Cytotoxic and morphological effects of microcystin‐LR, cylindrospermopsin, and their combinations on the human hepatic cell line HepG2

Abstract

Cylindrospermopsin (CYN) and Microcystin-LR (MC-LR) are toxins produced by different cyanobacterial species, which are found mainly in freshwater reservoirs. Both of them can induce, separately, toxic effects in humans and wildlife. However, little is known about the toxic effects of the combined exposure, which could likely happen, taking into account the concomitant occurrence of the producers. As both cyanotoxins are well known to induce hepatic damage, the human hepatocellular HepG2 cell line was selected for the present study. Thus, the cytotoxicity of both pure cyanotoxins alone (0-5 μg/mL CYN and 0-120 μg/mL MC-LR) and in combination for 24 and 48 h was assayed, as long as the cytotoxicity of extracts from CYN-producing and nonproducing cyanobacterial species. The potential interaction of the combination was evaluated by the isobologram or Chou-Talalay's method, which provides a combination index as a quantitative measure of the two cyanotoxins interaction's degree. Moreover, a morphological study of the individual pure toxins and their combinations was also performed. Results showed that CYN was the most toxic pure cyanotoxin, being the mean effective concentrations obtained ≈4 and 90 μg/mL for CYN and MC-LR, respectively after 24 h. However, the simultaneous exposure showed an antagonistic effect. Morphologically, autophagy, at low concentrations, and apoptosis, at high concentrations were observed, with affectation of the rough endoplasmic reticulum and mitochondria. These effects were more pronounced with the combination. Therefore, it is important to assess the toxicological profile of cyanotoxins combinations in order to perform more realistic risk evaluations.

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

“…Moreover, a risk assessment can be greatly influenced when diverging from individual toxin exposure to a multi-toxin exposure scenario. Gutiérrez-Praena et al [41] found an antagonistic effect of CYN and MC-LR when investigating the cytotoxicity of binary mixtures in comparison to the individual toxins in HepG2 cells. Hercog et al [42] observed a genotoxic potential of CYN/MC-LR mixtures comparable to that of CYN alone when using the micronucleus (MN) and comet assays in the same experimental model.…”

Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells

“…Likewise, two studies found no significant differences between the vehicle control and CYN-treated HepG2 cells at 0.5 μg/mL [ 28 ] and 2 μg/mL [ 29 ] after 24 h.…”

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

“…Although the simultaneous occurrence of cyanotoxins such as MCs and CYN is being reported more and more frequently [44,55], toxicological studies focusing on their potential interaction are very scarce [50,56]. Particularly, the genotoxicity of mixtures is of great interest for humans exposed to contaminated waters and food with cyanotoxins, due to the possible carcinogenic effects of MC-LR and pro-genotoxic activity of CYN.…”

“…Even though occurrence data show that MC-RR and other minority MCs are distributed worldwide becoming sometimes predominant, the congener MC-LR is widely distributed and the main focus of toxicological studies [8,64]. The ratio CYN/MC concentrations could oscillate between countries and continents, climatic conditions, or composition of the cyanobacteria communities [44,45], and in this study the ratio 1:10 CYN/MC-LR was chosen to compare or confirm previous toxicity studies in which combinations of CYN/MC-LR were assayed [49][50][51]. Furthermore, the International Conference on Harmonisation (ICH) S2 guidelines [90] and the OECD 474 [53] and OECD 489 [54] guidelines for the MN and Comet assay, respectively, recommend for the combined MN-comet assay the use of the highest dose and two additional lower doses [58] appropriately separated by less than √ 10 to prove dose-related responses [54].…”

“…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.…”