The human epithelial cell cytotoxicity assay for determining tissue specific toxicity: method modifications.
The Human Epithelial Cell Cytotoxicity (HECC) Assay (Meth Cell Sci, 22: 17-24, 2000) has been modified to include three additional cell lines and to allow protocol adjustments for slow growing cell lines. This manuscript presents methods using human epithelial cells from ten different normal human tissues including: skin, mammary, prostate, renal, bronchial, lung, oral, ecto-cervix, colon, and liver. The HECC Assay can also be used to evaluate other types of drugs, personal care products, environmental chemicals, and potential toxicants. Human epithelial cells at an early passage are seeded into multi-well dishes. The cells are exposed to multiple concentrations of each test agent. A preliminary assay using an exposure of five days at 1 mM (if soluble) and four log dilutions is used to determine the highest concentration for the HECC Assay. In the HECC Assay, cultures are exposed for three to four days. Following the exposure period, endpoint measurements for inhibition of growth, mitochondrial function, and PCNA (proliferating cell nuclear antigen) expression or albumin synthesis (hepatocytes) are made. Data are analyzed to determine the concentration that inhibited an endpoint by 50 percent (TC(50)) for each agent in each target epithelial cell line or culture and the data are compared to determine the relative sensitivity of each epithelial cell line to the test agent.
Elmore
Eugene
E
Department of Radiation Oncology and Chao Family Comprehensive Cancer Center, University of California, Medical Sciences I, B149, Irvine, CA 92697, USA. eelmore@uci.edu
Siddiqui
Shazia
S
Desai
Neeraj
N
Moyer
Mary Pat
MP
Steele
Vernon E
VE
Redpath
J Leslie
JL
D000418:Albumins / Q000187:drug effects / Q000378:metabolism; D000970:Antineoplastic Agents / Q000633:toxicity*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D003603:Cytotoxins / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D023381:Endpoint Determination / Q000379:methods*; D004734:Energy Metabolism / Q000187:drug effects / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism
D000418:Albumins; D000970:Antineoplastic Agents; D003603:Cytotoxins; D018809:Proliferating Cell Nuclear Antigen