Mol Cell Biochem (2015) 401:115–131 DOI 10.1007/s11010-014-2299-5

Alteration of hedgehog signaling by chronic exposure to different pesticide formulations and unveiling the regenerative potential of recombinant sonic hedgehog in mouse model of bone marrow aplasia Malay Chaklader • Sujata Law

Received: 16 September 2014 / Accepted: 27 November 2014 / Published online: 4 December 2014 Ó Springer Science+Business Media New York 2014

Abstract Chronic pesticide exposure-induced downregulation of hedgehog signaling and its subsequent degenerative effects on the mammalian hematopoietic system have not been investigated yet. However a number of concurrent studies have pointed out the positive correlation between chronic pesticide exposure induced bone marrow failure and immune suppression. Here, we have given an emphasis on the recapitulation of human marrow aplasia like condition in mice by chronic mixed pesticide exposures and simultaneously unravel the role of individual pesticides in the said event. Unlike the effect of mixed pesticide, individual pesticides differentially alter the hedgehog signaling in the bone marrow primitive hematopoietic compartment (Sca1 ? compartment) and stromal compartment. Individually, hexaconazole disrupted hematopoietic as well as stromal hedgehog signaling activation through inhibiting SMO and facilitating PKC d expression. On contrary, both chlorpyriphos and cypermethrin increased the sequestration and degradation of GLI1 by upregulating SU(FU) and bTrCP, respectively. However, cypermethrin-mediated inhibition of hedgehog signaling has partly shown to be circumvented by non-canonical activation of GLI1. Finally, we have tested the regenerative response of sonic hedgehog and shown that in vitro supplemented recombinant SHH protein augmented clonogenic stromal progenitors (CFU-F) as well as primitive multipotent hematopoietic clones including CFU-GEMM and CFU-GM of mixed pesticide-induced aplastic marrow.

M. Chaklader
S. Law (&) Stem Cell Research and Application Unit, Department of Biochemistry and Medical Biotechnology, Calcutta School of Tropical Medicine, 108, C.R Avenue, Kolkata 700073, West Bengal, India e-mail: [email protected]

It is an indication of the marrow regeneration. Finally, our findings provide a gripping evidence that downregulated hedgehog signaling contribute to pesticide-mediated bone marrow aplasia but it could be recovered by proper supplementation of recombinant SHH along with hematopoietic base cocktail. Furthermore, SU(FU) and GLI1 can be exploited as future theradiagnostic markers for early marrow aplasia diagnosis. Keywords Pesticide
Hedgehog signaling
Chlorpyriphos
Hexaconazole
Cypermethrin
Aplastic anemia

Introduction Presently, more than half of the global pesticide consumption has been found to encompass two well known groups-(i) Organpphosphate (OP) and (ii) Type-II Pyrethroid (PY). However, a number of OPs have been either banned or restricted in its uses. Despite banning, chlorpyriphos (CHP) remains the most popular and widely used OP. On the other hand, cypermethrin, a Type-II Pyrethroid, becomes more popular for the agro-household purposes over the chlorpyriphos. Both, chlorpyriphos (CHP) and cypermethrin (CYP) have been designed as neurotoxic agents to alter presynaptic cholinergic functions by inhibiting the enzyme acetylcholinesterase (AChE) in the insects. In spite of neurotoxicity, chlorpyriphos (organophosphate) and cypermethrin (pyrethroid) have also plethora of non-cholinergic toxicity via different cell signaling cascades including PKC, MAPK, EGF-EGFR, and Ca2 ? -AMPc [1–10]. However, in some cases cholinergic dose is more potent than non-cholinergic one. Cholinergic dose of Malathion (organophosphate) is such an example,

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which induces profuse anomalies in various extrahematopoietic organs and suppresses primary humoral immune responses in rodents than that of its non-cholinergic dose [11–15]. Like chlorpyriphos and cypermethrin, hexaconazole (HEX) is another promising crop protective pesticide of azole fungicide family. It posses potent clastogenic activity in rat bone marrow cells and reduces IL-17 production via RORa/c in helper T (Th)17 cell [16]. Here, most of the available laboratory animal studies are conducted under carefully controlled conditions with acute or relatively short-term exposures at concentrations considerably higher than those encountered in nature. Moreover, considerable variation exists in animal species used and in route, time, and level of exposure, as well as end point examined. Unfortunately, most of the pesticide-induced immunohematotoxic studies consider simple in vitro genotoxicity as an experimental end point instead of other modern approaches [17, 18]. In spite of these experimental uncertainties, it is clear that most of the pesticides are capable of altering the immune system. Similarly, mammalian hematopoietic system is not an exception because occupational protracted pesticide exposure induced hematotoxicity, and subsequent development of bone marrow aplasia (aplastic anemia) has also been well documented in a number of recent studies [19–24]. Unlike congenital and immune-mediated marrow aplasia, pathophysiology of pesticide-induced marrow aplasia has never been investigated more thoroughly than that of our present and previous works [25–27]. However, pesticide-induced bone marrow aplasia also reproduces the pathological features of classic aplastic anemia. It includes but not limited to adipocyte laden hypocellular bone marrow, peripheral blood pancytopenia, weight loss etc. Our contemporary studies have reported that chronic exposures to combined formulation (5 % aqueous solution) of cypermethrin, chlorpyriphos, and hexaconazole deliberately alters JAK-STAT and canonical Hedgehog-Gli signaling in the murine hematopoietic compartment during marrow aplasia development [26–28]. In addition, our investigation has also confirmed that pesticide-induced aplastic anemia mouse model severely suffers from immune suppression [25]. Primitive and definitive hematopoiesis has been reported to be controlled by hedgehog-gli signaling axis [29–31]. Hedgehog signaling is a well-conserved developmental signaling pathway from Drosophila to human beings. Till date, three mammalian hedgehog proteins (SHH, IHH, and DHH) have been identified to interact with cell surface receptor PATCH1 [32]. Binding of hedgehog ligands to PATCH stop the inhibition of its membrane bound signaling partner Smoothened (SMO), which in turn initiate the nuclear translocation and activation of the GLI family of transcription factors (GLI1, GLI2, and GLI3) [32, 33]. Following nuclear translocation, GLI protein initiate the

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transcription of plethora of target genes including Patch, Gli1, and Hhip. However, in absence of hedgehog stimulifree GLI proteins are subjected to sequestration by Suppressor of Fused or SU(FU). Thereafter, various kinases including GSK3b, PKC d come to phosphorylate sequestered GLI proteins for its subsequent degradation by bTrCP and inhibit the unwanted activation of hedgehog signaling [34]. Recently, a couple of studies have demonstrated that hedgehog signaling has no indispensable role in definitive hematopoiesis of conditionally deleted Smo-/- mice [35, 36]. In sharp contrast to these two controversial works, it has been found that constitutively Hh-activated Ptch-/? mice showed increased cycling and expansion of medullary hematopoietic stem cells (HSC) in a homeostatic condition [37]. On the other hand, Dierks et al. [38] has shown that Smo-/- murine cells have lost the colony forming ability at the second replating time, in contrast to the Ptch-/? murine cells (with activated Smo) having both regeneration and enhanced engraftment ability of bone marrow [38]. Moreover, a number of scientific works have also demonstrated the involvement of hedgehog signaling with leukemic hematopoiesis and lymphomagenesis [39–43]. The present investigation has been designed to explain the individual role of chloropyriphos, cypermethrin, and hexaconazole exposures in murine bone marrow hematopoietic as well as stromal compartment and interaction strategy of each individual pesticide with the canonical Hedgehog-Gli signaling axis during the course of pesticide mixture-induced bone marrow aplasia development.

Methods and materials Animals Swiss albino mice (Mus musculus Linnaeus.) of both sexes [N = 30 (12 weeks old and 23 to 24 g body weight) for mixed group, individual pesticide-treated groups, and control group] were selected from the inbred colony maintained under controlled room temperature (22 ± 2 °C) in the animal house of the Calcutta School of Tropical Medicine. The animals were fed on a standard recommended diet and water ad libitum, under standard conditions with a 12 h light dark period. Maximum six animals were housed in one cage containing sterile paddy husk as bedding throughout the experiment. The procedures followed were in agreement with the approved guide for the care and use of laboratory animals and Institutional Animal Ethical Committee (IAEC). Pesticides and method of exposure We used 5 % aqueous mixture of widely used agricultural pesticides [i.e., 100 ml of aqueous mixture contained 5 ml

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of 50 % (w/v) chlorpyriphos, 5 ml of 9 % (w/v) cypermethrin, and 5 ml of 5 % (w/v) hexaconazole] for the present study [25, 28]. Consideration and modeling the effect of pesticides either as combined or alone is coming from our experience with hematology outpatient department (OPD) at Calcutta School of Tropical Medicine and rigorous counseling of those acquired aplastic anemic patients having the background of chronic pesticide exposure in agricultural field [17]. In addition to combined formulation, we also tested the individual effect of 50 % (w/v) chlorpyriphos (Sigma), 9 % (w/v) cypermethrin (Sigma), and 5 % (w/v) hexaconazole (Sigma) aqueous solutions at 5 % concentration each for the experimental purposes. Here, we used aforementioned concentration of pesticide to keep the parity with agriculturally used local pesticides available in India and other Southeast Asian agrochemical markets. First two pesticides belonged to organophosphate and pyrethroid groups. The last one is a systemic fungicide of triazole family. Adult mice (12 weeks old) were divided into four groups and each group of mice assigned for each individual pesticide exposure e.g., Oraganophosphate (chlorpyriphos treated) group (CHP, N = 30), Pyrithroid (cypermethrin-treated) group (CYP, N = 30), Triazole fungicide (hexaconazole treated) group (HEX, N = 30), and combined formulation group (MIX, N = 30). Thereafter, each experimental animal group received inhalation and dermal exposure [by handheld glass atomizer (locally made), atomizing time being 5 min] to 10 ml of a 5 % aqueous solution of the aforesaid individual pesticide (e.g., CHP group received only chloropyriphos exposure, CYP group received only cypermethrin exposure, and HEX received exposure of hexaconozole only) and 5 % combined pesticide formulation for 6 h per day and 5 days a week up to 13 weeks. Another group i.e., control (N = 30) has received inhalation of and dermal exposure to the only aqueous solution without any trace of pesticide contamination for the same period of time.

Experimental design Altogether we have taken N = 21 animals for each pesticide group and for sham control (i.e., 7 animals/group for each of the three trials of experiment). We used the pooled bone marrow sample for the following long-term bone marrow stromal cell culture (LTBMSC), clonogenicity assay and flowcytometric experiments. The rest of the pesticide-treated animals (N = 9) and control animals (N = 9) were used in the bone marrow cellularity and scanning electron microscopic study purposes (3 animals per experiments). Here, pooled bone marrow samples were taken into consideration for each experiment.

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Peripheral blood hemogram We have randomly selected experimental (pesticide-treated groups) and control animals from respective cages for blood hemogram profiling after completion of 90 days. Since, the study has been related to hematopoietic machinery, we did the hemogram of each and every animal considered for the experiments and we follow the method as provided by Chatterjee et al. [25] and Chaklader et al. [28]. Isolation of bone marrow Mice of the chronic pesticide-exposed groups (showed progressive peripheral pancytopenia with other physical disparities) and the control group were sacrificed to isolate the long bones (Femur and Tibia). Bone marrow was carefully flushed out from the isolated bones by syringe containing RPMI-1640 (Sigma, USA) media supplemented with 10 % FBS (Lonza, Belgium). Bone marrow cellularity assessment Femoral marrow cells of control and pesticide-treated mice were aseptically collected in RPMI-1640 animal tissue culture media by flushing technique. Total cell counts of marrow single cell suspensions of both control and pesticide-treated animals were then performed in Hemocytometer chamber (Rohem India) by Erythrosin-B (SigmaAldrich, USA) dye exclusion method [28]. Ultrastructure of bone marrow microenvironment A small portion of the intact marrow tissue containing the total niche was macerated slowly in a physiological manner without damaging the inner cell mass and kept in 2.8 % glutaraldehyde overnight for fixation. To dry the tissue, it was repeatedly passed through 30 %, 50 %, 70 %, and 100 % gradient of alcohol and finally critical point drying was done. The samples were subjected to Scanning Electron Microscopic examination by S-5330 Hitachi SEM followed by coating with gold (Au) in IB-2 ion coater. Long-term bone marrow culture 29106 bone marrow cells/ml were subjected to culture in triplicate (for each of the normal and experimental groups) in six well culture plates (Corning, USA) containing 3 mL of RPMI-1640 (Sigma, USA) supplemented with 30 % FBS (Lonza, Belgium). The culture medium also consisted of 1 % Bovine Serum Albumin (BSA; Sigma, USA), 0.01 % (v/v) 2-mercaptoethanol (Sigma, USA). The cultures were incubated at 37 °C in an atmosphere of 5 %

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Table 1 Effect of pesticides on peripheral blood hemogram: Comparative peripheral blood hemogram showed a differential effect of combined and individual pesticides on the peripheral blood corpuscular count and hemoglobin concentration of the treated experimental animals Parameters

Control group Mean ± SD (A)

MIX group Mean ± SD (B)

CHP group Mean ± SD (C)

CYP group Mean ± SD (D)

HEX group Mean ± SD (E)

Hemoglobin (g/dl)

15.9 ± 2.20

7.0 ± 1.50*

10.76 ± 1.59*

12.45 ± 0.20*c

8.77 ± 1.26*b

a

Reticulocyte (%)

0.9 ± 0.15

0.23 ± 0.07*

0.59 ± 0.11*

1.20 ± 0.08*

Total RBC (x106/ ll) Total WBC

8.5 ± 0.70 6.0 ± 1.20

3.2 ± 0.60* 3.6 ± 0.30*

4.28 ± 0.90* 8.02 ± 1.23*a

5.22 ± 1.60*c 5.20 ± 0.50*

0.34 ± 0.10* 3.87 ± 2.60*d 4.21 ± 1.30*d

440 ± 13.92

169 ± 10.00*

180 ± 5.00*b

340 ± 2.00*

212 ± 5.00*

(x103/ ll) Platelets 3

(x10 / ll) PMNs/ul

1,402 ± 2.00

498.8 = 3.12*

824 ± 10.00*

572 ± 5.00*

632.57 = 5.21*

Lymphocytes/ll

4,320 ± 2.15

3,045 ± 3.60*

5,213 ± 8.00*

4,108 ± 10.50*

3,301 ± 8.32*

Monocytes/ll

220 ± 1.30

48 ± 1.50*

120 ± 5.50*

312 ± 2.00*

56 ± 1.50*

Basophiles/ll

58 ± 0.01

5 ± 0.05*

–

–

2 ± 0.01*

Abnormal cells/ll

–

4 ± 0.02*

1,863 ± 4.00*

208 ± 4.70*

22.43 ± 3.00*

Here, differential count is expressed in absolute number instead of percentage. Between groups comparison was performed by one-way ANOVA followed by post hoc Tukey test. Asterisks indicate the P values (* P \ 0.01; ** P \ 0.05). Labeling of particular data with a/b/c/d/e indicate insignificant difference with data of corresponding group. Error bars indicate mean ± S.D

CO2 in air. At every 72 h, the media was drained off to remove the nonadherent cells and fresh media (supplemented with 30 % FBS and 0.01 % (v/v) 2-mercaptoethanol) was added to the maintenance of the culture.

Cell-cycle study of marrow hematopoietic population The cell-cycle analysis of the bone marrow cells was performed using DNA QC particle (BD-Biosciences, USA) and according to Chatterjee et al. 2014 [27].

Clonogenecity assay CFU-F assay and hematopoietic progenitor cell colony forming assay This assay was performed according to the protocol of Chatterjee et al. (2013) [26]. The resulting colonies of both assay were then scored using an inverted microscope on the 7th, 11th, and 15th days of the culture. A little modification was done by supplementation of murine recombinant Sonic Hedgehog (100 ng/ml) (mrSHH, Biovision, USA) in the serum-free defined medium for the both assays to test the in vitro hematopoietic reconstitution potentiality of hedgehog signaling in hematopoietic degeneration following pesticide exposure. Evaluation of marrow LSK population To identify the murine hematopoietic progenitors, a PerCPCy5.5TM-conjugated Lineage (Lin) antibody cocktail (BD Pharmingen, USA) (CD3e, CD11b, CD45R/B220, TER119, and Ly-6G and Ly-6C), anti-mouse Sca-1-PE monoclonal antibody (BD-Bioscience, USA), and anti-CD117 antibody-FITC (BD-Bioscience, USA) were used in RBCdepleted bone marrow cells according to the protocol of Chaklader et al. (2012) [28].

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Surface and intracellular flowcytometric study of hedgehog signaling in Sca-1? compartment Sca1? compartment denotes murine stem cell compartment in bone marrow. Paraformaldehyde (PFA) prefixed bone marrow cells were subjected to permiabilization by chilled methanol (90 % v/v) treatment. Followed by permiabilization, respective primary antibodies [anti SHH antibody (H-160), anti IHH antibody(H-88), anti PTCH antibody (H-267), anti SMO antibody(N-19), anti GLI1 antibody(H-300), anti Su(Fu) antibody(C-15), anti GSK3b antibody(H-76), anti PKC-d antibody (C-20), and anti bTrCP antibody (H-85) were purchased from Santa Cruz Biotechnology, USA and anti HHIP antibody, anti DHH antibody, antiGLI3 antibodies from Abcam, UK.] were placed in respective tubes with cells from five different animal groups and incubated for one hour along with anti Sca-1antibody-PE (BD-Bioscience, USA). Following incubation with primary antibodies, secondary anti rabbit IgG –Alexa Fluor-488 antibody (Invitrogen, USA) was added in each respective tube and allowed to incubate for 45 min. Thereafter, samples were analyzed for protein expression in Sca-1 positive primitive hematopoietic population by BD-FACS Callibur (Becton–Dickinson, USA) using CellQuestpro software.

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A

B

C

D

E

Fig. 1 Ultra structural study of the bone marrow microenvironment: a Left most photographs of the first row represented the scenario of normal bone marrow microenvironment with healthy hematopoietic cellular distribution (yellow arrow). b Chronic mixed pesticide treatment caused profound changes in the medullary microenvironment of the animal concerned. Excessive stromal (mainly fibroblast) development is clearly visible in the given photograph and very few hematopoietic cellular colonies are present there with large prominent marrow sinuses (green arrow). In addition, cellular morphology is not perfectly well as we seen in control marrow. c Chloropyriphos exposed marrow microenvironment has excessive stromal elements

and low hematopoietic cellular content. d Cypermethrin exposure caused cellular deformity throughout the marrow microenvironment with numerous apoptotic pits (red asterisk). Abluminal wall (blue arrow) of the second lumen of CYP-treated marrow was morphologically deformed and lack of adevential reticular cell body. e Hexaconazole-exposed bone marrow microenvironment showed numerous apoptotic cells (red arrow marked) with excessive membrane shrinkage and blabbing. A large marrow sinus (green arrow) was visible in the lower left side of the electron micrograph. Scale bar = 20 lm. (Color figure online)

Immunocytochemical and flowcytometric study of hedgehog ligands and GLI1 expression in the bone marrow stroma

tochemical procedure, and permiabilization was done by a standard combination of detergents (0.2 %Tween 20 and 0.2 %Nonidet P-40 in 1X PBS). To prevent nonspecific binding of primary antibody, suitable blocker (0.5 %BSA, 0.5 % Gelatin, and 0.5 % goat serum dissolved in PBS) was used for 20 min. Overnight incubation with primary

Long-term bone marrow culture generated mesenchymal stroma cells were processed as per standard immunocy-

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Results Peripheral blood picture primarily ascertains the effect of pesticides on the bone marrow

Fig. 2 Bone marrow cellularity assessment: Comparative whole bone marrow cellularity analysis revealed that pesticide treatment severely reduced cell count in the treated marrow samples than that of the control marrow sample. In sharp contrast to the marrow cellularity of control- and cypermethrin-exposed animal, chronic mixed pesticide, hexaconazole, and chloropyriphos exposure significantly reduced marrow cellularity of the concerned animals. However, hypocellularization of mixed pesticide-exposed animal is mostly notable and it is sign of a bone marrow aplasia development. Here between group comparison was performed by one-way ANOVA followed by post hoc Tukey test and *P \ 0.01

antibodies [anti Shh antibody, anti Ihh antibody, anti Dhh antibody, and anti Gli1 antibodies were purchased from Santacruze Biotechnology, USA] (1:200) was completed before addition of 0.3 %H2O2 (Sigma) for inhibition of endogenous Peroxidase activity followed by HRP-conjugated anti rabbit IgG secondary antibody (1:1000) for chromogenic development with Delafield’s Hematoxylin (Merck) counter stain. Another set of the PFA-fixed stromal samples from those four experimental and one control groups were subjected to restricted tryptic digestion, subsequent washing and retrieval. These samples were then permiabilized and incubated with aforementioned primary antibodies against Hedgehog ligands and GLI1 transcription factor in respective tubes with anti CD45 antibody-PE (BD Bioscience, USA). Thereafter, secondary anti rabbit IgG –Alexa Fluor-488 antibody (Invitrogen, USA) was added in each respective tube. Followed by incubation and final wash samples were analyzed for protein expression in CD45-PE negative non-hematopoietic mesenchymal stromal population by BD-FACS Callibur (Becton–Dickinson, USA) using CellQuestpro software. Statistical analysis All the values of experiments were represented as mean ± SD (Standard Deviation). One-way ANOVA followed by Posthoc Tukey tests were used when differences between the groups were evaluated. For all comparisons, P B 0.05 were considered as significant difference.

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Peripheral blood hemogram of combined pesticide-treated mice (MIX) showed severe pancytopenia like human marrow aplasia cases along with downregulated hemoglobin concentration (7.0 ± 1.50 g/dl), significantly low neutrophil (*8.2 %), and reticulocyte count (*0.23 ± 0. 06 %) in comparison with control mice (hemoglobin15.9 ± 2.20 g/dl, neutrophil-23 %, and reticulocyte0.9 ± 0.15 %). Exposure to individual pesticide caused uniform reduction of various peripheral blood parameters in the treated animals with respect to control animals. Unlike CHP- and CYP-treated groups, hemoglobin concentration of HEX-treated group (8.77 ± 1.26 g/dl) was sharply downregulated nearly twofolds than that of control one (P \ 0.0001). In addition to the reduced hemoglobin concentration, total RBC (3.87 ± 2.00 9 106/ll) and reticulocyte counts (0.34 ± 0.10 %) were also reduced following Hexaconazole exposure. However, both CHP and CYP exposures also reduced total RBC count (CHP: 4.28 ± 0.90 9 106/ll and CYP: 5.22 ± 1.60 9 106/ll), whereas reticulocyte count was reduced by CHP (0.59 ± 0.11 %) not by CYP in comparison to the control RBC and reticulocyte counts. A moderate leukocytopenia (both lymphocytopenia and neutropenia) was observed in CYP- and HEX-treated animals than that of control- and CHP-treated animals. However, severe thrombocytopenia and monocytopenia were evident in CHP- and HEX-treated groups. Unlike control group, CYP-exposed group did not show severe thrombocytopenic features as we observed in CHP and HEX treatment. In conjunction with regular corpuscular counts, we observed high amount of abnormal cells in CHP- and CYP-treated animals than that of HEXtreated animals (Table 1). Altered ultrastructure of medullary microenvironment and cellularity hint the hematopoietic dysfunction followed by pesticide exposure Reasons of abnormality in peripheral blood hemograms of the chronically pesticide-exposed groups can be addressed by part through investigation of the bone marrow microenvironment and cellularity of the concerned groups. On the other hand, mix pesticide-treated marrow ultrastructure revealed the low abundance of hematopoietic cells and fibrotic change in the marrow microenvironment (Fig. 1b). In sharp contrast to the pesticide-treated marrows, ultrastructure of control marrow microenvironment was found to be sustainable for healthy hematopoiesis and the respective micrograph of control marrow showed a close

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10th Day

121

15th Day

20th Day

25th Day

CON

MIX

CHP

CYP

HEX

Fig. 3 Comparative long-term bone marrow culture and clonogenecity assay: Control long-term bone marrow culture successfully recapitulated the hematopoietic event at the end of the 25th day by supporting the development of numerous hematopoietic foci (blue arrow). Most of the hematopoietic development was found to be initiated in the vicinity of the stromal component (pink arrow). On contrary, chronic exposure of hexaconazole and chloropyriphos severely hampered the LTBMC in

comparison to the cypermethrin-treated marrow. Similarly mixed pesticide treatment caused noticeable apoptotic and degenerative activity in the stromal bed during the 25th day of the experiment. Hexconazole treatment reduced the cell sustenance capacity in the in vitro culture condition and caused cell death (yellow arrow). On the other hand, chloropyriphos exposure caused excessive large stromal fibroblast development with little hematopoietic activity. Scale bar = 5 lm. (Color figure online)

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association of stromal cells (having pseudopodes) and hematopoietic cells. Cell membrane topology of the control marrow was nearly smooth or with minimum parallel small ridges (Fig. 1a). On the contrary, CHP-exposed hematopoietic microenvironment was totally filled with bizarre abnormal stromal elements and apoptotic cells. A prominent marrow sinus was visible here in CHP-treated electron micrograph with the paucity of healthy hematopoietic cells (Fig. 1c). CYP-treated marrow showed low cellular distribution around two adjacently placed lumens and one sinus. Abluminal wall of the second lumen of CYP-treated marrow was morphologically deformed and lacked adventitial reticular cell body. A number of apoptotic pits were identified during the scanning process and a few were also present here in the micrograph of CYP-treated marrow (Fig. 1d). Ultrastructural study revealed that Hexaconazole-exposed murine bone marrow microenvironment was studded profusely with abnormal and apoptotic cells having prominent surface blebbing and ruffles (Fig. 1e). In conjunction with ultrastructural evidences, it was found that chronic exposures of MIX, HEX, and CHP caused a significant diminution of total marrow cellularity in the treated animals in comparison to the control marrow cellularity. However, CYP treatment was found not to be involved in marrow hypocellularity (Fig. 2). Abnormal stromal development indicate quantitative and qualitative damage of medullary hematopoietic machinery Long-term culture of control marrow showed confluent stromal microenvironment formation from the 10th day of culture with healthy fibroblasts and round stromal progenitors. On the other hand, combined pesticide exposure (MIX)-induced aplastic fibroblasts were elongated, stiff, and spindle shaped. Simultaneously, individual pesticidetreated explants exhibited the stromal development from the 15th day of culture. However, CYP-treated samples were successful to establish stromal matrix which supported the sustenance of hematopoietic progenitor cells (Fig. 4a). Unlike the stromal confluence of control and CYP-treated samples, HEX- and CHP-treated samples were failed to establish a proper stromal matrix in time. Reminiscence of numerous hematopoietic colonies was observed on the 25th day old culture of HEX-treated stroma but at the same time CHP-treated stroma showed discontinuous elongated stromal fibroblast with a few hematopoietic foci (Fig. 3). Sign of cellular apoptosis was clearly visible in case of combined pesticide-induced aplastic stromal microenvironment on day 25th onward. Altogether, it was evident that the control samples achieved confluent stromal matrix with multiple hematopoietic foci on the day 25th by recapitulating the

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Mol Cell Biochem (2015) 401:115–131 Fig. 4 Clonogenicity assay: a Hematopoietic mix clonogenicity c assay of individual and mixed pesticide-treated animals showed reduced number of clonal expansion as well progenitor differentiation activity in comparison to the control marrow. b Comparative clonogenicity assay of stromal fibroblasts (CFU-F) revealed that mixed pesticide treatment as well as individual hexaconazole and chloropyriphos treatment severely suppressed the CFU-F formation in comparison to the individual cypermethrin treatment. Here between groups comparison was performed by one-way ANOVA followed by post hoc Tukey test. Asterisks indicate the P values for number of CFU formation (*P \ 0.01; **P \ 0.05). Error bars indicate mean ± SD

hematopoiesis in vitro than that of individual and combined pesticide-treated stromal cultures. Hematopoietic and non-hematopoietic CFU development was markedly changed followed by pesticide exposures Chronic individual HEX treatment and combined pesticide (MIX) treatment showed uniform reduction of each and every kind of hematopoietic colonies including nonhematopoietic CFU-F (Fig. 4a, b). Both MIX and HEX treatment noticeably reduced most primitive and multipotent colonies of CFU-GEMM and most primitive erythroid colonies- BFU-Es than that of other two individual pesticides exposed groups. CHP-treated group showed moderate production of CFU-GMs and CFU-G. However, CYP treatment revealed no such serious toxicity on the hematopoietic progenitor compartment as well as in non-hematopoietic stromal fibroblast population which collectively formed hematopoietic stem cell niche (Fig. 4a, b). Reduced number of progenitors hints at both depletion of the primitive Lin-Sca-1 ? Kit ? cells in the marrow and alteration of cell-cycle pattern The numbers of gated LKS cells in the bone marrows were significantly reduced in MIX (0.48 ± 0.04) %, HEX (0.89 ± 0.01) %, and CYP (0.92 ± 0.85) %-treated animals in comparison to the LKS population of control (3.75 ± 0.32) % group. On the contrary, CHP-treated group showed a moderate diminution of LKS population (1.56 ± 0.52) % as compared to that of other two pesticide-treated groups (Table 2). Flowcytometric cell-cycle analysis established that a larger amount of quiescent (G0/G1) phase cells (*90 %) were present in MIX-, HEX-, and CYP-treated marrows than that of CHP-treated group which sheltered *75 % G0/G1 phase cells. Further analysis revealed that CHP treatment restricted *12 % cells in M phase and *13 % of cells in S phase of the cell cycle. However, no such restriction in M phase was documented in the marrow of MIX (0.3 %), CYP (*2.98 %), and HEX (*3. 16 %)

Mol Cell Biochem (2015) 401:115–131

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Table 2 Alteration of bone marrow Lin-Sca1 ? Ckit ? cells following pesticide exposure: Flowcytometric evaluation of bone marrow primitive hematopoietic stem cells (Lin-Sca1 ? Ckit ?) in control and individual pesticide-treated animals Animal groups

% of Lin-Sca1 ? Ckit ? cells

Control

3.75 ± 0.23

MIX group

0.48 ± 0.04*

CHP group

1.56 ± 0.52*

CYP group

0.92 ± 0.85*

HEX group

0.89 ± 0.01*

Between groups comparison was performed by one-way ANOVA followed by post hoc Tukey test. Asterisks indicate the P values for number of LSK (* P \ 0.01). Error bars indicate mean ± S.D

% of Cells in various stages of cell cycle

120 100

G0/G1 S G2/M

80 60 40 20 0

CON

MIX

CHP

CYP

HEX

Fig. 5 Flowcytometric cell-cycle analysis: Propidium iodide-based flowcytometric evaluation of cell-cycle pattern in control and different pesticide-treated animal groups reveal the effect of chronic toxicity of pesticide on hematopoietic machinery. Animal group wise cell-cycle stage specific distribution of bone marrow hematopoietic cells is as follow: Control:: G0/G1: 84.24 %, S: 11 %, G2/M:4.76 %; MIX:: G0/G1: 96 %, S: 3.7 %, G2/M:0.3 %; Chlorpyriphos (CHP):: G0/G1: 78.29 %, S: 12.6 %, G2/M:9.11 %; Cypermethrin (CYP):: G0/G1: 86.8 %, S: 10.22 %, G2/M:2.98 %; Hexaconazole (HEX):: G0/G1: 86.97 %, S: 9.87 %, G2/M:3.16 %. (Color figure online)

treated groups. HEX treatment showed less abundance of S phase cells and indicated low DNA synthesis activity in comparison to the CYP-treated group of animals. In sharp contrast to the individual pesticide-treated groups, animals of control group exhibited proper distribution of marrow cell population (Go/G1:84 %, S: 11 %, G2/M: 4.76 %) followed by cell-cycle analysis (Fig. 5). Chronic exposures to combined and individual pesticide differentially modulate hedgehog signaling in the hematopoietic stem-stromal compartment Chronic individual exposures to CHP, CYP, and HEX showed differential modulation of hedgehog signaling in the Sca1? compartment of the bone marrows of treated animals. MIX, HEX, and CYP exposures drastically downregulated the

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production of all three hedgehog ligands including Sonic (SHH), Indian (IHH), and Desert (DHH). However, CHP exposures did not affect IHH expression in comparison to SHH and DHH expression. PTCH and SMO expression reduced noticeably in the MIX- and HEX-exposed group compared to the PTCH and SMO expression of the other two groups. CHP and CYP upregulated the expression of downstream negative regulators e.g., SU (FU) and GSK3b compared to the HEX-exposed group. Interestingly, MIX and HEX treatment showed upregulation of PKCd and indifferent expression of GSK3b with respect to the control sample. Upregulation of SU (FU) and other two kinases phosphorylated and restricted the entry of transcription factor GLI1 into the nucleus and subjected it to bTrCP which ultimately stopped the signal execution. Our data revealed that bTrCP was significantly upregulated in the fungicide and combined groups in comparison to the other two (Fig. 6a, b; Table 3). Simultaneous observation of stromal immunocytochemistry unfolded that very low amount of GLI1 was accumulated outside of the nucleus in MIX- and HEXpretreated marrow stromal cells, whereas clear nuclear localization of GLI1 was evidenced from the control-, CHP-, and CYP-pretreated stroma. Moreover, our flowcytometric data of stromal cell also revealed that GLI1 protein expression was affected mostly in MIX- and HEXtreated group than that of CHP, CYP and control. In addition to GLI1 expression, the immunocytochemical and flowcytometric experiment also revealed that the expression pattern of three hedgehog ligands (SHH, IHH, and DHH) was noticeably downregulated in the stromal compartment of MIX, CHP, CYP, and HEX stroma in comparison to the control stream (Fig. 7a, b). In vitro supplementation of recombinant SHH (rmSHH) rescue stromal and hematopoietic precursors from combined pesticide-induced aplastic marrow sample In vitro supplementation of the rmSHH initially (7th and 11th day of culture) promoted every kind of hematopoietic CFU in the treated cultures of control and pesticideinduced aplasia in comparison to the untreated culture of previously mentioned two groups. The result was found to be statistically significant (Fig. 8a, b). However, the significant effect of rmSHH was limited to the most primitive progenitors which were involved in CFU-GEMM and CFU-GM formation. CFU-G, CFU-M, BFU-E, and CFU-E were not significantly responsive toward the supplementation of the rmSHH. Interestingly, the supplementation of rmSHH was found to increase the overall CFU number of control and pesticide-treated disease group in comparison to the rmSHH untreated control and disease culture at the end of the 15th day (Fig. 8b). Fibroblastic colony forming

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Fig. 6 Intracellular flowcytometric investigation of hedgehog signaling: Flowcytometric analysis of canonical hedgehog signaling in the Sca1 ? primitive hematopoietic population of the a mixed pesticide-exposed bone marrow and b individual pesticide-exposed bone marrow. Here ISO Isotype, NBM control marrow, PBM mixed pesticideexposed marrow, OPBM Chlorpyriphos-exposed marrow, PYBM Cypermethrinexposed marrow and FUBM Hexaconazole-exposed marrow. (Color figure online)

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assay of stromal precursors revealed that unlike control bone marrow, combined pesticide-induced aplastic marrow failed to establish a sufficient number of colonies in ex vivo condition. Supplementation of rmSHH augmented control CFU-F formation (P \ 0.01) in comparison to the untreated control and rescue the number of CFU-Fs in combined pesticide-induced aplastic marrow culture (P \ 0.01) than that of untreated aplastic culture. So rmSHH minimizes the toxicological suppression of different pesticide mixtures (Fig. 8c).

Discussion The present study proposes for the first time, the mechanistic and comparative insight of pathological alteration in the murine hematopoietic system followed by chronic individual exposures to chloropyriphos (CHP), cypermethrin (CYP), hexaconazole (HEX), and their combination (MIX). Chronic exposures to individual and combined pesticide formulation have been found to cause qualitative and quantitative damage of the cellular and sub-cellular physiology of the hematopoietic system. Our study unveiled that chronic MIX exposure caused recapitulation of human aplastic anemia like condition in mice through bone marrow failure, moderate to high pancytopenia and morphological abnormalities in the myelocytes. To underpin the exact reason of such pathophysiology, we assumed that MIX model can partly be useful for this study. Therefore, experimentation with each individual pesticidal component of MIX can only unearth the paradox. Subsequent experimentations with those components

Fig. 7 Stromal immunocytochemistry and flowcytometry: a Immu- c nocytochemical study of hedgehog signaling in the long-term bone marrow culture generated hematopoietic stromal cell population. Most of the hedgehog ligand expression is restricted into the cytoplasm of the stromal cell which is identified by DAB-HRP reaction of the corresponding antibodies mentioned in method sections. On the other hand, GLI1 expression is distributed in between cytoplasm and nucleus of the control, chlorpyriphos and cypermethrin-treated stroma. Here in the photographs, we can clearly observe the nuclear localization of GLI1 (black arrow) in the control stroma. Whereas mixed pesticide and hexaconazole treatment inhibits nuclear shuttling of the GLI1 transcription factors. Therefore, the respective cellular nuclei show the color of counter stain instead of brown coloration due to absence of DAB-HRP reaction of anti GLI1 antibody (red arrow). Scale bar = 50 lm. b Flowcytometric quantitative expression study of different hedgehog signaling related proteins expressed in long-term bone marrow cultured generated stroma of control, mixed pesticide-induced aplastic marrow and other individual pesticide-treated marrow. Here between groups comparison was performed by one-way ANOVA followed by post hoc Tukey test. Asterisks indicate the P values for MFI, which corresponds to expression of different proteins (*P \ 0.01; **P \ 0.05). Error bars indicate mean ± SD. (Color figure online)

revealed that HEX and CHP exposure caused reticulocytopenia in the treated animals. It caused low RBC count and reduced hemoglobin. On the contrary, reticulocyte counts of CYP-treated animals were roughly paralleled to those of control mice because the maturation process of reticulocyte to RBC was not affected by CYP. However, chronic CYP exposure hampered the sustenance of mature RBC and we observed low RBC: reticulocyte level in the group concerned. In addition, consistently moderate leucocytopenia and thrombocytopenia were documented in HEX- and CYPtreated groups, whereas CHP-treated group showed ectopic leukocytopenia.

Table 3 Mean fluorescence intensity (MFI) score corresponds the expression pattern of different protein components of the canonical hedgehog signaling pathway in Sac1 ? bone marrow compartment followed by the chronic exposure to combined and individual pesticides Markers

MFI of Control X ± SD (A)

MFI of MIX X ± SD(B)

SHH

95.74 ± 23.7

40.49 ± 9.2*

23.8 ± 9.19*

IHH

184.79 ± 10.9

48.19 ± 7.39*

100.50 ± 31.81*

39.5 ± 3.5*

DHH

238.05 ± 7.50

72.22 ± 1.68*

70.45 ± 2.19*b

62.5 ± 3.5*

88.5 ± 9.2**

HHIP

64.15 ± 2.26

16.88 ± 1.92*

18.5 ± 2.1*b

17.5 ± 2.46*b

14.5 ± 0.7**

42.00 ± 6.10*

32.5 ± 3.5*

30.2 ± 2.12*c

16.5 ± 0.7*

PTCH-1 SMO

180 ± 21.50 70.55 ± 5.65

17.7 ± 11.70*

MFI of CHP X ± SD (C)

MFI of CYP X ± SD (D)

b

32.0 ± 2.8*

MFI of HEX X ± SD (E)

45.47 ± 13.95*b

20.00 ± 2.0* a

GLI-1

155 ± 15.54

65.00 ± 20.50*

106.96 ± 13.2**

GLI-3

13.3 ± 5.00

9.50 ± 3.12a

11.00 ± 1.41a

26.5 ± 2.3*

17.11 ± 1.45

48.39 ± 2.50*

45.5 ± 3.5*

26.5 ± 2.0*

SU (FU) PKC d GSK 3b bTrCP

145.35 ± 45.5

a

10. 95 ± 1.5* 32.87 ± 3.6*

16.50 ± 2.2*d 60.75 ± 10.25** 13.5 ± 2.0abc 15.72 ± 0.3a

16.5 ± 1.90

24.43 ± 2.14*

10.0 ± 2.83**

13.1 ± 1.4

23.5 ± 3.0*b

17.34 ± 2.14 10.20 ± 1.59

10.11 ± 1.80** 21.10 ± 3.10*

16.0 ± 1.41ae 19.5 ± 0.7*

24.5 ± 2.5* 12.5 ± 0.8a

17 ± 1ac 28 ± 3.5*

Between groups comparison was performed by one-way ANOVA followed by post hoc Tukey test. Asterisks indicate the P values for MFI, which corresponds to expression of different proteins (*P \ 0.01; **P \ 0.05). Labeling of particular data with a/b/c/d/e indicate insignificant difference with data of corresponding group. Error bars indicate mean ± S.D

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Unlike CYP, reduced marrow cellularity of MIX-, HEX-, and CHP-treated group was correlated well with the peripheral blood picture and ultrastructural analysis. It adjoins extra evidence in support of the peripheral blood cytopenia, marrow hypocellularity and suppressed hematopoiesis. Ultrastructural and morphological analysis revealed the qualitative deterioration of the medullary hematopoietic cells and hematopietic microenvironment. Hypocellularity of the HEX- and CHP-treated marrow was further reassured through short-term bone marrow culture and cell release kinetics (data not shown). Using LTBMC, we further verified the functional aspect of the pesticide-exposed bone marrow microenvironment. LTBMC allow the formation of stromal adherent layers that are structurally and functionally similar to the in vivo hematopoietic microenvironment. Unlike the stroma derived from control LTBMC, functional evaluation of MIX-, HEX-, and CHP-exposed stroma revealed a stunted growth pattern with low sustenance. On contrary, similar condition was not identified in LTBMC of CYP group. Parallel to LTBMC, CFU-F assay was used to identify and quantify the clonogenic bone marrow stromal fibroblasts which were non-migratory, highly adherent precursor cells involved in hematopoietic microenvironment formation. It was evident that chronic HEX and CHP exposures along with MIX treatment severely retarded the number of clonogenic stromal precursors in the bone marrow of the respective animal groups. Qualitative as well as quantitative reduction of the clonogenic stromal precursor population also corroborated with the LTBMC generated stroma of these respective groups. Like LTBMC, cypermethrin treatment did not hamper in vitro CFU-F formation. So, like radiation or marrow ablation, chronic exposures to hexaconazole and chloropyriphos alone or in combination, causes marrow degeneration by destroying the hematopoietic microenvironment. However, possible additive effect of cypermethrin in combined formulation cannot be rule out immediately. In vitro hematopoietic commitment and clonogenicity assay revealed that exposures to either combined or individual pesticide reduced the quantum of primitive multilineage forming hematopoietic colonies like-CFUGEMM, CFU-GM, and BFU-E. Shortage of primitive multipotent progenitors was also found to affect the development of the unipotent progenitors which supported the observations of peripheral blood cytopenia (either unileneage cytopenia or pancytopenia) and hypocellularity in the marrow of pesticide-treated animals. Reduced number of primitive progenitors were the reflection of apoptosis and low self-renewal activity of LKS (LinSca1?Ckit?) population following chronic pesticide exposure. Self renewal, commitment and differentiation of the hematopoietic stem cells are only possible when they

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Mol Cell Biochem (2015) 401:115–131 Fig. 8 Effect of rmSHH on hematopoietic clonogenicity: a In first c row CFU-GEMM, CFU-GM, and BFU-E represents various primitive multipotent hematopoietic colonies generated during our differentiation experimentation. In the second row CFU-G, CFU-M, and CFUE represents various hematopoietic progenitor colonies. b Hematopoietic differentiation assay followed by in vitro supplementation of the recombinant sonic hedgehog (rmShh) in control and mixed pesticide-induced aplastic marrow. rmShh distinctly promotes the growth of primitive multipotent compartment including CFU-GEMM, CFU-GM and it has no growth promoting effect on the hematopoietic progenitor and precursor level including CFU-G and CFU-M. c In vitro supplementation of rmSHH directly promote the growth and proliferation of the stromal precursors through augmenting the count of the CFU-F in mixed pesticide-induced aplastic marrow and control samples than that of untreated one. Between groups comparison was performed by one-way ANOVA followed by post hoc Tukey test. Asterisks indicate the P values for number of CFU formation after rmSHH supplementation (*P \ 0.01; **P \ 0.05). Error bars indicate mean ± SD

break the dormancy and move from endosteal niche to perivascular niche of the bone marrow. However, MIX, HEX, and CYP treatment restricted the major portion of the medullary hematopoietic population in the dormant state (G0/G1 stage). Hence, the low differentiation and commitment of progenitors might be the reason of the aforementioned event. On the other hand, CHP treatment arrested the medullary hematopoietic cells in M phase and acted like mitotic blockers. Our in vitro flowcytometric experiments provide the compelling evidence for primitive hematopoietic cell intrinsic hedgehog signaling deregulation in pesticideexposed animal groups. We identified that different pesticide formulations either in combination or as individual preferentially target hematopoietic GLI1 transcription factor-heart of the hedgehog signaling network. Inhibition of GLI1 ultimately hampers intrasignaling transcriptional feedback mechanism of HH-GLI pathway. Proper nuclear translocation of GLI1 help to transcribe GLI1 itself (i.e., positive feedback mechanism) to rein the signaling cascade as well as transcribe PTCH1 (i.e., negative feedback mechanism) to limit the constitutive activation of the said signaling. Here, our experiments suggest that the process of GLI 1 antagonization is not same for all pesticide formulations. MIX treatment was found to suppress both positive and negative transcriptional feedbacks of GLI1 by upregulating SU(FU), PKCd and bTrCP. So the reason of reduced cytoplasmic GLI1 in the MIX group is overexpressed PKCd and bTrCP, which sequentially phosphorylate and degrade GLI1 proteins following its sequestration by SU(FU). In normal condition, bTrCP processed the GLI3 and GLI1 for further transcriptional repression [44]. However, our observation is similar to the recent findings, which revealed the bTrCP-mediated robust degradation of GLI2 instead of the processing [34]. Moreover, we also documented that MIX exposure caused downregulation of

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upstream positive regulator SMO but the reason was not clear from the model concerned. Subsequent experimentation with individual pesticide-treated model unveiled that chronic-protracted hexaconazole treatment drastically downregulates SMO expression in HEX group. The outcome of the said experimentation is concurrent with the study related to itraconazole (triazole fungicide)-mediated SMO repression in hedgehog driven tumor model [45]. In addition, choronic individual hexaconazole exposure was found to be involved in upregulation of PKCd and bTrCP. On the other hand, individual treatment group study also revealed that existence of chlorpyriphos in combined formulation was major responsible factor for upregulation of SU(FU) and subsequent GLI degradation. Role of cypermethrin is quite ambiguous with respect to hedgehog inhibition. Although cypermethrin try to suppress the GLI1 execution by upregulating GSK3b, it was circumvented by non-canonical activation of GLI1. Therefore, it has very subtle effect on hedgehog signaling during application with combined formulation. Like hematopoietic compartment, stromal Hh-Gli signaling is equally susceptible to chronic pesticide exposure. So, insufficient supply of microenvironmental paracrine hedgehog ligands and subsequent GLI1 decay inhibit autocrine-paracrine crosstalk of Hh-Gli signaling in between marrow hematopoietic stem and stromal compartment. To address the event, we attempted to rescue hematopoietic and non-hematopoietic stromal precursors in in vitro condition. In this experiment, we successfully altered stromal and hematopoietic degeneration by supplementing recombinant sonic hedgehog protein. However, recombinant sonic hedgehog protein was effective for primitive multipotent hematopoietic progenitors but it increased overall number of hematopoietic colonies in comparison to untreated sample. Our present result is consistent with the result of Bhardwaj et al. 2001 [39]. Finally, our findings provide compelling evidence that downregulated hedgehog signaling contribute to pesticidemediated bone marrow aplasia and suggest that further investigation of recombinant SHH as therapeutic option for targeting activation of hedgehog signaling in marrow aplasia be warranted. Furthermore, SU(FU) and GLI1 can be exploited as future theradiagnostic markers for early marrow aplasia diagnosis. Acknowledgments The authors are thankful to the Director, Calcutta School of Tropical Medicine. Conflict of interest The authors declare that they do not have any competing or financial interests. Funding This work is supported by the Council for Scientific and Industrial Research, Government of India (No. 37(1429)/10/EMRII).

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