Protective role of garlic against cadmium toxicity in rats ... · Mohamed, M.M. Metwally* and...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 22 No. 3 (July) 2009; 114 - 140

114

Protective role of garlic against cadmium toxicity in rats: Clinicopa-

thological and histopathological studies

By

Mohamed, M.M. Metwally* and Mohamed, A. Hashem**

*Pathology and **Clinical Pathology Departments, Faculty of Vet. Med.,

Zagazig University, Egypt.

SUMMARY

C admium (Cd) is an environmental and industrial pollutant of growing

concern that adversely affects various organs in human and animal.

This study was carried out to investigate the protective potentials of aque-

ous garlic extract (AGE) on Cd-induced toxicity in rats. Thirty male albino

Wistar rats were randomly divided into five groups. The control group (1)

received double distilled water; Cd group (2) received CdCl2 (1.5 mg/100 g

BW); AGE (0.5 ml/100 g BW) - treated group (3), and Cd plus AGE-

treated group (4). The tested doses were orally given to rats once daily for

4 weeks, but the 5th group was pre-treated with CdCl2 for 4 weeks, and

then treated by AGE for additional 4 weeks.

CdCl2 induced hepato-renal toxicity as indicated by a significant (P

<0.001) elevations in the levels of serum alanine aminotransferase (ALT),

aspartate aminotransferase, alkaline phosphatase (ALP), urea and

creatinine with a significantly (P <0.001) decrease in the levels of

creatinine- clearance in urine, serum proteins (P <0.01), and the activities

of the hepatic and renal antioxidant-enzymes when compared with the con-

trol. A significant (P < 0.05) decrease in the sperm concentration and mo-

tility (%), with increased numbers of dead and abnormal sperm, when com-

pared with the control. Furthermore, Cd induced multiple foci of hemor-

rhage, congestions, edema, coagulative necrosis and mononuclear-cell-

infiltrations in the liver, kidneys and testes. These alterations that associ-

ated with Cd-toxicity were significantly alleviated by garlic administration.

The protection level was better in rats of gp. (4) than gp. (5).

The present study provides evidence that the protective role of garlic

against Cd toxicity could be due to enhanced antioxidant defense and metal

chelating, therefore, garlic could be useful nutritional-supplement for alle-

viating the Cd-induced damage.

Referred byReferred by

Prof. Dr. Mohamed O. El-Shazly Professor of Pathology, Fac. Vet. Med., Cairo University

Prof. Dr. Adel Bakear Kholoussy Professor of Pathology, Fac. Vet. Med., Cairo University


115

INTRODUCTION

T he contamination of food and

feedstuff with heavy-metal-

ions represents problems for human

and animal's health, thus attracting

a world-wide attention (Satarug

and Moore, 2004 and El-Kady et

al., 2009). Cadmium (Cd) has been

involved in historic poisoning epi-

sodes of human and animal popula-

tions as a result of increased an-

thropogenic activities including in-

dustrial uses (such as electroplat-

ing, plastic production, pigments,

battery-manufactures and pesti-

cides), besides lifestyles (Muruga-

vel and Pari, 2007 and El-Kady et

al., 2009). The risk of Cd toxicity is

further increased because of its

long biological half-life (17–30

years), resulting in accumulating

and severe damaging effects, espe-

cially in the liver and kidneys

(Shukla and Kumar, 2009). The

pathobiochemical mechanism of

Cd-induced-cytotoxicity or damage

is mainly via induction of oxidative

stress (Pathak and Khandelwal,

2006 and Suru, 2008). One mecha-

nism by which Cd2+ ions can pro-

duce tissue-injury is the generation

of reactive oxygen species (ROS)

and lipid peroxidation, which in

turn depress hepatic and renal func-

tions (Shaikh et al., 1999 and

Thevenod, 2003). If these ROS

mediated stress events are not bal-

anced by repair processes, the af-

fected cells undergo apoptosis or

necrosis (Thevenod, 2003). The

epididymal-sperm-count is com-

monly used for measuring toxico-

logical lesions of the male repro-

ductive system (Strader et al.,

1996). Cd-exposure is strongly as-

sociated with reproductive toxicity

in both animal and human popula-

tions culminating in infertility and

cancer of the reproductive tissues

(El-Demerdash et al., 2004; Goy-

er et al., 2004 and Akinloye et al.,

2006). The pathogenesis of testicu-

lar damage and sperm-abnorm-

alities induced by Cd exposure is

generally ascribed to oxidative

damage (El-Demerdash et al.,

2004 and Yang et al., 2006). Rats

treated with Cd showed increased

plasma-activities of ALT and AST,

accompanied with a decrease in the

hepatic activities of glutathione, su-

peroxide-dismutase and catalase

(Suru, 2008 and Obioha et al.,

2009). Cd-intoxicated animals sho-

wed multiple foci of hemorrhage,

cloudy swelling, necrosis and

glomerular distension, besides indi-

vidual coagulative necrosis, mono-

nuclear cell infiltration, hepatic si-

nusoidal dilation, degenerative and

necrotic changes in spermatocytes

accompanied with decreased tes-

ticular sperm counts with a signifi-

cant increase in sperm abnormality

(Ahn et al., 1999; Pari et al.,

2007; Jihen et al., 2008 and

Manna et al., 2008).

Several chelating agents and

antagonists reduce the Cd-toxicity,

some of these agents exhibited side


116

effects, especially the medicinal

plants. Garlic (Allium sativum) is

versatile vegetable which is often

used in many dishes for flavor,

aroma and taste enhancement

(Stajner and Varga, 2003). It is a

good source of dietary phytochemi-

cals with proven antioxidant prop-

erties and ability to modulate the

detoxification systems (Nuutila et

al., 2003; Stajner and Varga, 2003

and El-Demerdash et al., 2005).

These functional effects could be of

great importance for their use in the

prevention and treatment of several

diseases (Lau, 1998; Banerjee et

al., 2001 and Ola-Mudathir et al.,

2008). The therapeutic and medici-

nal values of garlic are the subjects

of many researches. It has anticar-

cinogenic, hepatoprotective, antidi-

abetic, anti-platelet aggregation,

anti-biotic effects and may also act

as antidote for heavy metal poison-

ing (Agarwal, 1996; Lau, 1998

and El-Demerdash et al., 2005).

T his study was designed to

evaluate the protective effects

of garlic against the cadmium-

induced biochemical changes, al-

terations in sperm characteristics

histopathological alterations in

liver, kidney and testes.

MATERIAL AND METHODS

Preparation of extracts:

Garlic bulbs were purchased

from the local Market in Zagazig,

Egypt. Its botanical identification

was confirmed at the Field Crop

Department, Faculty of Agriculture,

Zagazig University, Egypt. Garlic

extraction was performed accord-

ing to Flora et al. (2009). Briefly,

garlic-bulbs were carefully un-

dressed, then crushed with distilled

water in a mixer (100 ml of dis-

tilled water per 100 g of garlic).

The resultant slurry was squeezed

through a double cheese cloth and

the filtrate was quickly frozen (0-

4°C) until used.

Animals and experimental de-

sign:

Thirty clinically healthy adult

male albino Wistar rats weighing

150 -200 g were obtained from the

breeding stock of Faculty of Veteri-

nary Medicine, Zagazig University,

Egypt. They were kept under stan-

dard laboratory conditions with ad

libitum access to food and water in

well-ventilated cages made of gal-

vanized zinc plates. All the animal

experiments were conducted in ac-

cordance with the Ethical Norms

on Animal Care and Use approved

by Faculty of veterinary Medicine,

Zagazig University, Egypt. Rats

were randomly divided into five

equal groups and were treated ac-

cordingly (Table, 1).


117

Table 1. Experimental design:

Groups Treatment

1 Only vehicle (double distilled water) (0.5 ml/100 g BW, once daily for 4 weeks)

2 Cd* only (CdCl2 in H2O) (1.5 mg/100 g BW, once daily for 4 weeks)

3 Aqueous garlic extract, 0.5 ml/100 g BW, once daily for 4 weeks

4

Co-administration of Cd (CdCl2 in H2O) (1.5 mg/kg BW) and garlic extract (0.5

ml/100 g) once daily, for 4 weeks.

5

Cd (1.5 mg/kg BW once daily for 4 weeks), followed by garlic extract (0.5

ml/100 g BW, once daily, for additional 4 weeks)

*Cd Chloride (CdCl 2 in H2O) 1000 mg Cd was obtained from Merck

Ltd., Cairo, Egypt.

The doses of garlic extract and CdCl2 administered by gavage and were

selected according to Ola-Mudathir et al. (2008).

Serum and tissue collection:

Blood samples were collected

from the retro-orbital venous-

plexus under diethyl ether anesthe-

sia at the end of the experimental

period and left to clot. The sera

were separated by cooled centrifu-

gation and stored at −20 °C until

analysed. The liver and kidneys

were immediately removed and

washed with chilled saline-

solution. Tissues were minced and

homogenized in ice-cold 1.15%

KCl (1g tissue/3 ml) in a Potter–

Elvehjem type homogenizer. The

homogenate was centrifuged at

5000 ×g for 20 min at 4 ºC, and

the resultant supernatant was used

for antioxidant enzyme assay

(Suru, 2008). Specimens were col-

lected from the liver, kidneys and

testes and immediately fixed in

10% buffered neutral formalin so-

lution. Five-micron thick paraffin

sections were prepared, stained by

Hematoxyline and Eosin and ex-

amined microscopically (Bancroft

and Stevens, 1996).

Biochemical analysis:

A-Measurement of hepatic pa-

rameters:

The activities of alanine

transaminase (ALT) and aspartate

transaminase (AST) were esti-

mated according to Reitman and

Frankel (1957). Serum alkaline

phosphatase activity was measured

according to Bowers and

McComb (1972). The serum total


118

protein (TP) was determined ac-

cording to Henry et al. (1974). Al-

bumin concentration was deter-

mined by the method of Doumas

et al. (1977). Globulin concentra-

tion was calculated as the differ-

ence between the total protein and

albumin.

B-Measurement of renal pa-

rameters:

The levels of creatinine and

urea, in serum, were estimated

spectrophotometrically, using com-

mercial diagnostic kits (Sigma Di-

agnostics (I) Pvt Ltd, Cairo,

Egypt). Creatinine- clearance as an

index of glomerular filtration rate

was calculated from serum

creatinine and a 24 h urine sample

creatinine levels.

C-Measurement of antioxidant

enzymes:

The protein content of the ho-

mogenized liver and kidneys was

estimated by the method of Lowry

et al. (1951), using the bovine se-

rum albumin as a standard. The ac-

tivities of the superoxide dismutase

(SOD), catalase (CAT) and glu-

tathione peroxidase (GPx) were

measured according to the methods

of Misra and Fridovich (1972),

Sinha (1971) and Paglia and Val-

entine (1967), respectively. The en-

zyme levels were determined using

commercial test kits according to

the manufacturer’ instructions.

Epididymal sperm characteris-

tics:

The epididymal spermatozoa

were obtained by mincing the right

and left epididymides with scissors

and scalpels into small pieces and

homogenized in a warmed Petri

dish, containing 5 ml physiological

saline and incubated at 37oC for 2

min (Linder et al., 1986). The sus-

pension was stirred, one drop was

placed on a warmed microscope

slide, and a 22 x 22 mm coverslip

was placed over the droplet. At least

10 microscopic fields were observed,

at 400 x magnification, using a light

microscope, and the percentage of

motile sperm was recorded. Other

slides were prepared and stained

with 1% eosin B and 5% nigrosine

in 3% sodium citrate dehydrate solu-

tion and examined at 400x magnifi-

cation. A total of 400 sperm cells

were counted for the assessment of

the live, dead and abnormal sper-

matozoa. The same suspension was

used for sperm counting, using a

Neubauer hemocytometer. Three

counts per sample were averaged

(Strader et al., 1996).

Statistical analysis:

The data were expressed as

means ±standard errors (SE). Differ-

ences between group means were

estimated using a one-way analysis

of variance (ANOVA) and the Dun-

can's Multiple Range Test was done

for multiple comparisons using the

SPSS 12.0 for Windows. Results


119

were considered statistically signifi-

cant at P <0.05.

RESULTS

Biochemical studies:

Cd - intoxicated rats, (gp. 2),

showed severe deviations in the he-

patic and renal parameters (Table,

2). A significant (P <0.001) in-

crease in the serum ALT, AST,

and ALP activities, besides a sig-

nificant (p <0.01) decrease in the

serum proteins (TP, albumin and

globulins) were found, when com-

pared with the control. The serum

enzymes were reduced and pro-

tein-profile was increased in gp.

(5). Gp. (4) showed that the ele-

vated parameters were closed to

the normal values. No significant

change in any of the parameters

was observed in gp. (3). A signifi-

cantly (P <0.001) increased level of

serum urea and creatinine with a

significantly (P <0.001) decreased

level of creatinine- clearance was

observed in gp. (2). Co-

administration of Cd and garlic ex-

tract gp. (4) did not affect the serum

urea and creatinine levels and

closely restored the level of

creatinine-clearance to the control

level. Gp. (5) showed a significant

(p <0.05) increase of serum urea,

without any significant change in

the serum creatinine and creatinine-

clearance levels when compared

with the control.

The activities of the hepatic

SOD, CAT and GPx were signifi-

cantly (p<0.001) reduced in rats of

gp. (2) as shown in table (3). The

activities of the aforementioned

enzymes were significantly (P <

0.05) enhanced in gp. (3). Chal-

lenging of garlic-treated rats with

CdCl2 gp. (4) was associated with

activation of the antioxidant en-

zymes towards the control levels.

However, treatments of CdCl2-

intoxicated rats with garlic extract

gp. (5) significantly (P<0.05) in-

creased the hepatic CAT activity

when compared with gp. (2), but

did not return to the normal level

of control.

The activities of the kidney

SOD, CAT and GPx were signifi-

cantly (P < 0.001) reduced in rats

of gp. (2) as shown in table (4).

Treatment with garlic extract alone

gp. (3) tended to significantly (P <

0.05) enhance the activities of

SOD, CAT and GPx when com-

pared with the control. Administra-

tion of garlic extract in combina-

tion with or following Cd signifi-

cantly (P < 0.01) increased the

level of these enzymes when com-

pared with Cd-intoxicated rats.


120

Table 2. Effect of garlic extract on some hepatic and renal parameters of

Cd-intoxicated rats.

Groups 1 2 3 4 5

ALT(IU/dl) 8.85 ±

1.29 c

24.80 ±

1.95 a

8.94 ±

1.40 c

9.23 ±

1.33 c

9.92 ±

1.60 bc

AST(IU/ dl) 22.10

±2.71 c

27.50 ±

2.85 a

21.64

±3.01 c

22.50 ± 3.

12 c

23.11±3

. 45 bc

ALP (IU/ dl) 19.54

±2.17 c

37.53 ±

3.44 a

19.24

±2.33 c

21.62 ±

2.50 c

22.00

±3.10 bc

TP (g/dl) 8.20 ±

1.33 a

5.10 ±

1.95 c

8.14 ±

1.40 a

8.11 ±

1.33 a

7.90 ±

1.60 ab

Albumin (g/dl) 4.70±

1.00 a

3.00±

0.55 c

4.51±

1.12 a

4.60 ±

1.30 a

4.30±

1.02 ab

Globulins (g/dl) 3.50±

0.85 a

2.20±

0.43 c

3.63±

1.04 a

3.41±

1.11 a

3.20±

1.00 ab

Urea (mg/ dl) 37.00

±4.13 c

62.23 ±

3.44 a

36.00

±3.80 c

40.00 ±

2.50 bc

45.4 ±

3.73 b

Creatinine (mg/

dl)

0.67 ±

0.15 c

1.2 ±

0.30 a

0.66 ±

0.20 c

0.69 ±

0.17 c

0.74 ±

0.23 bc

Creatinine clear-

ance (ml/min)

0.42±

0.03a

0.28±

0.01 c

0.40 ±

0.20 a

0.37 ±

0.17 ab

0.35±

0.23 ab

Values are expressed as means ±SE; n = 6 for each treatment group.

Groups indicated with different superscript letters (a, b, c, d) in the same

line are statistically significant (P < 0.05).


121

Table 3. Effect of Cd and garlic extract on the activities of the antioxidant

enzymes and protein levels in the liver of rats. Values are ex-

pressed as means ±SE; n = 6 for each treatment group.

Groups

Parameters

1 2 3 4 5

Protein (mg/ml ho-

mogenate)

27.50 ±

4.25

26.70 ±

3.77

28.12 ±

4.46

27.40 ±

3.60

27.11 ±

4.82

SOD (U/mg pro-

tein)

3.61 ±

0.25 b

1.15 ±

0.16 d

3.93 ±

0.43 a

3.40 b

±0.55

3.14 ±0.47

bc

CAT (µg/mg tis-

sue)

1.33 ±

0.12 b

0.83 ±

0.01 d

1.62 ±

0.15 a

1.15 ±0.55

bc

1.00 ±0.47

c

GPx (U /mg tis-

sue)

0.90 ±

0.05 b

0.42 ±

0.01 d

0.99 ±

0.04 a

0.86±

0.05 bc

0.80±

0.04 bc


line are statistically significant (P<0.05).

Table 4. Effect of Cd and garlic extract on the activities of the antioxidant

enzymes and protein levels in the kidney of rats. Values are expressed as

means ±SE; n = 6 for each treatment group.

Groups

Parameters

1 2 3 4 5

Protein

(mg/ml homogenate)

26.50 ±

3.12

23.70±

5.00

25.00 ±

3.88

24.13±

4.16

23.11 ±

3.34

SOD (U/mg protein) 1.68 ±

0.44 b

0.65 ±

0.27 d

1.80 ±

0.81 a

1.53 ±

0.65 bc

1.50 ±

0.53 bc

CAT (µg/mg tissue) 0.92 ±

0.14 b

0.44 ±

0.01 d

0.99 ±

0.15 a

0.87 ±

0.55 bc

0.81 ±

0.47 bc

GPx (U /mg tissue) 1.03 ±

0.05 b

0.50 ±

0.01 c

1.14 ±

0.04 a

1.00 ±

0.05 b

0.97 ±

0.04 b




122

Sperm characteristics:

Treatment of male rats with

CdCl2 gp. (2) caused a significant

(p < 0.05) decrease in sperm con-

centration and motility (%). The

dead and abnormal sperms were

increased when compared to the

control table (5). Treatment with

garlic-extract alone, gp. (3) or in

combination with CdCl2 gp. (4)

caused more significant (P < 0.05)

increase in semen quality, and

minimized the toxic effects of

CdCl2 than when Cd-intoxicated

rats were treated with garlic extract

gp. (5).

Table 5. Effects of garlic extract on epididymal sperm concentration

(Sperm conc., 106/ml), motility (%), dead (%) and abnormal

sperm (%) of Cd-intoxicated male rats. Values are expressed as

means ±SE; n = 6 for each treatment group.

Groups

Parameters 1 2 3 4 5

Sperm conc. 80.50 ±

6.44 a

44.60 ±

4.33 c

80.94 ±

7.54 a

76.13 ±

6.13 ba

63.55 ±

6.40 b

Motility 75.46 ±

4.00 b

35.00 ±

1.33 d

81.0 ±

5.41 a

75.40 ±

3. 12 b

70.0 ±

3. 15 bc

Dead 22.50 ±

1.10 c

47.60 ±

2.50 a

10.12 ±

1.10 d

27.55 ±

2.50 b

30.00 ±

3.10 b

Abnormal 12.40 ±

1.30 c

20.00 ±

1.70 a

8.0 ±

0.49 c

12.70 ±

1.33 b

15.30 ±

1.60 b



Histopathological results:

The light microscopic exami-

nation revealed a normal histologi-

cal structure of all organs in the

control gp. (1).

Animals treated with Cd gp.

(2) showed enlarged hepatocytes,

with severe hydropic degeneration

and coagulative necrosis (Fig. 1).

Fatty change was frequently ob-

served accompanied with conges-

tions and leukocytic infiltrations in

the portal areas (Fig. 2). Conges-

tions of the central veins and mul-

tiple hemorrhagic foci were ob-

served in many cases. Sinusoidal


123

dilatation was also evident (Fig. 3).

The kidneys showed congestion,

multiple hemorrhagic foci, accom-

panied with focal interstitial mono-

nuclear-cell-aggregations (Fig. 4).

The renal tubular epithelium

showed hyaline droplet formation,

cloudy swelling and coagulative

necrosis. These changes were ac-

companied with numerous swollen

glomeruli with proliferation of the

mesangial cells and presence of

eosinophilic material in the lumen

of some renal tubules (Figs. 5 &

6). Furthermore, numerous epithe-

lial casts, and tubular dilatation

were evident. The tests showed de-

generation and necrosis of some

seminiferous tubular epithelium,

with defoliation of many sperma-

tocytes into the lumen of seminif-

erous tubules. This was accompa-

nied with congestion and oedema

in the interstitial tissue (Fig.7).

Moreover, multiple hemorrhagic

foci were frequently noticed in the

interstitial tissue. The tail of the

epididymis showed marked deple-

tion in the numbers of spermato-

zoa. In addition some tubules con-

tained homogenous pink necrotic

particles with absence of sper-

matozoa (Fig. 8).

There was no significant dif-

ference in the histology of the

liver, kidneys and testes between

gp. (3) and gp. (1). However, the

hepatic tissues of the garlic-

treated-group showed few eosino-

philic cells infiltrations in the por-

tal areas (Fig. 9).

The co-administration of gar-

lic with Cd gp. (4) remarkably al-

leviated the lesions, induced by

Cd, where the hepatic coagulative

necrosis was absent and the con-

gestions and sinusoidal widening

was markedly reduced, but few

slightly enlarged hepatocytes with

light cytoplasm were observed

(Fig. 10). The portal areas showed

slight congestion and were infil-

trated with few mononuclears. The

kidnyes showed few swollen

glomeruli and minimal prolifera-

tion of the mesangial cells. The

number of the mononuclears, in

the interstitial tissue, became

scanty, but the congestion was ob-

vious (Fig. 11). Cloudy swelling

and vacuolar degeneration were

observed in the epithelium of some

proximal and distal convoluted tu-

bules. The testes of all animals in

this group were structurally similar

to the control, with only mild inter-

stitial oedema and few degenerated

spermatocytes (Fig. 12). The tails

of the epididymides were filled

with intact spermatozoa (Fig. 13).

Treatment of Cd-intoxicated

rats with garlic extract gp. (5) re-

sulted in a moderate improvement

in the lesions induced by Cd, but it

did not regain the control, as the

liver which revealed numerous

swollen hepatocytes with hydropic


124

degeneration accompanied with

slightly congested central veins

and mild sinusoidal dilatation.

Moreover, coagulative necrosis

was scanty and the portal areas

were infiltrated with moderate

numbers of leukocytes (Fig. 14).

The kidneys showed few swollen

glomeruli with proliferated mesan-

gial cells together with congested

capillaries and numerous minute

hemorrhagic foci (Fig. 15). Some

renal tubules showed cloudy swell-

ing and hyaline droplet formation

but coagulative necrosis was mini-

mal. The interstitial tissue revealed

a moderate number of mononu-

clear cells. The testes revealed

mild congestions, few round cell

infiltration and interstitial oedema,

accompanied with a mild degen-

eration of the germinal epithelium

(Fig. 16). The epididymis revealed

mild congestion and few leuko-

cytes in the interstitial tissue ac-

companied with mild depletion in

the spermatozoa. The histopa-

thological findings were graded in

tables (6, 7 and 8).

Table (6): Histopathological findings in liver of rats of different groups.

Groups

Lesions 1 2 3 4 5

Congestion and sinusoidal

dilatation - ++++ - ++ ++

Vacuolar and hydropic degen-

eration - ++++ - ++ +++

Fatty change - +++ - + ++

Apoptosis - +++ - + +

Mononuclear cell infiltrations - +++ + + ++

Hemorrhagic foci - ++ - - +


125

Table (7): Histopathological findings in kidneys of rats of different groups.

Groups

Lesions

1 2 3 4 5

Congestion - ++++ - + ++

Cloudy swelling - ++++ - + ++

Vacuolar and hydropic degen-

eration

- ++++ - + ++

Hyaline droplet formation - +++ - + ++

Hyaline and cellular casts - +++ - + +

Tubular necrosis - +++ - - +

Mononuclear cell infiltrations - +++ - + ++

Hemorrhagic foci - ++++ - + ++

Table (8): Histopathological findings in testes of rats of different groups.

Groups

Lesions

1 2 3 4 5

Congestion - ++++ - ++ ++

Tubular necrosis - ++++ - - +

Desquamations of spermato-

cytes

- +++ - - +

Interstitial edema - +++ - + ++

Mononuclear cell infiltrations - +++ - + +

Hemorrhagic foci - +++ - - +

(-) absence of the change in all rats of a group, (++++) a change often

found in all rats of a group, (+++) a change observed in almost all rats of

a group, (++) a change not so often observed in all rats of a group and (+)

a change which was rare within a group.


126

Figs. (1-4):

1- Liver of rat gp. (2) showing enlargement of the cell sizes, with severe hy-

dropic degeneration (arrow) and individual coagulative necrosis (arrow

heads), H&E., X.400.

2- Liver of rat gp.( 2) showing fatty change (arrow head), with congestion

(C), and leukocytic infiltrations (arrows), in the portal areas, H&E.,

X.400.

3- Liver of rat gp. (2) showing sinusoidal dilatation (arrows), H&E., X.400.

4- Kidney of rat gp. (2) showing congestions (C), hemorrhages (H), and fo-

cal interstitial mononuclear inflammatory cell aggregations (arrow),

H&E., X.400.


127

Figs. (5-8):

5- Kidney of rat gp. (2) showing cloudy swelling (arrow), and coagulative

necrosis (arrow heads), of the tubular epithelium, H&E., X.400.

6- Kidney of rat gp.(2) showing hyaline droplet formation (arrow head),

swollen glomeruli with proliferated mesangial cells (arrow), and presence

of eosinophilic material in the lumen of some renal tubules (E), H&E.,

X.400.

7- Testis of rat gp.(2) showing congestion (C), interstitial oedema (E), and

defoliation of many spermatocytes into the lumen of seminiferous tubules

(arrow ), H&E., X.400.

8- Tail of epididymis of rat gp. (2) showing marked depletion in the numbers

of spermatozoa (arrow), with presence of homogenous pink necrotic par-

ticles with absence spermatozoa in some tubules (arrow heads), H&E.,

X.50.


128

Figs. (9-12):

9- Liver of rat gp. (3) showing showed few eosinophilic cells infiltrations

(arrow), in the portal areas, H&E., X.400.

10- Liver of rat gp. (4) showing slightly enlarged hepatocytes with light cy-

toplasm (arrow),and marked reduction in the congestions (C), and sinu-

soidal dilatation (arrow head), H&E., X.400.

11- Kidney of rat gp. (4) showing scanty numbers of mononuclear cells in

interstitial tissue (arrow head),with marked congestion (C), of the renal

blood, H&E., X.50.

12- Testis of rat gp. (4) showing mild interstitial oedema (arrow), and few

degenerated spermatocytes (arrow head), H&E., X.400.


129

Figs. (13-16):

13- Tail of pididymis of rat gp. (4) showing epididymal tubules filled with

spermatozoa (arrows), H&E., X.100.

14- Liver of rat gp. (5) showing numerous swollen hepatocytes with hy-

dropic degeneration (arrow), scanty individual coagulative necrosis

(arrow heads), slightly congested central veins (C), and moderate portal

leukocytic infiltrations (L),), H&E., X.100.

15- Kidney of rat gp. (5) showing few swollen glomeruli with proliferated

mesangial cells (arrow heads), together with congested capillaries (C),

and numerous minute hemorrhagic foci (H), H&E., X.400.

16- Testis of rat gp. (5) showing mild congestion (C), and edema (arrow) in

the interstitial tissue accompanied with mild degeneration of the germi-

nal epithelium (arrow head), H&E., X.400.


130

DISCUSSION

C admium is a poisonous metal

that is widely used in differ-

ent industries. It promotes an early

oxidative stress and afterward con-

tributes to the development of seri-

ous biochemical, spermiotoxic and

pathological conditions because of

its long retention in some tissues.

The treatment strategies for Cd-

toxicity include chelation and anti-

oxidant therapies (Obioha et al.,

2009). The antagonism of the gar-

lic to Cd- toxicity was evaluated

on the basis of biochemical altera-

tions, alterations in sperm charac-

teristics and histopathological find-

ings in liver, kidneys and testes.

Our results indicated that Cd-

intoxication caused a significant in-

crease in the serum ALT, AST, ALP,

urea, creatinine and a decrease in

serum TP, albumin and globulins.

This was accompanied with a de-

crease creatinine-clearance levels in

urine and a decline in the levels of

SOD, CAT and GPx in the hepatic

and renal tissues. Similar results

were reported (El-Demerdash et

al., 2004; Pari and Murugavel,

2005; Chanta et al., 2007 and

Yadav and Khandelwal, 2009). A

key finding of the present study is

the significant increase in the mor-

phologically abnormal sperms and

dead sperms coupled with substan-

tial decrease in sperm count and

motility in rats intoxicated with Cd.

The same results were previously

reported (WHO, 1992; Yang et al.,

2006 and Ola-Mudathir et al.,

2008).

The disturbances in the activi-

ties of the enzymes (ALT, AST,

ALP) with serum urea and

creatinine, as well as creatinine-

clearance levels in urine repre-

sented biomarkers for hepatic and

renal damage (Chater et al., 2009;

El-Kady et al., 2009; Shukla and

Kumar, 2009). Furthermore, the

administration of Cd (3 mg/kg

BW/day) in rats for 3 weeks in-

duced renal damage, which was

evident by significantly increased

levels of serum urea and creatinine

with a significant decrease in

creatinine- clearance in urine (Pari

and Murugavel, 2005; Pari et al.,

2007).The decrease in serum pro-

teins may be due to the increased

excretion of high molecular weight

protein (proteinurea) (El-Deme-

rdash et al., 2004 and El-Kady et

al., 2009). Furthermore, the in-

creased serum creatinine and low-

ered creatinine-clearance points

out to renal damage (Jarup, 2002).

The decreased activities of the

antioxidants (SOD, CAT and GPx),

in the hepatic and renal tissues, indi-

cated a failure of the antioxidant-

defense-system to overcome the in-

flux of ROS on Cd. Our results

agree with Pari et al. (2007). The

significant decrease in the activities

of SOD and CAT in the Cd group


131

may be attributed to a direct inhibi-

tory effect on SOD and CAT activi-

ties via Cd–enzyme interaction

(Patra et al., 1999 and Casalino et

al., 2002). The GPx depletion may

be due to its enhanced utilization to

conjugate Cd, counteract ROS and

lipid peroxidative products (Singhal

et al., 1987). Similarly, Jurczuk et

al. (2004) reported that the exposure

to 50 mg Cd/l drinking water led to

a decrease in the activities of SOD

in the liver and CAT in the liver and

kidney, and an increase in the kid-

ney activity of SOD.

The gonadotoxic and spermi-

otoxic effects of Cd may be due to

a direct effect on the testes and

epididymides or by altering the

post-testicular events such as

sperm-progress, motility and/or

viability, which may culminate in

hypogonadism and infertility

(WHO, 1992 and Akinloye et al.,

2006). The observed spermato-

genic damage is consistent with

the report of El-Demerdash et al.

(2004) in rats and Acharya et al.

(2008) in mice. Generation of re-

active oxygen species (ROS) by

Cd-toxicity and consequent oxida-

tive damage may increase the mei-

otic errors and sperm deformation

(Acharya et al., 2008). The de-

creased sperm counts may be a di-

rect outcome of increased and con-

sistent lipid peroxidation (LPO)

and altered membrane properties

that led to germ cell death at dif-

ferent stages of development (Hew

et al., 1993). Increased sperm me-

mbrane LPO has been shown to

impede sperm progress motility,

and increase percent of the total

sperm abnormalities and cause a

dramatic loss in the fertilizing po-

tential of sperm (Sharma and Ar-

gawal, 1996 and El-Demerdash

et al., 2004). Cd disrupts the tight-

junctions between the Sertoli cells

and alters the Sertoli-germ cell-

adhesion, with consequent exfolia-

tion of immature cells into the lu-

men of seminiferous tubules and,

in turn, leads to a reduction of vi-

able sperm count in the epidi-

dymides (Hew et al., 1993).

Also, Cd intoxication resulted

in several structural changes in the

liver, kidneys and testes, where the

hepatic cells suffered vacuolar and

hydropic degenerations, fatty

change and coagulative necrosis.

The renal tubular epithelium

showed hyaline droplet formation,

cloudy swelling and coagulative

necrosis. The seminiferous tubules

revealed severe degeneration and

necrosis with partial loss of sper-

matogenic cells and the epidi-

dymides showed marked depletion

in the numbers of spermatozoa.

Moreover, leukocytic infiltration

and multiple hemorrhagic foci

were frequently observed in the

liver, kidneys and testes. Many in-

vestigators have noted similar or

more pronounced changes in the

hepatic, renal and testicular tissues


132

under Cd effect (Ahn et al., 1999,

Koyu et al., 2006, Jihen et al.,

2008, Pari et al., 2007 and Manna

et al., 2008). These lesions are

mainly due to oxidative stress

where the cytosolic Cd interacted

with mitochondria, peroxisomes,

and microsomes, resulting in ex-

cessive generation of ROS capable

of depleting endogenous antioxi-

dant status and causing oxidative

damage to biomolecules such as

membrane lipids, DNA and a vari-

ety of transport proteins, including

Na+/K+-ATPase, which results in

cell-death and organ-dysfunction

(Shaikh et al., 1999; Thevenod

and Friedmann, 1999; Tang and

Shaikh, 2001 and Wang et al.,

2004).

Rats received garlic alone gp.

(3) did not show any biochemical

or structural lesions with the ex-

ceptions of few leukocytic infiltra-

tions in the portal areas. Previ-

ously, several reports indicated that

treatment with garlic did not show

any toxicological or physiological

effects in rats (Ola-Mudathir et

al., 2008) and mice (Flora et al.,

2009). However, garlic administra-

tion significantly enhanced the ac-

tivities of SOD CAT and GPx in

the hepatic and renal tissues, and

improved the semen quality. These

benefits are mainly attributed to

the antioxidative activities of gar-

lic (Zeng et al., 2008) and sug-

gested that garlic influences the

host-detoxification processes (Das

and Saha, 2009).

The results showed that the he-

patic, renal and testicular protection

by garlic against Cd-induced toxic-

ity was evident, particularly when

administered in combination with

Cd. The alterations in the serum lev-

els of ALT, AST, ALP, proteins, urea

and creatinine, besides the creat-

inine- clearance levels in urine in-

duced by Cd were close to the nor-

mal values by the garlic extract ad-

ministration, especially in gp. (4).

The lowered trend in liver and renal

functional parameters implicated

that garlic alleviated the hepatic and

renal damage. Similar results were

reported by Nakagawat et al.

(2006) and Flora et al. (2009).

The levels of antioxidant en-

zymes (SOD, CAT and GPx), in the

hepatic and renal tissues, were in-

creased by garlic treatment, how-

ever, the hepatic CAT activity did-

n’t restore to the control level, es-

pecially in gp. (5). Similar results

were reported by (Nuutila et al.,

2003 and El-Beshbishy, 2008). The increase in these enzymes re-

duced the mitochondrial-generated-

reactive radicals from causing oxi-

dative stress and cellular damage

(Flora et al., 2009). The antioxida-

tive activities of garlic could be re-

lated to its contents of cysteine-

containing bioactive compounds,

diallyl sulfur compounds, seleno-


133

compounds and flavonoids (quer-

cetin, allixin, anthocyanins, and

kaempferol), which are known to

exert antioxidant effects (Banerjee

and Maulik, 2002). Moreover, it is

possible that garlic extracts could

have spared the consumption of

GPx, SOD, and CAT occasioned by

Cd-induced oxidative stress, and

enhance the endogenous antioxi-

dant status of the liver and kidneys

(catalase and SOD) (Banerjee et

al., 2001 and Massadeh et al.,

2007). The testicular protection

could be due to the antioxidant

properties of garlic (Nuutila et al.,

2003; Murugavel and Pari, 2007).

Also, the protective effects of garlic

may be related to their ability to

chelate/sequester Cd via formation

of Cd–flavonoid complexes

(Massadeh et al., 2007). Moreover,

Garlic is well documented for the

attenuation of oxidant- mediated

renal, hepatic and testicular tissue

damage induced by various agents

(Kabasakal et al., 2005; Pari et

al., 2007; suru 2008 and Obioha

et al 2009). Also garlic significan-

tly protected the testes, epididym-

ides and spermatozoa against Cd-

toxicity by reversing these altered

parameters and increasing the

semen quality. Rats exposed to Cd

for 4 weeks, garlic was effective in

ameliorating Cd induced bioche-

mical and morphological changes

in the liver, kidneys and epididymal

sperm (Yadav and Khandelwal,

2008). The histopathological results

were coincided with the above

findings, where the hepatic

coagulative necrosis was absent

and the congestions, sinusoidal

widening and leukocytic

infiltrations were markedly

reduced. The kidneys showed few

swollen glomeruli and proliferation

of the mesangial cells with minimal

mononuclear cells in the interstitial

tissue. Moreover, the testes of

group (4) were structurally similar

to the control with the exception of

only mild interstitial edema and

few degenerated spermatocytes.

The epididymal tubules were filled

with spermatozoa. These results are

in agreement with Pari et al.

(2007), Jihen et al. (2008), Manna

et al. (2008) and Ola-Mudathir et

al. (2008)

I t could be concluded that the

intoxication by Cd resulted in

severe toxic effects on the liver,

kidneys and testes. Garlic supple-

ment counteracted these toxic ef-

fects, thus it is recommended to

supplement the diet with garlic,

particularly in areas where Cd-

contamination is expected.

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