Effect of 1,8-dihydroxy-9-anthrone (anthralin) on rat hepatic ornithine decarboxylase activity in vivo

Intraperitoneal injection of the non-phorbol tumor promoter anthralin (1,8-dihydroxy-9-anthrone) in male rats resulted in an increase of hepatic ornithine decarboxylase (ODC) activity. Maximal activity was observed 8 h after promoter administration reaching levels about 30 times over control. The kinetics of anthralin dependent ODC induction differed markedly from that by either 12-O-tetradecanoylphorbol-13-acetate (TPA) or phenobarbital (PB) (Bisschop et al., Carcinogenesis 2 (1981) 1282). With anthralin a slow decrease of ODC back to control level is observed approximately within 22 h. In contrast, ODC induction mediated by other tumor promoters like TPA and PB decreased to control levels within 4-6 hours. Administration of a second dose of anthralin 8 h after the first dose prevented the activity decrease as normally observed after a single dose of a tumor promoter. This effect lasted at least 10 h. ODC activity induction occurred in a dose-dependent manner being linear from 10-2000 micrograms anthralin/kg body wt. Pretreatment of the animals either with actinomycin D or with cycloheximide completely blocked anthralin mediated ODC induction suggesting that de novo ODC-mRNA synthesis and subsequent translation is involved in this process.     

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.     

Inhibition of intercellular communication in rat hepatocytes by phenobarbital, 1, 1, 1-trichloro-2, 2-bis(p-chlorophenyl)ethane (DDT) and {gamma}-hexachlorocyclohexane (lindane): modification by antioxidants and inhibitors of cyclo-oxygenase

Several tumour promoting chemicals have been shown to inhibitintercellular communication (IC) through gap junctions in cellcultures. In the present investigation we studied the effectof the hepatic tumour promoters pheno-barbital (PB), 1, 1, 1-trichloro-2,2-(p-chlorophenyl)ethane (DDT) and     

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.     

致(促)癌物对大鼠肝、腺胃和直肠鸟氨酸脱羧酶的诱导

用大鼠作体内急性实验观察各种致(促)癌物对肝、腺胃及直肠等组织鸟氨酸脱羧酶(ODC)活性的诱导作用。结果发现:注射亚硝氨类致癌物[二乙基亚硝胺(DENA)及二甲基亚硝胺(DMNA)]24/小时可使肝中ODC活性分别升高12倍和3倍。促癌物苯巴比妥和巴豆油分别诱导4.5小时后,肝中ODC活性分别上升8.5倍和54倍;甲基胆蒽诱导24/小时后,肝中此酶活性上升6倍。分别灌入胃癌致癌物N—甲基—N—硝基—N—亚硝基胍(MNNG)或4—硝基—1—氧化喹啉(4—NQO)24小时后,大鼠腺胃粘膜ODC活性分别升高34倍和47倍。以促癌物脱氧胆酸钠保留灌肠3.5小时后,结肠和直肠粘膜ODC活性可诱导增高38倍。ODC与细胞增生及癌变关系密切,用致(促)癌物对ODC的诱导方法比较特异,可靠和迅速,因此可以考虑作为对肝、腺胃及结肠等消化道致(促)癌物检测筛选的生化指标。     

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.     

The induction of ornithine decarboxylase by phorbol 12-myristate 13-acetate or by serum is inhibited by antioxidants

The mechanism of action of tumor promoters may involve the modulationof gene expression, e.g., the induction of ornithine decarboxyiase(ODC). The tumor promoter phorbol-13-myristate-12-acetate (PMA)induces chromosomal damage via the intermediacy of active oxygenspecies which may trigger the activation of certain genes. Therefore,we have studied the effect of antioxidants on the inductionof ODC by PMA, medium change only and medium change plus PMAin mouse mammary tumor cells Mm5mt/Cl. CuZn-superoxide dismutase(SOD, a scavenger of superoxide radicals), catalase (CAT, ascavenger of hydrogen peroxide) and mannitol (a scavenger ofhydroxyl radicals) suppressed ODC induction under all threeconditions. The relative inhibitory potency of the antioxidantswas always SOD > CAT > mannitol > SOD + CAT. Maximalsuppression by SOD + CAT was {small tilde}50%. It is concludedthat active oxygen species play a role in ODC induction by factorscontained in serum and by PMA.     

Lack of promoting effect of clonazepam on the development of N-nitrosodiethylamine-initiated hepatocellular tumors in mice is correlated with its inability to inhibit cell-to-cell communication in mouse hepatocytes

The tumor-promoting ability of clonazepam (CZP), a widely usedbenzodiazepine anticonvulsant, was investigated in an in vivomouse liver tumor promotion assay and an in vitro mouse hepatocyteintercellular communication assay. The development of preneoplastichepatocellular foci of cellular alteration and hepatocellularneoplasms was studied in male B6C3F1 mice initiated, at 5 weeksof age, with a single i.p. injection of N-nitrosodiethylamine(NDEA; 90 mg/kg body weight) in tricaprylin, followed by administrationof either phenobarbital (PB; 0.05%) or CZP (0.068% or 0.136%)in diet beginning 2 weeks after carcinogen injection and continuingto 60 weeks of age. Several mice from each group were killedafter 9, 21, 33 or 53 weeks on test diet, and portions of liverand other organs were fixed In formalin and examined histotogically.Unlike PB, CZP did not promote the development of preneoplastichepatocellular foci or neoplasms (adenomas and carcinomas) inNDEA-initiated mice. Following limited (2 weeks) dietary exposureat 0.15%, CZP was a potent inducer of hepatic P450IIBl-mediatedalkoxyresorufin O-dealkylase activities. In contrast, the degreeof induction in hepatic tissue from mice fed 0.136% CZP for53 weeks was markedly lower than that in mice fed 0.05% PB for53 weeks. In the in vitro assay, diazepam, a strong tumor promoterin mouse liver, significantly inhibited mouse hepatocyte gapjunctional intercellular communication, while CZP had no significanteffect on this parameter. Thus, CZP, a drug structurally relatedto diazepam, is inactive as a liver tumor promoter in mice.     

Decreased levels of S-adenosylmethionine in the livers of rats fed phenobarbital and DDT

Chronic feeding of the liver tumour promoters phenobarbital(PB) and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT)to male weanling rats at levels of 0.05% in the diet for 1,3and 5 weeks reduced the hepatic S-adenosylmethionine (AdoMet)contents by 50%. Such reductions were inhibited by the simultaneousfeeding of choline chloride and were completely prevented bythe dietary administration of methionine. AdoMet levels >100µg/g liver were noted in rats receiving PB + methioninefor 1 week, DDT + methionine for 1 week or methionine alonefor 5 weeks. These results are consistent with the hypothesisthat the liver tumour promoting activities of PB and DDT mayoccur in part through a hepatic methyl insufficiency.     

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.     

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 skin tumor promoter-caused induction of epidermal ornithine decarboxylase in SENCAR mice by polyphenolic fraction isolated from green tea and its individual epicatechin derivatives.

Green tea, next to water, is the most popular and commonly consumed beverage in the world, especially in eastern countries. In prior studies we have shown that the polyphenolic fraction isolated from green tea (GTP) exerts antigenotoxic effects in various mutagenicity test systems (Mutat. Res., 223: 273-285, 1989) and that its topical application or oral feeding in drinking water protects against polycyclic aromatic hydrocarbon-induced skin tumor initiation and complete carcinogenesis in SENCAR and BALB/c mice [Cancer Lett., 42: 7-12, 1988; Carcinogenesis (Lond.), 10: 411-415, 1989] and UV B radiation-induced photocarcinogenesis in SKH-1 hairless mice [Carcinogenesis (Lond.), 12: 1527-1530, 1991]. In the present study we assessed the effect of skin application of GTP to SENCAR mice on 12-O-tetradecanoylphorbol-13-acetate (TPA) and other skin tumor promoter-caused induction of epidermal ornithine decarboxylase (ODC) activity. Topical application of GTP to mouse skin inhibited TPA-induced epidermal ODC activity in a dose-dependent manner. The inhibitory effect of GTP was also dependent on the time of its application relative to TPA treatment. Maximum inhibitory effect was observed when GTP was applied 30 min prior to topical application of TPA. GTP application to animals also inhibited the induction of epidermal ODC activity caused by several structurally different mouse skin tumor promoters. In order to identify which of the specific epicatechin derivatives present in GTP is responsible for these inhibitory effects, they were isolated from GTP and evaluated for their inhibitory effects against TPA-caused induction of epidermal ODC activity. Among these, (-)epigallocatechin-3-gallate (EGCG), which was the major constituent present in GTP by weight, exerted the maximum inhibition. EGCG also showed greater inhibitory effects against TPA-caused induction of epidermal ODC activity when compared with several other naturally occurring polyphenols. The results of this study suggest that GTP, specifically its epicatechin derivative EGCG, could provide anti-tumor-promoting effects against a wide spectrum of skin tumor promoters.     

Inhibition of tumor promoter-induced ornithine decarboxylase activity by tannic acid and other polyphenols in mouse epidermis in vivo

Naturally occurring plant phenols with antimutagenic and anticarcinogenic activities were tested for their abilities to inhibit the ornithine decarboxylase (ODC) response linked to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical applications of tannic acid (TA) inhibit remarkably and in a dose-dependent manner TPA-induced ODC activity in mouse epidermis in vivo. This inhibitory effect of TA is dependent on the time of its administration relative to TPA. The induction of epidermal ODC activity by 8.5 nmol of TPA is inhibited maximally when 20 mumol of TA are applied topically to the skin 20 min before the tumor promoter. Gallic acid and several of its derivatives inhibit the ODC response to TPA to a lesser degree than TA. Ellagic acid is the least effective inhibitor tested. TA also inhibits the ODC-inducing activities of several structurally different tumor promoters and the greater ODC responses produced by repeated TPA treatments. The ability of TA to inhibit by 85% the ODC marker of skin tumor promotion suggests that TA and other polyphenols may be effective not only against tumor initiation and complete carcinogenesis but also against the promotion phase of tumorigenesis.     

Structure-activity relationships for epidermal ornithine decarboxylase induction and skin tumor promotion by anthrones

The present study was designed to compare the skin tumor promotingand epidermal ornithine decarboxylase (ODC) inducing activitiesof various structural analogs of anthralin (1, 8-dihydroxy-9-anthrone)and chrysarobin (1, 8-dihydroxy-3- methyl-9-anthrone). Groupsof 30 SENCAR mice each were initiated with 7, 12-dimethylbenz[a]anthraceneand 2 weeks later promoted with once- or twice-weekly applicationsof various doses of these anthrone derivatives. Carbon-10 (C₁₀)-acylderivatives of anthralin were active skin tumor promoters inthe range of 25–440 nmol per mouse. 10-Acetylanthralinwas significantly more active than 10-myristoyl-anthralin atlow doses (e.g. 25 and 50 nmol per mouse) and nearly as potentas the unsubstituted compound. Higher doses ( 100 nmol per mouse)of this derivative were toxic, hence, reducing the final papillomaresponse. On a relative activity scale where anthralin is 1.0,these derivatives had activities that were 0.7 and 0.2, respectively.10, 10-Dipropylanthralin was totally inactive at the doses tested.C⁶-Substituted derivatives of chrysarobin demonstrated diversetumor promoting activities when tested in the range of 25–440nmol per mouse. On a relative activity scale where chrysarobinis 1.0, 6-methoxychrysarobin (physcion anthrone) was 0.9, whereas6-hydroxychrysarobin (emodin anthrone) had no activity. Chrysophanicacid (1, 8-dihydroxy-3-methyl-9, 10-anthraquinone) was alsoinactive as a tumor promoter at the doses tested. In general,the tumor promoting activities of these anthrone derivativescorrelated very well with their ability to induce epidermalODC after a single topical application indicating an importantrole for this enzyme in skin tumor promotion by anthones. Theability of C₁₀-substituted derivatives of anthralin to undergobase catalyzed oxidation in vitro correlated with both ODC inducingand tumor promoting activities. In addition, copper(II) bis(diisopropylsalicylate)was found to inhibit both ODC induction and skin tumor promotionby chrysarobin. These latter data, when taken together, suggesta role for oxidation at C₁₀ in skin tumor promotion by anthronederivatives.     

Induction of alkoxyresorufin O-dealkylases, epoxide hydrolase, and liver weight gain: correlation with liver tumor-promoting potential in a series of barbiturates

The effects of a series of barbiturates, of known and varyingliver tumor-promoting ability, on several short-term endpointsincluding liver weight and liver-to-body weight ratio increasesand induction of cytochromes(s) P-450 and epoxide hydrolaseactivities were examined. Male F344 rats (3 months of age) wereadministered barbiturates in the drinking water for 12 days.At the end of the treatment period they were killed, body andliver weights were taken, microsomal p-nitroanisole O-demethylationand epoxide hydration, and liver S-9 O-dealkylation of ethoxy-,pentoxy-andbenzyloxyresonifin were measured. The latter two substrateshave been shown to be preferentially metabolized by the majorphenobarbital inducible form of cytochrome(s) P-450 (P-450_band were employed since they offered a means of differentiatingmore dearly varying levels of P-450 induction. Exposure to sodiumbarbital (SB) and sodium phenobarbital (PB) resulted in significantincreases in liver weight and liver-to-body weight ratios. Inductionof cytochrome(s) P-450 and epoxide hydrolase activities by thevarious barbiturates depended on the functional groups on C₅.When ranked in terms of decreasing induction potency, the followingorder was obtained for each enzyme activity quantitated: PB,SB, sodium pentobarbital, amobarbital, hexobarbital and theC₅ substituted parent compound (barbituric acid). Thus, thebarbiturates were found to exhibit a spectrum of induction potendes,with PB and SB, the most potent liver twnor promoters, yieldingthe greatest degree of liver weight increase and induction ofcytochrome(s) P-450 and epoxide hydrolase activities.     

Ornithine decarboxylase activity and DNA synthesis in rats after long term treatment with butylated hydroxyanisole, sodium saccharin or phenobarbital

Ornithine decarboxylase (ODC) activity and DNA synthesis were measured in the forestomach, urinary bladder and liver of rats given diet containing 2% butylated hydroxyanisole (BHA), 5% sodium saccharin (SS) or 0.05% phenobarbital (PB) for 21 weeks. ODC activity was not increased significantly in any of these organs in any of the groups, but the labeling index (LI) of the forestomach epithelium was increased by BHA and PB, and that of the urinary bladder epithelium by SS. PB did not increase the LI of the liver. These results do not indicate any relation between the effects of promoters, and the ODC activity or DNA synthesis in non-initiated target organs.     

Characterization of the induction of ornithine decarboxylase activity by benzoyl peroxide in SENCAR mouse epidermis

The induction of epiderinal ornithine decarboxylase (ODC) activityby benzoyl peroxide (BPO) was characterized to evaluate theusefulness of this effect as a short-term marker of BPO-inducedmouse skin tumor promotion. The maximal induced levels of ODCspecific activity, after a single topical dose of BPO, were>2-fold higher when a cold scraping method was used to prepareepidermis rather than the commonly used heat treatment method.Therefore, the cold scraping method was used for all the workreported here. Application of a single 20 mg dose of BPO tothe dorsal skin of SENCAR mice caused a relatively small inductionof epidermal ODC activity, to a level <1/40 that inducedby a single 2 µg dose of 12-O-tetradecanoylphorbol-13-acetate(TPA). Furthermore, the time-courses of induction were differentafter single doses of TPA and BPO, with peak activities observedat 6 and 24 h after treatment respectively. In contrast, aftera total of five 20 mg doses of BPO were topically applied (onedose every 2 days), ODC activity was transiently induced toan average level >15 times after a single dose. Additionally,on this dosing regimen, the peak of ODC activity shifted to4 h after the last treatment, so that the time-course of ODCinduction resembled that after multiple applications of TPA.The extent of epidermal ODC induction by BPO was found to bea complex function of the frequency of dosing and the numberof treatments. However, when BPO treatments were administeredfrom 1 to 7 days apart, similar maximal induced levels of ODCactivity were eventually achieved after application of multipledoses. Importantly, the dose-response for the induction of ODCactivity by five doses of BPO (applied one dose every 2 days)was highly correlated with published data on the dose-responsefor tumor promotion by this organic peroxide, indicating thatODC induction is a good short-term marker of BPO-induced tumorpromotion in SENCAR mice.