Helen L. Gensler

Topical vitamin e inhibition of immunosuppression and tumorigenesis induced by ultraviolet irradiation

Ultraviolet (UV) irradiation of C3H/HeN mice induces skin cancer and an immunosuppression that prevents the host from rejecting antigenic UV-induced tumors. The capacity of topical vitamin E (dl-alpha-tocopherol) to prevent photocarcinogenesis or the immunosuppression induced by UV irradiation were assessed. Skin cancer incidence in UV-irradiated mice was 81% at 33 weeks after the first UV exposure; application to mice of 25 mg vitamin E three times per week for three weeks before UV irradiation, and throughout the experiment, reduced this incidence to 42% (p = 0.0065, log rank test). Immunoenhancement by vitamin E was assessed by comparing levels of immunosuppression by splenocytes from normal or UV-irradiated mice, with and without topical vitamin E treatment. Transfer of splenocytes from UV-irradiated mice to naive mice prevented the recipients from rejecting a UV-induced tumor challenge, whereas splenocytes from UV-irradiated mice treated with vitamin E did not prevent recipients from rejecting a similar tumor challenge. Phenotypic analysis of splenocytes used in the passive transfer assay, conducted with a biotin-avidin-immunoperoxidase technique, revealed that vitamin E treatment of mice undergoing UV irradiation prevented the UV-induced down regulation of Ia expression in splenocytes and increased the proportion of Lyt-2+ and L3T4+ splenocytes. Therefore, chronically applied vitamin E can effectively reduce cancer formation and immunosuppression induced by UV irradiation. Prevention of UV-induced down regulation of Ia expression may have contributed to this immunomodulation.

Prevention of photocarcinogenesis by topical administration of pure epigallocatechin gallate isolated from green tea.

Topical application of purified (-)-epigallocatechin-3-gallate (EGCG), a polyphenolic antioxidant isolated from green tea, inhibited photocarcinogenesis in BALB/cAnNHsd mice with no visible toxicity. Mice were treated with 0, 10, or 50 mg of EGCG in 200 microliters of acetone three times weekly for three weeks before ultraviolet (UV) treatments began and throughout the experiment. UV radiation consisted of five 30-minute exposures per week to banks of six FS40 Westinghouse sunlamps for 25 weeks. In the photocarcinogenesis study, mice received a total dose of approximately 2.1 x 10(6) J/m2. Skin cancer incidence in UV-irradiated mice was 96% at 28 weeks after the first UV treatment; EGCG at 10 or 50 mg reduced this incidence to 62% and 29%, respectively. UV-induced immunosuppression, assessed by the inability of UVB-irradiated mice to reject a syngeneic antigenic tumor, was not influenced by topical EGCG. Oral administration of 0, 100, or 500 mg of pure EGCG per liter of drinking water (approximately 0, 0.56, or 2.8 mg/day, respectively) did not decrease UV-induced skin tumor incidence, rate of primary tumor growth, or inability to reject antigenic tumors. Thus induction of skin tumors by UV radiation was significantly reduced by topical, but not by oral, administration of purified EGCG through a mechanism distinct from inhibition of photoimmunosuppression.

Oral niacin prevents photocarcinogenesis and photoimmunosuppression in mice

Topical nicotinamide (niacinamide) has demonstrable preventive activity against photocarcinogenesis in mice. To better understand how this vitamin prevents ultraviolet (UV) carcinogenesis, we tested systemic administration of another form of the vitamin, niacin, and its capacity to elevate cutaneous nicotinamide-adenine dinucleotide (NAD) content as well as to decrease photoimmunosuppression and photocarcinogenesis. BALB/cAnNTacfBR mice were fed the AIN-76A diet supplemented with 0%, 0.1%, 0.5%, or 1.0% niacin throughout the experiment. UV irradiation consisted of five 30-minute exposures per week to banks of six FS40 Westinghouse sunlamps for 22 weeks in the carcinogenesis experiments, yielding a total cumulative dose of approximately 1.41 x 10(6) Jm-2 of UV-B radiation. Dietary supplementation with 0.1%, 0.5%, or 1.0% niacin reduced the control incidence of skin cancer from 68% to 60%, 48%, and 28%, respectively, at 26.5 weeks after the first UV treatment. Two potential mechanisms by which niacin prevents tumor formation were identified. Photoimmunosuppression, critical for photocarcinogenesis, is measured by a passive transfer assay. Syngeneic, antigenic tumor challenges grew to an average of 91.6 +/- 19.7, 79.8 +/- 11.5, 41.9 +/- 11.7, or 13.2 +/- 4.1 mm2 in naive recipients of splenocytes from UV-irradiated mice treated with 0%, 0.1%, 0.5%, or 1.0% niacin supplementation, respectively, demonstrating niacin prevention of immunosuppression. Niacin supplementation elevated skin NAD content, which is known to modulate the function of DNA strand scission surveillance proteins p53 and poly(ADP-ribose) polymerase, two proteins critical in cellular responses to UV-induced DNA damage. These results clearly demonstrate a dose-dependent preventive effect of oral niacin on photocarcinogenesis and photoimmunosuppression and establish the capacity of oral niacin to elevate skin NAD levels.

Importance of the form of topical vitamin E for prevention of photocarcinogenesis

With increasing solar ultraviolet (UV)-B radiation reaching the Earths surface and the incidence of skin cancer rising steadily, there is an ever-increasing need to determine agents that modulate photocarcinogenesis and to understand the mechanisms underlying this modulation. Our laboratory has demonstrated that topical application of the dl-alpha-tocopherol form of vitamin E to mice prevents skin cancer and the immunosuppression induced by UVB irradiation. However, dl-alpha-tocopherol has limited stability at room temperature. The current study was designed to ask whether the thermostable esters of vitamin E, alpha-tocopheryl acetate, or alpha-tocopheryl succinate prevent skin cancer and immunosuppression induced in mice by UV radiation. In the alpha-tocopheryl acetate study, skin cancers developed in 70% of UVB-irradiated control mice and in 90%, 73%, and 90% of mice receiving topical applications of 12.5, 25, and 50 mg of dl-alpha-tocopheryl acetate, respectively. In the alpha-tocopheryl succinate study, skin cancer developed in 59.3% of control UVB-irradiated mice and in 82%, 100%, and 81.5% of mice treated with 2.5, 12.5, and 25 mg d-alpha-tocopheryl succinate, respectively. Thus neither alpha-tocopheryl acetate nor alpha-tocopheryl succinate prevented photocarcinogenesis. At 12.5 and 25 mg/treatment, alpha-tocopheryl acetate and alpha-tocopheryl succinate, respectively, enhanced photocarcinogenesis (p = 0.0114 and 0.0262, respectively, log rank test). On the basis of high-performance liquid chromatography analysis at 16-17 weeks after the first vitamin E treatment, the esterified forms of vitamin E applied epicutaneously accumulated in the skin, but the levels of free alpha-tocopherol remained low. Neither alpha-tocopheryl acetate nor alpha-tocopheryl succinate prevented the induction by UV radiation of immunosusceptibility to implanted syngeneic antigenic UV-induced tumor cells. Thus alpha-tocopheryl acetate or alpha-tocopheryl succinate not only failed to prevent photocarcinogenesis, but may have enhanced to process. Considering that alpha-tocopherol esters are included in many skin lotions, cosmetics, and sunscreens, further studies are needed to determine the conditions under which topical alpha-tocopheryl acetate and alpha-tocopheryl succinate enhance photocarcinogenesis.

Prevention of photoimmunosuppression and photocarcinogenesis by topical nicotinamide

Ultraviolet (UV) B irradiation leads to a potent immunosuppression of the capacity to reject syngeneic, antigenic tumors. If this immunosuppression is critical for the development of most skin tumors, then its prevention should result in prevention of photocarcinogenesis. We previously showed a correlation between the inhibition of photoimmunosuppression and prevention of photocarcinogenesis by dl-alpha-tocopherol, tannic acid, or alpha-difluoromethylornithine. The current study was designed to determine whether topical nicotinamide, the active form of vitamin B-3, or niacin, prevents immunosuppression and skin cancer in UV-irradiated mice. In a passive transfer assay for immunosuppression, splenocytes from UV-irradiated mice enhanced the growth of antigenic tumor challenges in recipient mice. Treatment of the UV-irradiated mice with 40 mumol of nicotinamide twice weekly starting two weeks before UV irradiation and throughout the experiment prevented this immunosuppression. UVB irradiation consisted of five weekly 30-minute exposures to banks of six FS40 Westinghouse fluorescent sunlamps. Mice received approximately 6.2 x 10(5) J/m2 in the passive transfer assays and 1.09 x 10(6) J/m2 in the photocarcinogenesis studies. Application of nicotinamide to UV-irradiated mice reduced skin tumor incidence from 75% to 42.5% (p = 0.016, Cox proportional hazards analysis). Thus topical nicotinamide prevented the immunosuppression and skin tumor induction by UVB irradiation.

Prevention of photocarcinogenesis by dietary vitamin E

Ultraviolet B (UV-B) irradiation of C3H/HeN mice induces skin cancer. In this study, the ability of dietary d-alpha-tocopheryl acetate to reduce photocarcinogenesis was tested in this murine model. Skin cancers developed in 67.5% of UV-B-irradiated mice by 31 weeks after the first UV exposure. Supplementation with 100 or 200 IU of d-alpha-tocopheryl acetate per kilogram of diet led to a reduction of the incidence to 46% and 19%, respectively. The latter value was significantly different from that found in mice fed the basal diet (p = 0.039, one-sided P value by log-rank test). Skin levels of alpha-tocopherol varied with the dietary dose of d-alpha-tocopheryl acetate. No toxicity was evident in unirradiated mice fed the vitamin E-supplemented diet, but 40% of the UV-B-irradiated mice fed 200 IU of vitamin E per kilogram of diet died by 31 weeks after the first UV-B treatment. Decreased relative spleen weight was observed in the UV-B-irradiated mice fed the vitamin E-supplemented diet. In summary, oral d-alpha-tocopheryl acetate prevented photocarcinogenesis, but at doses that were toxic to inbred C3H/HeN mice after exposure to 8.6 x 10(5) J/m2 of UV-B irradiation.

Inhibition of cyclobutane pyrimidine dimer formation in epidermal p53 gene of UV‐irradiated mice by α‐tocopherol

Mutations or alterations in the p53 gene have been observed in 50-100% of ultraviolet light (UV)-induced squamous cell carcinoma in humans and animals. Most of the mutations occurred at dipyrimidine sequences, suggesting that pyrimidine dimers in the p53 gene play a role in the pathogenesis of cutaneous squamous cell carcinoma. We previously showed that topical alpha-tocopherol prevents UV-induced skin carcinogenesis in the mouse. In the present study we asked whether topical alpha-tocopherol reduces the level of UV-induced cyclobutane pyrimidine dimers in the murine epidermal p53 gene. Mice received six dorsal applications of 25 mg each of alpha-tocopherol, on alternate days, before exposure to 500 J/m2 of UV-B irradiation. Mice were killed at selected times after irradiation. The level of dimers in the epidermal p53 gene was measured using the T4 endonuclease V assay with quantitative Southern hybridization. Topical alpha-tocopherol caused a 55% reduction in the formation of cyclobutane pyrimidine dimers in the epidermal p53 gene. The rate of reduction of pyrimidine dimers between 1 and 10 hours after irradiation was similar in UV-irradiated mice, regardless of alpha-tocopherol treatment. Therefore, the lower level of cyclobutane pyrimidine dimers in UV-irradiated mice treated with alpha-tocopherol than in control UV-irradiated mice resulted from the prevention of formation of the dimers, and not from enhanced repair of these lesions. Our results indicate that alpha-tocopherol acts as an effective sunscreen in vivo, preventing the formation of premutagenic DNA lesions in a gene known to be important in skin carcinogenesis.

Prevention byα-difluoromethylornithine of skin carcinogenesis and immunosuppression induced by ultraviolet irradiation

SummaryAdministration ofα-difluoromethylornithine (DFMO) to mice was found to inhibit both the cutaneous carcinogenesis and the immunosuppression induced by ultraviolet B (UVB) irradiation. BALB/cAnNTacfBR mice were given 1% F2MeOrn in their drinking water throughout the experiment. After 3 weeks, mice received UVB irradiation consisting of five 30-min exposures per week to banks of six FS40 Westinghouse sunlamps. In the photocarcinogenesis study, mice received a total dose of approximately 1273 kJ m−2. Skin cancer incidence in UV-irradiated mice was 38% 28 weeks after the first UV exposure; DFMO reduced this incidence to 9% (P= 0.025, log-rank test). Although DFMO has been demonstrated to be chemopreventive of chemical carcinogenesis, this is the first report that it is effective against cancers induced by a physical carcinogen. The immunosuppression induced by UVB irradiation prevents the host from rejecting antigenic, syngeneic UV-induced tumors, which normal mice can reject. The level of immunosuppression in UV-irradiated mice treated with DFMO was measured by a passive-transfer assay. Splenocytes from UV-irradiated mice to naive mice prevented the recipients from rejecting 20/24 UV-induced tumor challenges, whereas splenocytes from UV-irradiated mice treated with DFMO did not prevent recipients from rejecting such challenges (2/24 grew). The difference between these values was significant (P<0.001, two-sample test for binomial proportions). Phenotypic analysis of splenocytes used in the passive transfer, using a biotin-avidin-immunoperoxidase technique, revealed that DFMO treatment prevented the reduction of Ia expression normally seen in UV-irradiated mice. Thus, administration of DFMO reduced skin carcinogenesis and immunosuppression induced by UVB irradiation.

Prevention of photocarcinogenesis and UV-induced immunosuppression in mice by topical tannic acid.

Topical application of tannic acid, a phenolic antioxidant derived from plants, was found to inhibit the cutaneous carcinogenesis and the immunosuppression induced by ultraviolet B (UVB) irradiation with no visible toxicity. BALB/cAnNTacfBR mice were treated with 200 micrograms of tannic acid three times weekly for two weeks before UV treatments began and throughout the experiment. UVB irradiation consisted of five 30-minute exposures per week to banks of six FS40 Westinghouse sunlamps. In the photocarcinogenesis study, mice received a total dose of approximately 1.09 x 10(6) J/m2. Skin cancer incidence in UV-irradiated mice was 75% at 26 weeks after the first UV exposure; tannic acid reduced this to 42%. Immunosuppression induced by UVB irradiation normally prevents the host from rejecting antigenic syngeneic UV-induced tumors. Immunosuppression in these experiments was measured by a passive transfer assay. Tumor challenges grew to an average of 88 +/- 20, 36 +/- 11, and 20 +/- 8 mm2 in naive recipients of splenocytes from UVB-irradiated mice, nonirradiated control mice, and UVB-irradiated mice treated with tannic acid, respectively. Thus topical tannic acid treatment prevented the transfer of enhanced tumor susceptibility with splenocytes from UVB-irradiated mice.

Low level of U.V.-induced unscheduled DNA synthesis in postmitotic brain cells of hamsters: possible relevance to aging.

Abstract DNA repair was measured in brain and liver cells in terms of their ability to undergo unscheduled DNA synthesis (UDS) in response to u.v. radiation. The proportion of brain cells exhibiting u.v.-induced UDS decreased from 13.4 to 4.2% as hamsters aged from 4–8 days to 38–57 days and then remained at this low level at least to day 551, which is approx. 2 3 of the maximum life span of this strain of hamster. Repair synthesis in brain cells during this long period was approx. 9.6% of that found in adult lung cells, 22.4% of that found in adult kidney cells and, at most, 50% of that in adult liver cells. This suggests that the postmitotic brain has a low repair capacity compared to other tissues. If this rate of repair is less than the rate at which endogenous DNA damage occurs, then such damage would accumulate and perhaps contribute significantly to normal aging.