Toxicological Assessment of the Marine-Derived Cyclic Dipeptide Cyclo(Pro-Tyr) Using In Vitro, Zebrafish Embryo, and BALB/c Mouse Models

Cyclo(Pro-Tyr), a marine-derived cyclic dipeptide, has demonstrated anticancer activity, but its toxicological profile remains insufficiently characterized. The present study evaluated the preliminary toxicological effects of Cyclo(Pro-Tyr) using complementary in vitro cytotoxicity, zebrafish embryo developmental toxicity, and sub-acute oral toxicity in mice models. Cytotoxicity was assessed in 3T3-L1, HEK293, and HepG2 cells following 24 h exposure to Cyclo(Pro-Tyr) at 0–250 µM. Developmental toxicity was evaluated in zebrafish embryos exposed to 0–1000 µM for up to 96 h post-fertilization. A repeated-dose oral toxicity study was conducted in male and female BALB/c mice administered Cyclo(Pro-Tyr) at 100 or 500 mg/kg/day for 30 consecutive days, followed by hematological, serum biochemical, organ-weight, and histopathological assessments. Cyclo(Pro-Tyr) produced concentration-dependent reductions in cellular viability, with pronounced effects at the upper concentrations tested. In zebrafish embryos, exposure to 750 and 1000 µM was associated with increased mortality, reduced hatchability, developmental malformations, and reduced body length. In mice, repeated oral administration did not produce mortality, overt clinical toxicity, or significant changes in body weight or relative liver and kidney weights. However, dose-dependent alterations in selected hematological and serum biochemical parameters were observed, particularly at 500 mg/kg, accompanied by mild inflammatory and degenerative changes in hepatic and renal tissues. Overall, Cyclo(Pro-Tyr) exhibited concentration-dependent cellular and developmental toxicity at higher exposure concentrations and produced mild systemic alterations following repeated oral administration at the higher tested dose. These findings provide preliminary toxicological information relevant to the further preclinical evaluation of Cyclo(Pro-Tyr) as a marine-derived bioactive compound.