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[CANCER RESEARCH 46, 5696-5700, November 1986] Enhanced Release of Hydrogen Peroxide and Metabolites of Arachidonic Acid by Macrophages from SENCAR Mice following Stimulation with Phorbol Esters1 James G. Lewis and Dolph O. Adams Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710 ABSTRACT genotoxic ROIs,2 and lipid oxidation products (8-10). Leuko cytes stimulated with phorbol esters also suffer breakage of Chronic inflammation has long been associated with carcinogenesis. their DNA, produce soluble low molecular weight clastogenic Phorbol esters which are potent promoters of tumors in mouse skin are factors, and induce increased rates of sister chromatid exchange also potent inflammatory agents in skin and cause inflammatory cells to in cm-illtu red mammalian cells (11-14). We have shown that release large quantities of reactive oxygen intermediates and oxidized murine peritoneal macrophages, stimulated with phorbol esters lipid products. SENCAR mice have been bred for their sensitivity to the or particulate inflammatory signals, induce 5,6-ring saturated promotion of tumors by phorbol esters and CS7BL/6 mice have been thymine bases in the DNA of cocultured mammalian cells (15, shown to be resistant. We quantified the release of H2O2 and metabolites 16). This process was inhibited by both catalase and nordihy- of arachidi mic acid by macrophages obtained from SENCAR and C57BL/ droguaiaretic acid (an inhibitor of arachidonic acid metabolism) 6 mice, following exposure to phorbol esters and other stimulants. The and was enhanced by Superoxide dismutase and indomethacin basal level for secretion of 112(): in resident peritoneal macrophages was (an inhibitor specific for cyclooxygenases). These studies sug negligible in cells from both strains. Conversely, inflammatory macro phages from SENCAR mice, elicited by the injection of sterile casein, gested that both ROI and lipid oxidation products may play a secreted 4 times more IM >.•thanthe corresponding cells from C57BL/6 role in the induction of oxidative DNA damage by these chronic mice. Furthermore, cells from SENCAR mice required less than one- inflammatory cells. third the amount of phorbol ester to obtain 50% of the maximal response The contention that oxidizing agents produced by inflam than that required by cells from C57BL/6 mice. This difference was less matory cells play a role in carcinogenesis is also supported by when zymosan was used as a stimulant. Both resident and inflammatory studies conducted in vivo. Antiinflammatory agents are potent macrophages from SENCAR mice released more metabolites of arachi- inhibitors of the promotion of tumors in mouse skin by TPA donic acid than cells from CS7BL/6 mice when exposed to phorbol esters, (17, 18). Superoxide dismutase mimetic copper chelates also but macrophages from C57BL/6 mice released more metabolites when inhibit promotion by TPA (19). Retinoids which inhibit pro stimulated with zymosan. Few differences in the pattern of released motion by TPA also inhibit the release of ROI by inflammatory metabolites were noted between the strains of mice. There were large cells (10). Inhibitors of AA metabolism can enhance or inhibit differences in the relative amounts of individual metabolites released the promotion of tumors by TPA (17, 18). Finally, direct when different stimulants were used. The enhanced response to phorbol evidence for the participation of ROI in tumor promotion is esters of chronic inflammatory cells from SENCAR mice correlates with the observation that peroxides applied to initiated mouse skin the enhanced sensitivity to the promotion of tumors by phorbol esters in can promote tumors (20, 21). these animals. Species and strain differences in susceptibility to carcinogen esis is a well established observation (22). SENCAR mice have been bred for their sensitivity to the promotion of skin tumors INTRODUCTION by TPA following initiation with dimethylbenzanthracene (23). As early as 1860 Virchow (1) noted a relationship between In contrast, C57BL/6 mice are almost totally resistant to pro carcinogenesis and chronic irritation and inflammation. Initial motion of skin tumors by TPA (23) but are equally as sensitive studies by Friedwald and Rous showed that chronic wounding as SENCAR mice to complete carcinogenesis by multiple ex posures to dimethylbenzanthracene (23). These observations of skin could potentiate or promote tumors in skin which had suggest that the difference in tumor development observed in been exposed previously to carcinogen (2). Other investigators the two strains is restricted to the promotion phase of carcino using the blistering agent, croton oil, showed that this inflam genesis and the actions of TPA. Other strains of mice have matory agent was a potent promoter of tumors in initiated been shown to have sensitivities to promotion by TPA which mouse skin (3-5). Subsequent studies identified phorbol esters fall between these two extremes (24). as the active agents in croton oil and showed that these agents The purpose of the studies presented here is to determine if have a wide variety of effects on mammalian cells (6, 7). These there are qualitative or quantitative differences in secretion of effects, including the induction of general or selective cell ROI and oxidative products of AA by chronic inflammatory replication, induction of new gene products, altered cellular cells (macrophages) from these phorbol ester sensitive and differentiation, and changes in intercellular communication, resistant strains of mice which correlate with their different have all been implicated as a mechanism by which these agents susceptibilities to the promotion of tumors by phorbol esters. promote tumors (7). We report here that macrophages from SENCAR mice were Recently the inflammatory nature of phorbol esters and their significantly more sensitive to phorbol ester exposure and ex effects on inflammatory cells have been relinked to carcinogen hibited enhanced release of ROI and metabolites of AA. Fur esis. Studies conducted in vitro have shown that leukocytes thermore, these increased responses were selective for phorbol exposed to phorbol esters secrete large quantities of potentially esters because differences in responses were absent or greatly Received 2/26/86; revised 6/10/86; accepted 8/4/86. 2The abbreviations used are: ROÕ,reactive oxygen intermediate; AA, arachi The costs of publication of this article were defrayed in part by the payment donic acid; LTB4, leukotriene B4; LTC4, leukotriene C4; LTD4, leukotriene D4; of page charges. This article must therefore be hereby marked advertisement in PBDU, phorbol dibutyrate; PGE2, prostaglandin E2; PGF^ prostaglandin F¿,; accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I'M . calcium and phospholipid dependent protein kinase; 6-K I'GI-,,,. 6-keto- 1This work was supported in part by USPHS Grants ES02922, CA 29589, prostaglandin F,„;TPA, 12-O-tetradecanoylphorbol-13-acetate; HPLC, high per andCA 16784. formance liquid chromatography. 5696 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1986 American Association for Cancer Research. RELEASE OF OXIDANTS BY MACROPHAGES FROM SENCAR AND C57BL/6 MICE reduced when other stimuli for ROI secretion and AA metab olism were used. MATERIALS AND METHODS Animals. SENCAR mice which have undergone 16 generations of brother-sister inbreeding were obtained from the Biology Division, Oak Ridge National Laboratories. C57BL/6 mice were obtained from The Trudeau Institute (Lake Saranac, NY). Animals were all females be tween 6 and 9 weeks old, housed S/cage in an environment providing 12 h of light and darkness per day and provided water and food ad libitum. Macrophage Culture. All culture reagents were endotoxin free as judged by the Limulus lysate assay (Cape Cod Associates, Woods Hole, MA). Macrophages were lavaged from the unmanipulated peritoneal Fig. 1. Release of 11..<>_•byresident (A) and casein elicited macrophages from cavity or following the i.p. injection of sterile casein, and purified by SENCAR ( ) and C57BL/6 (——)miceexposed to varying concentrations of adherence as previously described (15). They were plated at a density PBDU. Peritoneal macrophages were lavaged from the peritoneal cavity and of 2-3 x 105/cm2 in 16mm (H2O2 release) or 30-mm (AA metabolism) cultured as described in "Materials and Methods." I !.,()•releaseover 60 min was tissue culture wells. Culture medium was RPMI 1640 containing pen measured by the horseradish peroxidase catalyzed oxidation of phenol red as icillin and streptomycin (100 units/ml) and 10% fetal calf serum. These described previously (26). Points, mean triplicate samples; bars, SE. Similar results were obtained in 3 separate experiments. techniques produced viable cultures of macrophages which were greater than 95% pure as judged by morphology, nonspecific esterase histo- 700, chemistry, and Fc receptor mediated phagocytosis of RBC (25). Quantification of HjOj. Macrophage cultures were exposed to various stimulants in Hanks' balanced salt solution for 60 min at 37'C and 600 IM)., release was measured by the horseradish perioxidase catalyzed oxidation of phenol red as described previously (26), by 60 min the response of macrophages is maximal and little increase in Ho release is observed after this time (27). Cellular protein was quantified by the method of Bradford (28) and data were expressed as nmol 11-()- per mg protein per h. PBDU was used instead of TP A as the phorbol ester in