Morphological Alteration in Mitochondria Following Diclofenac and
This study was conducted to identify and to compare
the mitochondrial morphological alterations in livers of rats treated
with various doses of diclofenac and ibuprofen. Hundred and forty-four
male Sprague Dawley rats were dosed with 3, 5 and 10 mg kg-1
diclofenac and ibuprofen in saline via intraperitoneal injection for 15
days. The control group was administered with saline in a similar manner.
Four rats were euthanised every 3 days until day 15. While 200 mg kg-1
diclofenac and ibuprofen-treated rats (n = 4) were euthanized 10 h post-treatment.
The livers were removed, cleaned and a section across the right lobe was
taken and fixed in 4% (v/v) glutaraldehyde for electron microscopy analysis
and the remaining samples were kept at -80°C for Western blot analysis.
Five milligram per kilogram and 10 mg kg-1 diclofenac-administered
rats for 15 days revealed the presence of enlarged mitochondria, irregular
and ruptured mitochondrial membranes. While rats administered with 10
mg kg-1 ibuprofen also showed the presence of mitochondria
with irregular membrane structure and ruptured membranes. Western blotting
analysis of mitochondrial fractions revealed the expression of cytochrome
c in all samples and complete absence of cytochrome c expression in the
cytosolic fraction of all samples after day 15. Analysis in 200 mg kg-1
diclofenac and ibuprofen-treated groups, revealed expression of cytochrome
c in both mitochondrial and cytosolic fractions. This observation indicates
that both diclofenac and ibuprofen may alter the morphology of mitochondria,
leading to cytochrome c release into the cytosol. Further studies needs
to be conducted to investigate on the activity of the mitochondria following
Hepatotoxicity is an adverse drug reaction associated with NSAIDs use.
Although its occurrence is less common compared to other NSAIDs-related
complications, hepatotoxicity is identified as the common cause for withdrawal
of some NSAIDs (Teoh and Farrell, 2003).
Diclofenac and ibuprofen are among the most widely used NSAIDs (Al-Nasser,
2000; Boelsterli, 2003). Both have been associated with some form of hepatotoxicity
(Bank et al., 1995; Al-Nasser, 2000; Rubeinstein and Laine, 2004;
Ponsoda et al., 1995; Masubuchi et al., 2002; Gomez-Lechon
et al., 2003a, b).
In the case of diclofenac, significant hepatotoxicity were seen in 1-5
per 100,000 patients consuming this drug (Garcia et al., 1994;
Tolman, 1998). Often, the onset of liver injury is characterized by anorexia,
nausea, vomiting occurring within 3 months (ranges from 1 to 11 months)
(Teoh and Farrell, 2003). Fever and rash have been reported in 25% patients
and reaction was severe with jaundice in 50% of the cases (Bank et
al., 1995). The hepatotoxicity is believed to be either due to metabolic
or immunologic idiosyncrasy (Boelsterli, 2003). 4-OH diclofenac and 5-OH
diclofenac yield following metabolism of diclofenac by CYP2C9 and CYP3A4
respectively were identified as toxicants that can undergo covalent binding
with various nonprotein or protein groups (Boelsterli, 2003). Many findings
also indicate that the metabolites are capable of causing apoptosis of
hepatocytes (Ponsoda et al., 1995; Masubuchi et al., 2002;
Gomez-Lechon et al., 2003a, b). This was identified to be related
to the ability of the drug to cause oxidative stress that is followed
by Mitochondrial Permeability Transition (MPT) (Masubuchi et al.,
2002). MPT was found to cause leakage of cytochrome c and other apoptotic
components from mitochondria into the cytosol, leading to activation of
caspase cascade. This event ends with apoptosis of hepatocytes.
Ibuprofen is known to be one of the safest NSAIDs available (Steward,
1992), as not many reports are available on ibuprofen-induced hepatotoxicity.
The side effects were identified to be related to the widespread use of
the drug (Al-Nasser, 2000). As with other `profen` drugs, acyl glucuronidation
is a major route for the biotransformation of ibuprofen (Li et al.,
2003) and it is well known that acyl glucuronides are reactive electrophiles
that undergo nucleophilic reaction with water, glucuronic acid and glutathione
(Grillo and Benet, 2002; Olsen et al., 2002). As with diclofenac,
recent study with ibuprofen was also found to induce apoptosis in cell
line (Campos et al., 2004). It could be related to the ability
of this drug to induce MPT, since it has been shown to target mitochondria
(Al-Nasser, 2000). The exact underlying mechanism is still not clear and
is under extensive study. This study was conducted to identify and to
compare the mitochondrial morphological alterations in the livers of rats
treated with various doses of diclofenac and ibuprofen.
MATERIALS AND METHODS
Diclofenac sodium and ibuprofen were purchased from Sigma-Aldrich. Glutaraldehyde
25% EM grade (Agar Scientific Limited, UK), Sodium cocadylate (Agar Scientific
Limited, UK), Osmium tetroxide (Agar Scientific Limited, UK). Ethanol
absolute (Merck, Germany), 1,2-propyleneoxid (Merck, Germany), Dodecyenyl
succinic Anhydride, DDSA (Agar Scientific Limited, UK), Benzyldimethylamine,
BDMA (Agar Scientific Limited, UK), Methyl Nadic Anhydride, MDA (Agar
Scientific Limited, UK), AGAR 100 Resin (Agar Scientific Limited, UK),
Acetone (Ajax Finechem, New Zealand), Lead citrate (Agar Scientific Limited,
UK), Uranyl acetate (Agar Scientific Limited, UK), grid 200 mesh (Agar
Scientific Limited, UK). Sucrose (Amresco, Ohio), EDTA (Sigma-Aldrich),
Glacial acetic acid (ICN Biomedicals, California), Tris-base (Sigma-Aldrich).
EDTA (Sigma-Aldrich), BCA protein assay kit (Pierce, USA). Thirty percent
Acrylamide solution (Biorad, USA), SDS (Amresco, Ohio), TEMED (Amresco,
Ohio), Ammonium Persulfate (Amresco, Ohio), Glycine (Amresco, Ohio), Methanol
(Systerm), Glycerol (ICN Biomedical, California), Bromophenol blue (Amresco,
Ohio), Dithioreitol, DTT (Amresco, Ohio), Casein (Sigma-Aldrich), NaCl
(Merck, Germany), Thimerosal (ICN Biomedicals, California), mouse monoclonal
Cytochrome c primary antibody (Santa Cruz Biotechnology Inc., USA), Hydrochloric
acid, HCl (ICN Biomedicals, California), rabbit anti-mouse HRP secondary
antibody (Zymed, California), Pageruler Prestained Protein ladder (Fermentas,
Canada), nitrocellulose membrane (Sigma-Aldrich), Supersignal West Pico
Chemiluminescent substrate (Pierce, USA).
Animal study: This study was conducted at Faculty of Medicine
and Health Sciences, Universiti Putra Malaysia. A hundred and forty four
male Sprague Dawley rats were acclimatised under control condition of
humidity with regular light/dark cycle and free access to food and water.
The rats were randomly distributed into groups of 3, 5 and 10 mg kg-1
diclofenac and ibuprofen, control group and 4 rats as untreated group.
The drugs were dosed intraperitoneally at 0.5 mL rat-1 day-1.
The control group was given saline in a similar manner. Four rats from
each group were euthanised every 3 days until day 15. While 200 mg kg-1
diclofenac and ibuprofen-treated rats (n = 4) were euthanized 10 h post-treatment.
Upon euthanisation, livers were removed, cleaned and weighed. A section
across the right lobe was taken and fixed in 4% (v/v) glutaraldehyde,
which was processed for transmission electron microscopy analysis. The
remaining samples were kept under -80°C for Western blot analysis.
Ultrastructural study: A section across the right lobe was removed,
cleaned and weighed in normal saline and sliced into small sections of
1 mm3 thickness. These sections were fixed in 4% (v/v) glutaraldehyde
for 24 h then post-fixed in 2% osmium tetroxide. The subsequent process
was carried out according to standard procedure for electron microscopy
analysis. Infiltration process was carried out with propylene oxide and
resin mixture. Then, the samples were embedded in freshly prepared resin
mixture. Ultrathin section was prepared and stained with uranyl acetate
and lead citrate and viewed using transmission electron microscope to
detect ultrastructural changes in mitochondria.
Detection of cytochrome c
Subcellular fractionation: Subcellular fractionation was carried
out using cold sucrose buffer (0.25 M sucrose, 15 mM Tris-base, 0.1 mM
EDTA, pH adjusted to 6.8 with glacial acetic acid). Liver samples from
each dose group were pooled, blotted dry, weighed, minced in 5 volumes
of sucrose buffer and homogenised at 1000 rpm (Ultra-Turaxx T8). The homogenate
was strained and 3 mL of aliquots were stored at -80°C. The remainder
of the homogenate was centrifuged at 600 x gav for 10 min (Eppendorf
5810 R). The supernatant was separated following centrifugation. The post
nuclear supernatants were centrifuged at 10,000 x gav for 20
min (Eppendorf 5810 R). The resulting supernatant was separated. The supernatant
corresponds to cytosolic fraction (with microsomes), 3 mL aliquots were
kept at -80°C. The pellet was washed twice by resuspending in one
volume of sucrose buffer and recentrifuging. The resulting pellet that
corresponds to mitochondrial fraction was kept at -80°C.
Protein assay: Protein concentrations of the samples were carried
out using the BCA protein assay reagent kit (Pierce), according to manufacturer`s
SDS-PAGE and immunoblotting: Protein samples were solubilised
by boiling for 3 min in buffer (125 mM stacking gel buffer, 10% (w/v)
SDS, 20% (v/v) glycerol, 0.02% (w/v) bromophenol blue, 0.2 M DTT). SDS-PAGE
was carried out using Mini PROTE-N® III Electrophoresis Cell apparatus
(Biorad) with 4% (w/v) stacking gel and 10% (w/v) resolving gel using
the discontinuous buffer system as described by Laemmili (1970). Protein
samples were loaded at 25 μg lane-1 and the gel was run
at 100 Volt for approximately 75 min in running buffer (0.025 M Tris,
0.192 M glycine, 0.1% (w/v) SDS, pH 8.3). Following electrophoresis, proteins
were electrophoretically transferred to a nitrocellulose membrane in transfer
buffer (15.7 mM Tris-base, 120 mM glycine and 20% (v/v) methanol, pH 8.3)
at constant voltage of 100 V for 1 h at 4°C using mini trans-blot
elctrophoresis cell (Biorad, USA).
Subsequent steps were carried out at room temperature with continuous
shaking. Following immunoblotting, the membrane was incubated overnight
in blocking buffer (154 mM NaCl, 2.8% (w/v) casein, 10 mM Tris-base and
0.02% (w/v) thimerosal, pH 7.6 with concentrated HCl) to block the non-specific
The nitrocellulose membrane was then incubated with mouse monoclonal
cytochrome c primary antibody diluted in wash buffer (154 mM NaCl, 0.5%
w/v casein, 1 mM Tris, 0.02% w/v thimerosal) (dilution 1:1000). After
four 10 min washes to remove unbound antibody, the membrane was then incubated
for 2 h with rabbit anti-mouse HRP secondary antibody (1:10,000 in wash
buffer). Following series of washes with wash buffer and TBS, the membrane
was incubated with Pierce Supersignal West Pico Chemiluminescent substrate
reagent for 5 min. The expression of protein bands were captured with
Flourochem 5500 (Alpha Innotech).
RESULTS AND DISCUSSION
Ultrastructural changes in mitochondria: Rats treated with 5 and
10 mg kg-1 diclofenac for 15 days showed presence of abnormal
mitochondria. The abnormalities observed in 5 mg kg-1 diclofenac
administered group include the presence of enlarged mitochondria (Fig.
1), irregular mitochondrial membrane (Fig. 2) and
ruptured mitochondrial membranes (Fig. 3). Similarly,
10 mg kg-1 diclofenac given rats revealed presence of enlarged
mitochondria (Fig. 4) and ruptured mitochondrial membranes
||Slight enlargement of mitochondria (EM) in 5 mg kg-1
diclofenac-treated groups after day 15, 8000x
||(→) indicates presence of irregular mitochondrial
membranes in 5 mg kg-1 diclofenac administered rats after
day 15, 20 000
Meanwhile, when rats were administered with ibuprofen, abnormal mitochondrial
ultrastructures were seen only with 10 mg kg-1 ibuprofen-dosed
group on day 15. The changes observed include, mitochondria with irregular
membrane structure (Fig. 6) and ruptured membranes (Fig.
7). Observations conducted on samples of both diclofenac and ibuprofen
treated groups at lower doses and at earlier time points showed normal
liver ultrastructure similar to saline-treated rats as shown in Fig.
||(→) indicates ruptured mitochondrial membranes
in 5 mg g-1 diclofenac-treated rats, 20 000
||Slightly enlarged mitochondria (EM) in 10 mg kg-1
diclofenac administered group after day 15, 8000x
||(→) indicates ruptured mitochondrial membrane
in 10 mg kg-1 diclofenac injected rat, 20 000
||(→) indicates presence of irregular mitochondrial
membrane in 10 mg kg-1 ibuprofen administered rats after
day 15, 8000x
||(→) shows ruptured mitochondrial membrane
in 10 mg kg-1 ibuprofen injected group, 8000x
||Intact mitochondria (M) in saline-treated rats at day
Expression of cytochrome c in mitochondrial fractions
of 3-10 mg kg-1 ibuprofen and diclofenac administered
rats on day 15
Absence of cytochrome c expression in liver cytosolic
fraction of 3-10 mg kg-1 ibuprofen and diclofenac-treated
rats on day 15
Western blotting analysis to detect cytochrome c expression in liver
samples: Western blotting analysis was carried out to detect the possible
expression of cytochrome c in the mitochondrial and cytosolic fractions
which indicates possible rupture in mitochondrial membranes.
Following subcellular fractionation of the liver homogenates, the expression
of cytochrome c was clearly observed in the mitochondrial fraction of
diclofenac and ibuprofen-treated groups as shown in Fig.
9. There was no expression of cytochrome c after 15 days of diclofenac
and ibuprofen treatment at 3-10 mg kg-1 as indicated in Fig.
10. However, with a single dose of 200 mg kg-1 diclofenac
and ibuprofen, cytochrome c is expressed in the cytosolic fraction as
revealed in Fig. 11.
Recent studies have identified the ability of diclofenac and ibuprofen
to induce apoptosis in various cell lines (Kusuhara et al., 1998;
Gomez-Lechon et al., 2003a; Masubuchi et al., 2002; Campos
et al., 2004) and mitochondria was found to play a pivotal role
in the mechanism (Gomez-Lechon et al., 2003a; Masubuchi et al.,
2002; Campos et al., 2004).
Presence of cytochrome c in liver homogenate (HI),
cytosolic fraction (CyI) and mitochondrial fraction (MtI) of 200
mg kg-1 ibuprofen-treated rats and in liver homogenate
(HD), cytosolic fraction (CyD) and mitochondrial fraction (MtD)
of 200 mg kg-1 diclofenac-treated groups 10 h post-treatment.
Saline (S), 3 mg kg-1 ibuprofen (I3), 5 mg kg-1
ibuprofen (I5), 10 mg kg-1 ibuprofen (I10), 3 mg kg-1
diclofenac (D3), 5 mg kg-1 diclofenac (D5), 10 mg kg-1
Multiple treatments for 15 days with diclofenac and ibuprofen, induced
mitochondrial swelling and mitochondrial membrane rupture. The changes
seen in this study suggested to be dose and frequency dependent (Kretz-Rommel
and Boelsterli, 1993a, b; Masubuchi et al., 1998). The observation
indicates that diclofenac is more potent compared to ibuprofen (Masubuchi
et al., 1998; Siraki et al., 2005) in inducing mitochondrial
changes. This is in consistent with IC50 of diclofenac for
inhibition of COX-1 of 0.26 in comparison to ibuprofen of 5.9 (Cryer and
Mitochondrial swelling and membrane rupture are believed to be due to
a phenomenon called Mitochondrial Permeability Transition (MPT). MPT refers
to an increase in mitochondrial membrane permeability to solutes with
molecular mass less than 1500 Da. The MPT is believed to be formed at
the contact sites between the inner and outer mitochondrial membranes.
MPT results in depolarization and chemical or solutes equilibration between
cytoplasm and mitochondrial matrix. This ultimately leads to disruption
and rupture of outer mitochondrial membrane (Benardi et al., 2006,
1999; Crompton, 1999).
Previously, it has been reported that the inability of mitochondria to
produce ATP as the major cause of diclofenac-induced hepatotoxicity (Ponsoda
et al., 1995; Bort et al., 1999). A recent study also revealed
the key role of mitochondrial dysfunction in the pathogenesis of diclofenac-induced
hepatic injury due to decrease in ATP caused by MPT (Masubuchi et al.,
2002). It has also been proved that inhibition of MPT with specific MPT
blockers prevents diclofenac-induced apoptosis both in human and rat hepatocytes
(Karpinich et al., 2002; Feldtrauer et al., 2002; Gomez-Lechon
et al., 2003a). Diclofenac-activated MPT was identified as the
consequence of oxidative damage to pre-existing membrane proteins, since
simultaneous incubation of diclofenac-added hepatocytes with antioxidants
prevented caspase activation and later apoptosis (Sokol et al.,
2001; Vrablic et al., 2001; Masubuchi et al., 2002; Gomez-Lechon
et al., 2003a).
Increase in intramitochondrial Ca2+ is also believed to be
another factor of MPT. It potentiates binding of cyclophilin D to the
matrix side of MPT pore in particular to Adenine Nucleotide Transporter
(ANT) (Crompton et al., 1998). Increase in intramitochondrial Ca2+
was related to the presence of diclofenac metabolites (Lim et
al., 2006). 4-OH and 5-OH diclofenac can be metabolized to respective
reactive intermediates (Shen et al., 1999; Tang et al.,
1999a, b; Poon et al., 2001) that generate Reactive Oxygen Species
(ROS). ROS is believed to inactivate critical sulfhydryl groups of Ca2+
pumps (Lim et al., 2006), which leads to increase in intramitochondrial
While for ibuprofen, similar observation to current study was made in
isolated mitochondria and was also related to MPT (Al-Nasser, 2000). It
was shown that exposure of liver mitochondria to low concentration of
ibuprofen resulted in increase swelling and loss of inner mitochondrial
membrane potential, which was inhibited by cyclosporine A (CsA); a well-known
MPT inhibitor. This indicates the ability of ibuprofen to induce MPT.
Due to detection of mitochondrial swelling and membrane rupture, cytochrome
c release from mitochondria into cytosol was assessed since apoptosis
was related to presence of cytochrome c in the cytosol (Gomez-Lechon et
Cytochrome c expression was detected in the mitochondrial fractions of
all samples under study. However, there was no detection of cytochrome
c expression in the cytosolic fractions of all doses. This is probably
due to the cytosolic fractions did not contain any detectable amount of
cytochrome c (Herde et al., 2000). This may also be explained by
the detection of only few ruptured mitochondria in 5 and 10 mg kg-1
diclofenac and 10 mg kg-1 ibuprofen given groups. This may
also be related to the use of monoclonal cytochrome c primary antibody
(ab), since this type of antibody is highly specific as compared to polyclonal
antibodies. However, the expression of cytochrome c was detected in both
mitochondrial and cytosolic fraction when animals were treated with 200
mg kg-1 of diclofenac and ibuprofen. The cytosolic cytochrome
c of diclofenac is more densely expressed than that of ibuprofen; indicating
diclofenac to be more potent compared to ibuprofen (Kretz-Rommel and Boelsterli,
1993a, b; Masubuchi et al., 1998) in inducing cytochrome c release
from mitochondria into cytosol.
Cytochrome c is involved in the formation of membrane potential for the
production of ATP (Robertson and Orrenius, 2000). The addition of cytochrome
c to cytosolic extract has been shown to be a determining factor in the
activation of caspase and later apoptosis (Liu et al., 1996; Zou
et al., 1997). Cytochome c release has been identified as the consequence
of mitochondrial swelling and membrane rupture (Benardi et al.,
1999). This observation indicates that both diclofenac and ibuprofen may
alter the morphology of mitochondria, leading to cytochrome c release
into the cytosol. Further studies needs to be conducted to investigate
on the activity of the mitochondria following both treatments.
The authors would like to acknowledge the Malaysian Toray Science Foundation
(MTSF) (Grant No. 54853) and the Ministry of Higher Learning Education
of Malaysia for financial assistance (Grant No. 55165) and Unit of Microscopy
and Microanalysis, Institute of Bioscience (Universiti Putra Malaysia)
for their technical assistance.
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