Ameliorative effects of phycocyanin against gibberellic acid induced hepatotoxicity

Accepted Manuscript Title: Ameliorative effects of phycocyanin against gibberellic acid induced hepatotoxicity Author: Mohamed M.A. Hussein, Haytham. A. Ali, Mona M. Ahmed PII: DOI: Reference:

S0048-3575(15)00041-3 http://dx.doi.org/doi:10.1016/j.pestbp.2015.02.010 YPEST 3802

To appear in:

Pesticide Biochemistry and Physiology

Received date: Accepted date:

27-10-2014 25-2-2015

Please cite this article as: Mohamed M.A. Hussein, Haytham. A. Ali, Mona M. Ahmed, Ameliorative effects of phycocyanin against gibberellic acid induced hepatotoxicity, Pesticide Biochemistry and Physiology (2015), http://dx.doi.org/doi:10.1016/j.pestbp.2015.02.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Ameliorative effects of Phycocyanin against Gibberellic acid induced hepatotoxicity Mohamed M.A. Hussein,a* Haytham. A.Ali a, Mona M. Ahmed b

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a

Department of Biochemistry, Faculty of Veterinary Medicine, Zagazig University, 44519,

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Egypt

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b

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Department of Forensic medicine and Toxicology, Faculty of Veterinary Medicine, Zagazig University, 44519, Egypt.

*

Corresponding author.

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Dr. Mohamed M. A. Hussein

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Department of Biochemistry, Faculty of Veterinary Medicine,

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Zagazig University, 44519, Egypt

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Tel :00201120736170

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Email : [email protected]

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Abbreviations: ALP, alkaline phosphatase; ALT, alanine amino transferase; AST, aspartate

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amino transferase ; CAT, catalase; cDNA, complementary deoxy ribonucleic acid ; CU-ZN

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SOD, superoxide dismutase; GA3, gibberellic acid; γGT, gamma glutamyl transferase; GPx,

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glutathione peroxidase; GSH, reduced glutathione; MDA, malondialdehyde; PGRS, plant

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growth regulators; RNA, ribonucleic acid; ROS, reactive oxygen species; RT-PCR, reverse

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transcriptase polymerase chain reaction.

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Highlights -The use of gibberellic acid as a plant growth regulator should take into concern due to its hepatotoxic effects through increase free radicals production that affects not only the activities of antioxidant enzymes but also on the mRNA level of these enzymes. Also, liver cell damage with release of liver enzymes in response to gibberellic toxicity. -Phycocyanin is a good natural product that ameliorates the hepatotoxic effects of gibberellic acid through its ability to scavenge free radical production and increase the activities of anti oxidant enzymes and their mRNA levels with improve liver cell integrity and decreasing levels of liver enzymes. Graphical Abstracts

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Abstract

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Gibberellic acid (GA3) was used extensively unaware in agriculture with sever dangerous

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effects on human health. The current study was designed to investigate the ameliorative

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effects of the co-administration of phycocyanin with GA3 induced oxidative stress and histo

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pathological changes in the liver. Forty male albino rats were randomly divided into four

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groups. Group I (control group) were received normal saline for 6 weeks, Group II (GA3 2

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treated group) were received 3.85 mg/Kg body weight GA3 once daily for 6 weeks, Group III

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(phycocyanin treated group) were received Phycocyanin 200 mg/ Kg body weight /day for 6

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weeks orally dissolved in distilled water and Group IV treated with GA3 and phycocyanin at

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the same doses of group 2&3. All treatments were given daily using intra- gastric intubation

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and continued for 6 weeks. Our results revealed significant down regulation of antioxidant

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enzymes activities and their mRNA levels (CAT, GPx and Cu-Zn,SOD) with marked

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elevation of liver enzymes and extensive fibrous connective tissue deposition with large

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biliary cells in hepatic tissue of GA3 treated rats while treatment with phycocyanin improved

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the antioxidant defense system, liver enzymes beside structural hepatocytes recovery in

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phycocyanin treated group with GA3. These data confirm the antioxidant potential of

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Phycocyanin and provide strong evidence to support the co-administration of Phycocyanin

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during using GA3.

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Keywords: Gibberellic acid, phycocyanin, antioxidant enzymes, RT-PCR

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1. Introduction

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The Plant growth regulators (PGRs) are widely used in Egypt to increase plant size,

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production and to increase plant availability all the year [1]. Gibberllic acid (GA3) is one of

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the most important growth stimulating plant hormone belongs to gibberellins that is

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used for promoting cell elongation, cell division and growth of many plant species[2].

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A growing amount of evidence indicates that GA3 alters the antioxidative

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systems in the rat's tissues as it induced oxidative stress leading to generation of free

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radicals and caused lipid peroxidation , The most important enzymes, superoxide dismutase

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(SOD), glutathione peroxidases (GPx) and catalase (CAT) were also affected [3]. GA3 exerts

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toxic effects on many soft organs including the liver [4]. GA3 treated cells lose their ability to

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scavenge reactive oxygen species (ROS) and this loss ultimately results in oxidative damage

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and cell death [5]. GA3 also induces microabscesses and hydropic degeneration in the liver

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and mononuclear inflammatory infiltration in the kidneys of laboratory mice [6].

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Phycocyanin is a biliprotein pigment found in blue-green algae Spirulina platensis,

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which have attracted attention because of their nutritional value and medicinal properties.

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This pigment has antioxidant, anti-inflammatory and hepatoprotective activity in different

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experimental models [7]. The antioxidant properties of Phycocyanin come from its ability to

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scavenge free radicals and also react with other oxidants of pathological relevance [8]. It is

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well known that ROS are main cause of many important pathological processes including

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inflammatory, neurodegenerative diseases, atherosclerosis, cancer and reperfusion injury [9].

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Antioxidant, free radical scavenging and other beneficial effects are reported for

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phycocyanin, could justify testing phycocyanin as a potential approach to alleviating the

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biochemical, molecular and pathological effects of GA3 on liver tissue.

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2. Materials and methods

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2.1. Chemicals

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Gibberellic Acid, phycocyanin, epinephrine, DTNB (5,5 Dithiobis 2-nitrobenzoic

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acid) and NADPH were purchased from Sigma Aldrich chemical Co., USA. Potassium

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dichromate, hydrogen peroxide, glacial acetic acid and EDTA were purchased from El-Nasr

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Company, Cairo, Egypt. Kits of alanine amino transferase (ALT), Aspartate aminotransferase

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(AST), Alkaline phosphatase (ALP) and gamma glutamyl transferase (γ GT) were obtained

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from spectrum kits, Egyptian company for biotechnology, Cairo, Egypt.

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2.2. Animal management

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Healthy adult forty male albino rats, weighting 120-150 g were obtained from the

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Animal House in Faculty of Veterinary Medicine, Zagazig University, Sharkia, Egypt. Rats

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were give free access to food and water with12h/12h dark light cycle. All animals were left

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2weeks for adaptation in standard cages under controlled conditions with free access to food

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and water. The European community Directive (86/609/EEC) and national rules on animal

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care have been followed.

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2.3 Experimental design

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After the period of acclimation, rats were randomly segregated into four groups (10 rats

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per group). Group I (control group) were received normal saline for 6 weeks. Group II (GA3

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treated group) were received 3.85 mg/Kg body weight GA3 once daily for 6 weeks [10]. 4

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Group III (phycocyanin treated group) were received Phycocyanin 200 mg/kg body

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weight/day for 6 weeks orally dissolved in distilled water [7]. Group IV treated with GA3 and

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phycocyanin at the same doses of group 2&3. All treatments were given daily using intra-

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gastric intubation and continued for 6 weeks.

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2.4 Sampling

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At the end of experimental period, rats from all groups were fasted for overnight and

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blood samples were collected into chilled non-heparinized tubes and centrifuged at 860 xg for

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20 min at 4°C. The separated sera were frozen at - 20°C for biochemical analysis. After the

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collection of blood samples, animals were sacrificed and samples from the liver tissue were

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accurately weighed and homogenized using tissue homogenizer (Potter–Elvehjem) using

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chilled potassium chloride (1.17%) for measure antioxidant status. To follow up changes in

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antioxidant enzymes gene expression, liver samples were collected in liquid nitrogen

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container until time of RNA extraction. Meanwhile, small parts of the liver were fixed in

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neutral formalin solution for histopathological examination.

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2.5. Biochemical analysis

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The sera were used for the determination of some liver enzymes such as ALT (EC 2.6.1.2)

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and AST (EC 2.6.1.1) activities using commercial assay kits according to the methods

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described by Breuer [11]. ALP (EC 3.1.3.1) and γ-GT (EC 2.3.2.2) were measured using

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commercial kits according to Moss et al [12]. The post mitochondrial fraction of liver was

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prepared according to Kim et al [13]. In brief, liver samples were homogenized by tissue

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homogenizer using chilled potassium chloride (1.17%). The nuclear debris were separated by

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centrifugation at 8000×g, 4◦C for5 min. Supernatant was again centrifuged for 20 min at

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10 500 × g, 4◦C to get the post-mitochondrial supernatant which was used to assay

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malondialdehyde (MDA) Lipid peroxidation marker , CAT( EC 1.11.1.6), SOD (EC 1.15.1.1),

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GPx (EC 1.11.1.9) and reduced glutathione (GSH). The level of MDA in liver homogenate

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was determined spectrophotometrically according to Nair and Turner [14]. The activity of

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SOD in the liver tissues was determined spectrophotometrically at wave length 480 nm by

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epinephrine method and expressed in units of enzymes activities per gram of tissues wet wt

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[15]. CAT activity in the liver was determined spectrophotometrically at wave length 570 nm

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according to the method of Sinha [16]. GPx activity was determined by the method of

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[17].GSH levels were determined according to the method of [18]. The optical densities of the

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given parameters were measured by Shimadzu type spectrophotometer (UV 120-02).

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2.6. Semi-quantitative RT-PCR

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Total RNA was extracted from liver with RNeasy Mini kit (Qiagen) following the

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manufacturer's instructions. The amount of extracted RNA was quantified and qualified using

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NanoDrop® ND-1000 Spectrophotometer, NanoDrop Technologies, Wilmington, Delaware

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USA. The purity of RNA was checked and ranged between 1.8 and 2.1, demonstrating the

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high quality of the RNA. The cDNAs were synthesized using Revert-Aid™ First Strand

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cDNA Synthesis Kit (Qiagen, Hilden, Germany) [19]. Equal amounts of the reverse

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transcriptional products (1 µg cDNA) were subjected to PCR amplification in a volume of

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50 µl using gene specific primers [20]. (Table I), 2X PCR Master Mix from (Fermentas,

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Cairo/Egypt) following the manufacturer instructions. β- actin was used as an internal control

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for PCR. Amplification was started with 5 min of denaturation at 94°C followed by 28 cycles

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consisted of 60 sec at 95°C, 55°C, 1 min; 72°C, 1 min for all genes were placed in 2720

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thermocycler (Applied Biosystems, USA). The final extension was for 7 min at 72°C. A single

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major band for each gene was detected by electrophoresis on a 1.5% agarose/ethidium

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bromide (0.25 µg /ml) gel. The intensity of the bands was compared to those collected from

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the control group using the public domain NIH Image program (National Institutes of Health,

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Bethesda, Maryland). mRNA expressions level of antioxidant enzymes were expressed as

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mean ± standard error (SE).

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2.7. Histopathological Examination

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The liver of male albino rats was collected from the different groups after 6 weeks. The

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samples were fixed in Bouin’s solution, then dehydrated in ascending grades of alcohols,

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cleared in xylene and embedded in paraffin. The samples were casted, then sliced into 5µm in

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thickness and placed on to glass slides. The slides were stained by general and specific stains

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[21].

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2.8. Statistical analysis

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Data are expressed as mean values ± SE of 10 rats. Statistical analysis was performed

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using one way analysis of variance (ANOVA) using SPSS statistical version 18 software

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package (SPSS, Inc, USA). Duncan’s test was used for making a multiple comparisons

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among the groups for testing the inter-grouping homogeneity.

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3. Results

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3.1. The changes in liver function enzymes in sera of experimental rats

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The results indicated that GA3 treated group showed significant increase in these

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enzymes (P< 0.05) as compared to control animals. However, co- administered of

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phycocyanin with GA3 showed significant decrease (P< 0.05) of ALT, AST, ALP and γ GT

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as compared to control rats (table 2).

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3.2. Oxidative and antioxidant status in liver homogenate of experimental rats

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role of phycocyanin after 6 weeks treatment as shown in table 3.GA3 administration showed

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significant increase in MDA level in comparison with control group meanwhile phycocyanin

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administration with GA3 in protective group return level near to control group. On the other

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hand the activity of catalase, GPx and GSH level showed significant marked decrease in GA3

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treated group when compared to control group but on treatment of phycocyanin with GA3

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these level return to normal level confirming the antioxidant of phycocyanin during GA3

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toxicity.

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The changes in antioxidant status in liver homogenate of rats treated with GA3 and

3.3. The changes in CAT, Cu-Zn SOD and GPx gene expression in experimental rats

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Present results revealed that GA3 produce statistically significant decrease (P˂ 0.05)

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in mRNA level of CAT, Cu-Zn SOD and GPx when compared to control values (Fig.1). The

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co administrated of phycocyanin with GA3 leads to return these mRNA level near to control

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group decreasing the devastating effect of GA3 toxicity.

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3.4. Histopathological findings

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The liver of control and phycocyanin treated groups showed normal hepatocytes with

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its sinusoidal architecture (Fig. 2) but on GA3 administrated group, extensive fibrous

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connective tissue was shown with scanty basophilic cytoplasm (Fig. 2). On the other hand co-

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administration of GA3 and phycocyanin showed scanty fibrous C.T. in portal tract with no

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evidence of cirrhosis (Fig 2). 26

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4. Discussion

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Although, GA3 has been caused alarming toxicity to mammalian system, it still used

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extensively in many countries [22]. For example, in Egypt it widely used to increase the

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growth of some fruits such as strawberries and grapes and some vegetables such as tomatoes,

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cabbages and cauliflower [23]. Therefore in the present study we cast the light on the role of

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natural component, phycocyanin in detracting the harmful impact of GA3 on the liver. As the

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liver is considered the key organ of metabolism and excretion is constantly endowed with the

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task of detoxification of xenobiotics, environmental pollutants and chemotherapeutic agents

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[24].

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On the level of liver enzymes, GA3 induced significant elevations of serum AST,

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ALT, ALP and γ-GT. This elevation of liver enzymes in GA3 treated group indicated the loss

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of functional integrity of liver cell membrane that allows cellular leakage [25-26].

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The histopathological changes showed sever hepatic damage in GA3 treated group

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with excessive fibrosis and destructed biliary cells membrane owed to the extensive release

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of reactive oxygen species (ROS) via oxidative stress as there was imbalance between the

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production of free radicals and antioxidant defense system [27]. Mean while, the co-

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administration of phycocyanin with GA3 ameliorate these biochemical hazards and improve

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cell integrity through its ability to scavenge free radicals and inhibit lipid peroxidation as its

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major water soluble antioxidant constituent in Spirulina with about 20 times more efficient

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than vitamin C [28-29].

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In fact, reactive oxygen species (ROS) can attack biomolecules, such as DNA, lipids,

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and thiols in proteins and glutathione leading to inactivation of enzymes, genotoxic damage,

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cell dysfunction, and cell death [30]. The antioxidant enzymes, CAT, SOD and GPx which

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play a significant role in protecting cells from oxidant stress have been shown to be sensitive

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indicators [31].

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It is widely accepted that the induction of antioxidant enzymes is a major strategy for

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protecting cells against a variety of endogenous and exogenous toxic compounds such as

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ROS and chemicals [32]. In seeking to improve health, phycocyanin is proposed to be safe

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for human and animal health [33].

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In the current study, administration of GA3 for 6 weeks was found to induce a state

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of oxidative stress represented by significant elevation of MDA with significant reduction of

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SOD, CAT and GPx enzymes activities and GSH level in hepatic tissue of treated rats in

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comparison to other groups. The accelerated level of lipid peroxidation marker (MDA)

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attributed to the formation of OH radicals that may react with lipids possibly by hydrogen

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abstraction leading to oxidative damage within the cell [34].

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The deleterious decreasing activities of hepatic CAT, SOD and GPx with surplus

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level of lipid peroxidation marker (MDA) in GA3 treated group either to increase superoxide

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and hydrogen peroxide radicals generation or to exhaustion of the enzymes which is the

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results of increase peroxidation status[10] .

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The co-administration of phycocyanin with GA3 treated group showed amelioration of

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hepatic antioxidant enzymes (CAT, SOD and GPx) activities near to the normal level and 8

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these results come in harmony with [35] who reported that the administration of phycocyanin

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to CCL4 treated mice could significantly elevate the activities of GPx and SOD through its

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ability to act as chain breaking antioxidant and as chemical scavengers of oxygen radicals as

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phycocyanin have ability to repair oxidizing radical directly and preventing the chain

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propagation step during lipid peroxidation.

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At the level of gene expression oral administration of GA3 showed significant down

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regulation of CAT, SOD and GPx mRNA in the liver tissue compared to control group and

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this down regulation was ameliorated by on co-administration of phycocyanin with GA3

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indicating the antioxidant property of phycocyanin. These results were on similar ground of

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[36] who suggested that the direct oxidative stress evidenced by increased lipid peroxidation

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and decreased GSH content and indirect pathway expressed by the down-regulation of

12

phospholipids, hydroperoxide and glutathione peroxidase gene expression in liver of

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aflatoxicosis in mice as Free radicals at low levels can induce antioxidant enzymes such as

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SOD, CAT and GPx however, when the free radicals are in excess of the antioxidant capacity

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it becomes inhibitory to these enzymes and factors.

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The result of local and global variation in the redox conditions within the cell may cause

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a drastic modulation of the oxidized / reduced ratio of signaling protein such as transcription

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factors that affect on antioxidant enzyme mRNA[37].

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The hepatoprotective effect of phycocyanin come from its ability to ameliorate ROS that

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leads to lipid peroxidation and cause damage to cell membrane through inhibit

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cyclooxygenase-2 which significantly inhibit liver microsomal lipid peroxidation and protect

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liver from oxidative stress [38].

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5. Conclusion

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The present study conclusively demonstrate the biochemical hazards of indiscriminate

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use of GA3 on hepatic tissue and the role of phycocyanin in withstand these challenges

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through scavenge free radicals and lessens to a great extent the severity of oxidative damage

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induced.

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Conflict of interest statement

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The author declares that there are no conflicts of interest.

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[35] A.M. Hassan, S.H. Abdel-Aziem, M.A. Abdel-Wahhab, Modulation of DNA damage

21

and alteration of gene expression during aflatoxicosis via dietary supplementation of

22

spirulina and whey protein concentrates. Ecotoxicology and Environmental Safety

23

79(2012) 294-300.

24 25

[36] Y. Morel, R. Barouki, Repression of gene expression by oxidative stress, Biochem J. 342(1999) 481–496.

26

[37] C.M. Reddy, V.B. Bhat, G. Kiranmai, M.N. Reddy, P. Reddanna, K.M. Madyastha,

27

Selective inhibition of cyclooxygenase-2 by C- phycocyanin, a biliprotein from Spirulina

28

platensis. Biochem. Biophys. Res. Commun. 277(2000) 599–603.

29 30 31

Fig.1. RT-PCR analysis of

CAT (a), Cu, Zn SOD (b) and

32

administration of either Phycocyanin or GA3 alone or together in rats. Phycocyanin and

33

GA3 were administered for 6 w e e k s as described in materials and methods. RNA

12

GPx (c) expression

Page 12 of 18

1

was extracted and reverses transcribed (1µg) and RT-PCR analysis was carried out for

2

these genes. Densitometry analysis was carried for 3 different rats.

3 4 5 6 7 8 9

Fig.2. Photomicrograph of male albino rat's liver of control (A) and phycocyanin (C) show;

10

normal hepatocytes and sinusoidal architecture x100 H&E. The liver of GA3 group (B) show;

11

focal replacement of portal area with extensive fibrous C.T. with large biliary cells and

12

destructed membrane with scanty basophilic cytoplasm while liver of rat's co administrated

13

of phycocyanin and GA3 (D) show; scanty fibrous C.T. proliferation (arrows) in the portal

14

tract forming incomplete bridge with no evidence of cirrhosis.

15 16 17 18 19 20 21 22 23 24 25 26 27

13

Page 13 of 18

1 Gene

Oligonucleotide sequences

Product length (bp) 272

Gene ID

CAT

F R

5'-GTCCGATTCTCCACAGTCGC-3' 5'-CGCTGAACAAGAAAGTAACCTG-3'

S50336.1

Cu-Zn SOD

F R

5'-ATGGGGACAATACACAAGGC-3' 5'-TCATCTTGTTTCTCGTGGAC-3'

225

AH004967.1

GPx

F R

5'-CACAGTCCACCGTGTATGCC-3' 5'-AAGTTGGGCTCGAACCCACC-3'

292

Z21917.1

ß-actin

F R

5'-CATAGCTCTTCTCCAGGGAG -3' 5'-AGGGTGTGATGGTGGGTATG -3'

600

NM_007393

2 3 4 5 6 7 8 9 10 11

Table 1. Primer sequences of rat's GPx, CAT, Cu Zn SOD and ß-actin with its product size F indicates forward primer; R indicates reverse primer.

12 13 14 15 16 17 18 19

14

Page 14 of 18

1 2 3 4 5 6 7 8 9 10

Table 2

11

Alanine aminotransferase (ALT), aspartate amino transferase (AST), alkaline phosphatase

12

(ALP) and gamma glutamyl transferase (γ GT) activities in serum of experimental rats

13 14

Treatment Control group

ALT (U/L) 34.27±1.96c

AST (U/L) 80.12±2.36c

ALP (U/L) 46.26±2.6c

γ GT (U/L) 12.21±0.6c

GA3 treated Group

120.23±2.41a

220.17±4.3a

117.3±4.15a

80.42±1.34a

Phycocyanin treated group

30.02±0.92d

74.19±3.29d

38.13±2.71d

10.3±1.21d

GA3+ phycocyanin 61.21±1.47b 99.19±6.82b Treated group Each value is a mean ±SE for group of n=10

10.3±1.22d

46.12±3.62b

15

a,b,c,d

16

different at (P˂ 0.05)

Means within the same column and bearing different superscripts are significantly

17 18 19 20 21 22

15

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1 2 3 4 5 6 7 8 9 10

Table 3

11

Malondialdehyde (MDA), reduced glutathione (GSH) level with activities of catalase (CAT),

12

superoxide dismutase (SOD) and glutathione peroxidase (GPx) in liver homogenate of

13

experimental rats Treatment

Control group

14 15

MDA (µmol/ gm tissue) 12.1± 1.36c

GSH (mg/gm tissue) 18.15±1.55b

CAT (µmol H2O2decompos ed /gm tissue) 109.6±2.51d

SOD (U/gm tissue) 20.23±1.07b

GPx (µmol NADPH /gm tissue) 18.7±1.02b

82.7±4.85c

14.25±1.01c

11.6±1.34d

GA3 treated group

28.13±1.46a 6.63±0.52c

Phycocyanin treated group

11.94±2.54c 21.17±1.85a 122.7±3.68a

30.3±1.33a

23.1±1.9a

GA3+ phycocyanin treated group

18.3±1.39b

19.89±2.15b

16.31±1.04c

17.14±1.69b 106.3±3.87b

Each value is a mean ±SE for group of n=10.

16

a,b,c,d

17

different at (P˂ 0.05).

Means within the same column and bearing different superscripts are significantly

18 19 20

16

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1 2 3 4

18

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