Toxicity of the cyanobacterial cyclic heptapeptide toxins microcystin-LR and -RR in early life-stages of the African clawed frog (Xenopus laevis)

Abstract

Numerous cyanobacterial species are capable of producing potent toxins, which have been known to cause intoxications and fatalities in wildlife, livestock and humans. Microcystis is amongst the most ubiquitously distributed blue green algal genus and almost invariably produces cyclic heptapeptide toxins called microcystins (MC). These toxins are highly persistent in water (several weeks). Highest concentrations are found in shallow littoral areas, the primary environment for aquatic early life-stage development. Therefore, the present study focussed on the potential embryotoxic effects of MC (MC-LR and -RR) in early life-stages of the amphibian Xenopus laevis. The endpoints chosen were mortality, malformation and growth inhibition. To achieve an improved dose-response relationship the uptake of MC was quantified simultaneously, using a radiolabeled derivative of MC-LR. As one of the best described molecular mechanisms of MC toxicity involves the specific inhibition of serine/threonine protein phosphatases-1 and -2A (PP), essential enzymes involved in the mechanisms of cell cycle regulation and maintenance of cellular morphology, the inhibition of PP in X. laevis exposed to MC was monitored. For this the presence of both PP-1 and PP-2A was confirmed by means of SDS-PAGE and immunoblotting. Second, the capacity of MC to inhibit X. laevis embryo-larval PP was corroborated by in vitro incubation of embryo-larval homogenates with MC-LR and -RR and subsequent determination of PP-inhibition. No increased mortality, malformation, or growth inhibition was observed even at the highest MC concentrations employed. MC had neither a demonstrable inhibitory effect on X. laevis PP-activity in vivo in the first 96 h of exposure. However, as of 96 and 120 h exposure a significant inhibition of PP activity was observed at the highest dose (2000 μg/l) in MC-LR and MC-RR exposed embryo-larvae, respectively. By the same token, no notable amounts of radiolabeled [3H]-MC-LR were taken up during the first 96 h, whereas a drastic increase in [3H]-MC-LR was observed after feeding of the larvae had commenced. The [3H]-MC-LR concentration was consistently found to be highest in the viscerothoracal sections of the larvae (2112±429 μg MC/kg dry weight after 120 h). The present findings indicate that transchorional/transdermal absorption of MC in X. laevis is minimal or absent and that oral uptake of MC with ambient water is necessary for the development of MC related toxicity. Furthermore, the comparison of the MC doses used in this study with the concentrations reported in surface waters indicate that early life-stages of amphibians (up to 5 days of development) are unlikely to be affected by cyanobacterial blooms producing MC-LR and -RR.