Journal of Photochemistry and Photobiology, B: Biology, 6 (1990) 2 3 7 - 2 4 7
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PHOTOCARCINOGENICITY OF PSORALENS USED IN PUVA TREATMENT: PRESENT STATUS IN MOUSE AND MAN* ANTONY R. YOUNG
Photobiology Unit, Institute of Dermatology, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, University of London, London (U.K.) (Received November 24, 1989; accepted D e c e m b e r 16, 1989)
Keywords. Psoralens, PUVA treatment, 8-methoxypsoralen, photocarcinogenesis, skin cancer, photosensitization.
Summary There is good evidence that 8-methoxypsoralen is photocarcinogenic in psoriatic patients undergoing long-term photochemotherapy (PUVA) in the U.S.A. However, this conclusion has not been supported by two m~jor European studies which have indicated that PUVA is a tumour promoter of damage initiated by other agents. Variation in PUVA treatment protocols in the U.S.A. and Europe may partly account for the different conclusions. There is much interest in the therapeutic potential of monofunctional psoralens. It is hoped that these may reduce long-term risk. Monofunctional and cross-linking psoralens have been shown to be photocarcinogenic in mouse skin. The relative risk of different compounds may be assessed in the mouse, but it is important to base comparisons on dose protocols that have been shown to be therapeutically effective.
1. Introduction The psoralens (furocoumarins) are photosensitizing drugs that have been used extensively with long-wave UV radiation (UVA; 3 1 5 - 4 0 0 nm) in photochemotherapy (PUVA) during the past 15 years. The most common use has been 8-methoxypsoralen (8-MOP) for the treatment of psoriasis [1, 2]. However, PUVA with 8-MOP has also been effective in the treatment of skin disorders as diverse as mycosis fungoides [3], vitiligo [4] and polymorphous light eruption [5]. More recently PUVA has been used in the extracorporeal *Paper presented at the Photomedicine Meeting organized by the F r e n c h Society of Photobiology, Paris, November, 1989.
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238 irradiation of lymphocyte-enriched blood for the treatment of cutaneous Tcell lymphoma [6]. The molecular basis of the therapeutic effects of PUVA is not known. The diversity of diseases that r e s pond to this treatment suggests that mechanisms vary. The most extensively studied aspect of psoralen photochemistry and photobiology is DNA photobinding [7-9]. A prerequisite of such binding is the formation of an unstable non-covalent "dark c o m p l e x " within the DNA molecule [7]. Dark complexation modifies the absorption properties of 8-MOP and other psoralens. Thus free 8-MOP shows an absorption maximum at about 300 nm (in the UVB range), whereas 8-MOP, "dark c o m p l e x e d " within DNA, shows an absorption maximum which is " r ed shifted" to about 315 nm [10]. On absorption of a photon, 8-MOP may undergo C4 cyclobutane addition across the 5,6 double bond of a pyrimidine base in DNA to form an 8-MOP monoadduct. This reaction may take place across either the 3,4 double bond of the pyrone ring or the 4',5' double bond of the furan ring of the psoralen molecule. Thus there are two types of lesion: the 3,4-monoadduct and the 4',5'-monoadduct. These adducts have quite different UVR absorption characteristics. The former shows virtually no absorption in the UVA range, but the latter has an absorption maximum at about 335 nm [8]. In the presence of UVA the 4 ' , 5 ' - m o n o a d d u c t may absorb a phot on and undergo a further C4 cyclobutane addition reaction across its 3,4 bond with the 5,6 double bond of a suitably placed pyrimidine base in the opposite DNA strand. This lesion is a DNA cross-link. 8-MOP, 5-methoxypsoralen (5-MOP), psoralen and trimethylpsoralen (TMP) are cross-linking, or bifunctional, compounds. Monofunctional psoralens do not progress be yond m o n o a d d u c t formation. Their molecular structure may prevent the necessary stereochemistry for reactions at both furan and pyrone double bond sites, such as with the angelicins and their synthetic derivatives. Alternatively, reactive sites may be inhibited by substituted terminal groups, such as with 3-carbethoxypsoralen (3-CP). The psoriatic epidermis is in a hyperproliferative state. Inhibition of DNA synthesis, whether by monoadducts or cross-links [11, 12] may well be the mechanistic basis of the therapeutic effect.
2. Photocarcinogenicity It has been clearly demonstrated, in bacterial, yeast and mammalian systems, that cross-linking and monofunctional psoralens are highly mutagenic in the presence of UVA radiation [ 1 3 - 1 5 ]. Mutagens must always be considered as potential carcinogens. 2.1. M o u s e s t u d i e s The photocarcinogenicity of both cross-linking and monofunctional psoralens has been studied. The latter have been studied because they may be less photomutagenic than the former [13].
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2.1.1. Cross-linking psoralens The photocarcinogenic potential of 8-MOP was first indicated in the mouse in 1957 [16]. This has been confirmed by several workers with UVA and solar simulating radiation (SSR) sources [ 1 7 - 2 0 ] . Studies on other crosslinking c o m p o u n d s have shown that 5-MOP [19, 20] and psoralen [19] are also photocarcinogenic. The study of Zajdela and Bisagni [19] showed a rank order of time-to-onset to first ear t u m o u r of psoralen > 8-MOP > 5-MOP (all topically applied at 0.025% in acetone in the presence of 365 nm radiation). The study of Young et al. [20] showed no difference between the c o m p o u n d s in the time-of-onset to first skin t u m o u r (topically applied at 0.01% and 0.03% to dorsal and flank skin in an oily vehicle and exposed to SSR). Trend analysis for time-to-onset to first t u m o u r has shown a d e p e n d e n c e on drug dose for 8-MOP and 5-MOP [ 2 0 - 2 2 ] . A d e p e n d e n c e of time-to-onset to first t u m o u r on SSR dose has been demonstrated with 8MOP [23].
2.1.2. Monofunctional psoralens Several monofunctional psoralens have been assessed. The first study was with 3-CP in XVIInc/Z mice [24]. Using 8-MOP as a positive control, there was no evidence of photocarcinogenicity with 3-CP after topical application to ear skin and e xpos ur e to 365 nm radiation. The lack of photocarcinogenicity with 3-CP was confirmed w hen topically applied to the dorsal skin of the hairless albino Skh-1 mouse [25]. A study of angelicin and its synthetic derivatives, 5-methylangelicin and 4,5'-dimethylangelicin, showed photocarcinogenicity of these c o m p o u n d s when applied topically to the skin of Skh 1 hairless albino mice in the presence of UVA radiation [25]. The synthetic derivatives were m or e carcinogenic than an 8-MOP positive control, but angelicin was less p o t e n t than 8-MOP. An International Agency for Research on Cancer (IARC) Working Group not ed that the com pounds tested were not pure and concluded that the evidence for the photocarcinogenicity of these agents was limited [26]. Photocarcinogenicity has also b e en demonstrated with pyridopsoralen (PP) and 7-methylpyridopsoralen (MPP) applied topically to the ear of the 17NCZ mouse prior to exposure to 365 n m radiation [27]. These c o m p o u n d s were less carcinogenic than 8MOP. It is not always clear from the studies described above ff comparisons between cross-linking and monofunctional c o m p o u n d s have b e e n made concurrently or historically. The latter are m uc h less reliable. It is essential that comparisons between c o m p o u n d s are made under similar conditions in the same mouse strain as it is well established that there is interstrain variation in photocarcinogenesis. The photocarcinogenic potential of cross-linking psoralens cannot specffically identify cross-links as the putative lesions as such agents also form monoadducts. It is evident that m o n o a d d u c t s have carcinogenic potential. A given n u m b e r of induced 8-MOP cross-links in epidermal DNA may have variable carcinogenic responses in different strains of mice [28] and may
240 not be relevant to risk a s s e s s m e n t if such lesions result in a g r e a t e r probability of e p i d e m a l cell lethality in situ.
2.1.3. W a v e l e n g t h dependence o f psoralen photocarcinogenesis The d e t e r m i n a t i o n of an action s p e c t r u m for l o n g - t e r m effects in the skin p r e s e n t s considerable practical difficulties. A first a p p r o a c h to this p r o b l e m has b e e n m a d e b y Young et al. [29] in the hairless albino mouse. This s t u d y has indicated that the action s p e c t r u m for skin c a n c e r with 8MOP is likely to be similar to that o b t a i n e d for acute effects on skin, s u c h as h u m a n e r y t h e m a [30], m o u s e s u n b u r n cell f o r m a t i o n [31] and inhibition of m o u s e e p i d e r m a l DNA synthesis [32], with p e a k activity in the 3 2 0 - 3 3 5 n m region. 2.2. H u m a n studies Knowledge of the DNA-damaging, m u t a g e n i c and c a r c i n o g e n i c p r o p e r t i e s of PUVA with 8-MOP has initiated careful and intensive follow-up studies in large n u m b e r s of psoriatic patients. Stern et al. [33] r e p o r t e d an increase in s q u a m o u s cell c a r c i n o m a in a 2.1 y e a r p r o s p e c t i v e multicentre s t u d y in the U.S.A. o f 1371 psoriatic patients u n d e r g o i n g PUVA t r e a t m e n t . An e x t e n s i o n of this study, r e p o r t e d on 1380 patients after a total of 5.7 years of follow-up, r e i n f o r c e d the original c o n c l u s i o n s with m o r e p o w e r f u l data [34]. T h e s e d e m o n s t r a t e d a clear relationship b e t w e e n cumulative UVA dose and incidence of s q u a m o u s cell c a r c i n o m a . F u r t h e r m o n i t o r i n g of 1380 patients, with a m e a n follow-up time of 10 years, c o n t i n u e s to s h o w a relationship b e t w e e n s q u a m o u s cell c a r c i n o m a and cumulative UVA e x p o s u r e as well as a m o d e s t dose relationship for basal cell c a r c i n o m a [35]. The o b s e r v a t i o n s of Stern and c o w o r k e r s [34, 35] have b e e n c o n f i r m e d b y F o r m a n et al. [36] in a r e t r o s p e c t i v e multicentre study in the U.S.A. No relationship b e t w e e n basal cell c a r c i n o m a and cumulative UVA dose was o b s e r v e d but t h e r e was a marked, but not significant, t r e n d for i n c r e a s e d s t a n d a r d m o r b i d i t y ratio o f s q u a m o u s cell c a r c i n o m a with cumulative UVA dose. However, the c o n c l u s i o n that PUVA is c a r c i n o g e n i c has not b e e n m a d e by o t h e r investigators in A m e r i c a and E u r o p e [ 3 7 - 4 2 ] . S o m e o f t h e s e studies have b e e n of s h o r t duration a n d / o r with relatively small n u m b e r s of subjects, and t h e r e f o r e are of limited value in the a s s e s s m e n t of l o n g - t e r m risk. In general, E u r o p e a n w o r k e r s have c o n c l u d e d that PUVA is not a c o m p l e t e skin carcinogen. S o m e investigators have c o n c l u d e d that PUVA is a " p r o m o t e r " of s q u a m o u s cell c a r c i n o m a initiated by p r i o r t r e a t m e n t with a g e n t s s u c h as ionizing radiation or arsenic [ 3 9 - 4 1 ] . To date t h e r e has b e e n no indication f r o m any of the studies that PUVA e n h a n c e s the risk of malignant m e l a n o m a . 3. P o s s i b l e r e a s o n s f o r t h e d i s c r e p a n c i e s b e t w e e n t h e A m e r i c a n and European follow-up studies The statistical a p p r o a c h to data analyses is i m p o r t a n t [43, 441. Stern [43] has e m p h a s i z e d the g r e a t e r p o w e r of p r o s p e c t i v e studies [ 3 3 - 3 5 , 3 9 - 4 1 ]
241 c o m p a r e d with a r e t r o s p e c t i v e a p p r o a c h [36, 42]. The A m e r i c a n a p p r o a c h to d a t a m a n a g e m e n t h a s b e e n to c o m p a r e t h e o b s e r v e d i n c i d e n c e of skin c a n c e r with the e x p e c t e d i n c i d e n c e d e t e r m i n e d f r o m a n age- a n d s e x - m a t c h e d n o r m a l p o p u l a t i o n . This w a s n o t originally c a r r i e d o u t in the m o s t e x t e n s i v e E u r o p e a n studies [39, 40], b u t o n e g r o u p f o u n d n o difference in n o n m e l a n o m a skin c a n c e r i n c i d e n c e b e t w e e n PUVA a n d non-PUVA p a t i e n t s t r e a t e d in a d e r m a t o l o g y d e p a r t m e n t [41]. 8 - M O P - e n h a n c e d p h o t o c a r c i n o g e n i c i t y in the m o u s e s h o w s a failure o f t i m e - d o s e r e c i p r o c i t y [23]. T h u s a g i v e n c u m u l a t i v e UVR d o s e is m o r e c a r c i n o g e n i c if g i v e n in s m a l l e r daily f r a c t i o n s o v e r a l o n g e r p e r i o d of time. This o b s e r v a t i o n is the b a s i s of a p r o p o s a l b y G i b b s et al. [45] t h a t the different c o n c l u s i o n s of A m e r i c a n a n d E u r o p e a n g r o u p s on PUVA carcinogenicity is r e l a t e d to different t r e a t m e n t p r o t o c o l s . The usual p r a c t i c e in the U.S.A. is to a d m i n i s t e r n o n - p h o t o t o x i c PUVA doses, w h e r e a s the E u r o p e a n a p p r o a c h is to m a x i m i z e a n d m a i n t a i n d o s e s at a n e a r p h o t o t o x i c level. The latter s y s t e m t e n d s to give m o r e r a p i d clearing with l o w e r c u m u l a t i v e UVA d o s e s [46]. E x t r a p o l a t i o n f r o m the m o u s e d a t a [23] s u g g e s t s t h a t it m a y also b e less c a r c i n o g e n i c . T h e m o u s e studies also indicate t h a t it m a y b e m i s l e a d i n g to c o m p a r e c a n c e r risk in t e r m s of c u m u l a t i v e UVA d o s e w h e n different d o s e p r o t o c o l s h a v e b e e n used.
4. S t r a t e g i e s f o r m i n i m i z i n g t h e s k i n c a n c e r risk w i t h 8-MOP 4.1. C o m b i n a t i o n t h e r a p y The a d m i n i s t r a t i o n o f e t r e t i n a t e with PUVA r e s u l t s in r e d u c e d clearing t i m e and n u m b e r o f t r e a t m e n t s with a 5 0 % r e d u c t i o n of c u m u l a t i v e UVA d o s e [47]. The u s e of e t r e t i n a t e in o n e E u r o p e a n s t u d y m a y h a v e b e e n a partial f a c t o r in the lack o f p h o t o c a r c i n o g e n i c i t y o f PUVA [39]. 4.2. S u n s c r e e n u s e PUVA p a t i e n t s are g e n e r a l l y a d v i s e d to w e a r e y e p r o t e c t i o n a f t e r d r u g i n g e s t i o n as p s o r a l e n s m a y p e r s i s t in p l a s m a for s e v e r a l h o u r s p o s t - i n g e s t i o n [48]. Animal studies h a v e s h o w n t h a t UVB s u n s c r e e n s ( c i n n a m a t e s w h i c h a b s o r b s h o r t e r - w a v e UVA) a n d UVB plus UVA s u n s c r e e n s significantly p r o t e c t skin f r o m 5-MOP p h o t o c a r c i n o g e n i c i t y in t h e m o u s e [21, 22]. U n d e r s o m e c o n d i t i o n s this p r o t e c t i o n is t o t a l [22]. S u n s c r e e n s also p r o t e c t a g a i n s t 5M O P - i n d u c e d p h o t o t o x i c i t y o f h u m a n skin [49] a n d m u t a g e n i c i t y in the y e a s t S a c c h a r o m y c e s c e r e v i s i a e [50]. S u n s c r e e n u s e m a y b e a d v i s e d to p r o t e c t sites n o t i n v o l v e d with psoriasis, in p a r t i c u l a r s c r o t a l skin [36]. T h e u s e o f s u c h s u n s c r e e n s a f t e r UVA e x p o s u r e w o u l d r e d u c e u n w a n t e d p s o r a l e n p h o t o a c t i v a t i o n b y a m b i e n t s o l a r r a d i a t i o n a n d p o s s i b l y skin c a n c e r risk. T h e r e m a y b e a n e e d for s p e c i a l l y f o r m u l a t e d s u n s c r e e n s for u s e with PUVA.
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4.3. A l t e r n a t i v e s to 8-MOP 4.3.1. Other cross-linking psoralens H6nigsmann et al. [51] r e por t ed the successful use of 5-MOP for the treatment of psoriasis. The main advantage of this drug is reduced nausea. 5-MOP, like 8-MOP, is a DNA cross-linking agent [9]. There are no extensive studies of 5-MOP photocarcinogenicity in human skin. One study indicated no increased risk of skin cancer in workers in the bergam ot fruit industry in southern Italy [52]; however an IARC Working Group noted limitations in this study [53]. 5-MOP is a skin phot ocarci nogen in the mouse [19-22]. Extrapolation of animal data of 5-MOP photocarcinogenicity to humans does not suggest that 5-MOP would pr e s ent a lower risk than 8-MOP. 4.3.2. Monofunctional psoralens Monoadducts are, in general, less mutagenic than cross-links [13]. It has been hypothesized that they may be less carcinogenic than cross-linking agents, but this has not been established from the mouse data described. In addition, this hypothesis pr e s uppos e s that carcinogenicity is related solely to DNA damage. There have been a few reports in the literature of clinical studies assessing the efficacy of clearing of psoriasis in small groups of patients. Dubertret et al. [24] r e por t ed the treatment of psoriasis with 3-CP as a promising non-photocarcinogenic alternative to 8-MOP. However, a Japanese group was not able to confirm the therapeutic efficacy of this c o m p o u n d [54]. More recent studies indicate that the pyridopsoralens may be effective in clearing psoriasis [27, 55]. However, the mean UVA dose for clearing with MPP is four times higher than with equimolar 8-MOP. The pyridopsoralens are three to four times less photocarcinogenic than 8-MOP [27], but this advantage may be lost if much higher doses are required for effective therapy. Other monofunctional agents have also been investigated. The 6-methylangelicins have been r epor t e d to have better therapeutic effect in psoriasis than 8-MOP but with equivalent photomutagenicity [56]. There has been recent interest in khellin, a monofunctional agent with a structure similar to psoralen. Good therapeutic activity for vitiligo has been report ed in association with genotoxicity [57]. As yet there are no photocarcinogenicity data on these agents. 4.4. M a x i m i z i n g spectral e giciency The action s pect r um for the clearing of psoriasis with 8-MOP, or any other psoralen, is not known. One study comparing 335 nm radiation with 365 nm radiation showed the former to be more effective [58]. In the absence of more specific data it seems reasonable to suppose that this action spect rum resembles that for acute effects of 8-MOP in the skin, including inhibition of epidermal DNA synthesis, with a maximum in the region of 3 2 0 - 3 3 5 nm [32].
243 Most UVA tubes used in PUVA have maxi m um emission at about 365 nm with very little output in the 3 2 0 - 3 3 5 nm region [59]. This may m ean that mu ch of the UVA radiation received by the skin is " r e d u n d a n t " with respect to 8-MOP photochemistry. UVA radiation induces DNA lesions in human skin i n s i t u [60] and is mutagenic and carcinogenic in mice [61]. Its acute effects on skin are de pe nde nt on oxygen [62] and there is evidence that it generates active oxygen species [63]. Such species may be important in t u m o u r p r o mot i on by PUVA [64]. Thus it is possible that the cumulative PUVA dose d ep ende nc e of squamous cell carcinoma observed in the American studies [ 3 4 - 3 6 ] may be partly due to UVA alone. In one experiment, 50°/0 of mice (Skh 1) e x p o s e d to 22 J cm -2 day -1 had skin tumours on day 265 after a cumulative UVA (peak emission at about 350 urn) dose of 5830 J cm -2 [65]. In another, using the same daily dose but with a different UVA source, 50% of mice had tumours on day 233 after a cumulative UVA (peak emission at about 3 5 0 - 3 8 0 nm) dose of 5126 J cm -2 [66]. Doses of this order would have be e n achieved in some of the high-dose American patients, but rarely in any of the E u r o p e a n patients. There are no animal investigations of UVA t i m e - d o s e reciprocity for skin cancer. However, if UVA is similar to UVB in this r es pe c t [23, 67], the American a p p r o a c h to PUVA could exacerbate the carcinogenic effects of UVA alone. Assuming that " r e d u n d a n t " UVA has little or no therapeutic benefit, it should be r e m o v e d from PUVA by using a radiation source that has an emission spectrum similar to that of the action spect rum for psoriasis clearing with 8-MOP. This may be particularly important when using other psoralens, e.g. MPP, that require higher cumulative UVA exposures for clearing. The determination of detailed action s pect ra for the clearing of psoriasis with a wide range of psoralens is a near impossible task. However, it may be a p p r o a c h e d by an appropriate model. Assuming that the primary mechanism of action of psoralens in psoriasis is the inhibition of DNA synthesis, it may be possible to use a simple i n vi t r o model such as the inhibition of growth in the yeast C. a l b i c a n s [68, 69]. The 8-MOP action spect rum for this end point, with peak activity at 3 2 0 - 3 3 5 um [10], is similar to that already described for skin [ 3 0 - 3 2 ] . Studies on PP and MPP showed peak activity at 340 um and 3 1 0 - 3 3 0 nm respectively [10].
5. C a r c i n o g e n i c i t y o f 8-MOP in t h e a b s e n c e o f UVA In general, mouse studies have b e e n designed to investigate the photocarcinogenicity of psoralens. The length of time that control groups, i.e. animals given psoralen but no UVR, have spent in the dark has b e e n determined by the time-to-onset of tumours in the UVR-treated groups. In m any cases this has b e e n a relatively short period in comparison with conventional 2 year chemical carcinogenesis studies. It has been widely assumed by the photobiology and dermatology community that 8-MOP in the absence of LWA does not present a carcinogenic risk. However, a recent 2 year study
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has shown 8-MOP to cause cancer of the kidney, Zymbal gland, subcutaneous tissue and lung in male, but not female, F344/N rats [70]. It must be stated that the doses of 8-MOP used were 3 7 - 7 5 times the daily human dose. The significance to humans is not known, but these data suggest that follow-up studies for non-skin neoplasia in PUVA patients may be necessary.
6. C o n c l u s i o n s
Animal studies provide clear evidence of the photocarcinogenic potential of cross-linking and monofunctional psoralens. The relationship between specific types of DNA lesion and carcinogenic potential is not known. Extensive follow-up studies of PUVA patients have provided conflicting results. Carcinogenicity of PUVA may depend on therapeutic protocol. Assessment of risk of new psoralens for PUVA must use dose protocols that have been shown to be clinically effective. There is a need for action spect rum data for psoriasis clearing or the establishment of a model for this purpose. UVA sources could be designed for maximum spectral efficiency with the elimination of 'redundant" UVA which may play a role in skin cancer.
References 1 J. A. Parrish, T. B. Fitzpatrick, L. T a n e n b a u m and M. A. Pathak, P h o t o c h e m o t h e r a p y of psoriasis with oral methoxsalen and longwave ultraviolet light, N. Engl. J. Med., 291 (1974) 1207-1211. 2 S. Rogers, J. Marks, S. Shuster, D. Vella Briffa, A. Warin and M. Greaves, Comparison of p h o t o c h e m o t h e r a p y and dithranol in the t r e a t m e n t of chronic plaque psoriasis, Lancet, March 3 (1979) 4 5 5 - 4 5 8 . 3 H. H. Roenigk, P h o t o c h e m o t h e r a p y for mycosis fungoides: long-term followup study, C a n c e r Treat Rep., 63 (1979) 6 6 9 - 6 7 3 . 4 B. Ortel, A. Tanew and H. H6nigsmann, Vitiligo treatment, Curr. Probl. Derm~tol., 15 (1986) 2 6 5 - 2 7 1 . 5 F. Gschnait, H. H6nigsmann, W. Brenner, P. Fritsch and K. Wolff, Induction of UV light tolerance by PUVA in patients with polymorphous light eruption, Br. J. Derrnatol., 99 (1978) 2 9 3 - 2 9 5 . 6 R. Edelson et al., Treatment of cutaneous T-cell l y m p h o m a by extracorporeal photochemotherapy, N. Engl. J. Med., 316 (1987) 2 9 7 - 3 0 3 . 7 F. Dall'Acqua, M. Terbojevich, S. Marciani, D. Vedaldi and M. Recher, Investigation on the dark interaction between furocoumarins and DNA, Chem. Biol. Interact., 21 (1978) 1 0 3 - 1 1 5 . 8 L. Musajo and G. Rodighiero, Mode of photosensitizing action of furocoumarins, in A. C. Giese (ed.), Photophysiology, Vol. VIII, Academic Press, New York, 1972, pp. 1 1 5 - 1 4 7 . 9 T. Sa E Melo, P. Morliere, R. Santus and L. Dubertret, Photoreactivity of 5-methoxypsoralen with calf thymus DNA upon excitation in the UVA, Photobiochem. Photobiophys., 7 (1984) 121-131. 10 S. A. Baydoun, N. K. Gibbs and A. R. Young, Action spectra and c h r o m o p h o r e s for lethal photosensitization of C a n d i d a a l b i c a n s by DNA m o n o a d d u c t s formed by 8-methoxypsoralen and monofunctional furocoumarins, Photochem. Photobiol., 50 (1989) 7 5 3 - 7 6 1 . 11 F. Bordin, F. Carlassare, F. Baccichetti, A. Guiotto, P. Rodighiero, D. Vedaldi and F. Dall'Acqua, 4,5-Dimethylangelicin: a new DNA-photobinding monofunctional agent, Photochem. Photobiol., 29 (1979) 1 0 6 3 - 1 0 7 0 .
245 12 F. P. Gasparro, C. L. Berger and R. L. Edelson, Effect of m o n o c h r o m a t i c UVA light and 8-methoxypsoralen on h u m a n lymphocyte r e s p o n s e to mitogen, Photoclermatology, 1 (1984) 10-17. 13 D. Averbeck, Photochemistry and photobiology of psoralens, Proc. Jim. Soc. Invest. Derrnatol., 8 (1984) 5 2 - 7 3 . 14 G. Abel, Chromosome damage induced in h u m a n lymphocytes by 5-methoxypsoralen and 8-methoxypsoralen plus UV-A, Murat. Res., 100 (1987) 6 3 - 6 8 . 15 M. B a n k m a n n and M. Brendel, Molecular dosimetry of 8-MOP+UVA-induced DNA photoadducts in S a c c h a r o m y c e s cerevisiae: correlation of lesion n u m b e r with genotoxic potential, J. Photochem. Photobiol. B, 4 (1989) 5 7 - 7 4 . 16 M. A. O'Neal and A. C. Griffin, The effect of oxypsoralen upon ultraviolet carcinogenesis in albino mice, C a n c e r Res., 17 (1957) 9 1 1 - 9 1 6 . 17 P.D. Forbes, R. E. Davies and F. Urbach, Phototoxicity and photocarcinogenesis: comparative effects of a n t h r a c e n e and 8-methoxypsoralen in the skin of mice, Food Cos'met. Toxicol., 14 (1976) 3 0 3 - 3 0 6 . 18 D. D. Grube, R. D. Ley and R. J. M. Fry, Photosensitizing effect of 8-methoxypsoralen on the skin of hairless m i c e - II: strain and spectral differences for tumorigenesis, Photochem. Photobiol., 25 (1977) 2 6 9 - 2 7 6 . 19 F. Zajdela and E. Bisagni, 5-Methoxypsoralen, the melanogenic additive in sun-tan preparations, is tumorigenic in mice exposed to 365 n m u.v. radiation, Carcinogenesis, 2 (1981) 1 2 1 - 1 2 7 . 20 A. R. Young, I. A. Magnus, A. C. Davies and N. P. Smith, A comparison of the phototumorigenic potential of 8-MOP and 5-MOP in hairless albino mice exposed to solar simulated radiation, Br. J. Dermatol., 108 (1983) 5 0 7 - 5 1 8 . 21 A. R. Young, N. K. Gibbs and I. A. Magnus, Modification of 5-methoxypsoralen phototumorigenesis by UVB sunscreens: a statistical and histologic study in the hairless albino mouse, J. Invest. Dermatol., 8P (1987) 6 1 1 - 6 1 7 . 22 A. R. Young, S. L. Walker, J. S. Kinley, S. R. Plastow, D. Averbeck, P. Morliere and L. Dubertret, Phototumorigenesis studies of 5-methoxypsoralen in b e r g a m o t oil: evaluation and modification of risk of h u m a n use in an albino mouse skin model, J. Photochem. Photobiol. B, in the press. 23 N. K. Gibbs, A. R. Young and I. A. Magnus, Failure of UVR dose reciprocity for skin tumorigenesis in hairless mice treated with 8-methoxypsoralen, Photochem. Photobiol., 42 (1985) 39--42. 24 L. Dubertret, D. Averbeck, R. Zajdela, E. Bisagni, E. Moustacchi, R. Touraine and R. Latarjet, P h o t o c h e m o t h e r a p y (PUVA) of psoriasis using 3-carbethoxypsoralen, a non-carcinogenic c o m p o u n d in mice, Br. J. Dermatol., 101 (1978) 379--389. 25 M. P. Mullen, M. A. Pathak, J. D. West, T. J. Harrist and F. DaU'Acqua, Carcinogenic effects of monofunctional and bifunctional furocoumarins, Natl. C a n c e r Inst., Monogr., 65 (1984) 2 0 5 - 2 1 0 . 26 IARC, m o n o g r a p h s on the evaluation of carcinogenic risk of chemicals to humans. Some naturally occurring and synthetic food components, furocoumarins and ultraviolet radiation. Angelicin and some synthetic derivatives, I A R C Sci. Pub£, 40 (1986) 2 9 1 - 3 1 5 . 27 L. Dubertret, D. Averbeck, E. Bisagni, J. Moron, E. Moustacchi, C. Bifiardon, D. Papadopoulo, S. Nocentini, P. Vigny, J. Blais, R. V. Bensasson, J. C. Ronfard-Haret, E. J. Land, F. Zajdela and R. Latarjet, P h o t o c h e m o t h e r a p y using pyridopsoralens, B i o c h i m i e , 57 (1985) 4 1 7 - 4 2 2 . 28 R. J. M. Fry, R. D. Ley a n d D. D. Grube, Photosensitized reactions and carcinogenesis, Natl. C a n c e r Inst., Monogr., 50 (1978) 3 9 - 4 3 . 29 A. R. Young, S. L. Walker a n d M. Garmyn, A first a p p r o a c h to an action s p e c t r u m for 8MOP phototumorigenesis in mice, J. Invest. Dermatol., 90 (1988) 1 7 5 - 1 7 8 . 30 D. J. Cripps, N. J. Lowe and A. B. Lerner, Action spectra of topical psoralens: a reevaluation, Br. J. Dermatol., 107 (1982) 7 7 - 8 2 . 31 A. R. Young and I. A. Magnus, An action s p e c t r u m for 8-MOP induced s u n b u r n ceils in mammalian epidermis, Br. J. Dermatol., 104 (1981) 5 4 1 - 5 4 8 .
246 32 K. H. Kaidbey, An action s p e c t r u m for 8-methoxypsoralen-sensitized inhibition of DNA synthesis i n vivo, J. Invest. Dermatol., 85 (1985) 9 8 - 1 0 1 . 33 R. S. Stern, L. A. Thibodeau, R. A. Kleinerman, J. D. Parrish and T. B. Fitzpatrick, Risk of cutaneous carcinoma in patients treated with oral methoxsalen p h o t o c h e m o t h e r a p y for psoriasis, N. Engl. J. Med., 300 (1979) 8 0 9 - 8 1 3 . 34 R. S. Stem, N. Laird, J. Melski and J. A. Parrish, Cutaneous squamous-cell carcinoma in patients treated with PUVA, N. Eng. J. Med., 310 (1984) 1156--1161. 35 R. S. Stern, R. Lange and m e m b e r s of the P h o t o c h e m o t h e r a p y Follow-up Study, Nonm e l a n o m a skin cancer occurring in patients treated with PUVA five to ten years after first treatment, J. Invest. Dermatol., 91 (1988) 1 2 0 - 1 2 4 . 36 A. B. Forman, H. H. Roenigk, W. A. Caro and M. L. Magid, Long-term follow-up of skin cancer in the PUVA -48 cooperative study, Arch. DerrnatoL, 125 (1989) 5 1 5 - 5 1 9 . 37 R. Lindskov, Skin carcinomas and treatment with p h o t o c h e m o t h e r a p y (PUVA), A c t a Dermatol. Venereol Stockholm, 61 (1981) 1 4 1 - 1 4 5 . 38 A. Eskelinen, K. Halme, A. Lassus and J. Idanpaan-Heikkila, Risk of cutaneous carcinoma in psoriatic patients treated with PUVA, P h o t o d e r m a t o l o g y , 2 (1985) 1 0 - 1 4 . 39 A. Tanew et al., N o n m e l a n o m a skin tumors in long-term p h o t o c h e m o t h e r a p y treatment of psoriasis, J. A m . Acad. Derrnatol., 15 (1986) 9 6 0 - 9 6 5 . 40 T. Hensler, E. Christophers, H. H6higsmann and K. Wolff, Skin tumors in the E u r o p e a n PUVA study, J. Am. A c a d . Der'rnatol., 16 (1987) 1 0 8 - 1 1 6 . 41 T. Henseler, Risk of n o n m e l a n o m a skin tumours in patients treated with long-termp h o t o c h e m o t h e r a p y (PUVA), in T. B. Fitzpatrick, P. Forlot, M. A. Pathak and F. Urbach (eds.), Psoralens: Past, P r e s e n t a n d F u t u r e o f P h o t o c h e m o p r o t e c t i o n a n d other Biological A c t i v i t i e s , J o h n Libbey Eurotext, Paris, 1989, pp. 3 7 7 - 3 8 6 . 42 T.-Y. Chuang, J. Tse and D. J. Cripps, A preliminary study on the link between PUVA and skin cancer, Int. J. D e r m a t o L , 28 (1989) 4 3 8 - 4 4 0 . 43 R. S. Stern, Risk a s s e s s m e n t of PUVA and cyclosporine, Arch. Dermatol., 125 (1989) 545-547. 44 R. W. Morris, Skin tumors in the E u r o p e a n PUVA study: eight-year follow-up of 1643 patients treated with PUVA for psoriasis, J. A m . Acad. Dermatol., 20 (1989) 297. 45 N. K. Gibbs, H. H6nigsmann and A. R. Young, PUVA t r e a t m e n t strategies and cancer risk, Lancet, January 18 (1986) 1 5 0 - 1 5 1 . 46 F. M. Carabott and J. L. M. Hawk, A modified dosage for increased efficiency in PUVA treatment of psoriasis, Clin. Exp. Dermatol., 14 (1989) 3 3 7 - 3 4 0 . 47 P. Fritsch and H. HSnigsmann, Combination phototherapy -- a critical appraisal, Curt. Probl. Dermatol., 15 (1986) 2 3 8 - 2 5 3 . 48 H. H6nigsmann, E. Jaschke, V. Nitsche, W. Brenner, W. Rausch Meier a n d K. Wolff, Serum levels of 8-methoxypsoralen in two different drug preparations: correlation with photosensitivity and UV-A dose requirements for photochemotherapy, J. Invest. Dermatol., 79 (1982) 2 3 3 - 2 3 6 . 49 L. Dubertret, D. Serraf-Tircazes, M. Jeaumougin, P. Moll~re, D. Averbeck and A. Young, Phototoxic properties of perfumes containing b e r g a m o t oil on h u m a n skin: photoprotective effects of UVA and UVB sunscreens, J. Photochem. Photobiol., submitted for publication. 50 D. Averbeck, S. Averbeck, L. Dubertret, A. Young and P. Moli~re, Genotoxicity of b e r g a p t e n and bergamot oil in S a c c h a r o m y c e s c e r e v i s i a e , J. Photochem. Photobiol., submitted for publication. 51 H. HSnigsmann, E. Jaschke, F. Gschnait, W. Brenner, P. Fritsch and K. Wolff, 5-Methoxypsoralen (bergapten) in p h o t o c h e m o t h e r a p y of psoriasis, Br. J. Dermatol., 101 (1979) 369-378. 52 G. Mezzadra, B. Gaurneri, C. Grupper and P. Forlot, Effects of chronic field exposure of h u m a n s to bergapten, in J. Cahn, A. Meybeck, P. Forlot, F. Urbach and C. Grupper (eds.), P s o r a l e n s i n Cosmetics a n d D e r m a t o l o g y , Pergamon, Paris, 1981, pp. 3 8 3 - 3 8 6 . 53 IARC, m o n o g r a p h s on the evaluation of the carcinogenic risk of chemicals to humans. Some naturally occurring and synthetic food components, furocoumarins and ultraviolet radiation. 5-Methoxypsoralen, IARC Sci. PubL, 40 (1986) 3 2 7 - 3 4 7 .
247 54 S. Kimura, N. Mizuno, S. Hirano and K. Yoshikawa, Topical application of 3-carbethoxypsoralen plus UVA in the t r e a t m e n t of psoriasis, J. Dermatol., 12 (1985) 2 5 1 - 2 5 7 . 55 A. Takashima, A. Sunohara and N. Mizuno, Comparison of the relative therapeutic efficacy of 7-methylpyridopsoralen and 8-methoxypsoralen in p h o t o c h e m o t h e r a p y in psoriasis treatment, J. D e r m a t o L , 15 (1988) 1 9 5 - 2 0 1 . 56 M. Cristofolini, G. Recchi, S. Boi, F. Piscioli, F. Bordin, F. Baccichetti, F. Carlassare, M. Tamaro, B. Pani, N. Babudri, A. Guiotto, P. Rodighiero, D. Vedaldi and F. Dall'Acqua, 6Methylangelicins: new monofunctional p h o t o c h e m o t h e r a p e u t i c agent for psoriasis, Br. J. Derrnatol., 122 (1990) 5 1 3 - 5 2 4 . 57 P. Morli~re, H. H6nigsmann, D. Averbeck, M. Dardalhon, G. Hiippe, B. Ortel, R. Santus and L. Dubertret, Phototherapeutic, photobiologic and photosensitizing properties of khellin, J. Invest. Dermatol., 90 (1988) 7 2 0 - 7 2 4 . 58 J. Brucke, A. Tanew and H. HSnigsmann, Action s p e c t r u m of photochemotherapy. Comparison of psoriasis treatment with 335 n m and 365 nm, in A b s t r a c t Book, lOth I n t e r n a t i o n a l Congress on Photobiology, J e r u s a l e m , Israel, 30 October-5 N o v e m b e r , 1088, p. 24. 59 B. L. Diffey, A. V. J. ChaUoner a n d P. J. Key, A survey of the ultraviolet radiation emissions of p h o t o c h e m o t h e r a p y units, Br. J. Dermatol., 102 (1980) 3 0 1 - 3 0 6 . 60 S. E. Freeman, R. W. Gange, J. C. Sutherland, E. A. Matzinger and B. Sutherland, Production of pyrimidine dimers in h u m a n skin exposed i n s i t u to UVA irradiation, J. Invest. Dermatol., 88 (1987) 4 3 0 - 4 3 3 . 61 L. Roza, R. A. Baan, J. C. van der Leun and L. Kligman, UVA hazards in skin associated with the use of tanning equipment, J. Photochem. Photobiol. B, 3 (1989) 2 8 1 - 2 8 7 . 62 M. Auletta, R. W. Gange, O. T. Tan and E. Matzinger, Effect of cutaneous hypoxia upon erythema and pigment responses to UVA, UVB and PUVA (8-MOP + UVA) in h u m a n skin, J. Invest. Dermatol., 86 (1986) 6 4 9 - 6 5 2 . 63 R. M. Tyrrell, UVA hazards in skin associated with the use of tanning equipment - a comment, J. Photochem. Photobiol. B, 4 (1989) 2 2 7 - 2 3 2 . 64 A. R. Young, Aspects of psoralen phototumorigenesis with emphasis on the possible role of t u m o u r promotion, B i o c h i m i e , 68 (1986) 8 8 5 - 8 8 9 . 65 H. van Weeldon and J. C. van der Leun, UV-A induced tumors in pigmented and albino hairless mice, in W. F. Passchier and B. F. M. Bosnjakovic (eds.), H u m a n E x p o s u r e to UVA R a d i a t i o n : R i s k s a n d R e g u l a t i o n s , Elsevier Science Publishers, Amsterdam, 1987, pp. 4 5 - 5 2 . 66 H. J. C. M. Sterenberg and J. C. van der Leun, Tumorigenesis by a longwave UVA source, Photochem. Photobiol., 51 (1990) 3 2 5 - 3 3 0 . 67 P. D. Forbes, H. F. Blum a n d R. E. Davies, Photocarcinogenesis in hairless mice: doseresponse and the influence of dose-delivery, Photochem. Photobiol., 34 (1981) 3 6 1 - 3 6 5 . 68 F. Daniels, A simple microbiological m e t h o d for demonstrating phototoxic compounds, J. Invest. Dermatol., 44 (1965) 2 5 9 - 2 6 3 . 69 N. K. Gibbs, An adaptation of the C a n d i d a a l b i c a n s phototoxicity test to demonstrate photosensitizer action spectra, P h o t o d e r m a t o l o g y , 4 (1987) 3 1 2 - 3 1 6 . 70 NIH, Toxicology and carcinogenesis studies of 8-methoxypsoralen in F344/N rats (gavage studies), National Toxicology Program, Research Triangle Park, NC 27709, 1989, N I H P u b l i c a t i o n No. 80-2814, U.S. Department of Health and H u m a n Services, Public Health Service, National Institutes of Health.
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PHOTOCARCINOGENICITY OF PSORALENS USED IN PUVA TREATMENT: PRESENT STATUS IN MOUSE AND MAN* ANTONY R. YOUNG
Photobiology Unit, Institute of Dermatology, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, University of London, London (U.K.) (Received November 24, 1989; accepted D e c e m b e r 16, 1989)
Keywords. Psoralens, PUVA treatment, 8-methoxypsoralen, photocarcinogenesis, skin cancer, photosensitization.
Summary There is good evidence that 8-methoxypsoralen is photocarcinogenic in psoriatic patients undergoing long-term photochemotherapy (PUVA) in the U.S.A. However, this conclusion has not been supported by two m~jor European studies which have indicated that PUVA is a tumour promoter of damage initiated by other agents. Variation in PUVA treatment protocols in the U.S.A. and Europe may partly account for the different conclusions. There is much interest in the therapeutic potential of monofunctional psoralens. It is hoped that these may reduce long-term risk. Monofunctional and cross-linking psoralens have been shown to be photocarcinogenic in mouse skin. The relative risk of different compounds may be assessed in the mouse, but it is important to base comparisons on dose protocols that have been shown to be therapeutically effective.
1. Introduction The psoralens (furocoumarins) are photosensitizing drugs that have been used extensively with long-wave UV radiation (UVA; 3 1 5 - 4 0 0 nm) in photochemotherapy (PUVA) during the past 15 years. The most common use has been 8-methoxypsoralen (8-MOP) for the treatment of psoriasis [1, 2]. However, PUVA with 8-MOP has also been effective in the treatment of skin disorders as diverse as mycosis fungoides [3], vitiligo [4] and polymorphous light eruption [5]. More recently PUVA has been used in the extracorporeal *Paper presented at the Photomedicine Meeting organized by the F r e n c h Society of Photobiology, Paris, November, 1989.
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238 irradiation of lymphocyte-enriched blood for the treatment of cutaneous Tcell lymphoma [6]. The molecular basis of the therapeutic effects of PUVA is not known. The diversity of diseases that r e s pond to this treatment suggests that mechanisms vary. The most extensively studied aspect of psoralen photochemistry and photobiology is DNA photobinding [7-9]. A prerequisite of such binding is the formation of an unstable non-covalent "dark c o m p l e x " within the DNA molecule [7]. Dark complexation modifies the absorption properties of 8-MOP and other psoralens. Thus free 8-MOP shows an absorption maximum at about 300 nm (in the UVB range), whereas 8-MOP, "dark c o m p l e x e d " within DNA, shows an absorption maximum which is " r ed shifted" to about 315 nm [10]. On absorption of a photon, 8-MOP may undergo C4 cyclobutane addition across the 5,6 double bond of a pyrimidine base in DNA to form an 8-MOP monoadduct. This reaction may take place across either the 3,4 double bond of the pyrone ring or the 4',5' double bond of the furan ring of the psoralen molecule. Thus there are two types of lesion: the 3,4-monoadduct and the 4',5'-monoadduct. These adducts have quite different UVR absorption characteristics. The former shows virtually no absorption in the UVA range, but the latter has an absorption maximum at about 335 nm [8]. In the presence of UVA the 4 ' , 5 ' - m o n o a d d u c t may absorb a phot on and undergo a further C4 cyclobutane addition reaction across its 3,4 bond with the 5,6 double bond of a suitably placed pyrimidine base in the opposite DNA strand. This lesion is a DNA cross-link. 8-MOP, 5-methoxypsoralen (5-MOP), psoralen and trimethylpsoralen (TMP) are cross-linking, or bifunctional, compounds. Monofunctional psoralens do not progress be yond m o n o a d d u c t formation. Their molecular structure may prevent the necessary stereochemistry for reactions at both furan and pyrone double bond sites, such as with the angelicins and their synthetic derivatives. Alternatively, reactive sites may be inhibited by substituted terminal groups, such as with 3-carbethoxypsoralen (3-CP). The psoriatic epidermis is in a hyperproliferative state. Inhibition of DNA synthesis, whether by monoadducts or cross-links [11, 12] may well be the mechanistic basis of the therapeutic effect.
2. Photocarcinogenicity It has been clearly demonstrated, in bacterial, yeast and mammalian systems, that cross-linking and monofunctional psoralens are highly mutagenic in the presence of UVA radiation [ 1 3 - 1 5 ]. Mutagens must always be considered as potential carcinogens. 2.1. M o u s e s t u d i e s The photocarcinogenicity of both cross-linking and monofunctional psoralens has been studied. The latter have been studied because they may be less photomutagenic than the former [13].
239
2.1.1. Cross-linking psoralens The photocarcinogenic potential of 8-MOP was first indicated in the mouse in 1957 [16]. This has been confirmed by several workers with UVA and solar simulating radiation (SSR) sources [ 1 7 - 2 0 ] . Studies on other crosslinking c o m p o u n d s have shown that 5-MOP [19, 20] and psoralen [19] are also photocarcinogenic. The study of Zajdela and Bisagni [19] showed a rank order of time-to-onset to first ear t u m o u r of psoralen > 8-MOP > 5-MOP (all topically applied at 0.025% in acetone in the presence of 365 nm radiation). The study of Young et al. [20] showed no difference between the c o m p o u n d s in the time-of-onset to first skin t u m o u r (topically applied at 0.01% and 0.03% to dorsal and flank skin in an oily vehicle and exposed to SSR). Trend analysis for time-to-onset to first t u m o u r has shown a d e p e n d e n c e on drug dose for 8-MOP and 5-MOP [ 2 0 - 2 2 ] . A d e p e n d e n c e of time-to-onset to first t u m o u r on SSR dose has been demonstrated with 8MOP [23].
2.1.2. Monofunctional psoralens Several monofunctional psoralens have been assessed. The first study was with 3-CP in XVIInc/Z mice [24]. Using 8-MOP as a positive control, there was no evidence of photocarcinogenicity with 3-CP after topical application to ear skin and e xpos ur e to 365 nm radiation. The lack of photocarcinogenicity with 3-CP was confirmed w hen topically applied to the dorsal skin of the hairless albino Skh-1 mouse [25]. A study of angelicin and its synthetic derivatives, 5-methylangelicin and 4,5'-dimethylangelicin, showed photocarcinogenicity of these c o m p o u n d s when applied topically to the skin of Skh 1 hairless albino mice in the presence of UVA radiation [25]. The synthetic derivatives were m or e carcinogenic than an 8-MOP positive control, but angelicin was less p o t e n t than 8-MOP. An International Agency for Research on Cancer (IARC) Working Group not ed that the com pounds tested were not pure and concluded that the evidence for the photocarcinogenicity of these agents was limited [26]. Photocarcinogenicity has also b e en demonstrated with pyridopsoralen (PP) and 7-methylpyridopsoralen (MPP) applied topically to the ear of the 17NCZ mouse prior to exposure to 365 n m radiation [27]. These c o m p o u n d s were less carcinogenic than 8MOP. It is not always clear from the studies described above ff comparisons between cross-linking and monofunctional c o m p o u n d s have b e e n made concurrently or historically. The latter are m uc h less reliable. It is essential that comparisons between c o m p o u n d s are made under similar conditions in the same mouse strain as it is well established that there is interstrain variation in photocarcinogenesis. The photocarcinogenic potential of cross-linking psoralens cannot specffically identify cross-links as the putative lesions as such agents also form monoadducts. It is evident that m o n o a d d u c t s have carcinogenic potential. A given n u m b e r of induced 8-MOP cross-links in epidermal DNA may have variable carcinogenic responses in different strains of mice [28] and may
240 not be relevant to risk a s s e s s m e n t if such lesions result in a g r e a t e r probability of e p i d e m a l cell lethality in situ.
2.1.3. W a v e l e n g t h dependence o f psoralen photocarcinogenesis The d e t e r m i n a t i o n of an action s p e c t r u m for l o n g - t e r m effects in the skin p r e s e n t s considerable practical difficulties. A first a p p r o a c h to this p r o b l e m has b e e n m a d e b y Young et al. [29] in the hairless albino mouse. This s t u d y has indicated that the action s p e c t r u m for skin c a n c e r with 8MOP is likely to be similar to that o b t a i n e d for acute effects on skin, s u c h as h u m a n e r y t h e m a [30], m o u s e s u n b u r n cell f o r m a t i o n [31] and inhibition of m o u s e e p i d e r m a l DNA synthesis [32], with p e a k activity in the 3 2 0 - 3 3 5 n m region. 2.2. H u m a n studies Knowledge of the DNA-damaging, m u t a g e n i c and c a r c i n o g e n i c p r o p e r t i e s of PUVA with 8-MOP has initiated careful and intensive follow-up studies in large n u m b e r s of psoriatic patients. Stern et al. [33] r e p o r t e d an increase in s q u a m o u s cell c a r c i n o m a in a 2.1 y e a r p r o s p e c t i v e multicentre s t u d y in the U.S.A. o f 1371 psoriatic patients u n d e r g o i n g PUVA t r e a t m e n t . An e x t e n s i o n of this study, r e p o r t e d on 1380 patients after a total of 5.7 years of follow-up, r e i n f o r c e d the original c o n c l u s i o n s with m o r e p o w e r f u l data [34]. T h e s e d e m o n s t r a t e d a clear relationship b e t w e e n cumulative UVA dose and incidence of s q u a m o u s cell c a r c i n o m a . F u r t h e r m o n i t o r i n g of 1380 patients, with a m e a n follow-up time of 10 years, c o n t i n u e s to s h o w a relationship b e t w e e n s q u a m o u s cell c a r c i n o m a and cumulative UVA e x p o s u r e as well as a m o d e s t dose relationship for basal cell c a r c i n o m a [35]. The o b s e r v a t i o n s of Stern and c o w o r k e r s [34, 35] have b e e n c o n f i r m e d b y F o r m a n et al. [36] in a r e t r o s p e c t i v e multicentre study in the U.S.A. No relationship b e t w e e n basal cell c a r c i n o m a and cumulative UVA dose was o b s e r v e d but t h e r e was a marked, but not significant, t r e n d for i n c r e a s e d s t a n d a r d m o r b i d i t y ratio o f s q u a m o u s cell c a r c i n o m a with cumulative UVA dose. However, the c o n c l u s i o n that PUVA is c a r c i n o g e n i c has not b e e n m a d e by o t h e r investigators in A m e r i c a and E u r o p e [ 3 7 - 4 2 ] . S o m e o f t h e s e studies have b e e n of s h o r t duration a n d / o r with relatively small n u m b e r s of subjects, and t h e r e f o r e are of limited value in the a s s e s s m e n t of l o n g - t e r m risk. In general, E u r o p e a n w o r k e r s have c o n c l u d e d that PUVA is not a c o m p l e t e skin carcinogen. S o m e investigators have c o n c l u d e d that PUVA is a " p r o m o t e r " of s q u a m o u s cell c a r c i n o m a initiated by p r i o r t r e a t m e n t with a g e n t s s u c h as ionizing radiation or arsenic [ 3 9 - 4 1 ] . To date t h e r e has b e e n no indication f r o m any of the studies that PUVA e n h a n c e s the risk of malignant m e l a n o m a . 3. P o s s i b l e r e a s o n s f o r t h e d i s c r e p a n c i e s b e t w e e n t h e A m e r i c a n and European follow-up studies The statistical a p p r o a c h to data analyses is i m p o r t a n t [43, 441. Stern [43] has e m p h a s i z e d the g r e a t e r p o w e r of p r o s p e c t i v e studies [ 3 3 - 3 5 , 3 9 - 4 1 ]
241 c o m p a r e d with a r e t r o s p e c t i v e a p p r o a c h [36, 42]. The A m e r i c a n a p p r o a c h to d a t a m a n a g e m e n t h a s b e e n to c o m p a r e t h e o b s e r v e d i n c i d e n c e of skin c a n c e r with the e x p e c t e d i n c i d e n c e d e t e r m i n e d f r o m a n age- a n d s e x - m a t c h e d n o r m a l p o p u l a t i o n . This w a s n o t originally c a r r i e d o u t in the m o s t e x t e n s i v e E u r o p e a n studies [39, 40], b u t o n e g r o u p f o u n d n o difference in n o n m e l a n o m a skin c a n c e r i n c i d e n c e b e t w e e n PUVA a n d non-PUVA p a t i e n t s t r e a t e d in a d e r m a t o l o g y d e p a r t m e n t [41]. 8 - M O P - e n h a n c e d p h o t o c a r c i n o g e n i c i t y in the m o u s e s h o w s a failure o f t i m e - d o s e r e c i p r o c i t y [23]. T h u s a g i v e n c u m u l a t i v e UVR d o s e is m o r e c a r c i n o g e n i c if g i v e n in s m a l l e r daily f r a c t i o n s o v e r a l o n g e r p e r i o d of time. This o b s e r v a t i o n is the b a s i s of a p r o p o s a l b y G i b b s et al. [45] t h a t the different c o n c l u s i o n s of A m e r i c a n a n d E u r o p e a n g r o u p s on PUVA carcinogenicity is r e l a t e d to different t r e a t m e n t p r o t o c o l s . The usual p r a c t i c e in the U.S.A. is to a d m i n i s t e r n o n - p h o t o t o x i c PUVA doses, w h e r e a s the E u r o p e a n a p p r o a c h is to m a x i m i z e a n d m a i n t a i n d o s e s at a n e a r p h o t o t o x i c level. The latter s y s t e m t e n d s to give m o r e r a p i d clearing with l o w e r c u m u l a t i v e UVA d o s e s [46]. E x t r a p o l a t i o n f r o m the m o u s e d a t a [23] s u g g e s t s t h a t it m a y also b e less c a r c i n o g e n i c . T h e m o u s e studies also indicate t h a t it m a y b e m i s l e a d i n g to c o m p a r e c a n c e r risk in t e r m s of c u m u l a t i v e UVA d o s e w h e n different d o s e p r o t o c o l s h a v e b e e n used.
4. S t r a t e g i e s f o r m i n i m i z i n g t h e s k i n c a n c e r risk w i t h 8-MOP 4.1. C o m b i n a t i o n t h e r a p y The a d m i n i s t r a t i o n o f e t r e t i n a t e with PUVA r e s u l t s in r e d u c e d clearing t i m e and n u m b e r o f t r e a t m e n t s with a 5 0 % r e d u c t i o n of c u m u l a t i v e UVA d o s e [47]. The u s e of e t r e t i n a t e in o n e E u r o p e a n s t u d y m a y h a v e b e e n a partial f a c t o r in the lack o f p h o t o c a r c i n o g e n i c i t y o f PUVA [39]. 4.2. S u n s c r e e n u s e PUVA p a t i e n t s are g e n e r a l l y a d v i s e d to w e a r e y e p r o t e c t i o n a f t e r d r u g i n g e s t i o n as p s o r a l e n s m a y p e r s i s t in p l a s m a for s e v e r a l h o u r s p o s t - i n g e s t i o n [48]. Animal studies h a v e s h o w n t h a t UVB s u n s c r e e n s ( c i n n a m a t e s w h i c h a b s o r b s h o r t e r - w a v e UVA) a n d UVB plus UVA s u n s c r e e n s significantly p r o t e c t skin f r o m 5-MOP p h o t o c a r c i n o g e n i c i t y in t h e m o u s e [21, 22]. U n d e r s o m e c o n d i t i o n s this p r o t e c t i o n is t o t a l [22]. S u n s c r e e n s also p r o t e c t a g a i n s t 5M O P - i n d u c e d p h o t o t o x i c i t y o f h u m a n skin [49] a n d m u t a g e n i c i t y in the y e a s t S a c c h a r o m y c e s c e r e v i s i a e [50]. S u n s c r e e n u s e m a y b e a d v i s e d to p r o t e c t sites n o t i n v o l v e d with psoriasis, in p a r t i c u l a r s c r o t a l skin [36]. T h e u s e o f s u c h s u n s c r e e n s a f t e r UVA e x p o s u r e w o u l d r e d u c e u n w a n t e d p s o r a l e n p h o t o a c t i v a t i o n b y a m b i e n t s o l a r r a d i a t i o n a n d p o s s i b l y skin c a n c e r risk. T h e r e m a y b e a n e e d for s p e c i a l l y f o r m u l a t e d s u n s c r e e n s for u s e with PUVA.
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4.3. A l t e r n a t i v e s to 8-MOP 4.3.1. Other cross-linking psoralens H6nigsmann et al. [51] r e por t ed the successful use of 5-MOP for the treatment of psoriasis. The main advantage of this drug is reduced nausea. 5-MOP, like 8-MOP, is a DNA cross-linking agent [9]. There are no extensive studies of 5-MOP photocarcinogenicity in human skin. One study indicated no increased risk of skin cancer in workers in the bergam ot fruit industry in southern Italy [52]; however an IARC Working Group noted limitations in this study [53]. 5-MOP is a skin phot ocarci nogen in the mouse [19-22]. Extrapolation of animal data of 5-MOP photocarcinogenicity to humans does not suggest that 5-MOP would pr e s ent a lower risk than 8-MOP. 4.3.2. Monofunctional psoralens Monoadducts are, in general, less mutagenic than cross-links [13]. It has been hypothesized that they may be less carcinogenic than cross-linking agents, but this has not been established from the mouse data described. In addition, this hypothesis pr e s uppos e s that carcinogenicity is related solely to DNA damage. There have been a few reports in the literature of clinical studies assessing the efficacy of clearing of psoriasis in small groups of patients. Dubertret et al. [24] r e por t ed the treatment of psoriasis with 3-CP as a promising non-photocarcinogenic alternative to 8-MOP. However, a Japanese group was not able to confirm the therapeutic efficacy of this c o m p o u n d [54]. More recent studies indicate that the pyridopsoralens may be effective in clearing psoriasis [27, 55]. However, the mean UVA dose for clearing with MPP is four times higher than with equimolar 8-MOP. The pyridopsoralens are three to four times less photocarcinogenic than 8-MOP [27], but this advantage may be lost if much higher doses are required for effective therapy. Other monofunctional agents have also been investigated. The 6-methylangelicins have been r epor t e d to have better therapeutic effect in psoriasis than 8-MOP but with equivalent photomutagenicity [56]. There has been recent interest in khellin, a monofunctional agent with a structure similar to psoralen. Good therapeutic activity for vitiligo has been report ed in association with genotoxicity [57]. As yet there are no photocarcinogenicity data on these agents. 4.4. M a x i m i z i n g spectral e giciency The action s pect r um for the clearing of psoriasis with 8-MOP, or any other psoralen, is not known. One study comparing 335 nm radiation with 365 nm radiation showed the former to be more effective [58]. In the absence of more specific data it seems reasonable to suppose that this action spect rum resembles that for acute effects of 8-MOP in the skin, including inhibition of epidermal DNA synthesis, with a maximum in the region of 3 2 0 - 3 3 5 nm [32].
243 Most UVA tubes used in PUVA have maxi m um emission at about 365 nm with very little output in the 3 2 0 - 3 3 5 nm region [59]. This may m ean that mu ch of the UVA radiation received by the skin is " r e d u n d a n t " with respect to 8-MOP photochemistry. UVA radiation induces DNA lesions in human skin i n s i t u [60] and is mutagenic and carcinogenic in mice [61]. Its acute effects on skin are de pe nde nt on oxygen [62] and there is evidence that it generates active oxygen species [63]. Such species may be important in t u m o u r p r o mot i on by PUVA [64]. Thus it is possible that the cumulative PUVA dose d ep ende nc e of squamous cell carcinoma observed in the American studies [ 3 4 - 3 6 ] may be partly due to UVA alone. In one experiment, 50°/0 of mice (Skh 1) e x p o s e d to 22 J cm -2 day -1 had skin tumours on day 265 after a cumulative UVA (peak emission at about 350 urn) dose of 5830 J cm -2 [65]. In another, using the same daily dose but with a different UVA source, 50% of mice had tumours on day 233 after a cumulative UVA (peak emission at about 3 5 0 - 3 8 0 nm) dose of 5126 J cm -2 [66]. Doses of this order would have be e n achieved in some of the high-dose American patients, but rarely in any of the E u r o p e a n patients. There are no animal investigations of UVA t i m e - d o s e reciprocity for skin cancer. However, if UVA is similar to UVB in this r es pe c t [23, 67], the American a p p r o a c h to PUVA could exacerbate the carcinogenic effects of UVA alone. Assuming that " r e d u n d a n t " UVA has little or no therapeutic benefit, it should be r e m o v e d from PUVA by using a radiation source that has an emission spectrum similar to that of the action spect rum for psoriasis clearing with 8-MOP. This may be particularly important when using other psoralens, e.g. MPP, that require higher cumulative UVA exposures for clearing. The determination of detailed action s pect ra for the clearing of psoriasis with a wide range of psoralens is a near impossible task. However, it may be a p p r o a c h e d by an appropriate model. Assuming that the primary mechanism of action of psoralens in psoriasis is the inhibition of DNA synthesis, it may be possible to use a simple i n vi t r o model such as the inhibition of growth in the yeast C. a l b i c a n s [68, 69]. The 8-MOP action spect rum for this end point, with peak activity at 3 2 0 - 3 3 5 um [10], is similar to that already described for skin [ 3 0 - 3 2 ] . Studies on PP and MPP showed peak activity at 340 um and 3 1 0 - 3 3 0 nm respectively [10].
5. C a r c i n o g e n i c i t y o f 8-MOP in t h e a b s e n c e o f UVA In general, mouse studies have b e e n designed to investigate the photocarcinogenicity of psoralens. The length of time that control groups, i.e. animals given psoralen but no UVR, have spent in the dark has b e e n determined by the time-to-onset of tumours in the UVR-treated groups. In m any cases this has b e e n a relatively short period in comparison with conventional 2 year chemical carcinogenesis studies. It has been widely assumed by the photobiology and dermatology community that 8-MOP in the absence of LWA does not present a carcinogenic risk. However, a recent 2 year study
244
has shown 8-MOP to cause cancer of the kidney, Zymbal gland, subcutaneous tissue and lung in male, but not female, F344/N rats [70]. It must be stated that the doses of 8-MOP used were 3 7 - 7 5 times the daily human dose. The significance to humans is not known, but these data suggest that follow-up studies for non-skin neoplasia in PUVA patients may be necessary.
6. C o n c l u s i o n s
Animal studies provide clear evidence of the photocarcinogenic potential of cross-linking and monofunctional psoralens. The relationship between specific types of DNA lesion and carcinogenic potential is not known. Extensive follow-up studies of PUVA patients have provided conflicting results. Carcinogenicity of PUVA may depend on therapeutic protocol. Assessment of risk of new psoralens for PUVA must use dose protocols that have been shown to be clinically effective. There is a need for action spect rum data for psoriasis clearing or the establishment of a model for this purpose. UVA sources could be designed for maximum spectral efficiency with the elimination of 'redundant" UVA which may play a role in skin cancer.
References 1 J. A. Parrish, T. B. Fitzpatrick, L. T a n e n b a u m and M. A. Pathak, P h o t o c h e m o t h e r a p y of psoriasis with oral methoxsalen and longwave ultraviolet light, N. Engl. J. Med., 291 (1974) 1207-1211. 2 S. Rogers, J. Marks, S. Shuster, D. Vella Briffa, A. Warin and M. Greaves, Comparison of p h o t o c h e m o t h e r a p y and dithranol in the t r e a t m e n t of chronic plaque psoriasis, Lancet, March 3 (1979) 4 5 5 - 4 5 8 . 3 H. H. Roenigk, P h o t o c h e m o t h e r a p y for mycosis fungoides: long-term followup study, C a n c e r Treat Rep., 63 (1979) 6 6 9 - 6 7 3 . 4 B. Ortel, A. Tanew and H. H6nigsmann, Vitiligo treatment, Curr. Probl. Derm~tol., 15 (1986) 2 6 5 - 2 7 1 . 5 F. Gschnait, H. H6nigsmann, W. Brenner, P. Fritsch and K. Wolff, Induction of UV light tolerance by PUVA in patients with polymorphous light eruption, Br. J. Derrnatol., 99 (1978) 2 9 3 - 2 9 5 . 6 R. Edelson et al., Treatment of cutaneous T-cell l y m p h o m a by extracorporeal photochemotherapy, N. Engl. J. Med., 316 (1987) 2 9 7 - 3 0 3 . 7 F. Dall'Acqua, M. Terbojevich, S. Marciani, D. Vedaldi and M. Recher, Investigation on the dark interaction between furocoumarins and DNA, Chem. Biol. Interact., 21 (1978) 1 0 3 - 1 1 5 . 8 L. Musajo and G. Rodighiero, Mode of photosensitizing action of furocoumarins, in A. C. Giese (ed.), Photophysiology, Vol. VIII, Academic Press, New York, 1972, pp. 1 1 5 - 1 4 7 . 9 T. Sa E Melo, P. Morliere, R. Santus and L. Dubertret, Photoreactivity of 5-methoxypsoralen with calf thymus DNA upon excitation in the UVA, Photobiochem. Photobiophys., 7 (1984) 121-131. 10 S. A. Baydoun, N. K. Gibbs and A. R. Young, Action spectra and c h r o m o p h o r e s for lethal photosensitization of C a n d i d a a l b i c a n s by DNA m o n o a d d u c t s formed by 8-methoxypsoralen and monofunctional furocoumarins, Photochem. Photobiol., 50 (1989) 7 5 3 - 7 6 1 . 11 F. Bordin, F. Carlassare, F. Baccichetti, A. Guiotto, P. Rodighiero, D. Vedaldi and F. Dall'Acqua, 4,5-Dimethylangelicin: a new DNA-photobinding monofunctional agent, Photochem. Photobiol., 29 (1979) 1 0 6 3 - 1 0 7 0 .
245 12 F. P. Gasparro, C. L. Berger and R. L. Edelson, Effect of m o n o c h r o m a t i c UVA light and 8-methoxypsoralen on h u m a n lymphocyte r e s p o n s e to mitogen, Photoclermatology, 1 (1984) 10-17. 13 D. Averbeck, Photochemistry and photobiology of psoralens, Proc. Jim. Soc. Invest. Derrnatol., 8 (1984) 5 2 - 7 3 . 14 G. Abel, Chromosome damage induced in h u m a n lymphocytes by 5-methoxypsoralen and 8-methoxypsoralen plus UV-A, Murat. Res., 100 (1987) 6 3 - 6 8 . 15 M. B a n k m a n n and M. Brendel, Molecular dosimetry of 8-MOP+UVA-induced DNA photoadducts in S a c c h a r o m y c e s cerevisiae: correlation of lesion n u m b e r with genotoxic potential, J. Photochem. Photobiol. B, 4 (1989) 5 7 - 7 4 . 16 M. A. O'Neal and A. C. Griffin, The effect of oxypsoralen upon ultraviolet carcinogenesis in albino mice, C a n c e r Res., 17 (1957) 9 1 1 - 9 1 6 . 17 P.D. Forbes, R. E. Davies and F. Urbach, Phototoxicity and photocarcinogenesis: comparative effects of a n t h r a c e n e and 8-methoxypsoralen in the skin of mice, Food Cos'met. Toxicol., 14 (1976) 3 0 3 - 3 0 6 . 18 D. D. Grube, R. D. Ley and R. J. M. Fry, Photosensitizing effect of 8-methoxypsoralen on the skin of hairless m i c e - II: strain and spectral differences for tumorigenesis, Photochem. Photobiol., 25 (1977) 2 6 9 - 2 7 6 . 19 F. Zajdela and E. Bisagni, 5-Methoxypsoralen, the melanogenic additive in sun-tan preparations, is tumorigenic in mice exposed to 365 n m u.v. radiation, Carcinogenesis, 2 (1981) 1 2 1 - 1 2 7 . 20 A. R. Young, I. A. Magnus, A. C. Davies and N. P. Smith, A comparison of the phototumorigenic potential of 8-MOP and 5-MOP in hairless albino mice exposed to solar simulated radiation, Br. J. Dermatol., 108 (1983) 5 0 7 - 5 1 8 . 21 A. R. Young, N. K. Gibbs and I. A. Magnus, Modification of 5-methoxypsoralen phototumorigenesis by UVB sunscreens: a statistical and histologic study in the hairless albino mouse, J. Invest. Dermatol., 8P (1987) 6 1 1 - 6 1 7 . 22 A. R. Young, S. L. Walker, J. S. Kinley, S. R. Plastow, D. Averbeck, P. Morliere and L. Dubertret, Phototumorigenesis studies of 5-methoxypsoralen in b e r g a m o t oil: evaluation and modification of risk of h u m a n use in an albino mouse skin model, J. Photochem. Photobiol. B, in the press. 23 N. K. Gibbs, A. R. Young and I. A. Magnus, Failure of UVR dose reciprocity for skin tumorigenesis in hairless mice treated with 8-methoxypsoralen, Photochem. Photobiol., 42 (1985) 39--42. 24 L. Dubertret, D. Averbeck, R. Zajdela, E. Bisagni, E. Moustacchi, R. Touraine and R. Latarjet, P h o t o c h e m o t h e r a p y (PUVA) of psoriasis using 3-carbethoxypsoralen, a non-carcinogenic c o m p o u n d in mice, Br. J. Dermatol., 101 (1978) 379--389. 25 M. P. Mullen, M. A. Pathak, J. D. West, T. J. Harrist and F. DaU'Acqua, Carcinogenic effects of monofunctional and bifunctional furocoumarins, Natl. C a n c e r Inst., Monogr., 65 (1984) 2 0 5 - 2 1 0 . 26 IARC, m o n o g r a p h s on the evaluation of carcinogenic risk of chemicals to humans. Some naturally occurring and synthetic food components, furocoumarins and ultraviolet radiation. Angelicin and some synthetic derivatives, I A R C Sci. Pub£, 40 (1986) 2 9 1 - 3 1 5 . 27 L. Dubertret, D. Averbeck, E. Bisagni, J. Moron, E. Moustacchi, C. Bifiardon, D. Papadopoulo, S. Nocentini, P. Vigny, J. Blais, R. V. Bensasson, J. C. Ronfard-Haret, E. J. Land, F. Zajdela and R. Latarjet, P h o t o c h e m o t h e r a p y using pyridopsoralens, B i o c h i m i e , 57 (1985) 4 1 7 - 4 2 2 . 28 R. J. M. Fry, R. D. Ley a n d D. D. Grube, Photosensitized reactions and carcinogenesis, Natl. C a n c e r Inst., Monogr., 50 (1978) 3 9 - 4 3 . 29 A. R. Young, S. L. Walker a n d M. Garmyn, A first a p p r o a c h to an action s p e c t r u m for 8MOP phototumorigenesis in mice, J. Invest. Dermatol., 90 (1988) 1 7 5 - 1 7 8 . 30 D. J. Cripps, N. J. Lowe and A. B. Lerner, Action spectra of topical psoralens: a reevaluation, Br. J. Dermatol., 107 (1982) 7 7 - 8 2 . 31 A. R. Young and I. A. Magnus, An action s p e c t r u m for 8-MOP induced s u n b u r n ceils in mammalian epidermis, Br. J. Dermatol., 104 (1981) 5 4 1 - 5 4 8 .
246 32 K. H. Kaidbey, An action s p e c t r u m for 8-methoxypsoralen-sensitized inhibition of DNA synthesis i n vivo, J. Invest. Dermatol., 85 (1985) 9 8 - 1 0 1 . 33 R. S. Stern, L. A. Thibodeau, R. A. Kleinerman, J. D. Parrish and T. B. Fitzpatrick, Risk of cutaneous carcinoma in patients treated with oral methoxsalen p h o t o c h e m o t h e r a p y for psoriasis, N. Engl. J. Med., 300 (1979) 8 0 9 - 8 1 3 . 34 R. S. Stem, N. Laird, J. Melski and J. A. Parrish, Cutaneous squamous-cell carcinoma in patients treated with PUVA, N. Eng. J. Med., 310 (1984) 1156--1161. 35 R. S. Stern, R. Lange and m e m b e r s of the P h o t o c h e m o t h e r a p y Follow-up Study, Nonm e l a n o m a skin cancer occurring in patients treated with PUVA five to ten years after first treatment, J. Invest. Dermatol., 91 (1988) 1 2 0 - 1 2 4 . 36 A. B. Forman, H. H. Roenigk, W. A. Caro and M. L. Magid, Long-term follow-up of skin cancer in the PUVA -48 cooperative study, Arch. DerrnatoL, 125 (1989) 5 1 5 - 5 1 9 . 37 R. Lindskov, Skin carcinomas and treatment with p h o t o c h e m o t h e r a p y (PUVA), A c t a Dermatol. Venereol Stockholm, 61 (1981) 1 4 1 - 1 4 5 . 38 A. Eskelinen, K. Halme, A. Lassus and J. Idanpaan-Heikkila, Risk of cutaneous carcinoma in psoriatic patients treated with PUVA, P h o t o d e r m a t o l o g y , 2 (1985) 1 0 - 1 4 . 39 A. Tanew et al., N o n m e l a n o m a skin tumors in long-term p h o t o c h e m o t h e r a p y treatment of psoriasis, J. A m . Acad. Derrnatol., 15 (1986) 9 6 0 - 9 6 5 . 40 T. Hensler, E. Christophers, H. H6higsmann and K. Wolff, Skin tumors in the E u r o p e a n PUVA study, J. Am. A c a d . Der'rnatol., 16 (1987) 1 0 8 - 1 1 6 . 41 T. Henseler, Risk of n o n m e l a n o m a skin tumours in patients treated with long-termp h o t o c h e m o t h e r a p y (PUVA), in T. B. Fitzpatrick, P. Forlot, M. A. Pathak and F. Urbach (eds.), Psoralens: Past, P r e s e n t a n d F u t u r e o f P h o t o c h e m o p r o t e c t i o n a n d other Biological A c t i v i t i e s , J o h n Libbey Eurotext, Paris, 1989, pp. 3 7 7 - 3 8 6 . 42 T.-Y. Chuang, J. Tse and D. J. Cripps, A preliminary study on the link between PUVA and skin cancer, Int. J. D e r m a t o L , 28 (1989) 4 3 8 - 4 4 0 . 43 R. S. Stern, Risk a s s e s s m e n t of PUVA and cyclosporine, Arch. Dermatol., 125 (1989) 545-547. 44 R. W. Morris, Skin tumors in the E u r o p e a n PUVA study: eight-year follow-up of 1643 patients treated with PUVA for psoriasis, J. A m . Acad. Dermatol., 20 (1989) 297. 45 N. K. Gibbs, H. H6nigsmann and A. R. Young, PUVA t r e a t m e n t strategies and cancer risk, Lancet, January 18 (1986) 1 5 0 - 1 5 1 . 46 F. M. Carabott and J. L. M. Hawk, A modified dosage for increased efficiency in PUVA treatment of psoriasis, Clin. Exp. Dermatol., 14 (1989) 3 3 7 - 3 4 0 . 47 P. Fritsch and H. HSnigsmann, Combination phototherapy -- a critical appraisal, Curt. Probl. Dermatol., 15 (1986) 2 3 8 - 2 5 3 . 48 H. H6nigsmann, E. Jaschke, V. Nitsche, W. Brenner, W. Rausch Meier a n d K. Wolff, Serum levels of 8-methoxypsoralen in two different drug preparations: correlation with photosensitivity and UV-A dose requirements for photochemotherapy, J. Invest. Dermatol., 79 (1982) 2 3 3 - 2 3 6 . 49 L. Dubertret, D. Serraf-Tircazes, M. Jeaumougin, P. Moll~re, D. Averbeck and A. Young, Phototoxic properties of perfumes containing b e r g a m o t oil on h u m a n skin: photoprotective effects of UVA and UVB sunscreens, J. Photochem. Photobiol., submitted for publication. 50 D. Averbeck, S. Averbeck, L. Dubertret, A. Young and P. Moli~re, Genotoxicity of b e r g a p t e n and bergamot oil in S a c c h a r o m y c e s c e r e v i s i a e , J. Photochem. Photobiol., submitted for publication. 51 H. HSnigsmann, E. Jaschke, F. Gschnait, W. Brenner, P. Fritsch and K. Wolff, 5-Methoxypsoralen (bergapten) in p h o t o c h e m o t h e r a p y of psoriasis, Br. J. Dermatol., 101 (1979) 369-378. 52 G. Mezzadra, B. Gaurneri, C. Grupper and P. Forlot, Effects of chronic field exposure of h u m a n s to bergapten, in J. Cahn, A. Meybeck, P. Forlot, F. Urbach and C. Grupper (eds.), P s o r a l e n s i n Cosmetics a n d D e r m a t o l o g y , Pergamon, Paris, 1981, pp. 3 8 3 - 3 8 6 . 53 IARC, m o n o g r a p h s on the evaluation of the carcinogenic risk of chemicals to humans. Some naturally occurring and synthetic food components, furocoumarins and ultraviolet radiation. 5-Methoxypsoralen, IARC Sci. PubL, 40 (1986) 3 2 7 - 3 4 7 .
247 54 S. Kimura, N. Mizuno, S. Hirano and K. Yoshikawa, Topical application of 3-carbethoxypsoralen plus UVA in the t r e a t m e n t of psoriasis, J. Dermatol., 12 (1985) 2 5 1 - 2 5 7 . 55 A. Takashima, A. Sunohara and N. Mizuno, Comparison of the relative therapeutic efficacy of 7-methylpyridopsoralen and 8-methoxypsoralen in p h o t o c h e m o t h e r a p y in psoriasis treatment, J. D e r m a t o L , 15 (1988) 1 9 5 - 2 0 1 . 56 M. Cristofolini, G. Recchi, S. Boi, F. Piscioli, F. Bordin, F. Baccichetti, F. Carlassare, M. Tamaro, B. Pani, N. Babudri, A. Guiotto, P. Rodighiero, D. Vedaldi and F. Dall'Acqua, 6Methylangelicins: new monofunctional p h o t o c h e m o t h e r a p e u t i c agent for psoriasis, Br. J. Derrnatol., 122 (1990) 5 1 3 - 5 2 4 . 57 P. Morli~re, H. H6nigsmann, D. Averbeck, M. Dardalhon, G. Hiippe, B. Ortel, R. Santus and L. Dubertret, Phototherapeutic, photobiologic and photosensitizing properties of khellin, J. Invest. Dermatol., 90 (1988) 7 2 0 - 7 2 4 . 58 J. Brucke, A. Tanew and H. HSnigsmann, Action s p e c t r u m of photochemotherapy. Comparison of psoriasis treatment with 335 n m and 365 nm, in A b s t r a c t Book, lOth I n t e r n a t i o n a l Congress on Photobiology, J e r u s a l e m , Israel, 30 October-5 N o v e m b e r , 1088, p. 24. 59 B. L. Diffey, A. V. J. ChaUoner a n d P. J. Key, A survey of the ultraviolet radiation emissions of p h o t o c h e m o t h e r a p y units, Br. J. Dermatol., 102 (1980) 3 0 1 - 3 0 6 . 60 S. E. Freeman, R. W. Gange, J. C. Sutherland, E. A. Matzinger and B. Sutherland, Production of pyrimidine dimers in h u m a n skin exposed i n s i t u to UVA irradiation, J. Invest. Dermatol., 88 (1987) 4 3 0 - 4 3 3 . 61 L. Roza, R. A. Baan, J. C. van der Leun and L. Kligman, UVA hazards in skin associated with the use of tanning equipment, J. Photochem. Photobiol. B, 3 (1989) 2 8 1 - 2 8 7 . 62 M. Auletta, R. W. Gange, O. T. Tan and E. Matzinger, Effect of cutaneous hypoxia upon erythema and pigment responses to UVA, UVB and PUVA (8-MOP + UVA) in h u m a n skin, J. Invest. Dermatol., 86 (1986) 6 4 9 - 6 5 2 . 63 R. M. Tyrrell, UVA hazards in skin associated with the use of tanning equipment - a comment, J. Photochem. Photobiol. B, 4 (1989) 2 2 7 - 2 3 2 . 64 A. R. Young, Aspects of psoralen phototumorigenesis with emphasis on the possible role of t u m o u r promotion, B i o c h i m i e , 68 (1986) 8 8 5 - 8 8 9 . 65 H. van Weeldon and J. C. van der Leun, UV-A induced tumors in pigmented and albino hairless mice, in W. F. Passchier and B. F. M. Bosnjakovic (eds.), H u m a n E x p o s u r e to UVA R a d i a t i o n : R i s k s a n d R e g u l a t i o n s , Elsevier Science Publishers, Amsterdam, 1987, pp. 4 5 - 5 2 . 66 H. J. C. M. Sterenberg and J. C. van der Leun, Tumorigenesis by a longwave UVA source, Photochem. Photobiol., 51 (1990) 3 2 5 - 3 3 0 . 67 P. D. Forbes, H. F. Blum a n d R. E. Davies, Photocarcinogenesis in hairless mice: doseresponse and the influence of dose-delivery, Photochem. Photobiol., 34 (1981) 3 6 1 - 3 6 5 . 68 F. Daniels, A simple microbiological m e t h o d for demonstrating phototoxic compounds, J. Invest. Dermatol., 44 (1965) 2 5 9 - 2 6 3 . 69 N. K. Gibbs, An adaptation of the C a n d i d a a l b i c a n s phototoxicity test to demonstrate photosensitizer action spectra, P h o t o d e r m a t o l o g y , 4 (1987) 3 1 2 - 3 1 6 . 70 NIH, Toxicology and carcinogenesis studies of 8-methoxypsoralen in F344/N rats (gavage studies), National Toxicology Program, Research Triangle Park, NC 27709, 1989, N I H P u b l i c a t i o n No. 80-2814, U.S. Department of Health and H u m a n Services, Public Health Service, National Institutes of Health.
