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, 2014, 949521

Genotoxicity of microcystin-LR in in Vitro and in Vivo Experimental Models

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Genotoxicity of microcystin-LR in in Vitro and in Vivo Experimental Models

Elsa Dias et al. Biomed Res Int.

Abstract

Microcystin-LR (MCLR) is a cyanobacterial toxin known for its acute hepatotoxicity. Despite being recognized as tumour promoter, its genotoxicity is far from being completely clarified, particularly in organs other than liver. In this work, we used the comet and/or the micronucleus (MN) assays to study the genotoxicity of MCLR in kidney- (Vero-E6) and liver-derived (HepG2) cell lines and in blood cells from MCLR-exposed mice. MCLR treatment (5 and 20 μM) caused a significant induction in the MN frequency in both cell lines and, interestingly, a similar positive effect was observed in mouse reticulocytes (37.5 μg MCLR/kg, i.p. route). Moreover, the FISH-based analysis of the MN content (HepG2 cells) suggested that MCLR induces both chromosome breaks and loss. On the other hand, the comet assay results were negative in Vero-E6 cells and in mouse leukocytes, with the exception of a transient increase in the level of DNA damage 30 minutes after mice exposure. Overall, the present findings contributed to increase the weight of evidence in favour of MCLR genotoxicity, based on its capacity to induce permanent genetic damage either in vitro or in vivo. Moreover, they suggest a clastogenic and aneugenic mode of action that might underlie a carcinogenic effect.

Figures

Figure 1
Figure 1
Viability of Vero-E6 and HepG2 cell lines exposed to MCLR (5 and 20 μM, 24 h) and assessed by the NR assay. H2O2 (400 mM, 1 h) was used as a positive control. Results are expressed as the mean percentage of absorbance values relative to the negative control (±SD) from three independent experiments tested in triplicate. ∗represents a statistically significant difference between the treated and the control cells (P < 0.05).
Figure 2
Figure 2
Micronucleus in cytokinesis-blocked Vero-E6 and HepG2 cells, following exposure to MCLR for 24 hours. Results are expressed as the mean frequency of micronucleated binucleated cells (MNBC) per 1000 binucleated cells (BC). Mean (±SD) were obtained from three (Vero-E6) or two (HepG2) independent experiments, using duplicate cultures. Mitomycin C (MMC, 0.1 μg/mL, 24 h) was used as the positive control of the assay. ∗represents a statistically significant difference between the treated and the control cells (P < 0.05).
Figure 3
Figure 3
Characterization of centromere-positive (cm+) and centromere-negative (cm−) micronuclei in untreated and MCLR-treated HepG2 cells (20 μM, 24 h) by FISH analysis using a human pancentromeric probe. (a) A binucleated cell with a cm+ (2 hybridization red signals) and a cm− MN, following MCLR treatment. (b) Absolute and relative frequencies of cm+ and cm− MN per 1000 binucleated (BC) HepG2 cells.
Figure 4
Figure 4
DNA damage evaluated by the comet assay in white blood cells from mice injected (ip) with 37.5 μg/kg bw of MCLR, at several timepoints after treatment. Results are expressed as the mean percentage of DNA in comet tails (±SD) of 6 animals per treatment condition. The positive control, ethylnitrosourea (ENU) (100 mg/Kg), induced a 2- to 6-fold increase in the percentage of tail DNA. ∗represents a statistically significant difference between the treated and the control cells (P < 0.05).
Figure 5
Figure 5
Results of the micronucleus assay in reticulocytes (Ret) from mice injected (i.p.) with 37.5 μg/kg bw of MCLR. Results are expressed as the mean frequency of micronucleated reticulocytes (MNRet) (±SD) from 6 animals at several timepoints (primary axis). The negative control consisted of samples collected at 0 h. The positive control, ethylnitrosourea (100 mg/Kg), stimulated the increase of MNRet after 48 h (13-fold) and 72 h (2-fold) of exposure. In the secondary axis the percentage of reticulocytes was included. ∗represents a statistically significant difference between the treated and the control cells (P < 0.05).

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