Toxicology 262 (2009) 175–183

Contents lists available at ScienceDirect

Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Aroclor 1254 induced cytotoxicity and mitochondrial dysfunction in isolated

rat hepatocytes
Hamdy A.A. Aly a,b,∗ , Òscar Domènech b
a
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt
b
Unit of Cellular and Molecular Pharmacology, Catholic University of Louvain B-1200 Brussels, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: Polychlorinated biphenyls (PCBs) are widespread persistent environmental contaminants that display a
Received 13 May 2009 complex spectrum of toxicological properties, including hepatotoxicity. Although Aroclor 1254 is ubiqui-
Received in revised form 21 May 2009 tous in the environment, its potential cytotoxic effect on rat hepatocytes and the mechanism underlines
Accepted 22 May 2009
its cytotoxicity are not fully investigated. Therefore, the present study was conducted to investigate: (1)
Available online 30 May 2009
the potential cytotoxicity of Aroclor 1254 in rat hepatocytes, and (2) characterization of the molecular
mechanisms involved in the Aroclor 1254-induced hepatotoxicity, particularly the role of mitochondria,
Keywords:
possibly a primary target in such event, could greatly explain the cytotoxic effect of Aroclor 1254 in rat
Hepatocytes
Mitochondria
hepatocytes. Hepatocytes were isolated from adult male albino rats and incubated for 24 h in a fresh
Aroclor 1254 media containing 0, 20, 30, 40, 50 or 60 ␮M of Aroclor 1254.
Oxidative stress At the end of incubation, hepatocytes and hepatocyte mitochondria were used for the assay. Our results
showed cytotoxicity of Aroclor 1254 in rat hepatocytes starting at a concentration of 30 ␮M as manifested
by increased lactate dehydrogenase (LDH) leakage, decreased cell viability (MTT assay) and increased lipid
peroxidation. As mitochondria are known to be one possible site of the cell damage, the effects of Aro-
clor 1254 on hepatocyte mitochondria was investigated. Aroclor 1254 induced reactive oxygen species
(ROS) generation in hepatocyte mitochondria, inhibited mitochondrial respiratory chain complexes I
and III and ␤-oxidation of free fatty acids, depletion of mitochondrial antioxidant enzymes GPx and GR
and the non-enzymatic antioxidant reduced glutathione, inhibited mitochondrial membrane potential
(␺m ), decreased mitochondrial aconitase and cardiolipin content, and elevated levels of cytochrome
P450 subfamily, CYP1A and CYP2B activities as indicated by ethoxyresorufin O-deethylase (EROD) and
pentoxyresorufin O-deethylase (PROD).
Therefore, we can conclude that Aroclor 1254 induced rat hepatocyte toxicity and our findings provide
evidence to propose that mitochondria are one of the most important and earliest cell targets in Aroclor
1254-mediated toxicity and delineate several mitochondrial processes at least, in part, by induction of
oxidative stress. These findings can be useful in future cytoprotective therapy approaches. Since mito-
chondrial events appear to be targeted in hepatocellular damage induced by Aroclor 1254, an antioxidant
therapy targeted to mitochondria may constitute an interesting strategy to ameliorate its toxicity.
© 2009 Published by Elsevier Ireland Ltd.

1. Introduction and on surfaces in homes and in factories (Sadeghi-Aliabadi et al.,

Polychlorinated biphenyls (PCBs) were widely used in a vari-
ety of industrial and consumer products for several decades before
their production was banned in the 1970s. As a result of their
extensive use and their chemical stability, PCBs are still ubiquitous
environmental contaminants that are frequently found as com-
plex mixtures of isomers and congeners in air, water, soil, dust
∗ Corresponding author at: Apartment No: 62, El-Nour Tower No: 1, El-Sefarate
Quartier, Nasr City, Cairo, Egypt. Tel.: +20 173809433.
E-mail address: hamdyaali@yahoo.com (H.A.A. Aly).
2007). PCBs mixtures contain various levels of chlorination, and
were sold under several trade names, including Aroclor, Clophen,
Fenchlor, Kanechlor, Phenoclor, and Pyralene (Larsen, 2006; Simon
et al., 2007). These compounds are highly lipophilic and accumu-
late easily in the lipid bilayer and fat deposits of the body (Safe,
1994; Parkinson et al., 1983). For these reasons, there is a toxico-
logical concern about possible risks associated with the exposure
of humans to PCBs (Silberhorn et al., 1990). PCBs are distributed
throughout the entire ecosystem, including soil, air and water
(Risebrough et al., 1968). They are absorbed through the skin, lungs
and GI tract and are transported by blood to liver and muscles (Baird,
1995). Exposure to these compounds results in various harmful

0300-483X/$ – see front matter © 2009 Published by Elsevier Ireland Ltd.

doi:10.1016/j.tox.2009.05.018
176 H.A.A. Aly, Ò. Domènech / Toxicology 262 (2009) 175–183
effects including reproductive toxicity, immune suppression, birth
defects, cancer, developmental and behavioral changes and liver
damage (Safe, 1994; Kimbrough, 1995; Adebusoye et al., 2008).
PCBs affect two enzyme systems responsible for production and
elimination of reactive intermediates: the xenobiotic-metabolizing
enzymes (XMEs) and the antioxidant enzymes (AOEs), respec-
tively. The effects of PCBs on the levels of XMEs, in particular
the cytochrome P450 (CYP) isozymes, and AOEs are well estab-
lished in liver (Twaroski et al., 2001a,b; Fadhel et al., 2002). A
mechanistic link between toxicity, induction of the CYP1A sub-
family, and binding to the aryl hydrocarbon (Ah) receptor has
been established for PCB congeners that are structurally related to
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Poland and Knutson,
1982).
Depending on their three-dimensional structure and their sub-
stitution pattern, PCB congeners bind to different cellular target
sites and cause adverse effects by a variety of different mechanisms
(Zhao et al., 2009). Structure–activity studies with individual PCBs
have demonstrated that congeners with chlorine atoms at the para
and meta, but not ortho, positions of both phenyl rings are able to
assume a dioxin-like conformation, bind the Ah receptor with high
affinity, and are efficacious inducers of CYP1A (Safe, 1994). PCB
congeners with multiple ortho chlorine substituents may bind to
or activate the constitutive androstane receptor (CAR) (Denomme
et al., 1983) and/or the pregnane X receptor (PXR) (Schuetz et al.,
1998). Other ortho-substituted PCB congeners interact with both
the aryl hydrocarbon (Ah-) receptor and CAR (Parkinson et al.,
1983). In addition, PCB congeners with multiple ortho substituents
sensitize ryanodine receptors (RyR) (Pessah et al., 2006), an inter-
action that has been shown to be enantiospecific in the case of PCB
136 (Pessah et al., 2009). The mono-ortho derivatives of the dioxin-
like PCBs have reduced Ah receptor-mediated activities and can
also induce CYP2B enzymes. Congeners possessing chlorine atoms
at the ortho positions of both phenyl rings are non-dioxin-like, do
not readily bind to the Ah receptor, and exhibit a pattern of toxic-
ity including carcinogenic, neurotoxic, behavioral, and endocrine
effects that differs from that of the coplanar PCBs (Giesy and
Kannan, 1998). Nevertheless, some di-ortho chlorinated PCBs are
extremely weak inducers of CYP1A enzymes, whereas other di-
ortho chlorinated congeners do not induce CYP1A enzymes, though
they induce other CYP enzymes (Ikegwuonu et al., 1996).
Data from many experimental studies with mixtures of these
compounds are consistent with an additive model (Van den Berg
et al., 1998). As a result of this generally accepted additivity, the
toxic equivalency concept was developed. It uses the relative effect
potency (REP) determined for individual PCB compounds for pro-
ducing toxic or biological effects relative to a reference compound,
usually 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The total toxic
equivalent (TEQ) is operationally defined by the sum of the prod-
ucts of the concentration of each compound multiplied by its TEF
value and is an estimate of the total 2,3,7,8-TCDD-like activity of the
mixture (Van den Berg et al., 2006).
Commercial PCB preparations, such as Aroclor 1254, are
described as mixed-type inducers as they contain dioxin-like,
mono-ortho, and di-ortho congeners and thus can induce both
CYP1A and CYP2B enzymes in laboratory animals (Ngui and
Bandiera, 1999). When induced, CYP isozymes may serve as a source
of reactive oxygen radicals and/or may catalyze the oxidation of a
broad range of endogenous and exogenous substances, including
PCBs themselves (Twaroski et al., 2001c). The cytochrome P450-
catalyzed oxidation of lower chlorinated biphenyls gives rise to
mono- and dihydroxy metabolites. The latter can autoxidize or can
be enzymatically oxidized to semiquinones and/or quinones (Song
et al., 2008a,b). Some PCB-quinones can undergo redox cycling, with
the formation of ROS, thus becoming another source of oxidative
stress (McLean et al., 2000).
Oxidative stress develops when there is an imbalance between
prooxidants and antioxidants ratio, and as results it leads to the
accumulation of oxidative damage molecules (Sakac and Sakac,
2000). A change in AOE activities is frequently used as an indica-
tor of the ongoing oxidative stress in a particular tissue (Andric et
al., 2003). Tharappel et al. (2002) have evidenced that PCB induced
toxic manifestations, which may be associated with production of
reactive oxygen species (ROS), thereby it leads to oxidative stress. It
has been reported that PCBs are completely carcinogens and tumor
promoters in the liver. Induction of oxidative stress has been pro-
posed as a mechanism for induction of their promoting activity in
liver (Fadhel et al., 2000).
Mitochondria are an important intracellular source of ROS as
well as a sensitive target for oxidative damage under certain
pathological conditions such as exposure to PCB. Mitochondrial
membranes are rich in the tetraacyl phospholipid cardiolipin.
Its integrity is important for efficient oxidative phosphorylation.
The increase in ROS generation may be related to mitochondrial
dysfunction. Moreover, there is a lot of evidence supporting the
proposal that the impairment of mitochondrial function is a key
event in ROS-signaling through apoptotic pathways (Roue et al.,
2003; Kirkland and Franklin, 2003; Shih et al., 2004; Liu et al., 2004;
Lambert et al., 2004).
Since liver is considered as the major site for detoxification of
toxic metabolites, this organ is considered as the potential site for
the PCB-induced damage. Williams et al. (1993) reported that PCBs
caused degradative changes such as cytoplasmic loss and periph-
eralization of cytoplasm and organelles in goat, rabbit, and duck
livers. Several studies have shown that PCBs increase lipid peroxi-
dation in rat liver (Fadhel et al., 2002). Further, PCBs cause several
toxic effects in the liver and the mechanisms of toxicity have not
been determined (Tharappel et al., 2008).
Studies of environmental and toxic effects of PCBs are ideally
performed with PCB mixtures reflecting the composition of envi-
ronmental PCB profiles to mimic actual effects and to account for
complex interactions among individual PCB congeners. Congener
profiles of PCBs found in environmental samples are complex and
frequently do not reflect the composition of commercial PCB formu-
lations (Zhao et al., 2009). As it is difficult to prepare environmental
PCB mixture that contain a significant number of congeners, this
study was conducted using Aroclor 1254.
Although the PCBs display a wide range of biological and toxico-
logical properties, the potential cytotoxic effect of Aroclor 1254 on
rat hepatocytes and the mechanism underlines its cytotoxicity are
not fully investigated. Therefore, the present study was conducted
to investigate: (1) the potential cytotoxicity of Aroclor 1254 in rat
hepatocytes, and (2) characterization of the molecular mechanisms
involved in the Aroclor 1254-induced hepatotoxicity, particularly
the role of mitochondria, possibly a primary target in such event,
could greatly explain the cytotoxic effect of Aroclor 1254 in rat
hepatocytes.
2. Materials and methods
2.1. Reagents
Aroclor 1254 (lot 6024), a commercial mixture of PCBs, and reagents for cell
culture were purchased from Sigma–Aldrich Chemical Company, St. Louis, MO, USA.
Other reagents were of analytical grade.
2.2. Isolation and culture of rat hepatocytes
Hepatocytes were isolated from adult male albino rats using the in situ col-
lagenase perfusion method (Seglen, 1976). Cell viability was determined by the
trypan blue exclusion method, and exceeded 90%. The isolated rat hepatocytes were
cultured, at a density of 106 cells/cm2 in six-well plates, in DMEM media supple-
mented with 10% FBS, 2 mM l-glutamine, 0.1 U/ml insulin, 0.1 ␮M dexamethasone
and 100 ␮g/ml kanamycin sulphate for 24 h at 37 ◦ C in a humidified atmosphere of
95% relative humidity with 95% O2 and 5% CO2 . Then, the cells were incubated for
 H.A.A. Aly, Ò. Domènech / Toxicology 262 (2009) 175–183
24 h in a fresh media containing 0, 20, 30, 40, 50 or 60 ␮M of Aroclor 1254. At the
end of incubation, hepatocytes were recovered, sonicated in 0.1 M Tris–HCl buffer,
pH 7.4 and centrifuged for 15 min at 240 × g (4 ◦ C). The supernatant was used for the
enzymatic and non-enzymatic assays in hepatocytes after estimating the protein
content according to the method of Smith et al. (1985).
The Aroclor doses selected in the present study 0, 20, 30, 40, 50 or 60 ␮M were
based on the findings presented by Thome et al. (1995) indicating that Aroclor 1254
at concentrations exceeding 25 ␮M (but not at lower concentrations) caused irre-
versible damage to cultured rat hepatocytes. Moreover, it is important to note that all
of the previously published studies of the effects of Aroclor 1254 on mitochondrial
functions utilized concentrations in the 10–200 ␮M range, which yielded a variety
of effects (Salvi and Toninello, 2001).
2.3. Isolation of mitochondria from hepatocytes
Mitochondria were isolated by differential centrifugation according to the modi-
fied protocol published by Choy et al. (2008). Briefly, hepatocytes were homogenized
in isolation buffer (Tris–HCl 1 mM, EGTA 1 mM, sucrose 70 mM, mannitol 220 mM
and HEPES 2 mM) and centrifuged for 10 min at 460 × g. The supernatant was col-
lected and further centrifuged for 10 min at 12,500 × g, then was decanted. The
middle part of the pellet was collected to mix with the isolation buffer and cen-
trifuged for another 10 min at 12,500 × g. The pellets containing mitochondria were
suspended in ice storage buffer (sucrose 250 mM, KCl 10 mM, and Tris–HCl 10 mM).
The mitochondria were used as such for the mitochondrial assay. The protein values
of mitochondria were measured by the method of Smith et al. (1985).
2.4. Cell viability
2.4.1. MTT assay
Cell viability and activity of mitochondrial electron transport chain, as indicator
for cytotoxicity, was determined by the capacity of cells to reduce MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan (Bruggisser et
al., 2002). This assay is based on the ability of living cells to (reductively) convert
the dissolved MTT (yellow) into the insoluble formazan (blue) in the presence of
the mitochondrial enzymes succinate dehydrogenase. The latter can be measured
colorimetrically and is proportional to the metabolic activity of the cells. At the end
of treatment of cells with Aroclor 1254, 0.5% MTT was added for 4 h at 37 ◦ C. The
reaction was stopped with 100 ␮l sodium dodecyl sulfate 20% and absorption was
measured at 550 nm.
2.4.2. Lactate dehydrogenase (LDH) leakage assay
LDH activity in hepatocytes was measured as an index of plasma membrane
integrity (Farber and Young, 1981). At the end of treatment with Aroclor 1254, the
medium was removed and measured for LDH activity by recording the absorbance
change at 340 nm in an assay medium containing 1 mM pyruvate, 0.15 mM NADH
and 0.1 M sodium phosphate (pH 8.0). The percentage of LDH release was expressed
as the fraction of LDH released into the culture medium relative to the total amount
of LDH (cells + medium) activity after cell lysis with 0.1% (v/v) Triton-X 100 (Ye et al.,
2007).
2.5. Lipid peroxidation (LPO)
The assay for lipid peroxidation in hepatocytes was performed following the
method described previously (Iqbal and Athar, 1998), by measuring the rate of
production of thiobarturic acid-reactive substances (TBARS) (expressed as malon-
dialdehyde equivalents). The amount of malonaldehyde formed was assessed by
measuring the optical density at 535 nm at 37 ◦ C. The results were expressed as
nmol MDA formed/mg protein.
2.6. Mitochondrial membrane potential (␺m )
The uptake and retention of the cationic fluorescent dye, rhodamine 123, in hep-
atocytes has been used for the estimation of mitochondrial membrane potential.
This assay is based on the selective accumulation of rhodamine 123 in active mito-
chondria by charge-facilitated diffusion. At the end of treatment with Aroclor 1254,
cells were incubated in a fresh medium containing 1.5 ␮M rhodamine 123 at 37 ◦ C for
30 min. Adherent hepatocytes were washed twice with PBS, harvested by trypsiniza-
tion followed by another wash with PBS. Fluorescence intensity was assessed at
excitation wavelength of 490 nm and emission wavelength of 520 nm. The capac-
ity of mitochondria to take up the rhodamine 123 was calculated as fluorescence
intensity of rhodamine 123 (% of control) (Shangari and O’Brien, 2004).
2.7. Detection of mitochondrial reactive oxygen species (ROS)
The production of mitochondrial ROS was measured by using the oxidation-
sensitive fluorescent dye 2 ,7 -dichlorodihydrofluorescin diacetate (DCFH-DA). The
membrane-permeable diacetate form of the dye can be hydrolyzed by cellular non-
specific esterases that cleave the acetate groups on DCFH-DA, thus trapping the
reduced form of the probe (DCFH) within the cell. ROS (hydrogen peroxide, as well
as other peroxides) in the cells oxidize DCFH, yielding the fluorescent product of
177
2 ,7 -dichlorofluorescein (DCF) (Vanden Hoek et al., 1997; Duranteau et al., 1998).
An increase in fluorescence intensity is used to quantify the generation of net mito-
chondrial ROS. At the end of treatment with Aroclor 1254, the mitochondria were
preloaded with 100 ␮M DCFH-DA dissolved in methanol and incubated for 30 min
at 37 ◦ C. The medium with the DCFH-DA was then removed, and the mitochondria
were washed, resuspended in ice-cold PBS. The formation of the fluorescent oxi-
dized derivative of DCFH, namely DCF, was measured using a spectrofluorimeter
with excitation wavelength at 485 nm and emission wavelength at 535 nm. ROS for-
mation was quantified from a DCF-standard curve and data were expressed as pmol
DCF formed/min/mg protein.
2.8. EROD and PROD assays
Mitochondrial ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-
deethylase (PROD) activities are commonly used as enzymatic markers for hepatic
CYP1A and CYP2B enzymes (Burke et al., 1994). The activity of these enzymes was
determined spectrofluorimetrically as described previously (Burke et al., 1985),
with some modifications. The reaction mixture contained 0.05 M Tris–HCl (pH
7.4), 2.6 ␮M ethoxyresorufin (EROD) or 9.6 ␮M pentoxyresorufin (PROD), NADPH-
regenerative system (1 mM NADP+ , 1 U/ml glucose-6-phosphate dehydrogenase,
4.4 mM glucose-6-phosphate, 5 mM MgCl2 , 1 mg BSA) and 50 ␮l of mitochondrial
extract in a total volume of 1 ml. The samples were incubated for 5 min at 37 ◦ C for
measurement of the EROD activity, and 25 min for measurement of the PROD activ-
ity. The reaction was initiated by adding 0.5 ml of NADPH-regenerative system and
terminated by adding 2 ml of methanol. After incubation, tubes were centrifuged
for 10 min at 10,000 × g to pellet the flocculated proteins. The supernatant was then
measured for fluorescence with excitation and emission wavelengths set at 544
and 590 nm, respectively. The same standard curve was used for both assays and
was constructed by measuring the fluorescence of resorufin at various concentra-
tions (0–70 nM). The enzymes activities for EROD and PROD were expressed as pmol
resorufin/min/mg protein.
2.9. Antioxidant enzyme assays
Glutathione reductase (GR) activity was measured according to the procedure
of Hsiao et al. (2001). Briefly, 10 ␮l of mitochondrial extract was added to a reac-
tion mixture (containing 0.99 ml of 100 mM potassium phosphate buffer pH 7.0,
1.1 mM MgCl2 , 5 mM GSSG and 0.1 mM NADPH) to trigger the NADPH conversion
reaction. Changes in absorbance were monitored by a continuous-recording spec-
trophotometer at 340 nm for 5 min at 25 ◦ C. The GR activity was expressed as nM
NADPH oxidized/min/mg protein. Glutathione peroxidase (GPx) activity was deter-
mined by the method of Paglia and Valentine (1967). The reaction mixture consisted
of 0.8 ml of 100 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM NaN3 ,
0.2 mM NADPH, 1 U/ml GR and 1 mM GSH. Fifty microliters of mitochondrial extract
were added to make a total volume of 0.9 ml. The reaction was started by the addi-
tion of 100 ␮l of 2.5 mM H2 O2 , and the conversion of NADPH to NADP was monitored
by a continuous recording spectrophotometer at 340 nm for 3 min. GPx activity was
expressed as nM NADPH oxidized/min/mg protein.
2.10. Glutathione (GSH) assay
GSH (gamma-l-glutamyl-l-cysteinylglycine) content was determined by a flu-
orimetric assay using o-phthalaldehyde (Hissin and Hilf, 1976). The reaction
mixture containing 0.1 M sodium phosphate buffer (pH 8.0), 5.0 mM EDTA, 10 ␮l o-
phthaldehyde (1.0 mg/ml) and 50 ␮l of mitochondrial protein. After incubation for
15 min at room temperature, the fluorescence intensity was recorded with excitation
and emission wavelengths of 420 and 350 nm, respectively. The reduced glutathione
content was expressed as nmol GSH/mg protein.
2.11. Aconitase activity
Aconitase activity was assessed in the mitochondrial fraction by measuring the
conversion of citrate to isocitrate, and subsequent production of ␣-ketoglutarate
was determined spectrophotometrically at 340 nm. The reaction mixture contained
50 mM Tris–Cl buffer (pH 7.4) containing 30 mM citrate, 0.2 mM NADP+ , 0.6 mM
MnCl2 , 1–2 unit(s) of isocitrate dehydrogenase and 50 ␮l of mitochondrial extract
in a total volume of 1 ml. The reaction was carried out at 25 ◦ C for 1 h. One unit of
aconitase activity is equivalent to the production of 1 ␮mol of isocitrate per min
(Chung et al., 2001).
2.12. Mitochondrial ˇ-oxidation
The ␤-oxidation of [1-14 C]palmitic acid by hepatocyte mitochondria was
assessed as described by Fréneaux et al. (1988) with some modifications. The
preincubation medium (900 ␮l of 70 mmol/l sucrose, 43 mmol/l KCl, 3.6 mmol/l
MgCl2 , 7.2 mmol/l potassium phosphate and 36 mmol/l Tris–HCl, pH 7.4) contained
0.2 mmol/l ATP, 50 ␮mol/l l-carnitine, 15 ␮mol/l coenzyme A, 50 ␮l mitochondrial
protein and 5 mmol/l acetoacetate. After 5 min of preincubation at 30 ◦ C the incuba-
tion mixture was brought to 1 ml by adding 100 ␮l of preincubation buffer containing
[1-14 C]palmitic acid (final concentration 40 ␮mol/l; 0.05 ␮Ci/ml). The incubations
178 H.A.A. Aly, Ò. Domènech / Toxicology 262 (2009) 175–183

were carried out at 30 ◦ C for 15 min with slow shaking. It was previously shown that
the reaction is in the linear phase at this time point (Spaniol et al., 2001). The reaction
was stopped by adding 200 ␮l of 20% perchloric acid (w/v) and by placing the tubes
on ice for 20 min. After centrifugation for 5 min at 12,000 × g, the 14 C-acid-soluble
products were counted in the supernatant. Such products represent mainly ketone
bodies and citric acid cycle intermediates (Morse et al., 1988).

2.13. Cardiolipin content

Fifty microliters of mitochondrial protein was incubated at 30 ◦ C for 45 min in

the presence of 10-N-nonyl-acridine orange 5 ␮M (NAO), in a medium containing
160 mM KCl and 10 mM Hepes–KOH, pH 7.4. The excess dye was washed out by cen-
trifugation and the mitochondrial pellet appropriately diluted in the same buffer.
Fluorescence was determined in a spectrofluorimeter operating at 485 nm (excita-
tion) and 535 nm (emission). Fluorescence was converted to relative fluorescence
units using quinine (1 mg ml−1 in 0.1N H2 SO4 , ex 360 nm, em 457 nm) as reference Fig. 1. Dose–response effect of Aroclor 1254 on CYP1A (EROD) and CYP2B (PROD)
(Petit et al., 1992; Gallet et al., 1995). in rat hepatocyte mitochondria. Values are mean ± SD (n = 3). Statistical anal-
ysis (ANOVA) for differences from corresponding controls: *p < 0.05; **p < 0.01;
2.14. Respiratory chain enzyme activities ***p < 0.001.

In order to determine the effect of Aroclor 1254 on complexes I and III more
precisely, these enzyme complexes were investigated individually in the hepatocyte
mitochondria. 3.2. Lipid peroxidation, mitochondrial membrane potential
(␺m ) and ROS generation
2.14.1. Complex I
The complex I (NADH–CoQ reductase) activity was measured in mitochondrial Table 2 demonstrates that, Aroclor 1254 significantly increased
particles obtained by three times of freezing and thawing of heptocyte mitochondria
dissolved in 50 mM phosphate buffer pH 7.2 as previously described (Petrosillo et al.,
lipid peroxidation, secondary products, MDA (malondialdehyde) in
2007). The assay mixture contained 3 mM sodium azide, 1.2 ␮M antimycin A, 50 ␮M hepatocytes in a dose-related manner as compared to correspond-
decylubiquinone and 50 mM phosphate buffer pH 7.2. The mitochondrial extract ing control while ␺m was significantly decreased in the same
(50 ␮l) was added to 3 ml of the assay mixture and the reaction was started by the manner as compared to untreated cells (Table 2). Aroclor 1254 sig-
addition of 60 ␮M NADH. The reaction was measured by following the decrease in
nificantly increased ROS generation in hepatocyte mitochondria
absorbance of NADH at 340 nm in a spectrophotometer. The specific activity of the
enzyme is expressed as nmol of NADH oxidized/min/mg of mitochondrial protein. in a dose-related manner as compared to corresponding control
(Table 2).
2.14.2. Complex III
Succinate cytochrome c reductase activity was measured spectrophotometri-
cally (Tisdale, 1967) at 25 ◦ C by following the reduction of oxidized cytochrome c 3.3. EROD and PROD
(electron acceptor) by the increase in absorbance at 550 nm. The reaction was ini-
tiated by the addition of 5 mM succinate to 3 ml of the standard reaction mixture, Mitochondrial EROD and PROD were measured in the current
as previously described, supplemented with 2 mM rotenone, 1 mM KCN, 54 mM of
study to determine the effect of A1254 on this CYP subfamily.
cytochrome c (III), and 50 ␮l of mitochondrial protein.
Aroclor 1254 (60 ␮M) increased significantly EROD (fivefold) and
PROD (fourfold) activities in comparison to corresponding controls
3. Results
(Fig. 1). The increase in the activity of these two enzymes was in a
dose-related pattern. These results suggest that the Ahr (CYP1A) is
3.1. Characterization of Aroclor 1254 toxicity
critical for Aroclor-induced mitochondrial superoxide production
in hepatocytes. In addition, CYP2B may be a target for Aroclor 1254
To characterize Aroclor 1254 toxicity, we evaluated the effect of
toxicity in hepatocyte mitochondria.
increasing concentrations of Aroclor 1254 on the viability of hep-
atocytes and LDH content. Table 1 shows that hepatocytes treated
with 20 ␮M of Aroclor 1254 for 24 h did not show any significant 3.4. Antioxidant status in hepatocyte mitochondria
effect on either cell viability or LDH release into medium. Starting
at 30 ␮M for 24 h, Aroclor 1254 caused a significant decrease in cell Aroclor 1254-significantly decreased the enzymatic antioxidant
survival in a dose-related manner. On the other hand LDH released GR and GPx activities and the non-enzymatic antioxidant GSH con-
into medium was significantly increased in the same pattern in tent of hepatocyte mitochondria as compared to corresponding
comparison to related controls. controls (Table 3).

Table 1
Dose–response effect of Aroclor 1254 on cell viability and LDH release.

Control 20 ␮M 30 ␮M 40 ␮M 50 ␮M 60 ␮M

Cell viability 93.67 ± 1.53 90.33 ± 2.52 85.67 ± 1.53* 83 ± 2** 79 ± 2.65*** 75.67 ± 4.93***
% of LDH released 27.33 ± 2.52 30.67 ± 3.06 36 ± 1* 38.67 ± 3.21** 42.33 ± 4.16*** 51.67 ± 2.52***

Values are mean ± SD (n = 3). Statistical analysis (ANOVA) for differences from corresponding controls: * p < 0.05; ** p < 0.01; *** p < 0.001.

Table 2
Dose–response effect of Aroclor 1254 on lipid peroxidation, reactive oxygen species production and mitochondrial membrane potential (␺m ) in rat hepatocytes.

Control 20 ␮M 30 ␮M 40 ␮M 50 ␮M 60 ␮M

Lipid peroxidation 1.63 ± 0.23 2.37 ± 0.15* 2.6 ± 0.1** 2.8 ± 0.2*** 3.57 ± 0.21*** 4.37 ± 0.32***
␺m (% of control) 100 ± 9.61 78.45 ± 4.61* 70.67 ± 5.33** 59.99 ± 4.61*** 49.22 ± 7.05*** 38.46 ± 7.05***
ROS generation 3.2 ± 0.2 3.97 ± 0.21* 4.23 ± 0.21** 6.23 ± 0.21*** 8.37 ± 0.25*** 10.37 ± 0.32***

Lipid peroxidation: nmol of MDA equivalent formed/mg protein; ␺m : fluorescence intensity of rhodamine 123 (% of control); ROS: pmol DCF formed/min/mg protein. Values
are mean ± SD (n = 3). Statistical analysis (ANOVA) for differences from corresponding controls: * p < 0.05; ** p < 0.01; *** p < 0.001.
 H.A.A. Aly, Ò. Domènech / Toxicology 262 (2009) 175–183 179

Table 3
Dose–response effect of Aroclor 1254 on ROS generation and antioxidant status of rat hepatocyte mitochondrial.

Control 20 ␮M 30 ␮M 40 ␮M 50 ␮M 60 ␮M

GPx 30.67 ± 2.08 24 ± 1*

22 ± 2.65 **
20.53 ± 2.84 **
17 ± 2***
14.67 ± 2.52***
GR 49.67 ± 2.08 42 ± 1.73* 40 ± 3* 36.93 ± 3** 33.83 ± 3.75*** 32.33 ± 2.52***
GSH 42 ± 2 34.67 ± 1.53* 32.33 ± 2.08** 28.67 ± 3.06*** 24.67 ± 2.52*** 21.17 ± 2.93***

GPx and GR: nmol NADPH oxidized/min/mg protein; GSH: nmol GSH/mg protein. Values are mean ± SD (n = 3). Statistical analysis (ANOVA) for differences from corresponding
controls: * p < 0.05; ** p < 0.01; *** p < 0.001.

Table 4
Dose–response effect of Aroclor 1254 on aconitase, ␤-oxidation and cardiolipin content in mitochondria of rat hepatocytes.

Control 20 ␮M 30 ␮M 40 ␮M 50 ␮M 60 ␮M

Aconitase 7.93 ± 0.4 6.7 ± 0.3*

5.97 ± 0.55 **
5 ± 0.03 ***
4.4 ± 0.46 ***
4 ± 0.5***
␤-Oxidation 0.68 ± 0.03 0.57 ± 0.07* 0.53 ± 0.04** 0.50 ± 0.03** 0.46 ± 0.02*** 0.41 ± 0.03***
Cardiolipin 34.33 ± 4.04 27 ± 1* 25 ± 2** 18.33 ± 1.53*** 15.33 ± 3.06*** 9.67 ± 1.53***

Aconitase: mU/mg protein; ␤-oxidation: [14 C] ␤-oxidation products (nmol/min/mg protein); cardiolipin: nmol/mg protein. Values are mean ± SD (n = 3). Statistical analysis
(ANOVA) for differences from corresponding controls: * p < 0.05; ** p < 0.01; *** p < 0.001.

Table 5
Dose–response effect of Aroclor 1254 on mitochondrial respiratory chain complexes I and III of rat hepatocytes.

Control 20 ␮M 30 ␮M 40 ␮M 50 ␮M 60 ␮M

Complex I 69 ± 3.61 57.33 ± 2.52** 48 ± 2*** 36.67 ± 4.16*** 26.67 ± 3.51*** 20.67 ± 3.06***
Complex III 248.38 ± 12.58 211.67 ± 7.64* 193 ± 12.12** 180.67 ± 11.02*** 165.67 ± 14.36*** 134 ± 14.42***

Complex I: nmol of NADH oxidized/min/mg protein; complex III: nmol of cytochrome c reduced/min/mg protein. Values are mean ± SD (n = 3). Statistical analysis (ANOVA)
for differences from corresponding controls: * p < 0.05; ** p < 0.01; *** p < 0.001.

3.5. Aconitase, ˇ-oxidation and cardiolipin content of hepatocyte
mitochondria
In order to evaluate the role of Aroclor 1254 in generating a
mitochondrial oxidative stress, we examined mitochondrial aconi-
tase activity, an enzyme that is inactivated by superoxide. Aconitase
inactivation has been suggested as a reliable marker for mitochon-
drial ROS production (Hausladen and Fridovich, 1994, 1996). Aroclor
1254-treated hepatocytes significantly decreased mitochondrial
aconitase activity in a dose-related manner as compared to cor-
responding control (Table 4). Aroclor 1254 impaired mitochondrial
formation of acid-soluble products, which reflects ␤-oxidation. The
content of the mitochondrial lipid cardiolipin was significantly
decreased in a dose-related pattern, probably due to oxidation pro-
cess (Table 4).
3.6. Mitochondrial respiratory chain complexes I and III
Complex I transfers electrons to complexes III and IV. Aroclor
1254 significantly inhibited state 3 oxidation, complexes I and III,
reflecting impairment of the function of the electron transport
chain and uncouple oxidative phosphorylation (Table 5).
4. Discussion
In the present study, Aroclor 1254, starting at 30 ␮M, seri-
ously damaged cell membrane integrity, decreased cell viability
and significantly increased LDH release. These morphological and
biochemical changes in hepatocytes manifested clear evidence
of the toxicity of Aroclor 1254 on hepatocytes. As mitochon-
dria are often a target of toxicity, an MTT test was performed
to assess cytotoxicity and therefore cell viability, which mainly
reflects the mitochondrial reductive activity (Bruggisser et al.,
2002).
Since reduction of MTT is dependent on the activity of the mito-
chondrial electron transport chain, impaired formazan production
from MTT is usually interpreted as indicative for mitochondrial
damage. To determine whether the reduced production of formazan
was indeed due to the impairment of mitochondrial function by
Aroclor 1254, the mitochondrial membrane potential was mea-
sured using the fluorescent dye rhodamine 123. The increase in
supernatant LDH activity directly correlates to the reduced amount
of formazan formed in MTT assay. These findings are in line with
Thome et al. (1995) who reported that Aroclor 1254 at concentra-
tions exceeding 25 ␮M causes irreversible damage to cultured rat
hepatocytes.
Several studies had suggested that increased cellular oxidative
stress be a critical underlying mechanism of PCB-mediated cell acti-
vation and dysfunction (Mariussen et al., 2002; Twaroski et al.,
2001c). Therefore, this toxic effect of Aroclor 1254 on hepatocytes
may be due to the increased lipid peroxidation, as shown in this
study, which was based on the critical role played by lipids in main-
taining membrane structure and function, and ultimately cellular
viability, and ROS production, as also shown in this study, which in
turn induces oxidative stress (Sridhar et al., 2004; Banudevi et al.,
2005). The increased lipid peroxidation may be due to increased
ROS or decreased redox status. These findings come in agreement
with Aly et al. (2009).
In addition to the cytotoxicity study on hepatocytes, the toxic
effect of Aroclor 1254 was investigated on isolated rat hepatocyte
mitochondria in order to characterize the molecular mechanisms
involved in the hepatotoxicity, focusing on the role played by the
mitochondria, a possible early target in this process.
Mitochondria are the main cellular source and target of ROS.
On the other hand, mitochondria possess an antioxidant defense
system that, under physiological conditions, decreases ROS pro-
duction or removes generated ROS (Czarna and Jarmuszkiewicz,
2006). Among, GPx, GR and reduced glutathione GSH are key com-
ponents of this mitochondrial antioxidant defense system (Droge,
2002).
ROS have been implicated in the pathogenesis of several liver
diseases (Petrosillo et al., 2007). ROS can alter vital cell compo-
nents like polyunsaturated fatty acids, proteins and nucleic acids.
To a lesser extent, carbohydrates are also the targets of ROS. These
reactions can alter intrinsic membrane properties like fluidity, ion
transport, loss of enzyme activity, inhibition of protein synthe-
180 H.A.A. Aly, Ò. Domènech / Toxicology 262 (2009) 175–183
sis, DNA damage, ultimately resulting in cell death (Halliwell and
Gutteridge, 1990). Oxidative stress develops when there is an imbal-
ance between prooxidants and antioxidants ratio, and as results it
leads to the accumulation of oxidative damaged molecules (Sakac
and Sakac, 2000).
The involvement of ROS in Aroclor 1254-induced toxicity was
assayed using the fluorescence probe DCFH-DA. This is a sensitive
and rapid method, which is well suited for detecting overall oxida-
tive stress after stimulation with ROS-forming compounds in low
concentrations (Myhre et al., 2000).
In the present study, ROS was shown to be induced by Aro-
clor 1254. These results come in accordance with Schilderman et
al. (2000), Banudevi et al. (2006) and Venkataraman et al. (2008)
who reported that Aroclor 1254 induced ROS production which can
damage the cellular elements in its target organs such as rat liver,
spleen, brain, seminal vesicle, prostate and epididymis. Moreover,
Aly et al. (2009) demonstrated that Aroclor 1254 induced ROS in rat
testicular mitochondria and impaired spermatogenesis. Although
mitochondria are likely the major site of ROS generation (James et
al., 2005; Talbot et al., 2004) in most cells, little is known about ROS
production within hepatocyte mitochondria. Here we report new
information concerning the mechanism leading to hepatocyte ROS
production at the mitochondrial level.
Respiratory chain complex I transfers the electrons to com-
plexes III and IV. Our study shows that, Aroclor 1254 inhibited
mitochondrial respiratory chain complexes I and III. This inhi-
bition of activity reflects mitochondrial impairment. Damaged
mitochondrial respiratory chain, as shown in the present study,
is considered an important source of ROS (Perez-Carreras et al.,
2003). It is conceivable that mitochondrial-mediated ROS gener-
ation leads to primary reactions and damages in the immediate
area surrounding where these ROS are produced, given that they
are a highly reactive and short-lived species. Therefore, as a major
source of ROS production, mitochondria also could be the major
target of ROS attack. This effect of ROS should be greatest at the
level of mitochondrial membrane constituents including the com-
plexes of the respiratory chain and phospholipid constituents rich
in unsaturated fatty acids, particularly cardiolipin (Martins et al.,
2008).
Beside the effect on the respiratory chain, ␤-oxidation of fatty
acids represents another target of mitochondrial toxicity. In the
present study, Aroclor 1254 inhibited mitochondrial ␤-oxidation.
Since the activity of the electron transport chain can be rate-limiting
for mitochondrial fatty acid metabolism (Latipää et al., 1986), the
inhibition of ␤-oxidation may be secondary to impaired function of
the electron transport chain by Aroclor 1254.
The findings of the current study so far have shown that
Aroclor 1254 inhibited the respiratory chain and mitochondrial
␤-oxidation, can lead to a decrease in the ␺m . Our data clearly
show that Aroclor 1254 caused mitochondrial dysfunction, char-
acterized by decreased ␺m which may subsequently depress
cellular viability and leading to cell death. Conditions favoring
increased mitochondrial generation of reactive oxygen species (e.g.,
the presence of high Ca2+ concentrations or when the mitochondrial
antioxidant defense mechanisms are compromised) may induce the
mitochondrial pore transition (MPT) and death of the cell (Hoek et
al., 1995). The consequence of ␺m disruption is the uncoupling
of the oxidative phosphorylation and the generation of superoxide
anion via the uncoupled respiratory chain (Wilson and Prochaska,
1990).
Another proposed mechanism for ROS generation and induction
of lipid peroxidation by Aroclor 1254 is the induction of cytochrome
P450 enzymes, with the subsequent release of ROS (Schlezinger et
al., 1999). We focused in this study to determine whether the CYP1A
and CYP2B are determinants in the pathway leading to Aroclor
1254-induced toxicity in the hepatocyte mitochondria. The CYP1A1
and CYP1A2 genes are controlled by the Ahr; consequently, follow-
ing dioxin exposure there is a robust accumulation of CYP1A1 and
CYP1A2 in mitochondria of mouse liver (Senft et al., 2002). Such
findings corroborate with our results as we had seen the elevated
levels of total CYP1A and CYP2B activities as indicated by EROD and
PROD, respectively. Moreover, Chu et al. (2008) demonstrated that
Aroclor 1254 induced microsomal enzyme activities in rat liver.
The cytochrome P450 monooxygenases sequentially transfer
two electrons to oxygen from mitochondrial adrenodoxin, with sub-
sequent formation of oxygenated substrate and water (Poulos and
Raag, 1992). Although electron transfer is normally a well-coupled
process, superoxide and H2 O2 may be released, particularly in the
presence of CYP1A1 ligands that are poorly metabolized. Burqin
et al. (2001) reported that Aroclor 1254 induced hepatic ethoxy-,
methoxy-, and pentoxyresorufin O-deethylase (EROD, MROD, and
PROD, respectively) activities in rat liver. Recent study demon-
strated that PCB-induced oxidative stress in rat kidney might be
attributable to induction of CYP1A1 (Lu et al., 2009). Further,
Bonfanti et al. (2009) reported that PCB induced hepatic CYP450
(total CYP450, CYP1A, CYP1B). Therefore, it is reasonable to sup-
pose that CYP-system may be implicated in Aroclor 1254 triggered
ROS production in hepatocyte mitochondria. The increase in ROS
generation would lead to irreversible damage to mitochondrial
membrane lipids and proteins resulting in mitochondrial dys-
function and ultimately in cell death (Kowaltowski and Vercesi,
1999).
The decreased levels of cardiolipin in the Aroclor 1254-treated
hepatocytes, as shown in this study, might also have accounted for
the alteration of the mitochondrial membrane lipid order, as car-
diolipin is a mitochondrial anionic phospholipid known to confer
stability and fluidity to the mitochondrial membrane (Fariss et al.,
2005). Moreover, cardiolipin also functions as a support for the elec-
tron transport chain, since it anchors complexes III and IV, forming
a supercomplex (Hauff and Hatch, 2006). Furthermore, cardiolipin
has been shown to be specifically required for the optimal function-
ing of mitochondrial complex I (Sharpley et al., 2006). Therefore, our
findings are in accordance with this proposition, since the oxida-
tion of cardiolipin could have caused disruption of the respiratory
chain, observed in this study. Further our result concerning cardi-
olipin corroborate with Zhou and Zhang (2005) who reported that
A1254 interfered with the integrity of cell membrane and caused
hepatocyte damage. Thus, it is reasonable that ROS-induced oxida-
tive damage to mitochondrial cardiolipin may be responsible, at
least in part, for the observed defect in complex I activity in Aroclor
1254-treated hepatocytes.
Since inhibition of the respiratory chain and cytochrome P450
can be associated with the generation of ROS (Kaufmann et al.,
2005), the redox status of the mitochondria of Aroclor 1254-treated
hepatocyte was assessed by measuring GR and GPx activities and
the reduced glutathione content.
Our results showed that in Aroclor 1254-treated hepatocytes, GR
and GPx activities in the hepatocyte mitochondria were decreased.
The decreased activities of GR and GPx may be due to excessive ROS
production in the mitochondria as shown in this study. Meanwhile,
the decreased activity in GR and GPx may be responsible for the ele-
vated lipid peroxidation. This study also showed that Aroclor 1254
(≥30 ␮M)-treated hepatocytes resulted in a significant decrease of
GSH content in mitochondria, which indicated that damage of cellu-
lar antioxidant systems was induced. Castell et al. (1997) suggested
that GSH depletion might ultimately lead to cell death by impairing
the cell defense against oxidative damage.
GSH is an important cellular antioxidant that plays a major role
in protecting cells against oxidative stress and involves in antiox-
idant defense of hepatocytes. GSH may also serve as a cofactor
for (a) several drug-metabolizing enzymes (i.e. GSTs) where it is
consumed, or (b) for antioxidant enzymes (i.e. GPX) where it func-
 H.A.A. Aly, Ò. Domènech / Toxicology 262 (2009) 175–183
tions as a redox partner (Uhlig and Wendel, 1992). Mitochondrial
GSH detoxifies free oxygen radicals physiologically generated by
the respiratory chain (Droge, 2002). During oxidation, GSH forms a
dimer, glutathione disulfide (GSSG), which, in turn, can be reduced
by the enzyme GR at the expense of NADPH (Bray and Taylor, 1993).
The decrease in GR and GPx activities and GSH levels in mitochon-
dria may possibly contribute to the Aroclor 1254-induced toxicity.
Enhanced generation of ROS and/or impaired antioxidant detoxifi-
cation functions contribute to an imbalance between oxidative and
reductive reactions, which alters thiol/disulfide redox (Moriarty-
Craige and Jones, 2004).
Mitochondria are the organelles of eukaryotic cells for carbohy-
drate metabolism, and mitochondrial oxidative phosphorylation is
the major energy transduction pathway. Aconitase activity, which
represents mitochondrial function, was measured by the turnover
of citrate to isocitrate as an index of cellular energy utilization. The
activity of aconitase enzyme was monitored in mitochondria. This
enzyme can be used to estimate indirectly the superoxide concen-
tration, since its activity is very sensitive to superoxide exposure
and its inactivation, as shown in this study, is a reliable marker for
mitochondrial superoxide production (Gardner, 2002; James et al.,
2005).
In summary, our results showed that Aroclor 1254 caused
hepatotoxicity by induction of oxidative stress in hepatocyte mito-
chondria, by promoting mitochondrial structural and functional
damage, inhibition of mitochondrial respiratory complexes I and
III and ␤-oxidation of fatty acids, reduction in ␺m , depletion of
antioxidant defense system with consequent redox status alter-
ation, decrease in mitochondrial cardiolipin and aconitase content,
increase in mitochondrial EROD and PROD activity and cellular
death. Therefore, we can conclude that Aroclor 1254 induced rat
hepatocyte toxicity and our findings provide evidence to propose
that mitochondria are one of the most important and earliest cell
targets in Aroclor 1254-mediated toxicity and delineate several
mitochondrial processes at least, in part, by induction of oxidative
stress. These findings can be useful in future cytoprotective ther-
apy approaches. Since mitochondrial events appear to be targeted
in hepatocellular damage induced by Aroclor 1254, an antioxidant
therapy targeted to mitochondria may constitute an interesting
strategy to ameliorate its toxicity.
Conflict of interest
None declared.
References
Adebusoye, S.A., Ilori, M.O., Picardal, F.W., Amund, O.O., 2008. Extensive biodegra-
dation of polychlorinated biphenyls in Aroclor 1242 and electrical transformer
fluid (Askarel) by natural strains of microorganisms indigenous to contaminated
African systems. Chemosphere 73, 126–132.
Aly, H.A.A., Domenech, O., Abdel-Naim, A.B., 2009. Aroclor 1254 impairs spermato-
genesis and induces oxidative stress in rat testicular mitochondria. Food Chem.
Toxicol., Mar 21.
Andric, N.L., Andric, S.A., Zoric, S.N., Kostic, T.S., Stojilkovic, S.S., Kovacevic, R.Z.,
2003. Parallelism and dissociation in the actions of an Aroclor 1260-based trans-
former fluid on testicular androgenesis and antioxidant enzymes. Toxicology
194, 65–75.
Baird, L., 1995. Toxic organic chemicals. In: Environmental Chemistry. WH Freeman
and Company, New York, pp. 252–274.
Banudevi, S., Arunkumar, A., Sharmila, M., Senthilkumar, J., Balasubramanian, K.,
Srinivasan, N., Aruldhas, M.M., Arunakaran, J., 2005. Diallyl disulfide-induced
modulation of a few phase I and II drug metabolizing enzymes on Aroclor 1254
toxicity in Rattus norvegicus liver and ventral prostate. J. Clin. Biochem. Nutr. 36,
59–65.
Banudevi, S., Krishnamoorthy, G., Venkataraman, P., Vignesh, C., Aruldhas, M.M.,
Arunakaran, J., 2006. Role of alpha-tocopherol on antioxidant status in liver,
lung and kidney of PCB exposed male albino rats. Food Chem. Toxicol. 44,
2040–2046.
Bonfanti, P., Colombo, A., Villa, S., Comelli, F., Costa, B., Santagostino, A., 2009. The
effects of accumulation of an environmentally relevant polychlorinated biphenyl
181
mixture on cytochrome P450 and P-glycoprotein expressions in fetuses and
pregnant rats. Chemosphere 75, 572–579.
Bray, T.M., Taylor, C.G., 1993. Tissue glutathione, nutrition, and oxidative stress. Can.
J. Physiol. Pharmacol. 71, 746–751.
Bruggisser, R., von Daeniken, K., Jundt, G., Schaffner, W., Tullberg-Reinert, H., 2002.
Interference of plant extracts, phytoestrogens and antioxidants with the MTT
tetrazolium assay. Planta Med. 68, 445–448.
Burke, M.D., Thompson, S., Elcombe, C.R., Halpert, J., Haaparanta, T., Mayer, R.T., 1985.
Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of sub-
strates to distinguish between different induced cytochromes P-450. Biochem.
Pharmacol. 34, 3337–3345.
Burke, M.D., Thompson, S., Weaver, R.J., Wolf, C.R., Mayer, R.T., 1994. Cytochrome
P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver.
Biochem. Pharmacol. 48, 923–936.
Burqin, D.E., Diliberto, J.J., Derr-Yellin, E.C., Kannan, N., Kodavanti, P.R., Birnbaum, L.S.,
2001. Differential effects of two lots of aroclor 1254 on enzyme induction, thyroid
hormones, and oxidative stress. Environ. Health Perspect. 109, 1163–1168.
Castell, J.V., Gomez-Lechon, M.J., Ponsoda, X., Bort, R., 1997. In vitro investigation of
the molecular mechanisms of hepatotoxicity. In: Castell, J.V., Gomez-Lechon, M.J.
(Eds.), In Vitro Methods in Pharmaceutical Research. Academic Press, London, pp.
375–410.
Choy, C.S., Cheah, K.P., Chiou, H.Y., Li, J.S., Liu, Y.H., Yong, S.F., Chiu, W.T., Liao, J.W.,
Hu, C.M., 2008. Induction of hepatotoxicity by sanguinarine is associated with
oxidation of protein thiols and disturbance of mitochondrial respiration. J. Appl.
Toxicol. 28, 945–956.
Chu, I., Bowers, W.J., Caldwell, D., Nakai, J., Wade, M.G., Yagminas, A., Li, N., Moir, D., El
Abbas, L., Håkansson, H., Gill, S., Mueller, R., Pulido, O., 2008. Toxicological effects
of in utero and lactational exposure of rats to a mixture of environmental con-
taminants detected in Canadian Arctic human populations. J. Toxicol. Environ.
Health A 71, 93–108.
Chung, H.C., Kim, S.H., Lee, M.G., Cho, C.K., Kim, T.H., Lee, D.H., Kim, S.G., 2001.
Mitochondrial dysfunction by gamma-irradiation accompanies the induction of
cytochrome P450 2E1 (CYP2E1) in rat liver. Toxicology 161, 79–91.
Czarna, M., Jarmuszkiewicz, W., 2006. Role of mitochondria in reactive oxygen
species generation and removal, relevance to signaling and programmed cell
death. Postepy. Biochem. 52, 145–156.
Denomme, M.A., Bandiera, S., Lambert, I., Copp, L., Safe, L., Safe, S., 1983. Polychlo-
rinated biphenyls as phenobarbitone-type inducers of microsomal enzymes.
Structure–activity relationships for a series of 2,4-dichloro-substituted con-
geners. Biochem. Pharmacol. 32, 2955–2963.
Droge, W., 2002. Free radicals in the physiological control of cell function. Physiol.
Rev. 82, 47–95.
Duranteau, J., Chandel, N.S., Kulisz, A., Shao, Z., Schumacker, P.T., 1998. Intracellular
signaling by reactive oxygen species during hypoxia in cardiomyocytes. J. Biol.
Chem. 273, 11619–11624.
Fadhel, Z.A., Lu, Z., Robertson, L.W., Glauert, H.P., 2000. Role of cytochrome P450
induction and lipid peroxidation in 3,3 ,4,4 -tetrachlorobiphenyl (PCB-77) and
2,2 ,4,4 ,5,5 -hexachlorobiphenyl (PCB-153)-induced toxicity in liver of male
rats. Toxicol. Sci. 54, 78.
Fadhel, Z., Lu, Z., Robertson, L.W., Glauert, H.P., 2002. Effect of 3,3 ,4,4 -
tetrachlorobiphenyl and 2,2 ,4,4 ,5,5 -hexachlorobiphenyl on the induction of
hepatic lipid peroxidation and cytochrome P-450 associated enzyme activities
in rats. Toxicology 175, 15–25.
Farber, J.L., Young, E.E., 1981. Accelerated phospholipid degradation in anoxic rat
hepatocytes. Arch. Biochem. Biophys. 211, 312–320.
Fariss, M.W., Chan, C.B., Patel, M., Van Houten, B., 2005. Role of mitochondria in toxic
oxidative stress. Mol. Interv. 5, 94–111.
Fréneaux, E., Labbe, G., Letteron, P., The Le Dinh, Degott, C., Genève, J., Larrey, D.,
Pessayre, D., 1988. Inhibition of the mitochondrial oxidation of fatty acids by
tetracycline in mice and in man: possible role in microvesicular steatosis induced
by this antibiotic. Hepatology 8, 1056–1062.
Gallet, P.F., Maftah, A., Petit, J.M., Denis-Gay, M., Julien, R., 1995. Direct cardiolipin
assay in yeast using the red fluorescence emission of 10-N-nonyl acridine orange.
Eur. J. Biochem. 15, 113–119.
Gardner, P.R., 2002. Aconitase: sensitive target and measure of superoxide. Methods
Enzymol. 349, 9–23.
Giesy, J.P., Kannan, K., 1998. Dioxin-like and non-dioxin-like toxic effects of polychlo-
rinated biphenyls (PCBs): implications for risk assessment. Crit. Rev. Toxicol. 28,
511–569.
Halliwell, B., Gutteridge, J.M.C., 1990. Role of free radicals and catalytic metal ions in
human disease: an overview. Meth. Enzymol. 186, 1–85.
Hauff, K.D., Hatch, G.M., 2006. Cardiolipin metabolism and Barth syndrome. Prog.
Lipid Res. 45, 91–101.
Hausladen, A., Fridovich, I., 1994. Superoxide and peroxynitrite inactivate aconitases,
but nitric oxide does not. J. Biol. Chem. 269, 29405–29408.
Hausladen, A., Fridovich, I., 1996. Measuring nitric oxide and superoxide: rate con-
stants for aconitase reactivity. Methods Enzymol. 269, 37–41.
Hissin, P.J., Hilf, R., 1976. A fluorometric method for determination of oxidized and
reduced glutathione in tissues. Anal. Biochem. 74, 214–226.
Hoek, J.B., Farber, J.L., Thomas, A.P., Wang, X., 1995. Calcium ion-dependent signalling
and mitochondrial dysfunction: mitochondrial calcium uptake during hormonal
stimulation in intact liver cells and its implication for the mitochondrial perme-
ability transition. Biochim. Biophys. Acta 1272, 93–102.
Hsiao, G., Lin, Y.H., Lin, C.H., Chou, D.S., Lin, W.C., Sheu, J.R., 2001. The protective
effects of PMC against chronic carbon tetrachloride-induced hepatotoxicity in
vivo. Biol. Pharm. Bull. 4, 1271–1276.
182 H.A.A. Aly, Ò. Domènech / Toxicology 262 (2009) 175–183
Ikegwuonu, F.I., Ganen, L.G., Larsen, M.C., Shen, X., Jefcoate, C.R., 1996. The regu-
lation by gender, strain, dose, and feeding status of the induction of multiple
forms of cytochrome P450 isozymes in rat hepatic microsomes by 2,4,5,29,49,59-
hexachlorobiphenyl. Toxicol. Appl. Pharmacol. 139, 33–41.
Iqbal, M., Athar, M., 1998. Attenuation of iron-nitrilotriacetate mediated renal
oxidative stress, toxicity and hyperproliferative response by the prophylactic
treatment of rats with garlic oil. Food Chem. Toxicol. 36, 485–495.
James, A.M., Cochemé, H.M., Smith, R.A., Murphy, M.P., 2005. Interactions of
mitochondria-targeted and untargeted ubiquinones with the mitochondrial
respiratory chain and reactive oxygen species. Implications for the use of
exogenous ubiquinines as therapies and experimental tools. J. Biol. Chem. 280,
21295–212312.
Kaufmann, P., Török, M., Hänni, A., Roberts, P., Gasser, R., Krähenbühl, S., 2005. Mech-
anisms of benzarone and benzbromarone-induced hepatic toxicity. Hepatology
41, 925–935.
Kimbrough, R.D., 1995. Polychlorinated biphenyls (PCBs) and human health: an
update. Crit. Rev. Toxicol. 25, 133–163.
Kirkland, R.A., Franklin, J.L., 2003. Bax, reactive oxygen, and cytochrome c release in
neuronal apoptosis. Antioxid. Redox. Signal. 5, 589–596.
Kowaltowski, A.J., Vercesi, A.E., 1999. Mitochondrial damage induced by conditions
of oxidative stress. Free Radical Bio. med. 26, 463–471.
Lambert, C., Apel, K., Biesalski, H.K., Frank, J., 2004. 2-Methoxyestradiol induces
caspase-independent, mitochondria-centered apoptosis in DS-sarcoma cells.
Int. J. Cancer 108, 493–501.
Larsen, J.C., 2006. Risk assessments of polychlorinated dibenzo-p-dioxins, polychlo-
rinated dibenzofurans, and dioxin-like polychlorinated biphenyls in food. Mol.
Nutr. Food Res. 50, 885–896.
Latipää, P.M., Kärki, T.T., Hiltunen, J.K., Hassinen, I.E., 1986. Regulation of palmitoyl-
carnitine oxidation in isolated rat liver mitochondria. Role of the redox state of
NAD(H). Biochim. Biophys. Acta 875, 293–300.
Liu, M.J., Wang, Z., Li, H.X., Wu, R.C., Liu, Y.Z., Wu, Q.Y., 2004. Mitochon-
drial dysfunction as an early event in the process of apoptosis induced by
woodfordin I in human leukemia K562 cells. Toxicol. Appl. Pharmacol. 194,
141–155.
Lu, C.F., Wang, Y.M., Peng, S.Q., Zou, L.B., Tan, D.H., Liu, G., Fu, Z., Wang,
Q.X., Zhao, J., 2009. Combined Effects of Repeated Administration of 2,3,7,8-
Tetrachlorodibenzo-p-dioxin and Polychlorinated Biphenyls on Kidneys of Male
Rats. Arch Environ Contam Toxicol., Apr 17.
Mariussen, E., Myhre, O., Reistad, T., Fonnum, F., 2002. The polychlorinated biphenyl
mixture Aroclor 1254 induces death of rat cerebellar granule cells: the involve-
ment of the N-methyl-d-aspartate receptor and reactive oxygen species. Toxicol.
Appl. Pharmacol. 179, 137–144.
Martins, N.M., Santos, N.A.G., Curti, C., Bianchi, M.L.P., Santos, A.C., 2008. Cisplatin
induces mitochondrial oxidative stress with resultant energetic metabolism
impairment, membrane rigidification and apoptosis in rat liver. J. Appl. Toxicol.
28, 337–344.
McLean, M.R., Twaroski, T.P., Robertson, L.W., 2000. Redox cycling of 2-(x’-mono,-
di,-trichlorophenyl)-1, 4-benzoquinones, oxidation products of polychlorinated
biphenyls. Arch. Biochem. Biophys. 376, 449–455.
Moriarty-Craige, S.E., Jones, D.P., 2004. Extracellular thiols and thiol/disulfide redox
in metabolism. Annu. Rev. Nutr. 24, 481–509.
Morse, R.M., Valenzuela, G.A., Greenwald, T.P., Eulie, P.J., Wesley, R.C., McCallum, R.W.,
1988. Amiodarone-induced liver toxicity. Ann. Intern. Med. 109, 838–840.
Myhre, O., Vestad, T.A., Sagstuen, E., Aarnes, H., Fonnum, F., 2000. The effects
of aliphatic (n-nonane), naphtenic (1,2,4-trimethylcyclohexane) and aromatic
(1,2,4-trimethylbenzene) hydrocarbons on respiratory burst in human neu-
trophil granulocytes. Toxicol. Appl. Pharmacol. 167, 222–230.
Ngui, J.S., Bandiera, S.M., 1999. Induction of hepatic CYP2B is a more sensitive indica-
tor of exposure to aroclor 1260 than CYP1A in male rats. Toxicol. Appl. Pharmacol.
161, 160–167.
Paglia, D.E., Valentine, W.N., 1967. Studies on quantitative and qualitative char-
acterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70,
158–169.
Parkinson, A., Safe, S.H., Robertson, L.W., Thomas, P.E., Ryan, D.E., Reik, L.M., Levin,
W., 1983. Immunochemical quantitation of cytochrome P-450 isozymes and
epoxide hydrolase in liver microsomes from polychlorinated or polybrominated
biphenyl-treated rats. A study of structure–activity relationships. J. Biol. Chem.
258, 5967–5976.
Perez-Carreras, M., Del Hoyo, P., Martin, M.A., Rubio, J.C., Martin, A., Castellano, G.,
Colina, F., Arenas, J., Solis-Herruzo, J.A., 2003. Defective hepatic mitochondrial
respiratory chain in patients with nonalcoholic steatohepatitis. Hepatology 38,
999–1007.
Pessah, I.N., Hansen, L.G., Albertson, T.E., Garner, C.E., Ta, T.A., Do, Z., et al., 2006.
Structure–activity relationship for noncoplanar polychlorinated biphenyl con-
geners toward the ryanodine receptor–Ca2+ channel complex type 1 (RyR1).
Chem. Res. Toxicol. 19, 92–101.
Pessah, I.N., Lehmler, H.-J., Robertson, L.W., Perez, C.F., Cabrales, E., Bose, D.D., et al.,
2009. Enantiomeric specificity of (−)-2,2 ,3,3 ,6,6 -hexachlorobiphenyl toward
ryanodine receptor types 1 and 2. Chem. Res. Toxicol. 22, 201–207.
Petit, J.M., Maftah, A., Ratinaud, M.H., Julien, R., 1992. 10-N-nonyl acridine orange
interacts with cardiolipin and allows the quantification of this phospholipid in
isolated mitochondria. Eur. J. Biochem. 209, 267–273.
Petrosillo, G., Portincasa, P., Grattagliano, I., Casanova, G., Matera, M., Ruggiero, F.M.,
Ferri, D., Paradies, G., 2007. Mitochondrial dysfunction in rat with nonalcoholic
fatty liver involvement of complex I, reactive oxygen species and cardiolipin.
Biochim. Biophys. Acta 1767, 1260–1267.
Poland, A., Knutson, J.C., 1982. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halo-
genated aromatic hydrocarbons: examination of the mechanism of toxicity.
Annu. Rev. Pharmacol. Toxicol. 22, 517–554.
Poulos, T.L., Raag, R., 1992. Cytochrome P450cam: crystallography, oxygen activation,
and electron transfer. FASEB J. 6, 674–679.
Risebrough, R.W., Rieche, P., Peakall, D.B., Herman, S.G., Kirven, M.N., 1968. Polychlo-
rinated biphenyls in the global ecosystem. Nature 220, 1098–1102.
Roue, G., Bitton, N., Yuste, V.J., Montange, T., Rubio, M., Dessauge, F., Delettre, C.,
Merle-Beral, H., Sarfati, M., Susin, S.A., 2003. Mitochondrial dysfunction in CD47-
mediated caspase-independent cell death: ROS production in the absence of
cytochrome c and AIF release. Biochimie 85, 741–746.
Sadeghi-Aliabadi, H., Chan, K., Lehmler, H.J., Robertson, L.W., O’Brien, P.J.,
2007. Molecular cytotoxic mechanisms of catecholic polychlorinated biphenyl
metabolites in isolated rat hepatocytes. Chem. Biol. Interact. 167, 184–192.
Safe, S.H., 1994. Polychlorinated biphenyls (PCBs): environmental impact, biochem-
ical and toxic responses, and implications for risk assessment. Crit. Rev. Toxicol.
24, 87–149.
Sakac, V., Sakac, M., 2000. Free oxygen radicals and kidney diseases—part I. Med.
Pregl. 53, 463–474.
Salvi, M., Toninello, A., 2001. Aroclor 1254 inhibits the mitochondrial permeability
transition and release of cytochrome c: a possible mechanism for its in vivo
toxicity. Toxicol. Appl. Pharmacol. 176, 92–100.
Schilderman, P.A., Mass, L.M., Pachen, D.M., Dekok, T.M., Kleinjans, J.C., Vanschooten,
F.S., 2000. Induction of DNA adducts by several polychlorinated biphenyls. Env-
iron. Mol. Mutagen. 36, 79–86.
Schlezinger, J.J., White, R.D., Stegeman, J.J., 1999. Oxidative inactivation of
cytochrome p-4501A (CYP1A) stimulated by 3,3 ,4,4 -tetrachlorobiphenyl: pro-
duction of reactive oxygen by vertebrate CYP1As. Mol. Pharmacol. 56, 588–597.
Schuetz, E.G., Brimer, C., Schuetz, J.D., 1998. Environmental xenobiotics and the anti-
hormones cyproterone acetate and spironolactone use the nuclear hormone
pregnenolone X receptor to activate the CYP3A23 hormone response element.
Mol. Pharmacol. 54, 1113–1117.
Seglen, P.O., 1976. Preparation of isolated rat liver cells. Methods Cell Biol. 13, 29–83.
Senft, A.P., Dalton, T.P., Nebert, D.W., Genter, M.B., Puga, A., Hutchinson, R.J., Kerzee,
J.K., Uno, S., Shertzer, H.G., 2002. Mitochondrial reactive oxygen production is
dependent on the aromatic hydrocarbon receptor. Free Radic. Biol. Med. 33,
1268–1278.
Shangari, N., O’Brien, P.J., 2004. The cytotoxic mechanism of glyoxal involves oxida-
tive stress. Biochem. Pharmacol. 68, 1433–1442.
Sharpley, M.S., Shannon, R.J., Draghi, F., Hirst, J., 2006. Interactions between
phospholipids and NADH:ubiquinone oxidoreductase (complex I) from bovine
mitochondria. Biochemistry 45, 241–248.
Shih, C.M., Ko, W.C., Wu, J.S., Wei, Y.H., Wang, L.F., Chang, E.E., Lo, T.Y., Cheng,
H.H., Chen, C.T., 2004. Mediating of caspaseindependent apoptosis by cadmium
through the mitochondria-ROS pathway in MRC-5 fibroblasts. J. Cell Biochem.
91, 384–397.
Silberhorn, E.M., Glauert, H.P., Robertson, L.W., 1990. Carcinogenicity of polyhalo-
genated biphenyls: PCBs and PBBs. Crit. Rev. Toxicol. 20, 440–496.
Simon, T., Britt, J.K., James, R.C., 2007. Development of a neurotoxic equivalence
scheme of relative potency for assessing the risk of PCB mixtures. Regul. Toxicol.
Pharm. 48, 148–170.
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D.,
Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein
using bicinchoninic acid. Anal. Biochem. 150, 76–85.
Song, Y., Buettner, G.R., Parkin, S., Wagner, B.A., Robertson, L.W., Lehmler, H.J., 2008b.
Chlorination increases the persistence of semiquinone free radicals derived
from polychlorinated biphenyl hydroquinones and quinones. J. Org. Chem. 73,
8296–8304.
Song, Y., Wagner, B.A., Lehmler, H.J., Buettner, G.R., 2008a. Semiquinone radicals
from oxygenated polychlorinated biphenyls: electron paramagnetic resonance
studies. Chem. Res. Toxicol. 21, 1359–1367.
Spaniol, M., Bracher, R., Ha, H.R., Follath, F., Krähenbühl, S., 2001. Toxicity of amio-
darone and amiodarone analogues on isolated rat liver mitochondria. J. Hepatol.
35, 628–636.
Sridhar, M., Venkataraman, P., Siva, D., Arunkumar, A., Aruldhas, M.M., Srinivasan,
N., Arunakaran, J., 2004. Impact of polychlorinated biphenyl (Aroclor 1254) and
vitamin C on antioxidant system of rat ventral prostate. Asian J. Androl. 6, 19–22.
Talbot, D.A., Lambert, A.J., Brand, M.D., 2004. Production of endogenous matrix
superoxide from mitochondrial complex I leads to activation of uncoupling pro-
tein 3. FEBS Lett. 556, 111–115.
Tharappel, J.C., Lee, E.Y., Robertson, L.W., Spear, B.T., Glauert, H.P., 2002. Regula-
tion of cell proliferation, apoptosis, and transcription factor activities during the
promotion of liver carcinogenesis by polychlorinated biphenyls. Toxicol. Appl.
Pharmacol. 179, 172–184.
Tharappel, J.C., Lehmler, H.J., Srinivasan, C., Robertson, L.W., Spear, B.T., Glauert,
H.P., 2008. Effect of antioxidant phytochemicals on the hepatic tumor promot-
ing activity of 3,3 ,4,4 -tetrachlorobiphenyl (PCB-77). Food Chem. Toxicol. 46,
3467–3474.
Thome, J.P., Roelandt, L., Goffinet, G., Stouvenakers, N., Kremers, P., 1995. Cytotoxic
effects of Aroclor 1254 on ultrastructure and biochemical parameters in cultured
foetal rat hepatocytes. Toxicology 98, 83–94.
Tisdale, H.D., 1967. Preparation and properties of succinic-cytochrome c reductase
(complex II and III). Methods Enzymol. 10, 213–215.
Twaroski, T.P., O’Brien, M.L., Larmonier, N., Glauert, H.P., Robertson, L.W., 2001a.
Polychlorinated biphenyl-induced effects on metabolic enzymes AP-1 binding,
vitamin E, and oxidative stress in rat liver. Toxicol. Appl. Pharmacol. 171, 85–93.
 H.A.A. Aly, Ò. Domènech / Toxicology 262 (2009) 175–183
Twaroski, T.P., O’Brien, M.L., Robertson, L.W., 2001b. In: Robertson, L.W., Hansen,
L.G. (Eds.), Recent Advances in the Environmental Toxicology and Health Effects.
Effects of PCB 77 (3,3 ,4,4 -Tetrachlorobiphenyl) on Glutathione Peroxidase
Activity and Regulatory Mechanisms. The University Press of Kentucky, pp.
317–319.
Twaroski, T.P., O’Brien, M.L., Robertson, L.W., 2001c. Effects of selected polychlo-
rinated biphenyl (PCB) congener on hepatic glutathione, glutathione-related
enzymes, and selenium status: implications for oxidative stress. Biochem. Phar-
macol. 62, 273–281.
Uhlig, S., Wendel, A., 1992. The physiological consequences of glutathione variations.
Life Sci. 51, 1083–1094.
Van den Berg, M., Birnbaum, L., Bosveld, A.T., Brunstrom, B., Cook, P., Feeley, M.,
Giesy, J.P., Hanberg, A., Hasegawa, R., Kennedy, S.W., et al., 1998. Toxic equivalency
factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ. Health
Perspect. 106, 775–792.
Van den Berg, M., Birnbaum, L.S., Denison, M., De Vito, M., Farland, W., Feeley, M.,
Fiedler, H., Hakansson, H., Hanberg, A., Haws, L., Rose, M., Safe, S., Schrenk, D.,
Tohyama, C., Tritscher, A., Tuomisto, J., Tysklind, M., Walker, N., Peterso, R.E.,
2006. The 2005 world health organization reevaluation of human and mam-
malian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol.
Sci. 93, 223–241.
Vanden Hoek, T.L., Li, C., Shao, Z., Schumacker, P.T., Becker, L.B., 1997. Significant levels
of oxidants are generated by isolated cardiomyocytes during ischemia prior to
reperfusion. J. Mol. Cell. Cardiol. 29, 2571–2583.
183
Venkataraman, P., Krishnamoorthy, G., Vengatesh, G., Srinivasan, N., Aruldhas, M.M.,
Arunakaran, J., 2008. Protective role of melatonin on PCB (Aroclor 1254) induced
oxidative stress and changes in acetylcholine esterases and membrane bound
ATPases in cerebellum, cerebral cortex and hippocampus of adult rat brain. Int.
J. Dev. Neurosci. 26, 585–591.
Williams, C.S., Jones, B.C., Nchege, O., Chung, R.A., 1993. Hepatocyte ultrastructure
following exposure to aroclors and pure polychlorinated biphenyls. J. Environ.
Pathol. Toxicol. Oncol. 12, 17–33.
Wilson, K.S., Prochaska, L.J., 1990. Phospholipid vesicles containing bovine heart
mitochondrial cytochrome c oxidase and subunit III-deficient enzyme: anal-
ysis of respiratory control and proton translocating activities. Arch. Biochem.
Biophys. 282, 413–420.
Ye, S.F., Hou, Z.Q., Zhang, Q.Q., 2007. Protective effects of Phellinus linteus extract
against iron overload-mediated oxidative stress in cultured rat hepatocytes.
Phytother. Res. 21, 948–953.
Zhao, H.X., Adamcakova-Dodd, A., Hu, D., Hornbuckle, K.C., Just, C.L., Robertson, L.W.,
Thorne, P.S., Lehmler, H.J., 2009. Development of a synthetic PCB mixture resem-
bling the average polychlorinated biphenyl profile in Chicago air. Environ. Int.,
Apr 16.
Zhou, C., Zhang, C., 2005. Protective effects of antioxidant vitamins on Aroclor 1254-
induced toxicity in cultured chicken embryo hepatocytes. Toxicol. In Vitro 19,
665–673.