Skin carcinogenesis: cholesterol-5alpha,6alpha-epoxide hydrase activity in mouse skin irradiated with ultraviolet light.

Cholesterol-5alpha,6alpha-epoxide hydrase activity was 96 percent greater in skin of hairless mice that were receiving suberythemic ultraviolet light irradiation for 15 weeks than in nonirradiated controls. This enzyme system, which metabolizes cholesterol-5alpha,6alpha-epoxide (a known carcinogen), appears to be substrateinducible and is apparently responsible for the concomitant reduction of the sterol carcinogen that occurs prior to tumor induction.

Effects of dietary constituents on ultraviolet light-mediated carcinogenesis.

The effects of several dietary supplements of antioxidants and enzyme inducers on ultraviolet light-mediated carcinogenesis were investigated. Glutathione (reduced) was without effect, but butylated hydroxytoluene, phenobarbital, and disulfiram all significantly suppressed the initiation and development of actinic lesions and tumors. On the basis of the present study and related previous ones, tumor inhibition appears to be due not to an umbrageous effect but rather to the induction of systemic physiological responses.

Relation of antioxidants and level of dietary lipid to epidermal lipid peroxidation and ultraviolet carcinogenesis.

It has become increasingly evident that both quantity and quality of dietary lipid can influence the developmental course of several major forms of cancer in experimental animals. Using the hairless mouse-ultraviolet (UV) model, we had previously demonstrated that unsaturated lipid compared to equivalent levels of hydrogenated lipid enhanced photocarcinogenesis with respect to both tumor latency and multiplicity. In the present study using the same model, we have examined the effect of unsaturated lipid level and antioxidants upon epidermal lipid peroxidation and UV carcinogenesis. Sixteen groups of 45 animals each were used in the study, representing all combinations of three design variables: (a) a semipurified diet containing 4, 2, or 0.75% corn oil or 4% soybean oil; (b) 2% (w/w) antioxidant supplement or no supplementation; and (c) an escalating regimen of UV radiation to a cumulative dose of 70 J/cm2 or no irradiation. The nonirradiated groups served as nutritional controls and as subjects for epidermal lipid peroxidation measurements. An approximate linear relationship between lipid level and tumor latency was observed, with 4% levels of unsaturated lipid producing maximum enhancement of photocarcinogenesis. Furthermore with increasing lipid level the numbers of tumors per animal increased. Antioxidants caused significant increases in tumor latency and decreases in tumor multiplicity but only at the highest lipid level used in these studies. Thiobarbituric acid values of epidermal homogenates also increased in relation to the level of dietary lipid intake. Epidermal thiobarbituric acid values from antioxidant supplemented animals were significantly lower regardless of lipid intake levels. From these data we conclude that (a) dietary lipid level has a direct effect upon the carcinogenic response to UV both in regard to tumor latency and tumor multiplicity; (b) antioxidants produce an inhibitory effect almost equal to the degree of exacerbation of carcinogenesis evoked by increasing lipid levels, at least for the range studied; and (c) dietarily administered antioxidants inhibit the formation of epidermal thiobarbituric acid reacting materials. These data strongly imply that free radical reactions, specifically lipid peroxidation, play a role in at least a part of the photocarcinogenic response.

A mode of action for butylated hydroxytoluene-mediated photoprotection.

Dietarily administered butylated hydroxytoluene (BHT) has previously been shown to inhibit UV radiation induction of carcinogenesis, erythema, and ornithine decarboxylase (ODC) activity. Butylated hydroxytoluene feeding also resulted in significant increases in epidermal absorption and it was suggested that BHT's photoprotective properties might be attributable to a diminution of UV radiation dose reaching respective target sites. To explore this possibility, the contribution of stratum corneum to BHT's photoprotective action was examined. SKH-Hr-1 hairless mice were fed diets containing 0.5% (w/w) BHT for 2 weeks prior to experimentation. Control animals received the unsupplemented ration. Stratum corneum from both groups was isolated and spectral transmission recorded. Transmission, between 280-320 nm, was approximately 65% greater through stratum corneum obtained from control animals compared with that of BHT-treated animals. Further evidence of the biologic significance of this BHT effect upon stratum corneum absorption was obtained when stratum corneum was first removed by tape-stripping, the animals irradiated with 0.45 J/cm2 of UVB, and epidermal ODC activity determined. BHT provided the usual inhibition of ODC activity induction in nonstripped animals, but ODC activity induction in BHT-treated, tape-stripped animals was restored to levels that did not significantly differ from controls. The protective effect exhibited by the stratum corneum could not be attributed to BHT-induced alteration of physical dimension, as neither the thickness of stratum corneum nor the number of stratum corneum layers, as determined from measurement of NaOH-distended frozen sections, differed from controls. Although the mechanism remains obscure, these data support the contention that systemically administered BHT results in diminished levels of UV radiation reaching potential epidermal target sites and delimits a large component of the photoprotective effect to the stratum corneum.

Antioxidant-mediated reversal of ultraviolet light cytotoxicity.

Several antioxidants were tested for their protective effect in Chinese hamster embryo cell against ultraviolet light (UVL)-irradiation. Ascorbic acid, DL-alpha-tocopherol, butylated hydroxytoluene, and reduced glutathione were all shown to reverse UVL-induced cytotoxicity. At concentrations tested, no protective effect was detected when any of these antioxidants were added prior to UVL irradiation.

Suppression of ultraviolet light-induced tumor formation by dietary antioxidants.

Dietary antioxidants were effective in reducing the number and severity of ultraviolet light-induced squamous cell carcinomas in skin of hairless mice. This and previous studies suggest that antioxidants may play a role in both inhibition of the primary event(s) involved in ultraviolet light-mediated tumor induction and the subsequent development of precancerous lesions into tumors.

Distribution of cholesterol-5alpha,6alpha-epoxide formation and its metabolism in mouse skin.

The distribution of cholesterol-5alpha,6alpha-epoxide in ultraviolet light-irradiated mouse skin was determined. Highest levels of cholesterol-5alpha,6alpha-epoxide were found in the epidermis and distributed rather evenly throughout all subcellular fractions. Low but potentially significant levels of the compound diffused from the epidermis. Metabolic studies demonstrated that cholesterol-5alpha,6alpha-epoxide was converted to cholestane-3ss,5alpha,6ss-triol by an enzyme, cholesterol-5alpha,6alpha-epoxide hydrase, which was localized in the epidermal fractions sedimenting at 10,000 and 100,000 X g. Comparative studies indicated a much higher capacity for metabolism of cholesterol-5alpha,6alpha-expoide in liver than in skin. The effect of the epoxide and its metabolic product on the skin is discussed.

Dietary modification of UV-induced epidermal ornithine decarboxylase.

The effect of several dietary antioxidant supplements upon ultraviolet light-induced ornithine decarboxylase activity was determined. Hairless mice received diets supplemented with either butylated hydroxytoluene, disulfiram, phenobarbital, glutathione (reduced), or a special antioxidant mixture for 2 weeks before irradiation with FS-20 fluorescent sun lamps. Epidermal ornithine decarboxylase activity, the induction of which is thought to be a necessary component of skin tumor promotion, was determined at designated post-irradiation periods. Significant inhibition of ornithine decarboxylase induction was found in epidermis from animals receiving diets containing butylated hydroxytoluene, the antioxidant mixture, or disulfiram whereas no significant effects were noted in animals receiving reduced glutathione or phenobarbital. Butylated hydroxytoluene, at physiological concentrations, had no effect upon ornithine decarboxylase activity when added directly to the reaction mixture. Nor did this compound, when provided in the diet of animals, evoke a notable effect upon 12-0-tetra-decanoylphorbol-13-acetate induced ornithine decarboxylase. The latter finding suggests that dietary butylated hydroxytoluene inhibition of ultraviolet light-induced ornithine decarboxylase is a response related directly to the degree of irradation insult rather than a general effect upon the processes associated with carcinogenic promotion.

Systemic protection by antioxidants against UVL-induced erythema.

An antioxidant supplemented diet provided marked systemic protection against ultraviolet light mediated erythema in hairless mice. Among the individual constituents of the diet, butylated hydroxytoluene was most effective whereas glutathione and vitamins C and E afforded negligible protection. The mixture of antioxidants, and butylated hydroxytoluene individually, demonstrated diminished, but significant, protection when applied topically. The safety of this systemic photoprotectant and its clinical relevance at present is unknown.

Reactivity of butylated hydroxytoluene.

Butylated hydroxytoluene (BHT) is a synthetic antioxidant that is widely used as an additive in foodstuffs to prevent spoiling. The physical-chemical properties of BHT and many related phenols have been examined previously although the mechanisms by which it exerts its antioxidant properties are poorly understood. The reactivity of BHT with singlet oxygen [O2(1 delta g)] and a number of radical species has been examined using the techniques of time resolved luminescence and pulse radiolysis. In benzene solution BHT reacted with O2(1 delta g) at a bimolecular rate constant of 1.3 x 10(6)M-1s-1. The one-electron oxidized, phenoxyl type BHT radical was generated using pulse radiolysis and the absorption spectrum showed a maximum at 400 nm. BHT reacts slowly with many radical species and upper limits for the bimolecular rate constant for reaction with several electron transfer processes are presented. The antioxidant role of BHT is discussed in terms of its reactivity, localization, and stability.

A new Brevibacterium sp. isolated from infected genital hair of patients with white piedra.

A new aerobic gram-positive non-sporeforming bacillus has been isolated from infected genital hair of patients with white piedra in association with Trichosporon beigelii. This species has been characterised morphologically, nutritionally, by DNA base composition, cell-wall analysis and cellular fatty-acid profile on the basis of 14 isolates. The G+C content of DNA is 63.05 mol%. Cell walls possess meso-diaminopimelic acid (Type IV) and the sugars glucose, galactose, xylose and ribose; mycolic acids are not present. The species has a distinct colonial and microscopic morphology, is strongly proteolytic and produces methanethiol. These findings and the cellular fatty-acid profile are compatible with the genus Brevibacterium. A new species is proposed based on the following characters: colonial and microscopic growth and morphology; conditions for rod-to-coccus cycle; ribose utilisation; and tellurite reduction. The type strain has been named Brevibacterium mcbrellneri E2cr (ATCC 49030). The strong proteolytic properties may be the mechanism of pathogenesis.

The mutagenicity of dinitrochlorobenzene.

A sample of dinitrochlorobenzene, determined by gas-liquid chromatography and mass spectrometry to be greater than 98% pure, was tested in the Salmonella typhimurium plate assay. The chemical evoked a marked mutagenic response at all concentrations tested (3 to 150 micrograms per plate). These results confute the suitability of this agent for the treatment of benign skin disorders.

Modification of membrane composition, eicosanoid metabolism, and immunoresponsiveness by dietary omega-3 and omega-6 fatty acid sources, modulators of ultraviolet-carcinogenesis.

Dietary sources of lipids containing predominantly n-3 or n-6 fatty acids (FA) have been examined for effect upon several potential pathophysiologic parameters. Epidermal, plasma, and red blood cell (RBC) membrane FA composition exhibited marked differences between animals fed the respective dietary lipid sources. Reduced levels of 18:1, 20:3 and 20:4 occurred in the n-3 FA fed animals which exhibited significantly higher levels of 20:5 and 22:6. Approximately equal levels of 18:2 were present in animals fed either diet. Despite marked differences in RBC membrane FA composition, only marginal effect upon osmotic fragility occurred. Lower levels of 20:3 and 20:4 found in n-3 fed animals could result from a deficit of elongase and/or delta 5-desaturase activity. Whether lower 20:4 levels in n-3 fed animals could rate-limit eicosanoid metabolism is unknown, but epidermal capacity to metabolise arachidonic acid in these animals was found to be closely related to n-6 FA intake. Animals fed n-3 FA exhibited markedly lower levels of plasma PGE2, even when the diet was supplemented with n-6 FA. In addition, UV-radiated animals receiving the n-3 FA source demonstrated a reduced (approximately 30%) response to inflammatory stimulus and a greater (4.5-fold) delayed hypersensitivity (DH) to dinitrochlorobenzene than animals fed the n-6 FA source. These data demonstrate that dietary lipid strongly influences tissue FA composition, eicosanoid metabolism, and, in the case of DH, at least one type of T-cell mediated immune response in UV-irradiated animals.

Glass and plastic photoluminescence: interaction with aqueous media and cultured mammalian cells.

Several types of glass and plastic materials were shown to exhibit intense photoluminescence when irradiated with UV. Water or phosphate buffered saline (PBS) contained within vessels of the respective materials when irradiated, also demonstrated relatively long-lived luminescence. A significant percentage (30%) of cultured mammalian cells were killed when exposed to UV-irradiated glass beads. The nature of the luminescence of water or PBS, or whether this or the photoluminescence of glass is directly responsible for cell toxicity, is unknown. However, we call attention to this phenomenon as a potential complicating factor in photobiological studies.

Protective role of butylated hydroxytoluene and certain carotenoids in photocarcinogenesis.

Butylated hydroxytoluene (BHT) and certain carotenoid pigments have been found to inhibit photocarcinogenesis in animal models. In addition, BHT protects against UV-B-induced erythema and UV-B induction of ornithine decarboxylase. Studies on the photoprotective mechanism(s) of BHT suggested that changes in the physico-chemical properties of the keratin of the stratum corneum layer of skin occurred, leading to increases in UV absorption of that tissue. These changes might be exerted via the anti-radical action of BHT that retards oxidation and prevents cross-linking of the keratin chains, resulting in a diminution of UV-B radiation reaching potential target sites. The carotenoids beta-carotene, canthaxanthin and phytoene also inhibit UV-B carcinogenesis. beta-Carotene and canthaxanthin are excellent quenchers of singlet oxygen, and all three pigments can quench free radicals. beta-Carotene and canthaxanthin have been shown to quench singlet oxygen/free radical reactions in the skin of porphyric mice, and these two pigments as well as phytoene have been found to quench excited species formed on irradiation of mouse skin by UV-B.

Influence of dietary omega-6, -3 fatty acid sources on the initiation and promotion stages of photocarcinogenesis.

To determine the segment along the carcinogenic continuum at which dietary lipid exerts its principal effect, six groups of 35 Skh-HR-1 hairless mice were placed on defined isocaloric diets containing either 0.75%, 12% corn oil or 12% menhaden oil as sources of omega-6 or omega-3 fatty acids, respectively. All animals received an 11 week course of UV-radiation from fluorescent sunlamps. Upon termination of UV, diets of some groups were crossed-over to either low fat, high fat, omega-6 or omega-3 fatty acid sources. The first tumor appeared at week 14. Life-table analysis of the tumor incidence curves and Wilcoxon tests of tumor multiplicity provided evidence that high corn oil diets significantly (P less than 0.01) enhance carcinogenic expression; that tumor enhancement by the omega-6 fatty acid source occurs during the post-initiation, or promotion, stage; that replacement with a low corn oil diet after UV-initiation will negate the exacerbating effect of high corn oil; and that an omega-3 fatty acid source inhibits UV-carcinogenesis even at high dietary levels, although not during the post-initiation stage.

Dietary fat modulates immunoresponsiveness in UV-irradiated mice.

Previous studies have shown that a high level of dietary lipid (corn oil) exacerbates UV-carcinogenic expression in hairless mice. Furthermore, it was demonstrated that this effect occurs at the postinitiation, or promotion, stage of UV-carcinogenesis--a stage believed to be modulated immunologically. Thus, we sought to examine the influence of dietary lipid on specific immune parameters at various times within a UV-carcinogenic protocol, with the purpose of detecting potential relationships to UV carcinogenesis. Hairless mice were fed either a high- (12%, wt/wt, corn oil) or low-fat (0.75%, wt/wt, corn oil) diet for 2 weeks prior to start of the UV or experimental protocols. Animals were sensitized to dinitrochlorobenzene (DNCB) hapten and delayed-type hypersensitivity (DTH) was assessed. Delayed-type hypersensitivity was significantly suppressed (P = 0.01) in the high-fat group, even before UV irradiation. Although both groups exhibited UV-induced suppression of this response, the high-fat group was totally suppressed after 3 weeks of UV, whereas the low-fat group exhibited reactivity through week 8. The splenic T-lymphocyte (Thy 1.2+) population had declined by about 50% at the time of UV termination (11 weeks). Dietary lipid exerted no apparent influence upon this T-cell population. However, after 6 weeks of UV, I-J+ cells (a marker shown to be acquired adaptively by suppressor T lymphocytes) began to increase. By week 15 (4 weeks post-UV) I-J+ cells had increased by about 65% in the high-fat group, twice the % increase that occurred in the low-fat group. When UV-induced tumors were transplanted to recipient animals receiving various periods (0, 6, 11 weeks) of UV irradiation, no significant differences in median tumor rejection times between the two dietary groups occurred at 0 or 6 weeks. After 11 weeks of UV, the low-fat group exhibited a tumor rejection time that was comparable to that of nonirradiated animals, i.e. 21 days. However, median tumor rejection time for the high-fat group was greater than 63 days, significantly (P = 0.01) longer than that of the low-fat group. Thus, suppression of tumor rejection by high fat occurred at a time when high fat had been shown to exacerbate carcinogenic expression and when I-J+ cells had markedly increased. These data demonstrate that level of dietary lipid modulates immunoresponsiveness in UV-irradiated animals and is compatible with the thesis that immune suppression may account for the exacerbation of carcinogenic expression elicited by high dietary fat.

Effect of dietary omega-3 and omega-6 fatty acid sources on PUVA-induced cutaneous toxicity and tumorigenesis in the hairless mouse.

Because of concern about psoralen-induced phototoxicity and photocarcinogenesis, we investigated the effects of dietary lipids in a mouse model in which 8-methoxypsoralen (8-MOP) and UVA (PUVA) therapy has been shown to be carcinogenic. SKH-Hr-1 hairless albino mice were fed diets containing either omega-3 or omega-6 fatty-acid sources (menhaden oil and corn oil, respectively). After 2 weeks on the diets, the mice were treated topically with 8-MOP and then exposed to UVA (5 J/cm2). Mice receiving the omega-3 fatty-acid source exhibited a marked decrease in inflammatory response and a more rapid repair, as expressed both grossly and microscopically. In support of the latter response, i.e. repair, ornithine decarboxylase activity was about 20% greater in animals receiving the omega-3 fatty-acid source. The effects of the dietary fatty acid sources on PUVA tumorigenesis were examined in long-term studies in which animals were treated topically with 0.01% 8-MOP thrice weekly after which they were exposed to UVA (1 J/cm2). These studies indicated that a dietary lipid rich in omega-3 fatty acid and known to exhibit anti-inflammatory properties can markedly ameliorate the course of PUVA toxicity but does not impede the course of PUVA tumorigenesis.

Influence of fish oil supplementation on the minimal erythema dose in humans.

A previous study using the hairless mouse model demonstrated that diets containing a fish-oil lipid source, which contained high levels of omega-3 fatty acids, markedly increased the minimum erythema dose (MED) when compared with diets containing other polyunsaturated fatty acids. To determine whether fish oil supplementation could produce a similar effect in humans, 20 subjects were randomized into two groups, a placebo group and a group receiving fish-oil supplements over a 4-week period. Results showed a small, but statistically significant, increase in MED in patients whose diet was supplemented with fish oil. Cholesterol and prostaglandin E2 levels were unchanged, while triglyceride levels were significantly decreased in the fish oil group. No significant changes in any of these parameters occurred in the placebo group.

Influence of dietary factors on actinically-induced skin cancer.

The first indication that high dietary fat intake could influence the development of ultraviolet (UV) radiation-induced skin cancer in experimental animals was reported in 1939. In the 1980s a series of animal studies showed that a high level of dietary fat intake markedly shortened the time between UV exposure and tumor appearance and increased the number of tumors that developed. Further, high levels of dietary fat affected skin cancer development at the promotional stage of UV-carcinogenesis, i.e., after the cancer causing dose of UV had been delivered. Perhaps more important, switching from a high-fat to a low-fat diet immediately after delivery of the UV-initiating dose negated the exacerbating effect of high fat intake. The latter finding suggested that dietary modification, even after a cancer-causing exposure to UV, might represent a potentially important intervention strategy in the prevention of non-melanoma skin cancer (NMSC) and provided the rationale for undertaking a dietary intervention trial. One hundred and fifteen skin cancer patients completed the 2-year clinical trial on the effect of a low-fat diet on occurrence of actinic keratosis (AK) and NMSC. Patients were randomly assigned to either continue their usual diet (control group, NI) or to adopt a diet with 20% of total caloric intake as fat (diet intervention group, DI). All patients were examined at 4-month intervals for new AK and NMSC. At baseline, the mean percent of caloric intake as fat was 40+/-4% in the NI group and 39+/-3% in the DI group. After 4 months of dietary therapy, the percent calories as fat had decreased to 21+/-7% in the DI group. The percent of calories as fat in the NI group did not drop below 37% during the study period. The cumulative number of new AK per patient from months 4 through 24 was 11.6+/-17 in the NI group and 3.2+/-6 in the DI group (P < 0.001). Numbers of new NMSC were analyzed in 8-month periods. There were no significant changes in NMSC occurrence in the NI group. However, NMSC occurrence in the DI group declined significantly (P < 0.02) in the last 8-month period. Patients in the DI group also had significantly (P < 0.01) fewer NMSC in the last 8-month period than did patients in the NI group (0.02 versus 0.26). Practical dietary advice, with respect to reduction of percent of calories as fat, could make an important contribution to the prevention and management of AK and NMSC.