J BIOCHEM TOXICOLOGY
Volume 11, Number 3, 1996
Inhibition of Mitochondrial Function in Isolated Rat
Liver Mitochondria by Azole Antifungals
Rosita J. Rodriguez and Daniel Acosta, Jr.*
Department of Pharmacology/Toxicology, University of Texas at Austin, Austin, TX 78712-1074
ABSTRACT: Ketoconazole is an imidazole oral antifungal agent with a broad spectrum of activity. Ketoconazole has been reported to cause liver damage, but
the mechanism is unknown. However, ketoconazole
and a related drug, miconazole, have been shown to
have inhibitory effects on oxidative phosphorylation
in fungi. Fluconazole, another orally administered antifungal azole, has also been reported to cause liver
damage despite its supposedly low toxicity profile. The
primary objective of this study was to evaluate the
metabolic integrity of adult rat liver mitochondria after
exposure to ketoconazole, miconazole, fluconazole,
and the deacetylated metabolite of ketoconazole by
measuring ADP-dependent oxygen uptake polarographically and succinate dehydrogenase activity spectrophotometrically. Ketoconazole, N-deacetyl ketoconazole, and miconazole inhibited glutamate-malate
oxidation in a dose-dependent manner such that the
50% inhibitory concentration (I50) was 32, 300, and 110
lM, respectively. In addition, the effect of ketoconazole, miconazole, and fluconazole on phosphorylation
coupled to the oxidation of pyruvate/malate, ornithine/
malate, arginine/malate, and succinate was evaluated.
The results demonstrated that ketoconazole and miconazole produced a dose-dependent inhibition of
NADH oxidase in which ketoconazole was the most
potent inhibitor. Fluconazole had minimal inhibitory
effects on NADH oxidase and succinate dehydrogenase, whereas higher concentrations of ketoconazole
were required to inhibit the activity of succinate dehydrogenase. N-deacetylated ketoconazole inhibited
succinate dehydrogenase with an I50 of 350 lM. In addition, the reduction of ferricyanide by succinate catalyzed by succinate dehydrogenase demonstrated that
ketoconazole caused a dose-dependent inhibition of
succinate activity (I50 of 74 lM). In summary, ketoconazole appears to be the more potent mitochondrial inhibitor of the azoles studied; complex I of the respiratory chain is the apparent target of the drug’s action.
q 1997 John Wiley & Sons, Inc.
Azole Antifungals, Mitochondria, NADH
Oxidase, Oxidative Phosphorylation, Rat Liver, Succinate Dehydrogenase.
KEYWORDS:
INTRODUCTION
*Received May 6, 1996.
Address correspondence to Dr. Daniel Acosta, Jr., Division of
Pharmacology & Toxicology, College of Pharmacy, The University
of Texas, Austin, TX 78712-1074. Tel: (512) 471-4736; Fax: (512) 4715002.
Ketoconazole (KT), miconazole (MC), and fluconazole (FL), shown in Figure 1, are antifungal agents in
a series of azole-derivatives with a broad spectrum of
activity against superficial and systemic mycoses. The
antifungal actions of these drugs are related to their
inhibition of cytochrome P-450-dependent 14a-demethylation of lanosterol or 24-methylenedihydrolanosterol in the biosynthetic pathway of ergosterol in
fungi (1). However, KT’s action is not limited to the
cytochromes of fungi because it also inhibits a number
of mammalian cytochromes; in contrast, FL is specific
for 14a-demethylase. Nonetheless, the literature
clearly indicates that KT and FL are toxic to the liver
(2–6). In addition, in vitro studies demonstrated that
KT was toxic to liver cells (7,8). It has also been reported that KT inhibited mitochondrial oxidative
phosphorylation in Candida albicans (9,10) and in rat
liver mitochondria (11). Similar effects were seen with
MC on isolated rat liver mitochondria (11). Presently,
the mechanism of azole-induced hepatic necrosis is unknown (12).
It has been reported that inhibition of mitochondrial NADH (nicotinamide adenine dinucleotide) oxidase was KT’s primary mechanism of toxicity in C. albicans (10), but higher concentrations of KT were
required to inhibit mitochondrial activity of succinate
oxidase (SDH) in rat liver. MC and clotrimazole directly damaged membranes of C. albicans cells at lower
concentrations than KT; however, KT’s inhibition of
respiratory function occurred at lower concentrations
than the other azole compounds studied (10). MC was
shown to exhibit uncoupling/inhibition patterns for
both glutamate/malate and succinate (11). Shigematsu
et al. (9) reported that KT blocked the transport of electrons in the respiratory chain of C. albicans under aerobic conditions with such substrates as NADH and
Journal of Biochemical Toxicology
q 1997 John Wiley & Sons, Inc.
0887-2082/97/030127-05
127
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RODRIGUEZ AND ACOSTA
Volume 11, Number 3, 1996
175–200 g were used. These rats were bred and maintained in the Animal Resources Center at the University of Texas at Austin. They were housed in temperature (258C)- and light cycle (12 h)-controlled rooms.
Water and feed were provided ad lib.
Preparation of Rat Liver Mitochondria
Rat liver mitochondria were isolated according to
the procedure of Chappell and Hansford (13). The final
rat liver mitochondrial suspension was placed into a
centrifuge tube on ice and was used 0.5–3 hours after
preparation.
Measurement of Oxidative Phosphorylation
FIGURE 1. Structures of ketoconazole, miconazole, fluconazole,
and deacetyl ketoconazole.
succinate. The addition of KT to C. albicans mitochondria without substrates resulted in strong reduction of
cytochrome a3, indicating that there was a specific interaction between KT and cytochrome c oxidase (9).
These results suggest that the primary site of KT inhibition on mitochondria isolated from C. albicans is at
the most distal portion of the respiratory chain. Because the aforementioned studies indicate that antifungals may inhibit mitochondrial function in fungi (9–
11), the objective of this investigation is to assess mitochondrial dysfunction in mammalian mitochondrial
preparations, rat liver, after exposure to KT, MC, FL,
and KT’s deacetylated metabolite by measuring oxidative phosphorylation polarographically and by
monitoring SDH activity spectrophotometrically.
MATERIALS AND METHODS
Materials
KT and deacetylated KT (DAK) were generous
gifts from Janssen Pharmaceutica (Beerse, Belgium). FL
was a gift from Pfizer Pharmaceuticals (Groton, CT).
Albumin fraction V, adenosine 58diphosphate (ADP),
carbonyl cyanide m-chlorophenylhydrazone (CCCP),
2,4-dinitrophenol (DNP), miconazole, and rotenone
were obtained from Sigma Chemical Co. (St. Louis,
MO). Thenoyltrifluoroacetone (TTFA) was obtained
from Aldrich Chemical Co. (St. Louis, MO). All other
reagents were of the highest available purity from commercial sources. Male Sprague-Dawley rats weighing
The respiration of the isolated rat liver mitochondria was measured polarographically with a Clarktype electrode in a 2 mL stirred vessel maintained at
308C. The signal was recorded with a HeathKit strip
Chart Recorder. The vessel contained a suspension of
mitochondria (4–6 mg protein) in 0.25 M sucrose; 10
mM potassium phosphate; 5 mg/mL albumin; 5 mM
MgSO4, pH 7.4, in a total volume of 2.2 mL. After 4–5
minutes temperature equilibration, the endogenous
rate of oxygen uptake was recorded before adding exogenous substrates in 50 lL through the capillary stem
of the oxygraph vessel. The substrate stock solutions
used were 0.385 M glutamate/0.115 M malate, 0.5 M
succinate, 0.385 M pyruvate/0.115 M malate; 0.385 M
ornithine/0.115 M malate; and 0.385 M arginine/0.115
M malate. After recording oxygen uptake for another
3–4 minutes, 0.8 lmoles ADP, pH 7.4 was added, and
oxygen uptake was recorded for another 3–6 minutes.
The effects of KT, MC, FL, and DAK on mitochondrial
substrate- and ADP-dependent oxygen uptake were
determined by adding the drugs to the oxygraph vessel to yield final concentrations above and below the
50% inhibitory concentration (I50). Stock solutions of
KT (18.8 mM) and DAK (20 mM) were prepared in 0.05
NHCl and MC (21 mM) and FL (33 mM) were prepared
in dimethyl sulfoxide (DMSO). The pH was checked
directly in the oxygraph chamber and adjusted to 7.4
before adding the mitochondria. The contribution of
complex I and II to oxygen uptake was evaluated by
adding the selective inhibitors rotenone and thenoyltrifluoroacetone. The percent inhibition of oxygen consumption was calculated from the slopes on the chart
before and after the addition of KT, MC, FL, and DAK.
Protein was measured by the Bio-Rade Protein Assay
using bovine serum albumin as a standard.
Ferricyanide Assay
The reduction of ferricyanide by succinate catalyzed by SDH was monitored spectrophotometrically
Volume 11, Number 3, 1996
AZOLES INHIBIT MITOCHONDRIAL FUNCTION
129
TABLE 1. The 50% inhibitory concentration (I50’s) of state 3 respiration of various azoles
(ketoconazole, deacetylated ketoconazole, miconazole, and fluconazole) with various
substratesa of the mitochondrial respiratory chain measured polarographically.
50% Inhibitory Concentration (I50) (lM)
Glutamate/Malate
Pyruvate/Malate
Ornithine/Malate
Arginine/Malate
Succinate
Ketoconazole
Deacetyl ketoconazole
Miconazole
Fluconazole
32
74
65
75
500
300
—
—
—
350
110
160
160
170
150
n.d.
n.d.
n.d.
n.d.
n.d.
a
The substrates final concentrations were 10 mM glutamate/3 mM malate; 10 mM pyruvate/3 mM; 10 mM arginine/3 mM malate; 10 mM ornithine/3 mM malate; and 12.5 mM succinate.
n.d. 4 not detectable.
by the method described by Singer (14). Activities were
expressed as a percentage of control (lmoles of ferricyanide reduced/min/mg protein) at 308C. Protein
was measured by the Bio-Rade Protein Assay using
bovine serum albumin as a standard.
RESULTS
The mitochondrial preparations exhibited excellent respiratory control ratios, and ADP consistently
stimulated glutamate/malate, pyruvate/malate, ornithine/malate, arginine/malate, and succinate oxygen
uptake. The respiratory control ratios were 6.6, 5.3, 4.0,
2.0, and 2.0 for glutamate/malate, succinate, pyruvate/malate, arginine/malate, and ornithine/malate,
respectively. In the presence of 0.4 mM ADP, both KT
and MC inhibited the oxidation of glutamate/malate
in a distinct dose-dependent manner, with KT being
more potent (Table 1). The inhibitory concentration (I50)
was 32 and 110 lM for KT and MC, respectively. These
imidazole antifungal drugs also inhibited pyruvate/
malate oxidation but at somewhat higher I50’s (74 lM
and 160 lM for KT and MC, respectively, Table 1). On
the other hand, FL up to 1 mM had essentially no detectable effect on these activities (data not shown).
Whereas KT and MC also inhibited succinoxidase (Table 1), the concentrations of KT required for 50% inhibition were much higher (I50 4 500 lM) than with
the NADH-linked substrates, MC was the more effective inhibitor. Moreover, the deacetylated metabolite of
KT required higher concentrations, 300 and 350 lM,
for 50% inhibition of glutamate/malate oxidase and
succinate oxidase, respectively (Table 1).
Because of these observations, other NADH oxidase-dependent substrates, arginine/malate and ornithine/malate, that enter the respiratory chain through
the urea cycle were evaluated. The results with arginine/malate and ornithine/malate are shown in Table
1. Once again, KT was more potent than MC with I50’s
of 75 and 65 lM for arginine/malate and ornithine/
malate, respectively, while the I50’s for miconazole were
170 and 160 lM for arginine/malate and ornithine/
malate, respectively. Thus, all of the NADH oxidase
substrates were sensitive to KT but not to the extent as
glutamate/malate. No uncoupling effect by KT, FL, or
DAK was observed in this study. On the other hand,
MC inhibited NADH oxidase and SDH equally. These
data were representative of three experiments. Positive
controls, such as 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, rotenone, an inhibitor of
NADH oxidase, and TTFA, a potent inhibitor of SDH,
demonstrated that the mitochondria reacted appropriately to the site-specific inhibitors and uncouplers
(data not shown). There was minimal inhibition (1%)
by DMSO on the oxidations catalyzed by mitochondria. The final concentration of DMSO with FL was
0.6% at the highest concentration and 1.0% with MC.
Because a high concentration of KT was required
to inhibit SDH polarographically, additional experiments were conducted to evaluate the effect of KT on
SDH activity by the ferricyanide assay. The reduction
of ferricyanide by succinate catalyzed by SDH was
monitored spectrophotometrically at 450 nm. KT produced a dose-dependent inhibition of SDH activity
with an I50 of 74 lM (Figure 2). TTFA was a potent
inhibitor of SDH activity, whereas rotenone had minimal effects.
DISCUSSION
In summary, ketoconazole, miconazole, and N-deacetyl ketoconazole directly inhibited both the NADH
and succinate oxidase enzymes necessary for mitochondrial oxidative phosphorylation in rat liver mitochondria. NADH oxidase, complex I, appears to be the
more sensitive component of the respiratory chain to
the inhibitory actions of ketoconazole in mammalian
mitochondria, in contradiction to a previous report
that states that the most distal portion of the respiratory chain (complex IV) was the primary site of keto-
130
RODRIGUEZ AND ACOSTA
FIGURE 2. Percent inhibition of succinate dehydrogenase (SDH)
activity (lmoles of ferricyanide reduced/min/mg mitochondrial
protein) by ketoconazole of rat liver mitochondria. Thenoyltrifluoroacetone (TTFA), 25 lM, was used as a positive control for the inhibition of SDH activity. Error bar represents the S.E.M. of three
experiments. A one-way completely randomized factor ANOVA resulted in significant dose interaction (P , 0.05).
conazole inhibition on oxidative phosphorylation in
fungi (9). Furthermore, succinate oxidase is also susceptible to the inhibitory effects of ketoconazole, miconazole, and N-deacetyl ketoconazole, as shown by
inhibition of oxidative phosphorylation and succinate
dehydrogenase activity. Thus, ketoconazole, as well as
its metabolite, N-deacetyl ketoconazole, and miconazole not only inhibit the NADH-linked complex but
also affect other complexes of the respiratory chain.
The interesting observation that fluconazole, a triazole,
did not have any detectable inhibitory effects on mitochondrial respiration may suggest that structural differences in the various azoles, particularly the imidazole ring, may result in a conformation conducive to
the inhibition of mitochondrial respiration at complex
I. Also, it is well known that ketoconazole is an inhibitor of cytochrome P450 in which it binds to the heme
portion of the cytochrome (15,16); thus, it is not surprising that it can also inhibit cytochrome c oxidase
(complex IV) in fungi (9). Furthermore, the significant
differences in the potency between ketoconazole and
its metabolite, N-deacetyl ketoconazole, on glutamate/malate oxidase (32 lM vs. 300 lM, respectively)
not only demonstrate that an imidazole ring may be
needed for the inhibition but that the lack of the acetyl
group results in a 10-fold decrease in mitochondrial
effects.
It has been shown that when ketoconazole is inserted into a lipid layer, the acetylated piperazine moi-
Volume 11, Number 3, 1996
ety is oriented toward the hydrophobic region and the
dichlorophenyl substituent is found in the hydrophilic
phase, thus having little effect on lipid organization
(17). Such an orientation is not supposed to affect the
lipid organization because the area occupied per drug
molecule (30 Å2) is low. In comparison, N-deacetyl ketoconazole caused complete reversal in the insertion
alignment of the molecule in artificial lipid membranes, thus having a destabilizing effect on the lipids
because the inversion resulted in an increase in the area
occupied per drug molecule (90 Å2) in the lipid layer
(17). Miconazole was also shown to exhibit a destabilizing effect such that it maintained its dichlorophenyl
groups in the hydrophobic phase, whereas the imidazole moiety was oriented in the hydrophilic phase
(17). The molecular mechanism responsible for these
effects has not been established. Thus, it is possible that
the mitochondrial membrane, which is also composed
of proteins and lipids, may undergo physical changes
in the outer and/or inner membrane, rendering it permeable to protons. Because the mitochondria play a
central role, not only in cellular respiration but also in
maintenance of intracellular pH and ion homeostasis,
the inhibitory effects of the imidazoles on mitochondrial respiration, particularly ketoconazole, may result
in an early cascade of toxicological damage to the cells,
resulting in cell death. Our laboratory has previously
reported that ketoconazole, unlike fluconazole, produced a distinct time and dose response in cultured rat
liver cells using various cytotoxic assays (7). The toxicity occurred after 2 hours with 115 lM ketoconazole
and 4 hours with 90 lM ketoconazole as evaluated by
lactate dehydrogenase leakage into the media of the
cultured hepatocytes (7). Thus, the immediate effect in
isolated mitochondria may be an early indication of a
cellular target of ketoconazole, which ultimately, after
several hours, results in a loss of cell viability. The lack
of inhibition of mitochondrial respiration after exposure to fluconazole correlates with our earlier cytotoxicity studies in which fluconazole did not produce
cytotoxic damage to the hepatocytes (7). Moreover, ketoconazole’s strong inhibition effect on the NADH oxidase in the TCA cycle can indirectly inhibit other biochemical events in the liver, in particular, urea
syntheses. Ornithine and arginine are carrier compounds for urea that is formed through the “ornithine
cycle” and that requires energy and utilizes ATP (18).
A toxicological consequence of the inhibition of urea
synthesis is increased ammonia formation that can ultimately result in hepatic coma (18,19). Interestingly,
the first reported fatality of ketoconazole is a patient
who died of hepatic coma (20). In summary, these mitochondrial respiration studies demonstrate that ketoconazole, miconazole, and N-deacetyl ketoconazole
directly affect the mitochondria by inhibiting ADP-de-
Volume 11, Number 3, 1996
pendent oxygen uptake at various sites in the respiratory chain that will ultimately result in a reduction
of ATP formation. Thus, these events occurring within
the mitochondria may be a possible explanation of the
mechanism of ketoconazole’s hepatotoxicity.
ACKNOWLEDGMENTS
We acknowledge Dr. Daniel M. Ziegler for his
helpful comments and suggestions. Part of this work
was presented at the 1995 meeting of the Society of
Toxicology [Toxicologist 15, 281 (1995)]. Rosita J. Rodriguez is a recipient of a NIGMS Pre-Doctoral Fellowship (1994–1996) sponsored by NIDDK Grant '1 F31
DK09063-01. Daniel Acosta, Jr. was a Burroughs
Wellcome Scholar in Toxicology.
REFERENCES
1. H. Vanden Bossche, P. Marichal, J. Gorrens, H. Geerts,
and P. A. Janssen (1988). Basis for the search for new antifungal drugs, Ann. N.Y. Acad. Sci., 544, 191–207.
2. P. A. Duarte, C. C. Chow, F. Simmons, and J. Ruskin
(1984). Fatal hepatitis associated with ketoconazole therapy, Arch. Intern. Med., 144, 1069–1070.
3. J. H. Lewis, H. J. Zimmerman, G. D. Benson, and K. G.
Ishak (1984). Hepatic injury associated with ketoconazole
therapy: analysis of 33 cases, Gastroenterology, 86, 503–
513.
4. B. H. C. Stricker, A. P. R. Blok, F. B. Bronkhorst, G. E. Van
Parys, and V. J. Desmet (1986). Ketoconazole-associated
hepatic injury—a clinicopathological study of 55 cases, J.
Hepatol., 3, 399–406.
5. M. A. Jacobson, D. K. Hanks, and L. D. Ferrell (1994). Fatal acute hepatic necrosis due to fluconazole, Am. J. Med.,
96, 188–190.
6. C. Wells and A. M. L. Lever (1992). Dose dependent fluconazole hepatotoxicity proven on biopsy and rechallenge, J. Infect., 24, 111–112.
AZOLES INHIBIT MITOCHONDRIAL FUNCTION
131
7. R. J. Rodriguez and D. Acosta (1995). Comparison of ketoconazole- and fluconazole-induced hepatotoxicity in a
primary culture system of rat hepatocytes, Toxicology, 96,
83–92.
8. K. N. Buchi, P. D. Gray, and K. G. Tolman (1986). Ketoconazole hepatotoxicity: an in vitro model, Biochem. Pharmacol., 35, 2845–2847.
9. M. L. Shigmatsu, J. Uno, and T. Arai (1982). Effect of ketoconazole on isolated mitochondria from Candida albicans, Antimicrob. Agents Chemother., 21, 919–924.
10. J. Uno, M. L. Shigematsu, and T. Arai (1982). Primary site
of action of ketoconazole on Candida albicans, Antimicrob.
Agents Chemother., 21, 912–918.
11. D. P. Dickinson (1977). The effects of miconazole on rat
liver mitochondria, Biochem. Pharmacol., 26, 541–542.
12. E. Bercoff, J. Bernuau, C. Degott, B. Kalis, A. Lamaire, H.
Tilly, B. Rueff, and J. P. Benhamou (1985). Ketoconazoleinduced fulminate hepatitis, Gut, 26, 636–638.
13. J. B. Chappell and R. G. Hansford (1972). Preparation of
mitochondria from animal tissues and yeasts. In Subcellular Components. Preparation and Fractionation, G. D. Birnie, ed., pp. 77–91, University Park Press, Baltimore.
14. T. P. Singer (1974). Determination of the activity of succinate, NADH, choline, and a-glycerophosphate dehydrogenases. In Methods of Biochemical Analysis, D. Glick,
ed., pp. 123–175, Wiley, New York.
15. J. J. Sheets and J. I. Mason (1984). Ketoconazole: a potent
inhibitor of cytochrome P-450-dependent drug metabolism in rat liver., Drug Metabol. Dispos., 12, 603–606.
16. A. D. Rodrigues, G. G. Gibson, C. Ionnides, and D. V.
Parke (1987). Interactions of imidazole antifungal agents
with purified cytochrome P-450 proteins, Biochem. Pharmacol., 36, 4277–4281.
17. R. Brasseur, C. Vandenbosche, H. Van den Bossche, and
J. M. Ruysschaert (1983). Mode of insertion of miconazole, ketoconazole, and deacylated ketoconazole in lipid
layers, Biochem. Pharmacol., 32, 2175–2180.
18. C. S. Davidson and G. J. Gabuzda (1969). Hepatic coma.
In Diseases of the Liver, L. Shiff, ed., pp. 378–409, J. B. Lippincot Co., Philadelphia.
19. S. P. Bessman and N. Pal (1976). The Krebs cycle depletion theory of hepatic coma. In The Urea Cycle, S. Grisolia,
R. Baguena, and F. Mayor, eds., pp. 83–89, Wiley New
York.
20. G. Cauwenbergh (1989). Safety aspects of ketoconazole,
the most commonly used systemic antifungal. Mycoses,
32, 59–63.