ISRAEL JOURNAL OF
VETERINARY MEDICINE
EFFECTS OF ALBENDAZOLE TREATMENT ON
LIPID PEROXIDATION OF HEALTHY AND Toxocara
canis INFECTED MICE
E. Yarsan1, S. Celik2, G. Eraslan1 and H. Aycicek3
Vol. 57 (2) 2002
1. Ankara University, Faculty of Veterinary Medicine, Department of
Pharmacology and Toxicology, 06110, Diskapi, Ankara, Turkey.
2. Mustafa Kemal University, Faculty of Veterinary Medicine,
Department of Biochemistry, 06110, Hatay, Turkey.
3. GŸlhane Military Medicine Academy, Faculty of Medicine, Department
of Parasitology, 06018, Ankara, Turkey.
Abstract
Toxocara canis is a nematode of the family Ascaridae. In this study, the effects of
albendazole treatment on lipid peroxidation in healthy and T. canis infected mice were
evaluated. Lipid peroxidation was evaluated by determining malondialdehyde(MDA) levels in
plasma and glutathione peroxidase, superoxide dismutase, and catalase activities in
erythrocytes. Enzymic analyses were performed on blood samples collected from mice on
days 15, 45, and 60 post-infection. Additionally the numbers of T. canis larvae were counted
in brain, liver, kidney, lung and muscles during the same periods. The data show that T.
canis infection stimulates lipid peroxidation as displayed by increased plasma MDA levels,
and decreased glutathione peroxidase, superoxide dismutase, and catalase activities in
erythrocytes. On the other hand albendazole application prevented lipid peroxidation
especially on days 45 and 60 post-infection and was accompanied by decreased T. canis
larval counts in tissues.
Introduction
Toxocara canis and T. cati are nematodes of the family Ascaridae, whose adult forms
inhabit the proximal small intestine of their respective mammalian definitive hosts, namely,
canids and felids. Toxocara canis completes its life cycle in dogs, with humans acquiring the
infection as accidental hosts. Following ingestion by dogs, the infective eggs hatch and larvae
penetrate the gut wall and migrate to various tissues, where they are encysted if the dog is
older than 5 weeks. In younger dogs, the larvae migrate through the lungs, bronchial tree, and
oesophagus; while adult worms develop and oviposit in the small intestine. In older dogs, the
encysted stages are reactivated during pregnancy, and infect puppies by the transplacental
and transmammary routes, where adult worms become established in the small intestine.
Thus, infective eggs are excreted by both lactating bitches and puppies. Humans are
accidental hosts who become infected by ingesting infective eggs in contaminated soil. After
ingestion, the eggs hatch and larvae penetrate the intestine wall and are carried by the
circulation through a wide variety of tissues (liver, heart, lung, brain, muscle, eyes). Although
the larvae do not undergo any further development in these organs, they can cause severe
local reactions that form the basis of toxocariasis. The two main clinical presentations of
toxocariasis are visceral larva migrans (VLM) and ocular larva migrans (OLM). The other
clinical features are fever, coughing, wheezing, anorexia, hepatomegaly and eosinophilia (1 5). Toxocariasis is treated with antiparasitic drugs, usually in combination with antiinflammatory medications. Among the antiparasitic drugs, diethylcarbamazine, albendazole
and mebendazole can be used (6 - 10).
The effects of T. canis infection on lipid peroxidation remain unknown, and there were no
studies on this subject previously. The present study was undertaken to clarify the effect of
parasitic infection and drug (albendazole) therapy in parasitic infection related to lipid
peroxidation event, and for this aim plasma malondialdehyde (MDA) levels, glutathione
peroxidase (GSH-Px), superoxide dismutase (Cu-Zn SOD), and catalase activities in
erythrocytes were determined.
Materials and Methods
Adult T. canis were collected from puppies’ faeces. Eggs were obtained from the uteri of
adult female worms and stirred by magnet for 10 minutes with 1% sodium hypochloride. This
suspension was filtered with a sieve having 150 µm pores. The filtrate was centrifuged at 500
G for 3 minutes, 3 times with 0,9 % NaCl solution to remove sodium hypochloride. The eggs
were incubated in 0,5 % formalin at 260C for 4 weeks to allow embryonic development (11).
The experiment was run in four groups; each group consisted of 21 mice, totally 84 mice.
Group 1 was used as control group; they were feed a commercial ration, not given
albendazole or T. canis. In Group 2 each mouse received 100 mg/kg body weight of
albendazole (Andazol susp. 2%, Biofarma Drug Corp. Istanbul, Turkey) in a single oral dose,
diluted in 0,1 ml of water for five consecutive days. Group 3 received infective T. canis eggs
(1500 infective eggs in 0,5 ml), and after inoculation of eggs, albendazole was given orally for
five consecutive days 100 mg/kg b.w. in a single dose, diluted in 0,1 ml of water for five
consecutive days. Group 4 received infective T. canis eggs (1500 infective eggs in 0,5 ml)
only (no albendazole application).
Embryonated eggs were placed in the stomach with a blunt needle. Before introducing the
embryonated eggs, the suspension was centrifuged at 5000 G for 3 minutes and washed 3
times in sterile distilled water to remove formalin. At the end of this process distilled water was
added to the remaining sediment to reach a concentration of approximately 1500
embryonated eggs in 0.5 ml. Each mouse in groups 3 and 4 was inoculated with 0.5 ml of egg
suspension.
Heparinised blood samples were collected from 7 mice each by cardiac puncture on days
15, 45, and 60 after egg inoculation. Samples were centrifuged immediately (3000 G, 15 min
at 40C). The plasma and erythrocytes were separated by centrifugation, and were stored at 700C until enzyme assays were performed. Therefore, on days 15, 45, and 60 the brain, liver,
kidney, lung and muscles were examined for somatic larvae. For counting the larvae, tissues
were removed and cut into 3 mm cubes. Sliced small particles were pressed between a
22x22 mm cover slip and a slide, and the areas were examined under a light microscope with
x50 magnification to count the larvae (12).
Malondialdehyde levels in plasma were determined spectrophotometrically by reaction with 2 thiobarbituric acid (TBA) as described by Yoshioka et al. (13). Cu-Zn SOD activity in erythrocytes
was measured by the previously detailed method of Fitzgerald et al. (14). Catalase in
erythrocytes was measured spectrophotometrically as described by Luck (15). Glutathione
peroxidase in erythrocytes was measured spectrophotometrically as described by Pleban et al.
(16). Haemoglobin levels were determined in haemolysed blood samples spectrophotometrically
(17).
The data were analysed by one-way analysis of variances (ANOVA) to detect significant
treatment effect; DUNCAN’s multiple range test was used to identify specific differences
between treatment means at a probability level of 5%.
Results
Malondialdehyde, haemoglobin levels, and GSH-Px, Cu-Zn SOD, catalase activities were
determined in plasma and erythrocytes collected from mice at day 15, 45, and 60 post
infection. The number of T.canis larvae were also counted in experimental groups during
these periods.
Plasma MDA levels increased significantly in treatment groups in comparison with the
control group. Especially these increases were found in Group 4, which was given infective T.
canis eggs, MDA levels also increased in the albendazole treated group (Group 2). However
MDA levels in Group 3 (albendazole and T. canis eggs together) increased compared with
Group 1 and Group 2, but decreased compared with Group 4. MDA levels for all treatment
groups are shown in Table 1.
Decreases in Cu-Zn-SOD activities were seen in Group 3 and especially Group 4 compared
with Group 1 and Group 2 (Table 2), but these were not statistically significant. On the other
hand catalase activities decreased in Group 4, compared with the other experimental groups
on days 15, 45, and 60. These differences were not statistically significant. The catalase
activities for all groups are shown in Table 3.
Significant differences were observed on days 45 and 60 for GSH-Px activities (Table 4).
Glutathione peroxidase decreased in these periods compared with the control group. The
maximum decreases were found in Group 4. Haemoglobin levels for all the groups are shown
in Table 5.
The number of T. canis larvae was examined in all groups on the days of 15, 45, and 60.
No larvae were counted in Groups 1 and 2 at these times. Also, T. canis larvae were counted
in Groups 3 and 4. Especially the decreases in the number of T. canis larvae were observed
in Group 3 compared with Group 4, attributable to the use of albendazole. These data are
shown in Table 6.
Discussion
In many parts of the World, several hundred million domestic dogs are infected with T.
canis. Adult worms reside in the gastrointestinal tract and produce eggs, which are
subsequently excreted in faeces into the environment. One T. canis adult female can produce
20,000 eggs per day and since intestinal parasite burdens range from one to several hundred
worms infective animals can contaminate the environment with millions of eggs per day (1, 2,
3, 4, 5). The treatment of toxocariasis is mostly supportive. The medical letter recommends
diethylcarbamazine, albendazole and mebendazole; for severe symptoms or eye lesions,
corticosteroids can be used additionally (6 - 10).
There is no report concerning the effects of toxocariasis and drug therapy on lipid
peroxidation, whereas a limited study has been done for other parasitic infection. Shaheen et
al. (18) studied the effect of praziquantel treatment on lipid peroxide and superoxide
dismutase activity in tissues of healthy and Schistosoma mansoni infected mice. They
showed lipid peroxide product levels were evaluated as MDA and SOD activity in plasma,
liver, spleen, intestine and kidney, and the MDA levels were affected by parasitic action and
drug therapy. Drug therapy and parasitic infection inhibited SOD activity in blood, spleen and
kidney.
Antioxidative metabolism and lipid peroxidation can be affected by various conditions and
substances, such as drugs, pesticides, stress and exercise. Lipid peroxidation is a
degenerative process which affects the polyunsaturated fatty acids of membrane
phospholipids. The general mechanism of this process involves the formation of toxic
aldehydes, which react with protein and non-protein substances and result in widespread
changes in cellular membranes. These degenerative processes can be prevented molecularly
(vitamin A, vitamin C, vitamin E, uric acid, ceruloplasmin) and enzymatically (Cu-Zn SOD,
GSH-Px, and catalase). Therefore lipid peroxidation can be estimated directly by
determination of reactive oxygen species (hydroxyl, superoxide anion, H 2O2 and singlet
reactive oxygen radicals) or MDA levels in plasma, tissues and erythrocytes (19, 20, 21).
In the present study, we evaluated the lipid peroxidation in mice; which were infected with
T. canis, and treated with albendazole. The estimation for lipid peroxidation was done by
determining MDA levels in plasma, and Cu-Zn SOD, GSH-Px, and catalase activities in
erythrocytes on days 15, 45, and 60. The results show that plasma MDA levels increased on
days 45 and 60 in T. canis-infected mice, compared with the control and other groups. Also in
albendazole-treated groups MDA levels increased on day 15, but after this period MDA levels
decreased in this group compared to Group 3 and 4. Lipid peroxidation can be measured
indirectly by the determining of Cu-Zn SOD, GSH-Px and catalase activities; in the present
study we determined all these parameters. Cu-Zn SOD, GSH-Px, and catalase activities in
erythrocytes decreased with infection. The decreases in GSH-Px activities on days 45 and 60,
were statistically significant. Therefore, Cu-Zn SOD and catalase activities were also
decreased in the T. canis infected group (Group 4). Similarly decreases in Group 2 and 3 also
determined for these enzymes, compared with control group. But these differences were not
important as in Group 4. Haemoglobin levels (Table 5) obtained in the study cannot be used
as a parameter to measure oxidative damage, however, it is a useful index to determine the
activity levels of enzymes such as SOD, catalase and GSH-Px, which are the characteristic
indicators of oxidative damages. Possible changes in haemoglobin levels are not directly
linked to oxidative metabolism, but are affected by various factors.
The effects of antiparasitic drugs on T. canis infection have been studied including
albendazole, levamisole, ivermectin, thiabendazole, mebendazole, and diethylcarbamazine
(6, 7, 8, 9, 10). These drugs affected various ratios in T. canis infection. Samanta (10) found
that ivermectin and albendazole were most effective. In another study Delgado et al. (8)
examined the effect of albendazole in experimental toxocariasis of mice. In that study effect of
albendazole was determined as 23,7 % at 100 mg/kg dose. In the present study we tested the
effect of albendazole at a single dose of 100 mg/kg on five consecutive days for T. canis
infection. In the study, it was determined that albendazole decreased T. canis larvae count in
the tissues. These decreases were shown especially on days 45 and 60 and the most larvae
were determined in the brain and muscles.
In the present study, it was proposed to reach various aims and for this reason, convenient
experimental groups were formed. Firstly, it is aimed to determine the effects of T. canis
infection, a commonly occurring parasitic infection, on oxidative metabolism. The results
derived from the experimental groups demonstrated that T. canis induce lipid peroxidation
and suppresses oxidative metabolism. Increase of MDA levels but decreases of SOD,
catalase and GSH-Px activities in the Group 4 (the group exposed to T. canis only) led us to
reach this conclusion. These parameters are basic requirements for oxidative damage, and
MDA is one of the most important metabolites occurring as a result of oxidative damage. The
other aim of this study was to determine if the antiparasitic drug prevented damage induced
by parasitic invasion. The results derived from the third group demonstrated that the drug
effectively prevents the oxidative damage in a time-dependent manner. Indeed, when the
parasite-given group was compared with the treated group, MDA levels and the other enzyme
activities were normalised. In addition, it was also aimed to determine if the drug reduces the
number of larvae in various tissues and to determine the curative efficiency of the drug. It was
demonstrated that the drug reduced the worm burden in a time dependent manner (Table 6).
This conclusion was reached by counting parasite larvae in brain, liver, kidney, lung and
muscles in the parasite and drug treated groups (3rd and 4th).
References
1. Lawrence, T. G. and Schantz, P. M.: Epidemiology and pathogenesis of zoonotic
toxocariasis. Epidemiol. Rev., 3: 230-250, 1981.
2. Lynch, N. R., Wilkes, L. K., Hodgen, A. N. and Turner, K. J.: Specifity of Toxocara ELISA
in tropical populations. Parasit. Immunol., 10: 323-337, 1988.
3. Magnaval, J. V., Galindo, V., Glickman, L. T. and Clanet, M.: Human Toxocara infection
of the central nervous system and neurological disorders: a case-control study. Parasitol, 115:
537-543, 1997.
4. Schantz, P. M.: Parasitic zoonozes in perspective. Int. J. Parasit., 21: 161-170, 1991.
5. Van Knapen, F., Buijs, J., Kortbeck, L. M. and Ljungstršm, I.: Larvae migrans syndrome:
toxocara, ascaris or both. Lancet., 340: 550-551, 1992.
6. Carillo, M. and Barriga, O. O.: Antelmentic effect of Levamisole hydrochloride or
ivermectin on tissue toxocariasis of mice. Am. J. Vet. Res. 48: 281-283, 1987.
7. Dafalla, A.A.: A study of the effect of diethylcarbamazine and thiabendazole on
experimental Toxocara canis infection in mice. J. Trop. Med. Hyg ., 75: 158-15, 1972.
8. Delgado, O., Botto, R. and Mattei, R.: Effect of albendazole in experimental toxocariasis
of mice. Ann. Tropical. Med. Parasitol., 6: 621-624, 1989.
9. Kondo, K.: Experimental studies on “Larva Migrans”. J. Kyoto. Pref. Univ. Med. 79: 3256, 1970.
10. Samanta, S.: Studies on some aspects of experimental infection of Toxocara canis
(Werner, 1782) larvae in albino mice. J. Vet. Parasit., 3: 163, 1989.
11. Oshima, T.: Standardisation of techniques for infecting mice with Toxocara canis and
observations on the normal migration routes of the larvae. J. Parasitol., 47: 652-656, 1961.
12. Sprent, J. F. A.: On the migratory behaviour of the larvae of various ascaris species in
white mice. I. Distribution of larvae in tissues. J. Infect. Dis., 90: 165-176, 1952.
13. Yoshioko, T., Kawada, K. and Shimada, T.: Lipid peroxidation in maternal and cord
blood and protective mechanism against activated-oxygen toxicity in the blood. Am. J. Obstet.
Gynecol., 135: 372-376, 1979.
14. Fitzgerald, S. P., Campell, J. J. and Lamant, J. V.: The establishment of reference
ranges for selenium. The Selenoenzyme Glutathione Peroxidase and Metalloenzyme
Superoxide Dismutase in Blood Fractions. The Fifth International Symposium on Selenium in
Biology and Medicine, Tennessee, 1992.
15. Luck, H.: Catalase. Methods in Analysis. In: Bergmeyer H. U. Ed.: Academic Press, Inc
NY and London, 1965.
16. Pleban, P. A., Munyani, A., Beachum, J.: Determination of selenium concentration and
glutathione Peroxidase activity in plasma and erythrocytes. Clin. Chem., 28: 311-316, 1982.
17. Fairbanks, V. F. and Klee, G. G.: Biochemical aspect of haematology. In: Tietz, N. W.
(Ed): Fundamentals of Clinical Chemistry. 3rd Ed. WB Sounders Company, Philadelphia,
1987.
18. Shaheen , A. A., Abd el-Fettah, A. A. and Ebeid, F. A.: Effect of praziquantel treatment
on lipid Peroxide levels and superoxide dismutase activity in tissues of healthy and
Schistosoma mansoni infected mice. Arzneimittelforschung, 1: 94-6, 1994.
19. Comporti, M.: Lipid peroxidation. Biopathological significiance. Molec. Aspects. Med. 14:
199-207, 1993.
20. Gromov, L. A., Seredi, P. I., Syrovatskaia, L. P. , Ovinova, G. V. and Filenenko, M. A.:
Free radical mechanisms of memory disorders of toxic origin and experimental therapy of the
condition. Patol. Fiziol. Exp. Ter., 4: 24-26, 1993.
21. Yarsan, E.: Lipid peroxidation and prevention process. J. Vet. Med. Univ of Yuzuncu
Yil., 9: 89-95, 1998.
1.
TABLES:
Table 1: Plasma Malondialdehyde levels for all treatment groups (nmol/ml).
Groups
Day 15
Day 45
Day 60
43.91±9.35 a
45.60±8.22 a
46.07±8.90
(36.23-54.26)
(36.23-54.26)
(36.23-54.26)
97.84±49.16 b
50.52±9.09 ab
47.42±22.58
(25.34-131.08)
(41.05-59.86)
(21.45-80.39)
115.48±10.93 b
48.95±7.14 ab
49.18±11.69
(102.78-128.75)
(36.85-56.60)
(39.18-62.04)
91.77±17.35 b
57.41±5.80 b
54.69±20.95
(76.97-122.37)
(48.82-61.57)
(30.67-80.18)
Group 1
Group 2
Group 3
Group 4
Table 2: Cu-Zn SOD activities in erythrocytes for all treatment groups (U/gHb).
Groups
Day 15
Day 45
Day 60
Group 1
181.93±37.69
184.25±47.02
192.61±51.18
(145.18-235.54)
(145.18-256.09)
(145.18-258.90)
248.28±141.45
191.57±110.99
162.23±35.58
(116.87-397.99)
(35.49-284.28)
(110.18-190.23)
191.00±17.95
136.49±42.02
159.61±9.05
(171.41-206.66)
(94.88±194.93)
(152.10-169.67)
171.03±94.37
92.66±36.19
147.18±42.43
(92.43-286.31)
(71.25-134.46)
(94.19-181.72)
Group 2
Group 3
Group 4
Table 3: Catalase activities in erythrocytes for all treatment groups (k/mgHb).
Groups
Day 15
Day 45
Day 60
Group 1
1108±500.48
836.21±299.74
886.36±252.24
(597.74-1889.71)
(526.76-1237.74)
(597.741237.74)
988.27±985.35
531.83±308.63
949.32±861.01
(139.03-2339.70)
(140.11-894.99)
(211.482111.44)
711.03±362.87
586.16±434.84
894.73±113.15
(292.53-938.07)
(320.72-1236.26)
(777.891003.80)
Group 2
Group 3
Group 4
657.53±534.64
504.43±129.68
657.24±455.28
(88.19-1088.05)
(401.99-694.09)
(180.901088.05)
Table 4: Glutathione peroxidase activities in erythrocytes for all treatment groups (U/gHb).
Groups
Day 15
Day 45
Day 60
Group 1
214.80±42.84
206.00±38.96 a
214.00±38.37 a
(140.00-245.00)
(140.00-236.00)
(140.00-245.00)
215.00±135.24
134.33±80.87 ab
194.25±33.49 ab
(94.00-361.00)
(78.00-227.00)
(170.00-242.00)
148.33±82.71
84.00±31.09 b
180.75±56.34 ab
(90.00-243.00)
(47.00-121.00)
(102.00-233.00)
153.50±56.41
73.00±98.75 b
135.50±66.56 b
(100.00-220.00)
(13.00-220.00)
(78.00-226.00)
Group 2
Group 3
Group 4
a,b. Means within the same columns with different letters are statistically significant (p<0.05).
Table 5: Haemoglobin levels for all treatment groups (g/dl).
Groups
Day 15
Day 45
Day 60
Group 1
13.59±9.56
10.08±8.50
12.96±4.61 a
(7.20-27.72)
(1.44-21.24)
(9.00-17.64)
Group 2
Group 3
Group 4
6.75±4.45
15.84±18.06
8.46±1.82 ab
(2.88-12.96)
(5.76-42.84)
(7.20-11.16)
8.16±2.04
30.94±35.29
6.48±3.17 ab
(6.48-10.44)
(6.48-15.84)
(2.52-10.08)
10.71±5.95
17.14±20.11
8.10±1.59 b
(5.40-16.56)
(7.56-83.52)
(6.84-10.44)
Table 6: T. canis larvae counts in tissues on days 15, 45, and 60.
Groups
Day 15
Day 45
Day 60
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Group 1
Group 2
Group
3
Brain
30.66±1.15
32.0)
(30.0-
26.66±1.15
28.0)
(26.0-
16.00±4.00
20.0)
(12.0-
Liver
N.T.
N.T.
1.33±0.5 (1.0-2.0)
N.T.
N.T.
N.T
1.33±0.5 (1.0-2.0)
N.T.
N.T.
22.0±2.0 (20.0-24.0)
18.66±1.15
20.0)
(18.0-
14.00±2.00
16.0)
(12.0-
68.66±2.51
71.0)
77.00±5.56
83.0)
(72.0-
72.00±21.0
92.0)
(50.0-
Kidney
Lung
Muscle
Group
4
Brain
(66.0-
Liver
5.0±3.0 (2.0-8.0)
5.56±1.52 (4.0-7.0)
5.56±0.57 (5.0-6.0)
3.0±1.0 (2,0-4,0)
2.0±1.0 (1.0-3.0)
N.T.
N.T.
N.T.
N.T.
35.0±3.60 (32.0-39.0)
55.66±3.21
58.0)
Kidney
Lung
Muscle
(52.0-
29.33±5.03
34.0)
(24.0-
a,b. Means within the same columns with different letters are statistically significant
(p<0.05).
a,b. Means within the same columns with different letters are statistically significant
(p<0.05).