Relation of Antioxidants and Level of Dietary Lipid to Epidermal Lipid Peroxidation and Ultraviolet Carcinogenesis1
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[CANCER RESEARCH 45, 6254-6259, December 1985] Relation of Antioxidants and Level of Dietary Lipid to Epidermal Lipid Peroxidation and Ultraviolet Carcinogenesis1 Homer S. Black,2 Wanda A. Lenger, Janette Gerguis, and John I. Thornby Photobiology Laboratory [H. S. B., W. A. L, J. G.] and Biostatistics Section [J. I. T.], Veterans Administration Medical Center, and Department of Dermatology [H. S. B.¡, Baylor College of Medicine, Houston, Texas 77211 ABSTRACT that dietary fat enhanced the incidence of coal tar-induced skin tumors. This effect of dietary lipid upon chemical carcinogenesis It has become increasingly evident that both quantity and was soon substantiated and numerous studies have since been quality of dietary lipid can influence the developmental course of conducted on the quantitative and qualitative composition of several major forms of cancer in experimental animals. Using the dietary lipids and their relationship to tumor enhancement (4-9). hairless mouse-ultraviolet (UV) model, we had previously dem Generally, these studies have shown that animals fed high-fat onstrated that unsaturated lipid compared to equivalent levels of diets develop tumors more readily than cohorts fed low-fat diets. hydrogenated lipid enhanced photocarcinogenesis with respect Tumors of which this effect has been most often observed are to both tumor latency and multiplicity. In the present study using those of the skin, mammae, and intestine. High levels of fat the same model, we have examined the effect of unsaturated appear to exert maximum influence upon the promotional stages lipid level and antioxidants upon epidermal lipid peroxidation and of carcinogenesis (10). UV carcinogenesis. Sixteen groups of 45 animals each were With regard to quality of fat, it is polyunsaturated lipids which used in the study, representing all combinations of three design generally enhance tumorigenesis. Recent studies indicate that variables: (a) a semipurified diet containing 4, 2, or 0.75% com small quantities of polyunsaturated fats when fed concomitantly oil or 4% soybean oil; (b) 2% (w/w) antioxidant supplement or with high levels of saturated fat enhance tumor formation as no supplementation; and (c) an escalating regimen of UV radia effectively as high levels of unsaturated lipid alone (7). These tion to a cumulative dose of 70 J/cm2 or no irradiation. The data suggest that polyunsaturated lipids are required for the nonirradiated groups served as nutritional controls and as sub most effective expression of specific chemically induced carci jects for epidermal lipid peroxidation measurements. An approx nogenesis. imate linear relationship between lipid level and tumor latency The impact of these observations concerning the effect of lipid was observed, with 4% levels of unsaturated lipid producing upon experimentally induced cancer has recently assumed maximum enhancement of photocarcinogenesis. Furthermore greater significance with the collection of epidemiológica! data with increasing lipid level the numbers of tumors per animal showing a positive correlation between dietary fat and human increased. Antioxidants caused significant increases in tumor mortality rates resulting from certain types of cancer, notably latency and decreases in tumor multiplicity but only at the highest mammary and colonie (11,12). Although a considerable literature lipid level used in these studies. Thiobarbituric acid values of is accruing that suggests that etiologies of the main human epidermal homogenates also increased in relation to the level of cancers stem largely from our life-styles (13), it seems ironic that dietary lipid intake. Epidermal thiobarbituric acid values from this potential relationship with respect to skin cancer has re antioxidant supplemented animals were significantly lower re ceived so little attention, especially as both initiator (UV) and gardless of lipid intake levels. From these data we conclude that modifier (diet) so profoundly manifest life-style. (a) dietary lipid level has a direct effect upon the carcinogenic Previously the only direct study of the relationship of diet to response to UV both in regard to tumor latency and tumor UV carcinogenesis was that of Baumann and Rusch (14) pub multiplicity; (b) antioxidants produce an inhibitory effect almost lished in 1939 in which they observed that animals fed high levels equal to the degree of exacerbation of carcinogenesis evoked of fat formed UV-induced tumors more rapidly than animals fed by increasing lipid levels, at least for the range studied; and (c) low-fat diets. More recently, we observed that the degree of dietarily administered antioxidants inhibit the formation of epi saturation of dietary lipids markedly influenced the UV carcino dermal thiobarbituric acid reacting materials. These data strongly genic response (15). Unsaturated lipid enhanced UV carcinogen imply that free radical reactions, specifically lipid peroxidation, esis both with respect to latency and multiplicity and it was play a role in at least a part of the photocarcinogenic response. suggested that unsaturated lipid might be required in photocar cinogenesis just as is the case for certain chemically induced cancers. In addition, the level of unsaturated lipid appeared to INTRODUCTION affect both the direction and magnitude of antioxidant-mediated modification of photocarcinogenesis (16). In the current study The influence of dietary lipid upon carcinogenesis first became we have examined the influence of antioxidants and dietary lipid apparent over 50 yr ago when Watson and Mellanby (3) observed level upon UV carcinogenesis and epidermal lipid peroxidation. Received 4/22/85; revised 7/24/85; accepted 8/16/85. 'Supported in part by USPHS Grant CA-20907 from the National Cancer Institute and by Veterans Administration Medical Center Research Funds. Prelimi MATERIALS AND METHODS nary reports of this work were presented at the Annual Meetings of the American Society for Photobiology, June 1983, Madison, Wl (1) and the American Association Animals and Diets. Three- to 4-mo-old female hairless (SKh-Hr-1) for Cancer Research, May 1984, Toronto, Canada (2). 2 To whom requests for reprints should be addressed, at Photobiology Labora mice were obtained from the Skin and Cancer Animal Colony, Temple tory, Veterans Administration Medical Center, 2002 Holcombe Boulevard, Houston, University, Philadelphia, PA. Upon receipt the animals were maintained TX 77211. on Wayne Lab-blox (Continental Grain Co., Chicago, IL) for a 2-wk CANCER RESEARCH VOL. 45 DECEMBER 1985 6254 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1985 American Association for Cancer Research. ANTIOXIDANTS, DIETARY LIPID, PEROXIDATION, AND UV CARCINOGENESIS Table 1 designated times. ADP and FeSO4 were added to some aliquots to give Semipurified experimental diets of varying lipid composition final concentrations of 0.73 and 0.65 mw, respectively (21). The reaction Diets" was halted by addition of 15% trichloroacetic acid to give a final concen tration of 5% trichloroacetic acid. The mixture was centrifuged at 2000 x g for 20 min, the supernatant was decanted, an equal volume of Casein (vitamin free) 27.40 27.40 27.40 27.40 0.67% 2-thiobarbituric acid was added, and the product was placed in a 4.00 2.00 0.75 Com oil (tocopherol stripped) boiling water bath for 10 min. The sample was cooled to room temper Soybean oil (cold pressed) 4.00 Com starch 55.90 60.30 63.00 55.90 ature and made alkaline by addition of 200 /<l of 5 M KOH (0.29 M, final Mineral mix6 6.00 6.00 6.00 6.00 concentration). Absorbance was read at 543 nm. Malonaldehyde Vitamin mixc 2.20 2.20 2.20 2.20 bis(diethylacetal) was used as a standard for quantitation of TBA reacting Celufil (nonnutritive) 2.10 2.10 2.10 2.10 materials. " Caloric density: Diets 1 and 4, 3.95 kcal/g; Diet 2, 3.86 Kal/g; Diet 3, 3.80 Peroxide values were determined by the method of Swoboda and Lea kcal/g. (20). At designated incubation times 1-ml aliquots of epidermal homog United States Biochemical Corp.; Phillips and Hart salt mixture. 0 United States Biochemical Corp.; vitamin mix minus ascorbic acid and to enate were acidified with 0.1 ml acetic acid and extracted with 1.5 ml copherol. chloroform followed by a second extraction of 1.0 ml. The pooled lipid extract was dried under N2, solubilized with 3 ml chlorofornracetic acid quarantine period after which they were divided into 16 groups of 45 (3:2, v/v), and transferred to a tube fitted with sidearm and stopcock. animals each. Control (non-UV) and UV-irradiated groups received the After deaeration with bubbling CO2, 80 ^l of iodide reagent (1.2 g powdered diets shown in Table 1. The custom diets were prepared by potassium iodide/ml H2O, freshly prepared) was added and the tube was United States Biochemical Corp., Cleveland, OH. In addition, similar sealed under slight positive pressure of CO2 and left standing for 1 h in groups received these diets supplemented with a 2% (w/w) antioxidant the dark. The mixture was then diluted with 7 ml of 0.5% aqueous mixture consisting of 1.2% ascorbic acid, 0.5% butylated hydroxytolu- cadmium acetate solution, stoppered, vortexed for 15 s, and the Diphasic ene, 0.2% DL-a-tocopheryl acetate, and 0.1% reduced glutathione. This system was separated by centrifugation (2000 x g for 15 min). Absor antioxidant mixture had previously been shown to inhibit UV carcinogen- bance of the aqueous supernatant was determined at 350 nm. Calibration esis, although the active principal is thought to be butylated hydroxytol- was accomplished using a series of reagent blanks consisting of various uene (17). Antioxidant-supplemented diets were mixed in a twin-shell dry volumes of 0.2 mw potassium iodate in 0.5% cadmium acetate solution. blender in our laboratory and all diets containing antioxidants were stored Protein was determined by the method of Lowry et al. (22). refrigerated under vacuum. Animals were fed ad libitum and housed 6- Statistics.