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72 °C for 5 min. Reaction products were stored at -80 °C prior exposure to AFB for 48 h [Table 2]. Other combinations did
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to electrophoresis. not have any statistically significant difference. Percentage
inhibition of the cells incubated for 24 h with FB showed
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Gel electrophoresis more cytotoxicity than those incubated for 48 h. On the
Amplified products together with a marker (100 bp DNA) were other hand, FB at the concentration of 200 μmol/L decreased
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resolved by gel electrophoresis 60 V/cm for 135 min on 2% cell viability to 41.6% [Figure 2b]. The protective effect of
agarose gel in TAE buffer containing 0.001 mg/mL ethidium 20 μg/mL A. hybridus extract was decreased by increasing FB
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bromide. Gels were photographed by a Gel Documentation dose to 100 μmol/L [Figure 2b]. A. hybridus extract at 40 μg/mL
system (Gensnap) equipped with its software (Synegen, UK). was more efficient at protection against all concentrations
of FB .
Band analysis 1
Gels of control and exposed DNA samples were run for each of Overall, AFB was more cytotoxic than FB for both exposure
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the 20 primers [Table 1]. A DNA ladder of 100 bps was also run periods [Figure 2a and b]. Exposure of H4IIE-luc cells to AFB
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in each gel. Bands in PCR products were analyzed by TotalLab led to a dose-and time-dependent decrease in cell viability. At
Quant (V11.5: TL100-LX59-7YF4-EX). The fluorimetric profiles 25 μmol/L AFB , viability was inhibited to 58.7% and 96.1% for
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of each amplification reaction were studied both qualitatively the 24 and 48 h exposure periods respectively. Pre-treating
and quantitatively by comparing profiles from the control and the cells to 40 μg/mL A. hybridus had a more protective effect
DNA exposed to the extracts. Each change observed in random than pre-treatment of 20 μg/mL [Figure 2a].
amplified polymorphic DNA (RAPD) profiles of the treated
groups (disappearances and appearance of bands in comparison The combination of AFB and FB was more cytotoxic than
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to the control RAPD profiles) was given the arbitrary score AFB alone which indicated that FB increased the cytotoxicity.
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of +1. The average was then calculated for each experimental This was true for both exposure periods [Figure 2a-c].
group exposed to the mycotoxins for varying time periods. However, this general trend was not corroborated by the
Genomic stability (%) was calculated as “100 - (100 a/n)” where EC values (EC = concentration by which viability was
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“a” is the average number of changes in DNA profiles and “n” declined to 50%) [Table 3]. They were in fact slightly greater
is the number of bands selected in control DNA profiles. [29] for the combined mycotoxins than exposure to AFB alone,
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meaning that 50% effect was reached at a greater mycotoxin
Statistical analysis concentration. Extract of A. hybridus alone (20-100 μg/mL) had
Values for EC and cell viability were statistically analyzed no significant influence on the viability of cells [Figure 2d].
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with the Graphpad Prism 4.02 Inc., (La Jolla, CA, USA).
Significance of differences among treatment groups was Table 1: Sequences of the primers used to amplify cell
determined with the Waller-Duncan k-ratio. All statements line of hepatoma (H4IIE-luc) cells
[30]
of significance were based on a probability of P < 0.05. Primer Sequence 5’-3’ Primer Sequence 5’-3’
D01 ACCGCGAAGG D11 AGCGCCATTG
D02 GGACCCAACC D12 CACCGTATCC
RESULTS D03 GTCGCCGTCA D13 GGGGTGACGA
D04 TCTGGTGAGG D14 CTTCCCCAAG
The extract was rich in polyphenols (total phenolic contents: D05 TGAGCGGACA D15 CATCCGTGCT
2181.2 mg/100 g dm, total carotenoids (113.6 mg/100 g dm) D06 ACCTGAACGG D16 AGGGCGTAAG
and β-carotene (18.4 g/100 g dm) [Figure 1a]. The results of D07 TTGGCACGGG D17 TTTCCCACGG
the lipid profile showed significant amounts of the fatty acids, D08 GTGTGCCCCA D18 GAGAGCCAAC
linolenic, linoleic and palmitic acids [Figure 1b]. The extract D09 CTCTGGAGAC D19 CTGGGGACTT
had moderate concentrations of palmitoleic, stearic, and D10 GGTCTACACC D20 ACCCGGTCAC
lignoceric acids whereas behenic, arachidic, and myristic acids
were found in low concentrations. The extract was rich in Table 2: Summary of the P values of the Wilcoxon
folic acid (72 mg/100 g dm), calcium, magnesium [Figure 1c], matched pair tests to compare viability of cells exposed
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iron, zinc, and selenium [Figure 1d]. to FB , AFB , and their mixture and those treated with
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A. hybridus extracts prior to 48 h mycotoxin exposure
Mycotoxins A. hybridus extract
Cytotoxicity of AFB , FB , and mixture with or without the
1 1 20 μg/mL 40 μg/mL
extract of A. hybridus on H4IIE-luc cell line as measured by the FB 0.04* 0.69
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tetrazolium dye-based MTT assay are shown in Figure 2a-d. AFB 0.5 0.08
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There was a significant difference in viability between cells FB + AFB 1 0.69 0.2
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treated with 20 μg/mL A. hybridus and those not treated before *P < 0.05. FB : fumonisin B ; AFB : afl atoxin B ; A. hybridus: Amaranthus hybridus
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Hepatoma Research | Volume 1 | Issue 3 | October 15, 2015 139