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Cytotoxic effects of oosporein isolated from endophytic fungus Cochliobolus kusanoi.

Ramesha A, Venkataramana M, Nirmaladevi D, Gupta VK, Chandranayaka S, Srinivas C - Front Microbiol (2015)

Bottom Line: After 24 h of exposure histopathological observations were made to know the effects of oosporein on target organs.Oosporein induced elevated levels of reactive oxygen species (ROS) generation and high levels of malondialdehyde, loss of mitochondrial membrane potential, induced glutathione hydroxylase (GSH) production was observed in a dose depended manner.Further, oosporein treatment to studied cell lines indicated significant suppression of oxidative stress related gene (Superoxide dismutase1 and Catalase ) expression, and increased levels of mRNA expression in apoptosis or oxidative stress inducing genes HSP70, Caspase3, Caspase6, and Caspase9 as measured by quantitative real time-PCR assay.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Biotechnology, Bangalore University Bangalore, India.

ABSTRACT
In the present study, oosporein, a fungal toxic secondary metabolite known to be a toxic agent causing chronic disorders in animals, was isolated from fungus Cochliobolus kusanoi of Nerium oleander L. Toxic effects of oosporein and the possible mechanisms of cytotoxicity as well as the role of oxidative stress in cytotoxicity to Madin-Darby canine kidney kidney cells and RAW 264.7 splene cells were evaluated in vitro. Also to know the possible in vivo toxic effects of oosporein on kidney and spleen, Balb/C mouse were treated with different concentrations of oosporein ranging from 20 to 200 μM). After 24 h of exposure histopathological observations were made to know the effects of oosporein on target organs. Oosporein induced elevated levels of reactive oxygen species (ROS) generation and high levels of malondialdehyde, loss of mitochondrial membrane potential, induced glutathione hydroxylase (GSH) production was observed in a dose depended manner. Effects oosporein on chromosomal DNA damage was assessed by Comet assay, and increase in DNA damage were observed in both the studied cell lines by increasing the oosporein concentration. Further, oosporein treatment to studied cell lines indicated significant suppression of oxidative stress related gene (Superoxide dismutase1 and Catalase ) expression, and increased levels of mRNA expression in apoptosis or oxidative stress inducing genes HSP70, Caspase3, Caspase6, and Caspase9 as measured by quantitative real time-PCR assay. Histopathological examination of oosporein treated mouse kidney and splenocytes further revealed that, oosporein treated target mouse tissues were significantly damaged with that of untreated sam control mice and these effects were in directly proportional to the the toxin dose. Results of the present study reveals that, ROS is the principle event prompting increased oosporein toxicity in studied in vivio and in vitro animal models. The high previlance of these fungi in temperate climates further warrants the need of safe food grain storage and processing practices to control the toxic effects of oosporein to humans and live stock.

No MeSH data available.


Related in: MedlinePlus

(A) Estimation of reactive oxygen species (ROS) production by DCFH-DA using spectrofluorimeter; (B) Dose dependent effect of oosporein on Mitochondrial membrane potential (MMP) in MDCK and RAW 264.7 celllines. Values represent mean ± SD of six parallel experiments. In the figure each series mean values followed by the different letter are significantly different according to DMRT at p < 0.05.
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Figure 2: (A) Estimation of reactive oxygen species (ROS) production by DCFH-DA using spectrofluorimeter; (B) Dose dependent effect of oosporein on Mitochondrial membrane potential (MMP) in MDCK and RAW 264.7 celllines. Values represent mean ± SD of six parallel experiments. In the figure each series mean values followed by the different letter are significantly different according to DMRT at p < 0.05.

Mentions: The intracellular formation of ROS was determined by using the DCFH-DA fluorescence technique. The exposure of cells to oosporein at concentration of 25–200 μM for 6 and 12 h resulted in increased ROS production with increase in concentration. MDCK cells treated with 25, 50, 100, and 200 μM oosporein for 6 h significantly increased ROS production and fluorescence intensity by 120, 140, 200, and 240%, respectively, whereas for 12 h by 150, 190, 300, and 350%, respectively. Increase in ROS production and fluorescence intensity by 120, 138, 190, and 220%, was observed in RAW 264.7 cells treated with various concentrations of oosporein for 6 h, and 155, 185, 318, and 365% for 12 h exposure (Figure 2A).


Cytotoxic effects of oosporein isolated from endophytic fungus Cochliobolus kusanoi.

Ramesha A, Venkataramana M, Nirmaladevi D, Gupta VK, Chandranayaka S, Srinivas C - Front Microbiol (2015)

(A) Estimation of reactive oxygen species (ROS) production by DCFH-DA using spectrofluorimeter; (B) Dose dependent effect of oosporein on Mitochondrial membrane potential (MMP) in MDCK and RAW 264.7 celllines. Values represent mean ± SD of six parallel experiments. In the figure each series mean values followed by the different letter are significantly different according to DMRT at p < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556033&req=5

Figure 2: (A) Estimation of reactive oxygen species (ROS) production by DCFH-DA using spectrofluorimeter; (B) Dose dependent effect of oosporein on Mitochondrial membrane potential (MMP) in MDCK and RAW 264.7 celllines. Values represent mean ± SD of six parallel experiments. In the figure each series mean values followed by the different letter are significantly different according to DMRT at p < 0.05.
Mentions: The intracellular formation of ROS was determined by using the DCFH-DA fluorescence technique. The exposure of cells to oosporein at concentration of 25–200 μM for 6 and 12 h resulted in increased ROS production with increase in concentration. MDCK cells treated with 25, 50, 100, and 200 μM oosporein for 6 h significantly increased ROS production and fluorescence intensity by 120, 140, 200, and 240%, respectively, whereas for 12 h by 150, 190, 300, and 350%, respectively. Increase in ROS production and fluorescence intensity by 120, 138, 190, and 220%, was observed in RAW 264.7 cells treated with various concentrations of oosporein for 6 h, and 155, 185, 318, and 365% for 12 h exposure (Figure 2A).

Bottom Line: After 24 h of exposure histopathological observations were made to know the effects of oosporein on target organs.Oosporein induced elevated levels of reactive oxygen species (ROS) generation and high levels of malondialdehyde, loss of mitochondrial membrane potential, induced glutathione hydroxylase (GSH) production was observed in a dose depended manner.Further, oosporein treatment to studied cell lines indicated significant suppression of oxidative stress related gene (Superoxide dismutase1 and Catalase ) expression, and increased levels of mRNA expression in apoptosis or oxidative stress inducing genes HSP70, Caspase3, Caspase6, and Caspase9 as measured by quantitative real time-PCR assay.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Biotechnology, Bangalore University Bangalore, India.

ABSTRACT
In the present study, oosporein, a fungal toxic secondary metabolite known to be a toxic agent causing chronic disorders in animals, was isolated from fungus Cochliobolus kusanoi of Nerium oleander L. Toxic effects of oosporein and the possible mechanisms of cytotoxicity as well as the role of oxidative stress in cytotoxicity to Madin-Darby canine kidney kidney cells and RAW 264.7 splene cells were evaluated in vitro. Also to know the possible in vivo toxic effects of oosporein on kidney and spleen, Balb/C mouse were treated with different concentrations of oosporein ranging from 20 to 200 μM). After 24 h of exposure histopathological observations were made to know the effects of oosporein on target organs. Oosporein induced elevated levels of reactive oxygen species (ROS) generation and high levels of malondialdehyde, loss of mitochondrial membrane potential, induced glutathione hydroxylase (GSH) production was observed in a dose depended manner. Effects oosporein on chromosomal DNA damage was assessed by Comet assay, and increase in DNA damage were observed in both the studied cell lines by increasing the oosporein concentration. Further, oosporein treatment to studied cell lines indicated significant suppression of oxidative stress related gene (Superoxide dismutase1 and Catalase ) expression, and increased levels of mRNA expression in apoptosis or oxidative stress inducing genes HSP70, Caspase3, Caspase6, and Caspase9 as measured by quantitative real time-PCR assay. Histopathological examination of oosporein treated mouse kidney and splenocytes further revealed that, oosporein treated target mouse tissues were significantly damaged with that of untreated sam control mice and these effects were in directly proportional to the the toxin dose. Results of the present study reveals that, ROS is the principle event prompting increased oosporein toxicity in studied in vivio and in vitro animal models. The high previlance of these fungi in temperate climates further warrants the need of safe food grain storage and processing practices to control the toxic effects of oosporein to humans and live stock.

No MeSH data available.


Related in: MedlinePlus