genotoxical, teratological and biochemical effects of

139Reprod. Nutr. Dev. 46 (2006) 139–156© INRA, EDP Sciences, 2006DOI: 10.1051/rnd:2006007

Original article

Genotoxical, teratological and biochemical effects of anthelmintic drug oxfendazole Maximum Residue

Limit (MRL) in male and female mice

Aida EL- MAKAWY, Hasnaa A. RADWAN, Inas S. GHALY, A. Abd EL-RAOUF

Cell Biology Department, National Research Center, Dokki, Giza, Egypt

(Received 30 May 2005; accepted 16 November 2005)

Abstract – Oxfendazole, methyl-5 (6)-phenylsulfinyl-2-benzimidazole carbamate, is a member ofthe benzimidazole family of anthelmintics. Anthelmintic benzimidazoles are widely used in meatproducing animals (cattle, sheep and pigs) for control of endoparasites. The extensive use ofveterinary drugs in food-producing animals can cause the presence of small quantities of the drugresidues in food. Maximum residue limit or “MRL” means the maximum concentration of residueresulting from the use of a veterinary medicinal product which may be legally permitted recognizedas acceptable in food. The FAO/WHO Expert Committee on Food Additives (1999) evaluations oftoxicological and residue data, reported that oxfendazole (MRL) has toxicological hazards on humanhealth. The toxicity of oxfendazole (MRL) was tested in male and female mice and their fetuses.Chromosomal aberrations, teratological examination and biochemical analysis were the parametersused in this study. The results show that oxfendazole MRL induced a mutagenic effect in all testedcell types. Also, oxfendazole exhibit embryotoxicity including teratogenicity. The biochemicalresults show that oxfendazole induced a disturbance in the different biochemical contents of all testedtissues. So, we must increase the attention paid to the potential risk of oxfendazole residues in humanbeings and should stress the need for careful control to ensure adherence to the prescribed withdrawaltime of this drug.

anthelmintic / oxfendazole / maximum residue limit / genotoxicity / embryotoxicity /teratogenicity / biochemical changes

1. INTRODUCTION

Antiparasitic agents represent an impor-tant class in veterinary medicine. Amongthem, anthelmintic benzimidazoles are widelyused in meat producing animals (cattle,sheep and pigs) for the control of endopar-asites. Some members of this class of drugshave teratogenic and embryotoxic effects in

animal species. In order to ensure humanfood safety, the European Union has setmaximum residue limits (MRL) for benz-imidazoles and their metabolites in animalproducts [1]. A Maximum Residue Limit or“MRL” means the maximum concentrationof residue resulting from the use of a veter-inary medicinal product which may belegally permitted recognized as acceptable

* Corresponding author: [email protected]

Article published by EDP Sciences and available at http://www.edpsciences.org/rnd or http://dx.doi.org/10.1051/rnd:2006007

http://www.edpsciences.org/rnd

http://dx.doi.org/10.1051/rnd:2006007

140 A. El-Makawy et al.

in food. This limit is either based on the typeand amount of residue considered to bewithout any toxicological hazard for humanhealth as expressed by the acceptable dailyintake (ADI), or on the basis of a temporaryADI that utilizes an additional safety factor[2]. Liver, kidney, muscle and fat are thetarget tissues for residue control. MRLrange from 10 to 1000 µg·kg–1 dependingon compound and matrix [1]. FAO/WHOExpert Committee on Food Additives [3]convened to evaluate the safety of residuesof certain veterinary drugs in food and torecommend maximum levels for such resi-dues. The Committee’s evaluations of tox-icological and residue data, reported thatoxfendazole (MRL) has toxicological hazardson human health. Oxfendazole, methyl-5 (6)-phenylsulfinyl-2-benzimidazole carbamate,is a member of the benzimidazole family ofanthelmintics. It is the sulphoxide metabo-lite of fenbendazole and it is possible thatfenbendazole owes much of its efficacyspectrum to oxfendazole. The probenzimi-dazole, febantel, when it is metabolized,also becomes fenbendazole, and finallyoxfendazole, which is believed to be theactive metabolite. The compound oxfenda-zole reaches peak blood levels more slowlyfollowing absorption from the intestinaltract than many of the older benzimida-zoles, thereby maintaining effective con-centrations for a longer time in both the serumand the intestinal tract increases its efficacy[4]. The heat stability of the anthelminticoxfendazole in water, cooking oil and asincurred residues in cattle liver was inves-tigated. It was found that cooking did notdestroy residues although it may affect thepoint of equilibrium between oxfendazole,oxfendazole sulphone, fenbendazole andsome other metabolites in incurred tissue[5]. The extensive use of veterinary drugsin food-producing animals can cause thepresence of small quantities of the drug res-idues in food. Certain drugs can be carriedover into the milk of lactating cows ordeposited in the tissues of an animal intendedfor slaughter [6, 7]. In recent years, therehas been widespread and increasing con-

cern that drugs, as well as environmentalchemicals, may present a potential hazardto mankind by causing gene mutations orchromosome aberrations. Genetic altera-tions in somatic tissues can have a numberof immediate effects upon the cellsinvolved, including cell death or transfor-mation into malignancy. Genetic alterationin germ cells leads to reproductive failureor genetic disorder in subsequent genera-tions that include heritable mutations low-ering the reproductive output of affectedpopulation [8]. So, the purpose of the presentstudy was to evaluate the competence ofoxfendazole maximum residue limit (MRL)on the incidence of genetic alteration inmale and female mice and their teratogeniceffect in embryos of treated parents.

2. MATERIALS AND METHODS

2.1. Drug

Tyvert, Oxfendazole 2.265% is a broadspectrum worm drench for cattle and sheep,manufactured by Pharmacia au Upjohn,Purs-Belgium. Oxfendazole CAS No.53716-50-0. The dose equal to Oxfenda-zole maximum residue limit in liver(1000 µg·kg–1) was used. Each animal gav-ages orally 0.01 mL daily.

2.2. Assay of genotoxicity in males

Twenty adult male Swiss mice weighing(20–30 g) obtained from the animal houseof the national research center were used toevaluate the oxfendazole (MRL) genotox-icity in male cells. The animals were ran-domly divided into two groups and hadfood and water ad libitum. The first groupwas given distilled water orally and servedas the control. The second group was orallygiven the dose of oxfendazole (MRL) dailyfor eight weeks. At the end of the time ofdrug administration, all animals wereinjected IP with a colchicine solution. Twohours later, the animals were sacrificed bycervical dislocation and the chromosomes

Toxicity of anthelmintic drug oxfendazole residues 141

of bone marrow cells and spermatocyteswere prepared according to Yosida andAmano [9], Brewen and Preston [10].

2.3. Assay of genotoxicity in females

Adult male and adult virgin females ofSwiss albino mice were maintained on feedand water ad libitum in the animal house ofthe national research center. Both male andfemale animals were divided into twogroups. The animals of the first group weregiven distilled water orally and served asthe control animals. The animals of the sec-ond group were orally given a dose ofoxfendazole (MRL) for four weeks. Afterthat, males and females were caged by ratio(1:1) according to the following groups:

Group I: control males and females. Group II: treated males and control females.Group III: control males and treatedfemales. Group IV: treated males and females.Successful mating was determined by

the presence of vaginal plugs on the nextmorning which was designed as day zero ofpregnancy. Drug oral administration in thesame manner continued until day 18 ofpregnancy.

2.4. Teratological examination

On day 18 of gestation, ten pregnantfemales of each group were sacrificed bycervical dislocation. The uterine horns wereopened and the numbers of alive, dead andresorbed fetuses were recorded accordingto Gleich and Frohberg [11]. The bodyweights of live fetuses were recorded just afterdissection. Also the fetuses were externallyexamined to determine any deformities. Forskeletal examination, fetal skeletons wereprepared and stained in Alizarin red-S accord-ing to the method described by Weesner [12].After staining, the skeletons were examinedunder the dissecting binocular microscope todetect any malformation in the bones of theaxial and appendicular skeletons.

2.5. Chromosome preparation

The chromosomes of bone marrow cellsof pregnant females were prepared by usingthe methodology of Yosida and Amano [9]and the chromosomes of fetal cells wereprepared according to the method ofRomagnano et al. [13].

The slides were stained with 10%Giemsa stain and 100 good metaphasespreads of each cell types for all animalswere examined microscopically to analyze thedifferent types of chromosomal aberrations.

2.6. Biochemical analysis

Total content of DNA, RNA, protein andenzymes in different tissues (liver, kidney,testes and embryos) of male and femalemice were determined. DNA contents weredetermined according to Dische [14] usingthe diphenylamine method; DNA givingblue color was read using a spectrophotom-eter at 600 nm. RNA content was deter-mined by using the Orcinol reagent methodaccording to Schneider [15] and read at660 nm. Protein content was determinedaccording to Gomall et al. [16] and read at545 nm. Enzyme activities of the liver(GOT and GPT) were determined accord-ing to Reitman and Frankel [17] at 545 nm.

2.7. Statistical analysis

Data of biochemical analysis andcytogenetical examination of males wereanalyzed by a paired- t -test. The data of thefetuses morphological examination andchromosome aberrations were analyzedstatistically by using one way analysis ofvariance (ANOVA).

3. RESULTS

3.1. Effect of oxfendazole maximum residue limit (MRL) in males

The data of the present study indicatedthat oxfendazole (MRL) oral administra-tion of male mice for eight weeks inducedchromosome aberrations in bone marrowand spermatocyte cells as shown in Figures 1


and 2. Table I and Figure 3 illustrate themean values and standard deviations of dif-ferent types of chromosomal aberrations inbone marrow cells of control and oxfenda-zole (MRL) treated male mice. The datashowed that oxfendazole caused a statisti-cally significant increase in the mean valuesof the gaps, endomitosis, and centric fusionsat (P ≤ 0.05) when compared with the con-trol. The results also revealed that oxfend-azole showed a statistically significantincrease in the mean values of fragments,deletions, and end to end associations, poly-ploidy and total chromosomal aberrations

at (P ≤ 0.01) than the control. However, thestatistical analysis of the other individualchromosomal aberrations showed non sig-nificant differences between the mean val-ues of the different treated groups and thecontrol. Table II and Figure 4 demonstratethat oxfendazole (MRL) treated male miceshowed a statistically significant increasein the mean values of autosomal univalent,x-y univalent, aneuploidy and total chro-mosomal aberrations at (P ≤ 0.01) whencompared with the control group, whereas,translocation, ring and polyploidy showednon significant differences.

Figure 1. Different types ofchromosomal aberrations indu-ced by oxfendazole (MRL) inbone marrow cells: (A) gap (g)and break (b) (B) deletion(C) end to end association(D) centric fusion (E) poly-ploidy (F) endomitosis.


3.2. Effect of oxfendazole maximum residue limit (MRL) in females

The mean frequencies of different indi-viduals and total chromosomal aberrationsinduced in female bone marrow cells of thetreated group and the standard deviationsare presented in Table III and Figure 5. Thedata showed that oxfendazole caused a sta-tistically significant increase in the meanvalues of endomitosis, centric fusion, frag-ments, deletions, end to end associations,polyploidy and total chromosomal aberra-tions at (P ≤ 0.01) when compared with thecontrol. Meanwhile, it showed a statisti-cally significant increase in the mean values

of the other types of chromosomal aberra-tions at (P ≤ 0.05).

3.3. Effect of oxfendazole maximum residue limit (MRL) on fetal cells

The data of Table IV and Figure 6 rep-resent the mean values of different individ-ual and total chromosomal aberrationsinduced by oxfendazole (MRL) in embryocells. The results indicate that oxfendazoleoral administration for males or femalesonly induced a statistically significantincrease in all individual and total chromo-some aberrations in the cells of theirembryos at (P ≤ 0.01) when compared withthe embryos of control males and females.

Figure 2. Different types ofchromosomal aberrations indu-ced by oxfendazole (MRL) inspermatocytes: (A) x-y univa-lent (B) x-y univalent (C) auto-somal univalent (D) hypoploidy(E) polyploidy.


Ta

ble

I. T

he m

ean

valu

es o

f diff

eren

t chr

omos

omal

abe

rrat

ions

indu

ced

by o

xfen

dazo

le m

axim

um re

sidu

e lim

it (M

RL

) in

bone

mar

row

cel

ls o

f mal

e m

ice.

Gro

ups

No. of

anim

als

Gap

Bre

akFr

agm

ent

Del

etio

nC

entr

omer

ic

atte

nuat

ion

End

omito

sis

Cen

tric

fu

sion

End

to

end

Rin

gH

ypo-

ploi

dyH

yper

-pl

oidy

Poly

-pl

oidy

Tota

l ch

rom

osom

alab

erra

tions

Con

trol

10M

± S

D0.

90±

0.8

80.

20±

0.4

20.

00±

0.0

00.

40±

0.5

22.

50±

0.8

50.

00±

0.0

00.

00±

0.0

00.

00±

0.0

00.

00±

0.0

00.

50±

0.7

10.

00±

0.0

00.

20±

0.4

24.

60±

1.0

7

Tre

ated

10M

± S

D1.

80*

± 0

.79

1.00

*±

0.8

20.

90**

± 0

.74

3.10

**±

1.6

04.

00±

1.6

30.

70*

± .8

20.

60*

± 0

.70

0.90

**±

0.7

40.

20±

0.4

20.

60±

0.9

70.

60±

0.9

71.

00**

± 0

.82

15.1

0**

± 2

.47

* Si

gnif

ican

t dif

fere

nce

at (

P ≤

0.0

5).

** S

igni

fica

nt d

iffe

renc

e at

(P

≤ 0

.01)

.

Tabl

e II

. The

mea

n va

lues

of d

iffe

rent

chr

omos

omal

abe

rrat

ions

indu

ced

by o

xfen

dazo

le m

axim

um re

sidu

e lim

it (M

RL

) in

sper

mat

ocyt

es o

f mal

e m

ice.

Gro

ups

No.

of a

nim

als

Aut

osom

alun

ival

ent

x-y

univ

alen

tT

rans

loca

tion

Rin

gH

ypop

loid

yH

yper

ploi

dyPo

lypl

oidy

Tota

l chr

omos

omal

ab

erra

tions

Con

trol

100.

80 ±

0.7

90.

40 ±

0.7

00.

00 ±

0.0

00.

00 ±

0.0

00.

90 ±

0.8

80.

00 ±

0,0

00.

00 ±

0.0

02.

10 ±

0.5

3

Tre

ated

103.

00**

± 0

.94

3,10

** ±

1.4

50.

20 ±

0.4

20.

30 ±

0.4

83.

70**

± 0

.82

1.60

** ±

0.8

40.

60 ±

0.8

412

.50*

* ±

1.7

8

* Si

gnif

ican

t dif

fere

nce

at (

P ≤

0.0

5).

** S

igni

fica

nt d

iffe

renc

e at

(P

≤ 0

.01)

.

Tabl

e II

I. T

he m

ean

valu

es o

f diff

eren

t chr

omos

omal

abe

rrat

ions

indu

ced

by o

xfen

dazo

le m

axim

um r

esid

ue li

mit

(MR

L) i

n bo

ne m

arro

w c

ells

of f

emal

e m

ice.

Gro

ups

No. of

anim

als

Gap

Bre

akFr

agm

ent

Del

etio

nC

entr

omer

ic

atte

nuat

ion

End

o-m

itosi

sC

entr

ic

fusi

onE

nd to

en

dR

ing

Hyp

o-pl

oidy

Hyp

er-

ploi

dyPo

ly-

ploi

dyTo

tal

chro

mos

omal

aber

ratio

ns

Con

trol

10M

± S

D1.

00±

089

0.66

± 0

.81

0.00

± 0

.00

0.50

± 0

.55

2.55

± 0

.85

0.00

± 0

.00

0.00

± 0

.00

0.00

± 0

.00

0.00

± 0

.00

0.60

± 0

.84

0.00

± 0

.00

0.25

± 0

.52

5.56

± 1

.97

Tre

ated

10M

± S

D1.

80*

± 0

.79

1.30

*±

0.9

51.

20**

± 0

.92

3.20

**±

1.4

34.

20*

± 1

.43

1.00

**±

0.9

41.

00**

± 0

.67

0.90

**±

0.7

40.

60*

± 0

.70

1.00

± 1

.05

0.80

*±

0.9

21.

10**

± 0

.74

17.9

0**

± 2

.42

* Si

gnif

ican

t dif

fere

nce

at (

P ≤

0.0

5).

** S

igni

fica

nt d

iffe

renc

e at

(P

≤ 0

.01)

.


3.4. Teratogenic effect of oxfendazole maximum residue limits (MRL)

3.4.1. External morphological examination

The mean values of different teratogenicvariables are tabulated in Table V and Fig-

ure 7. The results showed that oxfendazole(MRL) induced a statistically significantdecrease in the number of live fetuses in allgroups at (P ≤ 0.01) when compared withthe control, whereas, it induced a non sig-nificant increase in the number of resorbedfetuses (Fig. 8) in the groups in which males

Figure 3. Effect of oxfendazole (MRL) on bone marrow cells of male mice.

Figure 4. Effect of oxfendazole (MRL) on spermatocytes of male mice.


or females were treated only. However, inthe group of treated males and females,oxfendazole showed a statistically signifi-cant increase in the number of resorbed

fetuses at (P ≤ 0.01). Since, all fetuses ofthis group were completely resorbed. Mean-while, there was no significant differencebetween the number of hematoma (Fig. 9)

Figure 5. Effect of oxfendazole (MRL) on bone marrow cells of female mice.

Figure 6. Effect of oxfendazole maximum residue limit (MRL) on embryo cells of all experimentalgroups.


Tab

le I

V. T

he m

ean

valu

es o

f dif

fere

nt c

hrom

osom

al a

berr

atio

ns in

duce

d by

oxf

enda

zole

max

imum

res

idue

lim

it (M

RL

) in

embr

yo c

ells

of d

iffe

rent

expe

rim

enta

l gro

ups.

Gro

ups

No. of

embr

yos

Gap

Bre

akFr

agm

ent

Del

etio

nC

entr

omer

ic

atte

nuat

ion

End

o-m

itosi

sC

entr

ic

fusi

onE

nd to

en

dR

ing

Hyp

o-pl

oidy

Hyp

er-

ploi

dyP

oly-

ploi

dyTo

tal

chro

mos

omal

ab

erra

tions

Con

trol

50M

± S

D0.

22c

± 0

.51

0.10

c±

0.3

60.

00b

± 0

.00

0.20

c±

0.4

52.

48b

± 0

.95

0.00

c±

0.0

00.

00b

± 0

.00

0.00

b±

0.0

00.

00b

± 0

.00

0.30

b±

0.5

80.

00b

± 0

.00

0.10

c±

0.3

03.

38c

± 1

.60

Mal

e tr

eate

d50

M ±

SD

1.74

b±

0.9

40.

86b

± 1

.01

1.12

a±

1.1

21.

20b

± 1

.18

3.40

a±

1.4

60.

22b

± 0

.46

0.14

a±

0.3

50.

12a

± 0

.33

0.20

a±

0.4

00.

48ab

± 0

.71

0.12

a±

0.3

31.

00b

± 0

.90

10.5

4b±

3.7

2

Fem

ale

trea

ted

50M

± S

D2.

28a

± 0

.90

2.02

a±

0.8

71.

30a

± 0

.54

1.60

a±

0.9

33.

80a

± 1

.29

0.52

a±

0.6

10.

10a

± 0

.30

0.14

a±

0.3

50.

24a

± 0

.48

0.66

a±

0.7

50.

12a

± 0

.33

1.7a

± 0

.95

14.4

6a±

2.7

7

Mea

ns w

ith

diff

eren

t let

ters

wit

hin

each

col

umn

are

sign

ific

ant a

t 5%

leve

l.

Tab

le V

. Eff

ect o

f ox

fend

azol

e m

axim

um r

esid

ue li

mit

(MR

L)

on e

mbr

yos

of d

iffe

rent

exp

erim

enta

l gro

ups.

Gro

ups

No.

of

fem

ales

Aliv

e em

bryo

sR

esor

bed

embr

yos

Hae

mat

oma

Mea

n bo

dy w

eigh

t

Con

trol

10M

± S

D9.

40 ±

1.2

6 a

0.20

± 0

.42

b0.

50 ±

0.5

3 ab

2.70

8 ±

0.3

24 a

Mal

e tr

eate

d10

M ±

SD

7.10

± 3

.03

b1.

50 ±

2.0

1 b

0.80

± 0

.79

a1.

523

± 0

.135

b

Fem

ale

trea

ted

10M

± S

D7.

70 ±

1.9

7 ab

1.40

± 1

.15

b1.

00 ±

1.1

5 a

1.13

7 ±

0.1

15 c

Mal

e +

fem

ale

trea

ted

10M

± S

D0.

00 ±

0.0

0 c

8.70

± 2

.06

a0.

00 ±

0.0

0 a

0.00

± 0

.00

d

Mea

ns w

ith

diff

eren

t let

ters

wit

hin

each

col

umn

are

sign

ific

ant a

t 5%

leve

l.


in all treated groups and the control. How-ever, oxfendazole (MRL) showed a statis-tically significant decrease in the fetal bodyweights of all treated groups at (P ≤ 0.01)when compared with the untreated group.

3.4.2. Skeletal examination

Alizarin red–S stained 18th day of ges-tation oxfendazole treated male or femalefetal skeletons showed an obvious decrease

Figure 7. Teratogenic effect of oxfendazole (MRL) in all experimental groups.

Figure 8. Photograph of the uterus of a control pregnant female (A) and a uterus with resorbedembryos of an oxfendazole (MRL) treated female (B) on day 18 of gestation.


in the size and reduction in the ossificationof fetal skeletons as compared to those ofthe control group. Sacral vertebrae showedsevere lack of ossification and caudal ver-tebrae were completely non ossified. Thebones of the pelvic girdle of the controlfetuses, illium, ischium and pubis were wellossified, whereas in the skeletons of theoxfendazole (MRL) treated fetuses, thepubis bones were completely non-ossifiedand the ischium bones showed a lesserdegree of ossification (Fig. 10).

3.5. Biochemical results

The data of biochemical studies on dif-ferent parameters in all experimental tis-sues figure in Tables VI, VII and VIII. Thedata show that oxfendazole (MRL) affected

Figure 10. Photograph of fetal skeletons at day 18 of gestation showing (A) control fetus with wellossified bones (B) oxfendazole (MRL) maternally treated fetus showing complete non- ossificationof sacral and caudal vertebrae (ca.v) and pubis (pb) bones of the pelvic girdle (alizarine S- red stain).

Figure 9. Photograph of oxfendazole (MRL)maternally treated embryo on day 18 of gesta-tion showing an external Haematoma.


Tabl

e V

I. E

ffec

t of

oxfe

ndaz

ole

on to

tal p

rote

in, t

otal

DN

A, t

otal

RN

A a

nd li

ver

enzy

me

activ

ity o

f m

ale

mic

e.

Gro

ups

Pro

tein

mg·

g–1

DN

A m

g·g–

1R

NA

mg·

g–1

Liv

er e

nzym

es u

·L–1

Liv

erK

idne

yTe

stes

Liv

erK

idne

yTe

stes

Liv

erK

idne

yTe

stes

GO

TG

PT

Con

trol

9.66

± 0

.09

8.12

± 0

.32

7.12

± 0

.317

0.46

± 0

.017

0.37

± 0

.02

0.34

± 0

.02

0.28

3±

0.1

70.

26±

0.0

20.

23±

0.0

026

.20

± 1

.49

30.2

0±

1.7

1

Tre

ated

11.9

8**

± 6

.24

9.27

**±

0.1

09.

45**

± 0

.292

0.40

**±

0.0

100.

28**

± 0

.03

0.27

*±

0.1

60.

22*

± 0

.01

0.21

*±

0.0

20.

17*

± 0

.00

34.2

0*±

2.3

939

.40*

± 3

.18

* Si

gnif

ican

t dif

fere

nce

at (

P ≤

0.0

5). *

* S

igni

fica

nt d

iffe

renc

e at

(P

≤ 0

.01)

.

Tabl

e V

II. E

ffec

t of

oxfe

ndaz

ole

on to

tal p

rote

in, t

otal

DN

A, t

otal

RN

A a

nd li

ver

enzy

me

activ

ity o

f fe

mal

e m

ice.

Gro

ups

Pro

tein

mg·

g–1

DN

A m

g·g–

1R

NA

mg·

g–1

Liv

er e

nzym

es u

·L–1

Liv

erK

idne

yL

iver

Kid

ney

Liv

erK

idne

yG

OT

GP

T

Con

trol

9.01

± 0

.44

7.19

± 0

.10

0.45

± 0

.02

0.38

± 0

.02

0.26

± 0

.01

0.24

± 0

.01

24.6

0 ±

2.0

423

.40

± 2

.04

Tre

ated

11.7

28**

± 6

.24

9.09

**±

0.1

10.

28**

± 0

.02

0.28

0**

± 0

.01

0.19

**±

0.0

10.

20±

0.0

133

.20*

± 2

.39

35.8

0*±

3.1

8

* Si

gnif

ican

t dif

fere

nce

at (

P ≤

0.0

5). *

* S

igni

fica

nt d

iffe

renc

e at

(P

≤ 0

.01)

.

Tabl

e V

III.

Eff

ect o

f ox

fend

azol

e on

tota

l pro

tein

, tot

al D

NA

, tot

al R

NA

and

live

r en

zym

e ac

tivit

y of

par

enta

lly tr

eate

d em

bryo

s.

Prot

ein

mg·

g–1

DN

A m

g·g–

1R

NA

mg·

g–1

Liv

er e

nzym

es u

·L–1

GO

TG

PT

Con

trol

7.28

± 0

.27

0.32

± 0

.01

0.23

± 0

.01

20.8

0 ±

1.9

021

.00

± 1

.74

Fem

ale

trea

ted

8.06

* ±

0.1

20.

24 ±

0.0

16.

20 ±

0.0

121

.40

± 0

.98

22.6

0 ±

2.0

4

Mal

e tr

eate

d8.

98**

± 0

.25

0.22

** ±

0.0

10.

16 ±

0.0

127

.800

* ±

1.4

931

.20*

* ±

2.4

2

Mal

e F

emal

e tr

eate

d0.

00 ±

0.0

00.

00 ±

0.0

00.

00 ±

0.0

00.

00 ±

0.0

00.

00 ±

0.0

0

* Si

gnif

ican

t dif

fere

nce

at (

P ≤

0.0

5). *

* S

igni

fica

nt d

iffe

renc

e at

(P

≤ 0

.01)

.


the liver, kidney, testes and embryo tissuesand caused changes in all measured bio-chemical parameters of mice.

3.5.1. Biochemical changes in males

The data presented in Table VI and Fig-ure 11 showed that oral administration ofoxfendazole (MRL) caused a statisticallysignificant increase in the total protein con-tent of the liver, kidney and testes at (P ≤0.01) when compared with the control.However, the total content of DNA of thetissues was significantly decreased at (P ≤0.01) when compared to the control. Mean-while, a statistically significant decrease inthe level of total RNA content at (P ≤ 0.05)was observed. However, the levels of liverenzymes (GOT and GPT) of oxfendazole(MRL) male mice tissues were statisticallysignificantly higher (P ≤ 0.05) than thecontrol.

3.5.2. Biochemical changes in pregnant females and their embryos

The data of Tables VII and VIII,Figures 12 and 13 showed that the total

DNA and RNA content of the liver ofoxfendazole (MRL) pregnant females andtheir embryos, were significantly decreasedat (P ≤ 0.01) when compared with the con-trol. The DNA content of the kidney tissueswas statistically significant decrease at(P ≤ 0.05) when compared with the con-trol. Whereas, RNA content did not show asignificant change. A significant increase at(P ≤ 0.01) in the protein content of treatedpregnant female tissues and in embryosobtained from them was detected. Also,oxfendazole (MRL) significantly increasedthe liver enzyme (GOT and GPT) contentof females and their embryos at (P ≤ 0.01)as compared with the control.

4. DISCUSSION

The extensive use of veterinary drugs infood producing animals can cause the pres-ence of drug residues in food [7]. Certaindrugs can be carried over into the milk oflactating animals or deposited in the tissuesof animals intended for slaughter [6]. AMaximum Residue Limit is an amount of

Figure 11. Effect of oxfendazole (MRL) on different biochemical parameters in male mice.


residue that could remain in the tissue orfood product derived from a food-produc-ing animal that has been treated with a vet-erinary drug. This residue is considered topose no adverse health effects if ingesteddaily by humans over a lifetime [18]. In

recent years, there has been increasing con-cern that drugs may present a potential haz-ard to mankind by causing gene mutation orchromosome aberrations. Genetic altera-tions in somatic cells can include cell deathor transform into malignancy. Genetic

Figure 12. Effect of oxfendazole maximum residue limit (MRL) on different biochemical parame-ters in female mice.

Figure 13. Effect of oxfendazole maximum residue limit (MRL) on different biochemical parame-ters in embryo tissues.


alterations in germ cells lead to reproductivefailure or genetic disorder in subsequentgenerations [8]. In the present study, oraladministration of oxfendazole (MRL)induced a statistically significant increasein the frequencies of chromosomal aberra-tions in all tested cell types. This means thatanthelmintic oxfendazole has a possiblegenotoxic activity. Crebelli [19] reportedthat genotoxicity of benzimidazoles mayappear in DNA damage or involving mul-tiple interactions with non-DNA targets.Ardito et al. [20]) indicated that higher nontoxic concentrations of Thiabendazole causean increase in the sister chromatid exchangefrequency in lymphocyte culture. Theresults of the present study indicate thatoxfendazole induced a statistically signifi-cant increase in the frequencies of poly-ploidy in bone marrow and spermatocytecells of male mice and also in embryo cells. Theliterature reported that several anthelminticdrugs have been evaluated for genotoxicity.Crebelli et al. [21], Leopardi et al. [22],Natarajan et al. [23], Parry and Sors [24],Mailhes et al., [25] and Adler et al. [26]demonstrated that thiabendazole is capableof inducing numerical chromosomal changessuch as aneuploidy and polyploidy. At toxicdoses, thiabendazole increases the fre-quency of aneuploid in oocytes that impairovulation. Also, Schmid et al. [27] showedthat thiabendazole increases the frequencyof aneuploidy induction in the sperm oftreated male mice. Barale et al. [28] studiedthe cytogenetic effects of three benzimida-zoles, i.e., benomyl, methyl thiophanate andmethyl 2-benzimidazolecarbamate (MBC), inmouse bone marrow cells. Benomyl and MBCsignificantly induce micronuclei and aneu-genic flubendazole causes a dose and timedependent induction of polyploidy inChinese hamster lung cells. Mebendazoleinteracting with tubulin interferes with theassemblage of the mitotic apparatus in theparasite cells, thus causing a possible gen-otoxic activity leading to chromosomalmalsegregation [29]. Also, Okamura et al.[30] suggested that oxfendazole inducesapoptosis of meiotic spermatocytes, most

probably due to disruption of the microtu-bules, and degeneration of the Sertoli cells.Uppala et al. [31] indicate that chemicalscan induce cellular and chromosomal alter-ations, inducing early events in carcinogen-esis. This means that oxfendazole geneticalteration in somatic cells may lead tomalignancy. Our findings indicate that oraladministration of oxfendazole (MRL) toparents for eight weeks produced develop-mental toxicity in their embryos. This tox-icity appeared in the decrease in embryobody weights and the less ossified skele-tons. These results were in agreement withDelatour et al. [32] and Delatour [33] indi-cating that benzimidazole anthelminticoxfendazole, the active metabolite of fenb-antel, is embryotoxic and teratogenic in therat. Scholz and Baeder [34] demonstratedthat Fenbendazole showed fetotoxicity inrats and rabbits dosed by gavage. Embryo-toxicity was observed in the form of anincrease in the frequency of occurrence ofthe 13th ribs and delayed ossification of cra-nial bones in rabbits given a dose of63 mg·kg–1 of body weight/day. Lankaset al. [35] reported that thiabendazole isfoetotoxic in mice. Delatour et al. [36]reported that there is possible correlationbetween embryotoxicity and the plasmalevels of identified embryotoxic metabo-lites, but not with the levels of fetal tissuebound drug metabolites. Whittaker andFaustman [37, 38] reported that the benz-imidazole anthelmintic should be consid-ered as potential developmental toxicants.Since, they inhibit cell growth and differ-entiation of micromass culture of rat embryomidbrain and limb bud cells. In agreementwith our result, Capece et al. [39], Navarroet al. [40] reported that benzimidazoleinduces a significant increase of resorptions,a decrease of fetal body weights and anincrease in skeletal malformation. Mantovni[41] and Teruel et al. [42] indicated thatthe benzimidazole anthelmintic, albenda-zole, shows a dose related increase inembryolethality, growth reduction and areduction in the ossification process inthe rat. Cristofol et al. [43] studied the


disposition of albendazole and its metabo-lites, albendazole sulfoxide and sulfone inthe pregnant rat. The results indicated thatthe presence of albendazole and its metab-olites in the amniotic sacs and embryos atconcentration were higher than that in theplasma at the same times. A significant cor-relation was found between the rate ofdevelopmental toxicity and metabolite con-centration. Also, El-Makawy [44] men-tioned that embryotoxicity is mainly theresult of chromosome damage leading to aloss of genetic material. The data of the bio-chemical study in the present work showthat oral administration of oxfendazole(MRL) caused a significant increase in thetotal protein content of the liver, kidney andtestes and decrease in the total content ofDNA and RNA of tissues. Baliharova et al.[45] indicated that all benzimidazoles pro-voke an increase in the protein level of rathepatocytes. Also, oxfendazole (MRL) sig-nificantly increased the liver enzyme (GOTand GPT) content. This result was in agree-ment with that of Tada et al. [46] whoreported that thiabendazole showed anincrease in concentrations of GOT and GPTin male and female mice. Karmakar et al.[47] mentioned that hepatic enzymes sig-nificantly increased along with an increasedpercentage of chromosome aberrations inthe bone marrow. The results thereforestrongly suggest that oxfendazole exertsgenotoxicity in male and female mice.Also, oxfendazole exhibits embryotoxicityincluding teratogenicity. We can concludethat embryo developmental toxicity ofoxfendazole may be a result to the increasein the frequencies of different chromosomalaberrations and biochemical changes. So,we must increase the attention to the poten-tial risk of oxfendazole residues on humanbeings and should stress the need for carefulcontrol to ensure the adherence to the pre-scribed withdrawal time of this drug.

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