Behavioral response is a peculiar measure as it incorporates and reflects the organism’s overall functional status of its biological systems. It provides a non-damaging index of functional capacity that allows the tracing of a toxicity effect (Weiss, 1983). The mice responded to variants of doses administered. The weight loss was observed in all the four groups except in control (table 1). In another study, rats exposed to Cd in 21% protein diet have led to decreased body weight and growth (Mohamed et al., 1991). However, loss of appetite was observed only in the group exposed to group 4 (2mgCd + 1mgPb) and absent in all other groups. Meanwhile, all other parameters were not observed in all the four groups including control as presented in table 1. This could possibly be due to the fact that the study is an acute toxicity study within a short period of time.
Generally, the uptake, distribution, and the accumulation of metals in the organs and tissues are affected by several factors. These include metals’ characteristics and forms, route, dose and exposure duration, the ability of binding to ligands in the cells, and species sensitivity. The hematopoietic system is among the most vital organs to assess the toxicity. After oral administration, both Cd and Pb undergo intestinal absorption and are transported via blood. In the blood, they can be distributed via red blood cells and plasma proteins, mainly albumin (Swiergosz-Kowalewska et al. 2001; Timchalk et al. 2006).
The current study showed that experimental groups treated with all doses had produced a significant increase in the white cell indices with the exception of group II (2mg Cd b.w) as seen in Table 2.
As observed from the single Cd exposed group, similar toxicity impacts of Cd on LYM count were recorded following single treatment with Cd in rats (Mladenovic et al. 2014; Yildirim et al. 2018), then following Cd exposure for 14 days in male BALB/c mice (Karmakar et al. 2000), and after four weeks of administering CdCl2 to Wistar rats through drinking water (El-boshy et al. 2015; El-boshy et al., 2017). So also, LYM, GRA, and % GRA recorded values that were lower than control and not statistically significant in rats exposed to metal fumes (Sani & Abdullahi, 2019). This showed that single Cd dose exposure could lead to lymphopenia.
Information regarding the toxic effects of Pb on WBC indices is rather conflicting, some authors described an unchanged status of WBC (Omobowale et al. 2014; Cobbina et al. 2015), leucopenia (Sharma et al., 2010), low WBC count and % LYM (Sani & Abdullahi, 2019), and leukocytosis (Abdou et al., 2014). However, from Table 2 it is evident that Pb and Pb in the mixture with Cd produced elevated WBC indices in mice. The increase could be in response to inflammation, stress, allergy, etc. and could result in leukocytosis. In addition, moderate lymphocytosis and leukocytosis recorded in this study following the administration of Cd and Pb mixture could also be as a result of mobilization under inflammatory IL-6 and TNF-α from marginal neutrophil pools (Kataranovski et al., 2009; Djokic et al. 2014).
Group IV which was exposed to (2mgCd + 1mgPb/kg b.w) witnessed a non significant reduction (p > 0.05) in RBC and a significant reduction in HGB when compared with other groups, including control (p < 0.05) indicating possible effects of the metals’ mixture. Similarly, values of HCT for group IV which were exposed to (2mgCd + 1mg Pb) did not differ statistically from control but showed a significant difference compared to other groups (1mgPb only, 1mgCd only, and 2mgPb + 1mgCd/kg b.w). Our findings are in agreement with previous works using a variety of animal models, doses, and the exposure route where a reduction in HCT, RBC, and HGB were observed Sharma et al., 2010; Sharma et al., 2011; Abdou et al., 2014; Mladenovic et al., 2014; Elboshy et al., 2015; (El-boshy et al., 2017). It can be suggested that the decrease in RBC, HGB, and HCT could be as a result of intravascular hemolysis probably due to higher metals’ load when compared to control. Among the possible mechanisms of hemolysis brought about by metals’ toxicity is oxidative stress. Production of free radicals was associated with hemolysis of RBC and anemia in an acute study with Wistar rats as experimental model treated with 2 mg/kg b.w. CdCl2 via i.p. injection (Mladenovic et al. 2014). Similarly, exposure to Pb has resulted in the significant reduction in RBC count, HGB, and values (Terayama, 1993). The Pb is known to interfere with the biosynthesis of the heme group due to its inhibitory impacts on the enzymes associated with heme synthesis which was clearly reflected by the lower levels of HGB (blood protein) (El-Missiry, 2000).
The mean values of MCV, MCH, MCHC and RDWC of all the treatment groups were lower than control and not statistically significant (p > 0.05). The acute investigation did not significantly temper with MCV and MCHC, which is as reported by other authors (Cobbina et al., 2015; El-boshy et al., 2015; El- boshy et al., 2017).
Groups II & IV administered 1mgCd/kg b.w, and 2mgCd + 1mgPb/kg b.w respectively had higher PLT and PCT mean value and statistically significant (p < 0.05) as presented in Table 4. Thus, these possibly suggest thrombocytosis. Mean values for PLCR were less than control and statistically significant (p < 0.05). However, the values of MPV and PDW were not different from the control (p > 0.05).
Reports from studies were conflicting, wherein some pointed to PLT levels unchanged following subacute or acute treatment (Yuan et al., 2014; Cobbina et al., 2015; Curcic et al., 2017; Yildirim et al., 2018); others point to reduced PLT levels (Mladenovic et al., 2014; El-boshy et al., 2015; El-boshy et al., 2017), and some indicate an elevation of PLT count (Hounkpatin et al., 2013) as observed in the present study. This could result in thrombocytosis described by an excessive increase in PLT counts. Some possible explanation for this condition might be due to inflammation induced by administered single metals and in mixture, though not statistically different (p > 0.05). This is supported by the observations made for WBC count, or as reactive thrombocytosis, which changes and retire back to normal after the discharge of the pollutant.
Oxidative response markers which SOD and CAT were evaluated as stress endpoints. Highest SOD and CAT were observed in the group exposed to 2mgPb + 1mgCd/kg b.w and subsequently by 2mgCd + 1mgPb/kg b.w (see Table 5). Single metals’ doses of 1mgCd/kg b.w was higher than 1mgPb/kg b.w and all doses including in mixtures were higher significantly than the control (p < 0.05). The metals in mixture induce greater effect as pointed out by the SOD and CAT values with 2mgPb + 1mgCd/kg b.w dose impacting more oxidative stress than other corresponding dose (2mgCd + 1mgPb/kg b.w) and single metal doses (1mgCd/kg b.w and 1mgPb/kg b.w) as presented in Table 5.
SOD acts as an enzyme that ensures the conversion of oxidative molecules such as superoxide anions into oxygen and hydrogen peroxide (Bowler et al., 2012). The peculiarity of its actions elucidate the levels of O2− and H2O2, and hence, the likelihood of its central role in the defense mechanism. The higher level of SOD activity in metals-treated groups is a possible indication of heavy metal induced ROS generation. Also, the higher level observed in the treatment group exposed to a mixture of Pb and Cd (2mgPb + 1mgCd/kg b.w; 2mgCd + 1mgPb/kg b.w) showed a synergistic action in the generation of reactive species by both heavy metals.
In another supporting study, it was revealed that Mn, Pb and Cd associate with environmental conditions. Such relationship is majorly antagonistic (Markiewicz-Górkaet al., 2015).
The CAT on the other hand catalyzes the reduction of hydrogen peroxide to water and oxygen. In addition, CAT also reduces the product of SOD catalyzed reactions to non-toxic products of water and oxygen. It is therefore reasonable for the level of CAT to increase in the same pattern as SOD because they both catalyze successive steps of a reaction. The same observations were made in the present study as can be seen in all groups exposed to either individual Cd and Pb doses or in the mixture. To support this, CAT has been known to increase in conditions of extreme stress (Cuypers et al., 2010) and similar implications were realized in blood cells’ indices (see Table 2 to 4).
Saidi et al. (2013) conducted a study on the expression of antioxidant enzymes associated with heavy metals. It was revealed that there is a sequence of increase in SOD mRNA expression via exposure to Cd and co-treatment with H2O2. This therefore suggests that there is an improvement of the antioxidant system to eliminate ROS through exposure to Cd and H2O2. This finding was similar to the increased SOD value obtained from the current study.
So also, Javed et al. (2017) evaluated oxidative stress markers in Channa punctatus exposed to wastewater polluted with heavy metals by. They observed significantly higher levels of GST, SOD, CAT exposed to heavy metals when compared to fish from a reference site. In a study involving humans on the impacts of similar heavy metals on markers of oxidative stress markers, it was revealed that CAT and Cu2+/Zn2+SOD activities were greater in the workers having a higher creatinine-corrected urinary Hg concentrations than groups with lower (Perrin-Nadif, 1996). Similarly, there was a significant elevation of the CAT activity in the serum of glazers when compared with the controls. It was therefore shown that co-exposure to Pb and Cd could produce oxidative stress in glazers, leading to an increase in lipid peroxidation and altered antioxidant enzymes (Hormozi et al., 2018).
The increase in CAT of glazers might be as a result of activation of direct enzyme by Pb and Cd. This is as a repercussion of excessive production of ROS and the compensatory process engineered to balance the excess of LPO. These findings were supported by both experiments (Tandon et al., 2003; Gong et al., 2008) and occupational studies (Gurer and Ercal, 2000; Rendo´n-Ramı´rez et al., 2014) where an increase in blood CAT was realized following exposure to metals that include Cd or Pb.
As with other metals, Pb and Cd destroy several cellular components through the increased magnitude of oxidative stress. The toxic effect from such metals is multidimensional as rigorously prevent absorption of vital elements, disrupts the activity of enzymes, and deactivates antioxidant sulphydryl pools (Patrick, 2006).
So, Pb and Cd can induce oxidative damage in the body via elevation of ROS and altering the antioxidant defense system of the cell (Garc¸on et al., 2004; Patrick, 2006). These metals have the capacity to diminish the amount of vital cellular antioxidant molecules, majorly enzymes, thereby leading to lipid peroxidation and DNA damage (Valko et al., 2005). The antioxidant enzymatic defense system that involves CAT and SOD is improved as an alternative reaction to compensate the formation and effects of ROS. They are the active enzymes in removing the ROS produced during bioactivation of xenobiotics in the hepatic tissues (Sk and Bhattacharya, 2006) and the presence of CAT/SOD antioxidant system serves as the first line of defense against ROS (Van der Oost et al., 2003; Nwani et al., 2013; Ighodaro & Akinloye, 2018).
Several works have indicated the critical role played by oxidative stress in the toxicity of both Cd and Pb and the imbalance of prooxidant/antioxidant (Sugawara et al., 1991; Hunaiti and Soud, 2000; Wang and Fowler, 2008).
Some suggest that Cd could have a destructive effect on cellular enzymes and hence significantly depletes the level of antioxidants, especially SOD (Ogunrinola et al., 2016). Other studies have revealed that Pb can result to both a decrease and an increase in the serum levels of CAT and GPx (Sugawara et al., 1991; Chiba et al., 1996; Han et al., 2005). All these are indications that exposure to metals could result in a condition of oxidative stress as evidenced by the significant increase of oxidative stress markers.