BUTYLATED HYDROXYANISOLE INHIBITS TUMOR NECROSIS FACTOR-INDUCED CYTOTOXICITY AND ARACHIDONIC-ACID RELEASE

The mechanisms by which the antioxidant butylated hydroxyanisole (BHA) inhibits recombinant tumor necrosis factor alpha (rTNF-alpha)-induced cytotoxicity have been studied in WEHI 164 clone 13 (WEHI) and L929 f ibrosarcoma cells. When BHA was added simultaneously with rTNF-alpha, it completely inhibited rTNF-alpha cytotoxicity in the WEHI and L929 c ells. BHA also inhibited the toxicity when added 2 h after rTNF-alpha in WEHI cells, suggesting that BHA inhibits some late intracellular ev ent(s) in rTNF-alpha cytotoxicity. Pretreating WEHI cells with BHA for 4 h did not decrease the binding of rTNF-alpha to its receptors as me asured using flow cytometry. BHA inhibited rTNF-alpha toxicity in the presence of actinomycin D and cycloheximide, indicating that neither m RNA nor protein synthesis is necessary for the BHA effect. The antioxi dant butylated hydroxytoluene (BHT) and indomethacin did not inhibit t he rTNF-alpha-induced cytotoxicity nor the rTNF-alpha-induced release of [H-3]arachidonic acid. By comparison, BHA completely inhibited the rTNF-alpha-induced release of arachidonic acid, suggesting that BHA so mehow inhibits rTNF-alpha-induced activation of phospholipase(s). In W EHI cells, rTNF-alpha. increased the level of protein associated thiob arbituric acid reactive substances (TBARS) dose-dependently. BHA, but not BHT, blocked rTNF-alpha-induced cytotoxicity and rTNF-alpha-induce d accumulation of protein-associated TBARS, suggesting that rTNF-alpha cytotoxicity is correlated with protein-associated TBARS. In conclusi on, the results suggest that BHA. blocks some post receptor event in r TNF-alpha-induced cytotoxicity, and that activation of phospholipase(s ) coupled with the enzymatic formation of specific oxidized lipids cou ld be a pivotal event in rTNF-alpha-induced cytotoxicity.