DISCUSSION
Lead toxicity
Genotoxic Effects of Lead in PBLC:
The specific role of environmental and occupational exposure of heavy metal like lead in health-related consequences at cellular level was not well understood. The injurious effects of lead most certainly develop in a discrete cellular manner. Hence, we had applied genotoxicity and free radical toxicity analysis to find possible mechanism of lead induced cellular toxic effects in cultured lymphocytes. Lead genotoxicity is a complicated phenomenon, because it acts with both DNA as well as proteins (Flora, 2012; El-Ashmawy et al., 2006; Zakour et al., 1981). DNA damage is primarily repaired by different repair pathways; however, unrepaired or misrepaired DNA damage will lead to cytogenetic alterations (Sister chromatid exchanges, chromosomal aberrations and associations, micronuclei formation etc.). These cytogenetic end points assessed mainly in human peripheral blood lymphocytes for decades has been used as biomarkers to analyze genotoxic risks.
The Cell cycle proliferative index (CCPI) is only an indirect measure of cytotoxic/cytostatic effects and depends on the time after treatment. Cell cycle is a highly regulated event that controls the growth and differentiation of cells. In the case of studies without cytochalasin B is used for CCPI analysis; in the case of studies with cytochalasin B, it can be helpful to determine cytokinesis block proliferation index (CBPI). This indicates the number of cell cycles per cell during the period of exposure to cytochalasin B. So, CCPI and CBPI can be used to calculate cytotoxicity generated by toxic substances. Changes in cell cycle distribution might be associated with the apoptosis and differentiation of cells (Yedjou et al., 2015; Jakoby and Schnittger, 2004). CCPI has been considered as a sensitive endpoint to evaluate the mitotic changes caused by injury in the cell structure or function in different studies (Shah et al., 2016; Nair et al., 2004). A reduced CCPI can be interpreted as growth arrest at any moment during interphase or as loss of the capacity of the cells to proliferate or die. Hagmar et al., (1998) in their report from the European Study Group on Cytogenetic Biomarkers and Health (ESCH), clearly suggested that the frequency of Chromosomal aberrations (CAs) in lymphocytes can be a relevant biomarker for cancer risk in humans, reflecting either early biological effects of genotoxic carcinogens or individual cancer susceptibility. Increased cytogenetic damage has been shown to reflect enhanced cancer risk. Domingrez and co-workers (2002) had studied a time-dependent inhibition in DNA synthesis and cell growth in tissue culture exposed to Lead indicating exposure time is also important in cytogenetic anomalies. Cytotoxic effects through inhibition of cell division was found due to Lead by Ibrahim and co-workers (2006). The present study clearly demonstrated that lead is a potent genotoxic agent and its effects were dependent on the dose and exposure time, probably due to the increased free radicals and cellular inability to eliminate lead effectively from the body.
The present study also showed dose and duration dependent decrease in the index of CCPI and CBPI with increase in the Average Generation Time (AGT) and Population Doubling Time (PDT) in Lead treated cultures. Information on cell cycle kinetics, such as AGT and PDT, could be used as supplementary information in PBLC studies. AGT and PDT parameters are overall analysis that does not always reveal the existence of delayed subpopulations, and even slight increases in average generation time can be associated with very substantial delay in the time of optimal yield of micronuclei and aberrations. Moreover, there is a significant increase in frequency of cytokinesis block micronuclei (CBMN), sister chromatid exchanges (SCEs/Cell and SCEs/Chromosome), CAs and chromosomal associations (Acrocentric and Telomeric associations) which may responsible for lessens of cell function progressively from the time of exposure in peripheral blood lymphocytes culture (PBLC). The increase in the genotoxicity observed in the present study was corroborated with previous in vitro and in vivo studies (Shaik and Jamil, 2009; Shah et al., 2016; Aboul-Ela, 2002; Valverde et al., 2001; Nehez et al., 2000). In support to these results various scientists had demonstrated Lead induced dose-dependent increases in CBMN and SCEs in human lymphocytes, human melanoma and mouse bone marrow cells (Castilhos Ghisi et al., 2016; Kapka et al., 2007; Shah et al., 2016; Bonacker et al., 2005; Dhir et al., 1993; Poma et al., 2003). The analysis of SCEs by toxicant is proposed as a sensitive assay to analyze genetic damage related to mutation and cancer. The persistent of increased SCEs caused by lead, agrees with previously reported data which indicated demethylation derived epigenetic modifications and appearance of SCE eliciting sites in DNA which are revealed using BrdU (Morales-Ramírez, 2007; Ruiz-Hernandez et al., 2015). These may be a primary cause of chromosomal damage (CBMN and CAs) observed in the present study. Genotoxicity observed in this study where magnitude of exposure becomes more critical as the genotoxicity increases by time and dosages. This relationship of lead exposure and its genotoxicity may be dependent on the kinetics of lead metabolism. Lead acetate can induce breaks in DNA, as well as responsible for DNA protein cross link in in vitro condition (Shah et al., 2016; Wozniak and Blasiak, 2003). The inhibition of DNA repair may be one of the mechanism for genotoxicity induced by lead because lead ions can interact with DNA polymerase in DNA processing and repair and this alteration resulted in toxicity, which is not reversible as lead directly competes with key components like Zinc and Calcium (Hartwig, 1994; Simons, 1988). In vivo Lead acetate induced DNA damage and elevated rate of chromosomal and chromatid aberrations in somatic and germ cells was reported in mice (Aboul-Ela, 2002; El‐Ashmawy et al., 2006; Fracasso et al., 2002; Valverde et al., 2001). Similar findings for chromosomal aberrations and associations in human lymphocytes were also observed in the present study in both tested concentrations. A higher percentage of breaks were observed than gaps. The occurrence of frequency of associations was observed more significantly in both doses as compared with controls. The sticky molecular material would tend to hold the associated chromosomes together through mitosis. A high incidence of associations has often been considered as predisposing to an increased tendency of nondisjunction (Hanson, 1970; Jeno et al., 1999, Shaik, 2009).
Toxic Effects of Free Radicals by Lead in PBLC:
Even though lead toxicity has been subject of intense research over many decades, the mechanism responsible for its toxicity are still poorly understood. We had observed that lead increases genotoxicity in PBLC and these findings were in covenant with the increased free radical toxicity by lead in lymphocyte cultures.
Lead is an environmental toxicant that can induce oxidative stress via reactive oxygen species (ROS) generation, which has been reported as an important mechanism underlying lead toxicity (Flora, 2012; Gurer and Ercal, 2000; Pande and Flora, 2002; Kasperczyk et al., 2004; Farmand et al., 2005; Verstraeten et al., 2008; Wang et al., 2009; Martinez-Haro et al., 2011). Oxidative stress occurs when the generation of ROS exceeds the antioxidant system’s ability to defend cells against oxidized molecules. ROS is a term generally used to refer to free radicals derived from oxygen (e.g., superoxide anions [O2‑] and hydroxyl radicals [OH–]) or to non-radical species (e.g. hydrogen peroxide [H2O2]) (Halliwell and Cross, 1994).
Present study had determined dose and duration dependent toxic effects of lead and in these, free radicals play a primary role in degradation of macromolecules. Free radicals also affect other functions such as secretory process regulation, protein turnover, storage and inactivation of xenobiotics, defense mechanisms and cell death. Lead is known to induce oxidative stress by decreasing the activities of several antioxidant enzymes, creating an oxidizing environment and ultimately resulting in the imbalance of the pro-oxidants and antioxidants in the cells which is suggested to result in genotoxicity. It was concluded that reactive oxygen radicals formed from O2 and H2O2 (generated by Fenton reaction) are capable of damaging DNA (Shaikh, 2009; Jayaprakasha et al., 2002).
The most frequently used biomarkers for lead induced oxidative stress were: glutathione (GSH) (Siddiqui et al., 2002; Ahamed et al., 2006; 2008; 2011; Diouf et al., 2006; Jin et al., 2006; Cabral et al., 2012), lipid peroxidation (LPO) (Siddiqui et al., 2002; Ahamed et al., 2006; 2008;2011; Jin et al., 2006; Cabral et al., 2012; Wu et al., 2013), superoxide dismutase (SOD) (Jin et al., 2006; Diouf et al., 2006; Ahamed et al., 2008; Martinez et al., 2013; Wieloch et al., 2012; Wu et al., 2013 ), catalase (CAT) (Ahamed et al., 2006; 2008; 2011; Wieloch et al., 2012; Martinez et al., 2013), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) (Diouf et al., 2006; Jin et al., 2006; Ahamed et al., 2008). In this study, we had observed a dose and duration dependent reduction in the activities of these enzymes in lead treated lymphocytes.
Lead can generate oxidative stress as it can enter the mitochondria using the Ca2+ uniporter enhancing the dysregulation of Ca+2. This ability of lead to destabilize membranes potentials, which results in elevated LPO, could induce apoptosis or necrosis (Simons, 1988). The concentrations of malondialdehyde (MDA), one of the final product of LPO, commonly increase as the number of double bonds in the fatty acids increase due to lead exposure, thus making the cells more susceptible to damage than fatty acids with lesser double bonds (Ahamed and Siddiqui, 2007; Gurer and Ercal, 2000). In this study, we observed increased levels of LPO in lead treated lymphocytes along with a decrease in protein content may be due to destabilize membrane potential.
Glutathione is a tripeptide containing cysteine that has a reactive thiol group (-SH) (Nemsadze et al., 2009), which interacts directly with ROS or can be involved as a cofactor in the enzymatic detoxification reactions for ROS (Ding et al., 2000). The content of GSH was reduced after exposure of lead, indicating disturbance in defense pathway against free radicals. The notable inhibition of GSH could be closely associated with cell damage and reduction in the antioxidative defense mechanism. This may be due to the attack of free radicals on the fatty acid component of membrane lipids and utilization of GSH by increased ROS. Based on results obtained in this study, it has been recently reported that human lymphocytes grown in the presence of lead acetate exhibit a significant decrease in GSH level which leads to significant diminution of its ability to counteract against free radicals in cells (Flora, 2012).
The most prominent effect of lead treatment on human lymphocytes was a drastic reduction in intracellular enzymes, thus indicating that lead may interfere in the biochemical pathways in cell, with consequent lowering of cellular enzymatic stores (Ahamed and Siddiqui, 2007; Labrot et al., 1996; Gurer and Ercal, 2000). This is very well correlated with decreased total protein content of lead acetate treated lymphocytes and reduction in activity of various enzymes in the present study. It is already known that the antioxidant enzymes SOD, CAT, GPx, GR and GST involved in decreasing the free radical toxicity (Flora et al., 2012). Experimental studies have also shown divergent results regarding the association between lead with CAT and SOD activities. Mylroie and co-workers (1986) as well as Nehru and Kanwar (2004) identified a decrease in SOD activity in both cerebral end cerebellar regions in Wister strain rats exposed to lead acetate, suggesting that lead induced deficiency of copper, an essential metal for SOD activity, leading to a reduced capacity of eliminating ROS and resulting in oxidative damage which was in accordance to present study. Catalase activity also decreased in the presence of lead, possibly because lead interferes with iron absorption and heme biosynthesis (CAT contains heme as a prosthetic group) (Nehru and Kanwar, 2004). In contrast, the studies by Vaziri and co-workers (2003) and Farmand and co-workers (2005) showed upregulation of SOD and CAT activities in lead treated animals, wherein an increased level of H2O2 was produced because of increase in the SOD level (Matés et al., 1999). The higher concentration of H2O2 stimulated expression of the gene encoding the CAT enzyme, representing a compensatory response to oxidative stress.
Glutathione peroxidase is a selenium-containing enzyme that uses GSH as a reducing agent to transform H2O2 and lipoperoxides generated in the tissues to O2 and is also involved in the antioxidant system (Matés et al., 1999; Gurer and Ercal, 2000). Lead reacts with selenium, an essential element for GPx activity, leading to the formation of an insoluble complex (lead selenide) and reducing selenium uptake (Schrauzer, 1987; Whanger, 1992; Matés et al., 1999). In the present study decrease in GPx activity was observed may be due to decrease in the availability of selenium in presence of lead. In accordance to the present study Sivaprasad and co-workers (2003) GPx activity was also found lower in the erythrocytes of lead treated rats.
Lead induced chromosomal damage can be correlated with the elevated lipid peroxidation, depletion of GSH and production of ROS along with the diminution of overall antioxidant status (Flora, 2012; Fracasso et al., 2002). There have been reports on lead induced ROS and lead induced genotoxicity in this regard (Poma et al., 2003; Flora et al., 2012). The data of this study clearly indicated a significant relation of free radical generation and durations of lead exposure and its concentration. Toxic effects were observed severely in high dose long duration exposure as compared to low dose short duration exposure of lead acetate. In support to the previous findings (Dobrakowski et al., 2016; Hunaiti and Soud, 2000) present study, also proposes that as lead affects activity of enzymes like GR and GST at sensitive stages of cell cycle progression, it could be a contributing mechanism to genotoxic damages. Glutathione reductase (GR) contributes to cellular redox homeostasis, many using antioxidant metabolites like ascorbate, GSH or NADPH as substrates (Foyer and Noctor, 2005) and it is an important enzyme in glutathione redox cycle. GST belongs to the family of detoxifying enzymes and plays an important role in metabolism of toxic substances, catalysing reactions of binding xenobiotics with GSH. The decline in activity of GST resulted in abnormal glutathione cycle. GST catalyzes the conjugation between itself and a hydrophobic substrate possessing an electrophilic center. Hence, the declined activity of GR and GST resulted in abnormality in glutathione redox cycle ultimately responsible for oxidative stress.
Outputs revealed dose and duration dependent elevated free radical generation and consequent genotoxicity, hence free radical production probably the main cause of lead induced genotoxicity. The high degree of variability in the available data in different time duration and different levels of exposure as well as its vulnerability towards toxicity which makes the explanation of biomonitoring results quite complex but it clearly indicating that quenching the free radicals by some external intervention would exert a cytoprotective effects.
Study of Genotoxicity and Free Radical Abnormality in Occupationally Lead Exposed Workers:
Lead is still widely used in many industrial processes and is very persistent in the environment. We involved different factories/work exposure in our study sites, to analyze genotoxicity endpoints and free radical toxicity parameters as well as two genetic polymorphisms (ALAD and ACE gene polymorphism) related to lead toxicokinetic as susceptibility biomarkers in occupationally lead exposed workers Study population comprised 200 workers from Gujarat, India and 200 controls from the same area. The parameters analyzed related to lead exposure were blood lead levels (BLL) and analysis of delta-aminolevulinic acid dehydratase (ALAD) activity. Moreover, genotoxicity and free radical toxicity biomarkers were also analyzed.
Free Radicals Toxicity Study in Plasma of Lead Exposed Workers:
Plasma of occupationally lead exposed workers and unexposed healthy individuals was separated from blood and evaluated for free radical toxicity by lead. Along with an increase in free radical generation, there was simultaneous decrease in the activity of the antioxidant system could responsible for lead induced free radical toxicity in this study. Similarly, alteration in antioxidant enzymes activity and decrease in levels of antioxidant molecules have been observed by other researchers and had analyze lead induced free radical damage in both clinical and experimental studies (Vaziri et al., 2003; Wang et al., 2007). Several studies have shown that the effect of lead on antioxidant enzymes and other components of the antioxidant system, such as activity of SOD, CAT, GPx, GR, GST and level of GSH, depends on the level of lead in blood (BLL) (Kasperczyk et al., 2004; Ahamed et al., 2008; Mohammad et al., 2008; Kasperczyk et al., 2009; Grover et al., 2010; Wieloch et al., 2012).
Lead shows electron sharing capability and can form bond with sulfhydryl groups present in antioxidant enzymes, which are the most susceptible targets for lead. Lead inactivates GSH by binding to sulfhydryl groups present in it. This may be a primary cause for decline GSH level in present study. Similar decrease was observed in GSH levels in lead exposed subjects (Roels et al., 1975; Mohammad et al., 2008) as well as in different experimental studies (Wang et al., 2009; Martinez-Haro et al., 2011; Siddiqui et al., 2002; Ahamed et al., 2008; 2011). In contrast, in the study of Conterato and co-workers (2013), GSH levels were elevated in lead exposed workers with lower BLL but not in workers with higher BLL. A possible explanation is that GSH synthesis increases as a protective mechanism when the cells are oxidized. However, when oxidation is very high due to elevated concentrations of lead, GSH synthesis can’t protect against free radicals; subsequently, GSH levels tend to drop (Schafer and Buettner, 2001). The data were in agreement with other exposed group study in which GSH level was decreased significantly (Mohammad et al., 2008; Feksa et al., 2012; Kasperczyk et al., 2013) and were correlated with BLLs. However, some groups of scientists did not find significant changes in GSH levels with increase in BLL (Ahamed et al., 2006; Jin et al., 2006; Cabral et al., 2012) and increased level was found (Gurer-Orhan et al., 2004; Conterato et al., 2013).
Malondialdehyde (MDA) and thiobarbituric acid reactive substances (TBARS) are products of LPO that had produced in the presence of lead. Many studies (Akinhanmi et al., 2017; Al-Ubaidy et al., 2017; Ercal et al., 2001) have observed increased levels of MDA or TBARS related to higher BLL, suggesting an increase in LPO as a consequence of ROS. LPO is biomarker of oxidative stress and is one of the most investigated consequences of ROS on lipid membranes. In addition, lead can affect fatty acid composition by leading to elongation of the arachidonic acid (an essential unsaturated fatty acid in the membranes) and enhancing LPO (Yiin and Lin, 1995). These findings may explain the correlations identified between BLL and LPO. Jin and co-workers (2006) considered MDA the most sensitive oxidative stress biomarker because, when lead exposure induces overproduction of free radicals which can react directly with biological macromolecules (e.g., lipids, proteins, and DNA). Thus, it can lead to an increase in peroxides which was also observed as increased MDA level in current study. Regarding the occupationally exposed populations, this experimental study had asserted that higher BLL were associated with an increase in MDA or TBARS concentrations in plasma. The similar results were also observed in other studies of occupationally exposed individuals (Sandhir et al., 1994; Kaczmarek-Wdowiak et al., 2004; Jia et al., 2012). The free radicals generated in the present study by lead arrests the electrons from lipids inside the cell membranes and increases the LPO. Concentrations of MDA were significantly higher in subjects with higher BLLs in various studies (Siddiqui et al., 2002; Ahamed et al., 2006; 2008; Jin et al., 2006; Cabral et al., 2012; Wu et al., 2013; Ahamed et al., 2011). Apart from LPO, lead also causes hemoglobin oxidation, which directly causes RBC hemolysis and responsible for decrease in Hb. Activity of ALAD enzyme has been found lowered as compared to control individuals in the present study which can result in an increased concentration of substrate ALA. Elevated ALA levels generate hydrogen peroxide and superoxide radical and also interact with oxyhemoglobin, resulting in the generation of hydroxyl radicals (Patrick, 2006) causing oxidative stress. Similarly, lead inactivates enzymes like ALAD, GPx, GR and GST which further depresses the GSH content (Ahamed & Siddiqui, 2007). The same has been observed in the plasma of lead exposed workers in the present study, as compared to control individuals.
Other important antioxidant enzymes that also affected by lead were SOD and CAT. Decrease in SOD activity of plasma of occupationally lead exposed workers was noted in the present study which reduces the disposal of superoxide radical, whereas reduction in CAT impairs scavenging of superoxide radical. Apart from targeting the sulfhydryl groups, lead can also replace the zinc ions that serve as important co-factors for these antioxidant enzymes and inactivates them (Flora et al., 2007). Progression of all the above analyzed mechanisms makes things extremely vulnerable to oxidative stress and may responsible for cumulative lead mediated toxicity. SOD and CAT activity was substantially lower in plasma of subjects with higher BLL. Similarity in the outputs was also observed by various researchers in SOD and CAT activities in which they found significantly lower activities of the enzymes in groups exposed to lead (Wieloch et al., 2012; Sugawara et al., 1991; Patil et al., 2006; Mohammad et al., 2008; Grover et al., 2010; Conterato et al., 2013; Han et al., 2005).
GPx activity was found decrease in plasma of exposed population. Moreover, in support to the present study Diouf and co-workers (2006) also found the similar results and recognized negative correlation between GPx activity and BLL. In contrast to the present findings there were no significant changes in GPx activity associated with increased BLL by various researchers (Jin et al., 2006; Berrahal et al., 2007). This is might be due to differential mechanism of GPx activity in different toxic condition like duration and potency of free radicals.
Genotoxicity Study in PBLC of Lead Exposed Workers:
The present study indicates that workers occupationally exposed to lead, were employed for analysis of various genotoxicity and free radical parameters which showed clear evidence of abnormality in free radical defense ultimately responsible for chromosomal abnormality in their lymphocytes.
Earlier, studies on biomonitoring have performed in humans occupationally exposed to lead although relatively few studies have examined the genotoxic potential of lead and they offer some equivocal results, showed increased in chromosomal anomalies (Forni et al., 1976, Kentner, 1994). The data from individuals highly exposed to lead seem to support the hypothesis of an association between exposure to lead and genotoxicity (Flora, 2012). Studies on the induction of chromosomal anomalies both in vivo and in vitro by lead appear to depend on factors such as cell types, exposure duration and can also be influenced by synergistic effects.
The workers occupationally exposed to lead in different work environments, showed a clear evidence of genetic damage in peripheral blood lymphocytes culture when evaluated for genotoxicity testing. The determination of BLL revealed that the occupational exposure to lead was relatively high as compared to control individuals. Among the different genotoxicity testing protocols, the cell cycle proliferative index, sister chromatid exchanges and cytokinesis block micronuclei assay have increasingly been accepted as a reliable biomarker of chromosomal damage induced by various genotoxic agents like lead in occupational settings. Positive findings using these biomarkers indicated evidence of exposure to lead compounds had proved very reliable in assessing the genotoxic effects in occupational exposures (Bercés et al., 1993; Vaglenov, 1998; 1999).
Different genotoxic endpoints in the lymphocytes of workers (>30 µg/dL BLL) were assessed (CCPI, AGT, PDT, SCEs/Cell, SCEs/Chromosome, CAs, CBMN, CBPI) which reflecting different mechanisms of induction of genotoxicity. The results of the cytogenetic analysis demonstrated decreased frequency of cell cycle index, AGT and PDT along with increased frequencies of sister chromatid exchanges, chromosomal aberrations and micronuclei in the lymphocytes of occupationally lead exposed workers as compared with unexposed healthy individuals.
The present results were comparable with earlier studies such as increased frequencies of micronuclei in workers of different industries related to battery recycling, brass parts etc. (Madhavi, 2008; Naik et al., 2005; Flora, 2012). The results of present study estimate the genetic risk of lead by using various biomarkers and it is in correlation with many previous investigations (García-Lestón et al., 2010; 2012; Grover et al., 2010; Kasuba et al., 2010; Khan et al., 2010). Thus, all these data indicate that lead exposure in the workplace induces chromosomal alterations, DNA damage and mutagenicity.
Chromosomal aberration and sister chromatid exchange assays are among the most appropriate cytogenetic methods for detection of genomic aberrations and a combination of these two assays is recommended for monitoring a population frequently exposed to genotoxic agents (Mahata et al., 2004; Santovito et al., 2014). Furthermore, CBMN assay is an ideal method because, it is for assessing chromosomal damages and it is a fast and simple assay system broadly applied for in vitro and in vivo genotoxicity testing. Micronuclei are produced during mitosis via various mechanisms (acentric fragments, multicentric chromosomes, damaged kinetochores, spindle defects) and can be detected in the cytoplasm besides the cell nucleus as a small nucleus like particle with a high degree of certainty. It could be used as a biomarker for studies focused on genomic instability and initial biologic effects of genotoxic exposure in humans (Cavallo et al., 2007; Gourabi and Mozdarani, 1998; Hongping et al., 2006; Salimi and Mozdarani, 2015). The incidence of increased micronuclei, chromosomal aberrations as well as SCEs in lymphocytes of lead exposed workers in the present study can be the sign of elevated incidences of DNA damages in them. Groot de Restrepo and co-workers (2000) found enhanced DNA damages in leukocytes of workers with the higher BLL. Fracasso and co-workers (2002) also found significantly elevated levels of DNA breaks in lead exposed workers
Free Radical Toxicity Study in PBLC of Lead Exposed Workers:
As discussed in the PBLC of healthy individual study using different dosages of lead for two-time durations it was found that genotoxicity produced by lead, elevated free radical and oxidative stress are interdependent. Similarly, in PBLC study of occupationally exposed individual whose BLL is quite higher in the selected study population showed the lead induced genotoxicity and it can also be hypothesized that this genotoxic effect is because of generation of free radicals and depletion in antioxidants status of cells. Lymphocytes culture of occupationally lead exposed workers revealed declined in total protein and GSH level as well as considerable higher level of LPO as compared to unexposed healthy individual lymphocytes culture. Gurer-Orhan and co-workers (2004) had studied GSH/GSSG ratios in RBCs of lead exposed workers and noted decline in exposed population. Many researchers (Kasperczyk, 2005; Ding et al., 2000; Ito et al., 1985; Sugawara et al., 1991) had found increased MDA content which may be responsible for decrease in GSH concentrations and this finding is corroborate with present study.
Further evidence for oxidative damage in lead exposed workers arises from the decrease in SOD, CAT and GPx activity. Catalase has been suggested to provide an important pathway for H2O2 decomposition at higher steady state of H2O2, whereas GPx is believed to play a more important role in H2O2 decomposition under lower steady state of H2O2. Decreased catalase and GPx activity in lead exposed workers in present study can be explained as a diminishing mechanism against oxidative stress and it also distinguished in the previous findings on lead exposed workers (Sugawara et al., 1991; Patil et al., 2006). Several studies reported alterations in antioxidant enzyme activities such as SOD, CAT and GPx, and changes in antioxidant molecules, such as GSH in lead exposed animals (McGowan and Donaldson, 1986; Hsu, 1981; Sugawara et al., 1991; Patil et al., 2006) and workers (Monteiro et al., 1985; Ito et al., 1985; Sugawara et al., 1991; Chiba et al., 1996; Solliway et al., 1996; Kasperczy et al., 2004; Patil et al., 2006). Increase in LPO and depletion of GSH in lead exposed workers was might be due to reduced activity of GPx and the lack of increase in activity of GR and GST (Kasperczy et al., 2004). Similar explanation also considered for the results observed in the present study. Decrease in activities of these antioxidant enzymes could be due to massive production of free radicals, which override enzymatic activity (Hamed et al., 2010; Lodi et al., 2011; Gurer and Ercal, 2000; Malekirad et al., 2010). Activities may also be decreased directly by the interaction of lead with known metal cofactors of enzymes, such as zinc or copper. These declined have been also provoked by interaction of lead and sulfhydryl groups, which are known to be essential for enzymatic activity (Flora et al., 2004; Hamed et al., 2010; Sharma et al., 2010; Xia et al., 2010; Yu et al., 2008). In gist, lead can cause oxidative stress through various mechanisms, i.e. acceleration prooxidants formation and reducing the antioxidant defense system of cells via depleting glutathione, interfering with some essential metal cofactors which required for enzyme activity, inhibiting sulfhydryl-dependent enzymes or antioxidant enzymes activities and/or increasing the susceptibility of cells to oxidative attack by altering membrane integrity and fatty acid composition (Patil et al., 2006; De-Silva, 1981).
Amelioration of Lead toxicity:
Abetment of lead toxicity with rebalancing the impaired prooxidant/antioxidant ratio through supplementation of antioxidant nutrients are still not completely clear. However, evidences suggest significant protective effects of antioxidant nutrients like various herbal remedies. There is no substitute for eliminating exposure, but in a bad environment a good diet may help keep lead levels low. Certain foods actually facilitate lead removal which includes best natural chelators i.e. curcumin (from turmeric) and SAC (from garlic).
Ameliorative Effects of Curcumin against Lead Toxicity:
Curcumin is a yellow-colored polyphenolic compound and the principal active component of turmeric, which is obtained from the plant Curcuma longa. There are reports of antioxidant, radical scavenging and metal chelating effects of curcumin in metal toxicity (Sethi et al., 2009; Agarwal et al., 2010; Singh et al., 2010; Rao et al., 2008). Shukla and co-workers (2003) reported for the first time the protective effect of curcumin against lead-induced neurotoxicity in rats by showing significant improvement in the levels of various biomarkers of oxidative stress (GSH level, SOD and CAT activity) in different regions of the brain. Daniel et al. (2004) provided insight into the chelating properties of curcumin by showing a remarkable reduction in levels of lead in rat brains. The above-mentioned results on curative effects of curcumin on lead neurotoxicity were also successfully reproduced by Dairam and co-workers (2007) in male rats. In spite of these commendable properties, the major drawback associated with the use of curcumin is its low bioavailability. This is due to its poor aqueous dispersion and poor absorption from the intestine coupled with a high degree of metabolism of curcumin in the liver and rapid elimination in bile (Maiti et al., 2007).
Ameliorative Effects against Genotoxicity and Free Radicals Abnormality by Curcumin against Lead:
Turmeric is a routinely used Indian spice with a powerful medicinal compound called curcumin. Many researchers had found that curcumin made a remarkable impact on the various systems against many adverse effects. So, to evaluate the possible role of curcumin against lead induced genotoxicity, the curcumin was used as an antidote in present study. curcumin, had been studied in a wide range against many adverse effects in vitro and in vivo in various biological systems and models. Curcumin can significantly re-establish mitotic indices to normal as evidenced by elevated index of cell proliferation (CCPI), cytokinesis block proliferative index (CBPI) along with decreased Average Generation Time (AGT) and Population Doubling Time (PDT) in curcumin co-supplemented lead treated cultures in present study. Similar results were also found by Shah and co-workers in 2016. Kao and co-workers (2004) also observed elevated cell survival related with pretreatment of curcumin prior to H2O2 exposure. Curcumin can give protection against chemical carcinogenesis (Iqbal et al., 2003) and it can be the shield against chromosomal aberrations and fragments made by radiations (Thresiamma et al., 1998). Also in our study, supplementation of curcumin along with lead acetate significantly reduced the frequency of SCEs, Chromosomal Aberrations, Associations and Micronuclei (CBMN). Jayaprakasha and co-workers (2002) describe that the ameliorative properties of turmeric-oil and its elements may offer an explanation for their antimutagenic action. Among several reasons which make the curcumin a potent antioxidant; one is its content of polyphenolics, which encourage beneficial effects against ROS (Okuda and Hatano 1993; Qureshi et al., 1993). Moreover, possibility of chelation of lead by curcumin cannot be ruled out because the results obtained by Daniel and co-workers(2004) showed that there may be an interaction between curcumin and lead, with the probable creation of a composite among the metal and curcumin which may be ultimately responsible to decrease the adverse effects of lead.
The presence of sites of conjugation with bioconjugates containing glycine, alanine, and/or piperic acid in curcumin makes it more hydrophobic. As a result, curcumin get localized in the lipid bilayer membrane. Curcumin, being lipid soluble, reacts with the lipid peroxyl radicals and acts as a chain terminating antioxidant (Omayma et al., 2011). Curcumin has also been known to inhibit lead induced lipid peroxidation in rat liver (Grdina et al., 2002) as it possesses distinct structural motifs that are responsible for its antioxidant activity. The presence of electron donating groups like phenolic hydroxyl groups and a diketone structure in curcumin, it is responsible for the free radical scavenging activity and inhibiting lipid peroxidation (Jagetia and Rajanikant 2004; Jagetia and Reddy 2005; Nariya et al., 2017). Treatment of curcumin along with lead acetate on lymphocytes in the present study also resulted in decreased lipid peroxidation and improved antioxidant status, may be due to the antioxidant properties of curcumin and hence it prevents the damage to the lymphocytes against adverse effects of free radicals. Moreover, co-treatment of curcumin in lead treated lymphocytes in our study protect GSH depletion and restore the GSH content in the cells. Natarajan and co-workers (1996) suggest that curcumin stimulates the glutamylcysteinyl synthase, the rate limiting enzyme involved in the glutathione biosynthesis. This may be the reason to restore GSH after curcumin co-treatment in this study. The induction of the phase II enzyme system is an important event of the cellular response during which a diverse array of electrophilic and oxidative toxicants can be eliminated or inactivated before they cause damage to cellular macromolecules (Rajendra Prasad et al., 2005). In support to our result Park and Flovd (1992) have shown that curcumin significantly enhance the synthesis of antioxidant phase II enzymes such as SOD, CAT, G-Px and G-Rd in rat liver. Dinkova-Kostava and co-workers (2001) had also reported that curcumin and several other structurally related polyphenolic compounds induce the activities of phase II detoxification enzymes, which appear to be crucial in protection against carcinogenesis and oxidative stress. Curcumin, being a hydrophobic molecule, passes easily through plasma membrane into the cytosol (Kalpana and Menon 2004; Polasa et al., 2004) and this nature of curcumin can be beneficial against lead toxicity inside the cell. The presence of curcumin in the cytosol directly scavenges the free radicals like superoxide anion, hydroxyl radical and lipid peroxyl radicals, etc. and results in the formation of phenoxyl radicals (Chendil et al., 2004) produced by the electron transfer followed by the proton loss at the phenolic OH site as the phenolic OH group is the weakest bond to break. The phenoxyl radicals of curcumin thus produced are stabilized over the extended conjugation (Khafif et al., 2005). Studies have also shown that curcumin and related compounds have the potential to protect DNA against oxidative damage induced by singlet oxygen (Shimizu and Weinstein 2005). These findings also support results of present study.
Results obtained from current study suggest that curcumin could be one of the important antidote to ameliorate the free radical toxic effects of lead, thus potentially reducing their genotoxicity and tissue damage.
Ameliorative Effects of S-Allylcysteine against Lead Toxicity:
Garlic is a medicinal plant that has been an inseparable part of Indian culinary for over 5000 years. Besides its use as a condiment, it is credited to have remarkable therapeutic and pharamcological properties. Its active agent is allicin, which imparts its characteristic odor as well as medicinal properties (Sharma et al., 2010). Garlic can prevent oxidative stress by chelating lead ions and scavenging free radicals. Senapati and co-workers (2001) reported the prophylactic efficacy of garlic extract in reducing the lead burden from soft tissues. In another study, Pourjafar and co-workers (2007) further confirmed the ability of garlic to reduce the lead burden from the liver, kidney, blood and bone. The protective efficacy of aqueous garlic extract was studied against lead induced hepatic injury in rats. The results clearly indicated the ameliorative ability of garlic against hepatic injury caused by lead generated oxidative stress (Kilikdar et al., 2011).
Ameliorative Effects against Genotoxicity and Free Radicals Abnormality by S-Allylcysteine against Lead:
Garlic and its compounds exert strong biological activities including anti-oxidative, anti-inflammatory and anti-cancer activities. S-Allylcysteine (SAC) is an active ingredient and most abundant organosulfur compound of aged garlic extract as well as a promising therapeutic agent at many experimental and clinical levels (Amagase et al., 2001). In this study, the role of SAC against lead induced toxicity in human lymphocytes were evaluated.
The results clearly indicate that SAC modulates lead induced genotoxicity in a dose dependent manner in human lymphocytes in vitro. SAC can elevate lymphocyte proliferation (Ebrahimi et al., 2013), which is also evident by elevated index of CCPI and CBPI along with decreased AGT and PDT in SAC co-supplemented lead treated cultures in the present study. Also, our results are comparable with the earlier published works where the SAC has showed a significant decrease in chromosomal anomalies (e.g. CAs, CBMN, etc.) induced by various chemicals and drugs (Siddique and Afzal, 2005a, b; Yadav et al., 2006). This protection is presumed to arise from several mechanisms including quenching of free radicals and enhanced DNA repair (Milner, 2001). Also in this study, supplementation of SAC along with lead acetate significantly reduced the frequency of SCEs and CAs as well as Micronuclei which was in accordance to the previous study of Sowjanya and co-workers (2009). The efficiency of garlic is due to the presence of sulfur-containing amino acids and compounds having free carboxyl (C=O) and amino (NH2) groups in their structures, which act as anti-lead active substances (Borek, 2001; Chung, 2006).
The oxidative stress caused by lead was ameliorated using SAC by maintaining various enzymatic and non-enzymatic moieties in its functional forms. Supplementation of SAC along with lead acetate in cultured human lymphocytes has demonstrated an inhibition of LPO formation as well as normalized antioxidant parameters (GSH, GPx, GR, GST, SOD, Catalase) and also maintained protein level. Effects were based on several evidences which demonstrating antioxidant properties of SAC, such as its well-known capability to scavenge ROS (Kemper, 2000), to prevent LPO (Numagami and Ohnishi, 2001), and to block NFkB activation (Ide and Lau, 2001), a pro-apoptotic factor which is in accordance with this study.
In the present study, we found an increase in the levels of LPO due to lead induced toxicity but SAC readily stops LPO formation supporting its antioxidant activity. Other researchers also showed that SAC inhibit the lipid peroxidation in in vivo models (Numagami and Ohnishi, 2001; Sundaresan and Subramanian, 2003; Becerril-Chávez et al., 2017; Baluchnejadmojarad et al., 2017) which support the findings of this investigation. SAC contains a thiol group responsible for its antioxidant capacity because this nucleophile can easily donate its proton to an electrophilic species, thereby neutralizing them or making them less reactive (Colín-González et al., 2012). Glutathione is a metabolic regulator and putative indicator of health. GSH is the master antioxidant and protect cells from the harmful effects of free radicals. It also functions as a free radical scavenger and involved in the repair of biological damage caused by free radicals (Jones, 2008). SAC treatment in the present study showed a significant renovation in GSH content. SAC has also attenuated GSH level in various diseases and against various toxicants (Zarezadeh et al., 2017; Kattaia et al., 2017; El-Beih et al., 2017). Glutathione is a substrate for GPx, which plays a predominant role in removing excess free radicals and hydroperoxides. GPx plays a central role in the catabolism of H2O2 and the detoxification of endogenous metabolic peroxides and hydroperoxides which catalyzes GSH. The decreased activity of GPx after lead acetate treatment in this study might be due to the lowered level of GSH. A marked increase in GPx activity was observed in lead intoxicated lymphocytes when lead acetate co-supplemented with SAC and hence it was postulated that SAC reduced inactivation of GPx imposed by lead acetate. Mandal and co-workers (2012) had found the same effect of SAC against erythroid lead toxicity. SAC treatment also maintains the activity of GR during lead acetate toxicity in lymphocyte which enables the cell to sustain adequate levels of cellular GSH. Moreover, GST is an enzyme that catalyze the conjugation reaction of electrophilic hydrophobic compounds with GSH. SAC has remarkably restored GR and GST level to the normal against lead induced toxicity in the present study.
SAC was found scavenger of superoxide anion by increasing activity of SOD as observed in the present study. Consistently, Various scientists observed a protective effect of aged garlic extract against various adverse effect by affecting H2O2 or the O2•− generator system xantine/xanthine oxidase (Borek, 2001). Catalase reduces H2O2 production by dismutation reaction and prevents generation of hydroxyl radicals. The present study revealed that Catalase activity were significantly increased by SAC which was inhibited by lead acetate in cultured lymphocytes of human. In accordance with these findings the study of Sharma and co-workers (2010) had revealed the beneficial effects of Allium sativum extract and found increased activity of SOD and Catalase in mice.
