Combined Toxicity of Microcystins and N-methyl-N'-nitro-N-nitrosoguanidine on Esophageal Cancer Cell Line EC109

Objective To investigate the combined toxicity effect of microcystins (MC) and N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) on esophageal cancer cell line EC109.

Methods The inhibition rates (fa) of microcystins (MC-LR) (1 nmol/L, 10 nmol/L, 100 nmol/L, 1 μmol/L, and 10 μmol/L) and MNNG (5 pmol/L, 50 pmol/L, 500 pmol/L, 5 nmol/L, 50 nmol/L, 500 nmol/L, and 5 μmol/L) on EC109 cells were observed by MTT assay. Factorial design and the Chou-Talalay method (medianeffect principle) were used to evaluate the combination effect. Apoptosis was examined by flow cytometric assay.

Results Compared with the control group, when the exposure concentration was 50 pmol/L or more, the fa increased significantly in the cells treated with MNNG alone (all Ps < 0.05); whereas the fa decreased first and then increased in the cells treated with MC-LR alone, and except the 100 nmol/L group, significant differences were found in the rest treatment groups compared with the control group (all Ps < 0.05). Among all designed dosage combinations of MC-LR and MNNG, an increasing fa of EC109 cells was observed (r=0.961, 0.973, 0.950, 0.980, 0.959, 0.972, respectively, all Ps < 0.01) along with higher MNNG concentrations; while a reducing-and-thenin creasing pattern was found along with higher MC-LR, and the difference was statistically significant when compared with the control group (P < 0.05). The results of factorial design analysis of variance showed an interaction between the two toxins (P < 0.01). According to the Chou-Talalay method (combination index, CI), when fa=9.4%, CI=1, it indicated additivity; when fa < 9.4%, CI > 1, it in dicated antagonism; when fa > 9.4%, CI < 1, it indicated synergism. The results of flow cytometry showed that apoptosis was induced by MNNG or high concentrations of MC-LR, and the combination with high concentrations of MNNG and MC-LR was synergistic on in duction of apoptosis.

Conclusion When MC-LR is combined with MNNG, the effect is antagonistic at low concentrations and synergistic at high concentrations.