Lamiaa Fathy Asal
poultry
prof .Dr
Hassan saber zeweil
The biological role of antioxidants
for decreasing the damage
impact of free radicals in
rabbits
Introduction
What are Free Radicals?
A free radical is an unstable
molecule that loses one of
its electrons and therefore
becomes unbalanced and
highly reactive.
Free
radicals
damage
healthy cells and in turn
impair the body’s normal
functions.
How are Free Radicals
Produced?
• normal respiration
• metabolism
•
•
•
•
•
•
•
•
•
•
exposure to air pollutants
sun exposure
radiation from X-rays
drugs
viruses
bacteria
parasites
dietary fats
stress
injury
PHYSIOLOGICAL EFFECTS
• Free radicals have been implicated as
playing a role in the etiology of
cardiovascular disease, cancer,
Alzheimer's disease, and Parkinson's
disease.
• Harmful free radicals are toxic
molecules of oxygen that damage
every area of our bodies. One free
radical can initiate tens of thousands
of chain reactions that cause
tremendous harm (destroy cell
membranes, disrupt crucial processes
in the body, reprogram DNA, form
mutant cells, and more).
MEASUREMENT OF
FREE RADICALS
When a fatty acid is peroxidized it is
broken down into aldehydes, which are
excreted.
Aldehydes
such
as
thiobarbituric acid reacting substances
(TBARS) have been widely accepted as a
general marker of free radical
production. The most commonly
measured TBARS is malondialdehyde
(MDA)
ANTIOXIDANT
DEFENSES
•
Antioxidant means "against oxidation." Antioxidants work to protect
lipids from peroxidation by radicals. Antioxidants are effective
because they are willing to give up their own electrons to free
radicals. When a free radical gains the electron from an antioxidant
it no longer needs to attack the cell and the chain reaction of
oxidation is broken (4). After donating an electron an antioxidant
becomes a free radical by definition. Antioxidants in this state are not
harmful because they have the ability to accommodate the change in
electrons without becoming reactive. The human body has an
elaborate antioxidant defense system. Antioxidants are
manufactured within the body and can also be extracted from the
food humans eat such as fruits, vegetables, seeds, nuts, meats, and
oil. There are two lines of antioxidant defense within the cell. The
first line, found in the fat-soluble cellular membrane consists of
vitamin E, beta-carotene, and coenzyme Q (10). Of these, vitamin E is
considered the most potent chain breaking antioxidant within the
membrane of the cell. Inside the cell water soluble antioxidant
scavengers are present. These include vitamin C, glutathione
peroxidase, superoxide dismutase (SD), and catalase. Only those
antioxidants that are commonly supplemented (vitamins A, C, E and
the mineral selenium)
EXPERIMENTAL design
With vit c (1mMol )
• Positive control
• 1mMol AlCl3
• 5 mMol AlCl3
• 10 mMol AlCl3
• 15 mMol AlCl3
• 20 mMol AlCl3
With vit E(2mMol)
• Positive control
• 1mMol AlCl3
• 5 mMol AlCl3
• 10 mMol AlCl3
• 15 mMol AlCl3
• 20 mMol AlCl3
Without vit
• Negative control
• 1mMol AlCl3
• 5 mMol AlCl3
• 10 mMol AlCl3
• 15 mMol AlCl3
• 20 mMol AlCl3
Fig. 1. Changes in motility (%) and viability (%) of rabbit sperm at 0, 2 and 4 h of
incubation with AlCl3 alone or with vitamin C (Vit. C 1mM) (mean±S.E.). Asterisk (*)
indicates significant (p < 0.05) effect of treatment with AlCl3 alone compared to control
without Vit. C or in combination with Vit. C compared to control with Vit. C.
Fig. 2. Changes in motility (%) and viability (%) of rabbit sperm at 0, 2 and 4 h of incubation
with AlCl3 alone or in combination with vitamin E (Vit. E 2mM) (mean±S.E.). Asterisk (*)
indicates significant (p < 0.05) effect of treatment with AlCl3 alone compared to control
without Vit. E or in combination with Vit. E compared to control with Vit. E.
Fig. 3. Changes in TBARS levels and activities of SOD, CAT, ALT, AST and ACP in rabbit sperm
after 2 h incubation with AlCl3 alone or in combination with vitamin C (Vit. C; 1mM)
(mean±S.E.). Asterisk (*) indicates significant (p < 0.05) effect of treatment with AlCl3 alone
compared to control without Vit. C or in combination with Vit. C compared to control with
Vit. C.
Fig. 4. Changes in TBARS levels, activities of SOD,CAT, AST,ALT andACP in rabbit sperm
after 2 h incubation with AlCl3 alone or in combination with vitamin E (Vit. E 2mM)
(mean±S.E.). Asterisk (*) indicates significant (p < 0.05) effect of treatment with AlCl3
alone compared to control without Vit. E or in combination with Vit. E compared to
control with Vit. E.
Fig. 5. Changes in TBARS levels and activities of SOD, CAT, AST, ALT and ACP in rabbit
sperm after 4 h incubation with AlCl3 alone or in combination with vitamin C (Vit. C
1mM) (mean±S.E.). Asterisk (*) indicates significant (p < 0.05) effect of treatment with
AlCl3 alone compared to control without Vit. C or in combination with Vit. C compared to
control with Vit. C.
Fig. 6. Changes in TBARS levels, activities of SOD,CAT, AST,ALT andACP in rabbit sperm
after 4 h incubation with AlCl3 alone or in combination with vitamin E (Vit. E 2mM)
(mean±S.E.). Asterisk (*) indicates significant (p < 0.05) effect of treatment with AlCl3
alone compared to control without Vit. E or in combination with Vit. E compared to
control with Vit. E.
the obtained results showed that aluminium chloride exerted reproductive
toxicity in a dose and time-dependent manner on rabbit sperm in vitro.
Aluminium chloride increased TBARS level, and changed the activities of
antioxidant enzymes, transaminases and acid phosphatase. It also caused a
decline in sperm motility and viability more likely through other mechanisms
than oxidative injury, which require more investigation. The use of vitamin C
or vitamin E disturbing effects on free radicals level and enzymatic activity,
but not on motility and viability.
EXPERIMENTAL Diets
Control
6 rabbits
2.5 mg isoflavones /kg B.W
6 rabbits
5 mg isoflavones /kg B.W
6 rabbits
Fig.
1.
Changes
in
plasma
thiobarbituric
acid-reactive
substances
(TBARS)
and
glutathione
S-transferase
(GST)
during
treatment
of
male rabbits with either 2.5 or 5 mg/kg B.W. doses of isoflavone.
Table 1:Thiobarbituric acid-reactive substances (TBARS) and glutathion-Stransferase (GST)
activity in plasma, liver, testes and brain during treatment of male rabbits with either 2.5
or 5 mg/kg doses of isoflavones
Parameter
Isoflavones
Control
2.5 mg/kg
5 mg/kg
TBARS
(nmol/ml)
1.82±0.06a
1.16±0.07b
1.15±0.08b
GST (μmol/h)
0.53±0.007a
0.53±0.006a
0.53±0.005a
TBARS*
14.06±0.99a
11.5±0.15b
10.3±1.78b
GST**
1.04±0.007a
1.05±0.032a
1.04±0.020a
TBARS*
12.01±0.31a
7.25±0.1.60b
9.45±0.46ab
GST**
0.51±0.09a
0.51±0.011a
0.49±0.004a
TBARS*
36.1±2.67a
31.9±4.18ab
24.3±1.6b
GST**
0.49±0.004a
0.49±0.003a
0.49±0.003a
TBARS*
11.8±5.78a
7.3±4.05b
9.5 ±3.75ab
GST**
0.56±0.006a
0.54±0.013a
0.57±0.008a
Plasma
Liver
Testes
Brain
Kidney
Values are expressed as overall means±SE; n ¼ 6 for each treatment group.
abWithin row overall mean with different superscript letter differ significantly (P < 0:05).
*TBARS is expressed as nmol/g tissue.
**GST specific activity: lmol/h/mg protein.
Table 2: Plasma lipid and lipoprotein profiles during treatment of male rabbits
with either 2.5 or 5 mg/kg doses of isoflavones
Isoflavones
Lipids (mg/dl)
Control
2.5 mg/kg
5 mg/kg
TL
557±38.7a
459±15.4b
440±17.9b
Cholesterol
152±2.1a
122±1.9b
121±2.3b
TG
168.6±7.8a
139.1±4.8b
129.7±2.7c
HDL
34.9±0.33b
44.4±0.67a
45.5±0.89a
LDL
108±2.4a
87±2.0b
84±2.2b
VLDL
33.7±1.56a
27.8±0.97b
25.9±0.53b
LDL:HDL ratio
3.1 ±0.08a
2.0±0.06b
1.9±0.08b
Cholesterol (% TL)
30.1±2.79a
28.8±1.01a
29.2±1.13a
Values are expressed as overall means±SE; n ¼ 6 rabbits for each treatment group.
abcWithin row, means with different superscript letters differ significantly (P < 0:05).
TL: total lipids, TG: triglycerides, HDL: high density lipoprotein, LDL: low density lipoprotein, VLDL: very low density lipoprotein
Fig. 2. Changes in plasma total lipids, cholesterol and triglycerides during
treatment of male rabbits with either 2.5 or 5 mg/kg B.W. doses of isoflavone.
Fig. 3. Changes in plasma high density lipoproteins (HDL), low density
lipoproteins (LDL), very low density lipoproteins (VLDL) and LDL:HDL ratio during
treatment of male rabbits with either 2.5 or 5 mg/kg B.W. oses of isoflavone.
The results of the present study led to the conclusion that
isoflavone dosages (2.5 or 5 mg/kg body weight), which is more
than two or four times to the amounts consumed (40 mg) in many
Eastern nations (Adlercreutz et al., 1995), have beneficial effect on
plasma lipid and lipoprotein concentrations, TBARS level and
antioxidant activities in rabbits. Also, the 5 mg/kg dose of
isoflavones was more effective than the 2.5 mg/kg dose on lipids
and lipo-protein profiles, but more studies would help to confirm
the optimal amount required. Therefore, the animal’s diet must
contain an adequate level of soybean, which covers the
isoflavones requirements
EXPERIMENTAL Diets
Control
12 rabbits
Cadmium
12 rabbits
Cadmium
&Antioxidants
12 rabbits
Table 1 Serum parameters of rabbits received cadmium and
cadmium and antioxidants.
Parameters
Control
Cadmium
Cadmium
&Antioxidants
AST (U/L)
91.5 ±4.6a
205.9±8.9©
8.15±133.2a
ALT(U/L)
1.6± 0.22a
4.99±1.1©
2.37± 0.5a
GGT (U/L)
14.9± 1.3a
25.9±1.7©
15.5± 2.7a
LDH (U/L)
1303±69.9a
1177±40.5©
1225±81a
ALP (U/L)
1732±101a
1800±199 ©
1715± 211a
GST((nmol/ml
blood x10
0.66±0.04a
0.81±0.04 ©
0.59± 0.02a
Different superscripts in row indicates significantly different means at(P <0.05).
Table 2 Testicular parameters of rabbits received cadmium and
.
Cadmium and antioxidants
Parameters
Control
Cadmium
Cadmium and
antioxidants
Cd concen. (ug/gm
tissue)
0.35±0.04ª
39.33±6.66©
0.33±0.03ª
LPO (nmol/gm
tissue)
142.66±8.5ª
403.76±12.3 ©
139.45±7.6ª
AcP (IU/mg)
566.0±7.7ª
513.8±8.65©
592.22±32.8ª
ALP (IU/mg)
988.32±34.8 ª
867.4±16.6©
122.6±16.8ª
LDH (IU/mg)
2110±23.3ª
2860±43.2 ©
1992±36.7ª
AST (IU/mg)
59.9±2.0ª
48.9±2.33©
58.6±1.65ª
ALT (IU/mg)
16.6±4.3ª
12.7±8.8©
18.4±8.6ª
SOD (IU/mg)
165.5±22.5ª
99.8±13.3©
177.0±24.6ª
GST(IU/mg)
0.35±0.02ª
3.6±0.9©
0.30±0.9ª
Different superscripts in row indicates significantly different means at (P < 0.05)
Table 3.GSH-Px, GR, activities in red blood cells, GST in the
plasma and GSH in whole blood in the three
groups.
Parameters
Control
Cadmium
Cadmium and
antioxidant
GSHPx(nmol/mlblood
x10)
22.0 ± 3.6 ª
34.0 ± 7.4 ©
24.0 ± 2.02 ª
GR (nmol/ml blood
X10)
29.7 ± 4.3 ª
40.0 ± 5.8 ©
31.2 ± 6.4 ª
GST (nmol/ml
bloodX10
34.8 ± 3.2 ª
41.84 ± 1.97 ©
28.9 ± 3.31ª
GSH (nmol/mlX10
9.9 ± 0.6 ª
13.0 ± 1.72 ©
10.2 ± 1.7 ª
Different superscripts in row indicate significantly different means at(P < 0.05).
the antioxidant used is effective in combating celldamaging free radicals, which are
known to contribute towards testicular dysfunction. Studies have shown that
antioxidants are uniquely different from one another and each have a specific function
in the body. They are also synergistic, and will work most effectively when they are used
together. These combinations can perform a wide range of metabolic activities, free
radical scavenging and preventive actions. The present antioxidant formula used as a
blend of
antioxidants specifically designed to assist the body in overcoming a vast
array of physiological stressors and to help avert the effects of Cd-induced testicular
dysfunctions.