River Warri is an important river in the Niger Delta of southern Nigeria. The
economic importance of the river is predicated on its rich biota (Egborge 1986;
Egborge and Tawarri 1987; Opute 1991, Tetsola and Egborge 1991; Ikomi 1995)
that provide various kinds of fishes for human consumption, means of inland
water transport for most communities in the region and the site of a large port
at Forcados (Ikomi 2000). However, the river is highly polluted due to the
presence of oil / petrochemical complex (Egborge 1991), enormous oil
exploration activities in Warri and its environs and discharge of domestic and
other industrial effluent into the river (Egborge, 1991).
extent of pollution of Warri Rivers has been monitored by an array of the
scientific communities (Atuma and Egborge, 1986 Egborge 1991; 1994)culminating
in changes in water quality indices (Egborge and Benka – Coker, 1986; Ikomi
1993; 2000) and bioconcentratin of trace metals in fish (Kakulu et al 1997;
Ezemonye 1992; Agada, 1994).
in natural environment are often exposed to a variety of stressors that can
adversely affect their health. Thus, there is a need to develop tools to assess
environmental related stress in fish (Afonso et al 2003). Some of the
biondicators of stress in fish include increase in levels of plasma cortisol,
glucose and lactate (Barton and Iwama, 1991); induction of heat shock protein
(Iwama et al 1999); glucose and drug metabolizing enzymes (Isamah and Asagba,
induction of oxidative stress in fish by polluted environment is well
documented (Bainy et al 1996; DiGuilio et al 1989; Hai 1997). This study
reports on oxidative stress on Clarias heterobranchus from Warri River
in southern Nigeria.
Materials and Method
study area is Warri
River. It is located
between latitude 50211-60.001N and
longitude 50251E. The river took its source at Utagba –
Uno and flow towards south west through Eziokpor, Amai, Otorho – Abraka, Warri
and emptying into the sea at Forcados (Tetsola and Egborge, 1991).
fishes, Clarias heterobranchus were obtained from the upper, middle and near
the lower course of the Warri
River between August and
September, 2004. The fishes were caught using instrument made locally. They
were transported alive to the laboratory and allowed to stabilize for one week before
they were dissected to extract organs and tissues of interest. Similar fish of comparable size
were obtained from a commercial fish pond located in Abraka and were used
The fishes were sorted and duly identified by the Department of Zoology, Delta State
University, Abraka, Nigeria.
total of 30 samples of mature size-matched male C heterobranchus (average wet
weight 340+5g and average wet length 38.7+3.2cm) were collected
from each site. Males were chosen for this study in order to be sex specific
since biomarkers have been found to be sex related (Afonso et al 2003). All the
reagents used were of analytical grade.
OF EXTRACT FOR THE DETERMINATION OF LIPID PEROXIDATION
the isolated organs 0.5g were separated and homogenized with 10ml of ice-cold
0.05M phosphate butter pH 7.0 containing 1% (w/v) Triton X-100, excess
butylated hydroxyl toluene (BHT) and a few crystals of protease inhibitor,
phenylmethylsulfonyl fluoride using an MSE blender immersed in ice. Triton
X-100 solubilizes membrane-enclosed organells while BHT prevents in vitro
oxidation of lipid during homogenization. The extract was centrifuged at 7000g
for 20 min (40C). The supernatant (S1) was used for the
determination of lipid peroxidation by the method of Hunter et al (1963) as
modified by Gutteridge and Wilkins (1982).
AND ASSAY OF CATALASE
was measured with a similar (S1) fraction after addition of 1% (V/V)
of ethanol and incubation at 40C for 15 min. This treatment is
reported to reverse the inactivation of catalase, which takes place, by the
formation of compound 11 (Cohen et al, 1970). Catalase activity was determined
according to Beers and Sizer (1952) by measuring the decrease in the H202
concentration, at an absorbance of 240nm. An extinction coefficient for H202
of 40M-1 cm-1 (Abei, 1974) was used in the calculation.
EXTRACTION AND ASSAY OF SUPEROXIDE
aliquot of the supernatant (S1) was precipitated on ice with 0.30
volume of chloroform/ methanol (3:5v/v) stirred for 20min and centrifuged at
7000g at 40C. The obtained supernatant (S2) was used for
the assay of superoxide dismutase (SOD) activity, which was based on its
ability to inhibit the oxidation of epinephrine by superoxide anion (Aksnes and
Njaa, 1981). One unit of superoxide dismutase activity is defined as the amount
of enzyme required for 50% inhibition of the oxidation of epinephrine to
adrenochrome at 480nm per min (Misra and Fridovich, 1972). Manganese dependent
SOD was analyzed in the presence of 1mM NaCN to suppress Cu-ZnSOD activity and
the cytosolic Cu-ZnSOD activity was determined as the difference between total
and cyanide – sensitive enzyme activity (Crapo et al, 1978). The enzyme
activities were assayed with an SP 1800 UV/VIS Spectrophotometer.
The mean and standard
error of the mean (SE), for the various parameters, were compared for
statistical significant difference using the students –T test. P. values<0.05 were taken as being significantly different. RESULTS: Table 1 shows the level of lipid hydroperoxide in control and test fishes. Lipid peroxidation was highest in the intestinal tract followed by the liver and lowest in the gill in control fish. However, in the test fishes the brain had the highest level of lipid peroxidation, followed by the intestine and the liver. The muscle had the lowest value. Moreover, in most of the studied organs the level of peroxidation was significantly (P<0.05) different in control relative to test fishes. Similarly, the level of lipid peroxidation was higher in the fish collected downstream when compared to those obtained upstream. The activities of superoxide dismutase (SOD) in different organs of fish from different parts of the river are shown in table 2. The results indicate that the activities of SOD in fish from mid and lower part of the river is significantly (P<0.05) different from the control. Moreso, the activity of the enzyme is highest in the heart and followed by the liver. The activity of the enzyme is low and almost similar in the muscle, brain, and intestinal tract. Table 3 shows the activities of catalase enzyme in organs of catfish from Warri river and reference hatchery. The enzyme activity is highest in the heart, followed by the liver but very low in the brain. The activity of the enzyme in fish from the middle and lower part of the river is significantly (P<0.05) higher than fish from the upstream and reference hatchery, respectively. DISCUSSION: Oxidative stress biomarkers were studied in the muscle, liver, kidney, heart and intestinal tract of African catfish, C heterobranchus from Warri River and comparable fish from a local fish hatchery which served as control. Oxidative stress biomarkers were lipid peroxidation, catalase and superoxide dismutase activities. The results showed that lipid peroxidation was significantly (P<0.05) higher in all the organs/tissues from Warri River compared to fish from reference hatchery (table 1). Similarly, the level of lipid peroxidation products in fishes collected in the lower sector of the river were significantly (P<0.05) higher than those of fishes collected from the upper sector (table 2). This observation is consistent with the report of Fatima et al (2000) and Achuba (2002). Lipid peroxidation has been used as a measure of xenobiotic-induced oxidative stress in fish and these include lipid perroxidation in Atlantic croacks (Thomas et al 1993); Indian catfish (Parihar and Dubey, 1995) and Channel catfish (DiGuilio et al 1993). Moreover, increase in lipid peroxidation has been reported in fish exposed to polluted environment (Munkittrick et al 1998; 2000. Fatima et al 2000).Besides acting as a mediator in oxidative stress, higher levels of lipid peroxidation products can adversely affects cellular functions (Munkittrick et al 1998; 2000) and adduct with proteins and DNA which may predispose the cell to mutagenesis and carcinogenesis (Bailey et al, 1992; 1996). The activities of superoxide dismutase and catalase were higher in fish from Warri River relative to fish from reference hatchery (Table 2 and 3). Like lipid peroxidation, the activities of these antioxidant enzymes were higher in fishes collected from downstream compared to those obtained from upstream. This result is consistent with earlier observations (Achuba 2002; Fatima et al 2000; Livingstone, 2001).A Previous report indicated that in response to increased levels of reactive oxygen species and oxidative damage, cells will usually increase the accumulation of a number of enzymatic antioxidants (Downs et al, 2002). Cu/ZnSOD, and MnSOD are some of the markers of cellular responses to increased reactive species and have been found to accumulate in response to oxidative stress (Downs et al 2002). Similar responses have been reported in aquatic species in an environment with a history of exposure to xenobiotic causing oxidative stress (Rodriquez-Ariza et al 1995; Otto and Moon, 1996). Exposure to xerobiotics has been reported to greatly induce production of reactive oxygen species (Gokaoyr and Husay 1998; Livingstone 2001). Previous reports have implicated SOD and catalase as working in tandem to dismutate oxygen radicals at physiological conditions (Achuba and Osakwe 2003). SOD converts superoxide anions to hydrogen peroxide which is broken down to oxygen and water by catalase (Voet and Voet, 1990). It is, therefore, no surprise for the observed increase in catalase activities in all the studied organs (Table 2). Pollution-induced increase in the activity of catalase had been reported earliar by some investigators (Rodriquez-Ariaze et al 1993; Hasspieler et al, 1994, Isamah et al, 2000). In generally, total superoxide dismutase and catalase activities have been reported as a potent mediator in chemical stress in fish (Achuba and Osakwe, 2003). Simirnoff (1993) found that an increase in the capacity of antioxidant defense in response to an increased level of reactive oxygen represents an indirect measure of oxidative stress. Fatima et al (2003) reported a significant increase in extra-hepatic oxidative stress in tissues such as kidney and gill of fish exposed to pulp and paper mill effluents. The higher value of antioxidant enzyme activities in fishes collected from the middle sector of the river relative to fish from reference hatchery and from the upper sector predicts that the fish collected in these regions of Warri Rivers are experiencing oxidative stress. The difference in the activity of these enzymes is a function of its environment and this has been established by Izokun-Etoibhio et al (1990). It is relevant to conclude that a polluted environment could result in increase lipid peroxidation, superoxide dismutase and catalase activities in tissues of C heterobranchus. On the whole, the results presented suggest that environmental polution could act as a mediator in the induction of oxidative stress in C heterobranchus.