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In vitro changes of normal human keratinocytes (NHKs) derived from the oral mucosa after treatment with the chemical carcinogen 7,12 dimethylbenz[a]anthracene (DMBA; 5, 50, 200 ng/10 ml) were evaluated. NHKs were also treated with chemopreventive nutrient agents that previously had enhanced growth of epidermal and oral keratinocytes or suppressed growth of oral squamous cell carcinoma. These agents included the carotenoids beta-carotene and canthaxanthin and the retinoid retinyl palmitate (60 microM). Plating efficiency, growth in agarose (independent growth), viability [tetrazolium salt (MTT) assay], and proliferation ([3H]thymidine labeling) defined the growth of NHKs. The number of cornified cells and keratin expression (high-molecular-weight keratin) defined differentiation. gamma-Glutamyl transpeptidase, p53 expression, and tumorigenesis in mice defined oxidation and malignant transformation. Treatment with DMBA (50 ng/10 ml) was detected by autofluorescence; it produced an increase in pleomorphism and multinucleation and enhanced plating efficiency and the number of colonies grown in agarose. Chemopreventive treatment enhanced the number of colonies grown in agarose, but the MTT levels and [3H]thymidine incorporation-proliferation (24 h) were reduced. Chemopreventives also increased differentiation defined by the number of cornified cells and the expression of high-molecular-weight keratin-positive cells. Malignant transformation potential was depressed by reducing gamma-glutamyl transpeptidase and mutant p53 expression, whereas tumor suppressor p53 was enhanced. NHKs treated with DMBA and injected into nude mice (nu/nu: 1 x 10(6) cells/0.25 ml) produced tumor masses (3 of 3 animals), whereas the nutrient and DMBA groups produced smaller tumor masses, some with central ulcers (2 of 3 animals). Mock injection of untreated or nutrient-treated NHKs without DMBA treatment did not produce a tumor mass (0 of 3 animals). beta-Carotene, retinyl palmitate, and canthaxanthin increased differentiation and reduced transformation induced by DMBA in oral NHKs.

Using mouse skin papilloma as a model system, we examined whether the antitumorigenic activity of carotenoids was related to their provitamin A activity. Oral administration of canthaxanthin (CX) or beta-carotene at 200 mg/kg/day for 14 days significantly reduced the cumulative size of papillomas induced on the skin by 9,10-dimethyl-1,2-benzanthracene (p < 0.05), after the accumulation of these carotenoids in the tumors. The levels of a protooncogene, c-myc, were simultaneously suppressed in papillomas in carotenoid-treated mice. Because CX cannot be converted metabolically to retinoids, these results suggested that CX directly inhibited the growth of papillomas. Neither the accumulation of retinoids nor the expression of a retinoic acid-inducible gene, retinoic acid receptor-beta, was found in papillomas of CX- and beta-carotene-treated mice, suggesting that, like CX, beta-carotene might exert the tumor-suppressing effect without being converted to retinoids. Thus a certain antitumorigenic activity of carotenoids appears not necessarily to require their provitamin A activity.

To test whether beta-carotene or canthaxanthin can modulate the initiation of liver preneoplasia by diethylnitrosamine (DEN) in a sequential protocol of hepatocarcinogenesis, for three weeks male weanling rats were fed diets containing beta-carotene or canthaxanthin (300 mg/kg diet) or excess vitamin A (70,000 IU/kg diet) or were given beta-carotene by injection (9 injections at 10 mg/kg body wt ip). On Day 15, all rats were injected with 200 mg DEN/kg body wt ip; later they were submitted to 2-acetylaminofluorene treatment and to two-thirds hepatectomy, then to phenobarbital treatment, after which gamma-glutamyltranspeptidase- and placental glutathione-S-transferase-positive liver foci were histologically detected. Neither beta-carotene (fed or injected), canthaxanthin, nor an excess of dietary vitamin A had an influence on the number and size of preneoplastic liver foci, despite a significant incorporation and persistence in liver of both carotenoids, especially canthaxanthin, and of supplemental vitamin A. These results are in conflict with another report in which beta-carotene, given to rats during the initiation phase, was found to strongly inhibit DEN-induced hepatocarcinogenesis.

We observed that various retinoids (including all-trans-retinoic acid, 13-cis-retinoic acid, and the synthetic retinoid Ro-11-1430) have approximately the same ability to suppress ultraviolet light-induced transformation of C3H10T1/2 cells in vitro. Retinoids also suppress X-ray induced transformation in vitro. Ro-11-1430 has no effect when present for only 1 day after the X-ray exposure but does have a suppressive effect on radiation transformation when present for 5 or 10 days after irradiation. Ro-11-1430 has its major suppressive effect on X-ray transformation when present in irradiated cultures in the confluent stationary phase of growth. Natural beta-carotene (type IV) from carrots, but not synthetic beta-carotene, has the ability to suppress radiation (X-ray)-induced transformation when present for the entire transformation assay period. Natural beta-carotene is without effect on the transformation process when present in irradiated cultures only during confluence. For these retinoids, as well as beta-carotene and canthaxanthin, there is a highly significant suppressive effect on radiation transformation and radiation transformation enhanced by 12-O-tetradecanoylphorbol-13-acetate when the compounds are present at toxic levels; when nontoxic levels are utilized, these compounds have the ability to suppress the yield of transformed cells to approximately one-half of that observed in irradiated cultures in the absence of these compounds. A selective toxicity for transformed cells appeared to exist for the beta-carotene-treated F-17 cells. This apparent selective toxicity was not observed in another line of transformed cells. Cl 16 cells, or in human cells. We observed different uptake patterns of beta-carotene by nontransformed C3H10T1/2 cells, F-17 cells, and Cl 16 cells that may account for the observed apparent selective toxicity of one line of transformed cells (F-17 cells) to beta-carotene.

In a study of the effects of carotenoids, canthaxanthin (CA), beta-apo-8'-carotenal (BA), or beta-carotene in an extract of Spirulina-Dunaliella algae (AE) was fed at 0%, 0.1%, or 0.2% in a choline-deficient (CD) diet. In each of eight groups, 10 adult male Fischer 344 rats were fed diets with designated carotenoid sources and levels or a choline-sufficient diet for 12 weeks. Carotenoids altered some of the changes induced by the CD diet. Increases in enlargement of fatty livers and low plasma cholesterol levels occurred in rats fed 0.2% BA. Plasma retinol was further reduced 35% by BA or AE. BA and AE increased liver total vitamin A about 80% and 305%, respectively. Liver lipid peroxidation was enhanced and plasma alpha-tocopherol was reduced further by 1.0% AE. AE, BA, and CA (mg/g fat) depressed liver alpha-tocopherol about 49%, 67%, and 78%, respectively. The decreased liver alpha-tocopherol was concurrent with an increase in carotenoid stores of CA > BA > AE. Histopathological examination of sections of liver tissue by light microscopy showed fatty and cirrhotic changes in all rats fed CD diets. Histochemical evaluation based on a semiquantitative assay revealed a marked increase in peroxisome enzyme activity in the livers of all CD rats. None of the carotenoids appeared to have any effect on the development of morphological changes in the liver. Although carotenoids can function as antioxidants, they did not prevent changes observed in rats fed CD diets.

Four carotenoids, canthaxanthin, beta-carotene, 8H-apo-beta-carotenal, and 8'-apo-beta-carotene methylester were tested for their ability to suppress the mutagenicity of 1-methyl-3-nitro-1-nitrosoguanidine and benzo[a]pyrene (BP) in Salmonella typhimurium tester strain TA 100. The anticarcinogenic efficacy of the four carotenoids was further assessed in the BP-induced forestomach tumor model in female Swiss mice. The effect of dietary palm oil was also examined in BP-induced neoplasia in the female Haffkine Swiss mouse strain. Canthaxanthin, beta-carotene, 8'-apo-beta-carotenal, and 8'-apo-beta-carotene methylester showed a dose-dependent decrease in the mutagenicity compared with 1-methyl-3-nitro-1-nitrosoguanidine and BP in strain TA 100. In the BP-induced forestomach tumor model, all four carotenoids showed a similar significant anticarcinogenic effect. Dietary administration of palm oil showed a dose-dependent antitumor activity in the animals. Our results show that the intrinsic antimutagenic and anticarcinogenic properties of the carotenoids are not significantly influenced by their conversion to vitamin A.

The effects of β‐carotene, canthaxanthin, and extracts of tomato paste (containing lycopene) and orange juice (containing cryptoxanthin) on aflatoxin B1 (AFB1)‐induced mutagenesis in S. typhimurium TA 100 and TA 98 were investigated. Inhibition of mutagenesis was studied during and following completion of AFB, metabolism (i.e., after the addition of menadione), thereby permitting separate examination of the metabolic activation and phenotypic expression phases. Each experimental carotenoid, except lycopene, inhibited AFB1‐induced mutagenesis in both tester strains. Cryptoxanthin was the most potent inhibitor, being at least an order of magnitude more potent than the other carotenoids. Inhibition by β‐carotene and canthaxanthin was more prominent during the activation phase, whereas cryptoxanthin was more effective during the subsequent phenotypic expression phase. These inhibitory effects were not dependent on conversion to retinol.

To determine whether carotenoids can modulate xenobiotic-metabolizing enzymes in mice, catalytic activities of several phase I and phase II enzymes have been measured in liver microsomes and cytosol of male Swiss mice fed diets containing beta-carotene, beta-apo-8'-carotenal, canthaxanthin, or astaxanthin (300 mg/kg diet) or treated with 3-methylcholanthrene (3-MC) (3 times at 50 mg/kg ip) for 15 days. Canthaxanthin increased CYP 1A-dependent activities: ethoxyresorufin O-deethylase (EROD) was increased 3-fold, pentoxyresorufin dealkylase (PROD) was increased 2.5-fold, and methoxyresorufin O-demethylase (MROD) was increased 1.6-fold; these increases were much less than those induced by 3-MC, which induced EROD 49-fold, PROD 10-fold, and MROD 4-fold. 3-MC, but not canthaxanthin, also increased relative liver weight, liver P-450 content, NADH-cytochrome c reductase, and benzoxyresorufin dearylase. The three other carotenoids had little or no effect on phase I enzymes. Among the phase II enzyme activities, only NADPH-quinone reductase was slightly increased by 3-MC and carotenoids, except beta-carotene. Among the three carotenoids that have previously been found to be powerful CYP 1A inducers in the rat, i.e., canthaxanthin, astaxanthin, and beta-apo-8'-carotenal, only canthaxanthin shows some (weak) inducing effect of CYP 1A in the 3-MC-responsive Swiss mice, indicating that the mechanism of CYP 1A induction by carotenoids may not be the same as that by 3-MC. In addition, the fact that beta-carotene has no effect on the tested enzymes does not support the hypothesis that the modulation of xenobiotic metabolism is a possible mechanism for the antimutagenic and anticarcinogenic effects of beta-carotene, which have been demonstrated in several in vivo models in mice.

The carotenoids beta-carotene and canthaxanthin and the retinoid 13-cis-retinoic acid (13-RA) have inhibited oral carcinogenesis in the hamster cheek pouch (16 wks, 3 times/wk at 1.4 mg/kg) induced by an 0.5% solution of 7, 12-dimethylbenz[a]anthracene (DMBA). However, 13-RA at a higher dose (2.0 mg/kg per treatment) increased squamous cell carcinoma growth (Eur J Cancer Clin Oncol 24, 839-850, 1988). 13-RA, beta-carotene, and canthaxanthin administered to 60 hamsters (16 wks, 3 times/wk, 10 mg/kg) altered neovascularization characterized by immunohistochemistry for transforming growth factor-alpha (TGF-alpha) and factor VIII. 13-RA + DMBA resulted in more smaller-sized tumors, with a reduced volume and tumor burden (tumor controls, 185.9; 13-RA + DMBA, 151.0). The carotenoids reduced the number and the sizes of the carcinomas formed (beta-carotene, 60 tumors, 142.3 x 10(3) mm3; canthaxanthin, 30 tumors, 116.1 x 10(3) mm3). Factor VIII and TGF-alpha were expressed in high intensity at cancer sites of the 13-RA + DMBA and DMBA groups with50 and10 cells, respectively, per x 400 field. In contrast, beta-carotene- and canthaxanthin + DMBA-treated pouches showed20 and 5 cells, respectively, per x 400 field for factor VIII and TGF-alpha. These results suggest that 13-RA treatment may increase vascular growth, but the carotenoids also produced enhanced levels of endothelial cell growth and TGF-alpha compared with the untreated mucosa. The carotenoids may enhance tumor growth under the appropriate carcinogenic environment.

Astaxanthin, a carotenoid without provitamin A activity, enhances murine T-helper (Th) cell clone-mediated antibody (Ab) production with suboptimal antigen (Ag) challenges. It also suppresses interferon-gamma (IFN-gamma) production by cloned murine Th1 cells. beta-Carotene is less effective than astaxanthin. This study evaluates the effects of various carotenoids with various relative polarity, provitamin A activity, and antioxidant activity. Carotenoids tested include astaxanthin, cantaxanthin, zeaxanthin, lutein, and lycopene, and their effects were tested at a concentration at which astaxanthin's effect was most potent. A.E7 and CDC35 cells are used as representative type 1 and type 2 Th cell (Th1 and Th2) clones, respectively. In the Th1 clone, astaxanthin, but not other carotenoids, suppressed IFN-gamma production and increased the number of Ab-secreting cells with the use of primed spleen cells. With cultures of Th1 cells and unprimed spleen cells, astaxanthin and zeaxanthin augmented the number of immunoglobulin M Ab-secreting cells. In the cultures of Th2 clone and primed spleen cells, astaxanthin, but not other carotenoids, enhanced the number of Ab-secreting cells. With unprimed spleen cells, lycopene suppressed Th2 clone-mediated Ab production. Interleukin-5 production by the Th2 clone was not significantly altered with the carotenoids tested, irrespective of the use of unprimed or primed spleen cells. Carotenoid actions on Th cells may vary in each carotenoid and do not seem to be closely associated with carotenoid antioxidant activity or relative polarity.

It has been suggested that canthaxanthin (CX), beta-carotene, and other carotenoids may inhibit carcinogenesis via antioxidant activity. CX has been shown to inhibit lipid peroxidation in liposomes; this experiment was designed to assess the antioxidant activity of CX in biological membranes. Chicks were fed semipurified diets supplemented with placebo beadlets or CX beadlets (5 g beadlets/kg diet; 0.5 g CX/kg diet) for five weeks. Diets were deficient in vitamin E and selenium to maximize the power of detecting an antioxidant effect of CX and to induce exudative diathesis, which is a vitamin E and selenium deficiency disease of chicks. Dietary CX had no effect on the onset, incidence, or severity of exudative diathesis. Liver homogenates from CX-fed chicks exhibited significantly (p = 0.02) decreased formation of thiobarbituric acid-reactive substances over time in ferrous ion-induced peroxidations. Because dietary CX increased hepatic alpha-tocopherol content, subsequent peroxidations were conducted in membrane fractions prepared from placebo and CX livers matched for alpha-tocopherol content. In this system, CX was marginally but inconsistently protective against peroxidation at both 15 torr O2 (p = 0.12) and 150 torr O2 (p = 0.07). Nanomolar changes in hepatic alpha-tocopherol, unlike CX, substantially altered rates of peroxidation. These results suggest that under these conditions, dietary CX increased resistance to lipid peroxidation primarily by enhancing membrane alpha-tocopherol levels and secondarily by providing weak direct antioxidant activity.

The effects of feeding semipurified diets supplemented with excess retinyl palmitate (20,000 and 100,000 IU/kg), beta-carotene (250 mg/kg), and canthaxanthin (250 mg/kg) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced salivary gland tumors were studied in rats. None of the dietary supplements had a significant effect on tumor incidence. Tumor weights, however, tended to be lower in rats fed the dietary supplements compared with the controls. The incidence of tumor-bearing rats with large tumors was significantly lower in rats fed canthaxanthin than in the control rats. At termination of the experiment, the levels of vitamin A were higher in plasma, liver, normal salivary glands, and the tumor of rats fed diets supplemented with the higher level of retinyl palmitate. Feeding the canthaxanthin-supplemented diet had the opposite effect on tissue and plasma vitamin A levels. beta-Carotene supplementation was associated with higher vitamin A concentrations in the liver but not in plasma, salivary glands, or the tumor. The levels of beta-carotene were increased in tissues and plasma of rats fed the beta-carotene-supplemented diet. The results suggest that in this experimental model, the diet-induced modification of tissue or plasma vitamin A levels did not have an effect on tumor incidence.

An extract of Spirulina-Dunaliella algae was shown to prevent tumor development in hamster buccal pouch when a 0.1% solution of 7,12-dimethylbenz[a]anthracene (DMBA) in mineral oil was applied topically three times weekly for 28 weeks. The algae extract was delivered by mouth in continued dosages of 140 micrograms in 0.4 ml mineral oil three times per week. After 28 weeks, the animals given vehicle and untreated controls all presented gross tumors of the right buccal pouch. Animals fed canthaxanthin presented a notably and statistically significant reduction in tumor number and size compared with controls. Animals fed beta-carotene demonstrated a smaller but statistically significant reduction in tumor number and size. The algae animals presented a complete absence of gross tumors. However, microscopic sections of the buccal pouch in the algae group showed localized areas of dysplasia and early carcinoma-in-situ undergoing destruction.