Introduction by cleavage of esters as demonstrated in

Introduction

 T-2 mycotoxin, a low molecular
weight (250-500 daltons) non-volatile compounds produced by filamentous fungi
of the genera Fusarium,
Myrothecium, and
Stachybotrys 1. Predominantly F. sporotrichioides is responsible for
the production of T-2 toxin and HT-2 toxin. The most important factors that influence T-2 toxin production are
weather conditions and moisture content (13 to 22 %).  T-2 toxin is produced under a wide
temperature range (0 to 32 °C), with maximum production at temperatures below
15 °C 2,3. T-2 toxin is extremely heat stable and is the only mycotoxin known to
have been used as a biothreat weapon delivering via food or water sources, as well as, via
droplets, aerosols, or smoke from various dispersal systems and exploding
munitions 4,5.

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Chemically, T-2
toxin is a tetracyclic sesquiterpenoid with 12, 13 epoxytrichothec-9-ene
ring system 6
with  hydroxyl group at the C-3 position,
acetyloxy groups at C-4 and C-15 positions, Atom of hydrogen at C-7 position
and an ester-linked isovaleryl group at the C-8 position 7. The toxicity can
be reduced by cleavage of esters as demonstrated in different cell culture
experiments 8,9. T-2 toxin is well a known inhibitors of
protein, DNA and RNA syntheses known to, interfere with the metabolism of membrane
phospholipids, and increase the level of liver lipid peroxides 10.

The T-2 toxin is
known to cause acute and chronic toxicity, as well as alimentary toxic aleukia
(ATA) and Kashin-beck disease
(KBD) in humans and animals it has been shown to cause alteration of blood–brain barrier (BBB) 11-14. The toxicological effects of T-2
toxin have been summarized in Food and Agricultural Organization/World Health
Organization (FAO/WHO)  Expert Committee
on Food Additives and the Council for Agricultural Science and Technology
15,16. European Union (EU) states; T-2 toxin as a common contaminate to
cereals and cereals products and there are many reports from different regions
of  the world associating damage of
agricultural field associate T-2 toxin and it extends to humans and animals 17,18.
Sensitive and accurate analytical methods have been developed for measuring
mycotoxins in cereals and cereal based products in order to assess the risk of
humans and animal exposure 1-3.

T-2 toxin toxicity is the most severe compare to
other tricotysin. Major target organs include skin, liver, nerves etc., In the
present study, the fungal species growing on corn was identified as T-2 Toxin
producing organism by molecular characterization and T-2 Toxin identified by
FTIR analysis and toxicity of the toxin produced was evaluated in human
keratinocytes, hepatoma, and neuron cells invitro.

Materials
and method:

Potato
dextrose agar, Potato dextrose broth, glutaraldehyde
(Himedia Bangalore), methanol, ethyl acetate, ethanol, silica gel, (SRL,
Bangalore), T-2 toxin (Chempure Mumbai), ethidium bromide (Sigma Banglore) were
used.

Isolation
of Fusarium species from corn sample

Isolation
of Fusarium was carried out by standard
blotter method. Corn samples were collected
from different localities. Corn seeds were incubated for 3-5 days under moist
condition on petriplates. As the spore of fungi developed into mycelia, it was
transferred aseptically to sub-culture on potato dextrose agar to develop pure
colonies, and the colony morphology was examined under microscope.

Scanning
electron microscopy

After observation
under inverted microscope the sample subjected to scaning electron microscopy
(SEM). The mycelia were fixed
in glutaraldehyde in 0.1M PBS for 30 min., washed with 0.1M PBS and dehydrated by immersing in ice
cold ethanol for 10min. The dehydrated samples were smeared on silver stub like
a thin film, and were coated by cathodic spraying (Polaron gold). The SEM
observations were made using a ZEISS Instrument (EHT=15.00 kv, signal A=VPSE
G3).

Fusarium
species DNA Extraction

7days
old mycelium mat was collected and frozen at -800C and lyophilized. Lyophilized
sample were homogenised in  lysis buffer
and extraction was carried by phenol chloroform method. DNA was quantified in
nanodrop spectrophotometer, PCR was performed to amplify DNA 19.

 

Polymerase
Chain Reaction

The
PCR of fusarium was performed using universal primers (ITS/ITS4 primers).
Standard protocol was followed to amplify the DNA, after successful amplication
products were inspected by agarose gel  electrophoresis containing  ( 0. 01%) 
ethidium bromide and  observed under  UV. Gels were documented  using 
Gbox   (GE  Health Care,  
Mumbai).

Fusarium
culture for toxin production

Culture
preparation

Fusarium
sporotrichioides MTCC
1894 Venkataramana et al. 20 was
used as the test organism and was obtained from Department of Microbiology,
Defence Food Research Laboratory, Mysore. The fungus was purified and harvested
at 370C on potato dextrose broth (PDB).

Toxin
extraction and purification

The
Fusarium sporotrichioides was inoculated to
PDB broth and incubated for 10 days. After incubation the broth was filtered
and the filtrate was collected and mixed with equal volume of ethanol and
incubated for 24 hrs, then it was concentrated using flash evaporator.
The extracted toxin was stored in amber vial for further characterization.

Thin
Layer Chromatography (TLC)

T-2
toxin was extracted and detected by thin layer chromatographic method 21. The
plates were prepared by adding 50 ml of distilled water to 25g silica gel
powder and mixed uniformly to get homogenous mixture (slurry) and then drawn on
glass plates of 20 x 20cm using TLC applicator with thickness of 2mm and
allowed to dry. The coated plates were activated by drying in forced draft hot
air oven at 1100C for one to two hours. The activated plates were
then cooled to room temperature. Extracted toxin (crude) was spotted on TLC
plate with capillary tube and allowed to run using toluene: ethyl acetate:
formic acid (6:3:1) as mobile phase. After that the plate were dried, viewed
under UV light (366nm) and calculated Rf value.

High
performance liquid chromatography (HPLC)

Reverse
phase HPLC with C-18 column was used. The extracted toxin dissolved in methanol
was passed through membrane filters, Methanol: water (70:30) mixture was used
as mobile phase. 20 µl of the toxin was injected to HPLC column and made to run
through the column for 20 min. The flow rate of the column was adjusted to 0.7
ml and wavelength to 228nm. Toxin was detected in UV detector system. Standard
toxin was also injected to determine the appropriate peak and concentration of
toxin extracted 21.

Fourier
transformed infrared spectroscopy

             The functional groups of extracted
toxin were anlyzed using FTIR ( Nicolet 380 Thermo) based on the peak
values in the region of IR radiation. The toxin was passed into the FTIR, the
functional groups of the compounds were separated based on their peaks ratio.
The FTIR spectra were recorded in the absorption range between 4000 and
500cm?1.

Cell
Culture Studies

The Human skin cell line HaCaT, human liver cancer cell line HepG2 and human
neuroblastoma cell line SHSY5Y was procured from National Center for Cell
Sciences, Pune, India. The SHSY5Y and HaCaT cell lines were cultured in
DMEM/F-12 mixture and HepG2 cell line were cultured in MEM/F-12 mixture
supplemented with 10% FBS, antimycotic solution (Sigma, St.Louis, MO, USA).
Cells were maintained and incubated at 37°C in a humidified atmosphere and 5%
CO2 atmosphere 95% air at 370C. The three cell lines were treated
with different concentration of T-2 toxin (ng) for 24 hr.

Cell viability assay

To asses the changes in the metabolic activity of
the  cell MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used. The Tetrazolium dye used to measure the cell proliferation
and cell viability. The HaCaT, HepG2 and SHSY5Y cell were seeded in different
96 well plates and treated with T-2 toxin at different concentration. After
treatment incubated with MTT (0.5 mg/ml) at 370C for 4hrs and
the formed formazan crystals dissolved in DMSO. The absorbance measured at
540nm (VERSA max Hidex plate chameleon TM V (Finland) ). The cell viability
expressed as percent of control.

Lactate dehydrogenase (LDH) leakage
assay

Lactate dehydrogenase (LDH), leakage
a biomarker  of cellular cytotoxicity and
cytolysis. Cell injury was assessed by measuring the LDH efflux to the media
after cells has been treated for 24 hr. The protocol following
the manufacturers’ instructions (LDH-estimation
kit (Agappe-11407002, Mysore, India)). The
HaCaT, HepG2 and SHSY5Y cell were seeded in different 24 well plates and cell
count set as 1X104 cell/well. The cells were subjected to different
concentration of T-2 Toxin. After the treatment the cells were centrifuged at
2500Xg for 5min at 40C and supernatant was collected and measured the LDH
activity estimated.

Propidium iodide staining

The HaCaT, HepG2 and SHSY5Y cells
were seeded at 1X104 cell/well  into a six well plate. At 90% confluence,
the cells were treated with T-2 toxin and incubated for 24 hrs. After
incubation the cells were washed with PBS and fixed with 100% ethanol. The
fixed cells stained with 100ng/ml propidium iodide for 20 min and observed
under fluorescent microscope.

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