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Keywords:

  • pesticide;
  • bone marrow;
  • toxicity stem cell;
  • stromal cell;
  • aplastic anemia

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Long-term exposure of agriculturally used organochloride and organophosphate pesticides have been shown to cause long-lasting hematotoxicity and increased incidence of aplastic anemia in humans. The mechanisms involved in pesticide induced hematotoxicity and the features of toxicity that may play a major role in bone marrow suppression are not known. The aim of the present study was to investigate the hematological consequences of pesticide exposure in swiss albino mice exposed to aqueous mixture of common agriculturally used pesticides for 6 h/day, 5 days/week for 13 weeks. After the end of last exposure, without a recovery period, the strong hematotoxic effect of pesticide was assessed in mice with long-term bone marrow explant culture (LTBMC-Ex) system and cell colony forming assays. Bone marrow explant culture from the pesticide exposed group of mice failed to generate a supportive stromal matrix and did not produce adequate number of hematopoietic cells and found to contain largely the adipogenic precursors. The decreased cell colony numbers in the pesticide exposed group indicated defective maturational and functional status of different marrow cell lineages. As a whole, exposure of mice to the mixture of pesticides reduced the total number of bone marrow cells (granulocytes are the major targets of pesticide toxicity), hematopoietic, and non-hematopoietic progenitor cells and most of the hematological parameters. Replication of primitive stem/progenitor cells in the marrow was decreased following pesticide exposure with G0/G1-phase arrest of most of the cells. The progenitor cells showed decreased percentage of cells in S/G2/M-phase. The increased apoptosis profile of the marrow progenitors (Increased CD95 expression) and primitive stem cells (High Annexin-V positivity on Sca1+ cells) with an elevated intracellular cleaved caspase-3 level on the Sca1+ bone marrow cells provided the base necessary for explaining the deranged bone marrow microenvironmental structure which was evident from scanning electron micrographs. These results clearly indicate a strong, long lasting toxic effect of pesticides on the bone marrow microenvironment and different microenvironmental components which ultimately leads to the formation of a degenerative disease like aplastic anemia. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 84–97, 2014.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Pesticides are chemicals designed to kill a variety of pests, such as weed, insects, rodents, and fungi (Ramade, 1987; Kamsin, 1997). They can be characterized on the basis of their function as insecticidal, herbicidal, rodenticidal, and fungicidal and also on the basis of their chemical nature, i.e., organophosphates and organochlorides, S-triazines, and pyrethroids (Blondell, 1990; Bloom, 1993). Potentially hazardous environmental toxicants like pesticides display a broad spectrum of biological effects, being toxic not only to target organisms but also to humans. Chronic exposure to pesticide is known to lead toward progressive degeneration of the bone marrow (Aksoy, 1989; Whitney et al., 1995; Jamil et al., 2005; Law et al., 2006). The occupational exposure limits are presently based on the risk of aplastic anemia. The clinical evidences indicated pesticides as a primary inducer of the disease aplastic anemia (Broughton et al., 1990; Pasqualetti et al., 1991; Vojdani et al., 1992; Bhatia and Kaur, 1993). Pesticide, derivatives of bi-phenyl aromatic hydrocarbons, can produce toxic metabolites like phenols, catechols, hydroquinones, and several other polychlorinated biphenyl compounds that can accumulate within the bone marrow and increase the cellular toxicity (Rickert et al., 1979; Casida et al., 1983; Gassner et al., 1997; El-Gohary et al., 1999). The pesticides have shown to be hematotoxic even at lower concentrations for long-term exposure. Although the mechanisms of pesticide associated hematotoxicity remain unclear, the apparent selectivity of pesticide toxicity for hematopoietic tissue may be connected with its capacity to get accumulated by bone marrow several times greater than the other non-hematopoietic tissues (Andrews et al., 1979; Longacre et al., 1981; Abraham et al., 1985; Rinsky et al., 1987).

Pesticide induced cell damage has been demonstrated in many in-vitro and in-vivo studies. The investigation of the mechanism of pesticide toxicity has often focused on pesticide induced inhibition of cell replication (Fleming and Timmeny, 1993; Noble and Sina, 1993). Data on isolated parameters are difficult to use for identification of target cells of pesticide toxicity, as the bone marrow is very complex having close association and interdependence of various cell types in the microenvironment (Molineux et al., 1986; Testa et al., 1988; Zipori, 1989). Depression of growth factor production by cells of hematopoietic microenvironment and a variety of cellular disturbance throughout the hematopoietic system could be attributed to pesticide-induced toxicity. Bone marrow explants culture represents a near physiologic system for assessing the micro environmental defects and growth characteristics of the bone marrow cells in-vitro (Dexter et al., 1977; Harigaya et al., 1981; Weiss and Sakai, 1984). The proliferation and differentiation of hematopoietic stem/progenitor cells are dependent on the intimate contact and inter-dependence of the other matured hematopoietic cells and stromal cells. The stromal cells (essential matrix component) have found to play the pivotal role in maintaining normal hematopoiesis and healthy cell generation (Wolf, 1979; Lichtman, 1981; Hotta et al., 1985). No systematic study has yet been carried out to assess the long-term effects of pesticide toxicity on stromal cells regarding the bone marrow growth and cellular kinetics in ex-vivo culture system. Also the direct effects of pesticides on pluripotent stem and stromal progenitor cells have not been well characterized and documented.

In this present study, we have developed a murine model of pesticide-induced aplastic anemia to study the toxic effect of pesticide on bone marrow physiology and its various cellular components. The study focused on four major areas that include the effect of pesticide toxicity on the (1) in-vitro cellular generation from long-term bone marrow explant culture, (2) bone marrow primitive stem and stromal progenitor cell population and the cell cycle status of the stem/progenitor cells, (3) apoptosis profile of the bone marrow cells, and (4) bone marrow microenvironmental structure. We have used long-term bone marrow explant (LTBMC-Ex) culture system and colony forming assays to assess the maturational and functional defect of the bone marrow cell lineages. Phenotypic characterization of primitive marrow hematopoietic cells (Sca1+c-kit+, CD150+CD244−, and Tie2+) and non hematopoietic progenitor cells (Sca1+ CD44+, CD146+) (Nadri et al., 2007) were done to denote the receptor expression pattern in the pesticide exposed group of animals. The cell cycle status and expression of apoptotic markers were also analyzed to indicate the cell proliferation index and the degree of apoptosis in the bone marrow derived cells under the event of pesticide toxicity. Finally, the scanning electron microscopy (SEM) was used to demonstrate the associated changes in the bone marrow microenvironmental structure, if any, following pesticide exposure. As a whole, the above mentioned study was designed to unearth the cellular mechanisms involved in pesticide-induced toxicity as well as the pesticide-induced hematotoxicity that ultimately leads to a degenerative disease like aplastic anemia.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Animals

Inbred Swiss albino mice (6–8 weeks of age) were exposed to a mixture of commonly used agricultural pesticides that mainly included Alphamethrin, Cypermethrin, Profenofos, and Chloropyrofos. All the animals (n = 50) were routinely exposed to the aqueous mixture of pesticides [10% (w/v) Alphamethrin, 9% (w/v) Cypermethrin, 40% (w/v) Profenofos, and 50% (w/v) Chloropyrofos] for 6 h/day and 5 days/week up to 13 weeks (Chatterjee et al., 2010). Although, the procedure caused death of approximately 10% of the animals due to acute toxicity within the first week, others showed systematic adaptation to the condition. An initial drastic loss of body weight was compensated in the late phase. Initiation of the disease progression was routinely monitored through peripheral blood parameters study. In comparison with the experimental group, a set of normal control group (n = 50) received inhalation and dermal exposure of only aqueous solution without pesticide contamination. Both the groups of animals were left on normal diet and water ad libitum.

Hematological Parameters

Approximately 300 μL of blood sample was collected from each mice tail vein in a heparinized vial after the last day of exposure (i.e., after 13 weeks) to evaluate the hematological parameters. The hemoglobin concentration, white blood cell (WBC) count, red blood cell (RBC) count, platelets, reticulocytes, and neutrophil counts were determined using standard laboratory techniques.

Bone Marrow Culture

Long bones (femur, tibia, and fibula) isolated from both the experimental and normal control groups of mice were cut in both ends by scissors and the red-pulp region of the marrow was taken out by repeated flushing with RPMI-1640 media supplemented with 10% fetal bovine serum (FBS). Small 0.2 mm3 fragments of bone marrow explants were subjected to culture in triplicate (for each group) in 60-mm culture dish containing 4 mL of RPMI-1640 supplemented with 30% FBS. At different time course, the culture was monitored and cellular growth pattern was observed for both the normal and experimental group.

Leishman Staining of Bone Marrow

Bone marrow from both the normal and experimental groups of animals were stained with Leishman staining to observe the disease oriented changes of the bone marrow architecture under light microscopy (×400 magnification).

Adherent Cell Colony Forming Assay

Bone marrow cells were flushed from femurs and passed through a 100 mesh per inch stainless steel grid to produce a suspension of single cells. RBCs were lysed using RBC-depletion buffer (BD-Bioscience, USA). For the adherent cell colony formation, the bone marrow derived cells were suspended in RPMI-1640 media at a concentration of 4 × 106 cells/plate, supplemented with 30% FBS, 100 U/mL penicillin, 100 U/mL streptomycin, and 0.01% (v/v) mercaptoethanol. Four 35 mm × 10 mm petridishes each for the experimental and control groups were plated with 3 mL of media containing 4 × 106 cells and placed in a CO2 incubator at 37°C and 5% CO2. At every 72 h interval, the media was drained off and fresh media supplemented with 30% FBS and 0.01% (v/v) mercaptoethanol, added for the maintenance of the culture. After 8, 16, and 28, days the numbers of colonies were scored using an inverted microscope.

CFU-F Assay

Approximately 1–1.5 × 106 bone marrow derived cells were plated into a 75-mm petri-plate (Corning, USA) in triplicate in α-MEM with 30% FBS, L-glutamine, antibiotics (Streptomycin—100 U/mL and penicillin—100 U/mL), and 0.01% (v/v) 2-mercaptoethanol. At every 3rd day the media was drained off from each of the petri-plate and new media was added. For 2 weeks, the cultures were maintained at 37°C and 5% CO2. The resulting colonies were then scored using an inverted microscope.

Hematopoietic Progenitor Cell Colony Forming Assay

Hematopoietic progenitor cells were assayed in methylcellulose-based semisolid cultures. The culture medium consisted of 1.2% methylcellulose, 30% FBS, 1% BSA, and 100 ng/mL recombinant mouse (rm) stem cell factor (SCF) (Becton-Dickenson, USA), 50 ng/mL rm interleukin-3 (IL-3) (Becton-Dickenson, USA), and 50 ng/mL rm granulocyte-macrophage colony stimulating factor (GM-CSF) (Becton-Dickenson, USA). RBC depleted bone marrow cells were plated at a final concentration of 5 × 105 cells/mL in triplicate and the cultures were incubated at 37°C in an atmosphere of 5% CO2 in air. After 16 days of culture, colonies were scored in the same dish using an inverted microscope. Hematopoietic colonies were classified as follows: CFU-E, erythroid colonies containing 25–50 hemoglobinized cells; BFU-E, erythroid colonies containing more than 50 hemoglobinized cells grouped in one or several clusters. Myeloid colonies comprised the identifiable subpopulations of pure granulocytic colonies (CFU-G) and mixed type of colonies containing both the granulocyte and macrophage (CFU-GM) and erythroid and myeloid cells (CFU-GEMM).

Cell Cycle Analysis

The cell cycle status of the isolated bone marrow cells were analyzed by DNA QC particle kit (BD-Biosciences) containing propidium iodide (PI). Briefly, after RBC-depletion using RBC depletion buffer provided within the kit, approximately, 1 × 106 bone marrow cells were washed with phosphate buffer saline (PBS) and then fixed with 50% methanol for 15 min at room temperature. Then the cells were suspended in PBS containing RNAse (1 mg/mL, 20 μL) (Sigma, USA) and PI (10 mg/mL, 2.5 μL) and incubated for 30 min at room temperature. They were analyzed (an analysis gate was set on forward scattering (FSC) vs. side scattering (SSC) dot plot that include mostly the progenitor cell population) with BD FACS Calibur (Becton-Dickenson, USA) using Cell Quest Pro and Mod Fit LT (Variety Sales House, USA) software.

Flowcytometric Analyses of Cell Surface Marker Expression

Flowcytometry was used to detect the expression of combination of markers that are associated with hematopoietic stem cells (HSCs) (Sca1+ c-kit+, CD150+CD244−, and Tie2+) and stromal progenitor cells (Sca1+ CD44+, and CD146+) under normal and diseased condition. Bone marrow cells were washed and fixed with 3% paraformaldehyde (PFA). Then separately, in three different tubes each containing 1 × 106 PFA fixed cells were treated, respectively, with mouse anti-Sca1 PE and anti-c-kit FITC, CD 150 PE and CD244 FITC, and TIE2 PE conjugated monoclonal antibodies (BD-Biosciences, USA). In two other tubes, containing 1 × 106 PFA fixed cells each were treated with anti mouse Sca1 PE and CD44 FITC in combination and with CD146, respectively, (BD-Biosciences, USA) conjugated mAbs. All the tubes were incubated in dark at 37°C for 30 min. Finally, the cells were washed in PBS and analyzed by BD FACS calibur (Becton-Dickenson), using cell quest pro software.

Cellular Apoptosis Study

Two different aspects of cellular apoptosis profile was monitored in two sets of experiment: (1) expression of Fas(R) (CD95) on bone marrow cells and (2) Annexin-v expression by Sca1+ bone marrow cells.

For the first experiment, briefly 1 × 106 PFA fixed cells were incubated with 2 μL of mouse anti CD95 PE conjugated mAb (BD-Biosciences, USA) in dark at 37°C for 30 min. Finally, the cells were washed and analyzed (during the analysis an analysis gate was set on FSC vs. SSC dot plot that include mostly the progenitor cell population) by BD FACS Calibur.

For the second experiment, approximately, 1 × 106 PFA fixed cells were incubated with 2 μL of mouse anti Sca1PE mAb and 5 μL of (diluted with binding buffer) of FITC labeled mouse anti annexin-v mAb (e-biosciences, San Diego, USA). Excess fluorescence was then washed off with PBS and analyzed by BD FACS Calibur.

Cleaved Caspase-3 Expression on Sca1+ Marrow Cells

For the analysis of intracellular cleaved caspase-3 expression on Sca1+ marrow cells, approximately 1 × 106 cells were fixed with 1.5% paraformaldehyde. Cells were incubated in fixative for 10 min at room temperature and pelleted. They were then permeabilized by resuspending with vigorous vortexing in 500 μL ice-cold methanol per 106 cells and incubated at 4°C for 15–20 min. Cells were then washed twice in staining media (PBS containing 1% BSA) and resuspended in staining media at concentration 1 × 106 cells per 100 μL. Then the cells were incubated with anti-mouse Sca1 PE and unconjugated rabbit anti-mouse cleaved caspase-3 primary mAb in combination (Cell Signaling Technologies, USA) for 30 min at room temperature followed by the addition of goat anti-rabbit secondary antibody conjugated with Alexa Fluor-488 (Invitrogen, USA) and incubated further for 30 min at room temperature. The cells were washed with 15 volumes of staining media and pelleted. Finally, the pellet was resuspended in 100 μL of staining media and analyzed using BD FACS calibur.

Sample Preparation and Scanning Electron Microscopy

A small portion of the intact marrow tissue from each of the normal control and experimental groups were macerated slowly in physiological manner without damaging the inner cell mass and kept in 2.8% gluteraldehyde overnight for fixation. To dry the tissue it was repeatedly passed through 30%, 50%, 70%, and 100% gradients of alcohol and finally critical point drying was done. Then after coating with gold (Au) in IB-2 ion coater, the coated samples went through SEM examination using S-5330 Hitachi SEM and the photographs were recorded.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Peripheral Blood

In order to ascertain the clinical status of the disease following pesticide-induced toxicity, hemogram of the peripheral blood including hemoglobin concentration, WBC neutrophils, platelets, and reticulocytes counts were determined following standard laboratory techniques. The results (Table 1) showed a moderate depression in the hemoglobin concentration with uniformly reduced corpuscular counts, of which reticulocyte counts were significantly (p < 0.001) low in pesticide exposed groups (0.3%) compared to the normal (0.89%). The total WBC and neutrophil counts were also found to be significantly (p < 0.001) low in the experimental group compared to the normal control group.

Table 1. Peripheral blood hemogram profile
ParametersNormal Control Group (X ± SD)Pesticide Exposed Group (X ± SD)
Hemoglobin (g/dL)15.99 ± 2.2012.10 ± 1.10
WBC (×103/μL)6.2 ± 1.24.2 ± 0.19
RBC (×106/μL)8.44 ± 0.754.9 ± 0.79
Platelets (×103/μL)438 ± 14.22165 ± 12.32
Reticulocyte (%)0.89 ± 0.160.30 ± 0.05
Neutrophil (%)22.75 ± 2.2211 ± 1.22

Bone Marrow Culture

The bone marrow explants from the pesticide exposed animals [Fig. 1(B)] appeared to be distorted and fragile with predominant appearance of giant adipogenic precursors and lowered amount of cellular generation even after day 3 of culture compared to the normal [Fig. 1(A)] where healthy cell generation pattern was observed from the explant. At day 7 of culture, appearance of hematopoietic foci with close association of the stromal matrix was observed from the normal bone marrow explant [Fig. 1(C)]. The pesticide induced group [Fig. 1(D)] neither exhibits hematopoietic foci formation nor the appearance of stromal matrix with frequent appearance of giant immature stromal precursors. At day 14 of culture, the normal bone marrow explant [Fig. 1(E)] developed a complete network of mature stromal matrix, contained mainly the fibroblast-like cells upon which healthy cellular generation was observed. The pesticide exposed group [Fig. 1(F)] did not produce mature stromal matrix with predominance of immature fibroblasts. The healthy cell generation and colony formation was not observed in these groups, indicated a degenerated stromal microenvironment.

image

Figure 1. A: The bone marrow explant from the normal animal bone marrow showed healthy cell generation at day 3 of culture. B: Bone marrow explant from the pesticide exposed animal bone marrow appeared to be distorted and fragile with predominant appearance of (red arrow) giant adipogenic precursors at day 3 of culture. C: At day 7 of culture, appearance of hematopoietic foci (red arrow) with close association of stromal matrix was observed from the normal bone marrow explant. D: At day 7 of culture the bone marrow explant from the pesticide exposed group neither exhibited hematopoietic loci formation nor the appearance of stromal matrix with frequent presence of immature stromal precursors (red arrow). E: At day 14 of culture, the explant from the normal animal bone marrow produced a complete network of mature stromal matrix contained mainly the fibroblastic like cells. F: The pesticide exposed group failed to produce the mature stromal matrix with prevalence of immature fibroblasts (red arrow). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Bone Marrow Architecture

The typical bone marrow architecture was observed in normal as well as in experimental groups by bone marrow smear study. In the pesticide exposed groups of animals [Fig. 2(B)] the marrow architecture has found to be totally disorganized leaving scattered empty spaces with deformed stromal matrix. The cells present were significantly distorted in comparison with the normal [Fig. 2(A)].

image

Figure 2. A: Typical bone marrow architecture observed by Leishman staining of the bone marrow smear from the normal mice. The red arrow indicated the compact structure and cellular association of the bone marrow cells. B: Bone marrow architecture from the pesticide exposed group of mice had found to be totally disorganized leaving behind empty space with deformed stromal matrix. The cells present are significantly distorted. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Bone Marrow Adherent Cell Colony Formation

The quantitative distribution chart (Fig. 3) of bone marrow adherent cell colony numbers displayed that colony forming ability of the bone marrow adherent cell population from the pesticide exposed group of mice was always lowered in numbers compared to that of the normal control group throughout the days of culture. The distribution chart showed that at day 8 and 16 of culture, the experimental groups of animals had mean value of only 20% of the control mean. At day 28 of culture, however the colony formation increased to some extent in the experimental groups of animals with the value of 28% of the normal. The mean colony forming efficacy (p < 0.001) of the pesticide exposed animals was only 24% of the control mean.

image

Figure 3. The quantitative distribution chart of the bone marrow adherent cell colony numbers at different day's interval at 8th, 16th, and 28th days of culture from the normal and pesticide exposed group.

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CFU-F Assay

Fibroblastic colony forming efficacy of the pesticide exposed bone marrow cells was significantly (p < 0.01) decreased as compared to the normal (Fig. 4). The decreased fibroblastic colony numbers in the pesticide exposed group compared to the normal control essentially pointed towards the defects in functional maturation/terminal differentiation of the stromal precursors following pesticide-induced toxicity. With progression of days the CFU-F numbers were even more lowered in the pesticide exposed groups compared to the normal. The result represented the relative number of CFU-F formed by the bone marrow cells from the experimental pesticide exposed group as compared to the normal control and corresponded to the median ± S.D. levels observed from 15 (n = 15) samples studied.

image

Figure 4. The decreased fibroblastic colony numbers in the pesticide exposed group compared to the normal control. The result represented the relative number of CFU-F formed by the bone marrow cells from the experimental pesticide exposed group as compared to the normal control and corresponded to the median ± S.D. levels observed from 15 (n = 15) samples studied.

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Hematopoietic Progenitor Cell Colony Formation

Colony forming efficiency of the bone marrow derived hematopoietic progenitor cells depicted an overall significant (p < 0.001) decrease (Table 2) in colony formation in the pesticide exposed aplastic group compared to the normal control. CFU-G, CFU-GM, and CFU-GEMM documented a significant (p < 0.001) decrease in pesticide exposed aplastic group which indicated a suppression of the normal hematopoiesis in terms of stem/progenitor's ability in multilineage differentiation. CFU-E and BFU-E showed a significant (p < 0.001) decrease in mature erythrocyte formation in pesticide exposed group as compared to the normal. A total number of 15 animals (n = 15) were taken for each of the group and the results represented the relative number of CFUs in aplastic anemia as compared to the normal and corresponded to the mean ± S.D and p value < 0.05 was considered to be statistically significant.

Table 2. Hematopoietic progenitor cell colony formation
Colony TypesNormal Control (Mean ± SD)Pesticide Exposed Group (Mean ± SD)
CFU-G26 ± 215 ± 2
CFU-GM29 ± 118 ± 2
CFU-GEMM28 ± 113 ± 2
CFU-E40 ± 218 ± 2
BFU-E32 ± 116 ± 2

Cell Cycle Analysis

Cell cycle analysis of the bone marrow derived stem/progenitor cells (an analysis gate was set on FSC vs. SSC dot plot to include mostly the stem/progenitor cell population) (Fig. 5) showed an increased percentage of cells in the G0/G1 phase from the pesticide exposed experimental groups of animals (96.01%) compared to the normal group (86.78%). The cell fraction in the S/G2/M phase, however, was significantly (p < 0.01) reduced in experimental groups (4.8%) compared to the normal controls (9.16%) indicated an impairment in the cellular proliferation of the bone marrow stem/progenitor cells following pesticide exposure.

image

Figure 5. Cell cycle analysis of the bone marrow derived stem/progenitor cells showed an increased percentage of cells in the Go/G1 Phase from the pesticide exposed group (96.01%) compared to the normal (86.78%). The cell fraction in the S/G2/M phase however was significantly reduced in the pesticide exposed group (4.8%) compared to the normal (9.16%).

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Flowcytometric Analysis

Flowcytometric analysis of general forward scattering (FSC) vs. side scattering (SSC) characteristics of the whole bone marrow cells from the normal [Fig. 6(A)] and pesticide exposed groups [Fig. 6(B)] of animals unveiled the differences in the bone marrow cellularity in normal and diseased condition. Two different regions namely R1 (includes the lymphocytic precursors/ lymphocytes) and R2 (includes the granulocytic precursors/granulocytes) was set up to document the quantitative depression (p < 0.001) in the lymphocytic (R1 region) and granulocytic (R2) population in the pesticide exposed animals (R1—15.53%, R2—3.93%) compared to that of the normal (R1—22.68%, R2—25.41%). The results showed that the granulocytes are more vulnerable target of pesticide toxicity compared to the lymphocytes.

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Figure 6. General forward scattering (FSC) vs. side scattering (SSC) characteristics of the whole bone marrow cells from the normal (A) and pesticide exposed group (B). Two regions were selected namely R1 included the lymphocytes and R2 included the granulocytes. Dot plot revealed the vulnerability of granulocytes to the pesticide toxicity. Phenotypical characterization of Sca1+c-kit+, CD150+ and CD244−, and TIE2+ receptors expression in the bone marrow cells from normal (C–E) and pesticide exposed group (F–H) showed a quantitative depression in the primitive stem cell pool after pesticide exposure. Phenotypical characterization of non-hematopoietic progenitor markers (Sca1+ CD44+ and CD146+) expression from the normal (I,J) and pesticide exposed group (K,L) depicted a depletion of stromal cell population following pesticide exposure.

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Phenotypic characterization of hematopoietic progenitors through Sca1 and c-kit, CD150 and CD244, and TIE2 receptors expressions indicated highly downregulated Sca1+c-kit+ (1.09%), CD150+CD244− (3.76%), and TIE2+ (4.36%) cell populations in the pesticide exposed group of [Fig. 6(D,F,H)] compared to the normal controls (9.38%, 7.40%, and 7.36%, respectively) [Fig. 6(C,E,G)] (p < 0.001).

Further the phenotypic characterization of the bone marrow derived non-hematopoietic progenitors (Sca1+CD44+ and CD146+) cells demonstrated a decreased non-hematopoietic progenitors population in the pesticide exposed group (21.75% and 8.80%) [Fig. 6(J,L)] compared to the normal (33.38% and 16.38%) [Fig. 6(I,K)] (p < 0.001).

Cellular Apoptosis

Two different aspects of bone marrow cellular apoptosis profile was monitored,

  1. Increased CD95 (FasR) expression (p < 0.001) on the bone marrow derived stem/progenitor cell population (an analysis gate was set on FSC vs. SSC dot plot to include mostly the stem/progenitor cell population) was evidenced in the pesticide exposed group (13.71%) [Fig. 7(B)] compared to the normal (1.81%) [Fig. 7(A)] indicated an enhanced level of stem/progenitor cell apoptosis following pesticide induced toxicity.
  2. Annexin V positivity exhibited by Sca1+ primitive bone marrow cells in the experimental pesticide exposed group (4.23%) [Fig. 7(D)] was incurred to be significantly (p < 0.001) higher compared to the normal (0.49%) [Fig. 7(C)]. High annexin V positivity in the Sca1+ bone marrow cells indicated higher rate of premature senescence in the primitive bone marrow stem cells following pesticide toxicity.
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Figure 7. A: CD95 expression on normal bone marrow stem/progenitor cell population. B: Increased CD95 expression on the bone marrow stem/progenitor cell population from the pesticide exposed group showed a significant high level of apoptosis in the stem/progenitor population following pesticide exposure. C: Annexin V positivity exhibited by Sca1 positive bone marrow cells from normal control mice. D: Increased annexin V positivity on the Sca1 positive bone marrow cells from the pesticide exposed group of mice indicated premature senescence of the bone marrow stem cells following pesticide exposure.

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Cleaved Caspase-3 Expression on Sca1+ Marrow Cells

Flowcytometric analysis showed an elevated (p < 0.001) cleaved caspase-3 level on the Sca1+ bone marrow cells in the pesticide exposed group (9.32%) [Fig. 8(B)] compared to that of the normal (1.02%) [Fig. 8(A)], further confirming that the bone marrow progenitor cells undergo premature apoptosis following pesticide toxicity that ultimately resulted in severe degeneration of the bone marrow cellular compartment and an essentially empty marrow condition.

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Figure 8. A: Cleaved caspase-3 expression on the Sca1+ bone marrow cells from the normal control group. (B) Cleaved caspase-3 expression on the Sca1+ bone marrow cells from the pesticide exposed group. The increased cleaved caspase-3 expression on the Sca1+ bone marrow cells from the pesticide exposed group (9.32%) compared to the normal (1.02%) indicated an increased rate of apoptosis of the bone marrow progenitor cells following pesticide exposure.

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Scanning Electron Microscopy of Bone Marrow Microenvironmental Structure

SEM of normal bone marrow and the bone marrow from the pesticide exposed group of animals exhibited typical differences in their microenvironmental structure. The scanning electron micrographs of the normal bone marrow [Fig. 9(A)] represented a hematopoietic colony associated with the stromal cords. The compact cellular structures and intracellular association was also documented in the normal bone marrow microenvironment. Scanning electron micrographs from the pesticide exposed bone marrow [Fig. 9(B)] acquainted an overall scanty and degenerative marrow with deranged microenvironmental structure and distorted cellular components compared to the normal. No such stromal cord associated hematopoietic colony was observed throughout the field of pesticide exposed marrow.

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Figure 9. A: Scanning electron micrograph of the normal bone marrow microenvironment showed a compact cellular structure and presence of hematopoietic colony associated with the stromal cord. B: Scanning electron micrograph from the pesticide exposed bone marrow showed an overall scanty and degenerative marrow with frequent appearance of apoptotic pits within it. Stromal cords are completely absent throughout the field of the pesticide exposed marrow.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

In the present study, we have developed an animal model of pesticide-induced toxicity to evaluate the deleterious effects of long-term exposure of the environmental toxins-like pesticide on the bone marrow cellular physiology, specially, on the stem/progenitors and on the stromal cells which are part of the regulating microenvironment.

Pesticides can cause hematological disease and interfere with one or more hematopoietic lineages leading to neutropenia, agranulocytosis, thrombocytopenia, and anemia and in some cases aplastic anemia (El-Zayat et al., 2005). In our study, the peripheral blood cytopenic hemogram profile along with moderate depression of hemoglobin level apparently reflected the background physiology where the rate of hemoglobin synthesis did not get affected by pesticide toxicity as much as the rate of erythropoiesis, granulopoiesis, and lymphopoiesis.

The long-term bone marrow culture system has proved to be a useful tool for the characterization of hematopoietic microenvironment in-vitro. Pesticide exposure resulted in disorders with disturbed or impaired stroma which contribute to abnormal hematopoiesis (Bentley et al., 1982; Lenon and Micklem, 1986; Marsh et al., 1990). Results from our studies clearly demonstrated the deranged explant growth pattern with frequent appearance of giant adipogenic precursors and concomitant irregularities of stromal development and appearance of “immature stromal precursors” (toxicity induced inhibition of maturation) in the pesticide-induced group compared to the normal. The pictures correlated well with the above mentioned pesticide-induced bone marrow microenvironmental damage where the appearance of hematopoietic foci with close association of stromal matrix was observed on day 7 of culture in normal but the pesticide exposed group did not exhibit hematopoietic foci formation or the appearance of stromal matrix. At day 14 of culture, a deranged scattered microenvironmental bed with appearance of immature fibroblasts was observed in the pesticide-induced group pointed towards the developmental damage caused by pesticide toxicity to the stromal precursors that failed to generate a viable microenvironmental bed to sustain hematopoiesis.

The quantitative assay of adherent cell colony formation was used because adherent cells are vital to hematopoiesis in vitro and are representative of the microenvironment in vivo. The relevance of adherent cells to busulfan-induced marrow failure was indicated by Hays et al. and Anderson et al. (Hays et al., 1982; McManus and Weiss, 1984) matches well with our observation of sharp decrease of adherent cell colony formation in the pesticide exposed group. The decreased proliferative capacity and functional ability of the stromal precursors was well reflected on the CFU-F status of the bone marrow cells from the pesticide exposed mice. The CFU-F from the pesticide exposed bone marrow was significantly decreased compared to the normal CFU-F numbers which was directly correlated with the decreased proliferation and functional maturation/terminal differentiation of the stromal precursors. Inhibitory effects of the toxic pesticides on the hematopoietic system were also evidenced with significantly low CFUs forming ability by the marrow hematopoietic stem/progenitor cells from the pesticide exposed group of mice. The decreased values of CFU-E, BFU-E, CFU-G, and CFU-GM and CFU-GEMM essentially pointed towards a defect in the marrow hematopoietic stem/progenitor cell compartment in terms of functional maturation following pesticide toxicity. The defect could have originated either from the hematopoietic stem/progenitor cell level or from the non-hematopoietic stromal cell level depending on their physiological crosstalk in the hematopoietic microenvironment. Stem/progenitors from the pesticide exposed group showed cell cycle arrest at G0/G1 phase. The synthetic (S) and dividing (G2/M) phase data also exhibited a non-proliferative condition of the bone marrow stem/progenitor cells where most of the cell population were restricted to G0/G1 phase at higher level (96.01%) compared to the normal (86.78%). The inhibition of cell replication and restricted cell cycle pattern signified the background pesticide toxicity.

The flowcytometric analysis of whole bone marrow cells depicted the vulnerability of granulocytic population in pesticide-induced group than the lymphocytic population compared to the normal. Though our focus is mainly on stem and stromal progenitor cell population, yet we have documented the whole bone marrow cellular condition as a supportive base regarding our zone of interest and found that mainly the granulocytes fall prey to the pesticide-induced toxicity. A direct cytotoxic effect of pesticide was observed on the Sca1+c-kit+, CD150+CD244−, and TIE2+ hematopoietic progenitors cell populations. This quiescent primitive bone marrow cell population got affected regarding their receptor expression at the immature stage inside a blood bone barrier protected bone marrow microenvironment. We also observed a quantitative reduction in the non-hematopoietic marrow progenitor cell (Sca1+CD44+ and CD146+) populations in the pesticide exposed group in comparison with the normal which explained well the deranged stromal bed and adherent cell colony formation in the experimental group.

Apoptosis of highly proliferative tissue such as bone marrow always have a correlation with exposure to environmental toxins like pesticide. To denote the changes in the rate of apoptosis in the bone marrow cellular components during pesticide toxicity, we examined the level of various extracellular (CD95 and Annexin V) as well as intracellular (Cleaved Caspase-3) apoptotic marker expression on the Sca1+ bone marrow cells. Results depicted a significantly higher level of apoptotic receptor (CD95 and Annexin V) expression on the progenitor cells and a sharp rise in the intracellular cleaved caspase-3 level in the Sca1+ bone marrow cells following pesticide induced toxicity. The increased rate of apoptosis and premature senescence of the marrow cellular components induced by toxicity pointed towards an essentially empty marrow condition following pesticide exposure.

Further, we also looked into the scanning electron microscopic features of the very sophisticated tissue such as bone marrow after pesticide exposure and found huge deviations from the normal scenario-like complete absence of stromal cords throughout the field, presence of small andlarge apoptotic pits throughout the marrow microenvironment, uneven cellular distribution, and lack of compactness.

From all the above mentioned observations, we can conclude that the pesticide-induced toxicity in the bone marrow affects the primitive stem and stromal progenitor cells which misbalanced the cellular proliferation pattern and ultimately increased the cellular apoptotic machinery leading to the formation of a degenerative disease named “aplastic anemia.”

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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