Hepatotoxicity and oxidative stress induced by Naja haje crude … · 2015-09-17 · RESEARCH Open Access Hepatotoxicity and oxidative stress induced by Naja haje crude venom Saleh

Al-Quraishy et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2014, 20:42http://www.jvat.org/content/20/1/42

RESEARCH Open Access

Hepatotoxicity and oxidative stress induced byNaja haje crude venomSaleh Al-Quraishy1†, Mahamed A Dkhil1,2† and Ahmed Esmat Abdel Moneim2,3*†

Abstract

Background: Snake venoms are synthesized and stored in venom glands. Most venoms are complex mixtures ofseveral proteins, peptides, enzymes, toxins and non-protein components. In the present study, we investigated theoxidative stress and apoptosis in rat liver cells provoked by Naja haje crude injection (LD50) after four hours.

Methods: Wistar rats were randomly divided into two groups, the control group was intraperitoneally injected withsaline solution while LD50-dose envenomed group was intraperitoneally injected with venom at a dose of 0.025 μg/kgof body weight. Animals were killed four hours after the injection. Lipid peroxidation, nitric oxide and glutathione levelswere measured as oxidative markers in serum and liver homogenate. In addition, liver function parameters andactivities of antioxidant enzymes were determined.

Results: N. haje crude venom (0.025 μg/kg of body weight) enhanced lipid peroxidation and nitric oxide production inboth serum and liver with concomitant reduction in glutathione, catalase, glutathione reductase and glutathione-S-transferase activities. Superoxide dismutase and glutathione peroxidase activities were significantly increased in liver ofenvenomed rats. These findings were associated with apoptosis induction in the liver. In addition, N. haje crude venomcaused hepatic injury as indicated by histopathological changes in the liver tissue with an elevation in total bilirubin,serum alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transpeptidase, and alkaline phosphatase.

Conclusions: Based on the present results, it can hypothesized that N. haje crude venom is a potent inducer oftoxin-mediated hepatotoxicity associated with apoptosis in the liver.

Keywords: Naja haje venom, Hepatotoxicity, Oxidative stress, Apoptosis, Rats

BackgroundSnake venoms comprise complex mixtures that contain nu-merous different biological active compounds such as pro-teins, peptides and nucleotides. A number of these proteinsinteract with components of the human hemostatic systemproducing diverse effects [1].The Elapidae family of venomous snakes – found in

tropical and subtropical regions around the world – in-cludes cobras, mambas, sea snakes and coral snakes [2].Several species of cobras are natives to Africa, amongthem is the Egyptian cobra Naja haje (Linnaeus) foundfrom southern Egypt to northern South Africa [3]. The

* Correspondence: [email protected]†Equal contributors2Department of Zoology and Entomology, Faculty of Science, HelwanUniversity, Cairo, Egypt3Department of Biochemistry and Molecular Biology, Asturias Institute ofBiotechnology, University of Oviedo, 33006 Oviedo, SpainFull list of author information is available at the end of the article

© 2014 Al-Quraishy et al.; licensee BioMed CenCreative Commons Attribution License (http:/distribution, and reproduction in any mediumDomain Dedication waiver (http://creativecomarticle, unless otherwise stated.

venom of the Egyptian cobra consists mainly of neuro-toxins and cytotoxins [4].The venom of Egyptian cobra affects the nervous sys-

tem by blocking the transmission of nerve signals tomuscles and at later stages stopping those transmitted tothe heart and lungs as well, causing death due tocomplete respiratory failure. Envenomation causes localpain, severe swelling, blistering, necrosis and variablenon-specific effects [4]. Progress made over the past sev-eral decades has given rise to the identification of many ofthe venom components primarily responsible for these ef-fects including phospholipases A2 and metalloproteinases.The former induces local myonecrosis and lymphaticvessel damage, whereas snake venom metalloproteinases(SVMPs) are responsible for local hemorrhage, extracellu-lar matrix degradation, blistering and skin necrosis [5-7].In addition, both PLA2s and SVMPs promote an inflam-matory response that sets the stage for tissue repair and

tral Ltd. This is an Open Access article distributed under the terms of the/creativecommons.org/licenses/by/4.0), which permits unrestricted use,, provided the original work is properly credited. The Creative Commons Publicmons.org/publicdomain/zero/1.0/) applies to the data made available in this

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Al-Quraishy et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2014, 20:42 Page 2 of 10http://www.jvat.org/content/20/1/42

regeneration but, at the same time, may contribute to fur-ther tissue damage [8,9]. Insights into the molecular struc-ture of locally acting toxins has led to understanding theirstructure-function relationships and of the mechanismsinvolved in myonecrosis, hemorrhage, lymphatic vesseldamage and dermonecrosis [5-7,10-12].Reactive oxygen species are involved in the inflamma-

tory responses, thereby affecting the cellular physiologyand playing a significant role in the pathological condi-tions [13]. The free radicals, apart from being involvedin damaging cellular components, do play a significantrole in venom induced toxicity [14].Nevertheless, the effect of the venom of N. haje was not

sufficiently covered in the available literature. Thus, it is ofinterest to examine the possible damaging effect of LD50

of the crude venom on liver of rats, unveiling the molecu-lar mechanisms of venom-induced hepatotoxicity.

MethodsExperimental animalsAdult male Wistar albino rats weighing 180–200 g were ob-tained from The Holding Company for Biological Productsand Vaccines (VACSERA, Egypt). Animals were kept inwire-bottom cages in a room under standard condition ofillumination with a 12-hours light–dark cycle at 25 ± 1°C.They were provided with water and balanced diet adlibitum. We have followed the European CommunityDirective (86/609/EEC) and national rules on animalcare that are in accordance with the NIH Guide for theCare and Use of Laboratory Animals (available at http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf ).

Venom source and chemicalsTen specimens of N. haje were collected from the west-ern Nile delta in Egypt, in September. They were keptalive in the laboratory at the University of Helwan in in-dividual terrariums, fed fortnightly with mice and offeredwater ad libitum. Once a month, the snake venom wascollected by milking. Then, it was diluted in deionizedwater, centrifuged at 10,000× g for 15 minutes and pel-lets were discarded. The sample was vacuum dried andstored at −20°C. Before use, the venom was reconsti-tuted in saline solution, centrifuged at 3,000 rpm for tenminutes at 4°C and the supernatant was used in thepresent study. All solvents and chemicals used in thisstudy were of analytical grade and deionized water wasemployed as well.

Experimental protocolLD50 of N. haje crude venom was determined as de-scribed by Meier and Theakston [15]. To study the effectLD50 of the crude venom on liver of rats after fourhours, 12 adult male albino rats were randomly divided

into two groups with six. The first group served as con-trol and received intraperitoneally (IP) an injection of sa-line solution (0.2 mL saline/rat). The second group wasinjected IP with LD50 of N. haje venom in saline solution(25 μg/kg). Animals of the two groups were killed bycervical dislocation, and blood samples were collectedby cardiac puncture. Blood was allowed to stand for halfan hour and then was centrifuged at 500 g for 15 minutesat 4°C in order to separate serum and stored at −20°Cuntil analysis. Pieces of the liver were weighed and ho-mogenized immediately to give 50% (w/v) homogenatein ice-cold medium containing 50 mM Tris–HCl, pH,7.4. The homogenate was centrifuged at 500 g for tenminutes at 4°C. The supernatant (10%) was used for thevarious biochemical determinations.

Biochemical estimations

� Liver function test
Colorimetric determination of alanineaminotransferase (ALT) or aspartate aminotransferase(AST) was estimated by measuring the amount ofpyruvate or oxaloacetate produced by forming 2,4-dinitrophenylhydrazine according to the methodof Reitman and Frankel [16]. Moreover, serumγ-glutamyl transpeptidase (γGT) and alkalinephosphatase (ALP) were tested using kits purchasedfrom Biodiagnostic Co. (Egypt) according to themethod described by Szasz [17] and Belfield andGoldberg [18], respectively. Also, serum totalbilirubin (TB) was assayed according to Schmidtand Eisenburg [19].
� Determination of lipid peroxidation and nitric oxideLipid peroxidation (LPO) and nitrite/nitrate, as anindirect measure of nitric oxide (NO) production,were assayed colorimetrically in serum and liverhomogenate according to the method of Ohkawaet al. [20] and Green et al. [21], respectively. LPOwas determined by using 1 mL of trichloroaceticacid 10% and 1 mL of thiobarbituric acid 0.67% andwere then heated in a boiling water bath for30 minutes. Thiobarbituric acid reactive substanceswere determined by the absorbance at 535 nm andexpressed as malondialdehyde (MDA) formed. Nitricoxide was determined in acid medium and in thepresence of nitrite the formed nitrous acid diazotizedsulfanilamide is coupled with N-(1–naphthyl)ethylenediamine. The resulting azo dye has a brightreddish-purple color that can be measured at 540 nm.

� Estimation of glutathione and anti-oxidant enzymesGlutathione (GSH) level was determined in serumand liver homogenate by the method of Ellman [22],which is based on the reduction of Ellman’s reagent[5,5′ dithiobis (2-nitrobenzoic acid) DTNB] with

http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf




GSH in order to produce a yellow compound. Thereduced chromogen was directly proportional toGSH concentration and its absorbance wasmeasured at 405 nm. In addition, hepatic catalase(CAT) was determined colorimetrically according tothe method of Aebi [23]. The assay is based oncatalase-catalyzed reaction of a known quantity ofH2O2 with 3,5-dichloro-2-hydroxybenzene sulfonicacid (DHBS) and 4-aminophenazone (AAP) to forma chromophore, which has a color intensity inverselyproportional to the amount of catalase in the originalsample which can be measured at 510 nm. Superoxidedismutase (SOD) activity was assayed by the methodof Nishikimi et al. [24]. This assay relies on theability of the enzyme to inhibit the phenazinemethosulphate-mediated reduction of nitrobluetetrazolium dye. Also, activities of glutathione-S-transferase (GST), glutathione peroxidase (GPx)and glutathione reductase (GR) were determinedby the methods of Habig et al. [25], Paglia andValentine [26] and Factor et al. [27], respectively.

RT-PCR analysisTotal RNA was extracted from frozen liver samples ofsix rats following the Trizol reagent method [28]. Theextracted RNA was dissolved in water (diethylpyrocarbo-nate-treated) and stored at −70°C. Five μg of RNA wasused as template for cDNA production through incuba-tion with RevertAid™ H Minus Reverse TranscriptaseThermo Fisher Scientific Inc, Canada) for one hour at 45°C,in 10 lM random hexamers, 0.375 mM per dNTP, 3 mMMgCl2, 75 mM KCl, 50 mM Tris–HCl, pH 8.3, 10 mMdithiothreitol, and 40 units RNase inhibitor, followed byfive minutes at 70°C to inactivate the enzyme. Sampleswere incubated for 30 minutes at 37°C with 0.1 mg/mLRNAse. PCR amplification was performed in the pres-ence of 2 mM of MgCl2, 0.5 mM of each primer (Meta-bion International, Martinsried, Deutschland), 0.2 mMdNTPs, 2 U of Taq DNA polymerase (GoTaq™ DNAPolymerase, Promega Corporation) in a final volume of25 μL. Simultaneous amplification of the invarianthousekeeping gene GAPDH was performed. The se-quences of the primers were as follows:

� iNOS (S): 5′-GAAAGAACTCGGGCATACCT-3′.� iNOS (AS): 5′-GGCGAAGAACAATCCACAAC-3′.� GAPDH (S): 5′-CAAGGTCATCCATGACAACTT

TG-3′.� GAPDH (AS): 5′-GTCCACCACCCTGTTGCTGT

AG-3′

PCR conditions for iNOS consisted of 35 cycles of de-naturation at 95°C for 45 s, annealing at 63°C for 45 s,and extension at 72°C for 45 s. PCR conditions for

GAPDH were 25 cycles of denaturation at 95°C for 45 s,annealing at 60°C for 45 s, and extension at 72°C for45 s. Following the last cycle, the final extension wasperformed at 72°C for ten minutes for all PCR analyses.PCR products were visualized on a 2% agarose gel withethidium bromide staining. The expression of the testedenzyme was normalized to the expression of GAPDH ofeach sample and compared using TotalLab software.

Flow cytometryLiver tissue samples were prepared by manual disaggre-gation procedure. Briefly, a few drops of RPMI mediumwere added to tissue and then mixed until complete tis-sue disaggregation was achieved. Suspended cells werefiltered using a 50-μm pore size mesh and then centri-fuged at 1000 g for ten minutes. Cells were resuspendedin PBS, counted and washed by calcium buffer then cen-trifuged at 1500 g for five minutes. The pellet was resus-pended and then cells were counted. Annexin-PI apoptoticassay was carried out using BD Annexin V FITC Assay Kit(BD Biosciences, USA). FAC scan Becton-Dickinson (BD)flow-cytometer was used and data were analyzed using cellQuest software.

Western blotting analysisWestern blotting analysis was performed according to thestandard method. Briefly, cell lysates were prepared, sepa-rated on 12% sodium dodecyl sulfate polyacrylamide gelsand transferred onto nitrocellulose membrane (AmershamBiosciences, USA). Non-specific reactivity was blocked byincubating the membranes for two hours in 5% bovineserum albumin at room temperature. Membranes were in-cubated with primary antibody (SOD, GPx, GR Bax, Bcl-2or mitochondrial respiratory complexes namely, complex I,II, III and V) overnight at 4°C. After three washes for tenminutes with phosphate buffered saline tween-20 (PBST),the membranes were incubated at 37°C for one hour withthe appropriate secondary antibody (1:5000 dilution) andwashed three times with PBST. Reactive proteins were de-tected with the enhanced chemiluminescence (ECL) detec-tion system (Pierce), β-actin was used as an internal control.

Histopathological examinationConventional techniques of paraffin-wax sectioning andhematoxylin-eosin staining were used for histologicalstudies [29]. Pieces of fresh liver tissues were cut and fixedin neutral buffered formalin for 24 hours. Following fix-ation, livers were washed and processed through an as-cending series of ethanol, cleared in methyl salicylate andinfiltrated with wax at 57°C then embedded in paraffin.Sections of 5 μm were cut and stained with aqueoushematoxylin and alcoholic-eosin, then examined in aOlympus microscope at a magnification of 400 ×.

Table 1 Changes in liver function of rats induced by Najahaje venom after four hours

Parameters Control rats Intoxicated rats

Serum ALT (U/mL) 70.13 ± 2.17 75.26 ± 1.07

Serum AST (U/mL) 56.08 ± 0.74 95.50 ± 0.32*

Serum γGT (U/L) 3.88 ± 0.65 4.32 ± 0.32*

Serum ALP (IU/L) 55.40 ± 5.94 147.73 ± 8.97*

Serum TB (mg/dL) 0.64 ± 0.21 1.94 ± 0 .53*

Serum protein (mg/ dL) 7.20 ± 0.26 9.98 ± 0.40*

*significant changes at p < 0.05 with respect to the control group; values aremeans ± SE (n = 6).ALT: alanine aminotransferase, AST: aspartate aminotransferase, γGT: γ-glutamyl transpeptidase, ALP: alkaline phosphatase, TB: total bilirubin.

Table 2 Levels of serum and liver lipid peroxidation (LPO)and nitrite/nitrate (NO) of rats induced by Naja hajesnake venom after four hours


Serum LPO (nmol/mL) 32.37 ± 1.50 43.55 ± 1.05*

Liver LPO (nmol/g tissue) 1027.20 ± 27.07 1220.34 ± 40.09*

Serum NO (μmol/L) 47.33 ± 4.03 67.67 ± 3.60*

Liver NO (μmol/g tissue) 128.54 ± 5.86 191.82 ± 15.49*

*significant change at p < 0.05 with respect to the control group; values aremeans ± SE (n = 6).


Caspase-3 detection by immunochemistryImmunolocalization technique for caspase-3 was per-formed on 3- to 4-μm thickness sections according toPedrycz and Czerny [30]. For negative controls, the pri-mary antibody was omitted. In brief, mouse anti-caspase-3(diluted 1:250, Santa Cruz Biotechnology, USA), was incu-bated with sections for 60 minutes. Primary antibodieswere diluted in Tris buffered saline (TBS)/1% bovineserum albumin (BSA). Then a biotinylated secondary anti-body directed against mice immunoglobulin (BiotinylatedLink Universal – DakoCytomation kit, supplied ready touse) was added and incubated for 15 minutes, followed byaddition of horse radish peroxidase conjugated with strep-tavidin (DakoCytomation kit, supplied ready to use) alsoincubated for 15 minutes. At the sites of immunolocaliza-tion of the primary antibodies, a reddish to brown colorappeared after adding 3-amino-9-ethylcarbasole (AEC)(DakoCytomationkit, supplied ready to use) for 15 mi-nutes. Specimens were counterstained with hematoxylinfor one minute and mounted using the Aquatex fluid(Merck KGaA, Germany).

Statistical analysisThe obtained data were presented as means ± standarderror. Statistical analysis was performed using an un-paired Student’s t-test using a statistical package pro-gram (SPSS version 17.0). Differences among groupswere considered significant at p < 0.05.

Ethics committee approvalThe present study followed the European CommunityDirective (86/609/EEC) and national rules on animalcare that are in accordance with the NIH Guide for theCare and Use of Laboratory Animals (available at http://grants.nih.gov/grants/olaw/Guide-for-the-care-and-use-of-laboratory-animals.pdf ).

ResultsChanges in levels of serum parameters affected by thesingle IP injection of crude venom of N. haje after fourhours are shown in Table 1. Levels of AST, γGT andtotal billirubin were significantly increased (p < 0.05)when compared to untreated rats. However, the level ofALT was non-significantly changed. In addition, the totalserum protein level was significantly increased (38.6% atp < 0.05) four hours after N. haje venom injection.To check the oxidative stress status in liver in re-

sponse to N. haje crude venom, we measured LPO levelsin serum and liver homogenate. Results are displayed inTable 2 and showed that crude venom induced incre-ment in LPO production and NO generation in bothserum and liver homogenate. In addition, NO generationin serum and liver increased significantly by 42.97% and49.23%, respectively, when compared to control rats

(Table 2). Through RT-PCR analysis (Figure 1) an in-crease in iNOS expression was evident only in the enve-nomated group. iNOS transcription, however, wasconfirmed to accompany the previously observed in-crease in NO content in the envenomated group. iNOStranscription, however, was confirmed to accompany thepreviously observed increase in NO content in the enve-nomated group.In order to investigate the responses of the reactive

oxygen scavenging system of liver tissue after four hoursof exposure to N. haje venom, the level and the activityof enzymatic/non-enzymatic antioxidant system weremeasured (Table 3). The levels of GSH in serum andliver homogenate were significantly decreased (p < 0.05)by 35.26% and 38.66%, respectively. Moreover, activitiesof enzymatic antioxidant system were significantly di-minished (p < 0.05) in the liver, where the activity of GRdropped by 40.72%. Additionally, GST diminished by60.66% and CAT activity was decreased by 68.29% re-garding control animals. On the other hand, the SODand GPx activities were found to be significantly in-creased (p < 0.05) in liver tissue. SOD augmented from1.06 ± 0.03 in control (untreated rats) to 1.72 ± 0.08 U/gof tissue in treated rats with N. haje crude venom(Table 3).SOD, GPx, and GR expression in tested tissue was

modulated by N. haje crude venom. As observed bydensitometry, the reduction of GR in envenomated ratswas 38% in comparison with controls (Figure 2). SOD




Figure 1 Change in the expressions of inducible nitric oxidesynthase (iNOS) in the liver of male rats injected with Naja hajecrude venom. The expression of the tested enzyme was normalizedby comparison with the expression of GAPDH in each sample.


and GPx protein levels were also higher in the enveno-mated group. These results were supported by the activ-ities of the corresponding enzymes (Table 3).Figure 2 shows alterations in the activity of mitochon-

drial respiratory complexes. Complex II, III, III and V ac-tivities in the examined livers of envenomated rats weredecreased by 56%, 5%, 12% and 26%, respectively, p < 0.05when compared to the controls.Effects of N. haje crude venom on Bax and Bcl-2 protein

content in the liver are presented in Figure 2. N. hajevenom injection caused a significant (p < 0.05) increase inBax and a significant decrease in Bcl-2 protein content(p < 0.05).Hepatocytes were stained with both propodium iodide

(PI) and fluorescein isothiocyanate (FITC)-labeled annexinV (AV-FITC) in order to enable the analysis of apoptoticcells with flow cytometry. Necrotic cells were demon-strated by AV–/PI + or AV+/PI + staining, because whenmembrane integrity is lost PI enters cells and combineswith nucleic acids. Early apoptotic cells were demon-strated by AV+/PI– staining, because when AV combineswith phosphatidylserine, they translocate to the outer

Table 3 Changes in antioxidant state of rats induced byNaja haje snake venom after four hours


Serum GSH (mmol/mL) 1.56 ± 0.34 1.01 ± 0.15*

Liver GSH (mmol/g tissue) 92.42 ± 17.07 56.69 ± 2.46*

Liver GPx (U/g tissue) 1722.43 ± 69.54 2253.41 ± 71.24*

Liver GR (μmol/h/g tissue) 93.78 ± 10.75 55.59 ± 18.27*

Liver GST (μmol/h/g tissue) 0.61 ± 0.02 0.24 ± 0.01*

Liver SOD (U/g tissue) 1.06 ± 0.03 1.72 ± 0.08*

Liver CAT (U/g tissue) 0.41 ± 0.02 0.13 ± 0.01*

*significant change at p < 0.05 with respect to the control group; values aremeans ± SE (n = 6).GSH: glutathione, GPx: glutathione peroxidase, GR: glutathione reductase,SOD: superoxide dismutase, CAT: catalase.

leaflet of the plasma membrane during apoptosis. AV+/PI +stained cells were likely to be late apoptotic or necroticcells whereas AV–/PI– cells represented viable cells. Inthe control group, most liver cells were viable (Figure 3).When rats were exposed to N. haje venom, the number ofAV + cells were significantly increased (67.5%, p < 0.05).Liver section of control animals (Figure 4A) revealed

with normal cell structure, while liver sections of ratsinjected with LD50 of N. haje crude venom showed in-flammatory cell infiltration around the hepatic vein, dis-tended blood sinusoids, hepatocyte vacuolation andprominent van Kupffer cells (Figure 4B). Severe necrosisand apoptosis were also seen (Figure 4D). Figure 4E showssevere congestion in the central vein. Immunohistochemi-cal investigations for caspase-3 in hepatocytes of thecontrol and envenomated groups are represented inFigure 4C and F. In the envenomated group, the num-ber of caspase-3 positive immunostaining hepatocyteswas significantly increased, which proves the pro-apoptotic activity of N. haje crude venom (Figure 4F).

DiscussionSeveral studies analyzing snake venom effects on animalcells – from blood, marrow, muscle, liver, kidney andskin – showed different results, depending on the ex-perimental concentrations, exposure time, site of injec-tion, and the type of toxin [31,32]. The liver is a majorproducer of most serum proteins and their total levels inthe blood are regulated by the liver function. In thepresent study, the elevation of ALT, AST, γGT, ALP, andtotal bilirubin in envenomated rats could be attributedto the hepatocyte damage. Such imbalance has been re-ported by other researchers who analyzed snake venomeffects [33-35].The mechanism by which N. haje venom induces

cytotoxic effects is still not clear. To the best of our know-ledge, there are no available data regarding the involve-ment of oxidative stress induced by N. haje venomexposure in vitro. In order to evaluate the ability of N. hajevenom to produce oxidative stress, we choose to monitorone of the earliest responses of oxidative stress, which isthe increase of stress markers in liver homogenates.Levels of early markers of oxidative stress, including

antioxidant enzymes, may be altered in the presence oflower levels of oxidative stress. To this end, we havemonitored antioxidant enzyme activities. Our resultsclearly showed that N. haje venom enhances SOD andGPx activities (Table 3). The induction of the enzymaticantioxidant defenses after the exposure to N. haje venomcould be considered as an adaptive response; that is, acompensatory mechanism that enables cells to overcomethe damage caused.To further demonstrate the implication of oxidative

stress in venom induced toxicity, we decided to monitor

Figure 2 Expressions of β-actin, SOD, GPx, GR Bax, Bcl-2 and mitochondrial respiratory complexes proteins in the liver of rats injectedwith Naja haje crude venom. Values are means ± SD (n = 6). *Significant change at p < 0.05 regarding the control group.


Figure 3 Assessment of apoptosis in hepatic tissue of male rats injected with Naja haje crude venom. (A) Representative flow cytometrydot plot of FITC-annexin V/propidium iodide. (B) Table showing: viable cells, early apoptotic cells, and late apoptotic and necrotic cells.


LPO. Lipid peroxidation is one of the suggested cyto-toxic mechanisms of different venoms. The MDA is anend product of lipid peroxidation, considered as a latebiomarker of oxidative stress and cellular damage [36]. Itis generally considered as an excellent indicator of lipidperoxidation [37]. We have shown an increase of lipidperoxidation level that seemed related to N. haje crudevenom as inferred by the amount of MDA generated,confirming an increase of free radicals production. Thisfact emphasizes that the oxidative damage is induced bythe venom in the liver of rats (Table 2).

Figure 4 Histological and immunohistochemical investigations in thesections (HE stain, 400×). (C) Section from control group showing low affincrude venom showing high affinity to caspase-3.

In addition, venom phospholipase caused a disturb-ance of the cell membrane permeability with consequentinflux of Na+ and water [38]. Chethankumar andSrinivas [39] concluded that the exposure of cellularmembranes to N. haje venom phospholipase significantlydecreased the Na+/K+ ATPase activities, thereby alteringthe ionic gradients, disorganizing the membrane lipid bi-layer and eventually leading to cell death. According toMukherjee and Maity [40], the progression of hepaticcellular swelling together with the effect of the venomphospholipase on the membranous phospholipids during

livers of rats. (A) Control liver section. (B, D, E) Naja haje group liverity to caspase-3. (F) Section from the group treated with Naja haje


envenomation might be among the factors responsiblefor the rupture of hepatic cell membranes and the oc-currence of the observed cellular damage in the presentstudy.L-amino acid oxidases (LAAOs) are flavoproteins that

are able to catalyze the oxidative deamination of L-amino acids to produce the corresponding α-keto acidsalong with the concomitant release of hydrogen peroxide(H2O2) and ammonia. Although they occur in many dif-ferent organisms from invertebrates to vertebrates, theirfunctions in vivo are uncertain. LAAO is widely distrib-uted in venomous snakes including the viperids and ela-pids and is thought to contribute to their toxicity,possibly through H2O2 formed as a result of reoxidationof the transiently reduced FAD cofactor by molecularoxygen [41,42]. The enzyme is the major component ofsnake venoms, and in some species this enzyme aloneconstitutes approximately 30% of the total protein con-tent [42,43]. Furthermore, venom LAAO has beenshown to induce cell death in several mammalian celllines [44]. The effect was attributed to the formation oflocalized high concentrations of H2O2, a known reactiveoxygen species (ROS). It is interesting to note that theLAAO-induced apoptosis has been reported to be differ-ent from that caused by exogenous H2O2, suggestingthat the mode of delivery of H2O2 is an important factor.In addition, snake venom LAAOs appear to be cytotoxicagainst many organisms [45].Tempone et al. [46] suggested that cells submitted to

oxidative stress induced by LAAO generated H2O2 thatcould activate heat shock proteins and initiate cell mem-brane disorganization, DNA fragmentation, apoptosisand therefore cell death. Sun et al. [47] suggested thatthe generated peroxide activates the transcription ofsuch factors as the nuclear factor B, the activator protein1, Fas/Apo-1 and p53.Apoptosis is an extremely complex and sophisticated

process, involving many events, including the expressionof apoptosis-related genes. In general, apoptosis is athree-stage process that includes initiation, effector anddegradation periods. The initiation phase is largelydependent on cell type and apoptotic stimulus (e.g., oxi-dative stress, DNA damage, etc.). During the initiationphase, specific pro-apoptotic signal transduction path-ways or non-specific damage pathways are activated. Incertain instances, initiation phase may influence the effi-cacy of the effector and/or degradation phases. In the ef-fector phase, there is activation of proteases, nucleases,and other diffusible intermediaries that participate in thedegradation phase of DNA. Together, the effector anddegradation phases promote the ultrastructural featuresthat are suggestive of apoptosis. Finally, these steps arefollowed by rapid engulfment of the deceased cell byneighboring phagocytic cells [48].

Internal and external mitochondrial membrane perme-ability (MMP) changes led to disappearance of MMPand release of cytochrome c and other pro-apoptoticfactors into the cytosol. The release of pro-apoptotic fac-tors in the cytoplasm may initiate apoptosis cascade re-action, which includes activation of caspase-3 and othersubstances that trigger proteolytic enzymes and breakDNA into fragments [49]. Our data revealed that N. hajesnake venom induces apoptosis in hepatocytes throughincreased transcription of caspase-3 gene. These resultssuggest that N. haje venom components may increaseexpression of certain pro-apoptotic genes that lead tocell apoptosis.

ConclusionIn conclusion, despite advances in our understanding ofthe hepatotoxicity response to N. haje venom, much re-mains to be learned on the mechanisms involved in theinitiation and development of the hepatotoxicity eventstriggered by this venom. Particularly regarding the rangeof mediators involved, the regulatory steps associatedwith their production and action, and the actual typesand subtypes of receptors activated by the main media-tors. A deficit in our study is the usage of crude venom,therefore it is difficult to establish which componentlead to our results.

AbbreviationsCAT: Catalase; GPx: Glutathione peroxidase; GR: Glutathione reductase;GSH: Glutathione; GST: Glutathione-S-transferase; LPO: Lipid peroxidation;MDA: Malondialdehyde; NO: Nitric oxide; ROS: Reactive oxygen species;SOD: Superoxide dismutase.

Competing interestsThe authors declare that there are no competing interests.

Authors’ contributionsAll authors have read and approved the final manuscript.

AcknowledgmentsThe authors would like to extend their sincere appreciation to the Deanshipof Scientific Research at king Saud University for its funding this Researchgroup NO (RG -1435-002).

Author details1Department of Zoology, College of Science, King Saud University, Riyadh,Saudi Arabia. 2Department of Zoology and Entomology, Faculty of Science,Helwan University, Cairo, Egypt. 3Department of Biochemistry and MolecularBiology, Asturias Institute of Biotechnology, University of Oviedo, 33006Oviedo, Spain.

Received: 6 June 2014 Accepted: 11 September 2014Published: 15 September 2014

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doi:10.1186/1678-9199-20-42Cite this article as: Al-Quraishy et al.: Hepatotoxicity and oxidative stressinduced by Naja haje crude venom. Journal of Venomous Animals andToxins including Tropical Diseases 2014 20:42.

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