Industrial and natural resources of water have polluted
the water body with arsenic (As) compounds. The As is extremely poisonous
element and it can cause cancer in living bodies (Wang et al. 2006; Kumari et al.
2017; Zhang et al. 2019). So, water
contamination with As is becoming a serious
environmental and health issue and its harshness is increasing (Allen and Rana
2004). Animals and plants present in water bodies are infected directly by As in toxic ion (Authman et
al. 2015). This situation also causes human health risk when they use water
polluted with As and via food chain due to
bioaccumulation of As in living bodies (Yang et al. 2014; Javed 2015).
Using the biomarkers such as
oxidative stress is a way to estimate the presence of As
in a water body. The end phenomenon is formed by an alteration in the stability
between antioxidant defense systems and reactive oxygen species (ROS) of
individual (Barata et al. 2005). The
ROS are comprised of free radical of hydrogen peroxide (H2O2),
hydroxyl radical (OH) and the superoxide anion (O2) (Lushchak 2011).
The synthesis of OH radicals and increase in H2O2
concentration takes place due to deposition of As in
the tissues of fish (Atli et al.
2006). Various researches proved that introduction to diverse pollutants, which include
As, encourages the production of ROS in cell (Ruas et al. 2008), provoking an increase in peroxidation of lipids and
change in activity of various antioxidant enzymes including glutathione
peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) (Valko et al. 2005; Lushchak 2011).
The antioxidant defense
systems have vital role in stabilization of cell and conservation of stern
control of free radicals (Halliwell and Gutteridge 1989). In aquatic
toxicology, antioxidant actions of enzymes serve as sensitive biochemical
pointers and are broadly used to evaluate the fitness of animal (Gul et al. 2004). Bhattacharya and
Bhattacharya (2007) stated that two sub-lethal dosages of heavy metal on Clarias
batrachus presented rise in the activities of antioxidant enzymes such as
CAT, SOD and GPX. They also recorded a reduction in the activity of glutathione
reductase (GR) after one day of imposition of treatments which shows that oxidative damage
in fish was caused at initial stages. Superoxide (O-2), which is
one of the parental forms of intracellular ROS, is a
reactive molecule but it is possible to transform it to H2O2 by
SOD and then to oxygen and water by various enzymes such as CAT and GR (Kumari et al. 2014). So, observing the variation
in antioxidant enzymes activity such as CAT, GR and SOD will be an efficient
way of representing oxidative stress and variations in activity. Hence, this
study aimed to elucidate the As sub-lethal toxicity on
antioxidant defenses (GR, CAT, POD and SOD) and the ability of these enzymes to
reduce the oxidative stress in Cyprinus
carpio.
Materials and Methods
The fish C. carpio (15 ±
2 cm total length and 50 ± 1 g weight) were collected
from fish nursery Karor Lali Ehsan, Layyah Pakistan, were shifted to laboratory
in polyethylene bags with water that was oxygenated. The fish were acclimatized
in aquaria for 15 days under laboratory conditions prior to the experiment.
Stone diffusers attached to a motorized air compressor were used to ventilate
the aquaria. The pH was maintained between 6.6 and 7.5 and temperature of water
was 25 ± 2ºC. The fish were given food containing egg, raw brine shrimp pellets
and goat liver twice a day, and were exposed to various As
concentrations viz., 1.25, 1.65, 2.50, and 5.0 mg/L. One group was
consider as a control treatment where fish were not exposed to As. The 10 fish
for each concentration of As test were used. The fish
were exposed to As concentrations for 30 days. In the
experimental aquaria, water was replaced daily with fresh treatment of As. Five
replicates for each concentrations of arsenic were arranged under Complete
Randomized Design (CRD). After 30 days, the fish were anesthetized by immersion
in 50 mg/L tricaine methane sulphonate (MS-222) solution for 5–10 min before
they were killed by transection of the spinal cord.
Sample preparation for bioassay
Every test group of the sample liver, muscle, blood and
gill was made according to Habbu et al.
(2008). By using the iced cold solution of KCl 1.15%, the organs were washed,
blotted and weighed. For homogenization, the phosphate buffer (0.1 M; pH 7.2) was used. The laboratory
acid-washed sand was added to it before placing each organ in mortar and it was
followed by blending with mortar and pestle. After blending, the resulted
material was centrifuged (at 2500 rpm) for 15 mins. The antioxidant enzymes
were determined through UV-visible spectrophotometer from the supernatant
stored at -21°C (Habbu et al. 2008).
Lipid peroxidation
The data on peroxidation of lipids was calculated using
thiobarbituric acid (TBARS) and color reaction for malondialdehyde (MDA)
according to procedure by Placer et al.
(1966). In this method, tissues were homogenized in chilled 0.15 M KCl using a Teflon pestle to obtain
10% w/v homogenate. One ml of homogenate was incubated at 37°C (± 0.5) for two
hours. To each sample, 1 mL of 10% w/v trichloro acetic acid (TCA) was added.
After thorough mixing, the reaction mixture was centrifuged at 2000 rpm for 10 mins.
One mL of supernatant was then taken with an equal volume of 0.67% w/v TBA and
kept in a boiling water bath for 10 mins, cooled, and diluted with 1 mL of
distilled water. The absorbance of the color pink obtained was measured at 535
nm against a blank. The concentration of MDA was read from a standard
calibration curve plotted using tetra-methoxypropane (Sigma-Aldrich Co., St.
Louis, USA).
Determination of
hydrogen peroxide
Ferrous oxidation-xylenol orange method was used to
measure hydroperoxide content (HPC) (Jiang et
al. 1992). To 100 μL of
supernatant previously deproteinized with 10% trichloroacetic acid (TCA) was
added 900 μL of the reaction
mixture [0.25 mM FeSO4, 25
mM H2SO4, 0.1 mM xylenol orange and 4 mM butyl
hydroxytoluene in 90% (v/v) methanol], incubating for 60 min at room temperature.
Absorbance was read at 560 nm against a blank containing only reaction mixture.
A type curve was used to interpose result and result was stated as nM cumene hydroperoxide/mg protein.
SOD activity
determination
The method of determining the activity of SOD is
dependent upon spectrophotometric calculation of retarding impact of SOD on
autoxidation of 6-hydroxyidopamine (6-OHDA) (Heikkila and Cabbat 1976; Crosti et al. 1987). When quantity of enzyme
reduces the autoxidation of 6-OHDA by 50%, in 1 min at 37°C, then 1U of SOD
activity is recognized. Meanwhile, in this reaction, the curvature of rate of
autoxidation is steady in the 1–60 seconds, at 490 nm until the 60th
s of oxidation, the spectrophotometric calculation was done. In units per
milligram protein, results were stated.
CAT activity
determination
Aebi’s method (1984) was used to measure catalase
activity. The principle of the test depends upon rate constant of H2O2
determination by CAT enzyme. Usually, it is considered that one unit (IU) of
CAT enzyme is equal to the enzyme activity that was recognized in 1 μmoL of H2O2
at 37°C in 60 s. By detecting the variation on absorbance of sample and blank
for a min, spectrophotometrically at 240 nm, the CAT activity was calculated.
Consequences were stated as units per milligram protein.
Determination of soluble
protein
Bradford method (1976) was used to determine total
soluble protein content. A 75 μL
deionized water and 2.5 mL Bradford’s reagent [in 500 mL deionized water, 50 mL
H3PO4, 25 mL of 96% ethanol and 0.05 g Coomassie blue dye] was added to 25 μL of supernatant. The test tubes shook and rest of 5 min was
given before reading absorbance at 595 nm and exclamation on a bovine albumin
curve (Sigma-Aldrich, St. Louis).
Statistical analysis
STATISTIX 8.1 was used for statistical analysis of the
work. Graphs were presented as means ± standard deviation. By using LSD test,
pair wise judgment between experimental and control groups was done to
determine the statistical significance of difference between the groups (Pipkin
1984).
Results
The activity of superoxide dismutase (SOD) in blood,
liver, gill and muscle of C. carpio was
significantly affected by As exposure. The activities
of SOD were increased significantly by increasing the sub-lethal concentrations
of As. In blood and gill tissues, the SOD activities were statistically equal
in the all sub-lethal concentrations of As but
significantly high than the control group (Fig. 1). In liver, 75% higher SOD
activities were found in fish exposed to 2.50 mg L-1 sub-lethal
concentration of As followed by 67, 64 and 51% SOD
activities determined in fish infected with sub-lethal amounts of 5.0, 1.65 and
1.25 mg L-1 correspondingly when related to controlled group (Fig.
1). The higher SOD actions were observed in the muscles of fish exposed to 5.0
mg L-1, related with control group. In addition, SOD activities were
significantly decreased in the muscles tissues of fish by decreasing the
sub-lethal concentrations of As (Fig. 1).
Sub-lethal concentrations of As significantly affected the activities of peroxidase (POD)
in muscle, liver, gill and blood tissues of C. carpio.
A significant increase in POD activities was found in all tissues of fish that
were infected with various sub-lethal amounts of As in
comparison with the control group of fish. In blood and gill tissues of the
fish, POD activities reached to the maximum value when exposed to 1.65 mg L-1
sub-lethal concentration of As after that there was sudden decreased in
POD activities with increasing the concentration of As (Fig. 2). An increased
in POD activity was observed in the liver tissues of the fish with increasing
the sub-lethal concentrations of As. Significantly higher POD actions were
noted in the fish’s liver tissues of infected with maximum 5.0 mg L-1
level of sub-lethal concentration of As (Fig. 2). In muscle tissues of the
fish, the POD activities were statistically at par in the all sub-lethal
concentrations of As but significantly high than the
control group (Fig. 2).
Statistical analysis revealed
that catalase action within blood, liver, gill and muscle of C. carpio were responsive to various sub-lethal
concentration of As. The catalase activity was increased in all tissues of fish
infected with sub-lethal amounts of As. Levels of catalase activities were
statistically equal at all the sub-lethal concentrations of As but higher than
control group of the fish in all the tissues (Fig. 3). Glutathione reductase
activities were significantly affected by sub-lethal concentrations of As in all tissues viz blood, gills, livers and
muscles of C. carpio. In
blood, the activities of glutathione reductase were significantly increased by
increasing the level of sub-lethal concentrations of As. Significantly higher
glutathione reductase activity was recorded at 5.0 mg L-1 sub-lethal
concentration that was followed by the concentrations of 2.50, 1.65 and 1.25 mg
L-1 of As respectively, when comparison to the control group of the
fish (Fig. 4). In gill tissues, glutathione reductase activity was increased up
to 2.50 mg L-1 of As concentration after that there was a decreasing
trend with increasing the concentration of As whereas; minimum activity of
glutathione reductase was noted in the control group of fish where fish were
not exposed to As concentrations (Fig. 4). Related with control group, when
noteworthy, the higher glutathione reductase activity was determined within
liver and fish’s tissues of muscle infected with 2.50 and 5.0 mg L-1 of
As. In addition, glutathione reductase activities were seriously decreased
within liver and fish’s muscles tissues by decreasing the sub-lethal
concentrations of As (Fig. 4).
Statistical analysis of data
indicated that there was significant increase in lipid peroxidation level in
various fish’s tissues that were infected with the different sub-lethal
concentrations of As. In blood and muscles tissues, maximum level of lipid
peroxidation was recorded when fish was exposed to 1.65 mg L-1 of As
that was statistically at par with sub-lethal concentration of 2.5 and 5.0 mg L-1
of As when compared with control group of fish (Fig. 5). In gill and liver
tissues, 5.0 mg L-1 of As induced significantly higher level of
lipid peroxidation that was followed by sub-lethal concentration of 2.5 and
1.65 and 1.25 mg L-1 of As whereas; minimum lipid peroxidation level
was determined in control group of the fish (Fig. 5). The results showed that
sub-lethal concentrations of As significantly enhanced the hydrogen peroxide
stuffing’s in blood, liver, gill and muscles of C. carpio.
In all tissues of fish, hydrogen peroxide contents were increased with
increasing the concentrations of As. Significantly higher level of hydrogen
peroxide was recorded in every fish’s tissue infected with 5.0 mg L-1
when compared to the other sub-lethal concentrations and control group of the
fish (Fig. 6).
Discussion
The As is an aquatic ecological pollutant.
Tissues of aquatic organism may get deposited by the
heavy metals (Farombi et al. 2007; Rauf et al. 2009) including As. Thus, examination of tissues deposited
with toxic
Fig. 1: Effect of various sub-lethal concentrations of arsenic on superoxide dismutase activity in blood,
gills, liver and muscles of C. carpio. Values
represent mean ± SE (n = 3). Different small letters indicated that the means
are significantly different (P ≤ 0.05)
Fig. 2:
Effect of various sub-lethal concentrations
of arsenic on peroxidase activity in blood, gills, liver and muscles of C. carpio. Values represent mean ± SE (n = 3). Different
small letters indicated that the means are significantly different (P ≤
0.05)
Fig. 3: Effect of various sub-lethal concentrations of arsenic
on catalase activity in blood, gills, liver and muscles of C. carpio. Values represent mean ± SE (n = 3). Different
small letters indicated that the means are significantly different (P ≤
0.05)
Fig. 4: Effect of various sub-lethal concentrations of arsenic on glutathione reductase
activity in blood, gills, liver and muscles of C. carpio.
Values represent mean ± SE (n = 3). Different small letters indicated that
the means are significantly different (P ≤ 0.05)
Fig. 5: Effect of various sub-lethal concentrations of arsenic on lipid peroxidation in blood, gills, liver
and muscles of C. carpio. Values represent
mean ± SE (n = 3). Different small letters indicated that the means are
significantly different (P ≤ 0.05)
Fig. 6: Effect of various sub-lethal concentrations of arsenic on hydrogen peroxide in blood, gills, liver
and muscles of C. carpio. Values represent
mean ± SE (n = 3). Different small letters indicated that the means are
significantly different (P ≤ 0.05)
metals in
the water livings may be a sensible valuation for the health of human and
animal standards (Kumar and Banerjee 2012a). It is reported that after
introducing the As, a great concentration of As
deposition in blood, brain, muscle, skin, gills, and liver tissues of the
catfish were observed (Kumar and Banerjee 2012b). Actions of enzyme are
understood as biochemical pointers and extensively used to evaluate the
organism’s health in toxicology of water (Gul et al. 2004). It is due to excess production of ROS induced by the
metal toxicity (Flora et al. 2005).
Oxygen species that are reactive can result in
serious damage or injury to tissue of liver, gills, skin, muscle, brain and blood
(Patlolla and Tchounwou 2005). In current study, exposure of the C. carpio to
sub-lethal concentrations of As was found to cause an increased level of lipid
peroxidation in liver, gills, skin, brain, muscle and blood tissues, which is
an indicative of oxidative stress in infected animals related to control group
(Wang et al. 2004). The rise in lipid
peroxidation is because of retarding outcome on mitochondrial system of
transport of electron resulting in stimulated formation of intracellular ROS
(Stohs et al. 2001). A minor increase
in ROS production in exposed faunas to arsenite was sufficient to encounter a
vital rise in peroxidation of lipid (Zarazúa et al. 2006). The results indicate that as the concentration of As increases, fish tissues are prone to redox reactions, that
generate free radicals specifically ROS i.e.,
H2O2 (Patra et al.
2011). The highest H2O2formed by the incomplete reduce
oxygen, may induce alteration and may change some physiological responses of fish
(Varanka et al. 2004; Brucka-Jastrzebaska 2010).
ROS
formed due to oxidative stress is neutralized by antioxidant resistance scheme
(Gumustekin et al. 2005). The action
of antioxidant enzymes can be boosted or repressed under heavy metals
concentrations depending upon concentration and the extent of the applied
stress, along with the vulnerability of infected species (Kumari et al.
2017). In this, the enzyme action of SOD was enhanced in blood, liver, gill and muscles
of C. carpio in comparison to control group. The SOD enzyme is recognized for
providing cyto-protection against damage induced by ROS due to conversion of
superoxide radicals (O2-) produced in mitochondria and peroxisomes
to H2O2 (Olagoke
2008). Increased level of SOD under heavy metal toxicity in
blood, brain, muscle, skin, gills and liver results in decreased oxidative
stress within tissues due to harmful actions of the superoxide (Bharti et al. 2012). This indeed proved
in the study because when infected fishes were related to the control, there
was an increase in activity of CAT due to the As
metals. Same phenomena of an increase in CAT activity was stated by Otitoloju
and Olagoke (2011), Saliu and Bawa-Allah (2012) and Fatima and Ahmad (2005)
that as increasing concentration of heavy metals enhanced the activity of CAT. In
present study, GR level has been significantly increased in liver, gills, skin,
blood & muscle tissue of C. carpio exposed to sub-lethal amounts of As. The reduction of arsenate to
arsenite is due to electron donation by Glutathione. ROS is generated by As cell metabolisms, however mechanisms are not well known.
Toxic metals’ collaboration with the metabolism of GR is an important part of
response of different toxic metals (Hultberg et al. 2001). Tissues are also defended from oxidative stress by it
(Jifa et al. 2006). In this study, after 30 days, the action of
GST was reduced in the tissues of fishes’ liver that were infected with heavy
metals when related to control. This consequence is an agreement of conclusions
of Saliu and Bawa-Allah (2012) and Otitoloju and Olagoke (2011).
Conclusion
The findings of the current study reveal that As creates harmful effects by generating the ROS that damage
the cells of organs by lipid peroxidation. However, it was counter balanced by
the production of antioxidants such as SOD, POD and GRX in organs tissues of
carp fish with increasing the sub lethal As
concentrations. It was concluded that defensive nature and the adaptive
mechanism of cells against free radical play an important protective role to
cope with As stress.
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