Potent inhibitory effects of suicide inhibitors of P450 isozymes on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene initiated skin tumors

A single dose of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) or 2-ethynylnaphthalene (EN) was applied to the skin of SENCAR mice 5 min before an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P) and the development of skin tumors then promoted with biweekly topical applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). The application of EP strongly inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner. Application of 44 pmol of EP inhibited tumor initiation by 10 nmol of DMBA approximately 25%; application of 440 nmol of EP inhibited tumor initiation by 200 nmol of B[a]P approximately 51%. A high single dose of EP (4.4-44 mumol) nearly eliminated skin tumor initiation by either 10 nmol of DMBA or 200 nmol of B[a]P. Application of VP also inhibited the formation of skin tumors initiated by either DMBA or B[a]P in a dose-dependent manner, but higher doses of VP than of EP were required to produce comparable inhibitions. Application of 44 nmol of VP inhibited tumor initiation by 10 nmol of DMBA approximately 30%; application of 4.4 mumol of VP inhibited tumor initiation by 200 nmol of B[a]P approximately 56%. Application of EN yielded contrasting results. EN inhibited the formation of skin tumors initiated by 10 nmol of DMBA, but the observed dose-dependence was minimal; tumors were decreased about 40% by 3.3 mumol of EN and only about 65% by 132 mumol of EN. A high single dose of EN (132 mumol) increased both the mean number of tumors per mouse and the percentage of mice that developed tumors after initiation by 200 nmol of B[a]P. Topical application of 4.4 mumol of EP, 22 mumol of VP or 33 mumol of EN to the skin of SENCAR mice 5 min before a single initiation dose of 2.5 mumol of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) had a minimal inhibitory effect (14-28%) on the development of skin tumors produced by subsequent biweekly promotion with TPA. A single dose of 44 mumol of EP or 132 mumol of EN followed by biweekly applications of TPA did not produce skin tumors; however, a dose of 44 mumol of VP followed by promotion with TPA produced a low but significant number of skin tumors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibitory effect of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoylphorbol-13-acetate

The effects of topically applied curcumin, chlorogenic acid, caffeic acid, and ferulic acid on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal ornithine decarboxylase activity, epidermal DNA synthesis, and the promotion of skin tumors were evaluated in female CD-1 mice. Topical application of 0.5, 1, 3, or 10 mumol of curcumin inhibited by 31, 46, 84, or 98%, respectively, the induction of epidermal ornithine decarboxylase activity by 5 nmol of TPA. In an additional study, the topical application of 10 mumol of curcumin, chlorogenic acid, caffeic acid, or ferulic acid inhibited by 91, 25, 42, or 46%, respectively, the induction of ornithine decarboxylase activity by 5 nmol of TPA. The topical application of 10 mumol of curcumin together with 2 or 5 nmol of TPA inhibited the TPA-dependent stimulation of the incorporation of [3H]-thymidine into epidermal DNA by 49 or 29%, respectively, whereas lower doses of curcumin had little or no effect. Chlorogenic acid, caffeic acid, and ferulic acid were less effective than curcumin as inhibitors of the TPA-dependent stimulation of DNA synthesis. Topical application of 1, 3, or 10 mumol of curcumin together with 5 nmol of TPA twice weekly for 20 weeks to mice previously initiated with 7,12-dimethylbenz[a]anthracene inhibited the number of TPA-induced tumors per mouse by 39, 77, or 98%, respectively. Similar treatment of mice with 10 mumol of chlorogenic acid, caffeic acid, or ferulic acid together with 5 nmol of TPA inhibited the number of TPA-induced tumors per mouse by 60, 28, or 35%, respectively, and higher doses of the phenolic acids caused a more pronounced inhibition of tumor promotion. The possibility that curcumin could inhibit the action of arachidonic acid was evaluated by studying the effect of curcumin on arachidonic acid-induced edema of mouse ears. The topical application of 3 or 10 mumol of curcumin 30 min before the application of 1 mumol of arachidonic acid inhibited arachidonic acid-induced edema by 33 or 80%, respectively.     

Comparison of the tumor-initiating activity of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene in female SENCAR and CD-1 mice

The derivation of mice resistant and susceptible to skin tumorigenesisusing the initiation-promotion regimen is described. Dose-responserelationships for tumor-initiating activities of 7,12-dimethylbenz[a]-anthracene(DMBA) and benzo[a]pyrene (BP) in the susceptible line (SENCAR)are presented. A single topical dose of either 0.1, 1.0, 10or 100 nmol DMBA, followed one week later by twice weekly applicationsof 8.5 nmol 12–0-tetradecanoylphorbol-13-acetate (TPA)for 19 weeks, produced 0, 3.3, 4.9 and 23.1 papillomas per mouse,respectively. Single topical initiating doses of either 50,100 or 200 nmol BP produced 1.7, 3.8 or 7.8 papillomas per mouse,respectively, after 28 weeks of promotion with 8.5 nmol TPA.SENCAR mice were compared with CD-1 mice for the initiatingactivity of DMBA and BP. Initiating doses of 0.1, 1.0, 10 and100 nmol DMBA produced 0.6, 3.8, 7.0 and 24 papillomas per mouse,respectively, in SENCAR mice and in CD-1 mice produced 0, 0.2,3.0 and 5.6 papillomas per mouse, respectively, after 25 weeksof promotion with TPA. With BP as the initiator, 10, 50, 100and 200 nmol doses produced 0.9, 1.6, 3.8 and 8.3 papillomasper mouse, respectively, in SENCAR mice and in CD-1 mice produced0.1, 0.7,1.8 and 3.8 papillomas per mouse, respectively, after25 weeks of promotion with TPA. SENCAR mice were compared with CD-1 mice for possible differencesin the oxidative metabolism of DMBA using epidermal homogenatesas the enzyme source. Basal levels of monooxygenase activitytoward DMBA were similar in both mouse stocks. Epidermal monooxygenaseactivities following pre-treatment with inducers including DMBA,3-methylcholanthrene, dibenz[a,c]anthracene, Aroclor 1254 and2,3,7,8-tetrachlorodibenzo-p-dioxin, also were quite similarin both mouse stocks. High-pressure liquid chromatographic profilesof ethyl acetate/ acetone (2:1) extractable metabolites revealeda close similarity in the patterns as well as the rates of formationof specific metabolites. Metabolites of DMBA tentatively identifiedbased on cochromato-graphy with purified reference standardsincluded phenols, 12-hydroxymethyl-7-methylbenz[a]anthracene,7-hydroxymethyl-12-methylbenz[a]antnracene, 7,12-dihydroxymethylbenz[a]anthracene,(±)-trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a]anthraceneand (±)-trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene.The results suggested that differences in oxidative metabolismof DMBA were not responsible for the differences in sensitivityto tumor-initiation between SENCAR and CD-1 mice.     

12-O-tetradecanoylphorbol-13-acetate-induced rapid loss of persistent 7,12-dimethylbenz[a]anthracene-DNA adducts in mouse epidermis and dermis

Twice-weekly application to mouse skin of 10 nmol of the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), beginning at 3 weeks after topical application of 1.2 mumol of 7,12-dimethylbenz[a]anthracene (DMBA), was found to cause a rapid loss of persistent DMBA-DNA adducts from both epidermal and dermal DNA. This effect is thought to reflect TPA-induced proliferation of quiescent initiated skin cells containing persistent adducts and may be causally related to the irreversibility of the initial phase of tumor promotion, which is known to require cell proliferation.     

Tumor-initiating activity and carcinogenicity of dibenzo[a,l]pyrene versus 7, 12-dimethylbenz[a]anthracene and benzo[a]pyrene at low doses in mouse skin

Dibenzo[a,l]pyrene (DB[a,l]P) is an extremely potent carcinogenthat may be present in environmental samples. Dose-responsestudies were conducted at low doses in mouse skin by initiation-promotionand repeated application to compare its activity to that of7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B[a]P),DB[a,l]P-8,9-dihydrodiol and DB[a,l]P-11,12-dihydrodiol. FemaleSENCAR mice were initiated with 1 or 0.25 nmol of DB[a, l]P,DMBA, B[a]P or DB[a,l]P-11,12-dihydrodiol and promoted withphorbol ester acetate. At 1 nmol, DB[a, l]P induced 2.6 tumors/mouse,whereas DB[a,l]P-11,12-dihydrodiol and DMBA induced 0.17 and0.29 tumors/mouse respectively. At the low dose, DB[a,l]P induced0.79 tumors/mouse, but the other two compounds were virtuallyinactive. B[a]P, tested only at 1 nmol, was inactive. Thesethree compounds, as well as DB[a,l]P-8,9-dihydrodiol, were testedby repeated application twice weekly for 40 weeks at 1 and 4nmol per dose. In addition, DB[a,l]P, DMBA and B[a]P were alsotested at 8 nmol. At 8 and 4 nmol, DB[a,l]P induced malignanttumors in 91 and 70% of mice respectively. At 4 nmol DB[a, l]P-11,12-dihydrodiolelicited only benign tumors in 36% of mice. At 4 nmol DMBA inducedtwo carcinomas in one mouse and at 8 nmol it induced one papillomaand one sebaceous gland adenoma. B[a]P and DB[a,l]P-8,9-dihydrodiolwere inactive at all doses tested. These results demonstratethat DB[a, l]P is a much more potent carcinogen than DMBA, thearomatic hydrocarbon previously considered to be the most potent.Combination of these results with previous comparisons of DB[a,l]P,DB[a,l]P-11,12-dihydrodiol, DMBA and B[a]P at higher doses (E.L.Cavalieri et al. (1991) Carcinogenesis, 12, 1939–1944)shows clearly the interference of toxicity with the tumorigenicityof DB[a,l]P and its 11,12-dihydrodiol.     

Comparative dose-response tumorigenicity studies of dibenzo[alpha,l]pyrene versus 7,12-dimethylbenz[alpha]anthracene, benzo[alpha]pyrene and two dibenzo[alpha,l]pyrene dihydrodiols in mouse skin and rat mammary gland

Comparative studies were conducted of the tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a,l]pyrene (DB[a,l]P) versus 7,12-dimethyl-benz[a]anthracene (DMBA), the most potent recognized carcinogenic polycyclic aromatic hydrocarbon (PAH); benzo[a]pyrene (B[a]P), the most potent recognized carcinogenic environmental PAH; DB[a,l]P 8,9-dihydrodiol, the K-region dihydrodiol; and DB[a,l]P 11,12-dihydrodiol, precursor to the bay-region diolepoxide. The tumor-initiating activity of DB[a,l]P and B[a]P was compared in the skin of female SENCAR mice at doses of 300, 100 and 33.3 nmol. The mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) twice-weekly for 13 weeks. DB[a,l]P at all doses induced significantly more tumors than B[a]P at the corresponding dose, with a significantly shorter latency. Subsequently, the tumor-initiating activity of DB[a,l]P was compared in the skin of female SENCAR mice to that of DMBA, B[a]P, DB[a,l]P 8,9-dihydrodiol and DB[a,l]P 11,12-dihydrodiol at doses of 100, 20 and 4 nmol. The mice were promoted with TPA twice-weekly for 24 weeks. In addition, groups of mice were initiated with 100 nmol of DB[a,l]P, DMBA, B[a]P, DB[a,l]P 8,9-dihydrodiol or DB[a,l]P 11,12-dihydrodiol and kept without promotion. This experiment showed that in the mouse skin, DB[a,l]P and DB[a,l]P 11,12-dihydrodiol displayed similar tumor-initiating activity with a response inversely proportional to the dose, presumably due to the toxicity of the compounds. At the high dose they elicited tumors earlier than DMBA, though DMBA produced a much higher tumor multiplicity. At the low dose, DMBA, DB[a,l]P and DB[a,l]P 11,12-dihydrodiol exhibited similar tumorigenicities. DB[a,l]P 8,9-dihydrodiol was a marginal tumor initiator. Once again, DB[a,l]P was by far a much stronger tumor initiator than B[a]P. Female Sprague-Dawley rats were treated with 1.0 or 0.25 mumol of DB[a,l]P, DMBA or B[a]P by intramammillary injection at eight teats. DB[a,l]P at both doses was a more potent carcinogen than DMBA at the corresponding dose in the rat mammary gland. B[a]P was a marginal mammary carcinogen, eliciting only a few fibrosarcomas. Thus, these data suggest that DB[a,l]P is the strongest PAH carcinogen ever tested.     

Synthetic lipid second messenger sn-1,2-didecanoylglycerol: a complete tumor promoter in mouse skin

sn-1,2-Didecanoylglycerol, a synthetic lipid second messenger and model diacylglycerol, was evaluated as a complete skin tumor promoter in CD-1 mice. In addition, sn-1,2-dioctanoylglycerol, sn-1,2-didecanoylglycerol, the second stage tumor promoter mezerein, and the complete tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for their ability to stimulate epidermal protein kinase C activity in vitro. All four compounds stimulated epidermal protein kinase C activity utilizing lysine-rich histone as the phosphate acceptor substrate. sn-1,2-Dioctanoylglycerol and sn-1,2-didecanoylglycerol stimulated epidermal protein kinase C activity to a maximum velocity similar to that obtained when the enzyme was stimulated with TPA; however, about 1000 times greater concentration of the sn-1,2-diacylglycerols was required. sn-1,2-Didecanoylglycerol was evaluated as a complete skin tumor promoter in CD-1 mice utilizing a dosing regimen demonstrated to produce epidermal hyperplasia. Mice were initiated with 200 nmol 7,12-dimethylbenz[a]anthracene. One week later the mice received twice daily topical applications of 1 nmol TPA, 2 mumol sn-1,2-didecanoylglycerol or 5 mumol sn-1,2-didecanoylglycerol, 5 days/week. Additional initiated mice received twice weekly topical applications of 2 or 5 nmol TPA. Initiated mice treated with 5 nmol TPA twice weekly or with 1 nmol TPA twice daily for 5 days/week (cumulative weekly doses of 10 nmol TPA) responded similarly, based on the tumor incidence and the average number of tumors per mouse. Initiated mice treated with 2 or 5 mumol sn-1,2-didecanoylglycerol twice daily developed tumors in a dose-dependent manner. Initiated mice treated with 5 mumol sn-1,2-didecanoylglycerol twice daily developed many tumors, and at 20 weeks there was a 74% tumor incidence and an average of 6.0 tumors/mouse. At 20 weeks, 24% of the initiated mice treated with 2 mumol sn-1,2-didecanoylglycerol twice daily developed tumors, with an average of 1.1 tumors/mouse. Mice which were not initiated but treated twice daily with 5 mumol sn-1,2-didecanoylglycerol for 20 weeks did not develop any tumors. These data demonstrate that the representative synthetic lipid second messenger sn-1,2-didecanoylglycerol, like TPA, is a complete tumor promoter in DMBA-initiated mouse skin.     

Skin tumor initiating activity of therapeutic crude coal tar as compared to other polycyclic aromatic hydrocarbons in SENCAR mice

Crude coal tar (CCT), a complex mixture rich in polycyclic aromatic hydrocarbons (PAHs) including carcinogens such as benzo[a]pyrene (BP), is widely used therapeutically in dermatological practice, particularly in combination with ultraviolet radiation in the treatment of chronic dermatoses such as psoriasis. In this study we analyzed the tumor initiating activity of therapeutic CCT preparation (USP) in a two-stage model system (initiation and promotion) in SENCAR mice. The tumorigenicity of CCT was compared with other conventionally studied carcinogenic PAHs; 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), benzo[a]pyrene 7,8-diol (BP-7,8-diol) and benzo[a]pyrene (BP). A single topical application of an initiating dose of CCT (20 microliter), DMBA (39 nmol), MCA (746 nmol), BP-7,8-diol (352 nmol) or BP (396 nmol) was followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate (TPA) (3.24 nmol). The first tumor appearance in the CCT treated group was at 6 weeks on test as compared with DMBA and MCA (3 weeks) and BP-7,8-diol and BP (4-5 weeks). In a total of 20 animals after 8 weeks on test the DMBA, MCA, BP-7,8-diol, BP and CCT groups of mice showed 457, 176, 106, 76 and 46 tumors, respectively. After 11 weeks 100% of the mice in each experimental group had developed tumors and the number of tumors/mouse was 24.3, 15.0 9.8, 6.6 and 3.3 in the DMBA, MCA, BP-7,8-diol, BP and CCT groups, respectively. These studies provide first evidence that a single topical application of a therapeutic preparation of CCT possesses skin tumor initiating activity.     

Food restriction inhibits [3H] 7,12-dimethylbenz(a)anthracene binding to mouse skin DNA and tetradecanoylphorbol-13-acetate stimulation of epidermal [3H] thymidine incorporation

It has been known for many years that reducing the food intake of laboratory mice and rats inhibits the development of a broad spectrum of chemically induced and spontaneous tumors, but the mechanism of this effect is poorly understood. Food restriction of A/J mice for two weeks is now shown to inhibit the binding of topically applied [3H]7,12-dimethylbenz(a)anthracene (DMBA) to skin DNA by 50% and to abolish the stimulation of [3H]-thymidine incorporation in the epidermis produced by topical application of the tumor promoter tetradecanoylphorbol-13-acetate (TPA). Similar effects on the actions of DMBA and TPA are observed following topical application of the adrenal steroid, dehydroepiandrosterone (DHEA), a potent glucose-6-phosphate dehydrogenase (G6PDH) inhibitor, while food restriction for two weeks depresses epidermal G6PDH activity by 60%. It is suggested that both the inhibition of [3H]DMBA binding to skin DNA and the TPA stimulation in epidermal [3H]thymidine incorporation result from a reduction in the NADPH cellular pool as a result of G6PDH inhibition.     

The effects of weak or non-carcinogenic polycyclic hydrocarbons on 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene skin tumor-initiation.

Benzo[e]pyrene (B[e]P) inhibited 7,12-dimethylbenz[a]anthracene (DMBA) skin tumor-initiation in mice by 84%, whereas pyrene and fluoranthene inhibited DMBA initiation by 50 and 34%, respectively. However, B[e]P, pyrene and fluoranthene had either no significant effect or a slight enhancing effect on benzo[a]pyrene (B[a]P) skin tumor-initiation. In addition, B[e]P had essentially no effect on the initiating ability of (+/-)B[a]P-7 beta,8 alpha-diol-9 alpha,10 alpha-epoxide. As a tumor-initiator, B[e]P was found to have very weak activity at a 252 microgram/level (0.4 papillomas/mouse at 40 weeks) and no activity at 100 microgram. When given at a dose of 100 microgram twice weekly, B[e]P induced 2.1 papillomas/mouse at 30 weeks, and 25% of the mice had carcinomas at 40 weeks. However, B[e]P carcinogenic activity is weak when compared to B[a]P, which can induce a comparable tumor response at a dose of 5 microgram twice weekly. When B[e]P was tested as a tumor promoter at a dose of 100 microgram twice weekly after DMBA initiation, it induced 4.5 papillomas/mouse at 30 weeks and a 45% carcinoma incidence at 40 weeks, which was approximately twice as effective as B[e]P alone. The data show that B[e]P is a very weak tumor initiator, a weak complete carcinogen, a moderate tumor promoter, possibly a weak co-tumor-initiator when given with B[a]P, and a potent anit-tumor-initiator when given with DMBA. The anti-tumor initiating and co-tumor-initiating effects of B[e]P appear to be related to its ability to modify the conversion of the tumor initiator into an electrophilic intermediate(s) which are capable of covalently binding to DNA. In addition, B[e]P induced epidermal cellular proliferation which may be related to its promoting ability.     

Effect of ellagic acid and 3-O-decylellagic acid on the formation of benzo[a]pyrene-derived DNA adducts in vivo and on the tumorigenicity of 3-methylcholanthrene in mice

The effect of ellagic acid and its more lipophilic derivative,3-O-decylellagic acid, on the amount of DNA-bound adducts inthe epidermis or lung of CD-I mice treated with [³H]benzo-[a]pyrene([³H]B[a]P) was evaluated using several different treatmentprotocols. The i.v. administration of 50µmol/kg of ellagicacid or 3-O-decyIellagk acid either together with or 5 min beforea 0.2 µmol/kg i.v. dose of [³H]B[a]P did not inhibit theformation of pulmonary DNA-bound adducts. Feeding mice a dietthat contained 1% ellagic acid for 10 days or the i.p. administrationof 120 µmol/kg of ellagic acid 30 min before the i.v.administration of 0.2 µmol/kg of [³H)B(a)P did not inhibitthe formation of DNA-bound adducts in the lung. The applicationof 2500 nmol of ellagic acid or 3-O-decylellagic acid to mouseskin 5 min before the application of 2, 10 or 50 nmol of [³H]B[a]Phad little or no effect on the covalent binding of [³H]B[a]Pmetabolites to epidermal DNA. Feeding mice a diet containing1% ellagic acid for 10 days did not inhibit the formation ofepidermal DNA-bound adducts after a topical dose of 2 nmol of[³H]B[a]P. Similarly, the topical application of 2500 nmol ofellagic acid at 2 h, 1 h and 5 min before and at 10 min afterthe application of 2 nmol of [³H]B[a]P did not inhibit the formationof DNA-bound adducts, but the same dosing regimen of 3-O-decylellagicacid (total dose of 10 000 nmol) resulted in a modest inhibitionin the formation of DNA-bound adducts. The topical applicationof 1500 nmol of ellagic acid 1 h before the application of 1500nmol of 3-methylcholanthrene (3-MC) to CD-I or BALB/c mice twiceweekly did not inhibit the development of skin tumors. Our resultsindicate that ellagic acid and 3-O-decylellagic acid are noteffective in inhibiting [³H]B[a]P DNA adduct formation in mouseskin and lung and that ellagic acid does not inhibit 3-MC-inducedskin tumori-genesis in BALB/c or CD-I mice.     

Inhibitory effects of chlorophyllin on 7,12-dimethylbenz[a]anthracene-induced bacterial mutagenesis and mouse skin carcinogenesis.

Chlorophyllin (CHL), a water-soluble derivative of chlorophyll, has been used for the treatment of several abnormal human conditions without apparent toxicity. Recent studies have revealed that CHL has the excellent chemopreventive potential. In the present investigation, we have found the inhibitory activities of CHL against 7,12-dimethylbenz[a]anthracene (DMBA)-induced mutagenesis in Salmonella typhimurium TA100 and also on DMBA-initiated and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-promoted mouse skin tumor formation. The incidence and the multiplicity of skin tumors were not significantly decreased in mice by a single topical application of CHL prior to the DMBA treatment, but there was a marked suppression of papillomagenesis in mice treated with CHL during the promotional stage. Furthermore, the formation of DMBA-induced papillomagenesis was reduced in all mice that had received CHL for 6 weeks following treatment with TPA for 6, 18 and 24 weeks. These results indicate that CHL can inhibit both tumor promotion and the progression of papillomagenesis in the two-stage mouse skin carcinogenesis induced by DMBA and TPA.     

S/RV Cri-ba, a hairless mouse strain sensitive to skin tumorigenesis by suboptimal doses of 7,12-dimethylbenz[a]anthracene, initiation-promotion and two stage promotion protocols

Susceptibility of hairless inbred S/RV Cri-ba or Bare mice to skin tumor development with suboptimal doses of 7,12-dimethylbenz[a]anthracene (DMBA), DMBA-TPA two stage protocol and two stage promotion using 12-O-tetradecanoylphorbol-13-acetate (TPA) as sub-stage 1 promoter and mezerein (MEZ) or phorbol retinoate acetate (PRA) as substage 2 promoter was determined. A single application of 40 or 20 nmol DMBA induced 4-5 papillomas per mouse 40 weeks after initiation while no tumors appeared after similar treatment with 10 or 4 nmol DMBA. Dose response studies for DMBA initiation revealed that 10 nmol DMBA dose saturated the sites for initiation in the resting epidermis. In two stage promotion experiments, MEZ was found to be a potent stage 2 promoter, while PRA acted as a weak complete promoter.     

Inhibitory effects of topical application of low doses of curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion and oxidized DNA bases in mouse epidermis

The effects of topical applications of very low doses of curcumin (the major yellow pigment in turmeric and the Indian food curry) on 12-O- tetradecanoylphorbol-13-acetate (TPA)-induced oxidation of DNA bases in the epidermis and on tumor promotion in mouse skin were investigated. CD-1 mice were treated topically with 200 nmol of 7,12- dimethylbenz[a]anthracene followed one week later by 5 nmol of TPA alone or together with 1, 10, 100 or 3000 nmol of curcumin twice a week for 20 weeks. Curcumin-mediated effects on TPA-induced formation of the oxidized DNA base 5-hydroxymethyl-2'-deoxyuridine (HMdU) and tumor formation were determined. All dose levels of curcumin inhibited the mean values of TPA-induced HMdU formation in epidermal DNA (62-77% inhibition), but only the two highest doses of curcumin strongly inhibited TPA-induced tumor promotion (62-79% inhibition of tumors per mouse and tumor volume per mouse). In a second experiment, topical application of 20 or 100 nmol (but not 10 nmol) of curcumin together with 5 nmol TPA twice a week for 18 weeks markedly inhibited TPA- induced tumor promotion. Curcumin had a strong inhibitory effect on DNA and RNA synthesis (IC50 = 0.5-1 microM) in cultured HeLa cells, but there was little or no effect on protein synthesis.      

Relative tumor initiating activity of benzo[a]fluoranthene, benzo[b]fluoranthene, naphtho[1,2-b]fluoranthene and naphtho[2,1-a]fluoranthene on mouse skin

The tumor-initiating activities of benzo[a]fluoranthene (BaF), benzo[b]fluoranthene (BbF), naphtho[1,2-b]fluoranthene (NbF) and naphtho[2,1-a]fluoranthene (NaF) were evaluated on the skin of female CD-1 mice. Each of these polycyclic aromatic hydrocarbons was assayed at total initiation doses of 1.0 and 4.0 mumol/mouse. These hydrocarbons were applied in 10 subdoses administered every other day. Promotion commenced 10 days after the last initiator dose and consisted of thrice weekly application of 2.5 micrograms of tetradecanoylphorbol acetate for 20 weeks. BbF was the most potent tumor initiator inducing a 100% incidence of tumor-bearing mice with an average of 8.5 tumors per mouse at a total initiator dose of 1.0 mumol. NaF was slightly more active as a tumor initiator than either NbF or BaF. NaF induced a 90% incidence of tumor-bearing mice with an average of 5.9 tumors per mouse at a total initiator dose of 1.0 mumol. BaF and NbF at a total initiator dose of 4.0 mumol exhibited similar tumor-initiating activity with both inducing a 90% incidence of tumor-bearing mice with an average of 4.3 and 6.6 tumors per mouse, respectively. However, at a total initiator dose of 1.0 mumol, BaF and NbF induced a 95% and 65% incidence of tumor-bearing mice with an average of 3.3 and 2.5 tumors per mouse, respectively.     

Promoter independence as a feature of most skin papillomas in SENCAR mice

In the present study, the fate of individual papillomas induced by initiation-promotion on the backs of SENCAR mice was monitored after discontinuation of limited promoter treatment. Groups of 40 SENCAR mice each were initiated by a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) at 2, 1, 0.5, or 0.25 micrograms/mouse. Animals were promoted with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly during 10 weeks. At that time point, 10 papilloma bearing mice from each group were randomly selected to follow the growth of their existing tumors. Animals and their individual tumors were identified, charted, and photographed weekly. After an initial increase, the average number of papillomas/mouse remained constant after discontinuation of TPA in all the groups except the group receiving the highest DMBA dose (Group 1) and with highest tumor load. Twenty-one weeks after TPA was discontinued, only 10-20% of the papillomas had regressed and no statistically significant differences were found among the different DMBA dose groups. On the other hand, Group 1 showed the highest percentage of coalescing tumors which was apparently a function of tumor load. In addition, no differences were observed in the proportion of positive tumors with activating point mutations at codon 61 of the Ha-ras gene when comparing samples of papillomas from the highest DMBA initiation dose group (2 micrograms) versus the lowest DMBA initiation dose group (0.25 micrograms). Our present data suggest that papillomas induced with low doses of DMBA in SENCAR mice are no more TPA dependent than those induced by higher initiating doses. Furthermore, in SENCAR mice at the doses used in the present study (0.25-2 micrograms/mouse), the number of so-called "promoter dependent" papillomas represents only a small percentage of the total papillomas produced using the initiation-promotion protocol.     

Inhibition of the binding of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene to DNA in mouse skin epidermis by 1-ethynylpyrene

The effects of 1-ethynylpyrene (EP), 1-vinylpyrene (VP) and 2-ethynlnaphthalene (EN) on the covalent binding of 7,12-dimethylbenz[a]anthracene (DMBA) and of benzo[a]-pyrene (B[a]P) to the epidermal DNA in mouse skin were investigated. When applied topically, 5 min before an initiating dose of 10 nmol DMBA or of 200 nmol B[a]P, EP was an effective inhibitor of the formation of the covalent complexes of these procarcinogenic polycyclic aromatic hydrocarbons (PAHs) with the epidermal DNA. VP, applied under the same conditions, was a significantly less effective inhibitor of the binding of DMBA to DNA and showed even weaker inhibition of the binding of B[a]P. EN was ineffective as an inhibitor of the binding of either DMBA or B[a]P. These results establish that both the pyrene nucleus and the ethynyl substituent of EP contribute to the effective inhibition of the binding of DMBA and B[a]P to the epidermal DNA of mouse skin. No significant changes in the ratios of the anti- to the syndiol epoxide-DNA adducts of DMBA or of B[a]P were produced by doses of EP that produced inhibitions of the binding to DNA. At doses of VP that inhibited covalent binding of both DMBA and B[a]P, no changes in DMBA-DNA adduct distributions were observed but changes in the relative proportions of several B[a]P-DNA adducts were noted. These data are discussed in terms of the potential of aryl acetylenes to act as suicide inhibitors (mechanism-based inactivators) of cytochrome P450-dependent monooxygenase isozymes.     

Effects of ellipticine, flavone, and 7,8-benzoflavone upon 7,12-dimethylbenz[a]anthrancene 7,14-dimethyl-dibenzo[a,h]anthrancene and dibenzo[a]anthrancene initiated skin tumors in mice

Varying doses of ellipticine (EL), flavone (FL), or 7,8-benzoflavone(78BF) were applied to mouse skin 5 min before an initiatingdose of 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA), 47.5nmol 7,14-dimethylbenzo[a]anthracene (DDBA), or 200 nmol dibenzo[a,h]anthracene(DBA) and the development of skin tumors in the mice then promotedby topical applications of 2 µg 12-O-tetradecanoylphorbol-13-acetate(TPA). As expected, treatment with 78BF (37 nmol or 370 nmol)markedly inhibited the skin tumor initiation by DMBA (>70%).High doses of FL (4500 nmol) or EL (410 nmol) also inhibitedDMBA tumorigenesis (52% and 82%, respectively) but lower dosesof FL (450 nmol) or EL (4.1 nmol) stimulated DMBA tumorigenesis(>40%). As was the case with DMBA initiation, the higherdoses of FL or EL inhibited DDBA skin tumorigenesis and thelower doses of these two modifiers stimulated the DDBA tumorigenesis.In contrast with the results with DMBA initiation, treatmentwith 78BF (370 nmol or 3700 nmol) slightly enhanced DDBA tumorigenesis(22% and 6%, respectively). Treatment with EL and FL at alldoses tested stimulated DBA tumorigenesis (range 4–51%),while treatment with 370 nmol 78BF slightly stimulated DBA tumorigenesis(19%) and treatment with 3700 nmol slightly inhibited DBA tumorigensis(9%). The effects of a range of 78BF doses upon skin tumor initiationby 40 nmol DMBA were also investigated. While all doses of 78BFtested (0.37–370 nmol) inhibited the DMBA tumorigenesis,the dose response was not linear; treatment with 3.7 nmol 78BFresulted in more papillomas per mouse (12.20) than did treatmentwith either 0.37 nmol 78BF (8.70) or 37 nmol 78BF (5.97). Itis concluded that modifiers such as 78BF, FL and EL may havea variable, dose-dependent effect upon skin tumor initiationby carcinogenic polycyclic arylhydrocarbons. Some implicationsof this proposal are discussed.     

Tumor-initiating activity of the (+)-(S,S)- and (-)-(R,R)- enantiomers of trans-11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene in mouse skin.

A single administration of enantiomerically pure 11,12-dihydrodiols of dibenzo[a,l]pyrene (DB[a,l]P) on the back of NMRI mice and subsequent chronic treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) (initiation/promotion assay) revealed strikingly different carcinogenic activities of both enantiomers. Tumor-initiating activity of (-)-(11R,12R)-DB[a,l]P-dihydrodiol, which is the metabolic precursor of the (-)-anti-(11R,12S)-dihydrodiol (13S,14R)-epoxide, was exceptionally higher than the corresponding effect of (+)-(11S,12S)-DB[a,l]P-dihydrodiol, the metabolic precursor of (+)-syn-(11S,12R)-dihydrodiol (13S,14R)-epoxide. After topical application of 10 nmol (-)-11,12-dihydrodiol and promotion with TPA twice weekly for a further 18 weeks 93% of treated animals exhibited four to five tumors. In contrast, no neoplasms were observed after treatment with 10 nmol (+)-11,12-dihydrodiol, whereas in the group exposed to 20 nmol of this enantiomer only 13% of mice developed neoplasms (0.1 tumors/survivor). For DB[a,l]P, considered as the most potent carcinogenic polycyclic aromatic hydrocarbon to date, stereoselective formation of (+)-syn- and (-)-anti-11,12-dihydrodiol 13,14-epoxides via the corresponding enantiomeric 11,12-dihydrodiols has been found to be the principal metabolic activation pathway leading to DNA adducts and mutagenicity. Our study demonstrates that the striking difference in carcinogenic activity in mouse skin of (+)-(11S,12S)- and (-)-(11R,12R)-DB[a,l]P-dihydrodiol convincingly reflects the different genotoxicity, i.e. DNA binding and mutagenicity, of both enantiomers observed earlier.     

Metallothionein deficiency enhances skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate in metallothionein-null mice

To clarify the physiological role(s) of metallothionein (MT) in carcinogenesis, we studied the susceptibility of MT-null mice to chemically mediated carcinogenesis in the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage carcinogenesis model. The MT-null mice were subjected to a single topical application of DMBA (50 or 100 micro g/mouse) and, 1 week later, to promotion with TPA (10 micro g/mouse) twice a week for 20 weeks. At week 21, nearly all of the MT-null mice developed tumors in the skin, in contrast to only 10-40% of wild-type mice. No tumors were observed in MT-null or wild-type mice that were administered TPA alone. By using the PCR-restriction fragment length polymorphism and PCR-single strand conformation polymorphism methods, we found a transversion of A182 to T in codon 61 of c-Ha-ras in the papilloma tissue of MT-null mice and wild-type mice but failed to find any mutations in the c-Ki-ras and c-N-ras genes. In two-stage skin carcinogenesis induction by DMBA/TPA, p53 and p21(WAF1/Cip1) expression levels were found to be increased in MT-null mice compared with wild-type mice. As to an earlier change at the promotion stage triggered by TPA application, MT-null mice were found to have both hyperplasia of the epithelium and a marked degree of inflammation in the basal layer, indicating that the induced as well as endogenous MT acted as a protective factor against tumorigenesis. In conclusion, the present study has demonstrated that MT has antitumorigenic potential in both the initiation and promotion stages of the two-stage chemical skin carcinogenesis model.