The protective effect of N-acetylcysteine against cyclosporine A-induced hepatotoxicity in rats

N-ACETYLCYSTEINE AGAINST CYCLOSPORINE-A 15

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2008; 28: 15–20

DOI: 10.1002/jat

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2008; 28: 15–20Published online 27 April 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jat.1245

The protective effect of N-acetylcysteine againstcyclosporine A-induced hepatotoxicity in rats

Hasan Kaya,1 Ahmet Koc,2 Sadik Sogut,3,* Mehmet Duru,4 H. Ramazan Yilmaz,5 Efkan Uz5 andRamazan Durgut6

1 Department of Internal Medicine, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey2 Department of Histology and Embryology, Faculty of Veterinary, Mustafa Kemal University, Hatay, Turkey3 Department of Biochemistry, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey4 Department of Emergency Medicine, Faculty of Medicine, Mustafa Kemal University, Hatay, Turkey5 Department of Medical Biology, Faculty of Medicine, Suleyman Demirel University, Isparta, Turkey6 Department of Internal Medicine, Faculty of Veterinary, Mustafa Kemal University, Hatay, Turkey

Received 22 November 2006; Revised 13 January 2007; Accepted 12 February 2007

ABSTRACT: The immunosuppressive agent cyclosporine A (CsA) has been reported to exert measurable hepatotoxic

effects. One of the causes leading to hepatotoxicity is thought to be reactive oxygen radical formation. The aim of this study

was to investigate the effects of N-acetylcysteine (NAC) treatment on CsA-induced hepatic damage by both analysing

superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), aspartate aminotransferase (AST) and alanine transaminase

(ALT) activities with malondialdehyde (MDA) and nitric oxide (NO) levels, and using an histological approach. CsA ad-

ministration produced a decrease in hepatic SOD activity, and co-administration of NAC with CsA resulted in an increase

in SOD activity. MDA and NO levels increased in the CsA group and NAC treatment prevented those increases. A sig-

nificant elevation in serum AST and ALT activities was observed in the CsA group, and when NAC and CsA were co-

administered, the activities of AST and ALT were close to the control levels. CsA treatment caused evident morphological

alterations. Control rats showed no abnormality in the cytoarchitecture of the hepatic parenchyma. The co-administration

of NAC with CsA showed no signs of alteration and the morphological pattern was almost similar to the control group.

In conclusion, CsA induced liver injury and NAC treatment prevented the toxic side effects induced by CsA administration

through the antioxidant and radical scavenging effects of NAC. Copyright © 2007 John Wiley & Sons, Ltd.

KEY WORDS: cyclosporine A; N-acetylcysteine; hepatotoxicity; oxidant; antioxidant

complex (Mattila et al., 1990; Liu et al., 1991). It has

been found that the liver is more sensitive to CsA toxic-

ity, and hepatic function may be impaired even when the

blood levels of CsA are in the therapeutic range (Kahan

et al., 1984).

It was also reported that possible mechanisms of CsA

hepatotoxicity were through the production of the reac-

tive oxygen species (ROS), oxidative stress, depletion of

hepatic antioxidant system and increase in malondial-

dehyde (MDA) (Galan et al., 1999; Durak et al., 2004).

In particular, living structures having thiol groups are

important targets of oxygen radicals (Orrenius, 1993). As

is known, membrane lipid peroxidation is a common

mechanism of cell death, which was found to be in-

creased by CsA (Wolf et al., 1994). Similar observations

and suggestions implicating free radicals in the CsA-

induced hepatotoxicity were made by some other

researchers (Galan et al., 1995). It was reported that

increased oxidized glutathione concentrations could

modulate the activities of various regulatory enzymes and

might be a cause of the impaired hepatocellular functions

induced by CsA (Cadenas et al., 1983). GSH plays a

central role in protecting cells from oxygen-derived free

Introduction

Cyclosporine A (CsA), a fungal cyclic polypeptide, is

commonly used as an immunosuppressive agent

(Margreiter et al., 1983) and in treatment of the

autoimmune diseases (Kahan, 1992; Borel et al., 1996).

CsA significantly improves graft survival in cases of

renal, cardiac, pancreatic, bone marrow and hepatic trans-

plantations; however, recipients have to maintain therapy

for the rest of their lives. In addition, this drug is used

in the treatment of a variety of autoimmune diseases such

as idiopathic nephritic syndrome, inflammatory bowel

disease, psoriasis and rheumatoid arthritis (Berg et al.,

1986). Unfortunately, CsA induces several side effects

such as nephrotoxicity, cardiotoxicity, hypertension and

hepatotoxicity (Actis et al., 1995), because of its specific

inhibiting effect on signal transduction pathways of T cell

receptors through the formation of a CsA–cyclophylin

* Correspondence to: Dr Sadik Sogut, Mustafa Kemal University, Medical

Faculty, Department of Biochemistry, 31100, Hatay, Turkey.

E-mail: [email protected]

16 H. KAYA ET AL.


DOI: 10.1002/jat

radical (ODFR) injury and other activated toxic com-

pounds (Ross, 1980).

N-acetyl-L-cysteine (NAC), a potent antioxidant, may

serve as a precursor for glutathione synthesis (Bernard

et al., 1984). In addition, administration of NAC results

in a greater availability of glutathione for detoxification

of ODFR and other foreign substances (Meister and

Anderson, 1983). Besides, NAC is routinely used in clini-

cal practice in patients with acetaminophen overdose

(Buckpitt et al., 1979). The protective effects of NAC in

the cases of some organ injuries have also been demon-

strated (Fukuzawa et al., 1995; Menasche et al., 1992).

In consideration of the above mentioned facts, the aim

of the present study was to investigate the effects of

NAC treatment against CsA-induced hepatic damage

by using various oxidative/antioxidative markers such as

superoxide dismutase (SOD), glutathione peroxidase

(GSH-Px), MDA and nitric oxide (NO). Hepatic damage

was assessed by measuring serum AST and ALT activi-

ties and by histological analysis of liver sections as to

indices of damage and necrosis.

Materials and Methods

Animals and Experimental Procedures

In the experiments, male Wistar Albino rats weighing

230–270 g were used. The animals were fed ad libitum

commercial diet (standard rat ration, Aytekinler Yem

Sanayi, Konya, Turkey) and had free access to water.

This study was performed in accordance with Guide for

the Care and Use of Laboratory Animals, National Acad-

emy Press, Washington D.C. (1996). The animals were

housed under normal conditions in quiet rooms with

12:12 h light-dark cycle (7 a.m to 7 p.m). In this study,

the rats were randomly allocated to one of four groups,

and each group consisted of seven rats. The control group

received normal diet without any treatment. The second

group received only NAC (Sandoz Ltd, Basel, Switzer-

land) (11 days, 150 mg kg−1, i.m.) (Hsu et al., 2004)

(NAC group). The animals in the third group received

only CsA (Novartis, East Hanover, NJ, USA) daily (10

days, 15 mg kg−1 day−1, s.c.) (Schuurman et al., 1990)

(CsA group). The animals in the fourth group treated with

CsA (10 days, 15 mg mg−1 day−1, s.c.) and starting 1 day

before CsA injection were treated with NAC (11 days,

150 mg kg−1 day−1, i.m.) (CsA + NAC group). The animals

in all groups were anesthetized with ketamine hydro-

chloride (75 mg kg−1) and xylazine (8 mg kg−1) on day

10 of the CsA-administration, and then venous blood

was collected. After that, serum was separated. The liver

was also rapidly excised and sectioned vertically into two

pieces for microscopic examination and biochemical

analysis. The liver tissue and serum were stored at

−30 °C until biochemical analyses.

Liver Oxidant and Antioxidant Parameters

The tissues were homogenized in four volumes of

ice-cold Tris-HCl buffer (50 mM, pH 7.4) using a glass

Teflon homogenizer (Ultra Turrax IKA T25 Basic,

Germany) after cutting the livers into small pieces with

scissors (for 2 min at 5000 rpm). Measurements of MDA,

NO and protein levels were carried out at this stage. The

homogenate was then centrifugated at 5000 g for 60 min

in order to remove debris. The activity of GSH-Px

as well as the levels of protein were analysed by using

the clear supernatant fluid. The supernatant solution was

extracted with an equal volume of an ethanol/chloroform

mixture (5/3, volume per volume [v/v]). After centrifuga-

tion at 5000 g for 30 min, the clear upper layer (the

ethanol phase) was taken and used for the analysis of

SOD activity and protein assays. All preparation proce-

dures were performed at +4 °C.

Total (Cu-Zn and Mn) SOD (EC 1.15.1.1) activity

was determined according to the method of Sun et al.

(1988). The principle of the method was based on the

inhibition of nitroblue tetrazolium (NBT) reduction by

the xanthine–xanthine oxidase system as a superoxide

generator. GSH-Px (EC 1.6.4.2) activity was measured by

the method of Paglia and Valentine (1967). The MDA

level was determined by a method based on its reaction

with thiobarbituric acid at 90–100 °C (Esterbauer and

Cheeseman, 1990). NO has a half-life of only a few

seconds because it is readily oxidized to nitrite (NO2−)

and subsequently to nitrate (NO3−) that serves as an

index parameter of NO production. Samples were initially

deproteinized with Somogy reagent. The method for

homogenate, nitrite and nitrate levels was based on

Griess reaction. The total nitrite (nitrite + nitrate) was

measured by spectrophotometry at 545 nm after the

conversion of nitrate to nitrite by copperized cadmium

granules (Cortas and Wakid, 1990). The NO level was

expressed as micromole per liter. Protein assays were

made by the method of Lowry et al. (1951).

Serum AST and ALT

The activities of serum AST and ALT were assessed for

liver impairment as well as liver histology. The AST and

ALT activities were determined in serum samples with an

autoanalyser (Syncron LX 20, Ireland) by using commer-

cial Beckman Coulter diagnostic kits.

Histological Evaluation

For histopathological evaluation, different parts of liver

tissues were removed from rats and fixed in 10% neutral

buffered formalin solutions. The tissues were embedded

in paraffin. The paraffin blocks were cut in 5 μm thick



DOI: 10.1002/jat

Table 1. Enzyme activities of liver tissues in control, N-AC, CsA, and CsA + NAC groups in rats

Group (n = 7 SOD GSH-Px MDA NOin each group) (U mg−1 prot.) (U g−1 prot.) (nmol g−1 prot.) (nmol g−1 prot.)

Control (I) 0.236 ± 0.030 0.642 ± 0.188 16.5 ± 2.9 27.2 ± 5.3

NAC (II) 0.229 ± 0.035 0.660 ± 0.238 18.8 ± 4.1 27.5 ± 4.5

CsA (III) 0.142 ± 0.046 0.426 ± 0.103 29.1 ± 11.9 39.2 ± 7.9

CsA + NAC (IV) 0.205 ± 0.063 0.614 ± 0.314 18.1 ± 3.8 25.7 ± 3.7

P comparison table

I–II NS NS NS NS

I–III 0.001 NS 0.002 0.0001

I–IV NS NS NS NS

II–III 0.002 NS 0.008 0.001

II–IV NS NS NS NS

III–IV 0.016 NS 0.005 0.0001

Results were expressed as mean ± standard deviation.

SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; NO, nitric oxide; NAC, N-acetylcysteine; CsA, cyclosporine A; NS,

not significant.

slices and stained with hematoxylin-eosin (H & E), and

all of the sections were examined under light microscope

for characteristic histological changes.

Statistical Analysis

Data were analysed using a commercially available

statistics software package (SPSS® for Windows). Distri-

butions of the groups were analysed with one sample

Kolmogrov–Smirnov test. All groups showed normal

distribution, so that parametric statistical methods were

used to analyse the data. One-way ANOVA test was

performed and post hoc multiple comparisons were done

with LSD. Results were presented as mean ± SD. Values

of P < 0.05 were regarded as statistically significant.

Results

Hepatic damage was assessed by measuring serum AST

and ALT activities and by histological analysis of liver

sections as to indices of damage and necrosis. Measure-

ments of liver SOD and GSH-Px activities as well as MDA

and NO levels served as measures of oxidative stress.

Biochemical Results

Oxidant and Antioxidants

The activities of liver SOD and GSH-Px and the levels

of MDA and NO are presented in Table 1. The depletion

in SOD activity in the liver reflects indirectly the genera-

tion of ROS produced by CsA administration. CsA

produced a decrease in hepatic SOD content compared

with the control group (P < 0.001) and the NAC group

(P < 0.002). Co-administration of NAC and CsA abro-

gated the CsA-induced SOD decrease, compared with the

CsA group (P < 0.016). The activity of SOD was signifi-

cantly higher in the NAC treated groups compared with

the CsA group (P < 0.002).

Despite no significant difference among the groups,

the GSH-Px activities were decreased in the CsA group.

The tissue MDA level of the CsA group was increased

significantly in comparison with those of the control

(P < 0.002) and NAC groups (P < 0.008). NAC treat-

ment prevented this increase (P < 0.005). The NO level

was increased only in the CsA treated groups in compari-

son with the control group (P < 0.0001) and NAC group

(P < 0.001). The level of NO was significantly lower

in the CsA plus NAC group than that of the CsA group

(P < 0.0001).

Serum AST and ALT

In the present study, CsA-induced hepatotoxicity was

characterized by significant increases in serum ALT and

AST activities. Significant elevation in the mean serum

AST and ALT activities were observed in the CsA group,

compared with the control group and NAC group (P <0.0001) (Table 2). Co-administration of NAC with CsA

Table 2. Enzyme activities of serum in control, NAC,CsA and CsA + NAC groups in rats

Group AST (IU l−1) ALT (IU l−1)

Control (I) (n = 7) 57 ± 11 40.3 ± 3.7

NAC (II) (n = 7) 60.7 ± 9.6 41.1 ± 5.6

CsA (III) (n = 7) 221.4 ± 72 122.7 ± 30.7

CsA+NAC (IV) (n = 7) 56.3 ± 11.7 21 ± 6.5

P comparison table

I–II NS NS

I–III 0.0001 0.0001

I–IV NS 0.034

II–III 0.0001 0.0001

II–IV NS 0.027

III–IV 0.0001 0.0001

AST, aspartate aminotransferase; ALT, alanine transaminase. Results were

expressed as mean ± standard deviation.

18 H. KAYA ET AL.


DOI: 10.1002/jat

abrogated the CsA-induced ALT and AST increase com-

pared with the CsA group (P < 0.0001); AST and ALT

activities were close to that of the control group.

Light Microscopic Examinations

In both control (Fig. 1A) and NAC groups, hepatocyte

plates were normal, no ductal dilatation or proliferation or

inflammatory infiltration was observed, and the lobuli

were also regular in shape.

In the CsA group, cytoplasmic changes were observed

especially in the cells around the periportal regions.

Parenchyma around the periportal area showed histopa-

thological alterations including widespread cell swelling

and eosinophilic cell infiltration. The nuclei were moder-

ately pyknotic in many of these cells. In these areas, the

nuclei of some cells disappeared. Focal sinusoidal dilata-

tions were slightly more obvious in the CsA group than

those of the other groups. In addition, a mild congestion

and clusters of mononuclear cells in the surrounding

portal and periportal areas were seen. In some animals of

the CsA group, multifocal necrosis in both central vein

and surrounding areas were noticeable (Fig. 1 B1 and B2).

In the CsA plus NAC groups, marked decreases in

cytoplasmic changes of the hepatocyte and sinusoidal

dilatations around periportal areas were noticed, com-

pared with the CsA group. It was striking that the histo-

logical appearance of parenchyma in the CsA plus NAC

was quite comparable to that of the control and NAC

groups (Fig. 1C).

Discussion

Although several mechanisms have been suggested for

the CsA induced hepatic injury, it is not fully understood

and is still a matter of debate. Some researchers sug-

gested that free radical-mediated reactions might play

primary parts in the toxicity mechanism. These researchers

argue that CsA causes the formation of hydrogen perox-

ide to increase, the mechanism of which is not explained,

leading to a decrease in the ‘reduced glutathione/oxidized

glutathione ratio’ in rat hepatocyte cultures (Wolf et al.,

1994). The results showed that CsA induced hepatic

damage is induced through the action of oxygen free

radicals and lipid peroxidation. CsA is a drug most

frequently used in transplant surgery because of its potent

immunosuppressive action (Sigal and Dumont, 1993). It

has been shown that CsA causes hepatotoxicity in some

transplant recipients (Jazzar et al., 1994) and in some

animal models (Galan et al., 1999; Inselmann et al.,

1994). Previous studies have established that ROS pro-

duction and the oxidative stress situation are involved in

CsA hepatotoxicity (Andrés et al., 2000). Of the many

antioxidants, NAC was chosen, and its protective effects

Figure 1. (A) Light micrograph of rat liver sectionfrom control group. Central vein (CV). (B1) Liver ofcyclosporine A group. Rats were treated with15 mg kg−1 cyclosporine A. Note to sinusoidal dilation(arrow) and congestion (asterisk). Multifocal necroses(circled) are indicated in parenchyma around the cen-tral vein. (B2) Cluster of mononuclear cells (arrowhead)were observed in the multifocal necrosis. (C) Liverhistology after cyclosporine A + N-acetylcysteinetreatments of rats. Note the marked decrease incytoplasmic changes of hepatocytes and other findings.Portal area (PA). All images are stained withhematoxylin-eosin (H & E). This figure is available incolour online at www.interscience.wiley.com/journal/jat

www.interscience.wiley.com/journal/jat



DOI: 10.1002/jat

studied on CsA-induced hepatotoxicity (Andrés et al.,

2000; Andrés and Cascales, 2002). The data confirmed

that there is a relationship between oxidative stress

and hepatotoxicity. Regarding this point, some authors

suggested that CsA-induced oxidative stress was strictly

related to the biochemical parameters responsible for liver

toxicity (Hagar, 2004). In the present study, significant

increases in serum ALT and AST were observed in CsA-

induced hepatotoxicity. It is well known that CsA can

block the ‘permeability transition pore’ (Nicolli et al.,

1996), which results in an accumulation of mitochondrial

Ca2+ (Fournier et al., 1987) and an alteration in the

mitochondrial electron transport chain (Rezzani et al.,

2005). These events cause oxidative phosphorylation

uncoupling (Salducci et al., 1992) and a subsequent

increase in ROS production. The present study demon-

strated that CsA caused hepatic damage in rats, as indi-

cated by elevations of AST, ALT, NO and MDA levels

and by the depletion of SOD and GSH-Px activities, and

by damage and necrosis in the histological analysis of

liver sections. High AST and ALT activities indicated

that there was hepatic injury. Hagar (2004) showed that

CsA-induced hepatotoxicity (20 mg kg−1 body weight

daily for 21 days) results in high AST and ALT activities

in rats.

The possible protective properties of the antioxidant

agent NAC against CsA-induced hepatotoxicity were

studied in this work. NAC was chosen as it is one of the

most effective antioxidants. In addition, NAC has been

demonstrated to function as a direct antioxidant (Bernard

et al., 1984). When ROS begin to accumulate, hepatic

cells exhibit a defensive mechanism through various

antioxidant enzymes. The main detoxifying systems for

peroxides are catalase and GSH (Meister and Anderson,

1983). Catalase is an antioxidant enzyme, which destroys

H2O2, and can form a highly reactive hydroxyl radical

in the presence of iron as a catalyst (Gutteridge, 1995).

By participating in the glutathione redox cycle, GSH

together with GSH-Px convert H2O2 and lipid peroxides

to non-toxic products. Reduced activity of one or more

antioxidant systems due to the direct toxic effects of CsA

leads to an increased lipid peroxidation, oxidative stress

and hepatotoxicity. For example, Duruibe et al. (1989)

found that the total amount of liver GSH content

decreased in CsA-treated rats. Moreover, CsA induced

hepatotoxicity was exacerbated by GSH depletion

(Inselmann et al., 1994). In the current study, although

there was no significant difference among the groups, the

GSH-Px activities were decreased in the CsA group.

NAC has been demonstrated to function as a direct anti-

oxidant that scavenges or quenches oxygen free radicals

with glutathione synthesis (Bernard et al., 1984), the

inhibition of lipid peroxidation, and as an indirect antioxi-

dant that prevents the increase in membrane permeability

resulting from oxidant injury in many tissues including

liver (Fukuzawa et al., 1995; Menasche et al., 1992). It

is believed that increased oxidized glutathione concentra-

tions could modulate the activities of various regulatory

enzymes and might be a cause of the impaired hepatocel-

lular functions induced by CsA as reported by Cadenas

et al. (1983). The increased hepatic MDA level reported

in the CsA-treated group may also implicate oxidative

damage in the liver (Galan et al., 1999).

As seen from our results, antioxidant capacity was

significantly reduced in the hepatic tissues of the animals

treated with CsA as reported previously (Andrés et al.,

2000; Durak et al., 2002). This impairment may result

from enzymatic and/or nonenzymatic parameters. The

decreased activity of SOD as previously demonstrated by

Durak et al. (2002) may play a part in this event. In the

present study, the CsA plus NAC supplemented group

showed a significantly higher level of antioxidant capa-

city compared with the CsA group alone. Therefore, the

administration of NAC has a therapeutic role in prevent-

ing cyclosporine-induced hepatotoxicity as an antioxidant

agent.

Durak et al. (2004) found that when rats were treated

with 25 mg kg−1 day−1 (10 days, orally) CsA, mild con-

nective tissue proliferation in the periportal region and

severe hydropic degeneration in parenchymal cells were

observed in the hepatic tissues. On the contrary, in the

present study, no serious change was observed in the

CsA group, as mentioned above. These differences could

be related to the high dose effects. It was reported that

CsA treatment caused evident morphological alterations,

which included disorganization of hepatic parenchyma,

widespread cell swelling and congestion of sinusoids

(Rezzani et al., 2005). Consistently, our findings were

similar. The treatment with NAC (150 mg kg−1, i.m., for

11 days) considerably prevented the histological damage

induced by CsA injection. In some animals of the CsA

group, multifocal necrosis in both the central vein and

periportal areas were noticed in the present study. These

side effects associated with CsA treatment are numerous

and compromise the immunological system (Cohen,

2002).

It was demonstrated here that NAC treatment appears

to protect liver cells against CsA toxicity. NAC treatment

prevented lipid peroxidation, decreased the activities of

AST and ALT induced by CsA administration to near

control levels, indicating the protection of hepatocytes

from the toxicity. Also, NAC treatment caused prevention

of CsA dependent changes in light microscope evalu-

ations of the liver tissue. It may be thought that NAC

restored the balance between oxidants and antioxidants,

which was disturbed by CsA toxicity in the liver tissue.

In the light of biochemical and microscopic results, it

may be concluded that CsA induced liver injury and

NAC treatment prevented this toxic side effect through

its antioxidant and radical scavenging effects. However,

further investigation is needed to demonstrate the exact

mechanism of NAC in CsA-induced hepatotoxicity.

20 H. KAYA ET AL.


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The protective effect of N-acetylcysteine against cyclosporine A-induced hepatotoxicity in rats