*5.3. Pain-Inducing and Inflammatory Activities*

Immediate pain is the most distressing feature of envenomation by fish venoms, being disproportionate to the resulting wound in the absence of notable secondary tissue injury. In fact, counteracting pain is usually the main focus of treatment when the envenomation does not progress to systemic complications. Some experimental evidence points to the participation of cytolysins in the pain as well as in the local inflammatory process that rapidly develops on the site of injury caused by fish venoms.

The first association between fish cytolysins and these local effects was made when SNTX was first isolated in 1991 [12]. It was then observed that the toxin (0.15–0.4 mg, intraplantar injection—i.pl.) induced a dose-dependent edema (a minimum edema dose of 0.15 μg) within 1 h of injection into mouse hind paws, which lasted for more than 24 h and could not be blocked by pre-treatment with the antihistamine diphenhydramine (50 mg/kg, intraperitoneal injection—i.p.). No reference was made then as to the possible nociceptive effects induced by SNTX.

On the other hand, nocitoxin, a mildly hemolytic toxin isolated from the venom of the bullrout *Nothestes robusta*, was deemed to be the pain-causing protein in this venom, as only the fraction containing it could reproduce the algesic effect induced by the crude venom in human subjects [27]. There is, however, some doubt as to its falling into the fish cytolysin category, as it did not cross-react with the stonefish antivenom.

The nociceptive and inflammatory activities of Sp-CTx have been relatively well described when compared to those of other fish cytolysins. The first indication that Sp-CTx was at least partly responsible for the pain and swelling induced by the venom of *S. plumieri* came from the fact that these responses were blocked by the stonefish antivenom, which, as previously mentioned, reacted against a protein with the biochemical characteristics of this toxin [51]. The confirmation came in 2018, when [43] showed that the injection of Sp-CTx into mouse hind paws (0.1–3 μg, i.pl.) caused a dose-dependent nociceptive response and an intense, sustained, and also dose-dependent edema response that reached its peak within 60 min and persisted for up to 4 days.

Further insights into the mechanism underlying the local inflammatory reaction induced by fish cytolysins were obtained when these same authors reported that the swelling induced by Sp-CTx was reduced by pre-treatment with the bradykinin B2 receptor antagonist HOE-140 (100 nm/kg, i.p.) or the serine-proteases inhibitor aprotinin (8 mg/kg, i.p.) [43]. These findings indicate that the kallikrein-kinin system participates in this response, as had been already proposed when the inflammatory activity of the crude venom of *S. plumieri* was investigated a few years earlier [73]. However, these drugs did not prevent the edema response completely [43], suggesting that additional pathways might contribute to the onset of the inflammatory response. Moreover, the nociceptive activity induced by Sp-CTx could not be blocked by either drug, which indicates that it does not share the same mechanism described for the inflammatory activity [43].

That is, in fact, one of the biggest gaps in the study of fish venoms, and, by proxy, of fish cytolysins: the mechanisms behind the pain-inducing activity associated with these molecules remain largely unknown. It has been suggested that pore formation might be the underlying cause [43,55], but there is also evidence against this hypothesis. For instance, while investigating the pain caused by the lionfish *P. volitans* venom, [74] found that the heat-labile, protein components responsible for this activity appear to act specifically on neuropeptidergic nociceptors, although the identity of these components has yet to be determined.
