الفهرس 
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Abstract
Biochemical effect of Pirimiphos Methyl on lipid peroxidation and antioxidant enzymes in blood and tissues of rats and ameliorating role of certain antioxidants were investigated. This study was carried out on 120 male albino rats weighting 150 - 200 gm. Rats were housed in a separate metal cages under the same constant environmental and nutritional condition through the period of investigation.
The rats were randomly divided into eight groups as follows:
Group (1): Normal group: Consisted of 15 rats, served as control, fed on normal diet and received single dose of distilled water per os daily for 3 months.
Group (2): Pirimiphos methyl administered group: Comprised 15 rats, which were fed on normal diet and administered single dose of Pirimiphos methyl orally and daily for 3 months at a dose level of 50 mg/ Kg b.w (1/40 LD50).
Group (3): Rutin treated group: Composed of 15 rats, which were fed on normal diet and administered single dose of Rutin orally and daily for 3 months at a dose level of 45 mg/ Kg b.w.
Group (4): Rutin + POM treated group: Included 15 rats, which were fed on normal diet and administered single dose of Pirimiphos methyl orally and daily for 3 months at a dose level of 50 mg/ Kg b.w (1/40 LD50) followed by oral administration of Rutin at a dose level of 45 mg/ Kg b.w.
Group (5): Chlorophyllin treated group: Comprised 15 rats, which were fed on normal diet and administered single dose of Chlorophyllin orally and daily for 3 months at a dose of 27 mg/ Kg b.w.
Group (6): Chlorophyllin + POM treated group: Consisted of 15 rats, which were fed on normal diet and administered single dose of Pirimiphos methyl orally and daily for 3 months at a dose level of 50 mg/ Kg b.w. (1/40 LD50) followed by oral administration of Chlorophyllin at a dose level of 27 mg/ Kg b.w.
Group (7): Thyme treated group: Composed of 15 rats, which were fed on normal diet and administered single oral daily dose of Thyme for three months at a dose level of 45 mg/ Kg b.w.
Group (8): Thyme + POM treated group: Consisted of 15 rats, which were fed on normal diet and administered single dose of Pirimiphos methyl orally and daily for 3 months at a dose level of 50 mg/ Kg b.w (1/40 LD50) followed by oral administration of Thyme at a dose level of 45 mg/ Kg b.w.
Blood samples were collected by ocular vein puncture from all animal groups (control and experimental groups) 3 times along the duration of experiment and
a) Serum samples preserved at 20°C until used for determination of Ceruloplasmin and Ig E.
b) Plasma separated and preserved at 20°C until used for determination of (NO) and (GGT).
c) Erythrocytes washed after plasma separation for subsequent biochemical analysis:
1- (L-MDA). 2- (GSH). 3- (SOD).
4- (CAT). 5- (GPx). 6- (GR).
7- (GST). 8- (AChE).
Tissue samples:
a. Biochemical parameters in Brain tissue:
1- (L-MDA). 2- (GSH). 3- (CAT).
4- (AChE). 5- (MAO). 6- (GABA).
7- Phospholipids. 8- Cholesterol. 9-Free Fatty Acids.
b. Histopathological examination:
For histopathological examination, brain specimens were taken from, preserved in 10% buffered neutral formalin, stained and subjected for examination under light microscope by a pathologist without prior knowledge of the groups; Digital photos were taken to demonstrate the histological changes between the groups after different treatments.
The obtained results demonstrated that:
1- Erythrocytes L-MDA:
Pirimiphos methyl intoxicated rats induced significant increase in erythrocytes (L-MDA) concentration when compared to control group allover the experimental period.
Rutin administration in POM intoxicated rats caused significant decrease observed along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change along the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change along the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change along the periods of experiment as compared with control group.
2- Brain (L-MDA):
Pirimiphos methyl intoxicated rats induced significant increase in brain (L-MDA) concentration when compared to control group allover the experimental period.
Rutin administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant increase along the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant increase along the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change along the periods of experiment as compared with control group.
3- Plasma (NO):
Pirimiphos methyl intoxicated rats induced significant increase in plasma Nitric oxide (NO) concentration allover the periods of experiment when compared to control group.
Rutin administration in POM intoxicated rats caused significant decrease observed along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant decrease observed along the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant decrease observed along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
4- Erythrocytes (GSH):
Pirimiphos methyl intoxicated rats induced significant decrease in erythrocytes reduced glutathione (GSH) concentration when compared to control group allover the experimental period.
Rutin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant decrease allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Oral administration of chlorophyllin to normal rats exhibited non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant increase observed along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
5- Brain (GSH):
Pirimiphos methyl intoxicated rats induced significant decrease in brain reduced glutathione (GSH) concentration when compared to control group allover the experimental period.
Rutin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant increase observed along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
6- Serum Ceruloplasmin:
Pirimiphos methyl intoxicated rats induced significant increase in serum Ceruloplasmin concentration when compared to control group allover the experimental period.
Rutin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Oral administration of chlorophyllin to normal rats exhibited non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant increase observed along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant increase allover the periods of experiment as compared with control group.
7- Erythrocytes (SOD):
Pirimiphos methyl intoxicated rats induced significant increase in erythrocytes super oxide dismutase activity (SOD) activity along the periods of experiment when compared to control group.
Rutin administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change along the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant increase along the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant increase along the periods of experiment as compared with control group.
8- Erythrocytes (CAT):
Pirimiphos methyl intoxicated rats induced significant decrease in erythrocytes catalase activity (CAT) activity when compared to control group allover the experimental period.
Rutin administration in POM intoxicated rats caused significant increase observed allover the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change along the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase after 2 and 3 months as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant increase after 2 and 3 months when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change along the periods of experiment as compared with control group.
9- Brain (CAT):
Pirimiphos methyl intoxicated rats induced significant decrease in brain catalase activity (CAT) activity allover the periods of experiment when compared to control group.
Rutin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats a non significant change allover the periods of experiment as compared with control group.
Thyme administration in POM intoxicated rats caused significant increase observed along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
10- Erythrocytes (GPx):
Pirimiphos methyl intoxicated rats induced significant decrease in erythrocytes glutathione peroxidase (GPx) activity when compared to control group allover the experimental period.
Rutin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase after 3 months as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change along the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant increase after 2 and 3 months when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
11- Erythrocytes (GR):
Pirimiphos methyl intoxicated rats induced significant decrease in erythrocytes (GR) activity when compared to control group allover the experimental period.
Rutin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase observed along the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant increase observed along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
12- Erythrocytes (GST):
Pirimiphos methyl intoxicated rats induced significant decrease in erythrocytes (GST) activity along the periods of experiment as compared with control group.
Rutin administration in POM intoxicated rats caused significant increase allover the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change along the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase allover the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change along the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant increase in allover the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change along the periods of experiment as compared with control group.
13- Plasma (GGT):
Pirimiphos methyl intoxicated rats induced significant increase in plasma (GGT) activity allover the periods of experiment when compared to control group.
Rutin administration in POM intoxicated rats caused significant decrease after 2 and 3 months as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change along the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change along the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant decrease observed allover the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change along the periods of experiment as compared with control group.
14- Erythrocytes (AChE):
Pirimiphos methyl intoxicated rats induced significant decrease in erythrocytes AChE activity allover the periods of experiment as compared with control group.
Rutin administration in POM intoxicated rats caused significant increase after 2 and 3 months as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change along the periods of experiment when compared to control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase after 2 and 3 months as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Thyme administration in POM intoxicated rats caused significant increase when compared to POM intoxicated group after 2 and 3 months. Thyme administration in normal rats showed non significant change along the periods of experiment as compared with control group.
15- Brain (AChE):
Pirimiphos methyl intoxicated rats induced significant decrease in brain AChE activity allover the periods of experiment when compared to control group.
Rutin administration in POM intoxicated rats caused significant increase after 3 months as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change along the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused significant increase after 2 and 3 months as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused significant increase after 3 months when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change along the periods of experiment as compared with control group.
16- Brain MAO:
Pirimiphos methyl intoxicated rats induced non significant change in brain Monoamine Oxidase (MAO) activity allover the periods of experiment when compared to control group.
Rutin administration in POM intoxicated rats caused non significant change along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused non significant change along the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused non significant change along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
17- Brain GABA:
Pirimiphos methyl intoxicated rats induced non significant change in Brain Gamma amino butyric acid (GABA) concentration along the periods of experiment when compared to control group.
Rutin administration in POM intoxicated rats caused non significant change allover the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change along the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused non significant change allover the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change along the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused non significant change allover the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change along the periods of experiment as compared with control group.
18- Brain Phospholipids:
Pirimiphos methyl intoxicated rats induced non significant change in brain phospholipids concentration non significant change allover the periods of experiment when compared to control group.
Rutin administration in POM intoxicated rats caused non significant change along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused non significant change along the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused non significant change observed along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
19- Brain Cholesterol:
Pirimiphos methyl intoxicated rats induced non significant change in brain cholesterol concentration when compared to control group allover the experimental period.
Rutin administration in POM intoxicated rats caused non significant change along the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused non significant change along the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused non significant change along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
20- Brain Non esterified Fatty Acids:
Pirimiphos methyl intoxicated rats induced non significant change in brain Non esterified Fatty Acids concentration along the periods of experiment when compared to control group.
Rutin administration in POM intoxicated rats caused non significant change allover the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change along the periods of experiment as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused non significant change allover the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change along the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused non significant change when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
21- Serum Ig E:
Pirimiphos methyl intoxicated rats induced non significant change in serum Ig E concentration along the periods of experiment when compared to control group.
Rutin administration to POM intoxicated rats a non significant change allover the periods of experiment as compared with POM intoxicated group. Rutin administration in normal rats showed non significant change in serum the experimental period as compared with control group.
Chlorophyllin administration in POM intoxicated rats caused non significant change along the periods of experiment as compared with POM intoxicated group. Chlorophyllin administration in normal rats showed non significant change allover the periods of experiment when compared to control group.
Thyme administration in POM intoxicated rats caused non significant change along the periods of experiment when compared to POM intoxicated group. Thyme administration in normal rats showed non significant change allover the periods of experiment as compared with control group.
Histopathological Results
The brain of the control normal, Rutin, chlorophyllin and thyme adminsterd rat groups showing normal histological structure.
Pirimiphos Methyl intoxicated group:
The brain of rats chronically intoxicated with POM showing sever congestion of the meningeal and brain blood vessels with degenerative changes in the blood vessels wall in the form of vesiculation and injury in tunica intema. Perivascular hemorrhage and mild perivascular mononuclear infiltration were noticed.
The brain substances particularly, cerebrum and cerebellum showing focal areas of malacia in the form of clear irregular shaped areas scattered allover the brain substances.
The neurons in the brain of this group were suffered from degenerative changes. Tygrolysis, as the body of the neuron became rounded, swollen and loss of dentrites and contained Pyknotic nuclei. Some of the diseased neurons surrounded with glial cells (satillitosis) and may be engulved by some of them neuronophagia.
Pirimiphos methyl and Rutin group:
The brain of rats intoxicated with POM and administrated with Rutin showing congestion of the meningeal and brain blood vessels. Focal areas of hemorrhages among the brain substances were detected.
Focal areas of malacia which appeared as clear spaces scattered in the brain substances were seen. The neuron showing mild degenerative changes and mild demylination of the neuron.
Pirimiphos methyl and Chlorophyllin group:
The brain of rats intoxicated with POM and administrated with Chlorophyllin showing mild congestion of the brain blood vessels with the presence of focal areas of malacia. Focal areas of odema were detected as a faint homogenous eosinophylic areas in the brain substances. The neuron showing normal structure.
Pirimiphos methyl and Thyme group:
The brain of rats intoxicated with POM and administrated with Thyme showing congestion of the meningial and brain blood vessels with the presence of focal areas of hemorrhage among the brain substances. Mild areas of malacia were seen in the brain substances.
Conclusion and Recommendation
from the obtained results, it could be concluded that, chronic exposure to organophosphorous pesticides (Pirimiphos methyl) in rats caused significant increase in free radical production as erythrocytes and brain L-MDA and plasma Nitric oxide concentrations, in addition to significant decrease in activities of AChE, antioxidant enzymes as CAT, GR, GPx, GST and level of reduced glutathione - non enzymatic antioxidant - in erythrocytes and brain tissues, with significant increase in SOD and GGT activities.
The findings of the present study demonstrated that, administration of natural antioxidants as rutin, chlorophyllin and thyme provided an effective protection and powerful agents against oxidative damage induced by chronic exposure to POM in erythrocytes and brain tissues in rats, since these antioxidant compounds were able to ameliorate serum biochemical parameters, enzymatic and non-enzymatic antioxidant defense system and to prevent the oxidative stress as revealed by decreased lipid peroxidation in these tissues and exert a protective effect against POM induced peroxidative damage.
These results may contribute to a better understanding of the protective role of rutin, chlorophyllin and thyme, emphasizing the influence of these antioxidants in the diet for human health, possibly minimizing or preventing oxidative damage resulting from exposure to many pollutants including organophosphorous pesticides. Therefore, we recommended that, diet supplementation with natural antioxidants such as rutin, chlorophyllin and thyme is very important for protection of different body organs against oxidative stress or even inflammation or infections.
Since human life without pesticide exposure is practically inconceivable, supplementation with antioxidants remains a good strategy not only as a therapeutic tool but also as a preventive resource to reduce toxic effects of pesticides. So, we strongly support the use of rutin, chlorophyllin and thyme as pure active ingredients in pharmacological industry for production of new drugs used as therapeutics for protection against oxidative stress and which attenuate the undesirable and dangerous effects during hazardous exposure to organophosphorous compounds.