BUTYLATED HYDROXYANISOLE SPECIFICALLY INHIBITS TUMOR NECROSIS FACTOR-INDUCED CYTOTOXICITY AND GROWTH ENHANCEMENT Ole-L. Brekke,l M. Refaat Shalaby,2 Anders Sundan, Terje Espevik, 2 Kristian S. Bjervel The effect of commonly used food antioxidants on recombinant tumor necrosis factor cy (rTNF-a)-induced cytotoxicity, growth enhancement and adhesion has been evaluated. Butylated hydroxyanisole (BHA) and 4-hydroxymethyl-2,6-di-t-butylphenol (HBP) were the only two of nine antioxidants that completely inhibited rTNF-a-induced cytotoxicity in L929 and WEHI 164 fibrosarcoma cells. Ethoxyquin, propyl gallate and butylated hydroquinone only partially inhibited rTNF-a-induced cytotoxicity, while the antioxidants butylated hydroxytoluene (BHT), cw-tocopherol, ascorbic acid and thiodipropionic acid had minimal effects. The only difference between the molecular structure of the efficient HBP and the non-efficient BHT, is a hydroxymethyl group instead of a hydroxyl group on the phenolic ring. Neither BHA nor BHT inhibited the activation of NFB after 10 or 68 min challenge with rTNF-cu in L929 cells. BHA also inhibited rTNF-a-induced, but not rIL-lp-induced growth enhancement in FS-4 llbroblasts. Further, BHA blocked both rTNF-a-induced and rIL-lp-induced prostaglandin E, synthesis in FS-4 fibroblasts. BHA inhibited the rTNF-a-induced release of arachidonic acid in both FS-4 and L929 cells, suggesting that BHA inhibits cellular phospholipase(s). Neither cll-tocopherol nor BHA inhibited rTNF-a-induced adhesiveness of human endothelial cells. The results indicate that BHA is a specific and potent inhibitor of rTNF-a- and rTNF+induced cytotoxicity, as well as of rTNF-a-induced growth enhancement. TNF is a protein with a wide range of biological activities including killing of some sensitive cancer cells, l and stimulating the growth of fibroblasts.2 Recent observations indicate that TNF might be involved in the pathogenesis of meningococcal sepsis3 malaria4 and renal graft rejection5 as high serum concentrations of TNF have been found in these conditions. These findings have stimulated the development of TNF-a antagonists for certain disease From the Department of Clinical Chemistry’ and Institute of Cancer Research,2 Trondheim Regional Hospital, University of Trondheim, N-7006 Trondheim, Norway. Address correspondence to: Dr Ole-Lars Brekke, Department of Clinical Chemistry, Trondheim Regional Hospital, University of Trondheim, N-7006 Trondheim, Norway. Received 27 January 1992; accepted for p<blication 16 March 1992 0 1992 Academic Press Limited 1043-4666/92/040269+12 $05.00/O KEY WORDS: Arachidonic acidibutylated hydroxyanisolei cytotoxicityitumor necrosis factor CYTOKINE, Vol. 4, No. 4 (July), 1992: pp 269-280 states.6 TNF has also been used in treatment of human cancer patients.7 However, the toxicity associated with TNF treatment has been a problem. Indomethacin is able to inhibit TNF-induced tox- icity in vivo when injected 2 hours before TNFs and the toxic action of TNF in vivo is completely pre- vented by leukopenia and indomethacin.9 Inhibitors of the cyclooxygenase and lipoxygenase pathways,rO free-radical scavengers,10 dexamethasonero and the phospholipase A2 inhibitor quinacrine,rr are able to partially inhibit TNF-induced cytotoxicity in some cancer cells in vitro. The detailed biochemical mecha- nism(s) of TNF mediated toxicity are still, however, largely unknown, although previous reports indicate that the formation of hydroxyl radicals12 and oxidative damage probably are involved.13 Clark et al.14 showed that addition of the anti- oxidant butylated hydroxyanisole (BHA) in the diet enhanced the malaria infection in mice, suggesting that antioxidants like BHA may affect diseases where TNF is possibly involved. The present report therefore 269