Full Text - IDOSI Publications

Global Journal of Pharmacology 8 (4): 703-708, 2014
ISSN 1992-0075
© IDOSI Publications, 2014
DOI: 10.5829/idosi.gjp.2014.8.4.9457
Effect of Lead on Semen Characteristics and Some Enzymatic Activities in Serum
and Semen of Rabbits with A Reference to the Protective Effect of Vitamin C
G.A. Ali, W.S. El-Nattat and H.H. El-Khadrawy
Department of Animal Reproduction and A.I., National Research Centre, Dokki, Giza, Egypt
Abstract: The effect of lead acetate administration on testicular, hepatic and renal functions and the biomarker
effect for them were investigated in the present study with a trial of treatment by vitamin C. A total number of
35 rabbit bucks was divided into five groups. One served as the control group and four groups received low
and high doses of lead acetate (10.8 and 15 mg/kg. b. wt. respectively) orally. One low and one high
group received, in addition, 1 g vitamin C /L in drinking water. Superoxide dismutase (SOD), -glutamyl
transferase ( -GT), Aspartate amino transferase (AST), Alanine amino transeferase (ALT), cholinesterase,
acid phosphatase and Lactate dehydrogenase (LDH) activities were measured in both serum and semen. Also,
semen characteristics were determined. Results revealed that all the investigated enzymes (SOD, LDH, ALT and
acid phosphatase activities) in serum and semen were obviously affected by lead. Vitamin C was a good
antioxidant that recuperates the normal enzymatic status in both serum and semen. In conclusion, lead led to
testicular hypofunction, which is supported by the result of semen picture. The hazardous effect of lead led to
disturbance in the activities of enzymes under investigation such as SOD, -GT, LDH, AST, ALT,
cholinesterase and acid phosphatase. Vitamin C proved its antioxidant effect on recuperating the normal status
of enzymes in serum and semen. LDH and prostatic acid phosphatase are shown to be biomarker of testicular
dysfunction, while LDH, ALT may be used as biomarkers for hepatic and renal dysfunctions.
Key words: Lead
Vitamin C
Antioxidant
Male
Rabbits
INTRODUCTION
acid phosphatase [11]. So these enzymes are used as
biomarkers for approaching good diagnosis in case of
vital organs dysfunction. On the other hand, there is
another category of enzymes,
which
act on
superoxide radicals to eliminate its hazardous effects.
The inhibition of these enzymes by lead causes the
increase of superoxide radicals O2- and its
accumulation inside the cell leading to its death [12].
Vitamins A, E, D and C were recorded to have antioxidant
activities. Their antagonistic effects to superoxide
accumulation varied from one vitamin to another. It has
been found that levels of lead in serum is decreased due
to reduced absorption not due to increase excretion after
1000 mg vitamin C [13].
In this study, vitamin C was used to investigate its
antioxidant activity for decreasing the inhibitory effect of
lead acetate on serum and semen enzymes in New Zealand
rabbits and monitoring the suitable biomarker for its effect
on the vitality of the male.
Lead is one of the most toxic metals that induce wide
range of behavioral, bio-chemical and physiological
dysfunctions in human and animals [1]. A biomarker is a
term which is used to include, almost, any measurement
reflecting an interaction between a biological system and
environmental agent, which may be chemical, physical
or biological in nature. The identified biomarkers are three
classes; biomarkers of exposure, effect and susceptibility
[2].
Lead toxicity is retarding factor for the vitality
of reproductive and internal organs of all animals.
The effects of lead toxicity on reproductive organs
were screened in mature female and male rabbits [3, 4],
rats [5] and in human [6, 7, 8]. These effects were recorded
to induce leakage of enzymes from other vital
organs, as AST, ALT [9], aminoleviulinic acid
dehydrogenase (ALA-d) activities [10], ALT and total
Corresponding Author: Gamal A. Ali, Department of Animal Reproduction and A.I., National Research Centre,
Postal Code: 12622, Dokki, Giza, Egypt. E-mail: [email protected].
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Global J. Pharmacol., 8 (4): 703-708, 2014
MATERIALS AND METHODS
Enzymatic Analysis in Blood and Semen
In Erythrocytes: SOD is analyzed in whole blood using
chemical kits obtained from Randox, UK [15].
Experimental Design: A total number of 35 mature
male rabbits (2.5 kg average) were kept in the animal
house at National Research Centre. Animals were divided
into 5 equal numbered groups (n = 7). The first group is
adopted as the control group (treated with distilled water).
The second and third groups were orally dosed 10.8 mg
lead acetate/ kg b. wt. and considered as the low dose
groups. The fourth and fifth groups were dosed 15 mg
lead acetate/kg b. wt. and considered as the high dose
groups. All groups received their dosage orally dissolved
in distilled water using animal gavage. The four later
groups were treated for 5 consequent days /week and the
treatment expended for 8 weeks. The third and fourth
groups obtained, in addition to the lead acetate dose,
vitamin C (1g / liter drinking water every day without
disturbance). All lived animals were sacrificed on the day
after the last dose [11].
In Serum and Whole Blood:
AST, ALT and LDH were analyzed using kits from
Stanbio, Texas, USA [16, 17].
Cholinesterase [18], acid phosphatase[19] and -GT
[20] were analyzed using kits, Quimica Clinica
Aplicada S.A., Spain.
All enzymes were measured using Shimadzu UV
240 Spectrophotometer with different wavelengths
specific or each enzyme.
Statistical Analysis: Using one-way ANOVA, data were
analyzed to determine whether the effect of lead (Pb)
and vitamin C gave a significant difference, or not,
as compared with the control group (H0). The results were
accepted at a level of 95% confidence.
Semen Collection: Semen were collected from
bucks using rabbit artificial vagina (AV) and a teaser
female [14]. The collection was achieved from the end of
the fourth week till the end of the experiment for the
routine evaluation of both live sperm and sperm
abnormalities percentage using eosin analine stain and
before the slaughter immediately for enzymes
determination.
RESULTS
In Table (1) results of semen analysis revealed that
the lead treated groups show lower (P< 0.01) mass
motility, individual motility, sperm concentration / ml
semen and live sperm percentage, while they show higher
(P < 0.01) total primary sperm abnormalities percentage as
compared to the control group. Vitamin C was found to
improve the hazardous effects of lead in the third and fifth
groups.
Inhibition of SOD, in whole blood, increased
(P< 0.01) in the treated groups (2, 3 and 4) as compared to
the control group. At the same time, this activity
decreased (P< 0.01) in group 5 than in groups 2, 3 and 4.
Moreover, - GT activity, in serum increased (P<0.01) in
group 4 than in the control group, while it increased in
groups 2 and 4 than in groups 3 and 5, respectively
(Table, 2).
LDH, in serum, showed a decreased (P< 0.01)
activity in groups 2 and 4, while it increased (P< 0.01),
in semen, in groups 2, 3, 4 and 5 than control group
(Tables 2 and 3). Meanwhile, in serum, it is decreased (P<
0.01) in groups 2 and 4 than in groups 3 and 5,
respectively and it is increased (P< 0.01), in semen
samples, in groups 2 and 4 than in group 3 and 5,
respectively (Tables 2, 3).
Blood Sampling: While sacrificing animals, blood was
collected in sterilized capped tubes and sterilized
heparinized tubes for SOD determination. The tubes were
incubated at 37° C for 10 minutes in slope position, then
centrifuged at 3500 rpm for 10 minutes. Serum was
collected and immediately tested for the enzymes.
The whole heparinized blood was treated according
to the following method: 0.5 ml of blood was centrifuged
for 10 minutes at 3000 rpm and then the plasma was
aspirate off. Then the erythrocytes were washed four
times with 3 ml of 0.9% NaCl solution and centrifuged for
10 minutes at 3000 rpm after wash. The washed and
centrifuged erythrocytes were made up to 2 ml with cold
redistilled water, mixed and left to stand at +4 °C for
15 minutes. The lysate was diluted with 0.01 nmol/l
phosphate buffer pH 7 (Randox cat. No.SD 124), so that
the percentage inhibition falls between 10 and 60%.
A 25 fold dilution of lysate is recommended (final dilution
factor = 100) [15].
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Global J. Pharmacol., 8 (4): 703-708, 2014
Table 1: Semen characteristics in rabbits bucks after treatment with lead acetate alone or with the addition of vitamin C.
Treatment
--------------------------------------------------------------------------------------------------------------------------------------------------------Group I
Group II
Group III
Group IV
Group V
Control n=12
Low dosen=36
Low dose + Vit Cn=12
High dosen=28
High dose + Vit Cn=28
0.48a±0.056
0.69b±0.55
0.47a±0.014
0.39a±0.021
0.52ab±0.063
Concentration (x10 /ml)
120.00 ±6.89
a
87.50 ±5.59
a
77.00 ±2.71
70.40 ±3.99
106.86 b±6.71
Total concentration(x10 )
bc
53.80 ±3.62
c
58.23 ±5.24
ab
36.30 ±2.22
25.83 ±1.27
62.53c±9.33
Live %
94.33 ±0.25
82.53 ±0.32
90.26 ±0.50
74.21 ±0.51
818.83c±0.46
Mass motility(score 1-5)
4.5b±0.12
3.44a±0.13
4.00ab±0.25
3.57a±0.12
3.86a±0.22
Individual motility %
91.25b±0.90
76.25a±1.33
81.25a±3.09
79.64 a±1.31
80.71 a±2.32
Primary sperm abnormalities%
15.33a±0.55
25.16c±0.25
21.27b±0.41
34.31d±1.42
19.35 b±0.78
Biomarker
Volume(ml)
6
6
b
c
b
d
a
a
a
Same superscript are non-significantly different within row (P< 0.05) - Duncan test
Table 2: Some enzymes bio markers in blood of rabbits bucks after treatment with lead acetate alone or with the addition of vitamin C.
Treatment
--------------------------------------------------------------------------------------------------------------------------------------------------------Group I
Group II
Group III
Group IV
Group V
Biomarker
Controln=5
Low dosen=7
Low dose + Vit Cn=6
High dosen=7
High dose + Vit Cn=4
SODunits/ml (whole blood)
115.43a±18.27
210.64bc±32.08
187.58b±7.82
262.81 c±25.89
109.78 a±10.53
9.65ab±0.51
11.39bc±1.95
7.78a±0.80
14.09c±0.47
10.26ab±0.76
Lactate dehydrogenase(U/L)
518.66 ±22.87
349.19 ±12.30
559.71 ±21.04
355.32 ±7.68
532.6 b±20.60
AST(U/L)
81.33 ±3.55
95.37 ±2.95
80.82 ±3.07
81.86 ±4.47
72.79a±1.44
ALT(U/L)
88.04bc±2.84
100.09c±11.50
78.30ab±2.60
133.78d±9.06
61.46 a±4.25
Choline esterase (UL)
1049.44b±109.82
838.04ab±162.24
1051.23b±128.86
479.25 a±67.69
1090.89 b±166.16
Total acid Phosphatase (U/L)
59.51a±2.40
66.72b±1.97
84.91d±2.16
66.68 b±1.26
76.84 c±2.83
Prostatic acid Phosphatase(U/L)
1.82 ±0.33
3.31 ±0.43
8.00 ±0.56
3.00 ±0.43
7.45b±1.56
- GT(U/L)
b
a
a
a
b
b
a
a
b
a
a
a
Same superscript are non-significantly different within row (P< 0.05) - Duncan test
Table 3: Some enzymes bio markers in semen of rabbits bucks after treatment with lead acetate alone or with the addition of vitamin C.
Treatment
--------------------------------------------------------------------------------------------------------------------------------------------------------Group I
Group II
Group III
Group IV
Group V
Biomarker
Controln=5
Low dosen=7
Low dose + Vit Cn=6
High dosen=7
High dose + Vit C n=4
Lactate dehydrogenase(U/L)
954.75a52.98
3850.83c39.45
1646.90b30.45
4817.65d390.60
1788.63b5.07
AST(U/L)
420.79a62.50
1614.77c104.17
247.13a26.13
773.99b1.49
330.81a48.23
ALT(U/L)
43.32b2.12
44.20b7.56
38.60b3,15
23.18a4.87
16.50a0.93
Choline
esterase(UL)
195.50c25.60
119.9a4.93
156.40b3.41
101.66a3.41
106.87a7.88
Total acid Phosphatase(U/L)
124.74a5.40
113.86a12.24
107.01a9.53
171.20b14.70
99.76a10.84
Prostatic acidPhosphatase(U/L)
12.63a0.95
16.57a2.74
16.06a1.19
24.88b2.37
15.48a3.28
Same superscript are non-significantly different within row (P< 0.05) - Duncan test
AST in serum showed increase activity (P< 0.01) in
group 2 than control group (Table 2). At the same time,
its activity in semen is increased (P< 0.01) in both groups
2 and 4 than control group (Table, 3). Also, the activity of
AST in serum is increased (P< 0.01) in group 2 than group
3; and in groups 2 and 4 than group 3 in the semen
sample.
ALT activity in serum and semen are higher (P< 0.01)
in groups 4 and 2 and lower (P< 0.01) in group 5 in both
as compared to the control group. On the other hand,
its activity in serum, is higher (P< 0.01) in groups 2 and
4 than in groups 3 and 5, respectively. While, in the
semen, it showed a lower (P< 0.01) results in groups 4 and
5 than in other groups (Tables 2 and 3).
Regarding Cholinesterase activity, in serum, it is
decreased (P< 0.01) in group 4 than the control group,
third and fifth groups (Table, 2) and, in semen, in groups
2, 3, 4 and 5 than the control group (Table, 3). Meanwhile,
this activity is high (P< 0.01) in group 3 than other treated
groups (2, 4 and 5) (Table, 3).
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Global J. Pharmacol., 8 (4): 703-708, 2014
Total prostatic acid phosphatase activities increased
(P< 0.01) in serum in all treated groups as compared to
the control group. Moreover, the increment in both
groups 3 and 5 was clear (p<0.01) than in groups 2 and
4 (Table, 2). But, in semen it is increased (P< 0.01) in group
4 only than all other groups and control group (Table, 3).
present study, the results showed a significant increase
in the enzyme activity in case of high dose of lead acetate
(14.09 U/l) than the control group. This indicates that
there is a destruction of the enzyme in the store cells and
released in the blood [26]. Therefore, the activity in the
store cells like hepatocytes and renal tubular cells is
decreased [27]. As reported by Murray [26] that the cells
which are under deteriorating stress factors including
heavy metals, showed lower activities of SOD and,
GSH-Px. Moreover, this author added that a powerful
antioxidant can reverse the oxidative damage by bringing
about an improvement in the reductive status of the cell.
The present study proved that vitamin C has stored the
integrity of cells and reduced the leakage of enzymes
outside the damaged cells, in addition to its antioxidant
effect via an indirect way. Moreover, vitamin C may help
in decreasing the superoxide radicals’ accumulation inside
the cells although the SOD activity is inhibited by lead,
thence, it conserves the soundness of the cells in return,
no leakage of enzymes and no increased enzymes activity
in the serum than normal. This finding is in agreement
with Sivaprasad et al. [27]. In addition, vitamin C had a
significant anti-oxidant activity thereby protecting the
organs from the lead-induced toxicity [28].
LDH is a cytosolic enzyme which is released into
systemic circulation in case of damage of the liver, lung,
muscle, kidney, testicles or heart [29]. In the present
study, the decrease in activity of LDH in serum due to
lead toxicity and recuperation of activity again after
treatment with vitamin C; found to be in agreement with
the findings of Yagminas et al. [30]. Moreover,
the leakage of the enzyme in the semen from the testicular
and glandular tissues was, approximately twice to four
times than the treated groups with vitamin C and triple the
control group. This goes with Gulvic [31] who recorded a
reduced activity of LDH in the testicular tissue of the rat.
The results indicated that LDH is a sensitive and
convenient biosensor for detection of heavy metal salts;
and this is in agreement with Fennouh et al. [32].
The effect of lead on AST and ALT in the serum have
been discussed [11, 33]. Moreover, LC50 of lead acetate
(100 micromole) caused significant leakage of AST and
ALT in the medium of the liver cell culture [34]. So,
the leakage of cytoplasmic enzymes appear to be a
sensitive indicator of cell injury produced by heavy
metals. The results of this study indicated that lead
acetate led to increase the activity of the enzymes in both
serum and semen; while treatment with vitamin C removed
the effect of leakage. This was clear as the ALT activity in
serum and semen have been decreased.
DISCUSSION
In this study, results showed that lead has a
significant effect on semen characteristics in rabbit bucks
in both low and high dose groups of lead acetate,
while these adverse effects have been corrected in groups
treated with vitamin C (Table, 1). These results are in
agreement with the findings of Lähdetie [6] and El-Nattat
[4].
Most of the enzyme markers contain sulfhydryl
(-SH) group at the site of action [21], such group is a
target for metals, while others like some metallo-enzymes
have a prosthetic group that may be replaced by heavy
metals leading to inhibition of the enzymatic activity [2].
This suppression masks their physiological actions,
especially those enzymes related to superoxide radicals
O2- that accumul-ate inside the cells leading to the
deterioration and death of the cells. SOD is one of those
enzymes [22]. The results indicated that lead acetate
induced an exaggerated inhibitory effect for SOD. As lead
mimics Cd in replacing Zn in its site, this suggested the
interaction of lead with the Cu, Zn and Mn moieties.
Such interaction has been demonstrated where
lead replaced Zn to form Cu-Pb-SOD [23]. Vitamin C
ameliorates the inhibitory action of lead either by reducing
its absorption [13] or by removing ROS (reactive oxygen
system) once formed, thus prevent the radical chain
reaction. This observation supports the hypothesis that
SOD activity is stimulated by an increased superoxide
radical generation associated with the decline of SOD and
GSH-Px[24] generated by inhibitory action of lead, while,
the anti-oxidant refreshes the enzyme activity and
antagonizes the inhibitory effect of lead.
There are some plasma enzymes which have
unknown physiological function in blood, but only
provide a valuable diagnostic and prognostic clinical
evidence in case of dysfunction or diseases. Prostatic
acid phosphatase is one of these plasma enzymes [25].
The -GT, an enzyme that supports the transfer of
certain amino acids into the GSH, which reduces the
accumulation into the RBCs and other cells. It is present
in the plasma membrane of renal tubular cells and in
endoplasmic reticulum of the hepatocytes [26]. In the
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Global J. Pharmacol., 8 (4): 703-708, 2014
Cholinesterase is classified to true cholinesterase;
which is found in nerve tissue and in the red blood
cells; and pseudo cholinesterase which is present in
various tissues and in plasma or serum [35]. Since,
the heavy metal ions (mercury and lead) and their
organic compounds belong to non-competitive inhibitors
of enzymes; they may block the –SH groups that make
part of the catalytic site of the enzyme [36]. The
significant decrease of cholinesterase in both serum
and semen, in the large dose treated group, indicates that
the enzyme may behave the same behavior in both of
them.
Regarding the significant increase of both total and
prostatic acid phosphatase activities in serum of the lead
and lead + vitamin C treated groups which may indicates
presence of prostatic carcinoma; this is in agreement with
Krupp et al. [37]; who reported an increase of acid
phosphatase in serum due to carcinoma in prostate.
In conclusion, lead had led to testicular
hypofunction, which is supported by the semen picture
and the disturbances in the enzymes under investigation.
Vitamin C, as antioxidant, counteract the oxidant activity
of lead and improved its effects in both serum and semen.
LDH and ALT and acid phosphatase are good biomarkers.
6.
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