summary - agenciapyme a. said; saher, a. galal; zeinab, y. mohammed** and a. ... pended and slide...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 4 (December) 2008; 29 - 57

29

Protective effects of dietary dates against the toxicity of mercuric chloride in male albino rats

By Nagwa, A. Said*; Saher, A. Galal*; Zeinab, Y. Mohammed** and A.

A. Nada* *Immunity department- Animal Health Research Institute

** Pathology department- Animal Health Research Institute

SUMMARY

T he present study was planned to investigate the protective effect of dates on the adverse effect of mercuric chloride toxicity in rats.

Sixty rats were divided into four equal groups, group I served as a con-trol, group II fed basal diet and administrated 1/20 LD50 mercuric chlo-ride (2.29 mg/ kg b. w.), group III fed basal diet supplemented with 10% minced date only four weeks before exposure to mercuric chloride and the group IV fed basal diet with 10% minced dates four weeks before ex-posure to mercuric chloride, both mercuric chloride and dates were con-tinued along the experiment in this group. Referring to hematological pa-rameters, group II and III mercuric chloride induced significant decrease in RBCs count, hemoglobin content and PCV with normocytic normo-chromic anemia, significant decrease in WBC count but no significant changes were recorded in group IV. Group II showed significant in-creased in serum sodium, ALT, AST and urea while potassium, total pro-tein, albumin, globulin and testosterone showed significant decrease in rat serum. Significant increase of serum creatinine was detected at 90 days, the same results were showed in group III but in less density except creatinine showed non significant changes. In group IV, serum sodium potassium, creatinine and testosterone revealed no significant changes, but serum ALT, AST, total protein, albumin, globulin and urea were largely improved. Mercuric chloride was found to decrease phagocytotic percentage and phagocytic index of neutrophils in rats received mercuric chloride only (group II), but rats in group IV showed nearly value toward the control group. Serum immunoglobulins (IgG) revealed no significant changes in serum immunoglobulins levels (IgG) between groups at 45 days. At 90 days group II showed significant decrease in IgG values com-pared to control, whereas both III and IV did not show significant changes compared to control group. Slight increase in serum lysozyme activity in group II and group III at 45 days, whereas rats in group IV did not show significant change compared with control rats. Histopathologi-


30

cal findings revealed alterations in group II and III different in severity and extension, while group IV showed mild alteration in different exam-ined organs (livers, kidney and testes).

Referred byReferred by Prof. Dr. Jakeen K. A. El-Jakee Professor of Microbiology, Fac. Vet. Med.,

Cairo University Prof. Dr. Afaf A. El-khwas Professor of Pathology, Animal Health Re-

search Institute, Dokki

INTRODUCTION

E nvironmental pollution is a world wide problem. Heavy

metals are belonging to the most important pollutants. Heavy metals constitute some of most hazardous substance that can bioaccumulate, a process in which a chemical pol-lutant enters into the body of ani-mal and human and is low or not execrated but rather collected in their tissue (Ali and Amin, 2006).

Mercury is considered to be toxic to most organisms when pre-sent at appreciable levels. The total amount of mercury present in the environment as a result of natural erosion of the earth’s crust by wind and water far exceeds that caused by human activities. It disrupts most biological systems as a result of its affinity for sulfhydryl groups which are functional components of most enzymes and hormones (Hans et al., 1999).

Inorganic mercury is toxic to normal hepatic, neurological, re-productive, respiratory, immune, dermatological and developmental

sequels (Risher and Amler, 2005).

Many pathological manifes-tations caused by exposure to mer-cury and other heavy metal have been reported in general, substan-tial evidence exists to support the hypothesis that mammalian im-muno-compatent are sensitive to metals. Mercuric chloride is able to trigger immuno-dysregulation that leads either to auto immunity or immuno-suppression depending up on the genetic of the rat strain tested (Robert et al., 1995).

Recent evidences also show that mercury causes severe oxida-tive changes (Hansen et al., 2006), thus mercury proved to be a poten-tial oxidatant is the category of en-vironmental factories. Therefore, there is a need to provide protec-tion against mercury induced tox-icity.

Historically, plants have been used as folk medicine against various type of diseases. Remedies from plants sources have proved to be very popular in primary health


31

care. Dates, the fruit of date palm (phoenix dactylifera), represents an important highly nutritive source for human and animals. This has been attributed to con-stituents, of these constituents sugar, protein fat also contain some mineral Mg, iron, calcium, phosphorus, potassium and some vitamins as vit C, A, B complex, thiamine and nicotinic acid (AL-Shahhib and Marshall, 2003).

Some of properties of dates experimentally proved include an-tibiotic like effects on some bacte-ria (Abdel Salam, 1994), strength-ens immune system (Puri et al., 2000).

So, the purpose of this experi-ment is to study the use of date Tamr as protective and preventive agent against mercuric chloride toxicity as well as to study some immunological, hematological and biochemical alterations and histo-pathological changes in rats intoxi-cated with mercuric chloride.

MATERIALS AND METHODS

Dates: Dried dates were obtained

from Egypt Assuit type and minced into small pieces for addi-tion to ration

Chemical: Mercuric chloride was obtained from Merck Rohway, U S A.

Animals and experimental de-sign.

A total of 60 male albino rats weighing from 150 to 170 g were used. The rats were housed in plas-tic cages under good hygienic con-ditions and fed on balanced ration and water ad-libitum rats were di-vided into four equal groups: Group I: served as a control fed on

a commercial basal diet. Group II: rats fed basal diet and

administrated with 1/20 (LD50) mercuric chloride (2.29 mg/kg bw) in water. The lethal dose fifty of mercuric chloride was determined according to EL-Boushy et al. (2000).

Group III: rats fed on basal diet with 10% minced date as a pro-phylactic supplement 4 weeks before exposure to mercuric chloride (1/20 LD50) as group II.

Group IV: rats fed basal diet sup-plemented with 10% minced dates 4 weeks before exposure of mercuric chloride (1/20 LD50) and both mercuric chlo-ride and minced dates were con-tinued for all experiment (90 days).

Samples : Two blood samples were ob-

tained from retro-orbital venous plexus at 45 and 90 days through the experiment.

Blood on EDTA anticoagulant solution for hematological profile. Blood in dry clean tube for collec-


32

tion of serum to estimate bio-chemical and hormone assay.

Blood on heparin anticoagu-lant solution (20 IU /ml blood) for measuring phagocytic activity of neutrophils

After sacrificed rats and post mortem examination, tissue speci-mens from liver, kidney and testis of animals groups were collected and preserved in 10% neutral buff-ered formalin at the end of the ex-periment.

Hematological examinations: Hematological profile were

carried out using automated cell Counter, AL system, Germany (Feldman et al., 2000).

Serum biochemical analysis: Estimation of biochemical and

hormone assay included sodium and potassium which were deter-mined according to Oser (1979). Total protein according to Sonnen-writh and Jarette (1980), albumin according to Drupt (1974), ALT and AST according to Reitman and Frankle (1957), urea accord-ing to Patton and Crouch (1977), creatinine according to Hovot (1985) and testosterone hormone according to Wilson and Foster (1992).

Serum immunologlobulins IgG: Specific medial immunodiffu-

sion plates (The binding site, BINDA RID, Birmingham (IgG) in sera mg/dl) according to Mancini et al. (1965).

Measurement of lysozyme activ-ity:

Serum lysozyme activity was measured by agarose gel lyses as-say according to the method de-scribed by Schltz (1987). Briefly, lysophate were prepared by dis-solving 1% agarose in 0.06 M phosphate buffered saline at pH 6.3, 500 mg of Micrococcus lyso-deikticus in 5ml saline were added to 1 litre of agarose. Plates were poured then 25µl of serum samples and standard lysozyme were put in each well. After 18 hours the cleared zone diameter were meas-ured to both standard lysozyme and serum samples and concentra-tion was estimated.

Evaluation of phagocytic activity of neutrophils: Phagocytosis of polymor-

phonuclear cells using Candida al-bicans was performed according to (Wilkinson, 1981), briefly, 5 ml heparinized blood were collected in plastic tube, added to 2 ml dex-tran 6% (MW, 500,000, Sigma) and leucocytes rich plasma was re-moved, suspended in Hank's bal-anced salt solution. In a plastic tube, 0.25 ml Hank`s solution, 0.25 ml pooled normal serum, 0.25 ml heat killed Candida albicans (5X 106/ ml) and 0.25 ml leucocyte suspension (5 X 106 neutrophil/ ml) for 30 minutes after which it was centrifuged. The supernatant was removed leaving a droplet into which the sediment was resus-


33

pended and slide smears were pre-pared and stained with Giemsa stain. Hundred neutrophils were examined for estimation of phago-cytic percentage and index. Histopathological examination: Tissue specimens were previously fixed in 10% buffered formalin, processed for histopathological ex-amination (Bancroft et al., 1996).

Statistical analysis: Data obtained in this experi-

ment were statistically analyzed using analysis variance and com-paring between groups were per-formed using least significant dif-ference (LSD) at P<o.o5 according to Petrie and Watson (1999) and computerized according to SPSS (2002).

RESULTS

Hematological result: Tables (1 & 2) showed the

changes in hematological values in rats treated with mercuric chloride. In group II and III (the same result but in less degree or values, re-spectively).

Mercuric chloride induced significant decreased in RBC count, Hb content and PCV per-cent, while red cell indeces (MCV, MCH and MCHC) did not show any significant variation (normo-cytic normochromic anemia). Sig-nificant decreased in WBC count (leucopenia). In the same tables show non significant changes in haemogram in group IV.

Serum biochemical and hormone assay:

Serum biochemical and hor-mone values of rats are illustrated in Tables (3 & 4). Group II showed significant increases in serum so-dium, ALT, AST and urea, while serum potassium, total protein, al-bumin, globulin and testosterone were significant decreased.

Serum creatinine showed non significant changes at 45 days from beginning of the experiment but it significantly changes in-creased at 90 days. The same result were showed in group III but in less density, except creatinine showed non significant changes.

In group IV serum sodium, potassium, creatinine and testoster-one revealed non significant chan-ges. While serum ALT, AST, total protein, albumin, globulin and urea showed largely improved. Immunological findings:

Phagocytic percentage and phagocytic index of neutrophil re-vealed significant decrease in rats received mercuric chloride only (group II) at 45 and 90 days com-pared with control rats (group I) Also, dates pre-treated animals with mercuric chloride (group III) showed significant decrease in phagocytic index at 45 days com-pared with control animals (Table, 5). While group IV pre and post -treated date with mercuric chloride


34

revealed non significant change compared to control.

Serum immunoglobulins: Serum immoglobulins IgG

were illustrated in Table (6) and Fig. (1). There are no significant changes in serum immunoglobins levels IgG between groups at 45 days, but at 90 days rats received mercuric chloride (group II) sh-owed significant decrease in IgG value compared to control. Whe-reas both group II and group IV did not show significant changes compared to control group.

Serum lysozyme activity: Both mercuric chloride intoxi-

cated rats (group II) and dates pre-treated animals with mercuric chloride (group III) exhibited sig-nificant increases in serum ly-sozyme activity at 45 days with respect to control (group I).

Whereas dates pre- and post-treated animals with mercuric chloride (group IV) did not show significant changes compared with control animals (Table, 7). Pathological findings: Postmortem lesions:

The post-mortem changes ob-served in liver, kidney and testis of sacrified rats revealed enlarge-ment, congestion and petechial haemorrhage. In groups III showed similar post mortem but less in number and severity. While no gross lesions were detected in rats

of group IV.

Histopathological findings: Histopathological examina-

tion was carried out on liver, kid-ney and testis revealed alteration in groups II and III. The alteration in the two groups were more or less similar but they dif-ferent in severity and extension while group IV showed mild al-teration in different examined or-gans.

In group II the livers showed sever reaction as necrosis and atro-phy of hepatic cell. Hemorrhage and thrombosis formation in most hepatic vessels Fig (2). In the same group other rats liver showed focal necrosis with aggregation of few inflammatory cells (Fig, 3). Beside thrombosis and sever necrosis of hepatocyts, the liver showed granuloma formation consists of necrosis of liver cells and infil-trated with inflammatory cell (Fig, 4).

In group III microscopic ex-amination of liver showed conges-tion of hepatic sinusoids, haemor-rhage and atrophy of some hepatic cords (Fig, 5).

Moreover in group IV liver revealed mild hepatic necrosis with inflammatory cells infiltration (Fig, 6).

Regarding the kidney, the histopathological examination in group II revealed sever infiltration


35

of mononuclear inflammatory cells, as well as congestion of blood vessels accompanied by de-generation and necrosis of cells lining the renal tubules (Fig, 7 & 8).

Associated with degeneration and necrosis of cell lining renal tu-bules, the kidney showed forma-tion of renal cast inside the lumen of renal tubules (Fig, 9). The corti-cal vasculature appeared diluted and engorged with blood (Fig, 10). Another alteration found sever for-mation of different size of throm-bosis.

In group III histopathological examination of kidney showed moderate degree of congestion in renal blood vessels and atrophy of some glomeruli (Fig, 11). While group IV kidney showed mild de-generation of the lining tubular epithelium with few hyaline casts in some renal tubules (Fig, 12).

Microscopic examination of testicular tissue in group II re-vealed necrosis of spermatogonic cell layers (Fig, 13) with oedema and infiltration with mononuclear inflammatory cell in interstitial space. While in group III showed the same necrosis of spermato-genic cells beside congestion and interstitial oedema. In group IV showing nearly normal seminifer-ous tubules (Fig, 14).


36

Table (1): Mean values ± S.E. of hematogram of different experimental groups of rats at 45 days of the experiment.

Parameter

Group RBCs

x 106/ml

Hb

g/dl

PCV

%

MCV

fl

MCH

Pg

MCHC

g/dl

WBCs

x 103/ml

GI 6.13 ± 0.151a

14.83 ± 0.216a

44.0 ± 0.706a

71.98 ± 2.381

24.23 ± 0.65

33.75 ± 0.686

8.00 ± 0.217a

GII 5.0 ± 0.112b

12.33 ± 0.124c

35.0 ± 0.747c

70.64 ± 2.64

24.70 ± 0.82

35.66 ± 0.857

7.30 ± 0.194b

GIII 5.4 ± 0.105b

13.26 ± 0.185b

38.33 ± 0.957b

70.98 ± 2.10

24.55 ± 0.73

35.00 ± 0.759

7.55 ± 0.215b

GIV 6.0 ± 0.134a

14.56 ± 0.138a

42.33 ± 1.192a

70.27 ± 2.24

24.2 ± 0.71

34.43 ± 0.462

7.80 ± 0.354a

F-calculated 46.06# 47.16# 19.34# 1.321 1.245 1.329 42.216#

Probability 0.001 0.001 0.001 0.008 0.002 0.300 0.001

# Significant at P < 0.05

a, b, c insignificant different between similar litter at P < 0.05


37

Table (2): Mean values ± S.E. of haematogram of different experimental groups of rats at 90 days of the experiment.

Parameter

group RBCs

x 106/ml

Hb

g/dl

PCV

%

MCV

Fl

MCH

Pg

MCHC

g/dl

WBCs

x 103/ml

GI 5.86 ± 0.23a

15.0 ± 0.20a

46.3 ± 1.02a

79.07 ± 3.15

26.71 ± 0.89

32.66 ± 0.78

7.8 ± 0.27a

GII 3.8 ± 0.12c

10.16 ± 0.10c

30.33 ± 0.94c

80.14 ± 2.67

26.91 ± 0.87

33.48 ± 0.80

6.25 ± 0.19b

GIII 4.65 ± 0.10b

13.25 ± 0.21b

37.13 ± 0.80b

81.1 ± 2.51

28.5 ± 0.86

35.00 ± 1.10

7.5 ± 0.24a

GIV 5.5 ± 0.18a

14.6 ± 0.30a

44.0 ± 0.79a

80.12 ± 2.95

26.61 ± 0.70

33.2 ± 0.80

7.8 ± 0.33a

F-calculated 31.001# 106# 65.688# 2.130 2.243 3.744 12.148#

Probability 0.001 0.001 0.001 0.137 0.050 0.007 0.001


a, b, c insignificant different between similar litter at P < 0.05


38

Table (3): Mean values ± S.E. of serum biochemical and hormone assay of different experimental groups of rats at 45

days of the experiment.

Parameter

Group

Na

mEq/l

K

mEq/l

ALT

u/l

AST

u/l

T. Protein

g/dl

Albumin

g/dl

Globulin

g/dl

A:G

ratio

Urea

mg/dl

Creatinine

mg/dl

Testosterone

ng/ml

GI 149.3 ± 1.985a

6.95 ±0.091a

27 ± 0.593a

38.8 ±0.516a

6.16 ± 0.118a

3.86 ± 0.093a

2.3 ± 0.04a

1.68 ±0.03

20.5 ±0.635a

1.20 ± 0.042

2.55 ± 0.054a

GII 174.0 ± 2.056b

6.33 ±0.101b

62 ± 1.922d

79.6 ±1.835d

4.70 ± 0.151c

3.10 ± 0.074c

1.6 ± 0.13c

1.98 ±0.15

66.5 ±1.050d

1.50 ± 0.077

1.87 ± 0.033b

GIII 156.0 ± 2.208a

6.33 ±0.159b

46 ± 0.987c

59.8 ±0.832c

5.13 ± 0.174bc

3.33 ± 0.072bc

1.8 ± 0.13bc

1.89 ±0.14

47.0 ±1.019c

1.20 ± 0.037

2.00 ± 0.062b

GIV 148.7 ± 3.572a

7.15 ±0.13a

37 ± 0.663b

44.3 ±1.12b

5.75 ± 0.158b

3.52 ± 0.085b

2.18 ± 0.11b

1.80 ±0.09

34.3 ±0.824b

1.22 ± 0.043

2.44 ± 0.016a

F-calculated 73.96# 99.44# 85.83# 39.39# 62.23# 39.52# 7.933# 1..335 81.57# 2.93# 28.30#

Probability 0.001 0.001 0.001 0.001 0.001 0.001 0.002 0.298 0.001 0.001 0.001


a, b, c, d insignificant different between similar litter at P < 0.05


39

Table (4): Mean values ± S.E. of serum biochemical and hormone assay of different experimental groups of rats at 90

days of the experiment.

Parameter

group

Na (mEq/l)

K (mEq/l)

ALT (u/l)

AST (u/l)

T. Protein (gm/dl)

Albumin (gm/dl)

Globulin(gm/dl)

A:G ratio

Urea (mg/dl)

Creatinine(mg/dl)

Testosterone (ng/ml)

GI 152 ± 2.19a

6.8 ± 0.089a

28.4 ± 0.624a

38 ± 0.505a

6.5 ± 0.125a

3.86 ± 0.093a

2.66 ± 0.094a

1.454 ± 0.025a

22 ± 0.682a

1.25 ± 0.044a

2.55 ± 0.09a

GII 188.3 ± 2.23c

4.5 ± 0.072c

105 ± 3.255d

109.6 ± 2.526d

3.8 ± 0.122 d

2.7 ± 0.065 d

1.6 ± 0.050c

1.689 ± 0.019c

94.66 ± 1.495d

2.1 ± 0.038b

1.29 ± 0.04c

GIII 170 ± 1.92b

6.0 ± 0.151b

77.3 ± 1.658c

84.3 ± 1.173c

4.85 ± 0.164c

3.1 ± 0.067 c

1.8 ± 0.056c

1.725 ± 0.030c

64.33 ± 1.394c

1.46 ± 0.075a

1.70 ± 0.02b

GIV 158 ± 3.79a

7.0 ± 0.127a

48.6 ± 1.154b

62.3 ± 1.582b

5.60 ± 0.148b

3.74 ± 0.079b

2.3 ± 0.075b

1.561 ± 0.031b

47.22 ± 1.134b

1.3 ± 0.046a

2.30 ± 0.05a

F-calculated 34.2# 99.4# 309# 333# 62.2# 39.52# 42.79# 21.9# 62.7# 22.5# 39.12#

Probability 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001


a, b, c, d insignificant different between similar litter at P < 0.05


40

Table (5): Phagocytic percentage and index of rat neutrophils of different groups throughout the experiment.

Time Groups

45 days 90 days

% Index % Index

Control I 51.0±2.0A 1.26±0.03A 52±3.9A 1.25±0.05A

Toxic group II 39.0±1.0 1.13±0.03aB 43±1.8a 1.10±0.03a

Pro-treated + Toxic III 43.0±4.0 1.14±0.89ac 45±2.8 1.10±0.03

Treated + Toxic IV 52.0±4.0b 1.25±0.08bc 15±1.3 1.20±0.02

F-calculated 4.27 4.42 2.7 2.01

Table (6): Serum immunoglobulins IgG (mg/ml) in sera of albino rats.

Time Group

45 days 90days

Control I 15.5 ± 0.4 16.2 ± 0.5 A

Group II 16.0 ± 0.6 14.0 ± 0.9aB

Group III 16.4 ± 1.2 17.7 ± 1.3b

Group IV 17.0 ± 0.3 17.6 ± 0.6b

F-calculated 1.26 4.11*

Table (7): Serum lysozyme (µg/ml) of albino rats in different groups throughout the experiment.

Time Group

45 days 90 days

Control I 463 ± 33A 547 ± 14

Group II 598 ± 24aB 607 ± 27A

Group III 579 ± 27ac 607 ± 29B

Group IV 470 ± 34bc 523 ± 27ab

F calculated 5.85* 2.22*

* Significant at P< 0.05 using ANOVA test, Bb, Cc and Dd are signifi-cantly different between two comparison groups against Capital letters using LSD at P<0.05.


41

Fig (1): Shows radial immunodiffusion IgG of examined rat (RID IgG)

Fig (2): Liver of rat showing necrosis and atrophy of hepatic cell & thrombus formation of most the hepatic vessels group II (H & E X 200).

Fig (3): Liver of rat showing focal area of necrosis with aggregation of few in-flammatory cells group II (H& E X 400).


42

Fig (4): Liver of rat showing granuloma formation with necrosis of the hepatic cells and congestion of the hepatic sinusoid group II (H & E X400).

Fig (5): Liver of rat showing congestion and dilation of some hepatic sinusoids and atrophy of the hepatic cords group III (H & E X 100).

Fig (6): Liver of rat showing hepatic necrosis with inflammatory cells infiltra-tion group IV (H & E X 400).


43

Fig (7 & 8): Kidney of rat showing severe infiltration of mononuclear inflam-matory cells as well as degeneration and necrosis of cell lining the renal tubule group II. (H & E X 200).

Fig (9): Kidney of rat showing formation of renal cast inside the lumen of tu-bules accompanied by degeneration and necrosis of renal tubules group II. (H & E X 200).


44

Fig (10): Kidney of rat showing congestion and dilatation of some renal blood vessels. (H & E X 200).

Fig (11): Kidney of rat showing congestion of blood vessels, degeneration of some lining of epithelial tubules and atrophic glomeruli (H & E X 400).

Fig (12): Kidney of rat showing mild vascular degeneration of lining tubular epi-thelium with few hyaline casts in some renal tubules group IV (H & E X 100).


45

Fig (13): Testis of rat showing necrosis of the lining spermatogenic cell and degen-eration of some interstitial cells group II (H & E X 200).

Fig (14): Testis of rat showing congestion and degeneration of some lining sper-matogenic tubules, interstitial oedema and degeneration of some intersti-tial cells group III (H & E X 200)

Fig (15): Testis of rat showing nearly normal semineferous tubules Group IV (H & E x 200).


46

DISCUSSION

H eavy metal are considered as dangerous substances caus-

ing health hazards to human and animals through progressive irre-versible accumulation in their bod-ies as a result of a repeated con-sumption of small amounts of these metals. Mercury is capable of damaging the organism in many ways due to its high affinity to various tissues and its tendency to accumulates (Radwanska et al., 1993).

In Egypt mercury in relatively source metal that enters the fresh water via industrial processes (Abd EL-Hamid, 1994). Date fruits are important and readily source and as a good source of nu-tritionally important mineral, vita-mins, pholins, fibres, lipid and free from cholesterol (Mahmoud et al., 2003).

In our results mercuric chlo-ride induced a type of normocytic normochromic anemia in group II and group III, with different in de-gree and accompanied by leuco-penia. Many authors recorded ane-mia during mercuric chloride treat-ment as Lecavalier et al. (1994) and (Mohamed, 2004) who re-ported that there were normocytic normchromic anemia in rats and guinea pigs after administrated mercuric chloride in drinking wa-ter in different doses and duration. Normocytic normochromic anemia

due to depression of erythrogene-sis as in bone marrow hypoplasia as with poisoning (Bernard et al., 2000).

Leucopenia observed in our examination may be attributed to a number of metals that alter leuco-cytic count are known to markedly alter the equilibrium between the circulating and marginated com-partments. It produce apparent leu-copenia by increasing the size of the marginated pool (Wand and Colin, 1998).

These changes in haemogram may be attributed to affect of heavy metal directly on blood cell and indirectly on haemopiotic or-gans.

In our study the significant increase in serum sodium and de-creases in serum potassium levels in groups II and III may be due to the gradual development of renal function impairment as well as tu-bular damage (EL-Boushy et al. 2000), these alteration in sodium and potassium mainly attributed to the toxic effect of mercuric chlo-ride which seriously affect the cell membrane function leading to dis-turbance of the ion regulation.

The elevation in serum ALT and AST activity, the liver is con-sider as a second target depot for mercury in terms of total amount irrespective of administration routs. These elevation of the these enzymes attributed to severs dam-


47

age in liver cells which releasing the hepatic enzyme into serum (Ibrahim et al., 1991) and (Ga-mal, 1991) these finding was con-firmed histopathologically by de-generation and necrosis of liver cell.

The decrease in serum total proteins and albumin and globulin may be attributed to decrease in protein synthesis by hepatic cell and increase rate of excretion due to renal damage. Our results are similar to result recorded by EL-Boushy et al. (2000).

Significant increase of serum urea and creatinine in our result was indicating marked the nephro-toxic effect observed after expo-sure of inorganic mercury is as-sumed to be related to the fact that the kidney accumulates more mer-cury than other organ in the body (Osfar and Ibrahim, 2000 and Mohamed, 2004).

The observed reduction in se-rum testosterone level agree with the result of EL-Boushy et al. (2000). The decreased of testoster-one may be as result of direct dam-age of mercuric chloride in leydig cells (Robert et al., 1995).

The present investigation re-vealed that groups II received mer-curic chloride only intoxication causes significant decrease in phagocytic percentage and index of neutrophils compared to control

animals or groups IV. The princi-ple toxic effects of mercury in-volve interactions with large num-ber of cellular processes, including the formation of complexes with free thiols and protein thiol groups, which may lead to oxidative stress (Stacey and Kappus, 1982).

Our results agree with Soltys et al. (1997) who found that sub-chronic exposure to mercury to guinea pigs resulted in significant decrease in phagocytic activity of peritoneal macrophages through the experiment. Also, Worth et al. (2001) recorded mercuric chloride inhibited polarization and immu-noglobulin-mediated phagocytosis in a dose-dependant manner in hu-man neutrophils. Moreover, Hem-dan et al. (2007) found that expo-sure to mercuric chloride signifi-cantly reduced peripheral blood mononuclear cell vitality in vitro as well as elicited distinct effects on T-helper 1 (Th1) and T-helper 2 (Th2) cytokine expression depend-ing on cellular activation path-ways. Furthermore, Naz et al. (2008) found that subchronic ex-posure with mercuric chloride in rats results a decrease of 33% im-mune response as evident from an-tinuclear antibody (ANA) positive test.

The present results in group IV; toxicated with mercuric chlo-ride and treated with dates till the end of experiment showed nearly


48

values of phagocytic activity of neutrophils compared to control group that means ameliorative ef-fect of dates on adverse effect of mercuric chloride. Dates contain carbohydrate, fat, protein, vita-mins, fiber and at least 15 miner-als, including selenium which play an important role in immune func-tion and prevent cancer (AL-Shahhib and Marshall 2003). Dates possess strong antioxidant activity which provokes free radi-cal scavenging enzyme system. Antioxidants are compound that can delay or inhibit oxidation of lipids or other molecules by inhib-iting the initiation or propagation of oxidative chain reaction (Velioglu et al., 1998).

These results indicating sig-nificant immunomodulating effect of dates, which agree with the re-sults of Puri et al. (2000) who re-corded an enhancement of macro-phage activation and increased in haemagglutination antibody titres as well as plaque forming cell (PFC) counts in mice orally ad-ministrated date extract. The im-munostimulatory activity of dates may be attributed to presence of β-D-glucan in date flesh. Ishurd et al. (2002) isolated β-D-glucan from the fruit of dates. β- D- glu-can is and important candidate molecule of biological response modifier implicated in cancer, it can be obtained from other sources such as yeast, fungi, bacteria and

plants. Tokunako et al. (2000) mentioned that β- D- glucan has higher biological effect in activa-tion of alternative pathway of com-plement, induce interleukin-6 syn-thesis of macrophage in vitro, and act as adjuvant effect on antibody production.

Our results indicate that ame-liorative effect of dates on mercu-ric chloride toxicity. Host resis-tance is highly dependent on the first line of defence, phagocytes; there is not necessarily a direct correlation between serum im-muoglobuin levels and host resis-tance (Robert et al., 1995). Our data exhibited significant decrease of IgG at 90 day in mercuric chlo-ride toxicity (group II) compared to control. The present results agree with Shenker et al. (1993) who reported immunototic effect of mercuric chlorie on human lym-phocytes and monocytes; they sug-gest that mercuric chloride at low and high dose inhibit activation of human B cells to synthesize IgM and IgG. Contradiction of this re-sult, Lawreuce et al. (1983) who found that low dose of mercuric chloride enhance IgM production.

Bagenstose et al. (2001) and Zheng et al. (2005) reported that subtoxic doses of mercuric chlo-ride in SIJ mice resulting in in-creased production of serum im-munoglobulin IgE and IgG1, inter-leukin 4 and enhances the suscepti-


49

bility to Leishmaniasis.

Lysozyme are proteins of low molecular weight found in poly-morphonucelar leukocytes and mononuclear cells, lysozyme are considered as a member of innate humoral factors that elaborate from the body and showed dra-matic increase in concentration in response to infection or tissue in-jury (Weir, 1983). Lysozyme in serum can destroy the glucosidic bond in cell wall of Escherichia coli and Staphylococcus as a result of phagocytic activity (Guo et al., 2004). Our data revealed increased lysozyme activity in mercuric chloride group II and group III at 45 days. This increase may be at-tributed to activation effect of mer-curic chloride on immune cells or may be due to tissue injury as a re-sult of harmful effect of mercuric chloride. Zheng et al (2005) re-ported elevated production of B cell activating factor in susceptible mice administrated mercuric chlo-ride.

The histopathological lesions appeared approximately the same in group II, III and IV but differ in severity and extention.

The lesions observed in liver, the central vein and blood sinusoid were dilated and engorged with blood, some times considerable ar-eas of haemorrhage were observed. These changes were attributed to toxic effect of mercuric chloride on the endothelial cells lining

blood vessels [Hans et al. (1999) and Mohamed (2004)]. Several type of degenerative changes in hepatocytes and necrosis was ob-served which local or diffused in its distribution. These changes oc-curred most commonly due to the direct toxic effect of heavy metals on hepatocytes through the blood stream.

Kidney lesions were demon-strated by blood vessels were con-gested and haemorrhage. These le-sions attributed to direct toxic ef-fect of mercury on development and function of endothelial lining of renal blood vessels. A toxic tu-bular nephritis were observed as well as the epithelial lining of the renal tubules appear degenerated and some tubules contain hyaline cast these result agree with Hamza et al. (1994); Carlton and McGo-vin (1995).

The testis showed different alteration, these alteration mani-fested by different degree of de-generation and or necrosis in the lining spermatogenic tubules, in-terstitial oedema, congestion and infiltration of inflammatory cell. The lesions in the testis attributed to effect of mercuric chloride on endothelial cells of small vessels leading to its damage which give rise to increased capillary perme-ability, this lead to vascular escape of fluid and blood plasma sub-stance into interstitial which re-


50

sults oedema, decrease capillary blood flow ischemia and testicular degeneration (Robert et al., 1995) and EL-Boushy, et al. (2000).

From our result mention be-fore we found that the two groups treated with date (III and IV) have variation if compared with group treated mercuric only II. These variation due to date supplemented and his effect on toxicity of mercu-ric. In group III (pretreated with date) the values of hemogram, bio-chemical and alteration of histopa-thological are less degree of sever-ity than group II (moderate reac-tion), where the date improved the feed utilization and healthy condi-tion of animal and act as immu-nostimulant (Ali et al., 1999). Th-is improvement play role against mercury toxicity. By time this improvement was decreased by stopped supplementation of date so must be continuous supply of date through experiment time to avoid hazardous effect of date, these what done in group IV.

The improvement of hemo-gram, biochemical and histopa-tholgical studies which showed in results of group IV due to the ef-fect of date on mercuric toxicity which play role to prevent the haz-ardous effect of mercury.

Authors discussed the quality and nutritive value of date are in-fluence by their chemical composi-tion. They recorded that, the dates contain essential nutrient, high

sugar and moderates amount of protein and lipid. Also, they con-tain minerals (iron, manganese, copper, zinc, cobalt, sodium potas-sium, phosphorus, calcium and choline; and vitamins A, C, B co-mplex (Al-khouli et al., 1998; EL-Ghazoli and Hussin, 2003; and Mahmoud et al., 2003). More-over, the date contain antioxidant , vitamins and elements that play major role in prevention of heavy metals toxicity (Sharma et al., 2007 and Youseif et al., 2007).

The date contain vitamin C, which has strong antioxidant, hav-ing nucleophilic properties and binding to mercury ions to reduced mercury damage effect (Sato et al., 1997). EL-Sharkawy and EL-Nesr (2008) reported that, supple-mentation of vitamin C to mercuric chloride treated rabbits has effec-tively increase antioxidant en-zymes (glutathione peroxidase and superoxide dismutase) and reduced histopathological lesions. More-over Luck et al. (1995) found that ascorbic acid effects both integrity of tubular structure and functional-ity of the testes.

The dates contain vitamin A which has antioxidant effect as an-tioxidant substance degrade free radical damage proteins and re-move lipid hydroperoxide from membrane (Diplock, 1995). Vita-min A increase the immune re-sponse and or modulate the re-


51

sponse into the desired direction and thus may improve and prevent any cellular damage caused by oxygen radicals through the anti-oxidant effect of vitamin (Zaki and Zaki, 2006).

Date contain zinc and iron, they play important role in reducing nephrotoxic effect of murcury and prevent lipid peroxidation, respec-tively (Robert et al., 1995 and Si-lver and Waldan, 1997). Addition-ally, dates posses copper and co-balt which are promoting hemoato-poiesis (Yourgreatpet, 2004).

The constituents of dates im-prove the general healthy condi-tion of animal and can reduce the adverse effect of mercuric chloride toxicity. Dates today considered as food function (Ali et al., 1999).

I t was concluded that addition of dates to ration reduce adverse

effect of mercuric chloride toxicity on hematological, biochemical, some immunological and patho-logical changes in rats.

REFERENCES Abd-EL-Hamid, A. M. (1994):

“The Scientific Principles for Fish Production and Manage-ment.” 2nd Ed El-Wafa Li-brary, Cairo.

Abdel-Salam, N.A. (1994): “Trea-tment with date-Tamr and Ru-tab. Dar Al-Taliah Press Cairo.

Ali, B.; Bashar, A. and Alhad-

rani, G. (1999): “Reproducti-ve hormonal status of rats treated with date pits.” Food Chemistry, 66: 437-441.

Ali, O. I. and Amin, I. M. (2006): “Toxicological appraisal of some heavy metals level in water of EL-Ibrahemia canal in Beni-Seuf Governorate.” Egyptian J Comparative Pa-thology & Clinical Pathology, 19 (1): 25.

AL-Khouli, M; Hussein, F. and Sid Ahmed, A. (1998): “Analysis of fruits of some Egyptian date Palm cul-tivers.” Proc. Agri. Conf. for Date Palms. Marakesh 16-18: 327-333.

AL-Shahhib, W. and Marshall, R.J. (2003): “The fruit of date palm. Its possible use as the best food for future.” Int. J. Food Sci. Nutri., 54 (4): 247-259.

Bagenstose, L. M.; Mentink-Kane, M. M.; Brittingham, A.; Mosser, D. M. and Mon-esties, M. (2001): “Mercury enhances susceptibility to murine Leishmaniasis.” Para-site Immunol., 23 (12): 633-640.

Bancroft, J.; Stevens, A. and Tu-rnor, D. (1996): “Theory and Practice of Histological Tech-niques.” 4th ed. Chemical Liv-ing Stom, New York, London, Tokyo.

Bernard, F.; Joseph, G and


52

Nemi, C. (2000): “Schalm’s Veterinary Haematology” 5th ed. Lippinocott Williams and Welkens Co. Philadelphia, Blatimore, New York, Lon-don, Hong Kong, Tokyo.

Carlton, W. and McGovin, M. (1995): “Thomson`s Special Veterinary Pathology.” 2nd Ed Mosby Year Book Inc. 11830 Westlene Industrial Drive, St Louis Missouri, 63146.

Diplock, A. T. (1995): “Safety of antioxidant vitamin B and carotene.” Am. J. Clin. Nutr., (Suppl.): 15105.

Drupt, F. (1974): colormetric de-termine of protein. Pharma. Biol., 9: 777.

EL-Boushy, M. E.; Elhamamy, M. M. and Abbas, W. E. (2000): Experimental studies of chronic mercuric chloride toxicity on male albino rats. Egyptian J. Comp. Pathol. & Clinical Pathol., 13: 41-56.

EL-Ghazoli, M and Hussin, F. (2003): Effect of sun drying and mechanical drying on chemical composition of As-wan dry dates. Proc. The In-ternational Conference on Date Palm. In Saudi Ara-bia16-19 Sept., 333-350.

EL-Sharkawy, E. E. and EL-Nesr, N. A. (2008): Protective effect of ascorbic acid and tiron against mercuric chlo-ride induced oxidative stress

and neurotoxicity in rabbit. Assiut Vet. Med. J., 54: 129-158.

Feldman, B. F.; Zenkl, J. C. and Jain, N. C. (2000): "Schalm's Veterinary Haematology" 5th edition, LippencottWilliams Wilkens, Awolters Kluwer Company.

Gamal, I. M. (1991): “Liver and kidney functions in albino rats administrated methyl mercu-ric chloride.” M. D. Clinical Pathology. Collage of Vet. Med. Zagazig University.

Guo, S. Y.; Chen, Z.; Xia, C. and Yuan, J. M. (2004): “Effect of different types polyunsatu-rated fatty acids on immune function and PGE2 synthesis by peripheral blood leuko-cytes of laying hens.” Ani. Feed. Sci. Tech., 116: 249-257.

Hans, M.; Sigfried, S.; Roger, M. and Frank, W. (1999): “Toxi-cology.” Academic Press, Sandi ego, London, Boston, New York, Sydney, Tokyo, Toronto.

Hansen, J.; Zhang, H. and Jones, D. (2006): Differential oxida-tion of thioredoxin-1, thior-doxin-2 and flutathione by metal ions. Free Radic Biol. Medi., 1: 40 (1): 138.

Hamza, S.; Heguzy, A. and Bakry, H. (1994): “Some bio-chemical and pathological ef-


53

fects of mercuric chloride and methyl mercuric chloride in rabbit.” Egypt J. Vet. Sci., 21 (2): 261-271.

Hemdan, N. V. A.; Lehmann, I.; Wichmann, G.; Lehmann, J.; Emmrich, F. and Sack, U. (2007): “Immunomodulati-on of mercuric chloride in vi-tro: application of different cell activation pathways.” Clin. Exp. Immunol., 148 (2): 325-337.

Hovot, O. (1985): “Interpretation of Clinical Laboratory Tests. Determination of Creatinine.” Pp. 220-234, edited by Siest, G.; Hemj, J.; Schiele, F. and Yuong, D. Biochemical publi-cation.

Ibrahim, M.; Abd-allah, G. and Bahnsawy, M. (1991): “Toxic effects of environmental pol-lution by mercury on liver function.” J. Egypt Ger Soc-zool, 6 (A): 189-199.

Ishurd, O.; Sun, C.; Xioo, P.; As-hour, A. and Pan, Y. (2002): “A neutral B-D-glucan from dates of the date palm Phoe-nix dactyliferal.” Carbohy-drate Research, 337: 1225-1325.

Kaneko, J.; Haevey, W. and Brass, L. (1997): “Clinical Biochemistry of Domestic Animals.” 5th ed., Academic Press In San Diego London, New York.

Lawreuce, D. A.; Mitchell, D.

and Rudofsky, U. (1983): “Heavy metal modulation of lymphocytes and macrophage activity.” In Proceeding of the 13th Annual Conference on Environmental Toxicology, AFAMRL-TR-82-101, 63-79 National Technical Informa-tion Service Springfield, VA.

Lecavalier, P.; Vullenevue, C. and Valli, V. (1994): “Combi-ned effects of mercury and hexachlorabenzene in rat.” J. Environ. Sci. Health, 29, 951-961.

Luck, R.; Jeyasealan, I. and An-schales, R. (1995): “Mini re-view: Ascorbic acid and fertil-ity.” Biology Reprod., 52:262-266.

Mahmoud, H.; Mahady, T. and Foun, M. (2003): “Effect of Bagging and fruit theming treatment on yield and fruit quality of Zaghloul date under Aswan condition.” Proc. Of the International Conference on Dat Palm in Saudi Arabia. 16-19 Sept., 249-258.

Mancini, G.; Carbonora, A. O. and Hermans, J. F. (1965): “Immunochemical quantita-tion of antigens by single ra-dial immunodiffusion.” Im-munochem., 2: 235-254.

Mohamed, Z.Y. (2004): “Ultrast-ructural study of blood and tissue cells in laboratory ani-mals exposed to different pol-lutant.” M. V. Sc. Thesis, Fac.


54

Vet. Med., Cairo University Naz, H.; Baig, N.; Haider, S. and

Haleem, D.J. (2008): “Subc-hronic treatment with mercu-ric chloride suppresses im-mune response, elicits behav-ioural deficits and increase brain serotonin and dopamine metabolism in rats.” Pak. J. Pharm. Sci., 21 (1): 7-11.

Oser, B.L. (1979): “Hank’s Phys-iological Chemistry Estima-tion of Sodium and Potas-sium.” 1st ed., 337. McGraw Hill Publishing Company, New York.

Osfar, M. and Ibrahim, S. C. (2000): “Antitoxic effects of certain dietary nutrients aga-inst the contamination with mercuric chloride in New Zealand rabbits.” Egypt J. Comp. Path. & Clinical Path. , 13 (1): 99-113.

Patton, C. and Crouch, S. (1977): Enzymatic determination of urea. Anal. Chem., 49: 464-469.

Petrie, A. and Watson, P. (1999): “Statistics For Veterinary And Animal Science” 1st Ed., pp. 90-99, The Black Well Sci-ence Ltd, United Kingdom.

Puri, A.; Sahai, R.; Singh, K. L.; Saxena, R. P.; Jandon, J. S. and Saxena, K. C. (2000): “Immunostimulant activity of dry fruits and plant materials used in Indian traditional

medical system for mothers after child birth and invalids.” J. Ethnopharmacol., 71 (1-2): 89-92.

Radwanska, K.; Wazniak, F. and Siezieniewska, Z. (1993): “Influence of lead and mer-cury on the histological, ultra-strucural and histochemical picture of the liver of albino rats.” Ann. Univ. Mariae. Cure Sklodoaeska. Med., 48: 141-147.

Reitman, A. and Frankle, S. (1957): “A colorimetric meth-od for the deterimation of seru, glutamic oxaloacetic acid and glutamic pyruvic transminases.” Am. J. Clin. Path., 28: 56-67.

Risher, J. F. and Amler, S. N. (2005): “Mercury exposure: evaluation and intervention, The inappropriate use of che-lating agents in diagnosis and treatment of putative mercury poisoning.” Neurotoxicology 26 (4): 691-699.

Robert, A.; Curtitis, D. and Mi-chael, P. (1995): “Metal Toxi-cology.” 2nd Academic Press, San Diego, New York, Bos-ton, London, Sydney, Tokyo, Toronto.

Sato, K. Kusak, Y.; Zhang, O.; Okado, K. and Muraoka, R. (1997): “Direct effect of inor-ganic mercury on citrate up-take by isolated rat renal


55

brush border membrane vesi-cles.” Ind. Health, 35 (4): 456-460.

Sharma, P.; Shah, A.; Kumar, A. and Islam, F. (2007): “Role of combined administration of tiron and glutathione against aluminum induced oxidative stress in brain.” J. Trace Eel-ment Med. Biol., 21 (1) 63-70.

Shenker, B.J.; Berthold, P.; Ro-oney, C.; Vitale, L.; Debolt, K. and Shapiro, I. M. (1993): “Immunototic effect of mercuric compound of hu-man lymphocytes and mono-cytes. III Alteration in B- cell function and viability.” Im-munopharmacol. Immunototi-col., 15 (1): 87-112.

Silver, S. and Waldan, W. (1997): “Metal Ions and Gene Regula-tion.” Chapma and Hall, New York NY (in Press).

Stacey, N. H. and Kappus, H. (1982): “Cellular toxicity and lipid peroxidation in response to mercury.” Toxicology and Applied Pharmacology, 63 (1): 29-35.

Schltz, L. A. (1987): Methods in Clinical Chemistry. The C V Mosby. Lost Louis, 742-740.

Soltys, J.; Boroskova, Z. and Dvoroznakova, E. (1997): Effects of concurrently ad-ministrated copper and mer-cury on phagocytic cell activ-

ity and antibody levels in guinea pigs with experimental ascariaisis. J. Helminthol., 71 (4): 339-344.

Sonnenwrith, A. C. and Jarette, L. (1980): “Gradwal`s Clini-cal Laboratory Methods and Diagnosis.” 1st ed pp 258-259, CV Mobsy Co, St, Couis To-rontie, London.

SPSS (2002): “Statistical Package for Social Science.” SPSS for windows release 11.0.0, 12 June, 2002 Standard Version Copy right SPSS inc., 1989- 2002.

Tokunako, K.; Ohno, N.; Adachi, Y.; Tanaka, S.; Tamura, H. and Yadomae, T. (2000): “Immunophamacological ac-tivities of ware soluble (1-3) β-D-glucan, CSBG from Can-dia species.” International Im-munopharmacology, 22 (5): 383-394.

Velioglu, M. S.; Mazza, G.; Gao, L. and Oomah, B. D. (1998): “Antioxidant activity and to-tal phenolics in selected fruits, vegetables and grain products. J. Agriculture & Food Chem-istry, 46:4113-4116.

Wand, M. H. and Colin, G. R. (1998): “Fundamentals to Toxicologic Pathology” Aca-demic Press, New York, Syd-ney, Tokyo.

Weir, D.M. (1983): “Immunology: An out line for students of

summary - agenciapyme a. said*; saher, a. galal*; zeinab, y. mohammed** and a. ... pended and slide...

Documents

summary - agenciapyme a. said; saher, a. galal; zeinab, y. mohammed** and a. ... pended and slide...