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Altered canonical hedgehog-gli signalling axis in pesticide-induced bone marrow aplasia mouse model

The mechanistic interplay between pesticide exposure and development of marrow aplasia is not yet well established but there are indices that chronic pesticide exposure in some instances causes marrow aplasia like haematopoietic degenerative condition in human beings. Canonical Hedgehog (Hh) signalling has multiple roles in a wide range of developmental processes, including haematopoiesis. The present study was designed to explore the status of four important components of the canonical Hedgehog signalling cascade, the Sonic Hedgehog (Shh), Ptch1, Smo, and Gli1, in a mouse model of chronic pesticide-induced bone marrow aplasia. We used 5 % aqueous mixture of pesticides (chlorpyriphos, prophenophos, cypermethrin, alpha-methrin, and hexaconazole) for inhalation and dermal exposure of 6 hours per day and 5 days a week up to 90 days. Murine bone marrow aplasia related to chronic pesticide treatment was confi rmed primarily by haemogram, bone marrow cellularity, short term bone marrow explant culture for cellular kinetics, bone marrow smear, and fl ow cytometric Lin-Sca-1+C-kit+ extracellular receptor expression pattern. Later, components of hedgehog signalling were analysed in the bone marrow of both control and pesticide-treated aplastic groups of animals. The results depicted pancytopenic feature of peripheral blood, developmental anomaly of neutrophils, depression of primitive stem and progenitor population along with Shh, Ptch1, Smo and Gli1 expression in aplasia group. This investigation suggests that pesticide-induced downregulation of two critically important proteins - Ptch1 and Gli1 - inside the haematopoietic stem and progenitor cell population impairs haematopoietic homeostasis and regeneration mechanism in vivo concurrent with bone marrow aplasia.

A surge of numerous organophosphates, pyrethroids (analogs of naturally occurring phytopyrethrins), and thiazole fungicides replaced DDT after the ban in 1993. Depending on the physiological activity, synthetic pyrethroids are of two distinct types: Type I pyrethroids, which do not contain the α-cyano group, and Type II pyrethroids, which have the α-cyano group (1)(2)(3). Previous studies have affi rmed the genotoxic and/or cytotoxic effects of Type II pyrethroids such as cyfl uthrin, cypermethrin etc. (4)(5)(6)(7)(8)(9). The carcinogenic potential of pyrethroids was also reported on the murine model (9). A few recent studies revealed that besides blocking neurotransmission, both pyrethroids and organophosphate pesticides can cause immunosuppression and deregulation of many signalling molecules (10)(11)(12)(13)(14)(15). Organophosphates (derivatives of phosphonic acid) and systemic azole fungicides have profound cytotoxic activities, too (16,17). Despite a few epidemiological reports (18)(19)(20), we thus far have meagre understanding of the haematopoietic failure and the development of bone marrow aplasia by pesticide toxicity. Bone marrow aplasia represents a rare disorder of haematopoietic stem cell failure, often with severe life-threatening pathological consequences. More than half of the diagnosed cases of the aplastic anaemia are idiopathic, rest being acquired or inherited. Toxic chemicals, radiation, viral infections, and many other environmental factors are associated with frequent occurrence of acquired aplastic anaemia (18,(21)(22)(23)(24)(25)(26). Acquired bone marrow aplasia may arise due to one of the three events: (i) intrinsic stem cell defect due to cytotoxic chemicals, (ii) disturbed stem cell microenvironment or (ii) deregulated stem cell differentiation process. Our previous studies have confi rmed stem cell depletion in the pesticide exposed mouse model of marrow aplasia and in aplastic patients with history of chronic pesticide exposure (27)(28)(29).
The present study was planned to explore the probable mechanism of bone marrow aplasia development induced by chronic pesticide toxicity in mouse involving Hedgehog (Hh) signalling. Hedgehog-Gli signalling axis is an important cytosolic regulator that controls many fundamental processes from embryonic life to adulthood. Members of hedgehog family proteins are the key entities in growth patterning and morphogenesis of different regions within the body plan, from Drosophila to human beings. Intracellular Hedgehog signal transduction is executed through the binding of Hedgehog ligands to the transporter-like receptor, a tumour suppressor protein Patched 1 (Ptch1), which releases an inhibitory effect of its own on Smoothened (Smo), a seven pass transmembrane protein, and ultimately the signal goes down to the nucleus by Gli transcription factors thereby activating Hedgehog targeted genes (30)(31)(32)(33)(34)(35). Besides different activities, Hedgehog-Gli signalling axis controls primitive and defi nitive haematopoiesis during embryogenesis and postnatal condition (36)(37)(38)(39). In contrast, two contemporary articles reported that hedgehog signalling was dispensable for defi nitive haematopoiesis in conditionally deleted Smo -/in mice (40)(41). But, in support of Hh and definitive haematopoiesis, Trowbridge et al. (44) demonstrated that constitutively Hh activated Ptch -/+ in mice showed increased cycling and expansion of haematopoietic stem cells (HSC) under homeostatic conditions. On the other hand, Dierks et al. (45) showed that Smo -/in murine cells lost the colony forming ability at the second replating time, in contrast to the Ptch -/+ in murine cells where Smo was activated, which showed both regeneration and enhanced engraftment ability of the bone marrow result.
The involvement of Hedgehog signalling in definitive haematopoiesis ranges from normal haematopoiesis to differentiation and malignancy and these have been supported by numerous investigators (46)(47)(48)(49)(50)(51). Hedgehog activity specifi es cell fate either through long-range or short-range individual signalling and has also been associated with proliferative responses of target cells (31). Expression of Hedgehog in human haematopoietic population and cells comprising haematopoietic microenvironment suggests that Hedgehog proteins play a functional role in blood cells. Blood cells and haematopoietic microenvironment transduce Hedgehog signalling which has downstream Gli involvement (47). Therefore, the studies on self-renewal and expansion of HSC from bone marrow in pesticide-induced aplastic animal model were taken into consideration as an innovative experimental strategy to reveal the mechanism that lies beneath the Hh-Gli signalling pathway. In corroboration of the aforesaid studies related to hedgehog signalling and definitive haematopoiesis, we are the fi rst group to suggest that chronic long-term pesticide exposure downregulate Hh-Gli signalling axis, which accelerates bone marrow failure or aplasia in a mouse model.

Animals
Ten to twelve-week-old Swiss albino mice of both sexes (20 males and 20 females) for pesticide exposure purpose and 10 animals (5 males and 5 females) for control weighing 20 g to 24 g were selected from an inbred colony maintained under controlled room temperature (22±2) °C in the animal house of the Calcutta School of Tropical Medicine (India). Both male and female animals were equally used in all following assays. The animals were fed a standard recommended diet and water ad libitum, under standard conditions with a 12-hour light-dark period. Throughout the experiment, a maximum of six animals were housed in one cage containing sterile paddy husk as bedding. The procedures followed were in agreement with the approved guide for the care and use of laboratory animals, the Institutional Animal Ethical Committee (IAEC), and EU Directive 2010/63/EU.

Pesticide mixture preparation
In India, farmers and others use pesticides in a mixture and without any self-protection, so, we took a mixture of pesticides for the experimental purpose to mimic the normal exposure of individuals. We also observed and published the immunohaemotoxic effects of pesticide exposure where patients with aplastic anaemia, mainly farmers, used mixed pesticides (29). We used a 5 % aqueous mixture of DURSBAN (Chlorpyriphos 20 %; Dow Agrosciences, India), PROFEX SUPER (Prophenophos 40 % + Cypermethrin 4 %; Nagarjun Agrochemicals Ltd. India), STOP (alpha-Methrin 10 %; Biostadt, India), and Hexaconazole (Sigma-Aldrich, USA). The fi rst three easily available commercial pesticide preparations were of organophosphate and pyrethroid groups and were manufactured by local Indian agrochemical companies as mentioned above. All aforementioned chemicals were dissolved in aromatic hydrocarbon solvent as per information provided by the manufacturer on each pesticide container. The last one was a systemic fungicide rarely available in the local market as HEXACON SUPER (Hexaconazole 5.43 %). Due to its (HEXACON SUPER) paucity, we used Hexaconazole from Sigma-Aldrich, USA, in pure form and maintained the same concentration as in HEXACON SUPER.

Pesticide exposure
Adult mice (10 to 12 weeks old) were divided into two groups. Experimental animal group (N=40) received inhalation and dermal exposure [by handheld glass atomiser (local made), atomising time was 5 min] to 10 mL of 5 % aqueous solution of aforesaid pesticides mixture for 6 hours per day and 5 days a week up to 90 days (LD 50 of 10 % aqueous solution was found to be 20 days in our previous experiments). Control group (N=10) received inhalation and dermal exposure of aqueous saline solution without any trace of pesticide contamination over the same period of time (28). Altogether we took 24 exposed animals (8 animals per experiment) and 6 control animals (2 animals per experiment) for the following flow cytometric experiments which were repeated three times along with bone marrow cellularity assessment. The rest of the exposed animals (N=16) and control animals (N=4) were used for the bone marrow smear analysis and explant culture experiments. However, peripheral blood haemogram experiment was done for each and every animal as the investigation was related to the degeneration of haematopoietic machinery. Each haemogram experiment was performed with randomly selected fi ve control animals and twenty pesticidetreated animals at a time.

Blood haemogram profi le
We randomly selected experimentally pesticidetreated (N=20) and control (N=5) animals from respective cages at a time for blood haemogram profiling after completing 90-day exposure. Approximately 200 μL of blood was collected in heparinised vial by tail vein puncture from the animals. Total erythrocyte (RBC) count, total leukocyte (WBC) count, platelets, reticulocytes, differential WBC count, and haemoglobin estimation were performed manually as per standard laboratory protocol. Total RBC, WBC, and platelet count were performed by haemocytometer (Rohem, India) and binocular light microscope (Olympus Ch20i). Reticulocyte count was performed by Brilliant Cresyl Blue (Sigma) staining method and haemoglobin concentration was estimated by colorimetric (Systronics, India) cyanomethaemoglobin method. At the end, differential WBC count was done by Leishman staining (Himedia, India).

Bone marrow isolation and single cell preparation
The chronic pesticide-exposed experimental and control groups of mice were sacrifi ced to isolate the long bones (femur and tibia). Bone marrow was flushed out from the isolated bones by syringe (Dispovan 2 mL) containing RPMI-1640 (Sigma-Aldrich, USA) supplemented with 10 % FBS (Foetal Bovine Serum, Lonza, South American origin). Following bone marrow collection, a part of it was kept as undisturbed for explant culture and bone marrow smear analysis. Other part was subjected to single cell preparation for the assays such as total cellularity assessment, quantifi cation of bone marrowderived stem/progenitor cells, and status of hedgehog signalling in the bone marrow haematopoietic cells.

Bone marrow smears analysis
Bone marrows of the normal and exposed groups were Leishman stained (Himedia; India) and examined under light microscopy (1000x magnification; Olympus Ch20i) (52).

Bone marrow cellularity assessment
Femoral marrow cells were aseptically collected by fl ushing. Total cell counts of marrow single cell suspensions of both control and pesticide-exposed animals were then performed in Hemocytometer chamber (Rohem INDIA) by well known Erythrosin-B (Sigma-Aldrich, USA) dye exclusion method (53).
Bone marrow explants culture and cell release pattern study at (0, 24,48,72,96) hours Small portions of the isolated (described previously) marrow were cut into small pieces (0.2 mm 3 ) and subjected to culture in triplicate (for each of the normal and experimental groups) in 75 mm culture dish (Corning, USA) containing 3 mL of RPMI-1640 supplemented with 15 % FBS (Lonza, South American origin). The cultures were incubated at 37 º C inside CO 2 incubator and a time-course [(0, 24, 48, 72, 96) h] cell release pattern was monitored and calculated by the aid of haemocytometer (Rohem; India).

Flow cytometry Evaluation of murine bone marrow stem and progenitor population
To analyse haematopoietic stem cell population and signalling, 1x10 6 pooled cells from each group were fixed with 1.5 % para-formaldehyde (PFA, Ranbaxy, India) for 30 min at 37 °C in dark. To identify the murine haematopoietic progenitors, a PerCP-Cy5.5 TM conjugated Lineage (Lin) antibody cocktail (BD Pharmingen, USA) was used (CD3e, CD11b, CD45R/B220, TER-119, and Ly-6G and Ly-6C) in RBC depleted population for 20 min. This was followed by labelling with anti-mouse Sca-1-PE monoclonal antibody (BD-Bioscience, USA) and anti-CD117 antibody-FITC (BD-Bioscience, USA) in 1 % bovine serum albumin (Sigma), and dissolution in phosphate buffered saline (PBS) for another 30 min incubation. Furthermore, a single rapid wash was performed by PBS to remove unbound antibody from each sample. Samples were then analysed by BD-FACS Callibur (Becton Dickinson, USA) using CellQuest Pro software (v9.1 Becton-Dickinson, USA).

Flow cytometric analysis of Shh, Ptch1, Smo, and Gli1: components of canonical Hedgehog signalling pathway
Shh, Ptc1, and Gli1 proteins were stained by cell permeabilisation technique (27) in which 3x10 6 bone marrow haematopoietic cells were fi rst fi xed at room temperature in 1.5 % PFA and pelleted.

Statistical analysis
All the values of fl ow cytometric studies, cell release kinetics, and haemogram were represented as mean ± SD (Standard Deviation). Statistical analysis was performed by paired Student's t-test and the level of statistical signifi cance was P<0.05 and P<0.0001.

Bone marrow smear study
Bone marrow smear of control group of mice revealed normal haematopoietic cellular distribution (Figure 1a). On the other hand, pesticide-exposed bone marrow cell pool started to be replaced by stromal fi bres, large adipocytes, and empty spaces in contrast to the normal bone marrow (Figures 1b -1d).

Total bone marrow cellularity
Chronic pesticide exposure diminished (~2.5 fold) the total bone marrow cellularity of the pesticidetreated animals in comparison to the control group of animals. The difference in the bone marrow cellularity between control and pesticide-treated animals was statistically signifi cant (P<0.05) (Figure 2).

Figure 2 Total bone marrow cellularity study
Comparative whole bone marrow assessment of control and chronic pesticide-treated group of animals revealed a significant difference in its cellularity. Chronic pesticide exposure reduced the bone marrow cellular components approximately 2.5-fold in comparison to control group and observed difference was statistically verifi ed (P<0.05). Figure 1a shows the smear of a control bone marrow comprised of numerous haematopoietic cells at different stages of developmental cascade. In the pesticide-treated aplastic bone marrow (Figures  1b-1d), most of the spaces in smear are occupied by large adipocytes or stromal fi bres (arrow marked), which developed from pre-adipocytic fi broblasts and collagen, respectively, and very few haematopoietic cells are present there.   (Figures 3 and 4). However, the scenario of aplastic bone marrow explant was not same (Figures  3 and 4). At the beginning (0 h) and after 24 h of culture, very few cells were released from pesticide-

Hypocellularity was due to the shortage of stem/ progenitor cells in the bone marrow
The percentage of gated Lin -Sca-1 + C-kt + (LSK) cells, (0.47±0.05) % [i.e. (0.01±0.002) % of whole bone marrow] in the bone marrow, showed a nearly sevenfold decrease in the pesticide exposed animals (P<0.001) compared to control animals, (3.42±0.7) % [i.e. (0.07±0.05) % of whole bone marrow] ( Figure  5). It signifies that chronic pesticide exposures severely hampered the primitive haematopoietic stem cell compartment and its effects were manifested through hypocellular bone marrow and pancytopoenia in the treated group of animals.

Pesticide-mediated deregulation of canonical Hedgehog signalling: latent cause of bone marrow hypocellularity and degeneration
There was a severe depression in the expression of Ptch1 (Mean Fluorescence Intensity, MFI=320.  chemicals also harm a broad range of non-targeted organisms including humans. The widespread use of pesticides means we face a continuous risk of exposure to these compounds, which can have several adverse effects. In such cases previous research elucidated the mechanistic correlation of pesticide exposure and neurological disorders. Reports concerning the haematopoietic system, on the other hand, are scanty (4)(5)(6)(7)(27)(28)(29). Here we considered adult defi nitive haematopoietic system i.e. bone marrow as our organ of interest to study the mechanism of long term pesticide exposure through inhalation and haematopoietic failure. The present study revealed an overall diminution of peripheral haemogram parameters and it led us to propose an inference that chronic pesticide exposure hindered neutrophilopoiesis and distorted the morphology of nucleus along with leucopoenia. Low haemoglobin level and diminished total RBC were the ultimate results of impaired erythropoiesis and  (Figures 6a and 6b).
Smo, another transmembrane component and a positive regulator of Hedgehog signalling pathway also faced downregulation in aplastic condition in comparison with the control group (Figure 6c). Figure 6d shows further downregulation of Gli-1 expression in the aplastic bone marrow. Here, the flow cytometric expression estimation of Hh signalling components was performed on the basis of an analysis gate, which included mostly stem and progenitor population of scattergram. After gating MFI was determined by histogram analysis.  (P<0.0001) ( Table 2). Here, MFI of epitope-bound fl uorophore-tagged antibodies was directly proportional to the concerned protein expression in the subjected cells measured by fl ow cytometry (Figures 6a-6d, Table 2).

DISCUSSION
Agrochemicals like pesticides are designed to target agricultural pests but incidentally these generation of low number of reticulocytes, an immediate progenitor of mature RBC, which signifi ed the presence of aplasia in pesticide exposed mice. On the other hand, a declining state of haematopoietic cells was observed in aplastic marrow smear with scattered lymphocytes and numerous large adipocytes which developed from pre-adipocytic fi broblasts, a special feature of aplastic bone marrow (Figures 1b and 1c). Stromal collagenous fi bres were also evident in experimental bone marrow. These large adipocytes and stromal fi bres actually replaced the empty spaces of bone marrow produced by long-term pesticide treatment. Furthermore, chronic pesticide inhalation also reduced the total bone marrow cellular components in comparison to the control mice.
Time-bound and comparative bone marrow explant cultures of two aforesaid groups of animals represented a very authentic scenario regarding pesticide exposure and bone marrow cell release pattern under in vitro condition. Hypocellularity of pesticide-exposed bone marrow was further established by the present experimental effort. Unlike its normal counterpart, pesticide-exposed bone marrow explant released its cell pool very slowly and in low quantity over time. The low cell-releasing pattern was also an evidence of hypocellularity of bone marrow, which indicated the shortage of marrow haematopoietic cellular storage, low self-renewal, and proliferation activity. Bone marrow explant culture study also exhibited a phenomenon that the pesticide-exposed sample, following a 48-hour lagging period, starts releasing the haematopoietic cells which could not thrive under in vitro condition as compared to the normal counterpart. This finding reinforces the fact that chronic pesticide exposure hampers the cellular proliferation rhythm, which ultimately delays the cell release behaviour in comparison to normal bone marrow ( Figure 3).
The reduced proliferation, as well as depletion of haematopoietic stem and progenitor cells, added an extra dimension to the reason behind marrow hypocellularity, impaired neutrophilopoiesis, erythropoiesis, and ultimately the occurrence of peripheral blood pancytopoenia. Besides, it also explained how progressive loss of primitive Lin -Sca-1 + C-kit + (LSK) haematopoietic population in endosteal niche or bone marrow microenvironment of pesticideexposed mice failed to replenish the pancytopoenia in the vascular niche, as a result of which we observed a significant diminution in peripheral blood elements.
The reason behind the shortage of self-renewing, proliferating haematopoietic stem cells in the bone marrow of pesticide-induced aplastic mice is mostly unexplained. We tried to study the event by our fl ow cytometric analysis (Figure 6) of the expression pattern of important protein components related to the canonical Hedgehog-Gli1 signalling axis. As we were already aware that hedgehog signalling had a profound effect on embryogenesis and adult tissue homeostasis, we considered the expression status of Shh, Ptch-1, Smo, and Gli1, a transcription factor of the zinc fi nger family, which activates the hedgehog targeted genes in the haematopoietic stem-progenitor rich compartment. In 2009, Hoffamann et al. (40) and Gao et al. (41) separately reported that the Hh pathway was dispensable for the adult HSC maintenance and differentiation process. However, their fi ndings were not consistent with several studies by Trowbridge et al. (44) (42). Irrespective of their controversial studies, we observed sharp downregulation in Hh signalling cascade from the upstream regulator Ptch-1 to the downstream executor Gli-1 along with its ligands Shh in our pesticideinduced aplastic murine bone marrow haematopoietic compartment. It is known to us that Ptch-1 is a protooncogene which affects cell cycle as well as lineage commitment of haematopoietic stem cells (34). In our study, diminished expression of Ptch-1 in pesticide-induced aplastic bone marrow refl ected low conversion of primitive LSK population to more mature progenitors and blood corpuscles in peripheral circulation.
Diminution of bone marrow LSK population and downregulation of Smo expression in the pesticideexposed animals presented a parallel observation with the study of Zhao et al. who showed that Smo -/and cyclopamine (Smo inhibitor) treatment in mice drastically reduced bone marrow LSK population in comparison to the control (46). Downregulation of Smo expression in our study might be the direct possible manifestation of pesticide exposure. We suspect that the Hexaconazole, a systemic fungicide of the ′azole′ family used in our experiment, possibly acted as an inhibitor for Smo downregulation in pesticide-induced bone marrow failure because it has been found that Itraconazole, another clinically well known systemic fungicide of the azole family, showed Smo antagonism in a recent study on murine tumour (16). Smo failed to transduce the signal up to the fi nal downstream executor Gli1, which was seen as a usual depressive status in pesticide-induced aplastic bone marrow in contrast to its normal counterpart ( Figure  6).
Reduced Gli1 expression also affected the hedgehog pathway by downregulating the intracellular feedback loop of Ptch1 and Gli1. Under normal conditions, Gli1 protein helps to transcribe Gli1 by means of a positive feedback mechanism and also upregulates the signalling repressor Ptch1, a negative feedback that reins the Gli1 regulatory loop. It actually represents a signalling network composed of a positive transcriptional feedback loop embedded within a negative signalling feedback loop.
Our present experimental data revealed a steady dampening condition in the Gli1-Ptch1 feedback loop, in which downregulation of Gli-1 gene product automatically suppressed the Ptch-1 expression by direct action of pesticide-deregulated Smo repression. Henceforth, it is clear that chronic pesticide exposure also hampers the direct feedback loop between Gli-1 and Ptch-1 inside the bone marrow haematopoietic stem/progenitor cell population, in the context of Smo downregulation, which leads to a progressive degeneration of haematopoietic machinery. In these contexts, the altered internal regulatory loop between Ptch1-Smo-Gli1 of Hedgehog signalling crippled the self-renewal, proliferation, and differentiation capacity of haematopoietic stem cells and resulted in bone marrow suppression. However, future studies might be designed to further elucidate the role of Hedgehog signalling in the pathology of the pesticide-induced bone marrow toxicity by constitutive over-expression of the active components of the Hh signalling pathway in the stem cells. Only then could one determine whether this approach rescues the marrow failure observed in our established animal model.

CONCLUSION
At the end we conclude that the unresolved paradox of the development of intrinsic stem cell defect of acquired aplastic anaemia through chronic pesticide exposure lies in the deregulation of the canonical Hedgehog-Gli1 signalling cascade. Besides, commercially available pesticides are harmful not only to the nervous system of the non-target organisms but also to other developmentally important signalling cascades like Hedgehog signalling, which functions as "stem cell fate switch". Finally, our experiment suggests the possibility of using primarily Gli-1 as the biomarker for those patients who are chronically exposed to pesticides in agricultural fi eld and are either suffering from aplastic anaemia or having propensity towards the development of bone marrow aplasia.