الفهرس 
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Abstract
Biochemical effect of curcumin and TQ administration on biomarkers of oxidative stress, enzymatic and non-enzymatic antioxidant, in addition to DNA fragmentation, Bcl-2, CYP1A1, P53 and caspase-3 gene activity in lung cancer induced by B(a)P in male mice was investigated. One hundred and fifty male Swiss Albino Mice of 6-8 weeks old and each mice weighing 25-30 g were used in the experimental investigation of this study. Mice were obtained from the Laboratory Animals Research Center, Faculty of Veterinary Medicine, Benha University. The animals were housed in separated metal cages and kept at constant environmental and nutritional conditions throughout the period of the experiment. Fresh and clean drinking water was supplied ad-libitum. The animals were left for 14 days for acclimatization prior to the beginning of the experiment. Induction of lung cancer:
Benzo (a) Pyrene was freshly dissolved in corn oil to ensure the stability of the chemical just prior to use. Lung cancer have been induced in mice by a single intraperitoneal injection of [B(a)P] at a dose of (100 mg/kg body/weight) (Malhotra et al., 2014) .
Preparation and dosage of Curcumin: Curcumin was freshly prepared by dissolved in 7% DMSO solution then complete to 100 ml dist water just before treatment, and was administered orally at a dose of (100 mg/kg b.wt.) for 30 weeks. (Aggarwal et al., 2003).
Preparation and dosage of TQ:
TQ was freshly prepared by dissolving in traces of ethanol just before treatment, and was administered orally at a dose of (20 mg/kg b.wt).
Experimental design:
Mice were randomly divided into six main equal groups, 25 animals each, placed in individual cages and classified as follow:
Group (1): Control Normal Group: mice received no drugs, served as control non-treated for all experimental groups.
Group (2): [B(a)P] group: Mice administrated with a single dose of B(a)P (100 mg/kg.b.wt intraperitoneally (IP), served as the carcinogenic non-treated group.
Group (3): [B(a)P] +Curcumin treated group: Mice injected with a single dose of B(a)P (100 mg/kg.b.wt intraperitoneally) then treated with curcumin (100 mg/kg.b.wt/ day,orally) starting after 22 weeks of the experiment and continued till the end of the experimental periods (30 weeks) .
Group (4): [B(a)P] + Curcumin protected group: Mice received curcumin at a dose (100mg/kg .b.wt./ orally) on alternate days started from one day prior B(a)P injection as a single dose (100 mg/kg.b.wt/ IP) and were treated continuously with curcumin until 30 th. Weeks (end of experiment).
Group (5): [B(a)P] +TQ treated group: Mice injected with a single dose of B(a)P (100 mg/kg.b.wt. intraperitoneally) and treated with TQ (20 mg/kg.b.wt/ day, orally) starting after 22 week of the experiment and continued till the end of the experimental periods (30 weeks).
Group (6): [B(a)P] + TQ protected group: Mice received TQ orally at a dose (20 mg/kg .b.wt. /day after day) started one day before [B(a)P] injection as a single dose (100 mg/kg.b.wt) given intraperitoneal and treated continuously with TQ to 30 th. Weeks (end of experiment).
Sampling:
Blood samples and lung tissue specimen were collected once, 24 hours after the end of 30th weeks, from all animal groups (control and experimental groups).
Blood Samples:
Blood samples for serum separation were collected by ocular vein puncture at the end of the experiment in dry, clean, and screw capped tubes and serum were separated by centrifugation at 2500 r.p.m for 15 minutes .The clean, clear serum was separated by automatic pipette and received in dry sterile samples tube and kept in a deep freeze at -20oC until used for subsequent biochemical analysis .All serum samples were analyzed for the following parameters:
Serum samples
a. Gamma Glutamyl Transferase (γ-GT).
b. Adenosine Deaminase (ADA).
c. Haptoglobin (HPT).
d. Carcinoembryonic antigen (CEA).
Tissue Samples (lung):
a) For biochemical analysis:
At the end of the experiment, mice of each group were sacrified by cervical decapitation. The abdomen was opened and the lung and liver specimen was quickly removed and opened gently using a scrapper, cleaned by rinsing with ice-cold isotonic saline to remove any blood cells and clots, then blotted between 2 filter papers and quickly stored in a deep freezer at (-20 °C) for subsequent biochemical analysis. All lung tissue samples were analyzed for determination of:
L-malondialdehyde (L-MDA).
Antioxidant enzymes: Catalase (CAT), Superoxide Dismutase (SOD), Glutathione reductase (GR), Glutathione Peroxidase (GPx) and Glutathione-s transferase (GST).
Non-enzymatic antioxidant: Reduced Glutathione (GSH).
Nitric Oxide (NO).
In addition to:
DNA fragmentation.
Caspase – 3 genes.
Cyclo- oxygenase -2 (COX-2).
B cell lymphoma 2 (Bcl-2).
Tumor suppressor protein (P53).
Cytochrome P1A1 (CYP1A1).
b) For Histopathological Examination:
Lung specimens of mice were carefully examined by naked eyes for detection of any abnormalities. Lung specimens were taken from different parts. The specimens were preserved in 10% neutral buffered formalin solution and subjected for histo-pathological examination according to the technique described by (Bancroft and Stevens, 1996) as follows:
The obtained results summarized the followings:
1) Nitric Oxide (NO) concentration:
A significant increase in the lung tissue NO concentration was observed in B(a)P induced lung cancer in mice when compared with control normal group.
Treatment with curcumin and TQ to lung cancer induced mice resulted in a significant decrease in the lung tissue No concentration. Also, curcumin and TQ pre-treatment with B(a)P administered mice induced a significant decrease in lung tissue No concentration when compared with B(a)P non treated group.
Administration of curcumin and TQ to normal mice resulted in a significant increase in the lung tissue concentration when compared with control normal group.
2 - Lung L- Malondialdehyde (L-MDA):
A significant increase in the lung tissue L-MDA concentration was observed in B(a)P induced lung cancer in mice when compared with control normal group.
Treatment with curcumin and TQ to lung cancer induced mice resulted in a significant decrease in the lung tissue L-MDA concentration. Also, curcumin and TQ pre-treatment with B(a)P administered mice induced a significant decrease in lung tissue L-MDA concentration when compared with B(a)P non treated group.
Administration of curcumin and TQ to normal mice resulted in a significant increase in the lung tissue L-MDA concentration when compared with control normal group.
3) Lung superoxide dismutase activity (SOD)
A significant decrease in lung SOD activity was observed in B(a)P induced lung cancer in mice when compared with normal control group.
Treatment with curcumin and TQ in lung cancer induced mice resulted in a significant increase in the lung tissue SOD activity. However, curcumin and TQ pre-treatment with B(a)P treated mice caused significant increase in the lung tissue SOD activity when compared with B(a)P non-treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant decrease in the lung tissue SOD activity when compared with control normal group.
4) Glutathione reductase (GR) :
A significant decrease in lung GR activity was observed in B(a)P induced lung cancer in mice when compared with normal control group.
Treatment with curcumin and TQ in lung cancer induced mice resulted in a significant increase in the lung tissue GR activity. However, curcumin and TQ pre-treatment with B(a)P treated mice caused significant increase in the lung tissue GR activity when compared with B(a)P non-treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant decrease in the lung tissue GR activity when compared with control normal group.
5) Lung catalase (CAT) activity :
A significant decrease in the lung tissue CAT activity were observed in B(a)Pinduced lung cancer in mice when compared with normal control group.
Treatment with curcumin and TQ in B(a)P -induced lung cancer in mice significantly decreased the lung tissue CAT activity. Also, curcumin and TQ pre-treatment with B(a)P administered mice showed significant decrease in the lung tissue CAT activity when compared with B(a)P non -treated group.
Oral administration of curcumin and TQ in normal mice resulted in a significant increase in the lung tissue CAT activity when compared with control normal group.
6) Lung glutathione peroxidase activity (GPx):
A significant decrease in the lung tissue GPx activity was observed in B(a)P induced lung cancer in mice when compared with normal control group.
Treatment with curcumin and TQ in lung cancer induced mice caused a significant increase in the lung tissue GPx activity. Also, curcumin and TQ pre-treatment with B(a)P treated mice caused a significant decrease in the lung tissue GPx activity when compared with B(a)P non -treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant increase in the lung tissue GPx activity when compared with control normal group.
7) glutathione-S-transferase activity (GST):
A significant decrease in the lung tissue GST activity was observed in B(a)P-induced lung cancer in mice when compared with normal control group.
Treatment with curcumin and TQ in lung cancer induced mice caused a significant increase in the lung tissue GST activity. Also, curcumin and TQ pre-treatment with B(a)P treated mice caused a significant decrease in the lung tissue GST activity when compared with B(a)P non -treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant increase in the lung tissue GST activity when compared with control normal group.
8) Lung reduced glutathione concentration (GSH):
A significant decrease in the lung tissue GSH concentration was observed in B(a)P induced lung cancer in mice when compared with normal control group.
Treatment with curcumin and TQ in lung cancer induced mice resulted in a significant increase in the lung tissue GSH concentration. However, curcumin and TQ Pre-treatment with B(a)P administered mice resulted in a significant increase in the lung tissue GSH concentration when compared with B(a)P non -treated group.
Curcumin and TQ oral administration in normal mice resulted in a significant decrease in the lung tissue GSH concentration when compared with control normal group.
9) Cyclo-oxygenase-2 (COX-2) in lung tissue:
A significant increase in tissue COX-2 activities was observed in [B(a)P] -induced lung cancer in mice when compared with control group.
Treatment with curcumin and TQ in [B(a)P] -induced lung cancer in mice exhibited significant decrease in tissue COX-2 activities when compared with [B(a)P] non-treated group. Also, curcumin and TQ pr-treatment with [B(a)P] administered mice exhibited a significant decrease in tissue COX-2 activities when compared with [B(a)P] non-treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant decrease in tissue COX-2 activities when compared with normal control group.
10) Tumor suppressor protein (P53):
A significant increase in tissue P53 concentration was observed in B(a)P induced lung cancer in mice when compared with control group.
Tre atment with curcumin and TQ in B(a)P induced lung cancer in mice exhibited significant decrease in tissue P53 concentration when compared with B(a)P non-treated group. Also, curcumin and TQ pr-treatment with B(a)P administered mice exhibited a significant decrease in tissue P53 concentration when compared with B(a)P non-treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant decrease in tissue P53 concentration when compared with normal control group.
11) Lung caspase-3 gene activity
A significant increase in the lung tissue caspase-3 gene activity was observed in B(a)P induced lung cancer in mice when compared with control normal group.
Treatment with curcumin and TQ in lung cancer induced mice resulted in a significant decrease in the lung tissue caspase-3 gene activity and when compared with B(a)P non-treated group.
Curcumin and TQ pre-treatment with B(a)P administered mice caused a significant decrease in the lung tissue caspase-3 gene activity when compared with B(a)P non-treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant increase in the lung tissue caspase-3 gene activity the lung tissue showed a significant increase when compared with control normal group.
12) B cell lymphoma 2 (BCL2)
A significant increase in the lung tissue BCL2 concentration were observed in [B(a)P] -induced lung cancer in mice when compared with normal control group.
Treatment with curcumin and TQ in lung cancer induced mice resulted in a significant decrease in the lung tissue BCL2 concentration. However, curcumin and TQ pre-treatment with [B(a)P] administered mice resulted in a significant decrease in the lung tissue BCL2 concentration when compared with [B(a)P] non -treated group.
Curcumin and TQ oral administration in normal mice resulted in a significant increase in the lung tissue BCL2 concentration when compared with control normal group.
13) Cytochrome P1A1 in lung tissue
A significant increase in the lung tissue CyP1A1 concentration was observed in B(a)P induced lung cancer in mice when compared with normal control group.
Treatment with curcumin and TQ in lung cancer induced mice resulted in a significant decrease in the lung tissue CyP1A1 concentration. However, curcumin and TQ pre-treatment with B(a)P administered mice resulted in a significant decrease in the lung tissue BCL2 and CyP1A1 concentration when compared with B(a)P non -treated group.
Curcumin and TQ oral administration in normal mice resulted in a significant increase in the lung tissue CyP1A1 concentration when compared with control normal group.
14) DNA fragmentation percent
A significant increase in the lung tissue DNA fragmentation percent was observed in B(a)P induced lung cancer in mice when compared with control normal group.
Treatment with curcumin and TQ in lung cancer induced mice resulted in a significant decrease in the lung tissue DNA fragmentation percent when compared with B(a)P non-treated group.
Curcumin and TQ pre-treatment with B(a)P administered mice caused a significant decrease in the lung tissue with a significant decrease in the lung tissue DNA fragmentation percent when compared with B(a)P non-treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant increase in the lung tissue DNA fragmentation percent in the lung tissue showed a significant increase when compared with control normal group.
• Serum parameter
15- Serum Haptoglobin (HPT) concentration
A significant increase in serum HPT concentration was observed in B(a)P induced lung cancer in mice when compared with control normal group.
Treatment with curcumin and TQ in B(a)P induced lung cancer in mice resulted in significant decrease in serum HPT concentration when compared with [B(a)P] non-treated group. Also, curcumin and TQ Pre-treatment with B(a)P administered mice exhibited a significant decrease in serum HPT concentration when compared with B(a)P non-treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant increase in serum HPT concentration when compared with control normal group.
16) Serum Carcinogenic embryonic antigenic (CEA) concentration:
A significant increase in serum CEA concentration was observed in B(a)P induced lung cancer in mice when compared with control normal group.
Treatment with curcumin and TQ in B(a)P induced lung cancer in mice resulted in significant decrease in serum CEA concentration when compared with B(a)P non-treated group. Also, curcumin and TQ Pre-treatment with B(a)P administered mice exhibited a significant decrease in serum CEA concentration when compared with B(a)P non-treated group.
Oral administration of curcumin and TQ to normal mice resulted in a significant increase in serum CEA concentration when compared with control normal group.
17- Serum gamma-glutamyl transferase (GGT) activity
A significant increase in serum GGT activity was observed in B(a)P induced lung cancer in mice when compared with control normal group.
Treatment with curcumin and TQ in B(a)P induced lung cancer in mice resulted significant decrease in serum GGT activity when compared with B(a)P non-treated group. Also, curcumin and TQ pre-treatment with B(a)P injected mice induced significant decrease in serum GGT activity when compared with B(a)P non-treated group.
Oral administration of curcumin and TQ in normal mice resulted in a significant increase in serum GGT activities when compared with control normal group.
18 - Serum adenosine deaminase (ADA) activity
A significant increase in serum ADA activity was observed in B(a)P induced lung cancer in mice when compared with control normal group.
Treatment with curcumin and TQ in B(a)P induced lung cancer in mice resulted significant decrease in serum ADA activity when compared with B(a)P non-treated group. Also, curcumin and TQ pre-treatment with B(a)P injected mice induced significant decrease in serum ADA activity when compared with B(a)P non-treated group.
Oral administration of curcumin and TQ in normal mice resulted in a significant increase in serum ADA activities when compared with control normal group.
HISTOPATHOLOGICAL RESULTS
Group (1): control normal group
In this group the microscopical examination of the lung tissue revealed normal histological structure
Group (2): B(a)P induced lung cancer group
The lung of mice in this group is showing severe congestion and dilation of the pulmonary blood vessels and inters alveolar blood capillaries. Moreover, the bronchioles showing severe degree of hyperplasia with desquamation of epithelial cell lining. The Pei bronchial tissue showing severe degree of mononuclear leukocytic infiltration .the pulmonary blood vessels showing proliferation of the endothelial cells lining with infiltration of the blood vessels wall by neoplastic malignant cells. The neoplastic cell showing enlarged vesicular and hyper chromatic nuclei. The most common findings in this group represented by formation of well develop neoplasm either in the form of adenoma or adeno-carcinoma that arise mainly from the epithelial cell lining of the alveolar wall.
The normal lung tissues showing the presence of foci of adenoma. The neoplastic areas showing complete loss of the alveolar structure and its replacement by glandular papillary or cell mass of hyper-chromatic or vesicular nuclei. Adeno-carcinoma with multiple areas of dysplasia was seen among the neoplastic mass. Occasionally the neoaplastic cells with hyper-chromatic nuclei were seen in the alveolar septa.
Group (3): [B(a)P] +Curcumin treated group:
The lung of mice in this group showing severe congestion of the pulmonary blood vessels and inters alveolar blood capillaries. Moreover, focal neoplastic foci represented by aggregations of larg sized vesicular and hyper-chromatic nuclei were seen. The bronchioles showing severe degree of hyperplasia with severe proliferation of mononuclear leukocytic aggregations in the perbronchial area with formation of neoplastic cell beside the bronchiolar wall. Moreover, large neoplastic cells which showing hyper-chromatic vesicular nuclei were seen embedded in the pulmonary tissue.
Group (4):[B(a)P] +Curcumin protected group
The lung of mice of this group showing proliferation of the epithelial cell lining the alveolar septa but without formation either of complete or well developed neoplasm or aggregation of clear neoplastic mass as mentioned in the previous groups. The alveoli showing proliferation of the epithelial cell lining with severe leukocytic infiltration forming collapsed areas. Multiple areas of emphysema were seen beside these areas. Moreover, the blood vessels showing severe congestion with mildest proliferation of some neoplastic cells beside blood vessels wall. The bronchial epithelium showing mild degree of hyperplasia with proliferation of some areas and dysplasia in the pei- bronchial tissue. Moreover, the pulmonary blood vessels showing severe injury of lining epithelium with mild proliferation of some neoplastic cells.
Group (5): [B(a)P]+TQ treated group
The lung of mice in this group showing hyperplasia of the bronchiolar epithelium with severe congestion of pulmonary blood vessels. Moreover, the most of pulmonary blood vessels showing injury of the intema of blood vessels with thrombus formation. The alveoli showing degeneration and necrotic changes in the alveolar septa. The alveolar septa showing neoplastic cells with hyper chromatic and vesicular nuclei, neoplastic cell with multiple area of xemphysema and atelectasis and were seen scattered all over the pulmonary tissue.
Group (6):[B(a)P] + TQ protected group
The lung of mice of this group showing hyperplasia of the bronchiolar epithelium only with neoplastic growth. Moreover, multiple focal mononuclear aggregations were seen scattered all over the pulmonary tissue. The pulmonary blood vessels showing degenerative changes in lining endothelial cells with mild perivescular mononuclear cellular infiltration.