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.