Role of black seeds ( Nigella sativa ) in ameliorating carbon tetrachloride induced haematotoxicity in Swiss albino mice

1 Department of Zoology, Faculty of Science, Alexandria University, Alexandria, Egypt. 2 Department of Biology, Faculty of Applied Sciences, Um Al Qura University, Mecca, Saudi Arabia. 3 Department of Biological Sciences, College of Science, King Faisal University, Al-Hassa, Saudi Arabia. 4 Genetics Branch, Department of Botany, Faculty of Agriculture, Saba Basha, Alexandria University, Alexandria, Egypt.


INTRODUCTION
Carbon tetrachloride (CCl 4 ) is a haloalkane used in a variety of industrial and chemical applications.It has been widely used for its solvent properties, particularly in refrigerator fluids, as a propellant for aerosol cans, as a dry-cleaning agent in industry, as a household spot remover, as grain fumigant and as intermediate in the synthesis of chlorofluorocarbons.As a result of its widespread use, CCl 4 is a common contaminant of ground and surface waters where it persists for years.Therefore CCl 4 is now of greatest concern as an environmental contaminant (ATSDR, 1994;Guo et al., 2000).Nigella sativa L. is a plant of Ranunculaceae family that grows spontaneously and widely in several southern Mediterranean and Middle Eastern countries (Tariq, 2008).N. sativa seed has over 100 different chemical constituents, including abundant sources of all the essential fatty acids.Although it is the oil that is most often used medicinally, the seeds are a bit spicy and are *Corresponding author.E mail: amina_essawy@yahoo.com.often used whole in cooking curries, pastries and Mediterranean cheeses (Tariq, 2008).
The seeds of N. sativa, also known as black seed or black cumin, are often used as a spice but are also used extensively in the traditional medicine of many countries (Meddah et al., 2009).N. sativa has been used traditionally for the treatment of many diseases owing to the reported antiviral (Salem and Hossain, 2000), antibacterial (Kanter et al., 2003), anti-inflammatory (Hajhashemi et al., 2004), antidiabetic (Kanter et al., 2004), immunomodulatory (Tekeoglu et al., 2007), antischistosomiasis (El Shenawy et al., 2008), and hepatoprotective (El-Gharieb et al., 2010) activities.Furthermore, it was found that N. sativa extract has antitumor properties (Essawy et al., 1997;Worthen et al., 1998;Khan et al., 2003;Hussein et al., 2005;Mbarek et al., 2007), attenuates toxic side effects caused by several chemotherapeutic agents (Uz et al., 2008) and protects against gentamicin-induced nephrotoxicity (Yaman and Balikci, 2010).In addition, it prevents hippocampal neurodegeneration after chronic toluene exposure in rats (Kanter, 2008).From the experimental and clinical studies performed on N. sativa, it seems that most of its pharmacological actions are due to its antioxidant activity which is mainly due to its ability to scavenge free radicals and/or inhibit lipid peroxidation (Gupta et al., 2004).In spite of these observations, the protection offered by black seed or its components against toxicity in haematopoietic organs remains incomplete and needs further work to be elucidated.Thus, the objective of this study was designed to determine whether the administration of N. sativa into animals intoxicated with CCl 4 would improve the possible induced cellular damage in peripheral blood cells of Swiss albino mice.

Experimental animals
Ten weeks old laboratory male Swiss albino mice weighing about 25 g each, were obtained from breeding colony at University of Tanta, Egypt.Animals were housed in plastic cages in an animal room under controlled temperature (23±2°C), and 12 h photoperiod (12 h light/dark cycle), with a light from 0600 to 1800 h and darkness from 1800 to 0600 h.They were given free access to a commercial pellet diet and tap water, and allowed to acclimatize for two weeks before treatment.

Chemicals used
CCl4 (98.8% purity) was purchased from El-Nasr Pharmaceutical Chemical Company (Egypt).N. sativa seeds (black seed) were purchased from a local herb grocery (Egypt).Seeds were cleaned, air-dried and were then powdered mechanically to prepare a suspension in isotonic saline solution.The suspension (1.25 g powder of N. sativa + 100 ml isotonic saline) was freshly prepared and left a few minutes before administration.Olive oil (Laboratory grade) was obtained from Sigma Chemical Co.(St.Louis, MO).It had been used as a vehicle for carbon tetrachloride.

Experimental design
The animals were randomly divided into five experimental groups of 40 mice each: Group 1: Each animal had orally received 0.9% isotonic saline solution at a dose level 4 ml/kg b.wt.every other day for three successive weeks and served as a negative control group.
Group 2: Each animal had orally received olive oil at dose level of 4 ml/kg b.wt.every other day for three successive weeks and served as a positive control (vehicle).
Group 3: Each animal had orally received suspension of N. sativa at a dose level of 4 ml/kg b.wt.(50 mg/kg b.wt.) every other day for three successive weeks.
Group 4: Each animal had orally received CCl4 dissolved in olive oil at a dose level of 1.9 ml/kg b.wt.(¼ LD50) every other day for three successive weeks.
Group 5: Each animal had orally received suspension of N. sativa at a dose level of 4 ml/kg b.wt.(50 mg/kg b.wt.) every other day alternated with CCl4 at a dose level 1.9 ml/kg b.wt.(¼ LD50) for three successive weeks.Twenty four hours after the end of experimental period, unanaesthetized mice from both control and experimental groups were sacrificed by slaughtering.Peripheral blood samples were collected from the neck blood vessels.

Haematological studies
Anticoagulated blood samples were used for the determination of erythrocytic count, leucocytic count, haemoglobin content and haematocrit value according to Dacie and Lewis (1975).The platelets number was counted and calculated according to Baker and Silverton (1980).Well-spread blood films were stained by Leishman's stain, examined under oil immersion and the selected sections were photographed.

Preparation of the blood for transmission electron microscopy (TEM)
Following centrifugation of blood sample, plasma was removed gently with a pipette to avoid disturbing the buffy coat.About 2 ml of 4 F1G buffered with 0.1 M phosphate buffer was added drop by drop.The buffy coat was allowed to stand for 18 h at 4°C. 1 mm slices of the plug were cut into smaller pieces.Specimens were then postfixed in 2% OsO4 at 4°C for 2 h, dehydrated in graded series of ethanol and embedded in Epon-araldite mixture in labeled beam capsules.LKB ultramicrotome was used to obtain ultrathin sections (50 nm thick) which were picked upon 200 mesh naked copper grids.Grids were double stained with uranyl acetate for ½ h and lead citrate for 20-30 min.Scoping the grids was achieved by using Jeol 100 CX TEM.

Statistical analysis
Data were expressed as means ± SE.The results were computed statistically (SPSS software package, version 10) using one-way analysis of variance (ANOVA).Post-hoc test was performed for inter-group comparison using the LSD.Values of p<0.05 were considered statistically significant.

Effect of CCl 4 and/or N. sativa on blood picture findings
The erythrocytic count, haemoglobin content and haematocrit value decreased significantly (p<0.05) in CCl 4 intoxicated animals as compared to control (Table 1).In addition, MCV and MCH decreased insignificantly (p>0.05) while MCHC increased insignificantly (p>0.05) after administration of CCl 4 .Obvious thrombocytopenia was found in the same intoxicated animals.Marked leucopaenia in mice treated with ¼ LD50 of CCl 4 was detected.In differential WBC count, CCl 4 treatment affected the neutrophils mainly and resulted in a marked neutropenia (Table 2).Furthermore, significant (p<0.05)elevation in the percentages of lymphocytes and monocytes was quite evident.Co-administration of N. sativa and CCl 4 significantly prevented the changes recorded in erythrocyte parameters, platelet counts and total leucocyte counts, which showed insignificant  difference from the control values.However, it appeared that the animals treated with CCl 4 plus N. sativa still had lymphocytosis and monocytosis accompanied by mild neutropenia.

Effect of CCl 4 and/or N. sativa on morphological pattern observed in peripheral blood cells
Oral administration of CCl 4 induced profound alterations in the morphology of both erythrocytes and leucocytes.The erythrocytes in control mice were normochromic normocytic (Figure 1a).In mice treated with ¼ LD50 of CCl 4 the cells were hypochromic and showed poikilocytosis and anisocytosis.Erythrocytes with marked crenation and cup shaped cells, target cells, sickle cells, spherocytes, stomatocytes and elliptocytes were frequently observed (Figures 1b-d).The previously mentioned alterations were less profound in animals treated with CCl 4 plus N. sativa.
The cells had nearly normal haemoglobin content and regular contour, only some poikilocytic erythrocytes could be noticed (Figure 1e).Ultrastructurally, treatment with CCl 4 yielded obvious alterations in all types of leucocytes.The mature neutrophils of control animals appeared with more than one nuclear lobe (Figure 2a).Heterochromatin was located peripherally constituting a tick band subjacent to the nuclear membranes; the central area of the nuclear lobe was occupied chiefly by euchromatin.Treatment with ¼ LD50 of CCl 4 yielded obvious alterations in neutrophils.Neutrophils with extensive segmented nuclei, dilated nuclear envelope, abundant heterochromatin, numerous destructed immature specific and primary cytoplasmic granules and vacuoles were seen (Figures 2b-d).The alterations in neutrophils in mice treated with ¼ LD50 of CCl 4 plus N. sativa showed less severity, nuclear lobes were occasionally hyperchromatic (Figure 2e).Eosinophils with segmented nuclei and coarse eosinophilic granules with central dense crystals were encountered in the control specimens (Figure 3a).Administration of ¼ LD50 of CCl 4 resulted in drastic abnormalities in the eosinophils which exhibited severe chromatolysis, hyperchromatic nuclear lobes, vacuolated cytoplasm and destructed specific granules without central dense crystals (Figures 3b-d).Treatment with N. sativa attenuated the pathological alterations induced in eosinophils.Normal specific granules with distinct central dense crystals, a few dense primary granules were observed in the cytoplasm (Figure 3e).
Control animals displayed lymphocytes with typically spherical nuclei and scanty cytoplasmic organelles (Figure 4a).In animals intoxicated with CCl 4 , Lymphocytes showed various structural abnormalities including irregular shaped hyperchromatic nuclei with dilated or invaginated nuclear envelope, multiple nuclear vacuoles, highly vacuolated cytoplasm, destructed mitochondria and hypertrophied Golgi body (Figures 4bd).Treatment with N. sativa lessened the most drastic abnormalities in blood lymphocytes; only few cells showed degenerated areas of heterochromatin and cytoplasm containing few altered mitochondria (Figure 4e).Monocytes of the control animals were found to be large cells with large kidney shaped nuclei and cytoplasm containing numerous ribosomes, polysomes and small dense mitochondria (Figure 5a).After oral administration of ¼ LD50 of CCl 4 , monocytes showed several dramatic changes including both the nucleus and cytoplasm (Figures 5b-c).The nuclei had dilated nuclear envelope with irregular contour, obvious nucleoli, abnormal heterochromatin distribution and intranuclear inclusions.The cells also appeared with vacuolated cytoplasm containing vacuolated mitochondria or mitochondria with less distinct cristae and many destructed granules.In addition, the cell surfaces of monocytes had long filopodia.An improvement was noted in animals treated with CCl 4 + N. sativa where monocytes showed normal kidney shaped eccentric nuclei and cytoplasm containing mitochondria with distinct membranes and multiple groups of well developed Golgi apparatus (Figures 5d-e).A few dense granules and vacuoles could also be noticed.Worth mentioning is that no alterations could be detected in the shape of different cell types of peripheral blood in mice that received only N. sativa.

DISCUSSION
In the present study, oral administration of CCl 4 (¼ LD50) greatly affected all hematological parameters.It led to decrease in RBCs count, Hb, PCV and MCV values.Depletion in the number of RBCs count along with the Hb concentration was detected in mice treated with CCl 4 at a dose of 0.05 ml per mouse for five weeks (Mandal et al., 1998).According to Ballinger (2007), depletion in RBCs count and Hb content leads to iron deficiency anemia which is characterized by a microcytic hypochromic blood picture.The depression in RBCs count and Hb content recorded in the present work could be attributed to disturbed hematopoiesis, destruction of erythrocytes, and reduction in the rate of their formation and/or their enhanced removal from circulation.Tung et al. (1975) mentioned that, the reduction in the values of blood parameters (PCV, RBC and Hb) may be attributed to the hyperactivity of bone marrow, which leads to production of red blood cells with impaired integrity that are easily destroyed in the circulation.
Improvement of blood picture in mice treated with CCl 4 plus N. sativa may be considered as an expression of the role of N. sativa against CCl 4 induced microcytic hypochromic anemia.These results support that of Zaoui et al. (2002) who found that Hb and PCV levels were significantly increased by 6-17%, respectively in rats treated with fixed oil of N. sativa (1 ml kg -1 day -1 for 12 weeks).Also, Abdel-Wahhab and Aly (2005) reported that treatment with N. sativa oil (5 mg kg -1 b.w. for 30 consecutive days) showed an antioxidant activity and succeeded in the improvement of all haematological parameters in rats treated with aflatoxin-contaminated diet.Moreover, Kanter et al. (2005) and Demir et al. (2006) stated that treatment with N. sativa increased the lowered RBC, Hb, PCV, WBC count in cadmium treated rats.
In the current work, administration of CCl 4 induced significant decrease in total WBC (marked leucopaenia).This result supports the finding of Jirova et al. (1996) and Mandal et al. (1998) who stated that exposure to CCl 4 induced significant decrease in leukocyte count in peripheral blood of mice.Treatment with CCl 4 plus N. sativa lessened CCl 4 -induced changes in total and differential WBC count.In agreement, N. sativa improved the WBC count in rats treated with aflatoxin (Abdel-Wahhab and Aly, 2005) and cadmium (Demir et al., 2006).In addition, studies in mice and rats have shown that N. sativa extract significantly protects from cisplatininduced falls in leukocytes count (Nair et al., 1991;El-Daly, 1998) and influences leukocytes activities (Haq et al., 1995;Houghton et al., 1995).
In the present study, treatment with CCl 4 induced severe abnormalities in peripheral blood cells.Erythrocytes showed marked anisocytosis, hypocromasia and severe poikilocytosis including crenation, target cells, sepherocytes, stomatocytes, cup shaped cells, sickle shaped cells, elliptocytes, rouleaux formation and fragment, suggesting the occurrence of hemolytic and microcytic anemia.Similar abnormalities in blood cells were observed in the peripheral blood of rats treated orally with CCl 4 (2 ml kg -1 b.w.twice a week for up to 12 week) (Doi et al., 1991).The destruction of red cells reflects the failure in hepatocellular functions and the various morphological abnormalities in RBCs are probably due to the changes in the membrane cholesterol and phospholipid content and/or ratio (Sherlock and Dooley, 1993).According to Travlos et al. (1996), the presence of altered red cell morphology is consistent with erythrocyte damage and is presumed to be related to direct oxidative injury to the red cells by the chemicals or to the pitting function of the spleen.It may be assumed that the free radicals resulting from CCl 4 metabolism caused liver injury and a proportion of these free radicals librated from the liver into the blood and may affect the membranes of circulating red cells and induced a significant increase in the number of target cells (Doi et al., 1991).Improvement in erythrocytes in animals treated with N. sativa could be due to the important role of N. sativa in decreasing the oxidative damage in cell membrane.This is in agreement with Kanter et al. (2005) who reported that treatment with N. sativa oil (interperitoneal injection of 0.2 ml kg -1 b.w. for 30 days) reduced membrane destruction and haemolytic changes in erythrocytes of cadmium-treated rats.
Electron microscopy revealed various ultrastructural abnormalities in the leukocytes in the blood of mice treated with CCl 4 .These abnormalities included both the nucleus and cytoplasm.Neutrophils frequently appeared with irregular hypersegmented nuclear lobes and destructed/vacuolated cytoplasm with indistinct contour.Hypersegmented neutrophils were frequently noticed in patients with megaloblastic anemias (Sinha et al., 2006).Destructed specific granules with less electron density and less distinct central crystals were pronounced in eosinophils of mice treated with CCl 4 .Abnormalities in the size, shape and cytochemistry of eosinophilic granules have been reported in patients with myelocytic leukaemia and lymphoma (Mnathorpe et al., 1977;Anteunis et al., 1978).Moreover, some lymphocytes displayed less electron dense cytoplasm with dense destructed mitochondria.Most of these abnormalities were described by Pandolfi et al. (1982) in peripheral blood lymphocytes of patients with chronic lymphocytic leukemia.Ghadially (1985) suggested that these changes can be used as tentative markers for leukemias and lymphomas in man.Monocytes had highly irregular Ushaped nuclei with abnormal heterochromatin content and also had a few disintegrated mitochondria.Similar alterations in leucocytes have been previously described by El-Mofty et al. (2000a) and observed also in peripheral blood of mice treated orally with Nizoral at a dose level of 200 mg kg -1 b.w. for 12 months (El-Mofty et al., 2000b).Oral administration of N. sativa improved most of the ultrastructural changes induced in leucocytes after CCl 4 administration.This means that the black seed was able to constitute a live of defense, protecting the blood cells from the severe damaging effect of the toxins.
This investigation shows that N. sativa has the ability to protect the haematopoietic cells from the damaging effects of exposure to CCl 4 and this protection might be attributed to the antioxidative power of N. sativa.

Figure 4 .
Figure 4. Electron micrographs showing blood lymphocytes of mice; (a) control group, arrowhead indicates dense granules, arrows point to small cytoplasmic projections; (b) after treatment with ¼ LD50 of CCl4, lymphocyte with dilated nuclear envelope (arrow), arrowheads point to less dense granules; (c) after treatment with CCl4, note dilated nuclear envelope (double arrow) and invaginated nuclear envelope (thick arrow), arrowhead points to fragmented part of nucleus and arrows point to filopodia; (d) after treatment with CCl4, note the cell surface appears with microvilli (arrows); (e) after treatment with CCl4 plus N. sativa, lymphocyte with large spherical nucleus (N) showing slightly dilated nuclear envelope and lysis of heterochromatin at certain areas (arrowheads).Lc: lymphocyte, N: nucleus, Nu: nucleolus, M: mitochondria, G: Golgi apparatus, R: ribosomes, V: cytoplasmic vacuoles.Magnification: 10000x (a, d), 15000x (b), 13000x (c,e).

Table 1 .
Effect of CCl4 and/or N. sativa on erythrocyte values and platelet count in peripheral blood of Swiss albino mice.Values are expressed as mean ± SE, for three animals in each group; Values, within columns, with no common superscripts are statistically significant (p < 0.05); RBC: red blood cell count, Hb: haemoglobin content, PCV: haematocrit value, MCV: mean cell volume, MCH: mean cell haemoglobin, MCHC: mean cell haemoglobin concentration, Pl: platelet count. a

Table 2 .
Effect of CCl4 and/or N. sativa on leucocyte values in peripheral blood of Swiss albino mice.
bValues are expressed as mean ± SE, for three animals in each group; Values, within columns, with no common superscripts are statistically significant (p<0.05).