Introduction

The liver is recognizably the largest internal organ in the human body. The contemporary prevalent incidences of liver diseases is bothersome because of its unique role in: detoxification of xenobiotics introduced to the human body through various sources, as a location for xenobiotics detoxifying enzymes and as an important site for series of biochemical reactions. Pb is reportedly one of the known hepatotoxic agents known to man. There are reports demonstrating the interference of lead with internal organs like the liver (Flora et al., 2006). It is able to exhibit its toxicity because of its ability to affect virtually all organs or tissues through a mechanism that involves fundamental biochemical processes. Some of these mechanism s include the ability of Pb to inhibit or mimic the action of calcium which affects all calcium dependent processes and interact with proteins(ASTDR, 2005) and the  interference  with the synthesis of  heme, resulting in reduction in blood hemoglobin (ASTDR, 1999).   More concern for lead toxicity stems from the fact that lead and lead products used in various industries and lost into the environment, eventually end up in the aquatic environment (Sandhir and Gill, 1995) and subsequently the human body through consumption of aquatic animals like fish and crayfish.  Acute toxicity of Pb is workplace related and is quite uncommon, but chronic toxicity on the other hand is very common even at very low blood lead levels (Flora et al., 2006).

Warri River in the Niger Delta region of Nigeria is one of such aquatic environment, the river is  located on 5^o24’00’’N and 5^o.28’00’’E (USA, National Geospatial- intelligence Agency, 1994). Warri river is located in the Warri -South Local Government Area of Delta State and it is a harbor for major oil companies platform, a major sea port for the country.  Its bank is a site for activities like storage of bunkered crude oil and its products, illegal modular refineries, welding and fabrication, auto-mechanic workshops. The river joins two major rivers, Forcados and Escravos through the Jones creek in the lower Niger delta region. The major occupation of the indigents of the communities on shore the river is fishing and farming. Besides its use as a source of livelihood and aquatic food source, the still water end serves a source of drinking water to the communities on the river bank. Although, important measures have been adopted by regulatory authorities in the country to decrease or completely eliminate environmental lead exposure by promulgating policies supporting the use of  as unleaded gasoline and prohibiting processes like    lead smelting and coal combustion, there are speculations that the illegal make-shift refineries located on the bank of the river may not be in compliance.. Other measures adopted to reduce lead from the environment include: Removal of lead from paints, solder of canned foods and lead based solder in water system, battery recycling, grids and bearings, and glazed ceramics used for preparation and storage of foods. However, lead exposure is still a major environmental health problem in some specific communities. Currently, thousands of people obtaining sea foods and water from Warri River source may be at risk of exposure to this pollutant.

Albeit, lead toxicity is considered a widely explored area of research, studies on the evaluation of serum status of biochemical indices of liver injury and oxidative stress of  rats exposed to lead in the presence of some selected  metallic co-pollutants are far from being exhausted.

Materials and Methods

A total of 55 albino rats (of Wistar strain) weighing an average of 150±10g were used for this study. The rats were maintained under controlled environmental conditions as follows: 24⁰C-25.5⁰C; 24hours lighting. They were fed commercial rations of growers mash and potable tap water ad libitum and allowed 7 d  to acclimatize to the laboratory conditions,  temperature and humidity, before commencement of the study. The animals were exposed to the test metallic pollutants twice daily for 90 d.

The test metallic pollutants used in this study were soluble salts of the respective heavy metal. All metallic salts and epinephrine used in the study were obtained from May and Baker (Dagenham, UK). 2-thiobarbituric acid (Koch light laboratories Ltd, UK). Alkaline and acid phosphatase, ALT and AST kit were produced by Quimica Clinica Aplicada. (QCA, Spain). Total protein and bilirubin and glucose reagents were products of Randox Laboratories LTD, United Kingdom. All other chemicals used in this study were of Analytical grade 

Preparation of Laboratory Reconstituted Water (Obi et al., 2017)

The test water samples administered to the rats were prepared as follows

Pb and Fe contaminated water(Pb: 0.21mg/L; Fe: 1.60mg/L)

Aliquots (7.50ul) each of the respective stock solutions, Pb and Fe (0.033g/L and 0.03g/L of PbNO₃ and FeSO₄.7H₂O) were transferred to into 25 litres of deionized water

Pb and Ca contaminated water (Pb: 0.21mg/L;Ca:111.11mg/L)

Aliquots (750ul) each of the respective stock solutions (Pb(NO₃)₂ :0.033g/L and CaSO₄.12H₂O :4.13g/L)  were transferred into 25 litres deionized water

Pb and Cu contaminated water (Pb:0.21mg/L; Cu:0.012mg/L)

Aliquots (750ul) each of the respective stock solutions (Pb(NO₃)₂ :0.033g/L and CuSO₄.5H₂O :0.00070g/L)  were transferred into 25 litres deionized water

Pb and Mn Contaminated Water(Pb:0.21mg/L; Mn:0.22mg/L)

Aliquots (750ul) each of the respective stock solutions (Pb(NO₃)₂ :0.033g/L and MnSO₄.H₂O :0.0067g/L)  were transferred into 25 litres deionized water

Pb and Mg Contaminated Water (Pb:0.21mg/L; Mg:9.79mg/L)

Aliquots (750ul) each of the respective stock solutions (Pb(NO₃)₂ :0.033g/L and MgSO₄.7H₂O :0.10g/L)  were transferred into 25 litres deionized water

Pb and Zn Contaminated Water (Pb:0.21mg/L; Zn:0.059mg/L)

Aliquots (750ul) each of the respective stock solutions (Pb(NO₃)₂ :0.033g/L and ZnSO₄.7H₂O :0.003g/L)  were transferred into 25 litres deionized water

Pb and Co Metallic Pollutant Contaminated Water (Pb:0.21mg/L;Fe:1.60mg/L;Ca:111.11mg/L; Cu: 0.012mg/L; Mn:0.22mg/L; Mg: 9.79mg/L and Zn:0.059mg/L)

Aliquots (750ul) each of the respective stock solutions were transferred into 25 litres deionized water

Treatment of Animals

Treatment and management of animals were done according to the rules of local ethics committee of the Faculty of Life Sciences, University of Benin. The concentrations of Pb and metallic co- pollutants used were calculated on the basis of Warri River concentrations of the respective metals at the time of this study. A total of 55 rats were used for this study. The rats were divided

into eleven (11) groups of  5 rats each, and exposed  orally by gavage to Pb and the identified metallic co- pollutants reconstituted in deionized water as follows: Group 1 rats (control-I),received deionized water only (5ml H₂O/kg bd wt by gavage); Group II rats received Pti borehole water only (5ml H₂O/kg bd wt by gavage); Group III rats received Pb only stock (0.21mg/kg bd.wt) (5ml H₂O/kg bd wt) ;Group IV rats received Pb and Fe  stock solution(5ml H₂O/kg bd. Wt); Group V rats  received Pb and Ca stock solution(5ml H₂O/kg bd. wt); Group VI  rats received Pb and Cu stock solution(5ml H₂O/kg bd. Wt); Group VII  rats received Pb and Mn stock solution (5ml H₂O/ kg bd.wt); Group VIII rats received Pb and Zn  stock solution(5ml H₂O/ kg bd. wt); Group IX  rats received Pb and Mg stock solution(5ml H₂O/ kg bd .wt); Group X rats received Pb and all identified co- metallic pollutants (5ml/kg bd. wt)while Group XI rats received  Warri river water (5ml H₂O/kg bd wt). At the end of the study period (90d), the animals were sacrificed in accordance with the rules of the local ethics committee of the Faculty of Life Sciences, University of Benin. Blood samples were collected by cardiac puncture and  allowed to stand  for 10mins, the supernatant (serum) were collected and stored at -20^oC and assayed immediately. The liver was excised and stored at the same temperature until required for analysis. A 24hr urine sample was obtained and used for urine protein and glucose analysis.

Preparation of Tissue Homogenate

The liver was homogenized in ice-cold normal saline to obtain a 20% homogenate (1:4w/v). The homogenates were centrifuged at 4000rpm for 10minutes and the supernatant obtained were used for biochemical analysis. 

Gravimetry and Biochemical Assays

Body and liver organ weights were obtained using a Mettler electronic balance as outlined by Emmanuel et al., (2013).Formulation of test and control water was done according to the method of Asagba and Obi (2005). Preservation of tissue homogenate was done according to the method of (Walter and Scutt, 1974). Alkaline and acid phosphatase activities was estimated according to the method of Kind and King (1954) modified by Varley (1975). The principle of ALP was based on enzymatic end point, following formation of p-Nitro phenol, the rate of p-Nitro phenol formed was determined as ALP activity. While the activity of  ACP was determined by the formation of a red color complex from  the reaction of phenol and 4-Aminoantipyrine in an alkaline medium.  Alanine and aspartate amino transferases activity were estimated according to the method of Reitman and Frankel (1957) outlined by Sood (2006). The level of lipid per oxidation in the liver homogenate supernatant was estimated using the method of Buege and Aust (1978). The procedure involves the determination of thiobarbituric acid reactive substances (TBARs), which are indicators of lipid per oxidation. Values of TBARs are reported as Malondialdehyde (MDA) quantified using a molar extinction coefficient of 1.5 ×105 M-1cm-1 and expressed as micromole MDA per gram wet of tissue. A superoxide dismutase (SOD; EC 1.15.1.1) activity in the liver homogenate supernatant was estimated according to the method of Misra and Fridovich (1972). SOD activity is expressed as units per gram of liver tissue (one unit is the amount of the enzyme necessary to cause 50% inhibition of epinephrine oxidation for 60secs).while catalase(CAT; EC 1.11.1.6) activity was measured using the method of Cohen (1970), where decomposed hydrogen peroxide is measured by reacting it with excess of potassium tetraoxomanganate (VII)(KMnO4) and residual KMnO4 is measured spectrophotometrically at 480nm.the result was expressed as units  of enzyme activity/mg protein (U/mg protein)   Protein was determined by method of Lowry et al., 1951 while bilirubin was estimated according to the modified Jendrassik and Grof’s method outlined by Sood (2006). 

Statistical Analysis of Data

Statistical analysis was done using  one way Analysis of Variance (ANOVA) and Fischer’s protected least significant difference post-hoc testing or with unpaired student’s t test when appropriate. The Turkey-Kramer multiple comparison test was used to evaluate the differences between means. All statistical calculations were done using the graphpad instat statistical package. All data are expressed as mean ±SEM. Significant level was assigned at P<0.05

Presentation of Results

Influence of Pb in the presence of metallic co-pollutants on organ body wt ratios and MDA levels

The results of the effects of Pb and selected co-polluting metals on organ body weight ratio and malondialdehyde levels of the rat are presented in Table I.

Table 1: Effect of Pb in the presence of Metallic Co-Pollutants on liver weight/100gm Body weight and serum MDA level of the rat

^a(P>0.05); ^b= p(<0.05) relative to their respective grp1 value; ^c (P>0.05); ^d=p(<0.05) relative to their respective grp 2 values

^e(P>0.05); ^f=P<0.05) relative to their respective grp 3 values; ^g (P>0.05);  ^h=P<0.05)relative to their  respective grp 4 values ^I(P>0.05); ^j=P<0.05 relative to their respective grp5 values; ^k (P>0.05); ^l=P<0.05 relative to their respective grp 6 values ^m(P>0.05); ⁿ=P<0.05, relative to their respective grp7 values; ^o (P>0.05); ^p=P<0.05 relative to their respective grp 8 values ^q(P>0.05); ^r=P<0.05 relative to their respective grp9 values; ^s (P>0.05); ^t=P<0.05 relative to their respective group10 values

#= dose calculated based on Warri River concentrations of respective metals in a preliminary study.

Pb only exposed -rat group (Group III) showed a significant (P<0.05) decrease in liver organ/body weight ratio and a significant (P<0.05) increase in the products of lipid Peroxidation evident as malondialdehyde levels, relatives to the controls (Groups I, deionized water only exposed group and Group II: Pti borehole water -exposed group). Pb in combination with Cu (Pb+Cu), Fe (Pb+Fe), Zn (Pb+ Zn) and (Pb+ all co-polluting metals) caused significant (P<0.025) decreases in liver/body wt ratios and increases (P<0.025) in MDA levels in the respective rat groups relative to the controls. Also, Pb in the presence of Ca, Mn and Mg showed  significant (P<0.05) increases in liver body weight ratios and  decreases in levels of lipid peroxidation products of their respective rat group relative to the Pb-only group. There was a difference in liver body weight ratio and malondialdehyde (MDA) levels between the Warri river water-exposed rat group and the laboratory reconstituted river water group, although, the difference was not significant (P>0.05).

Influence of Pb in the presence of Metallic co-pollutants on serum SOD and CAT Activities

The results of the effects of Pb and metallic co-pollutants on superoxide dismutase (SOD) and catalase (CAT) activities are presented in Table 2.

Table 2:  Effects of Pb in the presence of  metallic co-pollutants on SOD and CAT activities

*See Table 1 for interpretations of alphabetical nomenclature

**bdfhjlnprt = values significantly (P<0.05) different from corresponding evaluated groups

*** acegikmoqs = values not significantly (P>0.05) different from corresponding statistically evaluated groups.

Exposure of rats to Pb only and Pb in the presence of Cu, Fe, Zn and combined metallic co-pollutants caused a significant (P<0.05) increases in SOD and CAT activities of the rat relative to the controls (Groups I and II). Pb in the Presence Ca, Mn, and Mg caused significant (P<0.05) decreases in SOD and CAT activities relative to the Pb-only exposed rat group. There was also significant (P<0.05) elevations in the activities of SOD and CAT of the river water exposed rat group relative to the controls (Groups I and II), and a slight decrease in SOD and CAT activities of the river water – exposed rat group relative to the laboratory reconstituted water. Although, it was not significant (P>0.05).

Influence of Pb in the presence of Metallic Co-Pollutant on serum AST and ALT Activities of the Rate 

The results of the effects of Pb and selected co-polluting metals of Warri River on plasma aspartate and alanine transaminases activities are represented on Table 3.

Table 3: Effects of Pb in the presence of metallic co-pollutants on serum AST and ALT Activities of the Rat

*See Table 1 for interpretations of alphabetical nomenclature

**bdfhjlnprt= values significantly (P<0.05) different from corresponding evaluated groups

*** acegikmoqs= values not significantly (P>0.05) different from corresponding statistically evaluated groups.

Pb, Pb+ Fe, Pb+ Zn, Pb+Cu and Pb+ all metallic co-pollutants -exposed rat groups caused significant increases in AST and ALT activities relative to their respective controls (Groups I and II). The groups of rats exposed to Pb in the presence of Ca (Pb+ Ca), Mn (Pb+ Mn) and Mg (Pb+ Mg) showed significant (P<0.05) decreases in the activities of AST and ALT relative to the Pb- only and Pb +Cu groups.

The Influence of Pb in the Presence of Metallic Co-pollutants on Serum Phosphatases Activities of the rat

The effects of Pb in the presence of metallic co-pollutants on serum phosphatases activities of the rat are presented on Table 4.

Table 4: Effects of Pb in the presence of metallic co pollutants on serum phosphatases activities of the rat.

*See Table 1 for interpretations of alphabetical nomenclature

**bdfhjlnprt= values significantly (P<0.05) different from corresponding statistically evaluated groups

***acegikmoqs= values not significantly(P>0.05) different from corresponding statistically evaluated groups.

The results of alkaline and acid phosphatase activities are presented in Table 4. ALT and AST activities were significantly(P<0.05) elevated  in the Pb only, Pb + Cu; Pb+ Fe and Pb +Zn groups relative to the control (dH₂O GroupI) and (Pti borehole H₂O, Group II). Pb+ Ca, Pb+ Mg and Pb+Mg–exposed rat groups reversed the effects by Pb-only, Pb+Cu, and Pb+Zn.

Influence of Pb in the presence of metallic co-pollutants on serum total protein and bilirubin levels 

The effects  of Pb in the presence of metallic co-pollutants on serum protein and bilirubin levels are represented on Table 5.

Table 5: Pb in the presence of metallic co-pollutants on serum total protein and bilirubin levels.

*See Table 1 for interpretations of alphabetical nomenclature

**bdfhjlnprt= values significantly (P<0.05) different from corresponding evaluated groups

*** acegikmoqs= values not significantly (P>0.05) different from corresponding statistical evaluated groups.

Table 5 shows the effects of Pb and  metallic co-pollutants on serum total protein, urine protein and bilirubin levels of the rat. Pb-only and, Pb + Cu, Pb+ Fe, Pb+ Zn and Pb+ metallic co pollutants –exposed rat groups (Groups III, IV, VI $IX) showed significant (P<0.05) decreases in concentrations of serum protein of the respective groups relative to the controls (Groups I and II).  There was a significant (P<0.05) increase in percentage(%) of  urine protein of the above mentioned groups relative to the controls (deionized water and Pti potable water groups) and  the Pb +Ca, Pb+Mn, and, Pb + Mg- exposed rat  groups. Pb + Fe –exposed rat group revealed a 92.46% protein  level  while Pb and Ca –exposed  rats  showed a 34.70%. protein level. Total bilirubin concentration of Pb only –exposed rats and Pb + Cu, Pb+ Fe and Pb+ all  metallic co pollutants was  significantly (P<0.05) elevated while the reverse was the case for the rat groups exposed to Pb + Ca, Pb+ Mg and, Pb+ Mn relative to the controls (deionized water and Pti borehole water groups). There was also a significant increase in direct bilirubin concentration of Pb only and, Pb + Cu, Pb+ Fe, Pb+ Zn, Pb+ metallic co-pollutant  and  river water-exposed  rat groups relative to their corresponding controls (Groups I and II).

Influence of Pb in the presence of metallic co-pollutants on blood and urine glucose

The effects of Pb in the presence of some selected metallic co-pollutants on blood and urine glucose are represented in Table 6.

Table 6: Effects of Pb and metallic co -pollutants on blood and Urine Glucose

*See Table 1 for interpretations of alphabetical nomenclature

**bdfhjlnprt= values significantly (P<0.05) different from corresponding evaluated groups

***acegikmoqs= values not significantly (P>0.05) different from corresponding statistical evaluated groups.

Table 6 shows the effects of Pb in the presence of metallic co-pollutants on blood and urine glucose concentration.

Rats exposed to Pb only, Pb + Cu, Pb+ Fe, and Pb+ Zn revealed a significant(P<0.05) increase in blood glucose and a corresponding increase in urine glucose concentrations  relative to the controls, deionized water and Pti borehole water groups. Pb + Ca, Pb + Mg and, Pb + Mn exposed rat groups showed,  significant (P<0.05) decreases  in plasma and urine glucose concentrations relative to the Pb only, Pb + Cu, Pb+ Fe and Pb+ all metallic co-pollutants. There was also a difference in the blood and urine glucose concentrations of river water and laboratory reconstituted Pb +metallic co-pollutants- exposed rats, albeit, it was not significant (P=0.065).

Discussion of Findings

The major consternation of this study was to evaluate some serum biochemical indices and the magnitude of oxidative stress occasioned by liver injury in rats exposed to Warri River levels of Pb in the presence of some selected  metallic co-pollutants.

Alterations in body weight change and organ/body weight ratio are frequently used as indicators of chemical toxicity (Der et al., 1976; Horiguchi et al., 1996; Ikatsu et al., 1998). The significant decrease in liver organ /body weight (Table I), of Pb only, and, Pb and Fe, Cu and Zn -exposed rat groups gives credence to the reports by Der et al., 1976; Horiguchi and his team (1996) and Ikatsu et al., 1998).This finding is in agreement with a previous work done by Obi and Fadairo, 2013, where Cd- exposed rats showed significant decline in organ/body weight ratio.

The body is known to protect itself from oxygen free radical toxicity by enzymatic antioxidant mechanisms (glutathione peroxidase , superoxide dismutase and catalase) and by non enzymatic antioxidant mechanisms like increase in certain proteins like albumin and bilirubin (Oboh et al., 2013), the significant (P<0.05) increases in catalase and superoxide dismutase activities, bilirubin levels of Pb-only rat group and Pb and Cu-exposed  rats  could be a strategy by the enzymatic and  non enzymatic antioxidant proteins to protect the liver organ against the possible free radical stimulating effect of Pb only and Pb in synergy with Cu, Fe and Zn. Our finding is in agreement with the previous work by Oboh et al., 2013. Our finding also showed a significant (P<0.05) statistical correlation between bilirubin levels (Table 3) and the levels of MDA (Table I), as bilirubin level was observed to increase along side with MDA levels in correspondence with the group of rats exposed to Pb-only and, Pb, in the presence of co-polluting metals like Fe, Cu and Zn.. This agrees with the previous works by Ahmed et al., 2005 and Oboh et al., 2013. Lipid Peroxidation is initiated by free radicals like superoxide and hydrogen peroxide. The significant (P<0.05) increase in the products of lipid Peroxidation in the plasma  of rats exposed to Pb only and Pb and Fe, Cu and Zn may not be surprising because of  report suggesting that transition metals catalyze highly reactive free radical form(Allisa and Fern, 2011).

ALT and AST activities are usually considered strong indicators of optimum liver function. Increased levels of ALT is found mainly in liver diseases like hepatitis and other hepatic diseases and a slight ALT elevation is also seen in myocardial infarction. ALT is found in variety of tissues but it is mainly found in the liver (Sood, 2006). AST is found mainly in the heart muscle, liver cells, skeletal muscle and kidneys (Sood, 2006). In this study, the significant (P<0.05) increases in the activities of the liver function enzymes (ALT and AST) in the plasma of rats exposed to Pb only, could suggest a leak in the membrane of hepatic cells of rats following the membrane damaging effect of Pb. This finding is consistent with the report of Sood, 2006.

Alkaline and acid phosphatases activities have been reported to be elevated when the liver all integrity is affected by either a disease process or a toxic substance (Emede and Igben, 2013). In this study, there was also an increased plasma ALP and ACP activities of the Pb only rat group. Our findings also agrees with the report of Henderson and Moss (2001) who reported  that serum or plasma ALP activities are of particular interest in the investigation of  two groups of conditions, namely bone disease associated with increased osteoblastic activities and hepatobilliary disease. Group III rats (Pb only-exposed) relative to control I (deionized water) and control II (Pti borehole water). The significant (P<0.05) increase in plasma ALP could be an indication of the onset of osteoblastic activities in bones of the Pb exposed rat group or, could also be due to disruption of the liver  parenchyma cells by Pb. This finding is also in agreement with the work of Brinkman et al., 1998.

Our present study was based on the hypothesis that the Pb and some selected metallic co pollutants of Warri river could result in elevation of some enzymes, non-enzymatic molecules and  lipid peroxidation products (MDA), usually use as biochemical markers of liver toxicity and oxidative stress  in order, to give a molecular rationale to the speculated increased incidences of liver diseases and liver related problems in the Niger delta region of Nigeria. Our findings revealed that Pb only and Pb in the presence of Cu, Fe, Zn, all co-polluting metals significantly induced the activities of  ALT, AST, ALP, ACP, SOD, catalase, and increased the levels of bilirubin and  glucose in Plasma and urine, caused a negative decrease in liver /body weight ratio, increased concentration urine protein, decrease Protein concentration of plasma, and a significant increase in the products of lipid per oxidation in the  rat,  relative to their  respective controls, deionized water and Pti borehole water.

Findings from this study shows that Pb only and, Pb +Cu, Pb+Fe – exposed rats also caused a significant (P<0.05) increase in plasma glucose and urine glucose concentration. The significant (P<0.05) increases in Plasma and Urine glucose could be as a result of other factors like impairment of kidney tubular  transport mechanism and morphology by these metals. Furthermore, malondialdehyde is a biomarker for measuring oxidative stress, and (Devasagayam et al.,2003; Maritime et al., 2003), demonstrated the role of oxidative stress  occasioned by high MDA levels in the pathology of diseases like diabetes and other related conditions, the significant(P<0.05) increases in plasma glucose and urine glucose of Pb- exposed and Pb + Cu,Pb+ Fe exposed rat groups could be due to the inability of the induced antioxidant enzymes and molecules (catalase, SOD and bilirubin) of same groups of rats to inhibit  the production of reactive oxygen species (ROS), which then resulted in significant increase in the formation of products of lipid peroxidation(MDA) in these groups of rats. Our findings is not in agreement with the previous reports of Halliwell, 2007; Hamid et al., 2010, who demonstrated that antioxidant molecules and enzymes are produce to counter the consequences of ROS generated from products of lipd peroxidation. This implies that exposure of rats to P-only, Pb + Cu, and Pb + Fe may have resulted in cellular responses leading to induction of antioxidant enzymes but it appears that the antioxidant enzymes and molecules were unable to antagonize the effect of Pb-only and in the presence of Cu and Fe metallic co-pollutants, from breaking down polyunsaturated fatty acids thereby resulting in the significant (P<0.05) high MDA of same groups of rats with high SOD, catalase and bilirubin observed in this study.

Overall, the overall effects of Pb only, and, Pb and Fe, Pb and Zn in the reduction of organ body weight ratio, increased ALP activities and increase plasma bilirubin concentration were synergistic as against our hypothesis of a remedial interaction. This does not align with a previous work of Ahamed et al., 2007, who demonstrated that essential elements like Ca, Zn, Fe and Selenium counteracted the negative effects of Pb. With the exemption of Ca, which, antagonized the effect of Pb in most biochemical markers of  liver toxicity evaluated. Fe and Zn were synergistic in their respective combined effects with Pb. Our finding is not in consonance with an earlier report referenced by Enuneku et al., 2013, who showed that binary mixture of Cd and Zn on mortality of biological specie was antagonistic instead of synergistic but agrees with the report of Shamar and Satyanaryan (2011), who demonstrated that co administration of Pb and Cu to vital tissues of the earth worm, caused more deleterious effect relative to when Pb was administered alone. Although some of our findings may not be in alignment with our hypothesis and some study reports, but there are reports demonstrating that transition metals, particularly the divalent ions such as Fe and Cu, are known to further catalyze highly, reactive free radicals forms (Allisa and Fern, 2011). Tagging along inconsistency in some of our findings especially as regards the reports on the antagonistic effects of some transition metals like zinc on Pb exposed rats, there is therefore, a need for a further study to equate the doses of these metallic co-pollutants to the levels of Pb and re-evaluate them on some of the biochemical markers evaluated in this study.

Acknowledgements

The authors wish to appreciate the following laboratories: Department of Biochemistry Laboratory, University of Benin; Chemical Pathology Laboratory, University of Benin; Benin-City and Analtrace Laboratory, Okwokwoko, Delta state, Nigeria.

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