S. Abdul Majeed

RETRACTED: Chitosan tripolyphosphate (CS/TPP) nanoparticles: Preparation, characterization and application for gene delivery in shrimp

The present study examines the use of CS/TPP nanoparticles for gene delivery in different tissues of shrimp through oral route. The viral gene of WSSV was used to construct DNA vaccines using pcDNA 3.1, a eukaryotic expression vector and the constructs were named as pVP28. The CS/TPP nanoparticles were synthesized by ionic gelation process and these particles were characterized. The structure and morphology of the nanoparticles were studied by field emission scanning electron microscopy (FE-SEM) and FTIR (Fourier Transform Infrared Spectra). The cytotoxicity of CS/TPP nanoparticles was evaluated by MTT assay using fish cell line. The expression of gene was confirmed by Immuno-dot blot, ELISA and RT-PCR analyses. The results indicate that DNA can be easily delivered into shrimp by feeding with CS/TPP nanoparticles.

In vitro assay for the toxicity of silver nanoparticles using heart and gill cell lines of Catla catla and gill cell line of Labeo rohita.

Silver nanoparticles (Ag-NPs) are used in commercial products for their antimicrobial properties. The Ag-NPs in some of these products are likely to reach the aquatic environment, thereby posing a health concern for humans and aquatic species. The silver nanoparticles were synthesized and characterized using, UV-vis spectra, Dynamic light scattering (DLS) and Transmission electron microscopy (TEM) analysis. Acute toxicity tests on fish were conducted by exposing Catla catla and Labeo rohita for 96h to AgNO3 and Ag-NPs under static conditions. The cytotoxic effect of AgNO3 and Ag-NPs in Sahul India C. catla heart cell line (SICH), Indian C. catla gill cell line (ICG) and L. rohita gill cell line (LRG) was assessed using MTT and neutral red (NR) assay. Linear correlations between each in vitro EC50 and the in vivo LC50 data were highly significant. DNA damage and nuclear fragmentation (condensation) were assessed by comet assay and Hoechst staining, respectively in SICH, ICG and LRG cells exposed to Ag-NPs. The results of antioxidant parameter obtained show significantly increased lipid peroxidation (LPO) level and decreased level of GSH, SOD and CAT in SICH, ICG and LRG cell lines after exposure to increasing Ag-NPs in a concentration-dependent manner. This work proves that fish cell lines could be used as an alternative to whole animals using cytotoxicity tests, genotoxicity tests and oxidative stress assessment after exposure to nanoparticles.

Comparison of in vitro and in vivo acute toxicity assays in Etroplus suratensis (Bloch, 1790) and its three cell lines in relation to tannery effluent.

Cell lines of Etroplus suratensis established in our laboratory were evaluated for their potential use as screening tools for the ecotoxicological assessment of tannery effluent. The cytotoxic effect of tannery effluent in three cell lines derived from eye, kidney and gill tissue of E. suratensis was assessed using multiple endpoints such as Neutral Red (NR) assay, Coomassie Blue (CB) protein assay and Alamar Blue (AB) assay. Acute toxicity tests on fish were conducted by exposing E. suratensis for 96 h to tannery effluent under static conditions. The toxic effect of tannery effluent on the survival of fish was found to be concentration and time dependent. The tannery effluent at the concentration of 15% caused 100% mortality at 96 h whereas the lower concentration (0.5%) caused 13.33% mortality. The cytotoxicity of tannery effluent was found to be similar in the three cell lines tested, independent of the toxic endpoints employed. EC(50) values, the effective concentration of tannery effluent resulting in 50% inhibition of cytotoxicity parameters after 48 h exposure to tannery effluent were calculated for eye, kidney and gill cell lines using NR uptake, AB and cell protein assays. Statistical analysis revealed good correlation with r(2)=0.95-0.99 for all combinations between endpoints employed. Linear correlations between each in vitro EC(50) and the in vivo LC(50) data, were highly significant p<0.001 with r(2)=0.977, 0.968 and 0.906 for AB(50), NR(50), and CB(50), respectively.

Development and characterization of cell line from the gill tissue of Catla catla (Hamilton, 1822) for toxicological studies.

Catla gill cell line (ICG) was established from gill tissue of Indian major carp (Catla catla), a freshwater fish cultivated in India. The cell line was maintained in Leibovitzs L-15 supplemented with 10% fetal bovine serum. These cells have been sub-cultured more than 55 passages over a period of 2 years. The ICG cell line consists predominantly of epithelial-like cells. The cells were able to grow at a wide range of temperatures from 24°C to 32°C with an optimum temperature of 28°C. The growth rate of gill cells increased as the fetal bovine serum (FBS) proportion increased from 2% to 20% at 28°C with optimum growth at the concentrations of 10% or 15% FBS. Amplification of mitochondrial gene 12s rRNA using primers specific to C. catla confirmed the origin of this cell line from C. catla. The cells were successfully cryopreserved and revived at passage numbers 25, 35, 45 and 55. The cytotoxicity of three metal salts (ZnCl(2), CuSO(4) and CdCl(2)) was assessed in ICG cell line using multiple endpoints such as MTT, Neutral Red assay, Alamar Blue assay and Coomassie Blue protein assay. Acute toxicity assay on fish were conducted by exposing C. catla for 96 h to three metal salts under static conditions. Statistical analysis revealed good correlation with r(2)=0.908-0.985 for all combinations between endpoints employed. Linear correlations between each in vitro EC(50) and the in vivo LC(50) data were highly significant.

Development and characterization of a new gill cell line from air breathing fish Channa striatus (Bloch 1793) and its application in toxicology: Direct comparison to the acute fish toxicity

A new cell line, Channa striatus gill (CSG), derived from the gill tissue of murrel, was established and characterized. The CSG cell line was maintained in Leibovitzs L-15 supplemented with 10% fetal bovine serum and has been subcultured more than 92 times. This cell line was able to grow in a range of temperatures from 22 to 32°C with optimal growth at 28°C. The plating efficiency was very high (52.21%) and doubling time was approximately 37h. The gill cell line was cryopreserved at different passage levels and revived successfully with 85% survival. Polymerase chain reaction amplification of mitochondrial 16S rRNA using primer specific to C. striatus confirmed the origin of this cell line from murrel. The cell line was further characterized by immunocytochemical analysis, chromosome number, transfection and mycoplasma detection. The cytotoxicity of endosulfan was assessed in CSG cell line using apoptosis assay, comet assay, mitochondrial alteration and five other endpoints such as Rhodamine 123 uptake, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red assay, Alamar Blue assay and Methylene Blue protein assay. Acute toxicity study on fish was conducted by exposing murrel for 96h to endosulfan under static conditions. Statistical analysis revealed good correlation with r(2)=0.972-0.997 among the five endpoints. Linear correlations between the in vivo lethal concentration 50 (LC50) and each in vitro effective concentration 50 (EC50) were highly significant. The present study highlights the development of a new gill cell line from an air breathing fish that could be used as an alternative in vitro tools for studying pesticide toxicity in fish.

In vitro cytotoxic, genotoxic and oxidative stress of cypermethrin on five fish cell lines.

The indiscriminate use of pesticides and herbicides to enhance crop production has aroused great concern, because these products are likely to reach the aquatic environment, thereby posing a health concern for humans and aquatic species. Cypermethrin (CYP), a type II pyrethroid insecticide, is widely used in agriculture and for other purposes. Therefore a study was conducted for the assessment of cytotoxic, genotoxic and oxidative stress of CYP in IEG, CB, ICG, LRG and CSG cell lines at 24h exposure. The cytotoxic effect of CYP in IEG, CB, ICG, LRG and CSG cell lines was assessed using MTT, NR, AB and CB assays. Linear correlations between each EC50 values, of CYP resulting in 50% inhibition of cytotoxicity parameters after 24h exposure to CYP were calculated for IEG, CB, ICG, LRG and CSG cell lines using MTT, NR, AB and CB assays. Statistical analysis revealed good correlation with R(2)=0.90-0.939 for all combinations between endpoints employed. The percentage of DNA damage was assessed by comet assay in IEG, CB, ICG, LRG and CSG cells exposed to CYP. The results of antioxidant parameters obtained show a significant increase in lipid peroxidation (LPO) level and decreased level of GSH, SOD and CAT in IEG, CB, ICG, LRG and CSG cell lines after exposure to increasing CYP in a concentration-dependent manner. This work proves that fish cell lines could be used not only for cytotoxicity and genotoxicity studies but also for studying oxidative stress when exposed to environmental contaminants such as pesticides and other pollutants.

Development, characterization and application of a new fibroblastic-like cell line from kidney of a freshwater air breathing fish Channa striatus (Bloch, 1793).

A new cell line, Channa striatus kidney (CSK), derived from the kidney tissue of murrel, was established and characterized. The CSK cell line was maintained in Leibovitzs L-15 supplemented with 10% fetal bovine serum and has been subcultured more than 140 times. This cell line was able to grow in a range of temperatures from 22 to 32°C with optimal growth at 28°C. The plating efficiency was very high (67.54%) and doubling time was approximately 29h. The kidney cell line was cryopreserved at different passage levels and revived successfully with 90-92% survival. Polymerase chain reaction amplification of mitochondrial 16S rRNA using primer specific to C. striatus confirmed the origin of this cell line from murrel. The cell line was further characterized by chromosome number, transfection and mycoplasma detection. A marine fish nodavirus was tested to determine the susceptibility of this new cell line. The CSK cell line was found to be susceptible to nodavirus and the infection was confirmed by cytopathic effect (CPE), reverse transcriptase-polymerase chain reaction (RT-PCR), immunodot blot, enzyme linked immunosorbent assay (ELISA), virus replication efficiency and real time RT-PCR. The present study highlights the development and characterization of a new kidney cell line from an air breathing fish that could be used as an in vitro tools for propagation of fish viruses and gene expression studies.

Comparison of betanodavirus replication efficiency in ten Indian fish cell lines.

Ten cell lines established from Indian marine, brackishwater and freshwater fish were tested for their susceptibility to fish nodavirus. In addition, the efficiency of betanodavirus replication was tested in these cell lines. Multiple vacuolation, a typical cytopathic effect for virus infection, was observed in infected SISK, SISS, SIGE and ICF cells. Infection of the different fish cell lines was confirmed by RT-PCR, immunodot blot assay and indirect ELISA. The virus concentration in culture supernatant collected from infected sea bass and grouper cell lines increased progressively from 103 at day 1 postinfection to 108 TCID50 ml−1 at day 9. The amount of virus in different cell lines was also quantified by real-time PCR. These results indicate the suitability of the SISK, SISS, and SIGE cell lines for fish nodavirus propagation for developing viral diagnostics and vaccines.

In vitro white spot syndrome virus (WSSV) replication in explants of the heart of freshwater crab, Paratelphusa hydrodomous

Explants from different organs of freshwater crab, Paratelphusa hydrodomous were prepared to establish an in vitro system for replication of white spot syndrome virus (WSSV) of shrimp. Heart explants were maintained for 53 days without any morphological changes in EX-CELL™ 405 medium with and without serum whereas the explants of eye muscle, gill, shell membrane and appendage muscle died within 15 days of culture period. The heart explants on different days of culture were exposed to WSSV for 10 days to study the viral replication. The infection of WSSV in explants of the heart was confirmed by PCR, RT-PCR, Western blot, histology, immunohistochemistry, bioassay and transmission electron microscopy. The WSSV was quantified by real-time PCR and indirect ELISA. The WSSV inoculum prepared from the heart explants of crab caused significant mortality in Penaeus monodon in challenge experiments and the results indicate that the WSSV which replicated in the heart explants of freshwater crab maintains its infectivity as in marine shrimp. The results indicate that the heart explants of P. hydrodomous would be a good alternative to whole animals for production of WSSV.

Antivenom activity of triterpenoid (C34H68O2) from Leucas aspera Linn. against Naja naja naja venom induced toxicity: antioxidant and histological study in mice.

The isolated and identified triterpenoid, 1-hydroxytetratriacontane-4-one (C34H68O2), obtained from the methanolic leaf extract of Leucas aspera Linn. was explored for the first time for antisnake venom activity. The plant (L. aspera Linn.) extract significantly antagonized the spectacled cobra (Naja naja naja) venom induced lethal activity in a mouse model. It was compared with commercial antiserum obtained from King Institute of Preventive Medicine (Chennai, Tamil Nadu, India). N. naja naja venom induced a significant decrease in antioxidant superoxide dismutase, glutathione (GSH) peroxidase, catalase, reduced GSH and glutathione-S-transferase activities and increased lipid peroxidase (LPO) activity in different organs such as heart, liver, kidney and lungs. The histological changes following the antivenom treatment were also evaluated in all these organs. There were significant alterations in the histology. Triterpenoid from methanol extract of L. aspera Linn. at a dose level of 75 mg per mouse significantly attenuated (neutralized) the venom-induced antioxidant status and also the LPO activity in different organs.