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. Semiquantitative reverse-transcriptase polymerase chain reaction was used to determine the mRNA content for four CYP3A genes (CYP3A2, CYP3A9, CYP3A18, and CYP3A23) in livers of control and EE(2)-treated female rats. EE(2) selectively induced CYP3A9 by 3.3-fold whereas the expression of CYP3A18 and CYP3A23 was slightly decreased; neither CYP3A2 mRNA nor CYP3A1 mRNA was detectable in these EE(2)-treated livers. To determine whether rat liver microsomal CYP3A9 was indeed responsible for the EE(2)-stimulated CyA metabolism, a recombinant CYP3A9 was heterologously expressed in Escherichia coli. When functionally reconstituted, this enzyme was active in metabolizing CyA preferentially to its AM9 and AM1c metabolites as compared with CYP3A4. These findings thus support the notion that the increased CyA-metabolizing capacity of EE(2)-treated female rat liver microsomes is due to the induction of the CYP3A9 enzyme.

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. CYP3A9 induction was different from other CYP3A isoenzymes, since phenobarbital was a more effective inducer than dexamethasone and clotrimazole. The results presented for CYP3A23 are those anticipated for CYP3A1, which may be an allelic variant of CYP3A23 not detected in these experiments. These data show that rat CYP3A genes are variably expressed depending on age, sex, or inducer type.

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. Although CYP3A was not detected in control leukocytes, in vivo treatment with known CYP3A inducers (dexamethasone, clotrimazole, phenobarbital, pregnenolone-16 alpha-carbonitrile) resulted in CYP3A leukocyte levels of 0.2-0.8 pmol/mg protein. This leukocyte induction was approximately 1000-fold lower than in induced liver. Interestingly, there was an apparent linear relationship between leukocyte and liver CYP3A contents (r2 = 0.748, n = 29). These results not only demonstrate for the first time that CYP3A is inducible in rat leukocytes after in vivo treatment with various CYP3A inducers, but also suggest that peripheral leukocytes could be used to assess induction in vivo.

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. The highest induction of CYP1A1 was obtained with a sample containing 274 mg 5- and 6-ring PAH/kg soil, resulting in a nearly 360-fold increase in the ethoxyresorufin deethylase (EROD) activity. In a semilogarithmic plot, a linear correlation was found between the 5- and 6-ring PAH concentration in the soil and the microsomal CYP1A1 content. As a model for the action of intestinal fluids, soil samples were extracted by bile acid solution. In these experiments, the selectivity in the solubilization of individual PAHs parallels that of toluene extraction, although the yield is lower than the latter and varies with the soil sample. The bioavailability of PAHs for microorganisms, but not for mammals, was shown to be considerably reduced in the presence of high total organic carbon (TOC) values of the soil samples. This may have implications for decontamination strategies, diminishing the effectiveness of biological decontamination in cases with high TOC values. The data suggest that CYP1A1 induction in rats is a parameter that may be useful in risk assessments of contaminated soils for mammalian species.

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., Gonzalez, F.J., Yamazoe, Y. and Kato, R. (1990) J. Biochem. 107, 718-725). Northern blots show the presence of cUT mRNA in livers of adult female and male rats. The transcription of the new gene is enhanced in either sex by pregnenolone-alpha-carbonitrile, dexamethasone, phenobarbital, and triacetyloleandomycin, known inducers of CYP3A gene expression.

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, 42.0 mumol ATP/min per mg). With increasing inhibitor concentrations, the apparent Km,2 increases as in competitive inhibition, while Vmax,1 decreases as in non-competitive inhibition. The Ki values derived for both types of inhibition are similar, but strongly different for 2- and 8-azido-AMP-PNP (4 microM and 460 microM, respectively). The decrease of the high-affinity Vmax is compensated by an increase in low-affinity catalysis, resulting in a constant sum of maximal velocities. These data can be described by a model where two sites interact with negative cooperativity in binding of substrate.

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. 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 2 beta-hydroxy-, 15 beta-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.

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. After addition of these subunits, F1(alpha beta gamma) is as active in reconstituting ATP-dependent transhydrogenase as five-subunit F1. The ATPase activity of F1 (alpha beta gamma) is inhibited by subunit epsilon in a 1:1 stoichiometry to the same extent (approximately equal to 90%) and with the same affinity (Ki = 0.2-0.8 nM) as reported earlier [Dunn, S.D. (1982) J. Biol. Chem. 257, 7354-7359]. In the presence of either delta or epsilon, F1(alpha beta gamma) binds to F1-depleted membrane vesicles and to liposomes containing the membrane sector (F0) of the ATP synthase to an extent commensurate with the F0 content. The binding ratios epsilon/F1 (alpha beta gamma) and probably also delta/F1 (alpha beta gamma) are close to unity. The specific, delta- or epsilon-deficient F1.F0 complexes presumably formed show ATPase activities sensitive to subunit epsilon but not to dicyclohexylcarbodiimide, and no energy-transfer capabilities. Binding studies at different pH values suggest that F1-F0 interactions in the presence of both subunits delta and epsilon are similar to a combination of those mediated by delta or epsilon alone.(ABSTRACT TRUNCATED AT 250 WORDS)

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 delta exhibit about 27% identity with residues 28-59 of precursor delta from N.crassa and in the first six residues is identical with delta from S.cerevisiae. Approximately half of bovine epsilon has been sequenced. Possibly significant sequence similarities exist between bovine gamma and epsilon and kinase-related gene and oncogene products. The bovine alpha subunit has a blocked NH2-terminus.

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. The possibility that F6, OSCP and subunit delta of E. coli F1 could have evolved from a common ancestral gene is supported by apparent gene duplication within the OSCP and subunit delta sequences.

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. Aurovertin alone inhibits 50% of the oxonol shift at 2 microM, and no further inhibition occurs at up to 12 microM. In combination with even weakly effective uncouplers, 4 microM aurovertin fully abolishes the oxonol response. No such effects are observed in the presence of oligomycin and uncouplers. No pH gradient is detectable by quenching of 9-amino-6-chloro-2-methoxyacridine; and nigericin is without effect on the oxonol response. Valinomycin is inhibitory even in the absence of added potassium, due to ammonium ions introduced during the purification steps. Thiocyanate inhibits the dye response by only 10-27%, depending on the preparation. The extent of the oxonol response depends on the ATP/ADP ratio rather than the phosphorylation potential. The dye response in the ATPase complex is 4-7-times less sensitive to bile salts than in submitochondrial particles. The inhibition by cardiolipin can be reversed by the addition of phospholipids. The possibility is discussed that the oxonol response in the ATPase complex reflects, at least in part, a more local, ATP-dependent and energy-related process.

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. Simultaneous administration of hexachlorobenzene and iron-dextran caused the porphyrin level to reach 25% of the amount induced by hexachlorobenzene alone. The iron concentrations in liver as well as in liver mitochondria are also decreased under these conditions, as compared to the effect of iron-dextran. In contrast, the effects of hexachlorobenzene combined with iron-dextran on mitochondrial oxidative phosphorylation and malondialdehyde content are greater than those of either hexachlorobenzene or iron-dextran. These data suggest that porphyrin accumulation per se causes little deleterious effect and that both agents administered together act synergistically in causing damage to the mitochondrial membrane.

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. Based on product analyses, irradiation of free or bound 2-azido-4-nitrophenolate with visible light results in the formation of nitrene intermediates with a singlet to triplet ratio of 6:1 to 9:1. 2-Azido-4-nitrophenolate and bovine serum albumin form a strong 1:1 complex (KD = 0.7 micron) which can be converted into a photoproduct with a covalent bond between the label and the protein. The acid dissociation constant of the protein-bound 2-amino-4-nitrophenol moiety is strongly pH dependent. Photoaffinity labeling of mitochondria by 2-azido-4-nitrophenolate follows a pattern expected from equilibrium binding studies using normal and lipid-depleted particles: polypeptides were found to bear 90-95% of the radioactive label, and 5-10% of the latter was bound to phospholipids. Two polypeptides (approximately 56 000 and 31 000 daltons) were associated with 60% of the label, indicating a high degree of specific photochemical labeling.

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 23.5 min and t1/2 beta of 8.5 hr. 5-Methyltetrahydrofolate appeared to be readily taken up by the tissues and was excreted in the urine primarily in the initial 4-8 hr. with minimal degradation in the process.