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 > propyl gallate > > BHT > ascorbic acid > or-tocopherol, which is similar to their ability to inhibit TNF-induced cell killing in this report. Recent reports suggest that TNF cytotoxicity in the presence of cycloheximide may be mediated by enzymes of the lipoxygenase pathways.25 This is supported by the observation that BHA, but generally not BHT, is known as a lipoxygenase inhibitor.26 It seems unlikely that the BHA inhibition of TNF-induced cytotoxicity is due to inhibition of the cyclooxygenase since indomethacin did not inhibit TNF-induced cytotoxicity in WEHI clone 13 cells (results not shown). Another possible explanation for the observed effects on TNF cytotoxicity, is that BHA has been reported to inhibit the cytochrome P-450 enzyme(s). Both BHA and BHT bind to the cytochrome P-450 enzyme, but BHA is known as a more potent inhibitor of the enzyme activity than BI-IT.27 The cytochrome P-450 enzyme is also involved in the synthesis of epoxy fatty acids ,*s but it has not been reported that such metabolites are involved in TNF-induced cytotoxicity. Previous reports have shown that both BHA and BHT can uncouple mitochondrial respiration in hepatocytes,*s BHT being somewhat more effective than BHA. It seems therefore unlikely that mitochondrial uncoupling is the mechanism of BHA inhibition of TNF-induced toxicity, although it is known that mitochondria are damaged early in TNF-induced cytotoxicity. Interestingly, BHA inhibited rTNF-a-induced but not rIL-lB-induced growth enhancement of FS-4 cells, suggesting that BHA does not inhibit rTNF-a-induced growth stimulation through an unspecific toxic or growth inhibitory effect. This observation also suggests that rTNF-a and rIL-1B have different signal pathways, which is in agreement with earlier reports.sOJr Although BHA could become toxic towards FS-4 cells in the presence of rTNF-or, but

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not in the presence of rIL-ll3, this seems unlikely since BHA protects against TNF cytotoxicity in the WEHI, L929 and U937 cells. BHA inhibited the synthesis of prostaglandin E, in both rTNF-cw- and rIL-l@-stimulated cells, indicating that inhibition of the cyclooxygenase could not explain the different effect of BHA on rTNF-a- and rIL-l@ induced growth enhancement. BHA inhibited both the rTNF-ol- and rIL-@&induced release of arachidonic acid from FS-4 cells, suggesting that BHA affects the activity of phospholipase(s) in these cells. Further, BHA, but not BHT inhibited the rTNF-induced release of arachidonic acid from L929 cells (results not shown). This suggests that BHA somehow affects the activity of cellular phospholipase(s), although it has not been reported that BHA directly inhibits these enzymes. Previous reports have shown that inhibitors of phospholipase(s) are able to inhibit TNF-induced cytot0xicity.l’ The observation that neither BHA nor tocopherol affected the TNF-induced adhesiveness in human endothelial cells, suggests that BHA selectively inhibits only some TNF effects, or that BHA selectively inhibits TNF effects in only some cell types. Since BHA did not block rTNF-a-induced adhesiveness of HEC (Table 4), the protection of BHA against TNF-induced cytotoxicity was probably not caused by inactivation of the cytokine and indicates that BHA does not inhibit TNF-induced synthesis of adhesive proteins in HEC. Clark et al.14 showed that BHA in the diet enhanced malaria infection in mice, suggesting that antioxidants like BHA may have selective effects also in vivo. Although both BHA and BHT are widely used as food additives, they are not regarded as completely harmless. BHT can produce lung damage,32 and both BHA and BHT increase the incidence of cancer in some animal models.33 However, both BHA and BHT are able to inhibit chemically induced carcinogenesis in vivo,33 indicating that these compounds can also have beneficial effects. In conclusion, this report indicates that antioxidants such as BHA and HBP, but not closely related antioxidants completely inhibit TNF-induced cytotoxicity in fibrosarcoma cells. BHA inhibits rTNF-a-induced, but not rIL-lB-induced growth in fibroblasts. The results indicate that BHA, BHT and structural analogues may be useful tools in studying the biochemical mechanism(s) of TNFinduced cytotoxicity.

TNF and butylated hydroxyanisoleI 211

MATERIALS AND METHODS Chemicals,

Media and Reagents

RPMI-1640, L-glutamine, FCS and Dulbecco’s PBS were obtained from Gibco Laboratories (Paisley, Scotland). MIT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), BHT, EDTA, Triton X-100, Tris, dl-atocopherol, heparin, collagenase and SDS were obtained from Sigma Chemical Co. (St. Louis, MO). Gentamicin sulfate was from Schering Corp (Kenilworth, NJ) and absolute ethanol was from Vinmonopolet A/S (Oslo, Norway). Citric acid monohydrate was obtained from E. Merck (Darmstadt, Germany). The antioxidants butylated hydroxyanisole (mixture of 2- and 3-BHA),4hydroxymethyl-2,6-di-t-butylphenol, ethoxyquin, butylated hydroquinone, thiodipropionic acid, propyl gallate and ascorbic acid were part of food additive kit-92 from Supelco (Bellefonte, PA). BHA purchased from Sigma had similar effects as BHA from Supelco. The C,, octadecyl columns (500 mg) were obtained from Bond Elut (Varian SPP., Harbor City, CA). Both [5,6,8,9,11,12,14,15sH]arachidonic acid and [5,6,8,11,12,14,15(n)-sH]prostaglandin E, were obtained from Amersham (Buckinghamshire, England). Unlabeled prostaglandins, hydroxyeicosatetraenoic acids and epoxy fatty acid standards for high-pressure liquid chromatography (HPLC) were obtained from Cayman Chemical Company (Ann Arbor, MI). Ethyl acetate of Uvasol quality and methanol of Lichrosolv quality was both obtained from E. Merck (Darmstadt, Germany). Hexane of HPLC quality was obtained from Fisons (FSA Laboratory Supplies, Loughborough, England). Acetonitrile and propan-2-01 of HPLC grade was purchased from Rathburn (Rathburn Chemicals Ltd, Walkerburn, Scotland). Acetic acid of HPLC grade was purchased from Pierce (Rockford, Ill.).

Cytokines Human rIL-1B with a specific activity of 7.7 x 107 U/mg protein, as determined in the thymocyte proliferation assay, was provided by Dr A. Shaw, Glaxo (Geneva, Switzerland). Human rTNF-a, human rTNF-B (lymphotoxin) and murine rTNF-o were generously provided by Genentech Inc. (South San Francisco, CA), and had specific activities of 7.6 x 107, 10 x 107 and 8 x 107 U/mg protein, respectively, as determined in a bioassay.34

Cell Culture WEHI 164 parental murine fibrosarcoma cells (termed WEHI parental cells) were obtained from Dr H.W. LSms Ziegler-Heitbrock (Institute of Immunology, University of Munich, F. R. G) . The highly TNF-sensitive WEHI 164 clone 13 cells (termed WEHI clone 13 cells) were previously isolated35 from the WEHI parental cells. U937 and HL-60 cells were obtained from American Type Culture Collection (Rockville, MD), and the L929 cell line from Dr D. Lovhaug (Norwegian Defence Research Establishment). Human FS-4 fibroblasts were kindly provided by Dr J. Vilcek (Department of Microbiology, New York School of Medicine, NY). HECs were obtained by collagenase

treatment of human umbilical cord veins as described.36 The identify of HECs was confirmed as detailed in a previous report.37 HECs were seeded at uniform density (0.15 x 106/well) in 24-well plates (Costar, Cambridge, MA) in FCS-M plus 20 mM Hepes and 90 pgiml heparin. All cell lines were grown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal calf serum, 2 mM L-glutamine and 40 ;Ig/ml gentamicin (FCS-M) in a humidified atmosphere of 95% air and 5% CO, at 37°C. Human rTNF was used in experiments with U937, FS-4 and HEC cells, while WEHI 164 and L929 cells received murine rTNF. Antioxidants were dissolved in ethanol, and the final ethanol concentration at ~0.05% (v/v) had no effect itself.

SWhromium

Release Assay

The chromium release assay was performed essentially as described.16 Non-adherent WEHI and L929 cells were obtained by seeding 1 x lo6 cells/20 ml FCS-M in 1400 mm microbiological petri dishes (Nunc A/S, Roskilde, Denmark) and cultured for 48 h. Thereafter, the cells were harvested and 4 x 106 cells in a total volume of 0.75 ml FCS-M were added to 150 pCi Na,siCrO, (Specific activity 11 GBq/mg Cr, Institutt for Energiteknikk, Kjeller, Norway) and the suspension incubated for 60 min at 37°C. The cells were washed three times in FCS-M (+4”C) and seeded into microtiter plates (Costar 3598) at 104 cells/well. Thereafter, 50 pl FCS-M containing TNF and 50 pl containing antioxidant were added and the cells incubated at 37°C in a total volume of 200 @/well. Unless otherwise indicated, supernatants were collected after 16 h incubation using a supernatant collecting system (Skatron AS, Lier, Norway). The radioactivity was measured using an LKB Wallac 1282 Compugamma counter (Turku, Finland). Percent specific lysis (% SL) was calculated as: 100 x (E-5)/( T-S), where E represents experimental 51Cr release, S represents spontaneous 51Cr release, and T represents total 5iCr release after lysis of cells with 0.5% (w/v) SDS. Spontaneous release after 16 h incubation from WEHI parental, WEHI clone 13 and L929 cells alone was between 18% and 28% of total release. Results are given as mean f SD of triplicates unless otherwise indicated.

Cell Survival Assay Cell survival was measured using the MTI assay described by Mosmann.38 Cells were trypsinized and 2 x 103 cells seeded per well in microtiter plates (Costar), using 100 pl FCS-M/well. After 44 h incubation, the medium was removed and cells received 50 pl FCS-M containing TNF and 50 pl containing antioxidant in a total volume of 100 @well. After 22 h incubation with TNF, the medium was again removed, and cells incubated for 4 h at 37°C in 100 pl FCS-M containing MIT (0.45 mg/ml). Thereafter, 50 l-11of the medium was removed and 100 pl 0.04 N HCl in propan-2-01 added to dissolve the formazan. The OD was measured on a Titertek Multiskan MCC/340 MK II microplate reader from Flow Lab. (Irvine, Scotland) in dual wavelength mode, with test and reference wavelengths set at 595 nm and 690 nm, respectively. Blanking was performed against wells containing FCS-M but no cells. Cell survival was calculated as: 100 x (OD in wells with TNF)/(OD in

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wells without TNF). Results are given as mean f. SD of quadruplicates. The MIT assay was not affected by 0.05% (v/v) ethanol or 100 pM of BHA (results not shown).

Viability and Cell Counting The viability of WEHI clone 13 and L929 cells after incubation with BHA and BHT was also determined by Trypan blue dye exclusion (0.2% W/v). Cells were counted using a hemocytometer or a Coulter ZF cell counter (Coulter Electronics Ltd, Dunstable Beds, Engl).

Assay of NF, B in L929 Cells L929 cells were seeded at 0.5 x 106cells in 60 mm petri dishes (Costar 3060) in FCS-M. After c. 48 h incubation, the cells received medium containing 100 PM BHA or BHT. After further 30 min incubation at 37°C , rTNF-a was added (1 rig/ml) and the incubation continued in the presence of BHA or BHT for 10 or 60 min. Thereafter, the cellswere put on ice, scraped and washed twice at +4”C in PBS (without calcium and magnesium). NF,$ was extracted and detected essentially as described by Meichle et al.39 applying a synthetic oligonucleotide probe derived from the HIVLTRN with the sequence S-GCTACAAGGGACTIT CCGCTGGGGACTITCCAGG-3’. Both strands were synthesized (Beckman DNA synthesizer), annealed and labeled with y[szP]-ATP applying Polynucleotid Kinase (Boehringer Mannheim). For competition experiments a 200-fold excess of unlabeled oligonucleotide or a mutated oligonucleotide40~41was mixed with the labeled probe.

Thymidine Incorporation Assay FS-4 cells were cultured in 75 cm2 flasks (Costar), trypsinized and seeded at 1 x 104 cells/well in microtiter (Costar) in 100 pl FCS-M. Approximately 22 h later, cytokine and the appropriate antioxidant were added in 50 pl FCS-M each. L929 and WEHI clone cells were seeded at 2 x 103 cells/well in microtiter plates, and after 48 h incubation, BHA or BHT were added in a total of 200 pl FCS-M/well. After the indicated incubation at 37°C 1 pCi of [methyl-sH]thymidine (NET-027, specific activity 6.7 Ci/mmole; New England Nuclear Corporation) was added to each well 4 h before cells were trypsinized, and harvested with a Skatron cell harvester (Skatron AS, Lier, Norway). Radioactivity was measured by liquid scintillation counting using an LKB Wallac 1211 Rackbeta counter. Results are given as cpm f SD of quadruplicates.

Cell Culture and Extraction of Arachidonic Acid Metabolites FS-4 or L929 cells were seeded in FCS-M at a density of 0.5 x 106 cells in 60 mm petri dishes, and incubated for 24 h. Thereafter, the medium was changed to 1% (v/v) fetal calf serum in RPMI-1640 (2 ml/well) containing 1 pCi sH-arachidonic acid/ml. After 24 h incubation, the cells were washed three times with FCS-M (37°C). Cytokine and antioxidants were added in 2 ml FCS-M and after 4 h or 22 h incubation, the supernatants were harvested and centrifuged for 10 min at 2000 rpm to remove loose

CYTOKINE, Vol. 4, No. 4 (July 1992: 269-280) cells. Arachidonic acid metabolites were extracted from the supernatants essentially as described by Powell42 using C,, octadecyl columns preactivated with methanol followed by water (pH = 3.0). The samples were acidified to pH = 3 using citric acid and thereafter passed through the C,, octadecyl cartridge using a Vat Elut SPS 24. The columns were then washed with n-hexane, and metabolites finally eluted with ethyl acetate. Recovery of sH-prostaglandin E, added to FCS-M was 88 & 4% (N = 3).

Analysis of Arachidonic Acid Metabolites by HPLC The samples were analysed by HPLC using a 5 pm Supelcosil LC-18 (15 cm x 4.6 mm) reversed phase column and a flow rate of 1 ml/min. The HPLC system consisted of a Hewlett Packard 1050 gradient pump (Avondale, PA), connected to a Shimadzu SPD-6A UV-detektor (Kyoto, Japan) equipped with an 8 pl flow cell, and a Radiomatic Flo-one Beta model A-110 on-line radioactivity detector (Westshore Blvd., Tampa, FL) equipped with a 0.5 ml flow cell. The gradient pump and the detectors were controlled and monitored by a Hewlett Packard Vectra QS/165 personal computer using Chem Station Software Hewlett Packard. A gradient between from solvent A (water:acetic acid, 100:0.03) and solvent B (acetonitrile:acetic acid, 100:0.03) was used to separate prostaglandins, hydroxy fatty acids, epoxy fatty acids and free fatty acids. The gradient started at 26% B, increased linearly up to 55% B after 29 min, thereafter 55% B to 44 min, increased linearly to 85% B after 49 min, and from 56 to 57 min increased linearly from 85% B to 100% B. Total run time was 66 min. Prostaglandin E, was identified by coelution with standards in two different gradients. The content of prostaglandin in each sample was calculated as percent of control by comparing the areas as determined on the Radiomatic detector. The detection limit of sH-arachidonic acid was c. 125 cpm, and a linear relationship was found between cpm injected and the area measured on the on-line radioactivity detector.

Human Endothelial Cell Adhesiveness Cultures of confluent HEC monolayers were treated with rTNF-c-w in the absence or presence of BHA or a-tocopherol for 4 h. Thereafter the cultures were washed twice with HBSS, and each culture received 250 pl of FCS-M containing 5 x 104 HL-60 cells prelabeled with siCr as previously described.16 After 1 h of incubation, non-adherent 5iCr-labeled HL-60 cells were removed and following four washes with HBSS, each culture received 500 pl-of 1% Triton X-100 to lyse the cells. The radioactivity was determined as the mean of four aliquots of cell lysate using a gamma counter as described above. The mean cpm, which reflects HL-60 adherence was expressed as a percentage of the total cpm in 5 x 1045iCr-labeled HL-60 cells.

Statistical Analysis Statistical analysis of data was performed by analysis of variance (ANOVA) using the SPSS-PC + version 4.01 (SPSSInc., Chicago, Ill.).

TNF and butylated hydroxyanisole

Acknowledgements

The technical assistance of Tora Bardal, Sylvia Nome Kvam, Mari Sorensen and Berit Stordal is gratefully acknowledged. Dr Harald Johnsen is acknowledged for help with the statistical analysis of data. This work was supported by grants from The Norwegian Cancer Society, The Cancer Research Foundation of Trondheim University Hospital and the Norwegian Research Council for Science and the Humanities.

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39. Meichle A, Schtitze S, Hensel G, Brunsing D, Kriinke M (1990) Protein Kinase C-independent activation of nuclear factor .B by tumor necrosis factor. J Biol Chem 265:8339-8343. 40. Nabel G, Baltimore D (1987) An inducable transcription factor activates expression of human immunodeficiency virus in T cells. Nature 326:711-713. 41. Duh EJ, Maury WJ, Folks TM, Fauci AS, Rabson AB (1989) Tumor necrosis factor (Y activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-,B sites in the long terminal repeat. Proc Nat1 Acad Sci USA 86~5974-5978. 42. Powell, WS (1987) High-pressure liquid chromatography in the analysis of arachidonic acid metabolites. In Benedetto C, McDonald-Gibson RG, Nigam S, Slater TF (eds) Prostaglandins and Related Substances, A Practical Approach, IRL PRESS, Oxford, p 76.