Comparison of the cytotoxic impact of chlorfluazuron on selected insect and human cell lines

Abstract

To gain new insight into the mechanism of selective cytotoxicity of benzoylureas as insecticides, the in vitro mode of action of chlorfluazuron was investigated on lepidopteran Tn5B1-4 and Sf-21 cells and human Hek293 and HepG2 cells. Chlorfluazuron inhibited the proliferation of Tn5B1-4 and Sf-21 cells with 50% inhibitory concentration values (IC50) of 4.96 µM and 1.12 µM at 48 h and 2.37 µM and 1.76 µM at 96 h, respectively, versus that of Hek293 and HepG2 cells with IC50 values >20 µM. When transferred to chlorfluazuron-free medium, lepidopteran Tn5B1-4 and Sf-21 cells had a postinhibitory recovery development period within 24 h followed by a suppressed increase in cell viability, but human Hek293 and HepG2 cells showed an accelerated increase over their control level. Chlorfluazuron affected Tn5B1-4 and Sf-21 cells, with ≥1.8-fold decreases in the ratio of cellular N-acetylglucosamine (GlcNAc) level and protein content and ≥1.5-fold increases in the mitotic index and G2 /M-phase arrest. Neither Hek293 nor HepG2 cells contained GlcNAc, and chlorfluazuron had no significant effects on the cell cycle and mitotic index of Hek293 and HepG2 cells. In conclusion, the differences between human and lepidopteran cell lines in the characteristic GlcNAc content, G2 /M arrest in the cycle progress, and mitotic index of cells in response to chlorfluazuron may contribute to the selective toxicity of chlorfluazuron to lepidopteran cells.

Triangulation of methods using insect cell lines to investigate insecticidal mode‐of‐action

“…Furthermore, it appears that avoiding the perimeter wells provided consistency, presumably by reducing evaporation influences 48 . Huang et al 55 . also reported that measuring growth inhibition and mortality of insect and human cells separately can elucidate a compound's mode‐of‐action as a retardant of growth rather than by cytotoxic means.…”

“…In this study we used D.mel ‐S2 cell lines to assess the reliability of an additional cell line, Sf9, in response to known insecticides of both a natural and nature‐equivalent, synthetic sources. In general, these insect cell lines have been shown to be useful in the elucidation of many insecticidal modes of action, 55, 76 since they offer a direct access to target sites that may otherwise be difficult to access in whole insect assays, or for pesticides whose efficacy may be compromised by biological activities such as physical exclusion, excretion, metabolic detoxification or cellular degradation 14 . However, careful consideration should be given to the appropriateness of each cell line and its ‘fit for purpose’ based on source tissue, for example embryonic, midgut or ovarian origin and their innate membrane properties 76 …”

“…Furthermore, it appears that avoiding the perimeter wells provided consistency, presumably by reducing evaporation influences. 48 Huang et al 55 also reported that measuring growth inhibition and mortality of insect and human cells separately can elucidate a compound's mode-of-action as a retardant of growth rather than by cytotoxic means. A further advantage to using this technique, besides its simplicity, is that cells grown as non-adhesive cultures can be assessed, which could otherwise pose issues for MTT assays which require repeated washing steps.…”