INTRODUCTION 

Chlorpyrifos (O, O-diethyl-O-(3, 5, 6-trichloro-2-pyridyl) -phosphorothioate) is a broad-spectrum organophosphate insecticide which is widely used in agriculture and in domestic use against harmful insects (Saulsbury et al., 2009). Liver is the organ where activation and detoxification of CPF takes place, while it is eliminated primarily through the kidney (Betancourt and Carr, 2004). Besides being potent anticholinesterase compound, CPF elicits a number of additional effects, including hepatic dysfunction, heamatological and immunological abnormalities, embryotoxicity, genotoxicity, neurotoxicity and neurobehavioral changes (Mehta et al., 2009). Many insecticides are hydrophobic molecules that bind extensively to biological membranes, especially phospholipids bilayers (Ogutcu et al., 2008), and they may damage membranes by inducing lipid peroxidation (LPO) (Kalender et al., 2010). Wheat germ is the component of wheat kernel with the highest nutritional value (Zhu et al., 2006). The germ makes up only 2%-3% of the wheat kernel and is the most nutritious part of the wheat kernel. Nutrients are concentrated in the germ, and it is rich in vitamins, minerals, protein, and fats. Wheat germ contains high levels of tocopherol and B vitamins. It is separated from the other wheat components by the milling process. In addition to the nutrients listed above, wheat germ can be subjected to fermentation with Saccharomyces cervisiae to yield the benzoquinones 2, 6-dimethoxybenzoquinone (DMBQ) and 2-methoxybenzoquinone. These benzoquinones are present in unfermented wheat germ as glycosides; yeast glycosides activity present during fermentation leads to release of the benzoquinions as a glycones.

 The wheat germ fermentation end-product, which is suitable for human consumption, is a dried extract standardized to contain methoxy-substituted benzoquniones (2- methoxy-benzoquinione and 2, 6-DMBQ) at a concentration of 0.04% (Philipp et al., 2007). The fermentation process of wheat germ increases the nutritional and therapeutic properties of wheat germ. In the case of FWGE fermentation produces biologically active quinones, which may be active principles. Quinones are carbonyl group molecules with a wide range of biological activity. One of their properties is to be able to attract and accumulate electrons in the carbon-hydrogen double bond. Benzo- and hydroquinones have antimicrobial activity. All of these compounds may modulate the immune system. The mechanism they share in common is suggested to be a free radical scavenging capability (Kenner, 2009). The present study aimed to investigate the biochemical effects of low and high doses of chlorpyrifos in experimental animals and the effect of FWGE supplementation in alleviation of the toxic effects of chlorpyrifos in rats

 MATERIALS AND METHODS

 Experimental animals: The healthy experimental animals used through-out the present work were 50 adult male albino Sprague-Dawely strains mean weight varied between 98g to 117g. They were obtained from El-Salam-Farm, Giza, Egypt. The animals were divided into 5 homogenous groups and housed individually in plastic cages fitted with a wire mesh bottoms and fronts in a room maintained at 25-30 °C with about 50% relative humidity. The room was lighted on a daily photo period of 12/12 hrs light / dark cycle. Then, they were allocated to the various experimental diets for 30 days. Experimental Design: 

The animals were divided into nine groups of rats (eight for each group). All rats offered a balanced diet prepared according to Reeves et al., (1993) for seven days for adaptation on the environmental conditions before starting the experiment. The experimental groups were fed on the balanced diet as control as well as the other tested groups fed on contaminated balanced diets with chlorpyrifos (CPF) at the two levels (25 and 50 mg/kg diet) or supplemented with treatment doses of FWGE and plus the different doses of CPF, as follows: Group (1) received normal balanced diet (control). Group (2) received normal balanced diet plus low dose (25 mg/kg diet) of CPF. Group (3) received normal balanced diet plus high dose (50 mg/kg diet) of CPF. Group (4) received normal balanced diet plus of CPF, supplemented with FWGE (3 g/kg diet). Group (5) received normal balanced diet plus high dose of CPF, supplemented with FWGE (3 g/kg diet). During the conditioning period and through out the experiment, food and water were provided ad libitum. At the end of the experimental period, the animals were fasted for 12hrs, and then anesthetized under diethyl ether and whole blood samples were taken from hepatic portal vein in three centrifuge tubes. The first tube contained ethylene diamine tetraacetic acid (EDTA) was used for haematological analysis. The second tube contained heparin then centrifuged for 10 minutes at 4000 rpm and plasma kept in plastic vials at -20 °C till used for the biochemical analysis. The third tube were left for 15 minutes at 37°C then centrifuged at 4000 rpm for 20 minutes for separating serum, and then serum were removed and kept in plastic vials at – 20 °C until analysis. 

Liver and kidney were separated, rinsed and washed by saline solution (NaCl 0.9%), then blotted on filter paper, weighed and calculated their relative weights. The livers were stored in 10% formalin saline 50% v/v for 24 hrs until microscopical examination is done. Bioche mical meas ure me nts: 1- Heamatological measurements of cellular fractions such as red blood cells (RBC'S), white blood cells (WBC'S), and platelets counts as well as erythrocyte indices, the constants depend on either erythrocyte size or hemoglobin content also determinations of blood hemoglobin and hematocrit levels. 2- Evaluation of some organ functions such as liver by determining some enzyme activities such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (γGT), as well as total proteins, albumin, globulins contents; A/G ratio and kidney function as measurements of creatinine and urea levels in serum. 3- Evaluation of some lipids peroxidation product as malondialdehyde and some antioxidant enzymes as superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST). 4- Determination of lipid profile in serum, as total lipids, total cholesterol, triacylglycerols, in addition to high density lipoprotein-cholesterol (HDLC), low density lipoprotein-cholesterol (LDL-C), very low density lipoproteincholesterol (VLDL-C) and LDL/HDL ratio. 5- Microscopical investigation will be carried out for normal and different experimental groups. Liver were dissected out and fixed instantaneously in 10% formalin saline 50% v/v for 24 hrs.

 The specimens were washed in tap water, dehydrated in ascending grades of ethanol, cleared in xylene, embedded in paraffin wax at melting point 55-60 °C. Sections of 6m thickness were prepared and stained with haematoxylin and eosin Harris (1990). Statistical analysis:- Statistical analysis was done by using SPSS 11.5 statistical software completely randomization design in factorial arrangement (ANOVA; F-test) and one way classification to determine least significant difference (L.S.D) (Dawson and Trapp, 2004). RESULTS 1. The effects of different experimental tested diets on red blood cells (RBC's), white blood cells (WBC's), platelets counts and hemoglobin, (Hb) hematocrit (Hct) values: Table (1) shows significant decreases between values of RBC's, WBC's, platelets counts and Hb and Hct values of control group and values of untreated groups G2 and G3. The increment 2.The effects of different experimental tested diets on mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC): From the results in table (2) the indices levels of MCV and MCHC, show significant difference in case of rats fed on low and high doses of CPF and the percent of change from control group levels was 3.16% and -3.65% for those fed on low of RBC's in treated groups fed on low CPF were 46.93% for G4 when compared to G2 and also 89.23%, for treated group G5 when compared to G3.

 The results of WBC's, Hb, Hct and platelets showed an increment in G4 and G5 when compared with G2 and G3, respectively. The FWGE enriched diet caused successful treatment for the defect that caused by CPF in RBC's, WBC's, Hb, Hct and platelets values.dose of CPF but it was 12.82% and -5.92% for those fed on high dose of CPF. There was a significant difference between the MCH of rats fed on low CPF and G4, while MCHC and MCV reveal no significant difference. The group fed on high CPF illustrates a significant difference in respect to G5 only for MCHC while MCH and MCV show insignificant difference.3.The effects of different experimental tested diets on serum alanine amino transferase (ALT), aspartate amino transferase (AST) and alkaline phosphatase (ALP) and gamma glutamyle transferase (γGT) activities: The results of table (3) revealed that CPF intake in untreated groups induced liver injury which is reflected by the significant increase in all hepatic serum enzyme activities than control and treated groups. There is a noticeable improvement in all studied enzyme activities by FWGE treatment as compared with G2 and G3. 4. The effects of different experimental tested diets on serum total proteins, Albumin, globulins level and A/G ratio: The results of table (4) demonstrated that, the administration of CPF either at low or high dose significantly decreased the levels of total protein, albumin and globulins. The improvement in serum total proteins and albumin levels was 5. The effects of different experimental tested diets on serum creatinine and urea levels: The results demonstrated that administration of CPF at low dose cause significantly increase in creatinine and urea levels and highly increased in case of administration of high dose of CPF.

 The results demonstrated that administration of FWGE (G4) to diet plus low dose CPF found in groups that received low dose of CPF plus FWGE diet (G4), then (G5) that administered high CPF plus FWGE diet when compared to control. The improvement was happened in globulins levels in all treated rats. Concerning A/G ratio, the effect of FWGE diet in reducing the impact of CPF especially at low doses was higher than that of the G5.caused a significant decrease in creatinine level by -38.99% when compared to G2. While administration of FWGE with diet plus high dose CPF caused a decrease in creatinine level (G5) by -51.62% when compared to high dose only of CPF (G3). Urea levels were improved in groups fed on FWGE when compared with untreated groups.6. The effects of different experimental tested diets on serum malondialdhyde (MDA) level and erythrocyte superoxide dismutase (SOD), plasma catalase (CAT), glutathione –S-transfrase (GST) activities: From the results in table (6), the level of MDA, SOD, catalase and GST were statistically significant differences in groups that received low and high dose of CPF alone when compared with control From the results of treated groups, there are some improvements in all enzymes exhibited in treated groups when compared with groups fed on CPF only. There were no significant difference between G4 (low CPF plus FWGE ) and control in MDA, SOD, Cat and GST activities by percent of change from control 8.27%, -5.54%, - 16.00% and -2.09% respectively. 

7.The effects of different experimental tested diets on serum total lipids, total cholesterol, triacylglycerols: The results presented in table (7) showed that the presence of CPF in diets either at low or high dose induce significant 8.The effects of different experimental tested diets on serum (HDL-C), (LDL-C), (VLDLC) and LDL-C/HDL-C ratio: From the results shown in table (8) it was clear that there were significant differences increase in the level of serum total lipids, total cholesterol and TG. From the results of treated groups, there are a noticeable improvements in all levels exhibited in treated groups by FWGE when compared with groups fed on CPF only. between G2, G3 and control by increasing the level of LDL-C, VLDL-C, and LDL/HDL ratio. FWGE supplementation reduced the serum levels of LDL-C, VLDL-C, and LDL-C/HDL-C ratio and increased serum level of HDL-C.DISCUSSION Chlorpyrifos (CPF), an organophosphorus insecticide is known to cause oxidative stress in different human and animal cells (Jett and Navoa, 2000). Chlorpyrifos is a lipophilic molecule which can easily pass through the cell membrane into the cytoplasm. Once inside the cell, CPF can generate a lot of damages. For these reasons, it is necessary to find solutions against this danger. Within this context, nature can provide us many substances that can attenuate this oxidative stress (Gupta, 2006). This study demonstrated that, CPF caused decrease in RBC count, Hb and Hct levels, which might be due to the effect of pesticide on blood forming organs suggesting the anaemic condition of the treated animals. 

The anemia may be due to the inhibition of erythropoiesis and hemosynthesis and to an increase in the rate of erythrocytes destruction in hemopoietic organs, as well as the leucocytosis observed in present study indicates an immune system to protect the rats against infection that might have been caused by chemical and also secondary infections, which may be contracted after the weakening condition of the rats. Leucocytosis, which may be directly proportional to the severity of the causative stress condition, may be attributed to an increase in leukocyte mobilization. The present treatment normalized the levels of blood cell counts, and other indices. The protective effects of FWGE are most likely due to its antioxidant potential. The present results are in harmony with the results of Akhtar et al. (2009) who reported that, the Hb and Hct levels were significantly decreased in animals exposed to CPF at 3, 6 and 9 mg/kg/d doses. Also Janeway, (2005) explained the reduction in the number of lymphocytes may be due to decreased production or rapid removal from circulation and subsequent destructtion. The reduction of lymphocytes is indicative of immunosuppressive effects of organophosphates which may require other studies to assess the levels of immunoglobulins. Moreover, the results obtained by Goel et al. (2006) confirmed that, decrease in leukocyte counts following intoxication with CPF could be attributed either to the slower rate of production of leukocytes or due to their inhibited release into the blood circulation. 

A significant decrease in the total bone marrow cell count was indicated. The results of Kazmi et al. (2003) showed the effect of sublethal dosage (120 mg/kg bw/day) of the organophosphate pesticide, CPF on the blood of adult male albino Sprague-Dawley rat under short-term conditions. CPF had no effect on hemoglobin. Red blood and white blood cell counts and packed cell volume showed a significant but transient decline. The mean corpuscular volume and mean corpuscular hemoglobin also increased transiently. Mean corpuscular hemoglobin concentrations showed persistent increase. It has been shown by Philipp et al. (2007) that FWGE , a fermented wheat germ extract standardized to methoxy-substituted benzoquinones, possesses cancer-fighting characteristics. Taken orally, FWGE can inhibit metastatic tumor also, FWGE was demonstrated to induce apoptosis in pancreatic carcinoma cells, T and B lymphocytic tumor cell lines, leukemia, melanoma, breast cancer and gastric carcinoma cells in vitro.