الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Pesticides, especially organophosphates have been extensively used in daily life and industry, and documented to be associated with increased incidences of lung toxicity and dysfunction. Chlorpyrifos (CPF), one of the organophosphates insecticides, was initially recommended as a low-toxicity agent, while recent scientific evidence suggests that accumulation of CPF is extremely detrimental to humans and animals, specifically to the respiratory system, where was associated with lungs inflammation and apoptosis which finally lead to death.
L-2-oxothiazolidine-4-carboxylate (OTC), known as cysteine precursors. OTC is transformed to cysteine in cells by 5-oxoprolinase and has been used in animal models and humans to increase GSH concentrations. In populations with low GSH status, OTC supplementation has been tested for its influence on GSH homeostasis, comparable to other cysteine prodrugs.
Undifferentiated biological stem cells have the ability to become specialised cells and can split through a process called mitosis to make new stem cells. Bone marrow-derived mesenchymal stem cells (MSCs) have the capacity to differentiate into bone, fat, cartilage, muscle, neurons, hepatocytes, skin, lung, and other cell types under the right in vivo circumstances.
The present study aimed to elucidate the therapeutic role of antioxidant, L-2-oxothiazolidine-4-carboxylate (OTC), and mesenchymal stem cells (MSCs) derived from the bone marrow (BM) in the restoration of lung structures and functions after pulmonary toxicity and dysfunctions induced by CPF exposure in male rats.
Forty eight adult male albino rats weighing 120±20g were used, and divided into 2 groups as follows:
group A (16 rats)
divided into 2 groups each of 8 rats:
1- Control group (GI): control group received distilled water daily.
2- Oxothiazolidine group (GII): received l-2-oxothiazolidine-4-carboxylate orally (100mg/kg/b.wt., daily for one month) at the beginning of MSCs transplantation.
group B (32 rats): were divided into 4 groups after oral administration of Chlorpyrifos 17.5mg/kg for one month):
1- Chlorpyrifos group (GIII): remain with no further treatment.
2- CPF+OXT group (GIV): toxic rats were treated with l-2-oxothiazolidine-4 carboxylate (100mg/kg/b.wt.) for one month by oral.
3- CPF+MSCs group (GV): toxic rats were treated with single intravenous injection of stem cells (2×106 cell) for one month.
4- CPF+OXT+MSCs group (VI): toxic rats were treated with both stem cells companies with l-2-oxothiazolidine-4-carboxylate.
Animals were sacrificed at the end of the experiment. The morphological investigation included total body and relative lungs weights.
The biochemical investigation included total protein and albumin level, total cholesterol, triglyceride (TG), lipoproteins (HDL and LDL). It was also measured tumor supressor protein (P53), interleukin-10 (IL-10) and Caspase-3. In addition, glutation peroxidase (GPx), total antioxidant capacity (TAC) and lipid peroxidation malondialdehyde (MDA) were measured in tissue. Further to inflammatory markers (CRP and ESR) and arterial blood gases (PO2 and PCO2) were measured in blood. The histological and immunohistochemical alterations in lungs tissue were examined.
The results obtained from the present investigation can be summarized as follows:
Morphological studies:
Detecting of mesenchymal stem cells:
In vitro, the cell culture showed mesenchymal stem cells (MSCs) as spindle and fibroblastic in morphology which later became the rounded cells.
MSCs showed negative immune reaction to CD34, but showed positive to CD29, and CD90.
PKH26 labeled stem cells in the lung tissue showing stronger red flourescence after treatment with both MSCs and OTC than after MSCs alone.
Total body and relative lungs weights:
The study showed a very highly significant decrease in body weight and increase in relative lungsweight in CPF-treated rats (GIII) compared to control group (GI), whereas all groups (CPF+OTC-treated group, CPF+MSCs treated group and CPF+OTC+MSCs-treated group) showed significant increase in total body weight and decrease in relative lungs weight compared to GIII.
A significant decrease occurred in relative lungs weight in CPF+OTC-therapeutic group (GIV), CPF+MSCs-therapeutic group (GV) and CPF+OTC+MSCs-therapeutic group (GVI).
Biochemical studies:
Protein profile:
The administration of CPF (GIII) induced an obvious depletion in both total protein and albumin levels in the serum as compared to control.
CPF+OTC-treated group (GIV) showed significant improvement in the level of both albumin and total protein, while OTC in the CPF+OTC group administered CPF followed by OTC (GV) revealed partial recovery in total protein.
CPF+MSCs group (GVI) showed significant increase in the levels of both total protein and albumin.
Lipid profile:
In CPF rats (GIII) a significant elevation of the levels of cholesterol, TG and LDL and significant reduction of HDL were recorded as compared to control groups. However, in the present investigation, an improvement occurred in lipid profile levels in CPF+OTC-group (GIV) and in the CPF+OTC-group (GV) in serum cholesterol, TG, HDL and LDL.
A marked improvement was recorded in CPF+OTC+MSCs-group (VI) in serum cholesterol, TG, HDL and LDL.
Cytokines:
Administration of CPF (GIII) resulted in disruption of some immune biomarkers reflected by the significant decrease in serum IL-10 level.
A partial improvement was observed in CPF+OTC-group (GIV) and CPF+MSCs-group (GV) in the parameter.
The combination of OTC and intravenous injection of MSCs into male rats in GVI showed marked restoration of cytokines (IL-10) levels as compared to control group.
Apoptotic markers:
Tissue tumor suppressor protein (P53) & Caspase-3 levels:
CPF rats (GIII) exhibited a significant elevation of P53 and Caspase-3 levels, in relation to the control rats (GI).
A considerable improvement for P53 and Caspase-3 levels was observed in CPF+OTC group (GIV) and CPF+MSCs group (GV) compared to the CPF group (GIII).
Moreover, treated with OTC + MSCs caused a great reduction in the P53 and Caspase-3 levels in (GVI) as compared to CPF rats (GIII).
Antioxidants and oxidative stress:
A significant depletion in the pulmonary GPx and TAC was designated, while a significant elevation was realized in MDA in CPF-treated rats (GIII) as compared with the control group (GI).
Partial improvement was shown in CPF+OTC-group (GIV) and in the CPF+OTC-group (GV) in pulmonary GPx, TAC and MDA.
The results revealed significant improvement in tissue GPx, TAC and MDA of CPF+OTC+MSCs-group (GVI).
Inflammatory Markers:
The present study showed a significant increase in the levels of CRP and ESR in rats exposed to CPF (GIII) as compared to control group (GI). However, a marked decrease was showen greatly in CPF+OTC-group (GIV) and in CPF+MSCs-group (GV).
The CPF+OTC+MSCs-group (GVI) revealed significant improvement in CRP and ESR.
Arterial blood gases:
The study revealed that rats treated with CPF (GIII) had markedly impaired arterial blood gases (ABG). CPF significantly lowered partial pressure of O2 (PO2) and significantly increased partial pressure of CO2 (PCO2) as compared to control group.
The administration of OTC (GIV) and MSCs (GV) in the present investigation indicated significant improvement as regards PO2 and PCO2.
A significant improvement was observed in CPF+OTC+MSCs- group (GVI) where the value was nearly returned to the control.
Histopathological studies Hx&E:-
The histopathological examination of lungs sections from the rats administered CPF revealed severe damage of its structure; the peri-bronchial blood vessels were severely congested with hyperplasia in the bronchial associated lymphoid tissue and peribronchial mononuclear cells infiltration. The bronchiolar epithelium was sloughed, and the wall was disrupted. Along with, there were obvious thickening in the interalveolar wall. Diffuse interstitial pneumonia was manifested by accumulation of mononuclear inflammatory cells in the interalveolar walls. In addition, focal aggregates of mononuclear inflammatory cells and distended alveoli were observed.
In rats treated with CPF as a toxic agent then followed by the administration of OTC (CPF+OTC-group, GIV) the histopathological changes were greatly reduced. This group showed recovery and preservation of alveolar structure except separating of small scattered foci of interstitial pneumonia, alveolar emphysema and appearance of extensive alveolar edema and hemorrhage in severely affected individuals.
The group exposed to CPF then treated with MSCs (CPF+ MSCs-group, GV) also limited the pulmonary destruction induced by CPF but only to some extent. A relatively some congested peri-bronchiolar blood vessels with some aggregations of mononuclear cells were noticed. Congested peri-alveolar blood capillaries, mild perivascular edema and alveolar emphysema were observed as well.
Lungs sections of rats that received CPF then followed by OTC+MSCs (CPF+OTC+MSCs-group, GVI) exerted the best action among the treated groups in maintaining lungs architecture. Focal mononuclear inflammatory cells aggregations were observed along with alveolar emphysema. Extensive peri-vascular edema and inflammatory cells infiltration was frequently observed.
Immune-histochemical studies:
Immune-staining for the proliferating cell nuclear antigen (PCNA):
Lungs of rat of the group received CPF showed extensive diffuse expression of PCNA positive cells in the bronchiolar epithelial lining, peri-bronchiolar tissue and interalveolar wall. Administration of OTC resulted in decrease in PCNA expression of the interalveolar wall cells. Treatment using MSC showed only mild expression of PCNA in lung tissue. The group co-administrated with OCT+ MSC showed relatively higher expression of PCNA positive cells compared to the other treated groups.
Conclusions:
from the morphological, biochemical and microscopical studies it can be concluded that chlorpyrifos (CPF) insecticide was proved to have toxic effect on adult male lungs which may lead to disorder of pulmonary functions thus death.
L-2-oxothiazolidine-4-carboxylate (OTC) at the dose of 100 mg/kg displayed a therapeutic effect against CPF-induced toxicity. While, intravenous transplantation of bone marrow mesenchymal stem cells (MSCs) proved to be beneficial as being a therapy after injury.
So, the results of this study indicated that treatment by OTC and with MSCs were more effective than therapy by OTC and MSCs, alon, in ameliorating CPF-induced lung damage and thus for improvement of pulmonary function and architecture as well as increased lungs work.