[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 these studies because the flatter dose curve of PBDU for stimulation of IM), release facilitated determinations of the dose required to elicit 50% of the maximal response. Quantification of AA Metabolism. Macrophages were obtained and cultured as described above and cultured with [3H]AA (0.5 ¿tCi/ml)in complete medium for 16 h. Following labeling the cultures were washed three times with Hanks' balanced salt solution and the medium was Fig. 2. Release of II..O, by casein elicited macrophages from SENCAR replaced with serumless ..minimal essential medium containing the ( ) and C57BL/6 ( ) mice over 60 min in response to varying concentra various stimulants. After 60 min of incubation at 37'C the medium was tions of zymosan. Macrophages were obtained and cultured and 11.().. quantified as described in the legend to Fig. 1. Points, mean of triplicate samples; bars, SE. collected and an aliquot was counted to determine total release of label. Similar results were obtained in 3 separate experiments. Medium from unstimulated control cultures was counted to determine spontaneous release. Other control cultures were washed, solubili/ed capacity of casein elicited, inflammatory macrophages to re immediately, and counted to quantify total label uptake. Total specific lease IM).- in response to PBDU. Casein elicited macrophages release of label was calculated by subtracting the spontaneous release from the total and dividing by the total label uptake, and expressed as from SENCAR mice released significantly more H2O2 at all the percentage of specific release. The duplicate samples were then doses tested (Fig. 1). At the largest dose tested (100 ng/ml), pooled, made 80% within respect to methanol, and evaporated to casein macrophages from SENCAR mice secreted greater than dryness in a centrifugal vacuum evaporator (Savant). They were then 4-fold more H2O2 per mg of cell protein (705 nmol H2O2/mg resuspended in 400 n\ of 30% methanol and the metabolites were protein/h) than macrophages from C57BL/6 mice (145 nmol separated by reverse phase HPLC using an Altex C|8 column, a IIO H2O2/mg protein/h). In addition, macrophages from SENCAR methanol gradient, and a flow rate of 1 ml/min according to the method mice required only one-third the amount of PBDU (8.5 ng/ml) of Henke et al. (28). Samples were collected each minute directly into than macrophages from C57BL/6 mice (26 ng/ml) to achieve scintillation vials containing 3.5 ml of Aqua Sol II (New England 50% proximal response (Fig. 1). Nuclear) and counted in a Packard Minaxi 4000 liquid scintillation counter. Individual metabolites were measured as a percentage of total To determine if the increased secretion of H2O2 by SENCAR identified metabolites released. Peaks were identified by authenticated macrophages was selective for phorbol ester stimulation, casein standards purchased from New England Nuclear. Ninety-five % of macrophages were exposed to zymosan, which is a particulate radioactivity was eluted in peaks which were identified by the standards. stimulant for H2O2 release (Fig. 2) (9). Overall levels of release The HPLC column was thoroughly washed with 13 MIMEDTA each by macrophages from both strains were lower with zymosan morning before use. than PBDU (Figs. 1 and 2). SENCAR macrophages released more H2O2 than macrophages from C57BL/6 mice in response RESULTS to zymosan, but the magnitude of the difference was less than that observed when PBDU was used. Macrophages from SEN- Release of IM):. We first determined the basal state of CAR mice released only twice the amount of H2O2 (370 nmol/ activation for IM)., secretion in resident macrophages from the mg/protein/h) as macrophages from C57BL/6 mice (175 nmol/ two strains. When these cells were exposed to PBDU H2O2 mg protein/h) when exposed to zymosan. In addition there was release from both SENCAR and C57BL/6 derived macro a sharp reduction in the 50% maximal effective dose. Macro phages was barely detectable (Fig. 1). We next measured the phages from SENCAR mice required 63 Mg zymosan/ml to 5697 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
obtain 50% maximal response and cells from C57BL/6 mice required 88 tig. Arachidonic Acid Metabolism. When resident macrophages, which have the highest capacity for AA metabolism, were exposed to TPA, cells from SENCAR mice released 3 times more label than cells from C57BL/6 mice (Fig. 3). This differ ence was larger (4-fold higher) when casein elicited macro phages were exposed to TPA (Fig. 3). Conversely, when resident macrophages were stimulated with zymosan, which is a more Residentkty Casein potent stimulus for the release of metabolites of AA than Fig. 5. Release of metabolites of AA from resident and casein elicited macro phorbol esters, cells from C57BL/6 mice released twice the phages (Miti) from SENCAR and C57BL/6 mice exposed to A23187 (10~* M). amount of label as cells from SENCAR mice (Fig. 4). When Experiments were conducted in the same manner described in the legend to Fig. 3. Columns, mean of duplicate samples; hum. range. Similar results were obtained casein elicited cells were exposed to zymosan, again, cells from in 3 separate experiments. C57BL/6 mice released more label than cells from SENCAR mice. Similar amounts of specific release were obtained from resident macrophages when the calcium ionophore A23187 was used as a stimulant (Fig. 5), but overall release was less in both types of macrophages (note the differences in the graph scale for percentage release). Casein macrophages from C57BL/6 o mice exposed to the ionophore A23187 secreted slightly more « 3- label than cells from SENCAR mice. Fig. 6 shows radiochromatograms of HPLC separations of authenticated standards and AA metabolites released by casein macrophages from SENCAR mice stimulated with TPA. 6-K PGF,„was the major metabolite released. PGE2, LTC4, LTB4, LTD4, and 5-hydroxyeicosatetranenoic acid were also released. Relative amounts of the various metabolites of AA released from different types of macrophages and stimulants from the
-Cfc
•/.SpecificReleaser\> Fig. 6. Radiochromatograms of the separation of authenticated standards of metabolites of AA (bottom) and metabolites released from casein elicited macro phages from SENCAR mice (top) by HPLC. Cells were cultured, prelabeled with 'I I A \. and stimulated, and the metabolites were extracted and separated by reverse phase HPLC as described in "Materials and Methods." Similar results were obtained in 3 separate experiments. 5 HETE, 5-hydroxyeicosatetraenoic acid.
Table 1 Metabolites ofAA released by macrophages stimulated with zymosan' 5-Hydroxy- sSfa eicosa-ResidentSENCARC57BL/6CaseinSENCARC57BL/66-Keto-PGF,a43.0*29.460.948.6tetraenoicPGE215.224.14.06.0PCF*,NDNDNDNDLTC422.136.931.239.6LTD,ND°ND1.42.1LTB416.35.31.11.5acid2.12.30.51.0AA1.11.80.81.3 Resident Casein Fig. 3. Release of metabolites of AA from resident and casein elicited macro phages (M
two strains of mice are given in Tables 1 to 3. Generally, the patterns of metabolite release were similar in macrophages from both strains depending upon the type of stimulant used. Differ ences were observed between stimulants. TPA induced more relative release of cyclooxygenase products in contrast to zym osan which induced release of similar amounts of cyclooxygen ase and lipoxygenase products (Tables 1 and 3). Conversely, Residen!M^ Casem the ionophore A23187 induced mostly the release of lipoxidase Fig. 4. Release of metabolites of AA from resident and casein elicited macro products especially from resident macrophages (Table 2). phages (M
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 Table 2 Metabolites ofAA released from macrophages stimulated with A23187" inflammatory signals, produced barely detectable levels of 5-Hydroxy- H2O2. After receiving a sterile inflammatory stimulus, macro eicosa- 6-Kelo- tetraenoic phages from both strains increased the capacity for release of PGF,„PGE2 LTC«LTD4 LTB4 acid AA H2O2 in response to PBDU. This response was markedly higher Resident in cells from SENCAR mice in both magnitude of H2O2 release SENCAR 4.6* 6.5 ND' 40.4 23.8 11.2 8.6 4.8 and sensitivity to PBDU (Fig. 1). This was not merely a function C57BL/6 4.6 8.7 ND 36.9 37.9 7.3 3.4 1.2 of dose. The release of H2O2 in both strains was maximal at Casein PBDU (50 ng/ml). The addition of more PBDU did not in SENCAR 25.4 ND ND 58.7 ND ND 7.6 8.4 crease H2O2 production in either strain. The observation that C57BL/6 15.4 14.1 ND 55.8 2.8 3.4 4.7 4.0 * Separated and quantified by HPLC as described in "Materials and Methods." differences in the amount of H2O2 released and sensitivity to * Percentage of total metabolites. Greater than 95% of all radioactivity eluted dose was significantly less when zymosan was used as a stimu in peaks which were identified with authenticated standards. lant suggests that the increase in response is due to an enhanced c ND, not detected. sensitivity to PBDU. Since, however, macrophages, unlike neu-
Table 3 Metabolites ofAA released by macrophages stimulated with TPA° trophils, must be activated for the ability to generate H2O2 before they are exposed to phorbol esters (9), the possibility of 5-Hydroxy- eicosa- differential levels of activation in response to inflammatory 6 Keto- tetraenoic signals still remains. PCF,. PGE2 PGFj. LTC4 LTD. LTB4 acid AA Metabolites of AA are potent signals which affect many ResidentSENCARC57BL/6CaseinSENCARC57BL/636.4*29.2584835.130.814.6ND9.712.1NDND5.510.58.741.8ND'ND2.9ND5.49.52.5ND2.21.85.2ND9.712.18.210.1cellular responses. They are important signals for inflammatory and immune responses, in the regulation of vascular tone and permeability, and in the regulation of thrombosis (30, 31). Given the diversity of the effects of these compounds and the fact that they interact synergistically with other inflammatory " Separated and quantified by HPLC as described in "Materials and Methods." mediators, it is not possible at this point to discern any specific * Percentage of total metabolites. Greater than 95% of all radioactivity eluted pattern in terms of potential biological responses of target cells in peaks which were identified with authenticated standards. ' ND, not detected. to the large range of products released by macrophages. The release of significant quantities of PGE2 and the lipooxidase products, especially LTB4, suggest a general vasodilative and amounts of LTB4 and PGE2 release in response to zymosan inflammatory character (30, 31). PGE2 is associated with vas- and A23187, respectively. In resident macrophages from SEN- odilation and increased vascular permeability and I I It, is CAR mice exposed to zymosan 16.3% of the total metabolism associated with chemoattraction of inflammatory cells. In of AA was LTB4 (Table 1). The corresponding cells from creases in the relative amounts of 6-keto-PGFia and decreases C57BL/6 mice released only 5.3% as LTB4 (Table 1). No PGE2 in the relative amounts of PGE2 and lipoxidase products re release was detected from casein macrophages obtained from leased from casein macrophages compared to resident cells SENCAR mice which were stimulated with A23187, whereas (Tables 1 and 3) may be a reflection of the change in the in the corresponding cells from C57BL/6 mice 14.1% of the function of the cells from sentinals to activated effector cells total release was PGE2 (Table 3). There were several apparent which then release inhibitory signals to turn off the inflamma differences between casein elicited cells exposed to TPA (Table tory response. 6-KetoF|„is the breakdown product of prosta- 3) but the overall release by cells from C57BL/6 mice was so cyclin and is associated with anticoagulant and cytoprotective low (Fig. 5) that the metabolites which were not detected may activities (30, 31). have been present but beneath the level of detection of the assay. It is important to keep in mind when evaluating any differ ences in the relative percentages of the different metabolites of AA released that the amount of metabolites released also varied DISCUSSION greatly between cell type and stimulus. For example, resident Macrophages from mice which are sensitive to the promotion macrophages from C57BL/6 mice exposed to TPA released of skin tumors by phorbol esters released significantly higher almost twice as much LTC4 as a percentage of total metabolism amounts of H2O2 and metabolites of AA in response to phorbol as the corresponding cells from SENCAR mice (Table 3). esters than cells from mice which are resistant to the promotion However, resident macrophages from SENCAR mice released of tumors by phorbol esters. Macrophages from sensitive mice 3 times the amount of total metabolites as cells from C57BL/ required less than one-third the amount of phorbol ester to 6 mice (Fig. 5) when exposed to TPA. Thus the net effect is elicit 50% maximal stimulation than cells from resistant mice. that more LTC4 is released from SENCAR macrophages than Furthermore these effects were selective for the response to cells from C57BL/6. Because there were very few differences phorbol esters. No difference or the opposite effect was observed in the types of metabolites released by macrophages from the in the metabolism of AA when two other classes of stimuli were two strains (Tables 1 to 3) the major difference was in total used. Macrophages from SENCAR mice exposed to zymosan release of metabolites. released more H2O2 than cells from C57BL/6 mice, but the Although we and others have shown that inhibition of AA amount of 11.•().•releasedand the magnitude of the differences metabolism can inhibit the induction of genotoxicity by leuko was less than with PBDU. Thus, two events hypothesized to cytes (12,13,16), the numerous other effects mediated by these mediate damage by inflammatory cells stimulated with phorbol products may play an important role in both the inhibition and esters occur at a higher level in the strain of mice which is the potentiation of tumor formation. Several studies conducted sensitive to phorbol ester tumor promotion. in vivo have shown that various inhibitors of AA metabolism Increase in the production of H2O2 by macrophages from can have significant stimulatory or inhibitory effects on the SENCAR mice is not simply a function of higher basal levels. promotion of skin tumors in the mouse by phorbol esters (17, Resident cells from both strains of mice, which had received no 18). In order to evaluate any contribution that metabolites of 5699 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
7. Boutwell, R. K. Some biological aspects of skin carcinogenesis. Prog. Exp. AA may make in carcinogenesis, the relevant products of AA Tumor Res., 4: 207-250, 1964. metabolism and the important cellular targets of these products 8. Scott, W. A., Pawlowski, N. A., Murray, H. W., Andreach, M., Zrike, J., must be determined. Furthermore, direct evidence that these and Cohn, Z. B. Regulation of arachidonic acid metabolism by macrophage activation. J. Exp. Med., IS: 1148-1160, 1982. products alone or in conjunction with ROI actually mediate 9. Nathan, C. F., and Root, R. K. Hydrogen perioxide release from peritoneal DNA damage in vivomust be obtained in order to determine if macrophages. Dependence on sequential activation and triggering. J. Exp. the mechanism of action of these products stems from their Med., 146: 1648-1662, 1977. 10. Troll, W., Witz, G., Goldstein, B., Stone, D., and Sugimera, T. The role of physiological functions, or as oxidants with genotoxic potential, free oxygen radicals in tumor promotion and carcinogenesis. In: E. Heckler or both. et al. (eds.), Carcinogenesis: a Comprehensive Survey, Vol. 7, pp. 593-597. New York, Raven Press, 1982. The enhanced responses of SENCAR cells to phorbol esters 11. Birnboim, H. C. DNA strand breakage in human leukocytes exposed to a may not be limited to inflammatory cells. Since it has been tumor promoter, phorbol myristate acetate. Science (Wash. DC), 215:1247- demonstrated that phorbol esters bind to and activate a PKC, 1249, 1982. 12. Emerit, I., and Cerotti, P. A. Tumor promoter phorbol-12-myristate-13- and the majority if not all of their effects may be mediated by acetate induces chromosomal damage via indirect action. Nature (Lond.) activation of this enzyme (32), it is conceivable that all re 293:144-146, 1981. 13. Emerit, I., and Cerutti, P. A. Tumor promoter phorbol 12-myristate 13- sponses governed by this protein may be higher in SENCAR acetate induces a clastogenic factor in human lymphocytes. Proc. Nati. Acad. mice. The opposite or similar responses of macrophages from Sci. USA, 79:7509-7513, 1982. both strains to other signals suggest that only when signal 14. Weitberg, A. B., Weitzman, S. A., Destrempes, M., Latt, S. A., and Stossel, T. P. Stimulated human phagocytes produce cytogenetic changes in cultured transduction linkages were circumvented by direct activation of mammalian cells. N. Engl. J. Med., 308: 26-30, 1983. PKC by phorbol esters were there increased responses in SEN- 15. Lewis, J. G., and Adams, D. O. Induction of 5,6-ring-saturated thymine bases in NIH-3T3 cells by phorbol ester-stimulated macrophages: role of CAR mice. Even so, these results do not indicate whether the reactive oxygen intermediates. Cancer Res., 45:1270-1275, 1985. differences in response to phorbol esters are a result of altera 16. Lewis,.!.(... Hamilton, T. A., and Adams, D. O. The effect of macrophage tions in PKC, or in systems regulated by PKC. Confirmation development on the release of reactive oxygen intermediates and lipid oxi dations, and their ability to induce oxidative DNA damage in mammalian of the role of PKC in these activities requires further investi cells. Carcinogenesis (Lond.), 7:813-818, 1986. gation. 17. Slaga, T. J., Fisher, S. M., Weeks, C. E., Nelson, K., Mamrack, M., and It is important to note that phorbol esters also have potent Klein-Szanto, A. J. P. Specificity and mechanism(s) of promotor inhibitors in multistage promotion. In: E. Heckler el al. (eds.), Carcinogenesis: A and varied effects on target cells. Recent studies have shown Comprehensive Survey, Vol. 7, pp. 19-34. New York: Raven Press, 1982. that phorbol esters promote the transformation of C3H10T'/2 18. Nakadate, T., Yamanoto, S., Iseki, H., Sonoda, S., Takemua, S., Ura, A., Hosoda, Y., and Kato, R. Inhibition of 12-O-tetradecanoyl-phorbol-13-ace- cells in culture without the presence of inflammatory cells. tate-induced tumor promotion by nordihydroguaiaretic acid, a lipooxygenase These same studies also suggested that intracellularly produced inhibitor, and p-bromophenacyl bromide, a phospholipase A. inhibitor. Gann, 73: 841-843, 1982. ROI may play an important role in transformation by phorbol 19. Kensler, K. W., Bush, D. M., and Kozumbo, W. J. Inhibition of tumor esters (33). Even so, the evidence for the participation of promotion by a biomimetic Superoxide dismutase. Science (Wash. DC), 221: 75-77, 1983. inflammation and inflammatory cells in vivo is considerable. 20. Slaga, T. J., Klein-Szanto, A. J. P., Triplet!, L. L., and Yotti, L. P. Skin The majority of compounds which inhibit the actions of phorbol tumor-promoting activity of benzoyl peroxide, a widely used free radical- esters on mouse skin are either antiinflammatory agents or generating compound. Science (Wash. DC), 213: 1023-1025, 1981. 21. Klein-Szanto, A. J. P., and Slaga, T. J. Effects of peroxides on rodent skin: agents which scavenge ROI or inhibit the production of ROI epidermal hyperplasia and tumor promotion. J. Invest. Dermatol., 79: 30- and lipid oxidation products by inflammatory cells (10,17-19). 34, 1982. In summary, we have shown that the release of two classes 22. Langenbach, R., Nesnow, S., and Rice, J. M. (eds.). Organ and species specificity in chemical carcinogenesis. New York: Plenum Publishing Corp., of compounds thought to play a role in the indirect action of 1983. phorbal esters is significantly higher in macrophages from 23. Römers.J. J., Newnow, S., and Slaga, T. J. Murine susceptibility to two- stage skin carcinogenesis is influenced by the agent used for promotion. SENCAR mice than in macrophages from C57BL/6 mice. This Carcinogenesis (Lond.), 5: 301-307, 1984. correlates well with the increased sensitivity of SENCAR mice 24. Hennings, H., Devor, D., Wenk, M. L., Slaga, T. J., Former, B., Colburn, N. H., Bouden, G. T., Elgjo, K., and Yuspa, S. H. Comparison of two-stage and resistance of C57BL/6 mice to the promotion of tumors epidermal carcinogenesis initiated by 7,12 dimethylbenz(a)anthracene or N- by phorbol esters. methyl-W-nitro-A'-nitrosoguanidine in newborn and adult SENCAR and BALB/c mice. Cancer Res., 41: 773-779, 1981. 25. Adams, D. O. Effector mechanisms of cytolytically activated macrophages. I. Secretion of neutral proteases and the effect at protease inhibitors. J. ACKNOWLEDGMENTS Immunol., 124: 286-292, 1980. 26. Pick, E., and Keizari, Y. A simple colorometric method for the measurement The authors gratefully acknowledge the expert technical assistance of hydrogen peroxide produced by cells in culture. J. Immunol. Methods, 38: of Jane Harrell. 161-165, 1980. 27. Johnson, P., Adams, D. O., and Hamilton, T. Regulation of the respiratory burst in murine peritoneal macrophages in different states of functional activation. Cell. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1986 American Association for Cancer Research. Enhanced Release of Hydrogen Peroxide and Metabolites of Arachidonic Acid by Macrophages from SENCAR Mice following Stimulation with Phorbol Esters
James G. Lewis and Dolph O. Adams
Cancer Res 1986;46:5696-5700.
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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1986 American Association for Cancer Research.