Cellular stress response, induction of micronuclei and DNA strand breaks in two common sh species of Rivers and Reservoirs in Ilorin, North Central, Nigeria.

Most rivers and reservoirs in the world are prone to pollution because of indiscriminate disposal of domestic, agricultural and industrial wastes into the water bodies. In this study, we investigated the ecotoxicological potential this could pose in sh species collected from major reservoirs and rivers in Ilorin, north central, Nigeria. Water samples were collected and the physicochemical parameters were examined from ve different sites; Unilorin reservoir, Asa reservoir, Apodu reservoir, Asa river (Unity) and Asa river (Harmony). We determined serum biochemical (AST, ALT, ALP, serum ALB), histopathological (gill, lungs), serum antioxidant enzyme responses (SOD, CAT, GPx, GR, GST) which serves as a biomarker for evaluating oxidative stress while micronucleus and comet assays were used to detect level of DNA damage in Tilapia zillii and Clarias gariepinus. The physicochemical parameters and heavy metal analysed (Pb, Mn, Cu, Ni, N, P, Fe, Cl, and Ca) in the ve different water bodies were below the permissible limits of WHO and USEPA except the DO, which was very low in the two rivers, indicating hypoxia. Our results showed signicant increase in biochemical and hematological proles, histopathological lesions in the gill and lungs, inductions of MN, NA and DNA single strand break in Tilapia zillii and Clarias gariepinus collected from Asa rivers compared to the Unilorin, Apodun and Asa reservoirs. This may be attributed to indiscriminate discharge of euents from nearby industries, agricultural and domestic wastes into the rivers.


Introduction
Water is a universal solvent capable of dissolving many substances to some varying degree. It is an invaluable resource as it offers a major support to both all ora and fauna. Reservoirs and rivers are very useful ecological resources that serve numerous human needs such as water conservation (irrigation water supply for domestic needs), ood control, hydroelectric power generation, etc (Mustapha, 2010).
There have been challenges of water conservation due to pollution. This could be attributed to the daily in ux of pollutants from domestic agricultural waste and industrial wastes which have adverse effects on ecosystem health (Kushwaha et al. 2012;Anifowoshe et al. 2018;Oladipo et al. 2018). Water pollution is recognized globally as a potential threat to both human and other animal population which interact with the aquatic environment; this causes gradual degradation to the quality of the water body (Sevenson et al. 1995;Anifowoshe et al. 2018;Anifowoshe et al. 2019).
Fish are of ecological importance and commercially valued in the Nigerian shing industries and around the world (Ita 1980;Oladipo et al. 2018). Thus, shes like Clarias gariepinus and Tilapia zillii are frequently and widely cultured in ponds and they also occur freely in Nigerian's natural fresh water. Hence, aqueous environment variability as well as physic chemical changes have great effect on the sh physiology (Musa and Omoregie 1999) Fishes are good indicators of biocontamination because their biochemical stress responses are quite similar to those of mammals (Mishra and Shukla 2003;Anifowoshe et al. 2019), as they accumulate pollutants directly from contaminated water.

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Fish have also been known as a good choice of animal for use in evaluating toxic potential of contaminants (Martin and Costa 2015) due to their ability to metabolize and store pollutants in water in various their organs. They also show fast responses to low concentration of toxicants and the responses are alike to that of vertebrates since sh possess similar vertebrate antioxidant enzymes used to nullify the harmful effects of ROS (Almeida et al. 2002;Klobucar et al. 2010).
Asa reservoir, Oyun reservoir and Apodu reservoir and the water bodies of the following rivers support a wide range of sh species and constitute the bulk of water being used in Ilorin metropolis, University of Ilorin and Malete town respectively in Kwara state (Omotosho 1998;Mustapha 2010;Oladipo et al. 2018).
Similarly, fresh sh and sh products were supply to the populace through it resources. However industrial, agricultural and domestic waste discharges and practices are carried out along the bank of the river. These discharges contaminate the water with heavy metals and cause devastating effect on ecological balance of recipient environment (Anifowoshe et al. 2018;.
This present study is an attempt to give an updated information on the current genotoxic status of major water bodies in Kwara state Nigeria by investigating the physicochemical parameters of the waterbodies, tissue lesions, oxidative stress and antioxidant response in the selected sh species and assessing the level of genotoxic damage induced in the selected sh collected from ve different waterbodies using micronucleus and comet assays.

Asa reservoir (ARe)
Asa reservoir lies at a point 5 kilometers south of Ilorin across river Asa between latitudes 8 25 -8 27 N and longitude 4 32 -4 34 E. The reservoir was constructed between May 1975 and January 1977, with the aim of meeting the ever-increasing demand of the villagers and nearby community for drinking, irrigation, and as a source of income through commercial shing for the rapidly growing population of Ilorin,the state capital of Kwara state. The major tributaries are rivers Iwonte, Jia, and Segbekuke. The reservoir is very large and wide with a maximum length of 20km, a breadth of 7km and a depth of 13m, with a storage capital of about 43 million cubic meters (Omotosho 1998 and 2016 with a depth of about 5m and a submerged area of about 650 km 2 . The main purpose of the dam is to supply the university communities water for dinking and irrigation. No industry is located on its course, thus, the water rarely received any wastes.

2.1.3Apodu reservoir (APRe)
Apodu reservoir in Apodu village, about 7 km away from Malete Town in Moro local government area of Kwara State, North Central Nigeria. The dam was constructed in the year 1980 and the re-impoundment of the dam was carried out in 2016. It lies between the longitude 8°45'25.9" N, 45'27.7" N and latitude 4°27'41.4"E, 4°27'35.5"E with 560m long and 400m wide and has a depth of 8.2m with its surface area of about 15 hectares. It is characterized by two seasons' i.e. dry season in which water ow rate decreases and rainy season in which there is continuous ow of water, it falls within the guinea savannah region of Nigeria. The main purpose of the reservoir is to provide water for drinking and irrigation for the surrounding communities (Anifowoshe et al. 2018;Oladipo et al. 2018;Oladipo et al. 2019).

Asa River (Unity) (ARiU)
Asa river is one off the major rivers in Ilorin Kwara state with the coordinate of 8°28'0"-8°31" N and 4°32'0"-4°34" E. Different companies like Dangote, Coca-cola, pharmaceutical and detergent industries are located near the river. Little agricultural activities like Ugwu and cassava plantation are also found near the river. The dumping of refuses, sewage disposal and indiscriminate discharge of e uents/chemicals from the above industries into the body of the river is common thereby making the water appear un t for drinking and domestic purposes (Kolawole et al. 2011).

Experiment
Two different sh species, Tilapia zillii and Clarias gariepinus were collected from the ve sites namely Unilorin reservoir, Apodu reservoir, Asa reservoir, Asa river (unity) and Asa river (Harmony) in the early hours of the day (6:00-8:30am). They were caught with the help of shermen using cast net, gill net and basket. The shes caught were sorted and identi ed into species level following Idodo-Umeh (2003) and Olaosebikan and Raji (1998) and also with the help of a sh taxonomist. T. zillii (scaled sh) and C. gariepinus (scale-less sh) were the two common sh species caught at the various sites, however, T. zillii shes were abundance and were used mostly in our experiment.
The sampled shes were transported transparent 50-L plastic aquaria with net cover (to allow ventilation and prevent the sh from jumping out) lled with water two-third volume to the Department of Zoology, University of Ilorin, Ilorin, Nigeria. The sh were allow recovered from stress before the collection of blood through the tail region or by caudal vein puncture for analyses. For each analysis, two different sh species (n=2) were used for our experiments.
Water samples from the reservoirs and rivers were assess for various parameters such as temperature, pH, electrical conductivity, dissolved oxygen (DO), total dissolved solids, total suspended solids, biochemical oxygen demand (BOD), chemical oxygen demand (COD) and heavy metals. Water parameters were measured using a Hanna portable waterproof tester, model HI 98129. The heavy metals were determined using a Hanna multiparameter bench photometer for laboratories, model HI 83200, and an AAS (Atomic Absorption Spectrophotometer) (model: Buck scienti c ACCUS-IS 211) was used.

Oxidative stress and tissue lesions analysis
To carry out the oxidative stress analysis, blood was taken from the T. zillii (n=3) from each site and kept in EDTA bottles prior to analysis and protocol by Reitman and Frankel, (1957) was followed with modi cation. The gills and liver tissue samples for lesion analysis were collected through dissection and xed. These tissue samples xed in buffered formal saline for a day (24 hours) were rinsed vigorously with distilled water and process for para n tissue embedding using the methods of Drury and Wellington (1980) and Gobinath and Ramanibai, (2014). The sections obtained from the organs were stained with hematoxylin and eosin (H & E) and observed using an Olympus light microscope using the standard procedure of Bancroft and Gamble (2008).

Haematological Analysis
Blood analysis was performed according to the methods described by Svobodova et al (2001), the blood samples collected were from T. zillii collected at the 5 sampling sites and taken to the laboratory for analyses such as red blood cells (RBC) pro les, white blood cells (WBC) pro les and platelets using standard method Sovio, and Oikari (2004) 2.5 Antioxidant Enzyme The use of 1 chloro 2, 4 dinitrobenzene as substrate was used for determination of Glutathione -Stransferase (GST) activity (Habig et al. 1974). The speci c activity of glutathione S-transferase was expressed as nmoles of GSH-CDNB conjugate formed/min/mg protein using an extinction coe cient of 9.6mM-1cm-1. The catalase activity (CAT) assay based on the breakdown of H 2 O 2 was performed as described by Clairborne (1995) and the absorbance was measured at 240nm (pH 7.0, 28°C) and expressed as unit/mg protein.
Superoxide dismutase activity (SOD) was determined by the method of Misra and Fridovich (1972) in which the assay depends on the auto oxidation of adrenalin due to the presence of superoxide anion, measured spectrophotometrically at 420nm and expressed as a unit/mg protein. The method of Paglia and Valentine (1967) was used for the assay of glutathione peroxidase. About 0.2ml of tissue homogenate was added to the mixture containing 0.2ml of buffer, 0.2ml of EDTA and 0.1ml of sodium azide. After well mixing, 0.1ml of reduced glutathione and 0.1ml of hydrogen peroxide were added before incubating in a water bath at 37°C for 10min. At the end of incubation period, 0.5ml of 10% TCA was added and centrifuged at 10000 rpm for 5min. 1.0ml of the supernatant was taken into a separate test tube and added 2.0ml Tris buffer and 50 µl DTNB. Immediately, the OD was measured at 412nm. While Glutathione reductase (GR, EC 1.6.4.2) activity was determined as described by Glatzle et al. (1974).

Micronucleus Assay
Blood was collected from the caudal region of the two sh species (T. zillii and C. gariepinus) [n=2] each using 2ml syringe and needle. The needle was inserted ventrally at the caudal region of the sh body until it pricks the vertebral column and then moved a little away so that the blood can ow into the syringe.
The dispenser pulled about 0.5 ml of blood and immediately a drop of blood was placed on a clean, grease free microscope slides to make a thin blood smear. The smeared slides were allowed to air-dry overnight in a dust free environment. The air-dried slides were xed in 70% absolute methanol for 20 minutes and air dried overnight. After drying the slide was stained with 10% Maygrunwald which was rinsed with distilled water and air dried overnight. Glass slide was subsequently stained with 5% giemsa, rinsed with distilled water and dried. The dried slide was then scored under light microscope by counting a total number of 2000 erythrocyte which were examined with oil immersion at 1000Xmagni cation for micronucleus (MN) and nuclear abnormalities (NA) as biomarkers of cytogenotoxicity (Carrasco et al. 1990;Ergene et al. 2007).

Comet Assay
A fully frosted slides were pre-coated with 1% normal-melting-point-agarose overnight, which formed the first/base layer. A mixture of 75 μl of 0.7% low-melting-point agarose (LMA) and 25μl of lymphocyte suspension was applied as the second layer. Cover slips were immediately placed over the second layer, and the slides were chilled on ice for 10 min to solidify the agarose. The cover slips were removed and a third layer of 90 μl 0.5% LMA was applied, the cover slips replaced, and the agarose allowed to solidify over ice for 10 min. All samples were done in triplicate. The slides were immersed in cold alkaline lysis solution for 2 h at 4°C. Then, slides were placed in chilled buffer for 20 min at room temperature in a horizontal electrophoresis tank pre-lled with cold alkaline electrophoresis buffer to loosen the tight double-helical structure of DNA for electrophoresis. Electrophoresis was then performed at 25 V, 300 mA for 20 min in electrophoresis buffer at 4°C. After electrophoresis, Tris buffer (0.4 M Tris, pH 7.5) was gently added drop-wise to neutralize excess alkali; the buffer was allowed to remain on the surface of slides for 5 min. This neutralizing procedure was repeated three times. The slides were then stained with 80 μl propidium iodide (2 μg/ml) for 10 min. All of the above procedures were performed in the dark to avoid additional DNA damage (Bajpayee et al. 2005). The comets were viewed using a Nikon 90i uorescence microscope, and images of 100 comets were collected for each concentration using a digital imaging system. Cells that overlapped were not counted. All the comet images were analyzed using Comet Assay Software Project (CASP, Wroclaw University, Poland) and the tail length (TL), % tail DNA (%TDNA), and Olive tail moment (OTM) were recorded to describe DNA damage to lymphocytes.

Statistical Analysis
The SPSS software package version 21.0 (SPSS 21.0) was used to evaluate the micronucleus abnormalities and the differences between the test groups. The differences were analysed by comparing them with the use of one-way ANOVA, and the level of statistical signi cance was estimated at p < 0.05 using the Duncan multiple range test (DMRT), the mean standard error was calculated.

Physicochemical parameters of the water
The physicochemical parameters (COD, BOD, DO, TDS, pH, conductivity & temperature) and heavy metals analysed in the water samples (Manganese, Copper, Chloride, Nitrate, Phosphate, Iron, Nickel, and Calcium) were mostly within the maximum level approved by standard organization except lead (Pb) that was above the standard level approved, the value was high at 2.683 mg/l in Asa reservoir which also recorded high value of Cu at 1.29mg/l. Also, Ca, Ni, and Cl were observed to be highest at Unilorin reservoir (Table 1). High values of COD, BOD, TDS and Conductivity were detected in Unity, Apodu reservoir and Asa river Harmony. Here, we observed that Asa reservoir seems to have low values of all the parameters measured when compared to the other four sites.

Biochemical and tissue lesions analyses
In table 2, there was a signi cant (p<0.05) increase the serum ALT, AST, ALP and albumin enzyme activities of T. zillii in Asa river Unity compared to other four sites. The AST enzyme activities in Asa river Unity and Asa river Harmony show higher activities compared to the reservoirs. Similarly, the ALT of Asa river Unity and Apodu reservoir also showing higher activities compare to other sites. Figure 2I representing the photomicrograph of the gills of T. zillii from the sampling sites. A represents photomicrograph of the gill of T. zillii from Unilorin reservoir which shows a high-power magni cation of the highly vascularized gill arch and outgrowing lamellae. Apodu reservoir (B) shows a high-power magni cation of the highly vascularized gill arch and their adjoining primary and secondary lamellae. It appears normal with long and well vascularized lamellar system with no pathological alteration. The primary lamellae appear short and supported in the central part by a cartilage with appreciable vascular supply. Figure 2I (C) shows highly vascularized gill arch and out growing lamellae of T. zillii from Asa reservoir with no signs of pathological alterations. The primary lamellae appear short and supported in the central part by a cartilage with appreciable vascular supply. Figure 2I (D) represents the photomicrograph of T. zilli gill from Asa river in Unity which shows a high-power magni cation (x400) of the gill arch and gill rakes. There appears to be some pathological alteration in the secondary lamella as they appear to be degenerating, distorted cartilage with epithelial lining. In Figure 2I (E) which represents the photomicrograph of the gills of T. zillii from Harmony, shows the gill arch and their adjoining primary and secondary lamellae. It appears distorted with as in the gill arch and perturbation of gill laments which appear to be degenerating and distorted.
Representative photomicrographs of the liver in Unilorin reservoir in Figure 2II (A) shows densely exhibited hepatocytes nuclei (black arrows) with normal staining characteristics and cellular disposition. Their general histomorphology appear characteristically normal with no apparent pathological alteration. In C; T. zillii liver from Asa reservoir shows densely distributed hepatocyte nuclei (black arrow). The micrograph appears characteristically normal with typical staining intensity and normal histoarchitectural manifestation. In D (Unity); the photomicrograph shows the central vein (black outline) and a high magni cation of the hepatocytes (black arrow) revealing the disposition of the nuclei, staining intensity and general histomorphological presentation. Histomorphological presentation of the liver showing typically sized halo spaced central vein surrounded by densely distributed hepatocytes. In E (Harmony), the T. zilllii liver shows densely distributed hepatocyte nuclei (black arrow). The micrograph appears characteristically normal with typical staining intensity and normal histoarchitectural manifestation.

Haematological analysis
Value of hematological parameters are presented in table 2 as mean value with ± standard deviation. Data presented in table 3A indicates signi cant increase (p<0.05) in RBC (red blood cell), HGB (haemoglobin) MCV (mean corpuscular volume) and MCH (mean corpuscular haemoglobin) in T. zillii from Asa river (Unity) compared to other sampling sites. There was also a signi cant increase in MCH and MCHC (mean corpuscular haemoglobin concentration) in sh from Asa river (Harmony). The differences for HCT (haematocrit) and RDW (red blood cell distribution width) were insigni cant across the 5 sites. In WBC pro les (WBC (white blood cell), LYM (lymphocyte), MID (mid-sized cells and GRAN (granulocyte)) measured, no signi cant difference (p<0.05) was observed across the 5 sites. However, T. zillii at Asa river (Unity) show a signi cant increase in GRAN when compare to others (Table 3B). PLT (platelet) MPV (mean platelet volume), PDW (platelet distribution width) PLCR (platelet larger cell ratio) and PCT (plateletcrit) measured show no signi cant difference (p<0.05) across the 5 sites. However, there was a signi cant increase in PLT and PDW in T. zillii at Asa river (Harmony) and PLT and PLCR at Asa river (Unity) ( Table 3C).

Antioxidant production
The trend of antioxidant enzymes responses in blood of T. zillii across the ve sampling sites were statistically different (p<0.05) in the order; SOD responses was highest in Unity river; followed by Unilorin; Harmony; Apodu and lowest response recorded at Asa reservoir; CAT responses was signi cantly highest in Unity; followed by Harmony; Unilorin; Apodu; and least signi cant in Asa reservoir; GPx responses was greatly induced in Apodu; followed by Asa reservoir; Unity; Harmony; and lowest in Unilorin; GR responses was signi cantly Unilorin; followed by Apodu; Harmony; Unity and Asa reservoir; GST responses was highest in Asa reservoir; followed by Apodu; Unity; Harmony and Unilorin (Table 4; Figure 3).

Micronucleus test
The peripheral blood erythrocyte of sh species (T. zillii & C. gariepinus) collected at different the sites show induction of micronuclei (MN) and other nuclear abnormalities (NA) which include; binucleated, nuclear bud, notched, lobed and blebbed. Site comparisons were conducted with each of the two sh species collected. The level of damage observed based on the frequency of MN & NA ( Figure 4I) are in the following descending order: (ARiU> (ARiH) ≥ (APRe) ≥ (URe) > (ARe) ( Figure 4II). It was observed that T. zillii showed a higher level of NA when compared to C. gariepinus in this study ( Figure 4II).

Comet assay
The level of DNA damage observed in the peripheral lymphocytes of shes obtained at the various sites were measured using the alkaline comet assay with parameters such as %tail DNA, olive tail moment and tail length as shown in Figure 5a and b. Signi cant differences (p < 0.05) were observed at some the sampling sites. The highest degree of DNA single strand break was detected in the blood cells of shes collected from Asa river Unity. No signi cant difference in the level of DNA single strand break was observed in shes collected at Unilorin and Asa reservoir. Over all the DNA damage observed between the two sh species can be said to be higher in T. zillii when compared to C. gariepinus. DNA damage was observed to be higher in sh species collected from the river sites when compared to the selected reservoirs. It was observed that at the reservoir sites, there is elevation in the level of DNA damage in Apodu reservoir. The level of DNA damage across the 5 sites goes in this order: Asa river unity >Apodu,> Asa river harmony,>Unilorin>Asa reservoir

Discussion
Most rivers and reservoirs in the world are prone to pollution because of indiscriminate disposal of domestic, agricultural and industrial wastes into the water bodies. The increased discharge of industrial e uents, domestic and agricultural wastes into waterbodies has raised a great concern as these e uents are toxic and can alter the ecology and genetic composition of aquatic animals. The physico-chemical parameters show low level of DO and high level of BOD in the rivers (Asa river Unity & Harmony) indicating hypoxia and slight nitrate content owing to runoff from agricultural farms around the area which employ the use of pesticides and fertilizers for farming. Low level of DO of the river and high BOD and COD values obtained from Asa river in Unity may be as a result of the industrial e uents as well as domestic wastes and run off some agricultural activities around the river bank. This a result of deoxygenation caused by the in uence of industrial e uents discharge into the water body. DO has been regarded as the most important parameter for assessing water quality because it in uences the fauna and ora distribution (Morrison 2001). High BOD and COD values are principal indicators of the presence of organic and inorganic pollutants respectively. The higher value of COD and BOD correlate with the work of (Kolawole et al. 2011) on this river which are implicative of water pollution. Changes in the water quality parameters have been linked to the activation of the antioxidant defense system in sh and other aquatic organisms living there and this may further elevate antioxidant responses in these organisms in the presence of environmental pollutants from agricultural, domestic, industries, land ll leachates among others leading to generation of reactive oxygen species (ROS) which result in oxidative stress in the biological organism like sh (Slaninova et al. 2009;Sevcikova et al. 2011).
The ALP, AST and ALT are enzymes produced in the liver and their increase in the blood indicates injury to the liver. The signi cant increased levels of oxidative enzymes in Asa river Unity indicate sh liver in ammation, injury, stress and disease and this might be attributed to high levels of conductivity, BOD, COD and decrease in DO which may have subjected the sh to undue stress. This correlates with ndings of Oshode et al. (2008) that reported high level of AST in assessed sh samples as a result of pollution of the water sites.
The gills, one of the vital organs and considered the primary target of contaminant as it remains in close contact with external environment (Camargo and Martinez 2007). The histopathological lesions observed in the gills especially at Asa river Unity and Harmony indicate that the sh respond to the effects of toxic agents present in the water. This could cause disturbance in the blood ow in the gills as observed by Stentiford et al. (2003). The organ most associated with the detoxi cation and biotransformation process is the liver which most affected by contaminants in the water. The liver histological presentation in Asa river Unity, Asa river Harmony and Apodu reservoir showed abnormal liver with vacuolation in architecture. This alteration may be a result of pollutant in the ecosystem as observed by 2020).
Blood is a tissue that is sensitive to any changes in our environment. It is made up of RBC, WBC, PLT and plasma. They are good bioindicators for assessing the quality of water (Kopp et al. 2013;Khan et al. 2015;Parrino et al. 2018;Osman et al. 2018;Anifowoshe et al. 2018;. Water contamination can cause changes in haematological parameters. In our study, a signi cant increase (p<0.05) in RBC, HGB, MCV, MCH, GRAN, PLT and PLCR in T. zillii from Asa river (Unity) was observed compared to other sampling sites. There was also signi cant elevation in MCH, MCHC, PLT and PDW in sh from Asa river (Harmony). This result is in line with Sahiti et al. (2018) wherein they reported slight differences in the values of HGB, MCHC and WBC and signi cant changes in RBC, MCV and MCH in blood of common carp (Cyprinus carpio) in two lakes of Kosovo. This difference in the RBCs may be due to low level of DO recorded in the rivers (Asa rivers (Unity/Harmony)). Blood parameters such as MCV, MCH and MCHC are important in diagnosing animal anaemia (Coles 1986). The increasing values of these indicators appear in the case of various anaemia.
The oxidative stress and antioxidants responses have been used as biomarkers to evaluate genotoxic potential of rivers in Nigeria like Ogun river,.Eleyele river and Asejiri river (Farombi et al.,2007;Arojojoye and Adeosun (2016a); Arojojoye et al. 2016b) but there is little information about the oxidative stress and antioxidant responses of sh in major reservoirs and rivers in Kwara state which provide drinking water and sh to the populace of Kwara state. The antioxidant defense system is usually activated when there is an imbalance in the Reactive Oxygen Species (ROS) level and antioxidant level in sh and this defense is quickly activated to checkmate oxidative stress that may be induced by increased level of pro-oxidants in the sh (Livingstone 2001). The SOD and CAT are usually the rst line of defense against oxidative stress and their activities are easily noticeable as increase in enzyme activity (McCord 1996).Antioxidants may be depleted in cells during exposure to environmental pollutants, but sometimes antioxidants level may increase to compensate the imbalance caused by oxidative stress (Arojojoye et al.2016a,b).
Increased SOD activities in Unity river and Harmony river may be a response to oxidative stress which could be caused by the low level of oxygen (low DO and high BOD) which may result in hypoxia. Decrease level of oxygen may have induced production of superoxide anions (which are the most dangerous free radicals) which was catalytically scavenged by SOD into H 2 O 2 and this a defense readily adopted by the antioxidant enzyme of the sh to reduce toxicity of oxygen. The results obtained in the SOD activities harmony and Unilorin reservoir correlate with Isamah et al.(2000) and Farombi et al. (2007) that attributes increased concentrations of SOD in sh to oxidative stress.
CAT is an important enzyme and acts on hydrogen peroxide in sh exposed to pollutants. CAT in conjunction with SOD are usually the rst defense enzymes and CAT levels generally increase in sh exposed to pollutants but decrease in CAT levels has been obtained polluted water bodies as reported by (Farombi et al.2007).The CAT levels were seen to decrease in representative sh species of Unilorin reservoir and Unity river and increased in Harmony river while the CAT values of Asa and Apodu reservoir were not statistically different. The reduced CAT may indicate that the sh is under oxidative stress as increased production of ROS is slowly beginning to overwhelm the CAT defense. The results in the CAT level is in line with Arojojoye et al. (2016) which established that increase in ROS production is correlated with decreased catalase activities and other antioxidant enzymes. The increased CAT activities in Harmony river is a typical response to environmental pollutants as CAT and SOD represents the rst line of defense against oxidative stress.
The GPx levels was highest in blood of representative sh species of Apodu reservoir followed by Asa reservoir and Unity river. The activity of GPx was decreased in Asa river (Harmony) with the lowest values observed in Unilorin reservoir. Elevated GPx levels suggests an adaptive and protective role of this enzyme against oxidative stress induced by the heavy metals or organic pollutants. The elevated GPx level supports the work of Lenartova et al. (1997) that found out that GPx activity of sh was 1.8-fold higher in polluted river. The decreased level of GPx may indicate a low level of pollution in Unilorin reservoir and a condition of self-puri cation of the Asa river (Harmony).
GR plays a role of catalytically reducing oxidized glutathione in order to maintain GSH/GSSG ratio. GR and GPx work together as increased level of GR may be indicative of oxidative stress and may require an increased level of GPX to counteract the effect of stress (Akpakpan et al. 2014). GR was observed to be highest in the blood of representative sh species of Unilorin reservoir, followed by Asa river (Harmony), Apodu reservoir, Asa river (Unity) and the lowest GR values were obtained in Asa reservoir. Increased GR may portray a situation of oxidative stress in sh, this is in line with the work of Pandey et al. (2003) that observed increased GR in sh with higher level of pollution.
GST was observed to reduce in the blood of representative T. zillii of Unilorin reservoir, Asa river (Harmony), Asa river (Unity), Apodu reservoir and elevated in Asa reservoir. The reduction can be attributed to increase in ROS production and according to Arojojoye et al. (2016) antioxidant levels may get depleted in cells during exposure to environmental pollutants. Next, we determined the level of DNA damaged using two different tests. In this study, micronucleus test which is a reliable and sensitive assay that is mostly consider as a biomarker of DNA damage showed induction of micronuclei and other nuclear abnormalities which increased mostly in T. zilli across all the ve sites when compared with C. gariepinus. Our result is in contrast with Ali et al. (2008) reports where the peripheral blood of C. gariepinus was shown to be very sensitive in formation of MN when compared to T. zillii which was shown to be sensitive in MN formation compared with two other tilapia species; Oreochromis niloticus, and Oreochromis aureus. This may be attributed to the fact that T. zillii might have accumulated more metals in it body systems and also indicates that its genome well tolerates such type of cytogenetic damage without apoptosis. The obtained results support the fact demonstrated by Kligerman (1982) that sh inhabiting polluted waters as in ARiU site in this present study have greater frequencies of micronuclei. The micronuclei frequencies may vary according to the season, stress, type of pollution and heavy metals.
The comet assay has been used successfully to investigate the effects of genotoxic pollutants on the integrity of DNA. It confers several advantages as a tool for genotoxic studies such as: ability to detect genotoxic damage at the single-cell level, suitability for most eukaryotic cell types; a small number of cells is required, faster and more sensitive than other available methods for detecting strand breaks, relatively low-cost and ability to detect early exposure response of genotoxins Frenzilli et al. 2009). Evaluation of genotoxicity of the ve sampling sites using the comet assay in sh was performed for the rst time in this present study across the ve sites. The percentage tail DNA(%tail DNA) is the parameter of choice for DNA damage assessment because comets can be observed with the same length but different uorescence intensities, this makes it ideal for description of DNA damage (Schnurstein and Braunbeck 2001). The (% tail DNA) in the ve reservoirs in descending order; Asa river (Unity)>Apodu reservoir> Asa river (Harmony)>Unilorin reservoir>Asa reservoir shows the reservoir with the highest and lowest DNA damage (Asa river (Unity) and Asa reservoir) respectively.
The Asa river (the upstream) which has its course extended towards the Unity area (mid-stream) and Harmony area (downstream) shows a remarkable trend. The Asa river(upstream) has the lowest DNA damage when compared to its courses (Unity and Harmony rivers). The low DNA damage at the upstream and high DNA damage at the midstream could be associated with the level of exposure to genotoxic substances. The increasing extent of genotoxicity in the middle of Asa River is a typical example showing that DNA damage in peripheral erythrocytes of C. gariepinus and T. zillii are closely linked to contaminant's level when a gradient of pollution exists along the river. The trend observed on the Asa water course corroborate with Hariri et al. (2018) in which DNA damage was highest in the midstream (Asara) of the Karai river because of high in ux of pollutants from sewage and industries and DNA damage was lowest at the upstream (Varangerud) because it was the region of minimum pollution.
The increase (%tail DNA and tail length) observed in Apodu reservoir compared to negative control can be attributed to the high nitrate pollution due to sewage disposal and runoff from neighbouring farms around the reservoir that utilize pesticides and fertilizers for its farming activities as reported by Anifowoshe et al. (2018). Exposure to contaminants of various sources may produce additive, synergistic or antagonistic interactive impacts on sh (Osman et al. 2012). Accumulation of DNA damage may occur either through an increase in the number of DNA-damaging events or a decrease in DNA repair (Cavas and Konen 2007). The results of the Apodu reservoir is in accordance with (Kushwaha et al. 2012) that revealed a signi cantly higher degree of DNA damage in comparison with baseline values in Channa punctatus and Mystus vittatus erythrocytes exposed to contaminated water of River Gomti, India. Unilorin reservoir has low %tail DNA when compared to negative control. This is probably because it is exposed to minimal pollutants as it is used for recreational activities and the location of the reservoir is not proximal to residential areas hence it receives little or no domestic waste, the only possible source of pollution would be runoff during rainy season or from its water course.
The results obtained from the integrated use of comet assay and antioxidant responses to evaluate genotoxic potential were in agreement in most of the sampling sites but contrasted in a few. The results of comet assay and antioxidant enzyme responses both pointed to the fact that Unity and Harmony rivers are polluted. This is similar to results obtained by Nwani et al. (2013) in which the comet assay and antioxidant enzyme responses were in agreement. There is a slight contrast in the Apodu and Unilorin reservoir. The DNA damage is low in Unilorin but the antioxidant responses were not as consistent and this may indicate that reservoir is probably exposed to minute pollutants that are triggering these responses. The Apodu reservoir which shows high DNA damage has moderate antioxidant responses. This is because the comet assay is a more sensitive test than the antioxidant response as it can detect toxins at low concentrations as reported by (Osman et al. 2012).Thus the comet assay in conjunction with the antioxidant enzyme responses helps to view the full picture of the genotoxic status of a water body.

Conclusion And Recommendations
The genotoxic potential and antioxidant response of ve water bodies was investigated using the integrated use of comet assay ad antioxidant enzymes responses. The water bodies investigated were observed to be currently at risk of pollution and if the anthropogenic activities on these water bodies remain unchecked, the ecology and the biotic life may deteriorate rapidly than being restored by selfpuri cation. It is highly recommended that the government should enforce laws to eliminate indiscriminate dumping of wastes into water bodies, enlightened the society of the damages of the acts and risk of consuming contaminated shes.