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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 292:982–987, 2000
Vol. 292, No. 3
Printed in U.S.A.
Possible Mechanism of Hepatocyte Injury Induced by
Diphenylamine and Its Structurally Related Nonsteroidal AntiInflammatory Drugs
YASUHIRO MASUBUCHI, SHOKO YAMADA, and TOSHIHARU HORIE
Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, Chiba, Japan
Accepted for publication November 11, 1999
This paper is available online at http://www.jpet.org
Hepatotoxicity is one of the adverse reactions of nonsteroidal anti-inflammatory drugs (NSAIDs) in their clinical use
(Lewis, 1984; Zimmerman, 1990; Rabinovitz and Van Thiel,
1992; Boelsterli et al., 1995). The toxicity also has been
observed experimentally as acute cell injury with freshly
isolated hepatocytes and cultured ones (Akesson and Akesson, 1984; Sorensen and Acosta, 1985; Castell et al., 1988;
Schmitz et al., 1992). Because biotransformation is an essential step to initiate the hepatotoxicity induced by a number of
drugs and chemicals, NSAID toxicity also has been of interest in connection with the involvement of the reactive metabolites. Carboxylic acid-containing NSAIDs were metabolized
into a common group of chemically reactive metabolites, acyl
glucuronides (Spahn-Langguth and Benet, 1992; Dickinson,
1993), whereas none of the glucuronide has been established
to be directly involved in hepatocyte injury. In contrast, a few
studies suggested that oxidative metabolism participated in
NSAID-induced hepatocyte injury (Kretz-Rommel and Boelsterli, 1993; Jurima-Romet et al., 1994). In line with the
involvement of the reactive metabolites, Bort et al. (1999)
recently reported that diclofenac metabolism into an N-hyReceived for publication July 29, 1999.
mitochondrial membrane potentials, which is known as one of
the characteristics for uncouplers of oxidative phosphorylation,
and also was caused by mefenamic acid and diclofenac. Incubation of hepatocytes with mefenamic acid, diclofenac, and
diphenylamine diminished cellular ATP content, followed by
leakage of lactose dehydrogenase from hepatocytes. Fructose,
a low Km substrate for glycolysis, partially protected against the
ATP depletion and hepatocyte injury induced by these compounds. Further addition of oligomycin, which blocks ATPase,
pronounced the protection against cell injury. These results
suggested that decreases in cellular ATP content, mainly
caused by uncoupling of mitochondrial oxidative phosphorylation, were responsible for acute hepatocyte injury induced by
diphenylamine and structurally related NSAIDs.
droxymetabolite was responsible for the cytotoxicity in addition to mitochondrial impairment by the parent compound.
Thus, the mechanism for hepatocyte injury by NSAID has
been extensively studied, whereas a common mechanism to
explain the toxicity of all of hepatotoxic NSAIDs has not been
provided.
We found that cytotoxic NSAIDs caused decreases in cellular ATP contents before leakage of lactate dehydrogenase
(LDH) from the cells (Masubuchi et al., 1998). It also was
shown that diphenylamine was one of the common “skeleton”
structures of NSAIDs to deplete cellular ATP and leak LDH.
In addition, diphenylamine itself, which is not an NSAID,
diminished ATP and caused hepatocyte injury. To further
elucidate the mechanism for ATP depletion by NSAIDs and
diphenylamine, we have studied the effects on mitochondrial
oxidative phosphorylation, the major source of cellular ATP,
whereas some of the acidic NSAIDs are well known as uncouplers of the oxidative phosphorylation (Mahmud et al.,
1996; Mingatto et al., 1996; Petrescu and Tarba, 1997). It was
found that diphenylamine was an uncoupler of mitochondrial
oxidative phosphorylation as well as mefenamic acid and
diclofenac (Masubuchi et al., 1999). Because the potent uncoupling leads to depletion of ATP content at the cellular
ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; LDH, lactate dehydrogenase; MPT, mitochondrial permeability transition.
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ABSTRACT
Diphenylamine is a common structure of nonsteroidal antiinflammatory drugs (NSAIDs) to uncouple mitochondrial oxidative phosphorylation and to cause a decrease in hepatocellular
ATP content and hepatocyte injury. The mechanism for acute
cell injury induced by diphenylamine and its structurally related
NSAIDs was investigated with rat liver mitochondria and freshly
isolated hepatocytes, focusing on the relation to the uncoupling
of oxidative phosphorylation. Incubation of mitochondria with
diphenylamine as well as mefenamic acid and diclofenac
caused pseudoenergetic mitochondrial swelling, indicating that
these compounds induce mitochondrial membrane permeability transition. Diphenylamine also caused changes in safraninebinding spectra to mitochondria that was energized by succinate oxidation. This spectral shift indicates the loss of
2000
Mechanism of Hepatocyte Injury by NSAIDs
level, it implies hepatotoxicity of these compounds, whereas
the cause-and-effect relation remains to be elucidated.
The purpose of this study was to clarify the role of the
uncoupling in cell injury induced by mefenamic acid, diclofenac, and their “skeleton” diphenylamine (Fig. 1) with freshly
isolated rat hepatocytes. In addition, we examined the ability
of these compounds to induce mitochondrial permeability
transition (MPT) and their effects on membrane potential to
further characterize the effects on mitochondrial function.
Materials and Methods
Fig. 1. Diphenylamine and its structurally related NSAIDs.
MgSO4, and 5 mM glucose. Then the perfusate was changed to the
same buffer described above except for containing 4 mM CaCl2 and
180 U/ml collagenase at a flow rate of 30 ml/min for 12 to 15 min. The
hepatocytes were released from the lobe by gentle agitation, and the
cell suspension thus obtained was filtered through a nylon mesh
(120) and centrifuged (50g, 2 min). The hepatocytes were resuspended in the Schwarz buffer (pH 7.4), which consists of 137 mM
NaCl, 5.2 mM KCl, 3 mM Na2HPO4, 0.9 mM MgSO4, 0.12 mM CaCl2,
5 mM glucose, and 15 mM HEPES, followed by centrifugation (50g,
2 min). The washing procedure was repeated twice and the hepatocytes were finally suspended in the same buffer. All the preparations
used in this study were ⬎85% viable as judged routinely by the
trypan blue exclusion test.
Incubation of Hepatocytes with Test Compounds. The
freshly isolated hepatocytes suspended in the above-mentioned
buffer (2 ⫻ 106 cells/ml) were preincubated at 37°C with or without
fructose (20 mM). After 30 min, a test compound, diphenylamine,
mefenamic acid, or diclofenac (500 ␮M), which was dissolved in
methanol to yield a final concentration of 1%, was added with or
without oligomycin (10 ␮g/ml). Fructose (10 mM) was added again 90
min after the onset of the incubation with the test drug. Aliquots of
the suspension were removed from the mixture 60 min after the
addition of the drug for assay of cellular ATP content and 180 min
after the addition for the assay of LDH leakage.
Assay of LDH Activity. LDH activities in the supernatant obtained by centrifugation (50g, 2 min) of the hepatocyte suspension
were assayed with LDH-UV-kit Wako as assessed by oxidation of
NADH (Wroblewski and La Due, 1955). Cytotoxicity was expressed
as a percentage of the total LDH activity, which was obtained from
the cells treated with 0.5% Triton X-100.
Assay of ATP Content. ATP contents were assayed by the HPLC
method of Jones (1981) with modifications. The hepatocyte suspension (1 ml) was mixed with 0.5 ml of 3 N HClO4, followed by addition
of 0.25 ml of 6 N KOH and 0.5 ml of 1 M Tris-HCl buffer (pH 7.4).
After centrifugation (2000g, 10 min), the supernatant with filtration
was applied to HPLC. The HPLC conditions were as follows: column,
Inertsil ODS (GL Sciences, Tokyo, Japan); mobile phase, 100 mM
potassium phosphate buffer (pH 6.0); flow rate, 1.0 ml/min; and
UV-detection, 259 nm.
Diclofenac Metabolism by Liver Microsomes. A 1-ml incubation mixture contained 0.5 mg of liver microsomal protein, 10 mM
glyceraldehyde-6-phosphate, 2 U glyceraldehyde-6-phosphate dehydrogenase, 1 mM NADPH, 10 mM MgCl2, 0.1 mM EDTA, and 25 ␮M
diclofenac in 0.15 M potassium phosphate buffer (pH 7.4). After
temperature equilibration without NADPH (37°C, 5 min), incubation
was started by adding NADPH, performed for 30 min, and terminated with 1 M ice-cold sodium phosphate buffer (pH 5.0). Unmetabolized diclofenac and its oxidative metabolites were extracted into
diethylether, the organic layer was evaporated to dryness, and the
residue was dissolved in methanol. The samples thus obtained were
subjected to following experiments for mitochondrial respiration.
Measurement of Respiration Rates. The rates of oxygen consumption were measured polarographically with a Clark-type oxygen electrode (Model GU-BMP; Iijima Electronics Mfg. Co., Ltd.,
Aichi, Japan). Respiration buffer (pH 7.4) contained 225 mM sucrose,
10 mM KCl, 5 mM MgCl2, 5 mM potassium phosphate, 0.5 mM
EDTA, and 20 mM Tris-HCl. Mitochondria (1 mg protein/ml) were
preincubated at 30°C in 1.6 ml of respiration buffer containing succinate (5 mM) as a respiration substrate. State 3 and state 4 respiration rates were measured after addition of the samples prepared as
described above in the presence (state 3) and after exhaustion (state
4) of ADP (87.5 ␮M). The respiratory control index was calculated as
the ratio of state 3/state 4 respiration (Chance and Williams, 1956).
Statistical Analysis. Statistical significance was calculated by
the Student’s t test.
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Chemicals. Diphenylamine, mefenamic acid, diclofenac sodium,
collagenase (type I), safranine O, fructose, and LDH-UV-Test-Wako
were purchased from the Wako Pure Chemical Ind. (Osaka, Japan).
Oligomycin was from the Sigma Chemical Co. (St. Louis, MO). ATP
sodium salt was from the Oriental Yeast Co., Ltd. (Tokyo, Japan). All
other chemicals and solvents were of analytical grade.
Preparation of Liver Mitochondria and Microsomes. Male
Wistar rats (2 months old) were obtained from Takasugi Experimental Animals (Saitama, Japan). The animals were housed in air conditioned rooms (25°C under a 12-h light/dark cycle for 1 week before
use). Food (commercially available pellet; Oriental Yeast Co., Ltd.)
and water were given ad libitum. Liver mitochondria fraction was
prepared according to the method described by Schneider and Hogeboom (1950) in a medium containing 0.25 M sucrose, 10 mM TrisHCl buffer (pH 7.4), and 0.5 mM EDTA. Liver microsomal fractions
were prepared according to the method of Omura and Sato (1964).
Protein concentrations were assayed by the method of Lowry et al.
(1951).
Measurement of Mitochondrial Swelling. Pseudoenergetic
mitochondrial swelling was evaluated spectrophotometrically according to Famaey (1973). Reaction mixture containing mitochondria
(0.15 mg/ml) and 0.15 M KCl in 30 mM Tris-HCl buffer (pH 7.5) was
preincubated at 30°C for 5 min. Absorbance at 520 nm was monitored after adding various concentrations of diphenylamine, mefenamic acid, and diclofenac with a Hitachi U-3200 spectrophotometer.
Initial slope for the decrease in the absorbance was measured as a
rate of the pseudoenergetic swelling.
Binding of Safranine to Mitochondria. Binding of safranine to
energetic mitochondria was monitored spectrophotometrically by
evaluating the spectral shift according to Colnna et al. (1973). Reaction mixture containing 200 mM sucrose, 2 ␮M rotenone, 20 mM
KCl, 5 mM succinate, and various concentrations of the test drugs in
10 mM Tris-HCl buffer (pH 7.2) was preincubated at 30°C for 5 min.
Safranine (12.5 ␮M) was added to the mixture and the absorption
spectra, in the range between 430 and 580 nm, were monitored.
Preparation of Isolated Rat Hepatocytes. Isolated hepatocytes were prepared from male Wistar rats by the collagenase perfusion method of Moldeus et al. (1978) with modifications. The liver
was isolated with perfusion of buffer (pH 7.2) consisting of 121 mM
NaCl, 6 mM KCl, 12 mM NaHCO3, 0.74 mM KH2PO4, 0.6 mM
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Masubuchi et al.
Vol. 292
Results
Fig. 3. Comparison of concentration dependence of mefenamic acid, diclofenac, and diphenylamine on induction of swelling in rat liver mitochondria. Mitochondrial fractions were incubated without substrate for
respiration in the presence of various concentrations of mefenamic acid
(F), diclofenac (E) and diphenylamine (䡺). Swelling was evaluated by
rate for decrease in absorbance at 520 nm, which were calculated from
initial slope for their decreases. Results are means ⫾ S.E. of three
different experiments. *P ⬍ .05, **P ⬍ .01, significantly different from
control obtained without drug.
Fig. 4. Effect of diphenylamine on the absorption spectra of safranine.
Mitochondria fractions were incubated with succinate in the absence (a)
or presence of diphenylamine at 25 (b), 100 (c), 250 (d), and 500 ␮M (e).
Safranine was added to the medium and absorption spectra were monitored in the range between 430 and 580 nm.
ture after metabolism of diclofenac did not affect the
respiration control. Because diclofenac in this sample was
eliminated to be less than one-third of the initial (data not
shown), the oxidative metabolites thus formed were indicated to lose the ability to uncouple mitochondrial oxidative
phosphorylation.
Fig. 2. Diphenylamine-induced swelling in rat liver mitochondria. Mitochondrial fractions were incubated without substrate for respiration in
the absence (a) or presence of diphenylamine at 10 (b), 25 (c), 50 (d), 100
(e), 250 (f), and 500 ␮M (g). Absorbance was monitored at 520 nm. Results
were from three experiments.
Discussion
We have found that diphenylamine as well as mefenamic
acid and diclofenac stimulate basal oxygen consumption
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Pseudoenergetic Mitochondrial Swelling. Incubation
of mitochondria with diphenylamine as well as mefenamic
acid and diclofenac caused time-dependent decreases in absorbance at 420 nm, suggesting that diphenylamine induces
pseudoenergetic mitochondrial swelling (Fig. 2). Rates of the
pseudoenergetic swelling induced by the compounds revealed
similar concentration-dependence, whereas effects of diphenylamine were pronounced compared with those of mefenamic acid and diclofenac (Fig. 3).
Binding of Safranine to Mitochondria. Diphenylamine
caused changes in safranine-binding spectra to mitochondria
that was energized by succinate oxidation, i.e., an increase in
absorbance and a shift in the wavelength of its maximum absorbance (Fig. 4). This safranine spectral shift indicates the
decrease in mitochondrial membrane potentials (⌬␸) and also
was caused by mefenamic acid and diclofenac (data not shown).
Protection by Fructose and Oligomycin against Hepatocyte Injury. Incubation of hepatocytes with mefenamic
acid, diclofenac, and diphenylamine diminished cellular ATP
contents, followed by LDH leakage. Fructose, a low Km substrate for glycolysis, partially protected against ATP depletion by the compounds used (Fig. 5). Further addition of
oligomycin, which blocks ATPase, pronounced the protection
against ATP depletion by mefenamic acid, resulting in full
protection. More pronounced results were obtained for protection against the LDH leakage induced by the compounds,
although considerable enzyme leakage was observed in the
control incubations for 3 h (Fig. 6). Namely, fructose partially
protected against the LDH leakage caused by mefenamic
acid, diclofenac, and diphenylamine, and additional effects of
oligomycin were observed for all of three compounds, resulting in nearly full protection.
Effect of Diclofenac Metabolism on Uncoupling Effect. Table 1 shows the effects of diclofenac and the ether
extract from the microsomal mixture after metabolism of
diclofenac on oxygen consumption by rat liver mitochondria
with succinate as a substrate. Diclofenac stimulated basal
oxygen consumption (state 4) and inhibited respiration stimulated with ADP (state 3), resulting in a marked decrease in
respiration control index, which are the typical characteristics of uncouplers of mitochondrial oxidative phosphorylation. However, the ether extract from the microsomal mix-
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Mechanism of Hepatocyte Injury by NSAIDs
985
TABLE 1
Effects of diclofenac and ether extract from microsomal mixture after
diclofenac metabolism on respiratory control (RC) in rat liver
mitochondria
Mitochondria (1 mg protein/ml) were incubated in 1.6 ml of respiration buffer
containing succinate (5 mM) as a substrate at 30°C. State 3 and state 4 respirations
were measured after addition of diclofenac (25 ␮M) or the ether extract from the
microsomal mixture before (diclofenac extract) and after (metabolite extract) metabolism of diclofenac. The RC was calculated as the ratio of state 3/state 4 respiration.
Results are means ⫾ S.E. of three different experiments.
Oxygen Consumption
Additions
RC
State 3
State 4
nmol/min/mg protein
Control
Diclofenac
Diclofenac extract
Metabolite
extract
Fig. 6. Effect of fructose and oligomycin on hepatocyte injury induced by
mefenamic acid, diclofenac, and diphenylamine. Hepatocytes were incubated under the same conditions as described in the legend for Fig. 5.
Abbreviations also correspond to those of Fig. 5. Viability of the hepatocytes was assessed by LDH leakage 180 min after the addition of the test
drug. Results are expressed as a percentage of the total LDH activity and
are means ⫾ S.E. of three different experiments. **P ⬍ .01, significantly
different from “Non” obtained without the test drug. ††P ⬍ .01, significantly different from “control” obtained with the corresponding drug but
without fructose.
(state 4 respiration) and inhibit ADP-stimulated respiration
(state 3), suggesting that these compounds are uncouplers of
mitochondrial oxidative phosphorylation (Masubuchi et al.,
1999). To further characterize the effect of diphenylamine on
the mitochondrial membrane, the ability to induced swelling
was tested. Incubation of mitochondria with diphenylamine
in the absence of a substrate caused a time-dependent decrease in absorbance at 520 nm (Figs. 2 and 3), suggesting
that diphenylamine as well as mefenamic acid and diclofenac
induce pseudoenergized mitochondrial swelling (Banos and
Reyes, 1989; Uyemura et al., 1997). Large-amplitude swelling is a typical phenomenon of MPT, which is due to the
9.7 ⫾ 0.42
23.0 ⫾ 2.35**
17.5 ⫾ 1.71*
10.5 ⫾ 1.84
5.6
1.4
1.8
4.7
* P ⬍ .05 and ** P ⬍ .01, significantly different from control.
opening of high-conductance permeability pores in the mitochondrial inner membrane (Bernardi et al., 1994). The MPT
was implicated as a causative factor in lethal cell injury, and
was recently noted for the relation to Reye’s syndrome (Trost
and Lemasters, 1996; Lemasters et al., 1998). Uncouplers of
oxidative phosphorylation are also inducers of the MPT.
However, because the pore opening causes the uncoupling of
oxidative phosphorylation, it is possible to cause apparent
uncoupling by the MPT. But it was considered that the uncoupling by diphenylamine did not result as a consequence of
the MPT, but the protonophoric ability, because cyclosporin
A, a specific inhibitor of the MPT (Broekemeier et al., 1989),
did not affect the observed uncoupling (Masubuchi et al.,
1999).
The electrochemical gradient, which plays an essential role
in the production of ATP, is comprised almost by the membrane potential (⌬␸). We thus measured the membrane potential according to a classical method, safranine binding to mitochondria (Famaey, 1973). Diphenylamine caused changes in
safranine-binding spectra to mitochondria that was energized
by succinate oxidation, i.e., an increase in absorbance and a
shift in the wavelength of its maximum absorbance (Fig. 4),
which also were obtained for mefenamic acid and diclofenac.
These spectral shifts indicate the decrease in mitochondrial
membrane potentials seem to be due to uncoupling of oxidative
phosphorylation (Moreno-Sanchez et al., 1999). Mitochondrial
depolarization as well as ATP depletion by the uncoupling is the
causative factor to impair hepatocytes (Byrne et al., 1999),
suggesting its involvement in cell injury. Moreover, it should be
emphasized that diphenylamine has similar properties to structurally related NSAIDs.
Fructose, but not glucose, provides cytoprotection against
cell damage associated with mitochondrial poisons and
prooxidants (Wu et al., 1990). Because fructose is an excellent substrate for the glycolytic pathway, which results in net
ATP production, fructose compensates for deficient mitochondrial ATP production and it was proposed as one of the
mechanisms for the cytoprotection. Thus, protective effects of
fructose were investigated against the hepatocyte injury
along with depletion of the ATP induced by mefenamic acid,
diclofenac, and diphenylamine. Fructose partially protected
against ATP depletion and subsequent LDH leakage (Figs. 5
and 6). Further addition of oligomycin, which blocks ATPase,
pronounced the protection against hepatocyte injury, whereas a
Downloaded from jpet.aspetjournals.org at ASPET Journals on August 9, 2017
Fig. 5. Effect of fructose and oligomycin on decrease in ATP content in
hepatocytes induced by mefenamic acid, diclofenac, and diphenylamine.
Freshly isolated rat hepatocytes were incubated with or without fructose
(F). After 30 min, a test drug, mefenamic acid (Mef), diclofenac (Dcf), or
diphenylamine (Dpa) was added with or without oligomycin (O). ATP
contents in the hepatocytes were assayed 60 min after addition of the
drug. Results are expressed as a percentage of the initial ATP content in
the hepatocytes and are means ⫾ S.E. of three different experiments.
*
P ⬍ .05, **P ⬍ .01, significantly different from “Non” obtained without
the test drug. †P ⬍ .05, ††P ⬍ .01, significantly different from “control”
obtained with the corresponding drug but without fructose.
53.8 ⫾ 2.31
31.7 ⫾ 2.51**
31.0 ⫾ 3.30**
45.0 ⫾ 5.13
986
Masubuchi et al.
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marked additional effect of oligomycin on ATP content was
observed only against the mefenamic acid-induced decrease.
Because uncouplers stimulate ATPase, resulting in enhancement of ATP hydrolysis, its inhibition by oligomycin seems to be
effective in compensation of cellular ATP content, which was
decreased by the potent uncoupler (Nieminen et al., 1994). The
effective cytoprotection by fructose and additional oligomycin
suggested that decreases in cellular ATP content, mainly
caused by the uncoupling of mitochondrial oxidative phosphorylation, were responsible for acute hepatocyte injury induced by
diphenylamine and structurally related NSAIDs.
Hepatocyte injury induced by diclofenac has been discussed in relation to oxidative metabolism (Kretz-Rommel
and Boelsterli, 1993; Jurima-Romet et al., 1994). Very recently, a couple of oxidative metabolites were identified as
the reactive metabolites of diclofenac (Shen et al., 1999; Tang
et al., 1999a,b). In relation to the hepatocyte injury by diclofenac, Bort et al. (1999) reported that N-5-dihydroxydiclofenac was a candidate responsible for cell injury by a futile
consumption of NADPH, in addition to the impairment of
ATP synthesis by diclofenac itself (Bort et al., 1999). Our
previous and present results suggested that the hepatocyte
injury by NSAIDs was not “metabolism-dependent” but
“structure-dependent” (Masubuchi et al., 1998), i.e., diphenylamine is one of the essential structures to cause hepatocyte injury, which is also necessary to inhibit mitochondrial
oxidative phosphorylation. Indeed, it was shown that oxidative metabolism of diclofenac lost the ability to uncouple
oxidative phosphorylation, confirming the lack of involvement of the metabolite (Table 1). However, because the consumptive oxidation of NADPH was suggested to disrupt the
balance of mitochondrial Ca2⫹ uptake and release, to lead to
net increase in mitochondrial Ca2⫹, and to result in opening
of the permeability transition pore and the onset of MPT
(Byrne et al., 1999), it is possible that N-5-dihydroxydiclofenac contributes to mitochondria impairment at the cellular
level. The findings could lead to speculation that not only
diclofenac but also diphenylamine and mefenamic acid converted to corresponding N-hydroxylamine metabolites, which
provide structural importance of diphenylamine to cause hepatocyte injury, whereas it is only speculative because metabolism of diphenylamine itself has been unknown. Therefore, the contribution of the metabolites to the mitochondrial
impairment cannot be excluded and is a further interest to
elucidate the molecular mechanism for the structural requirement. Regardless, it is concluded that the depletion of
cellular ATP induced by the uncoupling of mitochondrial
oxidative phosphorylation plays a crucial role in cytotoxicity
of diphenylamine and NSAIDs that have the diphenylamine
structure.
Vol. 292
2000
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Mechanism of Hepatocyte Injury by NSAIDs
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Send reprint requests to: Toshiharu Horie, Ph.D., Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Chiba University, 1–33 Yayoicho, Inage-ku, Chiba 263-8522, Japan. E-mail: [email protected]
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