Anti-mutagenesis and anti-promotion by apigenin, robinetin and indole-3-carbinol

We assessed the anti-mutagenic and anti-promotion propertiesof two flavones, apigenin and robinetin, and of indole-3-carbinol,because these compounds have been reported in vegetables, theconsumption of which has been associated with reduced ratesof cancer. However, the active components of these foods andtheir effects on carcinogenesis have not been established. Anti-mutagenicitywas determined in the Salmonella typhimurium assay by measuringthe effects of the test compounds on bacterial mutagenesis inducedby methyl-nitrosourea (MNU), methyl-n-nitro-N-nitrosoguanidine(MNNG), benzo[a]pyrene (BaP) or 2-aminoanthracene (2-AA). Inclusionof apigenin resulted in a 62% and a 43% inhibition of mutagenicitywith 13 nmol of 2-AA and 30 nmol BaP respectively. Robinetincaused an 87% inhibition of mutagenicity by 2-AA, but indole-3-carbinolhad little or no effect on the mutagenicity of any of the compounds.None of the three compounds inhibited mutagenesis by MNU orMNNG and none were mutagenic or toxic when tested in the absenceof mutagenic compounds at doses up to 20 µg/plate. Anti-promotionproperties were assessed by measuring the effects of apigenin,robinetin and indole-3-carbinol on induction of ornithine decarboxylaseactivity (ODC) in mouse epidermis by 17 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment of the skin half an hourbefore TPA with apigenin, robinetin, butylated hydroxyanisole,13-cis-retinoic acid (all at 50 µol) or di-fluoromethylornithine(1.6 µmol) inhibited ODC induction at 6 h after TPA by67–80%. Pretreatment with 50 µmol indole-3-carbinolcaused a 78% elevation in the TPA induction at this time. Doseresponse measurements were conducted with apigenin, indole-3-carbinoland robinetin. Inhibition by 30–90% of TPA-induced ODCwas observed at 6 h after TPA in mice pre-treated with 12.5–100µmol apigenin. Pretreatment with 37.5 or 50 µmolindole-3-carbinol or 0.5, 12.5 or 25 µmol robinetin resultedin elevated induction of epidermal ODC by TPA at 6 h after TPA.However, treatment with 50 or 100 µmol robinetin diminishedODC induction at 6 h after TPA. Treatment with 100 µmolapigenin or 50 or 100 µmol indole-3-carbinol in non-TPA-treatedmouse skin caused elevations in epidermal ODC. In comparingthe time course of ODC induction, indole-3-carbinol (50 µmol)pretreatment shifted the induction of epidermal ODC to earliertimes, in addition to elevating ODC induction by TPA. However,apigenin (50 µmol) pretreatment inhibited TPA-inducedODC activity at 4, 6 and 8 h, indicating no shift in ODC induction.In conclusion, indole-3-carbinol showed no potential for inhibitionof mutagenesis in the present study and presented potentialfor enhancement of promotion. In contrast, the potential ofapigenin and robinetin as inhibitors of the initiation and promotionphases of carcinogenesis merits further study.     

Inhibition of the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate in mouse skin by sphingosine sulfate

We investigated the effect of sphingosine sulfate on the inductionof ODC (ornithine decarboxylase) activity by TPA (12-O-tetradecanoylphorbol-13-acetate)in mouse skin. When applied topically to the shaved skin ofSENCAR mice at dosages of 10–40 µunol per animal,30 min before the superficial application of 8.5 nmol of TPA,sphingosine sulfate dramatically inhibited the induction ofODC activity by the tumor promoter. Significant inhibition ofTPA-induced ODC activity was observed at 4, 6 and 8 h afterTPA treatment in separate studies. The results indicate thatsphingosine sulfate is an effective inhibitor of ODC inductionby TPA in mouse skin.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity by lipoxygenase inhibitors: possible role of product(s) of lipoxygenase pathway

Induction of epidermal ornithine decarboxylase (ODC) by a topicalapplication of 12-O-tetradecanoylphorbol-13-acetate (TPA), atumor promoter, was inhibited by treatment of mouse skin withphenidone (3–90 µ mol/mouse), nordihydroguaiareticadd (30 µmol/mouse) or 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline(BW 755C, 30 µ/mouse), which are well-known lipoxygenaseinhibitors. Phenidone and BW 755C are also to be cyclooxygenaseinhibitors. Inhibition of TPA-induced ODC by indomethacin (1.12µmol/mouse), a selective cyclooxygenase inhibitor, wascounteracted by prostaglandin E₂(PGE₂) (140 nmol/mouse). Thiscounteracting effect of PGE₂ was reversed by the treatment ofmice with nordihydroguaiaretic acid (30 µmol/mouse) orphenidone (30 umol/mouse). ODC activity which was suppressedby nordihydroguaiaretic add or phenidone at a dose of 180 umol/mousewas not further inhibited by indomethadn (1.12 µmol/mouse).In addition, the counteracting action of PGE₂ (140 nmol/mouse)was not observed in mice treated with nordihydroguaiaretic acidor phenidone at a dose of 180 umol/mouse. Thus, the suppressiveeffect of nordihydroguaiaretic add or phenidone on the ODC inductionby TPA would be due to the inhibition of lipoxygenase. The abovefindings strongly suggest that not only cyclooxygenase product(i.e., PGE₂) but also lipoxygenase produces(s) are involvedin the mechanism of ODC induction in mouse epidermis, and alack of either cyclooxygenase product or lipoxygenase product(s)causes a failure of ODC induction by TPA.     

Regulation of ornithine decarboxylase gene expression in normal and transformed hamster embryo fibroblasts following stimulation by 12-O-tetradecanoylphorbol-13-acetate

We have compared the regulation of ornithine decarboxylase (ODC)gene expression in primary cultures of hamster embryo fibroblastsand in two independently transfonned hamster embryo cell lines.Previous studies have demonstrated that 12-O-tetradecanoylphorbol-13-acetate(TPA) can greatly potentiate the serum growth factor inductionof ODC enzyme activity in transformed cells, but not in normalhamster embryo fibroblasts. Treatment of either normal or transformedcells with both TPA and serum yielded greater accumulationsof ODC mRNA than with either treatment alone, which is consistentwith changes at the protein level. However, treatment of thetransformed cells with TPA and serum resulted in a greater increasein steady state levels of ODC mRNA than that observed usingnormal fibroblasts. The time course for the induction of ODCmRNA was similar for both normal and transformed cells withmaximal accumulations 4–8 h after treatment. Studies withactinomycin D further suggests that ODC mRNA is comparativelylong-lived in both normal and transformed cells. The accumulationof ODC mRNA after stimulation with TPA and serum is blockedby cycloheximide in normal hamster fibroblasts suggesting thatthis induction is dependent upon protein synthesis. In contrast,cydoheximide did not affect the accumulation of ODC mRNA undersimilar treatment conditions in transformed cells. This alteredregulation of ODC gene expression in transformed hamster embryofibroblasts cannot be explained by either gene rearrangementor the amplification of an ODC gene. These data suggest thattransformation of hamster embryo cells results in a loss ofcellular control over ODC gene regulation which includes analteration in the requirement for protein synthesis for ODCmRNA accumulation.     

Inhibitory effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced increase in ornithine decarboxylase mRNA in mouse epidermis

Application of 5 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA)to the skin of female CD-1 mice led to a rapid increase in theconcentration of epidermal ornithine decarboxylase (ODC) mRNAfrom an undetectable level in control mice to a high maximumlevel at 4–5 h after TPA administration. The concentrationof epidermal ODC mRNA then decreased rapidly during the next5 h. The time course for TPA-induced increases in epidermalODC enzyme activity paralleled very closely the time coursefor TPA-induced increases in ODC mRNA. Topical administrationof 1, 3 or 10µmol curcumin together with 5 nmol TPA inhibitedby 66, 81 and 91% respectively TPA-induced increases in epidermalODC enzyme activity measured 5 h later. In a parallel study,TPA-induced increases in the concentration of epidermal ODCmRNA was inhibited by 54, 85 and 82% respectively. Intraperitonealinjection of 10 or 30 µmol curcumin 1h before topicalapplication of 5 nmol TPA inhibited TPA-induced increases inepidermal ODC enzyme activity by 75 or 89% respectively. Ina parallel study, the induction of epidermal ODC mRNA was inhibitedby 53 and 65% respectively. The results indicate that curcumininhibits TPA-induced increases in epidermal ODC enzyme activityby inhibiting the synthesis and/or enhancing the breakdown ofODC mRNA.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion and ornithine decarboxylase activity by quercetin: possible involvement of lipoxygenase inhibition

Quercetin (30 µmol/mouse) markedly suppressed the effectof 12-O-tetradecanoylphorbol-13-acetate (TPA, 20 nmol/mouse)on skin tumor formation in the CD-1 mice initiated by 7,12-dimethylbenz[a]anthracene(200 nmol/mouse). TPA (20 nmol/mouse)-induced epidermal ornithinedecarboxylase (ODC) activity was also inhibited by quercetin(10–30 µmol/mouse), but it failed to inhibit thestimulation, of epidermal DNA synthesis by TPA. In addition,quercetin potently inhibited lipoxygenase from 105 000 g supernatantof epidermal homogenate of mice. The 50% inhibition of lipoxygenasewas observed by quercetin at 1.3 µM. These results suggestthat the inhibition of lipoxygenase by quercetin is one of themajor actions of the above agent to inhibit tumor promotionand TPA-induced ODC activity.     

Inhibition of mouse skin tumor promotion and of promoter-induced epidermal polyamine biosynthesis by methylglyoxal bis(butylamidinohydrazone)

Effects of methyiglyoxal bis(butylamidinohydrazone) (MGBB),a reversible inhibitor of ornithine decarboxylase (ODC) andS-adenosylmethionine decarboxylase (AdoMetDC), on 12-0-tetradecanoylphorbol-13-acetate(TPA)-induced increases of ODC and AdoMetDC activities, ODCmRNA level and polyamine contents in mouse skin were investigatedin connection with tumor formation. Formation of papillomasby applications of TPA to 7,12-dimethylbenz[a]anthracene (DMBA)-initiatedmouse skin was effectively inhibited by simultaneous topicalapplications of MGBB. MGBB also dose-dependently inhibited theability of TPA to induce increases of ODC activity, ODC mRNAlevel and the accumulation of putrescine and spermidine in mouseskin. Induction of AdoMetDC activity was not affected by thedrug. These inhibitory effects of MGBB on ODC induction andtumor promotion were more evident in multiple application experimentsthan with a single application of the drug.     

Palmitoylcarnitine reverses 12-O-tetradecanoylphorbol-13-acetate-induced refractory state for the TPA-caused ornithine decarboxylase induction in mouse epidermis

When a single topical application of 12-O-tetradecanoylphorbol-13-acetate(TPA) was performed 12 h before the second application, ornithinedecarboxylase (ODC) induction by the second application of TPAwas markedly suppressed (refractory state). However, at intervalsof 96 h between the first and the second application, the ODCactivity induced by the second application of TPA was higher(enhanced state) than the activity induced by the single application.When various antitumor promoting agents, i.e. p-bromophenacylbromide, nordihydroguaiaretic acid, quercetin, 1-tosylamide-2-phenylethylchloromethyl ketone, retinoic acid and palmitoylcarnitine, wereapplied concurrently with the first TPA application, the ODCinduction in the refractory state was restored only by palmitoylcarnitine,but not by other anti-tumor promoting agents. None of theseanti-tumor promoting agents affected the ODC induction in theenhanced state. Stearoylcarnitine also had the restorative effectbut was less effective than palmitoylcarnitine. Acetylcarnitineand palmitic acid were not effective. Pretreatment of mice withTPA 12 h or 96 h before the second TPA application resultedin the reduction or the increase in the V_max values of ODC bothfor ornithine and pyridoxal-5'-phosphate, respectively. Palmitoylcarnitinerestored these reduced V_max values to the control values. Twelvehours after TPA treatment, the epidermal protein kinase C activityof both cytosol and particulate fractions decreased moderately.At 96 h after TPA application, protein kinase C activities ofboth cytosol and particulate fractions were fully or at leastpartially restored to the control levels. Protein kinase C activitiesboth in the cytosol and the particulate fractions tended tobe restored by palmitoylcarnitine, but the effect was not alwaysreproducible. The TPA-induced refractory state and the enhancedstate for ODC induction appear to result from the changes inthe protein kinase C activities caused by TPA. However, it isnot known whether such changes in the protein kinase C activitiesare the major causes for the TPA-induced refractory and/or enhancedstate for ODC induction and whether or not the restorative effectof palmitoylcarnitine is due to its modulating action on proteinkinase C activity.     

TCDD-inducible plasminogen activator inhibitor type 2 (PAI-2) in human hepatocytes, HepG2 and monocytic U937 cells

Induction of PAI-2 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)has been studied in human primary hepatocytes, hepatoma HepG2cells and monocytic U937 cells, extending recent findings inhuman keratinocytes. PAI-2 represents a serpine-type proteaseinhibitor with wide-ranging implications in fibrinolysis, extracellularmatrix proteolysis, growth factor activation and carcinogenesis.PAI-2 was induced by >10^–9 M TCDD in hepatocytes andHepG2 cells and by >10^–10 M TCDD in U937 cells. Inthe latter cell line, PAI-2 induction by TCDD and by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been compared. TCDD appeared tobe less efficient than TPA as an inducer of PAI-2. In contrastto induction by TPA, PAI-2 induction by TCDD was found to bebiphasic, with an early peak of mRNA at 1–3 h and a latepeak at 12–24 h. A biphasic response was also seen atthe protein level although production of PAI-2 protein laggedbehind the corresponding mRNA. PAI-2 is known to contain AP-1sites, i.e. Jun/Fos protein-binding sites, in its promotor region.Hence, PAI-2 induction by TCDD has originally been conceivedto be due to an indirect response, secondary to the inductionof Jun/Fos proteins. Therefore, expression of jun/fos genesand their AP-1 activity were studied at the early phase of PAI-2induction by TCDD. TCDD did not increase mRNA of c-fos, c-jun,junB or junD (in contrast to TPA which markedly increased theexpression of c-fos and junB), nor did TCDD increase AP-1 activity.In conclusion, the findings suggest that PAI-2 induction byTCDD is not restricted to human keratinocytes but includes livercells and monocytic U937 cells. The induction mechanism is complexbut the early phase does not appear to involve Jun/Fos proteins.     

Ornithine decarboxylase induction by chemically complex liquids from two solvent refined coal processes

Studies with a variety of chemically purified substances havesuggested that induction of the enzyme ornithine decarboxylase(ODC) in mouse epiderinal cells may be a reliable indicatorof neoplastic transformation. In an effort to extend these observationson ODC to chemically complex materials, we examined ODC inductionby carcinogenic and non-carcinogenic mixtures and compared theseresults with tumorigenicity data for these materials. For thesestudies several boiling range fractions and several solvent-derivedsubfractions from two solvent-refined coal processes (SRC-Iand SRC-II) were evaluated for their ability to induce ODC.Single applications of heavy distillate (HD), the SRC-II high-boilingfraction and a potent mouse skin carcinogen, produced ODC inductionkinetics which were similar to that for 12-O-tetradecanoylphorbol-13-acetate(TPA). Both HD and TPA stimulated maximal ODC activity 3–5h after application, with epidermal ODC levels returning tobasal levels within 12 h. The magnitude of ODC induction aftermultiple applications of HD was not as great as that observedfor TPA. Single skin applications of TPA and HD also transientlyelevated hepatic ODC levels 27- and 7-fold, respectively; however,liver ODC activity did not increase following multiple applicationsof either chemical. Further, ODC induction by HD was also dose-dependent.Relative to controls, single applications of HD and processsolvent (boiling range >250°C) elevated ODC levels 145-to 205-fold, light distillate and light oil (boiling range <180°C)increased ODC levels 23- to 32-fold, and middle distillate andwash solvent (boiling range 180– 250°C) stimulated<2- to 8-fold increases in ODC. Single applications of threesolvent-derived subtractions of HD, which are complete carcinogens,induced 3- to 7-fold ODC elevations over background levels;multiple applications of two of these subtractions elevatedODC levels 10- to 22-fold. Of the complex mixtures evaluatedduring this study, all complete carcinogens induced ODC; however,the magnitude and temporal pattern of induction vaned with thematerial tested.     

sn-1,2-Didecanoylglycerol effectively induces epidermal ornithine decarboxylase but only weak hyperplasia in mouse skin

The abilities of sn-1,2-didecanoylglycerol (sn-1,2-DDG) to induceepidermal ornithinedecarboxylase (ODC) activity and epidermalhyperplasia were tested using SENCAR, DBA/2 and C57BL/6 mice.Following a single application of 5000 nmol of sn-1,2-DDG, ODCactivity reached a maximum at 4 h after treatment with a peakactivity of 6.03 nmol CO₂/6mg protein/60 min in C57BL/6, 1.50in SENCAR and 0.73 in DBA/2, respectively. The time course andmagnitude for induction of ODC activity after multiple treatmentswas very similar to that after a single application in thesethree mouse lines. Interestingly, the induced ODC activityinC57BL/6 was always higher than that in SENCAR and DBA/2 mouseepidermis regardless of the treatment protocol. Induction ofhyperplasia and dark basal keratinocytes (DCs) and changes inthe labeling index (LI) of basal keratinocytes in DBA/2 andC57BL/6 mice following treatment with sn-1,2-DDG were investigated.Multiple treatments (twice weekly for 2 weeks) of 5000 nmolsn-1,2-DDG did not induce substantial increases inepidermalthickness or DCs 24 or 48 h after the last treatment. In contrast,TPA induced a marked increase in epidermal thickness in DBA/2rather than CS7BL/6 and a considerably higher induction of DCsin DBA/2 (37.3 ± 2.2%) than in CS7BL/6 (9.6 ±2.5%) 48 h after the last treatment. The LIs after topical applicationof sn-1,2-DDG were elevated at 24 h, but returned to basal levelsby 48 h in both strains, whereas TPA treatment significantlyelevated the LI In both strains at 48 h after the last application.In addition, the effects of various doses and frequencies ofapplication of sn-1,2-DDG were investigated using SENCAR mice.Highdoses (20 000 nmol) or more frequent applications (5000nmol once daily for 7 days) of sn-1,2-DDG still produced onlyweak hyperplasia. These results suggest that the induction ofepidermal ODC activity can be dissociated from the inductionof epidermal hyperplasia and may provide an explanation forthe lack of complete promoting activity presently observed withmembrane permeable diacylglycerol derivatives.     

Tumor promoter-induced refractory state against ornithine decarboxylase induction by 12-O-tetradecanoylphorbol-13-acetate in mouse epidermis

More than one application of the potent tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), to mouse skin at intervals of more than 48 h led to a larger induction of ornithine decarboxylase (EC 4.1.1.17; ODC) than did a single application. In contrast, at intervals of less than 24 h, the first application of TPA appeared to induce a refractory state; the second application of TPA did not induce ODC. The extent of the inhibitory effect caused by the first application of TPA was dependent on the dose. The abilities of a series of phorbol esters to induce the refractory state correlated with their promoting abilities. However, both mezerein and ethylphenylpropiolate, potent hyperplastic agents with little or no promoting properties, induced the refractory state. On the other hand, pretreatment with TPA caused a refractory effect on ODC induction by mezerein but potentiated ODC induction by ethylphenylpropiolate. The epidermal cells escaped from the refractory state by repeated application of TPA at intervals of 24 h as well as at intervals of twice a week; that is, there was a full induction of ODC activity following a second application within 24 h of a prior application. TPA did not elicit production of detectable ODC-antizyme activity in mouse epidermis. Mixing of a soluble extract from mouse epidermis in the refractory state with that from TPA-stimulated epidermis gave essentially additive ODC activity. Elimination of ODC induction by topical application of retinoic acid or injection of cycloheximide concurrent with the first application of TPA did not restore the ability of a second application of TPA to induce ODC. These results suggest that the refractory effect on ODC induction by TPA does not result from feedback regulation of ODC.     

Acute effects of 12-Otetradecanoylphorbol-13-acetate, teleocidin B, or 2,3,7,8-tetrachlorodibenzo-p-dioxin on cultured normal human bronchial epithelial cells

Effects of teleoddin B, 12-O-tetradecanoylphorboi-13-acetate(TPA), phorbol, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and2,7-dichlorodibenzo-p-dioxin (DCDD) on normal human bronchialepithelial cell cultures were assessed by quantitation of cellularmorphology, clonal growth (population doublings per day), cross-linkedenvelope (CLE) formation and the enzymatic activities of arylhydrocarbon hydroxylase (AHH), ornithine decarboxylase (ODC)and plasminogen activator (PA). Toxicity was assessed by clonalgrowth assays. Teleocidin B and TPA had similar effects on growth,morphology and enzyme activities. When the cells were incubatedwith TPA or teleocidin B at concentrations of 1–100 nMfor 6 h, RNA synthesis was unaffected, but DNA synthesis decreasedand squamous differentiation, marked by an increase in cellsurface area and cross-linked envelope formation, was increased.TPA and teleocidin B also increased ODC activity in LHC-0 medium(a maintenance medium without epidermal growth factor) but causeda decrease of ODC activity in LHC-4 (a growth medium containingepidermal growth factor). Finally, TPA and teleocidin B eachcaused an increase of PA and a decrease of AHH activities inboth media. Phorbol, a non-promoting analogue of TPA, had noeffect on growth, morphology or biochemical assays. TCDD (100nM) caused a 15% decrease in cell growth when cells were incubatedin LHC-4, and this was accompanied by an increase in cell surfacearea, PA activity, and CLE formation. TCDD caused an increasein AHH and ODC activities when the cells were incubated in eitherLHC-0 or LHC-4 medium. DCDD did not alter cell growth, and itsmorphological and biochemical effects were similar to thoseof TCDD although less marked. In conclusion, results reportedhere are consistent with the hypothesis that an important propertyof some tumor promoters is their ability to induce terminaldifferentiation in normal, non-initiated epithelial cells.     

Inhibition by indomethacin and 5,8,11,14-eicosatetraynoic acid of the induction of rat hepatic ornithine decarboxylase by the tumor promoters phenobarbital and 12-O-tetradecanoylphorbol-13-acetate in vivo

The involvement of arachidonate metabolism in the inductionof rat hepatic ornithine decarboxylase (ODC) activity by thetumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) andphenobarbital (PB) was investigated. Pretreatment of the ratswith indomethacin or 5,8,11,14-eicosateraynoic acid dose dependentlyinhibited the induction of ODC by both tumor promoters. Bothinhibitors were more potent inhibitors of PB induction thanTPA induction of ODC. The data are consistent with an involvementof arachidonate cyclooxygenase products in the induction ofrat hepatic ODC by the tumor promoters.     

Inhibition of tumor promotion in benzo[a]pyrene-initiated CD-1 mouse skin by crocetin

The effects of topical application of crocetin on 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced promotion of skin tumors, hyperplasia, hydrogenperoxide, ornithine decarboxylase (ODC) and inflammation wereevaluated in female CD-1 mice. Topical application of crocetin(0.2 or 1.0 µmol) with TPA (15 nmol) twice weekly for20 weeks to mice previously initiated with benzo[     

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.     

Cytogenetic effects caused by phorbol ester tumor promoters in HeLa cells: mechanistic aspects

The effect of the convertogenic (‘first-stage’)tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) andthe nonconvertogenic (‘second-stage’) tumor promoterRPA (12-O-retinoylphorbol-13-acetate) on chromosomes was investigatedin HeLa cells which have previously been shown to exhibit aradiomimetic response to TPA. As in the case of mouse keratinocytes,only TPA had a significant clastogenic activity at non-cytotoxicconcentrations ranging from 10^–8 to 10^–6 M measuredafter 24 and 48 h. The values observed with RPA did not differsignificantly from values observed in the presence of the solvent(acetone, 0.2%). The response to TPA was saturable with respectto the dose of TPA. The chromosomal aberrations (mostly gapsand breaks) were predominantly of the chromatid type. Isochromatidaberrations were caused by a 24 or 48 h treatment with 10^–6M TPA. The aberrations appear as early as 6–8 h afterTPA application, i.e. as soon as the cells have recovered fromTPA-induced inhibition in G₂-phase. Even a 30 min exposure to10^–7 M TPA gives the same yield of aberrations as longertreatment, i.e. the response to TPA is ‘saturable’with respect to time. Both TPA and the non-clastogenic RPA causea temporal G₂-delay thus indicating that the G₂-inhibition isnot related to induction of chromosomal aberrations by TPA.The data are consistent with the hypothesis that TPA induceschromosomal aberrations via a receptor-mediated pathway.     

Inhibitory effects of glutathione level-raising agents and D-{alpha}-tocopherol on ornithine decarboxylase induction and mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

The constituent amino acids of reduced glutathione (GSH), GSHitself, and D--tocopherol inhibited 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced ornithine decarboxylase (ODC, L-omithine carboxy-lyase,EC 4.1.1.17 [EC] ) activity in mouse epidermis in vivo and in vitro.The inhibitory effects of cysteine (Cys), GSH and D--tocopherolon ODC induction were proportional to their abilities to decreasethe incidence of skin tumors in the initiation-promotion protocol.Moreover, the ability of the constituent amino acids of GSHand GSH to inhibit TPA-induced ODC activity correlated wellwith their ability to increase the ratio of GSW/oxidized glutathione(GSSG) in isolated epidermal cells. In vitro, various treatmentswith 1 mM GSH, 1 mM glutamic acid (Glu), 1 mM glycine (Gly),0.4 mM Cys and/or 0.2 mM cystine (CysCys) inhibited dramaticallythe sharp decline in the intracellular ratio of GSH/GSSG causedby 0.1 µM TPA. Since the inhibitory effects of Cys onboth the decrease in the ratio of GSH/GSSG and the inductionof ODC activity by TPA were greatly reduced by the inhibitorsof -glutamyl transpeptidase and -glutamylcysteine synthetase,it is suggested that some of the inhibitory effects of Glu,Cys and Gly on tumor promotion could result from their interferencewith the metabolism of the tripeptide GSH, a natural antioxidantwhich inhibits chemical carcinogenesis. The free radical scavengerD--tocopherol, which did not alter directly the intracellularratio of GSH/GSSG, also prevented completely the decrease inthe ratio of GSH/GSSG caused by TPA. These results, therefore,suggest that GSH level-raising agents and other antioxidantsmight inhibit by diverse means the effects of TPA on GSH metabolismand skin tumor promotion.     

Tumor promoting phorbol-ester derivatives increase ornithine decarboxylase activity and polyamine biosynthesis in the liver of the rat and mouse

The ability of the phorbol-ester tumor promoters to alter ornithinedecarboxylase (ODC) activity in the liver of the rat and mousewas determined. The injection of 12-O-tetradecanoyl phorbol-13-acetate(TPA, 100 µg, i.p.) led to a 250-fold increase in hepaticODC activity within 4 h of administration. This increase inODC activity required both RNA and protein synthesis and didnot occur when a variety of the non-tumor promoting phorbol-esterderivatives were administered to the rat. A distinct dose-dependentincrease in hepatic ODC activity could be observed at 4 h followingthe injection of increasing amounts of TPA (0–100 µg,i.p.). As little as 1.0 µg TPA (i.p.) administered toa rat resulted in a significant stimulation in the activityof ODC in the liver compared to the control unstimulated values.Both 200 µg and 500 µg TPA produced less of an elevationin hepatic ODC activity than did the optimal dose of 100 µg.In the mouse, the administration of 1 µg and 20 µgof TPA (i.p.) both led to a marked increase in hepatic ODC activityat 7 h and 4 h, respectively, following injection. A 4–5-foldincrease in putrescine levels occurred in the rat liver in abiphasic manner between 4–8 h and 16–24 h followingthe injection of TPA (100 µg). No alterations in eitherspermidine or spernine were observed during this period. Theadministration of 100 µg of TPA to the rat did not alterthe incorporation of [3H]thymidine into DNA in the liver comparedto untreated control animals. Under these identical conditionspartial hepatectomy led to a large increase in DNA synthesis.