Determination of the LD50

[1984] the median lethal dose of M. eupeus venom was found to be 1.45 mg/kg. The variation of LD50 of one kind of scorpion venom may due to affect of ...

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Determination of the LD50

CHAPTER 6

Determination of the LD50

148

Determination of the LD50

CHAPTER 6

Determination of the LDjo 1

LD 5 0 of crude scorpion venoms

The LD50 o f scorpions venoms were calculated using arithmetic method o f Karber and adapted by Alui and Nwude [1982]. The effect o f various doses o f crude venoms o f three species o f scorpions in Swiss albino mice after subcutaneous administration are shown in Table 6.1 to 6.3

The LD 50 o f crude venom o f^ . crassicauda in mice was found to be 0.27 mg/kg whereas for B. saulcyi and O. doriae, it was 1.725 and 0.14 mg/kg, respectively.

It was observed that majority o f death in animals occurred after 10-60 min o f administration o f venom. O. doriae caused mortality quickly while B. saulcyi envenomation produced mortality after some time. Signs o f intoxication before death included tremors, convulsions and increased urination.

Table 6.1: Lethal doses o f crude venom o f A. crassicauda for the determination o f the LD 50 after subcutaneous injection in Swiss albino mice using arithmetic method

Group

Dose (mg/kg)

Dose difference (m g/ kg)

No. o f Animal per group.

No. Dead

Mean Dead

Dose difference X

mean Dead

1

Control

-

5

0

0

0

2

0.1

0.1

5

0

0

0

3

0.2

0.1

5

3

1.50

0.150

4

0.4

0.2

5

4

3.5

0.7

5

0.8

0.4

5

5

4.5

1.8

LD 50 {A. crassicauda) = 0.8 - ^

Sum =2.65

= 0.27 mg/kg

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D eterm inatio n of th e LD50

Table 6.2: Lethal doses o f crude venom o f R. saulcyi for the determination o f the LDjo after subcutaneous injection in Swiss albino mice using arithmetic method

Group

Dose (mg/kg)

Dose difference (mg/ kg)

No. o f Animal per group.

No. Dead

IMean Dead

Dose difference X

mean Dead

1

Control

-

5

0

0

0

2

0.25

0.25

5

0

0

0

3

0.5

0.25

5

1

0.5

0.125

4

1.0

0.5

5

2

1.5

0.75

5

2.0

LO

5

3

2.5

2.5

6

4.0

2.0

5

5

4

8

LD5o(fi. saulcyi) = 4.0

Sum= 11.375

= 1.725 mg/kg

Table 6.3: Lethal doses o f crude venom o f O. doriae for the determination o f the LD50 after subcutaneous injection in Swiss albino mice using arithmetic method. Group

Dose (mg/kg)

Dose difference (mg/ kg)

No. o f Animal per group.

No. Dead

Mean Dead

Dose difference X

mean Dead

1

Control

-

5

0

0

0

2

0.05

0.05

5

I

0.5

0.025

3

0.1

0.05

5

2

1.50

0.075

4

0.2

0.1

5

4

3.0

0.3

5

0.4

0.2

5

5

4.5

0.9 Sum= 1.3

LDsoC 0 . doriae) - 0.401- ^ = 0.14 mg/kg

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D eterm in atio n of th e LD50

2 LD.so of selected fractions The LD50 o f selected venom fractions were calculated graphically by the method of Miller and Tainter [1944]. The effects o f various doses o f three fractions o f three species of scorpions in Swiss albino mice after subcutaneous administration are shown in Table 6.4 - 6 .6 . The LD50 o f FI 7 o f^ . crassicauda in mice was found to be 0.1 mg/kg (Fig.6 . 1) whereas for F7 o f B. saulcyi, it was 1.0 mg/kg (Fig.6.2). The LD50 o f F5 o f O. doriae was calculated to be 0.42mg/kg (Fig.6,3).

Table 6.4; Lethal doses o f F17 o f /i. crassicauda for the determination o f the LD50 after subcutaneous injection in Swiss albino mice using graphical method. Correction of% Dead

Probit

0

0

-

5

1

20

4.1584

-l.OOOOOOOOOOe+0

5

3

60

5.2533

0.2

-6.9897000434C-1

5

4

80

5.8416

0.4

-3.9794000867e-l

5

5

95

6.6448

Group

Dose (mg/kg)

Log dose

No. o f Animal /group

Dead

1

0

-

5

2

0.05

-1.30102999570+0

3

0.1

4 5

% Dead

Fig.6.1: The L D 5 0 o f F17 o f/i. crassicauda was calculated using graphical method. The graph o f percentage death (in probits) against log dose was plotted and the dose corresponding to probit 5 was read as LD 50 with 95% confidence intervals. 151

CHAPTER 6

Determination of the LD50

Table 6.5: Lethal doses o f F7 o f B. saukyi for the determination o f the LD50 after subcutaneous injection in Swiss albino mice using graphical method

Correction o f % Dead

Probit

0

0

-

0

5

3 .3 5 5

Group

Dose (mg/kg)

Log dose

No. o f Animal /group

Dead

1

0

-

5

2

0 .1 2 5

-9 .0 3 0 8 9 9 8 6 9 9 6 -1

5

% Dead

1

3

0 .2 5

-6 .0 2 0 5 9 9 9 1 3 3 8 -1

5

0

0

-

4

0.5

-3 .0 1 0 2 9 9 9 5 6 6 8 -1

5

1

20

4 .1 5 8 4

5

1

0 .0 0 0 0 0 0 0 0 0 0 8 + 0

5

3

60

5 .2 5 3 3

Fig.6.2: The LD 50 o f F7 o f B. saulcyi was c^culated using graphical method. The graph o f percentage death (in probits) against log dose was plotted and the dose corresponding to probit 5 was read as LD 50 with 95% confidence intervals.

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Determ ination of the LD50

Table 6 .6 : Lethal doses o f F5 o f O. doriae for the determination o f the LDso after subcutaneous injection in Swiss albino mice using graphical method.

Correction o f % Dead

Probit

0

0

-

5

1

20

4 .158 5

-6 .9 8 9 7 0 0 0 4 3 4 e-l

5

1

20

4.158 5

0.4

-3 .9 7 9 4 0 0 0 8 6 7 e-l

5

2

40

4.746 7

0.8

-9.6910013008e-2

5

4

80

5.841 6

Group

Dose (mg/kg)

Log dose

No. o f Animal /group

Dead

1

0

-

5

2

0.1

-l.OOOOOOOOOOe-i-0

3

0.2

4

5

% Dead

Fig.6.3: The LD50 o f F5 o f O. doriae was calculated using graphical method. The graph o f percentage death (in probits) against log dose was plotted and the dose corresponding to probit 5 was read as LD50 with 95% confidence intervals.

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D eterm ination of the LD50

CHAPTER 6

3 Discussion All scorpions are venomous but only 46-50 species have sufficient toxicity to provide mortality in humans [Bawaskar and Bawaskar, 2009]. The species of scorpion, venom composition and physiological reaction o f victim to the venom, are important factors to determine severity o f symptom after scorpion envenomation [Petricevich, 2010].

The median lethal dose (LD50) is a common test used to evaluate o f acute toxicity (short­ term poisoning potential) o f scorpion venoms. LD50 o f crude or purified toxins of scorpion venom is the amount of toxin, administer all at once, that after entering the body of animals usually by a subcutaneous administration and provide mortality in half of experimental animal group.

In this study LD50 o f lyophilized dried powder o f crude and three fractions of scorpions’ venoms by subcutaneous injection route in Swiss albino mice were analyzed using arithmetic and graphic methods. The median lethal dose of crude venom o f A. crassicauda, B. saulcyi and O. doriae scorpions were calculated to be 0.27, 1.725 and 0.14 mg/kg while LD50 value of FI 7, F7 and F5 were found to be 0.1, 1.0 and 0.42mg/kg respectively.

Ismail et al [1994] and Watt & Simard [1984] reported that LD50 of A. crassicauda venom in mice was 0.32±0.02 and 0.4 mg/kg respectively, whereas result of Ozkan etal. [2007] was shown

that the median lethal dose o f venom of this species was

1.1 mg/kg. Hassan [ 1984] reported that the LD50 o f natural crude venoms of B. saulcyi and O. doriae in mice was 1.01 and 0.19 mg/kg, respectively.

The LD50 o f scorpion venom can vary even if the venom was extracted by using one single method [Krifi etal., 1998]. For instance, Latifi and Tabatabai [1979] found that LD50 o f M eupeus scorpion venom from Iran was 1.36 mg/kg while in study o f Hassan [1984] the median lethal dose o f M. eupeus venom was found to be 1.45 mg/kg. The variation o f LD50 o f one kind o f scorpion venom may due to affect of many factors such 154

D eterm inatio n of th e LD50

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as: genetic variations, feeding, geographical and environmental affect on the expression levels o f venom [Shalabi et al., 2004]. Furthermore, method o f milking venom, variation in estimation o f protein in sample by using different methods, species o f animal that was used for test and calculation methods are other important factors that can affect result of LD 5 0 .

In consolation, although result o f this study have been demonstrated that the three peptide fractions isolated in this study induced higher mortality in cancer cells, in vitro, but in vivo FI7 and F7 exerted more toxicity and F5 was found to have lower toxicity in S. albino mice as compared to the crude venom. This property of F5 that induced more mortality in cancer cells and shown lower toxicity for normal cells may lead to introduce new peptide(s) with anticancer activities.

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