Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on newly synthesized proteins in mouse epidermis

We have analyzed the effects of treatment of mouse epidermis with the potent tumor promoter TPA on the profile of newly synthesized proteins. TPA was applied to the skin of the intact mouse, and either 3 or 24 hr later skin fragments were pulse-labeled in vitro with ³⁵S-methionine for 4 hr. The epidermal proteins were extracted and separated by two-dimensional gel electrophoresis. Over 200 individual proteins were resolved in acidic gels. At least 10 of these showed major (by a factor of 5 or more) increases or decreases in response to TPA; eight of these appear to be keratin proteins. Two-dimensional gel profiles of basic proteins synthesized by mouse epidermis resolved over 100 individual proteins. Only one of these showed a significant change in response to TPA. This 41 kd protein increased more than 100-fold within 24 hr after the application of TPA. Treatment of mouse skin with mezerein, a plant diterpene structurally related to TPA, produces an almost identical change in the pattern of proteins produced. Four agents that induce hyperplasia but are not potent tumor promoters, ethylphenylpropiolate, acetic acid, turpentine oil and the Ca⁺⁺ ionophore A23187, modulate the synthesis of only three of the keratin proteins. Thus the changes in protein profiles induced by TPA and mezerein are not simply the consequence of hyperplasia. In addition, application to mouse skin of a glucocorticoid that is a potent inhibitor of tumor promotion inhibits most of the changes in protein profiles induced by TPA. Taken together, these results indicate that TPA and mezerein induce early and marked changes in the profile of specific epidermal proteins. It seems likely that some of these changes are directly related to the process of tumor promotion.     

Epigenetic mechanism of tumor promotion. Interrelationship between inflammation and ornithine decarboxylase activity induced in vitro by the carcinogenic 12-O-tetradecanoyl-phorbol-13-acetate

Topical application of 2 micrograms 12-O-tetradecanoyl-phorbol-13-acetate (TPA) regularly induced two early events in mouse skin: inflammatory reaction localized in dermal compartment and stimulation of ornithine decarboxylase activity in epidermal cells, in relation to polyamine synthesis and cell division. These reactions were followed, after 48 hrs. by an epidermal hyperplasia. At this stage, another TPA treatment induced a strong ODC activity concurrent with severe inflammation of the dermis. Inhibition of the synthesis of inflammatory factors may antagonize TPA-induced ODC, but the protective potencies differs according to the evolutive stages of the cell. After the first TPA treatment the anti-inflammatory compounds dexamethasone and indomethacin effectively inhibited ODC activity. In contrast during the proliferative stage epidermal cell function may be less dependent on inflammatory factors; thus only partial protection was observed with the inflammatory inhibitors.     

Topically applied vitamin E prevents massive cutaneous inflammatory and oxidative stress responses induced by double application of 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice

Vitamin E (alpha-tocopherol) is a promising chemopreventive and pharmacologically safe agent, which can be exploited or tested against skin cancer. It is an established antioxidant with an ability to ameliorate the UV-induced skin damage and chemically induced inflammation in lungs. However, there are some conflicting reports about its role as a modulator of chemically induced promotion. We evaluated its efficacy in preventing the inflammatory and oxidative stress responses in a double 12-O-tetradecanoylphorbol-13-acetate (TPA) application tumor skin promotion protocol. Double application of TPA was undertaken to produce massive inflammatory and oxidative stress responses. Topical TPA treatment adversely altered many of the marker responses of stage I skin tumor promotion. Vitamin E application 30 min prior to TPA treatment (10 nmol) inhibited induction of hydrogen peroxide, myeloperoxidase (MPO) activity, xanthine oxidase (XO) activity and lipid peroxidation (LPO). Vitamin E also positively modulated altered antioxidants of mouse skin. Histological examination also revealed marked improvement. These results confirm the efficacy of vitamin E against early inflammatory and oxidative stress responses, which are hallmark of tumor promotion and provide rational basis for chemopreventive action of vitamin E in skin cancer.     

Effect of tumor promoting phorbol ester TPA on epidermal protein synthesis: stimulation of an elongation factor 2 phosphatase activity by TPA in vivo

Topical application of the phorbol ester TPA to mouse skin causes an increase in the amount of elongation factor 2 (EF-2), a factor in eukaryotic protein synthesis, in the epidermal cytosol (2- to 3-fold) and particulate fraction (7-fold). Furthermore, as a consequence of this TPA treatment the activity of an epidermal EF-2 phosphatase is stimulated. The EF-2 phosphatase has an apparent molecular weight of around 38,000 daltons. The enzyme activity is induced as early as 45 minutes after TPA treatment and remains at the elevated level for more than 17 hours. Both of the TPA-induced effects result in an increase in unphosphorylated, i.e. active EF-2 and can be suppressed by cyclosporine A.     

Early prostaglandin E synthesis is an obligatory event in the induction of cell proliferation in mouse epidermis in vivo by the phorbol ester TPA

Following the topical application of the phorbol ester TPA to mouse skin $\text{in vivo}$ a rapid increase of the prostaglandin E content after 10 and 60 minutes was observed. Pretreatment of mouse skin with indomethacin abolished the first PGE peak as well as the cellular proliferation induced by TPA. Both effects could not be prevented when indomethacin was applied 30 to 60 minutes after TPA treatment, suggesting that the early increase in epidermal PGE is an obligatory event in the course of the induction of epidermal cell proliferation by TPA. A small increase of epidermal PGE was also seen after treatment with the TPA-analogue “Ti₈”, whereas 4-O-methyl-TPA was inactive in this respect. “Ti₈”-induced epidermal cell proliferation could be partially inhibited by indomethacin, whereas 4-O-methyl-TPA-induced cell proliferation was insensitive to the drug.     

Regulation of the phosphorylation state and function of the epidermal growth factor receptor by vitamin K-3

Vitamin K-3 or 12-O-tetradecanoylphorbol 13-acetate (TPA) reduced the binding of epidermal growth factor (EGF) to its receptor by more than 90% in human foreskin fibroblasts. After the equilibration of fibroblasts with [32P]orthophosphate, vitamin K-3 or TPA markedly increased the amount of 32P found in the receptor; the increase was principally due to serine and threonine phosphorylation. By the use of two-dimensional tryptic phosphopeptide mapping, using a synthetic phosphopeptide as a standard, threonine-654 was identified as one of the residues whose phosphorylation state was elevated by vitamin K-3 or TPA. Because of the large amounts of EGF receptor present on A431 human carcinoma cells, these cells were used to study further the relationship between the phosphorylation state of threonine-654, the tyrosine phosphorylation state of the receptor, and the receptor's protein tyrosine kinase activity toward exogenous substrates. Vitamin K-3 and TPA both increased the amount of phosphate on threonine-654 in A431 cells. However, whereas receptor from TPA-treated cells lacked phosphotyrosine, vitamin K-3-treated cells contained receptor with markedly elevated levels of phosphotyrosine. The addition of vitamin K-3, TPA or EGF to intact A431 cells followed by homogenization of the cells and the assay of EGF receptor protein tyrosine kinase activity by the use of a synthetic peptide substrate resulted in marked decreases in apparent receptor kinase activity. Therefore, assuming that the activity measured in the peptide assay reflects the protein tyrosine kinase activity of the receptor in the intact cell, the activity of the EGF receptor kinase cannot be deduced from the amount of phosphotyrosine associated with the receptor.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornithine decarboxylase and protein kinase C by polyphenolics from grapes

Ornithine decarboxylase is the rate-limiting enzyme in the biosynthesis of polyamines, which are believed to play an essential role in diverse biological processes including cell proliferation and differentiation. We have previously reported [J. Bomser, K. Singletary, M. Wallig, M. Smith, Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds, Cancer Letters 135 (1999) 151-157] that pre-application of a grape polyphenolic fraction (GPF) to mouse skin epidermis inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, as well as 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated, TPA-promoted mouse skin tumorigenesis. The present studies were designed to further characterize the effect of time and dose of application of GPF on TPA-induced ODC activity and protein expression, and on protein kinase C activity in mouse skin epidermis. In addition, the effect of GPF on ODC kinetics in vitro was examined. Application of 5, 10, and 20 mg of GPF 20 min prior to treatment with TPA resulted in a significant decrease in epidermal ODC activity of 54, 53, 90%, respectively, compared with controls. Yet, ODC protein levels (Western blot) in the 10 and 20 mg GPF groups were significantly increased by 1.8 and 1.9-fold, respectively, compared with controls. A similar response was observed with the ODC inhibitor 2-difluoromethylornithine (DFMO), which served as a positive control. Application of grape polyphenolics (20 mg) at 60 and 30 min prior to treatment with TPA inhibited ODC activity by 62 and 68%, respectively, compared with controls (P<0.05). In contrast, application of grape polyphenolics (20 mg) at 60, 120 and 240 min after treatment with TPA resulted in no significant changes in ODC activity. A similar increase in epidermal ODC protein was observed in these GPF-treated animals, similar to that observed when GPF application preceded TPA. When applied to mouse skin prior to TPA, GPF was associated with a decrease in subsequent PKC activity compared with controls at 10 and 30 min following TPA treatment. The GPF-associated decrease in PKC activity preceded the decrease in ODC activity. In a separate in vitro study, kinetic analyses indicated that GPF is a competitive inhibitor of ODC activity. Collectively these data suggest that the grape polyphenolic fraction is effective as an inhibitor of ODC activity when applied before TPA, and that the magnitude of inhibition is independent of epidermal ODC protein content. In addition, GPF is a competitive inhibitor of ODC activity in vitro. The decrease in TPA-induced ODC activity due to GPF treatment is preceded by an inhibition of TPA-induced PKC activity. Thus, the polyphenolic fraction from grapes warrants further examination as a skin cancer chemopreventive agent that interferes with cellular events associated with TPA promotion.     

Initiator and promoter induced specific changes in epidermal function and biological potential

Mouse epidermal basal cells can be selectively cultivated in medium with a calcium concentration of 0.02–0.09 mM. Terminal differentiation and slouching of mature kcratinocytes occur when the calcium concentration is increased to 1.2–1.4 mM. When basal cell cultures are exposed to chemical initiators of carcinogenesis, colonies of cells that resist calcium-induced differentiation evolve. Likewise, basal cells derived from mouse skin initiated in vivo yield foci that resist terminal differentiation. This defect in the commitment to terminal differentiation appears to be an essential change in initiated cells in skin and is also characteristic of malignant epidermal cells. This model system has also provided a means to determine if basal cells are more responsive to phorbol esters than other cells in epidermis and to explore the possibility that heterogeneity of response exists within subpopulations of basal cells. The induction of the enzyme ornithine decarboxylase (ODC) was used as a marker for responsiveness to phorbol esters. ODC induction after exposure to 12-0-tetradccanoylphorbol-13-acetate (TPA) in basal cells is enhanced 20-fold over the response of a culture population containing both differentiating and basal cells. When basal cells are induced to differentiate by increased calcium, responsiveness to TPA is lost within several hours. In basal cell cultures, two ODC responses can be distinguished. After exposure to low concentrations of TPA or to weak promoters of the phorbol ester series, ODC activity is maximal at 3 hr. With higher concentrations of TPA, the ODC maximum is at 9 hr. These results arc consistent with the presence of subpopulations of basal cells with differing sensitivities to TPA. Other studies that use the enzyme epidermal transglutaminase as a marker for differentiation support this conclusion. In basal cell culture TPA exposure rapidly increases transglutaminase activity and cornified envelope development, reflecting induced differentiation in some cells. As differentiated cells arc sloughed from the dish, the remaining basal cells proliferate and become resitant to induced differentiation by 1.2 m M calcium. These data provide additional evidence of basal cell heterogeneity in which TPA induces one subpopulation to differentiate while another is stimulated to proliferate and resists a differentiation signal. Tumor promoters, by their ability to produce heterogeneous responses with regard to terminal differentiation and proliferation, would cause redistribution of subpopulations of epidermal cells in skin. Cells that resist signals for terminal differentiation, such as initiated cell, would be expected to increase in number during remodeling. Clonal expansion of the intitiated population could result in a benign tumor with an altered program of differentiation. In skin, benign tumors are the principal product of 2-stage carcinogenesis. Subsequent progression to malignancy may involve an additional step, probably a genetic alteration, that is independent of the tumor promoter.     

12-O-tetradecanoylphorbol-13-acetate and UV radiation-induced nucleoside diphosphate protein kinase B mediates neoplastic transformation of epidermal cells

The molecular changes associated with early skin carcinogenesis are largely unknown. We have previously identified 11 genes whose expression was up- or down-regulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin keratinocyte progenitor cells (Wei, S.-J., Trempus, C. S., Cannon, R. E., Bortner, C. D., and Tennant, R. W. (2003) J. Biol. Chem. 278, 1758-1768). Here, we show an induction of a nucleoside diphosphate protein kinase B (NDPK-B) gene in response to TPA or UV radiation (UVR). TPA or UVR significantly induced the expression of NDPK-B both in vivo hyperplastic mouse skin and in vitro mouse JB6 Cl 41-5a epidermal cells. Indeed, this gene was also up-regulated in TPA or UVR-mediated skin tumors including papillomas, spindle cell tumors, and squamous cell carcinomas, relative to adjacent normal skins. Functional studies by constitutive expression of nm23-M2/NDPK-B in TPA susceptible JB6 Cl 41-5a and TPA-resistant JB6 Cl 30-7b preneoplastic epidermal cell lines showed a remarkable gene dosage-dependent increase in foci-forming activity, as well as an enhancement in the efficiency of neoplastic transformation of these cells in soft agar but no effect on proliferation in monolayer cultures. Interestingly, stable transfection of the nm23-M2/NDPK-B del-RGD or G106A mutant gene in JB6 Cl 41-5a cells selectively abrogated NDPK-B-induced cellular transformation, implicating a possible Arg105-Gly106-Asp107 regulatory role in early skin carcinogenesis.     

Inhibition by epidermal extracts of the 12-O-tetradecanoylphorbol-13-acetate induced peak of ornithine decarboxylase activity in the mouse epidermis

The time course of induction of epidermal ornithine decarboxylase (E.C. 4.1.117) (ODC) activity following a single topical application of 17 nmoles of 12-O-tetradecanoylphorbol-13-acetate (TPA) on hairless mouse skin was established. Prior intraperitoneal (i.p.) administration of a crude epidermal extract prepared from hairless mouse epidermis led to a time-dependent, 50% inhibition of the peak level of TAP-induced ODC activity. Maximum inhibition was observed when the extract was injected 1.5 h before TPA treatment. The crude epidermal extract did not affect ODC activity in vitro. Following the administration of epidermal extracts, the inhibition of the TPA-induced ODC-response correlated positively with the presence of epidermal G2-chalone activity (determined by a stathmokinetic method) whereas myocardial, skeletal muscle, or heat-inactivated epidermal extracts with no epidermal G2-chalone activity, had no effect on TPA-induced ODC activity. These results indicate a possible relationship between ODC-activity and the control of mitotic rate by G2-chalone.     

Haemosiderin-like properties of free-radical-modified ferritin.

Evidence was sought that the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse epidermal ornithine decarboxylase (ODC, EC 4.1.1.17) activity involves both increased ODC mRNA and ODC protein. Application of 10 nmol of TPA to mouse skin led to a dramatic increase in soluble epidermal ODC activity which paralleled an increase in amount of enzymically active ODC protein as determined by gel electrophoresis of immunoprecipitated difluoromethyl[3H]ornithine-bound ODC. Application of TPA to mouse skin also resulted in an increase in ODC mRNA measured by dot-blot analysis using a radiolabelled cDNA probe. ODC mRNA induction preceded the increase in ODC activity by TPA. TPA-increased ODC mRNA displayed a single major band of 2.1 kilobases in size identified by the Northern blotting procedure.     

Hyperthermia and polyamine biosynthesis: decreased ornithine decarboxylase induction in skin and kidney after heat shock

The effect of hyperthermia treatments on ornithine decarboxylase (ODC) induction in mouse tissue was determined both in vitro and in vivo. In vitro, the addition of 12-0-tetradecanoylphorbol-13-acetate (TPA) to adult mouse skin pieces incubated at 37 degrees C in serum-free MEM led to a dramatic increase in epidermal ODC activity 5 hours following treatment. In contrast, incubation temperatures of 40 degrees C for the entire 5 hour incubation period rendered the skin pieces unresponsive to TPA for ODC induction. This inhibition of ODC induction was not the result of thermal skin kill, inactivation of TPA, or a general effect on epidermal protein synthesis. The inhibition of ODC induction could be reversed by switching the incubation temperature back to 37 degrees C. In vivo, raising the core body temperature in male mice to 41 degrees C for 1 hour resulted in a 78% decrease in kidney ODC activity. The kidney DNA synthesis and protein synthesis remained unaltered following the whole body hyperthermia treatments.     

Inhibition of phorbol ester-caused synthesis of mouse epidermal ornithine decarboxylase by retinoic acid

The mechanisms by which topically applied retinoic acid to mouse skin inhibits tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced epidermal ornithine decarboxylase activity were analyzed. Retinoic acid inhibition of the induction of epidermal ornithine decarboxylic activity was not the result of nonspecific cytotoxicity, production of a soluble inhibitor of ornithine decarboxylase, or direct effect on its activity. In addition, inhibition of TPA-caused increased ornithine decarboxylase activity does not appear to be due to enhanced degradation and/or post-translational modification of ornithine decarboxylase by transglutaminase-mediated putrescine incorporation. We found that retinoic acid inhibits the synthesis of ornithine decarboxylase caused by TPA. Application of 10 nmol TPA to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase activity which was paralled by increased [3H]difluoromethylornithine binding and an increased incorporation of [35S]methionine into the enzyme. Application of 17 nmol retinoic acid 1 h prior to application of 10 nmol TPA to skin resulted in inhibition of the induction of activity which accompanied inhibition of [3H]difluoromethylornithine binding and [35S]methionine incorporation into ornithine decarboxylase protein as determined by the tube-gel electrophoresis of the enzyme immunoprecipitated with monoclonal antibodies to it. Inhibition of ornithine decarboxylase synthesis was not the result of the inhibitory effect of retinoic acid on general protein synthesis. The results indicate that retinoic acid possibly inhibits TPA-caused synthesis of ornithine decarboxylase protein selectively.     

Effects of diverse intracellular thiol delivery agents on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione, and ornithine decarboxylase induction in isolated mouse epidermal cells treated with 12-O-tetradecanoylphorbol-13-acetate

Since the enhancement of the activity of the natural glutathione (GSH)-dependent antioxidant protective system of the epidermal cells appears to inhibit the oxidative challenge presumably linked to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA), we have compared the effectiveness of diverse intracellular thiol delivery agents as inhibitors of the effects of TPA on GSH metabolism and ornithine decarboxylase (ODC; L-ornithine carboxylase, EC 4.1.1.17) induction in isolated mouse epidermal cells. Here we report at a 2-mM concentration, the monoethyl and monomethyl esters of GSH, N-acetyl-L-cysteine, and L-2-oxothiazolidine-4-carboxylate are all significantly more effective than GSH in inhibiting the sharp decline in the intracellular ratio of reduced GSH/oxidized glutathione (GSSG), the prolonged decrease in GSH peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity, and the induction of ODC activity caused by 1 microM TPA. Moreover, diethyldithiocarbamate prevents totally the initial drop in the GSH/GSSG ratio of TPA-treated cells and is the most potent inhibitor of TPA-decreased GSH peroxidase activity in relation with its remarkable 98% inhibition of TPA-induced ODC activity, suggesting that the potential antitumor-promoting activity of this compound in mouse skin may be far superior to that previously demonstrated by GSH in the initiation-promotion protocol.     

HPLC analysis of vitamin E isoforms in human epidermis: correlation with minimal erythema dose and free radical scavenging activity

The content and composition of different vitamin E isoforms was analyzed in normal human skin. Interestingly the epidermis contained 1% alpha-tocotrienol, 3% gamma-tocotrienol, 87% alpha-tocopherol, and 9% gamma-tocopherol. Although the levels of tocotrienol in human epidermis appear to be considerably lower than reported in the hairless mouse, the presence of significant amounts of tocotrienol levels leads to speculation about the physiological function of tocotrienols in skin. Besides antioxidant activity and photoprotection, tocotrienols may have skin barrier and growth-modulating properties. A good correlation was found for epidermal alpha-tocopherol (r = 0.7909, p <.0003), gamma-tocopherol (r = 0.556, p <.025), and the total vitamin E content (r = 0.831, p <.0001) with the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging in epidermis, as assessed by electron paramagnetic resonance (EPR) spectroscopy. In human epidermis, alpha-tocopherol is quantitatively the most important vitamin E isoform present and comprises the bulk of first line free radical defense in the lipid compartment. Epidermal tocotrienol levels were not correlated with DPPH scavenging activity. The minimal erythema dose (MED), an individual measure for sun sensitivity and a crude indicator for skin cancer susceptibility, did not correlate with the epidermal content of the vitamin E isoforms. Hence it is concluded that vitamin E alone is not a determinant of individual photosensitivity in humans.     

An organ culture of adult mouse skin: An in vitro model for studying the molecular mechanism of skin tumor promotion

A simple method to culture explants of adult mouse skin in a modified Eagle's HeLa cell medium was developed in order to further study the biochemical responses to the tumor promoting phorbol esters. The skin explants remained viable for at least 48 hr, as determined by their ability to incorporate ³H-thymidine into DNA as well as to induce epidermal ornithine decarboxylase (EC 4.1.1.17) activity following 12-0-tetradecanoylphorbol-13-acetate addition. The time course of induction of ornithine decarboxylase activity by the tumor promoter was similar to that observed with intact mice. Furthermore, the addition of retinoic acid and indomethacin, the agents that are known to inhibit the induction of ornithine decarboxylase activity by topically applied TPA, also inhibited the induction of ornithine decarboxylase activity by TPA in skin explants.     

Lightening Effect on Ultraviolet‐Induced Pigmentation of Guinea Pig Skin by Oral Administration of a Proanthocyanidin‐Rich Extract from Grape Seeds

Antioxidants such as vitamins C and E have been reported to inhibit the progression of ultraviolet (UV) radiation‐induced pigmentation in the skin of hairless mice. However, little is known of the lightening effect of proanthocyanidin, a powerful polyphenolic antioxidant, on UV‐induced pigmentation of the skin. We investigated the lightening effect of oral administration of a proanthocyanidin‐rich grape seed extract (GSE) using guinea pigs with UV‐induced pigmentation. These pigmented guinea pigs were fed diets containing 1% GSE or 1% vitamin C (w/w) for 8 weeks. GSE‐feeding had an apparent lightening effect on the guinea pigs’ pigmented skin. Histologic evaluation demonstrated a decrease in the number of 3,4‐dihydroxyphenylalanine (DOPA)‐positive melanocytes as well as 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG)‐positive, Ki‐67‐positive, proliferating cell nuclear antigen (PCNA)‐positive melanin‐containing cells in the basal epidermal layer of the UV‐irradiated skin in GSE‐fed guinea pigs. In contrast, these parameters did not change in the skin of vitamin C‐fed or control guinea pigs. GSE inhibited the activity of mushroom tyrosinase and also inhibited melanogenesis without inhibiting the growth of cultured B16 mouse melanoma cells. In conclusion, we demonstrated that oral administration of GSE is effective in lightening the UV‐induced pigmentation of guinea pig skin. This effect may be related to the inhibition of melanin synthesis by tyrosinase in melanocytes and the reactive oxygen species (ROS)‐related proliferation of melanocytes.     

Effects of garlic and onion oils on glutathione peroxidase activity, the ratio of reduced/oxidized glutathione and ornithine decarboxylase induction in isolated mouse epidermal cells treated with tumor promoters

Garlic oil, onion oil and one of its constituents, dipropenyl sulfide, all increase, to diverse degrees, glutathione (GSH) peroxidase (GSH:H2O2 oxidoreductase, EC 1.11.1.9) activity in isolated epidermal cells incubated in the presence or absence of the potent tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). The stimulatory effects of these oils on epidermal GSH peroxidase activity are concentration-dependent and long-lasting, and thus, abolish totally the prolonged inhibitory effect of TPA on this enzyme. Moreover, garlic oil (5 micrograms/ml) inhibits by about 50% TPA-induced ornithine decarboxylase (ODC, L-ornithine carboxy-lyase, EC 4.1.1.17) activity in the same epidermal cell system. This concentration of garlic oil also increases remarkably GSH peroxidase activity and inhibits ODC induction in the presence of various nonphorbol ester tumor promoters. Since the same oil treatments inhibit dramatically the sharp decline in the intracellular ratio of reduced (GSH)/oxidized (GSSG) glutathione caused by TPA, it is suggested that some of the inhibitory effects of garlic and onion oils on skin tumor promotion may result from their enhancement of the natural GSH-dependent antioxidant protective system of the epidermal cells.