Oxidative stress and apoptosis are strongly involved in ricin-induced intestinal cell intoxication

Introduction: Ricin, a type 2 ribosome-inactivating protein, is a potent lethal toxin purified from castor bean seeds and has been classed as a chemical and biological weapon according to Schedule 1 by the OPCW. Ricin's cytotoxicity results from its lectin chain, which enables cell entry, and its active chain, which can depurinate different substrates, leading to multiple cell death pathways. Although the systemic toxicity of ricin has been extensively studied, its localized effects on the gastrointestinal tract remain a critical concern, particularly in cases of oral ingestion. The aim of this study is to elucidate ricin intoxication in human intestinal cell lines and determine its impact on the integrity of the epithelial barrier.

Methods: Ricin cytotoxicity in intestinal-derived HT29 and Caco-2 cells was evaluated in dose– and time–response experiments, with cell viability determined via an MTS-reduction assay. The effect of ricin on Caco-2 cell layer integrity was monitored by Trans-Epithelial-Electrical Resistance (TEER) measurements. The involvement of oxidative stress in ricin-treated cells was indirectly investigated by pretreating cells with reactive oxygen species scavengers. Cell death was determined through cytofluorimetric analysis of AnnexinV/PI positivity.

Results: Ricin showed high cytotoxicity toward HT29 and Caco-2 cells, with EC₅₀s ranging from 10 to 0.1 nM after 24-72 h of intoxication. Ricin strongly reduced TEER values in Caco-2 cells at 1-0.1 nM after 24 h of treatment. Ricin cytotoxicity (at 1 nM concentration) can be completely prevented by pre-incubating cells with antioxidant scavengers, demonstrating the involvement of oxidative stress in ricin cell death mechanisms. Moreover, ricin triggered only apoptosis without necrosis involvement.

Conclusions: Ricin has a potent cytotoxic effect on human intestinal cell lines that can be prevented by antioxidant scavengers. This study could pave the way for new pharmacological strategies to tackle ricin intoxication.