Biochemical parameters of epidermal aging in the hairless mouse and the relationship to UV-carcinogenesis

111

J. Photochem. Photobiol. B: Biol., 23 (1994) 111-118

Biochemical parameters of epidermal aging in the hairless mouse and the relationship to UV-carcinogenesis+ Homer

S. Black++, Judy Chiang,

Janette

Gerguis,

Wanda

Lenger,

John

I. Thornby

Photobiology Laboratory, Veterans Affairs Medical Center and Department of Dermatology, Baylor College of Medicine, Houston, TX 77030 (USA) (Received

July 19, 1993; accepted

January

10, 1994)

Abstract Epidemiological studies suggest that the incidence of cancer increases with age in both human and animal populations and that declining physiologic condition associated with aging might be responsible. Experimentally, the reverse has been most often observed, that is, older animals appear less susceptible to the induction of UVcarcinogenesis. Thus, we examined several biochemical parameters of epidermal macromolecular synthesis in hairless mice in an effort to gain insight into the role these processes play in physiological aging and their relationship to carcinogenesis. SKh-Hr-1 hairless mice were randomized into two groups &IV-irradiated and nonirradiated controls) and were two months of age at the start of irradiation and biochemical analyses. The UV group received 0.028 sunburn units (SBUs) daily (5 days wk-‘) for 16 months from 40 watt BZS-WLG lamps. Stratum corneum turnover rates @CR), cell label index (CLI), protein, DNA and RNA synthesis, and ornithine decarboxylase (ODC) induction were determined at monthly intervals over a period of two years. There were no age-related tendencies observed in SCR. CL1 increased with age. Chronic, low-dose UV had no effect upon either of these parameters. Epidennal capacity for DNA and protein synthesis increased with age from 2 months to 12-15 months at which time both parameters peaked and then began to decline. UV significantly reduced (P
Key words: Aging; Epidermis; Macromolecular (ODC) induction; UV-carcinogenesis

synthesis;

1. Introduction Epidemiological studies have demonstrated that the incidence of cancer increases with age in both human and animal populations. For example, Peto et al. [l] have shown that the probability that a human will develop cancer in the succeeding 5year period is 50 times greater at age 65 than at age 25. Furthermore, it has been shown that with equivalent sun exposure, individuals over 60 years of age are at a significantly greater risk of de‘Paper presented at the 21st Annual Meeting of the American Society for Photobiology, Chicago, IL, June, 1993. +‘Author to whom correspondence should be addressed.

loll-1344/94/$07.00 0 1994 Elsevier SSDZ 1011-1344(94)06986-Q

Sequoia. All rights reserved

DNA;

RNA;

Protein;

Ornithine

decarboxylase

veloping skin cancer than those under 60 [2]. However, experimental evidence to support the concept that a relationship exists between cancer and aging remains equivocal [3, 41. Forbes et al. [5] assert that tumor response reflects two distinguishable aspects of aging, i.e., . passage of time required by repeated exposure to the carcinogen, resulting in accumulated damage; and the “physiologic age” of the target tissue, which correlates with passage of time. Little is known of the latter. Whether the influence of physiologic age on carcinogenesis results from pathologic changes in the biochemical properties of the target tissue, or physiological properties of the organism (systemic factors, e.g., immunores-

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H.S. Black et al. I Relationship between epidemal aging and UV-carcinogen&s

ponse) is unknown. If predisposition to UV-carcinogenesis is not attributable to inherent properties of the skin per se, but dependent upon physiological factors of the host that change with age, then conventional reasoning would predict that the induction of cancer by carcinogenic agents should become progressively easier with advancing age [6]. This supposition appears to be in conflict with the experimental data from a wide range of UV-protocols, i.e., older animals appear less susceptible to UV-induction of cutaneous tumors [5, 7-91. Whereas the epidermis would be the logical tissue in which to pursue relationships between aging and UV-carcinogenesis, most studies have described dermal manifestations of cutaneous photoaging. Furthermore, the preponderance of experimental evidence that UV plays a role in aging comes from studies of cultured cells and although considerable evidence points to a decline in DNA repair and biosynthetic capacity as factors in both aging and carcinogenesis, corroboration of these results has not always been forthcoming [lo]. For example, the number of population doublings declines in cells derived from chronically sun-exposed sites whilst demonstrating an increased plating efficiency [ 111. Moreover, while decreased DNA excision repair has been related to increased age, fibroblasts derived from chronically sun-exposed areas demonstrate a higher capacity for macromolecular synthesis and DNA repair [12,13]. Thus, a number of paradoxes exist that not only cloud the potential relationship of aging and carcinogenesis, but also the role of macromolecular synthesis in these processes. Consequently, we undertook a chronologic study to examine several biochemical parameters of macromolecular synthesis in the epidermis of UV and non-irradiated hairless mice as a first step to gain insight into the role these processes might play in physiologic aging and their relationship to UV-carcinogenesis. 2. Experimental

methods

2.1. Animals

SKh-Hr-1 female, hairless mice were obtained, upon weaning, from the Skin and Cancer Animal Colony, Philadelphia, PA. An ear punch code was used to identify individual animals. The animals were housed six per cage and maintained, ad Zibitum, on Wayne Lab-blox (Allied Mills, Chicago, IL). Individual body weights were determined monthly. Animals were randomized into two groups

(UV-irradiated and non-irradiated controls) and were two months’of age at the start of irradiation and biochemical analyses. 2.2. Irradiation An irradiation rack of special design was employed [9]. The animals were irradiated unrestrained in their cages by a bank of two 4 ft Westinghouse BZS-WLG lamps positioned at a distance of 14 cm from the dorsal surface of the animals. The flux of each lamp bank was determined biweekly with a Model 2A Sunburn meter (Solar Light Co., Philadelphia, PA.). Systematic rotation of cages throughout the length of the respective lamp banks compensated for any variation in power output, the latter being minimal. The emission spectra of BZS-WLG and FS-40 lamps have been compared [9]. Animals were irradiated 5 days w-i for 16 months (352 days of irradiation) at an average h-radiance of 0.028 sunburn units (SBUs) per day. The total cumulative dose was 9.8 SBUs. This level of irradiance was expected to result in less than 10% tumor-bearing animals for the period of the study

WI. 2.3. Stratum comeum protein synthesis

turnover rate and epidemal

As atrophy is one of the few anatomical signs associated with epidermal aging, stratum corneum turnover rate (SCR) was employed as a quantitative tool to examine squamous epithelial renewal. Five animals from each group were weighed and, at 20 min post-UV, injected intraperitoneally (1 &i g -’ body weight) with 3H-leucine (SA 60 Ci mm01-~). At 72 hr post-UV (day 3), stratum corneum (SC) was sampled from three selected dorsal sites of two of the five animals from each group by evenly pressing Scotch brand cellophane tape onto the skin. The tape was removed and placed in scintillation cocktail, its radioactivity was determined, and quantitated as dpm cm-’ SC.The method was essentially that described by Downes et al. [15]. After tape-stripping, the animals were killed by cervical dislocation, and a dorsal flap of skin was excised from each mouse. Subcutaneous tissue was scraped away and the whole skin subjected to 55 “C heat treatment for 30 s [16]. The epidermis was isolated and homogenized in 4 ml cold 0.5 N HClO,. The homogenate was centrifuged at 2000g for 20 min and the supernatant discarded. The resulting pellet was resuspended and washed three times as described in isolation. The washed pellet was dissolved in 4 ml 0.5 M NaOH by

H.S. Black et al. / Relationship between epidermal aging and UV-carcinogenesis

heating for 35 min at 80-90 “C. The hydrolyzate was centrifuged at 10 OOOgfor 20 min and the supernatant recovered. Aliquots were taken for radioactivity and protein determinations [17]. Epidermal protein synthetic rates were expressed as dpm mg-’ protein. Stratum corneum was sampled from the remaining three animals of both groups on posttreatment days 4, 5 and 6. 2.4. Nucleic acid synthesis Epidermal nucleic acids were isolated by the Schmidt-Thannhauser-Schneider procedure [18]. Three animals from both groups were injected (1 &i g-’ body weight) with either tritiated uridine (SA 25 Ci mm01- ‘) or thymidine (SA 60 Ci rnm01-~) 20 min post-irradiation at the designated times. The animals were killed one hr post-injection and epidermal homogenates prepared as described previously. Preliminary studies had indicated that the low dose UV administered in this study did not result in elevated DNA synthesis as had been reported for higher UV exposures at similar postirradiation periods [19].

113

for 20 min, the supematants of the duplicate samples were combined. Aliquots were taken for radioactivity and DNA determinations. DNA was quantitated by the diphenylamine reaction [21]. 2.7. Cell label index (CLI) At the time of killing for DNA determinations, three punch biopsies were obtained from the dorsal skin. The tissue was fixed in buffered formalin, dehydrated, embedded, and sectioned at 4 pm. The sections were deparaffinized and the slides dip-coated with Kodak NTB-2 emulsion (Eastman Kodak Co., Rochester, NY). After 21 days exposure, the slides were developed in Kodak D19 solution and fixed in F-5 bath [22]. The sections were then stained with Mayers hematoxylin and counterstained with eosin. All epidermal cells labeled with >3 grains per nucleus were included for the index. A total of 900 cells per treatment were counted.

2.8. Omithine decarboxylase (ODC) induction Three animals at each of the time periods were irradiated from a bank of two Westinghouse FS20 sunlamps at a dose of 0.45 J cmm2. The relationship of ODC induction to UV in this dose 2.5. RNA range has been reported [23]. Emission energy Epiderinal homogenates were centrifuged at was measured with an Eppley circular thermopile 2000g at 4 “C for 15 min. Supematant was discarded, attached to a Keithley microvolt/ammeter. ODC the precipitate resuspended in 4 ml cold 1N HC104, assays were conducted at 28 h post-irradiation, a and recentrifirged. The precipitate was resustime at which UV-induced ODC activity has been pended and washed once in 4 ml of cold ethanol, shown to reach maximum levels [24, 251. In adafter which the precipitate was extracted at room dition, preliminary studies with young (9-12 wk) temperature with three times volume (12 ml) of and old (67-80 wk) animals were conducted at 3:l ethanol:diethyl ether. The extract was centri15 to 40 hr post-irradiation times to assure that fuged at 2000g for 15 min, the supematant disthe time-response for UV-induced ODC activity carded, and the precipitate re-extracted and cenhad not shifted with animal age. trifuged as previously. The precipitate was then Epidermal preparations from the three mice treated for 16-20 hours at 37 “C with 1M KOH. were pooled and homogenized in 3.1 ml of 50 The hydrolyzate was neutralized with 6N HC104, mM sodium phosphate buffer (pH 7.2) containing centrifuged at 2000g for 15 min and the supernatant 0.1 mM pyridoxal phosphate and 0.1 mM EDTA. recovered. The precipitate was washed with 4 ml The homogenates were centrifuged at 30 OOOgfor 0.5 N HC104, centrifuged, the supernatant re30 min to give a soluble supematant [26]. Individual covered, combined with the previous supernatant, assay mixtures contained 35 mM sodium phosphate and aliquots used for radioactivity and RNA de(pH 7.2), 0.2 mM pyridoxal phosphate, 4.0 mM terminations. RNA was quantitated by the orcinol dithiothreitol, 1.0 mM EDTA, 0.4 mM L-ornithine reaction for pentose [20]. with 0.5 &i of DL-[~-‘~C]ornithine hydrochloride 2.6. DNA (SA 59 mCi nnn01-~), and 100 ~1 of epidermal supematant in a final volume of 0.25 ml. The After initial preparation of the epidermal homixture was incubated at 37 “C for 60 min in mogenates, the samples were split prior to cenrubber-stoppered tubes with enter wells, each contrifugation. The centrifugate was washed twice with cold HClO, and once with cold ethanol. The taining 0.2 ml hyamine hydroxide. The reaction was halted by the addition of 0.5 ml 2 M citric supematant was discarded after each wash. The acid and incubated for an additional 60 min. The precipitate was hydrolyzed in 4 ml of 0.5 N HClO, at 90 “C for 15 min. After centrifugation at 2OOOg center well was then transferred to scintillation

114

H.S. Black et al. I Relationship between epidennal aging and W-carcinogenesiv

a-

cocktail and radioactivity was determined. All assays were conducted in triplicate and contained between 0.3 and 0.6 mg protein. Epidermal preparation and ODC determinations were routinely conducted between 1 and 4 pm to avoid circadian effects. 2.9. Statistics Second order polynomial models were fitted to the DNA, RNA, protein, and CL1 data. Differences, i.e., magnitude, between curves were tested using the Wilcoxon Signed Rank test. ODC activity was evaluated from 52 individual data points obtained in 14 experiments comparing various animal ages. The linear effect of age on ODC was estimated by regression, adjusting for differences among experiments. Tumor incidence was determined from a cumulative survival distribution (latency to tumor onset in weeks) estimated for the UV-treated group using the computer program LIFETEST in the Statistical Analysis System (SAS) library.

765 432

1 0'

0

6

I

I

I

12

18

24

AGE (Months) Fig. 1. Cell label index. The ordinate values represent the percentage of radiolabeled cells (> 3 grains per nucleus). A total of 900 cells were counted per treatment. Cl, dashed line - UVirradiated; 0, solid line - non-irradiated controls. 70000 -

l l

60000 -

3. Results Animals developed normally and remained in a general state of good health throughout the experimental period. No significant differences in body weight between groups occurred, nor were there significant differences in rate of weight gain. Body weights plateaued by month 10 and remained relatively constant thereafter. No tumor-bearing animals were used in any of the analyses. The first papilloma in the UV group appeared at eight months, the second at 11 months of age. The cumulative tumor incidence was only 0.146 at month 15, a time at which macromolecular synthetic rates had already reached peak values and which permitted the exclusion of tumor-bearing animals for analyses without introducing undue selection bias. Stratum comeum turnover times remained relatively constant throughout the two-year study, averaging 4.87 +0.96 S.D. and 4.56 f 1.09 days for control and UV groups, respectively. This value is in agreement with the 4-5 day SCR for this species reported by Downes et al. [15]. There were no discernible age-related tendencies, nor did chronic UV exposure alter this parameter. In what would seem to support this observation on epithelial renewal, increased age or UV exposure did not diminish the CL1 (Fig. 1). In Fig. 2, it can be seen that the level of DNA synthesis peaks at around 15 months after which

%I

l

;

50000 -

S g

40000 -

z F

30000 -

F l

.=a,

0

0

6

1

1

I

12

18

24

AGE (Months) Fig. 2. DNA synthesis. Ordinate values represent specific activity, i.e., dpm mg-’ DNA. Cl, dashed line - UV-irradiated; 0, solid line - non-irradiated controls.

it begins a decline that continues to the end of the experimental period. Chronic UV exposure significantly reduced (P< 0.04) the magnitude of DNA synthetic capacity, at peak periods of synthesis, but control levels had declined to those of UV-treated tissue by the end of the study. The rate profile of epidermal protein synthesis was similar to that of DNA, but UV-irradiation had no significant effect (Fig. 3). The RNA synthetic rate demonstrated a rather marked decline beginning at around 12-15 months of age and reached its lowest level at 24 months (Fig. 4). UV-irradiation had no significant impact. In accord with this general regression in RNA

115

H.S. Black et al. / Relationship between epidennal aging and UV-carcinogenesis

01 0

6

18

12

24

AGE (Months) Fig. 3. Protein synthesis. Ordinatevalues represent specific activity, i.e., dpm mg-’ protein. q, dashed line - UV-irradiated; 0, solid line - non-irradiated controls.

0

0

5

10

15

20

25

AGE (Months) 3500

Fig. 5. UV-induction of omithine decarbozylase activity. A standardized dose of UV was administered to animals of varying age. The regression line reflects a steady and signifkant reduction of inducibility with increasing animal age. Inset: spline plots of epidennal ODC activity/post-UV time responses of young (solid line) and aged (dashed line) animals.

0 r

a ,E E i+

3000

q

. 2500 I 2000

0 cl

l

0

4. Discussion

-

1500 1000 -

I 0

I

I

I

I

6

12

18

24

AGE (Months) Fig. 4. RNA synthesis. Ordinate values represent specific activity, i.e., dpm mg-’ RNA. 0, dashed line - UV-irradiated; 0, solid line - non-irradiated controls.

synthetic capacity with advancing age, UV-induction of ODC activity also significantly declined (P
Diminished epidermal thickness, accompanied with a reduced mitotic rate, is one of the clinically relevant changes commonly observed in aging human skin [27]. In general, the human epidermal turnover rate begins to decline after the age of 50 years [28]. A number of investigators have observed similar declines in proliferative capacity of aged rodent epidermis [29-311, and it is generally recognized that the proliferative capacity of cells declines with age. In cultured human diploid cells, the CL1 has been correlated with cell proliferation and has been used as a reproducible and quantitative measure of culture age [32]. Even here however, the decline in proliferative capacity is markedly influenced by alterations in population dynamics of the culture. Indeed, age-related changes in cell kinetics of rodent epidermis, complicated by body site-specific variation, reflect the difficulty in selecting a single biochemical index which is representative of physiological age. In the current study of hairless mouse epidermis, we observed no decline in the proliferative capacity in the two parameters examined, CL1 and SCR. These observations are in agreement with those of Iverson and Schjoelberg [33] who reported that the CL1 of epidermis from the Oslo strain of hairless mouse was low in animals less than two

116

H.S. Black et al. / Relationship between epidemal

months of age, increased up to five months, and remained relatively constant through 24 months. They did not find any evidence of a systematic decrease in epidermal cell proliferation with increasing age. Argyris [34] found a significant decrease in epidermal wet weight in aging Balb/c mice, and as no significant changes in age-related mouse epidermal cell proliferative parameters, as indicated by CL1 and mitotic index [35], were apparent, suggested that this effect, along with a thinner epidermal layer, might be due to a decrease in epidermal cell size. Interestingly, the decline in replicative capacity of aged cultured cells is accompanied by an increase in cell size [36]. Our data suggest that, at least in the hairless mouse, there is no general decline in the proliferative capacity of the epidermis during the first two years and that indices of aging derived from cultured cells are not reflective of what occurs in this animal. Danner and Holbrook [37] have recently emphasized that although some global parameters are similar, cells in vitro and in vivo demonstrate entirely different patterns of synthetic and degradative changes with age. Although there were no indications of diminished epidermal proliferative capacity with increasing age of hairless mouse epidermis, the profile of DNA synthetic rates indicate that the rate in 2-month old mice is relatively low, increases and reaches a peak around 15 months, after which there is a decline through the 24 month period. However, 24 month levels were higher than those of 2-month old animals (Fig. 2). Further, chronic low-dose UV significantly suppressed the peak level of DNA synthesis but it was no lower in UV-irradiated animals at 24 months than that of non-irradiated controls. Protein synthetic rates followed a similar pattern to that of DNA, but UV had no effect (Fig. 3). It is of interest that macromolecular synthesis, i.e., DNA and protein, were increasing or had peaked at times which others had shown animals to be most refractory to induction of UV-carcinogenesis (Fig. 6). It may be that nested within the rather broad parameters of synthetic capacity as we have measured in DNA and protein synthesis, and which the latter may represent general housekeeping products, are specific products of gene expression that are affected by age. Indeed, in cultured keratinocytes it has been shown that baseline expression of several differentiation-related genes are effected by age [38]. In support of the argument that expression of specific gene products may be diminished with age, profiles of RNA synthetic capacity indicate that rates decline after 12-15

aging and UV-carcinogenesis - 4000

-

DNA

------

PROTEIN

60000 - 3000

50000 -

z E

40000 - 2000

E

20000 10000 -

t

0 ve

0

0

Blumet al

v

Forbes et al

0

Black et al

0

Ebbesen 8 Kripka

- 1000

J.l,l,l,l,lll,l,l,lllllll 0

f (n z ii

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n

cn

2

4

6

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10

6 10

12 14 16 18 20 22 24

MONTH Immat.

1

Mature 1

Mlddle Age

1

Senile

Fig. 6. Relationship of epidermal macromolecular synthesis to experimental UV-carcinogenesis. Profiles of DNA (sold line) and protein (dashed line) synthetic rates represent those of nonirradiated animals. Symbols reflect ages when comparisons of sensitivityto UV-carcinogenesis were investigated in the respective studies: v, [5]; 0, [7]; Cl, [8]; 0 [9]. A representation of Bullough’s staging classification of mouse epidermal age [35] is shown below the abscissa to provide a perception of age to chronological time in months.

months, reaching their lowest levels at 2-yr of age (Fig. 4). Furthermore, a significant and steady reduction in ability to induce ODC activity occurred (Fig. 5). Complementary DNA probes for ODC expression have been cloned [39] and it will be important to determine whether reduced induction of this enzyme is related to gene expression or whether it could be the consequence of age-related differences in spectral transmission of the epidermis. The latter could as easily account for reduced induction of the enzyme [40] and was recognized some 50 years ago as a possible explanation for refractiveness to induction of UVcarcinogenesis [7]. In as much as ODC is believed to be closely associated with tumor promotion and thought to play a role in gene modulation, it is particularly interesting that induction of this enzyme should decline with age and exhibit diminished activity at times when mice have been reported to be most resistant to UV-induced carcinogenesis. Generally, the manifestations of neoplastic tissue are dependent upon increased rates of cell replication and decreased generation times [3] - in contraposition to the state found in the latter stages of aging in a number of tissues. However, our results suggest that in aging hairless mouse epidermis no decrease occurs in tissue regeneration

H.S. Black et al. I Relationship between epidennal aging and UV-carcinogenesir

times, no loss of ability to incorporate thymidine into DNA, nor a reduced protein synthesis. These data would indicate that epidermis is most refractory to UV-induced carcinogenesis at times when these parameters are greatest. Certainly our data contradict the argument that the most “effective fixation” of neoplastic transformation occurs with the highest rate of DNA synthesis. Finally, it is interesting to note the potential parallel between the experimental studies in which young animals have been shown to be more sensitive to UV-carcinogenesis and epidemiological studies which suggest that increased risk of both malignant melanoma and non-melanocytic skin tumors is associated with sun exposure experience in the first decades of life [41-44]. The demonstration of greater susceptibility of young mice to W-induced carcinogenesis may actually represent experimental support for the epidemiologic studies. In toto, both types of studies provide a clue to the time-frame in which mechanistic studies on the relationship of carcinogenesis and age should focus. In summary, we have found that specific parameters of epidermal proliferative capacity in the hairless mouse, i.e., SCRs, were not diminished with increasing age. Indeed, the CL1 increased with age. DNA and protein synthetic rates in the hairless mouse increased from 2 months of age, peaked between 12 and 18 months, after which the rates began to decline. The synthetic rates at 24 months remained above those observed at 2 months of age. RNA synthetic rates began to decline after 12 months of age. Induction of ODC activity declined with age. Chronic, low-dose UV had no significant effect upon any of the parameters examined except on DNA synthetic rate in which peak levels were diminished. It is apparent that certain parameters of epidermal proliferation and macromolecular synthesis in the aging hairless mouse do not correspond to those of cellular senescence in cultured cells. It also appears that epidermal proliferative and macromolecular synthetic capacities are lowest at ages when mice are most susceptible to the induction of UV-carcinogenesis. However, these data present several anomalies that make conclusions regarding the relationship of epidermal aging and UV-carcinogenesis highly speculative. Acknowledgment This research was supported by medical research funds from the U.S. Department of Veterans Affairs.

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and aging in mice and men, Br. J. Cancer, 32 (1975) 411-426. 2 P.P. Vitaliano and F. Urbach, The relative importance of risk factors in non-melanoma skin carcinoma&&. Dermatol., I16 (1980) 454-456. 3 H.C. Pitot, Carcinogenesis and aging - two related phenomena? Am. J. ParhoL, 87 (1977) -72. 4 H.S. Black, The homology of W-mediated cutaneous carcinogenic and aging processes, in E. Ben-Hur and I. Rosenthal (eds.), Photomedicine, Vol. 1, CRC Press, Boca Raton, FL, 1987, pp. 63-78. 5 P.D. Forbes, R.E. Davies and F. Urbach, Aging, environmental influences, and photocarcinogenesis, J. Invest. DermaroL, 73 (1979) 131-134. 6 R. Doll, Incidence of cancer in humans, in H.H. Hiatt, J.D. Watson and J.A. Winsten (eds.), Origins of Human Cancer, Cold Spring Harbor, NY, 1977 pp. 1-12. 7 H.F. Blum, H.G. Grady and J.S. Kirby-Smith, Relationships between dosage and rate of tumor induction by ultraviolet radiation, J. Nad. Cancer Inst., 3 (1942) 91-97. 8 P. Ebbesen and M.L. Kripke, Influences of age and anatomical site on ultraviolet carcinogenesis in BALB/c mice, J. Natl. Cancer Inst., 68 (1982) 691-694. 9 H.S. Black, V. McCann and J.I. Thomby, Influence of animal age upon antioxidant-modified W carcinogenesis, Photob&hem. Photobiophys., 4 (1982) 107-l 18. 10 B.A. Gilchrest, Skin and Aging Processes, CRC Press, Boca Raton, FL, 1984. 11 B.A. Gilchrest, Prior chronic sun exposure decreases the lifespan of human skin fibroblasts in vitro, J. Gerontol., 35 (1980) 537-541. 12 E.L. Schneider, Aging and cultured human skin fibroblasts, J. Invest. Dermarol., 73 (1979) 15-18. 13 E. Sbano, DNA repair after UV irradiation in skin fibroblasts from patients with actinic keratoses, Arch. Dermatol. Res., 262 (1978) 55-61. 14 F. Urbach, J.H. Epstein and P.D. Forbes, Ultraviolet carcinogenesis: experimental, global, and genetic aspects, in T.B. Fitzpatrick, M.A. Pathak, L.C. Harber, M. Seiji and A. Kukita (eds.), Sunlight and Man, University of Tokyo Press, Tokyo, 1974 pp. 259-283. 1.5 A.M. Downes, A.G. Matoltsy and T.M. Sweeney, Rate of turnover of the stratum comeum in hairless mice, J. Invest. Dermalol., 49 (1967) 400-405. 16 J.M. Marrs and J.J. Voorhees, A method of bioassay of an epidermal chalone-like inhibitor,I. Invest. Dermatol., 56 (1971) 174-181. 17 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folic phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 18 E. Volkin and W.E. Cohn, in D. Glick (ed.), Estimation of nucleic acids, in Methods of Biochemical Analysis, Vol. l., 1954 pp. 287-305. 19 J.H. Epstein, K. Fukuyama and W.L. Epstein, UVL induced stimulation of DNA synthesis in hairless mouse epidermis, J. Invest Dermatol., 51 (1968) 445-453. 20 A.H. Brown, Determination of pentose in presence of large quantities of glucose, Arch. B&hem., II (1946) 269-278. 21 K. Burton, A study of the conditions and mechanism of diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid, B&hem. I., 62 (1956) 315-323.

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