Cmcer Lettere. 41 WS8) 02-08 Elsevier Scientific Fkblishers Ireland Ltd

SENSITIVITY OF HWSkh HAIRLESS MICE TO INITIATION/ PROMOTION OF SKIN TUMORS BY CHEMICAL TREATMENT

H.H. STEINEL and R.S.U. BAKER

Genetic Toxicology Unit, National Inetitute of Occupational Health and Safety, Building A 27, UniueraityofSydney, NSW1006tAustralial

(Received 21 March 1988) (Accepted 28 March 1988)

SUMMARY

HRA/Skh hairless mice were investigated for their sensitivity to initiation and promotion by chemicals because of (al the known sensitivity of these mice to photocarcinogenesis, (b) their low background papilloma incidence (2/3000 mice under 1 year of age) and (cl ease of treatment and identification of tumors, in the absence of hair.

Employing a variety of treatments with 7,12dimethylbenz[u]nthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-12acetate (TPA) as promoter, it was found that the strain was susceptible to both initiation and promotion. Papilloma incidence was at least equivalent to that observed with other sensitive mouse strains. Following initiation with 2.56 pg DMBA, papilloma development was promoterconcentrationdependent, resulting in 22.5 papillomaslmouse at 20 weeks in animals administered 5 pg TPA. In the absence of DMBA initiation, TPA treatment was weakly carcinogenic in HRA/ Skh mice. This treatment induced a dosedependent increase in papillomas, one of which progressed to a keratoacanthoma-like tumor after 65 weeks. These results show that HRA/Skh mice are highly sensitive, not only to UV carcinogenesis, but also to chemical initiation and promotion of skin papillomas.

Key words: Hairless mice: Initiationtpromotion; Skin; 7,12Dimethyl- benz[abnthracene; 12O-Tetradecanoylphorbol-Xl-acetate.

INTRODUCTION

Carcinogenesis may be considered a multistage process during which the descendants of single normal cells become neoplastic in discrete sequential stages [I].

In order to further define the different steps in this process, considerable

CoWesponde9sce to: H.H. Steinel.

O2&288StBS/$02.50 0 1988 Elaevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland.

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attention has focussed on the initiation/promotion model of skin tumorigenesis in the mouse [2,7,9,10,11].

It was of interest to investigate chemical initiation and promotion in the hairless mouse, HRAISkh, because this strain has already proven to be highly susceptible to photocarcinogenesis [3] even when minimally erythemogenic doses of UV light have been used [4]. Furthermore, the hairless characteristic of HRAlSkh mice avoids problems associated with shaving and hair regrowth during tumor development.

MATEHIALS AND METHODS

Chemicals employed in this study were 7,12dimethylben~uhuithracene (DMBA; Sigma) and 12-O-tetradecanoylphorbol-13acetate (TPA; Sigma), as well as the 2 solvents, acetone (Ajax) and absolute ethanol (Merck), which served as negative controls.

Mature, inbred albino HRAlSkh mice at the age of 42-49 days were used in experiments. Mice of this strain (F23,19821, homozygous for the hairless (hr/hr) and albino (c/cl mutant genes, originated from Skh : HRl mice bred at Temple University ]3] and were a kind gift from the Department of Veterinary Pathology, University of Sydney. Animals were housed in plastic boxes (P. McGill & Co.) on vermiculite bedding (Borall and were maintained at 22 OC - 25OC under a 12-h daylight-night cycle. Lighting was produced by gold lamps (General Electrics, GEC F40GOl which emit minimal UV light. Animals were fed with mouse cubes (Allied Feeds) and water ad libitum. Cages, bedding and water bottles were changed twice weekly.

Control experiments included promoter controls (0.5, 1, 2 and 5 I.cg TPA/ treatment; without DMBA initiation) and initiator controls treated with a single dose of 2.56 or 25.6 pg DMBA alone or followed by applications of 50 pl acetone twice weekly or 10 4 ethanol daily. DMBA and TPA were applied in 50 fl acetone.

For initiation/promotion protocols, a single dose of 2.56 pg DMBA (10 nmol) was selected for initiation, followed 1 week later by biweekly TPA treatment, using 0.1, 1.2 and 5 rg (0.16,1.6,3.2 and 8.14 nmoll, respectively. Tumorigenesis was assessed by scoring the number of papillomaslmouse (tumor yield) and the percentage of mice with papillomas (tumor incidence) each week. Promoter treatment concluded after 20 weeks, but mice continued to be observed for a further 20 weeks, followed by a final pathological examination of selected papillomas, tumors and skin sections. All results represent the average of 2 experiments, each with 20 female animals per treatment.

RESULTS AND DISCUSSION

From observations in this laboratory, the background skin tumor incidence in HRA/Skh mice is very low, being 2 papillomas in 3000 stock animals younger than 1 year. No carcinomas of the skin were evident over this period. This

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TABLE 1

TUMOR YIELD FROM HRAlSkh MICE IN PROMOTER CONTROL EXPERIMENTS

Initiation Promotion regime regime-DMBA (amount/treatment)

PapiIiomas/mouse at various times

Week 15

Week 20

Week Week 30 40

0 0 0 50 pl acetone

0 0.5 TPA pg 0 1.0 TPA pg 0 2.0 TPA H 0 5.0 TPA pg

- - - - -

- iO25 ;025 ;025 - 0.025 0.05 0.025 0.025 0.1 0.16 0.1 02 0.21’ 0.24’ 0.16

Results are significantly different in the tw&aiIed Fisherexact test compared with controls, 0.5 and 1 rg TPA dose levels (P < 0.05). Results represent pooled data from 2 experiments w 40 mice.

agrees with the low incidence of skin tumors reported by F&eve et al. [S]. In general, the animals are robust and characterised by a normal healthy life span under open animal house conditions. Gallagher reported the survival of animals up to 900 days in some of his studies tpers. commun.1.

In addition to this low background incidence of tumors in untreated animals, repeated treatments with acetone also failed to induce any tumors over a 40- week period (Table 1). However, in the absence of initiation, repeated biweekly doses of TPA for up to 20 weeks resulted in a dose-dependent increase in papilloma yield and incidence. At week 20,211 of animals receiving 5 pg TPA/ treatment developed papillomas (Table 11. Following withdrawal of treatment, the incidence was reduced to 10% at week 40. At tieek 65, 1 papilloma developed into a keratoacanthoma-like tumor. These data are in agreement

TABLE 2

TUMOR YIELD FROM HRA/Skh MICE IN INITIATOR CONTROL EXPERIMENTS

Initiation regime-DMBA

Promotion regime (amount/treatment)

Papiliomas/mouse at various times

Week Week Week Week 15 20 30 40

2.56 ccg 0 0.025 - e.t: e.t. 2.58 @ 50 gi acetone 0.025 0.025 0.05 0.1 2.53 H ethanol 10 ~JI ethanol 0.027 0.027 0.027 0.081 25.0 pg 0 0.33 0.63 1.5 3.25 25.6 pg 50 d acetone 0.3 0.53 1.5 3.14

*Experiment terminated. Results represent pooled data from 2 experiments w 40 mice.

with TPA induction of skin tumors in SENCAR mice [lo], although the latter did not exhibit a dose response.

By contrast, a single dose of 25.6 rg DMBA was sufficient to induce papillomas in 26-2929% of mice at 20 weeks and in 100% of the animals by week 40 (Table 21. A single topical application of 2.56 pg DMBA yielded a near- background tumor incidence at 20 weeks (0.025 papillomaslmouse in 2.5% of the animals). Repeated acetone or ethanol treatments following DMBA initiation had little effect on tumor incidence or yield.

In initiation-promotion experiments, papilloma yield, incidence and time of appearance were related to the TPA concentrations applied. Following 2.56 pg DMBA initiation, TPA promotion yielded clear evidence of a dose response with tumor yields of 0.05, 4.36, 6.93 and 22.5 papillomaslanimal at 20 weeks, resulting from 0.1, 1,2 and 5 pg TPAltreatment, respectively (Fig. 1Al. At this time, there was essentially no difference in the papilloma yield at the lowest TPA concentration compared with initiated controls. After 30 weeks, a rapid increase in papillomas occurred in the group receiving the lowest TPA dose (0.1 pgl, resulting in 0.46 papillomaslmouse and 36% tumor incidence by week 40 (data not shown). By contrast, there was only a slight increase in papillomas in acetone-treated initiator controls, reaching 0.1 papillomaslmouse and 7.5% tumor incidence at 40 weeks (Table 21. Furthermore, ethanol-treated initiated controls displayed a similar yield and tumor incidence to those of the acetone- treated group at 40 weeks. Initiated animals treated with higher TPA doses of 1,2 and 5 pglmouse showed a net decrease in papilloma yield of 53%, 37% and 20%. respectively, over this period.

Figure 1B demonstrates the papilloma incidence in initiated/promoted mice up to week 20. Here, the tumor incidence reached 100% in the 2 and 5 pg TPA dose groups, 95% incidence in animals given 1 pg TPA, and only 5% incidence at the 0.1 pg TPA level. There was a correlation between the time of onset of papillomas and the TPA concentrations applied, with the time-to-first-tumor being 5, 6, 7 and 12 weeks at doses of 5, 2, 1 and 0.1 pg TPA/treatment, respectively.

In general, the papilloma yield appears to correlate with treatment-induced

0 0

D 5 ID IS 10 0 I 10 IS 10

Tlnu (wwksl Tblw (WnkS)

Fig. 1. Skin psptioma yield (A) and incidence (3) in DMBA-initiated HRA/Skh mice treated with 0.1 ~gcg(~).l~g(O),2~gcg(B)orS~TPA(O).

inflammatory response in the dermis. Absence of any observable inflammation, as in the initiated controls or with the lowest TPA dose used in this study (0.1 rgl, resulted in the absence or delay of promotion. The higher TPA doses also yielded a proliferative response detected in histological sections of the epidermis, as reported by other authors [9].

After the growth of a juvenile hair coat, hair follicle degeneration and coat loss is progressive and usually complete by 21 days post partum with only short “fuzzy” vibrissae remaining. Total hair loss may be expected by 9 weeks and the formation of follicular cysts resulting from follicle degeneration is seen lower in the dermis, but not in the epidermis where papillomas develop. Moreover, no hair follicles were found in histological sections of papillomas. Thus, tumors occur in HRAlSkh mice in the absence of hair follicles. This is in contrast to the report that papillomas arise exclusively from keratinocytes near hair follicles, following initiation/promotion [5]. Our results are also at variance with those of Giovanella and colleagues [6], who showed a low response by hairless mice to tumor initiation/promotion. Significantly, the HRAlSkh strain is derived from Skh : HRl mice which have been shown to be more sensitive to UV carcinogenesis than other hairless mice strains e.g. Skh : CRH stock, CQH/HeN-hr strain, HRSlJ strain, HR/De/HEler, Argonne hairless stock, and BALB/c Skh-ab strain [3]. It is possible that sensitivity to UV and chemical initiation/promotion may be linked in HRAlSkh mice. The appearance in an HRAlSkh line of a hairy revertant mouse which is also comparatively resistant to UV carcinogenesis (G. Greenoak, pers. commun.) suggests an association between the hr gene and skin tumor susceptibility in this mouse strain.

This investigation demonstrates that HRAlSkh mice are sensitive to skin tumor initiation and promotion as reported for other strains. The papilloma incidence, yield and time-to-first-tumor in these animals is equivalent to results with SENCAR mice treated similarly [lo]. Although these results were obtained with female mice, separate studies with male HRAlSkh mice have shown no evidence of sex differences. Thus, this strain appears to be suitable for studies of skin carcinogenesis, for assessing chemicals as possible tumor initiators or promoters, and for further investigations of the mechanism of skin tumor promotion.

ACKNOWLEDGEMENTS

This study was supported by a grant from the NSW State Cancer Council. The authors are grateful for considerable help from Mr. G. Greenoak and Dr. P. Canfield, Department of Veterinary Pathology, University of Sydney.

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