Original Research Article 1

2

Determination of Total Protein, Superoxide 3

Dismutase, Catalase Activity, and Lipid 4

Peroxidation in Soil Macro Fauna (Earthworm) 5

from Onitsha Municipal Open Waste Dump 6

7 8 . 9 10 ABSTRACT 11 12

Aims: Oxidants are substances which are toxic in high concentration, but at low doses can stimulate biological activities of living organisms. The effect of oxidants on cells is modulated by multiple interacting antioxidant defence mechanism. The present study evaluated the individual role of interaction of superoxide dismutase (SOD) and catalase in estimating the level of toxicity of municipal solid waste on soil organisms, and the rate of lipid peroxidation by the action of reacting oxygen species. Study design: Earthworm samples were collected from four different locations grouped into A, B, C, and D. Place and Duration of Study: Analysis was conducted at the Department of Biochemistry, Anambra State University, during March 2014 and May 2014. Methodology: Total protein estimation was determined by Lowry method while superoxide dismutase activity (SOD), catalase activity (CAT) and lipid peroxidation concentration was determined spectrophotometrically using Shimadzu UV-160 spectrophotometer. Results: A high increase in superoxide dismutase activity and catalase activity as a result of corresponding increase in lipid peroxidation was observed in group C and D (21.22±0.54 and 23.74±0.51) respectively compared to that of group A and B (4.88±0.54 and 7.24±0.31). As regards the above observation, group A and B can be classified as samples from the dormant part while group C and D can be classified as samples from the active part of the site. Conclusion: The increased level of superoxide dismutase and catalase activity may be as a result of increased concentration of reactive oxygen species (ROS) in waste dumps which can cause damage to soil organisms or even death and hence render the soil from the dump site unfit for agricultural purposes.

13 Keywords: Superoxide dismutase, catalase, lipid peroxidation, earthworm, oxidants, free 14 radicals. 15 16 1. INTRODUCTION 17

Exposure of living organisms to contaminated environment can have significant deleterious 18 consequences [1]. Environmentally induced stress frequently activate the endogenous 19 production of reactive oxygen species (ROS), most of which are generated as side products 20 of tissue respiration [2]. ROS includes the superoxide radical (•O2-), hydrogen peroxide 21 (H2O2), and the hydroxyl radical (•OH-), all of which affect crucial biomolecules by interacting 22 quickly and aggressively with polyunsaturated fatty acids, DNA and proteins due to there 23

unstable nature [3, 4]. Free radicals and other reactive oxygen species leads to oxidative 24 stress and have long been known to be mutagenic; these agents have more recently 25 emerged as mediators of the other phenotypic and genotypic changes that lead from 26 mutation to neoplasia [5, 6]. Furthermore, a number of factors play a role in contributing to 27 soil toxicity and one of them is the pH of the soil which plays a role in increasing the toxicity 28 of soil by increasing metal adsorption thus reducing bioavailability of fauna and flora [7]. 29

Oxidative stress however, is the imbalance between the production of free radicals and 30 antioxidant defence systems, either because of a defence deficit or an increase in the 31 production of oxygen reactive species [8]. This stress results in changes in the structure-32 function relationship in some organs, systems, or specialised cell groups [9,10]. Although, 33 under normal conditions free radicals and other reactive oxygen species are essential for life, 34 because they are involved in cell signaling and are used by phagocytes for their bactericidal 35 action. Cells are protected against the toxic effects of high concentrations of ROS by a 36 balanced level of endogenous enzymatic and non-enzymatic antioxidants. The antioxidant 37 system comprises several enzymes such as superoxide dismutase (SOD), catalase (CAT), 38 and Glutathione peroxidase (GPx) [11]. Superoxide radicals that are generated are 39 converted to H2O2 by the action of SOD, and the accumulation of H2O2 is prevented in the 40 cell by CAT and GPx [11]. David [12] reported that for every 10,000 electrons transferred 41 down the respiratory pathway in Escherichia coli cells; about 3 electrons end up on 42 superoxide instead of the proper place. 43

SOD works by taking two molecules of superoxide, strips the extra electron off of one, and 44 places it on the other. So, one ends up with an electron less, forming normal oxygen, and 45 the other ends up with an extra electron. The one with the extra electron then rapidly picks 46 up two hydrogen ions to form hydrogen peroxide which the cell must detoxify through the 47 help of CAT [12]. Notably, reactive oxygen species play a crucial role in the initiation of lipid 48 peroxidation which can cause oxidative damage to various biological macromolecules [13]. 49 Protective mechanisms that could scavenge the peroxidatively produced free radicals have 50 evolved within plants to keep these deleterious compounds to a minimum and these include 51 antioxidant enzymes such as superoxide dismutase and catalase [14]. 52 53 Lipid peroxides which results from hydroxyl radical-attack on the important polyunsaturated 54 fatty acids serves as hydroxyl radical markers caused by oxidative stress. The damage from 55 this oxidative stress in fatty acid rich structures, such as cell membranes, results in lose of 56 rigidity, integrity, and permeability [15]. This process is however referred to as lipid 57 peroxidation and can be determined from the carbonylic compounds called malondialdehyde 58 (MDA) [15]. The MDA is referred to as an index of lipid peroxidation [16]. 59

The earthworm (Eisenia fetida) is considered to be a representative of soil fauna [17]. 60 Earthworms are used as indicators for assessing the potential impact of chemicals on soil 61 organisms [18]. Biomarkers have been measured in earthworms while assessing their 62 exposure to contaminants in the soil through the measurement of antioxidant enzyme activity 63 [19]. E. fetida is characterised by its increased sensitivity to chemical agents compared with 64 other species of earthworms [20]. By definition, biomarkers are biochemical, physiological, 65 and histological reactions in organisms that can be used to determine exposure to and/or 66 toxic effects caused by chemicals [21]. The present study utilized biomakers in E. fetida to 67 ascertain the level of toxicity by examining the interaction of superoxide dismutase (SOD) 68 and catalase activities and the rate of lipid peroxidation of E. fetida samples collected from 69 four different locations in the same solid dump site located along Onitsha-Owerri road, 70 Anambra State, Nigeria. 71

72

2. MATERIALS AND METHOD 73

2.1 Sample Collection and Preparation 74

Earthworm samples were collected from four (4) different locations (A, B, C, and D) at the 75 Onitsha municipal solid waste dump site opposite National Metallurgical Institute along 76 Onitsha-Owerri road, Onitsha, Anambra State, Nigeria (Figure 1 and 2). Four earthworm 77 samples were collected from each location. The control earthworm samples were collected 78 from a waste dump - free farmland in Uli, Anambra State. All the samples were preserved in 79 isolation in ice/cold water before taking them to the laboratory for analysis. Each of the 80 earthworm samples were weighed, macerated and mixed with normal saline in the 81 proportion (1 g of earthworm = 10 ml of normal saline) and poured into a sample tube and 82 refrigerated. 83

84

Figure 1: Map of Anambra State, Nigeria showing the study area. 85

86

Study Area

87

88

Figure 2: The Municipal solid waste dump site.89

2.2 Total Protein Estimation90

The protein content for samples was estimated usin91 gram (1 g) of the sample was weighed and homogenized with 20 ml of 0.5 M NaOH. The 92 homogenate was poured into a centrifuge tube and centrifuge at 3500 rpm for 10 minutes. 93 The supernatant was collected in a tube. To 1.0 ml of the supernatant, 4ml of 94 was added to make up to 5 ml. A standard protein solution of 0.2 mg was prepared (bovine 95 albumin serum) in the same manner. Five (5) ml of alkaline solution was added to each tube 96 and then it was mixed properly and allowed to stand at room 97 This was followed by the addition of 0.05 ml of dilute Folin98 mixed immediately to give a blue colour. The absorbance was read at 750 nm against a 99 reagent blank using Shimadzu UV100 samples were extrapolated from the standard BSA curve.101

2.3 Determination of SOD Activity102

The activity of superoxide dismutase was determined by the103 [23]. This involves measuring the SOD i104 at pH 10.2 and at 30 ºC. One unit of superoxide activity is defined as the amount of SOD 105 necessary, to cause 50 % inhibition of epinephrine auto106 performed in 3.0 ml of 50 M 107 was then followed by the addition of 0.03 ml of epinephrine stock solution before taking the 108 absorbance reading at 480 nm for 3109 the reagents) was used for background correction.110

2.4 Determination of Catalase Activity111

Beers and Sizer [24] method was modified and used for the CAT activity assay. A mixture of 112 2.5 ml of phosphate buffer (pH 7.0), 2 ml of H113 made in each of the test tubes. The hydrogen peroxide (30 mM) was used as a substrate 114 and the decrease in H2O2 concentration at 22 °C was followed spectroscopical ly at 240 nm 115

Figure 2: The Municipal solid waste dump site.

2.2 Total Protein Estimation

The protein content for samples was estimated using the method of Lowry et al. [22gram (1 g) of the sample was weighed and homogenized with 20 ml of 0.5 M NaOH. The homogenate was poured into a centrifuge tube and centrifuge at 3500 rpm for 10 minutes. The supernatant was collected in a tube. To 1.0 ml of the supernatant, 4ml of distilled water was added to make up to 5 ml. A standard protein solution of 0.2 mg was prepared (bovine albumin serum) in the same manner. Five (5) ml of alkaline solution was added to each tube and then it was mixed properly and allowed to stand at room temperature for 10 minutes. This was followed by the addition of 0.05 ml of dilute Folin-ciotteau reagent to each tube and mixed immediately to give a blue colour. The absorbance was read at 750 nm against a

Shimadzu UV-160 spectrophotometer. The protein concentrations of the samples were extrapolated from the standard BSA curve.

2.3 Determination of SOD Activity

superoxide dismutase was determined by the method described by Magwere ]. This involves measuring the SOD inhibition of the auto-oxidation activity of epinephrine

at pH 10.2 and at 30 ºC. One unit of superoxide activity is defined as the amount of SOD necessary, to cause 50 % inhibition of epinephrine auto-oxidation. The analysis was

Na2CO3 buffer to which 0.02 ml of the sample was added. This was then followed by the addition of 0.03 ml of epinephrine stock solution before taking the absorbance reading at 480 nm for 3-5 minutes. A blank devoid of the sample (but have all

) was used for background correction.

2.4 Determination of Catalase Activity

] method was modified and used for the CAT activity assay. A mixture of 2.5 ml of phosphate buffer (pH 7.0), 2 ml of H2O2 solution and 0.5 ml of the samples wermade in each of the test tubes. The hydrogen peroxide (30 mM) was used as a substrate

concentration at 22 °C was followed spectroscopical ly at 240 nm

g the method of Lowry et al. [22]. One gram (1 g) of the sample was weighed and homogenized with 20 ml of 0.5 M NaOH. The homogenate was poured into a centrifuge tube and centrifuge at 3500 rpm for 10 minutes.

distilled water was added to make up to 5 ml. A standard protein solution of 0.2 mg was prepared (bovine albumin serum) in the same manner. Five (5) ml of alkaline solution was added to each tube

temperature for 10 minutes. ciotteau reagent to each tube and

mixed immediately to give a blue colour. The absorbance was read at 750 nm against a The protein concentrations of the

method described by Magwere oxidation activity of epinephrine

at pH 10.2 and at 30 ºC. One unit of superoxide activity is defined as the amount of SOD oxidation. The analysis was

buffer to which 0.02 ml of the sample was added. This was then followed by the addition of 0.03 ml of epinephrine stock solution before taking the

5 minutes. A blank devoid of the sample (but have all

] method was modified and used for the CAT activity assay. A mixture of solution and 0.5 ml of the samples were

made in each of the test tubes. The hydrogen peroxide (30 mM) was used as a substrate concentration at 22 °C was followed spectroscopical ly at 240 nm

for 1 min. The activity of the enzyme was expressed in units per mg of protein and 1 unit 116 equals the amount of an enzyme that degrades 1 mM H2O2 per minute. 117

2.5 Determination of Lipid Peroxidation 118

The thiobarbituric acid-reactive substances (TBARS) were measured using the modified 119 method of Du and Bramlage [25]. An aliquot of 1.0 ml of the incubated samples were added 120 to 0.5 ml of 25 % (w/v) TCA and 0.5 ml 1% TBA in 0.3 % (w/v) NaOH. The tubes were 121 heated at 90-95 0C in a water bath for 40 minutes and then cooled in ice water to reduce 122 turbidity. In this modified procedure, the absorbance was measured at 532 and 600 nm to 123 correct for the interference produced by TBARS sugar complexes. Measurements were 124 carried out in a Shimadzu UV-160 spectrophotometer. 125 126 127 2.6 Statistical Analysis 128

The data were expressed as mean±S.D. Statistical significance of differences among 129 treatments were determined by use of oneway analysis of variance and covariance 130 (ANOVA), followed by Tukey's pair-wise comparisons. 131

3. RESULTS AND DISCUSSION 132

3.1 Results 133

The result revealed that there was significantly higher concentration of protein in the control, 134 group A and group B to compare with the concentration of protein present in group C and 135 group D (Table 1). 136

Table 1. Total protein concentrations of groups A, B, C, and D 137

Groups Protein concentration Mean±SD (mg/ml) (mg/ml)

Control 13.515 13.51±0.08 13.510 13.605 13.420

A

14.006 13.12±1.77

10.879 12.637 14.945 B

5.055 6.15±2.40

5.934 4.066 9.560 C

2.198 1.92±0.23

1.978 1.648 1.868

D

1.429 1.54±0.24 1.319 1.538

1.868 * SOD Activity is presented as mean±SD (U/mg protein) of four determinations. 138 * Values are statistically significant at p<0.05 139

140

SOD Activity were significantly higher in group C and D (21.22±0.54 and 23.74±0.51) 141 respectively when compared to group A and B (4.88±0.54 and 7.24±0.31) (Table 2). This 142 could be as a result of low activity going on at the dormant site as compared to active site 143 which has high activity. 144

Table 2. SOD Activity of different groups 145

Groups Auto -oxidation rate (units/secs)

Percentage inhibition

SOD Activity (U/mg protein)

SOD Activity (mean±SD)

(U/mg protein) Control 0.07

0.06 0.07 0.08

32.520 32.424 32.520 33.324

0.6504 0.6010 0.6502 0.7000

0.6504±0.01

A 0.58 235.772 4.7154 0.53 215.447 4.3089 4.88±0.54 0.69 280.488 5.6098 0.60 243.902 4.8780 B 0.84 341.463 6.8293 0.92 373.984 7.4797 7.24±0.31 0.92 373.984 7.4797 0.88 357.724 7.1545 C 2.53 1028.455 20.5691 2.66 1081.301 21.6260 21.22±0.54 2.58 1048.780 20.9756 2.67 1085.366 21.7073 D 2.78 1130.081 22.6016 2.90 1178.862 23.5772 23.74±0.51 2.84 1154.472 23.0894 2.92 1186.992 23.7398 * SOD Activity is presented as mean±SD (U/mg protein) of four determinations. 146 * Values are statistically significant at p<0.05 147

Catalase activity of groups A to D was increasing with decrease in protein concentration 148 from groups A and D as shown in (Table 1 and 3).Catalase activity was significantly highest 149 in group D followed by group C while control group was the least. 150

151 152

153

154

155

156

157

158

159

160

Table 3. Catalase Activity of different groups 161

Groups Catalase velocity constant (K)

Catalase activity/mg protein

Catalase activity

(mean±SD) Control 0.0133

0.0132 0.0133 0.0135

0.0010 0.0009 0.0010 0.0011

0.001±0.00

A 0.0171 0.0012 0.0154 0.0014 0.001±0.00 0.0167 0.0013 0.0191 0.0013 B 0.0116 0.0023 0.0162 0.0027 0.003±0.00 0.0115 0.0028 0.0199 0.0021 C 0.0492 0.0223 0.0535 0.0270 0.027±0.00 0.0507 0.0308 0.0553 0.0296 D 0.0399 0.0279 0.0548 0.0415 0.035±0.01 0.0581 0.0378 0.0627 0.0336

* CAT Activity is presented as mean±SD (U/mg protein) of four determinations. 162 *Values are statistically significant at p< 0.05. 163 164

peroxidation concentration in group C and D (6.09±0.16 and 6.73±0.18 respectively) was 165 very high compared to group A and B (3.06±0.06 and 3.38±0.10).The differences were 166 significantly high at p<0.05. This correlation depicts that group C and D is the active site 167

while group A and B are the dormant site. 168

169

170

171

172

173

174

175

176

177

178

Table 4. Lipid Peroxidation concentration of groups A, B, C, and D 179 Groups A532-600nm Concentration

(mg/ml) Lipid Peroxidation

(mg/100g) Lipid peroxidation

(mean±SD) (mg/100g) Control 0.051

0.054 0.050 0.051

0.012 0.014 0.010 0.012

0.12 0.14 0.10 0.12

0.12±0.00

A 1.225 0.299 2.99 1.282 0.313 3.13 3.06±0.06 1.243 0.304 3.04 1.252 0.306 3.06 B 1.404 0.343 3.43 1.348 0.330 3.30 3.38±0.10 1.432 0.350 3.50 1.347 0.329 3.29 C 2.418 0.591 5.91 2.577 0.630 6.30 6.09±0.16 2.470 0.604 6.04 2.498 0.611 6.11 D 2.838 0.694 6.94 2.779 0.679 6.79 6.73±0.18 2.680 0.655 6.55 2.708 0.662 6.62

* Lipid Peroxidation is presented as mean±SD (mg/100g) of four determinations. 180 *Values are statistically significant at p<0.05 181 182

183

184 Figure 3. Overall Comparison of SOD Activity, CAT activity and Lipid Peroxidaion 185

186

187

188

3.2 Discussion 189

Analysis of Municipal solid wastes revealed that, some parts of the sites were found to be 190 dormant while some were active. Dormant sites indicated low catalase and superoxide 191 dismutase activity with corresponding low level of lipid peroxidation reaction 192 the active site which corresponds to high catalase and superoxide dismutase activity with 193 high lipid peroxidation. The result of protein concentration determination (Table 1) revealed 194 that there was low concentration of protein in group C an195 concentration of protein that was present in the control, group A and group B. This suggests 196 that ROS activities must have impacted negatively on the protein concentration197 increasing the oxidative stress in earthworm from group C and D thereby leading to 198 depletion of their protein concentration.199 activities observed in the control sample (0.65 U/mg protein), group A (4.200 protein) and B (7.24±0.31 U/mg protein) as compared to group C (21.22±0.54 U/mg protein) 201 and D (23.74±0.51 U/mg protein) wit202 oxidative stress in the active site C and D when203 effects of ROS were counteracted by the anti204 thereby restoring the balance and protecting the organism from the stress205 in catalase activities (Table 3) was seen in the contr206 dormant sites A and B (0.013±0.00 U/mg 207 compared to that of active site C and D (0.027±0.01 U/mg protein and 0.035±0.01 U/mg 208 protein respectively). 209 210 Lipid peroxidation activity (Table 4) in group A (3.06±0.06) and B (3.38±0.10) were relatively 211 high when compared to the control (0.12±0.00). The highest concentration was found in 212 group C (6.09±0.16) and D (6.73±0.18) at the active site of the solid waste dump. The resul213 was consistent with that reported by 214

Overall Comparison of SOD Activity, CAT activity and Lipid Peroxidaion concentration.

Analysis of Municipal solid wastes revealed that, some parts of the sites were found to be dormant while some were active. Dormant sites indicated low catalase and superoxide dismutase activity with corresponding low level of lipid peroxidation reaction in comparisonthe active site which corresponds to high catalase and superoxide dismutase activity with

The result of protein concentration determination (Table 1) revealed that there was low concentration of protein in group C and D to compare with the concentration of protein that was present in the control, group A and group B. This suggests

must have impacted negatively on the protein concentrationincreasing the oxidative stress in earthworm from group C and D thereby leading to depletion of their protein concentration. The result also reflect the low superoxide dismutase activities observed in the control sample (0.65 U/mg protein), group A (4.88±0.54 U/mg protein) and B (7.24±0.31 U/mg protein) as compared to group C (21.22±0.54 U/mg protein) and D (23.74±0.51 U/mg protein) with high SOD activities (Table 2). This suggests high

in the active site C and D when correlated with the fact that the damaging effects of ROS were counteracted by the anti-oxidant enzymes which remove the ROS, thereby restoring the balance and protecting the organism from the stress [7]. Similar result in catalase activities (Table 3) was seen in the control sample (0.001 U/mg protein), and the dormant sites A and B (0.013±0.00 U/mg protein and 0.003±0.00 U/mg protein respectively) compared to that of active site C and D (0.027±0.01 U/mg protein and 0.035±0.01 U/mg

on activity (Table 4) in group A (3.06±0.06) and B (3.38±0.10) were relatively high when compared to the control (0.12±0.00). The highest concentration was found in group C (6.09±0.16) and D (6.73±0.18) at the active site of the solid waste dump. The resulwas consistent with that reported by Oni and Hassan [7] who observed that there was

Overall Comparison of SOD Activity, CAT activity and Lipid Peroxidaion

Analysis of Municipal solid wastes revealed that, some parts of the sites were found to be dormant while some were active. Dormant sites indicated low catalase and superoxide

comparison of the active site which corresponds to high catalase and superoxide dismutase activity with

The result of protein concentration determination (Table 1) revealed d D to compare with the

concentration of protein that was present in the control, group A and group B. This suggests must have impacted negatively on the protein concentration by

increasing the oxidative stress in earthworm from group C and D thereby leading to low superoxide dismutase

88±0.54 U/mg protein) and B (7.24±0.31 U/mg protein) as compared to group C (21.22±0.54 U/mg protein)

2). This suggests high e fact that the damaging

oxidant enzymes which remove the ROS, . Similar result

ol sample (0.001 U/mg protein), and the protein and 0.003±0.00 U/mg protein respectively)

compared to that of active site C and D (0.027±0.01 U/mg protein and 0.035±0.01 U/mg

on activity (Table 4) in group A (3.06±0.06) and B (3.38±0.10) were relatively high when compared to the control (0.12±0.00). The highest concentration was found in group C (6.09±0.16) and D (6.73±0.18) at the active site of the solid waste dump. The result

] who observed that there was lipid

peroxidation activity in exposed worms versus the field control and that there was elevated 215 malon-dialdehyde (MDA) level in the test soils compared with the control. Thus, elevated 216 level of lipid peroxides strongly suggest hydroxyl radical activity (Figure 3), and reflect 217 oxidative damage [26]. Saturated fatty acids undergo less peroxidation than their 218 unsaturated counterparts. This could be dangerous because unsaturated fatty acids are 219 mainly essential fatty acid needed for living systems to function maximally. Indeed, 220 supplementation with polyunsaturated as opposed to saturated fatty acids results in a 221 statistically significant increase in lipid peroxidation in the plasma and liver [27-29]. However, 222 the study clearly showed the effects of contaminated dumpsite soils on E. fetida and may 223 explain the absence of earthworms from metal polluted dumpsites [7]. 224 225 From the above it can be deduced that refuse dumping could lead to the generation of 226 reactive oxygen species i.e free radicals and this may render soil from the dump site which 227 may be rich in nutrients unfit for agricultural purposes [30]. The prevention of oxidation is an 228 essential process in all the aerobic organisms, as decreased antioxidant protection may lead 229 to cytotoxicity, mutagenicity and/or carcinogenicity [31]. To adapt to this effect, earthworms 230 migrate to safer places which subsequently leads to loss of their beneficial functions in soil 231 aeration, drainage, enrichment of organic material leading to loss in soil fertility. The food 232 chain in the dumpsite is also affected because there could be decrease in the numbers and 233 distribution of their vertebrate predators [7]. Thus the loss of earthworms due to toxicity from 234 an area can impact on ecosystem [32]. 235

4. CONCLUSION 236

The exposures of living organisms to municipal solid waste dump sites are deleterious even 237 at low concentration because the living systems as seen in the earthworm tends to employ 238 its defence mechanism to annul the changes. Therefore it is conceivable that soil organisms 239 could suffer significant mortality when exposed to high concentration of municipal solid 240 waste. Further study on different dump sites should be carried out to ascertain the level of 241 toxicity and measures to check this effect should also be investigated. 242

CONSENT 243

Not applicable. 244

ETHICAL APPROVAL 245

Not applicable. 246

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