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modifications elicited in the genome. All primers used in amplification profiles. The nature of the RAPD reaction,
[50]
this study could detect changes in all treatments that might where the final products are the result of an exponential
be due to a latent phase required for the appearance of multiplication of the most abundant and stable fragments
adequate number of cells with genetic damage. co-amplified in the first cycles is the cause of the differences
in the concordance among replicates. In other words, it
Alterations observed in the present study included the is necessary that new annealing sites appear in a high
absence and/or presence of bands in all treatment groups. The proportion of the cell population to get a high reproducibility.
appearance and disappearance of bands might be associated The first new bands appeared at the high concentration of
with genetic rearrangements or clastogenic effects of the FB and/or AFB (D-7 1466 , D-7 ) suggested that the proportion
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525
toxicant. Such alterations in the genome might subsequently of cells with a new annealing primer-template was increased
interfere with binding of primers or amplification step. at the greater concentration. The RAPD assay is able to detect
[51]
Increases in band intensity and appearance of new PCR products mutation only if they occur in at least 2% of the DNA. A
[56]
have been attributed to conformational changes in DNA, which concentration-dependent effect was observed when the
[52]
might improve the access of primer(s) to the binding site(s). same chemical and the same cells were used. Similar results
Furthermore, enhancement and reduction of signal intensity were previously demonstrated a dose-dependent effect
of an amplified DNA fragment might be related to localized of the genotoxic action of mycotoxin when measured by
over- or under-amplification of that gene locus in the genome, micronuclei induction. [57]
which could result from changes at the chromosome level.
The combined use of in vitro systems and the RAPD technique
Instability in template DNA was observed in all treatments permits detection of alterations in DNA caused by multiple
which may be due to DNA damage. Although RAPD appears mechanisms with a sufficient degree of sensitivity. Alterations
to be instrumental in observing definitive changes, it requires were detected in an unspecific form by losses and/or gains
enough time and sufficient theoretical knowledge for initial of bands and variations in the amplification intensity.
standardization to obtain reproducible and unambiguous Nevertheless, when the objective is to establish the existence
results. Interpretation of molecular events responsible for of DNA damage, that is, for hazard identification in risk
differences observed in the RAPD pattern is not easy since assessment studies, the presence in the fingerprint of any of
different DNA alterations may induce similar types of changes. these abnormalities would be enough to identify a genotoxic
The RAPD is known to produce non-reproducible bands, effect. For example, the presence of one or both of the two
but once established and standardized, there are certain new bands in DNA extracts of cells treated either with a
additional benefits to using this method for early genotoxicity chemical or with an environmental sample can be considered
studies other than being fast. as a suitable genotoxicity biomarker of chronic exposure.

Differences in sensitivity were observed, depending on the The protective effects of A. hybridus extract against FB showed
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primer sequence. This observation suggests the mode of that the extract was more effective at its greater dose than
action of FB and/or AFB . The five primers used showed a at the lesser dose. In addition, the ability of the extract to
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greater alteration after the treatment and the appearance of eliminate the cytotoxic effects induced by AFB appeared less
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new bands in all the extracts-treated groups were produced effective compared with that induced by FB . The difference
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from those primers. The mechanism by which these toxins between AFB and FB -induced cytotoxic effects may be
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affect the sequence of DNA has been extensively supported in due to the stronger oxidative stress caused by AFB even
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the literature. Some of the AFB adducts have been shown at a lower concentration than FB . Several studies showed
[53]
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to be capable of inducing base substitution, frameshifts, the benefits of antioxidant compounds in the diet against
insertions and deletions at specific loci of the DNA. For the toxicity of mycotoxins. The inhibition of DNA and
[58]
example, AFB adduct induces G > T transversion at specific protein synthesis induced by AFB and FB were decreased by
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loci within p53. [54,55] The resulting alterations in DNA can pre-treatment of the CaCo-2 and Hep G2 cell lines with the
induce changes in the DNA sequence at specific places antioxidant cyaniding-3-0-β-glucopyranoside. The ability
[58]
generating different annealing primer-template sites. This of A. hybridus extract to inhibit the cytotoxic effects induced
[56]
is probably the reason why altered bands were always the by the mixture of AFB and FB was more pronounced at the
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same in most of the concentrations and the exposure periods higher concentration of the extract (40 μg/mL) than the lower
in both the qualitative and the quantitative analysis. concentration (20 μg/mL). Moreover, this protective effect was
smaller in the case of the mycotoxin mixture compared to that
Generation of new annealing primer-template sites would of FB only. These results were similar to those reported by
1
be in accordance with the presence of new bands in the Guerra et al. who suggested that the inhibitory action of
[59]
144 Hepatoma Research | Volume 1 | Issue 3 | October 15, 2015