Levels of retinyl palmitate and retinol in the skin of SKH-1 mice topically treated with retinyl palmitate and concomitant exposure to simulated solar light for thirteen weeks.

Retinyl esters account for more than 70% of the endogenous vitamin A found in human skin, and retinyl palmitate is one of the retinyl esters in this pool. Human skin is also exposed to retinyl palmitate exogenously through the topical application of cosmetic and skin care products that contain retinyl palmitate. To date, there is limited information on the penetration and distribution of retinyl palmitate and vitamin A within in the skin. In this study, the accumulation of retinyl palmitate and generation of retinol in the skin of male and female SKH-1 mice that received repeated topical applications of creams containing 0.0%, 0.1%, 0.5%, 1.0%, 5.0%, 10%, or 13% of retinyl palmitate 5 days a week for a period of 13 weeks were studied. Because products containing retinyl palmitate are frequently applied to sun-exposed skin, and because it is well established that exposure to sunlight and UV light can alter cutaneous levels of retinoids, mice in this study were additionally exposed 5 days a week to simulated solar light. The results showed that retinyl palmitate diffused into the skin and was partially hydrolyzed to retinol. The levels of retinyl palmitate in the skin of mice that were administered retinyl palmitate cream were higher than control values, and levels of both retinyl palmitate and retinol increased with the application of higher concentrations of retinyl palmitate in the cream. Our results indicate that topically applied retinyl palmitate may alter the normal physiological levels of retinyl palmitate and retinol in the skin of SKH-1 mice and may have a significant impact on vitamin A homeostasis in the skin.

Photoreaction, phototoxicity, and photocarcinogenicity of retinoids.

Sunlight is a human carcinogen. Many retinoid-containing cosmetics are used to protect damages caused by sunlight irradiation. Since retinol is thermally unstable and retinyl palmitate (RP) s relatively more stable, RP is also widely used as an ingredient in cosmetic formulations. In general, little is known about the photodecomposition of retinoids and the toxicity of retinoids and their photodecomposition products on the skin's responses to sunlight. This review focuses on the update information on photoreactions, phototoxicity, and photocarcinogenicity of the natural retinoids including retinol, retinal, retinoid acid (RA), retinyl acetate, and RP.

Basal cell proliferation in female SKH-1 mice treated with alpha- and beta-hydroxy acids.

Alpha- and beta-hydroxy acids are compounds that have been used extensively in cosmetic and dermatological formulations. Clinical and qualitative effects of alpha- and beta-hydroxy acids have been well characterized, but little is known about their mechanism of action or acute and chronic biochemical effects. In the present study, we examined the acute proliferative effects of glycolic and salicylic acids on cell proliferation in the epidermis of SKH-1 female mice, using BrdU incorporation as a marker of epidermal proliferation. In preliminary experiments, we observed an increase in the rate of proliferation after 3 days of treatment with 10% glycolic acid-containing cream and this was sustained throughout a 6.5-week (treatment 5 days/week) time course compared with untreated control animals. After each treatment with cream containing glycolic acid there was a wave of proliferation that was maximal 12 to 16 h (significant at p < 0.05) after treatment, followed by a subsequent increase in epidermal thickness at 18 to 20 h (significant at p < 0.05). The effects of the concentration and pH level of glycolic acid- and salicylic acid-containing creams on the rate of proliferation and increases in skin thickness in SKH-1 epidermis were also investigated. We observed a dose-dependent increase in epidermal proliferation of animals treated with either glycolic or salicylic acid. A similar time-dependent response was observed in the epidermal thickness in animals treated with salicylic acid, but not with glycolic acid. Differences in pH (3.5 or 4.0) had no significant effect on either epidermal proliferation or skin thickness. The data that we present here should be useful in characterizing not only the beneficial but also the adverse effects that occur following acute or chronic usage of alpha-hydroxy acids.

beta-Carotene uptake and effects on intracellular levels of retinol in vitro.

The purpose of the present study was to determine beta-carotene uptake and resultant effects on intracellular levels of retinol in cell lines of varied origin. Human skin fibroblasts, mouse embryonic fibroblasts, rabbit corneal epithelial cells, and rat liver cells were studied. Cells were cultured in medium supplemented with beta-carotene in a water-dispersible beadlet formulation. At selected intervals, cells and media were sampled and analyzed by high-performance liquid chromatography for beta-carotene and retinol content. beta-Carotene was taken up by all four cell lines. An increase in the intracellular levels of retinol was concomitant with beta-carotene uptake in all cell lines. The uptake of beta-carotene and the increase in intracellular retinol were highest in the two fibroblast cell lines. Incubation with media supplemented with crystalline beta-carotene, dissolved in tetrahydrofuran, resulted in significantly lower beta-carotene uptake and intracellular retinol levels. We view these results as a demonstration that a wide variety of cells, cultured in vitro, are able to convert beta-carotene to retinol. Therefore, beta-carotene's provitamin A activity should be carefully considered when the protective effects of beta-carotene in vitro are interpreted.

Rapid liquid chromatographic method to distinguish wild salmon from aquacultured salmon fed synthetic astaxanthin.

Analytical methods are needed to determine the presence of color additives in fish. We report a liquid chromatographic (LC) method developed to identify the synthetic form of the color additive astaxanthin in salmon, based on differences in the relative ratios of the configurational isomers of astaxanthin. The distributions of configurational isomers of astaxanthin in the flesh of wild Atlantic and wild Pacific salmon are similar, but significantly different from that in aquacultured salmon. Astaxanthin is extracted from the flesh of salmon, passed through a silica gel Sep-Pak cartridge, and analyzed directly by LC on a Pirkle covalent L-leucine column. No derivatization of the astaxanthin is required-an important advantage of our approach, which is a modification of our previously described method. This method can be used to distinguish between aquacultured and wild salmon. The method has general applicability and can also be used to identify astaxanthins derived from other sources such as Phaffia yeast and Haematococcus pluvialis algae.

In vitro photooxidation of nucleic acids by ultraviolet A radiation.

Although ultraviolet A radiation (UVA, 315-400 nm) has been shown to induce oxidative stress in mammalian cells and skin, the critical chromophore(s) and molecular target(s) involved have not been identified. We examined the role of oxidative damage to nucleic acids induced by UVA. To assess photooxidation of cellular DNA and RNA by UVA, we irradiated human skin fibroblasts with up to 765 kJ/m2 UVA. Cellular DNA and RNA were isolated immediately and enzymatically hydrolyzed to nucleosides. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a primary oxidation product in DNA, and 8-oxo-7,8-dihydroguanosine (8-oxoG), resulting from hydroxylation of guanine in RNA, were measured by HPLC with electrochemical detection. We determined that irradiation of skin fibroblasts with levels of UVA that produced moderate toxicity also resulted in significant levels of guanine hydroxylation in RNA. Lower levels of photooxidation were observed in DNA. These results suggest that measurement of guanine hydroxylation in nucleic acids, particularly in cellular RNA, may be a powerful tool for investigating the photobiological activity of UVA.

Oxidative damage to nucleic acids photosensitized by titanium dioxide.

The semiconductor TiO2 is known to have photobiological activity in prokaryotic and eukaryotic cells. Applications of this photobiological activity have been suggested including sterilization of waste water and phototherapy of malignant cells. Here, several model and cellular systems were used to study the mechanism of photocatalysis by TiO2. Treatment of TiO2 (anatase, 0.45 microns), suspended in water containing a spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), with UV radiation (320 nm) resulted in an electron spin resonance (ESR) signal characteristic of the hydroxyl radical. Irradiation of solutions containing calf thymus DNA and TiO2 with UVA (320-400 nm) radiation resulted in hydroxylation of guanine bases. The degree of hydroxylation was dependent on both UVA fluence and amount of TiO2 in suspension. Human skin fibroblasts, preincubated 18 h with 10 micrograms/cm2 TiO2 and then UVA-irradiated (0-58 KJ/m2), showed dose dependent photocytoxicity. RNA, isolated from similarly treated fibroblasts, contained significant levels of photooxidation, measured as hydroxylation of guanine bases. However, no oxidative damage was detectable in cellular DNA. These results suggest that nucleic acids are a potential target for photooxidative damage sensitized by TiO2, and support the view that TiO2 photocatalyzes free radical formation.

Effects of alpha-tocopherol and beta-carotene on hepatic lipid peroxidation and blood lipids in rats with dietary iron overload.

The ability of dietary antioxidants to reduce lipid peroxidation induced by iron overload was examined in weanling male Sprague-Dawley rats. Animals were fed ad libitum a modified AIN-76A diet (control) or control diet with 0.5% alpha-tocopherol acid succinate, 0.5% crystalline trans-beta-carotene, or 0.5% alpha-tocopherol acid succinate + 0.5% trans-beta-carotene for four weeks. In the following four-week period, the animals received the above diets with 10,000 micrograms Fe/g; a control group continued to receive 35 micrograms Fe/g, and a high-iron group received 10,000 micrograms Fe/g with no antioxidants. After four weeks of dietary supplementation with alpha-tocopherol. Beta-carotene or alpha-tocopherol + beta-carotene, liver concentrations of alpha-tocopherol and beta-carotene increased significantly (p < 0.001). Liver lipid peroxidation, measured by the lipid-conjugated diene assay, increased significantly from 0.012 mumol/mg of lipid in the controls to 0.021 mumol/mg of lipid in animals receiving the high-iron diet. However, lipid peroxidation was significantly reduced in all animals fed the antioxidants, with the group fed alpha-tocopherol + beta-carotene having a lower level than the high-iron group. Total serum cholesterol was elevated in animals fed a high-iron diet and in animals fed the high-iron diet with alpha-tocopherol. In contrast, total serum cholesterol levels in the two groups of animals receiving the diets containing high iron with beta-carotene alone or high iron with beta-carotene + alpha-tocopherol were significantly reduced to the level of the control group. High-density lipoprotein cholesterol also decreased to baseline in the animals receiving beta-carotene alone. Modulation of lipid peroxidation by alpha-tocopherol or beta-carotene may be an important mechanism for reducing oxidative stress.

Furocoumarin-photosensitized hydroxylation of guanosine in RNA and DNA.

Guanosine hydroxylation was used as a marker for assessing photooxidation of DNA and RNA sensitized by monofunctional and bifunctional furocoumarins. DNA or RNA, treated with sensitizer and UVA light, was enzymatically hydrolyzed, dephosphorylated and then analyzed by reversed-phase HPLC with electrochemical detection. Hydroxylated guanosine, i.e. 8-hydroxy-2'-deoxyguanosine (8-OHdG) or 8-hydroxyguanosine (8-OHG), was quantitated. 3-Carbethoxypsoralen (3-CP) was found to be an efficient photosensitizer for oxidation of guanosine in DNA, resulting in conversion of up to 0.4% of guanosine residues to 8-OHdG. In contrast, dramatically lower levels of guanosine hydroxylation were observed in 3-CP-photosensitized RNA. Psoralen was found to be a more efficient photosensitizer than angelicin in both DNA and RNA. Additional studies of oxidation of 3-CP-photosensitized DNA indicated that double-stranded DNA is 10 times more susceptible to photooxidation than single-stranded DNA, implicating 3-CP binding to DNA as an important mechanistic step in photooxidation of guanosine. The effects of D2O and degassing with argon on photooxidation of guanosine in DNA sensitized by 3-CP were inconsistent with a mechanism involving 1O2. In addition, chelation of adventitious metal ions present in preparations of DNA photosensitized by 3-CP had no effect on hydroxylation of guanosine.

beta-Carotene inhibition of chemically induced toxicity in vivo and in vitro.

In the past several years there has been a great deal of interest in the antioxidant beta-carotene and other micronutrients for their protective potential against various toxic insults. Two studies concerning the protective effects of beta-carotene, which were conducted in our laboratory, are reported here. The first involved the role of beta-carotene in modifying two-stage skin tumorigenesis initiated by 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by phorbol 12-myristate 13-acetate (PMA, TPA). In this study, the protective effects of two types of dietary beta-carotene, a beadlet formulation and crystalline beta-carotene, were compared in two strains of mice (Skh:HR-1 and CR:ORL Sencar). Mice were maintained on food fortified with 3% beta-carotene or on control diets. Mice receiving the beta-carotene-supplemented diets had fewer tumours than mice in the control groups. However, only in the Skh strain of mice was this difference statistically significant. In the second study, an in vitro experiment, BALBc 3T3 mouse fibroblasts were used to determine beta-carotene's accumulation in cells and the ability of these cells to metabolize beta-carotene to vitamin A. This in vitro model was also used to show a beta-carotene protective effect towards 8-MOP phototoxicity. These studies contributed to the increasing evidence of in vivo and in vitro protection by beta-carotene against chemically induced toxicity.

The protective but nonsynergistic effect of dietary beta-carotene and vitamin E on skin tumorigenesis in Skh mice.

Various epidemiological and experimental studies have indicated that beta-carotene and vitamin E protect against a variety of cancers. This investigation determined whether a synergistic protective effect could be observed against chemically induced skin tumorigenesis in Skh mice by combining these two antioxidants in the diet. Forty-five mice were used in each of four diet groups. Control animals were fed standard mouse chow. Three other groups received the chow supplemented with one of the following: 0.5% beta-carotene, 0.12% vitamin E (added as d-alpha-tocopheryl succinate), or 0.5% beta-carotene + 0.12% vitamin E. Mice were topically treated with a single application of the initiator 7,12-dimethylbenz[a]anthracene and promoted with multiple applications of phorbol 12-myristate 13-acetate. Mice were observed for tumors each week for 27 weeks after initiation. The protective effect of each diet was determined by the decrease in the number of skin tumors in supplemented diet groups compared with that of the control diet group. Decreases in the number of cumulative tumors at Week 27 were 32% for beta-carotene-, 25% for vitamin E-, and 21% for beta-carotene+vitamin E-supplemented diet groups. However, differences in the number of tumors among the three groups supplemented with beta-carotene and/or vitamin E were not statistically significant. Thus, although protection was provided by the individual supplements, there was no synergistic effect for a decrease in the number of chemically induced skin tumors by the simultaneous dietary administration of beta-carotene and vitamin E.

beta-Carotene uptake, metabolism, and distribution in BALB/c 3T3 cells.

Although a growing number of epidemiological studies indicate that dietary beta-carotene has anticarcinogenic activity, the mechanism(s) of beta-carotene protection remains to be definitively established. In this context, in vitro studies of beta-carotene have been, and continue to be, valuable. We examined the following critical features in designing an in vitro system for studying the protection action of beta-carotene: 1) form of beta-carotene used for cellular uptake, 2) cellular metabolism of beta-carotene, and 3) subcellular distribution of beta-carotene. It was determined that beta-carotene added to medium in a water-dispersible formulation is readily taken up by BALB/c 3T3 cells and is located predominantly in cellular membranes. Cellular uptake of beta-carotene added to medium in an organic solvent is greatly reduced. It was also found that intracellular retinol increased significantly after a three-day exposure of BALB/c 3T3 cells to media containing beta-carotene. This result suggests that the ability to metabolize beta-carotene to retinoids is not limited to cells of intestinal origin. The results and methodology described here will be useful in the rational design of in vitro assays for elucidating the mechanism(s) of beta-carotene protective effects at the cellular level.

Antitumor activity in skin of Skh and Sencar mice by two dietary beta-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.

Difference in topical and systemic reactivity of psoralens: determinations of epidermal and serum levels.

Topical application of 8- and 5-methoxypsoralen (8-MOP, 5-MOP), psoralen, and 4,5',8-trimethylpsoralen (TMP) to the epilated backs of albino guinea pigs followed by UV irradiation at 320-400 nm resulted in elicitation of similar phototoxicity. However, the systemic phototoxicity determined after oral administration of these psoralen derivatives differed significantly. Serum and epidermal levels of 8-MOP, 5-MOP, and TMP were determined 2 hours post administration. We found a linear relationship between serum and epidermal levels of 8-MOP, but the levels of the other psoralen derivatives were significantly lower. We believe that the reduced oral phototoxicity of 5-MOP and TMP relative to 8-MOP in the guinea pig is due to its reduced levels in the epidermis.

Psoralen phototoxicity: correlation with serum and epidermal 8-methoxypsoralen and 5-methoxypsoralen in the guinea pig.

Serum and epidermal concentrations of 8-methoxypsoralen and 5-methoxypsoralen 2 hours after oral administration to guinea pigs were determined by high-performance liquid chromatography. A linear relation was found between the serum and epidermal concentrations of 8-methoxypsoralen. In addition, a relation was found between serum concentrations of 5-methoxypsoralen and 8-methoxypsoralen and the appearance of phototoxicity. The lower phototoxicity of orally administered 5-methoxypsoralen as compared to 8-methoxypsoralen in the guinea pig appears to be due to its reduced concentrations in the epidermis, the primary site of the phototoxic events.