The widespread use of FVB/N mice for the establishment of transgenic lines containing active oncogenes suggested the importance of testing the parent FVB/N mice for sensitivity to experimental carcinogenesis. After initiation of mouse skin by a single treatment with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion by 20 weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), the skin tumor incidence was compared in FVB/N mice, TPA-sensitive (SENCAR and CD-1) and TPA-resistant mice (BALB/c and C57BL/6). Initiation by 25 micrograms DMBA followed by promotion with a low dose of TPA (2 micrograms/week) induced one or more papillomas in only 25% of FVB/N mice, compared with 100% in SENCAR, 53% in CD-1, 17% in BALB/c and 0% in C57BL/6 mice. At a more effective dose of TPA (5 micrograms/week), FVB/N mice initiated by 5, 25 or 100 micrograms DMBA developed 3.4, 6.9 and 11.8 papillomas per mouse. In contrast, the incidence of squamous cell carcinomas (SCCs) (17-18/30 mice) did not increase with DMBA dose. TPA promotion of non-initiated mice induced only six papillomas, but three progressed to SCCs, a high rate of malignant conversion. Skin tumor induction by 20 weekly treatments with 10 micrograms DMBA produced few papillomas, but 50.0% of the papillomas progressed to carcinomas in FVB/N mice, compared with 9.15% in SENCAR, 37.5% in CD-1, 23.1% in BALB/c and 15.0% in C57CL/6 mice. The first carcinomas appeared after 14 weeks in FVB/N, 24 weeks in SENCAR, 26 weeks in CD-1 and C57BL/6 and 34 weeks in BALB/c mice. Thus, FVB/N mice develop an unusually high incidence of SCCs after treatment with repeated DMBA, DMBA initiation-TPA promotion and even TPA alone.

SENCAR mice were used to determine the effects of the provitamin A compound beta-carotene on papilloma formation and the conversion of papillomas to carcinomas in a two-stage protocol with one application of the initiator 7,12-dimethylbenz[a]anthracene (DMBA, 20 micrograms) and 20 weekly applications of the promotor 12-O-tetradecanoylphorbol-13-acetate (TPA, 2 micrograms). A purified vitamin A-free diet was supplemented with beta-carotene at four levels (0.6, 6, 60 and 600 micrograms/g of diet) for female mice and two levels (60 and 600 micrograms/g) for male mice. Dietary supplementations of beta-carotene did not result in significant changes in body weight and survival of female and male mice. However, papillomas developed more rapidly and papilloma incidence (% mice with papillomas) reached its maximum (100%) sooner in male mice fed 600 micrograms of beta-carotene/g of diet than those fed 60 micrograms/g. There were smaller differences in papilloma incidence among the dietary groups in female mice, but the papilloma incidence again reached 100% sooner in mice fed 600 micrograms of beta-carotene/g of diet. Female and male mice fed 600 micrograms of beta-carotene/g of diet had significantly higher papilloma yields (average number of papillomas/mouse) than other dietary groups and a very low percentage of these papillomas converted to carcinomas in these mice. Thus, beta-carotene at 600 micrograms/g inhibited the conversion of papillomas to carcinomas in both sexes. In addition, papilloma yields were higher in female mice and these papillomas regressed more quickly than those in the corresponding groups of male mice. In conclusion, dietary beta-carotene caused differential effects on papilloma and carcinoma yields and sex-dependent differences in papilloma formation in female and male SENCAR mice treated with DMBA and TPA in a two-stage carcinogenesis protocol.

We have previously reported that high dietary retinoic acid (RA; 30 micrograms/g diet) inhibits carcinoma formation in a two-stage skin carcinogenesis protocol, using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiator and 12-O-tetradecanoyl phorbol-13-acetate (TPA) as the tumor-promoter in female SENCAR mice. We next asked whether switching the diets from high to control levels of RA and vice versa would influence carcinoma formation. Mice at 3 weeks of age were initiated with DMBA (20 micrograms) once, followed by 20 weekly applications of TPA (2 micrograms). At 3 weeks of age mice were weaned onto a diet containing either 3 (control) or 30 (high) micrograms RA/g diet. Half of the mice from either dietary group were switched to the other diet at 20 weeks of age, when papilloma formation was at its peak. These four groups are designated RA 3 micrograms, RA 30 micrograms, RA 3/30 micrograms and RA 30/3 micrograms groups. As previously found, papilloma formation (including incidence and yield) was not significantly affected by dietary treatment. However, high dietary RA inhibited carcinoma formation; specifically cumulative carcinoma incidence (18.5-23.1% versus 50%) and yield (0.19-0.23 versus 0.68) were significantly lower (P < 0.05) in the high dietary RA treatment groups than the RA 3 micrograms control group, as was the carcinoma conversion efficiency (2.1-3.8% versus 9.4%). The beneficial effect on carcinoma formation was still evident when excess RA was given late during the carcinogenesis process (i.e. the RA 3/30 micrograms group). Moreover, a residual effect of excess RA was also seen after the dietary RA was switched to the control level at 20 weeks of age, when papilloma yield was highest (i.e. the RA 30/3 micrograms group). It is therefore concluded that the chemopreventive effect of high dietary RA on skin carcinogenesis induced by a two-stage carcinogenesis protocol with DMBA and TPA resides mainly at the step of conversion from benign papillomas to malignant carcinomas.

Previously we have shown that dietary retinoids are essential for papilloma formation induced by either an initiation-promotion or a complete skin carcinogenesis protocol. The present study was conducted to further determine the effect of dietary retinoic acid (RA) on papilloma formation and the conversion of papillomas to carcinomas. Skin tumors were induced in 3 week old female SENCAR mice by an initiation-promotion protocol with one application of 20 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA), followed by 20 weekly applications of 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA). Mice were fed RA at one of the three doses: 0.3 (nutritionally marginal dose), 3 (near physiological) and 30 (pharmacological) micrograms/g of diet. Mice fed 30 micrograms of RA/g of diet had the same survival rate as the other two groups despite a lower body weight and all three groups had similar papilloma incidence, which reached 100% at age 18 weeks. Mice fed 3 micrograms of RA/g of diet had the highest papilloma yield (approximately 14 papillomas/mouse) of all groups and it peaked between weeks 18 and 38 of age. These papillomas later regressed such that mice from all three groups had about the same papilloma yield at week 44 of age. Mice fed 30 micrograms of RA/g of diet failed to develop any visible carcinoma, while mice fed 0.3 or 3 micrograms/g showed 1.9% conversion of papillomas to carcinomas. Therefore, dietary RA at 30 micrograms/g of diet inhibited the conversion of papillomas to carcinomas without affecting papilloma incidence. In addition, dietary RA at 30 and 0.3 micrograms/g of diet lowered papilloma yield.

Using 7,12-dimethylbenz[a]anthracene as the initiator and 12-O-tetradecanoyl-13-acetate as the tumor promoter on the dorsal skin of Sencar mice, we previously showed that pharmacological dietary all-trans-retinoic acid and beta-carotene inhibit the conversion of papillomas to carcinomas in a two-stage system of chemical carcinogenesis. The purpose of this study was to determine the influence of dietary retinoic acid and beta-carotene on retinoid and beta-carotene concentrations in skin and other tissues. We were unable to measure tissue retinoic acid because of the relatively limited amount of tissue available for analysis and the fast rate of metabolism. Different dietary levels of retinoic acid or beta-carotene did not influence total retinol of skin, papilloma, and carcinoma tissues, which all showed a concentration of approximately 1 +/- 0.5 microgram/g wet wt. Equally refractory to dietary retinoic acid or beta-carotene was serum retinol concentration. In contrast, dietary retinoic acid protected loss of liver retinol and retinyl palmitate, and beta-carotene caused an increase in beta-carotene and retinyl palmitate in liver but did not affect serum and liver retinol. We further investigated metabolic and functional aspects of retinoic acid in cultured mouse epidermal keratinocytes (LC-8 cells) and found that these cells actively metabolized [10,11-14C]retinoic acid to polar compounds. Isomers of retinoic acid were a minor product in the presence of cells and the major product when incubated in serum-containing medium in the absence of cells. From the functional point of view, exposure of LC-8 cells to 3 x 10(-6) M all-trans-retinoic acid (RA) caused a 75-fold induction in tissue transglutaminase and an approximately 9-fold induction in 10(-6) M RA at three days of culture. We conclude that retinoic acid spares endogenous retinol and that beta-carotene greatly enhances liver retinyl palmitate levels. Moreover we show that although mouse epidermal cells metabolize retinoic acid at a very high rate, they respond functionally by induction of tissue transglutaminase activity. Because this enzyme has been suggested to be involved in programmed cell death, we are presently investigating the possibility that it may be involved in the inhibition of carcinogenesis in mice fed pharmacological doses of RA.