Evaluation of DNA damage in spinal cord and mutagenic effect of a Phα1β recombinant toxin with analgesic properties from the Phoneutria nigriventer spider

Abstract

Phα1β peptide isolated from the venom of the Phoneutria nigriventer spider has shown higher analgesic action in pre-clinical studies than ω-conotoxin MVIIA peptide used to treat severe chronic pain. In view of the great potential for the development of a new Phα1β-based drug, a Phα1β recombinant form (CTK 01512-2) has been studied for efficacy and safety. The aim of this study was to evaluate cytotoxic, genotoxic and mutagenic effects of a Phα1β recombinant form and compare it with native Phα1β and ω-conotoxin MVIIA. Cytotoxicity was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colourimetric assay in L929 mouse fibroblast cells (0.5-10.0 μmol/L). Genotoxic and mutagenic activities were analysed using the alkaline comet assay in peripheral blood and spinal cord, and the micronucleus test in bone marrow from Wistar rats treated by intrathecal injection of CTK 01512-2 (200, 500 and 1000 pmol/site), native Phα1β (500 pmol/site) and ω-conotoxin MVIIA (200 pmol/site). CTK 01512-2 decreased the cell viability of the L929, showing IC 50 of 3.3 ± 0.1 µmol/L, while the Phα1β and ω-conotoxin MVIIA did not show cytotoxicity (IC 50 > 5.0 µmol/L). Native and recombinant Phα1β forms induced DNA damage in the spinal cord, but not in peripheral blood. CTK 01512-2 at 1000 pmol/site increased the micronucleus frequency suggesting mutagenic effects. In conclusion, the recombinant form has cytotoxic, genotoxic and mutagenic effects, evidenced in doses five times above the therapeutic dose. K E Y W O R D S comet assay, cytotoxicity, DNA damage, micronucleus, Phα1β, toxins

Analgesic effects of Phα1β toxin: a review of mechanisms of action involving pain pathways

“…Evaluation of the potential cytotoxic, genotoxic, and mutagenic effects of Phα1β by different methods showed that Phα1β (500 pmol/site) induced DNA damage in the spinal cord but not in peripheral blood [82]. In conclusion, the native toxin showed a good safety profile with transient signs of clinical toxicity [81] and genotoxic effects only in SNC [82] at doses five times higher than those used to obtain the analgesic effect. The results demonstrate that Phα1β produces analgesia after single or continuous i.t.…”

“…Serum biochemical data in male rats revealed a significant reduction within the physiological limits of species related to urea, AST, ALT, ALP, and hepatic and renal congestion [81]. Evaluation of the potential cytotoxic, genotoxic, and mutagenic effects of Phα1β by different methods showed that Phα1β (500 pmol/site) induced DNA damage in the spinal cord but not in peripheral blood [82]. In conclusion, the native toxin showed a good safety profile with transient signs of clinical toxicity [81] and genotoxic effects only in SNC [82] at doses five times higher than those used to obtain the analgesic effect.…”

A Review on Genotoxic and Genoprotective Effects of Biologically Active Compounds of Animal Origin

“…Positive control animals received hydrogen peroxide, and negative control animals received phosphate-buffered saline; there were five animals per group. The increased micronucleus frequency in bone marrow cells suggested mutagenic effects [ 36 ].…”

Neuroprotective effect of CTK 01512‐2 recombinant toxin at the spinal cord in a model of Huntington's disease

“…CTK 01512-2 might therefore display a good safety profile. In addition, de Souza et al (2019) have shown that this compound has low cytotoxic, genotoxic and mutagenic effects on the spinal cord. Finally, when injected intrathecally, CTK 01512-2 acts locally, without causing serious side effects.…”