TOXICOLOGY AND APPLIED PHARMACOLOGY
ARTICLE NO.
144, 1–11 (1997)
TO978121
Furan-Mediated Uncoupling of Hepatic Oxidative Phosphorylation
in Fischer-344 Rats: An Early Event in Cell Death1
Cheryl A. Mugford, Mark A. Carfagna,2,3 and Gregory L. Kedderis4
Chemical Industry Institute of Toxicology, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, North Carolina 27709-2137
Received May 31, 1996; accepted November 18, 1996
Furan is the parent compound of a large class of chemicals
used in industry and manufacturing. Furan is a volatile organic liquid used as an intermediate in the synthesis of many
chemical and pharmaceutical agents and as a solvent for
resins and lacquers. In addition, furan and furan-containing
compounds are produced as by-products in the distillation
of certain agricultural products such as oat hulls and corn
cobs (Ulbricht et al., 1984). Furan and compounds containing the furan ring are also found in a variety of foods,
with coffee and canned meat containing the highest concentration of furan (Maga, 1979). Because humans are exposed
to furan compounds by a variety of sources and routes, it is
important to examine the risks of exposure.
Furan is a rodent hepatotoxicant and hepatocarcinogen
(Wiley et al., 1984; NTP, 1993). In vitro studies with rat
liver microsomes have shown that furan and its analogs are
metabolized by cytochrome P450 to a reactive ene-dialdehyde metabolite, cis-2-butene-1,4-dial (Ravindranath et al.,
1984; Burka et al., 1991; Chen et al., 1995). The toxicity
of furan correlates with the formation of cis-2-butene-1,4dial (Masuda et al., 1984). A cancer bioassay performed by
the National Toxicology Program (NTP) demonstrated that
furan is a potent rodent carcinogen (NTP, 1993). Administration of furan in corn oil by gavage to both sexes of F-344
rats (2, 4, or 8 mg furan/kg) and B6C3F1 mice (8 or 15
mg furan/kg) 5 days per week for 2 years produced dosedependent hepatocellular carcinomas in female F-344 rats
and in male and female B6C3F1 mice following oral administration (NTP, 1993).
The mechanism of furan-induced carcinogenesis has not
been established. Furan-induced hepatocarcinogenesis does
not appear to be mediated by a DNA-reactive metabolite
because furan is not mutagenic in in vitro genotoxicity assays
and does not elicit a DNA repair response in vivo (Mortelmans et al., 1986; Wilson et al., 1992). Furan produces
hepatotoxicity under the conditions of the bioassay (Wilson
et al., 1992). A sharp increase in the percentage of hepatic
nuclei in S-phase was observed 48 hr after rats or mice were
treated with toxic doses of furan (Wilson et al., 1992). Cell
proliferation in both species was preceded by cytotoxicity,
as indicated by increases in serum enzyme activities as early
Furan-Mediated Uncoupling of Hepatic Oxidative Phosphorylation in Fischer-344 Rats: An Early Event in Cell Death. Mugford,
C. A., Carfagna, M. A., and Kedderis, G. L. (1997). Toxicol. Appl.
Pharmacol. 144, 1–11.
Furan is a potent rodent hepatotoxicant and carcinogen. The
present study was done to examine the effects of furan on hepatic
energy metabolism both in vivo and in vitro in male F-344 rats.
Furan produced concentration- and incubation time-dependent
irreversible reductions in ATP in freshly isolated F-344 rat hepatocytes. Furan-mediated depletion of ATP occurred prior to cell
death and was prevented by including 1-phenylimidazole, a cytochrome P450 inhibitor, in the suspensions. Male F-344 rats were
treated with furan (0–30 mg/kg, po) and killed 24 hr later to
prepare hepatic mitochondria. Furan produced dose-dependent
increases in state 4 respiration and ATPase activity. Both of these
changes were prevented by 1-phenylimidazole cotreatment. In a
separate series of experiments, mitochondria were prepared from
isolated rat hepatocytes following incubation with furan (2–100
mM) for 1–4 hr. Furan produced incubation time- and concentration-dependent increases in state 4 respiration and ATPase activity. Furan-mediated mitochondrial changes were prevented by
adding 1-phenylimidazole to the hepatocyte suspensions. These
results indicate that the ene-dialdehyde metabolite of furan uncouples hepatic oxidative phosphorylation in vivo and in vitro. In vitro
studies using an isolated hepatocyte suspension/culture system
demonstrated that the concentration response for furan-mediated
mitochondrial changes in suspension corresponded with the concentration response for cell death after 24 hr. Including 1-phenylimidazole or oligomycin plus fructose in hepatocyte suspensions
prevented furan-induced cell death after 24 hr in culture. The
results of this study indicate that furan-induced uncoupling of
oxidative phosphorylation is an early, critical event in cytolethality
both in vivo and in vitro. q 1997 Academic Press
1
Presented in part at the 32nd Annual Meeting of the Society of Toxicology, New Orleans, LA, March 15–19, 1993, and at the 35th Annual Meeting
of the Society of Toxicology, Baltimore, MD, March 5–9, 1995.
2
Supported in part by an Individual National Research Service Award
(ES 05560) awarded by the National Institute of Environmental Health
Sciences.
3
Present address: Lilly Research Laboratories, A Division of Eli Lilly
and Company, 2001 West Main Street, Greenfield, IN 46140.
4
To whom correspondence should be addressed.
1
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Copyright q 1997 by Academic Press
All rights of reproduction in any form reserved.
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MUGFORD, CARFAGNA, AND KEDDERIS
as 12 hr after furan administration. These results have led
to the hypothesis that furan-induced hepatocarcinogenesis is
related to cytolethality, which leads to compensatory cell
proliferation and contributes to subsequent tumor formation.
However, the relationship between furan-induced cytolethality, cell proliferation, and tumor formation is unknown. Furthermore, the mechanisms of furan-induced cell death have
not been identified.
Previous studies in our laboratory have demonstrated that
furan pharmacokinetics can be predicted from studies with
isolated hepatocytes (Kedderis et al., 1993; Kedderis and
Held, 1996). Studies have shown that substitution of the
kinetic parameters determined in hepatocytes in vitro into
the physiologically based pharmacokinetic model for furan
accurately simulated in vivo pharmacokinetics. Metabolic
constants (Km and Vmax) measured in isolated hepatocytes in
suspension were extrapolated to the whole animal based on
the cellularity of the liver (Kedderis et al., 1993). Carfagna
et al. (1993) developed an isolated hepatocyte suspension/
culture system that mimics both the time course and dose
dependence for furan-induced cell death observed in vivo.
Exposing isolated hepatocyte suspensions to furan (2–100
mM) for up to 4 hr in suspension and placing the cells in
culture for 24 hr to allow the expression of cytolethality
provide a realistic exposure scenario comparable to what is
observed in the whole animal (Carfagna et al., 1993). Our
previous work with this system suggested that the isolated
hepatocyte suspension/culture system is a useful model for
investigating and characterizing the early cellular responses
and changes that occur in rodent hepatocytes following exposure to cytotoxic concentrations of furan.
In the present study, we investigated the mechanisms of
furan-mediated cell death in F-344 rats. We examined the
effects of furan exposure on hepatic energy metabolism in
intact F-344 rats given toxic doses of furan and in freshly
isolated hepatocytes exposed to cytotoxic concentrations of
furan in vitro. Qualitatively and quantitatively similar results
were obtained in vivo and in vitro, illustrating that isolated
hepatocytes are an appropriate model for studying the mechanisms of furan-mediated cytolethality. The results from this
study have identified an early, critical mitochondrial lesion
that is responsible for furan-mediated cytolethality both in
vivo and in vitro.
MATERIALS AND METHODS
Animals. Male F-344 rats (200 – 300 g) were obtained from Charles
River Laboratories (Raleigh, NC). Rats were housed in polycarbonate
cages with compressed fiber bedding under conditions of a 12-hr light –
dark cycle at 22 { 27C (mean { SD) and 55 { 5% relative humidity.
Animals were allowed free access to food (NIH-07) and water and were
acclimated to the animal facility for at least 10 days prior to use. All
procedures were approved by the CIIT Institutional Animal Care and
Use Committee and conducted under federal guidelines for the use and
care of laboratory animals.
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Chemicals. Furan (ú99% pure) containing 0.025% butylated hydroxytoluene as a polymerization inhibitor was purchased from Aldrich Chemical
Company (Milwaukee, WI). 1-Phenylimidazole was also purchased from
Aldrich Chemical Company. Williams’ Medium E, Hanks’ balanced salt
solution, without calcium and magnesium, fetal bovine serum, and L-glutamine were obtained from Gibco (Grand Island, NY). Insulin/selenium/transferrin, dexamethasone, gentamicin, Triton X-100, Percoll, and ATP standard
and ATP assay reagents were purchased from Sigma (St. Louis, MO).
Collagenase (type IV) was purchased from Boehringer Mannheim (Indianapolis, IN). Reagents for lactate dehydrogenase (LDH) were obtained from
Roche (Nutley, NJ). All other chemicals were of reagent grade and purchased from commercial sources.
Furan treatment. Male F-344 rats were given furan (0, 8, 15, or 30
mg/kg, po in corn oil) and euthanized 24 hr later. Hepatic mitochondria
were isolated by differential centrifugation according to the methods of
Greenwalt (1974) and Niranjan and Avadhani (1980). Mitochondria were
prepared in buffer containing 2 mM Hepes, pH 7.4, 222 mM mannitol, 70
mM sucrose, and 1 mM EDTA.
When preparing the furan dosing solution, the solution was chilled and
handled in a ventilated hood. In all studies, furan-treated animals were
placed in cages in a ventilated hood and housed separately from other
animals. To examine the role of cytochrome P450-dependent biotransformation in furan-induced mitochondrial injury, some rats were given 1-phenylimidazole (50 mg/kg, ip, in saline) prior to the administration of furan.
Hepatocyte isolation. Hepatocytes were isolated from male F-344 rats
according to the procedure outlined by Seglen (1976) and modified by
Kedderis et al. (1988). Briefly, hepatocytes were isolated by in situ perfusion
with EGTA-containing, calcium-free media followed by a collagenase solution containing calcium. Preparations were enriched with viable cells by
centrifugation through Percoll (Kukongviriyapan and Stacey, 1988). Hepatocytes were suspended in Williams’ Medium E containing 10 mM Hepes
buffer, pH 7.3. Hepatocyte viability was determined by LDH release using
Roche Kit No. 43622 and a Cobas Fara II autoanalyzer (Roche). Total LDH
activity was determined by comparing an aliquot of cells solubilized with
1% Triton X-100 to the amount of LDH leaking into the medium from the
cells. Only cell preparations with initial viability ú80% (õ20% LDH leakage into the medium) were used for experiments.
Hepatocyte incubation and culture conditions. Because of the volatility of furan, all incubations were performed in sealed, screw-top 25-ml
Erlenmeyer flasks with Teflon/silicone septa. Separate flasks were prepared
for each concentration and time point of furan exposure. Each incubation
contained 2 1 106 cells/ml in Williams’ Medium E containing 10 mM
Hepes, pH 7.3, in a total volume of 3 ml. Cells were gassed with 5% CO2
in air and the flasks immediately sealed. After a 10-min preincubation
period at 377C, furan was added through the septa using a gas-tight syringe
as described by Carfagna et al. (1993). Hepatocytes were incubated with
furan at concentrations ranging from 2 to 100 mM for 1 to 4 hr with reciprocal
shaking (60 cycles/min) at 377C. Furan medium concentrations were calculated using the two-compartment model as described by Kedderis et al.
(1993). Several incubations at each concentration of furan and time of
exposure contained 200 mM 1-phenylimidazole (dissolved in medium) to
investigate the role of cytochrome P450 in furan-induced changes in hepatic
energy metabolism. Several incubations also contained 20 mM fructose and
1.0 mg/ml oligomycin to investigate the role of furan-induced mitochondrial
changes in cell death. After incubation, LDH content was measured as
described earlier. Under the described incubation conditions, all hepatocyte
suspensions maintained ú80% viability (loss of õ20% of total LDH). ATP
concentration in the hepatocyte samples was measured using a bioluminescence assay (Sigma Chemical Company) and quantitated by comparing to
known standards of ATP using a Dynatech Laboratories microtiter plate
luminometer (Model ML 300) (Van Dyke et al., 1969).
Following incubation with furan in suspension, an aliquot of cells was
removed and placed in culture. Cells were allowed to attach to 6-well
culture plates (3 1 104 hepatocytes/cm2) in the presence of 5% fetal bovine
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FURAN-MEDIATED UNCOUPLING OF OXIDATIVE PHOSPHORYLATION
3
serum for 90 min at 377C in air/CO2 (95/5). Cells were washed and cultured
in medium containing 2 ml of Williams’ medium E supplemented with 2
mM L-glutamine, 10 ng/ml dexamethasone, 50 mg/ml gentamicin, 5 mg/ml
insulin, 5 mg/ml transferrin, and 5 ng/ml selenium. Culture medium was
removed after 24 hr of culture to measure LDH leakage from the hepatocytes. Two milliliters of fresh media containing Triton X-100 was added
to each well, and the cells were collected by scraping after 15 min at room
temperature. Viability after 24 hr in culture was calculated by dividing the
amount of LDH in the cells attached to the plate by the total LDH (medium
plus plate).
At the end of each incubation, several hepatocyte incubations at each
concentration and time point were pooled to prepare mitochondria according
to the methods of Greenwalt (1974) and Niranjan and Avadhani (1980) as
described earlier, with the modification of using a glass-on-glass, hand-held
homogenizer. Approximately 20 passes of the homogenizer were required
to disrupt the cells.
Effects of furan on hepatic energy metabolism. Mitochondrial respiration (2 mg mitochondrial protein/ml) was measured using a Clark-type
electrode in a YSI Model 53 oxygraph system at 257C (Estabrook, 1967).
Respiration buffer contained 2 mM Hepes, 222 mM mannitol, 70 mM sucrose, 1 mM EDTA, 1 mM potassium phosphate, and 1 mM magnesium
chloride. State 4 respiration was quantitated after the addition of 3 mM
succinate, and state 3 respiration was measured with 300 mM ADP (Estabrook, 1967). Mitochondrial ATPase activity (5 mg mitochondrial protein/
ml) was measured by quantitating phosphate release from ATP according
to the method of Baginski et al. (1967).
Statistical analysis. Experiments were conducted with at least three
animals per dose or treatment or at least three different hepatocyte preparations at each concentration and time of furan exposure. All data are expressed as means { SE. Statistical analyses were performed using StatView
software (Abacus Concepts). Means were compared using analysis of variance with a repeated t test when F was significant (Sokal and Rolf, 1981).
The level of statistical significance was p £ 0.05.
RESULTS
Effects of furan on ATP concentration in isolated hepatocytes in suspension. Concentration- and time-dependent
decreases in cellular ATP concentration were observed in
isolated hepatocytes exposed to furan in suspension (Figs.
1A and 1B). The concentration response of ATP depletion
in isolated hepatocytes in suspension following 1 hr of incubation demonstrated that 2 and 10 mM furan did not affect
cellular ATP concentration (Fig. 1A). However, incubating
isolated hepatocytes with 100 mM furan reduced cellular ATP
content to approximately 55% of control (Fig. 1A). The
viability of both control and furan suspensions, as measured
by LDH leakage from the cells into the medium, was ú80%
under these experimental conditions (data not shown).
Incubation of isolated hepatocytes with 100 mM furan produced a time-dependent loss of greater than 90% of cellular
ATP by 4 hr (Fig. 1B). The viability of both control and
furan suspensions, as measured by LDH leakage from the
cells into the medium, was ú80% at all times of incubation
(data not shown).
Furan-mediated loss of ATP in the isolated hepatocyte
suspensions was irreversible. After incubating hepatocytes
with 100 mM furan in suspension for 2 hr, several vials were
vented to remove furan, and hepatocytes were reincubated
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FIG. 1. Concentration dependence and time course of furan-induced
depletion of ATP concentration in isolated rat hepatocytes incubated with
furan in suspension. Hepatocytes were incubated with furan (2–100 mM)
in suspension for 1 hr (A). Hepatocytes were incubated with 100 mM furan
in suspension for 1–4 hr (B). Open circles are data from cells incubated
with furan, and closed circles are data from cells incubated with furan plus
200 mM 1-phenylimidazole. Cell viability at the end of each incubation was
ú80%. Each point represents the mean { SE of three hepatocyte preparations. Control ATP values were 19.3 { 1.9 nmol ATP/1 1 106 cells.
at 377C with reciprocal shaking for 2 additional hr. ATP
depletion in hepatocytes exposed to furan under these conditions was the same as the ATP reduction seen in hepatocytes
incubated with 100 mM furan for 4 hr (data not shown).
Furan-mediated depletion of ATP was prevented by including the cytochrome P450 inhibitor 1-phenylimidazole in
the hepatocyte suspensions (Figs. 1A and 1B). ATP concentrations in hepatocyte suspensions coincubated with furan
and 1-phenylimidazole were not different from control incubations, indicating that cytochrome P450-dependent metabolism is required for furan-mediated depletion of ATP.
Furan-mediated changes in hepatic energy metabolism.
Studies were designed to investigate the mechanisms of furan-mediated irreversible depletion of ATP observed in isolated rat hepatocytes. Since mitochondrial respiratory pro-
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MUGFORD, CARFAGNA, AND KEDDERIS
FIG. 2. Simulation of oxygen uptake curves by either (A) control mitochondria prepared from male rats treated with corn oil or from isolated
hepatocytes incubated without furan or (B) mitochondria isolated from rats treated with furan (30 mg/kg, po) or from isolated hepatocytes incubated
with furan (100 mM) in suspension as described under Materials and Methods. Arrows indicate the addition of either 3 mM succinate to measure state 4
respiration or 300 mM ADP to quantitate state 3 respiration to the mitochondrial preparations.
cesses produce the majority of the hepatocytes’ ATP, an
impairment in mitochondrial respiration would be reflected
in a reduction in the ATP content of cells. We examined the
effects of furan on mitochondrial respiration in rats dosed
with furan and in freshly isolated hepatocytes exposed to
furan in vitro.
Administration of furan (30 mg/kg, po) to male F-344
rats produced a change in the shape of the oxygen uptake
curve generated from mitochondria prepared from these
animals (Fig. 2). Tightly coupled control mitochondria
prepared from animals given corn oil alone had a slow
rate of oxygen uptake in the presence of substrate (3 mM
succinate) and absence of ADP (Fig. 2A). The addition
of 300 mM ADP to the mitochondria caused an immediate
increase in the rate of oxygen consumption (Fig. 2A). In
contrast, treatment of male rats with furan (30 mg/kg, po)
caused a very rapid uptake of oxygen in the presence of
succinate and the absence of ADP (Fig. 2B). The rate of
oxygen uptake following furan treatment was no longer
dependent on the concentration of ADP present in the
mitochondrial preparation (Fig. 2).
Administration of furan to male F-344 rats produced a
dose-dependent increase in state 4 (succinate-stimulated)
respiration (Fig. 3). State 4 respiration was increased from
control levels of 28 nmol/min/mg protein to 60 nmol/min/
mg protein in male rats treated with 15 mg furan/kg (Fig.
3). Animals treated with 30 mg furan/kg showed a greater
than threefold increase in state 4 respiration as compared to
control animals (Fig. 3). In contrast to the marked effects
on state 4 respiration, state 3 (ADP-stimulated) respiration
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was not changed at any dose of furan treatment (Fig. 3).
The rate of state 3 respiration was maintained at approximately 84 nmol/min/mg protein at all doses of furan that
were examined (Fig. 3).
Furan treatment also caused a decrease in the respiratory
FIG. 3. Effect of furan on oxygen uptake in mitochondria isolated from
rats treated in vivo. Rats were given furan (0–30 mg/kg, po) and euthanized
24 hr later to prepare hepatic mitochondria. Mitochondrial respiration was
measured as described under Materials and Methods. Open circles show
state 4 respiration (succinate-induced), and closed circles show state 3
respiration (ADP-induced). Each point represents the mean { SE of three
animals.
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FURAN-MEDIATED UNCOUPLING OF OXIDATIVE PHOSPHORYLATION
control index (RCI), which is the ratio of the rate of oxygen
uptake when ADP is in plentiful supply (state 3 respiration)
to the rate of oxygen uptake in the absence of ADP (state
4 respiration) (Lehninger, 1975). Under experimental conditions similar to those of these studies, normal, tightly coupled
rat liver mitochondria exhibit an RCI of about 5 (Nakagawa
and Tayama, 1995; Smith et al., 1989). In the present studies,
mitochondria prepared from male F-344 rats treated with
corn oil alone had a RCI of about 4 (Fig. 3). Increasing the
dose of furan caused a reduction of the RCI to about 2 after
15 mg/kg and to less than 1 following a toxic dose of 30 mg
furan/kg. This dose-dependent reduction in the RCI provides
further evidence that toxic doses of furan cause mitochondrial injury.
Because treatment of male F-344 rats with toxic doses
of furan appeared to cause an impairment in mitochondrial
function as evidenced by changes in mitochondrial oxygen
uptake measurements, we measured mitochondrial ATPase
activity. Normal, tightly coupled mitochondria have very
little ATP hydrolyzing activity (Lehninger, 1975). Accompanying the furan-induced effects on state 4 respiration was a
dose-dependent increase in ATPase activity (Fig. 4). Mitochondrial ATPase activity in animals treated with corn oil
was approximately 4 mmol/Pi/mg protein (Fig. 4). ATPase
activity in animals receiving 15 mg furan/kg was increased
to 6 mmol/Pi/mg protein. Administration of 30 mg furan/kg
produced a greater than twofold elevation in ATPase activity
as compared to animals receiving the corn oil vehicle alone
(Fig. 4).
These data indicate that treatment of male F-344 rats with
FIG. 4. Dose dependence of furan-induced increases in ATPase activity
in mitochondria prepared from rats treated with furan in vivo. Rats were
given furan (0–30 mg/kg, po) and euthanized 24 hr later to prepare hepatic
mitochondria. ATPase activity was measured as described under Materials
and Methods. Each point represents the mean { SE of three animals.
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5
FIG. 5. Effect of furan on oxygen uptake in mitochondria prepared
from isolated rat hepatocytes. Hepatocytes were isolated from male F-344
rats and incubated with furan (2–100 mM) in suspension for 1 hr. After each
incubation, mitochondria were prepared, and respiration was quantitated as
described under Materials and Methods. Open circles show state 4 respiration (succinate-induced), and closed circles show state 3 respiration (ADPinduced). Each point represents the mean { SE of three or four hepatocyte
preparations.
furan produced a dose-dependent mitochondrial lesion consistent with the effects of an irreversible uncoupler of oxidative phosphorylation. Characteristically, uncouplers stimulate the rate of oxygen uptake by mitochondria in the absence
of ADP and increase the ATP-hydrolyzing activity in mitochondria (Lehninger, 1975).
We incubated isolated hepatocytes with cytotoxic concentrations of furan to determine if furan produced the same
mitochondrial lesion in vitro that was observed in vivo.
Changes in oxygen uptake measurements from mitochondria
prepared from hepatocytes incubated with furan were similar
to the changes observed in vivo. The shape of the oxygen
uptake curve generated from mitochondria prepared from
isolated F-344 rat hepatocytes incubated with 100 mM furan
was different than the shape of the curve of the rate of
oxygen uptake by control mitochondria (Fig. 2). The effect
of furan on mitochondrial respiration in vitro was similar to
the furan-induced changes observed in vivo (Fig. 2). The
mitochondria prepared from isolated hepatocytes following
incubation with furan showed a dramatic increase in the rate
of oxygen uptake in the presence of substrate (3 mM succinate) and the absence of ADP. Exposing isolated hepatocytes
to furan in suspension caused the rate of oxygen uptake to
no longer be dependent on the concentration of ADP present
in the reaction medium.
Incubation of isolated hepatocytes with furan produced a
concentration-dependent increase in state 4 respiration but
caused no changes in state 3 respiration (Fig. 5). This effect
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MUGFORD, CARFAGNA, AND KEDDERIS
was the same as the respiratory changes observed in mitochondria prepared from animals treated with furan in vivo
(Fig. 3). State 4 respiration in mitochondria prepared from
hepatocytes incubated with 2 mM furan for 1 hr was similar
to the rate of oxygen uptake in control mitochondria (Fig.
5). Incubations containing 10 mM furan produced a slight
increase in state 4 respiration as compared to control mitochondria (Fig. 5). However, incubation of isolated hepatocytes with 100 mM furan for 1 hr increased mitochondrial
state 4 respiration to approximately three times control respiratory rates (Fig. 5). In contrast, state 3 respiration was not
affected by any of the concentrations of furan we examined
(Fig. 5).
Accompanying the furan-induced concentration-dependent increase in state 4 respiration was a decrease in the
RCI (Fig. 5). Control mitochondria exhibited an RCI of 4.5,
while mitochondria prepared from isolated hepatocytes incubated with 100 mM furan for 1 hr had an RCI of about 1
(Fig. 5). These data indicate that exposure to furan causes
similar mitochondrial changes both in vivo and in vitro.
We also examined the effects of furan on mitochondrial
ATPase activity in vitro. Incubating hepatocytes with furan
produced concentration- and time-dependent increases in
ATPase activity (Figs. 6A and 6B). Mitochondrial ATPase
activity in hepatocyte suspensions incubated with 2 and 10
mM was similar to ATPase activity in control incubations
(Fig. 6A). However, 100 mM furan increased ATPase activity
to about twice control activity within 1 hr of incubation (Fig.
6A). Mitochondria prepared from hepatocytes incubated
with 100 mM furan for 2 hr showed about a twofold increase
in ATPase activity (Fig. 6B). ATPase activity measured in
mitochondria isolated from hepatocytes incubated with 100
mM furan for 4 hr was about three times control (Fig. 6B).
Role of biotransformation in changes in hepatic energy
metabolism. Because furan-induced cytolethality and ATP
depletion (Fig. 1A and 1B) were mediated by cytochrome
P450-dependent metabolism, we investigated the role of biotransformation in furan-induced mitochondrial changes with
the nonspecific cytochrome P450 ligand 1-phenylimidazole.
Furan-induced increases in both state 4 respiration and ATPase activity were mediated by cytochrome P450-dependent
metabolism (Table 1). Pretreatment of male rats with 1phenylimidazole (50 mg/kg, ip) prevented furan-induced increases in state 4 respiration (Table 1). ATPase activities in
animals pretreated with 1-phenylimidazole were comparable
to animals treated with corn oil vehicle (Table 1).
The role of biotransformation in furan-mediated mitochondrial changes observed in vitro was examined by including the cytochrome P450 inhibitor 1-phenylimidazole in
some of the incubations. The addition of 200 mM 1-phenylimidazole to the hepatocyte suspensions prevented furan-mediated mitochondrial changes (Table 2). Furan-mediated increases in state 4 respiration in mitochondria prepared from
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FIG. 6. Concentration and time dependence of furan-induced increases
in ATPase activity in isolated rat hepatocytes. Hepatocytes were isolated
from male F-344 rats and incubated with furan (2–100 mM) in suspension
for 1 hr, and mitochondria were prepared as described under Materials and
Methods (A). Hepatocytes were isolated from male F-344 rats and incubated
with 100 mM furan in suspension for 1–4 hr, and mitochondria were prepared as described under Materials and Methods (B). Cell viability at the
end of each incubation was ú80%. Each point represents the mean { SE
of three or four hepatocyte preparations.
isolated hepatocytes were prevented by including 1-phenylimidazole in the incubations (Table 2). State 4 oxygen uptake
rates in mitochondria prepared from hepatocyte suspensions
containing 1-phenylimidazole were similar to control incubations (21.1 { 3.3 vs 18.3 { 1.8 nmol/min/mg protein).
These results provide further evidence that furan-induced
mitochondrial changes are mediated by cytochrome P450dependent metabolism.
Furan-induced cytolethality in isolated hepatocyte suspension/culture system. We used the isolated hepatocyte
suspension/culture system developed by Carfagna et al.
(1993) to investigate the role of the furan-induced mitochondrial lesion in cell death. The concentration response for cell
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7
TABLE 1
Role of Biotransformation in Furan-Induced Mitochondrial
Injury in Male F-344 Rats Treated with Furan in Vivoa
Incubation
State 4 respiration
(nmol/min/mg protein)
ATPase activity
(mmol/Pi/mg protein)
26.6 { 0.5
90.7 { 6.3*
3.5 { 0.02
11.3 { 0.18*
32.9 { 0.7
5.1 { 0.02
Control
Furan
Furan plus
1-phenylimidazole
a
Rats were given furan (30 mg/kg, po) or furan (30 mg/kg, po) plus 1phenylimidazole (50 mg/kg, ip) as described under Materials and Methods.
Mitochondria were prepared 24 hr following treatment, and respiration and
ATPase activity were quantitated as described under Materials and Methods.
Data are expressed as the means { SE of three animals.
* Significantly different from mitochondria isolated from control animals,
p õ 0.05.
death after 24 hr in culture corresponds with the concentration response for increases in ATPase following incubation
with furan in suspension (Figs. 8A and 8B). Incubation of
isolated rat hepatocytes with 100 mM furan for 4 hr produced
a significant increase in ATPase activity as compared to
control (Fig. 8A). The viability of the cells exposed to 100
mM furan in suspension for 4 hr was reduced to about 40%
after 24 hr in culture (Fig. 8B).
The addition of 1-phenylimidazole (200 mM) to the isolated hepatocyte suspensions prevented furan-induced cytolethality in the suspension/culture system (Fig. 8B). Viability
after 24 hr in culture of hepatocytes incubated with 10 and
100 mM furan in suspension for 4 hr was 70 and 42% of
control, respectively (Fig. 8B). The addition of 200 mM 1phenylimidazole prevented furan-induced cytolethality at
these time points. These data indicate that furan-induced
cytolethality at 24 hr is dependent upon cytochrome P450dependent metabolism.
TABLE 2
Effect of Furan (100 mM) on Mitochondrial Respiration
in Isolated Rat Hepatocytesa
Incubation
State 3 respiration
(nmol/min/mg protein)
State 4 respiration
(nmol/min/mg protein)
63.3 { 2.9
60.7 { 3.8
18.3 { 1.8
54.6 { 2.7*
62.7 { 1.8
21.1 { 3.3
Control
Furan
Furan plus
1-phenylimidazole
a
Hepatocytes were incubated with furan (100 mM) or furan (100 mM)
plus 1-phenylimidazole (200 mM) for 1 hr. After incubation, mitochondria
were prepared, and respiration was quantitated as described under Materials
and Methods. Data are expressed as the means { SE of three hepatocyte
preparations.
* Significantly different from mitochondria prepared from control incubations, p õ 0.05.
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FIG. 7. Effect of fructose (20 mM) and oligomycin (1 mg/ml) on furanmediated ATP depletion in isolated rat hepatocytes incubated with furan
for 1–4 hr. Open circles are data from cells incubated with furan (100 mM)
alone, and closed circles are data from cells incubated with furan (100 mM)
plus fructose (20 mM) and oligomycin (1 mg/ml). Cell viability at the end
of each incubation was ú80%. Each point represents the mean { SE of
three or four hepatocyte preparations. Control ATP values were 18.9 { 2.1
nmol ATP/1 1 106 cells.
The role of furan-mediated uncoupling of oxidative phosphorylation in cytolethality was investigated by including
fructose and oligomycin in some of the hepatocyte incubations. Fructose protects against chemically induced mitochondrial lesions by providing an increase in glycolytic ATP
production (Nieminen et al., 1990; Cannon et al., 1991),
while oligomycin blocks F1F0ATPase activity to prevent the
destruction of ATP generated by glycolysis (Rouslin, 1983).
The addition of fructose and oligomycin prevented furanmediated reductions in ATP (Fig. 7). Including fructose and
oligomycin in the incubations also modified the viability
curves of the cells that were incubated with furan in suspension (Fig. 8B). Hepatocytes incubated with both fructose and
oligomycin had a higher viability than hepatocytes incubated
with furan alone (Fig. 8B). Increasing energy supplies by
adding oligomycin and fructose to the damaged cells improved the 24-hr viability of the cells exposed to furan.
The observation that both oligomycin and fructose provided
protection against furan-induced cytolethality indicates that
furan-induced mitochondrial changes play a role in the cytotoxicity of this compound.
DISCUSSION
Furan is a potent cytotoxic, non-DNA reactive rodent carcinogen. The mechanism of furan-induced carcinogenesis is
related to cell death followed by compensatory cell prolifera-
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MUGFORD, CARFAGNA, AND KEDDERIS
FIG. 8. Comparison of the concentration dependence of furan-induced
increases in ATPase activity in isolated hepatocytes after incubation with
furan in suspension for 4 hr (A) with furan-induced cytolethality after 24
hr in the suspension/culture system (B), which also shows the effect of 1phenylimidazole and fructose plus oligomycin on furan-induced cytolethality in the isolated suspension–culture system. Isolated hepatocytes were
incubated with furan (2–100 mM) in suspension for 4 hr (A). After incubation in suspension, an aliquot of cells was removed and placed in culture
for 24 hr as described under Materials and Methods (B). (B) Open triangles
represent cells incubated with furan alone, open circles represent cells incubated with furan plus 200 mM phenylimdazole, and closed circles represent
cells incubated with furan plus fructose and oligomycin. Each point represents the mean { SE of three or four hepatocyte preparations.
tion and tumor formation. However, the relationship between
these events is unknown. In the present study, we examined
early mechanisms of furan-mediated cell death in intact rats
exposed to toxic doses of furan and in isolated rat hepatocytes treated with cytotoxic concentrations of furan in suspension and placed in culture for 24 hr to examine cytolethality. One advantage of the present study is that the isolated
suspension/culture system employed allows for accurate in
vivo/in vitro comparisons (Carfagna et al., 1993). This sys-
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tem combines the advantage of freshly isolated hepatocytes,
which retain metabolic capacity comparable to that of the
liver in vivo, with the utility of short-term culture which
allows examination of cytolethality. We identified early mitochondrial changes that play a critical role in furan-induced
cell death in male F-344 rats both in vivo and in vitro.
Incubation of isolated hepatocytes with physiologically
relevant concentrations of furan (2–100 mM) produced irreversible concentration- and time-dependent reductions in
ATP concentration that preceded cell death (Figs. 1A and
1B). This observation suggested that furan-induced changes
in mitochondrial function may play a role in the cytotoxicity
of this compound. Mitochondria produce about 95% of the
total ATP required in eukaryotic cells and therefore play a
central role in the maintenance of the energy supply of the
cell. We examined mitochondrial changes both in intact rats
given toxic doses of furan and in isolated rat hepatocytes
incubated with physiologically relevant cytotoxic concentrations of the chemical.
Furan produced a dose-dependent uncoupling of oxidative
phosphorylation in vivo. Administration of toxic doses of
furan to rats produced an increase in state 4 respiration and
an increase in ATPase activity, which is consistent with
an uncoupler of oxidative phosphorylation (Figs. 3 and 4).
Similar effects were observed in isolated hepatocytes exposed to toxic concentrations of furan in suspension. Furan
produced a concentration-dependent increase in state 4 respiration and ATPase activity (Figs. 5 and 6) that preceded cell
death (Fig. 8B). The role of furan-induced mitochondrial
changes in cell death is further supported by the protection
provided by fructose plus oligomycin (Fig. 8B). This treatment allows hepatocytes to maintain ATP levels and prevents the subsequent irreversible changes that would have
led to cytolethality. These data demonstrate that a furanmediated mitochondrial lesion is an early event in furaninduced cytolethality both in vivo and in vitro.
Furan-mediated mitochondrial changes observed both
in vivo and in vitro were prevented by pretreating rats with
1-phenylimidazole or by including 1-phenylimidazole in
the incubations, indicating that cytochrome P450-dependent bioactivation of furan is required to cause mitochondrial changes (Tables 1 and 2). These results indicate that
uncoupling of oxidative phosphorylation is caused by cytochrome P450-dependent metabolism of furan to cis-2butene-1,4-dial (Burka et al., 1991; Chen et al., 1995).
This toxic metabolite is generated by cytochromes P450
located in the endoplasmic reticulum and must be transported, perhaps by a mitochondrial shuttle, to a critical
site in the mitochondria to cause uncoupling of oxidative
phosphorylation. There is also the potential for furan to
be metabolized within the mitochondria by cytochrome
P450 to produce cis-2-butene-1,4-dial.
The requirement for cytochrome P450-dependent me-
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FURAN-MEDIATED UNCOUPLING OF OXIDATIVE PHOSPHORYLATION
tabolism for generation of the reactive metabolite that
disrupts mitochondrial function and eventually causes cytolethality is consistent with previous studies in which
the toxicity of furan observed in vivo correlated with the
formation of a reactive metabolite generated by cytochrome P450-dependent metabolism (Masuda et al.,
1984). Carfagna et al. (1993) have also shown that the
cytolethality of furan in the isolated hepatocyte suspension/culture system is dependent upon the production of
a cytochrome P450-generated metabolite.
Furan-induced mitochondrial changes in isolated hepatocytes occurred prior to the expression of toxicity in vitro.
The cell viability of all suspensions following incubation
with toxic concentrations of furan was greater than 80%
despite irreversible uncoupling of oxidative phosphorylation.
Therefore, furan-mediated depletion of ATP in isolated rat
hepatocytes is not a consequence of cell death, nor does the
depletion induce immediate cytolethality. Other investigators have shown that hepatocytes can lose up to 85–95% of
total cellular ATP and still maintain viability (Cannon et
al., 1991; Nieminen et al., 1994). Thus, furan-induced ATP
depletion alone is not responsible for cell death. The profound ATP depletion must initiate a cascade of irreversible
events that lead to cell death.
Mitochondria are a frequent intracellular target for a variety of cytotoxic chemicals (Rush et al., 1985; Burcham and
Harman, 1991; Smith et al., 1989; Hill et al., 1992; Nakagawa and Tayama, 1995). The severity of the mitochondrial
damage determines whether the cells are able to repair the
damage and survive or are irreversibly damaged and committed to die. Chemical-induced mitochondrial injury usually
occurs very early in the toxicologic process (Smith et al.,
1989; Nakagawa and Tayama, 1995), similar to the time
frame we have reported for furan in the present study. For
example, toxic concentrations of propyl gallate produce
rapid, concentration-dependent losses of ATP in rat hepatocytes that precede cell death (Nakagawa and Tayama, 1995).
Uncoupling of hepatic oxidative phosphorylation plays a
role in the toxicity of several peroxisome proliferators that
are also nongenotoxic rodent carcinogens (Keller et al.,
1991, 1993). Both clofibrate and 2-ethoxyethanol cause a
concentration-dependent increase in state 4 respiration and
ATPase activity in rat liver mitochondria (Mackerer et al.,
1973; Katyal et al., 1972; Keller et al., 1991). 2-Ethoxyethanol also produces a concentration-dependent decrease in calcium uptake by isolated mitochondria (Keller et al., 1991).
Keller et al. (1993) have also demonstrated that Wyeth14,643 causes a dose-dependent increase in state 4 respiration in rats in vivo and an increase in intracellular calcium
in cultured hepatocytes. Keller et al. (1993) hypothesized
that uncoupling of oxidative phosphorylation depletes the
ATP supply for ion pumps, causing a disruption of ion gradients which can cause an increase in intracellular calcium.
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9
They suggested that the elevation in calcium could activate
growth factors resulting in a sustained increase in cell replication. Increases in intracellular calcium concentration also
activate phospholipases, proteases, and endonucleases that
can be involved in chemical toxicity (Nicotera et al., 1992).
Studies with acetaminophen, a hepatotoxicant that causes
centrilobular necrosis, have shown that mitochondrial dysfunction plays a role in cell death. Toxic doses of acetaminophen inhibit hepatic mitochondrial respiration in rats and
mice both in vivo and in vitro (Esterline et al., 1989; Meyers
et al., 1988; Burcham and Harman, 1991). These mitochondrial changes occur prior to the expression of hepatotoxicity
(Meyers et al., 1988). The link between early mitochondrial
dysfunction and cell death was made by the observation that
toxic doses of acetaminophen in mice cause an increase
in calcium accumulation in the nucleus and an increase in
intracellular calcium (Ray et al., 1990; Shen et al., 1992).
This increase in calcium activates a series of enzymes that
can irreversibly commit a cell to death. An early event is
the activation of calcium-dependent endonucleases that fragment DNA causing a loss of large genomic DNA both in
vivo and in vitro (Ray et al., 1990; Shen et al., 1992). A
later event in this cascade involves the activation of calciumdependent phospholipases that can damage cellular membranes causing the functional expression of hepatic necrosis
(Ray et al., 1990). These studies demonstrate that compromised ATP levels in a cell can fatally disrupt energy-dependent transport, leading to the activation of calcium-dependent hydrolytic enzymes involved in cell death.
A wide variety of cytotoxic, non-DNA reactive chemicals
have been shown to deplete hepatocyte ATP and cause DNA
double-strand breaks prior to cell death (Elia et al., 1993,
1994). The DNA double-strand breaks occur within several
hours after chemical intoxication, and are considered to be
one of the earliest indicators of impending cell death (Elia
et al., 1993, 1994). The induction of hepatic DNA doublestrand breaks by such a diverse group of chemicals, from
detergents to alkylating agents, is consistent with the interpretation that DNA is an important cellular target for cell
death (Bradley et al., 1987; Corcoran and Ray, 1992; Elia et
al., 1993, 1994). Although DNA fragmentation has generally
been interpreted as a hallmark of apoptosis (Eastman, 1993;
Alison and Sarraf, 1995), many of the chemical agents demonstrated to induce DNA double-strand breaks in hepatocytes are not known to produce apoptosis in vivo. The available data suggest that DNA is an important target for chemically induced cell death by mechanisms leading to either
apoptosis or necrosis.
Preliminary studies in our laboratory have shown that furan induces DNA double-strand breaks in treated hepatocytes (Mugford and Kedderis, 1996). The DNA damage appears after depletion of ATP but before cell death, as has
been described by Elia et al. (1993, 1994) for a variety of
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MUGFORD, CARFAGNA, AND KEDDERIS
toxicants. While furan has recently been shown to induce
apoptosis in treated mice (Goldsworthy et al., 1996), the
predominant hepatic lesion is midzonal and centrilobular
necrosis (Wilson et al., 1992). Studies are currently in progress to characterize furan-induced apoptosis and its relationship to necrosis. The DNA double-strand breaks induced
by furan may be involved in mutation induction through
misrepair of the DNA damage. Several studies have shown
that misrepair of DNA damage can lead to genotoxicity
(Bradley et al., 1987; Bryant and Riches, 1989). Since DNA
repair processes require energy, furan-mediated ATP depletion may also compromise the capacity of the cell to repair
DNA damage. Studies are in progress to characterize the
mechanisms involved in furan-induced DNA double-strand
breaks and the role of these lesions in mutation induction.
ACKNOWLEDGMENT
We thank Carmen L. Laethem for her excellent technical assistance.
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