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the medium was removed and replaced with medium 12.5 pmol/L master mix (×2) (Thermo Fisher Scientific,
containing C. olitorius extract at either of two concentrations Carlsbad, CA, USA), 1.0 units of Supertherm Taq polymerase
(20 or 40 μg/mL) and incubated for another 24 h. The medium and 50 pmol/L primer. The PCR reactions were carried out in
was replaced with medium containing varying concentrations a thermocycler (Bio-Rad C1000, Bio-Rad, Hercules, CA, USA),
of AFB (50, 25, 2.5, 0.25, 0.025 μmol/L) dissolved in methanol, programed with a first denaturation for 5 min at 95 °C, followed
1
or of FB (200, 100, 10, 1, 0.1 μmol/L) dissolved in methanol. by 40 cycles for 30 s denaturation at 95 °C, 30 s annealing at
1
A combination of the already mentioned concentrations of 37 °C and 1 min extension at 72 °C. Final extension at 72 °C for
AFB and FB were also tested: 50 μmol/L AFB +200 μmol/L 5 min was allowed before holding at 4 °C for 5 min. Reaction
1 1 1
FB ; 25 μmol/L AFB + 100 μmol/L FB , and so on. Exposure products were stored at -80 °C prior to electrophoresis.
1 1 1
to mycotoxins was carried out in triplicates. Cells in six wells
in each plate were exposed only to the aqueous extract of Gel electrophoresis
C. olitorius and cells in 11 wells were not exposed to anything Amplified products together with marker (100 bp DNA)
except the growth medium. were resolved by gel electrophoresis (60 V/cm for 135 min)
on 2% agarose gel in tris-acetate-EDTA buffer containing
To determine the viability, based on metabolic activity of 0.001 mg/mL ethidium bromide purchased from Sigma
cells, a colorimetric assay was performed using the yellow Chemical Co. (St. Louis, MO, USA). Gels were photographed by
dye 3-(4,5-dimethyltiazol-2yl)-2,5-diphenyl tetrazolium Gel Documentation System (Gensnap) software (Synegen, UK).
bromide (MTT, Montigny-le-Bretonneux, France). In this
assay, MTT is converted to formazan (blue) by mitochondrial Band analysis
reductase enzymes in living cells. A final concentration of The gels for control and exposed DNA were run for each
[40]
500 μg/mL MTT was added to each well and incubated for of the 20 primers [Table 1]. A DNA ladder of 100 bp was
30 min. Blue formazan crystals that were formed by reduced also run in each gel. The bands for PCR products were
MTT were dissolved with dimethylsulfoxide and absorbance analyzed by TotalLab Quant (V11.5: TL100-LX59-7YF4-EX).
by the formazan was measured spectrophotometrically The fluorimetric profiles of each amplification reaction were
at 560 nm. The amount of blue formazan produced is studied both qualitatively and quantitatively by comparing
proportional to the amount of viable cells, and the percentage profiles from control and DNA exposed to the extracts. Each
of viable to dead cells was calculated by comparison change observed in random amplification of polymorphic
with a control (untreated and solvent control). Viability DNA (RAPD) profiles of treated groups (disappearances and
among various C. olitorius treatments described above appearance of bands in comparison to the control RAPD
were compared to the viability of cells treated only with profiles) was given the arbitrary score of +1. The mean was
mycotoxins by applying the same protocol described before, then calculated for each experimental group exposed to
but omitting aqueous extract of C. olitorius. the mycotoxins for varying time periods. Template genomic
stability (%) was calculated as “100 - (100a/n)” where “a” is
Extraction of DNA the average number of changes in DNA profiles and “n” is the
Harvested cells were washed with PBS to remove the number of bands selected in control DNA profiles. [42]
nonadherent dead cells. The adherent cells were removed by
trypsinizing (0.25% trypsin, 0.1% versene EDTA; purchased from Statistical analysis
Thermo Scientific, Rockford, IL, USA) and activity was stopped All data were statistically analyzed with the Graphpad
by addition of media. The cell suspension was centrifuged at Prism 4.02 Inc. (La Jolla, CA, USA). The significance of the
3,000 g for 5 min at room temperature. Genomic DNA was
extracted from cells according to the Qiagen instruction manual Table 1: Sequences of the primers used to amplify DNA
and concentrations determined spectrophotometrically by use of H4IIE-luc rat hepatoma cells
of the NanoDrop ND-1,000 Spectrophotometer. Purity of DNA Primer Sequence 5’-3’ Primer Sequence 5’-3’
was assessed by examining the 260/280 nm ratio. [41] D01 ACCGCGAAGG D11 AGCGCCATTG
D02 GGACCCAACC D12 CACCGTATCC
D03 GTCGCCGTCA D13 GGGGTGACGA
Random amplifi cation of polymorphic DNA-polymerase
chain reaction analysis D04 TCTGGTGAGG D14 CTTCCCCAAG
D05
CATCCGTGCT
D15
TGAGCGGACA
Amplification of DNA fragments was carried out using an D06 ACCTGAACGG D16 AGGGCGTAAG
ICycler (Bio-Rad, Herts, UK) thermal cycler using 20 primers D07 TTGGCACGGG D17 TTTCCCACGG
from the Operon Biotechnologies (BioCampus Colonge D08 GTGTGCCCCA D18 GAGAGCCAAC
Nattermannalle, Germany). PCR amplification was conducted D09 CTCTGGAGAC D19 CTGGGGACTT
in 25 μL reaction volumes containing 10 ng genomic DNA, D10 GGTCTACACC D20 ACCCGGTCAC

Hepatoma Research | Volume 1 | Issue 2 | July 15, 2015 77