The balance between the therapeutic and toxicological effects of a compound is a very important measure of the usefulness of a pharmacological drug. Therefore, the determination of the potential mutagenic effect of any drug under development is mandatory .
In previous studies, Medola et al.  showed that HK not only had no genotoxic effect, but also was effective in reducing the chromosome damage induced by DXR, by the rat peripheral blood micronucleus test. Recently, Resende et al.  assessed the possible genotoxic activity of HK and its influence on the activities of two known mutagenic agents (DXR and methyl methanesulfonate - MMS), in the micronucleus test with Chinese hamster lung fibroblast V79 cells. HK alone had no genotoxic effect under the conditions tested, but it reduced the chromosome damage caused by MMS. The reduction in DXR-induced clastogenicity was observed at lower concentrations. At higher concentrations, HK acted as a potentiator of DXR-induced clastogenicity, with the observation of a significantly higher frequency of micronuclei in the combined treatment when compared to the positive control.
To complement the above results, the genotoxic∕ mutagenic activities of HK, and its influence on the activities of known mutagenic agents, were assessed by comet and Ames test in this study. According to Witte et al. , experience with genetic toxicology testing over the past few decades has demonstrated that no single test method is capable of detecting all types of genotoxic effects. Therefore, the potential for a chemical to cause genotoxicity is typically determined by using a battery of in vitro and in vivo tests.
Through the comet assay, the first and extremely important observation was the absence of DNA strand breaks; moreover, there were no gene mutations by the Ames test in the presence and absence of metabolic activation. The performance of assays for to assess mutagenicity, as well as other risks, is essential, given the potential consumption of HK by the population. The absence of genotoxic∕ mutagenic effects by HK on V79 cells in the comet test and against S. typhimurium bacterial strains in the Ames test is a positive step towards ensuring its safe use in medicine. Considering the possible use of HK as an antichagasic drug, a lack of mutagenic effects in animal cells and bacteria is highly relevant.
On the other hand, the influence of HK on DXR-induced DNA damage depends on the experimental conditions used and draws attention to the synergistic effect that HK may have when combined with other drugs. In the comet test, the lower concentrations of HK (0.5; 1.0 and 2.0 μM) significantly reduced the extent of DNA damage induced by DXR. However, the higher concentrations of HK (32; 64 and 128 μM), when combined with DXR, showed a higher rate of class 2 damage than in the cells treated with DXR, which was statistically significant at concentrations of 32 and 128 μM. However, the extent of DNA damage did not differ significantly from the frequencies observed in the DXR treatment. These results are consistent with Resende et al. , who assessed the influence of HK, at the same concentrations, on DXR-induced genotoxicity.
The chemical structure of DXR favors the generation of free radicals and the compound can bind to iron and form complexes with DNA, inducing DNA damage. Some studies have demonstrated that oxidative damage is probably related to this formation of free radicals accompanied by a reduction in antioxidant capacity . Thus, at low concentrations, HK might possibly interfere in the intercalation of DXR with DNA or scavenge the generated free radicals. However, at higher doses, HK may increase the oxidative stress generated by DXR, since qualitative HPLC analysis showed that no new compound is formed after the incubation of a mixture of DXR and HK. HK may act as a “janus” compound, i.e., exerting an antioxidant effect at lower concentrations and a pro-oxidant effect at higher concentrations . The synergistic effect also was observed when HK was combined with NOPD in the strain TA97a in the absence of metabolic activation in the Ames test, reinforcing the hypothesis that the HK may act as a “janus” compound.
In the antimutagenicity evaluated by Ames test, HK exhibited a protective effect in more than one test strain and acted against various mutational mechanisms. Among the antimutagenic activity against directly acting mutagens, a moderate effect was found only against frameshift mutations induced by NOPD in the TA98 strain, with the highest %; of inhibition at concentration of 39.0 μg/ plate (26%;).
HK did not affect the SA-induced mutagenicity in strain TA100, MMC in strain TA102 or NOPD in strain 97a.
The protection of the bacterial genome against directly acting mutagens may be due to the rapid elimination of mutagens from the bacteria, before their interaction with the DNA . HK may facilitate or stimulate the bacterial transmembrane export system to eliminate the mutagens; it may also interfere with the uptake of mutagens into bacteria [29, 30].
The activity displayed by HK was profoundly increased by incorporating the microsomal fraction (S9), which is a mammalian metabolic activation system, into the culture medium. The results of this experiment show that HK inhibited BaP, AFB1, 2-AF and 2-AA mediated mutagenesis. The microsomal fraction of rat liver, containing mixed-function oxidase (MFO) and the cytochrome-based P450 metabolic oxidation system, can activate BaP to an active mutagen, benzoapyrene-7,8-diol-9,10-epoxide . The mutagenicity of BaP was significantly reduced in a dose-dependent manner by 31 to 59%; by HK.
This diol epoxide exerts its carcinogenic activity by alkylating nucleosides on DNA molecules at their bay region. The reaction occurs primarily with the purine bases, deoxyguanosine and deoxyadenosine, in DNA . As a result, bulky stable and depurinating DNA adducts are formed [33, 34]. Insufficient removal of these DNA adducts prior to replication creates hot spots in the gene and can result in deactivation of tumor suppressor genes or activation of oncogenes leading to tumor initiation [35, 36].
There are at least two possible mechanisms by which HK could decrease BaP-DNA adduct formation: by interacting with reactive intermediates or by interfering with the action of microsomal enzymes . However, more studies are needed to confirm these ideas.
HK also reduced the frequency of mutations induced by the fungal toxin, AFB1, in TA100 with metabolic activation, resulting in the highest percent inhibition of mutagenicity (89%;). The S. typhimurium tester strain TA100 reveals base-pair-substitution point mutations .
Aflatoxins, a group of potent mycotoxins with mutagenic, carcinogenic, teratogenic, hepatotoxic and immunosuppressive properties, are of particular importance because of their adverse effects on animal and human health. Aflatoxins are produced as secondary metabolites by fungi of various species of Aspergillus (A. flavus, A. parasiticus and A. nomius) that grow on a variety of food and feed commodities. AFB1, which is the most toxic aflatoxin, is metabolized mainly in the liver to AFB1-8,9-exo-epoxide and 8,9-endo-epoxide. The exo-epoxide form of AFB1 binds to DNA to form the predominant 8,9-dihydro- 8-(N7-guanyl)-9-hydroxy AFB1 adduct, leading to a more stable imidazole ring-opened AFB1–formamidopyrimidine adduct. The pseudo-half-life for loss of 8,9-dihydro-8- (N7-guanyl)-9-hydroxy AFB1 is short, but AFB1–formamidopyrimidine adducts are stable, accumulate for several days and remain detectable for several weeks . This aflatoxin is of particular interest because it is a frequent contaminant of many food products and one of the most potent naturally occurring mutagens and carcinogens known .
HK also induced a strong antimutagenic effect, significantly diminishing the mutagenicity of 2-AF in TA102 with metabolic activation, in a dose-dependent manner, by 42 to 52%;. 2-AF is converted in rat liver, via N-hydroxy metabolites, to the reactive carcinogenic ester 2-acetylaminofluorene-N-sulfate, which can attack guanine residues in nucleic acids . The inhibition of 2-AF induced mutagenicity may be mediated through the inhibition of the MFO (in the S9 fraction) or inactivation of the activated reactive ester of 2-AF. The S. typhimurium tester strain TA102 is normally used to detect mutagens that cause oxidative damage and base-pair-substitution mutations . In this case, antimutagenic activity can be partially ascribed to antioxidant activity. This speculation is further supported by the significant antimutagenic effect that the lower concentrations of HK demonstrated against DXR in the comet test, as well as that against mutagens needing metabolic activation, where free radical generation is anticipated.
In this study, the antimutagenic property of HK related to its ability to modulate the xenobiotic-metabolizing enzymes in the liver, either by preventing the metabolic activation or by altering the enzymatic activity in the detoxification pathway to induce the disposal of the known mutagen , was again demonstrated by the results obtained with the mutagen 2-AA in strain TA97a with metabolic activation, where 67%; inhibition was observed.
In general, inhibitors of mutagenesis can act in one of several ways: by inhibiting the interaction between genes and biochemically reactive mutagens; inhibiting metabolic activation of indirectly-acting mutagens by inactivation of metabolizing enzymes, or interacting with the pro-mutagens to make them unavailable for the enzymatic process .