Toxicological

Communication

Biosci. Biotech. Res. Comm. 10(2): 165-172 (2017)

The protective effect of chlorogenic acid on arsenic

trioxide induced hepatotoxicity in mice

Javad Ghahhari

, Gholamhassan Vaezi

, Gholamhossein Riazi

, Ali Abbasi

, Mona

Modanloo

and Mohammad Shokrzadeh

Department of Biology, Damghan Branch, Islamic Azad University, Semnan, Iran

Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

Department of Pathology, Sari Branch, Islamic Azad University, Sari, Iran

Pharmaceutical Sciences Research Center, Mazandaran University of Medical Sciences, Sari, Iran.

Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical

Sciences, Sari, Iran

ABSTRACT

Several studies have shown that chronic exposure to arsenic trioxide risk factor for many cancers such as lung, liver,

kidney and bladder. Free radicals in various ways such as lipid peroxidation, protein oxidation and DNA damage that

leads to many diseases. Chlorogenic acid, an antioxidant plant and prevent many diseases caused by oxidative stress

such as cancer. In this study, the protective effect of chlorogenic acid on arsenic trioxide-induced liver toxicity were

studied. Biochemical parameters including Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate

aminotransferase (AST), and Glutathione (GSH) were assessed for liver damage.In compare to positive control group

(arsenic trioxide, 10 mg/kg), the serum levels of ALP, ALT, and AST have signi cantly decreased (p<0.05) and the

level of GSH has signi cantly increased (p<0.05) in the groups administered with chlorogenic acid (10, 50, and 100

mg/kg).The results showed that chlorogenic acid has signi cant protective effects against the hepatotoxicity caused

by arsenic trioxide.

KEY WORDS: ALANINE AMINOTRANSFERASE, ALKALINE PHOSPHATASE, ARSENIC TRIOXIDE, ASPARTATE AMINOTRANSFERASE,

GLUTATHIONE

165

ARTICLE INFORMATION:

*Corresponding Author:

Received 1

March, 2017

Accepted after revision 19

June, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

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166 THE PROTECTIVE EFFECT OF CHLOROGENIC ACID ON ARSENIC TRIOXIDE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Javad Ghahhari et al.

INTRODUCTION

Exposure to arsenic trioxide take place on variety ways,

for example, fuel, drinking water, pesticides, air, and

food that is a threat to human health (Holt et al., 2009;

Mittal et al., 2012). Researches have shown that expo-

sure to arsenic can cause acute and chronic effects on

different organs of the body, like cardiovascular sys-

tem, digestive system, respiratory system, and nervous

system(Centeno et al., 2002; Abernathy et al., 2003).

Kidney and liver are the most important organs for met-

als and have the highest level of metallothionein bind-

ing to metals (Sarvankumar et al., 2011). Liver is an

important site for the metabolism of arsenic trioxide and

chronic exposure to arsenic trioxide cause liver disease

(Flora et al., 2007). Arsenic toxicity is due to its ability

to react with sulfhydryl groups in proteins and enzymes

and can be replaced phosphorus in many biochemical

reactions (Stevens et al., 2010, Šeruga and Tomac, 2014

and Aoyama and Nakaki, 2015).

Damages caused by arsenic in the liver are deter-

mined by changes in liver enzymes, including Alkaline

phosphatase (ALP), Alanine aminotransferase (ALT), and

Aspartate aminotransferase (AST) (De Azevedo et al.,

2008). Free radicals can lead to various injuries at cells,

such as DNA damage, inhibition of mitochondrial respi-

ratory chain enzymes, and membrane lipid peroxidation

(Umamaheswari and Chatterjee, 2008). The free radicals

by creating oxidative stress and toxic oxidative leads to

various diseases (Halliwell and Whiteman, 2004). Anti-

oxidants by neutralizing free radicals prevent damages

caused by free radicals in the body organs (Gradecka

et al., 2001). Glutathione synthesis of glutamate, cysteine,

and glycine has done with the help of two enzymes,

including glutamylcysteine synthetase and glutathione

synthetase. The liver is an important location for manu-

facturing and exporting of glutathione (Wu et al., 2004).

Glutathione is caused failure of reactive oxygen species

such as lipid peroxyl radical, peroxynitrite, hydroxyl

radical, and other free radicals (Fang et al., 2002). Glu-

tathione de ciency leads to oxidative stress. Oxidative

stress plays an important role in various diseases such

as cancer, diabetes, Alzheimer’s disease, kwashiorkor,

seizures, liver disease, and Parkinson’s disease (Wu et

al., 2004) .

Researches have shown that phenolic compounds as

natural antioxidants play an important role in protect-

ing the body’s cells against the toxic oxidative (Bralley et

al., 2008). Chlorogenic acid as a polyphenol compound

is an ester between caffeic acid and quinic acid, and

there is in many foods like coffee and apple. Chlorogenic

acid is a strong antioxidant that has pharmacological

properties like anti cancer and antibacterial (Um et al.,

2006; Das et al., 2012). The present study evaluates the

protective effects of chlorogenic acid on the liver toxic-

ity caused by arsenic trioxide in vitro by assessing the

biochemical parameters including Alkaline phosphatase

(ALP), Alanine aminotransferase (ALT), Aspartate ami-

notransferase (AST), and Glutathione (GSH).

MATERIAL AND METHODS

42 male mice (27 ± 2 g) procured from the animal house

of the Mazandaran University of Medical Sciences, Sari,

Iran. They were maintained in a controlled environment

(12 h light/dark cycles) and temperature (28 ± 1°C).

The mice were fed with drinking water and standard

diet.

Chlorogenic acid was obtained from Sigma-Aldrich

Company (USA). Arsenic trioxide was purchased from

Merck Company (Germany).Mice were divided in 7

groups and 6 mice in each group. In these experiments,

the effects of intraperitoneal administration of different

doses of chlorogenic acid on the biochemical param-

eters of the kidney were investigated. The  rst group

was administered with normal saline (0.9%) (10 mg/

kg) as control, the second group was administered with

arsenic trioxide (10 mg/kg) as positive control, the third

group was administered with chlorogenic acid (100 mg/

kg) as negative control, and the fourth to the seventh

groups were administered with different doses of chlo-

rogenic acid (5, 10, 50, 100 mg/kg), then after 2 hours

the fourth to the seventh groups were administered with

arsenic trioxide (10 mg/kg) (Sabath and Robles-Osorio,

2012).

Blood samples were collected of heart with the

syringe, then transferred to centrifuge tubes, and cen-

trifuged at 4000 rpm for 15 minutes. After centrifuging,

the serum was separated and stored in the refrigerator

(Tapio and Grosche, 2006). The level of serum Alkaline

phosphatase (ALP), Alanine aminotransferase (ALT),

and Aspartate aminotransferase (AST) were measured

on a chemistry auto analyzer (Tchounwou et al., 2004).

Homogenized 0.1 g of liver tissue sample with 1ml assay

buffer (EDTA) was taken then, its contents were trans-

ferred to centrifuge tubes and 0.5 ml EDTA was added

to those. In the next step, 1.5 ml TCA 10% was added to

centrifuge tubes. Centrifuge samples for 15 min at 3000

rpm. Remove 1 ml of supernatants and place in new

tubes. Then, was added 2.5 ml Tris buffer (0.4 M) and 0.5

DTNB. Absorbance was measured of solutions at 412 nm

with spectrophotometry (Saeedi Saravi and Shokrzadeh,

2008; Shokrzadeh et al., 2015). The data were analyzed

with SPSS 16 software. Statistical analysis of data was

carried out with one way analysis of variance and Tukey

test. The differences were considered signi cant at p <

0.05.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE PROTECTIVE EFFECT OF CHLOROGENIC ACID ON ARSENIC TRIOXIDE 167

Javad Ghahhari et al.

RESULTS AND DISCUSSION

The serum level of ALP has signi cantly increased

(p<0.05) in the groups administered with of chlorogenic

acid (5, 10, 50, and 100 mg/kg) when compared to the

control group (normal saline, 10 mg/kg) (Figure 1). The

serum level of ALP has signi cantly decreased (p<0.05)

in the groups administered with of chlorogenic acid (10,

50, and 100 mg/kg) when compared to the positive con-

trol group (arsenic trioxide, 10 mg/kg) but didn’t show

signi cant difference in dose of 5 mg/kg (Figure 2). The

serum level of ALP has signi cantly increased (p<0.05)

in the groups administered with of chlorogenic acid (5,

10, 50, and 100 mg/kg) when compared to the negative

control group (chlorogenic acid, 100 mg/kg) (Figure 3).

The serum level of ALT has signi cantly increased

(p<0.05) in the groups administered with of chlorogenic

acid (5, 10, 50, and 100 mg/kg) when compared to the

control group (normal saline, 10 mg/kg) (Figure 4). The

serum level of ALT has signi cantly decreased (p<0.05)

in the groups administered with of chlorogenic acid (10,

50, and 100 mg/kg) when compared to the positive con-

trol group (arsenic trioxide, 10 mg/kg) but didn’t show

signi cant difference in dose of 5 mg/kg (Figure 5). The

serum level of ALT has signi cantly increased (p<0.05)

in the groups administered with of chlorogenic acid (5,

10, 50, and 100 mg/kg) when compared to the negative

control group (chlorogenic acid, 100 mg/kg) (Figure 6).

The serum level of AST has signi cantly increased

(p<0.05) in the groups administered with of chlorogenic

acid (5, 10, 50, and 100 mg/kg) when compared to the

control group (normal saline, 10 mg/kg) (Figure 7). The

serum level of AST has signi cantly decreased (p<0.05)

in the groups administered with of chlorogenic acid (10,

50, and 100 mg/kg) when compared to the positive con-

trol group (arsenic trioxide, 10 mg/kg) but didn’t show

signi cant difference in dose of 5 mg/kg (Figure 8). The

serum level of AST has signi cantly increased (p<0.05)

in the groups administered with of chlorogenic acid (5,

10, 50, and 100 mg/kg) when compared to the negative

control group (chlorogenic acid, 100 mg/kg) (Figure 9).

The level of GSH has signi cantly decreased (p<0.05)

in the groups administered with of chlorogenic acid (5,

10, 50, and 100 mg/kg) when compared to the control

group (normal saline, 10 mg/kg) (Figure 10). The level of

GSH has signi cantly increased (p<0.05) in the groups

administered with of chlorogenic acid (10, 50, and 100

mg/kg) when compared to the positive control group

FIGURE 1. The serum level of ALP has signi cantly increased (p<0.05)

in the groups administered with of chlorogenic acid (5, 10, 50, and

100 mg/kg).

FIGURE 2. The serum level of ALP has signi cantly decreased (p<0.05)

in the groups administered with of chlorogenic acid (10, 50, and 100

mg/kg).

168 THE PROTECTIVE EFFECT OF CHLOROGENIC ACID ON ARSENIC TRIOXIDE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Javad Ghahhari et al.

FIGURE 3. The serum level of ALP has signi cantly increased (p<0.05)

in the groups administered with of chlorogenic acid (5, 10, 50, and 100

mg/kg).

FIGURE 4. The serum level of ALT has signi cantly increased (p<0.05)

in the groups administered with of chlorogenic acid (5, 10, 50, and 100

mg/kg).

FIGURE 5. The serum level of ALT has signi cantly decreased (p<0.05)

in the groups administered with of chlorogenic acid (10, 50, and 100

mg/kg).

(arsenic trioxide, 10 mg/kg) but didn’t show signi cant

difference in dose of 5 mg/kg (Figure 11). The level of

GSH has signi cantly decreased (p<0.05) in the groups

administered with of chlorogenic acid (5, 10, 50, and 100

mg/kg) when compared to the negative control group

(chlorogenic acid, 100 mg/kg) (Figure 12).

Arsenic compounds are one of the pollutants of envi-

ronment which are a serious threat to human health.

Millions of people around the world through drinking

water are exposed to arsenic compounds (Sabath and

Robles-Osorio, 2012). Exposure to arsenic and its com-

pounds can have dangerous effects on health (Tapio and

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE PROTECTIVE EFFECT OF CHLOROGENIC ACID ON ARSENIC TRIOXIDE 169

Javad Ghahhari et al.

FIGURE 6. The serum level of ALT has signi cantly increased (p<0.05)

in the groups administered with of chlorogenic acid (5, 10, 50, and 100

mg/kg).

FIGURE 7. The serum level of AST has signi cantly increased

(p<0.05) in the groups administered with of chlorogenic acid (5, 10,

50, and 100 mg/kg).

FIGURE 8. The serum level of AST has signi cantly decreased (p<0.05)

in the groups administered with of chlorogenic acid (10, 50, and 100

mg/kg).

Grosche, 2006) and can lead to many types of cancers,

such as skin, liver, kidney, lung, intestine, and bladder

(Tchounwou et al., 2004). Inorganic arsenic compounds

as carcinogenic compounds are known. Epidemiological

studies have shown that these compounds are related

to the types of cancers like liver, kidney, and bladder

(Gradecka et al., 2001). Researches have shown that con-

sumption of drinking water containing inorganic arsenic

compounds is related to liver diseases such as liver can-

cer (Islam et al., 2011). In clinical studies of the liver,

liver enzymes, including Alkaline phosphatase (ALP),

Alanine aminotransferase (ALT), and Aspartate ami-

Javad Ghahhari et al.

170 THE PROTECTIVE EFFECT OF CHLOROGENIC ACID ON ARSENIC TRIOXIDE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 9. The serum level of AST has signi cantly increased (p<0.05)

in the groups administered with of chlorogenic acid (5, 10, 50, and 100

mg/kg).

FIGURE 10. The level of GSH has signi cantly decreased (p<0.05) in

the groups administered with of chlorogenic acid (5, 10, 50, and 100

mg/kg).

FIGURE 11. The level of GSH has signi cantly increased (p<0.05) in the

groups administered with of chlorogenic acid (10, 50, and 100 mg/kg).

notransferase (AST) are evaluated. The enzymes levels

of ALT and AST are an indicator to assess the integrity

of liver cells and the enzyme level of ALP represents the

perfect synthesis of albumin and bile by the liver (Islam

et al., 2011). Our research has shown that the serum lev-

els of ALP, ALT, and AST have signi cantly increased

(p<0.05) in all groups administered with of chlorogenic

acid when compared to the control and negative control

groups. Antioxidants are including two major catego-

ries, enzymatic and non enzymatic. Enzymatic antioxi-

dants like catalase, glutathione peroxidase, and super-

oxide dismutase, which are produced by the body and

non enzymatic antioxidants like  avonoids, tannins,

and carotenoids that obtained from plants (Lee et al.,

2004). Phenolic compounds as antioxidants in plants are

caused by the elimination of free radicals. The antioxi-

dant properties of phenolic compounds are related to

the characteristic of the redox (oxidation and reduction)

that reduce free radicals. Also, they are caused chelat-

ing of metals (Hsu, 2006). There is chlorogenic acid

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE PROTECTIVE EFFECT OF CHLOROGENIC ACID ON ARSENIC TRIOXIDE 171

Javad Ghahhari et al.

as a polyphenolic compound widely in various plants

(Šeruga and Tomac, 2014).

Studies, on the health bene ts of consuming chloro-

genic acid found in foods have shown that this antioxi-

dant to reduce diseases such as cancer, stroke, obesity,

diabetes, and Alzheimer’s disease (Zhao et al., 2012). The

serum levels of ALP, ALT, and AST have signi cantly

decreased (p<0.05) in the groups administered with of

chlorogenic acid (10, 50, and 100 mg/kg) when com-

pared to the positive control group (arsenic trioxide, 10

mg/kg). It sounds that the antioxidant property of chlo-

rogenic acid plays an important role in the protection of

cells against free radicals caused by arsenic trioxide. The

free radical is an atom or molecule that has an unpaired

electron. The cells use of oxygen for produce energy that

lead to the production of free radicals such as super-

oxide anion, hydroxyl, and peroxyl that are known as

reactive oxygen species (ROS) and mainly produced by

mitochondria. Free radicals can lead to a wide range of

toxic oxidative reactions like membrane lipid peroxi-

dation, the failure of enzymes and proteins that could

cause cell death (Umamaheswari and Chatterjee, 2008).

Free radicals cause oxidative stress that plays an impor-

tant role in the development of many diseases such as

diabetes, cardiovascular, and cancer (Bandyopadhyay et

al., 1999). The studies show that many metals such as

iron (Fe), lead (Pb), and copper (Cu) with modulating

the redox (oxidation and reduction) in cell are causing

the level change of thiols like glutathione in the cells

(Tchounwou et al., 2002). The glutathione (GSH) as a

thiol compound and important in cells have antioxidant

role and are caused destroy of free radicals. The glu-

tathione (GSH) is in various body organs such as liver,

kidney, brain, pancreas, and heart and highest levels of

that found in the liver (Aoyama and Nakaki, 2015).

In this study, the level of GSH has signi cantly

decreased (p<0.05) in the groups administered with of

chlorogenic acid (5, 10, 50, and 100 mg/kg) when com-

pared to the control and negative control groups that

was speci ed arsenic trioxide reduced GSH in the liver

but the level of GSH has signi cantly increased (p<0.05)

in the groups administered with of chlorogenic acid

(10, 50, and 100 mg/kg) when compared to the posi-

tive control group that was identi ed chlorogenic acid

as an antioxidant reduces free radicals and increases

the glutathione level of the liver. The glutathione (GSH)

with formation reversible disul de bonds between thiols

of protein can inhibit the oxidation of proteins during

oxidative stress (Giustarini et al., 2004). The glutath-

ione (GSH) as an antioxidant have an important role

in balancing of intracellular redox processes (Aoyama

and Nakaki, 2015). Overall, our results showed that chlo-

rogenic acid as a powerful and important antioxidant

had signi cant protective effects against the toxicity of

arsenic trioxide.

ACKNOWLEDGMENT

We appreciate Of all colleagues who have worked on this

project with us include the Laboratory of Toxicology in

the Faculty of Pharmacy of Mazandaran University of

Medical Sciences and Noor medical laboratory.

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