Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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AMELIORATIVE EFFECT OF PUMPKIN SEED OIL ON
ALTERATIONS IN THYROID GLAND FUNCTIONS INDUCED BY
CHLORPYRIFOS IN ADULT MALE RATS
Nabeel Mahdi Abed*,Jassim M. A. Alkalby**
* College of Veterinary Medicine, University of Thi-Qar.
** Department of Physiology, Pharmacology, and Chemistry College of Veterinary Medicine,
University of Basrah.
Keywords: Chlorpyrifos, Pumpkin Seed oil, Thyroid functions
Corresponding Author:
ABSTRACT
This study carried out in College of Veterinary Medicine /University of Basrah to
evaluate the effect of different doses of pumpkin seed oil (PSO) on Thyroid functions in
Chlorpyrifos (CPF) treated male rats. Fifty adult male rats were used and randomly divided
into five equal groups, 10 rats per each group: Group 1(control) administered orally corn
oil(1ml /kg.bw) , whereas first treated group administered CPF 1/20th LD50 (6.7 mg/kg bw).
Group 3, 4 and 5 were administered CPF (6.7mg /kg.bw) plus PSO(20, 40, and 80 ) mg/kg.bw
respectively. The treatments were given once daily by oral gavages for 8 weeks. At the end of
the experiment, animals were sacrificed and blood samples were obtained for evaluation of
thyroid hormones (T4 and T3 ) and tissue samples from thyroid gland kept in 10% neutral
formalin for histopathological examination.
The results indicated that male rats treated with Chlorpyrifos demostreated a significant
increase (P<0.05) in thyroid stimulating hormone (TSH) and a significant decrease (P<0.05) in
thyroxin(T4) and triiodothyronine (T3) hormones compared with control group.
Histopathological examination of CPF treated males showed that thyroid gland revealed
destruction of thyroid follicles with infiltration of macrophages on the site of follicular
destruction. On other hand groups treated with 20,40 and 80 mg pumpkin seed oil induced
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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improvement in above cited parameters and histological changes in dose dependent manner.
INTRODUCTION
The environment is a heavily contaminated with many chemicals, which interact with each
other in such a way that modify their toxic response in humans and animals(1). According to
WHO, 3 million cases of pesticide poisoning occur every year, resulting in more than 250,000
deaths (2). Pesticide such as Chlorpyrifos (CPF) can cause adverse effects by interfering with
the body’s hormones or chemical messengers. These substances (pesticides) are therefore
called hormone disruptors or endocrine disruptors (3). Organophosphate (OP) compounds are
the most widely used insecticides accounting for 50% of global insecticidal use (4).
Chlorpyrifos (CPF), an organophosphate insecticide (OP) that is widely used in agriculture,
horticulture and public health has been shown to disrupt the activities of endocrine organs(5).
The thyroid gland is the most commonly affected endocrine gland as it is considered a
sensitive target to organophosphate insecticide (OPI) leading to affection of thyroid function
(6).Thyroid hormones are important in several physiological processes such as metabolism
and normal growth development and any imbalance in their levels could lead to a wide range
of clinical conditions (7).
Pumpkin seed oil is used as nutritional supplements for natural source of proteins, essential
fatty acids, polyunsaturated fatty acids, omega 3, 6 and 9, Beta-carotenes, lutein, vitamins
such as carotenoids and β- and γ-tocopherols, phytosterols, chlorophyll, and trace elements,
such as zinc and selenium (8). The essential trace mineral zinc in pumpkin seeds acts as an
antioxidant which is attributed to its ability to neutralize free radical generation or directly
engross the iron or copper binding sites of lipids, proteins, and DNA molecules(9). However,
pumpkin seed extracts have been used as an adjuvant for immunomodulation, reproductive
health and therapeutic purposes for wide ranges of disease conditions(10). In addition, omega
3 and 6 essential fatty acids in pumpkin seed oil are important for healthy brain and body
functioning as well as preventing and improving bladder and prostate problems(11). The aim
of this study carried out to investigate the effect of different doses of pumpkin seed oil on
thyroid hormones levels and histological changes in chlorpyrifos- induced thyroid
dysfunction of adult male rats.
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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MATERIALS AND METHODS
Experimental Animals:
Forty adult male rats weighting (280- 290 gm) and twenty healthy adult fertile female rats
weighting (270-280 gm) were used in this study. The animals were kept in the animal house for
acclimatization for fifteen days before the beginning of the experiments. The animals were
maintained under optimum conditions (25±2°C) and (12/12 hours light/dark) cycle throughout
the study, with standard pellets and tap water ad libitum.
Extraction and Evaluation of Pumpkin Seed Oil (PSO):
Pumpkin seeds (Cucurbita pepo L.) brought from local market in the province of Basrah .
Seeds were selected according to their condition, damaged seeds were discarded and those
seeds which were in good condition cleaned. After cleaning, the sand and foreign materials
were removed. The dried pumpkin seeds were ground to a fine powder using a grinder. The oil
was extracted with n-hexane (1:4 w/v) by soxhlet device and agitation in a shaker at room
temperature in the dark for 36 h. The solvent was evaporated at 40 °C to dry it . The extracted
oil was stored in sealed and dark bottles until analysis(12). Pumpkin oil content was analyzed
by GC- MS , Collage of Agriculture ,University of Basrah. GC-MS analysis showed 20 oil
constituents, Linoleic acid,Oleic acid(omega-9 fatty acid,omega-3 fatty acid) ,Palmatic acid,
Stearic acid , Ascorbic acid and tocopherol.
Experimental Design:
After acclimation period, 50 male rats were divided into 5 groups with 10 animals in
each as the following: Group 1 (Control group): Animals of this group administered corn
oil (0.2 ml/kg)/day orally by gavages. Group 2 (CPF treated group): Animals of this
group administered orally CPF 1/20th LD50 (6.7 mg/kg BW),/day by gavages (13). Group
3 (CPF + 20 PSO): Animals of this group administrated orally CPF (6.7 mg) + pumpkin
seed oil (20 mg)kg BW daily orally by gavages. Group 4 (CPF + 40 PSO): Animals of
this group administer orally CPF (6.7 mg) + pumpkin seed oil (40 mg)/kg BW daily orally
by gavages Group 5 ( CPF + 80 PSO): Animals of this group administer orally CPF (6.7
mg) + pumpkin seed oil (80 mg)/kg BW daily orally by gavages. The experimental was
continued for 8 weeks .
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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Blood and Specimens Collection :
At the end of the experiment, animals of each group were anaesthetized by chloroform
and sacrificed. Blood sample were collected from the heart via the cardiac puncture of
sacrificed animals by using 5cc sterile syringe and dropped in plain without anticoagulant
tubes and serum samples were isolated from blood by centrifugation at 3000rpm for 15 min.
and separated in eppendroff tubes and stored at -20°C until used for hormonal analysis.
Thyroid gland was removed and kept in 10% formal saline for histological study.
Hormonal assay:
T3 and T4 concentrations were assayed by using ELISA kits from Monobind Inc.lake forest
CA 92630; USA, Product code : l25-300. TSH also was measured by using ELISA kit from
(Calbiotech Inc. a life science company, USA), Product Code: TS227T.
Histopathological study:
Samples of the thyroid gland were taken and fixed in 10% formalin and then embedded in
paraffin and sectioned at thickness of 6 micrometers and then carried on glass slides, stained
with hematoxylene-eosin and examined under a light microscope(14).
Statistical Analysis:
Data are expressed as mean values ± SD. Statistical analysis was performed using one-way
analysis of variance (ANOVA) to assess significant differences among treatment groups. The
criterion for statistical significance was set at P <0.05. All statistical analyzes were
performed using SPSS statistical version 8 software package (SPSS Inc., USA).
RESULTS
The results of thyroid function tests revealed a significant increase (P ≤ 0.05) in TSH
concentration and significant decrease (P ≤ 0.05) in serum concentrations of T3 and T4 in CPF
treated group compared with control group, as seen in table (1). On the other hand the group of
CPF treated with different doses of PSO revealed a significant decrease (P ≤ 0.05) in serum TSH
concentration compared with CPF treated group. The results showed a significant decrease (P ≤
0.05) in serum concentration of TSH in CPF group treated with 20 mg / kg. bw PSO compared
with CPF group but still significantly higher (P ≤ 0.05) than those of control group. Whereas no
significant differences were observed in TSH concentrations in CPF groups treated with 40 and
80 mg / kg. bw PSO respectively compared with control group. A significant decrease (P ≤ 0.05)
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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in T3 concentration was recorded in CPF group treated with 20 mg / kg. bw PSO compared with
control group. While no significant differences were observed in T3 concentration in CPF groups
treated with 40 and 80 mg/ kg.bw PSO compared with control. On the other hand, significant
decrease (P ≤ 0.05) in T4 concentration was noted in CPF groups treated with 20 and 40 mg / kg.
bw PSO compared with control group, however no significant difference was recorded in T4
concentration in CPF group treated with 80 mg / kg. bw PSO compared with control group.
Table (1): Effect of PSO on TSH, T4 and T3 Concentrations in Male Rats treated
With CPF.
Results of Histopathological Examination:
Thyroid gland of control male rats showed normal architecture composed of normal thyroid
follicles with normal epithelium as shown in figure (4-1). While histopathological section in
thyroid gland of adult male rats treated with CPF for 8 Weeks (fig. 4-2) revealed marked
destruction of thyroid follicles with infiltration of macrophages on the site of follicular
destruction. Figure (4-3) Thyroid gland of CPF+20 mg PSO treated group , preservation of
thyroid follicles with infiltration of macrophages in the follicles in which most of the contain no
colloid. Figure (4-4) Thyroid gland of CPF+40 mg PSO treated group, reveal preservation of
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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thyroid follicles in which most of follicles contain colloid secretion of the gland, while
histopathological section in thyroid gland of adult male rats treated with CPF + 80 mg PSO for 8
Weeks (fig. 4-5) reveal well preserved thyroid follicles (F) in which the follicles contain colloid
secretion of the gland.
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DISCUSSION
The present study demonstrated that CPF treated rats showed hypothyroidism which was
evidenced biochemically by a significant decrease in serum T3 and T4 levels. Hypothyroidism in
CPF treated rats was reported also by some investigators (15) ; (16) and (17). Some OPs disrupt
endocrine system, some thyroid disruptors inhibit thyroperoxidase; thereby they change ability
of follicular cell in producing T4 and then T3, even at sufficient iodine concentration(18). Many
chemical compounds have high structural similarity to T4 and T3thereby they disrupt the binding
of thyroid hormones to their receptors or transfer proteins, resulting in subclinical
hypothyroidism(19). (20) showed that CPF administration to rats for 12 weeks induced reduction
in thyroid hormones which may be attributed to the excessive production of reactive oxygen
spices and free radicals in the central nervous system and related glands, including the
hypothalamic–pituitary axis (21). The elevation in TSH in CPF group reflects the response of
this hormone to the lowered T3 and T4 concentrations. TSH stimulates the thyroid glands to
increase the elaboration of T3 and T4. However, the failure of the thyroid glands to respond to
increased TSH stimulation for the elaboation of T3 and T4 in the CPF group may have been due
to peroxidative damage to the gland (17).
A significant improvement in the levels of thyroid hormones was observed in animal groups
treated with different doses of pumpkin seed oil which may be attributed to vitamin E and
phenolic compound found in pumpkin oil which acts as a potent antioxidant by removing free
radicals and inhibition fat peroxidation and thus preserving the cell membrane. (22). showed that
the supplementation with Iron, Zinc, Vit.E and Vit.C significantly increased the concentration of
thyroid hormones and also caused a significant decrease in TSH level compared with control
positive group. The marginal decrease in serum T3 and T4 with a significant increase in the
concentrations of TSH in the group co-administered with CPF were ameliorated by Vitamin .C
partly due to its antioxidant properties (1). A combination with vitamin C and E has been
demonstrated to protect against CPF-induced toxicity(23). Since it has the ability to augment the
activities of other antioxidants. (24), reported that administration of antioxidant vitamin C or E
partially attenuated the toxicity induced by exposure to CPF. The results of above studies are
consistent with the results of current study because the PSO contains both vitamin E and C and
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
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the essential mineral elements that play an important reducing the oxidative stress resulted from
exposure to CPF.
Histopathological examination of thyroid gland in CPF treated male rats for 8 weeks showed
marked destruction of thyroid follicles with infiltration of macrophages on the site of follicular
destruction (Fig.4-2). These results are correlated with those reported by (25) who found that
degenerated follicles with decrease colloid, obvious exfoliation of the follicular epithelial cells
and vascular congestion in CPF treated rats as compared with control. Similarly (13) reported
that male rats treated with CPF showed decrease in the size of follicles and amount of colloid,
degeneration of follicular cells and congestion of blood vessels .In addition ,(20) showed that
male rats treated with 6.75 mg/kg b.w. CPF for 12 weeks revealed damaging effects in thyroid
tissue, thickened collagen fibers between the follicles. (17) reported that CPF treated rats showed
hypothyroidism that was evidenced by decreased amount of colloid. The present results
confirmed with the reduction of serum T3 and T4 and elevation of serum TSH in CPF-treated
rats.
CONCLUSION
The results of this study revealed that oil extracted from pumpkin seeds is an important source
rich in many active substances such as vitamin C and vitamin D and metal elements such as zinc
which play an important role as antioxidants relieving the harmful effects of exposure to
chlorpyrifos.
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