Orally Administered Venom of Naja Naja Alters Protein ... · Snake bite pose a major health risk in...

Orally Administered Venom of Naja Naja Alters

Protein Metabolic Profiles in the Liver of Albino

Rats

M. Malleswari1, P. Josthna

2, and P. Jacob Doss

1

1Dept. of Zoology, S.V. University, Tirupati

2Sri Padmavathi Mahila University, Tirupati

Email: [email protected]

Abstract—Recent studies indicate that the venom of cobra is

active even when it is orally administered. However, studies

pertaining to the oral intoxication of Naja naja venom have

not been extensively studied. Hence, in the present

investigation we report the effect of oral intoxication of Naja

naja venom in protein metabolic profiles in the liver of

Albino rat. We have selected 1/50th LD50 dose (7µg/ kg body

weight) in the present investigation and this dose was

administered orally. Group I (3h) received single dose,

Group II (6h) received 2 doses with an interval of 3h and

Group III (12h) received 3 doses daily with an interval of 3h.

At the end of the experiment (i.e. 12 h after administration

of venom) the animals were sacrificed and different Protein

metabolic profiles were studied in liver. All the parameters

studied in the present investigation except total proteins

showed an increment and this increment was more

pronounced in 12h. Histopathological studies revealed the

damage of liver. The degree of severity was more in 12h.

The present study indicates that snake venom ingested

orally, it severally alters the protein metabolic profiles in

the liver and therefore, snake venom should not be ingested

in any form.

Index Terms—snake naja naja venom, oral administration,

protein metabolic profiles, histopathology.

I. INTRODUCTION

Snake venom is a complex mixture of many

substances, such as toxins, enzymes, growth factors,

activators and inhibitors with a wide spectrum of

biological activities (Theakston, 1983; Rahmy and

Hemmaid, 2000). They are also known to cause different

metabolic disorders by altering the cellular inclusions and

enzymatic activities of different organs. Snake bite is an

important cause of mortality and morbidity and it is one

of the major health problems in India. Snake bite often

results in puncture wounds inflicted in human beings.

Although, the majority of snake species are non-

venomous rather than venomous, snakebite remains an

important medical problem in both developing and

developed countries (Kasturiratine et al., 2010). Snake

bite pose a major health risk in many countries, with the

global snake bites exceeding 5,000,000 per year

Manuscript received August 1, 2014; revised December 22, 2014.

(Kasturiratine et al., 2010). Behavioral tolerance of

individual animal to snake Naja naja venom influences

several protein metabolic profiles, there by modifying the

general metabolic state of the animal. Protein metabolism

could be one of the major physiological events involved

in the compensatory mechanism under stress condition.

Histology, the study of micro anatomy of specific tissues,

has been successfully employed as a diagnostic tool in

medical and veterinary science. Exposure of animals to

contaminated water also causes severe pathological

changes at the tissues level. Snake venom enters the body

via internal digestive system after oral administration.

Venom is not subjected initially either to the detoxifying

reactions of the liver or to excrete via the biliary system.

Compounds transported by oral feeding effect can be

distributed to all parts of the body in their unmetabolised

form.

There are numerous publications on the clinical use of

cobra venom by injection as an analgesic in addition to

numerous laboratory studies (Reid, 2011). The snake

Naja naja venom is orally administered in countries like

China for the treatment of pain, arthritis and cancer and

orally administered cobra neurotoxins have been found to

be effective in controlled trials. The present study was

undertaken to establish the protein metabolic profiles and

pathology of cobra venom administered orally in Albino

rats ranging from 3hrs, 6hrs, 12hrs.

II. MATERIALS AND METHODS

The protocol was approved by Institutional Animal

Ethics Committee, S.V. University (Resolution

No.10/20122013/(i)a/CPCSEA/IAEC/SVU/PJD-MM/ dt.

1-2-2012). Lyophilized powder of Naja naja venom was

obtained from Irula Snake Catchers, Industrial co-

operative society, Vadanamelli, Tamilnadu. Healthy adult

Albino rats of same age (100 ± 10 days) and weight (150

± 10 g) were procured from Indian Institute of Sciences,

Bangalore. Rat feed was supplied by Sai Durga feeds and

foods, Bangalore. All the animals were divided into four

groups having six animals each. Animals of Group I

received saline by oral administration (Control). Group II

animals received a concentration of 1/50th

of LD50 orally.

Group III animals received 2 doses orally with an interval

International Journal of Life Sciences Biotechnology and Pharma Research Vol. 4, No. 1, January 2015

10©2015 Int. J. Life Sci. Biotech. Pharm. Res.

of 3h and Group IV received 3 doses with an interval of

12h.

The total protein content was estimated by the method

of Lowry et al. (1951). Free amino acid content was

estimated by the method of Moore and Stein (1954) as

described by Colowick and Kaplan (1957). Protease

activity was estimated by the method of Moore and Stein

(1954) considering the amount of free amino acids

liberated from the protein substances as a measure of

proteolytic activity. The activity of aspartate

aminotransferase (AST) was assayed by the colorimetric

method of Reitman and Frankel (1957) as described by

Bergmeyer and Bernt (1965). The activity of alanine

aminotransferase (ALAT) was assayed by the

colorimetric method of Reitman and Frankel (1957) as

described by Bergmeyer and Bernt (1965). The activity

of GDH was assayed by the method of Lee and Lardy

(1965). Ammonia was estimated by the method of

Bergmeyer (1965). Urea was estimated by the

diacetylmonoxime method as described by Natelson

(1971).

A. Statistical Treatment

The data was subjected to statistical treatment. One

way analysis of variance (ANOVA), and S-N-K tests

were performed using SPSS (ver. 21) in the personal

computer and p < 0.01 was considered as statistically

significant.

III. RESULTS

A. Total Protein Assay

Testing for total protein activity in the liver of Albino

rat exposed to snake Naja naja venom showed a

statistically significant decrease in the protein- peptide

accumulation in the experimental groups when compared

to control group. Decreased protein content might also be

attributed to the destruction or necrosis of cellular

function and consequent impairment in protein synthetic

machinery. The depletion of protein level induces to

diversification of energy to meet the impending energy

demands during the toxic stress. The disturbance of renal

function by the venom and the hemorrhage usually

associated with snake bites are the acute factors for the

observed hypoproteinemia. The increased vascular

permeability due to the toxic action of the venom

contributes to the loss of protein in the tissues. The

elevation in free amino acid content in the present

investigation is consistent with the decreased protein

level, enhanced protease activity and transaminase

activity during snake Naja naja venom exposure to 3 h,6

h and12 h (Table I).

B. Free Aminoacids

Peptide aminoacid assay in the liver of Albino rat

exposed to snake Naja naja venom results showed a

statistically significant higher amounts of aminoacids in

the dilysate compared to the control group. This increase

might be considered as the operation of the stress

phenomenon at the tissue level. The increase in FAA

content is a clear indication of step up proteolysis and

fixation of ammonia into keto acids resulting in amino

acid synthesis. The elevated free amino acid levels

indicate altered protein homeostasis and nitrogen

imbalance due to snake Naja naja venom exposed to 3 h,

6 h and 12 h (Table I).

TABLE I. CHANGES IN THE PROTEIN METABOLIC PROFILES IN THE LIVER OF ALBINO RATS EXPOSED TO SNAKE NAJA NAJA VENOM.

Parameter Control 3 h 6 h 12 h F ratio

Total protein (mg/gm wet wt of tissue) 150.534

± 9.504

133.422

± 13.551 (-11.367)

118.327

8.482 (-21.395)

90.806

5.485 (-39.677)

29.330

Free amino acid content (μ moles of

tyrosine/gm wet wt of tissue)

10.686

±2.513

11.656

±1.065

9.080

13.995

±1.401

30.973

15.067

±1.593

41.004

11.047

Protease activity (μ moles of tyrosine/mg protein/hr)

1.182

±0.125

1.326

±0.116 12.215

1.472

±0.132 24.528

1.640

±0.100 38.772

14.820

Aspartate aminotransferase (μ moles of pyruvate formed/mg protein/hr)

1.516

±0.103

1.658

±0.167

9.360

1.849

±0.057

21.947

2.006

±0.157

32.321

28.159

Alanine aminotransferase (μ moles of

pyruvate formed/ mg protein/h)

1.856

±0.144

1.662

±0.090

-10.430

1.413

±0.153

-23.846

1.121

±0.136

-39.611

40.729

Glutamate dehydrogenase (μ moles of formazon formed/mg protein/h)

0.251

±0.011

0.274

±0.011 9.282

0.324

±0.007 28.902

0.406

±0.092 61.621

25.687

Ammonia levels (μ moles of ammonia/g

wet wt of tissue)

10.986

±1.192

12.568

±0.551

14.399

14.673

±1.301

33.554

16.184

±1.979

47.307

16.391

Urea levels (μ moles of urea /g wet wt of tissue)

3.345 ±0.260

3.716 ±0.699

11.079

4.114 ±0.521

22.999

4.766 ±0.877

42.469

4.564

Values are expressed in Mean ± SD of six individual observations. Values in parenthesis indicate % change cover control. Mean values with the same

superscript do not significantly differ among themselves through S-N-K test. *P < 0.01



C. Protease Activity

Protease activity results in the liver of Albino rat

exposed to snake Naja naja venom showed a statistically

significant increase in the dialysate compared to the

control group. Increase in acidic protease activity might

be due to increase in number and size of lysosomes.

Proteases cause structural organization in different tissues

and cause disassembly of intact myofibrils during

metabolic turnover of myofibrillar proteins. The

breakdown of proteins dominates over synthesis under

enhanced proteolytic activity exposed to 3 h, 6 h and12 h

(Table I).

D. Alanine and Aspartate Aminotransferase Activity

ALAT and AST activity results showed a statistically

significant increase in the dialysate compared to the

control group. The increase may be due to shunting of

amino acids into TCA cycle through oxidative

deamination and active transamination. It has been

suggested that stress conditions in general induce

elevation in the transamination pathway. Increased AST

and ALAT activities may be due to disruption of

mitochondrial integrity or increased synthesis of enzymes.

The aspartate and alanine aminotransferases which

function as a strategic link between carbohydrate and

protein metabolisms are known to alter under severe

pathological conditions exposed to 3 h, 6 h and12 h of

snake Naja naja venom (Table I).

E. Glutamate Dehydrogenase Activity

GDH activity results showed a statistically significant

increase in the dialysate compared to the control group.

The increase indicates its contribution to enhance

glutamate oxidation during snake Naja naja venom

toxicity. Glutamate dehydrogenase (GDH) plays a crucial

role in the cells affected by a variety of effectors of

protein metabolism in the cells. Besides, GDH helps in

supplying keto acids to the TCA cycle in order to

compensate the energy crisis in different tissues during

snake Naja naja envenomation to 3 h,6 h and12 h (Table

I).

F. Ammonia

The activity of results of ammonia showed a

statistically significant increase in the dialysate compared

to the control group. Most ammonia is detoxified at its

site of formation, by amination of glutamate to glutamine,

which is mainly derived from muscle and used as an

energy source by enterocytes. When the activity of

ammonia are significantly increased, supplies of α-

ketoglutarate in cells of the CNS may be depleted,

resulting in inhibition of the TCA cycle and production

of ATP during snake Naja naja envenomation to 3 h 6 h

and12 h (Table I).

G. Urea

Urea activity results showed a statistically significant

increase in the dialysate compared to the control group.

The increase in the urea activity was due to the

catabolism of proteins usually results in the production of

some of the unwanted nitrogenous end products like, urea,

and uric acid. The urea cycle also functions in removing

excess bicarbonate, which are derived from oxidative

metabolism and thereby helping in regulating the acid-

base balance during snake Naja naja envenomation to 3h,

6 h and 12 h (Table I).

Figure. 1. Control rat liver showing Hepatocytes (H), with centrally

placed prominent Nucleus (N) with Sinusoids (S) and Central Vein (CV)

H & E. 100 X.

Figure. 2. In higher magnification 3 hrs Snake Naja naja venom administrated rat liver showing, Central Vein Congestion (CVC), and

Dilated Sinusoids (DS), Dilated and Engorged Hepatic Portal Vein

(DEHPV). H & E. 400 X.

Figure. 3. 6 hrs Snake Naja naja venom administrated rat liver showing Diffused Necrotic Areas (DNA), and Severe Degenerative Changes in

Central Vein (SDCV), Inflammatory foci (IF). H & E. 100 X.

Figure. 4. 12 hrs of Snake Naja naja venom administrated rat liver showing Sinusoidal Haemorrhage (SH), Focal Necrotic Areas (FNA),

and Amyloid Precipitation (AP), Hepatocyte Cell With Prominent Nucleus (HCPN). H & E. 400 X.



H. Histology

Histological analysis throughout this study was used to

visually conform the in vitro findings (Fig. 1-Fig. 4). The

venom studies showed various degrees of tissue damage

which exhibited marked muscle cell destruction. The

microscopic examinations revealed that oral intoxication

of the venom induced histopathological lesions in liver.

The degree of severity was more in 12h. The microscopic

examinations revealed that snake Naja naja venom

induced histopathological lesions in liver, for 3 hrs, 6hrs

and 12 hrs of venom administration. The degree of

severity differed from 3 hrs to 12 hrs of envenomation of

snake Naja naja venom and it was more in the later

period than former. In 3 hrs snake Naja naja venom

administration in Albino rat showed the, central vein

congestion, dilated sinusoids, dilated and engorged

hepatic portal vein were observed. In 6 hrs of snake Naja

naja venom administration in Albino rat showing

diffused necrotic areas, severe degenerative changes in

central vein, Inflammatory foci. In 12 hrs of snake Naja

naja venom administration in Albino rat showing

sinusoidal haemorrhage, focal necrotic areas, and

amyloid precipitation, hepatocyte cell with prominent

nucleus were observed.

I. Discussion

The results indicate changes in protein metabolism and

associated enzyme systems after the administration of

snake Naja naja venom in Albino rat of the liver. The

physiological and biochemical activities in the Albino

rats were completely disturbed after the oral

administration of snake Naja naja venom. Rabie, et al.,

(1972), reported changes in the enzymatic activities of

mammalian tissues could be one of the mechanisms by

which venomous snakes produce harm. The venom may

either act by activating or inhibiting enzyme activities in

the cell or destruction of the cell organelles with

liberation of particular enzymes (Moustafa et al., 1974).

This has been observed in rabbits injected with scorpion

venom, (Ismail, 1978). Mohamed et al., (1981) explained

the measurements of tissue enzyme activities are

important in assessing the state of the liver. Severe

hepatocellular injuries, necrosis of hepatocytes and acute

renal damage in liver were observed in rats after

Echiscarinatus venom envenomation.

The total protein content of the tissues of the liver

decreased with envenomation. The disturbance of renal

function by the venom and the hemorrhage usually

associated with snake bites are the acute factors for the

observed hypoproteinemia. The increased vascular

permeability due to the toxic action of the venom (Meier

and Stocker 1991) contribute to the loss of protein in the

tissues and also observed that hemorrhages in vital

organs together with increased vascular permeability

were observed in the majority of viper and pit viper

envenomation. Such increased vascular permeability,

together with renal damage would further aggravate the

accompanying hypoproteinemia and hypo albuminaemia.

The increase in ALP activity in snake envenomated rats

might be attributed to the destruction of liver cells

(Abdel-Nabi et al., 1993). Al Jammaz et al., (1994)

studied the effect of Walterinnesia aegyptia and

Echiscoloratus venom on solute levels in the plasma of

Albino rats and observed a rise in plasma and urea level

In the present study, the elevation in urea levels was in

consonance with increased proteolytic activity, enhanced

transamination and elevated levels of ammonia during

snake Naja naja venom intoxification. Rahmy, et al.,

(1995) reported increase in the amount of ALAT in the

liver, kidney, brain, heart because it is more specific to

liver cells. Increase in the levels of AST was found to

induce severe myonecrosis and fatal myocardial injury.

Marsh et al., (1997) suggested that viper venom might

bring about a typical pattern of hemorrhage in tissues.

Thus, it is likely that such hemorrhage might have

contributed to the decrease in the tissue protein observed

in the present work. Abdel-Nabi et al., (1997), Marsh et

al., (1997), Fahim et al., (1998), reported that there is a

reduction of total proteins in envenomated rats. The

observed effects upon those parameters might suggest

that the snake venom could have disturbed protein

synthesis in hepatocytes due to cellular damage together

with haemorrhages in liver leading to protein loss.

Mukherjee and Maity (1998) reported the progression of

hepatic cellular swelling together with the effect of the

venom phospholipase on the membranous phospholipids

during envenomation might be among the factors

responsible for the rupture of hepatic cell membranes and

the occurrence of the observed cellular damage in the

present study. Hanafy et al., (1999) studied the

pathological symptoms from the venom of Cerastes

cerastes and said that the hyperaemia around the central

vein, focal mononuclear inflammation of white blood

cells, bleeding in sinusoids around the portal area,

cellular swelling, cellular necrosis, nuclear pyknosis and

presence of foci of damaged hepatic cells invaded with

inflammatory cells and hepatocyte necrosis tissue on the

liver. The appearance of vacuoles within the hepatocytes

of the envenomed rats might indicate venom interference

with mitochondrial and microsomal function that leads to

disruption of lipoprotein and lipids accumulation. Ali et

al., (2000) detected hepatocyte degeneration,

inflammation, necrosis, fibrosis, regeneration, and cell

infiltration. Rahmy and Hemmaid et al., (2000) reported

that snake (Naja haje) envenoming causes cellular

swelling, cytoplasmic granulation and vacuolization in

addition to intrahepatic hemorrhage, liver necrosis and

activation and hyperplasia of the Kupffer cells and this

activation of these cells might represent a defence

mechanism of detoxification induced by the venom

correlated with the degree of injury to the hepatic tissue

which increases autophagy throughout the hepatic tissue.

Maria et al., (2003) and Fox et al., (2008) studied the

effects of snake venoms in cells or tissues from the

organs of rodents, like liver, kidney and muscle showed

varying results, depending on the experimental

concentrations, exposure time, site of injection, the

species of the snake and the composition of the venom.

Girish et al., (2004) reported the pathology of

envenomation includes both local and systemic effects



such as neurotoxicity, myotoxicity, cardiotoxicity,

coagulant disorders, hemorrhagic, hemolytic and edema

forming activities. Adzu et al., (2005) studied the liver

injury is among the common and most serious symptoms

of cobra snake envenoming and these changes included

congestion of intrahepatic blood vessels, increase in

number of Kupffer cells, inflammatory cell, hydropic

degeneration, variable degrees of cellular swelling,

cytoplasmic changes, cellular necrosis and cellular

damage. Chang et al., (2005) made a study on venom

from Agkistrodon halys, a Pallas-type venomous snake,

the fibrotic activity of rat livers was examined and

hepatocellular damage was detected. Amino acids may

not only act as precursors for the synthesis of essential

proteins, but also contribute towards gluconeogenesis,

glycogenesis and keto acid synthesis (Murray et al.,

2007). The elevation in free amino acid content in the

present investigation is consistent with the decreased

protein level, enhanced protease activity and

transaminase activity during snake Naja naja venom

exposure to 3hrs, 6hrs, and 12hrs. GDH catalyzes the

reversible reaction of oxidative deamination of glutamate

to α-ketoglutarate and ammonia and plays an important

role in the catabolism and biosynthesis of amino acid

(Murray et al., 2007). The elevation in the Glutamate

dehydrogenase activity indicates its contribution to

enhanced ammonia levels and glutamate oxidation during

snake Naja naja venom toxicity. Increased free amino

acid levels and their subsequent transamination results in

greater production of glutamate, thus increasing the

intracellular availability of substrate, glutamate for

consequent oxidative deamination reaction through GDH.

Besides the elevation of transaminases, GDH helps in

supplying keto acids to the TCA cycle in order to

compensate the energy crisis in different tissues during

snake Naja naja envenomation. Though ammonia is

essential for the synthesis of important compounds such

as purines, pyramidines and non-essential amino acids, it

also play a key factor in acid-base regulation and is toxic

in non-physiological concentrations and excess ammonia

therefore has to be disposed off (Murray et al., 2007).

Abdel Ghani et al., (2009) who attributed these changes

to a hepatotoxic effect of the Naja nigricollis venom and

it is more likely to be described as cytoplasmic changes

of some snake toxins. The elevation in urea levels was in

consonance with increased proteolytic activity, enhanced

transamination and elevated ammonia levels during snake

Naja naja venom toxicosis. Increased levels of urea

under snake Naja naja venom stress reveal that the rats

might have adapted to the biosynthesis of urea as a major

pathway of detoxification of ammonia. Probably this

pathway may be beneficial to animals in detoxification

and physiological compensation or adjustment to various

exogenous and endogenous toxicants. The blood urea

level in viper bite cases increased significantly after the

sixth hour. Since anti-venom does not decrease the blood

urea to normal, dialysis is required for normalization of

urea level (Pradeep kumar and Basheer, 2011). Saleh

Quraishy et al., (2014) studied the snake venom effects

on animal cells from blood, bone marrow, muscle, liver,

kidney and skin showed different results, depending on

the experimental concentrations, exposure time, site of

injection, and the type of toxin. Assi and Naser. (1999),

Murray et al.,(1988), Porth (1990) reveal symptoms

similar to hepatitis, liver cirrhosis and muscular

dystrophy. Mohammed et al., (1981) reported the Naja

haje venom induced a significant increase in liver AST

activity which may be due to destruction of hepatic

cellular organelles and intracellular liberation of these

enzymes. Felig (1975) reported a decrease in the levels of

ALT could be explained by the glucose-alanine cycle in

which pyruvate produced from the glucose is transmitted

to alanine. AI-Sadoon et al., (2011) reveal that ᵧ-GT

enzyme is produced in many tissues including those of

liver in W.aegyptia venom. Ueno and Rosenberg (1996)

reported a elevation in the activity of ALP in the liver in

animals treated with W.aegyptia venom. Mohammed et

al.,(1978) revealed that the LD 50 of N.haje venom of the

liver sections taken from the envenomated animals

demonstrated pyknotic nuclei, clumped chromatin and

inflammatory cellular infiltrations. Rahmy and Hemmaid

(2000) observed that an injected sub-lethal intramuscular

dose of N.hage venom caused alterations in the liver total

protein. Refael and Sarkar, (2009), Evangelista et al.,

(2010) reported the disturbance in the protein synthesis in

the hepatocytes could be due to cellular damage. Fahim

(2001) discussed that the venom altered gluconeogenesis

mechanism especially in liver favouring the usage of the

key aminoacids and resulting in the augmentation of

serum glucose level. Sajevic et al., (2011) reported

inflammation and vacuolation, in pyknotic cells as well

as fatty change or steatosis represents the intra

cytoplasmic accumulation of triglycerides (neutral fats)

as observed in liver sections of W.aegyptia venom.

Mirakabadi, et al., (2006) revealed the elevated activities

of ALT, ALP and AST have been reported due to

envenoming with animals venom. Adzu et al., (2005)

revealed harmful effects of venom on hepatocytes and

induction of degenerative changes the liver. Aznaurian

and Amiryan (2006) made a study and executed by

focused on the damage caused by Montivipera raddei

venom on the rabbit tissues including the liver. Ali et al.,

(2000) detected hepatocyte degeneration, inflammation,

necrosis, fibrosis, regeneration, and cell infiltration in the

Hydrophis cyanocinctussea snake venom on liver tissue.

Chang et al., (2005) studied on the venom from

Agkistrodon halys, a Pallas-type venomous snake, the

fibrotic activity of rat liver was examined and

hepatocellular damage was detected.

Since crude venom was injected, differences in venom

composition could have contributed to the above

differences. Necrosis of liver tissue produced by snake

Naja naja venom suggests the possibility of a cardiotoxin

like substance. This is supported by the fact that

cardiotoxin in cobra venom has a higher affinity towards

the liver. The present study suggests that the snake Naja

naja venom induces moderate histopathological changes

in liver after its oral intubation. These changes are

intiated at early stages of the envenomation and may be

associated with a behavioral or functional abnormality of



those organs during envenomation. Moreover, these

damages may lead to permanent sequelae. As

considerable caution should be exercised in extrapolating

experimental studies in animals to human envenomation,

it would be interesting to determine whether snake Naja

naja venom acts similarly in Human victims.

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