Use of THP-1 cells to identify prohaptens.
Chipinda-I; Ruwona-TB; Templeton-SP; Siegel-PD
Toxicologist 2010 Mar; 114(1):298
Animal based testing is currently used for screening of potential allergenic chemicals. Recent efforts have been directed at the development of non-animal based alternative tests for the identification of skin sensitizers including the use of in vitro cell activation assays. Upregulation of activation and costimulatory markers in antigen presenting cells are key events in the sensitization process and have been reported to serve as indicators of skin sensitization. Prohapten identification remains a limitation due to the lack of bioactivation of prohaptens in these cell lines. The present study evaluated the effect of hapten and prohapten exposure on cell surface marker expression (CD86, CD54, CD44 and CD40) on THP-1 cells. Cells were exposed to the prohaptens benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA) and carvone oxime (CVO) at concentrations ranging from 1-10 uM for 24 and 48 hours. The direct-binding hapten, dinitrochlorobenzene (DNCB), was used as a positive control. Bioactivation of prohaptens was achieved by addition of a rat liver microsomal (S9) cocktail to the cell cultures. Flow cytometry data demonstrated a consistent dose-dependent increase of surface expression of the Tcell costimulatory molecule CD86 when cells were dosed with a hapten or prohapten in the presence of S9. Expression of the adhesion molecule CD54 (ICAM-1) and the antigen presenting cell costimulatory molecule CD40 were also significantly elevated in both hapten and prohapten (+ S9) treated cells, however S9 alone also upregulated CD54. CVO and DMBA, but not BaP, induced inconsistent increases in the adhesion molecule CD44. In conclusion, modification of in vitro cell culture assays to include co-incubation with microsomes enhances identification of prohaptens and allows them to be clearly distinguished from haptens.
Airborne-particles; Biochemistry; Biological-effects; Cell-biology; Cellular-reactions; Chemical-hypersensitivity; Chemical-reactions; Exposure-assessment; Exposure-levels; Exposure-methods; Inhalation-studies; Laboratory-animals; Laboratory-testing; Microbiology; Microchemistry; Microscopic-analysis; Microscopy; Molecular-biology; Particulate-dust; Particulates; Skin; Skin-exposure; Skin-sensitivity; Statistical-analysis; Toxic-effects; Toxins
The Toxicologist. Society of Toxicology 49th Annual Meeting and ToxExpo, March 7-11, 2010, Salt Lake City, Utah