Study of the Influence of Gamma Radiation on Certain Pharmacological and Biochemical Actions of quinquestriatus

Thesis submitted for the partial fulfillment of the Master Degree in Pharmaceutical Sciences (Pharmacology and Toxicology)

By

Heba Abd El-Monem Mohamed Abd Rabo B. Pharm. Sci. (Faculty of Pharmacy, Cairo University) 2006 Pharmacist in National Center for Radiation Research and Technology Atomic Energy Authority

Under the Supervision of

Dr. Sanaa A. Kenawy Dr. Esmat A. Shaaban Professor of Pharmacology Professor of Pharmacology and Toxicology and Toxicology Department of Pharmacology Department of Pharmacology Faculty of Pharmacy National Center for Radiation Cairo University Research and Technology Atomic Energy Authority

Faculty of Pharmacy Cairo University 2012

سورة البقرة , األية ۲٥٥

Acknowledgements

First of all, thanks to ALLAH for giving me everything to accomplish this work and support to achieve my goal.

I would like to express my deepest appreciation and deepest gratitude to Dr. Sanaa A. Kenawy, Professor of Pharmacology & Toxicology,Faculty of Pharmacy, Cairo University, for her keen supervision and continuous encouragement. She offered deep experience, unlimited support, valuable suggestions and sincere advice. It was a great honor to work under her supervision. To her, I owe a great deal of gratitude and thanks.

I am sincerely grateful and I have no adequate word to Dr. Esmat A. Shaaban, Professor of Pharmacology & Toxicology, National Center for Radiation Research & Technology, for her suggestion for the research point, generous helps, supervision, encouragement, practical support throughout this study and valuable guidance which assisted me greatly in completing this work.

I greatly appreciate the help and co-operation of Dr. Aber M. Amin, Assist. Prof. of Biochemistry & Labeled Compound, Hot Lab Center, Atomic Energy Authority, for her kindness, valuable technical advice, effort and deep experiences to stimulate me and push me forward.

Special thanks are also to Dr. Magd El-Din Raieh, Professor of Radiochemistry, Hot Lab Center, Atomic Energy Authority, for effective advice, valuable discussion, fruitful comments, continuous encouragment and fatherhood sensation.

This work was possible through the moral support and facilities given by Dr. Mona A. El-Ghazaly, Professor of Pharmacology & Toxicology, head of National Center for Radiation Research & Technology, so my sincere gratitude for her aid.

A special word of thankfulness is directed to my professors and colleagues in the National Center for Radiation Research & Technology, for their friendly support and concern. Endless thanks to all who helped me even just a kind word thought this work.

No words can express my gratitude to my family, great thanks and appreciation will not be enough to express my feeling to my mother, my father, my brother, my sister, for their patience and encouragement throughout the progress of this work.

Heba Abd El-Monem Mohamed Abd Rabo

Contents Subject Page

List of Tables……………………………………………...... i List of Figures…………………………………………………..…… ii List of Abbreviations…………..………………………….………… iv Introduction ……………….. …………………………………………. 1 Composition of Leiurus quinquestriatus venom…………………… 5 The pharmacological actions of irradiated L.q scorpion 8 venom……………………………………………………………… Potential use of the scorpion venom ……………….……………… 12 Antivenin ………………………………………………………….. 14 Mechanism of antivenin ………………………………………….. 14 Treatment of scorpion poisoning …………...... 16 Radiation………………………………………………………….. 19 Types of ionizing radiations………...... 20 Radiation dose units………………………………………………. 21 Use of radiation in medicine………………………………..…….. 22 Effect of radiation on proteins……………………………….. 23 Labeling of scorpion venom……………………………………… 25 Radiochemistry of iodine……………………………………..….. 25 Iodine isotopes………………………………...... 25 Radioiodination methods………….…...…..…...... 28 Electrophilic aromatic substitution ……………………………….. 28 Purification of radioiodinated compounds...... 31 Aim of the Work……………………………………………….……. 32 Materials and Methods…………………………………………….. Materials.…………………………………………………….…… 33 Experimental design…………………………………………...... 36 Methods………………………………………………………….. 39

Median lethal dose (LD50)…………………………………….. ... 39 Immunological properties (double immunodiffusion technique).. 40 Labeling of scorpion venom…………………………………… 42 Iodination of scorpion venom…………………………………. 43 Determination of radiochemical yield ………………………… 43 Study of the factors affecting the labeling yield………………. 44 In-vitro stability of the125I- labeled scorpion venom………….. 45 In-vivo biodistribution of the125I- labeled scorpion venom in mice 45 Determination of plasma lipid peroxides (MDA) …...... 46 Determination of blood glutathione (GSH) …………………..… 48

Contents (cont.) Subject Page

Determination of serum alanine aminotransferase (ALT) activity 51 Determination of serum aspartate aminotransferase (AST) activity 53 Determination of serum cholesterol concentration …………...... 55 Determination of serum triglycerides concentration …………….. 56 Determination of serum HDL–cholesterol concentration ……….. 58 Determination of serum LDL–cholesterol concentration …….….. 59 Determination of serum creatine kinase (CK) …………………… 59 Determination of serum creatine kinase isoenzyme (CK-MB)….. 61 Determination of serum lactate dehydrogenase activity (LDH)….. 62 Statistical Analysis………………………………………………… 64 Results………………………………………………………………... 65

Discussion…………………………………………………………….. 106

Summary and Conclusions………………………………….………. 118

References……………………………………………………………. 122

١ ..……………………………………………………Arabic Summary

List of Tables

Table No Title Page

(i): Nuclear properties and uses of some radioiodine 26 (1): Determination of LD50 for native Leiurus quinquestriatus (L.q) scorpion venom (1mg/ml) in male mice…………..…………………………………………. 66 (2): Determination of LD50 for 1.5 KGy γ-irradiated Leiurus quinquestriatus (L.q) scorpion venom (1mg/ml) in male mice…………………………………………………….. 67 (3): Determination of LD50 for 3 KGy γ-irradiated Leiurus quinquestriatus (L.q) scorpion venom (1mg/ml) in male mice……..……………………………………………… 68 (4): LD50 for native, 1.5 and 3 KGy γ-irradiated Leiurus quinquestriatus (L.q) scorpion venom…………………. 69 (5): The In-vitro stability of 125I-L.q scorpion venom……… 80 (6): Biodistribution pattern of 125I- L.q scorpion venom in different organs or body fluids in normal mice at different time intervals……..………………………….. 82 (7): Brain/ Blood ratio of 125I- L.q scorpion venom in normal mice at different time intervals………………………… 83 (8): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on lipid peroxides (MDA) and blood glutathione (GSH) level in rats…………………………………………………….. 86 (9): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in rats…..…………………………… 90 (10): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on total cholesterol, triglycerides, HDL-cholesterol and LDL- cholesterol levels in rats …...…………………………. 96 (11): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on creatine kinase, creatine kinase-MB and lactate dehydrogenase activities in rats ……………………………………..… 102

i

List of Figures

Figures Title Page No.

(i): Leiurus quinquestriatus scorpion ….. ……………… 2 (ii): Different types of electromagnetic radiation...……… 19 (iii): Abilities of three different types of ionizing radiatian to penetrate solid matter.….………………………… 20 (iv): Electrophilic radioiodination of tyrosine residue of a protein or peptide chain…………………………………….. 29 (v): N-chloro compounds commonly employed as oxidants for electrophilic radioiodination to terminate electrophilic radioiodination reactions………………… 30 (vi): Standard curve for malondialdehyde (MDA)…………. 47 (vii): Standard curve for blood glutathione (GSH)…………. 50 (viii): Standard curve for alanine aminotransferase (ALT) activity………………………………………………….. 52 (ix): Standard curve for aspartate aminotransferase (AST) activity………………………………………………….. 54 (1): Effect of 1.5 KGy & 3 KGy γ-irradiation on (LD50) of native Leiurus quinquestriatus scorpion venom (L.q)……………………………………………. 70 (2): Immunodiffusion reaction of horse serum polyvalent antivenin with native (non-irradiated), 1.5 KGy & 3 KGy γ- irradiated Leiurus quinquestriatus (L.q) scorpion venom……………………………………………………. 72 (3): Radiochemical yield of 125I-L.q scorpion venom as a function of scorpion venom amount……………………. 75 (4): Radiochemical yield of 125I-L.q scorpion venom as a function of chloramine-T concentration………………... 76 (5): Radiochemical yield of 125I- L.q scorpion venom as a function of reaction temperature………………………. 77 (6): Radiochemical yield of 125I- L.q scorpion venom as a function of reaction time……………………………….. 78 (7): Radiochemical yield of 125I- L.q scorpion venom as a function of pH of reaction mixture……………………... 79 (8): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on plasma lipid peroxides (MDA) in rats………………………………. 87

ii

(9): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on blood glutathione (GSH) in rats…………………………………………… 88 (10): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum alanine aminotransferase (ALT) activity in rats………………... 91 (11): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum aspartate aminotransferase (AST) activity in rats……………….. 92 (12): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum cholesterol level in rats…………………………………. 97 (13): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum triglycerides level in rats……………………………….. 98 (14): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum HDL- cholesterol level in rats………………………………... 99 (15): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum LDL- cholesterol level in rats……………………………….. 100 (16): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum creatine kinase (CK) activity in rats…………………………….. 103 (17): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum creatine kinase (CK-MB) activity in rats…………………...... 104 (18): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum lactate dehydrogenase (LDH) activity in rats……………..….. 105

iii

List of Abbreviations

ALT Alanine aminotransferase AST Aspartate aminotransferase ANOVA Analysis of Variance ΔA Absorbance difference ADP Adenosine 5’-diphosphate CAT Chloramine-T CP Creatine phosphate CK Creatine kinase Cpm Count per minute DTPA Disodium salt of ethylene diaminetetraacetic acid Eβ+ Energy of positron Gy Gray γ-irradiation Gamma irradiation gp Group g Gram GSH Glutathione G6P Glucose-6-phosphate G6P-DH Glucose-6-phosphate dehydrogenase HDL High density lipoprotein HK Hexokinase h Hour i.p intraperitoneal i.v intravenous Kev Kilo electron volt KGy Kilo Gray L.q Leiurus quinquestriatus LDH Lactate dehydrogenase LDL Low density lipoprotein LD50 Median lethal dose mg Milligram ml Milliter μg Microgram μl Micro liter μmol Micromole mmol Milli mole Mev Million electron volt MBq Mega Becquerel MDA Malondialdehyde min Minute mM Millimolar

iv

nmol Nanomole NAC N-acetylcysteine NADP+ Reduced nicotinamide-adenine dinucleotide phosphate NADPH Nicotinamide-adenosine dinucleotide phosphate n Neutron Nm Nano meter Na2EDTA Diethylenetriaminepenta acetic acid Na2HPO4 Disodium hydrogen phosphate OH• Hydroxyl radical P Proton PLA2 Phospholipase A2 ROS Reactive oxygen S.E Standard error S.D Standard deviation TBA Thiobarbituric acid TBARS Thiobarbituric acid reactive substances TCA Trichloroacetic acid Te Telirum U/ml Unit per milliliter V/V Volume per Volume W / V Weight per volume Xe Xenon

v

Introduction

Scorpions

Unlike the amphibians, birds, mammals and reptiles, scorpions have no back bone, as they are . Scorpions belong to the phylum arthropoda, their bodies are segmented and their legs are jointed. They belong to the class arachnida, because they have eight legs, two pedipalps, two chelicerae and a body composed of eighteen segments. The most important species of family are Buthotus tamulus, Leiurus quinquestriatus, Androctonus crassicauda, , serrulatus, and Centruroides suffusus (Minton, 2010).

Scorpions are found in East Africa, , India, as well as Central and South America. Classification of scorpions from Africa and Middle East, has been summarized by Vachon, (1966).

Unlike snakes, all scorpions are venomous. The venom is injected by means of a stinger found at the tip of the telson, the terminal structure of the tail. The smallest adults may range from approximately 2 to 3 cm and the largest between 15 to 25 cm (Bucherl, 1971).

Scorpions have quite variable lifespans. The age range appears to be approximately 4–25 years. Scorpions prefer to live in areas where the temperatures range from 20 °C to 37 °C, but may survive from freezing temperatures to the desert heat (Hadley, 1970; Hoshino et al., 2006).

The (Leiurus quinquestriatus), is a member of the Buthidae family. It is also known as Palestine yellow scorpion, Omdurman scorpion, Israeli desert scorpion and numerous other colloquial names. To eliminate confusion, especially with potentially dangerous species, the scientific name is normally used to refer to them. The name Leiurus quinquestriatus roughly translates into English as "five-striped smooth-tail". Other species of the Leiurus are often referred to as "deathstalkers" as well (Werness, 2004; Minton, 2010).

- 1 - Introduction

The body of a scorpion is divided into two segments as shown in figure (i): the cephalothorax (also called the prosoma) and the abdomen/opisthosoma. The abdomen consists of the mesosoma and the metasoma (Gary and Polis, 1990).

1. Cephalothorax or Prosoma. 2. Abdomen or Mesosoma. 3. Tail or Metasoma. 4. Claws or Pedipalps. 5. Legs. 6. Mouth parts or Chelicerae. 7. Pincers or Chelae. 8. Moveable claw or Manus.

9. Fixed claw or Tarsus. 10. Sting or Telson. 11. Anus.

Figure (i): Leiurus quinquestriatus scorpion (Gary and Polis, 1990; Minton, 2010).

Cephalothorax/prosoma: the scorpion's “head”, comprises the carapace, eyes, chelicerae (mouth parts), pedipalps (claw) and the three to four pairs of walking legs.

Mesosoma: the abdomen's front half, is made up of six segments. The first segment contains the sexual organs as well as a pair of vestigial and modified appendages forming a structure called the genital operculum. The second segment bears a pair of featherlike sensory organs known as the pectines; the final four segments each contains a

- 2 - Introduction pair of book lungs. The mesosoma is armored with chitinous plates, known as tergites on the upper surface and sternites on the lower surface (Minton, 2010).

Metasoma: the scorpion's tail, comprises six segments. The first tail segment looks like a last mesosoma segment. The last contains the scorpion's anus and bears the telson (the sting). The telson, in turn, consists of the vesicle, which holds a pair of venom glands and the hypodermic aculeus, the venom-injecting barb (Minton, 2010).

Cuticle: this makes a tough armor around the body. In some places it is covered with hairs that act like balance organs. An outer layer that makes them fluorescent green under ultraviolet light is called the hyaline layer. Newly molted scorpions do not glow until after their cuticle has hardened. The fluoresent hyaline layer can be intact in fossil rocks that are hundreds of millions of years old (Minton, 2010).

On rare occasions, scorpions can be born with two metasoma (tails). Two-tailed scorpions are not a different species, but rather a genetic abnormality (Prchal and Steve, 2008).

When a scorpion stings, it ejects the venom at its volution. The venom ejection is violent and rapid, conditioned to the voluntary contraction of both muscle layers of the gland and is favored especially by the rapid movements of the curved tail (Balozet, 1971).

Accidents caused by scorpion stings are a relatively common event in subtropical and tropical countries and can cause lethal envenomation in , especially in children (Ismail, 1995). The signs of the scorpion envenomation are determined by scorpion species, venom composition, and the victim’s physiological reaction to the venom. The symptoms of the sting start immediately with a few minutes after the sting and usually progress to a maximum severity within 5 h. At this period the massive release of neurotransmitters results in sweating, nausea, and vomiting (Mebs, 2002). The victims usually have the major signs, with the most common being mydriasis, hypersalivation, dysphagia, and restlessness. They may exhibit signs and symptoms involving the central nervous system, stimulation of the autonomic nervous system, and occasionally, respiratory and heart failure, and even death.

After stings by dangerous scorpions from different parts of the world the signs and symptoms are similar (Murthy and Krishna, 2002). The victims of scorpion envenoming that presented multi-system-organ

- 3 - Introduction failure are characterized by changes in hormonal environment with a massive release of counter-regulatory hormones, such as catecholamine, glucagon, cortisol, angiotensin-II, and with decreased levels of insulin and an increase blood glucose level. The grading of these scorpions envenomation depends on local signs and whether or not neurological signs are predominant.

The simplest method to obtain scorpion venom is to kill the scorpions, cut up the telsa, crush them, make a maceration in physiological saline solution, or in distilled water and purify the solution (Shaaban, 1990).

The scorpion venom:

Pure venom, or most of the secretion of the venom gland, can be obtained by provoking contraction of the muscles of the gland with high frequency electric current (Balozet, 1971; Ismail et al., 1973; Massaud, 1993). The fresh, natural venom has a pH from neutral to alkaline. The vacuum-dried venom is of white grayish colour, hygroscopic and may be stored perfectly in a vacum dessicator over calcium chloride, at room temperature and in the dark, without apparent change of activity after many months (Bucherl, 1971) or when refrigerated for many years (Balozet, 1971).

The sensitivity of various species to scorpion venom is extremely variable (Miranda et al., 1964). The very important, factor in the degree of sensitivity, is the age of the test animal. Young are much more sensitive than adults.

All the parenteral routes of injection have been used. The intravenous route yields the most severe reactions. The subcutaneous, intramuscular and intraperitoneal routes give equivalent results. The intraperitoneal route seems the most convenient and gives the most constant results. The venom is inactive when deposited on mucous membranes and in the digestive tract (Anderson, 1983; Shaaban, 1990).

The symptoms of envenomation in mice, appear almost immediately after injection; and are manifested initially as cries, great agitation, vertical leaps, great aggressiveness and violent battles. This period of excitation is followed by a period of inactivity, salivation and lacrimation; with bleeding from the mouth and eyes. New phases of agitation may be repeated. When the dose is lethal, paralysis and

- 4 - Introduction contractions appear; the posterior limbs are extended back, respiration becomes short and superficial and death of the animal varies from 2 minutes to 3 hours. This period of time, however, is not inversely proportional to the dose (Balozet, 1971; Ismail et al., 1974). In the rat, the period before death is prolonged, but does not exceed 6-7 hours. Tears mixed with blood are more frequent in the rat.

In all species, if death is not too rapid; hyperglycemia, glycosuria, hypertension and cardiac arrhythmias are observed. No lesion, other than congestion at the site of injection (after subcutaneous administration) or peritoneal bleeding (after intraperitoneal administration), is seen at auopsy (Ismail et al., 1975a). It seems that the clinical effects of envenomation by different species of scorpions appear essentially similar (Ismail et al., 1980).

The deathstalker is regarded as a highly dangerous species because its venom is a powerful cocktail of , with a low LD50. While a sting from this scorpion is extremely painful. It normally would not kill an otherwise healthy adult . However, young children, the elderly, or infirm (such as those with a heart condition or those who are allergic) would be at much greater risk. Any envenomation runs the risk of anaphylaxis, a potentially life-threatening allergic reaction to the venom. If a sting from Leiurus quinquestriatus does prove fatal, the cause of death is usually pulmonary edema (Emteris et al., 2006).

Composition of Leiurus quinquestriatus scorpion venom:

Fischer and Bohn, (1957), found that scorpion venom is composed mainly of proteins. There are no free amino-acids except 1 or 2 % lysine. The scorpion venom is milky white in colour. It contains twenty different kinds of amino-acids (Emteris et al., 2006). The toxic materials are the proteins made from these amino-acids. The number of different kinds of protein known from scorpion ranges from five to sixteen, depending on the species. Some of these proteins are toxic, and the venoms from the most dangerous scorpions contain five or more toxic proteins (Anderson, 1983). The toxic proteins in scorpion venom consist of a single polypeptide chain, cross linked by four bridges. These chains contain from 56 to 78 amino-acids. The kinds of toxic proteins present differ from species to another. Indeed, scorpions of the same species collected in different geographical areas have venoms that vary in their chemical composition.

- 5 - Introduction

The location of the disulfide bridges in scorpion is quite different from that in snake . The two types of toxins do not cross immunologically (Shaaban, 1990).

Components of the deathstalker's venom include chlorotoxin, , scyllatoxin, type 1, 2 and 3. The peptide chlorotoxin, has shown potential for treating human brain tumors (Liliana et al., 1998). There has also been some evidence to show that other components of the venom may aid in the regulation of insulin and could be used to treat diabetes (Emteris et al., 2006).

Scorpion venom is a highly antigenic compound, made up of several proteins and enzymes that posses neurotoxic, cardiotoxic, nephrotoxic, and hemotoxic effects. The rapid action of these agents presents major issues. Treatment of patients with systemic symptoms should be initiated as soon as possible. Lethal dose is measured in milligrams injected per kilogram of body weight of the victim; thus, lower body weight is related to increased morbidity and mortality (Ben- Abraham et al., 2000). Most reported deaths occur within 24 hours and are related to respiratory and cardiovascular collapse.

Rochat et al., (1967), isolated five neurotoxins from the venom of Leiurus quinquestriatus. Paper chromatography and fluorometry allowed Adam and Weiss, (1958) to establish the existence of 5- hydroxytryptamine in the venom of Leiurus quinquestriatus and to estimate its concentration as 2 - 4.5 μg/mg venom.

Scorpion venoms generally contain few or no hemolysins; although there are some exceptions. The venom of Leiurus quinquestriatus is devoid of hemolysin (Balozet, 1951), any proteolytic action (Hossany, 1919), and is devoid of all blood coagulating activity (Emteris et al., 2006).

Scorpion venom is composed of a variety of substances including mucus, polynucleotides, small organic molecules, salts and non toxic proteins (Meves et al., 1984). The venom of scorpions also contains a number of basic polypeptides and neurotoxins (Rosenberg and Coull, 1982; Watt et al., 1984). Toxicity in scorpions is variable, even within the same species and from place to place. It also varies with the size, age, nutrition of the scorpion as well as climatic conditions (Gueron et al., 1980). The clinical effect of the venom is also affected by the body mass and the general health of the patient (Yarom, 1970; Watt and Simard, 1984). The very young and eldery are most susceptible to

- 6 - Introduction developing systemic symptoms of envenomation. Diabetic and hypertensive patients are also at higher risk of developing systemic toxicity.

Scorpion venom is a powerful stimulant of the autonomic nervous system. The primary action of venom is through both sympathetic and parasympathetic postganglionic stimulation. In most circumstances, the sympathetic response predominates, resulting in what has been described as a ''sympathetic storm'' (Yarom, 1970). There is also a direct stimulant effect on the heart (Ismail et al., 1972).

There are sufficient experimental and clinical evidences that venoms from different scorpion species release catecholamines from the sympathetic nervous system and stimulate the cardiac adrenergic endings (Gueron et al., 1980).

Cardiovascular manifestations are due to the direct effects of excess circulating catecholamines and cholinergics from autonomic hyperstimulation. The sympathetic branch of the autonomic nervous system usually predominates, resulting in hypertension and tachycardia, and in cases of severe envenomation, dysrhythmias, left ventricular failure and pulmonary edema (Emteris et al., 2006). Parasympathetic predominance may result in bradycardia, various grades of AV blocks, and non-cardiovascular manifestations such as priapism and hypersalivation. The cardiovascular effects of severe toxicity are the primary cause of death (Yarom, 1970; Amitai et al., 1984; Soomro et al., 1998). They can be divided into different syndromes including hypertension, pulmonary edema with hypertension, and rhythm disturbances.

The most significant electrocardiographic changes caused by scorpion venom include myocardial ischemia, anterior or inferior myocardial infarction, and malignant dysrhythmias. Myocardial infarction has been confirmed to occur in the setting of severe envenomation, through enzyme elevation (Ismail, 1995).

The mechanism of poisioning by scorpion venom is simple. It consists, primarily, of neurotoxic action on the nerve centres; and, secondarily, of local action of 5-hydroxytryptamine. It can be distinguished from poisoning by serpent venoms, which possess several enzymes, and thus produce a more complex effect (Balozet, 1971). Scorpion venoms inhibit nerve transmission at the pre-synaptic site of

- 7 - Introduction neuromuscular junctions; and also inactivate, or slow, sodium channels of excitable membranes (Mozhayeva et al., 1980).

The symptoms of scorpion poisoning in man and experimental animals, show that the action of resembles that of parasympathomimetics (muscarine, acetylcholine, pilocarpine and eserine) and sympathomimetics (adrenaline). The best analogy is with the action of nicotinic compounds, which produce bradycardia and hypotension following tachycardia, hypertension, stimulation of the adrenal medulla (to secrete adrenaline), hyperglycemia, polypnea and mydriasis (El-Asmar et al., 1974; Ghazal et al., 1975).

Most of the effects of the toxin can be explained by its very marked selective action on the sympathetic and parasympathetic autonomic centres in the hypothalamus. Excitations of these areas produce hypertension (arterial hypertension) accompanied by tachycardia that is due to release of catecholamines from adrenal glands and postganglionc nerve endings, increase of the peripheral resistance due to the action of catecholamines on alpha adrenergic receptors, and increasing the cardiac contractility or releasing renin from the kidneys due to the catecholamines action, also, on beta adrenergic receptors (Gueron et al., 1980).

Mydriasis, sweating, hyperglycemia and gastro-intestinal inhibition, all are reactions of the sympathetic nervous system. If the anterior region of the hypothalamus is stimulated, the reactions are of the parasympathetic type as contraction of the bladder, increased gastro- intestinal motility, bradycardia and vasodilatation. Stimulation of certain other hypothalamic areas results in secretion of adrenaline (Ghazal et al., 1975).

The pharmacological actions of irradiated L.q scorpion venom:

The toxicity of the L.q scorpion venom belonging to the family Buthidae, was attributed to the effect of small proteins containing 57-78 amino-acids, cross linked by four disulfide bridges (Zlotkin et al., 1978; Shaaban, 1990). They found that, the mammal and insect toxins have three disulfide bridges at homologous positions; while the fourth bridge was different. It is likely that, the position of the disulfide bridges is the same for all the scorpion neurotoxins, active on mammals. Neurotoxins are present in the majority of scorpion venoms and some of them have

- 8 - Introduction also in their composition autacoids such as serotonin in L.q (Ismail et al., 1975b).

Effect of gamma irradiation on venom solution could be attributed to its known effects on protein molecules, as venoms are mainly protein in nature, as well as, ionizing radiation can change the molecular structure and the biological properties of protein molecules (Boni- Mitake et al., 2001). This can occur by two forms: direct process by which ionizing radiation interacts directly on target molecules and an indirect process by which the product generated by water radiolysis, like - - + - e , O2 , H and OH interact with target molecules and can modify the biological activity of protein and peptides by reacting with certain sites or groups in the molecule (Garrison, 1987; Casare et al., 2006). These radicals act by removing hydrogen, breaking disulfide bonds, promoting deamination as well as inducing the formation of intramolecular and intermolecular covalent bonds (Alexander and Hamilton 1962; Halliwell and Gulteridge, 1989). These structural changes result in a decrease or loss of the enzymatic and biological activities of the proteins (Gallacci et al., 2000).

Loss of function of protein by irradiation is not usually due to breaking peptide bonds, or otherwise, disrupting the primary skeletal structure of peptide chain. It may result from a break in the hydrogen or disulfide bonds which in turn can result in a disorganization of the internal relationships of side chain groups, or an exposure of amino-acid groups, resulting in change in biological activity (Hayes and Francis, 2001).

It has been shown that gamma irradiation is an effective technique for attenuating venom toxicity and maintaining venom immunogenicity in Naja haje and Cerates cerastes venoms (Shaaban, 2003; Abib and Laraba-Djebari, 2003). The double immunodiffusion test of native (non-irradiated) and gamma irradiated venom against the specific antivenin, showed similar pattern. The visible lines obtained in the immunodiffusion reactions were identical and joined smoothly at the corners. This indicated that, the antigenic response was not changed as judged by the capacity of irradiated venom to react with the antivenin. These results demonstrate that the ability of the venom antigens to react with its corresponding antibodies was maintained in spite of being exposed to radiation.

The mechanisms by which generation of free radicals and lipid peroxidation occurs in scorpion envenomation are complex and may

- 9 - Introduction depend on a multitude of interacting factors. Scorpion venoms contain neurotoxins that act on a number of ionic channels resulting in alteration of ionic transport and cytosolic calcium overload (Harvey et al., 1992). This elevates AMP concentration and increases its catabolism, and subsequently leads to generation of free radicals. Ultimately, these free radicals attack membrane phospholipids causing their peroxidation. Occurrence of lipid peroxidation in biological membranes eventually causes impairment of membrane functioning, decreased fluidity, and inactivation of membrane-bound receptors, all of which may culminate in multiple organ dysfunction (Bhaumik et al., 1995; Love, 1999; Al-Omar et al., 2004), a feature encountered in scorpion envenomation (Meki et al., 2003). Alternatively, it is known that scorpion venoms, by their action mainly on sodium channels, enhance release of various neurotransmitters such as adrenaline and noradrenaline (Azevedo et al., 1979; Ismail, 1995; Meki et al., 2003). It has been reported that catecholamines can induce the generation of free radicals which may lead to oxidative stress and apoptosis (Khorchid et al., 2002).

There are few reports on the effect of gamma rays on scorpion venoms concerning lipid peroxidation in general (El-Alfy et al., 2007). The authors showed revealed that experimental envenomation by L.q venom was accompanied by free radical generation and depletion of antioxidant defense system. Such elevation in oxidative stress parameters correlate with the cardiac injury biochemical markers as well as hemodynamic manifestations following scorpion envenomation (El-Alfy et al., 2007).

Rats envenomated by native L.q venom exhibited marked depletion of glutathione peroxidase as well as glutathione reductase activities in cardiac tissue. Such decreased activity implicates low cardiac glutathione content, which is a key biomarker of oxidative stress (Meister and Anderson, 1983). Reduced GSH is an endogenous antioxidant that acts among the first line of defense system against pro- oxidant status (Halliwell and Gulteridge, 1999). It seems probable that, these results might be a secondary event following the increase in lipid peroxides, as lipid peroxidation is seemingly an obligatory consequence of life which is compensated by the antioxidative defence systems which include; enzymes (glutathione peroxidase, superoxide dismutases and catalase) or low molecular compounds (ascorbic acid, carotinoids and tocopherols) (Nigam and Schewe, 2000).

- 10 - Introduction

The serum transaminase level is most widely used as a measure of hepatic injury, due to its ease of measurement and high degree of sensitivity. It is useful for the detection of early damage of hepatic tissue and requires less effort than that required for a histologic analysis, moreover without sacrifice of the animals (Harper et al., 1977; Ray et al., 2006). L.q venom caused destruction of the hepatocytes as a result of venom injection accompined with disturbances in the hepatic and renal functions of the envenomated animals through severe hepatocellular injuries and necrosis of hepatocytes.

The variations in serum cholesterol and triglycerides could be due to the damage of hepatocytes by the venom making them unable to phosphorylate the large amounts of fatty acids, together with the destruction of cell membranes of other tissues (El-Asmar et al., 1979). This suggests that those variations could be time dependent. It has been found that there were changes in lipid profile levels in rats post envenomation in comparison with the control group.

The injection of scorpion Leiurus quinquestriarus venom alters the liver lipid composition. Serotonin, which is present in high concentration in the venom of Leiurus quinquestriatus (Adams and Weiss, 1958) is known to have a lipolytic effect through increasing cyclic AMP concentration (Mansour et al., 1960; Levine et al., 1964). Scorpion venom was reported to release catecholamines (Henriques et al., 1968; Ismail et al., 1972) these factors may cause lipolysis in the adipose tissues. The mobilization of the fatty acids from the adipose tissues may lead to their esterification in the liver with the formation of neutral fat and phospholipids. The free fatty acids liberated by the venom would result in an increased level of acetyl CoA (Ashmore and Weber, 1968). This increase could lead to an increase in the synthesis of cholesterol.

Scorpion venoms comprise a complex mixture of mucopolysaccharides, amino acids, lipids, hyaluronidase, phospholipase, low molecular weight molecules as serotonin and /or histamine releasers, but mostly significantly neurotoxic peptides (El- Asmar, 1984; Possani et al., 1999). Such peptides prolong the of excitable cells by deferring the sodium channel inactivation (Courard et al., 1982, Weisel-Eichler and Libersat, 2004). These result in an increase in the presynaptic release of neurotransmitters including catecholamines (Ito et al., 1981), acetylcholine (Gwee et al., 2002), glutamate (Massensini et al., 1998), and GABA (Purali, 2003), leading to the observed signs of toxicity on the cardiovascular system.

- 11 - Introduction

Moreover, it is documented that the cardiovascular effects are the major factors that may lead to death following both exipermental and human scorpion envenomation (Ismail, 1995; Cupo and Hering, 2002). Even though the mechanism of venom –induced cardiovascular effects is strongly believed to be a consequence of the increase of neurotransmitter release evoked by the action of toxins on voltage- sensitive sodium channels (Rogers et al., 1996; Chen and Heinmann, 2001). The enzymes of the venoms were shown to be responsible for several observed biological, pharmacological and toxicological effects associated with the envenomation process (Kini, 1997). Potential use of the scorpion venom:

The key ingredient of the venom is a scorpion toxin protein. Short chain scorpion toxins constitute the largest group of potassium (K+) channel blocking peptides; an important physiological role of the KCNA3 channel, also known as KV1.3, is to help maintain large electrical gradients for the sustained transport of ions such as Ca2+ that controls T lymphocyte (T cell) proliferation. Thus KV1.3 blockers could be potential immunosuppressants for the treatment of autoimmune disorders (such as rheumatoid arthritis, inflammatory bowel disease and multiple sclerosis) (George et al., 2004).

Furthermore, the venom of Uroplectes lineatus is clinically important in dermatology (Rapini et al., 2007).

Chlorotoxin is a 36- peptide found in the venom of the deathstalker scorpion (Leiurus quinquestriatus) which blocks small- conductance chloride channels (Debin and Strichartz, 1991). The fact that chlorotoxin binds preferentially to glioma cells has allowed the development of new methods, that still are under investigation, for the treatment and diagnosis of several types of cancer (Deshane et al, 2003).

Scientists have used synthetic venom, called TM-601, to deliver small doses of radioactive iodine to brain tumor cells in human patients. Unlike other forms of radiation treatment for cancer, the small protein targets brain tumor cells without harming neighboring healthy tissue or other organs (Fong, 2006).

Jafari, (2005), found that the treatment is based on the discovery that the venom in the Israeli yellow scorpion (Leiurus quinquestriatus)

- 12 - Introduction contains a protein that binds selectively to the glioma cells. The procedure uses a compound called TM-601, a synthetic version of the venom protein attached to a radioactive substance called 131I that kills the glioma cells. When injected into the bloodstream, the radioactive scorpion venom protein travels to the brain and attaches to the glioma cells, with the 131I releasing radiation that kills the cells.

Around the world, research is being done on the potential effectiveness of scorpion venom as a cancer-fighting tool. Scientists have taken a synthetic version of venom of Leiurus quinquestriatus, also known as the Giant Yellow Israeli scorpion, labeled it with radioactive iodine and found it to be an effective delivery vehicle for targeted radiotherapy against glioma. The synthetic venom binds to glioma cells and has an unusual ability to pass through the blood-brain barrier that blocks most substances from reaching brain tissue from the bloodstream. The synthetic venom is used primarily as a carrier to transport radioactive iodine to glioma cells, but there is data that suggests that it may also slow down the growth of tumor cells (Hockaday et al., 2005).

When the venom-derived drug, called chlorotoxin, was injected into the brain, it targets cancerous cells known as glioma and blocks their ability to spread basically; it paralyzes the tumor as it does the cockroach’s muscles. In clinical trials, the chlorotoxin injection carries radioactive iodine, which like radiation therapy, destroy cancer cells. But unlike chemotherapy and radiation, attack on glioma cells but does not harm the surrounding healthy brain tissues Jafari, (2005).

Omran, (2003) studied cytotoxic and apoptotic effects of scorpion Leiurus quinquestriatus venom on 293T and C2C12 eukaryotic cell lines. The study proved that the venom from scorpions of the Buthidae family, particularly Leiurus quinquestriatus, produced apoptosis in human cells. It was shown that scorpion venom produces both apoptosis (cellular suicide) and necrosis (cellular death through trauma). The key issue is concentration, since at certain concentrations, scorpion venom resulted in apoptosis. In other, larger concentrations, cytotoxicity shifted to necrosis.

- 13 - Introduction

Antivenin

Polyvalent scorpion antivenin Equine registered by the ministry of health is a refined and highly purified preparation containing the F(ab')2 fraction of the immunoglobulin raised against scorpion venoms. The antivenin is prepared by hyperimmunizing healthy arabian horses using gradually increasing doses of local scorpion venoms and immunomodulators (Eric and Ryan, 2007).

Polyvalent scorpion antivenins are highly specific in neutralizing the venoms of yellow scorpions, black scorpions (Androctonus crassicauda), and a variety of other scorpions found in the Middle East and North Africa. The antivenin also has a wide spectrum of activity and can neutralize the venoms of many of the scorpions, including arenicola, Butus mimax, Buthus occitanus, Leiurus quinquestriatus hebreus, and , found in that part of the world (Cheng et al., 2006).

Mechanism of the antivenin:

Antivenin acts to neutralize the poisonous venom and causes the venom to be released from the receptor site. Thus, the receptor sites that were previously blocked by venom are now free to interact with the acetylcholine molecule and normal respiration resumes. The spent antivenin and the neutralized venom are then excreted from the body (Gutierrez et al., 2003).

Parenteral administration of horse- and sheep-derived antivenin constitutes the cornerstone in the therapy of envenomations induced by animal bites and stings. Depending on the type of neutralizing molecule, antivenins are made of: (i) whole IgG molecules, (ii) F(ab)2 immunoglobulin fragments or (iii) Fab immunoglobulin fragments. Because of their variable molecular mass, these three types of antivenins have different pharmacokinetic profiles. Fab fragments have the largest volume of distribution and readily reach extravascular compartments. They are catabolized mainly by the kidney, having a more rapid clearance than F(ab)2 fragments and IgG. On the other hand, IgG molecules have a lower volume of distribution and a longer elimination half-life, showing the highest cycling through the interstitial spaces in the body. IgG elimination occurs mainly by extrarenal mechanisms. F(ab)2 fragments display a pharmacokinetic profile intermediate between those of Fab fragments and IgG molecules (Gutierrez et al., 2003).

- 14 - Introduction

Such diverse pharmacokinetic properties have implications for the pharmacodynamics of these immunobiologicals, since a pronounced mismatch has been described between the pharmacokinetics of venoms and antivenins. Some venoms, such as those of scorpions and elapid snakes, are rich in low-molecular-mass neurotoxins of high diffusibility and large volume of distribution that reach their tissue targets rapidly after injection (Gutierrez et al., 2003). In contrast, venoms rich in high- molecular-mass toxins, such as those of viperid snakes, have a pharmacokinetic profile characterized by a rapid initial absorption followed by a slow absorption process from the site of venom injection. Such delayed absorption has been linked with recurrence of envenomation when antibody levels in blood decrease. This heterogeneity in pharmacokinetics and mechanism of action of venom components requires a detailed analysis of each venom-antivenin system in order to determine the most appropriate type of neutralizing molecule for each particular venom.

Besides having a high affinity for toxicologically relevant venom components an ideal antivenin should possess a volume of distribution as similar as possible to that of the toxins being neutralized (Gutierrez et al., 2003). Moreover, high levels of neutralizing antibodies should remain in blood for a relatively prolonged time to assure neutralization of toxins reaching the bloodstream later in the course of envenomation, and to promote redistribution of toxins from extra vascular compartments to blood (Gutierrez et al., 2003).

Most investigators believe that the antivenin should be administered immediately after scorpion sting, otherwise the venom will produce irreversible lesions (Balozet, 1971). However, according to Ismail et al., (1983), the ineffectiveness of serotherapy with scorpion antivenin injected several hours after scorpion sting was then suggested to be due to the rapid rate of distribution of the venom to the tissues and the expected much slower rate of distribution of the antivenin.

First aid for scorpion stings is generally symptomatic. It includes strong analgesia, either systemic (opiates or paracetamol) or locally applied (such as a cold compress). Hypertensive crises are treated with anxiolytics and vasodilators (Adriaan et al., 2009). Furthermore, the victim should be immobilized and should be made to follow the below first aid plan to reduce further complications.

The important first aid measures to be considered include washing the scorpion sting site under cold water if possible, topical application

- 15 - Introduction of an ointment like antihistamine or analgesics, applying ice packs on the area to decrease the pain and swelling, application of antiseptic ointment at the area, and administration of tetanus injection, if possible application of a sterile pad over the sting site (Adriaan et al., 2009).

Treatment of scorpion poisoning:

Hassan and Mohamed, (1940), prescribed the combination of a sympatholytic drug (ergotoxin) with a parasympatholytic drug (atropine), as an antidote in experimental envenomation with the venom of leiurus quinquestriatus. Mohamed, (1942) used the combination for the immunization of animals producing serum. Mohamed and EL-Karemi, (1953), replaced atropine by bellafoline and erogtoxin by ergotamine tartarate for human treatment.

Mohamed et al., (1954), advised the use of corticoids, which cause retention of sodium chloride and water and favor the elimination of potassium. Thus, they oppose certain actions of the venom.

Barbiturates, morphine and morphine derivatives are contraindicated, as they have inhibitory effects on the respiratory center. Artificial respiration can be of great help when respiratory paralysis appears probable. Certain techniques should not be done these include slashing the sting site to let the venom out, application of a tourniquet (If applied on the wrong site may cause further complications like reducing the blood supply to the part, and sucking the venom out (Freire-Maia et al., 1978).

In adults there is a fatality rate from scorpion stings of 0.25 to 1.8 %; which can rise to 1 to 25 % in children up to five years of age (Bucherl, 1971). All severe cases of poisoning must be treated with anti scorpion serum. Specific or polyvalent anti-scorpion serum has to be given as early as possible. Even as interval of 2 h after the accident can make the success of the treatment questionable. The total dose of the serum has to be applied at once; one half intravenously and the other half by subcutaneous or intradermal route. The serum dose must be large enough to neutralize at least 2 mg of the dry scorpion venom.

Some investigators (Campos et al., 1980; Friere-Maia and Compos, 1987) recommended certain treatment of scorpion poisoning.The mild cases of intoxication were treated with symptomatic measures and/or antivenin, whereas the severe cases were treated with symptomatic measures, support of vital functions and intravenous

- 16 - Introduction injection of dipyrone and/or anesthesia of the site of the sting with lidocaine. Vomiting was treated with metoclopramide and correction of the fluid and electrolyte disturbances. Hyperthermia was controlled with dipyrone and/or tepid water sponging.

Anti-scorpion serum was first produced by Told in Cairo in 1909. Later it was produced in Johansburg, Algiers (Sergent, 1936, 1938), Tunisia (Renoux and Juminer, 1958) and Bombay (Deoras, 1961).

Scorpion antivenins are rather specific. The antivenin against Buthus minax was ineffective against Leiurus quinquestriatus venom and that against Androctonus amoreuxi venom was ineffective against Buthus minax and Leiurus quinquestriatus venoms (El-Asmar et al., 1973; Ismail et al., 1975b).

The LD50 decreases progressively as a function of the time elapsed between the injection of the venom and that of the serum and attains at the limit, of the LD50 of the venom alone (without serum). This limits mark the moment from which the serum is without action; because the venom has already produced irreversible lesions. However, Ismail et al., (1980), explained the ineffectiveness of serotherapy with scorpion antivenin injected several hours after scorpion sting, to be due to the rapid rate of distribution of the venom to the tissues and the expected much slower rate of distribution of the antivenin. Ismail et al., (1983) had separated and purified immunoglobulin fraction from the antivenin for Androctonus amoreuxi venom and showed that its neutralizing properties about 28 times greater than that of the crude antivenin.

The pharmacokinetics of the effects of radioactively labeled antivenin made for Androctonus amoreuxi venom showed that the venom is very rapidly distributed to the tissues, with an estimated half life of 5.6 minutes. The peak tissue concentrations of the venom were reached within 37 minutes. The immunoglobulin (antivenin) would take about 40 times longer to reach peak tissue concentration. This explains the ineffectiveness of scorpion given 15 minutes following the injection of scorpion venom experimentally (Ismail et al., 1983). It confirms the ineffectiveness of serotherapy with antivenin injected several hours after scorpion sting, due to differences of distribution half lives.

Scorpions, usually, inoculate the venom into the interstitial space, and not directly into the blood circulation. Therefore, the antivenin should be injected directly intravenously, as soon as possible, to neutralize the

- 17 - Introduction circulating venom, and venom which is being absorbed from the site of the sting (Magalhaes, 1938). Moreover, it seems likely that antivenin or immunoglobulins injected intravenously could act at the tissue level the following day (Ismail et al., 1983).This possible effect of antivenin would be important to neutralize some persistent effects of scorpion venom, such as sinus tachycardia, leukocytosis and hyperglycemia (Campos et al., 1980).

Antivenins have been traditionally tested according to their capacity to neutralize the lethal effect of scorpion venoms. However, it has been demonstrated that neutralization of lethality does not necessarily correlate with neutralization of specific pharmacologic or enzymatic activities (Gutierrez et al., 1981; WHO, 1981; Ownby et al., 1983).

According to Ownby et al., (1985), the addition of antimyotoxin serum to antivenin, in equal proportions, greatly improves the neutralization of the myotoxic activity of Cortalus viridis venom; and it does not decrease the ability of antivenin to neutralize hemorrhage and lethality.

- 18 - Introduction

Radiation

Radiation is defined as the emission and propagation of energy in the form of waves or particles through space or matter. It has two basic types, ionizing and non-ionizing figure (ii). The basic difference between these two types is the amount of energy they possess. Ionizing radiation has higher energy and has the ability to break chemical bonds, causing ionization of atoms and production of free radicals that can result in biological damage. Non-ionizing radiation doesn't have enough energy to cause ionization but disperses energy through heat and increased molecular movement (Zaider and Rossi, 1985; Hall, 2000).

Figure (ii): Different types of electromagnetic radiation (Hall, 2000).

- 19 - Introduction

Types of ionizing radiations:

Ionizing radiations have received their names owing to their ability of ionizing atoms and molecules in an irradiated substance. They are classified into: alpha particles (α), beta particles (β), gamma (γ) and x- rays.

Figure (iii): Abilities of three different types of ionizing radiatian to penetrate solid matter. Alpha particles (α) are stopped by a sheet of paper while beta particles (β) are stopped by an aluminium plate. Gamma radiation (γ) is dampened when it penetrates matter (Ng, 2003).

Any elementary event of interaction of radiation with a substance consists of absorption of a photon by valence electrons. As a result the atom or molecule becomes excited and this can be accompanied by the loss of electrons. In this case, the remaining part of the atom or molecule acquires a positive charge and becomes a positive ion.

All ionizing radiations are divided in their nature into electromagnetic and particulate (or corpuscular) ones. Electromagnetic radiations include x-rays from x-rays tubes. The gamma rays of radioactive elements and the x-rays of electron accelerators are produced when highly accelerated electrons are directed at special x-ray target (Branley and Dank, 1979; Ng, 2003).

Gamma and x-rays are electromagnetic radiations that cause the ejection of electrons from the atoms through which they pass, resulting in ionization and are accordingly referred to as ionizing radiation (Upton and Wald, 1994). .

- 20 - Introduction

Visible light and radio waves are also electromagnetic radiations, but they don't ionize matter because they are characterized by a longer wave length (lower frequency) (Ng, 2003).

Particulate radiations include charged particles as beta particles (electrons, positrons), alpha particles (helium nuclei) or uncharged particles as neutrons. Electromagnetic photons are chargeless bundles of energy that travel in a vacuum at the velocity of light (3 х 108 m/sec) as x-rays and γ-rays. The source of ionizing radiation is either artificial (man-made) or from the natural background radiation, these include; cosmic and solar sources, terrestrial sources (uranium and its decay product thorium, radium and radon) and internal sources resulting from naturally occurring radioactive potassium-40, carbon-14, lead-210 and other isotopes which are deposited inside human bodies from birth or due to ingestion or inhalation (Cockerham et al., 1994).

Gamma radiation is the most penetrating type of radiation and usually accompanies beta and some alpha particles. Both x-rays and gamma rays are photons, or small quantized packets of pure electromagnetic energy just like light, but with a much higher non visible frequency. Although their origins are distinctly different, the ways in which they interact with matter are exactly the same. Specifically, gamma ray has a shorter wavelength and it originates from a nuclear interaction (Cobalt 60 and Cesium 137) whereas x-ray originates from electronic or charged particle interactions at metal surface in a vacuum (Upton and Wald, 1994).

Radiation dose units:

The international commission of radiological units and measurement (ICRU) introduced the system international units to express radiation dose. The variety in units used to measure radiation and radioactivity depends on the purpose of the measurements. The system international unit of radioactivity is the Becquerel (Bq), the unit of absorbed dose is the Gray (Gy) and the unit of Equivalent dose is the sievert (Sv) (Lorris et al., 1984).

1) Units of radioactivity:

Becquerel (Bq) and curie (Ci) are units for quantity of radioactivity, where the curie is the old unit and equal 3.7 X 1010 disintegrations per second. While, 1 Bq equal to 1.0 disentigration per second.

- 21 - Introduction

Ci = 3.7X1010 Bq

2) Units of radiation absorbed dose:

The system unit absorbed dose is the Gray (Gy) which represents one joule of energy absorbed per kilogram of material.

There is another unit of absorbed dose still in use called rad (abbreviation of radiation absorbed dose). One rad corresponds to the deposition of 100 ergs of energy in a material per gram of that material by ionizing radiation.

1 rad = 100 erg/g 1 Gy = 1 joule/kg = 100 rad

3) Units of biological equivalent dose:

Biological equivalent dose reflects the biological effects of radiation. The equivalent dose to a tissue is found by multiplying the absorbed dose by a dimensionless radiation weighting factor '' quality factor'' which is dependent upon radiation type, where radiation weighting factor of gamma radiation and x-rays and electrons is equal to one, while this factor is equal to 20 for alpha particles.

Sievert (Sv) is the system international derived unit of equivalent dose, when absorbed dose is measured in gray. Another unit is still in common use, is rontgen equivalent man (rem), when absorbed dose is measured in rad.

1 Sv = 100 rem

Uses of radiation in medicine:

Radiation and radioactive substances are used for diagnosis, treatment and research. X-rays, for example, pass through muscles and other soft tissue but are stopped by dense materials. This property of X- rays enables doctors to find broken bones and to locate cancers that might be growing in the body. Doctors also find certain diseases by injecting a radioactive substance and monitoring the radiation given off as the substance moves through the body. Radiation used for cancer treatment is called ionizing radiation because it forms ions in the cells of the tissues it passes through as it dislodges electrons from atoms.

- 22 - Introduction

This can kill cells or change genes so the cells cannot grow. Other forms of radiation such as radio waves, microwaves, and light waves are called non-ionizing. They don't have as much energy and are not able to ionize cells (Raieh and EL-Mashri, 1995).

Gamma radiation is often used to kill living organisms, in a process called irradiation. Applications of this include sterilizing medical equipment (as an alternative to autoclaves or chemical means), removing decay-causing bacteria from many foods or preventing fruit and vegetables from sprouting to maintain freshness and flavor (Raieh and EL-Mashri, 1995).

Despite their cancer-causing properties, gamma rays are also used to treat some types of cancer, since the rays kill cancer cells also. In the procedure called gamma-knife surgery, multiple concentrated beams of gamma rays are directed on the growth in order to kill the cancerous cells. The beams are aimed from different angles to concentrate the radiation on the growth while minimizing damage to surrounding tissues (Raieh and EL-Mashri, 1995).

Gamma rays are also used for diagnostic purposes in nuclear medicine in imaging techniques. A number of different gamma-emitting radioisotopes are used. For example, in a PET scan a radiolabled sugar called fludeoxyglucose emits positrons that are converted to pairs of gamma rays that localize cancer (which often takes up more sugar than other surrounding tissues). The most common gamma emitter used in medical applications is the nuclear isomer technetium-99m which emits gamma rays in the same energy range as diagnostic X-rays. When this radionuclide tracer is administered to a patient, a gamma camera can be used to form an image of the radioisotope's distribution by detecting the gamma radiation emitted (see also SPECT). Depending on what molecule has been labeled with the tracer, such techniques can be employed to diagnose a wide range of conditions (for example, the spread of cancer to the bones in a bone scan) (Raieh and EL-Mashri, 1995).

Effect of radiation on proteins:

It is generally recognized that energy absorbed from ionizing radiation can inactivate biological material in two ways. A direct effect occurs when ionization is produced in the molecule itself. This is the case when a pure compound is irradiated in dry state. When a compound is irradiated in a solution form, the indirect effect join the

- 23 - Introduction direct one. This indirect effect results from the reactions between the studied molecules and the products of radiation interaction with water or other solvents. Highly reactive compounds, so called free radicals, are formed which undergo numerous reactions amongst themselves, with dissolved gas and with other molecules in the solution (Skalka and Antoni, 1970).

The following equations represent the radiolysis of water: + - H2O → H2O + e + + ◦ H2O + H2O → H3O + OH + - ◦ ◦ H2O + e → H2O + H + OH ◦ ◦ H + O2 → HO2 ◦ ◦ HO2 + HO2 → H2O2 + 2O

Certain changes may be induced in the conformation of proteins, by ionizing radiation, and the changes occurring in their biological activity may be studied simultaneously. It is possible to modify the structures of proteins and, by doing so, to alter their existing biological activity, or to initiate new useful biological functions. The biocatalytic and hormonal activities and, possibly, the antigenic property, may be influenced in this way (Antoni, 1973). A decrease and loss of such biological properties as the enzymatic, hormonal, toxic and immunological functions of proteins may occur (Skalka and Antoni, 1970). . Proteins are, as a rule, heat sensitive biopolymers. So they can not be sterilized by heat but only by other procedures as by filtering. By choosing a filter with pores of adequate diameter, the pathogen germ will be filtered out with the exception of viruses (Antoni, 1973).

Protiens are, in general, radio-sensitive. However, within the protein molecule itself, there are preferentially sensitive sites and structural elements that are the aromatic amino-acid such as tyrosine, phenylalanine, and tryptophan. On the other hand, doses of the order of mega rads are required to split the peptide bonds (Antoni, 1973).

Most probably, there exist preferentially radio-sensitive sites within the protein molecule. This supposition seems to be confirmed by the observation that those proteins, which have two or even more different biological functions, lose only one of these as the effect of exposure to a

- 24 - Introduction given radiation dose, while they invariably retain the other function or functions which will be impaired by the action of higher doses only. The enzyme proteins activity can be inactivated by lower doses, than can be antigenic property (Antoni, 1973).

Chemical analysis of the irradiated proteins revealed damage to amino-acids, in side chains and the production of new groups such as carboxyl and amido groups, by splitting the C-N bonds in amino-acids, splitting of SS group with occurrence of SH group to another SS group. There is also evidence of the splitting of peptide bonds, and the formation of intra-molecular and inter-molecular cross-links (Alexander et al, 1960; Hunt and Williams, 1964).

After irradiation, there is a change in a number of physico-chemical properties of proteins. Because of the unfolding of the molecule, some new groups become accessible for specific reaction, or some groups disappear, owing to a rearrangement of the molecule (Antoni, 1973). . Irradiation may also lead to a decrease in antigenicity and, in few cases; the irradiated proteins have been reported to acquire a new antigenic properties (Hunt and Williams, 1964).

Labeling of scorpion venom Selection of the proper isotope to be used in preparation of radiopharmaceutical is important because it should have suitable half life to avoid unwarranted harmful exposure to radiation and suitable photon energy. The two most proper isotopes that fulfill these two precautions are 123I and 99mTc. The selection of either of them depends on the structure of the compound to be labeled.

Radiochemistry of iodine:

Iodine isotopes: There are thirty three known isotopes of iodine ,110I to 142I, all of them are radioactive except the naturally occurring 127I which is stable. Twelve radioisotopes from the rest radioisotopes of iodine are produced by fission and their half lives are ranging from 1.5 seconds (139I) to 16 million years (129I) (Ledrer et al., 1967).

- 25 - Introduction

Among these thirty two radioactive iodine isotopes, four are of special interest, namely 123I,124I,125I and 131I. Table (1) summarizes some of the nuclear properties of these four isotopes.

Table (i): Nuclear properties and uses of some radioiodine (Brown and Firestone, 1986).

Main γ half- Radionuclide Mode of ray Application life decay energy (T ) 1/2 (KeV) Electron Single photon 13.2 emission computed 123I capture 159 hour tomography (EC) (SPECT) Positron 4.18 Positron emission 124I emitter 603 tomography day (β+ ) (PET)

Radioimmunoassay 59.4 35.5 (RIA), Auger 125I EC day electron therapy Beta decay 8.02 Radiotherapy of 131I emitter 364 day the thyroid cancer (β- )

125I can be produced at the reactor according to the following nuclear reaction: (EC, β+) 124 Xe gas (n,γ) 125 Xe → 125 I (IAEA, 2003)

It can be also being produced at the cyclotron from 125 Te as;

125 Te (P, n) 125 I or 125 Te (d, 2n) 125 I (IAEA, 1971)

125I emits gamma rays and Auger electrons. Auger-emitters emit low-energy electrons with a short path length in the range of 0.06-14 μm, which is too short to reach the DNA of a tumor cell unless the radionuclide is internalized into the cell (Behr et al., 2000).

The chemical and biological effect of the Auger electrons lead to double strand breaks of the DNA helix (Sedelnikova et al., 2002).

- 26 - Introduction

It is well known that 125I radionuclide is able to cause radiotoxic effects due to the low energy Auger electrons possessing a sub-cellular range of action. The charge transfer associated with the released electron leaves the resulting 125Te atom with a mean net charge of +8. The deposition of these ionizing particles in or close to the DNA molecule cause extensive damage to the genetic apparatus. In contrast, its toxicity is minimal when it decays at a distance from the DNA. For this reason; 125I has large applications in cancer research (El- Ghany et al., 2002).

131I was discovered by Livingood and Seaboreg in 1938 (Choppin and Rydberg 1980) 131I is produced via a reactor with high purity and specific activity from tellurium dioxide according to the following nuclear reaction: β- β- 130Te (n.γ) 131Te → 131I → 131Xe 25min. 8.02d

131I can also recover from uranium fission products:

235U (n, f) 131I

The half-life of 131I is 8.02 days that is sufficiently long to permit distribution of labeled compounds but limits its use for in-vivo applications.131I is used in high doses in the treatment of the thyroid cancer and may be used in a small dose in the treatment of the overactive thyroid. The β- emission of 606 KeV of 131I prohibits its use in situations where the radiation dose to the patient must be minimized but this medium energy beta emission can kill cells and it has proven to be useful in the therapy of the thyroid cancer (Hinkle et al., 1981; Humm, 1986).

A labeled compound is a compound which has one of its atoms or larger structural units substituted in a way, which distinguish that atom or unit from others.

Pharmaceuticals are substances given to patients and also defined as drugs while, radiopharmaceuticals (RPs) are drugs that contain one or more radioactive atoms and are used for diagnosis and treatment. So the (RPs) is a combination of the radionuclide, which provides a detectable signal, and a ligand, which determines the biodistribution (Michael and Willson, 1998).

- 27 - Introduction

Radioiodination methods:

The radioiodination method can be classified as one of the following:

1- Isotopic exchange. 2- Nucleophilic substitution. 3- Electrophilic aromatic substitution. 4- Addition to double bond. 5- Demetalation. 6- Conjugation labeling.

Electrophilic Aromatic Substitution:

Substitution of electrophilic radioiodine, the functional equivalent of I+, for hydrogen of an electron rich aromatic compound is one of the best methods for radioiodination. It was initially developed for radioiodination of the tyrosine residues in proteins as presented in figure (iv). The technique is used extensively for labeling both large and small molecules (Seevers and Counsell, 1982; Coenen et al., 1983; Wilbur, 1992). O O

NH NH *I+ " *I* " + + H

I*

OH OH

Figure (iv): Electrophilic radioiodination of tyrosine residue of a protein or peptide chain (March, 1985).

The reactions are efficient, take place under mild conditions, and provide high yields at either carrier-added or no- carrier-added levels. The position of labeling is dictated by the overall activating profile and substitution pattern of the aromatic ring (March, 1985). Strong electron donating substituents (OH, NH2) faster rapid reactions, and direct

- 28 - Introduction radioiodine incorporation to ortho-positions or para-positions. Aromatic rings with moderately activating groups (OR, NHCOR, SR) may be readily labeled, depending on other functionality. Simple alkyl groups are weakly activating, and electron withdrawing substituent’s (NO2, CO2H, and CONH2) are deactivating.

Radioactive iodine is usually obtained as iodide, the (I-) state, requires oxidation prior to use in electrophilic reactions. Treatment of radioiodide with a mild oxidant in situ is the method of choice for generation of electrophilic radioiodine. One should use reagents that are readily separated from the radiolabeled product and that cause minimal oxidative damage or side product formation .The most common oxidants are N-chloro compounds as clearly shown in figure (v), which promote radioiodination of activated aromatic rings within a few minutes.

One can add a reducing agent, such as sodium metabisulfite, to quench the reaction, convert residual radioiodine to non-volatile iodide form, and minimize oxidative damage (Wilbur, 1992). Techniques have been developed to reduce harmful effects that might also be caused by a reducing agent. Peroxygenated reagents, notably hydrogen peroxide and peracetic acid. The principal advantage of using peroxygenated reagents is elimination of chlorinated side products, which can result from the use of N-chloro-amines (Moerlein et al., 1988).

Oxidative enzymes (such as lactoperoxidase), together with added H2O2, provide another useful method for electrophilic radioiodination. The principal value lies in radiolabeling of proteins and monoclonal antibodies, although small molecules can also be labeled (Okamoto, 1980).

- 29 - Introduction

SO N-Na+ SO N(Cl) 2 2 2 Cl H C H C 3 3 Chloramine-T Dichloramine-T

O O Cl Cl N N N Cl N N Cl Cl O O Iodogen N-Chlorosuccinimide

polystyrene SO2N-Na+

Cl

Iodo-Bead

Figure (v): N-chloro compounds commonly employed as oxidants for electrophilic radioiodination to terminate electrophilic radioiodination reactions (Moerlein et al., 1988).

- 30 - Introduction

Purification of radioiodinated compounds:

Many radioiodination techniques are used in the preparation of iodine labeled compounds. Unreacted iodide and non desirable radioiodinated species (radiochemical impurities) must be removed from the solution. For micro scale preparation, paper or thin layer radiochromatography can be applied. For large scale production, when high activities are used, column chromatography is used for purification of iodo-compounds from undesirable radiochemical impurities. Anion exchange resins Dowex-1, Dowex-2 and Amberlite IR-400 were the most widely used. Free iodine and inorganic iodide are adsorbed by the resin. Optimum separation is achieved by choosing the proper eluent. (El-Ghany et al., 2002)

The high performance liquid chromatography (HPLC) has been developed as an efficient tool for the purification of radioiodinated compounds (Meyer, 1982). Different materials are used for packing the columns such as silica gel, octadecyl silane and dextran. The iodinated compound is separated from the reaction mixture on HPLC column according to its retention time by a solvent (HPLC grade). This separation is monitored by detectors such as UV detectors and γ- detectors. According to the signals of the detectors, the eluted fractions of the desired iodinated compounds can be collected by fractional collector, evaporated and the residue dissolved in an isotonic saline. The product is sterilized using a bacterial millipore filter (0.22 μm) (Celerier et al., 1997).

- 31 -

Aim of the work

Deathstalker (Leiurus quinquestriatus (L.q)) is a scorpion belonging to family Buthidae. It is the most dangerous species in East africa because its venom contains powerful neurotoxins with low LD50.

The aim of the present study was to obtain an effective method for reducing the toxic effect of venom in immunized animals and for preparing better toxoids and vaccines.

This study was undertaken to evaluate the effect of gamma irradiation on L.q scorpion venom. This was carried out by studying the toxicological, immunological properties as well as pharmacological, and biochemical characteristics of the venom before and after exposure to gamma radiation.

In order to achieve the aim of the present work, the lethal dose fifty (LD50) and iodination of L.q scorpion venom using the chloramine-T method, as well as its immunogenic properties were determined before and after gamma irradiation (1.5 KGy and 3 KGy).

Moreover, few studies about the effects of scorpion venom on oxidative stress biomarkers have been evaluated. Thus, the present study was constructed to investigate the effect of native and 1.5 KGy gamma irradiated L.q scorpion venom on rat plasma lipid peroxides, and blood glutathione levels.

Furthermore, some biochemical parameters were selected to be investigated in this study to evaluate the effect of 1/20 the LD50 of native or irradiated (1.5 KGy) L.q scorpion venom. These effects included the activities of certain enzymes: alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase-MB (CK-MB), and the levels of total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, in order to differentiate between the effects of native and irradiated venom.

- 32 -

Materials and Methods

Materials:

Venom:

Lyophilized crude scorpion venom of Leiurus quinquestriatus (L.q) was obtained from the laboratory animal unit of Medical Research Center, Faculty of Medicine, Ain Shams University. The venom was pooled by electrical stimulation of scorpion. It was dried and kept in the refrigerator at 4 ˚С till used.

Antivenin:

Egyptian polyvalent antivenin produced in horses prepared against L.q, Androctonus ameoreuxi, Androctonus crassicauda, Androctonus aeneas, Androctonus australis, Scorpiomarus palmatus and Buthus occitanus was used. It was obtained from the Holding Company for Biological Products and Vaccines (VACSERA), Agouza, Cairo, . The polyvalent antivenin was kept at 4˚С till used.

Irradiation facilities:

Gamma irradiation of L.q scorpion venom was carried out at the National Center for Research and Radiation Technology, Cairo, Egypt; using a cobalt-60 gamma cell 220, manufactured by the Atomic Energy of Canada (AECL). The radiation dose rate was 1.42 Gy per second. In this study, a saline solution of Leiurus quinquestriatus scorpion venom (in aqueous state), was subjected to radiation dose levels of 1.5 or 3 KGy.

Radioactive material

125I was purchased from Radiochemical Center-Amersham-England diluted with NaOH when required (specific activity= 15.5 mCi/µg).

- 33 - Materials and Methods

Animals:

1. White male Swiss albino mice, weighing 20-25 g were used for LD50 experiments & weighing 30-35 g for experiments of iodinated venom, the animals were obtained from the Institute of Ophthalmology (Giza, Egypt).

2. Adult male albino Wistar rats, weighing 120-150 g, were obtained from the Institute of Ophthalmology (Giza, Egypt). The animals were kept under suitable laboratory conditions throughout the period of investigation. They were allowed free access to food consisting of standard pellet obtained from El-Nasr Chemical Company (Cairo, Egypt) and water ad libitum.

The study was carried out according to the approval of the Ethics Committee for Animal Experimentation at Faculty of Pharmacy, Cairo University, and in accordance with the guidelines set by the EEC regulations (revised directive 86/609/EEC) at the National Center for Radiation Research and Technology.

Chemicals and reagents:

All the chemicals, reagents and reagent kits used in the present study and their sources are shown in the following list:

Item source

Acetic acid Riedel-de Haen

Chloroform ADWIC, Egypt

Citric acid BDH laboratory, England

Chloramine-T:(N-chloro-p-toluene Aldrich Chemical Company, sulfonamide sodium salt) Germany

Disodium hydrogen phosphate ADWIC, Egypt (Na2HPO4)

Disodium salt of Ethylenediamine- ADWIC, Egypt tetraacetic acid (Na2EDTA)

- 34 - Materials and Methods

Disodium hydrogen phosphate MERCK, Darmstadt, Germany. Na2HPO4. 2H2O

Ellman's reagent [5,5-dithiobis(2- Sigma Chemical Company, USA nitrobenzoic acid)]

Ethanol (absolute) ADWIC, Egypt

Ethanol (C2H5OH) Riedle, De-Haen-Seelze Hannover- Germany GSH standard Sigma, St. Louis, Mo, USA

Metaphosphoric acid Fluka, USA

Malondialdehyde (1,1-3,3- ICN Biomedicals, Germany tetramethoxypropane

Methanol CH3OH (99.5%) BDH Chemicals LTD, England.

N-butanol 99% Fluka (Riedel-de Haen)

Sodium chloride ADWIC, Egypt

Sodium citrate Sigma Chemical Company, USA

Sodium metabisulphite (Na2S2O3) BDH LTD England. Pellets,Riedel-DeHaenSeelze Sodium hydroxide (NaOH) Hannover Germany.

Sodium dihydrogen phosphate BDH Chemicals LTD England. NaH2PO4. H2O

Na azide (S.d. Fine-Chem. Ird. Biosar)

Nobel agar (Difeco. Lab. Detroit. Mich.)

Thiobarbituric acid Acros Organics, USA

Trichloroacetic acid ADWIC, Egypt

AST kit and ALT kit Diamond Diagnostics, Egypt

Cholesterol-liquizyme kit Spectrum Diagnostics, Egypt

- 35 - Materials and Methods

CK-liquid reagent kit & CK-MB kit Spectrum Diagnostics, Egypt

HDL cholesterol-precipitant kit Spectrum Diagnostics, Egypt

LDH-liquizyme kit Spectrum Diagnostics, Egypt

Triglycerides-liquizyme kit Spectrum Diagnostics, Egypt

Experimental design:

Mice were used for determination of median lethal dose (LD50) of native, 1.5 KGy and 3 KGy gamma irradiated L.q scorpion venom.

Mice were classified into three sets, each set consists of 6 groups; each group included 6 animals. The procedure was carried out according to Spearman and Karber method (Finney, 1964) as follows:

Set1: was used to determine the LD50 of native L.q scorpion venom.

Set 2: was used to determine the LD50 of irradiated (1.5 KGy) L.q scorpion venom.

Set 3: was used to determine the LD50 of irradiated (3 KGy) L.q scorpion venom.

The 1.5 KGy γ-radiation is the selected dose to carry out the biochemical experiments of this study, as it gets rid of venom toxicity while maintains immunogenicity.

One over twenty of LD50 of native and 1.5 KGy γ-irradiated L.q scorpion venom were the selected doses to carry out the biochemical experiments of this study in rats.

Rats were used for measuring the biochemical parameters; since, the LD50 of the used venom was measured in mice; the equivalent rat dose was calculated according to Paget and Barnes, (1964) to be used in the following biochemical experiments.

- 36 - Materials and Methods

Rats were classified into three sets each set was sub classified into 3 groups each consisted of 8 animals as follows:

Set 1: normal non-envenomated rats that received saline i.p and served as normal group.

Set 2: rats that received a single dose 1/20 LD50 of native L.q scorpion venom (0.133 mg/kg i.p) and served as native group.

Set 3: rats that received a single dose 1/20 LD50 of 1.5 KGy γ-irradiated L.q scorpion venom (1.230 mg/kg i.p) and served as irradiated group.

The 3 groups of were used for collection of each set blood samples were collected at 0, 1& 4 h. Blood samples (2-3ml) were withdrawn via the retro-orbital vein under light anesthesia using heparinized capillary tubes (Cocchetto and Bjornsoon, 1983). The blood samples were divided into 3 aliquots. The first part was used for estimation of MDA. The second part was collected in dry clean test tubes containing EDTA and left for 1h then centrifuged at 3000 r.p.m at 4 C° for 15 minute to separate plasma to be used for determination of GSH level. The third part was left at room temperature for separation of serum and used for estimation of the cardiac enzymes, liver enzymes and lipid profile. At the end of the experiment animals were sacrificed by decapitation, organs were isolated for required estimations. The used dead animals were frozen at -20 C° till being incinerated.

- 37 - Materials and Methods

The following parameters were determined:

I. Median Lethal Dose LD50 (Finney, 1964).

II. Immunological Properties (Ouchterlony, 1948).

III. Labeling of the L.q scorpion venom according to Hunter and Greenwood, (1962).

IV. Biochemical parameters:

1. Oxidative stress biomarkers:

A) Plasma lipid peroxides measured as malondialdehyde (MDA) (Yoshioka et al., 1979). B) Blood glutathione (GSH) (Beutler et al., 1963).

2. Liver enzymes:

A) Serum aspartate aminotransferase (AST) activity (Tietz, 1976). B) Serum alanine aminotransferase (ALT) activity (Tietz, 1976).

3. Lipid profile:

A) Serum total cholesterol level (Ellefson and Carawy, 1976). B) Serum triglycerides level (Bucolo and David, 1973). C) Serum HDL-cholesterol level (NCEPR, 1995). D) Serum LDL-cholesterol level (calculated).

4. Cardiac enzymes:

A) Serum creatine kinase (CK) activity (IFCC, 1989). B) Serum creatine kinase-MB (CK-MB) activity (IFCC, 1989). C) Serum lactate dehydrogenase (LDH) activity (Dito, 1979).

- 38 - Materials and Methods

List of the used instruments:

1. Centrifuge, Hettich, MIKRO 22, Germany. 2. Incubator (37˚С), Heraeus, Hanau. 3. Water bath, Greenfield, Oldham, England. 4. Spectrophotometer, UNICAM 8625, UV/VIS, England. 5. Precision electronic balance: model HA 120 M-A.D Company . limited, Japan.

6. PH meter: Model 601A-digital, Orien research, USA. 7. Stirring hot plate: Model 210T, Thrmix, Fisher, USA. 8. Ionization Chamber: Model CRC-15R, Capintec, USA. 9. Poly state constant temp. Circulator: model 12101-15, USA. 10. Gamma-counter: Scaler Rate meter SR7, Nuclear Enterprises LTD, USA. 11. Instant Thin Layer Chromatography (ITLC) - SG-5x20 cm, Gellman Instrument Company, Ann Arbor, Michigan.

Methods:

I. Median lethal dose (LD50):

Toxicity of L.q scorpion venom was determined before and after exposure to 1.5 KGy or 3 KGy gamma radiation. The LD50 of native, 1.5 & 3 KGy gamma irradiated venom was calculated by the Spearman and Karber method (Finney, 1964).

Procedure:

1. Mice were intraperitoneally injected with various concentrations of the venom. 2. Both LD0 (dose inducing zero % mortality) and LD100 (dose inducing 100 % mortality) were first determined. 3. Log LD0 and LD100 were calculated. 4. The logarithmic interval (log LD0 – log LD100) was divided into several equal intervals. 5. Doses at these logarithmic intervals were calculated by taking the anti logarithms. 6. Each dose was injected in a group of six mice.

- 39 - Materials and Methods

7. After injection, mice of each group were placed in suitable cages supplied with the usual laboratory food and water ad libitum. 8. After 24 h from injection, the number of dead animals in each group was recorded. 9. The LD50 and its 95 % confidence limits were determined from the following formulas:

M = Xk + 1/2 d – d r1 / N

2 2 Variance (Vm) = d /N (N-1) Σ r1 (N – r)

95% Confidence limits = M ± 1.96 x S.E

Where:

M = log LD50. Xk = log LD100. d = logarithmic interval of doses. r1 = total number of dead animals in all groups. N = number of animals per group. r = number of dead animals in each group.

II. Immunological properties (double immunodiffusion technique):

The immunodiffusion technique was carried-out as described by Ouchterlony, (1948).

Principle:

Double immunodiffusion in which a concentration gradient is established for both antigen and antibody, the initial concentration of antigen and antibody is critical. Each molecule in the system achieves a unique concentration gradient with time. When the leading front of antigen and antibody diffusion overlap, the reaction begins but formation of a precipitin line does not occur until moderate antibody excess is achieved. A precipitin band may form and be dissolved many times by incoming antigen before equilibrium is established and the position of the precipitin band is stabilized. It permits direct comparison of two or more test materials and provides a simple and direct method used to determine

- 40 - Materials and Methods whether the antigens in the test specimens are identical, cross-reactive, or non identical.

Reagents:

1 - Nobel Agar 1.3% (Difco lab., Detroit. Michigan). 2 - Sodium chloride solution (0.9%). 3 - Sodium azide to retard bacterial growth (0.05%). 4 - Amidoschwartz stain solution (0.5%); this solution was prepared by dissolving 0.5 g of amidoschwartz in 100 ml of 5% acetic acid. 5 - Decolorizing solution: (methanol: acetic acid 9:1).

Procedure:

1. The nobel agar and sodium azide were dissolved in sodium chloride solution (0.9 %), melted in a boiling water bath,(autoclaved) and poured on glass slides (5x5 cm), and allowed to harden for about 5 minutes.

2. Five wells were made; the four peripheral wells (0.5 cm in diameter) were located 4 mm from the edge of the central well, on the agar slide using a glass cutter and plugs are sucked out.

3. Antivenin (20 μl) were placed in the central well and 20 μl of saline, native (non-irradiated), 1.5 KGy and 3 KGy gamma irradiated venom solution were placed in peripheral wells (venom concentration was 20mg/ml).

4. The slides were incubated at room temperature, in a humid chamber, for 20 - 72 h.

5. After incubation the slides, were washed using normal saline for 24 - 48 h. with changing the saline for three times.

6. The slides were dried in the incubator at 37˚С, stained with 0.5% amidoschwartz stain solution for 7 minutes and the excess stain was removed by decolourising solution (methanol : acetic acid 9:1) dried in air and photographed.

- 41 - Materials and Methods

III. Labeling of L.q scorpion venom:

Principle:

Halogens have the maximum tendency to attract electrons towards themselves but this character decreases on descending from F to I due to the decrease in the electronegativity and also the decrease in the ionization energy, so iodine exhibits maximum non-metallic character, i.e. iodine can easily lose electrons more readily and form unipositive (I+)or tripositive iodine (I+3) (Satya et al., 2003).

The electronegativity of iodine (2.66) is approximately equal to the electronegativity of carbon (2.55), so the polarization of C-C bond is similar to C-I bond, and basically the iodine atom occupies a similar volume to that of methyl or ethyl group, as a result iodine can substitute it in the organic molecule without disturbing the polar configuration (Chan et al., 1976).

Reagents:

 Preparation of buffers:

1) Citric acid buffer (pH 2):

NaOH solution 1ml 2M was added to 10 ml 2M citric acid and the volume was completed to 100 ml using bi-distilled water.

2) Citric acid buffer (pH 4):

NaOH solution 13ml 2M were added to 10 ml 2M citric acid and the volume was completed to 100 ml using bi-distilled water.

3) Phosphate buffer 0.2 M, (pH 7):

1- Disodium hydrogen phosphate (Na2 H PO4.2H2O) (35.61 g) were dissolved in 1 liter bi-distilled water to get 0.2 M Na2HPO4 solution (solution A).

2- Sodium dihydrogen phosphate (NaH2PO4 .H2O) 27.61 g were dissolved in 1 liter bi-distilled water to get 0.2 M NaH2PO4.H2O solution (solution B).

- 42 - Materials and Methods

3- Solution A (30.5 ml) were added to solution B (19.5 ml) and complete the volume to 100 ml using bi-distilled water.

4) Bicarbonate buffer (pH 9):

1- Sodium bicarbonate 2.1 g were dissolved in 500 ml bi-distilled water to get 0.05 M bicarbonate solution (solution A).

2- Sodium hydroxide 2 g were dissolved in 500 ml bi-distilled water to get 0.1 M sodium hydroxide solution (solution B).

3- Solution A 50 ml were added to 5 ml from solution B and the volume was completed to 100 ml using bi-distilled water.

5) Bicarbonate buffer (pH 11): 1- Sodium bicarbonate 2.1 g were dissolved in 500 ml bi-distilled water to get 0.05 M bicarbonate solution (solution A).

2- Sodium hydroxide 2 g were dissolved in 500 ml bi-distilled water to get 0.1 M sodium hydroxide solution (solution B).

3- Solution A 50 ml were added to 22.7 ml from solution B and complete the volume to 100 ml using bi-distilled water.

Iodination of L.q scorpion venom:

Iodination of L.q scorpion venom was carried out according to the method of Hunter and Greenwood, (1962). In a 1 ml volume reaction vial, that could be tightly closed by a screw cap, 200 µl of phosphate buffer 0.2M, pH 7, were introduced a suitable amount of scorpion venom was placed and an appropriate quantity of chloramine-T (freshly prepared in water ) was added. For labeling the previous solution 10 µl of Na125I (3-5MBq) was added and then the reaction mixture was kept at room temperature for 30 min. The reaction was quenched by the addition of sodium metabisulphite.

Determination of the radiochemical yield:

The radiochemical yield and the in-vitro stability of 125I-scorpion venom were determined using instant thin layer chromatography (ITLC). The instant thin layer chromatography (ITLC) was done using ITLC silica gel strips developed in n-butanol : water : acetic acid (30%) (4:2:1,

- 43 - Materials and Methods

V/V/V) (Asikoglu et al., 2000) The strips were dried and cut into 1 cm long segments, and the radioactivity was measured using a well-type NaI (Tl) detector connected with a single channel -counter (SR-7). In the present work, the ITLC strips were previously impregnated with Na2S2O3 (20mg/ml, 0.12M) to reduce all the unreacted iodine and also to inhibit the oxidation of the radioiodide to a volatile form. The free iodide 125 remained at the origin with R f = 0 - 0.1 while the I-SV migrated with the solvent front with R f = 0.9.

Radiochemical yield % = Activity of labeled product X 100 Total activity

Study of the factors affecting the labeling yield:

1- Effect of substrate amount:

This factor was studied to get the minimum concentration of scorpion venom required to obtain maximum labeling yield. Different amounts (50, 100, 150, 200, 250 and 300 µg) of scorpion venom were used. The minimum concentration at which the maximum labeling yield was considered the optimum concentration (Ismail, 2005; Coenen et al., 1983; El-Tawoosy, 1997), the other factors were kept constant.

2- Effect of the oxidizing agent chloramine- T (CAT) amount: Studying the concentration of CAT required to give maximum labeling yield is a very important factor because excess CAT may lead to oxidation of the scorpion venom itself. Different amounts of CAT (50, 100, 150, 200, 250 and 300 µg) were used (Mcfarlane, 1958). The concentration of CAT at which maximum labeling yield was considered the optimum concentration, while the other factors were kept constant.

3- Effect of the reaction temperature: The effect of the reaction temperature on the labeling yield was studied in the range from room temperature to 100 ˚C (Mcfarlane, 1958). The experiment was performed by using the selected amount of scorpion venom followed by the selected amount of chloramine-T and 200 µl of 0.5 M phosphate buffer (pH 7) in a reaction vial. For each temperature the radiochemical yield was estimated to determine the appropriate temperature at which the maximum labeling yield will be obtained. The other factors were kept constant.

- 44 - Materials and Methods

4- Effect of reaction time: The same experiment was performed while changing the reaction time (5, 15, 30, 45 and 60 min) while keeping all the other factors constant to estimate the minimum reaction time leading to maximum labeling yield (Mcfarlane, 1958; Knust et al., 1990).

5- Effect of pH on the reaction medium: The same experiment was performed while changing pH of the reaction medium (2, 4, 7, 9 and 11) using the buffer corresponding to each pH value (Cynthia et al., 1979). While keeping all the other factors constant to estimate the optimum pH of the reaction medium leading to maximum labeling yield.

In-vitro stability of the125I- labeled scorpion venom: The in-vitro stability of the labeled scorpion venom was studied. The reaction mixture was prepared with the conditions which gave the best radiochemical yield, and took a spot of the labeling mixture on a TLC at different time intervals (1, 2, 4, 8 , 12, 24h) then chromatography was carried out.

In-vivo biodistribution of the125I- labeled scorpion venom in mice: Organ distribution of the125I- labeled scorpion venom was carried out in a group of three male Albino mice. Each animal was injected in the tail vein with 0.2 ml solution containing 3.7 MBq of 125I- scorpion venom. Mice were put in metabolic cages for the recommended time then sacrificed. Time dependent pharmacokinetic studies were carried out at 10 min, 1 h, 2 h, and 4 h post injection. The organs as well as other body parts and fluids were collected. Activity in each organ was counted and expressed as a percentage of the injected activity per organ. The weights of blood, bone and muscles were assumed to be 7, 10 and 40% of the total body weight respectively (Rhodes, 1974). Correction was made for background radiation and physical decay during experiment Organ cpm % Uptake / organ = ------x 100 Standard cpm

- 45 - Materials and Methods

IV. Biochemical parameters:

A) Determination of plasma lipid peroxides:

Lipid peroxides were determined in plasma as thiobarbituric acid reactive substances (TBARS) according to the method of Yoshioka et al., (1979).

Principle:

Lipid peroxidation products were estimated by the determination of the level of thiobarbituric acid reactive substances (TBARS) that were measured as malondialdehyde (MDA). The latter is a decomposition product of the process of lipid peroxidation and is used as an indicator of this process.

The principle of the assay depends on a colorimetric determination of a pink pigment product, resulting from the reaction of TBARS with thiobarbituric acid in an acidic medium, at high temperature. The resultant colour product was extracted in n-butanol, and the absorbance was measured at 535 nm.

Reagents:

1- Trichloroacetic acid (TCA) 20 % in distilled water.

2- Thiobarbituric acid (TBA) 0.67 % in distilled water. Warming as well as the use of a magnetic stirrer is necessary for preparation.

3- n-Butanol.

4- Standard solutions: serial dilutions of MDA in concentrations ranging from 10 to 60 nmol/ml were prepared by dissolving 1,1-3,3- tetramethoxypropane in distilled water. The latter when dissolved in water is hydrolysed to produce MDA.

Procedure:

In a centrifuge tube, 2.5 ml of 20 % trichloroacetic acid and 1 ml of 0.67 % thiobarbituric acid were added to 0.5 ml sample (plasma or standard). The mixture was heated for 30 min. in a boiling water bath followed by rapid cooling, then 4 ml n-butanol was added and mixed

- 46 - Materials and Methods vigorously. The n-butanol layer was separated by centrifugation at 3000 rpm for 10 min. The absorbance of the pink colored product was measured at 535 nm against blank containing 0.5 ml distilled water instead of the sample.

Calculations:

The concentration of TBARS in the plasma sample was expressed as nmol/ml and was calculated using the standard curve constructed with the prepared serial dilutions of MDA figure (vi).

1.2 y = 0.0184x - 0.0947 2 1 R = 0.9862

0.8

0.6

0.4 Absorbance atnm Absorbance 535 0.2

0 0 10 20 30 40 50 60 70 MDA concentration (nmol/ml)

Figure (vi): Standard curve for malondialdehyde (MDA).

- 47 - Materials and Methods

B) Determination of blood glutathione (GSH):

GSH in blood was determined according to the method described by Beutler et al., (1963).

Principle:

The method depends on the fact that both Pr-SHs and non-Pr-SHs (mainly GSH) groups react with Ellman's reagent [5,5-dithiobis (2-nitrobenzoic acid)] to form a stable yellow colour of 5-thio-2- nitrobenzoic acid, which can be measured at 412 nm. In order to determine GSH level in blood, precipitation of Pr-SHs groups by meta- phosphoric acid is necessary before the addition of Ellman's reagent.

Reagents:

1- Precipitating solution was prepared by dissolving 1.67 g of meta- phosphoric acid, 0.2 of Na2EDTA and 30 g of NaCl in 100 ml of bidistilled water. This solution is stable for 3 weeks at 4 ºC. A fine precipitate of Na2EDTA may be formed and does not interfere. The addition of Na2EDTA is only necessary when water supplies contain appreciable concentration of metallic ions.

2- Phosphate solution (0.3 M) was prepared by dissolving 42.59 g of Na2HPO4 in one liter of bidistilled water. This solution is stable and if crystals develop during storage at 4 ºC, heating may redissolve them.

3- Ellman's regent was prepared by dissolving 40 mg of [5,5- dithiobis(2- nitrobenzoic acid)] in 100 ml of 1% sodium citrate. Sodium citrate has been selected for convenience, since its pH is appropriate for both the solubility and stability of the reagent.

4- Standard solution: serial dilutions of GSH in concentrations ranging from 9 to 75 µg /ml were prepared by dissolving 7.5 mg of GSH in 100 ml of 1% meta-phosphoric acid.

- 48 - Materials and Methods

Procedure:

An aliquot of 0.1 ml of blood sample was haemolysed by the addition of 0.9 ml bidistilled water. To the haemolysate, 1.5 ml of the precipitating solution was added, mixed and allowed to stand for 5 min. Centrifugation at 1000 x g was carried out for 15 min.

To 1 ml of the resulting supernatant, 4 ml of phosphate solution was added followed by 0.5 ml of Ellman's regent. The absorbance was measured within 5 min. at 412 nm. The blank solution was prepared with 4 ml phosphate solution, 1 ml diluted precipitating solution with bidistilled water (3: 2 V/V) and 0.5 ml of Ellman's regent.

To 1 ml of the prepared standard GSH serial dilutions, 4 ml phosphate solution and 0.5 ml of Ellman's regent were added and the absorbance was measured at 412 nm against blank, containing 1 ml bidistilled water instead of the standard solution. A standard curve for GSH was constructed by plotting absorbance of the samples versus their concentrations figure (vii).

Calculations:

Blood GSH was expressed as mg % and was calculated from the following formula: AT 1 GSH (mg %) = × n × × 100 × Dilution factor AS 1000

Where: AT = Absorbance of the test sample. AS = Absorbance of the standard sample. n = Concentration of the standard as µg /ml. Dilution factor = 2.5/0.1 = 25

- 49 - Materials and Methods

0.8 0.7 y = 0.009x + 0.0152 R2 = 0.9949 0.6 0.5 0.4 0.3 0.2

Absorbance412at nm. 0.1 0 0 20 40 60 80 GSH Concentration µg /ml

Figure (vii): Standard curve for blood glutathione (GSH).

- 50 - Materials and Methods

2. Liver enzymes:

A) Determination of serum alanine aminotransferase (ALT) activity:

ALT was determined in serum. The ALT colorimetric assay was performed using a test reagent kit according to the method of Tietz, (1976).

Principle: ALT 2-oxoglutarate + DL-alanine L-glutamate + pyruvate

The formed pyruvate is measured by monitoring the concentration of pyruvate hydrazone formed with 2,4-dinitrophenylhydrazine.

Reagents:

1- Reagent 1: phosphate buffer pH 7.4 100 mmol/L Buffer: DL-alanine 80 mmol/L Substrate: 2-oxoglutarate 4.0 mmol/L

2- Reagent 2 (color reagent): 2,4- dinitrophenylhydrazine 4.0 mmol/L

3- NaOH 0.4 mol/L

All reagents were stable up to the expiry date when stored at 2-8 ºC.

Procedure:

In a test tube, 100 µl of sample were added to 0.5 ml of reagent 1, mixed and incubated for exactly 30 minutes at 37 ºC, then 0.5 ml of reagent 2 was added, mixed and incubated for exactly 20 minutes at 20- 25 ºC followed by addition of 5 ml of NaOH, mixed and the absorbance of sample (fresh serum) was measured after 5 minutes at 546 nm. against reagent blank containing 100 µl of distilled water instead of the sample. The color intensity was stable for 60 minutes.

- 51 - Materials and Methods

Calculation:

The activity of ALT in serum was determined as U/L using ALT standard curve figure (viii).

Figure (viii): Standard curve for alanine aminotransferase (ALT) activity.

- 52 - Materials and Methods

B) Determination of serum aspartate aminotransferase (AST) activity:

AST was determined in serum. The AST colorimetric assay was performed using a test reagent kit according to the method of Tietz, (1976).

Principle: AST 2-oxoglutarate + L-aspartate L-glutamate + oxaloacetate

The formed oxaloacetate is measured by monitoring the concentration of oxaloacetate hydrazone formed with 2,4- dinitrophenylhydrazine.

Reagents:

1- Reagent 1: phosphate buffer pH 7.2 100 mmol/L Buffer: L-aspartate 100 mmol/L Substrate: 2-oxoglutarate 4.0 mmol/L

2- Reagent 2 (color reagent): 2,4- dinitrophenylhydrazine 2.0 mmol/L

3- NaOH 0.4 mol/L

All reagents were stable up to the expiry date when stored at 2-8 ºC.

Procedure:

In a test tube, 100 µl of sample were added to 0.5 ml of reagent 1, mixed and incubated for exactly 30 minutes at 37 ºC. Reagent 2 0.5 ml was added, mixed and incubated for exactly 20 minutes at 20-25 ºC followed by addition of 5 ml of NaOH, mixed and the absorbance of sample (fresh serum) was measured after 5 minutes at 546 nm against reagent blank containing 100 µl of distilled water instead of the sample. The color intensity was stable for 60 minutes.

- 53 - Materials and Methods

Calculation:

The activity of AST in serum was determined as U/L using AST standard curve figure (ix).

Figure (ix): Standard curve for aspartate aminotransferase activity.

- 54 - Materials and Methods

3. Lipid profile: A) Determination of serum cholesterol concentration: Cholesterol concentration was determined according to the method of Ellefson and Carawy, (1976).

Principle: The cholesterol present in the sample originates a colored complex according to the following reaction:

Cholesterol esterase Cholesterol esters + H2O cholesterol +fatty acids. Cholesterol oxidase Cholesterol +O2 cholest-4-en-3-one + H2O2 Peroxidase 2H2O2 + phenol + 4-amino-antipyrine quinoneimine dye +

4 H2O The intensity of the formed colour is proportional to the cholesterol concentration in the sample.

Reagents: 1- R1 : pipes p H 7 50mmol/L Buffer : phenol 30mmol/L 2- R2 : sodium cholate 5.0mmol/L

Enzymes: cholesterol esterase (CHE) >250 U/L cholesterol oxidase (CHOD) >250 U/L peroxidase (POD) >2.0 KU/L 4- amino-antipyrine 1.0 mmol/L

Sodium azide 8.0 mmol/L

Standard cholesterol 200 mg/dL (ST)

- 55 - Materials and Methods

Procedure: Preparation: working reagent (WR): were dissolved the content of one vial R2 enzymes in one bottle of R1 buffer. Cap and mix gently to dissolved contents.

1- Appropriate set of test tubes was labeled 3 tubes for each sample, the standard, the blank and the test. 2- WR (1ml) was added in all the tubes. 3- The standard (10 μl) was added in the standard tube. 4- The sample (10 μl) was added in the sample tube. 5- The mixture was mixed and incubated for 5 min. at 37 °C or 10 min. at room temperature 6- Absorbance (A) of the samples and standard was read against blank at 546 nm within 30 minute.

Calculation:

(A)sample × 200 (standard conc.) = Serum cholesterol conc (mg /dL). (A)s tandard

B) Determination of serum triglycerides concentration: Triglycerides concentration was determined according to the method of Bucolo and David, (1973).

Principle: Enzymatic colorimetric test: lipoprotein lipase Triglycerides + 3H2O Glycerol +Fatty acids Glycerol + ATP glycerokinase G- 3- p + ADP Mg2+ glycerol-3-p-oxidase G-3-P + O2 DAP + H2O2 peroxidase H2O2 + 4-amino atipyrine + 4-chlorophenol quinoneimine dye + 4 H2O

56 Materials and Methods

Reagents:

1- R1: Standard triglyceride (ST) 200 mg/dL

2- R2: Pipes buffer pH 7 50mmol/L 4- Chlorophenol 6 mmol/L Magnesium aspartate >0.5mmol/L Lipase >10 K U/L Glycerol kinase >750 U/L Glycerol- 3- p- oxidase >3.5 K U/L Peroxidase >2.0 K U/L 4- aminoantipyrine 1.0 mmol/L ATP 1.0 mmol/L Sodium azide 8.0mmol/L Procedure: 1- Appropriate set of test tubes was labeled, 3 tubes for each sample, the standard, the blank and the test. 2- The standard (10 μl) was added in the standard tube. 3- The sample (10 μl) was added in the sample tube. 4- The WR (1000 μl) was added to all tubes, mixed, measured after incubating at 37°C for 5 minutes or 10 minutes at 20° C to 25°C within 60 minutes absorbance of sample was read against reagent blank at 546 nm. Calculation: Asample Serum triglycerides conc. (mg / dL) = × 200 As tandard

57 Materials and Methods

C) Determination of serum HDL–cholesterol concentration: HDL-cholesterol was determined according to the method of NCEPR, (1995).

Principle: Low density lipoproteins (LDL) and very low density lipoproteins (VLDL) are precipitated quantitatively by the addition of phosphotungestic acid in the presence of magnesium ions. After centrifugation, the cholesterol concentration in the HDL (high density lipoproteins) fraction that remains in the supernatant was determined.

Reagents: Reagent (R) Phosphotungstate 0.52 mmol/L Magnesium chloride 30 mmol/L Reagents also contain non- reactive stabilizers and surfactants. Procedure: 1- Precipitation Into centrifuge tubes: Reagent (0.5 ml) was added to the specimen (0.2 ml), mixed and incubated for 10 minutes at room temperature. Then centrifuged for 10 minutes at 4000 rpm, then the supernatant was collected carefully.

2- Cholesterol- Liquizyme a- Appropriate set of test tubes was labeled 3 tubes for each sample, the standard, the blank and the test. b- Distilled water (50 μl) was added in the blank tube. c- HDL- cholesterol standard (50 μl) was added in the standard tube. d- The sample supernatant (100 μl) was added in the sample tube. e- The cholesterol reagent (1ml) was added in all tubes.

58 Materials and Methods f- The mixture was mixed, incubated for 10 minutes at 20-25°C or 5 minutes at 37°C. The absorbance of the sample (A sample) and standard (A standard) was measured against reagent blank at 546 nm, within 60 minutes. Calculation:

HDL cholesterol conc. (mg/dL) = A sample × 570

D) Determination of serum LDL–cholesterol concentration: LDL cholesterol in mg/dL was calculated as follows: LDL-cholesterol = total cholesterol- triglycerides + HDL-cholesterol. 5 4. Cardiac enzymes:

A) Determination of serum creatine kinase (CK):

Creatine kinase was determined according to the method of IFCC, (1989). Principle:

Creatine kinase (CK) catalyszes the phosphorylation of ADP in the presence of creatine phosphate to form ATP and creatine. The catalytic concentration is determined from the rate of NADPH formation, measured at 340 nm, by means of the hexokinase (HK) and glucose-6- phosphate dehydrogenase (G6PDH) coupled reaction 1, 2.

Creatine phosphate + ADP CK creatine +ATP

ATP + glucose HK ADP + glucose-6-phosphate

G6PDH Glucose-6-phosphate + NADP+ 6-phosphogluconate

+ NADPH + H+

59 Materials and Methods

Reagents: 1- Reagent 1 (pH 6.7) (Buffer /Coenzyme)

Imidazol 125 mmol/L D-Glucose 25 mmol/L N-Acetyl-L- 25 mmol/L Magnesium acetate 12.5 mmol/L NADP 2.5 mmol/L EDTA 2 mmol/L 2- Reagent 2 (Enzymes)

ADP 15.2 mmol/L AMP 25 mmol/L P1.P5-di(adenosine-5-) penta- 103 mmol/L phosphate glucose-6-phosphate 9 KU/L dehydrogenase (G6PDH) Creatine phosphate 250 mmol/L Hexokinase (HK) 3 KU/L

Procedure: 1. R1 (5ml) was mixed with 1 ml of R2 (working solution), stability: 2 week at 2-8˚С or 48 h at (15-25˚С). 2. Into a thermostatized cuvette; 1ml of working solution was pipetted and 50μl serum was added. 3. The mixture was mixed and incubated for 3 min. 4. The initial absorbance (A) of the sample was estimated at 1 min intervals from zero to 3 min. 5. (∆ A/min) which are the difference between absorbance and the average absorbance were calculated.

Calculation: CK (U/L) = ∆ A/min x 3333

60 Materials and Methods

B) Determination of serum creatine kinase isoenzyme (CK- MB):

Creatine kinase-MB was determined according to the method of IFCC, (1989).

Principle:

A specific antibody inhibits the M subunits of CK-MM and CK-MB, and thus allows determination of the B subunit of CK-MB (assuming the Absence of CK-BB or CK-1). CK-MB catalytic concentration, which corresponds to half of CK-MB concentration, is determined from the rate of NADPH formation, measured at 340 nm, by means of the hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) coupled reactions 1, 3.

Creatine phosphate + ADP CK Creatine +ATP

ATP + Glucose HK ADP +Glucose-6-phosphate

G6PDH Glucose-6-phosphate + NADP+ 6-phosphogluconate

+ NADPH + H+

Reagents: 1- Reagent 1 (pH 6.7) (Buffer /Coenzyme)

Imidazol 125 mmol/L D-Glucose 25 mmol/L N-Acetyl-L-Cysteine 25 mmol/L Magnesium acetate 12.5 mmol/L NADP 2.5 mmol/L EDTA 2 mmol/L

2- Reagent 2 (Enzymes)

ADP 15.2 mmol/L AMP 25 mmol/L P1.P5-di(adenosine-5-) penta- 103 mmol/L phosphate glucose-6-phosphate 9 KU/L dehydrogenase (G6PDH)

61 Materials and Methods

Creatine phosphate 250 mmol/L Hexokinase (HK) 3 KU/L

Procedure: 1. R1 (4ml) was mixed with 1 ml of R2 (working solution), stability: 2 week at 2-8˚С or 48 h at (15-25˚С). 2. Into a thermostatized cuvette; 1ml of working solution was pipetted and 50μL serum was added. 3. The mixture was mixed and incubated for 3 min. 4. The initial absorbance (A) of the sample was estimated at 1 min intervals from zero to 3 min. 5. (∆ A/min) which are the difference between absorbance and the average absorbance were calculated.

Calculation: CK-MB (U/L) = ∆ A/min *6666

C) Determination of serum lactate dehydrogenase activity (LDH):

The LDH catalyses the reaction between pyruvate and NADH + H+ with the formation of lactate and NAD.

Pyruvate + NADH+ H+ LDH L-Lactate + NAD+ The decrease in absorbance due to the oxidation of NADH to NAD was measured at 340 nm. It is proportional to the activity of LDH in the sample (Dito, 1979).

62 Materials and Methods

Reagents: 1- Reagent 1 (R1 Buffer)

Phosphate buffer (pH 7.5) 50 mmol/L Pyrovate 0.6 mmol/L Azide 8.0 mmol/L

2- Reagent 2 ( R2 Coenzyme)

NADH >0.18 mmol/L Sodium azide 8.0 mmol/L

Procedure: 1. The working solution was prepared according to the number of

tests required by mixing 9 volumes of reagent 1 (R1) and 1 volume

of reagent 2 (R2). 2. Working solution (1 ml) was pipetted into cuvette at 37 ˚С and add to it 20 μL of specimen. 3. The mixture was mixed, read initial absorbance after 30 seconds, 1, 2 and 3 minutes and start timer simultaneously. 4. The mean absorbance change was determined per minute (∆ A/min).

Calculation: LDH activity was calculated according to the following formula:

(U/L) = 8095 x ∆ A 340 nm /min.

63 Materials and Methods

Statistical Analysis:

Values were calculated as mean ± Standard Error (S.E) of the mean. Comparison between different groups was carried out by one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test using Instat software, version 3 (Graph pad Software, Inc., San Diego, USA). The p value was set at ≤ 0.05.

The figures were drawn using instant software program (Microsoft Office Excel 2003).

64 Results

The median lethal dose (LD50):

The present LD50 for leiurus quinquestriatus (L.q) scorpion venom before and after irradiation by 1.5 KGy or 3 KGy are shown in tables (1, 2 & 3) and illustrated in figure (1).

The LD50 of the native (1mg/ml) and 1.5 KGy & 3 KGy γ-irradiated (1mg/ml) L.q scorpion venom were 0.394 mg/kg, 3.517 mg/kg and 7.5 mg/kg respectively.

The present study revealed that the toxicity of L.q scorpion venom was reduced 11 and 20 times following exposure to gamma radiation 1.5 KGy and 3 KGy respectively, as compared to its native venom.

The present LD50 and 95% confidence limits for L.q scorpion venom before and after irradiation by 1.5 KGy or 3 KGy are shown in table (4).

Thus, there was a dose dependent increase in the LD50 after gamma irradiation and decrease in toxicity for L.q scorpion venom figure (1).

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Results

Table (1): Determination of LD50 for native Leiurus quinquestriatus (L.q) scorpion venom (1mg/ml) in male mice (N=6).

Log dose Dose Number of dead animals (mg/kg) (r)

- 0.720 0.190 0 - 0.620 0.239 1 - 0.520 0.301 2 - 0.420 0.379 0 - 0.320 0.477 5 - 0.220 0.601 6

Native Leiurus quinquestriatus (L.q) scorpion venom (1mg/ml) was injected i.p in different doses into male mice. Both LD0 which causes zero % mortality (0.190 mg/kg) and LD100 which causes 100 % mortality (0.601 mg/kg) were first determined, logarithm LD0 and LD100 was calculated. Doses at equal logarithmic intervals were chosen between these two doses. Each dose was injected in a group of six mice. After 24 h from injection the number of dead animals was recorded in each group. The LD50 was calculated by the formula of Spearman and Karber method.

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Results

Table (2): Determination of LD50 for 1.5 KGy γ-irradiated Leiurus quinquestriatus (L.q) scorpion venom (1mg/ml) in male mice (N=6).

Log dose Dose Number of dead animals (mg/kg) (r)

0.379 2.394 0 0.479 3.014 4 0.579 3.794 4 0.679 4.777 5 0.779 6.014 4 0.879 7.571 6

1.5 KGy γ-irradiated Leiurus quinquestriatus (L.q) scorpion venom (1mg/ml) was injected i.p in different doses into male mice. Both LD0 which causes zero % mortality (2.394 mg/kg) and LD100 which causes 100 % mortality (7.571 mg/kg) were first determined, logarithm LD0 and LD100 was calculated. Doses at equal logarithmic intervals were chosen between these two doses. Each dose was injected in a group of six mice. After 24 h from injection the number of dead animals was recorded in each group. The LD50 was calculated by the formula of Spearman and Karber method.

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Results

Table (3): Determination of LD50 for 3 KGy γ-irradiated Leiurus quinquestriatus (L.q) scorpion venom (1mg/ml) in male mice (N=6).

Log dose Dose Number of dead animals (mg/kg) (r)

0.479 3.014 0 0.579 3.790 2 0.679 4.770 6 0.979 9.527 4 1.079 11.994 5 1.179 15.100 4 1.279 19.010 6

3 KGy γ-irradiated Leiurus quinquestriatus (L.q) scorpion venom (1mg/ml) was injected i.p in different doses into male mice. Both LD0 which causes zero % mortality (3.014 mg/kg) and LD100 which causes 100 % mortality (19.010 mg/kg) were first determined, logarithm LD0 and LD100 was calculated. Doses at equal logarithmic intervals were chosen between these two doses. Each dose was injected in a group of six mice. After 24 h from injection the number of dead animals was recorded in each group. The LD50 was calculated by the formula of Spearman and Karber method.

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Results

Table (4): LD50 for native, 1.5 and 3 KGy γ-irradiated Leiurus quinquestriatus (L.q) scorpion venom.

Venom LD50 (mg/kg) 95% Confidence limits

Native L.q 0.394 0.265-0.948 1.5 KGy γ-irradiated 3.517 0.049-5.388 L.q 3 KGy γ-irradiated L.q 7.5 0.652-1.106

Three sets of mice, each consisting of several groups, were used for estimation of LD50. For each set, both LD0 and LD100 were first determined, logarithm LD0 and LD100 was calculated. Doses at equal logarithmic intervals were chosen between these two doses. Each dose was injected in a group of six mice. Mortality was recorded 24 h following the injection of the venom and LD50 was calculated.

- 69 -

Results

LD50

8 7 6 5

4

(mg/kg)

50 3

LD 2

1 0 0 0.5 1 1.5 2 2.5 3 3.5 Dose of gamma radiation (KGy)

Figure (1): Effect of 1.5 KGy & 3 KGy γ-irradiation on median lethal dose (LD50) of native Leiurus quinquestriatus (L.q) scorpion venom.

- 70 -

Results

Immunological properties (double immunodiffusion technique):

The result of double immunodiffusion test of native (non-irradiated), 1.5 KGy and 3 KGy gamma irradiated venom against the commercial polyvalent Egyptian antivenin, showed similar patterns of precipitin bands.

Four distinct lines were observed with the non-irradiated, as well as, the two dose levels 1.5 KGy & 3 KGy gamma irradiated L.q scorpion venom figure (2). The visible lines were identical and joined smoothly at the corners, indicating that, they have the same antigenic determinant i.e., no change in the antigenic properties was observed after gamma irradiation of the L.q scorpion venom at the two dose levels used.

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Results

S: saline (upper well). AV: polyvalent antivenin of Sc.v (central well). V1: native L.q Sc.v (right well) V2: irradiated (1.5 KGy) L.q Sc.v (lower well). V3: irradiated (3KGy) L.q Sc.v (left well).

Figure (2): Immunodiffusion reaction of horse serum polyvalent antivenin with native (non-irradiated), 1.5 KGy & 3 KGy γ-irradiated Leiurus quinquestriatus (L.q) scorpion venom.

Double immunodiffusion was performed utilizing 1.3% Nobel Agar in 0.9% NaCl immunodiffusion plates. 20µl of saline (S), native (L.q), 1.5 KGy and 3 KGy (L.q) γ-irradiated Leiurus quinquestriatus venom solution were placed in peripheral wells (venom concentration was 20 mg/ml), whereas, central one filled with 20µl of antivenin (AV), the slides were incubated at room temperature in humid chamber for 20 - 72 h then washed using saline, dried in the incubator at 37ºС, stained, dried in air and photographed.

- 72 -

Results

The labeling technique:

Factors affecting the labeling yield and determination of radiochemical purity of the produced compounds:

1- Effect of substrate amount: The influence of scorpion venom amount on the radiochemical yield (%) of 125I-scorpion venom using chloramine-T as oxidizing agent has been investigated. As shown in figure (3), increasing the amount of scorpion venom was accompanied by an increase in the radiochemical yield of 125I-scorpion venom, where it reached about 95 % at 200g of scorpion venom (optimum concentration).

2- Effect of chloramine-T concentration: Chloramine-T is a strong oxidizing agent commonly used in the iodination of organic compounds. Below 0.87mM chloramine-T, the percent radiochemical yield of 125I-scorpion venom was low (85 %). Above 0.87mM, chloramine-T concentration has little effect on the radiochemical yield of 125I- scorpion venom. The maximum yield of 125I-scorpion venom was obtained at 0.87mM chloramine-T as shown in figure (4) under the conditions of the experiment.

3- Effect of reaction temperature:

The radioiodination reaction of scorpion venom with 125I was carried out using 200g scorpion venom and 0.87Mm chloramine-T at different temperatures, figure (5). The radiochemical yield of 125I-scorpion venom was found to be about 68% at 25ºC and increased to about 75% and 96% on increasing the reaction temperature of the mixture to 40ºC and 60ºC respectively at 30 min reaction time. By raising the reaction temperature to 100ºC, decrease in the radiochemical yield of 125I-scorpion venom was observed indicating the instability of the labeled compound and its denaturation.

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Results

4- Effect of reaction time:

The relationship between the reaction time and the radiochemical yield of 125I- scorpion venom is shown in figure (6). It is clear that the radiochemical yield of 125I- scorpion venom increased from 75.8% to 95.4% by increasing the reaction time from 5 min to 30 min at 60ºC. Extending the reaction time to 60 min caused slight decrease in the radiochemical yield of 125I- scorpion venom.

5- Effect of pH of the reaction mixture:

The effect of alteration of the pH of reaction mixture on the radiochemical yield of 125I- scorpion venom was investigated in the pH range from 2- 11 figure (7). At pH 2, the radiochemical yield of 125I- scorpion venom was relatively low (63%). The maximum radiochemical of 125I- scorpion venom yield (95%) was obtained around pH 7. By increasing the pH to 9 and 11, a decrease in the radiochemical yield of 125I- scorpion venom was observed.

- 74 -

Results

100

90

80

70

60

50

40

30

20 Radiochemicalyield (%) 10

0 0 50 100 150 200 250 300 L.q scorpion venomSV amount (μg)

Figure (3): Radiochemical yield of 125I- L.q scorpion venom as a function of scorpion venom amount.

Different amounts of scorpion venom in μg (50, 100, 150, 200, 250 & 300 µg) were used to get the optimum amount required to obtain maximum labeling yield while the other factors were kept constant.

- 75 -

Results

100

90

80

70

60

50

40

30

20 (Radiochemical yield %) yield (Radiochemical 10

0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 (Chloramine-T concentration, ﴾ mM)

Figure (4): Radiochemical yield of 125I- L.q scorpion venom as a function of chloramine-T concentration.

The optimum amount of scorpion venom (200g) was used, using different concentrations of the oxidizing agent chloramine-T (0.2, 0.42, 0.87, 1.1 & 1.3 mM) to get the optimum concentration of chloramine-T required to obtain maximum labeling yield while the other factors were kept constant.

- 76 -

Results

100

90

80

70

60

50

40

30

20 Radiochemical yield % yield Radiochemical

10

0 0 10 20 30 40 50 60 70 80 90 100 110 Reaction temperature,oC

Figure (5): Radiochemical yield of 125I- L.q scorpion venom as a function of reaction temperature.

The optimum amount of scorpion venom (200g) and chloramine-T (0.87mM) were used at different reaction temperature (25, 40, 60, 80, 100 ºC) on the labeling process to get the optimum reaction temperature required to obtain maximum labeling yield while the other factors were kept constant.

- 77 -

Results

100

90

80

70

60

50

40

30

20 Radiochemical yield % yield Radiochemical

10

0 0 10 20 30 40 50 60 ﴿ Reaction time, ﴾ min

Figure (6): Radiochemical yield of 125I- L.q scorpion venom as a function of reaction time.

The optimum amount of scorpion venom (200g), chloramine-T (0.87mM) and reaction temperature (60 ºC) were used at different reaction time (5, 15, 30, 40, 60 min) on the labeling process to get the optimum reaction time required to obtain maximum labeling yield while the other factors were kept constant.

- 78 -

Results

100

80

60

40

Radiochemical yield % yield Radiochemical 20

0 0 1 2 3 4 5 6 7 8 9 10 11 12 pH of reaction mixture

Figure (7): Radiochemical yield of 125I- L.q scorpion venom as a function of pH of reaction mixture.

The optimum amount of scorpion venom (200g), chloramine-T (0.87mM), reaction temperature (60 ºC) and reaction time (30 min) were used at different pH values (2, 4, 7, 9, 11) on the labeling process to get the optimum reaction medium required to obtain maximum labeling yield while the other factors were kept constant.

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Results

In –vitro stability of the labeled 125I-L.q scorpion venom:

The stability of 125I-L.q scorpion venom was studied in order to determine the suitable time for injection to avoid the formation of the undesired products. These undesired radioactive products may be accumulated in non-target organs. Table (5) shows the stability of 125I- L.q scorpion venom up to 24h.

Table (5): The In-vitro stability of 125I- L.q scorpion venom

125I- L.q scorpion Time post labeling (h) Free iodine,% venom,% 1 95.80  0.85 3.20  0.15 2 95.00 0.60 4.00  0.15 4 94.30 0.65 5.70  0.11

8 93.70*@  0.65 6.30  0.05

12 91.90*@  0.81 7.11  0.10

24 90.30*@0.45 9.700.15

Each value represents Mean ± S.E (n = 3)

Statistical analysis was carried out by one way ANOVA followed by Tukey-kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from preceding value at p ≤ 0.05.

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Results

Labeling of irradiated (1.5 and 3 KGy) L.q scorpion venom:

Labeling of irradiated (1.5 and 3 KGy) scorpion venom was unsuccessful using this method of iodination (chloramine-T method).

Biodistribution of 125I- L.q scorpion venom in normal mice: When 125I- L.q scorpion venom was injected into normal mice via intravenous route, the tracer was distributed all over the body organs and fluids. This route of biodistribution depends mainly on the scorpion venom compound. The activity in the intestine and urine which is equal to 9.43 and 12.70 % respectively at 1 h post injection suggests the rapid excretion of the labeled compound. The activity present in bone which is equal 2.90 % at 1 h post injection reflects its high clearance from this organ. Other organs uptakes were normal and do not show significant variation as indicated in table (6).

All the obtained data demonstrate that the tracer was distributed rapidly throughout the body after intravenous injection (10 min), and cleared rapidly through the hepatobiliary system (4 h). The liver was the organ with the highest radioactivity that was quickly excreted into the intestinal tract. The presence of activity in the urinary bladder suggests the excretion of the tracer through the kidneys to some extent. The low activity located in the thyroid gland indicates that 125I- L.q scorpion venom is stable in-vivo against biological decomposition.

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Results

Table (6): Biodistribution pattern of 125I- L.q scorpion venom in different organs or body fluids in normal mice (n=3) at different time intervals.

% of the i.v injected dose of 125I-L.q Sc.v (200μg) /organ or Whole organ or body fluid at different time intervals post injection body fluid 10min 1h 2h 4h

* *@ * @ # Blood 28.50 ± 0.40 20.80 ± 0.14 12.00 ± 0.36 5.80 ± 0.03

* *@ * @ # Bone 3.30 ± 0.06 2.90 ± 0.02 2.10 ± 0.04 1.80 ± 0.03 * *@ * @ # Muscle 3.10 ± 0.04 4.50 ± 0.04 3.70± 0.04 1.10 ± 0.02 * *@ * @ # Brain 1.40 ± 0.02 1.20 ± 0.02 0.90 ± 0.01 0.70 ± 0.02 * *@ * @ # Lungs 1.90 ± 0.02 0.60 ± 0.02 0.50 ± 0.02 0.11± 0.01 * * * Heart 1.31 ± 0.02 0.22 ± 0.02 0.12± 0.01 0.11± 0.01 * @ * @ # Liver 10.40 ± 0.02 12.60 ± 0.02 10.50 ± 0.02 8.30 ± 0.02 * *@ * @ # Kidneys 11.30 ± 0.03 29.08 ± 0.01 22.31 ± 0.02 11.60 ± 0.02 * * * @ # Spleen 1.22 ± 0.02 0.28 ± 0.01 0.43 ± 0.02 0.24 ± 0.02 * *@ * @ # Intestine 4.21 ± 0.02 9.43 ± 1.40 14.50 ± 0.02 14.91 ± 0.02 * *@ * @ # Urine 6.21± 0.08 12.70± 0.09 16.52± 0.23 34.50± 0.02 * * * Thyroid gland 0.91±0.03 1.12±0.04 1.00±0.04 1.22±0.02

Each value represents Mean ± S.E (n = 3)

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from 10 min at p ≤ 0.05. @ Significant difference from 1h at p ≤ 0.05. # Significant difference from 2h p at ≤ 0.05.

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Results

Table (7): Brain/ Blood ratio of 125I- L.q scorpion venom in normal mice at different time intervals.

% 125I-L.q scorpion venom distribution/organ (g) or body Organ or body fluid fluid at different time intervals post injection (g) 10min 1h 2h 4h Brain * *@ * @ # 3.50 ± 0.06 3.00 ± 0.03 2.20 ± 0.02 1.71 ± 0.02 * *@ * @ # Blood 16.33±0.03 11.92 ±0.06 6.91 ± 0.02 3.33 ± 0.03

Brain/Blood 0.20 0.25 0.32 0.52

Each value represents Mean ± S.E (n = 3)

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from 10 min at p ≤ 0.05. @ Significant difference from 1h at p ≤ 0.05. # Significant difference from 2h at p ≤ 0.05.

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Results

Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on plasma lipid peroxides (MDA) in rats:

The results are shown in table (8) and illustrated in figure (8).

The normal level of MDA in non-envenomated value of plasma MDA was shown to be 18.23 ± 1.047 nmol/ml.

The native Leiurus quinquestriatus (non-irradiated) scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (0.133 mg/kg) significantly increased plasma MDA value after 1 h to 22.16 ± 0.558 nmol/ml, while didn’t cause significant difference in plasma MDA value after 4 h of envenomation 20.53 ± 0.503 nmol/ml, respectively compared to the normal non-envenomated value.

In contrast, the 1.5 KGy γ-irradiated Leiurus quinquestriatus scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (1.230 mg/kg) 1h and 4h after envenomation significantly decreased plasma MDA value to 17.630 ± 0.543 nmol/ml, 16.58 ± 0.920 nmol/ml, respectively compared to the native L.q (non-irradiated) scorpion venom value. However, no significant changes were observed when compared to normal non envenomated value.

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Results

Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on blood glutathione (GSH) in rats:

The results are shown in table (8) and illustrated in figure (9).

The normal non-envenomated level of blood GSH was shown to be 6.50 ± 0.566 μg/ml.

The native Leiurus quinquestriatus (non-irradiated) scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (0.133 mg/kg) significantly increased blood GSH level after 1 h to 12.25 ± 1.098 μg/ml, and after 4 h to 11.13 ± 0.639 μg/ml (71.2%), respectively compared to the normal non- envenomated level.

However, the 1.5 KGy γ-irradiated Leiurus quinquestriatus scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (1.230 mg/kg), 1h and 4h after envenomation significantly decreased blood GSH level to 5.62 ± 0.263 μg/ml and 4.00 ± 0.327 μg/ml, respectively compared to the native L.q (non-irradiated) scorpion venom level. But no significant changes were observed when compared to the normal non envenomated level.

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Results

Table (8): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on lipid peroxides (MDA) value and blood glutathione (GSH) level in rats.

Groups Normal Native L.q scorpion venom γ-irradiated (1.5 KGy) L.q scorpion non (non irradiated) venom envenomated (0.133 mg/kg) (1.230 mg/kg) Parameters i.p i.p 1h 4h 1h 4h

MDA * @ @ (n mol/ml) 18.23±1.05 22.16±0.56 20.53±0.50 17.63±0.54 16.58±0.92

* * @ @ GSH 6.50±0.57 12.25±1.09 11.13±0.64 5.63±0.26 4.00 ± 0.33 (μg/ml)

Three groups of animals each consisting of 8 rats were used. They received saline (0.1ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of MDA value and GSH level.

Each value represents Mean ± S.E (n = 8)

Statistical analysis carried out by one way ANOVA followed by Tukey-kramer Multiple Comparisons Test: * Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (8): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on plasma lipid peroxides (MDA) in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of MDA value.

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (9): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on blood glutathione (GSH) in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of GSH level.

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Effect of native (0.133 mg/kg i.p) and 1.5 KGy gamma irradiated (1.230 mg/kg i.p) Leiurus quinquestriatus (L.q) scorpion venoms on serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in rats:

The results are shown in table (9) and illustrated in figure (10 & 11).

The normal non-envenomated activity of ALT and AST were shown to be 37.49 ± 3.151 U/L and 56 ± 5.340 U/L, respectively.

The native L.q (non-irradiated) scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (0.133 mg/kg) L.q significantly increased ALT & AST activities 1 h after envenomation to 57.72 ± 5.079 U/L & 96.61 ± 5.158 U/L, and 4 h after envenomation to 87.25 ± 5.770 U/L & 78.25 ± 4.190 U/L respectively compared to the normal non-envenomated activities.

In contrast, The 1.5 KGy γ-irradiated L.q scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (1.230 mg/kg) L.q significantly decreased in ALT & AST activities 1h after envenomation to 27.62 ± 0.577 U/L & 45.50 ± 2.600 U/L and 4h after envenomation to 27.81 ± 0.898 U/L & 47 ± 2.360 U/L respectively compared to the native L.q (non-irradiated) scorpion venom activities. However, no significant changes were observed when compared to the normal non envenomated activities.

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Results

Table (9): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in rats.

Groups Normal Native L.q scorpion venom (non γ-irradiated (1.5 KGy) L.q scorpion non irradiated) venom envenomated (0.133 mg/kg) (1.230 mg/kg) Parameters i.p i.p

1h 4h 1h 4h

ALT * * @ @ (U/L) 37.49±3.15 57.72±5.08 96.61±5.16 27.62±0.58 27.81±0.89

* * @ @ AST 56.00±5.34 87.25±5.77 78.25±4.19 45.50±2.60 47.00±2.36 (U/L)

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of ALT and AST activity.

Each value represents Mean ± S.E (n = 8)

Statistical analysis was carried out by one way ANOVA followed by Tukey-kramer Multiple Comparison Test: * Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (10): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum alanine aminotransferase (ALT) activity in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of ALT activity.

Statistical analysis carried out by one way ANOVA followed by Tukey-kramer Multiple Comparisons Test:

*Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (11): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venom on serum aspartate aminotransferase (AST) in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of AST activity.

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum cholesterol level in rats:

The results are shown in table (10) and illustrated in figure (12).

The normal non-envenomated level of serum cholesterol was shown to be 53.03 ± 3.343 mg/dl.

The native Leiurus quinquestriatus (non-irradiated) scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (0.133 mg/kg) significantly decreased serum cholesterol level after 1 h to 39.12 ± 2.060 mg/dl, while didn’t cause significant difference in serum cholesterol level 4 h after envenomation 46.37 ± 3.792 mg/dl, respectively compared to the normal non-envenomated level.

In contrast, the 1.5 KGy γ-irradiated Leiurus quinquestriatus scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (1.230 mg/kg) 1h and 4h after envenomation significantly increased serum cholesterol level to 62.75 ± 1.882 mg/dl and 59.66 ± 2.739 mg/dl, respectively compared to the native L.q (non-irradiated) scorpion venom level, but no significant changes were observed when compared to the normal non envenomated level.

Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum triglycerides level in rats:

The results are shown in table (10) and illustrated in figure (13).

The normal non-envenomated level of serum triglyceride was shown to be 66.20 ± 6.024 mg/dl.

The native Leiurus quinquestriatus (non-irradiated) scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (0.133 mg/kg) significantly decreased serum triglycerides level after 1 h to 47.84 ± 2.172 mg/dl, and after 4 h to 29.74 ± 2.324 mg/dl, respectively compared to the normal non- envenomated level.

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Results

In contrast, the 1.5 KGy γ-irradiated Leiurus quinquestriatus scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (1.230 mg/kg) 1h and 4h after envenomation significantly increased serum triglycerides level to 75.53 ± 2.110 mg/dl, 65.24 ± 3.628 mg/dl, respectively compared to the native L.q (non-irradiated) scorpion venom level. However, no significant changes were observed when compared to the normal non envenomated level.

Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum HDL-cholesterol level in rats:

The results are shown in table (10) and illustrated in figure (14).

The normal non-envenomated level of serum HDL-cholesterol was shown to be 29.73 ± 1.560 mg/dl.

The native Leiurus quinquestriatus (non-irradiated) scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (0.133 mg/kg) significantly decreased serum HDL-cholesterol level after 1 h to 22.93 ± 0.604 mg/dl, and 4 h after envenomation to 18.96 ± 0.563 mg/dl, respectively compared to the normal non envenomated level.

In contrast, the 1.5 KGy γ-irradiated Leiurus quinquestriatus scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (1.230 mg/kg) significantly decreased serum HDL-cholesterol level after 1h to 12.25 ± 0.273 mg/dl, and 4h after envenomation to 14.85 ± 0.397 mg/dl, respectively compared to the native L.q (non-irradiated) scorpion venom level. However, significantly decreased serum HDL-cholesterol level after 1h to 12.25 ± 0.273 mg/dl, and 4h after envenomation to 14.85 ± 0.397 mg/dl, respectively compared to the normal non envenomated scorpion venom level.

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Results

Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum LDL-cholesterol level in rats:

The results are shown in table (10) and illustrated in figure (15).

The normal non-envenomated level of serum LDL-cholesterol was shown to be 69.521 ± 3.386 mg/dl.

The native Leiurus quinquestriatus (non-irradiated) scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (0.133 mg/kg) significantly decreased serum LDL-cholesterol level after 1 h to 52.48 ± 2.435 mg/dl, while didn’t cause significant difference in serum LDL- cholesterol level 4 h after envenomation 59.38 ± 4.061 mg/dl, respectively compared to the normal non envenomated level.

In contrast, the 1.5 KGy γ-irradiated Leiurus quinquestriatus scorpion venom injected i.p with a dose equals to the 1/20 of LD50 (1.230 mg/kg) 1h and 4h after envenomation didn’t cause significant difference in serum LDL- cholesterol level 59.96 ± 1.888 mg/dl, 61.47 ± 3.144 mg/dl, respectively compared to the native L.q (non-irradiated) scorpion venom level, but, no significant changes were observed when compared to the normal non envenomated level.

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Results

Table (10): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on total cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol levels in rats.

Native L.q scorpion venom (non γ-irradiated (1.5 KGy) L.q scorpion Groups Normal irradiated) venom (non e nvenomated) (0.133 mg/kg) (1.230 mg/kg) i.p i.p Parameters 1h 4h 1h 4h Total cholesterol * @ @ (mg/dl) 53.03±3.34 39.12±2.06 46.37±3.79 62.75±1.88 59.66±2.74

Triglycerides * * @ @ (mg/dl) 66.20±6.02 47.84±2.17 29.74±2.32 75.53±2.11 65.24±3.63

HDL-cholesterol * * * *@ (mg/dl) 29.73±1.56 22.94±0.60 18.97±0.56 12.25±0.27 14.85±0.39

LDL-cholesterol * (mg/dl) 69.52±3.39 52.49±2.44 59.39±4.06 59.96±1.89 61.47±3.14

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of total cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol levels.

Each value represents Mean ± S.E (n = 8) Statistical analysis was carried out by one way ANOVA followed by Tukey-kramer Multiple Comparison Test: * Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (12): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum cholesterol in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of total cholesterol level.

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (13): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum triglycerides in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of triglycerides level.

Statistical analysis was carried out by one- way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (14): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum HDL-cholesterol in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of HDL-cholesterol level.

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (15): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum LDL-cholesterol in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of LDL-cholesterol level.

Statistical analysis was carried out by one- way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Effect of native (0.133 mg/kg i.p) and 1.5 KGy gamma irradiated (1.230 mg/kg i.p) Leiurus quinquestriatus (L.q) scorpion venoms on serum creatine kinase (CK), creatine kinase isoenzyme (CK- MB) and lactate dehydrogenase (LDH) activities in rats:

The results are shown in table (11) and illustrated in figure (16, 17& 18).

CK, CK-MB and LDH activities of normal (non-envenomated) rats were 794.80 ± 57.270 U/L, 1042 ± 57.270 U/L and 283.50 ± 0.990 U/L, respectively.

The group injected i.p with native Leiurus quinquestriatus (non- irradiated) scorpion venom (a dose equals to the 1/20 of LD50 (0.133 mg/kg)) showed significant elevation in CK, CK-MB and LDH activities 1 h after envenomation by 1321 ± 83.940 U/L, 432 ± 32.200 U/L & 1568 ± 83.940 U/L, and 4 h after envenomation by 1626 ± 156.600 U/L, 623.70 ± 2.178 U/L and 1873 ± 156.600 U/L, respectively compared to the normal non envenomated activities.

Moreover, the group injected i.p with 1.5 KGy γ-irradiated Leiurus quinquestriatus scorpion venom (a dose equals to the 1/20 of LD50 (1.230 mg/kg)) showed significantly decreased in CK, CK-MB and LDH activities 1 h after envenomation by (788.80 ± 57.270 U/L, 275.30 ± 1.022 U/L and 1033 ± 57.270 U/L) and 4 h after envenomation by 790.30 ± 57.270 U/L, 272.40 ± 1.053 U/L and 1036 ± 57.270 U/L compared to the native L.q (non-irradiated) scorpion venom activities. However, no significant changes were observed when compared to the non envenomated activities.

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Results

Table (11): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on creatine kinase, creatine kinase-MB and lactate dehydrogenase activities in rats.

Groups Normal Native L.q scorpion venom (non γ-irradiated (1.5 KG )L.q scorpion (non envenomated) irradiated) venom (0.133 mg/kg) (1.230 mg/kg) Parameters i.p i.p 1h 4h 1h 4h

Total CK * * @ @ (U/L) 794.80±57.27 1321.00±83.94 1626.00±156.60 788.80±57.27 790.30±57.27

CK –MB * * @ @ (U/L) 283.50±0.99 432.00±32.20 623.70±2.18 275.30±1.02 272.40±1.05 LDH * * @ @ (U/L) 1042.00±57.27 1568.00±83.94 1873.00±156.60 1033.00±57.27 1036.00±57.27

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of creatine kinase, creatine kinase-MB and lactate dehydrogenase activities.

Each value represents Mean ± S.E (n = 8)

Statistical analysis was carried out by one way ANOVA followed by Tukey-kramer Multiple Comparison Test: * Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (16): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum creatine kinase (CK) in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of CK level.

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (17): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum creatine kinase isoenzyme (CK-MB) in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of CK-MB level.

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Results

Figure (18): Effect of native and 1.5 KGy gamma irradiated Leiurus quinquestriatus (L.q) scorpion venoms on serum lactate dehydrogenase (LDH) in rats.

Three groups of animals each consisting of 8 rats were used. They received saline (0.1 ml), native (0.133 mg/kg i.p) and 1.5 KGy γ-irradiated (1.230 mg/kg i.p) L.q scorpion venoms respectively. Blood was collected 1 and 4 h after envenomation for estimation of LDH level

Statistical analysis was carried out by one way ANOVA followed by Tukey- kramer Multiple Comparisons Test:

* Significant difference from normal group at p ≤ 0.05. @ Significant difference from the corresponding native L.q scorpion venom group at p ≤ 0.05.

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Discussion

In the present study the toxicological, pharmacological, immunological and biochemical effects of the Leiurus quinquestriatus (L.q) scorpion venom; non-irradiated and gamma-irradiated at two dose levels, were investigated.

In the present study the toxicity of the native L.q scorpion venom was reduced 11 and 20 times following the exposure to 1.5 KGy & 3 KGy gamma rays respectively. Thus, progressive increase in LD50 after gamma irradiation indicates decrease in its toxicity.

According to Bennacef-Heffar and Laraba-Djebari (2003), it was shown that when, Vipera lebtina venom was irradiated with 1 KGy and 2 KGy, there was significant decrease in the toxicity four and nine times, respectively.

Effect of gamma irradiation on venom solution could be attributed to its known effects on protein molecules, as venoms are mainly protein in nature, as well as, ionizing radiation can change the molecular structure and the biological properties of protein molecules (Boni-Mitake et al., 2001). This can occur by two forms: direct process by which ionizing radiation interacts directly on target molecules and an indirect process by - - + - which the product generated by water radiolysis, like e , O2 , H and OH interact with target molecules and can modify the biological activity of protein and peptides by reacting with certain sites or groups in the molecule (Garrison, 1987; Casare et al., 2006). These radicals act by removing hydrogen, breaking disulfide bonds, promoting deamination as well as inducing the formation of intramolecular and intermolecular covalent bonds (Alexander and Hamilton 1962; Halliwell and Gulteridge, 1989). These structural changes result in a decrease or loss of the enzymatic and biological activities of the proteins (Gallacci et al., 2000).

It was known that, the toxicity of the scorpions venom belonging to the family Buthidae, was attributed to the effect of small proteins, with an approximate mol wt of 7000, containing 57-78 amino-acids, cross linked by four disulfide bridges (Zlotkin et al., 1978; Shaaban, 1990). It was found that, the mammal and insect toxins, have three disulfide bridges at homologous positions; while the fourth bridge was different. It is likely that, the position of the disulfide bridges is the same for all the scorpion neurotoxins that are active on mammals.

One believes that, the effects of radiation could, lead to a break of the disulfide bridges, with the resultant attenuation of the toxicity of the

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Discussion venom. This finding was in agreement with that of Yang, (1970) who demonstrated, the integrity of the disulphide bonds in cobrotoxin (toxic protein from Formosan cobra). The reduction of the disulphide bonds to sulphydryl, by B. mercaptoethanol, leads to loss of toxicity of cobrotoxin.

Loss of function of protein by irradiation is not usually due to breaking peptide bonds, or otherwise, disrupting the primary skeletal structure of peptide chain. It may result from a break in the hydrogen or disulfide bonds which in turn can result in a disorganization of the internal relationships of side chain groups, or an exposure of amino-acid groups, resulting in change in biological activity (Hayes and Francis, 2001).

Shaaban, (1990) demonstrated that, irradiation of Androctonus amoreuxi scorpion venom with gamma rays in dry state (15 & 30 KGy) decreased its lethal and toxic activity while retaining its antigenicity.

It has been shown that gamma irradiation is an effective technique for attenuating venom toxicity and maintaining venom immunogenicity in in Naja haje and Cerates cerastes venoms (Shaaban, 2003; Abib and Laraba-Djebari, 2003).

The double immunodiffusion test of native (non-irradiated), 1.5 KGy and 3 KGy gamma irradiated venom against a commercial polyvalent Egyptian antivenin, showed similar pattern. The visible lines obtained in the immunodiffusion reactions were identical and joined smoothly at the corners. This indicated that, the antigenic response was not changed as judged by the capacity of irradiated venom to react with the antivenin. These results demonstrate that the ability of the venom antigens to react with its corresponding antibodies was maintained in spite of being exposed to radiation doses of 1.5 KGy and 3 KGy.

These results are in agreement with the data of other studies that reported that ionizing radiation is able to detoxify Androctonus amoreuxi without affecting the immunogenic properties (Shaaban, 1990). It has been shown that higher doses of radiation (15 KGy and 30 KGy) destroyed practically all the venom toxicity though keeping its immunogenicity.

Fatani et al, (2010) also observed that, the Egyptian and Saudi were tested against L.q scorpion venom, both showed one prominent long thick band, with Saudi exhibiting 3 prominent shorter bands and the Egyptian 3 fainter bands. Egyptian and saudi antivenoms

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Discussion cross-reacted with L.q scorpion venom indicating production of high amounts of specific antibodies and strong antigen-antibody complexes.

Furthermore, it has been reported that immunodiffusion studies revealed identity between gamma irradiated (15 KGy) and native Naja haje and Cerastes cerastes venoms since the antigenic response was not changed as judged by the capacity of irradiated venoms to react with horse polyvalent antivenin (Shaaban, 2003).

However, Kume and Matsuda, (1995) the radiation who studied induced changes in the structural and antigenic properties of egg albumin and bovine serum albumin, suggested that the main part of conformation- dependent antigenic structures (conformation epitopes) is easily lost by radiation, but some antigenicity, which is mostly due to the amino acid sequence-dependent antigenic structures (sequential epitopes) remains, even at high doses.

The present results suggested that, treatment with gamma radiation is a promising tool for Leiurus quinquestriatus scorpion venom detoxification without affecting their immunogenic properties.

The present experiment that was carried out for producing the optimum radiochemical yield of 125I-scorpion venom using chloramine-T as oxidizing agent has been investigated. Increasing the amount of scorpion venom was accompanied by an increase in the radiochemical yield of 125I-scorpion venom, where it reached about 95% at 200g of scorpion venom. By increasing the amount of venom above 300g, the radiochemical yield remains constant because the entire generated iodonium ions in the reaction are captured at that concentration of scorpion venom (Coenen et al., 1983).

Chloramine-T is a strong oxidizing agent commonly used in the iodination of organic compounds. It is able to oxidize the I- to I+ for electrophilic substitution in scorpion venom. Below 0.87mM chloramine- T, the percent radiochemical yield of 125I-scorpion venom was low (85 %) which may be due to the insufficiency of chloramine-T concentration to oxidize all the iodide ions present in the solution. Above 0.87mM, chloramine-T concentration has little effect on the radiochemical yield of 125I-scorpion venom. The maximum yield of 125I-scorpion venom was obtained at 0.87mM chloramine-T under the conditions of the experiment. Increasing the amount of oxidizing agent above 0.87mM lead to a decrease in the radiochemical yield due to the formation of

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Discussion undesirable oxidative side reactions like chlorination, polymerization and denaturation of substrate (Mcfarlane, 1958).

The reaction temperature in most electrophilic substitution reactions played an important role, as it was very important in the build up of a new covalent bond between the iodonium ion and the carbon atom in the molecule.

The radioiodination reaction of scorpion venom with iodine-125 was carried out using 200g scorpion venom and 150g chloramine-T at different temperatures. The radiochemical yield of 125I-scorpion venom was found to be about 68% at 25ºC and increased to about 75% and 96% on heating the reaction mixture to 40ºC and 60ºC respectively at 30 min reaction time. This is due to the fact that the leaving hydronium ion required some energy to break the C-H bond and to initiate the introduction of the radioactive iodonium ion into the scorpion venom. By raising the reaction temperature to 100ºC, decrease in the radiochemical yield of 125I-scorpion venom was observed indicating the instability of the labeled compound.

The radiochemical yield of the present 125I-scorpion venom increased from 75.8% to 95% by increasing the reaction time from 5 min to 30 min at 60ºC. Extending the reaction time to 60 min caused slight decrease in the radiochemical yield of 125I-scorpion venom. According to (Mcfarlane, 1958; Knust et al., 1990) this can be attributed to the long exposure of substrate (scorpion venom) to the oxidizing agent which causes oxidative side reactions like chlorination, polymerization and denaturation of substrate.

The effect of alteration of the pH of reaction mixture on the radiochemical yield of the present 125I-scorpion venom was investigated in the pH range from 2- 11. At pH 2, the radiochemical yield of 125I- scorpion venom was relatively low (63.7%) as a result of the predominance of iodine monochloride (ICl) species which have lower oxidation potential than hypochlorus acid (HOCl) species (Cynthia et al., 1979) The maximum radiochemical yield of 125I-scorpion venom (95.4%) was obtained around pH 7. The chloramines-T method has an optimum labeling efficiency at approximately pH 7 and labeling efficiency was reduced at higher or lower pH values. (Rayudu, 1983) By increasing the pH to 9 and 11, a decrease in the radiochemical yield of 125I-scorpion venom was observed. This may be attributed to the decrease in iodonium ion which is responsible for the electrophilic substitution reaction (Saccavini and Bruneau, 1984).

- 109 -

Discussion

The present radioiodination reaction of venom with iodine-125 was carried out using 200g venom and 150g chloramine-T at 60ºC for 30 min. The maximum radiochemical of 125I-venom yield was obtained around pH 7.

The current study of the distribution of 125I-labeled L.q venom showed the highest contents of radioactivity in liver and kidney. Radioactivity of the brain indicates that 125I-scorpion venom was stable in-vivo against biological decomposition.

In this study labeling irradiated (1.5 & 3 KGy) scorpion venom was unsuccessful. However, Boni-Mitake et al., (2006) successfully labeled both native and irradiated venom using modified method and studied distribution of 125I-labeled crotamine in mice tissues. Both native and irradiated (2 KGy) proteins were labeled with 125I using chloramines-T method after purified crotamine from venom by gel filtration followed by purifying ion exchange chromatography, using high performance liquid chromatography (HPLC). Native and irradiated crotamine were rapidly absorbed and they appear to have hepatic metabolism and renal elimination. It is possible that the irradiated crotamine was metabolized and eliminated faster than the native crotamine, probably by means of structural alterations induced by gamma irradiation, which could possibly explain the reduced toxicity of the irradiated protein.

It has been previously established that the pathophysiological manifestations of human envenomation can be modeled in animals by the injection of the scorpion toxins (Andrade et al., 2004). In fact, scorpion venoms consist of a complex mixture of active substances, mainly proteins that are often responsible for the observed pharmacological and toxicological symptoms that follow the envenomation by scorpion sting.

The current experiments assessed the impact of gamma radiation of L.q scorpion venom on the effects induced by the venom on some oxidative stress biomarker, liver enzymes, lipid profile & cardiac enzymes. Exposure of scorpion venom in aqueous solution to a dose of 1.5 KGy gamma radiation was suitable for attenuating venom.

The present study revealed that native L.q scorpion venom caused significant increase in normal plasma MDA. These results were in agrement with those reported by Fatani et al., (2006) and El-Alfy et al., (2007) that defined the role of oxidative stress following experimental envenomation by L.q scorpion venom by measuring of thiobarbituric

- 110 -

Discussion acid-reactive substances (TBARS) that demonstrated enhanced level of lipid peroxidation.

Hypothetically, the mechanisms by which generation of free radicals and lipid peroxidation occurs in scorpion envenomation are complex and may depend on a multitude of interacting factors. One of these factors is that scorpion venoms contain neurotoxins that act on a number of ionic channels resulting in alteration of ionic transport and cytosolic calcium overload (Harvey et al., 1992). This elevates AMP concentration and increases its catabolism, and subsequently leads to generation of free radicals. Ultimately, these free radicals attack membrane phospholipids causing their peroxidation. Furthermore, occurrence of lipid peroxidation in biological membranes eventually causes impairment of membrane functioning, decreased fluidity, and inactivation of membrane-bound receptors, all of which may culminate in multiple organ dysfunction (Bhaumik et al., 1995; Love, 1999; Al-Omar et al., 2004), a feature encountered in scorpion envenomation (Meki et al., 2003). Alternatively, it is known that scorpion venoms, by their action mainly on sodium channels, enhance release of various neurotransmitters such as adrenaline and noradrenaline (Azevedo et al., 1979; Ismail, 1995; Meki et al., 2003). It has been reported that catecholamines can induce the generation of free radicals which may lead to oxidative stress and apoptosis (Khorchid et al., 2002).

These changes in lipid peroxidation due to the lethal effects of scorpion venom are attributed to a direct correlation between degree of lipid peroxidation and phospholipase A2 induced phospholipids hydrolysis (Sevanian et al., 1989). On the other hand, inhibition of phospholipase A2 significantly reduced lipid peroxidation (Borowitz and Montgomery, 1989). Phospholipid hydrolysis by PLA2 enzyme release arachidonic acid in turn its metabolism results in the formation of potentially toxic reactive oxygen species (ROS) and lipid peroxidation (Adibhatla et al., 2003).

Previous studies demonstrated the effect of gamma radiation on the Vipera lebetina and that phospholipase A2 activity was abolished in the irradiated venom (Bennacef-Heffar and Laraba-Djebari, 2003).

The present investigation explored the gamma irradiated venom effect on lipid peroxides levels. There was a significant decrease in MDA levels in rats envenomated with 1.5 KGy γ-irradiated L.q venom compared to the rats envenomated with the equal dose of native venom.

- 111 -

Discussion

There are few reports on the effect of gamma rays on scorpion venoms concerning lipid peroxidation in general. The current study revealed that experimental envenomation by L.q scorpion venom was accompanied by free radical generation and depletion of antioxidant defense system. Such elevation in oxidative stress parameters correlate with the cardiac injury biochemical markers as well as hemodynamic manifestations following scorpion envenomation (El-Alfy et al., 2007).

In the present study, normal blood GSH was significantly increased by the native L.q scorpion venom, while unaffected by the γ-irradiated (1.5 KGy) one. It seems probable that, these results might be a secondary event following the increase in lipid peroxides, as lipid peroxidation is seemingly an obligatory consequence of life which is compensated by the antioxidative defence systems which include; enzymes (glutathione peroxidase, superoxide dismutases and catalase) or low molecular compounds (ascorbic acid, carotinoids and tocopherols) (Nigam and Schewe, 2000).

Rats envenomated by native L.q scorpion venom exhibited marked depletion of glutathione peroxidase as well as glutathione reductase activities in cardiac tissue. Such decreased activity implicates low cardiac glutathione content, which is a key biomarker of oxidative stress (Meister and Anderson, 1983). Reduced GSH is an endogenous antioxidant that acts among the first line of defense system against pro-oxidant status (Halliwell and Gulteridge, 1999).

The venom-evoked increase in GSH in rat observed in this study is an intriguing observation and is in harmony with the work of Yadav et al., 1997), who observed elevated GSH concentration in the rat following alloxan-induced diabetes. The authors attributed its elevation to its increased activity in the heart and decreased efflux into the blood stream to neutralize the superoxide anions and counteract oxidative stress. It is known that reduced GSH is a tripeptide involved in a wide range of metabolic functions, primarily aimed at providing protection against toxic compounds and free radical species. Usually, reduced GSH is regenerated and GSH levels are maintained (Simmons et al., 1990; Langley and Kelly, 1992; Erin et al., 1996; Al-Omar et al., 2004).

Both an increase and a decrease in GSH levels have been described following exposure of rats to a variety of stressors. For example, the levels of GSH were significantly decreased in several organs and body fluids following cold-restraintelicited stress, alloxan-induced diabetes and aluminum toxicity, with depletion of GSH while combating free radical

- 112 -

Discussion generation as its cause (Simmons et al., 1990; Erin et al., 1996; Abd El- Fattah et al., 1998). Alternatively, the venom-evoked increase in GSH levels in the present study could be explained by enhanced apoptosis due to stress following venom injection (Sedlak and Lindsay, 1968; Omran and Abdel-Rahman, 1992; El-Asmar, 1984; Abd El-Fattah et al., 1998), which would increase availability of amino acid substrates for GSH synthesis.

The previously mentioned finding is in harmony with the study of Fatani et al., (2006) who reported that, the enhanced rate of thiobarbituric acid reactive substances (TBARS) was accompanied by a concomitant increase in the rate of reduced glutathione in rats.

The serum transaminase level is most widely used as a measure of hepatic injury, due to its ease of measurement and high degree of sensitivity. It is useful for the detection of early damage of hepatic tissue and requires less effort than that required for a histologic analysis, moreover without sacrifice of the animals (Ray et al., 2006).

The present activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) underwent a highly significant increase following envenomation with native L.q compared to the normal non- envenomated. However, these enzyme activities were unaffected by the γ-irradiated (1.5 KGy) L.q scorpion venom due to the destruction of the hepatocytes as a result of venom injection accompined with disturbances in the hepatic and renal functions of the envenomated animals through severe hepatocellular injuries and necrosis of hepatocytes.

It seems that serum AST and ALT are the most sensitive markers employed in the diagnosis of hepatic damage due to their location in the cytoplasm and hence released into the circulation after cellular damage (Pradeep et al., 2007).

The results concerning effect of native and irradiated L.q scorpion venom on liver enzymes are in agreement with prior reports (El-Missiry et al., 2010) that used Echis pyramidium venom. There were elevation in the serum concentration of AST, ALT and ALP in animals four hours post envenomation in comparison with the control group. Furthermore, Shaaban and Hafez, (2003) reported that intra peritoneal injection of sublethal dose of Naje haje venom in rats induced significant elevation in the activities of AST, ALT and ALP as compared to control. These results suggest that tissue destruction occurs in most of the organs secondary to venom injections. The increase in enzymatic activity of the

- 113 -

Discussion serum was attributed to the release of enzymes from liver, kidney and heart. Organ damage is followed by an increase in levels of AST, ALT and ALP (Omran et al., 1997).

Measurements of serum enzyme activities are important in assessing the state of the liver, heart and other organs; and fluctuations indicate damage of those organs (Mohamed et al., 1981). In this respect, severe hepatocellular injuries, necrosis of hepatocytes and kidney have been suggested to be the result of the significant rise in serum ALT, AST and ALP levels of rats after Echis carinatus venom injection (Abdel-Nabi and Rahmy, 1992).

In addition, Tiets, (1983), reported that although kidney, heart and skeletal muscles have significant amount of ALT, liver damage is more likely to be the source of the elevation of serum ALT levels because it is more specific to liver cells.

Elevated activity of lactic dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the blood serum was observed, indicating myocardial and liver damage after Mesobuthus tamulus gangeticus venom administration. The main causes of altered permeability of myocardial and liver cells are circulatory hypoxia, metabolic disorders and inflammation (Chaubey and Upadhyay, 2010).

Normally serum transaminase levels are low, but after extensive tissue injury, these enzymes are liberated into the serum and the levels of serum transaminase, were reported to be increased following damage to skeletal muscles, myocardial muscles and liver (Harper et al., 1977). It was suggested that, tissue destruction occurred in most of the organs secondary to venom injections. The increase in enzymatic activity of the serum might be due to release of the enzymes from liver, kidney and heart (Metzler, 1977).

Measurement lipid profile of both venom before and after gamma irradiation (1.5 KGy) showed that, native L.q scorpion venom caused significant decrease in serum cholesterol, triglyceride, HDL-cholesterol & LDL-cholesterol compared to normal non-envenomated.

The variations in serum cholesterol and triglycerides in the present study could be due to the damage of hepatocytes by the venom making them unable to phosphorylate the large amounts of fatty acids, together with the destruction of cell membranes of other tissues (El-Asmar et al.,

- 114 -

Discussion

1979). This also suggests that those variations could be time dependent. The results are in agreement with a prior report (Ibrahim and Al- Jammaz, 2003) that used Echis coloratus venom. It has been found that there were changes in lipid profile levels in rats post envenomation in comparison with the control group.

In contrast, the γ-irradiated (1.5 KGy) L.q scorpion venom significantly decreased serum HDL-cholesterol compared to normal non- envenomated and native non-irradiated scorpion venom, while caused significant increase in serum total cholesterol and triglycerides compared to native non-irradiated scorpion venom value.

The injection of L.q scorpion venom alters the liver lipid composition. Serotonin, which is present in high concentration in the venom of Leiurus quinquestriatus (Adams and Weiss, 1958) is known to have a lipolytic effect through increasing cyclic AMP concentration (Mansour et al., 1960; Levine et al., 1964). Scorpion venom was reported to release catecholamines (Henriques et al., 1968; Ismail et al., 1972), these factors may cause lipolysis in the adipose tissues. The mobilization of the fatty acids from the adipose tissues may lead to their esterification in the liver with formation of neutral fat and phospholipids. The free fatty acids liberated by the venom would result in an increased level of acetyl CoA (Ashmore and Weber, 1968). This increase could lead to an increase in the synthesis of cholesterol.

Mohamed et al., (1972) reported a significant lipolysis in in vitro studies with liver slices using Buthus quinquestriatus venom, while the present study was able to demonstrate an increase in the total lipid in the liver of rats treated with scorpion venom.

Scorpion venoms comprise a complex mixture of mucopolysaccharides, amino acids, lipids, hyaluronidase, phospholipase, low molecular weight molecules such as serotonin and /or histamine releasers, but mostly significantly neurotoxic peptides (El-Asmar, 1984; Possani et al., 1999). Such peptides prolong the action potential of excitable cells by deferring the sodium channel inactivation (Courard et al., 1982, Weisel-Eichler and Libersat, 2004). These result in an increase in the presynaptic release of neurotransmitters including catecholamines (Ito et al., 1981), acetylcholine (Gwee et al., 2002), glutamate (Massensini et al., 1998), and GABA (Purali, 2003), leading to the observed signs of toxicity on the cardiovascular system.

- 115 -

Discussion

Moreover, it is documented that the cardiovascular effects are the major factors that may lead to death following both exipermental and human scorpion envenomation (Ismail, 1995; Cupo and Hering, 2002). Even though the mechanism of venom –induced cardiovascular effects is strongly believed to be a consequence of the increase of neurotransmitter release evoked by the action of toxins on voltage-sensitive sodium channels (Rogers et al., 1996; Chen and Heinmann, 2001). The enzymes of the venoms were shown to be responsible for several observed biological, pharmacological and toxicological effects associated with the envenomation process (Kini, 1997).

The present study indicated that the native L.q scorpion venom caused a highly significant increase of lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme (CK-MB) compared to the normal control while unaffected by the γ-irradiated (1.5 KGy) L.q scorpion venom when compared to normal control. These results indicate a decrease in the myotoxicity of the γ-irradiated L.q venom. The obtained results are in agreement with those previously reported by other investigators (El-Missiry et al., 2010). Native Echis pyramidium venom caused a highly significant increase of lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme (CK-MB) compared to the normal control .

Gutierrez and Lomonte, (1995) reported that the myotoxic effect of venoms of the genus Bothrops is particularly important, not only because it may lead to permanent tissue loss, disabling the victim, but also because it may induce severe cutaneous lesions on the animals chronically exposed to the venom during the immunization process.

Souza et al., (2002) investigated the ability of gamma radiation from 60Cо (2 KGy) to attenuate the toxic effects of Bothrops jararacussu venom on mouse neuromuscular preparations in vitro. It was concluded that 60Cо gamma radiation is able to abolish both the paralyzing and the myotoxic effects of Bothrops jararacussu venom on mouse neuromuscular junction. These findings support the hypothesis that gamma irradiation could be an important tool to improve antisera production by reducing toxicity while preserving immunogenicity.

Irradiation of crotoxin was shown to result in its aggregation and generation of low molecular weight breakdown products (Rogero and Nascimento, 1995). The aggregate presented no toxicity, no phospholipase activity and no ability to promote creatine kinase (CK) release into muscle tissue.

- 116 -

Discussion

Sofer et al., (1991) reported that the enzymatic activity of CK-MB isoenzyme and total CK were elevated following envenomation by the scorpion L.q in children. They speculated that the myocardial lesions are too small to cause heart failure in most cases, but they may account for the cardiovascular changes frequently seen in scorpion envenomation.

This assumption was also confirmed by the findings of Hering et al., (1993) and Correa et al., (1997), who reported an increase in the of CK and LDH in patients stung by scorpion and showed cardiomyopathy picture.s

It has been reported that the intraperitoneal injection of a sublethal dose of Naja haje venom in rats induced a significant elevation in the activities of LDH and CK as compared to normal control Shaaban and Hafez, (2003).

Interestingly, the present 1.5 KGy γ irradiated Leiurus quinquestriatus venom caused a non significant change (when used at a dose equal to that used for the native venom) of ALT, AST, LDH, CK and CK-MB compared to the normal control. This was in contrast to the native Leiurus quinquestriatus venom that induced a highly significant increase of ALT, AST, LDH, CK and CK-MB compared to the normal control or the 1.5 KGy γ irradiated Leiurus quinquestriatus venom.

In conclusion, the present results indicated that L.q scorpion venom is a highly toxic venom. It produced a significant increase in oxidative stress biomarkers, liver enzymes, cardiac enzymes and lipid profile disturbance, whereas exposing venom to 1.5 KGy gamma rays showed venom detoxification and loss of their biological properties without affecting their immunogenicity. Study of the distribution of 125I-labeled L.q scorpion venom showed the highest levels of radioactivity in liver and kidney. In addition, radioactivity of the brain indicates that 125I- scorpion venom is stable in-vivo against biological decomposition.

The present data revealed that, gamma irradiation of venom with 1.5 KGy dose offers an effective method for reducing the chronic toxic effect of venom in immunized animal for preparing better toxoids and vaccines.

- 117 -

Summary and Conclusions

This study was undertaken to evaluate the effect of gamma radiation on L.q scorpion venom. This was carried out by studying the toxicological, immunological properties and biochemical changes of the venom before and after exposure to gamma radiation.

Leiurus quinquestriatus scorpion venom is a highly toxic venom. Exposing venom to 1.5 KGy gamma rays showed venom detoxification and loss of their biological properties without affecting their immunogenicity.

The present data revealed that, gamma irradiation of venom with 1.5 KGy dose offered an effective method for reducing the toxic effect of venom in immunized animal while, maintaining immunogenicity.

Experimental design:

Saline solutions of Leiurus quinquestriatus scorpion venom were subjected to integral radiation dose levels (1.5 KGy and 3 KGy).

Mice were used for determination of median lethal dose (LD50) of native, 1.5 KGy and 3 KGy gamma irradiated Leiurus quinquestriatus scorpion venom. Mice were classified into three sets, each set included 6 groups; each group included 6 animals.

The 1.5 KGy γ-radiation is the selected dose to carry out the biochemical experiments of this study, as it gets rid of venom toxicity while maintains immunogenicity.

One over twenty of LD50 of native and 1.5 KGy γ-irradiated L.q scorpion venom were the selected doses to carry out the biochemical experiments of this study in rats.

Rats were used for measuring the biochemical parameters; since, the LD50 of the used venom was measured in mice; the equivalent rat dose was calculated according to Paget and Barnes, (1964) to be used in the following biochemical experiments.

Rats were classified into three sets each set consisted of 3 groups each group consisted of 8 animals. The 1st set of normal non-envenomated rats that received saline i.p and served as normal group. The second set of rats that received a single dose of 1/20 LD50 of native Leiurus quinquestriatus scorpion venom (0.133 mg/kg i.p) and served as native group. The third set

- 118 - Summary and Conclusions

of rats received a single dose 1/20 LD50 of 1.5 KGy γ-irradiated Leiurus quinquestriatus scorpion venom (1.230 mg/kg i.p) and served as irradiated group.

Blood samples were collected at 1 and 4 h to estimate; plasma lipid peroxides (MDA), blood glutathione (GSH), total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol levels and the activities of certain serum enzymes; alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), creatine kinase isoenzyme (CK-MB) as well as lactate dehydrogenase (LDH).

Moreover, the distribution of 125I-labeled L.q scorpion venom was studied in male Swiss mice tissue using chloramine-T method by being injected intravenously. At various time intervals (10 min, 1, 2 & 4 h), urine and blood were collected and the animals were sacrificed. Brain, lungs, heart, liver, kidneys, spleen, intestine, bone and muscle were isolated in order to determine the radioactivity content.

The following are the most important results of the present study:

1) The LD50 of the native (1mg/ml), 1.5 KGy & 3 KGy γ-irradiated (1mg/ml) Leiurus quinquestriatus scorpion venom were 0.394 mg/kg, 3.517 mg/kg, 7.5 mg/kg respectively.

2) The toxicity of the native L.q scorpion venom was reduced 11 and 20 times following the exposure to 1.5 KGy and 3 KGy gamma rays respectively. It has been shown that gamma irradiation is an effective technique for attenuating venom toxicity.

3) Immunogenicity was evaluated by performing the double immunodiffusion test for native (non-irradiated), 1.5 KGy and 3 KGy gamma irradiated venom against a commercial polyvalent Egyptian antivenin showed similar precipitin bands. Indicating that, the antigenic response was not changed as judged by the capacity of irradiated venom to react with the antivenin. The immunodiffusion technique showed identity between native and irradiated samples.

4) The radioiodination reaction of venom with iodine-125 was carried out using 200g venom and 0.87 mM chloramine-T at 60ºC for 30 min. The

- 119 - Summary and Conclusions maximum radiochemical yield of 125I-venom was obtained at around pH 7.

5) Distribution of 125I-labeled L.q venom showed the highest contents of radioactivity in liver and kidney. Radioactivity of the brain indicates that 125I-scorpion venom is stable in-vivo against biological decomposition. Labeling irradiated (1.5 and 3 KGy) scorpion venom was unsuccessful using this method of iodination (chloramine-T method).

6) Native L.q scorpion venom caused significant elevation in plasma (MDA) and blood GSH level 1 & 4 h after injection compared to the normal control. However, the 1.5 KGy γ-irradiated L.q scorpion venom caused significant reduction in plasma (MDA) and blood GSH levels 1 & 4 h after injection compared to the native L.q (non-irradiated) scorpion venom level.

7) The native L.q scorpion venom caused significant increase in liver enzymes (serum AST& ALT) activities 1 & 4 h after envenomation compared to the normal control. However, the 1.5 KGy γ-irradiated L.q venom 1 & 4 h after envenomation caused significant decrease in liver enzymes (serum AST& ALT) activities compared to the native L.q (non- irradiated) scorpion venom activity.

8) Native L.q scorpion venom caused significant reduction in lipid profile levels 1 & 4 h after envenomation compared to the normal control. In contrast, the 1.5 KGy γ-irradiated L.q scorpion venom 1 & 4 h after envenomation caused significant increased of total cholesterol & triglycerides levels compared to the native L.q (non-irradiated) scorpion venom level. However, there was a significant decrease of HDL- cholesterol level compared to the native L.q venom level.

9) Native L.q scorpion venom caused significant increase in cardiac enzymes activities 1 & 4 h after envenomation compared to the normal control. However, the 1.5 KGy γ-irradiated L.q scorpion venom 1 & 4 h after envenomation caused significant decrease in cardiac enzymes activities compared to the native L.q (non-irradiated) scorpion venom activity.

In conclusion, the present results indicated that L.q scorpion venom is a highly toxic venom. It produced a significant increase in oxidative stress biomarkers, liver enzymes, cardiac enzymes and significant decrease in lipid profile levels compared to the normal control, whereas exposing venom to 1.5 KGy gamma rays showed venom detoxification

- 120 - Summary and Conclusions and loss of their biological properties without affecting their immunogenicity. Study of the distribution of 125I-labeled L.q scorpion venom showed the highest levels of radioactivity in liver and kidney. In addition, radioactivity of the brain indicates that 125I-scorpion venom is stable in-vivo against biological decomposition.

The present data revealed that, gamma irradiation of venom with 1.5 KGy dose obtain an effective method for reducing the toxic effect of venom in immunized animal for preparing better toxoids and vaccines.

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