Tumors of the skin in the HRASkh mouse after treatment with 8-methoxypsoralen and UVA radiation

FUNDAMENTALANDAPPLIEDTOXICOLOGY

16,92-102(1991)

Tumors of the Skin in the HRA/Skh Mouse after Treatment with 8-Methoxypsoralen and UVA Radiation JUNE K. DUNNICK,*,’ P. DONALD FoRBEs,t SCOT L. EUSTIS,* JERRY F. HARDISTY,~ AND DAWN G. GOODMAN~ *National Institute of Environmental Health Sciences. Research Triangle Park, North Carolina 27709; t Temple University Health Sciences Center, Philadelphia, Pennsylvania 19122; $.Experimental Pathology Laboratories, Research Triangle Park, North Carolina 27709; and gPATHC0. Inc.. Gaithersburg, Maryland 20879

Received

March

2, 1990; accepted

September

11, 1990

Tumors of the Skin in the HRA/Skh Mouse after Treatment with I-Methoxypsoralen and UVA Radiation. DUNNICK,J.K.,FORBES,P.D.,EUSTIS,S.L.,HARDISTY,J.F.,ANDGOODMAN, D. G. (199 1). Fundam. Appl. Toxicol. 16,92-102. GMethoxypsoralen (8-MOP) with and without UVA radiation was administered to HRA/Skh mice (36 animals per treatment group) three times a week in the feed for a total dose of 9-80 mg/kg/week for 52 weeks. Most of the animals at the top dose of 8-MOP with UVA radiation had developed skin toxicity and/or skin tumors by 52 weeks. The skin lesions seen after treatment with 8-MOP and UVA radiation were characterized as squamous cell hyperplasia, squamous cell papilloma, and squamous cell carcinoma and are similar to what has been reported in humans after exposure to 8-MOP and UVA. Squamous cell hyperplasia and acute inflammation of the cornea were also seen in some of the treated female mice. Oral administration of 8-MOP and UVA did not result in a carcinogenic response to other organ systems.There were no increases in skin neoplasms after 8-MOP or UVA radiation alone. 8-MOP given in combination with UVA was carcinogenic to the skin of mice at dose levels similar to those used to treat psoriasis in humans. 0 1991 Society of Toxicology.

treatment had been used in a collaborative clinical trial sponsored by the National Institute of Arthritis, Musculoskeletal and Skin Diseases from 1974 to 1982. After a 5.7-year follow-up, patients receiving 8-MOP and UVA radiation had a 12-fold increase in the rate of squamous cell carcinoma compared to control groups; no substantial dose-related increase was noted for basal cell carcinoma (Stern et al., 1984). The objective of this study was to evaluate the toxicity and carcinogenicity of PUVA treatment and to examine all major organ sites for target organ toxicity. Previous studies with 8-MOP/UVA had concentrated primarily on skin toxicity after topical application. HRA/ Skh mice were exposed to an oral dose of 8MOP followed one-half hour later by UVA at

Oral administration of 8-methoxypsoralen (8MOP) followed by exposure to ultraviolet radiation (primarily UVA, 320-400 nm; PUVA therapy) is used in the treatment of vitiligo and psoriasis (Stern et al., 1984; Swinyard and Pathak, 1985). Exposure to 8-methoxypsoralen also occurs through ingestion of common vegetables such as parsnip, carrots, and parsley (Pathak et al., 1962; Ivie et al., 1981; Ceska et al., 1986). Recently PUVA has also been used in the treatment of cutaneous T-cell lymphoma (Edelson et al., 1987). PUVA therapy was approved for use in the United States for the treatment of psoriasis in 1982, but this ’ To whom correspondence should be addressed at NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709.

0272-0590191 $3.00 CopyrigJd 0 199 I by the Society of Toxicology. All rights of reproduction in any form reserved.

92

I-METHOXYPSORALEN

2 J/cm2 for 52 weeks. The high exposure level was set just below the level at which severe skin toxicity was seen in 13-week studies (Dunnick et al., 1987). The HRA/Skh mouse is an inbred hairless mouse strain sensitive to ultraviolet light effects (Mann, 197 1a,b; Forbes, 198la,b; Smith et al., 1982). METHODS

AND

MATERIALS

I-Methoxypsoralen (Car.. No. 298-8 I-7; Lot 2 1900; MW 2 16.18) was obtained from Elder Pharmaceuticals (Bryan, OH) and was found to be greater than 99% pure. g-MOP was mixed in NIH-07 feed (Zeigler Bros., Inc., Gardners, PA) using a Hobart C-100 mixer: half-inch-diameter feed pellets were prepared (Dyets, Inc. Bethlehem, PA) containing the following concentrations of g-MOP: 0, 100, 250, and 625 ppm corresponding to doses of 0,0.46, 1.15, and 2.9 mmol/kg.

Male and female HRA/Skh mice were obtained from the Animal Services Division, Skin and Cancer Hospital, Temple University Health Sciences Center (Philadelphia, PA). Animals were 7-8 weeks of age at the start of dosing. The animals were housed individually in stainless steel wire mesh cages (3 X 3 X 31 in.; Harford Manufacturing Co., Aberdeen, MD), 72 mice per rack (Forbes et al., 1982). Animal cages were rotated one position clockwise on the rack each week. A 12-hr room light cycle was provided using gold fluorescent lamps. Temperature was maintained at 76-80°F and humidity at 48-60%. Tap water and NIH-07 chow (Zeigler Bras., Inc, Gardners, PA) were available ad libitum except during the treatment periods. All animals were checked daily for morbidity and mortality. Moribund animals were killed and necropsied. Clinical signs, body weights, and feed consumption were recorded weekly.

93

AND UVA RADIATION TABLE 1 GROUPSIN

8-MOP/UVA

STUDYIN

HRA/Skh MOUSE

g-MOP concentration (ppm in diet) UVA level (J/cm*)

0

100

250

625

0 2 (glass filtered) 2

A E I

B F J

C G K

D H L

For treatment Weeks 3-52, animals were fed in the same manner followed 3 hr later by exposure to 0 or 2 J/cm* surface area (5-min exposure). At the end ofthe treatment period of 52 weeks, surviving animals were kept on study for an additional 28 weeks without treatment. Radiation Treatment Phototherapy lamps (black fluorescent light, Code No. FR74T12 PUVA) were obtained from GTE Sylvania (Danvers, MA). The spectrum output was characteristic of near ultraviolet light, primarily 320-400 nm with a peak at 354 nm, and less than 2% radiation below 320 nm. Exposures were controlled by the Mu3 dosimetry system (Model 3A, Solar Light Co., Philadelphia, PA) (Forbes el al., 1982). During the UV radiation period, a rack of animal cages was placed in front of a vertical bank of 36 lamps; animals were allowed to move freely during the radiation period. Mice received either unfiltered UV radiation or UV radiation filtered through 6-mm plate glass to remove short wave UV light between 280 and 320 nm. After the radiation treatment, mice were returned to feed and water ad libitum. Treatment consisted of a total of 150 pulse feedings and 144 UV radiations over a 52-week period. Pathology

Experimental Groups Male and female mice were randomized into 12 treatment groups as described in Table 1. The study was conducted using four replicates of 9 animals for a total of 36 animals/sex/dose group. For treatment with g-MOP, animals were fasted over night for 16 hr and then allowed access to feed pellets for 1 hr, and the feed pellet was weighed before and after dosing. For the first 2 weeks of study animals received feed pellets with or without g-MOP three times a week (Monday, Wednesday, and Friday).

Moribund animals and animals surviving to the end of the study were killed with carbon dioxide gas and gross necropsy examinations were performed. Tissues were preserved in neutral buffered 10% formahn, embedded in paraffin, sectioned, and stained with hematoxyhn and eosin. The following tissues were examined microscopically: gross lesions and tissues masses,skin (inguinal, back, and ears), mandibular and/or mesenteric lymph nodes, salivary glands, stemebrae and marrow, thyroids, parathyroids, small intestine, cecum, colon and rectum, liver, gallbladder, prostate/testes/epididymis or ovaries/uterus,

94

DUNNICK

ET AL.

TABLE

OF HRA/Skh

SURVIVAL

Week

A

B

C

D

2

E

F

MICE

G

H

I

J

K

L

36 34 32 29 18 I1

36 34 31 9 2 0

36 35 34 31 21 10

36 35 35 28 18 10

36 35 33 21 19 11

36 35 31 7 3 I

36 36 36 36 34 25

36 35 34 1 0 0

36 34 33 33 32 27

36 34 34 32 28 22

36 36 36 35 33 29

36 36 33 1 1 1

Males 0 12 24 52 64 80

36 33 30 22 20 13

36 35 35 28 22 14

36 34 32 22 17 11

36 32 31 26 17 12

36 35 35 25 17 9

36 35 34 26 23 18 Females

0 12 24 52 64 80

lungs heart, cress, eyes,

36 30 29 26 25 21

36 33 33 31 27 23

36 36 36 35 30 24

36 36 36 32 28 23

36 35 35 34 32 25

and mainstem bronchi, nasal cavity and turbinates, esophagus, stomach, brain, thymus, trachea, panspleen, kidneys, adrenals, urinary bladder, pituitary, and mammary gland.

RESULTS

36 35 35 35 30 25

ble 2). This decreased survival was attributed ’ to severe skin toxicity. Survival in the control and lower dose groups at Week 80 was 2850% in males and 60-80% in females, suggesting that in this strain of mice the female has a longer life span than the males.

Body Weight and Survival

Skin-Nonneoplastic

Body weight effects after dosing with 8-MOP or UV radiation alone or with 8-MOP in combination with UV radiation were not statistically significant, except in female mice exposed to 625 ppm 8-MOP followed by UV radiation. At Week 52 mean body weights of female mice at 625 ppm/8-MOP were approximately 86% of the mean body weights of the control groups. Body weights of all other dosed groups were within f 10% of the respective control groups throughout the course of the study. Decreased survival in comparison to controls and lower dose groups was seen in the 625 ppm 8-MOP and UV radiation groups (groups H and L) after 35 weeks of dosing (Ta-

Skin was the primary target organ after dosing with 8-MOP and UV radiation. Dose-related lesions were not seen in the other organ systems examined, except for the eyes in high dose female mice. An increase in the incidence of squamous cell hyperplasia and acute inflammation was seen in the cornea of some female mice exposed to 8-MOP at 625 ppm with UVA radiation (Table 3). An increase in the incidence and severity of skin hyperplasia (Figs. 1 and 2) was seen in males and females exposed to 8-MOP at 625 ppm and UV radiation (Table 3). Skin sections were routinely taken from the back, the inguinal region, and the ears. The data presented

Lesions

19 (54%) 1.0 0 0 0 0 0 2

1 1

0

4

12 (39%) 1.1 0 0 0 0

0

0

35

3 (9%) 1.3 0 0 0 0

5 (15%) 1.0 0 0 0 0

31

35

B 100

33

A 0

0 3

15 (42%) 1.0 0 0 0 0

36

0

0

7 (20%) 1.0 0 0 0 0

35

C 250

36

1

0

1 1

0 2

MOUSE

31

G 250

32

H 625

36

0

1

0 5

35

3

0

36

1

0

10 (30%) 1.0 0 0 0 0

33

I 0

0 3

4 11

0 3

21 (63%) 29 (83%) 24 (67%) 1.3 2.8 1.1 0 7 (20%) 0 0 29 (83%) 0 0 19 (54%) 0 0 6 (17%) 0

34

1

0

4 (I 1%) 14 (45%) 27 (84%) 1.0 1.0 2.5 0 0 4(13%) 0 0 14 (44%) 0 0 8 (25%) 0 0 4(13%)

35

F 100

2 J/cm2 filtered

UV radiation

IN THE HRA/Skh

19 (56%) 21 (58%) 1.1 1.1 0 0 0 0 0 0 0 0

34

Female

6

0

9 (26%) 1.0 0 0 0 0

34

Male

E 0

EYE LESIONS

6 (17%) 1.2 0 0 0 0

35

D 625

AND

14 (39%) 1.1 0 0 0 0

SKIN

36

1

0

10 (28%) 1.1 0 0 0 0

36

K 250

0 4

0 4

25 (71%) 24 (67%) 1.2 1.5 0 0 0 6 (17%) 0 2 (6%) 0 0

35

0

1

9 (25%) 1.0 0 0 0 0

36

J 100

2 J/cm2 unfiltered

8 11

36 (97%) 2.9 9 (24%) 34 (92%) 12 (32%) 9 (24%)

36

6

0

31 (89%) 2.2 11 (31%) 12 (35%) 5 (14%) 0

35

L 625

Note. Grading scale of skin hyperplasia: 1, minimal, two to four epithelial cell layers; 2, mild, four to six epithelial cell layers; 3, moderate, six to nine epithelial cell layers; 4, marked, more than nine epithelial cell layers.

Nuclear atypia Dermis-hyaline change Cleft/vesicle Ulcer Eye Squamous cell hyperplasia cornea Acute inflammation cornea

No. examined Skin, back Squamous cell hyperplasia

Nuclear atypia Dermis-hyaline change Cleft-vesicle Ulcer Eye Squamous cell hyperplasia (cornea) Acute inflammation cornea

No. examined Skin, back Squamous cell hyperplasia

Group: 8-MOP (ppm):

None

NONNEOPLASTIC

TABLE 3

96

DUNNICK

ET AL.

FIG. 1. Normal skin from the back. Epithelium is one to two cell layers thick. Dilated hair follicles and granulomatous inflammation deep in dermis is characteristic of HRA/skh mice. H&E. X30.

in this table are for skin from the back, but similar lesions were seen in other regions. The skin lesions were most severe on the dorsal surfaces. Females tended to be more affected than males. The severity of skin hyperplasia in females (but not in males) at the next lower exposure level of 250 ppm 8-MOP and UV radiation was greater than that in controls. Other skin lesions seen after 625 ppm g-MOP and UV radiation included nuclear atypia, hyaline change, and cleft/vesicles, and in females, ulceration. Nuclear atypia of the skin was characterized as enlargement of nuclei (two to four times normal) in the basal layer of the epithelium, with irregular condensed nuclear membranes, and prominent nucleoli. In animals with cleft/ vesicles there appeared to be a focal separation of the epithelium from the dermis. In small lesions, the basal layer of epithelium often ap-

peared frayed and small granules of basophilic material were attached to the outer layer of the dermis. In larger lesions, the space was filled with an amorphous or faintly fibrillar eosinophilic material which was PAS positive. A few animals at 625 ppm S-MOP and UV radiation had hyalinization or increased eosinophilia of the collagen fibers. With an elastic stain, there were increased numbers of elastic fibers in the dermis. Condensation of the dermis just below the basement membrane could best be seen with this stain, although elastic fibers were not present in this area. Skin-Neoplastic

Lesions

Skin neoplasms were seen in the high dose groups (625 ppm 8-MOP with UV radiationgroups H and L) including both the unfiltered

%METHOXYPSORALEN

AND UVA RADIATION

97

FIG. 2. Moderate squamous cell hyperplasia of the skin from the back. Epithelium is six to nine cell layers thick. Dermis is condensed particularly under the basement membrane. H&E. X30.

and the filtered UV radiation groups. There was also a marginal increase in tumors in females at the next lower exposure level (groups G and K, Table 4). Animals were freely moving during the exposure to UVA and skin tumors occurred primarily on the dorsal surfaces, the hindquarters, the back, and the head. After 52 weeks of treatment, surviving animals were kept on study for an additional 28 weeks without dosing to determine if continued treatment with 8-MOP and UVA was necessary for skin neoplasms to be present. Most of the animals in the 625 ppm 8-MOP/ UV radiation groups had died before Week 80, and many of these animals had skin neoplasms (Table 4). In the next lower exposure level (250 ppm 8-MOP/UV radiation) l/l 1 males (group G), O/l 1 males (group K), 5/25 females (group G), and 7/29 females (group

K) had either skin papillomas or carcinomas at Week 80. The squamous cell papillomas were exophylic masses with a roughened, irregular surface. Microscopically, the papillomas consisted of an arborized core of fibrovascular tissue contiguous with the dermis and covered by a thickened, often hyperkeratotic squamous epithelium. The epithelial cells, although more numerous and crowded, were orderly in their arrangement, resembling normal squamous epithelium. In most instances, the papillomas were pedunculated with the arborized projections arising from a single stalk, although occasionally papillomas were more broad-based or sessile. Squamous cell papillomas were usually solitary. Squamous cell carcinomas, in situ (Fig. 3), were usually small nodules, one to several

a 36 animals/group.

Squamous cell papilloma Squamous cell carcinoma, in situ Squamous cell carcinoma Squamous cell papillomas or carcinomas

Squamous cell papilloma Squamous cell carcinoma, in situ Squamous cell carcinoma (tumors) Squamous cell papillomas or carcinomas

Group: &MOP (ppm):

0 0

2 (6%)

1 (3%)

2 (6%)

1 (3%)

0 0

1 (3%)

0

2 (6%)

0

1 (3%)

1 (3%)

1 (3%)

B 100

0

A 0

None

3 (9%)

0 2 (6%)

1 (3%)

1(3%)

0

0

1 (3%)

C 250

INCIDENCE

3 (9%)

0 1(3%)

2 (6%)

1(3%)

0

0

1 (3%)

D 625 E 0

2 (6%)

2 (6%)

0 0

0

0

0

0

1 (3%)

0 0

1(3%)

Female a

2 (6%)

0

0

Male”

F 100

6 (17%)

1(3%) 1(3%)

4(11%)

1(3%)

0

0

1 (3%)

G 250

2 J/cm’ filtered

UV radiation

SKIN LESIONS IN THE HRA/Skh

2 (6%)

OF NEOPLASTIC

TABLE 4

31 (86%)

4(11%) 27 (75%)

0

24 (67%)

15 (42%)

7 (19%)

2 (6%)

H 625

MOUSE

3 (9%)

0 0

3 (9%)

2 (6%)

0

0

2 (6%)

I 0

4(11%)

1(3%) 2 (6%)

1 (3%)

1(3%)

0

0

1 (3%)

J 100

11 (30%)

4(11%) 6 (17%)

1 (3%)

5 (14%)

2 (6%)

0

3 (9%)

K 250

2 J/cm* unfiltered

22 (61%)

5 (14%) 17 (47%)

0

24 (67%)

14 (39%)

8 (22%)

2 (6%)

L 625

8-METHOXYPSORALEN

AND UVA RADIATION

FIG. 3. Squamous cell carcinoma in situ. There is cellular atypia, dysplasis, and individual cell keratinization within the thickened epithelium. H&E. X90.

millimeters in size. Microscopically these le- tumors were characterized by downward prosions were discrete nodular lesions in which jecting sheets, nests, and anastamosing cords the epithelial border was smooth and sharply of neoplastic squamous cells that extended demarcated from the dermis. Within the ep- into the dermis. Foci of keratinization were ithelium, there was a disorderly arrangement present in clusters of neoplastic cells. Individof cells and often the nuclei were atypical. In- ual cell keratinization and nuclear atypia were dividual cell keratinization was common as common. The carcinomas varied from well differentiated to anaplastic squamous cell tuwere mitotic figures. Often individual epithemors. In a few tumors, the cells had a spindle lial cells were anaplastic. Marked hyperkeraappearance, resembling a fibrosarcoma. Some tosis was common. Within underlying dermis there was often an inflammatory base con- of the invasive carcinomas appeared to arise sisting of plasma cells and lymphocytes. Squa- from the in situ carcinomas. Invasive carcinomas were often multiple and were found in mous cell carcinomas, in situ, were frequently multiple and were often seen in animals with animals with in situ carcinomas and hyperplasia. squamous cell carcinomas. Squamous cell carcinomas (Fig. 4) were DISCUSSION nodular masses with irregular surfaces, often When 8-methoxypsoralen was administered appearing to be covered with a thick, hard scaly material. Ulceration was common. These by the oral route three times a week at doses

100

DUNNICK

from 9 to 80 mg/kg/week followed by UV radiation, the primary toxic response was seen in the skin where severe hyperplasia and ulcers occurred particularly at the high dose of 8MOP. This skin toxicity led to decreased survival by Week 52 at 8-MOP doses of 625 ppm (approximately 80 mg/kg/week). In the high dose group there were some decreases in body weights relative to the control groups, but in the other dosed groups the body weight was unaffected by treatment. These studies showed that 8-MOP in males (625 ppm) and in females (250 and 625 ppm) with UVA radiation (2 J/cm2) produced skin tumors in the HRA/Skh mouse at levels of drug and UVA radiation comparable to those used in PUVA therapy (Table 5). Skin toxicity in female mice was more severe than in males and this was also observed in 13-week studies

ET AL. TABLE5 COMPARISONOF ANIMAL AND HUMAN DOSE OF &METHOXYPSORALEN (g-MOP)

mg/h

Animal dose (mm)

mmol/kg feed

body wt/week

100 250 625 Average human doseb

0.46 1.15 2.9

9 21 80 1.8

mg/m’ body surface area/week” 21 63 240 67.0

’ Calculations for body surface area based on Freireich et al. ( 1966).

b Physicians Desk Reference (1988).

in the HRA/Skh mouse treated with 8-MOP and UVA radiation (Dunnick et al., 1987). Skin tumors occurred at dose levels where skin

FIG. 4. Squamous cell carcinoma. The neoplasm is infiltrating the dermis and the overlying epithelium is hyperplastic. H&E. X22.

I-METHOXYPSORALEN

toxicity, including hyperplasia and nuclear atypia, were seen. In the lower dosed groups, skin tumors persisted even after a 28-week period without dosing. Treatment-related eye lesions were seen in high dose female mice. A thorough examination of all other major organ systems was conducted and other target organ toxicity was not observed. Removal of wavelengths below 320 nm, approximately 2% of the total spectrum output, had no significant effect on tumor development in these experiments. Other mouse studies have concentrated on the study of toxicity of topically applied 8MOP and UV radiation, and these studies showed skin tumors after treatment (Grube et al., 1977; Lunggeren et al., 1981; Young et al., 1983; IARC, 1980, 1987). The studies described in this paper administered 8-MOP by oral route, the route of administration used in the treatment of psoriasis and vitiligo, and also demonstrated that, by this route of administration, skin is the primary target organ when 8-MOP is used in combination with UV radiation. In a study of 8-MOP without UV radiation in the F344/N rat where exposure was for a 2-year period, dosed males developed an increased incidence of kidney tumors and Zymbal gland tumors and a marginal increased incidence of lung and subcutaneous tumors (Dunnick, 1988). The rat 2-year study was at dose levels 5- 10 times human dose levels when compared on a milligrams of drug per square meter of surface area basis, while in the 8MOP/UV radiation study in mice, reported here, drug levels were comparable to human dose levels of a milligrams per square meter of surface area basis. In both the rat and mouse studies the top exposure level was set at the maximum tolerated dose, but the maximum tolerated level is lower when 8-MOP is given in combination with UV radiation because of the development of skin toxicity. Skin toxicity is not seen when 8-MOP is used alone. The HRA/Skh mouse is an animal that is

AND

UVA

RADIATION

101

sensitive to the effects of 8-MOP/UVA radiation at levels which cause similar effects in man, and 8-MOP and UVA cause tumors of the skin in both the mouse and man. The female mouse is more sensitive than the male to developing skin lesions as has previously been seen in the 13-week study of 8-MOP and UVA radiation (Dunnick et al., 1987). ACKNOWLEDGMENTS The in-life phases of this study were conducted at Temple University facilities, and the pathology by Experimental Pathology Laboratories (terminal necropsies) and by PATHCO, Inc. (histopathology).

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