The Protective Effects of Garlic Extract Against Acetaminophen-Induced Oxidative Stress and Glutathione Depletion
Acetaminophen, the most commonly sold over-the-counter antipyretic analgesic, is capable of causing severe and sometimes fatal hepatic damage in humans and experimental animals. The incidence of liver injury due to acetaminophen overdose, either with suicidal intent or by accident, is increasing. Garlic is among those medicinal plants famous for its different health protective effects. In this study, the protective effects of garlic extract on acute acetaminophen-induced liver injury were investigated using freshly isolated rat hepatocytes. The hepatocytes were isolated from Sprague-Dawley male rats by a two step collagenase model. Formation of Reactive Oxygen Species (ROS) and Glutathione (GSH) depletion were studied after addition of acetaminophen to cell suspensions. The effects of garlic extract on prevention of ROS formation as well as GSH depletion was investigated and compared with the effects of N-Acetyl Cysteine (NAC) as the standard treatment. Reactive oxygen species formation was assessed by a spectrofluorometry method and garlic extract was shown to be as effective as NAC in decreasing ROS formation induced by acetaminophen. Glutathione (GSH) levels of hepatocytes were determined using HPLC. Garlic extract was effective in preventing GSH depletion significantly (p<0.05). It is concluded that garlic extract has an antioxidant effect and can protect hepatocytes from GSH depletion following NAPQI production.
For many centuries, garlic (Allium sativum) has been widely used as
a flavor and as one of the nutrients which prevent different types of diseases.
Earlier studies have shown that garlic possesses a wide range of biological
activities, such as anti-atherosclerotic, antihypertensive, anticancer, immune-modulator,
antimicrobial and potential anti-aging effects (Agarwal,
1996; Banerjee et al., 2003; Amagase,
2006). The main organosulfur compound in intact garlic bulbs, 7-glutamylcysteines,
can be hydrolyzed and oxidized into alliin and then to allicin, which has been
mistaken as the active component of garlic. Allicin is unstable and decomposed
into the odorous compounds, i.e., Allyl Mercaptan (AM), diallyl sulfide (DAS),
diallyl disulfide (DADS) and diallyl trisulfide (DATS) (Amagase
et al., 2001; Xu and Simon Cho, 2000; Zeng
et al., 2008).
It has been shown that high doses of the analgesic drug acetaminophen (APAP)
produce centrilobular liver necrosis in human and other susceptible species
(Ruepp et al., 2001; Sumioka
et al., 2004; Aleksunes et al., 2008).
Acetaminophen is primarily metabolized by sulfation and glucuronidation, but
with an increasing dose rate, these pathways become saturated and a greater
proportion of the drug is available for oxidation by the microsomal cytochrome
P-450 system (Amar and Schiff, 2007). N-Acetyl-P-benzoquinone
Imine (NAPQI) is the product of this pathway which is thought to be responsible
for the subsequent hepatic damage (Aleksunes et al.,
N-Acetyl-P-benzoquinone Imine (NAPQI) is a highly reactive electrophile and
is detoxified in liver by either reduction to the parent compound, acetaminophen,
or conjugation at the meta-position with glutathione, which both reactions consume
GSH (Ruepp et al., 2001). The importance of glutathione
in acetaminophen toxicity is further emphasized by the large body of evidence
which indicates that interventions which increase GSH content can dramatically
reduce acetaminophen and NAPQI-induced hepatic injury (Mitchell,
1977; Ruepp et al., 2001).
Oxidative stress is also considered to be involved in the induction of hepatotoxicity
by APAP. Oxidation of APAP by CYPs may generate Reactive Oxygen Species (ROS).
Hydrogen peroxide and superoxide are produced during metabolic activation of
APAP in the mixed function oxidase system (Amimoto et
Some in vivo studies have mentioned the antioxidant and hepatoprotective
effects for garlic (Park et al., 2005; Zeng
et al., 2008; El-Shenawy and Hassan, 2008),
but the cellular effects of garlic extract on the rat isolated hepatocytes with
acetaminophen have not been studied before. Here, for the first time, we have
shown that GE is able to prevent ROS formation and GSH depletion caused by APAP
administration in hepatocytes freshly isolated from β-naphtoflavon-treated
male Sprague-Dawley rats.
MATERIALS AND METHODS
This research project was conducted from 2007 to 2009, in the Drug Applied Research Center and Faculty of Pharmacy in Tabriz University of Medical Sciences.
Chemicals: Bovine Serum Albumin, collagenase A from clostridium histolyticum and HEPES were obtained from Roche diagnostics (Indianapolis, IN) acetaminophen (APAP; 4-acetamidophenol) from Sigma-aldrich (St. Louis, MO), N Acetyl-Cysteine (NAC) from Acros Pharmaceuticals, Di-Chloro-Fluorescin (DCF) and GSH from FLUKA, Beta-Naphto-Flavon (BNF), Heparin sodium salt grade 1-A, Trypan Blue 0.4% solution, Methanol, MgSO4 and other buffer salts, were obtained from Merck (Germany). All other chemicals were of the highest grade commercially available.
Animals: Male Sprague-Dawley rats (200-250 g) were obtained from the laboratory of Animals Research Center of Tabriz University of Medical Sciences. The rats were housed in an air-conditioned room, under controlled temperature of 23±1°C, relative humidity of 36±6% and 12 h light/12 h dark conditions for 1 week before starting the experiments. They were allowed to feed with standard laboratory chow and tap water ad libitum. Procedures involving animals and their care were conducted in conformity with the NIH guidelines for the care and use of laboratory animals. The whole procedure was approved by the Animal Ethics Committee and the Research Council of Vice-Chansellor for Research Affairs at Tabriz University of Medical Sciences, under the license number 5/4/6704 on 2007. 10.28.
Preparation of extract: Fresh garlic (Allium sativum L.) was
purchased from a retail food store (Tabriz, Iran) and identified by botanists
in the herbarium of Tabriz University. On the day of experiments, the garlic
bulbs were peeled, weighed and ground to obtain a fine juice. It was then homogenized
in deionized water. The homogenized mixture was filtered through cheesecloth.
Garlic extracts of lower concentrations were prepared by dilution of this solution
with media used for cell suspensions (Baluchnejadmojarad
et al., 2003).
Determination and preparation of allicin from garlic extract: In order
to determine and isolate the allicin content in garlic, aqueous extract was
assayed using analytical HPLC (Vargas et al., 2008).
Separation of allicin from extract was performed using a Spherisorb ODS2 column
(4.6x250 mm, 5 μm, waters, Ireland) and methanol (75%) and water (phosphate
buffer pH = 3), as a mobile phase, with a flow rate of 1 mL min-1,
detecting allicin at the wavelength of 254 nm. Retention time for allicin was
5.8 min. Pure allicin was obtained on the basis of analytical HPLC from a further
preparative HPLC. The achieved allicin solution was dried with freeze drier
and the resulting allicin powder was used for all experiments. Allicin content
of garlic bulbs was quantified with analytical HPLC mentioned above as 80 mg.
Preparation of hepatocytes: Hepatocytes were isolated from male Sprague-Dawley
rats by a two-step collagenase perfusion, as described previously (Moldeus
et al., 1978; Eghbal et al., 2004).
The first step involves the perfusion of a calcium-free buffer. The second step
is circulation of a calcium-supplemented buffer containing collagenase. The
initial perfusion facilitates desmosomal cleavage and further dispersion of
liver cells. The addition of Ca2+ to the enzyme solution ensures
adequate collagenase activity. After isolation, the cells were suspended (106
cells mL-1) in Krebs-Henseleit buffer containing 12.5 mM HEPES and
incubated under a stream of 95% O2 and 5% CO2 in continuously
rotating round-bottomed 50 mL flasks at 37°C. Cell viability was measured
by Trypan blue exclusion method. The hepatocytes used in this study were at
least 85-90% viable immediately after isolation.
Determination of ROS formation: The dichlorofluorescein-diacetate (DCFH-DA) was dissolved in methanol and was added to the cell suspensions at the same time as APAP. The final concentration of DCFH-DA in cell suspensions was 1 μM. The samples were taken at special time intervals and the fluorescence intensity was measured at the excitation wavelength 485 nm and the emission wavelength of 530 nm. Results were expressed as the fluorescent intensity per 106 cells.
Measurement of intracellular GSH: Intracellular GSH in isolated hepatocytes
was measured in deproteinized samples (5% metaphosphoric acid) after derivatization
with iodoacetic acid and 1-fluoro-2,4-dinitrobenzene, by HPLC (Reed
et al., 1980), using a μBondapak NH2 column (Water
associates, Milford, MA).
The procedure is based upon the initial formation of s-carboxymethyl derivatives
of free thiols with iodoacetic acid followed by conversion of free amino groups
to 2, 4-dinitrophenyl derivatives by reaction with 1-fluoro-2,4-dinitrobenzene
(FDNB). Determination of nanomole levels of GSH is possible with this method.
Briefly, 0.8 mL of the cell suspension was spun at 50 g for 40 sec and the cell
pellet was resuspended in 0.8 mL of fresh medium. 0.2 mL of 25% metaphosphoric
acid was added to the sample and after 10-60 min the sample was centrifuged
at 100 g for 5 min. 0.5 mL of supernatant and 0.05 mL of iodoacetic acid were
mixed in 200-300 mg of sodium bicarbonate. The mixture was sealed and left in
the dark and room temperature for 1 h. Then 0.5 mL of FDNB solution (1.5% v/v
in ethanol) was added to the sample and the sample was sealed. The sample was
left in the dark for 4 h at room temperature and then analyzed by HPLC (Khan
and OBrien, 1997; Eghbal et al., 2004).
Experimental protocols: The animals of test group received 3 I.P injections
of BNF (80 mg kg-1) during 72 h before starting the experiments;
while the animals of positive control and negative control groups received Corn
oil and no chemicals, respectively (Marvasi et al.,
2006). On the day of experiments, after induction of anesthesia using sodium
pentobarbital, the liver cells were isolated via a two step model. The cells
were allowed to get adapted with the incubation conditions for 20 min before
the addition of compounds to the incubation mixture. The cells were then exposed
to different concentrations of GE and/or NAC 30 min before, at the same time
with APAP and 30 min after APAP addition. Aliquots of the cells were taken at
different time points (0, 60, 120, 180 and 0, 30, 60, 90, 120 and 150 min) for
determination of ROS formation and GSH levels respectively.
Statistical analysis: All the data were expressed as Mean±SEM statistical software SPSS14.0 was used for statistical analysis. All the data were analyzed using one-way Analysis of Variance (ANOVA) for models with repeated measurements, followed by TUKEYs post hoc tests. The differences were considered significantly at p<0.05 level.
The viability of hepatocytes treated with all chemicals used in this study was examined by trypan blue exclusion method. The results showed that none of the treatments were hepatotoxic, except for APAP which was cytotoxic, when added to the hepatocytes isolated from BNF pre-treated rats, while it did not show any significant hepatotoxicity in rats without pretreatment with BNF. Hepatocytes were incubated in Krebs-Henseleit solution, pH 7.4 at 37°C under the atmosphere of 95% O2/5% CO2. The samples were taken at mentioned time intervals and cell death was assessed by trypan blue exclusion test.
Effects of garlic extract and NAC on acetaminophen-induced ROS formation:
We measured the production of ROS in isolated hepatocytes by measuring DCF fluorescence.
The probe 2, 7-dichlorofluorescin has been used as an indicator
of reactive oxygen species formation and oxidative stress (Eghbal
et al., 2004). The principle of this assay is that DCFH-DA diffuses
through the cell membrane and is enzymatically hydrolyzed by intracellular esterases
to nonfluorescent dichlorofluorescin (DCFH). In the presence of ROS, this compound
is rapidly oxidized to highly fluorescent dichlorofluorescein (DCF) (LeBel
et al., 1992; Eghbal et al.,
2004). In order to determine, the possible effects of the chronologic treatments,
three protocols were applied. Briefly, after preparation and incubation of cell
suspensions, garlic extract and/or NAC was added to suspensions 30 min before,
at the same time with and 30 min after addition of APAP solution (500 μM).
The results show significant differences between APAP and control group (Fig.
1, 2) which means APAP could induce a high amount of ROS
formation. Figure 1 indicates the protective effect of GE
against APAP induced ROS formation; especially when the extract added 30 min
before and at the same time with APAP. It can be seen from Fig.
3 that NAC, as the standard treatment, is completely protective even if
administered 30 min after APAP.
||The effect of Garlic Extract on acetaminophen-induced ROS
formation (three protocols), ROS formation induced in isolated hepatocytes
by APAP. Garlic extracts were added at 3 different time intervals according
to the time point of APAP addition. ROS formation was measured at 4 time
points. The APAP addition time was equal to 0time point. Values represent
Mean±SEM and are at least from 3 independent experiments, *Shows
significant difference (p<0.05) with APAP group, **Shows significant
difference (p<0.001) with APAP group, ***Shows significant difference
(p<0.001) with control group (BNF treated cells)
||The effects of NAC on acetaminophen-induced ROS formation
(three protocols), ROS formation induced by APAP in isolated rat hepatocytes
pretreated with BNF. Hepatocytes maintained under the constant flow of 95%
O2/5% CO2 .Garlic extracts were added at 3 different
time intervals according to the time point of APAP addition. ROS formation
was measured at 4 time points. The APAP addition time was equal to 0 time
point. Values represent Mean±SEM and are at least from 3 independent
experiments, **Shows significant difference (p<0.001) with APAP group,
***Shows significant difference (p<0.001) with control group (BNF treated
||The effects of APAP, GE and NAC on GSH levels of isolated
rat hepatocytes, APAP-induced GSH depletion in isolated rat hepatocytes
and protective effects of Garlic extract as well as NAC. Hepatocytes (106
cells mL-1) were isolated from rats pretreated with BNF and maintained
under the constant flow of 95% O2/5% CO2 atmosphere.
GSH levels were determined at 6 time points according to the time point
of APAP addition by the method of Reed et al. (1980). Values represent
Mean±SEM and are at least from 3 independent experiments, **Shows
significant difference (p<0.05) with APAP group, ***Shows significant
difference (p<0.001) with APAP group
Effects of garlic extract and NAC on acetaminophen-induced GSH depletion:
In order to determine the effect of APAP on GSH depletion, GSH content of the
hepatocytes was assessed. The standard calibration curve (Fig.
3) was obtained using GSH depleted cells (treated with 200 μM Bromoheptane).
Glutathione standard solutions of 4 different concentrations were added to the
cell suspensions 30 min after their incubation with bromoheptane. Concentrations
used for GSH were 10, 20, 40 and 80 nM.
As shown in Fig. 3, APAP (500 μM) caused a time-dependent GSH depletion in hepatocytes isolated from BNF-pretreated rats with a significant decrease in GSH levels after 60 min of addition. Normal hepatocytes which were isolated from rats without any pretreatment did not show GSH depletion in presence of APAP. Furthermore, there were not any significant differences between 3 timed protocols (described for ROS formation) in GSH levels (data not shown). GE prevented GSH depletion with a high percentage, especially after 60 min which was comparable to the effect of NAC.
APAP has been used effectively and safely by a large number of patients for
its analgesic and antipyretic effects. At its regular dose in the body, APAP
is mainly detoxified either by glucuronide conjugation or sulfation.
At overdose, it is also metabolized by CYP2E1 to form NAPQI that depletes GSH.
Saturation of the detoxification pathways which causes excess of NAPQI to be
formed consequently leading to extensive depletion of GSH and therefore cell
death (Burke et al., 2006). Present finding is
in accordance with these results as APAP was not able to induce toxicity without
the induction of CYP2E1 with BNF. Glutathione (GSH) plays an important role
in protecting cells from electrophilic compounds and free radicals such as reactive
oxygen species generated during cellular metabolism. Reduced glutathione can
act as a reductant, reducing hydrogen peroxide and lipid hydroperoxides directly
to H2O, a reaction catalyzed by GSH-Px.
Depletion of intracellular GSH, under conditions of continuous intracellular
oxidative stress, leads to oxidation and damage of lipids, proteins and DNA
by the reactive oxygen species (Kaplowitz, 2000; Nordberg
and Arner, 2001). Oxidative stress (along with nitrosative stress) is one
of the proposed mechanisms (Jaeschke, 1990) and depletion
of cellular GSH in the liver cells is known to play an important role in APAP
toxicity (Mitchell et al., 1973). Glutathione
(GSH) is used by cells to detoxify reactive intermediates and is critical for
elimination of many drugs, such as APAP. Depletion of GSH can lead to tumor
cell death in vitro, especially in cells generating high levels of oxyradicals
(Wolchok et al., 2003). It is also well known
that NAC is used as an antidote in acetaminophen induced toxicity mainly because
it restores the GSH content in the liver (Vina et al.,
1980; Mitchell et al., 1985,). In a earlier
reported study, Mitchell et al. (1985) investigated
GSH status to cellular unity in cultured hepatocytes over time after chemical
exposure. Their findings indicate that depletion of GSH to 20% of normal levels
qualifies cells for significant drug/chemical induced injury implying that GSH
depletion precedes cell toxicity. Previous studies have shown that GSH content
decreases after APAP overdose in animal livers (Jaeschke,
1990; Amimoto et al., 1995).
Present results support this idea, because APAP addition caused hepatocytes
to lose their GSH content after 30 min. This GSH loss was significantly prevented
by GE (p<0.05) and NAC (p<0.001) (Fig. 3). Difference
between GE and NAC might be due to the presence of different ingredients in
GE which necessitates the isolation and purification of the active compounds.
It should be mentioned that, the control group which was just pre-treated with
BNF did not show significant decrease in GSH levels during 3 h. This study examines
the hypothesis that the toxicity of acetaminophen might occur as a result of
ROS formation as well as GSH depletion and that; garlic extract could protect
the hepatocytes from these processes. In the present study, administration of
AAP resulted in decreased GSH and increased ROS levels indicating that an oxidative
stress is present in the cells exposed to AAP.
ROS formation is an important mechanism in various cytotoxicities. The one-electron
oxidation of APAP by CYPs may generate Reactive Oxygen Species (ROS). Hydrogen
peroxide and superoxide are produced during metabolic activation of APAP in
the mixed function oxidase system (Sumioka et al.,
2004). It has also been reported that APAP overdose results in a significant
decreases in antioxidant enzyme activities such as catalase and glutathione
peroxidase (Sumioka et al., 2004). Thus, ROS
formation could have a critical role in manifestation of acetaminophen overdose
In this study, the amount of ROS formation was shown to increase significantly
60 min after APAP inclusion which is completely inhibited by NAC and to a lesser
amount by GE (Fig. 1, 2).
Allicin which is one of the major but unstable components of GE (Amagase
et al., 2001) was shown to be cytotoxic (data in-press). Compounds
such as allicin which are present in garlic extract can cover the effects of
pure antioxidant components of GE like DATS (Zeng et
al., 2008) and allyl mercaptan which possess antioxidant activities.
The antioxidant and hepatoprotective activities of allyl mercaptan have been
studied by our research group and the results will be published in the near
Present results are in accordance with the other studies showing garlic to
be protective in vivo against hepatotoxicity induced by acetaminophen
(Hu et al., 1996) carbon tetrachloride (Fanelli
et al., 1998), or cyclophosphamide (Das et
al., 1993). Present data indicate that GE is able to protect the cells
against consequences of APAP induced cytotoxicity. This protection might be
as a result of an ability of garlic components to inhibit phase 1 enzymes or
induce phase 2 enzymes, GE antioxidant activity and/or its interaction with
NAPQI to protect cellular GSH. Further research is needed to clarify the subject.
This study was supported by Drug Applied and Biotechnology research centers in Tabriz University of Medical Sciences, Tabriz, Iran. The authors want to thank Dr. S. Dastmalchi for his kind supports.
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