Differential effects of route of T-2 toxin exposure on hepatic oxidative damage in mice.

T-2 toxin is the most toxic among mycotoxins and poses a potential health hazard for both humans and animals. At high doses, T-2 toxin can cause shock-like syndrome that can result in death. We evaluated the effect of time course and route of exposure on hepatic oxidative damage in mice and it is only such study so far to compare the effects of dermal and subcutaneous exposure of T-2 toxin. Mice were exposed to 1 LD50 of T-2 toxin either by percutaneous (5.94 mg/kg body weight) or subcutaneous (1.54 mg/kg body weight) route and sacrificed at 0, 1, 3, and 7 days postexposure. Analysis of a number of serum biochemical variables, antioxidant enzymes activity, gene and protein expression by immunoblot assay showed time and route dependent effects of T-2 induced hepatic oxidative damage. Time dependent increase in protein carbonyl content and protein oxidation was seen in serum and liver. Results of our study may provide possible mechanism for developing medical countermeasures against T-2 toxin.

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous.

Complete genome sequencing and evolutionary analysis of Indian isolates of Dengue virus type 2.

Dengue is the most important arboviral infection of global public health significance. It is now endemic in most parts of the South East Asia including India. Though Dengue virus type 2 (DENV-2) is predominantly associated with major outbreaks in India, complete genome information of Indian DENV-2 is not available. In this study, the full-length genome of five DENV-2 isolates (four from 2001 to 2011 and one from 1960), from different parts of India was determined. The complete genome of the Indian DENV-2 was found to be 10,670 bases long with an open reading frame coding for 3391 amino acids. The recent Indian DENV-2 (2001-2011) revealed a nucleotide sequence identity of around 90% and 97% with an older Indian DENV-2 (1960) and closely related Sri Lankan and Chinese DENV-2 respectively. Presence of unique amino acid residues and non-conservative substitutions in critical amino acid residues of major structural and non-structural proteins was observed in recent Indian DENV-2. Selection pressure analysis revealed positive selection in few amino acid sites of the genes encoding for structural and non-structural proteins. The molecular phylogenetic analysis based on comparison of both complete coding region and envelope protein gene with globally diverse DENV-2 viruses classified the recent Indian isolates into a unique South Asian clade within Cosmopolitan genotype. A shift of genotype from American to Cosmopolitan in 1970s characterized the evolution of DENV-2 in India. Present study is the first report on complete genome characterization of emerging DENV-2 isolates from India and highlights the circulation of a unique clade in South Asia.

Molecular and virological investigation of a focal chikungunya outbreak in northern India.

Chikungunya (CHIK) fever is one of the most important arboviral infections of medical significance. The objective of the present study is to identify and characterize the etiology of a focal febrile arthritis outbreak from Gwalior, northern India, during October-November 2010. A detailed virological (isolation) and molecular (end-point RT-PCR, quantitative RT-PCR, and nucleotide sequencing) investigation of this outbreak was carried out by collecting and studying 52 clinical samples and 15 mosquito pools from the affected region. The investigation revealed the presence of CHIK viral RNA in 29% of clinical samples and 13% mosquito pool by RT-PCR. The quantification of CHIK viral RNA in samples varied from 10(2.50) to 10(6.67) copies/mL, as demonstrated through quantitative RT-PCR. In addition, six CHIK viruses were isolated from RT-PCR positive samples. The nucleotide sequences of partial E1 gene of five representative CHIK viruses were deciphered, which revealed that all the viral strains from this outbreak belong to the recently emerging ECS African genotype. Identification of Chikungunya virus ECSA African genotype as the etiology of the present outbreak confirms the continued circulation of the novel genotype, since 2006, in India. The identification of CHIK virus in Aedes aegypti also confirmed it as the major vector in northern India.

Development of a pilot-scale production process and characterization of a recombinant Japanese encephalitis virus envelope domain III protein expressed in Escherichia coli.

Japanese encephalitis virus (JEV) is the most important cause of encephalitis in most Asian regions. JEV envelope domain III (JEV EDIII) protein is involved in binding to host receptors, and it contains specific epitopes that elicit virus-neutralizing antibodies. A highly immunogenic, recombinant JEV EDIII protein was expressed in Escherichia coli. In order to take this vaccine candidate for further studies, recombinant JEV EDIII protein was produced employing a pilot-scale fermentation process. Recombinant JEV EDIII protein expressed as inclusion bodies (IBs) was solubilized in 8 M urea and renatured by on-column refolding protocol in the presence of glycerol. A three-step purification process comprising of affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. About ~124 mg of highly purified and biologically active EDIII protein was obtained from 100 g of biomass. Biological function of the purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant JEV EDIII protein in combination with compatible adjuvant is highly immunogenic and elicit high-titer neutralizing antibodies. Thus, recombinant JEV EDIII protein produced at large scale can be a potential vaccine candidate.

Monoclonal antibody-based antigen capture immunoassay for detection of circulating non-structural protein NS1: implications for early diagnosis of Japanese encephalitis virus infection.

An early diagnosis of Japanese encephalitis (JE) is important for timely clinical management and epidemiological control in areas where multiple flaviviruses are endemic. The NS1 antigen has an advantage over IgM enzyme-linked immunosorbent assay (ELISA) for early confirmatory diagnosis of Japanese encephalitis virus (JEV) infection due to its proliferation on the surface of the host cells in the acute phase of infection. In this study, the development and evaluation of JE-specific NS1 antigen capture ELISA is described using high-affinity monoclonal antibody specific to the recombinant NS1 protein for early diagnosis of JE. The gene encoding NS1 protein was cloned and expressed in the pQE30UA expression vector followed by purification of the recombinant protein by affinity chromatography. A sandwich ELISA for antigen detection was developed using purified rabbit IgG antibody and mouse monoclonal antibody as the capture and detector antibody, respectively. The application of JE NS1 antigen ELISA for early diagnosis was evaluated with 120 acute phase sera and 80 CSF samples. The comparative evaluation of the JE NS1 antigen ELISA by real-time RT-PCR revealed 97% concordance with a sensitivity and specificity of 97% and 98%, respectively. The JE NS1 antigen was detectable in the blood from the first day up to day 9 after the onset of symptoms. These findings suggest that the JEV NS1 antigen capture ELISA may help early diagnosis of JE infection.

Transcriptomic profile of host response in Japanese encephalitis virus infection.

BACKGROUND: Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy with the highest mortality rate of 30-50%. The purpose of this study was to understand complex biological processes of host response during the progression of the disease. Virus was subcutaneously administered in mice and brain was used for whole genome expression profiling by cDNA microarray. RESULTS: The comparison between viral replication efficiency and disease progression confirms the active role of host response in immunopathology and disease severity. The histopathological analysis confirms the severe damage in the brain in a time dependent manner. Interestingly, the transcription profile reveals significant and differential expression of various pattern recognition receptors, chemotactic genes and the activation of inflammasome. The increased leukocyte infiltration and aggravated CNS inflammation may be the cause of disease severity. CONCLUSION: This is the first report that provides a detailed picture of the host transcriptional response in a natural route of exposure and opens up new avenues for potential therapeutic and prophylactic strategies against Japanese encephalitis virus.

Yeast expressed recombinant Hemagglutinin protein of novel H1N1 elicits neutralising antibodies in rabbits and mice.

Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

Phylogenetic studies reveal existence of multiple lineages of a single genotype of DENV-1 (genotype III) in India during 1956-2007.

BACKGROUND: Dengue virus type 1 (DENV-1) have been mostly circulating silently with dominant serotypes DENV-2 and DENV-3 in India. However recent times have marked an increase in DENV-1 circulation in yearly outbreaks. Many studies have not been carried out on this virus type, leaving a lacunae pertaining to the circulating genotypes, since its earliest report in India. In the present study, we sequenced CprM gene junction of 13 DENV-1 isolated from Delhi and Gwalior (North India) between 2001-2007 and one 1956 Vellore isolate as reference. For comparison, we retrieved 11 other Indian and 70 global reference sequences from NCBI database, making sure that Indian and global isolates from all decades are available for comparative analysis. RESULTS: The region was found to be AT rich with no insertion or deletion. Majority of the nucleotide substitutions were silent, except 3 non-conservative amino acid changes (I --> T, A --> T and L --> S at amino acid positions 59,114 and 155 respectively) in the Indian DENV-1 sequences, sequenced in this study. Except two 1997-98 Delhi isolates, which group in genotype I; all other Indian isolates group in genotype III. All Indian genotype III DENV-1 exhibited diversity among them, giving rise to at least 4 distinct lineages (India 1-4) showing proximity to isolates from diverse geographic locations. CONCLUSION: The extensive phylogenetic analysis revealed consistent existence of multiple lineages of DENV-1 genotype III during the last 5 decades in India.

Multiple signal transduction pathways in okadaic acid induced apoptosis in HeLa cells.

Okadaic acid (OA) is the major component of diarrhetic shell fish poisoning toxins and a potent inhibitor of protein phosphatase 1 and 2A. We investigated the signal transduction pathways involved in OA induced cell death in HeLa cells. OA induced cytotoxicity and apoptosis at IC50 of 100nM. OA treatment resulted in time dependent increase in reactive oxygen species and depleted intracellular glutathione levels. Loss of mitochondrial membrane permeability led to translocation of bax, cytochrome-c and AIF from mitochondria to cytosol. The cells under fluorescence microscope showed typical apoptotic morphology with condensed chromatin, and nuclear fragmentation. We investigated the mitochondrial-mediated caspase cascade. The time dependent activation and cleavage of of bax, caspases-8, 10, 9, 3 and 7 was observed in Western blot analysis. In addition to caspase-dependent pathway AIF mediated caspase-independent pathway was involved in OA mediated cell death. OA also caused time dependent inhibition of protein phosphatase 2A activity and phosphorylation of p38 and p42/44 MAP kinases. Inhibitor studies with Ac-DEVO-CHO and Z-VAD-FMK could not prevent the phosphorylation of p38 and p42/44 MAP kinases. Our experiments with caspase inhibitors Ac-DEVD-CHO, Z-IETD-FMK and Z-VAD-FMK inhibited capsase-3, 8 cleavages but did not prevent OA-induced apoptosis and DNA fragmentation. Similarly, pretreatment with cyclosporin-A and N-acetylcysteine could not prevent the DNA fragmentation. In summary, the results of our study show that OA induces multiple signal transduction pathways acting either independently or simultaneously leading to apoptosis.

Oxidative damage and gene expression profile of antioxidant enzymes after T-2 toxin exposure in mice.

T-2 toxin is one of the most potent trichothecenes, and on exposure causes severe human and animal diseases. We investigated the dose- and time-dependent effect of T-2 toxin on certain biochemical variables, oxidative damage in terms of antioxidant enzyme activity, and gene expression profile in mice. Mice treated intraperitoneally with either 1 LD50 or 2 LD50 dose (5.61 and 11.22 mg/kg body weight, respectively) of T-2 toxin showed significant alterations in hepatic alanine amino transferase, aspartate amino transferase, and lactate dehydrogenase. Significant changes in hepatic lipid peroxidation, depletion of glutathione (GSH), and expression of heat shock protein-70 indicated oxidative damage. We also evaluated the activity of antioxidant enzymes and compared the gene expression profile by quantitative real-time reverse transcriptase-polymerase chain reaction. Except for glutathione reductase (GR), there was a significant increase in activity of glutathione-S-transferase (GST), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase at 1 LD50 dose. At 2 LD50 dose, SOD showed decrease in activity, whereas GST, GPx, and catalase showed significant increase. In contrast, gene expression profile showed downregulation in GR, GPx, GST, and catalase at 1 LD50 dose. At 2 LD50 dose except GSH synthetase, all other genes were downregulated. The results clearly show oxidative stress as one of the mechanisms of T-2 toxin-mediated toxicity.

Development and evaluation of a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection.

Dengue (DEN) and chikungunya (CHIK) have emerged as the 2 most important arboviral infections of global significance. The similarities in clinical presentations, their circulation in the same geographic area, and the transmission through the same vector necessitate an urgent need for the differential diagnosis of these 2 infections. So far, no single assay is reported for differential diagnosis of these 2 infections. In this study, we report the development and evaluation of a 1-step single-tube duplex reverse transcription polymerase chain reaction (D-RT-PCR) assay by targeting E1 gene of CHIK and C-prM gene junction of DEN virus (DENV), respectively. The sensitivity of this assay was found to be better than conventional virus isolation and could detect as low as 100 copies of genomic RNA, which is equivalent to respective virus-specific RT-PCR. The evaluation was carried out with 360 clinical samples from recent CHIK and DEN outbreaks in India. This assay could also be able to detect dual infection of CHIK and DEN in 3 patients. The phylogenetic analysis based on the nucleotide sequencing of D-RT-PCR amplicon could precisely identify the genotypes of all the serotypes of DENV and CHIK viruses (CHIKV). These findings demonstrate the potential clinical and epidemiologic application of D-RT-PCR for rapid sensitive detection, differentiation, and genotyping of DENV and CHIKV in clinical samples.

Production and characterization of recombinant dengue virus type 4 envelope domain III protein.

Dengue fever, a mosquito-borne viral disease has become a major worldwide public health problem with a dramatic expansion in recent years. Cultivation process for production of recombinant dengue virus type 4 envelope domain III (rDen 4 EDIII) protein in Escherichia coli was developed for its diagnostic use as well as for further studies in immunoprophylaxis. The dissolved oxygen level was maintained at 20-30% of air saturation. The culture was induced with 1mM of isopropyl beta-d-thiogalactoside when dry cell weight was 13.78 g l(-1) and cells were further grown for 4h to reach 17.31 g l(-1) of culture. The protein was overexpressed in the form of insoluble inclusion bodies. The rDen 4 EDIII protein was purified by affinity chromatography and analyzed by SDS-PAGE. The final yield of purified rDen 4 EDIII protein in this method was approximately 196 mg l(-1) of culture. The purified protein was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue infected human serum samples. These results show that the product has the potential to be used for the diagnosis of dengue infection or for further studies in vaccine development. This production system may also be suitable for the high yield of other recombinant dengue proteins.

Production of IgM specific recombinant dengue multiepitope protein for early diagnosis of dengue infection.

Dengue virus infections have recently undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Early detection of dengue infection based on the identification of antibodies has emerged as a practical and reliable means of diagnosis of dengue fever. The recombinant dengue multiepitope (rDME-M) protein specific to IgM in E. coli was produced in a 5-L fermentor for use in diagnostic purpose. After fermentation, dry cell weight was approximately 11.8 g/L of the culture. The rDME-M protein was purified under denaturing conditions using single-step nickel nitrilotriacetate (Ni-NTA) affinity chromatography. The final yield of purified rDME-M protein from this method was approximately 68.5 mg/L of the culture. The purity of rDME-M protein was checked by SDS-PAGE analysis, and the reactivity of this protein was further checked by Western blotting and enzyme-linked immunosorbent assay (ELISA). The purified protein was used as an antigen in the development of an in-house dipstick ELISA and evaluated with a panel of 80 patient sera, characterized using commercially available tests for detection of dengue antibody. The results were in excellent agreement with those of IgM capture ELISA (Pan-Bio) and rapid immunochromatography (IC) test (Pan-Bio). These results show that the in-house dipstick ELISA using rDME-M protein can be used as a promising kit because of its comparable sensitivity, specificity, field applicability, and low cost.

Bacterially expressed and refolded envelope protein (domain III) of dengue virus type-4 binds heparan sulfate.

An arboviral infection like dengue fever/dengue hemorrhagic fever (DHF) with high morbidity and mortality rate are extensively prevalent in several parts of the world. Global efforts have been directed towards development of vaccine for prevention of dengue. However, lack of thorough understanding about biology and pathogenesis of dengue virus restricts us from development of an effective vaccine. Here we report molecular interaction of domain III of envelope protein of dengue virus type-4 with heparan sulfate. A codon optimized synthetic gene encoding domain III of dengue virus type-4 envelope protein was expressed in Escherichia coli and purified under denaturing conditions, refolded and purified to homogeneity. Refolded Den4-DIII was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified protein was recognized in Western analyses by monoclonal antibody specific for the 6x His tag as well as the H241 monoclonal antibody. The in vitro refolded recombinant protein preparation was biologically functional and found to bind cell free heparan sulfate. This is the first report providing molecular evidence on binding of dengue-4 envelope protein to heparan sulfate. We developed a homology model of dengue-4 envelope protein (domain III) and mapped the possible amino acid residues critical for binding to heparan sulfate. Domain III envelope protein of dengue virus is a lead vaccine candidate. Our findings further the understanding on biology of dengue virus and will help in development of bioassay for the proposed vaccine candidate.

Production, purification and characterization of recombinant dengue multiepitope protein.

Dengue is an acute mosquito-borne viral disease of humankind. Dengue fever, dengue haemorrhagic fever and dengue shock syndrome have become global public health problems in recent years. rDME-G (recombinant dengue multiepitope protein that can specifically detect IgG) was produced in a 5-litre fermenter in Escherichia coli for use in diagnosis. The culture was induced with 1 mM isopropyl beta-D-thiogalactoside and cells were further grown for 4 h before harvesting. After fermentation, dry cell weight resulted in approx. 16.2 g/l. The rDME-G protein was purified from inclusion bodies using affinity chromatography. The final yield of purified rDME-G protein from fermentation resulted in approx. 168 mg/l of pure biologically active rDME-G protein. The purity of rDME-G protein was checked by SDS/PAGE analysis and the reactivity of this protein was further determined by Western blotting. The purified protein was used to develop an in-house dipstick ELISA and tested using a panel of 60 patient sera characterized using the commercially available tests for detection of dengue antibody. We compared our results with IgG-capture ELISA (Pan-Bio, Windsor, QLD, Australia) and rapid IC (immuno-chromatography) test (Pan-Bio). By using rDME-G protein as an antigen, in the dipstick ELISA, the results were in excellent agreement with commercial rapid IC test and IgG capture ELISA. These results show that the product has a promising potential to be used for diagnosis of dengue in both laboratory- and field-based detection systems with minimum cost and a high degree of sensitivity and specificity.

Evaluation of abrin induced nephrotoxicity by using novel renal injury markers.

Abrin is a potent plant toxin analogous to ricin that is derived from the seeds of Abrus precatorius plant. It belongs to the family of type II ribosome-inactivating proteins and causes cell death by irreversibly inactivating ribosomes through site-specific depurination. In this study we examined the in vivo nephrotoxicity potential of abrin toxin in terms of oxidative stress, inflammation, histopathological changes and biomarkers of kidney injury. Animals were exposed to 0.5 and 1.0 LD50 dose of abrin by intraperitoneal route and observed for 1, 3, and 7 day post-toxin exposure. Depletion of reduced glutathione and increased lipid peroxidation levels were observed in abrin treated mice. In addition, abrin also induced inflammation in the kidneys as observed through expression of MMP-9 and MMP-9/NGAL complex in abrin treated groups by using zymography method. Nephrotoxicity was also evaluated by western blot analysis of kidney injury biomarkers including Clusterin, Cystatin C and NGAL, and their results indicate severity of kidney injury in abrin treated groups. Kidney histology confirmed inflammatory changes due to abrin. The data generated in the present study clearly prove the nephrotoxicity potential of abrin.

Reemergence of dengue virus type-3 (subtype-III) in India: implications for increased incidence of DHF & DSS.

BACKGROUND: Dengue virus infection has recently taken endemic proportion in India implicating all the four known dengue serotypes. There was a major dengue outbreak in northern India including Delhi in October- December, 2003 and again in 2004. We have carried out a detailed investigation of the 2004 outbreak by Serosurveillance, RT-PCR, nested PCR, virus isolation and genotyping. We also report the molecular epidemiological investigation of these outbreaks. RESULTS: The serological investigation of 162 suspected serum samples using an in-house dengue dipstick ELISA revealed 11%-IgM, 51%-IgG and 38%-both IgM and IgG antibody positivity. The RT-PCR analysis revealed presence of dengue RNA in 17 samples. Further subtyping and genotyping by nested PCR and nucleotide sequencing of C-prM gene junction revealed the association of subtype III of dengue virus type 3 in the outbreak. CONCLUSION: The sudden shifting and dominance of the dengue virus serotype-3 (subtype III) replacing the earlier circulating serotype-2 (subtype IV) is a point of major concern and may be attributed to increased incidence of DHF and DSS in India.

Activity and gene expression profile of certain antioxidant enzymes to microcystin-LR induced oxidative stress in mice.

Microcystins are cyclic heptapeptide toxins produced by certain strains of Microcystis aeruginosa and microcystin-LR (MC-LR) is the most toxic among the 70 variants isolated so far. These toxins have been implicated in both human and livestock mortality. In the present study we investigated the microcystin-LR induced oxidative stress in mice in terms of its effect on activity and gene expression profile of certain antioxidant enzymes and expression of heat shock protein-70 (HSP-70). Mice were treated with 0.5 LD50 (38.31 microg/kg) and 1 LD50 (76.62 microg/kg) and the biochemical variables were determined at 1, 3, 7 days and 15, 30, 60 and 120 min post-exposure for 0.5 and 1 LD50 dose, respectively. A significant time-dependent increase in HSP-70 expression over control was observed at 1 LD50 dose. The toxin induced significant increase in liver body weight index, hepatic lipid peroxidation and depletion of GSH levels at 1 LD50 compared to control group. There was significant decrease in the activity of antioxidant enzymes glutathione peroxidase (GPX), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione-S-transferase (GST) at 1 LD50. Except catalase, there was no effect on other antioxidant enzymes at 0.5 LD50 dose. In contrast to activity of antioxidant enzymes the gene expression profile did not show any significant difference compared to control at 1 LD50. GR showed significant decrease in expression at 1, 3 and 7 days in animals dosed with 0.5 LD50 MC-LR. The results of our in vivo study clearly show the oxidative stress induced by MC-LR, and a correlation with activity and regulation at gene expression level of antioxidant enzymes.

Mechanism of ricin-induced apoptosis in human cervical cancer cells.

The mechanism of ricin-induced apoptosis in human cervical cancer cell line HeLa was studied. The present study demonstrated that ricin induces apoptosis of human cervical cancer cells (HeLa) in a time dependent manner with an IC(50) for cell viability of 1 microg/ml. Ricin treatment resulted in a time dependent increase in LDH leakage, DNA fragmentation, percent apoptotic cells, generation of reactive oxygen species and depletion of intracellular glutathione levels. DNA agarose gel electrophoresis showed typical oligonucleosomal length DNA fragmentation. Additionally, DNA diffusion assay was performed to confirm DNA damage and apoptosis. Ricin activated caspase-3 as evidenced by both proteolytic cleavage of procaspase-3 into 20 and 18 kDa subunits, and increased protease activity. Caspase activity was maximum at 4h and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the 85 kDa cleavage product. Ricin-induced caspase-3 activation also resulted in cleavage of DNA fragmentation factor-45 (DFF45/ICAD) and DFF40 or caspase-activated DNase in HeLa cells. Activation of caspase-3, cleavage of PARP and DNA fragmentation was blocked by pre-treatment with caspase-3 specific inhibitor Ac-DEVD-CHO (100 microM) and broad-spectrum caspase inhibitor Z-VAD-FMK (40 microM). Ricin-induced DNA fragmentation was inhibited by pre-treatment with PARP inhibitors 3-aminobenzamide (100 microM) and DPQ (10 microM). Our results indicate that ricin-induced cell death was mediated by generation of reactive oxygen species and subsequent activation of caspase-3 cascade followed by down stream events leading to apoptotic mode of cell death.