Semiautomated quantification of cytotoxic damage induced in cultured insect cells exposed to commercial Bacillus thuringiensis biopesticides.
A convenient in vitro bioassay based on semiautomated quantification of live-cell reduction of tetrazolium dyes--3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-corb  oxanilide sodium salt (XTT)--to formazan was developed and used to evaluate cytotoxic effects of two commercial insecticides (BT) derived from Bacillus thuringiensis subsp. kurstaki (Btk). Comparison of two target insect cell lines MG1 (Trichoplusia ni, cabbage looper midgut) and Sf9 (Spodoptera frugiperda, fall army worm oocyte) revealed similar cell-dependent responses in mitochondrial-associated electron transport activity. The 50% inhibition of formazan production (ID50) obtained by exposing these cells to 20 microM 2,4-dinitrophenol or 5 mM sodium azide occurred in the range 10(-7)-10(-6) International Units (IU) of BT cell(-1) 24 h(-1). Damage to cell adhesion and cytoarchitecture, revealed by light and electron microscopic analysis, increased with BT exposure and dose. MTT was superior to XTT as a cytotoxic indicator in kinetic studies related to spores, a major component of BT. Unless blocked by antibiotic (gentamicin), vegetative growth resulting from spore germination was the major cause of toxicity. The ID50 exposure time using vegetative Btk cells was approximately 0.1-0.2 times that required for BT spores, with or without intact parasporal proteins present. This difference in exposure is an indirect measure of the time required for spores to germinate and produce vegetative cells. The assay methodology developed here, if linked with suitable target and non-target animal cell types, should have broad application for conducting standardizable estimates of cytotoxic potential of any microbe-based biotechnology product.
Tayabali
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AF
Mutagenesis Section, Department of Health Canada, Ottawa, Canada.
Seligy
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VL
D001413:Bacillus thuringiensis / Q000166:cytology / Q000187:drug effects*; D001681:Biological Assay; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D004396:Coloring Agents / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport / Q000187:drug effects; D005562:Formazans / Q000737:chemistry / Q000378:metabolism*; D007306:Insecticides / Q000008:administration & dosage / Q000633:toxicity*; D007928:Lethal Dose 50; D010944:Plants; D013045:Species Specificity; D013170:Spores; D013778:Tetrazolium Salts / Q000737:chemistry / Q000378:metabolism*; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism*
D004396:Coloring Agents; D005562:Formazans; D007306:Insecticides; D013778:Tetrazolium Salts; D013844:Thiazoles; C079782:MTT formazan; C022616:thiazolyl blue