In Vitro Mutagenic and Genotoxic Assessment of a Mixture of the Cyanotoxins Microcystin-LR and Cylindrospermopsin

Abstract

The co-occurrence of various cyanobacterial toxins can potentially induce toxic effects different than those observed for single cyanotoxins, as interaction phenomena cannot be discarded. Moreover, mixtures are a more probable exposure scenario. However, toxicological information on the topic is still scarce. Taking into account the important role of mutagenicity and genotoxicity in the risk evaluation framework, the objective of this study was to assess the mutagenic and genotoxic potential of mixtures of two of the most relevant cyanotoxins, Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN), using the battery of in vitro tests recommended by the European Food Safety Authority (EFSA) for food contaminants. Mixtures of 1:10 CYN/MC-LR (CYN concentration in the range 0.04–2.5 µg/mL) were used to perform the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium, the mammalian cell micronucleus (MN) test and the mouse lymphoma thymidine-kinase assay (MLA) on L5178YTk± cells, while Caco-2 cells were used for the standard and enzyme-modified comet assays. The exposure periods ranged between 4 and 72 h depending on the assay. The genotoxicity of the mixture was observed only in the MN test with S9 metabolic fraction, similar to the results previously reported for CYN individually. These results indicate that cyanobacterial mixtures require a specific (geno)toxicity evaluation as their effects cannot be extrapolated from those of the individual cyanotoxins.

Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats

“…No oxidative damage was observed in the pyrimidine and purine bases of DNA, in agreement with the negative results found in the in vivo experiment performed at the same doses with CYN alone in male rats [43]. Moreover, it corroborates the results reported in vitro on Caco2 cells exposed to CYN/MC-LR mixtures [49], and the scarce results obtained from in vitro enzyme-modified comet assay for CYN in HepG2 cells [69] and in Caco-2 cells [57]. This suggests that oxidative stress could have a minor role in the CYN mediated genotoxicity [22,69].…”

“…Generally, the results of this study show no synergism or antagonism between CYN and MC-LR in the assayed combinations and concentrations in rats, because the effects are similar to the in vivo exposure to CYN alone [43]. Moreover, these findings confirm the in vitro experiments performed with the CYN/MC-LR mixture through a battery of tests, in which MN and comet assays were included [49].…”

“…A few studies dealt with the toxicological profile of CYN and MC-LR combinations. Thus, the in vitro assessments of the potential interactions of CYN and MC-LR are very scarce [48][49][50][51], and only two focus on their genotoxicity. Hercog et al [48] described an induction of DNA sb by MN and comet assays at 24 h after the treatment of HepG2 cells with CYN/MC-LR combinations, but to a lesser extent for only CYN.…”

“…The genomic instability detected by the cytokinesis block micronucleus assay was, however, comparable to the individual CYN. Recently, Diez-Quijada et al [49] applied a battery of in vitro tests in several cell lines, including bacterial systems, and they described genotoxic effects only in the MN test, when the metabolic fraction S9 was used. To the best of our knowledge, no in vivo studies have been yet performed to assess the genotoxicity of CYN/MC-LR mixtures and they are necessary to elucidate the contradictory results obtained in vitro.…”

Ecotoxicological investigation of cyanobacterial crude extracts to Daphniamagna under subchronic test conditions

“…Daphnia spp.) due to their toxicities and ubiquitous distribution (Díez-Quijada et al, 2019). Therefore, the adverse effects of the microcystin-producing strain of cyanobacteria on zooplankton have extensively investigated (Liang et al, 2017).…”