Hydrolyzable tannins: potent inhibitors of hydroperoxide production and tumor promotion in mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate in vivo.

The anti-oxidant and the anti-tumor-promotion activities of several hydrolyzable tannins (HTs), including a commercial tannic-acid (TA) mixture, were examined in mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in vivo. A single application of TPA gradually increases the hydroperoxide (HPx)-producing activity of the epidermis, which is maximally stimulated at 3 days and returns to control levels at 9 days. Pre-treatments with TA and ellagic acid (EA) strongly inhibit, in a dose-dependent manner, this HPx response to TPA. Total inhibition by TA lasts for about 16 hr, beyond which it is substantially reduced but not completely lost. TA can also reduce the level of epidermal HPx when it is applied 36 hr after the tumor promoter. EA is an antioxidant 10 times more potent than TA and n-propyl gallate (PG), which are equally effective against TPA-induced HPx production. Gallic acid is the least effective of the HTs in inhibiting HPx formation. TA also inhibits the production of HPx induced by several structurally different tumor promoters and the greater HPx responses produced by repeated TPA treatments. When applied 20 min before each promotion treatment, twice a week for 45 weeks, several HTs inhibit the incidence and yield of papillomas and carcinomas promoted by TPA in initiated skin. Overall, TA is more effective than EA and PG in inhibiting skin-tumor promotion by TPA, suggesting that the anti-oxidant effects of HTs are essential but not sufficient for their anti-tumor-promotion activity.     

Inhibition of multistage tumor promotion in mouse skin by diethyldithiocarbamate

Diethyldithiocarbamate (DDTC) injected i.p. inhibits remarkably and in a dose-dependent manner 12-O-tetradecanoylphorbol-13-acetate (TPA)-decreased glutathione (GSH) peroxidase and TPA-induced ornithine decarboxylase (ODC) activities in mouse epidermis in vivo. DDTC is more potent in inhibiting these effects of TPA than 16 other antioxidants, free radical scavengers, thiol-containing compounds, and reduced glutathione (GSH) level-raising agents, even though some of these treatments are applied directly to the TPA-treated skin. DDTC also inhibits the effects of several structurally different tumor promoters and the greater GSH peroxidase and ODC responses produced by repeated TPA treatments. The inhibitory effects of DDTC on TPA-decreased GSH peroxidase and TPA-induced ODC activities are additive with those of Na2SeO3 and D-alpha-tocopherol (vitamin E). Interestingly, DDTC is a more effective inhibitor when it is administered after TPA, suggesting that DDTC may supplement, facilitate, and/or enhance the activity of the natural GSH-dependent detoxifying system protecting the epidermis against the oxidative challenge presumably linked to the tumor-promoting activity of TPA. When tested in the initiation-promotion protocols, DDTC inhibits to the same degree complete tumor promotion by TPA and stage 2 tumor promotion by mezerein, in relation with its identical inhibition of the GSH peroxidase and ODC responses to both TPA and mezerein. Moreover, the inhibition of the first stage tumor-promoting activity of TPA by DDTC may be attributed to its ability to inhibit TPA-induced DNA synthesis, a postulated component of the conversion phase of skin carcinogenesis when TPA is used as a stage 1 tumor promoter.     

Antitumor-promoting activity of oligomeric proanthocyanidins in mouse epidermis in-vivo

The flavanoid catechin and heterogenous samples of oligomeric proanthocyanidins extracted from various sources were compared for their ability to inhibit the biochemical and biological effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis in vivo. Topical applications of catechin fail to alter the hydroperoxide response to TPA but inhibit the induction of ornithine decarboxylase (ODC) activity and, to a lesser degree, the stimulation of RNA, protein, and DNA synthesis caused by this tumor promoter. Under similar conditions, condensed tannins (CTs) from guamuchil, loblolly pine, and southern red oak barks inhibit to various degrees all these biochemical markers of TPA promotion. The most effective antioxidant, loblolly pine bark CT, also inhibits TPA-induced ODC activity and macromolecule synthesis to a much greater degree than catechin or the other CTs tested. Pecan nut pith CT, however, has no inhibitory activity in this system. Pretreatments with 4 and 12 mg of loblolly pine bark CT remarkably inhibit the incidence and yield of skin tumors promoted by TPA in initiated mice, whereas similar doses of catechin are ineffective. Loblolly pine bark CT inhibits the 2nd rather than the 1st stage of tumor promotion. In contrast to their monomer units, therefore, some naturally occurring polyflavanoids have antioxidant activities and may be valuable against tumor propagation but their efficacy may vary considerably depending on their origin and structure.     

Characterization of the hydroperoxide response observed in mouse skin treated with tumor promoters in vivo

The production of hydroperoxides is rapidly increased and remains at 200-280% of the control 1-24 h after the second daily application of 17 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin in vivo. The levels of hydroperoxides are increased 1.63-, 2.64-, 4.07-, and 4.31-fold 18 h after one, two, three, or four applications of TPA at 24-h intervals, respectively. The hydroperoxide response to TPA observed in whole skin reflects almost entirely the increased hydroperoxide-producing activity of the epidermis. Such hydroperoxide responses are triggered to various degrees by the anthrone derivatives and the phorbol esters and diterpene with complete and/or stage 2 tumor-promoting activities but not by the agents with only inflammatory, hyperplastic or stage 1 tumor-promoting activities. However, the Ca2+ ionophores A23187 and ionomycin are potent inducers of hydroperoxide formation. Several discrepancies are observed between the hydroperoxide response to TPA and the known effects of the tumor promoter on ornithine decarboxylase (ODC) induction. In contrast to the refractory state against ODC induction caused by TPA treatments repeated at intervals of less than 48 h, the time interval required for recovery of the hydroperoxide response to TPA in TPA-pretreated skins is only 5 h. The stimulatory effects of A23187, ionomycin and various diacylglycerols (DAGs) on hydroperoxide production do not correlate with their ODC-inducing activities. The increasing susceptibilities of C57BL/6, CF-1, and SEN-CAR mice to skin tumor promotion correlate with their hydroperoxide responses but not with their ODC responses to TPA. alpha-Difluoromethylornithine (DFMO) and other inhibitors of TPA-induced ODC activity fail to alter hydroperoxide production whereas the compounds that inhibit the hydroperoxide response to TPA, such as fluocinolone acetonide, have no or only minimal inhibitory activity against ODC induction. This would suggest that the hydroperoxide response to TPA does not require ODC induction and may not be essential for ODC induction. The hydroperoxide response to TPA is mimicked, but to a lesser degree, by the activator of protein kinase C, 1,2-dioctanoyl-sn-glycerol, and inhibited by verapamil, trifluoperazine, and palmitoylcarnitine. Populations of TPA-treated keratinocytes, therefore, may be responsible not only for ODC activation but also for hydroperoxide production. However, these two responses, which involve, at least in part, Ca2+ mobilization and protein kinase C activation and play important roles in the mechanism of skin tumor promotion, do not appear to be correlated.     

Inhibition of mouse skin tumor promotion by tenuazonic acid.

Tenuazonic acid (TA) was topically applied to the interscapular region of Swiss albino mice at different doses before the application of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Skin from the painted area was examined for ornithine decarboxylase (ODC) enzyme estimation. It was observed that TA inhibited TPA induced ODC activity. The inhibitory effect of TA was also found in mouse skin tumor promotion in the two stage initiation promotion protocol. There was a remarkable delay in the latency period and decrease in the number of tumors developed and the percentage of tumor bearing animals after TA treatment.     

Modulation by adriamycin, daunomycin, verapamil, and trifluoperazine of the biochemical processes linked to mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

The antitumor antibiotics Adriamycin (ADR) and daunomycin (DAU) were tested for their ability to alter some of the molecular events linked to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). When applied topically to mouse skin, DAU is a more effective inhibitor of the basal level of epidermal DNA synthesis than ADR. However, these drugs alone are unable to inhibit the sequential induction of RNA, protein, and DNA synthesis caused by TPA in mouse epidermis in vivo. Moreover, ADR enhances substantially the induction of epidermal ornithine decarboxylase (ODC) activity by TPA. In vitro, the incorporation of [3H]DAU into isolated epidermal cells resembles more that of the HL-60 cells resistant to vincristine than that of the parental cell line. TPA does not alter the incorporation of [3H]DAU into epidermal cells. The Ca2+ antagonists verapamil (VRP) and trifluoperazine (TFP) enhance significantly the amount of [3H]DAU associated with the epidermal cells after 1 h. When applied shortly before TPA in vivo, VRP and TFP inhibit TPA-induced ODC activity at 5 h and TPA-induced DNA synthesis at 17 h. Moreover, the combinations of Ca2+ antagonists and anthracycline antibiotics administered before TPA inhibit synergistically these ODC and DNA responses to the tumor promoter. When they are applied at various times after TPA treatment, the same combinations of ADR or DAU and VRP or TFP fail to alter TPA-induced RNA and protein synthesis but still exert synergistic inhibitory effects on the peak of DNA synthesis observed 17 h after TPA. However, the chronic administration of ADR and DAU alone or in combination with VRP prior to the peak of TPA-induced DNA synthesis 16 h after each promotion treatment with TPA fails to alter the promotion of skin papillomas in the two-stage protocol of mouse skin carcinogenesis. In contrast, when administered alone or in combination with DAU prior to each TPA treatment, VRP inhibits skin tumor promotion and reveals the antitumor-promoting activity of DAU. These results point to the modulatory role of Ca2+ in the action of ADR and TPA and demonstrate the refractory nature of mouse epidermis to cancer chemotherapy by anthracycline antibiotics. However, ADR and DAU may be effective against skin tumor promotion if they are applied in combination with Ca2+ antagonists and at a time when they can inhibit the inductions of both ODC activity and DNA synthesis by TPA.     

Inhibition of mouse skin tumor promotion and of promoter-stimulated epidermal polyamine biosynthesis by alpha-difluoromethylornithine

Application of the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin leads to a manifold induction of ornithine decarboxylase (ODC) activity within 5 hr and an increased accumulation of putrescine. The relevance of these TPA-induced changes to the mechanism of tumor promotion was investigated using alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO applied to mouse skin (0.3 mg in 0.2 ml of solvent) or administered in the drinking water (1%) in conjunction with skin tumor promotion by TPA inhibited the formation of mouse skin papillomas by 50 and 90%, respectively. TPA-induced ODC activity and the accumulation of putrescine were almost completely inhibited. DFMO given in the drinking water decreased spermidine levels, but DFMO treatment by any route did not alter the spermine levels of mouse epidermis. DFMO decreased TPA-induced hyperplasia by 25 to 40%, and the TPA-caused increases in DNA synthesis and mitotic index were inhibited by 60 and 50%, respectively. Therefore, in mouse epidermis, enhanced cell proliferation can be dissociated from ODC induction and the accumulation of putrescine. At the tested dose levels and routes of administration, DFMO did not inhibit the inflammatory response to TPA in several tissues. These results provide evidence for an essential role of ODC induction and the accumulation of putrescine in tumor promotion by TPA and add strength to the proposal that DFMO may be a promising drug for the prevention and treatment of cancer in human beings.     

Antitumor-promoting activities of hydrolyzable tannins in mouse skin.

Ellagic acid and gallic acid and its derivatives, applied topically to female CF-1 mice 20 min before each 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment inhibit the inductions of epidermal ornithine decarboxylase activity, hydroperoxide production and DNA synthesis caused by this potent tumor promoter in relation with their abilities to inhibit the promotion of skin papillomas and carcinomas in the two-step initiation-promotion protocol. Because of its potency against TPA promotion, tannic acid, which is already known to inhibit tumor initiation, may inhibit the multistage process of carcinogenesis.     

Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds

The anti-tumor promoting activity of a polyphenolic fraction from grape seeds (GSP) was examined in CD-1 mouse skin epidermis. Specifically, the ability of this fraction to inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion and two markers of promotion in mouse skin, ornithine decarboxylase (ODC) and myeloperoxidase (MPO) activities, was evaluated. Pretreatment of mouse skin with 5, 10, 20 and 30 mg of GSP resulted in a dose-dependent reduction in TPA-induced epidermal ODC activity of 27, 37, 48 and 70%, respectively, compared to controls. In addition, pretreatment of mouse skin with 1, 5, 10 and 20 mg of GSP resulted in a significant 43, 39, 54 and 73% inhibition of MPO activity, respectively, compared to controls. In 7,12-dimethylbenz[a]anthracene (DMBA)-initiated CD-1 mice, biweekly treatment of mouse skin with 5, 10, and 20 mg of GSP 20 min prior to TPA application resulted in a 30, 40, and 60% inhibition of final skin tumor incidence, respectively, compared to controls. In addition, the final number of tumors per mouse in the 5, 10 and 20 mg GSP-treated animals was decreased 63, 51, and 94%, respectively, compared to controls. These studies indicate that GSP possesses anti-tumor promoting activity when applied to CD-1 mouse skin prior to treatment with TPA. The mechanism of this tumor inhibition is due, in part, to a GSP-associated inhibition of TPA-induced epidermal ODC and MPO activities. Thus, GSP warrants further evaluation as a skin cancer chemopreventative agent.     

Characterization of the tumor-promoting activity of m-chloroperoxybenzoic acid in SENCAR mouse skin and its inhibition by gallotannin, oligomeric proanthocyanidin, and their monomeric units

m-Chloroperoxybenzoic acid (CPBA),which induces ornithine decarboxylase activity as much as 12-O-tetradecanoylphorbol-13-acetate (TPA), was tested for its ability to induce DNA synthesis, hydroperoxide (HPx) production, and tumor promotion in mouse epidermis in vivo. After an early inhibition, CPBA stimulates DNA synthesis, a response which is maintained between 16 and 72 h and maximal after two treatments. CPBA at 0.6-5 mg stimulates DNA synthesis more than other organic peroxides, and nearly as much as TPA. The HPx-producing activity of the epidermis is maximally stimulated 48 h after two CPBA treatments at a 24-h interval. However, the HPx response to CPBA is much smaller than that to TPA. Aleppo gall tannic acid (AGTA) and loblolly pine bark condensed tannin (LPCT) inhibit both the DNA and HPx responses to CPBA. In contrast, their respective monomeric units, gallic acid (GA) and catechin (Cat) inhibit the DNA response to CPBA but fail to alter CPBA-stimulated HPx production. Although it is more potent than benzoyl peroxide, CPBA is a complete tumor promoter much weaker than TPA and even less effective than mezerein (MEZ). CPBA in stage 1 cannot enhance like TPA the tumor-promoting activity of MEZ in stage 2. And in contrast to that of MEZ, the very weak tumor-promoting activity of CPBA is not enhanced after stage 1 treatment with TPA. At equal mg doses, AGTA, GA, LPCT, and Cat pretreatments all remarkably inhibit complete skin tumor promotion by CPBA. In spite of their antioxidant activities, AGTA post-treatments have no or very little inhibitory effects on the development of skin tumors by CPBA during 2-stage or complete tumor promotion.     

Anti-tumor promoting action of phthalic acid mono-n-butyl ester cupric salt, a biomimetic superoxide dismutase

Skin tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by a concurrent and topical application of phthalic acid mono-n-butyl ester cupric salt (PAMBCu) in CD-1 mice initiated with 7,12-dimethylbenz[a]anthracene. PAMBCu inhibited TPA-caused epidermal ornithine decarboxylase (ODC) induction and ear edema formation, i.e. skin inflammation. However, neither PAMBCu nor superoxide dismutase (SOD) inhibited TPA-caused ODC induction in primary cultured mouse epidermal cells. 7-Bromomethylbenz[a]anthracene (BrMBA) is known to be a non-TPA type of tumor promoting agent. Epidermal ODC induction and inflammation caused by BrMBA were not inhibited by a concurrent application of PAMBCu. When mice were topically treated twice with PAMBCu, i.e. concurrently with and 7 h after BrMBA treatment, BrMBA-caused ODC induction was markedly suppressed. The same dose regimen of PAMBCu, however, failed to inhibit tumor promotion and inflammation caused by BrMBA. PAMBCu showed SOD-mimetic activity in superoxide generating systems, i.e. xanthine-xanthine oxidase reaction and TPA-stimulated polymorphonuclear leukocytes (PMN). Mono-n-butyl phthalate, which lacks SOD-mimetic activity, failed to inhibit TPA-caused ODC induction and skin inflammation. Therefore, inhibition by PAMBCu of TPA-caused tumor promotion, epidermal ODC induction and inflammation may be attributable to its SOD-mimetic activity. The results also support the contention that a superoxide anion of non-epidermal cell origin, such as PMN and macrophages, plays a role (probably some enhancing role) in in vivo ODC induction and tumor promotion caused by TPA. Failure of PAMBCu to inhibit BrMBA-caused tumor promotion suggests that superoxide anion generation is not involved in the tumor promoting action of this agent and that the anti-tumor promoting action of PAMBCu is dependent on the nature of the tumor promoting agents.     

Induction of mouse epidermal ornithine decarboxylase activity and skin tumors by 7,12-dimethylben

Application of a single large dose (3.6 micromol) or smaller weekly repeated doses (0.2 micromol) of 7,12-dimethylbenz[a]anthracene (DMBA) to the skin of CD-1 mice led to a 20 to 50-fold increase in epidermal ornithine decarboxylase (ODC) (EC 4.1.1.17) activity as well as tumor formation. Retinoic acid (0.17-68 nmol), a potent inhibitor of both the induction of ODC activity and tumor formation by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), failed to inhibit both the induction of ODC activity and tumor formation by DMBA. In contrast, 7,8-benzoflavone (367 nmol), which did not inhibit the induction of ODC activity by TPA, effectively inhibited the induction of ODC activity as well as the formation of skin tumors caused by DMBA. These results indicate that (a) the mechanism of the induction of ODC activity and tumor formation by a complete carcinogen appears to be different from that of the tumor promoter TPA, (b) DMBA-induced ODC activity may be an important component of the mechanism of DMBA carcinogenesis, and (c) the protective effect of retinoic acid on skin carcinogenesis is not universal; it inhibits skin tumor formation by some agents and not by others.     

Effects of combined treatments with selenium, glutathione, and vitamin E on glutathione peroxidase activity, ornithine decarboxylase induction, and complete and multistage carcinogenesis in mouse skin

Several structurally different tumor promoters altered to various degrees both glutathione (GSH) peroxidase (EC 1.11.1.9) and ornithine decarboxylase (ODC, L-ornithine carboxy-lyase, EC 4.1.1.17) activities in mouse epidermis in vivo. At 5 h after their application to the skin, the complete tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the stage 2 promoter mezerein were the most potent in inhibiting GSH peroxidase activity and inducing ODC activity. In comparison, the effects of anthralin, phorbol-12,13-didecanoate, benzoyl peroxide, H2O2, and phorbol-12,13-dibenzoate were much smaller, whereas the nontumor promoter phorbol, the hyperplastic agent ethyl phenylpropiolate, and the stage 1 promoter 4-O-methyl TPA did not alter GSH peroxidase and ODC activities. Various treatments including i.p. injections of 40 micrograms of Na2SeO3 and 100 mumol of GSH and/or topical applications of 40 mumol of D-alpha-tocopherol (vitamin E) 20 or 15 min, respectively, before tumor promoter treatment inhibited in an additive manner the effects of either TPA or mezerein on both GSH peroxidase activity and ODC induction. Moreover, these Na2SeO3, GSH, and/or vitamin E treatments inhibited in the same additive manner the tumor-promoting activity of TPA in the initiation-promotion protocol. However, when tested in the 2-stage promotion protocol with 4 doses of TPA followed by twice weekly applications of mezerein, Na2SeO3 plus vitamin E and GSH plus vitamin E treatments inhibited remarkably the tumor-promoting activity of mezerein but were ineffective in the first stage of promotion. The sequence and magnitude for the effects of 7,12-dimethylbenz[alpha]anthracene (DMBA) on GSH peroxidase and ODC activities were very different from those of the tumor promoters. In contrast with their antitumor-promoting activity, the treatments with Na2SeO3 plus vitamin E and GSH plus vitamin E failed to inhibit the carcinogenicity of a single large dose of DMBA and even enhanced the induction of skin tumors by repeated applications of subcarcinogenic doses of DMBA. These results suggest that the promoting component of DMBA carcinogenesis may be different from that of TPA. Moreover, the anticarcinogenicity of Na2SeO3, GSH, and vitamin E may be linked to their ability to facilitate or enhance the activity of the natural GSH-dependent antioxidant protective system of the epidermal cells during the later stages of skin tumor promotion.     

Camptothecin post-treatments inhibit the biochemical events linked to the tumor-promoting component of carcinogenesis in mouse epidermis in vivo

20(S)-Camptothecin (CPT), a topoisomerase I inhibitor specifically toxic toward S-phase cells, was tested topically for its ability to inhibit the biochemical markers of skin tumor promotion. CPT has no or very little inhibitory effect on the covalent binding of an initiating dose of 7,12-dimethylbenz-[a]anthracene (DMBA) to DNA at 24 hr, but CPT post-treatments remarkably inhibit stimulations of DNA synthesis caused by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 16 hr and a carcinogenic dose of DMBA at 7 days. CPT is a much more potent inhibitor if it is applied 10–14 hr after TPA or 4–6 days after DMBA, when DNA synthesis starts being stimulated after the periods of early inhibition caused by TPA and DMBA. When applied 12 hr after the tumor promoter, the ability of 3–3,000 nmol of CPT to inhibit TPA-stimulated DNA synthesis at 16 hr is dose-dependent. A single dose of 500 nmol of CPT inhibits the entire time course for the stimulation of DNA synthesis observed 16–64 hr after TPA. CPT also reduces the various DNA responses to chronic TPA treatments and structurally different non-TPA-type tumor promoters. CPT may indirectly decrease the ornithine decarboxylase-inducing activity of multiple TPA treatments because it can inhibit the stimulation of RNA synthesis by this compound. However, CPT fails to alter TPA-stimulated hydroperoxide production in relation to its inability to inhibit TPA-stimulated protein synthesis. On an equal dose basis, topotecan and 10-hydroxycamptothecin are more and less effective than CPT, respectively, whereas 10,11-methylenedioxycamptothecin is much more potent than its parent compound at inhibiting the DNA response to TPA. A single dose of 400 nmol of CPT has no effect on tumor initiation when applied 4 hr before or 1 hr after a single subcarcinogenic dose of DMBA. In contrast, 400 nmol of CPT chronically applied 1 hr before or 24 hr after each treatment with TPA remarkably inhibits the complete tumor-promoting activity of this agent. CPT post-treatments also inhibit the respective activities of TPA and mezerein in the 1st and 2nd stages of skin tumor promotion. © 1996 Wiley-Liss, Inc.     

Inhibitory effect of silymarin, an anti-hepatotoxic flavonoid, on 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity and mRNA in SENCAR mice

In recent years, considerable emphasis has been placed on identifyingnew cancer chemopreventive agents which could be useful forhuman populations. Silymarin, an anti-oxidant flavonoid isolatedfrom artichoke, has been shown to possess siginificant activityagainst hepatotoxicity and other pharmacological and physiologicaldisorders. Since many antioxidants inhibit tumor promotion,and because ornithine decarboxylase (ODC) is a well known biochemicalmarker of tumor promotion, we assessed the effect of skin applicationof silymarin on 12-O-tetradecanoylphorbol-13-acetate (TPA) inducedepidermal ODC activity and ODC mRNA levels in SENCAR mice. Applicationof silymarin at doses of 0.5–18 mg (1–37 µmol)/mouseprior to that of TPA (2.5 µg) treatment resulted in significantinhibition of TPA-induced epidermal ODC activity in a dose-and time-dependent manner. Northern blot analysis revealed thattopical application of silymarin at the dose of 2 mg/mouse resultedin almost complete inhibition of TPA-induced epidermal ODC mRNA.In other studies, silymarin also showed significant inhibitionof epidermal ODC activity induced by several other tumor promoters,including free radical-generating compounds. Our data suggestthat silymarin could be a useful anti-tumor promoting agentcapable of ameliorating the tumor promoting effects of a widerange of tumor promoters.     

Induction of epidermal ornithine decarboxylase activity in mouse skin exposed to biogenic silica fibers.

The present study demonstrates that biogenic silica fibers (BSF), previously shown to promote skin tumors in mice and more recently to promote the induction of mesotheliomas when injected into the pleural cavity of rats, rapidly induces epidermal ornithine decarboxylase (ODC) activity in SENCAR mice following topical application. The time course for induction of epidermal ODC by BSF was very similar to that observed following topical treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). Maximal ODC activity was observed 4-6 h following treatment with BSF. Cycloheximide (70 mg/kg i.p.) partially inhibited (61%) the induction of ODC by BSF at 5 h. In addition, retinoic acid (RA, 5 micrograms per mouse given 30 min before BSF) effectively inhibited BSF-induced ODC by 68%, while indomethacin (100 micrograms per mouse 2 h before BSF) had little or no effect. Copper(II) bis(diisopropylsalicylate) (2 mumol 30 min before BSF), an effective inhibitor of TPA-induced ODC activity and tumor promotion, also had little or no effect on BSF-induced ODC. The work described in this paper suggests that BSF induces epidermal ODC by a very specific mechanism that exhibits both similarities and differences with that of the phorbol ester, TPA. Nevertheless, this response strongly supports the conclusion that BSF is an effective tumor promoter in mouse skin and that ODC induction is an integral part of the mechanism of action of this environmental promoter.     

Ability of the Ca2+ ionophores A23187 and ionomycin to mimic some of the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on hydroperoxide production, ornithine decarboxylase activity, and DNA synthesis in mouse epidermis in vivo

When applied topically to the skin twice at a 48-h interval or thrice at 24-h intervals, 17 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 mumol of A23187 or ionomycin induce the same 3-fold increases of hydroperoxide (HPx) production in mouse epidermis. In contrast, these doses of Ca2+ ionophores applied once or twice at a 48-h interval produce only 3-8% of the 16- or 34-fold inductions of epidermal ornithine decarboxylase (ODC) activities caused by similar TPA treatments. However, these various Ca2+ ionophore treatments mimic entirely the stimulatory effects of TPA on epidermal DNA synthesis at 16 h and produce from 30 to 70% of the DNA responses to TPA at 32 h. Interestingly, the Ca2+ ionophore and TPA treatments applied thrice at 24-h intervals still produce above maximal or submaximal DNA responses, in spite of their very weak ODC-inducing activities or refractoriness against ODC induction. Treatment with alpha-difluoromethylornithine plus methylglyoxal bis-(guanylhydrazone) (1.25 mumol each), which inhibits the activity of the polyamine-synthesizing enzymes, does not block the HPx and DNA responses to TPA. Conversely, 1.6-25-nmol doses of fluocinolone acetonide inhibit both TPA-induced HPx production and DNA synthesis, without affecting ODC induction. The results suggest that the magnitudes of Ca2+ ionophore- and TPA-induced DNA synthesis may be linked to HPx production rather than ODC induction. Each of these three responses appears to be essential but not sufficient for tumor promotion. A23187 may be a poor or incomplete skin tumor promoter because it lacks sufficient ODC-inducing activity and cannot fully maintain the prolonged stimulation of DNA synthesis required for hyperproliferation.     

Enhanced induction of epidermal ornithine decarboxylase activity in C57BL/6 compared to DBA/2 mice by protein kinase C-activating skin tumor promoters: relevance to genetically mediated differences in promotion susceptibility.

Previous work from our laboratory demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) or a synthetic diacylglycerol induced significantly higher epidermal ornithine decarboxylase (ODC) activity in C57BL/6 than in DBA/2 mice. To understand further the genetic basis for this strain difference, two tumor promoters were evaluated for their effects on epidermal ODC activity: teleocidin, which activates protein kinase C (PKC); and 1,8-dihydroxyl-3-methyl-9-anthrone (chrysarobin), which does not. In addition, the ODC induction response in B6D2F1 offspring and BXD recombinant inbred (RI) strains was examined following multiple treatments with TPA. A single topical application of teleocidin to mouse dorsal skin led to the hyperinduction of epidermal ODC activity in C57BL/6 mice. In contrast, while chrysarobin induced epidermal ODC activity, no significant differences in the magnitude of this response were observed in SENCAR, DBA/2 or C57BL/6 mice. Consistent with our previous findings, the magnitude of ODC induction by teleocidin in these three mouse lines (C57BL/6 greater than SENCAR greater than DBA/2) did not correlate with their susceptibility to tumor promotion by TPA (SENCAR greater than DBA/2 greater than C57BL/6). ODC activity induced by multiple application of TPA in B6DF1 mice, whose susceptibility to phorbol ester tumor promotion is inherited as an incomplete dominant trait, was comparable to that induced in C57BL/6 mice at all the doses examined. Cluster analysis of TPA-induced ODC activity in BXD RI strains allowed us tentatively to group them into four or five phenotypes and to estimate a minimum of two genetic loci controlling TPA-induced ODC activity. Furthermore, in BXD RI strains, there was no apparent relationship between the magnitude of ODC induction and responsiveness to tumor promotion or sustained hyperplasia. Collectively, these results suggest that hyperinducibility of ODC in response to PKC-activating tumor promoters is inherited as an autosomal dominant trait, and that genetic determinants for ODC induction, at least in C57BL/6 and DBA/2 mice, appear completely independent of those controlling tumor promotion susceptibility.     

Cytotoxicity and absence of mutagenic activity of vomitoxin (4-deoxynivalenol) in a hepatocyte-mediated mutation assay with V79 Chinese hamster lung cells

The effects of naturally occurring sweetening agents, which inhibited the induction of Epstein-Barr virus-associated early antigen (EBV-EA) induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and related compounds on the induction of ornithine decarboxylase (ODC) by TPA is examined. Application of glycyrrhetinic acid or steviol to mouse skin 1 h before TPA treatment showed a remarkable decrease in TPA-induced ODC activity. Post-treatment with glycyrrhetinic acid or steviol 1 h after application of TPA also resulted in a considerable depression in the induction of ODC activity. Neither glycyrrhetinic acid nor steviol alone induced epidermal ODC activity. These results suggest that glycyrrhetinic acid and steviol interfere with the process of induction of epidermal ODC by TPA treatment of mouse skin. cis-Abienol, frullanolide and norambreinolide, which have a partially similar structure in the moiety with glycyrrhetinic acid or steviol, were tested. cis-Abienol and frullanolide showed an inhibitory effect when applied 1 h before TPA treatment, but norambreinolide was not effective. A relationship between suppression of ODC activity and inhibition of EBV-EA induction is discussed.