Abstract
The depletion degree of reduced glutathione is a critical indicator for assessing the toxicity of alkylating agents. In the present research, we have developed a novel method to evaluate the glutathione (GSH) depletion induced by a series of alkylating agents and the protective effect of various active thiol compounds based on a high-content cell analysis system. The cytotoxicity of some alkylating agents was first assessed using the CCK-8 assay. The results showed that bis(2-Choroethyl) methylamine (nitrogen mustard, HN2) and 1,2-bis(2-chloroethythio) ethane (Q) exhibited the highest cytotoxicity, with IC(50) values of 14.45 μM and 23.27 μM, respectively. The cytotoxicity of 2-choroethylchoromethylsufide (CECM) and bis(2-choroethylthioethyl) ether (T) was comparable to that of bis(2-choroethyl) sulfide (HD), and bis(2-choroethylthiomethyl) ether (CEMEE) showed the lowest cytotoxicity. At the same exposure dose, Q exhibited the strongest GSH depletion ability, followed by HD > CECM > CEPR(1,3-bis(2-Chloroethylthio)-n-propane) > CEBU(1,4-bis(2-Chloroethylthio)-n-butane) > CEPE(1,5-bis(2-Chloroethylthio)-n-pentane) > CEME(bis(2-Chloroethylthio) methane) > T(bis(2-Choroethylthioethyl) ether) > CEMEE, and the depletion ability of nitrogen mustard compounds followed the order HN2 > HN1(bis(2-Choroethyl) ethylamine) > HN3(tri(2-Choroethyl) amine). In addition, the protective effect of four active thiol compounds was investigated. The results revealed that reduced glutathione ethyl ester (GSH-MEE) was most effective in preventing GSH depletion, whereas glutathione monoethyl ester (MEE) showed the highest efficacy in restoring GSH levels. The proposed method holds significant potential for analyzing the damaging effects of various alkylating agents and screening protective drugs.