Dithranol (Anthralin)-induced Skin Irritation in C57BL/6, NMRI and SENCAR Mice

Pharmacology & Toxicology 1991, 69, 96-104.

Dithranol (Anthralin)-induced Skin Irritation in C57BL/6, NMRI and SENCAR Mice

Matti Viluksela' and Veli-Matti Kosma'

'Department of Pharmacology and Toxicology, University of Helsinki, Siltavuorenpenger 10 A, SF-001 70 Helsinki, and *Department of Pathology, University of Kuopio, P.O.Box 6, SF-70101 Kuopio, Finland

(Received February 4, 1991; Accepted February 28, 1991)

Abstract: Dithranol-induced skin irritation was compared in C57BL/6, NMRI and SENCAR mice, the strains representing different sensitivity to tumour promotion. Skin irritation was assessed using ear thickness and skin weight measurements, visual estimation of back skin irritation and histopathology. Both single and repeated applications of dithranol caused a delayed skin irritation resulting in the maximal response between 7-11 days after the beginning of the treatment. Contrary to the findings with 12-0-tetradecanoyl-phorbol-13-acetate (TPA), C57BL/6 mice were the most sensitive and SENCAR mice the most resistant to the dithranol-induced skin irritation up to 30 days from the beginning of the treatment. NMRI mice were intermediate. Differences were found in the ear swelling, epidermal hyperplasia, amount of inflammatory cell infiltrate and skin ulceration. During repeated treatment of about 40 days, however, the responsiveness of SENCAR mice increased over that of C57BL/6 and NMRI mice. SENCAR mice had also more epidermal hyperplasia than the other strains at the end of the 74 day period of 3 times weekly applications. The magnitude of epidermal hyperplasia after long term treatment seems to correlate with the sensitivity to tumor promotion in the different mouse strains.

Among the commonly used laboratory animals mice are the most sensitive species to chemical skin carcinogenesis (Stenback 1980; Slaga & Fischer 1983). The susceptibility of different mouse strains to both complete and initiation- promotion type epidermal carcinogenesis has been shown to vary considerably (Stenback 1980; DiGiovanni et al. 1980, 1984; Hennings et al. 1981; Ashman et al. 1982; Sla- ga & Fischer 1983; Slaga 1986), emphasizing the role of genetic factors in the regulation of sensitivity. It has been suggested that the strain differences in skin carcinogenesis would be based on the differences in the promotion stage, and would also depend on the promoting agent (Slaga & Fischer 1983; DiGiovanni et al. 1984; Reiners et al. 1984; Slaga 1984).

The primary mode of action of tumour promoters is not completely understood. The most intensively studied class of skin tumour promoters is the phorbol esters, especially 12-0-tetradecanoyl-phorbol-13-acetate (TPA), the most ac- tive derivative of this class. Interaction of phorbol esters with their specific receptor, the protein kinase C (PKC), is believed to be involved with the promotion response (Driedger & Blumberg 1980; Ashendel et al. 1983; Diamond 1984; Solanki & Slaga 1984). Anthrone derivatives, like the antipsoriatic drugs dithranol (anthralin, 1,8-dihydroxy-9- anthrone) and chrysarobin (1,8-dihydroxy-3-methy1-9-anthrone) are the next most potent tumour promoters after phorbol esters, and other compounds capable of interacting with PKC (Slaga 1984; DiGiovanni et al. 1987). Dithranol and other anthrones are not able to interact directly with the phorbol ester receptor (Leach & Blumberg 1985; Di- Giovanni et al. 1987). Regardless of the differences in the primary mode of action and the magnitude of promoting response, all known skin tumour promoters, although

chemically diverse, share a common feature of producing inflammation and hyperplasia in the skin (Slaga 1983; Dia- mond 1984; Gschwendt et al. 1984; Klein-Szanto 1984; Le- wis & Adams 1987; Naito et al. 1987). Also the cellular and ultrastructural changes induced by chemically unrelated tumour promoters in epithelial cells seem to be analogous (Trosko et al. 1982; Arenholt et al. 1987). On the other hand, the different modes of action of various tumour promoters might also result in varying sensitivity between mouse strains.

Direct comparisons of the sensitivity to tumour promotion between mouse strains have been almost entirely based on studies with phorbol ester tumour promoters. The comparative data on other promoters is more limited. SENCAR mice have been selectively bred for sensitivity to skin tumour initiation by 7,12-dimethylbenz(a)anthracene (DMBA) followed by TPA promotion. They seem to be extremely responsive for the initiation-promotion type carcinogenesis in general, as well as for the complete carcinogenesis (DiGiovanni et al. 1980; Slaga & Fischer 1983; Slaga 1986; Viluksela et al. 1986). NMRI mice represent a strain of intermediate sensitivity to promotion (Mannisto et al. 1984; Hennings & Yuspa 1985; Viluksela et al. 1986). C57BL/6 mice, on the contrary, have been shown to be highly resistant to the TPA promotion, but sensitive to the promotion by the free radical-generating compound benzoyl peroxide (Reiners et al. 1984).

Studies on TPA have demonstrated that the extent of inflammatory changes and hyperplasia, even after a single dose, correlates well with the sensitivity to tumour promotion in various mouse strains (Sisskin et al. 1982; Lewis & Adams 1987; Naito et al. 1987). This difference seems not to be mediated by PKC (Wheldrake et al. 1982; Yamamoto

DITHRANOL-INDUCED SKIN IRRITATION IN MICE 97

et al. 1988; Hirabayashi et al. 1990). It is, therefore, of interest to examine if the treatment with tumour promoters, which do not interact with PKC, would result in analogous differences in inflammatory response in sensitive and resistant mouse strains.

The objective of the present study was to compare the nature and time course of the inflammatory response after single and repeated application of dithranol in mouse strains of different susceptibility. Together with histopathology, repeated ear swelling measurements, which enable the quantitative survey of the time course of skin reactions, and reflect mainly edema and hyperplasia of the ear skin, have been utilized.

Materials and Methods Chemicals. Dithranol (mol. wt. 226.2, purity 98.8%) was purchased from Bayer (Leverkusen, FRG) and purified by recrystallizing from a mixture of acetic acid and water (90: 10). Dithranol was dissolved in acetone (BDH Ltd., Poole, England, > 99.5%) immediately before use.

Animals. All animals used were female mice between 6 and 8 weeks of age. Outbred SENCAR mice were obtained from Harlan Sprague Dawley (Indianapolis, U.S.A.), inbred C57BL/6N mice from Charles River Wiga (Sulzfeld, FRG) and outbred NMRI mice from the National Laboratory Animal Center, University of Kuopio (Kuopio, Finland) and from Charles River Wiga (Sulzfeld, FRG). The mice were acclimatized for at least one week before experiments. The animals were housed in groups of 5-10 in Macrolon 111 polycar- bonate cages in a thermostatically controlled room at 20k I" and at relative humidity of 50k 10%. Aspen chips were used as bedding. The room was artificially illuminated on a time schedule of 14 hr light and 10 hr dark. Pelleted rat/mouse feed (Altromin 1314, Chr. Petersen A/S, Ringsted, Denmark) and tap water were available ad libitum.

Experimental procedures. In ear swelling studies 5 pl of dithranol solution was applied to the inner surface of the left ear by an automatic pipette (Finnpipette, Helsinki, Finland). The right ear was used as a control ear and treated only with acetone. The thicknesses of both ears of the mice were measured before treatment and at different time points after dithranol application using a Mitutoyo Model 7309 spring load micrometer (Tokyo, Japan) as described by Lahti & Maibach (1985). For histopathology the mice were killed by cervical dislocation and both ears preserved in 10% buffered formalin.

In back skin irritation studies the backs of the mice were shaved with an electric razor before treatment and thereafter as needed. A 50 )rl dose of dithranol solution was applied to the shaved skin by an automatic repetitive pipette (Eppendorf multipette, Hamburg, FRG). Control animals received only acetone. Histological samples were taken from the application site. For quantitation of the skin response a skin plug from the application site was obtained using a punch (0 5 mm), and weighed.

Single dose studies. 3.5 mM Dithranol. Groups of 18 mice of each strain were used. A single dose of 3.5 mM dithranol was applied to the ears and the ear thickness measured once daily for 4 days after exposure. Histo- logical samples were taken from 6 mice of each strain at 24,48 and 96 hr.

50 rnM Dithranol. A single dose of 50 mM dithranol was applied to the ears of 10 mice of each strain. This treatment has been earlier used to produce a definite ear swelling in NMRI mice (Viluksela, 1991). The ear thickness was measured daily during the first work week and less often thereafter up to day 74. Histological samples

from the ears of 6 separate mice per strain were taken 9 days after the exposure (at the time point of maximum ear swelling response). The same concentration of dithranol was applied to the shaved back skin of 10 mice of each strain. Acetone was applied to 5 control mice per strain. Histological and skin punch samples were taken from 5 mice per test group at 24 and 48 hr following exposure.

Repeated dose studies. 1.1 mM Dithranol once weekly. This treatment protocol and concentration (220 nmol/mouse in 200 pI acetone) has been estimated to be the optimum for promoting papillomas with chrysarobin in SENCAR mice (Kruszewski et al. 1987). Groups of 6 mice of each strain were used. 1.1 mM dithranol was applied to the ears of the mice 5 times and the ear thickness measured daily (excluding weekends). Histological samples were taken 48 hr after the last dosing, on day 30.

3.5 mM Dithranol three times weekly for I0 weeks. An identical treatment protocol was used in the tumor-promotion studies by Van Duuren et al. (1978), Mannisto e t al. (1984) and Viluksela et al. (1986). Groups of 6 mice of each strain were used. 3.5 mM dithranol was applied to the ears of the mice three times weekly for 10 weeks. Ear thickness was also measured three times weekly, just before each application. Histological samples were taken from both ears of each animal 48 hr after the last dose. Using the same dosing protocol the back skin of 10 test mice and 5 control mice of each strain were exposed to dithranol and acetone, respectively, for 10 weeks. Skin irritation at the application site was estimated visually just before each application and the severity of irritation evaluated as follows: 0 =no irritation, 1 =mild redness, 2 =moderate redness, 3 =skin ulceration. Histological and skin punch samples were taken from all the animals 48 hr after the last dose.

Histology and guantitation. After fixation in 10% buffered formalin the skin samples were dehydrated, embedded in paraffin wax, cut into the thickness of 5 pm, stained by the van Gieson method and examined using light microscope. Epidermal thickness (except the horny layer) was measured using the IBAS 1 + 2 image analyzer (Kontron, FRG) operated in an interactive mode at an objective magnification of x 40 (Olympus Vanox T light microscope). Fif- teen-20 areas of a section from each block were measured to give an even coverage of the whole section. The quantitative histology data presented represents mean& S.E.M. of the group, and these figures were calculated from one skin section (15-20 different areas) per animal. Inflammatory infiltrates were evaluated as follows: O = negative, 1 =scanty, 2=moderate, 3 =extensive. In certain cases the intermediate impressions were obvious, and the values 0.5, 1.5 and 2.5 were used. The incidence of epidermal ulceration was evaluated as well.

Statistics. The ear thickness and epidermal thickness data was tested for homogeneity of variances using the Bartlett's test. If the groups were homogeneous (at the level of 0.01) comparisons between the strains were performed by one-way analysis of variance (ANOVA) followed by Scheffe's test. If the groups were heterogen- eous or in the case of inflammatory infiltrate data, the comparisons were made using the nonparametric Kruskal-Wallis test followed by the distribution-free multiple comparison test.

Results

The mean pretreatment ear thicknesses (mm x lo-** S.E.M.) of the different mouse strains were (right and left ear, respectively): C57BL/6 23.5 0.15, 23.5 0.15; NMRI 24.9k0.18, 24.9Ik0.17; SENCAR 26.5k0.24, 26.4Ik0.24 (n = 30).

Single dose studies. 3.5 m M Dithranol. Acute ear swelling response after a single dose of 3.5 mM dithranol was strongest in C57BL/6 mice

98 MATTI VILUKSELA AND VELI-MATTI KOSMA

Dlthranol3.5 mM

150c 100 0 1 2 3 4

Time (days)

Dlthranol50 mM B

250

200

150

1 00

0 10 28 74 Time (days)

Fig. 1. Mean ear swelling responses (test ear thickness/control ear thicknessx IO0kS.E.M.) in various mouse strains after a single 5 p1 dose of 3.5 mM (A) and 50 mM (B) dithranol. Statistics: *P<0.05, **P<O.Ol, ***P<O.OOl C57BLi6 versus NMRI and SENCAR (A); C57BL/6 versus SENCAR (B; other significant differences in B: C57BL/6 versus NMRI on days 1 (P<O.Ol), 14 and 28 (P<O.OS), and NMRI versus SENCAR on days 4 (P<0.05) and 7 (P<O.OOl).). A: n=18 (day l), n=12 (day 2), n = 6 (days 3 and 4); B: n=10.

(fig. 1 A). It was milder and nearly equal in NMRI and SENCAR mice. The histopathology was quite similar in all the strains 24 hr after the treatment (table 1). Only a scanty inflammatory infiltrate of polymorphonuclear leukocytes (PMNLs) was seen in all groups. Later dermal infiltration of both PMNLs and mononuclear cells was more prominent in C57BL/6 mice than in the other strains. Ulceration was observed only in C57BL/6 mice. Epidermal thickness was slightly increased in all the groups, but the differences between the strains were negligible.

50 m M Dithranol. In the ear swelling measurements a peak response was observed 24 hr (48 hr in NMRI) after application, followed by a temporary regression (fig. 1 B). The second and intensive peak response arose 7-8 days following the application, after which the response gradually faded. The latter peak response was strongest in C57BL/6 mice, slightly lower in NMRI mice, and rather faint in SENCAR mice. The differences between C57BL/6 and SENCAR mice were the most significant. Histopathology 9 days after application (during the second peak irritation)

Table I . Effects of a single application of dithranol on the skin of C57BL/6, NMRI and SENCAR mice.

Treatment Epidermal thickness (pm) Inflammation

Concentration Time Strain Control Test Change (%) Cell infiltrate Ulceration (YO) 3.5 mM (ear) 24 hr C57BL/6

NMRI SENCAR

NMRI SENCAR

NMRI SENCAR

NMRI SENCAR

NMRI SENCAR

NMRI SENCAR

48 hr C57BL/6

96 hr C57BL/6

50 mM (back skin) 24 hr C57BL/6

48 hr C57BL/6

50 mM (ear) 9 days C57BL/6

19.4k 0.6 19.8k0.7 21.2k0.8 19.3k0.4 19.7 k 0.6 20.5 k 0.4 19.6 k 0.8 18.7 k0.2 20.1 k 0.7

22.2k0.4 22.6 f 1.2 25.620.7 19.9k0.2 20.3 20 .3 21 .O k0.5

23.8k0.9 24.3 k0.7 28.0k0.9 28.4k 1.2 28.5f 1.1 28.8k0.8 28.4 k 1.2 28.4 k I .O 29.8 k 1.2 55.1 *2.8**" 36.5k3.9 43.6+ 1.8 74.1 5 5. I ***' 38.8f6.5 37.5 k4.4 72.3k2.7 48.0k2.5 52.351.7

124 123 133 147 144 141 146 152 149 248 161 170 334 171 I46 363**ah 238 25 1

1 .o 0 0.6 0 0.7 0 1 .4*a 0 0.9 0 0.9 0 2.0*b 17 1.6 0 1.1 0 2.1 80 2.0 0 1.8 0 2,8*"**b 100 2.0 80 2.0 100 2pa**b 67 I .4 0 I .3 0

Epidermal thickness represents mean+S.E.M. of the group (5-10 animals per group), and these figures were calculated from one skin section (15-20 different areas) per animal. Inflammatory cell infiltrate represents a mean of the group, and was evaluated as follows: O = negative, 1 =scanty, 2 =moderate, 3 =extensive. Statistics: *P < 0.05, **P < 0.01, ***P < 0.001, "Significantly different from NMRI mice. bSignificantly different from SENCAR mice.


Fig. 2. Photomicrographs of the ear skin 9 days after a single application of 50 mM dithranol. A: control (SENCAR), B: C57BL/6, C: NMRI, D: SENCAR. Pronounced hyperplasia is seen in C57BL/6 mice (B). Note also an intense inflammatory infiltrate of mononuclear cells in the dermis. Epidermal thickness is quite similar in NMRI (C) and SENCAR (D) mice. van Gieson, original magnification x 400.

revealed epidermal hyperplasia in all groups (table 1, fig. 2). Hyperplasia was most prominent in C57BL/6 mice differing significantly from both NMRI and SENCAR mice. The inflammatory infiltrate, consisting of mononuclear cells and PMNLs, was also more pronounced in C57BL/6 mice than in NMRI and SENCAR mice, but there were no qualitative differences between the strains. Furthermore, epidermal ulceration was observed only in C57BL/6 mice.

Application of dithranol to the back skin of the mice caused an increase in the skin weight at 24 hr and 48 hr. Similarly with the changes in ear thickness, the increase was most extensive in C57BL/6 mice (mean changes 24 hr and 48 hr after dithranol, respectively: C57BL/6 164% and 163%; NMRI 144% and 124%; SENCAR 143% and 109%; C57BL/6 differed significantly from NMRI (P < 0.01) and SENCAR (P < 0.001) mice at 48 hr). In histopathology epi-

dermal hyperplasia was seen in all groups, mostly in C57BL/6 mice (table 1). Hyperplasia occurred in conjunc- tion with epidermal ulceration, necrosis and dermal infiltration of PMNLs. Also the inflammatory infiltration was most extensive in C57BL/6 mice at 48 hr. It must be empha- sized that due to epidermal necrosis in all strains it was quite laborious to measure the epidermal thicknesses after this particular treatment (24 hr and 48 hr after 50 mM dithranol).

Repeated dose studies. 1.1 m M Dithranol once weekly. Lower concentration of dithranol caused a relatively faint peak ear swelling response at 24 hr only in C57BL/6 mice, whereas in NMRI and SENCAR mice the response slowly increased during the first week (fig. 3). The maximal peak response at day 10 (3

100 MATTI VILUKSELA AND VELI-MATTI KOSMA

Dithranol 1.1 mM - C57BL/6 - NMRI - SENCAR 250

150

. _ _ loo.#, , , , , , , , ' , , , , , , ( , , , , . , , , , , . 0 " 10 A 20" A 30 A Time (days)

Fig. 3. Mean ear swelling responses (+S.E.M.) during five once weekly application of 1.1 mM dithranol. C57BL/6 mice were significantly different from NMRI and SENCAR mice (P < 0.001 on days 1 and 3-11, P<O.O1 on days 2 and 14-30). n=6.

days after the second application) was very outstanding in C57BL/6 mice. It was much weaker and occurred earlier (at day 8) in NMRI and SENCAR mice. The next applications were followed by slight and transient increases in ear thickness. The swelling response was clearly stronger in C57BL/6 mice than in NMRI and SENCAR mice through- out the study. Histopathology revealed epidermal hyperplasia in all the groups 48 hr after the fifth dose of dithranol (table 2, fig. 4 A, C, E, G). C57BL/6 mice differed significantly from NMRI and SENCAR mice. There was also more inflammatory infiltration of mononuclear cells and PMNLs in C57BL/6 mice than in the other strains, but no qualitative differences between the strains.

3.5 m M Dithranol three times weekly. In the ear swelling study, application three times a week revealed a peak ear swelling response between days 9 and 11, which was strongest in C57BL/6 mice (P<O.054.001 on days 4-14) and mildest in SENCAR mice (fig. 5 A). After an application

period of about 40 days, the ear swelling response in SEN- CAR mice was more pronounced than in the other strains. At the end of the study the response was mildest in NMRI mice. In histopathology all groups had epidermal hyperplasia (table 2, fig. 4 B, D, F, H), but it was strongest in SENCAR mice (54.3 pm versus 46.9 pm in C57BL/6 mice and 40.3 pm in NMRI mice). The dermal infiltrate consisted of mononuclear cells and its magnitude was nearly similar in all groups.

In the back skin irritation study with the same dosing schedule, the visual estimation of the skin irritation did not show any clear differences between the strains during the first three weeks. However, from day 30 to the end of the 74-day dosing period, the strongest response was seen in SENCAR mice (fig. 5 B). The visual comparison of irritation and redness of the skin between the strains was limited by the fact that C57BL/6 mice are pigmented while the other strains are not. Mean skin weight at the end of the study was most elevated in SENCAR (168%) mice, followed by NMRI (160%) and C57BL/6 (151%0) mice. In histopathology the extent of epidermal hyperplasia decreased in the same order (table 2). Scanty inflammatory infiltrates were seen only in 7 out of 10 SENCAR mice and in 1 out of 10 C57BL/6 mice. In SENCAR mice the infiltrate consisted of PMNLs, suggesting acute inflammation.

Discussion

The time course and characteristics of dithranol-induced delayed skin irritation in the mouse ear model have been described recently (Viluksela 1991). The present study dem- onstrates that there are considerable sensitivity differences in skin irritation between mouse strains. Among the strains used, C57BL/6 mice were the most sensitive and SENCAR mice the most resistant to the short-term dithranol-induced skin irritation. The difference was evident both in the single dose and, even more, in the repeated dose studies. Repeated application of low dithranol concentrations caused the most definite differences in skin irritation between C57BL/6 mice

Table 2. Effects of multiple application of dithranol on the skin of C57BL/6, NMRI and SENCAR mice 48 hr after the last application.

Treatment Epidermal thickness (pm) Inflammation Concentration Time Strain Control Test Change (YO) Cell infiltrate Ulceration (YO) 1.1 mM (ear) I x weekly C57BL/6 19.6k0.3 53.1 k4.2 271**a*b 1.6 0

30days NMRI 20.6k0.4 34.0k0.8 165 1.0 0 SENCAR 19.9k0.5 36.0+ 1.9 182 1 .O 0

3.5 mM (ear) 3 x weekly C57BL/6 21.2k1.1 46.9k2.9 222 1 .O 0 74 days NMRI 19.7k0.3 40.6k2.0 207 0.5 0

SENCAR 20.9 f 1.2 54.3 & 8.4 259 0.8 0 3.5 mM (back skin) 3 x weekly C57BL/6 23.8+ 1.2 65.5k7.7 276 0.05 0

74days NMRI 24.3 f 1.1 67.9 k 3.6 277 0.0 0 SENCAR 21.6k0.7 71.6k3.8 331 0.6**+8*C 0

Epidermal thickness represents mean5S.E.M of the group (5-10 animals per group), and these figures were calculated from one skin section (15-20 different areas) per animal. Inflammatory cell infiltrate represents a mean of the group, and was evaluated as follows: O=negative, 1 =scanty, 2=moderate, 3 =extensive. Statistics: *P<O.O5, **P<O.Ol, ***P<O.OOl. "Significantly different from NMRI mice. bSignificantly different from SENCAR mice. "Significantly different from C57BL/6 mice.


Fig. 4. Photomicrographs of the ear skin 48 hr after the last application of 1.1 mM dithranol once weekly for 30 days (left panel; A,C,E,G) and 3.5 mM dithranol three times weekly for 74 days (right panel; B,D,F,H). A: control (NMRI), B: control (C57BL/6), C and D: C57BL/6, E and F: NMRI, G and H: SENCAR. In C57BL/6 mice the extent of epidermal hyperplasia and dermal inflammatory infiltrate is slightly more increased on day 30 (C) than on day 74 (D). In NMRI mice more hyperplasia is seen on day 74 (F) than on day 30 (E). SENCAR mice have clearly more hyperplasia on day 74 (H) than on day 30 (G). van Gieson, original magnification x 400.

102 MATTI VILUKSELA AND VELI-MATT1 KOSMA

and the other strains, especially during the maximal response on days 4-21. However, when the treatment period was continued for more than 40 days, the situation was reversed and SENCAR mice exhibited the strongest response in the long-term treatment.

Previous studies in C57BL/6 mice with various tumour promoters have not indicated any general susceptibility of these mice to skin irritation. However, in accordance with the present findings with dithranol, this strain has been reported to be sensitive to skin ulceration by DMBA (Thomas et al. 1973). The primary reason for this sensitivity to ulceration is not known. There are no obvious strain differences in skin structure, which could explain the difference in susceptibility.

C57BL/6 mice has been shown to be extremely resistant to phorbol ester skin irritation (Lewis & Adams 1987; Naito

A Dithranol3.5 mM n

8 200

C

D

8 -

150

6 6 100 0 20 40 60

B

3-1 C 0 E

h O 2 L

L I-

C

Y 1 0 O m

I I I I

0 20 40 60 Time (days)

Fig. 5. A Mean ear swelling responses during three times weekly application of 3.5 mM dithranol for 10 weeks. Statistically significant differences (P <0.05-O.001) between C57BL/6 and SENCAR mice on days 4-14, 53, 56 and 60, between C57BL/6 and NMRI mice on days 4-1 I , 18,21,25-30 and between SENCAR and NMRI mice on days 49-63 and 74 (n=6). B: Mean skin irritation scores during three times weekly application of 3.5 mM dithranol for 10 weeks (scoring: O=no irritation, 1 =mild redness, 2=moderate redness, 3=skin ulceration). n = 10 in test groups, 5 in control groups).

et al. 1987) and tumour promotion (DiGiovanni et al. 1984; Reiners et al. 1984) in comparison with SENCAR or DBA/2 mice. This resistancy of C57BL/6 mice is believed to be based on the lower inflammatory responses and reactive hyperplasia in the skin (Lewis & Adams 1987; Naito et al. 1987), reduced generation of free radicals (superoxide anion) in epidermal cells (Fischer et al. 1986), and reduced release of hydrogen peroxide as well as oxidized metabolites of arachidonic acid from macrophages (Lewis & Adams 1986). On the other hand, the available data suggests that the strain differences in susceptibility would not be due to the differences in the oxidative metabolism of carcinogens, specific binding of TPA, PKC activity or downregulation, distribution of epidermal PKC isozymes or induction of ornithine decarboxylase (Wheldrake et al. 1982; DiGiovanni et al. 1988b; Yamamoto et al. 1988; Hirabayashi et al. 1990).

When comparing skin reactions between C57BL/6 mice and the sensitive DBA/2 mice after four weekly applications of chrysarobin. Naito et al. (1987) observed a slightly greater epidermal hyperplasia, but considerably lower infiltration of PMNLs in C57BL/6 mice. Interestingly, the infiltration of PMNLs after TPA treatment was also negligible in C57BL/6 mice as compared with SENCAR mice (Lewis & Adams 1987) or DBA/2 mice (Naito et al. 1987). These findings, suggesting the insignificant role of inflammatory infiltration in the skin reaction of C57BL/6 mice, were opposite to our findings in dithranol irritation. A single application of both low and high concentrations of dithranol, as well as five weekly applications of the low concentration resulted in definitely more inflammatory infiltration in C57BL/6 mice than in the other two strains. The same was essentially true with the epidermal hyperplasia.

In spite of their resistance to TPA, C57BL/6 mice have been reported to be sensitive to tumour promotion by benzoyl peroxide, a free radical generating compound, and to complete carcinogenesis by DMBA and benzo(a)pyrene (Reiners et al. 1984). Furthermore, benzoyl peroxide promotion was reported to induce mainly papillomas in SEN- CAR mice, but almost exclusively carcinomas in C57BL/6 mice, whereas promotion by TPA resulted in equally low incidence of carcinomas in the both strains (Reiners et al. 1984). Recently DiGiovanni et al. (1987 & 1988b) presented new data on the sensitivity of C57BL/6 and SENCAR mice to the promotion by chrysarobin. Interestingly, chrysarobin was capable to promote tumors also in C57BL/6 mice, although less than in SENCAR mice. However, in these studies with both benzoyl peroxide and chrysarobin 40 times higher doses of DMBA were used for initiation of C57BL/6 mice than in SENCAR mice. Further studies would be therefore needed to clarify the relative sensitivity of C57BL/6 mice to tumour promotion by anthrones, and the role of prominent early inflammation in the tumour promotion.

In the short-term studies the responses ofNMRI mice were mainly intermediate in strength, but after long repeated treatment, NMRI mice were the least responsive. Preliminary studies in Balb/c mice (unpublished data) showed that their


ear swelling response after a single application of 50 mM dithranol was quite similar with that of NMRI mice. Previous studies on Balb/c mice have led to varying estimations of their relative susceptibility to the promotion by croton oil or TPA (Stenback 1980; Hennings et ai. 1981; Ashman et ai. 1982). Promotion by dithranol, however, was not shown to produce any papillomas in Balb/c mice (Ashman et al. 1982). Surpris- ingly, SENCAR mice were most resistant in the short dithran- 01 treatments, but during the longer treatment, they respond- ed more strongly than the other strains. In terms of hyperplasia and tumour yield, our previous findings in NMRI and SENCAR mice (Mannisto et ai. 1984; Viluksela et al. 1986) seem to correlate with our present results on the inflammatory changes during the long term treatment. Initiation-promotion treatment with DMBA and dithranol induced clearly more papillomas in SENCAR mice than in NMRI mice, indi- cating a correlation between sustained hyperplasia and tumour promotion (cf. Sisskin et ai. 1982; Kruszewski et a/. 1989).

Epidermal toxicity leading to regenerative hyperplasia has been suggested to be closely associated with the tumour promotion by anthrones (Kruszewski et al. 1987 & 1989). However, severe epidermal toxicity seems not to represent the optimal level of toxicity for tumor promotion. The studies of Kruszewski et al. (1987) showed that the highest dose of chrysarobin was not the most optimal one for tumour promotion. Moreover, treatment of SENCAR mice with chrysarobin using the optimal tumour promoting protocol of one weekly application was shown to produce less toxic manifestations, but more epidermal hyperplasia than the two weekly applications (Kruszewski ef al. 1989). Even the repeated application of dithranol for five times using the once weekly protocol (1.1 mM) was not sufficient to produce the differences in hyperplasia which would correlate with the extent of tumour promotion, as it seems obvious that C57BL/6 mice would not be more sensitive to anthrone tumour promotion than SENCAR mice (cf. Di- Giovanni et al. 1988b). Thus, the development of sustained hyperplasia by dithranol seems to be a relatively long process. The magnitude of initial inflammatory changes may not be similarly important for tumour promotion (or the strength of these changes may be more than optimal, leading to reduced responsiveness). Modulation of tumour promotion by drugs is not necessarily related with changes in the inflammatory process. Retinoic acid, an inhibitor of tumour promotion by dithranol (Dawson et al. 1987) and chrysarobin (DiGiovanni et ai. 1988a), was not able to decrease the dithranol-induced skin irritation in NMRI mice (Viluksela 199 I). On the other hand, anti-inflammatory ster- oids, which are effective inhibitors of dithranol irritation, are also effective inhibitors of chrysarobin tumour promotion (DiGiovanni et al. 1988a).

The dissimilar susceptibility of C57BL/6 mice to TPA and anthrone induced skin reactions may be based on the differences in the mechanisms and time course of action between these classes of compounds. Dithranol is able to generate free radicals (Martinmaa et al. 1978), but it does

not bind to PKC (Leach & Blumberg 1985; DiGiovanni et al. 1987). Also the time course of induction of epidermal ornithine decarboxylase and hyperplasia are delayed in comparison with TPA (DiGiovanni et al. 1985; Kruszewski et ai. 1986 & 1989; Naito et al. 1987). In addition, anthrones produce significant epidermal toxicity leading to regenerative hyperplasia (DiGiovanni et ai. 1987; Kruszewski el al. 1989). Anthrones are also more efficient in producing skin carcinomas than TPA (DiGiovanni et ai. 1985) suggesting differences in tumour progression. It also seems obvious that the genetic factors regulating the sensitivity of skin to different classes of tumour promoters would be at least partly different.

In conclusion, contrary to the findings with TPA, C57BL/6 mice were the most sensitive and SENCAR mice the most resistant to dithranol-induced acute skin irritation. During repeated treatment of about 40 days, however, the responsiveness of SENCAR mice increased over that of C57BL/6 and NMRI mice. The extent of epidermal hyperplasia after long-term treatment seemed to correlate with the sensitivity to tumour promotion in the different mouse strains.

Acknowledgements This study was supported by grants from the Finnish

Drug Research Foundation and Pharmacal Research Foundation.

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Dithranol (Anthralin)-induced Skin Irritation in C57BL/6, NMRI and SENCAR Mice