Gel microdrop flow cytometry assay for low-dose studies of chemical and radiation cytotoxicity

Toxicology. 2001 Mar 7;160(1-3):5-10. doi: 10.1016/s0300-483x(00)00432-7.

Abstract

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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • CHO Cells / drug effects
  • CHO Cells / radiation effects
  • Cell Survival / drug effects*
  • Cell Survival / radiation effects*
  • Clone Cells
  • Colony-Forming Units Assay / methods
  • Cricetinae
  • Dose-Response Relationship, Drug
  • Dose-Response Relationship, Radiation
  • Flow Cytometry / methods*
  • Gamma Rays*
  • Gels
  • Humans
  • Mice
  • Quinoxalines / toxicity*

Substances

  • Gels
  • Quinoxalines
  • 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline