Probit analysis of comparative assays on toxicities of lead chloride and lead acetate to in vitro cultured human umbilical cord blood lymphocytes

This work describes that cytotoxicity of lead chloride and lead acetate to in vitro cultured lymphocytes from human umbilical cord blood, using four monitoring methods namely, trypan blue staining, acridine orange/ethidium bromide staining, 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide (MTT) and neutral red uptake assays; lead genotoxicity to lymphocytes was monitored by comet assay. The MIC value in each method was invariably 300 mg/L for PbCl2. Lethal concentration25 (LC25) values were almost in an agreeable range: 691.83 to 831.76 mg/L; LC50 values in each method were almost in the range: 1174.9 to 1348.9 mg/L; LC100 values were in the range: 3000 to 3300 mg/L, for lead chloride. Similarly, The MIC value in each method were invariably 150 mg/L; LC25 values were almost in the range: 295.12 to 371.53 mg/L; LC50 values were in the range: 501.18 to 588.84 mg/L; LC100 value was 1500 mg/L in all assays, for lead acetate. The comet assay also indicated that the LC100 values were 3300 mg/L lead chloride and 1500 mg/L lead acetate. Thus, both cytotoxicity and genotoxicity were recorded at 3300 mg/L lead chloride and 1500 mg/L lead acetate with lymphocytes.

Moreover, leaching of water soluble Pb compounds in soil in the rainy season causes the Pb-sorption by biota (Sharma et al., 2010, Moussa et al., 2008, Campbell et al., 2004. From studies on lead toxicity with Wistar rats by injecting lead acetate solution at 15 mg/kg body weight, a significant decrease in the level of superoxide dismutase activity and increase in plasma bilirubin level were recorded (Berrahal et al., 2007, Rogers et al., 2003. A report in rat system showed that the ingestion of Pb induced stimulation in glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase activity; the cholinesterase activity was inhibited, while hyperglycemia was induced in lead acetate toxicity; in blood, metallic Pb reduced hemoglobin contents and red blood cell (RBC) count as well as the plasma levels of T 3 , T 4 and white blood cell count had decreased (Ewis et al., 2012). In Wistar rats the relative retention of lead acetate by the issues was determined as follows: lungs>spleen>stomach>kidney>

Introduction
Without any role in metabolism, lead or Plumbum (Pb, Group 2b) is a toxic heavy metal. Effluents from industries dealing with paper, food canning, battery and cosmetics as well as mining industry, oil refineries and coal mine establishments mainly add Pb compounds to the total environment. Of all Pb compounds produced in the United States, for example, a 4% (80,000 tons/year) approximately is made into bullets; consequently, 58,300 tons/year Pb approximately from shot and munitions get deposited onto landscape through shooting activities (USEPA, 2001). Pb is present in environments in soluble, PbCl 2 , CH 3 COOPb (lead acetate) and PbCO 3 , as well as insoluble PbS, Pb 3 (PO 4 ) 2 , PbO and PbSO 4 forms, apart from spillage as ore powders from establishments dealing with Pb-minerals, hydrocerussite, cerussite and massicot. exposure to lead acetate reported to cause adverse effects on neurons of zebrafish embryos (Zhang et al., 2012).
A variety of toxic effects caused by Pb compounds had been identified during gestation and lactation in animals and humans (Bunn et al., 2001). Lead exposure was demonstrated to occur both through respiratory and gastro-intestinal tracts (Park et al., 2008). About a 60% of ingested lead was reported to be absorbed by empty stomach, but along with food a lesser amount (15-20%) was absorbed. Obviously, blood is the inadvertent route of the metal mobility to brain, liver, bone marrow and testis. Furthermore, child-neuro development and adult brain cells were recorded to be affected by lead toxicity (Bellinger, 2008). It was reported that because of the exposure of the mother to pollutants, the fetus in womb was affected by lead poisoning with the eventual impairment of the natural development of immune system in the postnatal life (Bishoyi & Sengupta, 2006). Ultimately, Pb gets stored in bone inducing decrease in bone mineral density (Lee et al., 2010). Monitoring of levels of renal thioredoxin reductase-1 activity in rats exposed to 25 mg/kg lead acetate was recorded to induce renal damage (Conterato et al., 2014). And it was demonstrated that lead acetate at 1 and 1.5 g/L caused male sterility in rats in a dose dependent manner (Wang et al., 2013). Increase of low density lipoprotein cholesterol, atherogenetic index and coronary heart disease index levels with exposure to lead acetate in broiler chicken were, 45.24%, 100%, 16.66% compared to the control group, in a study (Karimi et al., 2013).
Apart from gastro-intestinal disturbances in humans, lead toxicity causes restlessness, irritability, headache, hepatic and renal damage, hypertension, hallucination and encephalopathy; in blood basophilic stippling and decreased hemoglobin synthesis occur due to Pb (Anonymous, 2007). Age related impairments in behavioral functions had been linked to lead poisoning that caused changes in neurotransmitter levels in brain tissue (Chand et al., 2014). Lead induced biochemical and structural changes occur in liver causing cataclysmic alterations by oxidative stress, apoptosis and mitogen activated protein kinase (Mujaibel & Kilarkaje, 2013, Samaraghandian et al., 2013. Particularly, oxidative stress had been reported to be one of several important possible mechanisms in lead toxicity (Shalan et al., 2005) with the generation of hydroxyl radical, hydrogen peroxide, superoxide anion and lipid peroxidase, etc. (Soltaninejad et al., 2003).
A single report on the use of human umbilical cord mesenchymal stem cells had been recorded monitoring lead trioxide toxicity at <10 mM; lead acetate too had toxic effect on the self-renewal, multipotent differentiation potential and hematopoiesis-promoting function of mesenchymal stem cells from umbilical cord blood (UCB) (Sun et al., 2012). Pb had been reported to have toxicity to hematopoietic system by interfering with hemoglobin synthesis and erythrocyte morphology. In this study, cell count, 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide (MTT) assay, apoptosis assay, osteogenic differentiations were recorded to be affected by lead trioxide. Alkaline phosphatase enzyme was lower than control group (Sun et al., 2012). DNA damage by lead acetate exposure was reported in the rabbit model at 15 mg/kg; infraction of the kidneys and intranuclear specific inclusion bodies in liver and kidney were detected (Ahmed et al., 2012).
This work describes toxicity of PbCl 2 and lead acetate to in vitro cultured lymphocytes from human UCB. Cyto-toxicity was monitored using trypan blue (TB) and acridine orange/ethidium bromide (AO/EB) staining, MTT and neutral red uptake (NRU) assays; genotoxicity was monitored by comet assay. Cord blood being a waste blood, its use in experiment in 'predictive toxicity', such as herein, should not warrant ethical issues. Predictive toxicology is an offshoot of pharmacology describing concepts related to toxic effects of newer chemicals and prospective drugs in model systems to predict health-risk assessments, before institutional recommendation as a drug and other uses. The underlying method could well be adopted for environmental toxicants, for planning well-being of human health. Differentiated cells obtained from a mass of lymphocytes could be able to mimic some actions related to chemical kinetics operative naturally in metabolism as a response to the chemical. Thus, this would help examine the probable role of some toxic chemicals in human system. Lymphocytes help the defense mechanism of the body against infectious agents, due to their ability to distinguish the body's own cells from the foreign ones.

Collection of lymphocytes
With an aliquot of 100 or 250 μL 1,000 IU heparin (HiMedia, Mumbai), in a sterile 15 or 50 mL size falcon tube (Tarson, Kolkata), the UCB sample was collected according to volume, immediately after the delivery of an infant and was stored at 4 °C till use. Lymphocytes were isolated immediately or within at the best 24 hrs after the collection of UCB. For the isolation of lymphocytes, the collected UCB sample was diluted with phosphate buffered saline (PBS) solution in 1:1 proportion. The  mixture was loaded carefully into a centrifuge tube for over-layering on Histopaque (Sigma, Mumbai) for the separation of lymphocytes, which was one-third the total volume of the mixture. The mixture was centrifuged at 1,800 rpm for 25 min at 22-24 °C; and four heavy to light layers, RBC, Histopaque, buffy coat and plasma were seen ( Figure 1). Mononuclear cells in the buffy coat layer were taken out carefully from the tube. To the separated cells, another aliquot of PBS in the 1:1 ratio was added, mixed gently and re-centrifuged at 2,000 rpm for 5 min. The pellet of lymphocytes was taken for culturing and subsequently cell counts were done using a haemocytometer.

Growth of lymphocytes
After separation, UCB-derived lymphocytes were diluted to the density of 1×10 6 cells/mL with a required volume of Dulbecco's modified Eagle's medium (DME medium-lowglucose, HiMedia, Mumbai), and were loaded into a 6-well culture plate (Tarson); DME medium contained 15% fetal bovine serum (Sigma), 1% penicillin-streptomycin and 1% sodium pyruvate, along with graded concentrations of PbCl 2 or lead acetate solution for growth. The stock solution was prepared by dissolving 1000 mg of PbCl 2 or lead acetate (Sigma) in aliquots of 100 mL of triple distilled water for the final concentration of 10,000 mg/L, and the stock solution was stored at 4 º C, for further use. The volume of 2 mL in total mixture was maintained in each well of the culture plate; the cells were incubated with different concentrations of PbCl 2 (0, 300, 600, 900, 1200, 1500, 1800, 2100, 2400, 2700, 3000 and 3300 mg/L), or lead acetate (0, 150, 300, 450, 600, 750, 900, 1050, 1200, 1350 and 1500 mg/L) in an incubator at 37 °C in 5% atmospheric CO 2 concentration for 24 hrs for growth ( Figure 2).

Assessment of cyto-and genotoxicity
The viability of lymphocytes grown in the presence of PbCl 2 and lead acetate was assessed using two staining procedures, with TB and AO/EB, using a phase-contrast (Magnus, New Delhi) and fluorescent microscope (Magnus), respectively. MTT assay and NRU assay were also carried out as the standard procedures for monitoring the live cell density. Comet assay was done for assessing Pb-induced nuclear damages.

TB staining
TB solution was prepared in PBS at the concentration of 0.4 g/mL. For the study of cell viability to the in vitro grown mass of lymphocytes, TB solution was added at the 1:1 ratio; the mixture was kept in an incubator for 2 min at 37 °C and cells were observed under the phase-contrast microscope at the 400× magnification. The live cells remained unstained, whereas the nuclei of the dead cells were blue in appearance, as TB enters dead cells and stains the nuclei to blue, as it is a membrane permeable dye.

AO/EB staining
The AO/EB solution was prepared in PBS at the concentration of 100 μg/mL and is applied to cultured lymphocytes after a 24 hrs of incubation in presence of graded concentrations of any of the two Pb compounds. When observed under the fluorescent microscope at 400×, green colour indicated live cells, whereas cells with orange and red colour were apoptotic and necrotic cells, respectively ( Figure 3). AO is taken by both live and dead cells and emits green fluorescence. EB is only taken up by cells when the integrity cytoplasmic membrane is lost and stains nucleus orange. Hence live cells, apoptotic cells and necrotic cells were green, orange and red in appearance respectively. Toxicity values were obtained with different concentrations of PbCl 2 and lead acetate (Figure 4). Percent lethality (PL) values of the third repeated experiment were transformed to probit values by Finney's method, which were potted against corresponding log 10 values of PbCl 2 and lead acetate concentrations, as exemplified before (Rath et

MTT assay
The MTT stock solution was prepared at the concentration of 5 mg/mL in PBS. After 24 hrs of toxin treatment in 6-well culture plate, 80 μL of MTT stock solution was added to each well to study the toxicity effect. The plate was kept in an incubator (37 °C, 5% CO 2 ) for 4 hrs and it was found that media containing cells and toxin got converted to blue colour after incubation with MTT. The mixture was gently centrifuged at 1,000 rpm for 10 min at 22 °C. The supernatant was removed and the pellet was dissolved in 1 mL aliquot of 100% dimethyl sulfoxide (DMSO) and the suspension was kept in an incubator for 1 hr. Optical density 570 of the suspension with the purple colour was measured with a spectrophotometer. Percentage of cell density was calculated as follows: 100 × (OD sample -OD blank ) / OD control MTT in DMSO solution was taken as the blank. Probits of observed lethality percentage values and log 10 concentration values of chemical were used for analysis of toxicity.

NRU assay
Neutral red solution was prepared in serum-free DME medium at the concentration of 100 μg/mL. After 24 hrs growth of lymphocytes in the presence of a toxin in a 6-well culture plate, an aliquot of 200 μL of neutral red dye solution was added to each well. The plate was kept in an incubator (37 °C, 5% CO 2 ) for 3 hrs. Indeed, lysosomes of viable cells absorb the dye and the dead cells remain unstained. Thereafter, an aliquot of 1 mL of separately prepared neutral red desorption solution (1% acetic acid, 50% ethanol and 49% distilled water) was added to each well for removal the dye from lysosomes of live cells. Optical density 540 of the washout with a spectrophotometer was measured that assessed live cell density whose percentage values were calculated.

Comet assay
Single cell gel electrophoresis was carried out to study DNA damage of the cells treated with different concentrations of PbCl 2 and lead acetate. Lymphocytes cultured with different concentrations of a Pb-chemical were harvested and used in the alkaline comet assay technique. Slides were coated with 1% agarose and were allowed for air drying. Pellets of lymphocytes, obtained by centrifugation of cultured cells were washed with PBS, and the pellet was mixed with three times the cell volume of the pellet with low melting point agarose (LMPA) 1% in sol state. The mixture of cells and LMPA sol was placed over the agarose coated slide that was kept at 4 °C for 10 min until the slide got dry. The dried slides were submerged into a pre-cooled lysing solution of the mixture of 0.4 M Tris HCl, 0.8 M dithiothreiotl (DTT) and 1% sodium dodecyl sulphate (SDS) of pH 7.5 and the mixture was kept at 4 °C in dark for about 30 min. The slides were subsequently removed and placed in another lysing solution with 0.4 M Tris HCl, 2 M NaCl, 1% SDS, 0.05 M EDTA at pH 7.5 for 30 min. The slides were placed in tris borate EDTA (TBE) buffer, which contained 89 mM Tris, 89 mM Boric acid and 2.5 mM EDTA at pH 8.3 for 10 min. The slides were transferred to a horizontal gel electrophoretic chamber with TBE buffer. Electrophoresis was carried out at 20 V and 12 mA for 12.5 min. After the electrophoresis, the slides were washed with 0.9% NaCl for 2.5 min, and the   second electrophoresis was carried out at 20 V, 12 mA for 4 min in alkaline solution, which contained, 0.03 M NaOH and 1 M NaCl. The slides were placed in a neutralizing solution with 0.4 M Tris HCl for 5 min, and again the slides were washed in TBE buffer. After 5 min, the slides were stained with EB solution and were observed under the fluorescence microscope at 400×, for scoring the comets. Probits of observed lethality percentage values calculated from percent values of observed comets due to PbCl 2 or lead acetate treatment; probits analyses of toxicity data were done.

Results
Percent lethality (PL) values recorded from data sets of tests with TB, AO/EB, MTT and NRU assays were transformed to their probits were used to construct respective plots against Pb concentrations in log scale ( Figure 3) Figure 5, Table 4).

AO/EB staining
Treatment of cells with different concentrations of PbCl 2 for 24 hr resulted in a decreasing pattern of living cell counts ( Figure 6). The number of dead cells gradually increased from the level of 300 to 2700 mg/L level, and it was found that there were no cells at the 3000 mg/L level. In case of lead acetate, the decreasing pattern of live cell count started from 150 to 1350 mg/L level, whereas   Figure 5, Table 4).

MTT assay
The cell density at OD 570 gradually decreased from the level of 300 mg/L to 3000 mg/L and it was found that there were no cells at the 3300 mg/L PbCl 2 level. Experimentally, the MIC value was 300 mg/L, whereas the HPC was 3000 mg/L, and the LC 100 value was 3300 mg/L PbCl 2 .   there were no cells at 1500 mg/L. Experimentally, the lethality was seen at 300 mg/L, which was recorded as the MIC value, the HPC was 2700 mg/L, and the LC 100 value was 3000 mg/L for PbCl 2 ; and for lead acetate the MIC,  Figure 5, Table 4).

NRU assay
Cell density, monitored at OD 540 gradually decreased from the level of 300 to 3300 mg/L PbCl 2 . Experimentally, the MIC value was 300 mg/L; whereas the HPC was 3000 and the LC 100 was 3300 mg/L PbCl 2 . Log 10 values of PbCl 2 concentrations extrapolated from the probit plot yielded log 10 values 2.85, 3.10 and 3.30, for values of LC 25, LC 50 , and LC 75 , respectively (Figure 4,  Figure 5, Table 4).

Comet assay
It was observed that, comet tail was found increasing in cells treated with 300 mg/L to 3000 mg/L PbCl 2 and 150 to 1500 mg/L for lead acetate (Figure 7); DNA fragmentation index (DFI) too increased with increasing gradations of both lead chemicals (Tables 1 and 3 Figure 5, Table 4).

Discussion
This work demonstrated that four methods of monitoring cytotoxicity of lymphocytes, namely TB staining,  It was recorded that each assay method monitored for cytotoxicity were in same range monitored for a chemical; but, separate ranges for each chemical were noted. Cytotoxicity and genotoxicity values due to each chemical were in the same range for a chemical. Many toxicology test systems are undependable in animal systems as the exemplary mouse or rabbit or guinea pig systems are not true representative of a human body. Therefore, there are large gaps in the mirror of toxicity models with animals (Tzimas 1994); nonetheless, all popular animal models are mammals. Further, the requirement of a high number of animals in toxicology are so much for chemicals like 30,000 or more, which discourages the use of animals in assay systems (Gilbert 2010). Therefore the use of pluripotent stem cells (PSCs) and related cell lines in toxicology had been well-recognized (Rosler et al., 2004). It is consensus that, the animal system has a holistic approach in toxicity studies that is unavailable in cell cultures -a fact that supports the use of whole animal models.
However, animals tests can never be adequately standardized like human cellular systems, as the experiment should have growing cells under controlled conditions -in vitro human cell lines. In fact, the use of PSCs and their derivatives in toxicology has been well recognized (reviewed by Laustriat et al., 2010). PSCs have unique capacity of self-renewal in differentiation with advantages over somatic cells and those could be grown in vitro, as permanent cell lines. Moreover, surplus embryos from an in vitro fertilization unit could lend to the initiation of individual cell lines (Loser et al., 2010), during toxicity testing. Reprogramming murine fibroblasts by viral transfer of 4 genes, associated with pluripotency could be induced, thus PSCs could be created (Takahashi & Yamanaka, 2006). These workers could demonstrate that these specialized somatic cells could be reversed in PSCs in vitro.
The use of induced pluripotent stem cells (iPSCs) is a reliable technology for the development of different cell lines. The use of iPSC along with bio-monitoring data could enable a proper understanding of environmental metal/chemical toxicity. Thus, the iPSC technology has a tremendous potential for the development of predictive toxicology. Developmental toxicity has slowly become an important area of future assessment (Laustriat et al., 2010). Indeed, we are unaware of rising frequencies of many ailments, for example, cancer, autism, changes in pubertal timings are the areas not clearly known, with problem from environmental pollution, for example. In fact, if the toxicity pathways that induce DNA damage and affect DNA repair and other associated genetic process, predictive toxicology would bring new ideas in cancer mechanism due to chemicals of environmental concerns that could aid in the identification and classification of carcinogens (Grimsrud & Andersen, 2010). By the by, toxic effect on immune pathways could be elucidated (Guzik et al., 2001). Genotoxicity testing of 131-radioiodine a drug used for treatment of patients with thyroid diseases with cultured human lymphocytes was recently described (Hosseinimehr et al., 2013), as a work in predictive toxicology.
It has been consensus that hematopoietic /progenitor cells are more in UCB compared to bone marrow blood, as the later is involved in adults. Thus, this toxicity work using cord blood lymphocytes overrides the use of bone marrow stem cells in studies with predictive toxicology. In a study with human UCB, it was estimated that contents of nucleolar cells, granulocyte macrophage, erythroid and multipotent progenitor cells are mainly viable for at least three days under 4 °C or 21 °C (Broxmeyer et al., 1989). This study involves storage of the cells at 4 °C and further work at 22 °C. Thus, essential constituents of cord blood relating to stem cell would not be destroyed during the period of the study. Moreover, microarray technology involving environmental toxicants for understanding the underlying toxicity has been defined; to identify toxicant with specific genetic markers, such studies with heavy metals have been initiated (Bartosiewicz et al., 2001). Indeed, metals cause oxidative stress in cells, which is well studied in microarray system.

Conclusion
Lead acetate with the LC 100 value around 1500 mg/L was more toxic than lead chloride with the LC 100 value around 3300 mg/L to human lymphocytes. LC 50 values obtained by probit analyses were accordingly different for each chemical. Cyto-toxicity and Geno-toxicity values due to each chemical were in the same range for a chemical.