Neurogenic Component of Phorbol Ester-Induced Mouse Skin Inflammation Arpad S/Allasi and Peter M

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[CANCER RESEARCH 49. 6052-6057, November I. 1989] Neurogenic Component of Phorbol Ester-induced Mouse Skin Inflammation Arpad S/allasi and Peter M. Blumberg1

Molecular Mechanisms of Tumor Promotion Section. Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892

ABSTRACT induced a transient erythema lasting for only 1-2 h, in contrast to the 24-h duration for PMA-induced erythema (11, 12) and Tumor-promoting phorbol esters are potent inflammatory agents for mouse skin, and the potential mechanistic role of inflammation in tumor that RTX did not bind to protein kinase C or induce protein promotion is under active investigation. We have shown previously that kinase C-mediated responses at biologically effective concentra resiniferatoxin, a uniquely irritant phorbol-related diterpene, acts as a tions (13, 14). We have recently identified the mechanism of capsaicin analogue to induce and then to block neurogenic inflammation. RTX action (15, 16). We find that RTX is an ultrapotent (103- We report here that pretreatment of CD-I mice with resiniferatoxin 104 fold more potent) analogue of capsaicin. Capsaicin, the blocked the early (3 h) erythema and edema (6 h) in response to phorbol pungent component in red pepper, activates and then desensi 12-myristate 13-acetate (I'M \). whereas the edema at later times (12- tizes polymodal nociceptors (17). This neural pathway mediates 24 h) was only partially blocked. Since the efficiency of resiniferatoxin pretreatment decreased as a function of time if I'M \ was applied 24, 48, neurogenic inflammation and pain perception via afferent C fibers. Denervation abolishes the inflammatory response to or 96 h after resiniferatoxin administration, the late edema response to I'M A may be a combination of increasing edema of nonneurogenic origin RTX, measured by either Evans blue extravasation or water and the recovering neurogenic response due to the decreasing desensiti- content of skin (15), as described previously for capsaicin (19). zation. For other phorbol esters, 12-deoxyphorbol mono- and diesters, In this report, we use desensitization of mice to RTX to and mezerein, differing kinetics of edema and differing degrees of block explore the possible contribution of neurogenic inflammation ade of edema following resiniferatoxin pretreatment were observed, as to the edema in response to phorbol esters and related deriva expected from the discrepancies between their inflammatory and tumor- tives. Consistent with RTX not being a tumor promoter, desen promoting activities. PMA-induced skin hyperplasia, unlike edema, was sitization of at least the early phase of neurogenic inflammation not inhibited by resiniferatoxin pretreatment, suggesting that the early did not inhibit the hyperplastic response of mouse skin to component of neurogenic inflammation was not essential for hyperplasia phorbol ester treatment. under our conditions. Distinction between inflammatory mechanisms may help to clarify the role of inflammation in tumor promotion. MATERIALS AND METHODS INTRODUCTION Female CD-I mice between 6 and 7 weeks of age were obtained from The phorbol esters are the most extensively studied class of Charles River Laboratories (Wilmington, MA). Animals were given mouse skin tumor promoters ( 1). Their biochemical mechanism food and water ad libitum during the course of the experiments. RTX was obtained from Chemicals for Cancer Research, Inc. is thought to be through activation of their receptor, protein (Chaska, MN), and from Chemsyn Science Laboratories (Lenexa, KS); kinase C, which mediates one arm of the phosphatidylinositol capsaicin was from Polysciences (Warrington, PA). PMA, PDBu, and signal transduction pathway (2-5). In skin, phorbol ester treat mezerein were purchased from Sigma Chemical Co. (St. Louis, MO); ment initiates a series of acute responses, including erythema, the 12-deoxyphorbol derivatives dPP and dPPA were from LC Services edema, infiltration of polymorphonuclear leukocytes, and hy (Woburn, MA). perplasia (6). The relationship of these acute responses to the Inflammation was characterized qualitatively by observing erythema promoting activity of the phorbol esters has been an area of (ear reddening) and quantitatively by measuring tissue swelling (ear extensive investigation. plug weights). Compounds were dissolved in acetone and applied with Inflammation, quantitated by ear reddening (7), has been the the aid of a microliter pipet to the inner surface of the right ear in a standard assay for detecting phorbol esters and related deriva volume of 20 u\. The left ear was treated with solvent as a control. At tives during purification from the Euphorbiaceae and the Thy- the indicated times after application, the animals were killed by cervical dislocation and the ears were removed. Tissue plugs were obtained melaeaceae and provides the only available quantitative, in vivo from the tips of each ear with a 6-mm punch and were weighed (18). measure of potency for most phorbol derivatives. Structure- Solvent alone caused no ear edema. activity analysis suggests that inflammatory activity correlates For determination of hyperplasia, mice in the resting phase of the with tumor promotion but that this correlation is only partial. hair growth cycle were clipped. Two days later the compounds in a Whereas all tumor-promoting phorbol esters are inflammatory, volume of 0.2 ml acetone were applied to the back skin. The animals some derivatives are highly inflammatory but either nonpro- were killed by cervical dislocation at times between 4 and 72 h after moting or weakly promoting (8). The kinetics of erythema application, the back skin was removed, fixed in formalin, sectioned, differ, moreover, among derivatives (9). These discrepancies and stained with hematoxylin-eosin. In other experiments, compounds have been postulated to reflect an ill-defined combination of in 20 n\ of acetone were applied to the ears, and hyperplasia was then examined as for back skin. pharmacokinetics and different contributions through two in For desensitization of neurogenic inflammation, RTX or capsaicin flammatory pathways (10). was applied as indicated either topically in acetone or s.c. under the The strongest evidence for independent inflammatory path ways was that RTX,2 a 20-homovanillyl resiniferonol derivative. back skin in 10% ethanol/10% Tween 80/80% physiological saline.

Received 3/27/89; revised 7/17/89; accepted 8/3/89. RESULTS The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in RTX applied topically to the ears of CD-I mice induced ear accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Bldg. 37-3B25, reddening, as reported previously (11, 12), together with a fast National Cancer Institute. Bethesda, MD 20892. and transient tissue swelling. The edema could be conveniently 2The abbreviations used are: RTX, resiniferatoxin; PMA. phorbol 12-myris- quantitated by a modification of the procedure of Gschwendt tate 13-acetate; PDBu, phorbol 12,13-dibutyrate; dPP. 12-deoxyphorbol 13- phenyl acetate; dPPA, 12-deoxyphorbol 13-phenyl acetate 20-acetate: ED50, 50% et al. (18). Edema reached a peak at 30 min following RTX effective dose; ID50, 50% inhibitory dose. treatment and then subsided, approaching baseline by 3 h (Fig. 6052

tion, we also examined the effect of pretreatment with capsaicin. Topical capsaicin treatment (1 mg/ear) abolished PMA-induced inflammation in a fashion similar to RTX pretreatment (data not shown). The decreasing inhibition of edema as a function of time after RTX treatment could be explained either by a time-dependent decrease in the efficiency of desensitization with RTX or by an increasing contribution to the edema of a nonneurogenic in flammatory pathway for PMA. To distinguish between these possibilities, we treated with RTX 4, 24, 48, and 96 h before PMA application and assayed edema at a fixed time, 6 h after PMA application (Table 1). Inhibition was 76% at 24 h and decreased gradually thereafter. It should be noted, however, that there was significant variation, consistent with previous results of others in desensitization to capsaicin in other species (20, 21). Comparison of the 76% inhibition of the early (6 h) Fig. 1. Time course of neurogenie edema in response to RTX or xylene. RTX edema response to PMA 24 h after RTX pretreatment with the [10 >ig(16 nmol) in acetone] [â€¢]orxylene (20 M'][O] was applied to the ears of 40% inhibition of the late (24 h) edema response to PMA mice and edema was measured as a function of time as indicated. At 4 h (arrow) the RTX-pretreated animals were treated again with either RTX (â€¢)or xylene applied 4 h after RTX pretreatment suggests that the late edema (O). Values were calculated from the combined ear punch weights for 5 animals response to PMA represents a combination of both neurogenic for each time point. A second experiment gave similar results. Points, mean; bars, and nonneurogenic mechanisms. We are currently exploring SEM. other desensitization protocols to resolve this issue definitively. In addition to examining the duration of desensitization, we also determined how short a pretreatment interval with RTX 300 - sufficed to inhibit the early edema response (6 h) to PMA. Prior or simultaneous addition of RTX was effective, whereas dimin ished inhibition was seen if the RTX application was delayed until 1-3 h after PMA (Table 1). These data are consistent with establishment of a desensitized state by RTX within 2-3 h (see Fig. 1) and a similar time interval being required before the 200 induction of neurogenic inflammation by PMA (see Fig. 2). The concentration of RTX required to inhibit the early (6 h) edema response to PMA was determined for RTX administered either topically or s.c. 4 h before PMA (Fig. 3). The ED50 for topical application of RTX was 8 x 10~7g/kg (1.3 nmol/kg) or 3 x IO"8 g (0.048 nmol)/ear. The ED50 for s.c. administered

12468 12 24 48 RTX was 8 x IO"6 g/kg (13 nmol/kg), a factor of 10 higher. Time (hrs) Fig. 2. Effect of RTX pretreatment on the time course of ear edema following This latter ED50 is similar to that which we had obtained for PMA treatment. The ears of mice were pretreated with 10 fig (16 nmol)/ear of desensitization of neurogenic inflammation in Sprague-Dawley RTX (â€¢)orwith control solvent (O). Four h later 10 nmol/ear PMA were applied rats by RTX administered s.c. (15). The dose of topically and ear punch weights were determined as a function of time. Values were calculated from the combined ear punch weights for 5 animals at each tinn-point. applied RTX which we used routinely for desensitization in the Three further experiments gave similar results. Points, mean; bars, SEM. experiments described in this study, 10 pg (16 nmol)/ear, is 320-fold the ED50 for desensitization. 1). In other experiments, we confirmed that edema did not Since our data suggested that the early inflammatory re reappear at later times (12-72 h). The loss of response to RTX sponse to PMA was neurogenic, whereas perhaps one-half of reflects desensitization of the RTX pathway. A second appli the late response (24 h) was nonneurogenic, we wished to cation of RTX at 4 h failed to induce ear reddening or edema. determine whether the two responses showed similar or distinct Treatment of the desensitized mice with xylene, a second well characterized agent acting through the neurogenic pathway Table 1 Inhibition of PMA-induced edema as a function of time (19), was likewise ineffective. of RTX treatment Mice were treated with RTX. 10 jig (16 nmol)/ear, at the indicated times The development of edema in response to treatment of con relative to treatment with PMA at 10 nmol/ear. Edema was measured 6 h after trol mice with PMA at 10 nmol/ear showed quite different PMA treatment. Mean values were calculated from the combined ear punch kinetics from the edema in response to RTX (Fig. 2). Little weights for 10-20 animals in 2-4 experiments for each treatment condition. In parentheses the lowest and the highest values are given. edema was observed over the first 2 h. Edema then increased Time (h) of treatment markedly by 4 h, remained elevated for 24 h, and then declined. with RTX relative to The maximal level of edema (250-300% of control) was very PMA-96-48-24-3-2-10123Edema (% of PMAcontrol)64(31-96)47(10-76)24 much greater than that observed for RTX (140% of control). Desensitization of neurogenic inflammation by topical appli (4-60)3 cation of RTX to the ears 4 h before PMA treatment almost (0-9)5(0-11)4(0-14)10(3-18)41 fully prevented edema induced at early times, with diminishing effect by 12-24 h (Fig. 2). Thus, inhibition of edema was 94% at 4 h and approximately 40% at 24 h. (26-71)47 As confirmation that the action of RTX on the PMA-induced (38-62)71 (52-96) edema was through desensitization of neurogenic inflamma 6053

the edema increased to approximately a plateau level at 4 h. In contrast, the edema in response to dPP was maximal by 30 min 100 and declined thereafter (Fig. 5Q; the response to PDBu (Fig. 5A) and mezerein (Fig. 5B) were intermediate. On the other hand, the duration of edema induced by PMA was much longer than that for the other derivatives. As was the case for PMA, RTX pretreatment abolished most 50 of the early edema in response to dPP, PDBu, and mezerein and had much less effect at later times. The response to dPPA was unusual, in that the appearance of edema was slow and at no time did desensitization to RTX have a prominent effect (Fig. 5D). Analysis of late times was clouded by apparent enhanced toxicity of the phorbol ester in the RTX-pretreated 10Â« 10Â« 10s 10' RTX |g kg] animals. These results suggest that the neurogenic response Fig. 3. Inhibition of PMA-induced edema as a function of the dose of RTX may help protect against phorbol ester toxicity. used for pretreatment. RTX was applied topically to the right ears of mice (O) or To verify the comparability of the doses used for the different was injected s.c. under the back skin (â€¢)atthe indicated dosages. Four h later 10 derivatives, we examined their dose dependencies for edema nmol PMA were applied to the right ears and edema was measured 6 h after PMA application. Values were calculated from the combined ear punch weights formation, measured at 6 h (Fig. 6). The doses extrapolated for S animals at each RTX dosage. A second experiment gave similar results. A from the ID50 values all appeared near the top of the dose- dosage of 10 /ig (16 nmol)/ear corresponds to 4 x 10"* g/kg (640 nmol/kg). Points, mean; bars, SEM. response curves. The lower edema observed for dPPA in the time course appears to reflect the lower maximal induction of edema, not an insufficient dose. Dose-response curves for the various derivatives were not measured for response times other than 6 h. To determine the effect of RTX pretreatment on PMA- induced hyperplasia, we applied 30 nmol PMA to the back skin or 10 nmol to the ears of control mice or mice desensitized with RTX administered 4 h before. Pathohistological sections were prepared from animals 4, 24, 48, and 72 h after treatment. As illustrated for sections from the back skin at the 72-h time point (Fig. 7), RTX pretreatment reduced neither the hyperpla sia nor the degree of infiltration of inflammatory cells induced by PMA. Indeed, perhaps associated with the greater toxicity visually apparent in the pretreated ears at 48 h, enhanced infiltration was apparent in the back skin at 24 h. In the case of animals treated with RTX alone, the only histological re sponse at 2 h was dilation of blood vessels and increased 1 10 100 thickness of the subcutis. The dilation of the blood vessels PMA dose applied (nmole ear) visible histologically persisted for 24 h, despite the rapid dis Fig. 4. Comparison of dose dependency for induction of early and late ear edema by 12-CMetradecanoylphorbol-I3-acetate in control and RTX-pretreated appearance of erythema, and slight infiltration of inflammatory animals. RTX at a dose of 10 /ig (16 nmol) (â€¢,A)or control solvent (O. A) was cells became apparent. Similar effects of RTX pretreatment on applied to the right ears of mice, and PMA at the indicated doses was administered hyperplasia were observed for ears as for back skin. 4 h later. Ear edema was determined 6 h (A. A) or 24 h (O, â€¢¿)afterPMA application. Values were calculated from the combined ear punch weights for 5 animals at each experimental condition. A second experiment gave similar results. Points, mean; bars, SEM. DISCUSSION The complexity of phorbol ester-induced inflammation is dose dependence on PMA concentration. In fact, similar PMA hardly unexpected, given the central role of protein kinase C in dose-response curves were obtained for edema at 6 h, for the biological regulation. Our results presented here indicate that RTX-resistant component of the edema observed at 24 h, and RTX can be used to probe at the whole animal level the for the total edema observed at 24 h in the absence of RTX involvement of neurogenic inflammation. pretreatment (Fig. 4). The dose of PMA routinely used in the The existence of at least two mechanisms of phorbol ester- experiments described in this study, 10 nmol/ear, was submax induced inflammation had been postulated previously, based on imal for edema at 6 h but was chosen to minimize toxicity at the rapid kinetics and desensitization observed for RTX. Our 48-72 h. emerging understanding of RTX action, however, suggests that The time course for edema formation in response to PMA in RTX and closely related derivatives substituted at C-20 with control and RTX-pretreated animals was compared with those homovanillyl analogues are unique, in that the homovanillyl for several other phorbol esters and related derivatives. The substitution inhibits interaction at protein kinase C (26) and is compounds mezerein (22, 23), PDBu (24), dPP (9, 10), and essential for conferring capsaicin-like activity (12, 26, 27). In dPPA (10, 25) were chosen on the basis of differences in their contrast, all of the other derivatives are active on protein kinase inflammatory and tumor-promoting activities. For better com C, either as measured directly or as determined by activation of parison, each compound was used at the same dose as for PMA protein kinase C-mediated responses. Their induction of neu times its relative ID50 (50% inflammatory dose in the ear rogenic inflammation may therefore reflect the involvement of reddening assay) compared to PMA. Marked differences were protein kinase C as an intermediate in this pathway. In support observed (Fig. 5). PMA began to cause edema only at 2 h, and of this model, Burgess et al. (28) have reported recently that 6054

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Fig. 5. Induction of ear edema by phorbol esters and related diterpenes as a function of time and of RTX pretreatment. Control mice (O) or mice pretreated 4 h before with 10 Mg (16 nmol)/ear of RTX (â€¢)were treated with (A) 40 nmol/ear PDBu, (B) 10 nmol/ear mezerein, (Q 22 nmol/ear dPP, or (D) 67 nmol/ 48 8 12 24 48 ear of dPPA, and edema was then determined Time (hrs) as a function of time. Values were determined for the ear punch weights of 5 animals for each treatment condition in each experiment. Points, average for 2 experiments; bars, range. , 300 Ears of RTX-pretreated mice showed en hanced toxic effects (necrosis) by 48 h follow ing dPP administration or by 24 h after dPPA treatment; we did not determine ear plug weights in these mice.

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4 8 12 24 48 24 Time (hrs) Time (hrs)

itive evaluation will require complete inhibition of the late neurogenic response. Approaches are currently being assessed for achieving complete long-term desensitization. Other factors dictating caution in extrapolation of the present results to tumor promotion are possible differences in response between back skin and ears of mice and the lower dose of PM A typically used in promotion protocols. The involvement of inflammation in tumor promotion by the phorbol esters has been controversial. On the one hand, many antiinflammatory agents are inhibitors of tumor promotion (32), prostaglandins play a role in the mitogenic response of I epidermal cells to the phorbol esters (33), and differences in arachidonic acid metabolism represent one of the few charac terized differences between Sencar and NMRI mice (34). On the other hand, significant discrepancies exist between the inflammatory activity of phorbol esters as quantitated by ery thema and their tumor-promoting activity. In light of the diver gent kinetics of edema formation and of the different contri butions of early neurogenic inflammation, rÃ©Ã©valuationofthe 0.1 1 10 100 Dose applied (nmole ear) relationship between the nonneurogenic component of the in Fig. 6. Dose dependency for induction of edema by phorbol esters and related flammatory response and tumor promotion may be of interest. derivatives. Mezerein (O), PDBu (â€¢),dPP (A), and dPPA (A) were applied to the A difficulty in the separate analysis of the involvement of right ears of mice and edema was measured 6 h later. Values were calculated from neurogenic and nonneurogenic inflammation is that the two the ear punch weights for 5 animals at each experimental condition in a single experiment. Points, mean; bars, SEM. processes are not mutually independent. Rather, the early neu rogenic component of the phorbol ester response appeared to protect against phorbol ester toxicity, as reflected in greater phorbol ester treatment caused membrane depolarization and toxicity in the RTX-desensitized animals. This toxicity may calcium influx in cultured dorsal root ganglion cells, the target influence the attainable promotion response either positively or cells for capsaicin action. Moreover, capsaicin pretreatment negatively, and a difference in tumor yield for different deriva abolished the depolarization of ventral roots in response to tives might reflect different degrees of protection by the differ PDBu administration (29). ing proportions of early neurogenic inflammation. The ability to induce chronic hyperplasia represents one of the better correlations with tumor-promoting activity (30, 31). The retention of the hyperplastic response upon inhibition of ACKNOWLEDGMENTS the early neurogenic inflammation suggests that the neurogenic We thank Stuart H. Yuspa for critical reading of the manuscript. pathway may not be necessary for tumor promotion, but defin For the histological studies, fixation of the tissues, sectioning, and 6055

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Fig. 7. Effect of RTX pretreatment on the induction of hyperplasia by PMA. Mice were treated topically with 100 ng (160 nmoi)/mouse of RTX in 200^1 acetone or with solvent only, applied to the back skin. Four h later 30 nmol PMA or control solvent were applied to the back skin, and skin histology was determined 72 h after this second treatment. A, solvent, solvent; B, solvent. PMA; C, RTX, solvent; D, RTX, PMA.

staining were performed by Pathology Associates, Inc., Ijamsville, MD. relations of polyfunctional diterpenes of the daphnane type. I. Revised Animal care for the long-term experiments was provided by Immuquest, structure for resiniferatoxin and structure-activity relations of resiniferonol and some of its esters. J. Nat. Prod., 4S: 347-354, 1982. Rockville, MD. 13. Driedger, P. E., and Blumberg, P. M. Different biological targets for resini feratoxin and phorbol 12-myristate 13-acetate. Cancer Res., 40: 1400-1404, 1980. REFERENCES 14. Delclos, K. B., Nagle, D. S., and Blumberg, P. M. Specific binding of phorbol ester tumor promoters to mouse skin. Cell, 19: 1025-1032, 1980. Szallasi, A., and Blumberg, P. M. Resiniferatoxin, a phorbol-related diter 1. Hecker, E. Cocarcinogenic principles from the seed oil of Croton tiglium and 15. from other Euphorbiaceae. Cancer Res., 28: 2338-2349, 1968. pene, acts as an ultrapotent analog of capsaicin, the irritant constituent in red pepper. Neuroscience, 30: 515-520, 1989. 2. Blumberg. P. M. Protein kinase C as the receptor for the phorbol ester tumor promoters. Sixth Rhoads Memorial Award Lecture. Cancer Res., 48: 1-8, 16. de Vries, D. J., and Blumberg, P. M. Thermoregulatory effects of resinifer atoxin in the mouse: comparison with capsaicin. Life Sci., 44: 711-715, 1988. 3. Ashendel, C. L. The phorbol ester receptor: a phospholipid-regulated protein 1989. kinase. Biochim. Biophys. Acta, Â«22:219-242, 1985. 17. Buck, S. H., and Burks, T. F. The neuropharmacology of capsaicin: review 4. Nishizuka. Y. The role of protein kinase C in cell surface signal transduction of some recent observations. Pharmacol. Rev., 38: 179-226, 1986. and tumor promotion. Nature (Lond.). 308: 693-698, 1984. 18. Gschwendt, M., Kittstein, W., Furstenberger, G., and Marks, F. The mouse 5. Nishizuka, Y. Studies and perspectives of protein kinase C. Science (Wash. ear edema: a quantitatively Ã©valuableassayfor tumor promoting compounds DC), 233: 305-312. 1986. and for inhibitors of tumor promotion. Cancer Lett., 25: 177-185, 1984. 6. Klein-Szanto. A. J. P. Morphological evaluation of tumor promoter effects 19. Jancso. N., Jansco-Gabor, A., and Szolcsanyi, J. Direct evidence for neuro- on mammalian skin. In: T. J. Slaga (eds.). Mechanisms of Tumor Promotion, genie inflammation and its prevention by denervation and by pretreatment Vol. 2, pp. 41-72. Boca Raton, FL: CRC Press, 1984. with capsaicin. Br. J. Pharmacol. Chemother., 31: 138-151, 1967. 7. Hecker, E., Immich, H., Bresch, H., and Schairer, H. U. Ãœberdie Wirkstoffe 20. Jancso, N. Desensitization with capsaicin and related acylamides as a tool des Crotonols. VI. Entzundungsteste am Mauseohr. Z. Krebsforsch.. 68: for studying the function of pain receptors. In: Pharmacology of Pain, Vol. 366-374. 1966. 9, pp. 33-55. Elmsford, NY: Pergamon Press, 1968. 8. Furstenberger. G., and Hecker, E. Zum Wirkungsmechanismus cocarcino- 21. Szolcsanyi, J. Capsaicin-sensitive chemoceptive neural system with dual gener Pflanzeninhaltsstoffe. Planta Med.. 22: 241-266. 1972. sensory-efferent function, in: L. A. Chahl, J. Szolcsanyi, and F. Lembeck 9. Hergenhahn. M., Furstenberger, G., Opferkuch, H. J., Adolf, W., Mack, H., (eds.), Antidromic Vasodilatation and Neurogenic Inflammation, pp. 27-52. and Hecker, E. Biological assays for irritant tumor-initiating and -promoting Budapest: Akademiai Kiado, 1984. activities. I. Kinetics of the irritant response in relation to the initiation- 22. Hecker. E. Structure-activity relationships in diterpene esters irritant and promoting activity of polyfunctional diterpenes representing figliane and cocarcinogenic to mouse skin. In: T. J. Slaga. A. Sivak, and R. K. Boutwell some daphnane types. J. Cancer Res. Clin. Oncol., 104: 31-39. 1982. (eds.), Carcinogenesis. Vol. 2, pp. 11-48. New York: Raven Press, 1978. 10. Schmidt, R. J.. and Evans, F. J. Skin irritant effects of esters of phorbol and 23. Slaga, T. J., Fischer, S. M., Nelson, K., and Gleason, G. L. Studies on the related polyols. Arch. Toxicol., 44: 279-289, 1980. mechanism of skin tumor promotion: evidence for several stages in promo 11. Schmidt, R. J., and Evans, F. J. Investigations into the skin-irritant properties tion. Proc. Nati. Acad. Sci. USA, 77: 3659-3663. 1980. of resiniferonol ortho esters. Inflammation, 3: 273-280, 1979. 24. Thielmann, H. W., and Hecker, E. Beziehungen zwischen der Struktur von 12. Adolf, W., Sorg, B., Hergenhahn, M., and Hecker, E. Structure-activity Phorbolderivaten und ihren entzÃ¼ndlichenand tumorpromovierenden Eigen- 6056

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6057

Arpad Szallasi and Peter M. Blumberg

Cancer Res 1989;49:6052-6057.

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