Gel microdrop flow cytometry assay for low-dose studies of chemical and radiation cytotoxicity.
Low-level cytotoxicity may affect low-dose dose-response relations for cancer and other endpoints. Conventional colony-forming assays are rarely sensitive enough to examine small changes in cell survival and growth. Automated image-analysis techniques are limited to ca. 10(4) cells/plate. An alternative method involves encapsulation of single proliferating cells into ca. 35-75-microm-diameter agarose gel microdrops (GMDs) that are randomly grouped, differential exposure of these groups, culture at 37 degrees C for 3-5 days, and finally GMD analysis by flow cytometry (FC) to determine the ratio of GMDs containing multiple versus single cells as a measure of clonogenic survival. This GMD/FC assay was used to examine low-dose cell killing induced by a cooked-meat mutagen/rodent-carcinogen (MeIQx) in DNA-repair-deficient/metabolically-sensitive CHO cells. Results of conventional colony-forming assays using up to 30 replicate plates indicate a shouldered, threshold-like dose-response; in contrast, those obtained using the GMD/FC assay suggest "hypersensitivity"-like nonlinearity in dose-response. The GMD/FC assay was also applied to human A549 lung cells after GMD-encapsulation and gamma radiation followed by culture for a total of 4 days, to examine survival after exposure to > or =100 cGy delivered at a relatively low dose rate (0.18 cGy/min). Dose-response for clonogenic growth was again observed to be reduced with apparent nonlinear suggesting hypersensitivity between 0 and 50 cGy, insofar as doses of 5 and 10 cGy appear to be ca. fivefold more effective per unit dose than the 50- or 100-cGy doses used. The GMD/FC assay may thus reveal low-dose dose-response relations for chemical and radiation effects on cell proliferation/killing with implications for low-dose risk assessment.
Health and Ecological Assessment Division (L.396), Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550-9900, USA. bogen@linl.gov
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Bogen
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Enns
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Hall
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Keating
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Weinfeld
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Murphy
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Wu
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Panteleakos
D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects* / Q000528:radiation effects*; D002999:Clone Cells; D003114:Colony-Forming Units Assay / Q000379:methods; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry / Q000379:methods*; D005720:Gamma Rays; D005782:Gels; D006801:Humans; D051379:Mice; D011810:Quinoxalines / Q000633:toxicity*
D005782:Gels; D011810:Quinoxalines; C036990:2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline