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ORIGINAL ARTICLE
Toxicological evaluation of the effect of
phenol-contaminated water on the liver of
albino rats
Aanuoluwa James Salemcity1,2, TiƟlope Ayoola Iyanda3, Olakunle Oladimeji1, Ayobami MaƩhew Olajuyin1
Departments of 1Biochemistry and 3Chemistry, University of Ibadan, Ibadan, Oyo State, 2Department of Biochemistry,
Salem University, Kogi State, Nigeria
A B S T R A C T
Phenol is a constituent of coal tar and is formed during the natural decomposition of organic materials. The effect of phenol-contaminated
water on the liver of rats was investigated. Activities of some liver enzymes, alkaline phosphatase, acid phosphatase, alanine
transaminase, aspartate transaminase, and gamma-glutamyl transpeptidase were determined alongside some serum indices of liver
function such as serum bilirubin, albumin, globulin and serum enzymes. The total bilirubin of rats treated with phenol-contaminated
water was observed to be 8.4 ± 0.8 g/l while that of control rats was 5.6 ± 0.5 g/l. Serum albumin of test rats was found to be 15 ± 2 g/l
while that of control rats was 7 ± 3 g/l. Activity of all the enzymes studied reduced significantly in the liver of test rats compared with
the control (P < 0.05). However, serum enzymes activity, with the exception of serum aspartate transaminase, of test rats increased
significantly (P < 0.05) relative to that of test rats. It is viewed that phenol-contaminated water is hazardous to health as it may be
responsible for the leakage of enzymes into the serum and may impair liver function as portrayed by reduced serum globulin and albumin.
Keywords: Enzyme activity, phenol, toxicological evaluation, water
INTRODUCTION
Human life, as with all animal and plant life on the
planet, is dependent upon water. Often, water that is
of a sufficiently high quality at the point of collection
is contaminated before it is used because it has to be
carried and stored before use or because of unhygienic
practices. Contaminants carried in water are dependent
on solid waste composition and on the simultaneously
occurring physical, chemical and biological activities
within landfill.[1]
Phenol is identified as a contaminant that is used in the
manufacture of phenolic resins, production of germicidal
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paints, pharmaceutical products, dyes, and disinfectant.
Potential sources of phenol exposure include: The
residential wood burning, cigarette smoke and presence
of phenol in liquid manure. Increased phenol has been
reported in sediments and ground water associated
with industrial pollution and this is due to an increase
in the use of synthetic chemicals, e.g. insecticides and
herbicides, which can easily run off from agricultural
land and industrial discharge into surface waters.[2]
Previous research reported that the phenol has serious
health effect. For instance, phenol solutions are corrosive
to the skin and eyes, while phenol vapor can irritate the
respiratory tract. Also, drinking water with extremely
high concentrations of phenol has caused muscle
tremors, difficulty walking, and death in animals.[3]
Quick Response Code:
Website:
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DOI:
10.4103/2348-3334.153257
The paucity of information on the effects of
phenol-contaminated water (0.06 mg/l) on animal
health [4] has necessitated the present study. The
present study investigated the hepatotoxic effect of
phenol-contaminated water on albino rats.
Corresponding Author: Dr. Aanuoluwa James Salemcity, Department of Biochemistry, Salem University, Kogi State, Nigeria.
E-mail: xityglory@gmail.com
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Salemcity, et al.: Effect of phenol contaminated water on liver
MATERIALS AND METHODS
The experimental water for the study was collected from
the water supply system of Adekunle Ajasin University,
Akungba-Akoko, Ondo State, Nigeria and the study
was conducted in the Department of Biochemistry of
the same university after prior permission from the
institutional animal ethics committee. Physiochemical
properties of all water sample were determined in
accordance with standard methods [5] and Atomic
Absorption Spectrophotometer (Buck 210VGP) was
used for the determination of heavy metals.
Experimental design
Twenty Albino rats (Rattus norvegicus) were obtained
from the Animal Holding Unit of the Department of
Biochemistry of University of Ilorin, Nigeria whereas the
animals were transported to the Animal Holding Unit
of the Department of Biochemistry, Adekunle Ajasin
University, Akungba-Akoko, Nigeria where the study
was conducted. The experimental sample size was
determined using power analysis[6] and animals were
assigned into two main groups of 10 animals each.
Each group was further classified into two sub-groups
detailed thus:
Group A: Rats placed on tap water (control)
Group B: Rats placed on water contaminated with
phenol (0.05 mg/l)
Group a: Treated as animals in Group A
Group b: Treated as animals in Group B
The phenol-contaminated water was prepared in
the laboratory with concentration of 0.05 mg/l. The
concentration of this contaminant is more than 10 times
the permissible limits[7] and typifies the concentration
found in the open runoffs.[8]
The feeding exercise lasted over a period of 65 days,
a long-term standard for rats.[8] All animals were kept
in a wooden cage and fed ad libitum after 10 days
acclimatization.
BLOOD SAMPLE COLLECTION AND ENZYME
ASSAYS
The rats were fasted overnight, anesthetized in a
jar containing chloroform and sacrificed by jugular
puncture. The blood was collected in an anticoagulant
free bottle and centrifuged at 3500 rpm for 15 min using
refrigerated centrifuge RC650s, and the serum obtained
was preserved at −8°C until required for use.
130
Liver samples were weighed and homogenized (1:10, w/v)
with ice-cold 50 mM Tris-HCl, (pH 7.4) using Teflon
and homogenizer (Janke and Kunkel, Germany). The
homogenates were centrifuged at 3500 rpm for 15 min
and the supernatant stored at −8°C until use. The
protein concentration in the tissue of experimental
rats was determined with Biuret assay using BSA as
standard.[9]
The activity of ALP in serum and tissues of experimental
rats was determined following the method described by
Bessey et al.[10] as modified by Wright et al.[11] A volume
of 0.1 M carbonate buffer (2.2 cm3) was dispensed
into test-tubes, 0.1 cm3 MgSO4.7H2O was added and
0.2 cm3 of either the homogenate or serum (sample)
obtained from the experimental animals was added
to the test tubes. Distilled water (0.2 cm3) was added
to the blank instead of the sample. The mixture was
incubated at 37°C for 10 min after which 0.5 ml of
10 mM PNPP was added. The mixture was further
incubated at 37°C for 10 min in water bath. The
reaction was stopped by adding 2 ml of 1 N NaOH
solution and the absorbance was read at 400 nm on a
Spectrophotometer.
The activity of ACP in serum and tissues of experimental
rats was determined following the method described by
Bessey et al. as modified by Wright et al. A volume of
0.1 M acetate buffer (1.3 cm3) was dispensed into two
test-tubes labeled blank and sample. Sample (0.2 cm3)
obtained from experimental rats was added to the
sample test-tube while the same volume of distilled
water was added to the blank instead of the sample. The
mixture was incubated at 37°C for 10 min after which
0.5 cm3 of 10 mM PNPP was added. The mixture was
further incubated at 37°C for 30 min in water bath, and
the reaction was stopped by adding 2.0 ml of 1 N NaOH
solution. The absorbance was thereafter read at 400 nm
on a spectrophotometer.
The activity of aspartate aminotransferase (AST)
in the serum and tissues of experimental animals
was determined following the procedure reported
by Reitman and Frankel[12] as modified by Schmidt
and Schmidt.[13] A calibration of AST standard curve
was first carried out by dispensing various volumes
(0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 cm3) of pyruvate
standard into different test-tubes. The volumes were
then made up to 1 cm3 with AST buffered substrate.
The mixture was shaken and incubated for 30 min
at 37°C. 0.001 M 2,4-dinitrophenylydrazine (1 cm3)
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Salemcity, et al.: Effect of phenol contaminated water on liver
was added to each of the test-tubes. The mixture was
allowed to stand for 20 min at 25°C after which 5 cm3 of
0.4 N NaOH was added. The absorbance of the mixture
was read at 546 nm on a spectrophotometer and was
plotted against the corresponding concentration of
pyruvate.
The activity of alanine aminotransferase (ALT) in
the serum and tissues of experimental animals was
determined following the procedure reported by Reitman
and Frankel as modified by Schmidt and Schmidt.
The activity of gamma-glutamyl transpeptidase (GGT)
was determined following the method described by
Tietz.[14] Sample (0.2 ml) and distilled water (0.2 ml)
was pipetted into different cuvettes (for sample and
blank respectively) and 2 ml of reagent (tris buffer,
glycylglycine, L-γ-glutamyl-p-nitroanilide and
surfactants) was added to each. The reactants were
mixed thoroughly, and the absorbance was read, against
blank, at 400 nm at time 0, 1, 2 and 3 min.
The bilirubin in the serum was determined by method
described by Tietz.[15] Sample/distilled water (1.5 ml) was
dispensed into test-tubes, 0.4 ml diazoA and diazo B was
added to the sample and blank test-tubes respectively.
Absorbance was read after 5 min at 540 nm.
RESULTS
The effect of phenol-contaminated water on the liver of
albino rats was studied in this research. Various indices
of liver function (bilirubin, albumin and globulin) were
determined in the serum of rats as shown in Table 1.
reduction in weight of group B rats began to manifest
from day 25.
Figure 2 shows the liver/body weight ratio of rats placed
on phenol-contaminated water over a period of 65 days.
The liver/body weight ratio of test group B rats was
significantly lower (P < 0.05) than that of the control
group A rats.
Figure 3 shows the specific activity of ALP of liver and
serum of rats placed on phenol-contaminated water
over a period of 65 days. The specific activity of ALP
of liver of rats in group B was found to be significantly
lower (P < 0.05) than that of group A. Conversely, the
specific activity of ALP of serum of rats is observed to
be significantly higher (P < 0.05) in test group B relative
to control group A.
Figure 4 shows the specific activity of ACP of liver and
serum of rats placed on phenol-contaminated water
over a period of 65 days. The specific activity of ACP
of liver of rats was found to be significantly lower
(P < 0.05) in test group B relative to control group A
whereas the specific activity of ACP of serum of rats
is also significantly higher (P < 0.05) in test group B
compared to control group.
Figure 5 shows the specific activity of ALT of liver and
serum of rats placed on phenol-contaminated water
over a period of 65 days. The specific activity of ALT
of liver of rats was observed to be significantly lower
(P < 0.05) in test group compared to control group A.
Also, the specific activity of ALT of serum of rats is
significantly higher (P < 0.05) in test group B relative
to control group A.
Generally, the indices of liver function namely
bilirubin (direct and total), albumin and globulin in
test group of rats were observed to be significantly
higher (P < 0.05) than those of control group A.
230
180
Table 1: Effect of phenol-contaminated water on some
indices of liver function in the serum of rats
Group
of rats
A
B
Total
Albumin
(g/l)
Globulin (g/l)
Direct
Bilirubin (u/mol/l)
1.2±0.03
3.2±0.2*
5.6±0.5
8.4±0.8*
15±2
7±3*
10±1
5±0.7*
A
B
Weight (g)
Figure 1 shows the growth pattern of rats placed on
phenol-contaminated water over a period of 65 days.
The weight of rats in group B were significantly
lower (P < 0.05) than those of rats in group A. This
130
80
30
0
Tabulated results are means of five determinations±SD * Values are significantly
different (P<0.05)
5
10
15
20
25
30 35 40
Time (Days)
45
50
55
60
65
Figure 1: Growth pattern of rats placed on phenol-contaminated water over a
period of 65 days. Plotted results are means of five determinations ± standard
deviation (A and B are the group of rats)
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Salemcity, et al.: Effect of phenol contaminated water on liver
7
0.043
a
Specific Activity (U/mg Protein)
0.042
b
Liver/Bd. Wt. Ratio
0.041
0.04
0.039
a
0.038
0.037
0.036
6
A
B
5
b
4
3
2
b
0.035
1
0.034
0
0.033
A
B
a
Liver
Serum
Rat Tissues
Group of Rats
Figure 2: Liver/body weight ratio of rats placed on phenol-contaminated water
over a period of 65 days. Plotted results are means of five determinations
± standard deviation bars with same notations are not significantly different
(P > 0.05) (A and B are the group of rats while a and b are the bar notations)
18
Serum
b
0.5
a
0.4
0.3
0.2
0.1
0
a
A
B
b
14
12
10
8
6
4
A
Group of Rats
B
Figure 4: Specific activity of ACP of liver of rats placed on phenol-contaminated
water over a period of 65 days. Plotted results are means of five determinations
± standard deviation bars with same notations are not significantly different
(P > 0.05) (A and B are the group of rats while a and b are the bar notations)
Figure 6 shows the specific activity of AST of liver and
serum of rats placed on phenol-contaminated water over
a period of 65 days. The specific activity of AST of liver
of rats is significantly lower (P < 0.05) in test group B
compared to control group A. In contrast, there exists
no significant difference (P < 0.05) between the specific
activity of AST in the serum of group A and group B rats.
Figure 7 shows the specific activity of GGT of liver and
serum of rats placed on phenol-contaminated water
over a period of 65 days. The GGT specific activity
of the liver is significantly lower (P < 0.05) in test
group B compared to control group A. In contrast, the
GGT specific activity of the serum was observed to be
significantly higher (P<0.05) in test group B than that
of control group A.
132
16
Specific Activity (U/mg Protein)
Specific Activity (U/mg Protein)
0.6
Figure 3: Specific activity of ALP of liver of rats placed on phenol-contaminated
water over a period of 65 days. Plotted results are means of five determinations
± standard deviation bars with same notations are not significantly different
(P > 0.05) (A and B are the group of rats while a and b are the bar notations)
2
0
a
Liver
b
Serum
Rat Tissues
Figure 5: Specific activity of alanine aminotransferase of liver of rats placed on
phenol-contaminated water over a period of 65 days. Plotted results are means
of five determinations ± standard deviation bars with same notations are not
significantly different (P > 0.05) (A and B are the group of rats while a and b are
the bar notations)
DISCUSSION
Some indices of liver function in the serum of rats
namely bilirubin, albumin and globulin have responded
to phenol-contaminated water [Table 1]. Bilirubin
protects tissues against oxidative damage caused by
free radical and other reactive oxygen species. Baranano
et al.[16] reported that the statistical analysis of people
with high normal bilirubin levels in the blood shows
that they have a lower risk of developing cardiovascular
diseases. Mild rises in bilirubin may be caused by an
increased breakdown of red blood cells while a very high
level is also caused by severe liver failure with cirrhosis.
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Salemcity, et al.: Effect of phenol contaminated water on liver
25
90
80
A
B
a
Specific Activity (U/mg Protein)
Specific Activity (U/mg Protein)
30
b
20
15
10
a
A
B
70
60
b
50
40
30
10
5
a
a
0
Liver
0
b
a
20
Serum
Rat Tissues
Liver
Serum
Rat Tissues
Figure 6: Specific activity of aspartate aminotransferase of liver of rats placed
on phenol-contaminated water over a period of 65 days. Plotted results are
means of five determinations ± standard deviation bars with same notations are
not significantly different (P > 0.05) (A and B are the group of rats while a and
b are the bar notations)
Hemoglobin transports oxygen from the lungs or gills
to the rest of the body such as muscle. Decrease of
hemoglobin with the absolute decrease of red blood cells
leads to symptoms of anemia. It was reported that the
decrease in hemoglobin can cause renal failure. Human
serum albumin maintains oncotic pressure, transport
thyroid hormones and fatty acids to the liver. Low level
of human serum albumin causes hypoalbuminemia.
A report showed that glycosylation has the potential to
alter the biological structure and function of the serum
albumin protein.[17]
The observation on body weight [Figure 1] may be
associated with the high concentrations of phenol
in the phenol contaminated water which may make
utilization of the nutrients inefficient or complexing
with the nutrients that should have been available for
use by the rats causing malabsorption of the nutrients
in the gastrointestinal tract. Dolk et al.[18] reported that
stunted growth is observed in children who consume
contaminated water containing high levels of organics.
Liver-body weight ratio of experimental rats [Figure 2]
revealed that, within the period of the experiment, the
concentration of phenol in the contaminated water
may have affected the liver– body weight ratios of the
animals in those groups. However, this may be because
the level of phenol in contaminated water is several
times higher than the safe level for drinking water
and have the potential to produce desirable effects on
tissues, particularly liver.
Figure 7: Specific activity of gamma-glutamyl transpeptidase of liver of rats
placed on phenol-contaminated water over a period of 65 days. Plotted results
are means of five determinations ± standard deviation bars with same notations
are not significantly different (P > 0.05) (A and B are the group of rats while a
and b are the bar notations)
Alkaline phosphatase is essential for the hydrolysis
of nucleotides of ATP, ADP and AMP in human liver.
It is a marker enzyme for the plasma membrane and
endoplasmic reticulum. Reduction in the liver ALP
specific activity and subsequent increase in the serum
of rats with phenol-contaminated water [Figure 3] may
seem to be due to malfunctioning of the liver membrane.
The reduction could also be attributed to inhibition of
the enzyme by the contaminated water as is the case with
the consumption of metasulfate[19] or inactivation of the
enzyme molecules in situ.[17] Furthermore, the membrane
of the liver may have been damaged by the pollutants
present in the phenol contaminated water thereby
allowing the leakage of ALP into the extracellular fluid.
The specific activity of acid phosphatase of liver of
rats placed on phenol-contaminated water is shown in
Figure 4. ACP is a lysosomal enzyme and very hydrolytic
in nature. It occurs when there is a fall in cytosol pH due to
apoptosis. ACP is also needed to trigger specific chemical
reactions and hence, used to monitor carcinogenicity in
the prostate.[20,21] Reduction in ACP activity in the liver
of the test rats may be as a result of the interactions
between the constituents of the contaminated water and
the components of the membrane causing the leakage
of the enzyme into the serum of the extracellular fluid,
hence, the high value observed in the serum. This leads
to malfunctioning of the liver.
The specific activities of the transaminases (AST and
ALT) of liver tissue and serum over phenol- contaminated
water are presented in Figures 5 and 6. Transaminases
are used to monitor the activity of the liver especially
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Salemcity, et al.: Effect of phenol contaminated water on liver
in the serum and also, it predicts liver diseases. The
reduction in the specific activities of AST and ALT in the
liver implies that constituents of phenol-contaminated
water may be inhibiting the enzymes or causing their
leakages into the serum. Previous study (Schimidt and
Schmidt, 1963) reported that elevated serum level of
ALT may be due to leakage of enzymes from the liver
and may significantly impair liver function. Conversely
affected by the treatment suggests the inhibition of
the enzyme rather than leakage of enzymes from
the cell into the extracellular space. Kumar et al.[22]
reported that AST and ALT activities reduction in the
liver is inversely proportional to the concentration of
pollutants, e.g. ammonia in a polluted or contaminated
water.
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The data generated from this study suggest that:
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Cite this article as: Salemcity AJ, Iyanda TA, Oladimeji O, Olajuyin AM.
Toxicological evaluation of the effect of phenol-contaminated water on the
liver of albino rats. CHRISMED J Health Res 2015;2:129-35.
Source of Support: Nil. Conflict of Interest: No.
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