Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
464
AMELIORATIVE EFFECT OF MELATONIN, VIT.C ALONE AND THEIR
COMBINATION ON LIVER AND KIDNEY FUNCTIONS IN ACRYLAMIDE
INTOXICATEDOF ADULT MALE RATS
Nawras A. Alwan; Jassim M. A. Alkalby and Eman Aboud Al-Masoudi
Department of Physiology, Pharmacology and Chemistry, College of Veterinary Medicine,
University of Basrah,Basrah,Iraq
Key words: Acrylamide, Melatonin, Vitamin C.
ABSTRACT
The present study was designed to determine the ameliorative effect of melatonin (Mel),
vitamin C (Vit.C) alone and their combination on liver and kidney functions in acrylamide (ACR)
intoxicated rats(administration ACR for 45days). Forty eight adult male rats were divided randomly
into two main groups. Control group (no=20) subdivided into two groups: groupI: ten animals of
control administration distal water and group II:ten animals give Mel(5mg/kgBW) for 21 days.
second group: the ACR treated group(n=40) subdivided into ACR+distal water orally, ACR+Mel
(5mg/kg BW/day), ACR+Vit.C (200 mg/kg BW/day), ACR+Mel (5mg)+Vit.C (200 mg)/kg BW/day
for 21 days. The result revealed that no significant differences in serum AST, ALT and ALP
enzymes levels between control group treated with Mel and control group. A significant reduction in
serum AST, ALT and ALP levels were recorded in all treated groups compared with ACR-non
treated group in which the above cited parameters still significantly higher compared with control.
No significant differences were recorded in serum total protein, urea and creatinine concentrations
between control+Mel treated group compared with control. A significant improvement in serum total
protein, urea and creatinine concentrations were recorded in all treated groups compared with ACRnon
treated group.
INTRODUCTION
Acrylamide is odorless, white crystalline solid at room temperature and have chemical
formula: C3H5NO and structure H2C=CHCONH2 , acrylamide is α, β-unsaturated vinyl monomer of
polyacrylamide (CH2CH-CONH2), it is a water soluble substance (1and2). Acrylamide monomer has
been reported to be formed in certain foods cooked in high temperature, levels of acrylamide as 3500
μg/Kg have been reported in potato chips and french fries, it was first detected in certain foods in
April 2002 and the toxicity studies on animals indicate the results that acrylasmide is carcinogenic, in
rodent caused toxic effects on the reproductive and nervous systems while in the human causes only
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
465
neurotoxicity (3).Metabolism of acrylamide occurs by two ways either by conjugation with
glutathione or oxidation to glycidamide. Melatonin secretion is not only in blood and also in all types
of bodily fluid which including: saliva, cerebrospinal fluid, aqueous humor of the anterior chamber,
follicular fluid and also in breast milk. Melatonin receptors are distributed in all tissues and organs
and also present so far been confirmed in the brain (including the SCN), pituitary gland, spinal cord,
retina, thymus, spleen, liver, heart, kidney, adrenal gland, lungs, testes, ovaries, blood vessel,
osteoblasts and lymphocytes (4 and 5).Melatonin is a direct scavenger of radical oxygen and nitrogen
species (OH, O2
- and NO) (6). The melatonin is a most effective lipophilic antioxidant has been
proven to be twice as active as vitamin E and C, which differs from other classic antioxidants, it has
amphiphilic properties and proved to be better protected against mitochondrial oxidative stress (7).
Stimulator actions of melatonin on the synthesis of another important antioxidant intracellular,
glutathione (8) and also its protection of antioxidative enzyme from oxidative damage (9). The
present study was aimed to determine the ameliorative effect of Mel, Vit.C and their combination on
ACR induced liver and kidney disfunctions by biochemical and histological changes.
MATERIALS AND METHODS
Animals and housing: sixty adult male rats were used in this study. They were kept in animal house
under constant environmental condition for 2 weeks to acclimatization before beginning of the
experiment. Food and drinking water were provided ad libitum throughout the experiment.
Experimental Design: rats were divided randomly into two groups as follows:
1-Control group (n=20): adult male rats administered distalled water daily by gavage for 45 days.2-
Treatment group(n=40): adult male rats administrated ACR (5mg/kg BW/day) for 45 days by
gavage.
At the end of experimental period the animals of each group were divided into the following
subgroups:The control group was divided into two equal groups: G1(-ve control) (n=10)
administrated distal water by gavage for 21 days.G2 (+vecontrol)(n=10) administrated (5 mg/kg
BW/day) orally for 21 days by gavage. The ACR group (n=40) divided into four equal
ubgroups:ACR+distal water (G3): administrated distal water.ACR+Mel (G4): administrated Mel
5mg/kg BW/day.ACR+Vit.C (G5): administrated Vit.C (200 mg/kg BW/day).
ACR+Mel+Vit.C(G6): administrated both Mel (5mg)+Vit.C (200 mg)/kg BW/dayby gavage for 21
days.
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
466
Collection of blood samples:At the end of treatmentt peroid the animals were anesthized using
diethyl ether and sacrificed. The blood samples were collected directly by cardiac puncture into clean
dry testtube and serum were separated and stored at -20˚C until used for hormonal analysis. The liver
and kidney were excised directly and fixed in neutral buffered formalin 10% for histological study
according to Luna (10).
Biochemical Measurments: Some biochemical measurements were done on the serum after
separation by using special enzymatic kits as follow:
Serum Aspartate aminotransferase (AST) and Serum Alanine aminotransferase (ALT)
Estimation(U/I):Aspartate and alanine aminotransferase is measured by monitoring the
concentration of oxaloactate hydrazone formed with 2,4-dinitrophenyl-hydrazine (11).
Serum Alkaline Phosphatase (ALP) measurment(U/I): This estimation was done by using the
colorimetric determination of alkalin phosphatase activity (12).
Total Protein Measurement: Colometeric method described by Young (13) and Titez (14). By
using the biuret reagent contains sodium potassium tartrate to complex cupric ions and maintain their
solubility in alkaline solution
Urea Measurement : Urea is hydrolyzed in the presence of water and urease to produce ammonia
and n dioxide (15).
Serum Creatinine Measurment:Creatinine is endogenously produced and released to body fluids at
a stable rate and its plasma and serum levels are maintained within narrow limits, it can be measured
as an indicator of glomerular filtration rate (GFR) (16).
RESULTS
The results as demonstrated in the table (1) showed that serum ALT concentration increased
significantly (P<0.05) in ACR-non treated group compared with control and other treated groups
after 21 days of treatment. While no significant differences were observed between ACR+Mel,
ACR+Vit.C and ACR-Mel+Vit.C treated groups compared with the control. No significant
differences were recorded in serum ALT concentration between ACR+Mel and ACR-Vit.C treated
groups compared with control. While serum ALT concentration still significantly (P<0.05) higher in
ACR+DW group compared with control and other treated group. Serum AST concentration was
significantly (P<0.05) higher in ACR-non treated group compared with control and other treated
groups, while no significant differences were recorded in control+Mel and ACR+Mel treated groups
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
467
compared with control. The low significant (P<0.05) value of serum AST concentration was
recorded in ACR+Mel+Vit.C treated group compared with control and all other treated groups. No
significant differences were observed in serum ALP concentrations between all treated groups and
control at the end of treatment peroid except in ACR-non treated group in which ALP concentration
still significantly (P<0.05) higher than those of control and all other treated groups.
Table (1): Effect of Mel, Vit.C alone and their combination on serum liver enzymes of ACRtreated
adult male rats (M±SD.): (n=10)
Values expressed in small letters mean significant differences at (P<0.05) levels.
The data in table (2) revealed the serum total protein concentration that no significant
difference was observed between control+Mel, ACR+DW, ACR+Mel, ACR+Vit.C and
ACR+Mel+Vit.C treated groups compared with control. While serum total protein concentration still
significantly (P<0.05) lower in all treated groups compared with control. After 21 days of treatment
serum urea concentration indicated no significant differences between control+Mel and ACR+Mel
treated groups compared with the control. However significant (P<0.05) elevation in serum urea
concentrations were recorded in ACR+DW, ACR+Vit.C and ACR+Mel+Vit.C treated groups
compared with control. While serum urea concentrations still significantly higher in ACR+DW
group compared with control and all other treated groups. Finally serum creatinine concentrations
revealed that no significant differences were observed between control+Mel and ACR+Mel
compared with control. However in ACR-non treated and ACR+Vit.Ctreated groups serum
creatinine concentrations were significantly higher compared with control and other treated groups.
The creatinine concentrations still significantly higher compared with control and other treated
groups.
Parameters
Groups
ALT
(U/l)
AST
(U/l)
ALP
(U/l)
G1(Control) 18.38±3.37c 21.98±2.10 c 11.44±0.72 b
G2(Control+Mel) 19.95±4.88c 22.17±3.04 c 12.00±0.47 b
G 3(ACR +DW) 31.73±4.82a 39.66±1.21 a 18.09±1.38 a
G4(ACR+Mel) 20.21±4.91bc 22.16±1.65 c 10.84±0.61b
G5(ACR+Vit.C) 21.35±2.56bc 25.25±3.60 b 14.14±0.63b
G6(ACR+Mel+Vit.C) 24.03±1.24b 18.48±1.54d 11.54±1.25 b
LSD 4.08 3.08 1.16
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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Table (2): Effect of Mel, Vit.C alone and their combination on serum total protein, urea and
creatinine concentrations in ACR-treated adult male rats (M±SD): (n=10)
Parameters
Groups
Total
protein
g/l
Urea
mg/dl
Creatinin
Mbn kg/l
G1 (Control) 8.39±0.51a 62.39±7.86 d 4.23±0.50c
G2(Control+Mel) 7.87±0.59ab 61.95±8.87d 3.59±0.78cb
G 3(ACR+DW) 7.33±0.82b 107.72±11.43a 6.84±0.47a
G4 (ACR+Mel) 7.31±0.67b 65.51± 5.40d 3.58±0.70cb
G5(ACR+Vit.C) 7.36±0.61b 84.32±4.59b 5.47±1.04b
G6(ACR+Mel+Vit.C) 7.72±0.63b 76.01±3.93c 2.72±0.64d
LSD 0.67 8.31 0.85
Values expressed in small letters mean significant differences at (P<0.05) levels.
DISCUSSION
The results of the present study as illustrated in the table (1) revealed that no significant
differences were observed in serum ALT, AST and ALP enzymes in normal Mel. treated group for
21 days compared with control. These results are in accordance with Bhatti et al. (17) and Sulaiman
(18) who found that no significant differences were observed in adult male and female rats treated
with 10 mg/kg BW of Mel compared with control group. Similarly Ebaid et al. (19) mentioned that
adult male rats treated with Mel 10mg/kg BW/day for 3 weeks revealed no significant differences in
plasma ALT, AST and ALP levels compared with control. The results also indicated that the ACRnon
treatment group the levels of serum ALT, AST and ALP enzymes still significantly higher at the
end of the experiment which may be occurred either due to the toxic effects of ACR. on liver tissue
and other organs which not regenerated at the end of experiment or the period of withdrawal (21
days) not enough to regeneration takes place.
While in ACR+Mel, ACR+Vit.C and ACR+Mel+Vit.C treated groups the levels of serum
ALT, AST and ALP enzymes showed non significant differences compared with control. These
results are in line with those of Sulaiman (18) who concluded that the intoxicated of both adult male
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
469
and female rats with both chlorpromazine and lipopolysacchride revealed a significant increase in
ALT, AST and ALP enzymes compared with control. While pretreatment with 10 mg/kg BW of Mel
cause significant reduction in serum ALT and ALP levels and nonsignificant reduction in serum AST
level compared to the control group. Similarly fluoride intoxicated adult female rats showed a
significant increase in serum ALT, AST and ALP enzyme levels compared with control and the
cotreatment with Mel 10 mg/kg BW. for 28 days resulted in a significant elevation of these
parameters compared with fluoride intoxicated female rats but still significantly higher than that of
control group (20).
The results are also in agreement with Ebaid et al. (19) who demonstrated that adult male rats
treated with carbon tetrachloride (1 mg/kg) for 15 days cause significant elevation in serum liver
enzymes AST, ALT and ALP levels while Mel treatment for 3 weeks resulted in improvement of
these parameters compared to CCl4 intoxicated group but still significantly higher compared with
control.The results of ACR-groups treated with Vit.C consistent with Mongi et al. (21) who
indicated that Wistar rats administrated deltamethrin (1.28 mg/kg) resulted in a significant increase
in serum AST, ALT and ALP. While pretreatment with Vit.C (200mg/kg) normalize the above cited
parameters. Similarly Hussein et al. (22) showed a significant elevation in serum AST and ALT in
adult male rats treated with fenvalerate in dose of (2-8mg/kg) for 30 days compared with control.
However the administration of Vit.C causes significant decrease in serum AST and ALT activity
compared with control. Shawky et al. (23) showed a significant increase in serum levels of AST,
ALT and ALP enzymes in Lead acetate treated male mice compared with control. However
coadministration of Vit.C resulted in ameliorating the above cited parameters compared with the
Lead intoxicated group but still significantly higher compared with control. Soliman (24) mentioned
a significant elevation in serum AST, ALT and ALP activity in adult male rats treated with ACR
compared with control. The coadministration of Vit.C cause significant reduction of these parameters
compared to the group of control. The present results also coincidence with Elzoghby et al. (25) who
found that administration of Vit.C to malathion intoxicated male rats resulted in significant
reduction of serum AST, ALT and ALP toward the normal values compared with the control group.
In agreement with these results Ahmadizadeh et al. (26) reported that adult male rats exposed to
different doses (200, 400 and 600 mg/kg) syrene cause significant elevation in serum AST, ALT and
ALP levels in a dose dependent manner. While pretreatment with Vit.C (200mg/kg BW) I/P cause a
significant reduction in these parameters compared with syrene group but still significantly higher
than those of controls. Vit.C is water soluble (hydrophilic) and considered one of important
antioxidant trapping the free radicals in extracellular fluid and protecting the biomembranes from
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
470
peroxidation damage. On other hand the co-treatment of Mel and Vit.C causes an improvement in
serum AST, ALT and ALPlevels in male rats treated for 21 days compared with ACR-untreated
group, but still significantly higher than that of control these results are inconsistent with Soliman
(23) who I indicated that the ACR-intoxicated adult male rats showed a significant elevation in
serum stand ALT levels these parameters begin to improve after treatment with Mel, Vit.C and both
Mel and Vit.C but still significantly higher than that of control.The present results as illustreated in
table (2)indicated that no signifciant differences were observed in serum total protein, urea and
creatinine concentrations between Mel-treated group compared with control. Moreover no significant
difference was recorded in serum total protein between Mel treated male rats and control (27 and
17). Our resultes are also parallel to those recorded by Bharti& Srivastava (20) and Ebaid et al. (19)
who found that no significant differences were recorded in plasma total protein, creatinine and urea
between Mel treated female rats compared with control.
In ACR-non treated group which administrated distal water for 21 days showed a significant
increase in serum urea and creatinine compared with control while serum total protein showed no
significant differences compared with control. A significant restoration of above parameters to
normal values were recorded in ACR+Mel treated group compared with ACR-non treated and
control groups. These results were matched with Bharti & Srivastava (20) who demonstrated that
Mel treatment of fluoride intoxicated female rats lead to significant reduction of serum urea and
creatinine levelscompared with the control. In agreement with the results of the present study Ismail
(28) showed a significant increase in serum urea and creatinin in adult male rats intoxicated with the
malathion compared with the control. While no significant difference in serum total protein was
observed. The administration of Vit.C to malathion intoxicated group resulted in restoration of above
parameter to near normal value. However Ismail & Ismail (29) mentioned the mercury intoxicated
adult rats showed a significant increase in serum urea concentration and non significant changes in
serum creatinine concentration compared with the control. Administration of Vit.C with mercury
kept the urea level within its normal range compared with the control. In the same line Elzoghby et
al. (25) demnstrated a significant increase in serum BUN and creatinine and a signifiicant decrease in
serum total protein concentrations in adult male rats intoxicated with malathion compared with
control. However coadministration with Vit.C lead to restoration of these parameters toward normal
values compared with control. A similar results were recorded by Ahmadizadeh et al. (26) in styrene
indicated toxicity in adult male rats treated with Vit.C,the present results also observed that ACR
group treated with combination of Mel and Vit.C cause improvement in serum urea and creatinine
concentrations compared with control and ACR-non treated groups. The results consistent with those
Basrah Journal of Veterinary Research,Vol.15, No.3,2016
Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
471
obtained by Soliman (23) who found a significant increase in serum urea and creatinin
concentrations compared with control. But administration of Mel, Vit.C alone and their combination
lead to restoration of all these parameters toward the normal values but still significantly lower than
those of the control. The data are in accordance with Quiroz (30) and Ebaid et al. (19) who reported
that a significant increase in serum urea and significant decrease in serum total protein levels in adult
male rats treated with carbontetrachloride while administration of Mel, Vit.C alone and their
combination resulted in restoration of all above cited to their normal levels. The administration of
Vit.C protect the liver from the effect of free radicals resulted from oxidative stress. Vit.C also cause
improvement of total protein concentration due to reduction in apoptotic properties to white blood
cells due to its antiooxidant properties (31).
التأثیرالوقائی للمیلاتونین وفیتامین ج لوحدھما و کلاھما على وظائف الکبد والکلیة فی ذکور الجرذان
البالغة المسممة بالاکریلاماید
نورس عبدالالھ علوان ، جاسم محمد احمد الکلبی ، ایمان عبود المسعودی
فرع الفسلجة والادویة والکیمیاء ، کلیة الطب البیطری، جامعة البصرة،البصره،العراق
الخلاصة
تم تصمیم ھذه الدراسة لتحدید التأثیر التحسینی للمیلاتونین وفیتامینج وحدھما وکلاھما معاً على وظائف الکبد والکلى فی
جرذان الذکور البالغة المسممة بالاکریلامید (جرعت بالاکریلامید لمدة 45 یوم) ثم تم تقسیم جرذان الذکور البالغة ( 60 جرذ)
: G بشکل عشوائی إلى مجموعتین رئیسیتین. مجموعة السیطرة (عدد= 20 ) قسمت إلى مجموعتین فرعیة: المجموعة الأولى 1
عشرة حیوانات اعطیت میلاتونین ( 5 ملغم / :G عشرة حیوانات من مجموعة السیطرة جرعة بماء مقطر والمجموعة الثانیة 2
عدد = 40 ) قسمت ) ACR کجم من وزن الجسم) لمدة 21 یوما. المجموعة الرئیسیة الثانیة: المجموعة التی جرعت بالاکریلامید
کما یأتی إلى الاکریلامید + ماء مقطر عن طریق الفم ومجموعةالاکریلامید + میلاتونین ( 5ملغم/ کغم من وزن الجسم / یوم)،
ومجموعة الأکریلامید+ فیتامین ج ( 200 ملغم/ کغم من وزن الجسم / یوم)،ومجموعة الأکریلامید + میلاتونین ( 5ملغم)+ فیتامین
ج ( 200 ملغم)/ کغم من وزن الجسم / یوم ولمدة 21 یوما. وبینت النتیجة عدم وجود فروق معنویة فی مستویات الانزیمات
فی مصل الدم بین مجموعة السیطرة التی عولجت بالمیلاتونین ومجموعة السیطرة. وتم تسجیل انخفاض معنوی ALP وALT وAST
فی جمیع المجموعات المعالجة مقارنة مع مجموعة الأکریلامید +ماء ALP فی مصل الدم، ومستویات ALT و AST فی مستویات
مقطر والمجامیع المعالجة بالمذکور أعلاه لا تزال أعلى بکثیر بالمقارنة مع مجموعة السیطرة. لم تسجل أی اختلافات معنویة فی
تراکیز البروتین الکلی والیوریا والکریاتینین فی مصل الدم بین مجموعة السیطرة + میلاتونین مقارنة مع مجموعة السیطرة. وتم
تسجیل تحسن کبیر فی تراکیز البروتین الکلی والیوریا والکریاتینین فی المصل لجمیع المجموعات المعالجة مقارنة مع مجموعة
الأکریلامید +ماء مقطر.
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Proceeding of 5th International Scientific Conference,College of Veterinary Medicine
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