ABSTRACT
Pesticides have increased crop yield but severely impacted ecosystems and non-target organisms. Flubendiamide, a new generation pesticide, targets insect larvae but also affects non-target organisms. This study examines the effects of lowest observed effect concentration of technical grade flubendiamide (0.5⯵g/µL) flubendiamide on chick liver, focusing on cytochrome P450 (CYP) enzyme expression, oxidative stress, and liver damage. Chick embryos treated with flubendiamide showed significant alterations in CYP mRNA and protein levels, indicating increased toxicant accumulation. Elevated CYP3A4, CYP1A1, CYP1A2, and CYP2C19 levels were noted, suggesting enhanced biotransformation and detoxification processes. However, increased oxidative byproducts led to oxidative stress, as evidenced by decreased glutathione (GSH) levels and elevated superoxide dismutase (SOD) and catalase activities. DCFDA staining confirmed increased hydrogen peroxide (H2O2) levels, indicating heightened reactive oxygen species (ROS). Liver function tests revealed significant increases in serum ALP, ALT, and AST levels, indicating acute liver damage. Histopathological analysis showed structural liver damage, including expanded sinusoidal spaces, impaired portal veins, and compromised hepatocyte architecture. These findings underscore flubendiamide's potential hepatotoxicity in non-target organisms, emphasizing the need for cautious pesticide use to minimize environmental impacts.
ABSTRACT
Thiourea, a widely used agrochemical, is known to inhibit the activity of thyroid peroxidase, a key enzyme in the biosynthetic pathway of thyroid hormones. Thyroid insufficiency compromises the basal metabolic rate in warm-blooded organisms and embryonic development in vertebrates. In this study, we looked for developmental defects by exposing the zebrafish embryos to an environmentally relevant dose of thiourea (3 mg/mL). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to validate thiourea's presence in the treated zebrafish embryos. Structural anomalies like bent tail and pericardial edema were noticed in 96-h post-fertilization (hpf) larvae. On histological examination, underdeveloped swim bladder was noticed in 96 hpf larvae exposed to 3 mg/mL thiourea. The treated larvae also failed to follow the characteristic swimming behavior in response to stimuli due to defective swim bladder. Swim bladder being homologous to the lung of tetrapod, the role of Bmp4, a major regulator of lung development, was studied along with the associated regulatory genes. Gene expression analysis revealed that thiourea administration led to the downregulation of bmp4, shh, pcna, anxa5, acta2, and the downstream effector snail3 but the upregulation of caspase3. The protein expression showed a similar trend, wherein Bmp4, Shh, and Pcna were downregulated, but Cleaved Caspase3 showed an increased expression in the treated group. Therefore, it is prudent to presume that exposure to thiourea significantly reduces the expression of Bmp4 and other key regulators; hence, the larvae fail to develop a swim bladder, a vital organ that regulates buoyancy.
ABSTRACT
The northern house gecko Hemidactylus flaviviridis exhibits appendage-specific responses to injuries. The autotomized tail regenerates, whereas the severed limb fails to regrow. Many site-specific cellular processes influence tail regeneration. Herein, we analyzed the epithelial-mesenchymal transition contrast in the lizard's amputated appendages (tail and limb). Morphological observations in the healing frame indicated the formation of regeneration blastema in the tail and scar formation in limb. Histology of the tail showed that epithelial cells closer to mesenchyme appeared less columnar and loosely packed, with little intercellular matrix. Whereas in the limb, the columnar epithelial cells remained tightly packed. Collagen deposition was seen in the limb at the intersection of wound epithelium and mesenchyme, favoring scarring by blocking the epithelial-mesenchymal transition. Markers for epithelial-mesenchymal transition were assessed at transcript and protein levels. The regenerating tail showed upregulation of N-cadherin, vimentin, and PCNA, favoring epithelial-mesenchymal transition, cell migration, and proliferation, respectively. In contrast, the scarring limb showed persistently elevated levels of E-cadherin and EpCAM, indicating retention of epithelial characteristics. An attempt was made to screen the resident epithelial stem cell population in both appendages to check their potential role in the epithelial-mesenchymal transition (EMT), hence the differential wound healing. Upregulation in transcript and protein levels of Nanog and Sox2 was observed in the regenerating tail. Fluorescence-activated cell sorting (FACS) provided supporting evidence that the epithelial stem cell population in tail remained significantly higher than in limb. Thus, this study focuses on the mechanistic role of the epithelial-mesenchymal transition in wound healing, highlighting the molecular details of regeneration and scarring events.
Subject(s)
Epithelial-Mesenchymal Transition , Extremities , Lizards , Regeneration , Tail , Animals , Lizards/metabolism , Epithelial-Mesenchymal Transition/physiology , Extremities/physiology , Regeneration/physiology , Amputation, SurgicalABSTRACT
Duchenne muscular dystrophy (DMD) is the most common form of progressive childhood muscular dystrophy associated with weakness of limbs, loss of ambulation, heart weakness and early death. The mutations causing either loss-of-expression or function of the full-length protein dystrophin (Dp427) from the DMD gene are responsible for the disease pathology. Dp427 forms a part of the large dystroglycan complex, called DAPC, in the sarcolemma, and its absence derails muscle contraction. Muscle biopsies from DMD patients show an overactivation of excitation-contraction-coupling (ECC) activable calcium incursion, sarcolemmal ROS production, NHE1 activation, IL6 secretion, etc. The signalling pathways, like Akt/PBK, STAT3, p38MAPK, and ERK1/2, are also hyperactive in DMD. These pathways are responsible for post-mitotic trophic growth and metabolic adaptation, in response to exercise in healthy muscles, but cause atrophy and cell death in dystrophic muscles. We hypothesize that the metabolic background of repressed glycolysis in DMD, as opposed to excess glycolysis seen in cancers or healthy contracting muscles, changes the outcome of these 'growth pathways'. The reduced glycolysis has been considered a secondary outcome of the cytoskeletal disruptions seen in DMD. Given the cytoskeleton-crosslinking ability of the glycolytic enzymes, we hypothesize that the failure of glycogenolytic and glycolytic enzymes to congregate is the primary pathology, which then affects the subsarcolemmal cytoskeletal organization in costameres and initiates the pathophysiology associated with DMD, giving rise to the tissue-specific differences in disease progression between muscle, heart and brain. The lacunae in the regulation of the key components of the hypothesized metabolome, and the limitations of this theory are deliberated. The considerations for developing future therapies based on known pathological processes are also discussed.
Subject(s)
Glycogenolysis , Muscular Dystrophy, Duchenne , Humans , Child , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/pathology , Costameres/metabolism , Costameres/pathology , Dystrophin/genetics , Dystrophin/metabolism , Muscles/metabolism , Muscles/pathology , Sarcolemma/metabolism , Sarcolemma/pathology , Muscle, Skeletal/metabolismABSTRACT
Cyclooxygenase-2 (COX-2), a member of the Cyclooxygenase family, initiates the biosynthesis of prostanoids that regulates various cellular functions. Our pilot attempt revealed that the administration of etoricoxib, an inhibitor specific for COX-2, induces abnormal looping in the chicken heart. The present study attempts to reveal the mechanistic details of etoricoxib-induced abnormal cardiac looping. The activity of COX-2 was inhibited by administering 3.5 µg of etoricoxib into the egg's air cell on day zero of incubation. The gene and protein expression patterns of key mediators of heart development were then analyzed on day 2 (HH12) and day 3 (HH20). Reduced COX-2 activity altered the expressions of upstream regulators of organogenesis like Wnt11, BMP4, and SHH in the etoricoxib-exposed embryos. The observed expression shifts in the downstream regulators of myocardial patterning (MYOCD, HAND2, GATA4, GATA5, and GATA6) in the treated embryos corroborate the above results. In addition, the reduction in COX-2 activity hampered cardiomyocyte proliferation with a concomitant increase in the apoptosis rate. In conclusion, the collective effect of altered expression of signaling molecules of myocardial patterning and compromised cardiomyocyte turnover rate could be the reason behind the looping defects observed in the heart of etoricoxib-treated chick embryos.
Subject(s)
Chickens , Myocardium , Animals , Chick Embryo , Cyclooxygenase 2/genetics , Etoricoxib , Myocytes, CardiacABSTRACT
Across the animal kingdom, lizards are the only amniotes capable of regenerating their lost tail through epimorphosis. Of the many reptiles, the northern house gecko, Hemidactylus flaviviridis, is an excellent model system that is used for understanding the mechanism of epimorphic regeneration. A stage-specific transcriptome profile was generated in the current study following an autotomized tail with the HiSeq2500 platform. The reads obtained from de novo sequencing were filtered and high-quality reads were considered for gene ontology (GO) annotation and pathway analysis. Millions of reads were recorded for each stage upon de novo assembly. Up and down-regulated transcripts were categorized for early blastema (EBL), blastema (BL) and differentiation (DF) stages compared to the normal tail (NT) by differential gene expression analysis. The transcripts from developmentally significant pathways such as FGF, Wnt, Shh and TGF-ß/BMP were present during tail regeneration. Additionally, differential expression of transcripts was recorded from biological processes, namely inflammation, cell proliferation, apoptosis and cell migration. Overall, the study reveals the stage-wise transcriptome analysis in conjunction with cellular processes as well as molecular signaling pathways during lizard tail regeneration. The knowledge obtained from the data can be extrapolated to configure regenerative responses in other amniotes, including humans, upon loss of a complex organ.
ABSTRACT
The role of COX-2 induced PGE2 in the site-specific regulation of inflammatory mediators that facilitate disparate wound healing in the tail and limb of a lizard was studied by analysing their levels during various stages of healing. The activity of COX-2 and concentration of PGE2 surged during the early healing phase of tail along with the parallel rise in EP4 receptor. PGE2-EP4 interaction is corelated to early resolution (by 3 dpa) of inflammation by rising the antiinflammatory mediator IL-10. This likely causes reduction in proinflammatory mediators viz., iNOS, TNF-α, IL-6, IL-17 and IL-22. Conversely, in the limb, COX-2 derived PGE2 likely causes rise in inflammation through EP2 receptor-based signalling, as all the proinflammatory mediators stay elevated through the course of healing (till 9 dpa), while expression of IL-10 is reduced. This study brings to light the novel roles of IL-17 and IL-22 in programming wound healing. As IL-17 reduces in tail, IL-22 behaves in reparative way, causing conducive environment for scar-free wound healing. On the contrary, synergic elevation of both IL-17 and Il-22 form a micro-niche suitable for scarred wound healing in limb, thus obliterating its regenerative potential.
Subject(s)
Cyclooxygenase 2/metabolism , Cytokines/metabolism , Extremities , Lizards , Tail , Wound Healing/physiology , Animals , Cyclooxygenase 2/genetics , Cytokines/genetics , Female , Gene Expression Regulation/physiology , MaleABSTRACT
A recent study from our lab revealed that the inhibition of cyclooxygenase-2 (COX-2) exclusively reduces the level of PGE2 (Prostaglandin E2) among prostanoids and hampers the normal development of several structures, strikingly the cranial vault, in chick embryos. In order to unearth the mechanism behind the deviant development of cranial features, the expression pattern of various factors that are known to influence cranial neural crest cell (CNCC) migration was checked in chick embryos after inhibiting COX-2 activity using etoricoxib. The compromised level of cell adhesion molecules and their upstream regulators, namely CDH1 (E-cadherin), CDH2 (N-cadherin), MSX1 (Msh homeobox 1), and TGF-ß (Transforming growth factor beta), observed in the etoricoxib-treated embryos indicate that COX-2, through its downstream effector PGE2, regulates the expression of these factors perhaps to aid the migration of CNCCs. The histological features and levels of FoxD3 (Forkhead box D3), as well as PCNA (Proliferating cell nuclear antigen), further consolidate the role of COX-2 in the migration and survival of CNCCs in developing embryos. The results of the current study indicate that COX-2 plays a pivotal role in orchestrating craniofacial structures perhaps by modulating CNCC proliferation and migration during the embryonic development of chicks.
ABSTRACT
Temporal expression patterns and activity of two cyclooxygenase (COX-1 and COX-2) isoforms were analysed during early chick embryogenesis to evaluate their roles in development. COX-2 inhibition with etoricoxib resulted in significant structural anomalies such as anophthalmia (born without one or both eyes), phocomelia (underdeveloped or truncated limbs), and gastroschisis (an opening in the abdominal wall), indicating its significance in embryogenesis. Furthermore, the levels of PGE2, PGD2, PGF2α, and TXB2 were assessed using quantitative LC-MS/MS to identify which effector prostanoid (s) had their synthesis initiated by COX-2. COX-2 inhibition was only shown to reduce the level of PGE2 significantly, and hence it could be inferred that the later could be largely under the regulation of activated COX-2 in chick embryos. The compensatory increase in the activity of COX-1 observed in the etoricoxib-treated group helped to maintain the levels of PGD2, PGF2α, and TXB2. Though the roles of these three prostanoids in embryogenesis need to be further clarified, it appears that their contribution to the observed developmental anomalies is minimal. This study has shown that COX-2 is functionally active during chick embryogenesis, and it plays a central role in the structural configuration of several organs and tissues through its downstream effector molecule PGE2.
Subject(s)
Avian Proteins/metabolism , Chick Embryo/embryology , Cyclooxygenase 2/metabolism , Dinoprostone/metabolism , Animals , Avian Proteins/genetics , Chick Embryo/abnormalities , Chick Embryo/drug effects , Chick Embryo/metabolism , Chickens , Cyclooxygenase 2/genetics , Cyclooxygenase 2 Inhibitors/pharmacology , Gene Expression Regulation, Developmental/drug effectsABSTRACT
Exposure to chlorpyrifos-cypermethrin combination during early development resulted in defective looping and ventricular noncompaction of heart in domestic chicken. The study was extended to elucidate the molecular basis of this novel observation. The primary culture of chicken embryonic heart cells showed a concentration-dependent loss of viability when challenged with this combination of technical-grade insecticides. Comet assay, DNA ladder assay, and analyses of appropriate markers at transcript and protein levels, revealed that chlorpyrifos-cypermethrin combination induced cell death by activating apoptosis. Parallelly, the tissues derived from control and experimental group hearts were checked for apoptotic markers, and the result was much similar to that of the in-vitro study. Further analysis showed that chlorpyrifos-cypermethrin combination deranged the expression pattern of the transcriptional regulators of cardiogenesis, namely TBX20, GATA5, HAND2, and MYOCD. This, together with heightened apoptosis, could well be the reason behind the observed structural anomalies in the heart of chlorpyrifos-cypermethrin poisoned embryos.
Subject(s)
Chlorpyrifos/toxicity , Embryonic Development/drug effects , Gene Expression Regulation, Developmental/drug effects , Heart/drug effects , Insecticides/toxicity , Pyrethrins/toxicity , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Survival/drug effects , Cell Survival/genetics , Cells, Cultured , Chick Embryo , Chickens , Chlorpyrifos/administration & dosage , Comet Assay , DNA Damage , Dose-Response Relationship, Drug , Female , Heart/embryology , Insecticides/administration & dosage , Myocardium/cytology , Myocardium/pathology , Pyrethrins/administration & dosageABSTRACT
New chemotherapeutic agents with minimum side effects are indispensable to treat non-small-cell lung cancer (NSCLC) since the mortality rate of patients suffering from NSCLC remains high despite receiving conventional medication. In our previous study, many coumarin derivatives were screened for their anticancer properties in A549, an in vitro NSCLC model. One of these, 4-flourophenylacetamide-acetyl coumarin (4-FPAC), induced cytotoxicity at a concentration as low as 0.16 nM. Herein, initially, the cytotoxic potential of 4-FPAC was tested on a noncancerous cell line NIH3T3 and was found safe at the selected dose of 0.16 nM. Further, we investigated the mechanism by which 4-FPAC induced cytotoxicity and arrested the progression of cell cycle as well as metastasis in A549. Results of ethidium bromide/acridine orange (EtBr/AO), 4,6-diamidino-2-phenylindole, comet, and lactate dehydrogenase assays revealed that 4-FPAC caused cytotoxicity via reactive oxygen species-induced p53-mediated mechanism, which involves both extrinsic and intrinsic pathways of apoptosis. Dichlorodihydrofluorescein diacetate, rhodamine 123, and AO staining confirmed the involvement of both mitochondria and lysosome in inducing apoptosis. However, flow cytometric analysis revealed that it causes cell cycle arrest at the G0/G1 phase by modulating p21, CDK2, and CDK4 expression. Aggregation, soft-agar, clonogenic, and scratch assays as well as gene expression analysis collectively confirmed that 4-FPAC minimizes the metastatic property of A549 by downregulating Snail, matrix metalloproteinase 9, and interleukin-8. Additional studies reaffirmed the above findings and substantiated the role of PI3K/AKT in achieving them. The cell-type-specific selective cytostatic and antimetastatic properties shown by 4-FPAC indicate its potential to emerge as a drug of choice against NSCLC in the future.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , G1 Phase/drug effects , Neoplasm Metastasis/prevention & control , Proto-Oncogene Proteins c-akt/metabolism , Reactive Oxygen Species/metabolism , Resting Phase, Cell Cycle/drug effects , Signal Transduction/drug effects , A549 Cells , Apoptosis/genetics , Comet Assay , HumansABSTRACT
Lizards restore their lost tail by the recruitment of multipotent cells which are selectively differentiated into varied cell types so as to sculpt a new tail. The precise coordination of the events involved in this complex process requires crosstalk between many signaling molecules and differential regulation of several mediators that facilitate the achievements of various milestones of regeneration. Fibroblast growth factor-2 is one such signaling molecule which activates a number of intracellular signaling pathways. Herein, the regulatory role of FGF2 during tail regeneration in Hemidactylus flaviviridis was investigated. Upon inhibition of FGFR using SU5402, the FGF2 levels were found to be significantly reduced at both transcript and protein level. Further, the compromised levels of the gelatinases, namely MMP2 and MMP9 in the tail tissues of treated lizards indicate that FGF2 regulates the activity of these enzymes perhaps to facilitate the recruitment of multipotent mesenchymal cells (blastema). The in vivo 5BrdU incorporation assay showed a lower cell proliferation rate in FGF2 signal inhibited animals during all the proliferative stages of regeneration studied. This observation was substantiated by decreased levels of PCNA in treated group. Moreover, from the combined results of Caspase-3 localization and its expression levels in the regenerates of control and SU5402 treated lizards it can be deduced that FGF2 signal regulates apoptosis as well during early stages of regeneration. Overall, the current study indicates beyond doubt that FGF2 signaling plays a pivotal role in orchestrating the matrix reorganization and cell cycle turnover during lizard tail regeneration.
Subject(s)
Cell Cycle , Extracellular Matrix/pathology , Fibroblast Growth Factor 2/metabolism , Lizards/metabolism , Regeneration , Reptilian Proteins/metabolism , Signal Transduction , Tail/physiology , AnimalsABSTRACT
Pesticides despite being agents that protect the plants and humans from noxious pests, are infamous for their potential to cause detrimental health issues in nontargeted species. In order to ascertain the latter, a set of experiments were conducted by exposing early chick embryos to a widely used combination insecticide (Ci, 50% chlorpyrifos and 5% cypermethrin). The results revealed a myriad of congenital defects pertaining to craniofacial development such as anophthalmia, microphthalmia, exencephaly as well as deformed beak and cranial structures. These teratological manifestations could be attributed to the Ci induced alteration in the titre of major regulators of neurulation and ossification. Therefore, the mRNA and/or the protein level expression pattern of genes which are reported to be involved in the craniofacial development were studied at selected time points of embryonic development. The analysis of the result showed that there have been significant alternations in the expression patterns of the signalling molecules such as SHH, WNTs, CDH1, CDH2, L1CAM, PAX6, HOX, PCNA, GLI3, BMP7, FGF8, GLIs, SOX9, RUNX2, DLX5, COL10A1, CASPASE3 etc. on embryonic days 2, 4 and/or 10. Concurrently, on day 10, whole-mount skeletal staining and biochemical estimation of hydroxyproline were carried out in the cranial tissues of the embryos. The overall result of the current study indicates that exposure to Ci during early development impede the crucial regulatory signals that orchestrate the morphogenesis of cranial neural crest cells thereby hindering the normal progression of neural tube and endochondral ossification which collectively lead to craniofacial dysmorphism in domestic chicks.
Subject(s)
Craniofacial Abnormalities/chemically induced , Insecticides/toxicity , Signal Transduction/drug effects , Signal Transduction/genetics , Animals , Beak/abnormalities , Brain Chemistry/drug effects , Chick Embryo , Chickens , Chlorpyrifos/toxicity , Craniofacial Abnormalities/mortality , Craniofacial Abnormalities/physiopathology , Female , Gene Expression Regulation, Developmental/drug effects , Hydroxyproline/metabolism , Pregnancy , Prenatal Exposure Delayed Effects , Pyrethrins/toxicity , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
Studies using fish fin as a model to understand the nuance of epimorphosis are gaining interest of lately. This study illustrates for the first time the daily changes in the tissue architecture of regenerating tail fin of Poecilia latipinna. Wound epithelium is formed within 24 hpa that eventually gets stratified into apical epithelial cap by 48 hpa. In the subsequent day, proliferating cells accumulate in front of each fin-ray marking the beginning of blastema. Distally these cells express signs of cartilage condensation by 4 dpa. However, ossification and subsequent transformation of actinotrichia to lepidotrichia was observed on 5 dpa. Subsequently, the regenerate grew at variable rate until it achieved the original size on 25 dpa. This result would serve as a worthwhile standard reference for further explorative studies that demand manipulation of a regulatory signal at a defined time point.
Subject(s)
Animal Fins/growth & development , Poecilia/growth & development , Regeneration , Animals , Bone Development , Cartilage/growth & development , Cell Proliferation , Epithelium/growth & development , MorphogenesisABSTRACT
Bone morphogenetic proteins play a pivotal role in the epimorphic regeneration in vertebrates. Blastema formation is central to the epimorphic regeneration and crucially determines its fate. Despite an elaborate understanding of importance of Bone morphogenetic protein signaling in regeneration, its specific role during the blastema formation remains to be addressed. Regulatory role of BMP signaling during blastema formation was investigated using LDN193189, a potent inhibitor of BMP receptors. The study involved morphological observation, in vivo proliferation assay by incorporation of BrdU, comet assay, qRT-PCR and western blot. Blastemal outgrowth was seen reduced due to LDN193189 treatment, typified by dimensional differences, reduced number of proliferating cells and decreased levels of PCNA. Additionally, proapoptotic markers were found to be upregulated signifying a skewed cellular turnover. Further, the cell migration was seen obstructed and ECM remodeling was disturbed as well. These findings were marked by differential transcript as well as protein expressions of the key signaling and regulatory components, their altered enzymatic activities and other microscopic as well as molecular characterizations. Our results signify, for the first time, that BMP signaling manifests its effect on blastema formation by controlling the pivotal cellular processes possibly via PI3K/AKT. Our results indicate the pleiotropic role of BMPs specifically during blastema formation in regulating cell migration, cell proliferation and apoptosis, and lead to the generation of a molecular regulatory map of determinative molecules.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Poecilia/physiology , Animal Fins/drug effects , Animals , Bone Morphogenetic Proteins/genetics , Cell Cycle , DNA Fragmentation , Extracellular Matrix , Female , Male , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RegenerationABSTRACT
Currently, scientists show keen interest in the drugs that inhibit multiple kinases, LDN193189, being an example. It combats certain cancers in vitro as well as in vivo, making it a prerequisite for researchers to study the toxic potential of this drug in animal models. As most of the drugs metabolized by liver cause hepatic injury, LDN193189-induced hepatotoxicity was examined using a teleost fish, Poecilia latipinna. As a prelude, calculation of LD50 showed a value of 95.22 mg/kg body weight and three doses were decided based on it for further evaluations. All these groups were tested for antioxidant enzyme levels and were significantly raised for mid- and high-dose group. Similar trend was recorded for ALP, AST, and ALT levels. Furthermore, some key indicators of drug metabolism in liver were tested for their expression in response to LDN193189 treatment. Among these, Cyt-C, CYP3A4, CYP1B1 and CYP1A1 were elevated in mid- and high dose, except CYP21A1, which declined remarkably. Moreover, histological profile of the liver reflected high degree of inflammation due to drug treatment, but this was found only at high dose. In summary, LDN193189, at 2.5 mg/kg body weight, did not cause any adverse hepatotoxicity, rendering it safe for use as an anti-proliferative agent - an activity for which it has already shown promising results in the same animal model. The low-dose group previously studied for its anti-proliferative property showed no adverse effect in liver, whereas the mid- and high dose induced moderate or severe hepatotoxicity in P. latipinna.
Subject(s)
Antineoplastic Agents/toxicity , Chemical and Drug Induced Liver Injury/etiology , Pyrazoles/toxicity , Pyrimidines/toxicity , Animals , Antineoplastic Agents/administration & dosage , Antioxidants/metabolism , Chemical and Drug Induced Liver Injury/pathology , Dose-Response Relationship, Drug , Female , Lethal Dose 50 , Liver/drug effects , Liver/pathology , Male , Poecilia , Pyrazoles/administration & dosage , Pyrimidines/administration & dosageABSTRACT
Chick embryonic cells can be used to develop an easy and economical in vitro model for conducting studies on the disease muscle dystrophy (MD). For this, the limb myoblasts from 11th day chick embryo were isolated and cultured. To this muscle cell culture, anti-dystroglycan antibody (IIH6) was added so as to target the α-dystroglycan and disrupt the connection between the cytoskeletal proteins and the extracellular matrix (which is a characteristic feature of MD). Cells were allowed to differentiate further and the morphometrics and mRNA expression were studied. The IIH6-treated muscle cells displayed changes in morphometry, contractibility, and also atrophy was observed when compared to the control cultures. Concomitant gene expression studies showed an upregulation in TGF-ß expression, while the muscle sculpture genes MYOD1, MYF5, LAMA2 and MYOG were downregulated resembling the MD in vivo. This simple and cost-effective method can be useful in studies to further understand the disease mechanism and also in conducting initial studies on effect of novel pharmacological agents.
Subject(s)
Muscle Cells/pathology , Muscular Dystrophies/pathology , Animals , Caspase 3/metabolism , Cell Death , Cell Shape , Cell Survival , Cells, Cultured , Chick Embryo , Disease Models, Animal , Dystroglycans/metabolism , Gene Expression Regulation , Muscular Dystrophies/genetics , MyoD Protein/genetics , MyoD Protein/metabolismABSTRACT
Lizards are unique in having both-regeneration competent (tail) as well as non-regenerating appendages (limbs) in adults. They therefore present an appropriate model for comparing processes underlying regenerative repair and nonregenerative healing after amputation. In the current study, we use northern house gecko Hemidactylus flaviviridis to compare major cellular and molecular events following amputation of the limb and of the tail. Although the early response to injury in both cases comprises apoptosis, proliferation, and angiogenesis, the temporal distribution of these processes in each remained obscure. In this regard, observations were made on the anatomy and gene expression levels of key regulators of these processes during the healing phase of the tail and limb separately. It was revealed that cell proliferation markers like fibroblast growth factors were upregulated early in the healing tail, coinciding with the growing epithelium. The amputated limb, in contrast, showed weak expression of proliferation markers, limited only to fibroblasts in the later stage of healing. Additionally, apoptotic activity in the tail was limited to the very early phase of healing, as opposed to that in the limb, wherein high expression of caspase-3 was observed throughout the healing process. Early rise in VEGF-α expression reflected an early onset of angiogenesis in the tail, while it was seen to occur at a later stage in case of the limb. Moreover, the expression pattern of transforming growth factor beta members points toward a pro-fibrotic response being induced very early in the amputated limb. Collectively, these results explain why regenerating appendages are able to heal without scars and if we are to induce scar-free healing in nonregenerating limbs, what interventions can be envisaged. This is crucial to the field of regenerative medicine since it is the initial stages of repair following amputation, which decide whether the appendage will be restored or only covered with a scab.
Subject(s)
Amputation, Surgical , Cicatrix/pathology , Extremities/physiopathology , Lizards , Regeneration/physiology , Tail/physiology , Wound Healing , Animals , Cell Proliferation/physiology , Extremities/blood supply , Immunomodulation , Models, Animal , Neovascularization, Physiologic/physiology , Regenerative Medicine , Reptilian Proteins/genetics , Reptilian Proteins/metabolism , Tail/blood supply , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Pesticide exposure to the non target groups especially during embryonic development has quite often resulted in congenital malformations. A commercially available combination insecticide (Ci, 50% chlorpyrifos and 5% cypermethrin) is known to induce ventral body wall defects (VBWDs) wherein abdominal viscera protrude out of the ventral body wall. Herein, an attempt was made to understand the mechanistic insight into Ci induced VBWDs. For this, before incubation, the chick embryos were dosed with the test chemical and then at different developmental stages of incubation, they were monitored for the changes in the expression of certain genes, which are indispensable for the ventral body wall closure since they regulate the cell fate, proliferation and survival. Concurrently, histopathological changes during the embryonic development were examined to corroborate the above observations. The results of mRNA profiling revealed a significant downregulation of Shh on day 4 and upregulation on day 10, while bmp4, Pitx2, E-cadherin, Wnt11, Wnt6, Pxn, MyoD1, Caspase-3, AHR, Cyp3A4, showed a significant upregulation on day 4 and/or on day 10. N-cadherin, fgf8, bmp1 showed no significant changes. The possible means by which these skewed expression patterns of regulatory molecules culminated into the VBWD are discussed.
ABSTRACT
Epimorphic regeneration is a process allowing the animal to regain its lost structure which depends on the resident pluripotent stem cells as well as de-differentiation of existing cells to form multi-potent stem cells. Many studies have been done to understand the appendage regeneration mechanism. The animal model used since decades is an urodele amphibian the axolotl. However, this ability is also seen in some members of reptiles, mainly lizards which on autotomy of tail regain the same by forming a replica of its lost tail. Lizards being closer to mammals are of greater interest and cannot be neglected. Hence, a stage specific protein profiling was undertaken in order to find the peptides playing a major role in epimorphosis. 2-DGE being basic tool for creating a protein profile was used. With advent of newer modern technology, label-free analysis which uses MS/MS was also performed. The study reports the peptides involved in apoptosis, inflammation and ECM remodelling across the stages of lizard tail regeneration for the first time. Apart from these peptides, structural protein, enzymes involved in metabolism have also been highlighted in the current study to give a bigger picture of the processes and the specific peptides required for tail regeneration.