Changes in some blood parameters in lactating female rats and their pups exposed to lead : effects of vitamins C and

The present work was designed to examine the changes in some blood parameters in lactating rats treated with lead acetate (10 mg /kg B.W. orally) and its interaction with vitamin E (600 mg/kg diet) or vitamin C (100 mg/kg B.W. orally) during lactation period (20 days) and their pups. Administration of lead acetate to the female lactating rats caused a significant decrease in packed cell volume (PCV), hemoglobin concentration (Hb), red blood cell count (RBC), body weight, mean corpuscular heamoglobin concentration (MCHC) whereas the white blood cells count (WBC), total proteins, the percentage of monocyte and mean corpuscular volume(MCV) significantly increased administration of lead acetate to female lactating rats produced a significant decrease in PCV, Hb, RBC, MCHC, body weight, and the percentage of the neotrophils in their pups. But the WBC count, total proteins, the percentage of lymphocyte, monocyte, MCV a significantly increased in their pups. Treatment dams with vitamin E concomitantly with lead acetate increased the PCV, Hb, MCHC, whereas percentage of monocyte significantly decreased, PCV, Hb, RBC, the percentage of neutrophils a significantly increased, whereas WBC count, the percentage of lymphocyte decreased significantly in their pups of this group of dams. Treatment dams with vitamin C concomitantly with lead acetate significantly increased the PCV, MCV, whereas percentage of monocytes significantly decreased, but Hb, PCV and RBC significantly increased in their pups. It could be concluded that treatment female lactating rats with vitamin E or C concomitantly with lead acetate exert an antioxidant effect on blood constituent in dams and their pups and vitamin E more effective than vitamin C.


Introduction
Lead is non-essential heavy metal widely distributed in the environment, chronic exposure to low levels of this agent is one of the problems of public health, due to its toxicity, Source of lead exposure may include air, water, food, soil (1), breast milk can however be pathway of maternal excretion of lead and this toxin impact most severely on the newborn (2,3).Lead produce toxicological and pathological effects on central nervous system, peripheral nerves, kidneys and hematopoitic system (1), as well as infertility (4).Lead is known to induce oxidative stress leading to tissue damage (5), and lipid peroxidation by inhibiting the synthesis of some antioxidant enzymes such as phospholipids hydroperoxidase glutathione peroxidase (PHGPX) (6).
It has long been known that heamatopoiesis and heme synthesis affected by lead poisoning (1).
Lead can cause damage in the erythrocyte, originating defective cells, preventing them from carrying oxygen also it produce high blood pressure that increases the risk of heart attack (7).
An increase in oxidative damage to body cells and tissues is balanced by powerful enzymatic and non enzymatic antioxidant defense system that scavenge and suppress the formation of reactive oxygen species (ROS) and protect cells from lipid peroxide -mediated damage (8) e.g.(Carotinoids, vitamin E & C, Flavonoids, GSH-Peroxidase, Soperoxide -dismutase, Catalase) (9).Vitamins C&E are great powerful antioxidants, protect cells from oxidation, vitamin C is biological reducing agent, vitamin E act by giving up of its electrons to the electron deficient free radicals making it more stable (10).Both vitamins C & E helping to prevent degenerative disease such as cardiovascular diseases (10).

Materials and methods
Twenty female albino rats (dams) obtained from the animal house of Veterinary Medical College, University of Mosul, at (3-4) months of age, they were housed in polypropylene cages under controlled conditions of temperature (18)(19)(20) c° and lighting (10 hours light /14 hours dark).The dams were supplied a standard pellet diet and tap water ad libitum.
The dams and their pups were randomly divided at the first day of parturition in to four groups (5 dams /group).The first group received tap water (1ml/kg BW) during lactation period(20 days) serve as control.The second group received lead acetate (10 mg /kg B.W. orally) during lactation period (20 days), lead acetate dissolved in distalled water and given at volume of 1ml/kg (4).The third group received lead acetate (10 mg /kg B.W) concomitantly with feeding of vitamin E (600 mg / kg diet) during lactation period (11).The fourth group received lead acetate (10 mg /kg B.W. orally) concomitantly with vitamin C (100 mg /kg B.W.orally) during lactation period (12).
At the end of the experiment (21day) body weights of dams and their pups were recorded, blood samples collected from dams and their pups from the eye vein and placed in tubes which contain anticoagulant (EDTA) for complete blood picture.Also blood samples were collected into clean dry centrifuge tubes allowed to clot, serum separated after centrifugation at 1500 rpm for 15 minute.Hemoglobin concentration was determined by Sali method (13).Red and White blood cells count were counted per cubic millimeter of blood by Hemocytometer (13).Packed cell volume determined by microhematocrite capillary tube and Microhematocrite centrifuge (12000 circle / minute) for 10 minutes (13).Differential Leucocytes count (DLC) Thin blood film was prepared and stained with Gimza (13).Mean corpuscular volume (MCV), mean corpuscular Hemoglobin (MCH) and mean corpuscular Hemoglobin concentration (MCHC) were calculated (13).Determination of total protein by using Biuret method (14).

Statistical analysis
Our data were analyzed statistically using one -way analysis of variance.Group differences were determined using Duncan multiple range test.Statistical significance was considered at (P<0.05) (15).

Results
Administration of dams with lead acetate during the lactation period (20days) produced a significant decrease (P<0.05) in body weight, packed cells volume, hemoglobin concentration and red blood cells count as compared with the control group (Table 1), whereas a significant increase (P<0.05) in the white blood cells count and total proteins compared with control group.Treatment dams with vitamin E or C concomitantly with lead acetate during lactation period caused a significant increase (P<0.05) in packed cell volume compared with lead acetate group, and closed to control value group.Treatment with vitamin E or C did not affect significantly in body weight, red blood cells count, white blood cells count and total proteins compared with lead acetate group.Treatment with vitamin E and lead acetate caused a significant increase (P<0.05) in the hemoglobin concentration.But vitamin C did not affect significantly in hemoglobin concentration compared with group that treated with lead acetate alone.
Table (2) shows a significant increase (P<0.05) in monocytes percentage in lead acetate group compared with control group.No significant differences occurred between groups in the percentage of neutrophiles, lymphocytes, esinophiles and basophiles.
Treatment of dams with lead acetate caused a significant increase (P<0.05) in the mean corpuscular volume (MCV) and significant decrease (P<0.05) in mean corpuscular heamoglobin concentration (MCHC) (Table 3).Treatment with vitamin E and lead acetate produced a significant increase (P<0.05) in the mean corpuscular heamoglobin concentration (MCHC) as compared with lead acetate group.Treatment with vitamin C produced significant increase (P<0.05) in MCV as compared with lead acetate group.
Table (1) Body weight, packed cell volume, hemoglobin concentration, red blood cell count, white blood cell count and total proteins in lactating female rats treated with lead acetate and its interaction with vitamin E and C. Treatment of dams with lead acetate during the lactation period produced a significant decrease (P<0.05) in body weight, packed cell volume, hemoglobin concentration and red blood cell count accompanied with a significant increase in white blood cell count and total proteins in their pups compared with pups of control group (Table 4).Treatment of dams with vitamins E or C produced a significant increase (P<0.05) in the packed cell volume, hemoglobin concentration and red blood cell count of their pups as compared with the pups of the lead acetate group.Treatment of dams with vitamin E concomitantly with lead acetate produced a significant decrease (P<0.05) in white blood cell count of their pups.
Administration of dams lead acetate produced significant decrease (P<0.05) in the percentage of neutrophils accompanied with a significant increase (P<0.05) in the percentage of lymphocytes and monocytes in their pups compared with pups of control group (Table 5).Treatment the dams with vitamin E and lead acetate concomitantly produced significant increase (P<0.05) in the percentage of neutrophils, with significant decrease in the percentage of lymphocyte in their pups compared with lead acetate group.
Table (4) The body weight, packed cell volume, hemoglobin concentration, red blood cell count, white blood cell count and total proteins in pups from dams treated with lead acetate and its interaction with vitamin E or C. Mean corpuscular heamoglobin concentration decreased significantly (P<0.05)(Table 6), whereas the mean corpuscular volume significantly increased (P<0.05) in pups from lead acetate treated dams as compared with control group.

Discussion
The results of the present study demonstrated that lead acetate administration to female lactating rats during result in significant decrease in body weight, PCV, Hb, RBC count, MCHC and a significant increase in WBC count, MCV, total proteins in the dams and their pups compared with control group.This finding therefore corroborates similar findings reported by Teijon et al (16), Mugahi et al (17) and Khan et al (18) in mice, whom observed a significant decrease in body weights, erythrocytes number, hemoglobin concentration in rats that injected with lead as acetate acetic acid.This decrease in the body weights can be due to gradual toxicity that lead causes in the animals (16).Lead acetate is a toxic metal known to exert oxidative stress on multiple organs (19), it can be transferred from the maternal to the infant through milk when there is maternal exposure to lead (3).Anemia is one of the early manifestations of lead poisoning, it result from reduction of the life span of circulating erythrocyte as well as by inhibition the body's ability to make heamoglobin by interfering with several enzymatic steps in hem pathway (1,16).Ferrochelatase, which catalyze the insertion of iron into protoporphyrin IX, is quite sensitive to lead (1).Erythrocyte Na-K-ATPase is some what inhibited by lead suggesting a loss of cell membrane integrity this may account for the shortened lifespan of erythrocytes (1), also lead can cause damage in the erythrocytes originating defective cells that are eliminated by spleen and their hemolysis (16).
Table (5) The percentage of neutrophils, lymphocytes, eosinophils, basophiles and monocytes in pups from dams treated with lead acetate and its interaction with vitamin E or C. In the present study the increase in the mean corpuscular volume and the decrease in the mean corpuscular hemoglobin concentration of dams that treated with lead acetate and their pups may result from the toxic effect of lead acetate that affect on red blood cells count and heamoglobin concentration, because the validity of these indexes is influenced by the value of red cell count, hemoglobin concentration and packed cell volume (13).

Monocytes
In the present study oral administration of lead acetate caused significant increase in the white blood cell count and monocytes percentage in the dams and their pups, on the other hand treatment of dams with lead acetate produced significant increase in the lymphocyte percentage and significant decrease in the neutrophils percentage in their pups.Opposite results has been reported by other investigators (16), suggested no changes in the white blood cell count in rats treated with lead.Gheng and others (20) Have reported that lead increases lipopolysacharide (LPS), lead to liver injury and over expression of monocytes and macrophages, lipopolysacharide causes liver injury at high doses but non injurious inflammation at low doses, this may result in the increase of white blood cell count.These different findings reported in the peripheral blood cell is probably result from effect of lead on progenitor cells (20).
Concomitant administration of vitamin E and lead acetate during the lactation produced significant increase in the packed cell volume, heamoglobin concentration, mean corpuscular heamoglobin concentration, and significant decrease in the monocytes percentage of dams.In addition treatment of the female rats with vitamin E and lead acetate produced significant increase in the packed cell volume, heamoglobin concentration, red blood cell count, neutrophils percentage, and significant decrease in white blood cell count, lymphocyte percentage of their pups.Similar results has been reported by (21), suggested that administration of vitamin E to dams exposed to oxidative stress by cadmium chloride during lactation decreased the adverse effects produced by cadmium chloride on blood parameters in weaned pups.The main function of vitamin E is as a chain -breaking free radical trapping antioxidant in cell membranes and plasma lipoproteins, it react with lipid peroxide radicals formed by peroxidation of polyunsaturated fatty acids before they can establish chain reactions (10), vitamin E play a role in the protecting tissue from oxidative damage especially at the early stages of life, it transported to the infant during the lactation and pregnancy (22).Murray and others (10) reported that deficiency of vitamin E produce anemia in the infant by hemolysis of red blood cells and decrease in the heamoglobin production.Vitamin E deficiency enhances the susceptibilities of animals to hemolytic effect of lead poisoning (23), vitamin E useful in order to protect membrane-lipids and to prevent protein oxidation produced by lead intoxication (24).
On the other hand vitamin C given with lead acetate in the group 4 produced significant increase in the packed cell volume in the dams, in the pups vitamin C treatment result in significant increase in the packed cell volume, heamoglobin concentration, red blood cell count.Recent studies by (21) has shown that treatment of dams with vitamin C and cadmium chloride during the lactation period produced significant increase in heamoglobin concentration, packed cell volume in the dams and their pups.Ascorbic acid is great antioxidant and helps in the protecting the body against pollutants (25).The effects of vitamin C on blood parameters and anemia may result by increasing the iron absorption in gastrointestinal tract (GIT) and enhances iron bioavailability (26).Wang and others (27) reported that vitamin C is effective in reversing reactive oxygen species (ROS) induced mouse embryo toxicity.As an antioxidant vitamin C protect folate, vitamin E and polyunsaturated substance from destruction by oxygen as they move throughout the body (28).On the other hand vitamin C did not reverse some of blood parameters like white blood cell count, total protein, MCV, MCHC in the dams and pups this may result from the dose of vitamin C, rout of administration, duration of treatment, individual variations between the animals.

Table ( 3
) Mean corpuscular volume, mean corpuscular heamoglobin and mean corpuscular hemoglobin concentration in lactating female rats treated with lead acetate and its interaction with vitamin E or C. Values were expressed as means ± S. E., Values with different letters in column are significantly different (P<0.05),Number of animals 5 female rats / group.
(2)d acetate (10mg/kgB.W.orally) +Vitamin C (100mg/kg B.W. orally) Values were expressed as means ± S. E., Values with different letters in column are significantly different (P<0.05),Number of animals 5 female rats / group.Table(2)The percentage of neutrophils, lymphocytes, eosinophils, basophiles and monocytes in lactating female rats treated with lead acetate and its interaction with vitamin E or C.

Table ( 6
Values were expressed as means ± S. E., Values with different letters in column are significantly different (P<0.05),Number of animals 22 pups / group.) The mean corpuscular volume, mean corpuscular hemoglobin\ and mean corpuscular heamoglobin concentration in pups from dams treated with lead acetate and interaction with vitamin E or C.