SENCAR Mouse Skin Research Articles

Effects of dietary calcium and vitamin D3 on tumor promotion in mouse skin

The effects of low, adequate, and supplemental intake of calcium and vitamin D3 on 12-O-tetradecanoylphorbol-13-acetate (TPA) skin tumor promotion were examined. Administration of the experimental diets was started one week before the first TPA application to the 7,12-dimethylbenz[a]anthracene-initiated dorsal skin of female Sencar mice. Neither dietary calcium in a range from 0.15% to 2.0% of the diet as calcium carbonate nor vitamin D3 ranging from 200 to 4,000 IU/kg diet resulted in modulation of the skin papilloma response in terms of incidence, number per mouse, or size distribution of tumors. There were also no effects of the varied levels of calcium or vitamin D3 on mouse body weights, serum calcium, or TPA induction of epidermal ornithine decarboxylase activity. These results indicate that dietary administration of a wide range of doses of calcium or vitamin D does not alter the serum calcium levels and, therefore, does not appear capable of altering skin tumor promotion. These results are in contrast to reports that demonstrate antineoplastic activity for both calcium ion and active hormonal vitamin D, either in control of epidermal cell proliferation and/or differentiation or inhibition of carcinogenesis.

Characterization of covalently modified deoxyribonucleosides formed from dibenz[a,j]anthracene in primary cultures of mouse keratinocytes

Identification of various deoxyribonucleoside adducts formed in primary cultures of mouse keratinocytes exposed to dibenz[a,j]anthracene (DB[a,j]A) is presented. A preliminary analysis of the DNA adducts formed from 7-methyldibenz[a,j]anthracene (7MeDB[a,j]A) also is presented. Cultures of keratinocytes obtained from dorsal skins of female SENCAR mice were exposed to 0.5 microgram of tritium-labeled hydrocarbons/mL of medium for 24 h. The total DNA binding was 2.23 +/- 0.54 and 5.28 +/- 0.97 pmol of hydrocarbon/mg of DNA for DB[a,j]A and 7MeDB[a,j]A, respectively. These binding values represented the radioactivity associated with the modified deoxyribonucleosides separated from the normal deoxyribonucleosides on Sephadex LH-20 columns following enzymatic digestion of isolated DNA. Treatment of keratinocytes with DB[a,j]A produced adduct peaks corresponding to marker adducts derived from trans addition of both deoxyguanosine as well as deoxyadenosine residues to the (+) enantiomer of the anti-diol epoxide where the deoxyadenosine adducts were predominant. In addition, DNA adduct peaks corresponding to markers of trans and cis addition, respectively, of deoxyguanosine and deoxyadenosine to the (+)-syn-diol epoxide were also noted in these chromatograms. A major DNA adduct in cells exposed to DB[a,j]A was tentatively identified as resulting from the addition of deoxyadenosine to DB[a,j]A-5,6-oxide. Several other later eluting DNA adduct peaks, not corresponding to any of the marker adducts, were also present in these chromatograms. In comparison, when cells were exposed to the more biologically potent 7-methyl analogue, at least 12 DNA adduct peaks were consistently observed in HPLC chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the antioxidant/prooxidant status of murine skin following topical treatment with 12-O-tetradecanoylphorbol-13-acetate and throughout the ontogeny of skin cancer. Part I: quantitation of superoxide dismutase, catalase, glutathione peroxidase and xanthine oxidase

The activities of several enzymes involved in reactive oxygen production and detoxification were quantified in murine skin during the ontogeny of chemically induced skin cancer. Relative to solvent-treated controls, the specific activities of epidermal superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) were reduced approximately 45, approximately 60 and approximately 24% respectively, 24 h after the fourth or tenth topical application of 1 microgram of 12-O-tetradecanoylphorbol-13-acetate (TPA) to the dorsal skin of SENCAR mice. The specific activity of epidermal xanthine oxidase (XO) increased approximately 350% during the same period. SOD and CAT specific activities in papillomas and carcinomas generated in an initiation-promotion protocol were approximately 15 and approximately 40% respectively of the activities measured in age-matched, non-treated mice. CAT and SOD activities were also significantly suppressed in the skin adjacent to the papillomas for several weeks following the cessation of TPA promotion, but eventually recovered to the levels measured in age-matched controls. XO specific activities in papillomas and squamous cell carcinomas (SCC) were approximately 85-350% greater than the activities determined in skin adjacent to the tumors. The increases in XO and the decreases in SOD and CAT activities measured in the tumors were independent of continued treatment with TPA, and thus characteristic of the tumor phenotype. GPX activities in papillomas were comparable to normal, untreated skin, but reduced approximately 22-41% in SCC. Collectively, these studies demonstrate that TPA orchestrates changes in the activities of several enzymes involved in reactive oxygen metabolism that are characteristic of the papilloma and SCC phenotype.

Potent inhibitory effects of suicide inhibitors of P450 isozyines on 7, 12-dimethylbenzℵaℵanthracene and benzoℵaℵpyrene 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)

Carcinogenicity study of fecapentaene-12 diacetate on skin painting in SENCAR mice

To investigate the effects of both diol esterification and coadministration with antioxidant on the tumorigenicity of fecapentaene-12 (FP-12) preparations, diacetylfecapentaene-12 (DAFP-12) in dimethylsulfoxide (DMSO) was applied to SENCAR mouse skin with or without the stabilizer, vitamin E, twice/week for 5 weeks, following which all animals were promoted for up to 25 weeks by weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). While positive controls receiving 7,12-dimethylbenz[a]anthracene (DMBA) instead of DAFP-12 in a similar protocol all developed papillomas (average of 23/animal), papilloma incidence in mice given DAFP-12 did not differ significantly from that of the vehicle control. We conclude that DAFP-12 shows little or no tumor initiating activity for mouse skin even when coadministered with vitamin E.

Metabolism of xenobiotics during percutaneous penetration: Role of absorption rate and cutaneous enzyme activity

The role of absorption rate and enzyme activity on cutaneous metabolism of topically applied xenobiotics was assessed by determining the simultaneous percutaneous penetration/metabolism of benzo[ a]pyrene (B[ a]P) and 7-ethoxycoumarin (7-EC) in intact, metabolically viable skin of Sencar mice, hairless guinea pigs, and humans. In addition, specific activities of aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin deethylase (ECDE) were determined in cutaneous microsomal fractions. Both compounds were readily absorbed but only minimally metabolized. Sencar mouse and hairless guinea pig skin absorbed 55–60% of the applied B[ a]P dose and metabolized only 6 and 3%, respectively, of that absorbed. Human skin absorbed 31% of the applied dose and B[ a]P metabolism was not detectable. All three species absorbed 60–80% of the applied 7-EC dose. Sencar mouse and hairless guinea pig skin metabolized 1.3 and 1.2% of the absorbed dose, respectively, and human skin metabolized only 0.05%. When 7-EC absorption was increased to the maximum possible rate, its metabolism by Sencar mouse and hairless guinea pig skin was also substantially increased. In human skin, a much smaller increase in 7-EC absorption rate was possible and no increase in 7-EC metabolism occurred. Thus relatively slower absorption of 7-EC and B[ a]P by human skin may limit cutaneous metabolism of these penetrating compounds. Specific activities of AHH and ECDE were significantly lower in human skin than in Sencar mouse and hairless guinea pig skin, suggesting that low enzyme activity contributes as well to a low rate of metabolism by human skin compared to other species. Thus absorption rate and cutaneous enzyme activity are interrelated determinants of the extent of cutaneous metabolism of B[ a]P and 7-EC occurring during their percutaneous penetration, and slow absorption and low enzyme activity limit cutaneous metabolism of B[ a]P and 7-EC in human skin in particular.

Inhibition of Benzoyl Peroxide-Mediated Tumor Promotion in 7,12-Dimethylbenz(a)anthracene-Initiated Skin of Sencar Mice by Antioxidants Nordihydroguaiaretic Acid and Diallyl Sulfide

Benzoyl peroxide (BPO), a free radical generating compound, is widely used in topical medications prescribed for acne vulgaris and in cosmetic products. It has been shown to possess tumor-promoting activity in murine skin initiated with chemical carcinogens such as 7,12-dimethylbenz(a)anthracene (DMBA). In the present study we assessed the effect of the antioxidants nordihydroguaiaretic acid (NDGA) and diallyl sulfide (DAS) against BPO-mediated tumor promotion in murine skin. Pretreatment of Sencar mice with NDGA and DAS prior to skin application of BPO resulted in a time- and dose-dependent inhibition of epidermal ODC induction caused by BPO. Tumor initiation was achieved by a single topical application of DMBA (10 micrograms/animal) to Sencar mice. Ten days later tumor promotion was begun by twice-weekly topical application of BPO (20 mg/animal). The anticarcinogenic effects of NDGA (25 mumol/mouse) and DAS (20 mumol/mouse) were evaluated by administering these agents topically 60 min prior to each BPO application. After 26 weeks on test, the number of benign papillomas/mouse were 0.10 +/- 0.07 and 2.15 +/- 0.30 in the NDGA and DAS pretreated group of animals as compared to 4.40 +/- 1.14 in animals receiving BPO alone. After 51 weeks on test, the number of squamous cell carcinomas/mouse were 0.00 +/- 0.00, 0.35 +/- 0.10 in the NDGA and DAS pretreated group of animals as compared to 0.65 +/- 0.12 in animals receiving BPO alone. From these data we suggest that the antioxidants NDGA and DAS can abrogate the tumor-promoting effects of BPO in murine skin and that NDGA is substantially more effective than DAS in this regard.

Antitumor activity in skin of Skh and Sencar mice by two dietaryβ‐carotene formulations

There is currently a great interest in the protective potential of beta-carotene and other micronutrients against carcinogenesis. We investigated the role of beta-carotene in modifying 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted, two-stage skin carcinogenesis. We were interested in comparing the protective effects of two types of dietary beta-carotene, a beadlet formulation and crystalline beta-carotene, in two strains of mice (Skh:HR-1 and CR:ORL Sencar). Mice were maintained throughout the study on one of these 3% beta-carotene-fortified diets or on control diets. In Week 11 after the start of the diets, the DMBA/TPA treatment regimen was begun. The resulting skin tumors were counted weekly. In addition, serum and skin levels were monitored for beta-carotene at the time of chemical initiation and at the termination of the study. A decrease in the number of cumulative tumors in the beta-carotene-fed animals compared with the appropriate control groups was observed in both strains of mice. However, statistical evaluation of the data revealed that the decrease was significant only in Skh mice. This phenomenon might be explained by the inherent sensitivity of Sencar mice to the two-stage carcinogenesis treatment regimen. The mechanism of the protective effect found in this study is still not clear. Recent data suggest that a vitamin A pathway is not probable but that a direct 1O2 and/or radical-quenching property of the parent beta-carotene molecule may be involved. This study also demonstrates that two-stage-induced skin tumorigenesis can be modified by both types of beta-carotene-fortified diets.

Modulation of the co-promoting activity of gamma interferon in SENCAR and C57BL/6 mouse skin by difluoromethyllornithine and the scheduling and duration of interferon treatment

The murine skin multistage carcinogenesis model was used to characterize the co-promoting and tumor progressing activities of i.p. administered recombinant DNA-derived murine gamma interferon (rMuIFN-gamma). The dorsal skins of female SENCAR mice were topically initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice a week for 20 weeks with 1 microgram of 12-O-tetradecanoylphorbol-13-acetate (TPA). Doses of rMuIFN-gamma that had no effect on papilloma multiplicities when administered 1 day prior to TPA treatment increased the numbers of papillomas per mouse by 33-38% when administered immediately prior (zero time) to TPA application. A minimum of 6 weeks of co-treatment with TPA and rMuIFN-gamma (zero time) were necessary for demonstration of rMuIFN-gamma-dependent co-promotion. The ad libitum administration of either 0.25 or 1% (w/v) solutions of alpha-difluoromethylornithine (DFMO) in the drinking water inhibited by 90% the TPA-dependent elevation of epidermal ornithine decarboxylase activity but had minimal effect on papilloma multiplicities in TPA-promoted mice. However, both doses of DFMO completely suppressed rMuIFN-gamma-dependent co-promotion. Carcinoma incidence and multiplicities by weeks 46-48 of the promotion-progression period were statistically indistinguishable for initiated mice treated with TPA, TPA + DFMO, TPA + IFN-gamma or TPA + DFMO + IFN-gamma. Similarly, i.p. administration of rMuIFN-gamma to papilloma-bearing mice in a tumor progression study, with and without simultaneous topical TPA treatment, did not affect carcinoma latency or carcinoma multiplicities. C57BL/6 mice initiated with DMBA developed few papillomas (0.2 paps/mouse) after 19 weeks of TPA promotion. The i.p. administration of rMuIFN-gamma to C57BL/6 mice at the time of TPA treatment, at doses that were co-promoting in SENCAR mice, did not increase papilloma multiplicities. Collectively, our studies suggest that the co-promoting activity of rMuIFN-gamma is exceptionally sensitive to inhibition by DFMO and dependent upon the scheduling and duration of rMuIFN-gamma treatment, and the mouse strain/stock employed for the studies.

Conversion of Xanthine Dehydrogenase to Xanthine Oxidase Occurs During Keratinocyte Differentiation: Modulation by 12-O-Tetradecanoylphorbol-13-Acetate

The distributions of xanthine dehydrogenase (XD) and xanthine oxidase (XO) in subpopulations of murine keratinocytes differing in their stages of terminal differentiation were determined by enzymatic analyses. Keratinocytes were isolated from the skins of female SENCAR mice that had been treated 72 h earlier with either acetone or 12-O-tetradecanoylphorbol-13-acetate (TPA). The ratio of XO/(XD + XO) specific activities was used as an index of the XD to XO conversion. The XO/(XD + XO) ratios for basal cell, suprabasal cell, granular cell plus squamae, and horny sheet preparations isolated from acetone- or TPA-treated mice were 0.35, 0.35, 0.45, 0.75 and 0.28, 0.29, 0.58, and 1.0, respectively. Total XD + XO and XO specific activities in each subpopulation derived from TPA-treated mice were approximately twice the values measured in their control counterparts. Suspension culturing of basal cell keratinocytes in methylcellulose induced terminal differentiation and a conversion of XD to XO. The kinetics of keratin disulfide crosslinking and the XD to XO conversion were similar and preceded cornification. Collectively, these studies demonstrate that the conversion of XD to XO occurs primarily during the later stages of keratinocyte terminal differentiation. Furthermore, the increases in XO activity measured in epidermal homogenates after TPA treatment are due to TPA-dependent increases in 1) the relative proportions of keratinocytes undergoing differentiation, 2) tissue XD content, and 3) increased conversion of XD to XO.

Induction of metallothionein mRNA by tumor promoters in mouse skin and its constitutive expression in papillomas

A single topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) was found to induce mRNA of a metallothionein (MT) gene or genes in the skin of Sencar mice, and papillomas produced by repeated applications of TPA were shown to have elevated levels of MT mRNA. Induction of MT mRNA was maximal 4-8 h after application of TPA and returned to the control level 24 h later. A dose-dependent increase of MT mRNA was observed with doses of TPA of 1-5 micrograms. Of the other promoters tested, phorbol-12, 13-didecanoate, mezerein, and the ionophore A23187 also induced MT mRNA, but 4-O-methyl-TPA and benzoyl peroxide did not. Phorbol and 4 alpha-phorbol-12,13-didecanoate, which are not promoters, also did not induce MT mRNA. Retinoic acid and 1 alpha, 25-dihydroxyvitamin D3, inhibitors of tumor promotion, did not induce MT mRNA themselves or inhibit the induction of MT mRNA by TPA. In C57BL/6 promotion-resistant mice, TPA caused only slight induction of MT mRNA. These data suggest a correlation between induction of MT mRNA and epidermal hyperplasia. The constitutive elevation of MT mRNA levels in papillomas may be due to the loss, during the process of tumor promotion, of some mechanism regulating MT gene expression.

Histologic Alterations Produced by Chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) in SENCAR Mouse Skin: Relationship to Skin Tumor Promoting Activity

Histologic changes induced in SENCAR skin following a single treatment with chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) exhibited differences in time course from that observed with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although not significantly different, maximum elevations in epidermal thickness, total number of nucleated epidermal cells, and dark basal keratinocytes (DCs) induced by 220 nmol chrysarobin occurred at 96 h after treatment, while those induced by 3.4 nmol TPA occurred at 48 h. Both compounds elicited comparable inflammatory responses. Twice-weekly applications of chrysarobin for 2.5 weeks induced a moderate hyperplasia, increase in total nucleated epidermal cells, and increased DCs at 48 and 96 h after the last treatment, with a higher value for these parameters occurring at 48 h. Interestingly, the magnitude of these changes was similar to that observed after a single application. In contrast, twice-weekly applications of TPA induced a dramatic, potentiated induction of epidermal hyperplasia and DCs. Once-weekly applications of chrysarobin led to a potentiated induction of both hyperplasia and DCs compared to the twice-weekly treatment regimen and also more effectively promoted epidermal papillomas in previously initiated SENCAR mice. Skin sections from mice treated with chrysarobin displayed overt signs of epidermal toxicity including altered basal cell morphology and a decreased number of basal cells per 125 micron of basement membrane. Hyperplasia induced by multiple but not single treatments with chrysarobin and TPA correlated quantitatively with their papilloma promoting activity. In addition, the data suggest that epidermal toxicity may play a role in tumor promotion by anthrones.

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 induction of ODC (ornithine decarboxylase) activity by TPA (12-O-tetradecanoylphorbol-13-acetate) in mouse skin. When applied topically to the shaved skin of SENCAR mice at dosages of 10-40 mumol per animal, 30 min before the superficial application of 8.5 nmol of TPA, sphingosine sulfate dramatically inhibited the induction of ODC activity by the tumor promoter. Significant inhibition of TPA-induced ODC activity was observed at 4, 6 and 8 h after TPA treatment in separate studies. The results indicate that sphingosine sulfate is an effective inhibitor of ODC induction by TPA in mouse skin.

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

The induction of epidermal ornithine decarboxylase (ODC) activity by benzoyl peroxide (BPO) was characterized to evaluate the usefulness of this effect as a short-term marker of BPO-induced mouse skin tumor promotion. The maximal induced levels of ODC specific activity, after a single topical dose of BPO, were greater than 2-fold higher when a cold scraping method was used to prepare epidermis rather than the commonly used heat treatment method. Therefore, the cold scraping method was used for all the work reported here. Application of a single 20 mg dose of BPO to the dorsal skin of SENCAR mice caused a relatively small induction of epidermal ODC activity, to a level less than 1/40 that induced by a single 2 micrograms dose of 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the time-courses of induction were different after single doses of TPA and BPO, with peak activities observed at approximately 6 and approximately 24 h after treatment respectively. In contrast, after a total of five 20 mg doses of BPO were topically applied (one dose every 2 days), ODC activity was transiently induced to an average level greater than 15 times after a single dose. Additionally, on this dosing regimen, the peak of ODC activity shifted to approximately 4 h after the last treatment, so that the time-course of ODC induction resembled that after multiple applications of TPA. The extent of epidermal ODC induction by BPO was found to be a complex function of the frequency of dosing and the number of treatments. However, when BPO treatments were administered from 1 to 7 days apart, similar maximal induced levels of ODC activity were eventually achieved after application of multiple doses. Importantly, the dose-response for the induction of ODC activity by five doses of BPO (applied one dose every 2 days) was highly correlated with published data on the dose-response for tumor promotion by this organic peroxide, indicating that ODC induction is a good short-term marker of BPO-induced tumor promotion in SENCAR mice.

N-Nitrosocimetidine as a modifier of chemically-initiated tumors in mice

N-Nitrosocimetidine (NCM) is a nitrosation product of cimetidine, a commonly-prescribed pharmaceutical agent. In spite of its known genotoxicity, NCM has failed to cause tumors in assays with rats and mice, but has given indications of enhancing or suppressive effects on tumor development. This possiblity was tested by administering NCM topically to the skin or in the drinking water to mice in which tumors had been initiated by treatment with chemical carcinogens. Sencar mouse skin papillomas initiated by 7,12-dimethylbenzanthracene (DMBA) and promoted by 12-O-decanoylphorbol-13-acetate (TPA), progressed more rapidly to carcinoma on mice given treatment during stage 3 (after TPA) with NCM (1 mg/week) or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG, 120 g/week) than on stage 3 acetone controls. Oral NCM (1 g/l drinking water) did not have this effect but rather suppressed development of keratoacanthomas, as did stage 3 MNNG or TPA. Primary lung tumors initiated in BALB/c mice by i.p. injection of urethane; and tumors of forestomach, lung, mammary, lymphoid and skin tissues caused in ( C57BL 6 × DBA 2 )F 1 mice by oral DMBA were not markedly affected by NCM given in drinking water (1000–1800 ppm) until 14–16 months of age. These results confirm NCM's general lack of activity as an in vivo toxicant, but show that under certain circumstances it may enhance or suppress tumor development

Inhibition of the skin tumorigenicity of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene by tannic acid, green tea polyphenols and quercetin in Sencar mice

The effect of pretreatment of skin of Sencar mice with topically applied tannic acid, quercetin and green tea polyphenols (GTP) on the skin tumor initiating activity of (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPDE-2) has been evaluated. The animals were pretreated with the plant phenols (tannic acid and quercetin (3000 nmol) or GTP 24 mg/mouse) for 7 days after which they received a single topical application of 200 nmol of BPDE-2 as the initiating agent. Beginning 7 days following initiation animals received twice weekly applications of 3.24 nmol of 12- O-tetradecanoyl phorbol-13-acetate (TPA). Tannic acid and GTP afforded significant protection against skin tumor induction. These inhibitory effects were verified both by prolongation of the latency period and subsequent development of tumors. Quercetin, on the other hand, afforded only moderate protection. Each phenolic compound was found to be highly effective in accelerating the disappearance of BPDE-2 from aqueous medium. Our results suggest that tannic acid and GTP have substantial potential for protecting against the skin tumorigenic response to BPDE-2 and the mechanism of inhibition may involve inactivation of the reactive carcinogenic moiety.

Effects of retinoids and inhibitors of arachidonic acid metabolism on tumor-promoter-induced soft agar colony formation of mouse epidermal cells and rat urinary bladder cells.

Effects of retinoids and inhibitors of arachidonic acid metabolism on tumor‐promoter‐induced soft agar colony formation of mouse epidermal cells and rat bladder cells were evaluated. Topical application of retinoic acid, an anti‐tumor‐promoter, to female SENCAR mouse skin inhibited 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced soft agar colony formation of mouse epidermal cells, an event proposed to be essential for tumor promotion. Effects of dietary retinyl acetate, nordihydroguaiaretic acid (NDGA) and quinacrine hydrochloride on colony formation of rat bladder cells were then examined. Male Fischer 344 rats were given 0.05% N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine for 3 weeks, followed immediately by the administration for 9 weeks of 5% sodium saccharin supplemented with or without 0.05% retinyl acetate, 0.1% NDGA or 0.01% quinacrine hydrochloride. Saccharin‐induced colony growth was significantly inhibited by the administration of retinyl acetate or NDGA, suggesting that these two agents have anti‐tumor‐promoting effects on rat bladder carcinogenesis. Thus, the colony‐forming assay might be useful for early detection of anti‐tumor‐promoters of skin and bladder.

Qualitative and quantitative effects on epidermal Langerhans (Ia+) and Thy-1+ dendritic cells following topical application of phorbol diesters and mezerein.

Repeated twice-weekly applications of promoting doses of 12-O-tetradecanoylphorbol-13-acetate (TPA) to the dorsal skin of female adult SENCAR mice led to a reduction in numbers per unit area of epidermal Thy-1+ dendritic cells. Although no parallel effect was observed on Ia+ Langerhans cells, a concurrent reduction of dendritic morphology of both cell types was observed. Topical administration of TPA (2 micrograms) twice weekly for 4 or 8 weeks led to reductions in Thy-1+ cell numbers of 52 and 61%, respectively, whereas a single treatment was without effect. Similar effects were observed in animals initiated with 10 nmol 7,12-dimethylbenz[a]anthracene suggesting that the response was specific to the promotion, rather than the initiation, phase of two-stage tumorigenesis. All initiated animals bore tumors after 16 promoter treatments. In comparison with the potent promoter TPA, the weak overall, but stage-specific promoters 4-O-methylTPA and mezerein showed no effects on number or morphology of either dendritic cell type. These findings are therefore consistent with an important role for quantitative and qualitative alterations in epidermal non-keratinocytes of immune function in tumor promotion.

EFFECTS OF 9 MUTAGENIC PYROLYSATES, 5 FOODSTUFFS, 4 ANTI-TUMOR DRUGS AND 6 HEAVY METALS IN A TWO-STAGE MOUSE SKIN MODEL

Nine mutagenic heterocyclic amines, 5 foodstuffs, and 4 anti-tumor drugs were tested for tumor initiating activity in a two stage skin carcinogenesis model using 12-O-tetradecanoyl phorbol-13-acetate (TPA) as the promoter. All compounds were topically applied twice weekly for 5 weeks on the dorsal skin, and then followed by similar TPA administration for 47 weeks. Test chemical groups with TPA promotion induced skin tumors in 5 to 35% of the mice, wheter no tumors appeared in the groups treated with test chemicals alone or TPA alone. Statistical analysis according to either Fisher exact test or Peto trend test revealed significant differences for tumor appearance in the Trp-P-1, Trp-P-2, MeAαC, Phe-P-1, AF-2, and BHA followed by TPA groups as compared to the TPA alone. Zinc Chloride, chromic chloride, cadmium chloride, nickel chloride, mercuric chloride, and arsenic acid were tested for possible tumor promoting activity in the skin of emale SENCAR mice pretreated with 7, 12-dimethylbenz (a) anthracene (DMBA). At a dose of 200 μg applied twice weekly for 26 weeks, none of the metal compounds tested enhanced skin tumor development. They also showed no tumor-developing potency when continuously applied in the smae manner without DMBA initiation. The results indicated that Trp-P-2 and AF-2 have an initiating activity, and Trp-P-1, MeAαC, Phe-P-1 and BHA show possible initiating potency in two-stage mouse skin model, but the heavy metal compounds tested do not have any tumor-promoting or tumorigenic activity in the mouse skin.

The influence of a hyperthermia treatment on chemically induced tumor initiation and progression in mouse skin.

A single hyperthermia treatment given near the time of initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz[a]anthracene (DMBA) increased the number of initiated cells in the skin of SENCAR mice subjected to a two-stage tumorigenesis protocol. In animals subsequently promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA), a 44 degrees C, 30-min hyperthermia treatment given just before, just after or 24 h before MNNG initiation increased the average papilloma frequency by 40-50%. In the groups of animals that received a hyperthermia treatment just before MNNG initiation, tumor latency was reduced by 40-60%. Treatment with MNNG in the absence of hyperthermia produced two classes of initiated cells. One type, formed in low yield, was independent of TPA promotion and formed tumors with a high probability of progression to malignancy. The other type was promotion dependent, and formed in relatively high yield but produced tumors with a probability of progression to carcinomas approximately 10-fold less than promotion-independent initiated cells. A single hyperthermia treatment given just before or just after MNNG initiation increased the yield of both promotion-dependent and promotion-independent initiated cells, and consequently increased the yield of carcinomas. In animals given a single hyperthermia treatment 24 h prior to initiation (to induce thermotolerant skin cells), MNNG exposure resulted in an increased yield of promotion-dependent initiated cells but no change in the yield of promotion-independent initiated cells. Hyperthermia treatment of DMBA-initiated skin increased the yield of initiated cells (promotion-dependent) only when given just after exposure to the initiator. The extra initiated cells produced by hyperthermia treatment of MNNG or DMBA exposed skin had the same probability of progression to carcinomas as initiated cells produced by the same initiation in the absence of hyperthermia. As noted previously for DMBA-initiated mice, hyperthermia given at the time of each application of TPA promoter also suppressed the formation of papillomas initiated by MNNG. Only the promotion and progression of promotion-dependent initiated cells, and not of promotion-independent cells, was suppressed. The results show that a single hyperthermia treatment near the time of exposure to an alkylating agent increased the number of both promotion-dependent and promotion-independent initiated cells and, as a consequence, increased the risk of carcinogenesis associated with that exposure.(ABSTRACT TRUNCATED AT 400 WORDS)