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Folic acid period, the media was replaced with media containing either
Folic acid was quantified at the South African Bureau of 20 or 40 μg/mL A. hybridus and incubated for another 24 h
Standards (Pretoria, South Africa). A standard method for the which was followed by the mycotoxin exposure routine, but
microbiological assay of folic acid in foods and pharmaceutical for only the 48 h period. The controls included (1) 11 wells
products was followed according to Barton-Wright and with cells and nutrient medium only for the duration of the
[24]
AOAC. [25] entire experiment (when media was replaced, their media
was replaced with fresh nutrient medium) and (2) 6 wells with
Quantifi cation of fatty acids cells and plant extract containing media only. The mycotoxin
Fatty acids were identified and quantified by use of gas exposures were dosed in triplicate.
chromatography coupled with mass spectrometry system
with split-less injection. An Agilent 6890 gas chromatograph The viability of H4IIE-luc cells was determined using the
ported to a 5973 mass selective detector (CA, USA) was used methyl thiazol tetrazolium (MTT) salt assay in which the
according to the method described by van der Walt et al. [17] mitochondria of live cells metabolize the yellow MTT
[27]
into blue formazan. A final concentration of 500 μg/mL
Mineral and trace element analysis MTT was incubated for 30 min and blue formazan crystals
Minerals and trace elements were quantified by use of dissolved with dimethyl sulfoxide. The absorbance was
an Agilent 7500c inductively coupled argon plasma mass measured spectrophotometrically at 560 nm. The amount
[18]
spectrometer as described by van der Walt et al. Three of formazan gives an estimation of the proportion of
separate samples were analyzed and values were reported viable cells. The percentage of viable to dead cells was
as the mean ± SD in mg/100 g dm. calculated by comparison with a control (untreated and
solvent control). The MTT assay assessed the viability of
Cytotoxicity measurements H4IIE-luc cells that were subjected to the two A. hybridus
The mammalian model was rat hepatoma cells (H4IIE-luc) that extract concentration treatments compared to the viability
had been transfected stably with a firefly luciferase reporter of cells that were not treated with A. hybridus extracts prior
gene under control of the dioxin response element and thus to mycotoxin exposure.
the aryl hydrocarbon receptor mechanism. These cells had
[26]
originally been developed as a reporter gene assay to detect DNA extraction
and semi-quantify the levels of certain groups of persistent Cells were harvested by first washing away non-adherent
organic pollutants. Since these cells are essentially still dead cells with PBS before trypsinizing (0.25% trypsin, 0.1%
[26]
mammalian cells, they were useful to assess whether extracts versene ethylenediaminetetraacetic acid) adherent cells.
of selected A. hybridus can be protective against AFB and FB Enzyme activity was stopped by the addition of media. The
1 1
or their mixture. cell suspension was centrifuged for 5 min (300 g) at room
temperature. The genomic DNA was extracted from the cells,
Cells were seeded with a density of 1.0 × 10 cells/mL media according to the Qiagen instruction manual. The concentration
4
in the inner 60 wells of a 96-well microplate. Growth medium of DNA was determined by photometry (NanoDrop ND-1000
was Dulbecco’s modified Eagle’s medium (Sigma, D2902) Spectrophotometer) and the purity of the DNA was judged by
supplemented with 0.044 mol/L NaHCO and 10% fetal bovine examining the ratio of absorbency at 260/280 nm. [28]
3
serum (Gibco). The volume in each well was 250 μL. The
outer cells received 250 μL Dulbecco’s phosphate buffered Random amplifi ed polymorphic DNA-polymerase chain
saline (PBS) to create a homogenous microclimate across all reaction analysis
wells containing cells and incubation conditions were 37 °C Amplification of DNA fragments was carried out on an
in a humidified 5% CO :air mixture. The plates were seeded ICycler (Bio-Rad, UK) thermal cycler using 20 primers purchased
2
and after an initial 24 h incubation medium was replaced from the Operon Biotechnologies (BioCampus Cologne
with medium containing varying concentrations of AFB (50, Nattermannalle, Germany). PCR amplification was conducted
1
25, 2.5, 0.25, 0.025 μmol/L) and FB (200, 100, 10, 1, 0.1 in a 25 μL reaction volume containing 10 ng genomic DNA,
1
μmol/L) dissolved in methanol. A combination of the already 12.5 pmol Master mix (2X) (Fermentas Life Science, USA), 1.0
mentioned concentrations of AFB and FB were also tested: units of Supertherm Taq polymerase, and 50 pmol primer. PCR
1
1
50 μmol/L AFB plus 200 μmol/L FB ; 25 μmol/L AFB plus reactions were carried out in a thermocycler (Bio-Rad C1000)
1
1
1
100 μmol/L FB ; and so on. Two exposure periods 24 h and 48 h programmed with initial denaturation period for 5 min at
1
were investigated. In order to evaluate the protective effect 95 °C, followed by 40 cycles denaturation (95 °C for 30 s),
of extracts of A. hybridus, this experiment was repeated with primary annealing at 37 °C for 1 min and extension at 72 °C.
the following adjustments: After the initial 24 h incubation Amplification was terminated by a final extension period of
138 Hepatoma Research | Volume 1 | Issue 3 | October 15, 2015