studied in two reactions catalyzed by this system, i.e., ATP-dependent spectral shift of oxonol VI, and ATP-Pi exchange activity.

Effects of hexachlorobenzene and iron loading on rat liver mitochondria.

The effects of hexachlorobenzene treatment and simultaneous iron-overload on the iron and porphyrin content of rat liver and rat liver mitochondria have been examined. In order to assess damages to the mitochondrial membrane occurring with these treatments, the content of malondialdehyde and selected functional properties of mitochondria were compared with those from control animals. Prolonged intake of hexachlorobenzene (8 weeks) resulted in a strikingly increased level of porphyrins together with a moderate increase in iron concentration.

Radiochemical synthesis and photochemical properties of the uncoupler 2-azido-4-nitrophenol, a versatile photoaffinity labeling reagent.

2-Amino-4-nitrophenol was tritiated in an acid-catalyzed hydrogen exchange reaction. Radioactive 2-azido-4-nitrophenol with a specific radioactivity up to 21 mCi/mmol was synthesized from 2-amino-4-nitrophenol by diazotization and azide coupling. The photochemical properties of the uncoupler, 2-azido-4-nitrophenol, were studied as free solute and as ligand bound to uncoupler binding sites in bovine serum albumin and mitochondria.

Pharmacokinetics of 5-methyltetrahydrofolate.

The pharmacokinetic behavior of the reduced folate coenzyme, 5-methyltetrahydrofolate, was studied in the rat. Following oral administration the coenzyme was well absorbed from the intestinal tract and was bound to serum proteins. Plasma disappearance occurred in two exponential phases with a t1/2 alpha of 23.

Mitochondrial ATP-Pi exchange complex and the site of uncoupling of oxidative phosphorylation.

Five enzyme complexes, which are concerned with electron transport and oxidative phosphorylation, have been isolated from beef heart mitochondria. Enzyme complexes I, II, III and IV are the electron transfer complexes discovered in 1961. Complex V is an energy-conserving complex.