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differences among treatment groups was determined with exposure, respectively [Table 3]. At the lesser concentration,
two-way analysis of variance. The assumptions of parametric FB did not cause measurable cytotoxicity. However, the
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statistics were confirmed. Normality was confirmed by the MTT assay revealed cytotoxicity at the greater concentration
Kolmogorov-Smirnov test and homogeneity of variance (200 μmol/L) although all doses studied were less than those
was confirmed by use of Levine’s test. All statements of required to obtain an EC .
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significance were based on a probability of P ≤ 0.05.
Only 5 of 10 oligonucleotide primers, primers D07, D09, D13,
RESULTS D15, and D16, used to measure responses of molecular-genetic
parameters of cells among various treatments, gave
The results of cell viability assay revealed that H4IIE-luc cells detectable bands [Figure 3]. A total of 75 DNA sequences,
that were treated with both concentrations (20 and 40 μg/mL) ranging from 144 to 2,000 bp, were observed. All of the bands
of C. olitorius extract were statistically significantly more were “polymorphic” given 100% polymorphism for control
viable after 24 h exposure than the ones that were not treated cells and the other treatments for the 2 time periods using
with the plant extract. However, there was no significant all primers. Quantitative analysis of these bands, expressed
protection by the plant extract against FB after 48 h of as a percentage of band loss, showed a time-dependent
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exposure. Furthermore, C. olitorius extract could not protect relationship [Figure 3 and Table 4]. Similarly, in the case of
the cells from the AFB concentration series or the combination losses of bands after the shorter period of exposure (24 h),
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exposure (AFB + FB ) irrespective of the exposure period 12 of 75 bands (16%) had disappeared [Figure 3a]. At the
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(24 or 48 h) [Table 2]. A dose-dependent decrease of cell viability longer duration of exposure (48 h), 21 of 75 bands (28%)
after exposure to increasing amounts of AFB was observed only had disappeared [Figure 3b]. Protective effects of C. olitorius
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after 48 h [Figure 1a]. After 24 h exposure, the response was not extract were observed after 24 h, when 25 of 75 bands (33.3%)
linear (hormetic effect). Except for the lowest concentration,
cytotoxicity was more pronounced after 48 h. However, Table 2: Summary of Wilcoxon matched pair tests to
protective effects of the C. olitorius extract were observed after compare the viability of rat hepatoma H4IIE-luc cell line
both 24 h and 48 h of exposure to AFB . After 48 h, viability, treated with C. olitorius extract
1 Mycotoxins Exposure time
expressed as a percentage of H4IIE-luc cells affected by FB , C. olitorius extract
1 concentrations
was approximately 40% less than that of cells exposed to FB
1 20 μg/mL 40 μg/mL
alone. After 48 h, there was also no dose-dependence, but FB 24 h 0.04* 0.04*
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cytotoxicity was less pronounced. Protective effects of 20 or 48 h 0.69 0.89
40 μg/mL C. olitorius extract were observed. After both 24 and AFB 1 24 h 0.9 0.5
48 h of exposure, production of MTT formazan was greater in 48 h 0.35 0.89
the presence of both concentrations of C. olitorius extract at FB + AFB 1 24 h 0.69 0.5
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all tested doses of FB compared to those in the absence of 48 h 0.22 0.08
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C. olitorius extract [Figure 1b]. No significant differences were *P ≤ 0.05. AFB : afl atoxin B ; FB : fumonisin B ; C. olitorius: Cochorus olitorius
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1
1
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found between 20 and 40 μg/mL of C. olitorius extract after Table 3: EC values of AFB , FB , and AFB + FB alone or
1
1
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1
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24 h of exposure. in combination with the C. olitorius extract after 24 and
48 h and exposure measured by the MTT bioassay using
Incubation of H4IIE-luc cells with AFB + FB for 24 h resulted H4IIE-luc rat hepatoma cells
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in greater cytotoxicity to cells as measured by the MTT assay, Mycotoxin and/or plant Time Cytotoxicity (EC )
50
with significant toxicity at the sum of the two mycotoxin extract treatments exposure (h) H4IIE-luc
concentrations 12.5 and 125 μmol/L [Figure 1c]. The cells FB 1 24 ND
ND
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were least viable when they were exposed to the mixture of AFB 24 6.90
250 μmol/L mycotoxin. Addition of C. olitorius extract to cells 1 48 1.95
resulted in slightly greater viability. At lesser concentrations FB + AFB 24 14.5
1 1
of AFB (1.25 μmol/L) + FB (12.5 μmol/L), protective 48 6.8
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effects of aqueous extracts of C. olitorius on viability of cells FB + C. olitorius (20 μg/mL) 24 542.8
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was greater relative to the cells that did not receive plant FB + C. olitorius (40 μg/mL) 24 26646
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extract [Figure 2]. AFB + C. olitorius (20 μg/mL) 24 4.32
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AFB + C. olitorius (40 μg/mL) 24 2.42
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FB + AFB + C. olitorius (20 μg/mL) 24 18.5
The EC values for AFB were 6.9 and 1.8 after 24 and 48 h 1 1
50 1 FB + AFB + C. olitorius (40 μg/mL) 24 21.77
of exposure, respectively. When C. olitorius extract was 1 1
AFB1: afl atoxin B ; FB : fumonisin B ; C. olitorius: Cochorus olitorius;
1
1
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added, the EC values were 4.3 and 2.49 after 24 or 48 h of ND: not detectable; MTT: methylthiazole tetrazolium
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78 Hepatoma Research | Volume 1 | Issue 2 | July 15, 2015