Effects of lipoxygenase and cyclooxygenase inhibitors on the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate and isoproterenol in mouse tissues in vivo

I.p. administration of 12-O-tetradecanoylphorbol-13-acetate(TPA) (400 µg/kg) caused a remarkable increase in ornithinedecarboxylase (ODC) activity in CD-1 mouse liver (8.3-fold),spleen (17.8-fold), kidney (4-fold), lung (7.7-fold) and brain(2.7-fold). TPA induced an increase in ODC activity in liver,spleen and kidney in a dose-dependent manner (100–800µg/kg). The putrescine contents of these tissues werealso increased by TPA injection. BW755C, an inhibitor of cyclooxygenaseand lipoxygenase, prevented the TPA-induced increase in ODCactivity in liver, spleen and kidney in a dose-dependent manner.AA861-a selective lipoxygenase inhibitor, also showed the inhibitionof TPA-induced increase in ODC activity in these tissues. Significantinhibition was observed either by BW755C or AA861 at the doseof 30 mg/kg. On the other hand, indomethacin, a selective cyclooxygenaseinhibitor, enhanced the TPA-induced increase in ODC activityin these tissues dose-dependently. Significant enhancement wasobserved at 3 mg/kg for liver and spleen and 1 mg/kg for kidney.The subcutaneous administration of isoproterenol (1 mg/kg) causedan increase in ODC activity in both liver (11-fold) and spleen(3.4-fold). Both AA861 and BW755C failed to inhibit the isoproterenol-inducedincrease in ODC activity in these tissues. These results indicatethat product(s) of lipoxygenase pathway play an important rolein ODC induction caused by TPA in liver, spleen and kidney,while the lipoxygenase pathway does not play an essential rolein the isoproterenol-induced increase in ODC activity.     

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.     

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.     

Increased endogenous N-nitrosamine and nitrate formation by induction of nitric oxide synthase in rats with acute hepatic injury caused by Propionibacterium acnes and lipopolysaccharide administraction

In rats treated i.v. with heat-killed Propionibacterium acnes(100 mg/kg body wt), followed 5 days later by an i.v. dose ofEscherichia coli lipopolysaccharide (LPS, 1 mg/kg body wt),acute hepatic cell necrosis was accompanied by significant inductionof nitric oxide (NO) synthase activity in the liver. Endogenousnitrosation of thiazolidine 4-carboxylic acid (TCA, 50 µmol/rat)administered by three different routes (i.v., i.p. and p.o.)5 h after LPS injection to the P.acnes-treated rats was assessedby analysing its nitrosated product (NTCA) excreted in 24 hurine. The amounts of NTCA formed in vivo after i.v., i.p. andp.o. administration of TCA were 4.07 ± 1.00, 5.79 ±2.15 and 58.3 ± 20.7 nmol/rat (n = 5–10) respectively,which were about 5-, 10- and 8-fold greater than those excretedby rats which had not been treated with P.acnes and LPS butreceived TCA by the same route. Nitrate concentration in plasmaand NO synthase activity in the liver started to increase within2.5 h after LPS injection, reached a maximum at 7.5 h and remainedat high levels for serveral further hours. Levels of nitriteand nitrate in gastric contents were also increased significantlyafter LPS administration. The co-administration of N     

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.     

Effect of polycyclic aromatic compounds and phorbol esters on ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in mouse liver

Single i.p. injections of 3-methylcholanthrene (MC; 50 mg/kg) administered to inbred C57BL/6 mice or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 micrograms/kg) to DBA/2 mice gave an increase in the hepatic activities of ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) with peaks occurring by 12 and 48 hr, respectively. A single i.p. dose of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 micrograms/kg) enhanced the activity of ODC about 70-fold within 12 hr in C57BL/6 mice and 18-fold within 24 hr in DBA/2 mice without affecting AHH activity markedly. 4-O-Methyl-12-O-tetradecanoylphorbol-13-acetate (100 micrograms/kg) raised ODC activity to about 25% of the TPA-treated value in C57BL/6 mice; in DBA/2 mice, TPA and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate induced ODC activity to roughly the same level. Benzo(e)pyrene (50 mg/kg) failed to affect ODC and AHH activities significantly in either strain. The inducing effect of TPA on ODC activity was potentiated by a simultaneous administration of MC to C57BL/6 mice; combined TPA and TCDD to DBA/2 mice exerted an additive effect on hepatic ODC activity. Difluoromethylornithine administered i.p. effectively inhibited the induction of ODC activity elicited by TPA, MC, or TCDD either alone or in various combinations but did not interfere with AHH induction. These data indicate that different regulatory factors are involved in the ODC induction process elicited by TPA and polycyclic aromatic compounds and that MC and TCDD may induce ODC activity by different mechanisms. The results also confirm our earlier findings in rat skin and cells in culture which suggest that the ODC and AHH induction processes can occur independently of each other. Additionally, there is a strain-related difference in sensitivity with regard to ODC-inducing activity of TPA in the livers of C57BL/6 and DBA/2 mice.     

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.     

Comparison of enhancement of GGTase-positive foci and induction of ornithine decarboxylase in rat liver by barbiturates

The induction of ornithine decarboxylase (ODC) by barbituratesand the ability of barbiturates to enhance neoplastic progressionof chemically initiated cancer was examined in rat liver. Allseven barbiturates induced ODC with barbital (7.7 fold increase)and phenobarbital (5.7 fold increase) demonstrating the mostpotent activity. Maximum induction of ODC by phenobarbital wasobtained in 18 h. Barbital (500–5000 p.p.m.) and phenobarbital(500 p.p.m.) administered in the drinking water enhanced theappearance of diethylnitrosamine (DENA)-initiated -glutamyltranspeptidase(GGTase)-positive foci. Amobarbital, hexabarbital and pentabarbitaldid not enhance the appearance of GGTase-positive foci. In theabsence of previous initiation by DENA, the enhancing regimenof the barbiturates did not cause the appearance of GGTase-positivefoci. Barbiturates induced ODC activity in rat liver and enhancedthe incidence of DENA initiated GGTase-positive foci.     

Elevated levels of ornithine decarboxylase and polyamines in the kidneys of estradiol-treated male hamsters

The effects of 17 ß-estradiol (E₂) on the levels ofornithlne decarboxytase (ODC), S-adenosylmethionine decarboxylase(SAMDC), and of the polyamines putrescine, spermidine and sperminein the kidneys of castrated male hamsters were determined. Thei.p. injection of E₂ into male hamsters led to renal ODC levelsthree times above the control levels 6–12 h after treatment.Similarly, the renal ODC levels in hamsters treated with chronicdoses of E₂ for 60–180 days were 13–1.9 times thecorresponding enzyme levels in control, sham-operated animals.With a series of estrogen analogues, there was a direct correlationbetween the rise in renal ODC in vitro and the binding to renalestradiol receptor sites in vitro. The hamster kidney levelsof the polyamines putrescine, spermidine and spermine all declinedduring the 180-day experimental period. A single i.p. injectionof E led to a 70% increase in renal SAMDC activity 12 h aftertreatment. However, the administration of E for 180–270days was without effect on the normal age-dependent declinein SAMDC levels noted in kidneys. These results indicate that,like other carcinogens and promoters, E₂ increases the levelsof ODC and of polyamines in its target tissue and that the risein ODC is mediated by a specific estradiol-binding protein.     

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.     

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.     

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.     

Relation between induction of rat hepatic ornithine decarboxylase activity by tumor promoters 12-O-tetradecanoylphorbol-13-acetate and phenobarbital and levels of the polyamines putrescine, spermidine and spermine, in vivo; differential effects of retinyl-acetate

A single i.p. injection of 12-O-tetradecanoylphorbol-13-acetate(TPA) induced a transient increase in the levels of rat liverputrescine, spermidine and spermine. These polyamine concentrationscontinuously increased until 6 h, immediately following administrationof the tumor promoter. Phenobarbital (PB) induced an increaseof the putrescine and spermidine concentrations during the 8h post administration studied, while spermine reached a plateauafter 4 h. When retinyl-acetate (RA) was injected one hour priorto TPA, the increases of the polyamines were considerably inhibited.This treatment with RA partially inhibited further increaseof putrescine and spermine by PB. These findings are discussedin respect to the concomitant ornithine decarboxylase (ODC)activities, we have previously reported. I.p. injection of RAalone caused an elevation of ODC activity as well as putrescineand spermidine concentration within 2 h of exposure.     

Tamoxifen: evidence by 32P-postlabeling and use of metabolic inhibitors for two distinct pathways leading to mouse hepatic DNA adduct formation and identification of 4-hydroxytamoxifen as a proximate metabolite

Exposure to pentachlorophenol (PCP) strongly intensifies theformation of mouse hepatic DNA adducts elicited by oral administrationof tamoxifen (TAM), as previously shown by ³²P-postlabeling.To explain this effect, PCP was proposed to interfere with thedetoxication by sulfate conjugation of an as yet unidentifiedhydroxylated proximate TAM metabolite. A comparison of the presentand earlier results shows that the hepatic TAM adduct patternin female ICR mice depended on the route of administration ofTAM (120 µmol/kg), with oral administration primarilyeliciting formation of more polar adducts (termed group I adducts),while after i.p. administration less polar adducts (group II)predominated over group I adducts by a factor of 17.5. All theseadducts were also formed in female Sprague–Dawley ratsafter i.p. dosing with TAM, but total adduct levels were 3.5-to 5-fold higher than in mice. After four daily i.p. treatments,TAM adducts accumulated in mouse liver DNA in a non-linear fashion.Adduct levels were 30–50 times lower in mouse kidney andlung than in liver. The phenolic metabolite 4-hydroxy TAM (120µimol/kg) exclusively led to formation of polar (groupI) hepatic adducts, and this process was stimulated 8-fold bycoadministration of PCP (75 µimol/kg). Co-administrationof PCP with the parent compound led to an 11-fold enhancementof group I adduct formation; simultaneously, levels of groupII adducts were suppressed 6-fold. Another inhibitor of sulfateconjugation, 2,6-dichloro-4-nitrophenol, unlike PCP, had noeffect on group I adducts, but it reduced group II adduct formation2.2-fold. The PCP metabolite 2,3,5,6-tetrachlorohydroquinone(75 µimol/kg) did not significantly affect any major TAMadduct, suggesting that PCP itself was the active compound.Similar to group II TAM adducts, the formation of hepatic safrole–DNAadducts was inhibited in female ICR mice by both sulfotransferaseinhibitors, consistent with the proposal that metabolic     

SHORT COMMUNICATION: Partial hepatectomy of rats 3 weeks before or simultaneously with 2-aminofluorene injection can affect the amounts of adducts induced in hepatic DNA

The influence of partial hepatectomy on the level of 2-amino-fluorene(2-AF) induced DNA adducts in rat liver was studied. We foundthat partial hepatectomy performed either 3 weeks before orsimultaneously with the injection of 2-AF affected the amountsof adducts in rat hepatic DNA compared to controls. The levelof DNA adducts in rats that were treated with 2-AF and simultaneouslyhepatectomized was higher (19.9 fmol/µg DNA) than in non-hepatectomizedones (14.4 fmol/µg DNA) when measured 48 h after 2-AFadministration. In rats treated with the carcinogen 3 weeksafter hepatectomy the level of DNA adducts was significantlyhigher than in non-hepatectomized rats when measured 15 daysafter the injection of 2-AF (10.9 fmol/µg DNA and 5.9fmol/µg DNA respectively). The high level of DNA adductsin that group of hepatectomized animals was correlated witha relatively lower rate of 2-AF-induced radioactive thymidineincorporation into hepatic DNA (in comparison to non-hepatectomizedrats).     

DNA damage produced by N-nitrosomethyl(2-oxopropyl)amine (MOP) in hamster and rat pancreas: a role for the liver

Utilizing the technique of alkaline elution analysis, the abilityof N-nitrosomethyl(2-oxopropyl)amine (MOP), a potent pancreaticcarcinogen, to damage pancreatic DNA in rats and hamsters wasexamined. Pancreatic DNA isolated from hamsters exposed for1 h to MOP given i.p. at doses of 7–60 mg/kg showed dose-relatedDNA damage. A similar dose-response was observed in the pancreasof rats receiving 20 — 180 mg MOP/kg, suggesting thathamsters were 2–3 times more sensitive than rats. In contrastto the results obtained in vivo, functionally viable acinarcells from both rat and hamster pancreas, when exposed in vitroto levels of MOP comparable to those in vivo (20–180 µ/ml),failed to show dose-related DNA damage. Acinar cells from hamsterspretreated with 5, 6-benzoflavone, an inducer of cytochromeP-450 activity, showed greatly enhanced drug-metabolizing capability,but again no DNA damage was observed upon exposure to MOP. Mincedhamster or rat pancreas also failed to show DNA damage in responseto MOP treatment. When hamsters in which hepatic blood supplywas interrupted by ligation were given 60 mg/kg MOP i.v. andsacrificed 15 min later, damage to pancreatic and liver DNAwas comparable to that observed in ligated controls which hadreceived saline only. Administration of MOP to sham-operatedanimals led to extensive DNA damage in both pancreas and liverat 15 min. Analysis by h.p.l.c. showed an almost Mold increasein the amount of MOP present in the pancreases of the liver-ligatedanimals as compared to the sham-operated unligated animals.MOP was absent from the liver of the ligated animals. Theseexperiments strongly suggest that DNA damage by MOP to the pancreaticacinar cells and probably to other pancreatic cell types, aswell, requires metabolic activation by the liver.     

Distribution of catalase and its modulation by 12-O-tetradecanoylphorbol-13-acetate in murine dermis and subpopulations of keratinocytes differing in their stages of differentiation

Topical treatment of female SENCAR mice with 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced both dermal and epidermal catalase-specificactivities 38% and 51% within 6 h and 18 h of promoter application,respectively. Dermal catalase activity recovered to controllevels within 72 h of treatment whereas epidermal catalase activityremained suppressed. Activity measurements were also made infour subpopulations of keratinocytes prepared by Percoll gradientcentrifugation that differed in their stages of differentiation.Catalase-specific activity increased with keratinocyte maturityand ranged from 45–54 U /mg protein for basal cell preparationsto 252 U /mg protein for granular-squamous cell preparations.Pretreatment of the epidermis for 16 –18 h with TPA (2µg) uniformly reduced catalase-specific activity 46 –52% in all keratinocyte subpopulations prepared by Percoll gradientcentrifugation. Similarly, plots of catalase units per cellversus extracted protein per cell suggested 55 –60 % decreasesin catalase activity in basal and spinous cell keratinocytesof TPA treated epidermis. Furthermore, catalase-specific activityin homogenates of whole epidermis (144 –182 units /mgprotein) was most similar to the activity of the granular/squamouskeratinocyte subpopulation. Collectively, these studies suggestthat: (i) TPA reduces the capacity for H₂O₂ detoxification bycatalase throughout the epidermis; and (ii) activity measurementson unfractionated epidermal preparations may not be representativeof the basal cell keratinocyte population.     

Dissociation between induction of ornithine decarboxylase and oxidative burst by phorbol esters in a macrophage cell line

In order to investigate the correlation between stimulationof superoxide generation and induction of ornithine decarboxylase(ODC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) we haveused the macrophage cell line J774.16 and a clone derived fromthis line that, by contrast with the parental line, is unableto generate superoxides in response to TPA. No difference wasobserved between the normal and the defective cells, with respectto ODC induction by TPA over a wide range of TPA concentrations(0.2–5.0 µg/mI). Similar results were obtained comparingresident and caseinate-elicited mouse peritoneal macrophages.Although resident macrophages did not generate superoxides inresponse to TPA, they did not differ from superoxide-generating,caseinate-elicited macrophages with respect to ODC induction.These data suggest a dissociation between the stimulation ofthe oxidative burst by TPA and a growth factor-like effect suchas ODC induction.