**6. Conclusions**

Fish cytolysins stand out among other animal toxins because they alone can reproduce the main in vitro and in vivo effects induced by the venoms in which they are contained. These multifunctional toxins account for the extreme pain and inflammatory events experienced by the victims of envenomation by fish and affect the neuromuscular and cardiovascular systems in a significant way, potentially leading to death. Not to mention the species-specific hemolytic activity that, although seemingly irrelevant where envenomation is concerned, characterizes this type of toxin.

Although intriguing, this multifunctionality also adds to the complexity surrounding the mechanisms of action underlying the different activities displayed by these toxins (Figure 5). At this point, it is established that fish cytolysins destroy cells by forming non-selective pores in the membrane, but how much this pore-forming ability actually contributes to the pharmacological activities induced by these molecules is a matter for discussion.

Ample evidence points to fish cytolysins affecting the cardiovascular and neuromuscular systems through the modulation of signaling pathways that might vary in different tissues and species. However, that does not exclude the possibility that these toxins might also be able to form pores in the membrane of the cells that compose these systems. Based on the data gathered so far, we propose it to be a function of dose, time of exposure, and naturally—the presence of proper recognition sites in the aforesaid membranes. Future studies on fish cytolysins should take this possibility into account.

All in all, much has been done regarding the investigation of biochemical and pharmacological features of fish cytolysins, considering how very labile and complex these molecules are. Nevertheless, as exposed in this review, there are still considerable gaps and contradictions, especially where what we believe to be their multi-mechanistic mode of action is concerned. The major role played by fish cytolysins in the envenomation process, added to the many questions raised by their multifunctionality, fully justifies the quest for a better understanding of how these molecules act.

**Figure 5.** Fish cytolysins: multifunctional toxins. The various pharmacological activities displayed by multifunctional fish cytolysins and what has been determined regarding the mechanisms of action underlying each activity.

> **Author Contributions:** Conceptualization, F.V.C., J.B.C. and S.G.F.; investigation, F.V.C., H.B.F., J.B.C. and S.G.F.; resources, J.B.C. and S.G.F.; writing—original draft preparation, F.V.C., H.B.F., J.B.C. and S.G.F.; writing—review and editing, F.V.C. and S.G.F.; supervision, J.B.C. and S.G.F.; funding acquisition, S.G.F. All authors have read and agreed to the published version of the manuscript.

> **Funding:** This research was funded by CAPES, postdoctoral fellowship to FVC; Instituto Butantan, Ph.D. fellowship to HBF; and INCTTox (573790/2008-6) and CAPES Toxinologia (063/10- 23038006280/20+11-07), grants to SGF.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
