A Model System for the Evaluation of the Role of Cholesterol -Oxide in Ultraviolet Carcinogenesis

Homer S. Black and David R. Douglas Cancer Res 1972;32:2630-2632.

Updated Version

Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/32/12/2630

Citing Articles

This article has been cited by 3 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/32/12/2630#related-urls

E-mail alerts Reprints and Subscriptions Permissions

Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, contact the AACR Publications Department at permissions@aacr.org.

Downloaded from cancerres.aacrjournals.org on September 24, 2012 Copyright 1972 American Association for Cancer Research

[CANCER RESEARCH 32, 2630-2632,

December 1972)

A Model System for the Evaluation of the Role of Cholesterol a-Oxide in Ultraviolet Carcinogenesis1
Homer S. Black and David R. Douglas
Departments of Dermatology [H. S. B., D. R. D./ and Biochemistry [H. S. B.], Baylor College of Medicine [H. S. B., D. R. D./, and Veterans Administration Hospital fff. S. B./, Houston, Texas 77025

SUMMARY

Cholesterol a-oxide (cholestan-5a,6a-epoxy-3j3-ol), known to be carcinogenic, was formed in the skin of hairless mice in response to UV radiation. The levels of compound formed were dose dependent. Three criteria, namely, comparable effects by both the primary incitant (ultraviolet light) and cholesterol a-oxide, the same predisposing specificity, and a direct relationship between the ability of the primary incitant to form tumors and produce cholesterol a-oxide, are suggested as the minimum required to establish a causal involvement of this compound in the etiology of UV carcinogenesis.
INTRODUCTION

the midquadrant of each side was removed. The skin was trimmed, and s.c. tissue was scraped away. The skin specimens were incubated for 24 hr at 4in a mixture of 0.5 ml Tween 80 solution (86.6 mg/100 ml Tween 80 in l%ethanol); 10ml of Krebs-Ringer phosphate buffer, pH 7.4; and 10 tCiof cholesterol-4-14 C (specific activity, 61.7 mCi/mmole). Some tissues were incubated exactly as described, except that labeled cholesterol was omitted from the incubation medium. After the incubation period, the tissues were removed and thoroughly rinsed with Tween 80 solution and deionized water to remove surface contamination by labeled cholesterol. The specimens were blotted dry and weighed, after which they were irradiated for periods of 15, 30, or 60 min with a mercury arc lamp (1.43 X 10s ergs/sq cm/sec of exposure). Conditions of irradiation have been described in detail elsewhere (8). Controls were maintained under the same experimental conditions, except for irradiation. Following irradiation, the specimens were minced with a razor blade and homogenized in a Model 10 Polytron equipped with a saw-toothed generator. Homogenization was accom plished in 10 ml deionized water, followed by a 2nd homogenization cycle to which were added 10 ml chloroform:methanol (2:1 v/v). The total lipids were extracted 4 times with chloroform:methanol. The pooled lipid extract was washed 3 times with an upper-phase salt solution, according to the method of Folch et al. (6). After being washed, the total lipid extract was reduced in volume under N2 and streaked on TLC2 plates (SilicAR-7GF, 20 x 20 cm, 250 /um). The plates were developed in 1,2-dichloroethane, and the polar material remaining at the origin of the plate was eluted. This material was rechromatographed in chloro form racetone (9:1 v/v). A band, corresponding to the RF of reference cholesterol a-oxide, was eluted and prepared for radio-gas-liquid Chromatographie analysis. A stream splitter (30:1 split ratio) was inserted before the detector, and effluent from the Chromatographie column was collected at 2-min intervals. The effluent was collected in small glass cartridges packed with 3% DC-200 silicone fluid on 100 to 200 mesh Florisil. The same preparative procedure was followed for those samples incubated in the absence of labeled cholesterol except that, after elution of the band from the 2nd TLC plate, the material was dried under N2 and esterified with acetic anhydride-14C. The dried eluant was allowed to react overnight at room temperature in the presence of pyridine

UV radiation is known to produce a number of acute effects in both rodent and human skin, including effects upon nucleic acids, proteins, and lipids, as well as upon their metabolism and upon the normal mitotic cell cycle (2, 4, 5, 9). Although some of these responses have been thoroughly studied, thus far no positive experimental relationship between these effects and their etiological role in UV carcinogenesis has been established. The presence of cholesterol a-oxide was recently demon strated in human skin irradiated with UV (3). This compound was simultaneously reported to be present in serum from patients who exhibited elevated blood cholesterol levels (7). Although sterol epoxides apparently occur rarely in nature, previous studies with cholesterol a-oxide have shown that this particular epoxide possesses carcinogenic properties (1). This property, as well as the known carcinogenic effects of UV and the fact that cholesterol a-oxide is formed in skin in response to UV radiation, suggests that the compound might be involved in the etiology of skin cancer. The purpose of this paper is to report the formation of cholesterol a-oxide in the skin of hairless mice irradiated with UV and to suggest criteria that could provide more definitive evidence for the involvement of cholesterol a-oxide in UV carcinogenesis.
MATERIALS AND METHODS

Albino hairless mice were decapitated, and a flap of dorsal skin from the shoulder to the hip and extending ventrally to

1Supported in part by the Morrison Trust of San Antonio, Texas; by with acetic anhydrideC (1:1, v/v). The reaction was halted the American Cancer Society; and by USPHS Research Grant 2The abbreviations used aie: TLC, thin-layer Chromatographie; GLC, CAI3464-01 from the National Cancer Institute. gas-liquid chromatography. Received June 15, 1972; accepted August 23, 1972.

2630

CANCER RESEARCH

VOL. 32

Downloaded from cancerres.aacrjournals.org on September 24, 2012 Copyright 1972 American Association for Cancer Research

A UV-induced Sterni Carcinogen by the addition of an equal volume of water and then was extracted 3 times with a volume of heptane equal to the total reaction mixture. The heptane extract was dried under N2 and made to the desired volume for GLC. A Victoreen Model 4000 Chromatograph equipped with a flame ionization detector was used for GLC determinations. The glass column, 6 ft x 3 mm i.d., was packed with 3% OV-1 on 100 to 120 mesh Gas-Chrom Q [column temperature, 225; flash heater and detector temperature, 300;carrier gas (N2) flow rate, 17 ml/min]. Radioactivity measurements were made with a Packard Model 3375 liquid scintillation spectrometer. Mean counting efficiency for 14C was 78%. The percentage S.D. of sample counts was always 2.5 or less. Quenching was determined the automatic external standardization method.
RESULTS AND DISCUSSION

by

Previous studies with human skin demonstrated that the techniques used in the present investigation were adequate to separate many of the polar photooxidation products of cholesterol (3, 8). The total lipid fraction was applied to silica gel-coated TLC plates and was developed with 1,2-dichloroethane. The polar
FORMATIONOF PRODUCTS_ POLAR PHOTO-OXIDATION 3o3o oa.
20ot0EQ_i

nQ1 U<C_iOU-*
^>->V^^1

nP_1610<XX^> ^

^1^^jj'S//}1rS/SW,
10 20
IN MINUTES

LEVELS OF CHOLESTEROL-OXIDE IN PHOTO-OXIDATION PRODUCTS 30 rx o

30

40

28

TIME

20
o o:

12

14

Chart 2. Gas Chromatographie record and radioactivity profile. The GLC tracing is that of nonlabeled reference cholesterol a-oxide acetate ester. The reference compound was added to the radioactive esterified fraction of skin irradiated for 60 min. The histogram represents levels of radioactivity in sequential fractions of the GLC effluent. The concentration of cholesterol a-oxide ester, based on the specific activity of the acetic anhydride, is approximately 1 jug/g skin.

10

material that remained at the origin of the plates containing the cholesterol-derived photoproducts was eluted, and radioactivity levels were determined (Chart 1). Photoproducts were further analyzed by cochromatography with reference 0 15 30 45 60 TIME OF U V IRRADIATION oxidation products of cholesterol and development in a (MINUTES) chloroform: acetone system. The photoproduct fraction Chart l. Levels of polar photooxidation products were determined as migrating with the same RF as reference cholesterol a-oxide the percentage radioactivity remaining at the origin of the 1st TLC. Levels of cholesterol a-oxide were determined by GLC as the was eluted and analyzed by radio-gas-liquid chromatography. percentage of cholesterol a-oxide in the polar photooxidation product A comparison of the radioactivity profiles eluted from the fraction. GLC column clearly indicates the presence of this compound
o
TL O

DECEMBER

1972

2631

Downloaded from cancerres.aacrjournals.org on September 24, 2012 Copyright 1972 American Association for Cancer Research

Homer S. Black and David R. Douglas as a component of the photooxidized constituents derived of the compound under question. Wheeler and Luke further from the cholesterol-14C taken up by mouse skin (Chart 1). stated that the rule of "Occam's razor" be satisfied in assuming The levels of both polar photoproducts and cholesterol a-oxide formed in mouse skin are functions of the light dose that was received. In preliminary studies, cholesterol a-oxide was shown to be less susceptible to photooxidation than cholesterol. Therefore, one would not expect that the compound would be destroyed at a rate comparable to its formation from cholesterol. It could be argued that dependence of the identification of cholesterol a-oxide upon the uptake of radiolabeled parent compound distracts from the supposition that cholesterol a-oxide is, indeed, formed in skin from the naturally occurring sterol. It has been demonstrated that many radiolabeled organic compounds are more subject to autooxidation. Additional evidence for the identification of cholesterol a-oxide was obtained, as well as proof of origin of this compound from naturally occurring sterols, when those samples incubated in the absence of radiolabeled cholesterol were isolated and labeled by esterification with radioactive acetic anhydride (Chart 2). Thus it may be concluded that cholesterol a-oxide is formed in the skin of hairless mice in response to non-ionizing UV radiation. The usefulness of the hairless mouse previously was demonstrated in the study of UV carcinogenesis (5). Whereas the formation of a compound with demonstrated carcinogenic properties in this animal may be of biological interest, the mere presence of such a compound does not necessarily implicate it in the disease etiology. One must establish critical criteria which can, at least, correlate the observed response to the developmental sequence of the disease. The following criteria fulfill the basis for proof of causal involvement of a naturally occurring compound in disease etiology and are modifications of those criteria suggested by Wheeler and Luke (10) for the causal involvement of microbial toxins in plant disease: (a) primary incitant (UV) and the UV-induced compound both must be capable of causing tumor formation; (b) UV and the UV-induced compound must exhibit the same predisposing specificity; (c) the ability of UV to cause tumor formation varies directly with its ability to induce formation that only a single substance produced all the disease symptoms. We have omitted this criterion. It seems too stringent in lieu of the fact that, at best, only a positive correlation can be obtained in regard to the role played by the substance in causation of disease symptoms. In conclusion, we have demonstrated tht UV will induce the formation of cholesterol a-oxide in the skin of hairless mice. The role of this compound in the etiology of UV carcinogenesis can be critically evaluated by application of the suggested criteria. With further modification, these criteria might successfully be applied in the evaluation of other light-induced acute effects in the etiology of UV carcinogenesis.

REFERENCES 1. Bischoff, F. Carcinogenic Effects of Steroids. Advan. Lipid Res., 7: 165-244, 1969. 2. Black, H. S. An Experimental Basis for Carcinogenic Effects of Ultraviolet Radiation. Space Life Sci., in press. 3. Black, H. S., and Lo, W.-B. Formation of a Carcinogen in Human Skin Irradiated with Ultraviolet Light. Nature, 234: 306-308, 1971. 4. Blum, H. F. Caicinogenesis by Ultraviolet Light. Princeton, N. J.: Princeton University Press, 1959. 5. Epstein, J. H. Ultraviolet Carcinogenesis. Photophysiology, 5: 235-273, 1970. 6. Folch, J., Less, M., and Stanley, G. H. S. A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues. J. Biol. Chem.,226: 497-509, 1957. 7. Gray, M. F., Lawrie, T. D. V., and Brooks, C. J. W. Isolation and Identification of Cholesterol a-Oxide and Other Minor Sterols in Human Serum. Lipids, 11: 836-843, 1971. 8. Lo, W.-B., and Black, H. S. Formation of Cholesterol-derived Photoproducts in Human Skin. J. Invest. Dermatol., 58: 278-283, 1972. 9. Urbach, F. (ed.) The Biologic Effects of Ultraviolet Radiation. Oxford, England: Pergamon Press, Inc., 1969. 10. Wheeler, H., and Luke, H. H. Microbial Toxins in Plant Disease. Ann. Rev. Microbiol., 1 7: 223-242, 1963.

2632

CANCER RESEARCH

VOL. 32

Downloaded from cancerres.aacrjournals.org on September 24, 2012 Copyright 1972 American Association for Cancer Research