Susceptibility of clinical isolates of Candida spp. to terconazole and other azole antifungal agents

Terconazole is a triazole ketal derivative with potent, broad-spectrum antifungal activity. We investigated the in vitro activity of terconazole, miconazole, and clotrimazole, against 94 clinical isolates of Candida spp.: C. albicans (n = 68), C. tropicalis (n = 18), and C. parapsilosis (n = 8). In vitro susceptibility testing was performed using a broth microdilution method. The minimal inhibitory concentrations of terconazole were less than those of miconazole against C. albicans and C. parapsilosis but higher against C. tropicalis. Terconazole was more active than clotrimazole against C. parapsilosis and less active against C. albicans and C. tropicalis. Terconazole inhibited the uptake of 14C-labeled glucose, leucine, and hypoxanthine into C. albicans and caused the rapid release of intracellular K+. Based on these studies, terconazole has promising anticandidal activity and warrants further in vitro and in vivo investigation.     

Anticandidal activities of terconazole, a broad-spectrum antimycotic.

Terconazole is a new triazole ketal derivative with broad-spectrum in vitro and in vivo antifungal activities. This study further characterizes the effects of terconazole in vitro on yeast cell growth, viability, and morphology. Terconazole inhibited the growth of Candida albicans ATCC 44859 in a concentration-related manner, but with modest effects noted at levels from 10(-8) to 10(-5) M when the yeast was grown on media favoring the cell form. The inhibitory potency of terconazole on yeast cell viability varied with the strain and species of Candida tested. The susceptibility of C. albicans ATCC 44859 to terconazole was markedly enhanced when the yeast was grown on Eagle minimum essential medium, which favors mycelium formation. The effects of terconazole on the morphology of yeast cells (grown on Eagle minimum essential medium) were shown by phase-contrast and electron microscopy. There is a progression of changes, from loss of mycelia formation at 10(-8) M terconazole through complete necrosis at 10(-4) M.     

A new triazole, voriconazole (UK-109,496), blocks sterol biosynthesis in Candida albicans and Candida krusei.

Voriconazole (UK-109,496) is a novel triazole derivative with potent broad-spectrum activity against various fungi, including some that are inherently resistant to fluconazole, such as Candida krusei. In this study we compared the effect of subinhibitory concentrations of voriconazole and fluconazole on sterol biosynthesis of fluconazole-resistant and -susceptible Candida albicans strains, as well as C. krusei, in an effort to delineate the precise mode of action of voriconazole. Voriconazole MICs ranged from 0.003 to 4 microg/ml, while fluconazole MICs ranged from 0.25 to >64 microg/ml. To investigate the effects of voriconazole and fluconazole on candidal sterols, yeast cells were grown in the absence and presence of antifungals. In untreated C. albicans controls, ergosterol was the major sterol (accounting for 53.6% +/- 2.2% to 71.7% +/- 7.8% of the total) in C. albicans and C. krusei strains. There was no significant difference between the sterol compositions of the fluconazole-susceptible and -resistant C. albicans isolates. Voriconazole treatment led to a decrease in the total sterol content of both C. albicans strains tested. In contrast, exposure to fluconazole did not result in a significant reduction in the total sterol content of the three candidal strains tested (P > 0.5). Gas-liquid chromatographic analysis revealed profound changes in the sterol profiles of both C. albicans strains and of C. krusei in response to voriconazole. This antifungal agent exerted a similar effect on the sterol compositions of both fluconazole-susceptible and -resistant C. albicans strains. Interestingly, a complete inhibition of ergosterol synthesis and accumulation of its biosynthetic precursors were observed in both strains treated with voriconazole. In contrast, fluconazole partially inhibited ergosterol synthesis. Analysis of sterols obtained from a fluconazole-resistant C. albicans strain grown in the presence of different concentrations of voriconazole showed that this agent inhibits ergosterol synthesis in a dose-dependent manner. In C. krusei, voriconazole significantly inhibited ergosterol synthesis (over 75% inhibition). C. krusei cells treated with voriconazole accumulated the following biosynthetic intermediates: squalene, 4,14-dimethylzymosterol, and 24-methylenedihydrolanosterol. Accumulation of these methylated sterols is consistent with the premise that this agent functions by inhibiting fungal P-450-dependent 14alpha-demethylase. As expected, treating C. krusei with fluconazole minimally inhibited ergosterol synthesis. Importantly, our data indicate that voriconazole is more effective than fluconazole in blocking candidal sterol biosynthesis, consistent with the different antifungal potencies of these compounds.     

Posaconazole is a potent inhibitor of sterol 14alpha-demethylation in yeasts and molds

Posaconazole (POS; SCH 56592) is a novel triazole that is active against a wide variety of fungi, including fluconazole-resistant Candida albicans isolates and fungi that are inherently less susceptible to approved azoles, such as Candida glabrata. In this study, we compared the effects of POS, itraconazole (ITZ), fluconazole (FLZ), and voriconazole (VOR) on sterol biosynthesis in strains of C. albicans (both azole-sensitive and azole-resistant strains), C. glabrata, Aspergillus fumigatus, and Aspergillus flavus. Following exposure to azoles, nonsaponifiable sterols were extracted and resolved by liquid chromatography and sterol identity was confirmed by mass spectroscopy. Ergosterol was the major sterol in all but one of the strains; C. glabrata strain C110 synthesized an unusual sterol in place of ergosterol. Exposure to POS led to a decrease in the total sterol content of all the strains tested. The decrease was accompanied by the accumulation of 14alpha-methylated sterols, supporting the contention that POS inhibits the cytochrome P450 14alpha-demethylase enzyme. The degree of sterol inhibition was dependent on both dose and the susceptibility of the strain tested. POS retained activity against C. albicans isolates with mutated forms of the 14alpha-demethylase that rendered these strains resistant to FLZ, ITZ, and VOR. In addition, POS was a more potent inhibitor of sterol synthesis in A. fumigatus and A. flavus than either ITZ or VOR.     

Terconazole - a new broad-spectrum antifungal

Terconazole, a new triazole ketal, is found to be highly active in vitro on a wide range of yeasts and mycelium-forming fungi. The in vitro activity depends largely on the medium used. In vitro it is a potent antifungal agent in preventing the morphogenetic transformation of the yeast into the (pseudo-)mycelium form of Candida albicans. In vivo terconazole is highly active in topical treatment of various experimental models of dermatophytosis and candidosis. It also possesses moderate oral broad-spectrum activity. No side effects were observed.     

Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

Sterol delta22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14alpha-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol delta22-desaturase activity in a reconstituted system with NADPH-cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 microM and a Vmax of 0. 59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol delta22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol delta22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.     

Effects of itraconazole on cytochrome P-450-dependent sterol 14 alpha-demethylation and reduction of 3-ketosteroids in Cryptococcus neoformans.

As in other pathogenic fungi, the major sterol synthesized by Cryptococcus neoformans var. neoformans is ergosterol. This yeast also shares with most pathogenic fungi a susceptibility of its cytochrome P-450-dependent ergosterol synthesis to nanomolar concentrations of itraconazole. Fifty percent inhibition of ergosterol synthesis was reached after 16 h of growth in the presence of 6.0 +/- 4.7 nM itraconazole, and complete inhibition was reached at approximately 100 nM itraconazole. This inhibition coincided with the accumulation of mainly eburicol and the 3-ketosteroid obtusifolione. The radioactivity incorporated from [14C]acetate in both compounds represents 64.2% +/- 12.9% of the radioactivity incorporated into the sterols plus squalene extracted from cells incubated in the presence of 10 nM itraconazole. The accumulation of obtusifolione as well as eburicol indicates that itraconazole inhibits not only the 14 alpha-demethylase but also (directly or indirectly) the NADPH-dependent 3-ketosteroid reductase, i.e., the enzyme catalyzing the last step in the demethylation at C-4. This latter inhibition obviates the synthesis of 4,4-demethylated 14 alpha-methylsterols that may function at least partly as surrogates of ergosterol. Eburicol and obtusifolione are unable to support cell growth, and the 3-ketosteroid has been shown to disturb membranes. The complete inhibition of ergosterol synthesis and the accumulation of the 4,4,14-trimethylsterol and of the 3-ketosteroid together with the absence of sterols, such as 14 alpha-methylfecosterol and lanosterol, which can partly fulfill some functions of ergosterol, are at the origin of the high activity of itraconazole against C. neoformans. Fifty percent inhibition of growth achieved after 16 h of incubation in the presence of 3.2 +/- 2.6 nM itraconazole.     

Differential inhibitory effects of protoberberines on sterol and chitin biosyntheses in Candida albicans.

The anti-Candida potentials of 12 Korean medicinal plants were explored: methanol extracts from Coptis rhizoma and Phellodendron amurense caused significant inhibition of growth of Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis. The predominant active components of the extracts were the protoberberines berberine and palmatine; the most potent inhibition of growth was exhibited by berberine on C. krusei (MIC <4 mg/L) and palmatine on C. parapsilosis (MIC 16 mg/L). Both berberine and palmatine inhibited the in-vivo rate of incorporation of L-[methyl-14C]methionine into C-24 of ergosterol in C. albicans (50% inhibition concentration (IC50 values), 25 microM and 300 microM, respectively); this result suggests that sterol 24-methyl transferase (24-SMT) is one of the cellular targets for the antifungal activity of the protoberberines. In-vitro 24-SMT activity in microsomes from the yeast growth form of C. albicans was inhibited by both berberine (inhibition constant (Ki) 232 microM) and palmatine (Ki 257 microM) in a non-competitive manner; inhibition of 24-SMT was more marked for the mycelial form than for the yeast growth form of this organism. Palmatine inhibited chitin synthase from both the yeast and mycelial growth phases of C. albicans in a non-competitive manner (Ki 780 microM). The effects of protoberberines, extracted from established medicinal plants, on both sterol and cell wall biosyntheses in pathogenic fungi indicate that the potential of these compounds, or their semi-synthetic derivatives, as a novel class of antifungal agents should be investigated more fully.     

Effects of terconazole and other azole antifungal agents on the sterol and carbohydrate composition of Candida albicans

The effects of terconazole, a triazole antifungal, on the sterol and carbohydrate composition of Candida albicans was compared with that of three imidazoles: clotrimazole, miconazole, and butoconazole. Exposure of C. albicans to terconazole resulted in a profound depletion of ergosterol with a corresponding increase in lanosterol content versus control cells. Carbohydrate analysis revealed a significant (245%) increase in chitin and a minimal effect on glucan and mannan in terconazole-treated cells. Similar effects on sterol and carbohydrate composition were observed with clotrimazole and miconazole. Butoconazole had a similar effect on sterol composition but had no effect on carbohydrate composition. The decreased ergosterol and increased lanosterol content is consistent with 14 alpha-demethylase inhibition by terconazole and the other azoles. The increase in cell wall chitin is most likely due to deregulation of chitin synthesis secondary to ergosterol depletion in the cell membrane. Because both chitin and ergosterol are critical components of the fungal cell, perturbation of the production and localization of these components by terconazole is likely to contribute to the selective toxicity of this compound for C. albicans and other fungi.     

Strong Antifungal Activity of SS750, a New Triazole Derivative, Is Based on Its Selective Binding Affinity to Cytochrome P450 of Fungi

SS750 [(R)-(-)-2-(2,4-difluorophenyl)-1-(ethylsulfonyl)-1,1-difluoro-3-(1H-1,2,4-triazol-1-yl)-2-propanol] is a new triazole, and its potential as an antifungal agent was evaluated by in vitro and in vivo studies. In a comparison of the MICs at which 50% of isolates are inhibited (MIC(50)s) for all strains of Candida species and Cryptococcus neoformans tested, SS750 was four times or more active than fluconazole and had activity comparable to that of itraconazole. The most important advantage of SS750 was that, when the MIC(90)s were compared, SS750 had 64 and 32 times greater antifungal activities than fluconazole against Candida krusei and Candida glabrata, respectively, which are intrinsically less susceptible to fluconazole. In cyclophosphamide-immunosuppressed mouse models of systemic and pulmonary candidiasis caused by C. albicans, oral SS750 prolonged the number of days of survival of infected animals in a dose-dependent manner and was 4 and > or =64 times more potent than fluconazole and itraconazole, respectively. In a safety profile, SS750, like fluconazole, had less of an affinity for binding to mammalian cytochrome P450 compared with that of ketoconazole, despite its strong affinity for binding to fungal cytochrome P450. The mechanism for the increased in vitro antifungal activity of SS750 against C. krusei is partially due to the potent inhibitory activity (3.7 times versus that of fluconazole) of C. krusei cytochrome P450 sterol 14alpha-demethylase; SS750 showed a strong affinity for binding to cytochrome P450 of C. krusei, indicating that SS750 acts by inhibiting the cytochrome P450 sterol 14alpha-demethylase of fungal cells.     

Cytochrome P-450 complex formation in rat liver by the antibiotic tiamulin.

Tiamulin is a semisynthetic diterpene antibiotic frequently used in farm animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds. It has been suggested that this is caused by a selective inhibition of oxidative drug metabolism via the formation of a cytochrome P-450 metabolic intermediate complex. In the present study, rats were treated orally for 6 days with tiamulin at two different doses: 40 and 226 mg/kg of body weight. For comparison, another group received 300 mg of triacetyloleandomycin (TAO) per kg, which is equivalent to the 226-mg/kg tiamulin group. Subsequently, microsomal P-450 contents, P-450 enzyme activities, metabolic intermediate complex spectra, and P-450 apoprotein concentrations were assessed. In addition, effects on individual microsomal P-450 activities were studied in control microsomes at different tiamulin and substrate concentrations. In the rats treated with tiamulin, a dose-dependent complex formation as evidenced by its absorption spectrum and an increase in cytochrome P-4503A1/2 contents as assessed by Western blotting (immunoblotting) were found. The effects were comparable to those of TAO. Tiamulin induced microsomal P-450 content, testosterone 6 beta-hydroxylation rate, erythromycin N-demethylation rate, and the ethoxyresorufin O-deethylation activity. Other activities were not affected or decreased. When tiamulin was added to microsomes of control rats, the testosterone 6 beta-hydroxylation rate and the erythromycin N-demethylation were strongly inhibited. It is concluded that tiamulin is a potent and selective inducer-inhibitor of cytochrome P-450. Though not belonging to the macrolides, the compound produces an effect on P-450 similar to those of TAO and related compounds.     

Comparison of D0870, a new triazole antifungal agent, to fluconazole for inhibition of Candida albicans cytochrome P-450 by using in vitro assays.

D0870 was 12 to 15 times more active than fluconazole in experiments to determine the MIC for growth arrest for two isolates of Candida albicans. A biochemical comparison of in vitro sterol biosynthesis in cell extracts showed only a twofold superiority of D0870 over fluconazole. A large differentiation (10-fold) in 50% saturating concentrations obtained by examining the binding of the azoles to microsomal P-450 was observed in a type II binding spectrophotometric assay, possibly reflecting the differential affinity for more than one P-450 enzyme. Additional mechanisms besides affinity for the target enzyme sterol 14 alpha-demethylase, such as differential intracellular accumulation of drug, may contribute to the differences in antifungal activity.     

Bioactivation of 8-methoxypsoralen and irreversible inactivation of cytochrome P-450 in mouse liver microsomes: modification by monoclonal antibodies, inhibition of drug metabolism and distribution of covalent adducts

The in vitro bioactivation of 8-MOP was studied in liver microsomes of male CD-1 mice. In 10-min incubations with 40 microM [14C]8-MOP, covalent binding (mean +/- S.D.) was 1.8 +/- 0.4, 3.1 +/- 0.6 and 5.4 +/- 0.4 nmol/mg protein, respectively, in microsomes from mice pretreated for 3 days with vehicle, phenobarbital or beta-naphthoflavone (BNF). A monoclonal antibody (MAb 1-7-1), which recognizes isozymes of cytochrome P-450 induced by 3-methylcholanthrene (P1-450 and P3-450), selectively inhibited the metabolism of 8-MOP (-57%) and covalent binding of its metabolites (-40%) in microsomes from mice pretreated with BNF, but had no effect in microsomes of mice pretreated with phenobarbital or vehicle. Monoclonal antibody 2-66-3, which recognizes the major isozymes of rat cytochrome P-450 induced by phenobarbital and unknown isozymes in the mouse, enhanced the covalent binding of 8-MOP metabolites in microsomes of mice pretreated with vehicle (+74%), phenobarbital (+44%) or BNF (+31%) without affecting the disappearance of 8-MOP. Preincubation of liver microsomes from BNF-pretreated mice with 40 microM 8-MOP decreased the activity of 7-ethoxycoumarin de-ethylase in a time-dependent manner. Preincubation with 40 microM 8-MOP for 10 min decreased the Vmax from 3.4 to 1.2 nmol/min/mg protein and increased the Michaelis constant from 46 to 90 microM, thus demonstrating mixed competitive and noncompetitive inhibition of 7-ethoxycoumarin de-ethylase. Cysteine trapped three-fourths of the reactive intermediates of 8-MOP but was ineffective in preventing the irreversible inhibition of 7-ethoxycoumarin de-ethylase activity or the 45% spectral loss of cytochrome P-450. Cysteine was ineffective probably because it did not prevent the irreversible binding of metabolites of 8-MOP to cytochrome P-450. There was no spectral evidence that 8-MOP formed cytochrome P-420 or metabolite-intermediate complexes with cytochrome P-450. These findings support the hypothesis that irreversible inactivation of cytochrome P-450 by 8-MOP is caused by modification of the apoprotein by reactive metabolites.     

In vitro and in vivo effects of the antimycotic drug ketoconazole on sterol synthesis.

Ketoconazole, an orally active antimycotic drug, is a potent inhibitor of ergosterol biosynthesis in Candida albicans when added to culture media which support yeast or mycelial growth or to cultures containing outgrown mycelium. This inhibition coincides with accumulation of sterols with a methyl group at C-14 and can thus be attributed to an interference with one of the reactions involved in the removal of the 14 alpha-methyl group of lanosterol. When administered to rats infected with C. albicans, ketocanazole also inhibits fungal synthesis of ergosterol. A six-times-higher dose is required to effect cholesterol synthesis by rat liver.     

Azole susceptibility and hyphal formation in a cytochrome P-450-deficient mutant of Candida albicans.

A cytochrome P-450-deficient mutant of Candida albicans, strain D10, was employed to study the mode of action of imidazole antifungal agents. This mutant accumulates exclusively 14-alpha-methylsterols, resulting in a sterol profile which mimics that of azole-treated wild-type strains. Since the widely accepted primary effect of imidazoles is the inhibition of cytochrome P-450-mediated demethylation of the ergosterol precursor lanosterol, strain D10 and its wild-type revertant, strain D10R, were grown in the presence of concentrations of clotrimazole, miconazole, and ketoconazole known to inhibit demethylation. The growth of strain D10 was unaffected by these antifungal agents, while that of strain D10R was significantly reduced. At higher azole concentrations (which are known to exert a direct, disruptive action on the cell membrane), the growth of both strains was immediately and completely inhibited by clotrimazole and miconazole. Ketoconazole was membrane disruptive only for strain D10; this is the first report of a direct membrane effect for this drug. Because hyphal formation has been implicated in the pathogenesis of C. albicans and because it has been shown to be inhibited by azoles, the hypha-forming capability of strain D10 was examined. Strain D10 was shown to be seriously defective in hyphal formation, suggesting that this function may be dependent on the 14-alpha-demethylation of lanosterol. The results of this study suggest that inhibition of lanosterol demethylation per se is neither fungicidal nor fungistatic, although the growth rate is reduced. In addition, the substitution of 14-alpha-methylsterols for ergosterol results in defective hyphal formation and in a cell that is more susceptible to membrane-active agents such as ketoconazole.     

Erythromycin breath test as an assay of glucocorticoid-inducible liver cytochromes P-450. Studies in rats and patients.

The major P-450IIIA gene family member present in human liver is HLp which, like its rat liver orthologue P-450p, is inducible by glucocorticoids and catalyzes erythromycin N-demethylation. To develop a practical method to estimate the amounts of HLp in patients [14C]N-methyl erythromycin was injected into rats that had been pretreated with dexamethasone or with inducers of other forms of cytochrome P-450. The rate of demethylation of this substrate, measured simply as 14CO2 in the breath, correlated well with the concentrations of immunoreactive P-450p protein (r = 0.70), holocytochrome P-450p (r = 0.70), or with erythromycin N-demethylase activity (r = 0.90) determined in the liver microsomes prepared from each rat. Next, [14C]N-methyl erythromycin was administered to 30 patients and there was a sixfold interindividual variation in breath 14CO2 production seemingly unrelated to medications, smoking status or age. However, the average breath test values were twofold greater in female as compared to male patients (P less than 0.01). Breath 14CO2 production rose in patients retested after treatment with the P-450IIIA inducers dexamethasone (P less than 0.05) or rifampicin (P less than 0.05) and was decreased after treatment with the HLp inhibitor triacetyloleandomycin (P less than 0.05). We conclude that the erythromycin breath test provides a convenient assay of P-450IIIA cytochromes in rats and in some patients.     

Mechanism of Fluconazole Resistance in Candida krusei

The mechanisms of fluconazole resistance in three clinical isolates of Candida krusei were investigated. Analysis of sterols of organisms grown in the absence and presence of fluconazole demonstrated that the predominant sterol of C. krusei is ergosterol and that fluconazole inhibits 14α-demethylase in this organism. The 14α-demethylase activity in cell extracts of C. krusei was 16- to 46-fold more resistant to inhibition by fluconazole than was 14α-demethylase activity in cell extracts of two fluconazole-susceptible strains of Candida albicans. Comparing the carbon monoxide difference spectra of microsomes from C. krusei with those of microsomes from C. albicans indicated that the total cytochrome P-450 content of C. krusei is similar to that of C. albicans. The Soret absorption maximum in these spectra was located at 448 nm for C. krusei and at 450 nm for C. albicans. Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not greater than that of C. albicans. Thus, there are significant qualitative differences between the 14α-demethylase of C. albicans and C. krusei. In addition, fluconazole resistance in these strains of C. krusei appears to be mediated predominantly by a reduced susceptibility of 14α-demethylase to inhibition by this drug.     

Selective inhibition of rat hepatic microsomal cytochrome P-450. II. Effect of the in vitro administration of cimetidine.

The purpose of this study was to determine whether the differential inhibition of rat hepatic microsomal cytochrome P-450 in adult male rats by in vivo cimetidine administration is observed when the drug is administered in vitro. Cimetidine, at concentrations of up to 10 mM, did not affect the catalytic function of cytochrome P-450IIA1 as measured by testosterone 7 alpha-hydroxylase activity. In contrast, it did inhibit activities that are specific for cytochromes P-450IIC11 (testosterone 2 alpha-hydroxylase activity), P-450IIB1/2 (testosterone 16 beta-hydroxylase activity) and P-450IIA1/2 (testosterone 2 beta- and 6 beta-hydroxylase activities), with IC50 values in the range of 1.0 to 7.4 mM. To further investigate the inhibition of cytochrome P-450 enzymes by in vitro cimetidine, preincubation experiments were performed. Hepatic microsomes were preincubated with a low concentration (0.05 mM) of cimetidine and 1 mM NADPH for 15 min before the initiation of substrate (testosterone) oxidation. Under these conditions, cimetidine resulted in the inhibition of the enzyme activities specific for cytochrome P-450IIC11, but it had no effect on those specific for cytochromes P-450IIA1, P-450IIB1/2 and P-450IIIA1/2. This differential inhibition by in vitro cimetidine required the presence of NADPH in the preincubation medium, suggesting that a catalysis-dependent process is involved. Therefore, preincubation of hepatic microsomes with NADPH and a relatively low concentration (0.05 mM) of cimetidine in vitro results in a pattern of inhibition of cytochrome P-450 enzymes similar to that which occurs after the in vivo administration of cimetidine reported in the previous study (Chang et al., 1992).     

Effects of beta-naphthoflavone administration to pregnant rats on steroid hormone biosynthesis and metabolism in ovarian microsomes

Administration of low doses of beta-naphthoflavone (beta-NF) to pregnant rats on days 7 to 14 of gestation has been associated with extensive fetal mortality. Studies were conducted to evaluate the effects of beta-NF administration on several extrahepatic cytochrome P-450-dependent enzymes responsible for steroid biosynthesis in the ovary. Pregnant rats received beta-NF (15 mg/kg) for 2-, 4- or 6-day periods before study on day 15 of gestation. At this stage of pregnancy no adverse effects on maternal weight gain, percent resorptions or fetal body weight were observed after beta-NF treatment. Cytochrome P-450 content of ovarian microsomes was not altered by beta-NF treatment. Estrogen biosynthesis in ovarian microsomes (aromatase activity), assayed as conversion of [14C]testosterone to 17 beta-estradiol, was increased nearly 2-fold in the beta-NF-treated dams. Incubation of ovarian microsomes with [14C]progesterone yielded 20 alpha-hydroxy progesterone and an unidentified compound as major products. There was no evidence of cytochrome P-450-dependent conversion of progesterone to androgens at this stage in pregnancy. In contrast to the effects of beta-NF on aromatase activity, progesterone catabolism was unaltered after administration of beta-NF for a 4-day period. The absence of a change in 20 alpha-hydroxy steroid dehydrogenase activity suggests that corpus luteum function is unaltered after beta-NF exposure. These data indicate that beta-NF administration during midgestation is associated with a selective alteration in in vitro ovarian steroidogenesis and further suggest a mechanism for beta-NF related in utero toxicity.     

Sterol composition of Cryptococcus neoformans in the presence and absence of fluconazole.

Analysis of the sterol compositions of 13 clinical isolates of the pathogenic yeast Cryptococcus neoformans obtained from five patients with recurring cryptococcal meningitis showed that, unlike Candida albicans, the major sterols synthesized by this yeast were obtusifoliol (range, 21.1 to 68.2%) and ergosterol (range, 0.0 to 46.5%). There was considerable variation in the sterol contents among the 13 isolates, with total sterol contents ranging from 0.31 to 5.9% of dry weight. The isolates from the five patients who had relapses had different total sterol contents and compositions in comparison with those of the pretreatment isolates, indicating either that the sterols had been changed by therapy or that the patients were infected with new isolates with different sterol compositions. Growth of the cryptococcal isolates in the presence of subinhibitory concentrations of fluconazole (0.25x the MIC) significantly altered the sterol content and pattern. The total sterol content decreased in nine isolates and increased in four isolates in response to pretreatment with fluconazole. Fluconazole had no consistent effect on ergosterol levels. In contrast, fluconazole caused a decrease in obtusifoliol levels and an increase in 4,14-dimethylzymosterol levels in all isolates. These results indicate extensive diversity in sterol content, sterol composition, and sterol synthesis in response to subinhibitory concentrations of fluconazole in C. neoformans strains. We propose that fluconazole inhibits the sterol synthesis of C. neoformans by interfering with both 14 alpha-demethylase-dependent and -independent pathways. No correlation between the sterol compositions of C. neoformans isolates and their susceptibilities to fluconazole was found.