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Mutation Research, 39 (1976) 29--74 © Elsevier/North-Holland Biomedical Press

M O L E C U L A R A N D G E N E T I C B A S I S O F F U R O C O U M A R I N R E A C T I O N S

BARRY R. SCOTT 1, MADHU A. PATHAK 2 and GEORGES R. MOHN s

1 National Institute o f Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 2 7709; 2Department o f Dermatology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114 (U.S.A.) and S Zentrallaboratorium fiir Mutagenitfftspriifung, 78 Freiburg 1, Br, Breisacher Strasse 33 (West Germany)

(Received May 4th, 1976) (Accepted June 24th, 1976)

C o n t e n t s

I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 O c c u r r e n c e a n d n o m e n c l a t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 F u r o c o u m a r i n s a n d r a d i a n t e n e r g y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5

N o n - i o n i z i n g r a d i a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 5 G e n e r a l c o n s i d e r a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 S o m e f a c t o r s t h a t a f f e c t f u r o c o u m a r i n p h o t o s e n s i t i z a t i o n . . . . . . . . . 36

A b s o r p t i o n s p e c t r a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 R e l a t i o n s h i p b e t w e e n s t r u c t u r e a n d p h o t o s e n s i t i v i t y . . . . . . . . . . . 37 D o s e a n d c o n c e n t r a t i o n o f p h o t o s e n s i t i z e r . . . . . . . . . . . . . . . . . . . 3 8 T e m p e r a t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 H y d r o g e n i o n c o n c e n t r a t i o n ( p H ) . . . . . . . . . . . . . . . . . . . . . . . . . 40 A c t i v i t y is o x y g e n i n d e p e n d e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 R a d i a t i o n v a r i a b l e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 E r y t h e m a l t h r e s h o l d o f h u m a n s u b j e c t s . . . . . . . . . . . . . . . . . . . . . 41

V i t i l i g o . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 8 - M e t h o x y p s o r a l e n s e n s i t i z a t i o n b y i o n i z i n g r a d i a t i o n . . . . . . . . . . . . . . 43

B i o c h e m i c a l e f f e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4 P h a r m a c o l o g i c a l a c t i o n a n d m e t a b o l i c a l t e r a t i o n . . . . . . . . . . . . . . . . . . 44 I n h i b i t i o n o f D N A s y n t h e s i s - - p so r i a s i s t r e a t m e n t . . . . . . . . . . . . . . . . . 46 I n h i b i t i o n o f t e m p l a t e a c t i v i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 8

I n a c t i v a t i o n a n d t o x i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 C a r c i n o g e n i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 T e r a t o g e n i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 M u t a g e n i c i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 R e p a i r o f d a m a g e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 P r o p o s e d m e c h a n i s m o f a c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 0

30

Molecular complexing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Photobinding of furocoumarin to DNA and bases . . . . . . . . . . . . . . . . . 61 Cross-linking between opposite strands of DNA . . . . . . . . . . . . . . . . . . . 62

Genetic hazards to man . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Introduct ion

Ancient Hindus, Turks, Egyptians and other orientals have known about some of the biological effects of furocoumarins for more than 3000 years [99]. In particular, the Indian sacred book, "Atharva Veda" [32] and the old Budd- hist Bower manuscript [125] both mentioned treatment of leukoderma using a poultice application of material from a plant that is now classified as Psoralea corylifolia. Another important plant, Ammi majus (Umbelliferae), a weed found in the Nile Valley, has also been employed for centuries as a "cure" for leukoderma (vitiligo). In the 13th century, Ibn E1 Bitar gave a description of the usefulness of this plant for leukoderma in his famous book, "Mofradat E1 Adwiya" [130]. Deleterious effects of the furocoumarins were also mentioned in a German Fairy tale of the 18th century [120]. In this tale, the central char- acter, "Li t t le Muck", suffers from photodermatit is after ingesting figs and ex- posing himself to sunlight. However, it was not until 1834, that the first furocoumarins, 5-methoxypsoralen, was isolated by Kalbrunner from bergamot oil [101]. Seventy-seven years later the most commonly used medicinal furo- coumarin today, 8-methoxypsoralen, was isolated by Thoms [265]. Finally in 1933, Sp~ith and Holzen [254] established its chemical structure and reported on its successful synthesis. Only in 1972 was the crystal and molecular structure of 8-methoxypsoralen established [260].

Another important information-stone in the history of the furocoumarins was made available when in 1931, Phyladelphy [225] recognized the impor- tance of sunlight in reactions of these compounds with biological materials. Kuske [157] then established the relationship between the chemical com- ponents of certain plant tissues and the development of phytophotodermati t is (phyto = plant, photo = light). By studying the time factor, the causative agent, and the nature of photoreaction produced by fig extracts (Ficus carica), Pasti- nice sativa, Angelica officinalis, Ruta graveolens and Heracleurn mantegazzianum with his associate Mickelmann, he succeeded in presenting evidence that the photosensitizing substances responsible for phytophotodermati t is were furo- coumarins. At about the same time, Jensen and Hansen [138] were able to es- tablish the wavelengths (320--400 nm) responsible for phytophotodermati t is . This lead to the development of an artifical UV light source by Kuske in 1940 [157,158] and its use in bio-assay for detecting other photosensitizing agents [184]. Finally, a Woods-Horn Filter was added to the artifical UV light source to filter out the shorter wavelengths of light (less than 3200 •) in the 1950's [216].

Interestingly, even though these compounds have been used in folk-medicine for a long time, it was only in the first half of this century that the pharmo- kinetic properties of these compounds were clinically studied. One of the first

31

clinical trials using substances isolated by Fahmy and Abu-Shady [1,93], from the grey-green powder used by Egyptian herb doctors was conducted by E1 Mofty [87] in 1948. As a result of these trials it became apparent that furo- coumarins might possibly be developed as drugs to increase the melanin pro- duction and hence, increase the tolerance of human skin to sunlight. Because of the important basic and clinical implications of this hypothesis, a fairly exten- sive research effort was begun in the 1950's into the botanical sources of furo- coumarihs, their mechanism of action, their toxicity in animals and man, the development of high intensity monochromatic UV light source and the effect of furocoumarins on the incidence of solar or UV light-induced skin cancer in man and mice. Besides accomplishing many of these objectives, it was also de- termined that these compounds were very useful in the t reatment of pig- mentary disorders such as vitiligo (leukoderma), psoriasis and to increase the tolerance of skin to solar radiation in people who easily sunburn.

Several aspects of the biological and chemical properties of furocoumarins have already been reviewed individually (historical [97], new clinical uses [126], photochemist ry [48,70,185,221] , photosensitization [174,182,187, 189,191,192,207,221] , occurrence and uses [223,244] , isolation and identifica- tion [233], biological action [243], and genetic effects [290]). Thus, the pur- pose of the present review is to give a summerized general overview, with par- ticular emphasis on the genetic toxicology of these compounds.

Occurrence and nomencalture

Furocoumarins are distributed both in the natural environment and the syn- thetic environment (see Table I). In nature the source of these compounds have world-wide distribution and belong to about eight families of plants.Whereas, in the synthetic environment, many natural and synthetic furocoumarins have been made in the laboratory. The furocoumarins, certain isomers of which are called psoralens, belong to a group of heterocyclic compounds that are consid- ered to be derivatives of coumarin (benz-a-pyrone or 1,2-benzopyrone). The fusion of a pyrone ring with a benzene nucleus can give rise to a class of heter- ocyclic compounds known as benzopyrones (Fig. 1). The two distinct types of benzopyrones that are commonly recognized are the coumarins (benzo<~- pyrones, e.g. umbelliferone) and the chromones (benzo-~/-pyrones, e.g. khellin). They differ in the position of the carbonyl group.

Furocoumarins are synthesized when the furan ring is built on a suitably sub- st i tuted coumarin derivative. A furan ring can be fused on a suitably substi- tu ted coumarin molecule in 12 different ways and each of the resulting com- pounds can become the parent of a family of psoralen-like derivatives [100,

O (a) (b)

Fig. 1. Chemica l s t ruc tu re of benzo-c~-pyrone (a) and benzo- 'y -pyrone (b).

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TABLE I

OCCURRENCE OF FUROCOUMARINS IN MAN'S ENVIRONMENT

Source F u r o c o u m a r i n s Refe rences

Genera l a c c o u n t ( rev iew) Var ious Plants

Genera l d i s t r ibu t ion all over the wor ld ( reviews and list). F o o d sources: figs

pars ley frui ts of A m m i majus a nd Ficus caricia

Leaves of Ficus caricia

Psoralea subacaulis Ru ta m o n t a n a

R o o t s of Herac leum candidans

Psorallea su beaulis Psorallea plica ta

Ru ta graveolens R u t a bracteosa

Flowers of Psoralea subcaul is Seeds o f Herac leum candidans

Psoralea subcaul is Psoralea psoraloidea

Genera l p lan t tissues of Ruta graveolens

Herac leum g igan teum Psoralen drupacea

Mold m e t a b o l i s m ( inc luding reviews)

Cosmet ics inc luding s u n t a n p repa ra t i ons a nd eau de co logne (oil of b e r g a m o t )

Medical t r e a t m e n t of L e u k o d e r m a Psoriasis

Chemica l synthes is

O the r sources - - Psoberan

Var ious

X a n t h o t o x i n , Bergap ten an d I m p e r a t o r i n

Psoralen and Bergap ten Psoralen Psora len and Bergap ten Bergap ten an d o thers Psoralen Psoralen and Angel ic in Paoralen, Bergap ten and X a n t h o t o x i n Psoralen Be rgap ten an d o thers Psoralen Psoralen X a n t h o t o x i n an d Bergap ten Bergap ten Var ious

Var ious

Be rgap ten

2 1 1 ,2 2 3

1 8 9 , 1 9 5 , 2 1 1

1 8 9 ,2 8 9 1 8 9 ,1 3 7 93 79 3 , 4 3 , 2 3 0 , 2 3 6

23 249

19

23 2

110 1 6 0 , 4 2 23 12

21 22 2 3 1 ,1 5 9

109 156

2 8 8 , 2 4 3 , 2 4 2 , 2 4 5

81 ,228

See sec t ion p. 35 1 8 , 1 9 5 , 2 2 9 , 2 4 8 See sec t ion p. 46 1 0 , 4 1 , 1 4 5 , 1 4 6

Var ious der iva t ives 285

Var ious 84 ,85

101,145,219] . Several of these compounds (see Fig. 2). (e.g. psoralen, iso- psoralen, isopseudopsoralen, allopsoralen) have been synthesized, although, in nature, rings fused in a linear fashion (e.g. psoralen) are more common than in one of several possible angular modes. Most of the naturally occurring furo- coumarins so far described (e.g. psoralen, 8-methoxypsoralen, 5-methoxypso- ralen, angelicin, halfordin, isohalfordin) have been found to be derivatives of either psoralen ((7H-furo(3,2-g)(1)benzopyran-7-one) or angelicin and rarely resemble the synthetic isopseudopsoralens. Abou t 90 coumarins compose one of the most important groups of natural products. All but six or so are derived

~o ~ o (a) (b)

33

~o~o °

(d ) Cc)

(e)

Fig. 2. The structure of i sopsoralen (a), pseudopsora len (b), psoralen (e), t w o i sopseudopsora lens (d,e). The term pseudopsora len is designated on ly for conven ience .

OCH 3

(a) (b )

OH (c) (d)

CH 3

~0 CH3-~O CH3 CH 3

(e) ( f )

~ O

(g) Fig. 3. S t r u c t u r a l f o r m u l a e o f t h e m o s t c o m m o n f u r o c o u m a r i n s . P s o r a l e n (a) ; 8 - m e t h o x y p s o r a l e n (xan- t h o t o x i n ) (b) ; b e r g a p t e n (c) ; x a n t h o t o x o l (d) ; 8 - m e t h y l p s o r a l e n (e): t r i m e t h y l p s o r a l e n (f); ange l i c in (g).

34

from plants and the others have been isolated from microorganisms, including fungi. Natural furocoumarins have been isolated from five major plant families. The important families of plants that contain furocoumarins are: (a) Umbel- liferae (e.g. parsely, parsnip, celery) Ammi majus, Angelica archangelica, (b) Rutaceae (e.g. bergamot fruits, lime, gas plant, cloves, common rue); (c) Le- guminosae (psoralea corylifolia, Xanthoxylum flavum), (d) Moraceae (e.g. Ficus caria or fig); (e) Orchidaceae. Over 28 different furocoumarins have been isolated from natural sources. Of these psoralen, 8-methoxypsoralen and 4,5', 8-trimethylpsoralen are used clinically in the t reatment of vitiligo and have been extensively investigated in terms of their therapeutic usefulness, photo- sensitivity and melanogenic properties.

Several chemical nomenclatures for the furocoumarins are in use. The syn- onyms issued by EMIC of the most important members of the group are:

Common name Synonyms

Trimethylpsoralen

Angelicin

P s o r a l e n

M e t h o x y p s o r a l e n

CAS P r e f e r r e d I n d e x N a m e : 7 H - F u r o ( 3 , 2 - g ) ( 1 ) B e n z o p y r a n - 7 - o n e , 2 , 5 , 9 - t r i m e - e t h y l - ( 9 C I )

T r i s o r a l e n Tr io x y s a l e n T r i o x s a l e n 6 - H y d r o x y - b e t a , 2 , 7 - t r i m e t h y l - 5 - b e n z o f u r a n a c r y l i c ac id , d e l t a - l a c t o n e 4 , 5 , 8 - t r i m e t h y l p s o r a l e n 2 ' , 4 , 8 - t r i m e t h y l p s o r a l e n 4 , 5 ' , 8 - t r i m e t h y l p s o r a l e n 5 - B e n z o f u r a n a c r y l i e ac id , 6 - h y d r o x y - b e t a , 2 , 7 - t r i m e t h y l - , d e l t a - l a c t o n e 5 ' , 4 , 8 - t r i m e t h y l p s o r a l e n

CAS P r e f e r r e d I n d e x N a m e : S t i g m a s t - 5 - e n - 3 - 0 1 , ( 3be t a ) - (9CI ) S t i g m a s t - 5 - e n 3 b e t a - o l

C i n c h o l S t i g m a s t e r o l , 22 , 2 3 - d i h y d r o - b e t a - S i t o s t e r o l B E T A - S i t o s t e r o l C u p r e o l S i t o s t e ro l s 2 4 x i - E t h y l c h o l e s t - 5 - e n - 3 b e t a - o l S K F 1 4 4 6 3 b e t a - S i t o s t e r o l S i t o s t e r o l B h a n n o l Q u e b r a c h o l 2 2 , 2 3 - D i h y d r o s t i g m a s t e r o l a l p h a - D i h y d r o f u c o s t erol

CAS P r e f e r r e d I n d e x N a m e : 7 H - F u r o ( 3 , 2 - g ) ( 1 ) b e n z o p y r a n - 7 - o n e - ( 9 C I ) 2 - P r o p e n o i c ac id , 3 - ( 6 - h y d r o x y - 5 - b e n z o f u r a n y l ) - , d e l t a - l a c t o n e 5 - B e n z o f u r a n a c r y l i c ac id , 6 - h y d r o x y - , d e l t a - l a c t o n e P s o r a l e n F i c u s i n F u r o ( 4 ' , 5 ' : 6 , 7 ) c o u m a r i n

CAS P r e f e r r e d I n d e x N a m e : 7 H - F u r o ( 3 , 2 - g ) ( 1 ) b e n z o p y r a n - 7 - o n e , 9 - m e t h o x y - (9Cl)

8-Methoxypsoralene Xanthotoxin Meladinin Methoxsalen Methoxa°Dome Meloxine 8 - M e t h o x y - ( f u r a n o - 3 ' . 2 ' : 6 . 7 - c o u m a r i n ) O x s o r a l e n 5 - B e n z o f n r a n a e r y l i c a~id, 6 - h y d r o x y - 7 - m e t h o x y - , d e l t a - l a c t o n e A m m o i d i n 8 -MOP ~ ,

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Common n a m e

Bergapten

Synonyms

CAS Preferred In d e x N a m e : 7H-Furo(3,2-g)(1)benzopyran-7-one,4-methoxy- (9CI)

5-Benzofuranacrylic acid, 6-hydroxy-4-methoxy-, de l ta - lac tone 5 - M e t h o x y p s o r a l e n Majudin Bergaptan Heracl in o f Gu tz e i t Bergaptene

The most explicit of these, adopted by Sp~ith [255,256] is based on the conven- tional numbering of both the coumarin structure and the furan ring from the respective hetero atom with the designations of prime numbers used in the furan ring. This numbering system for psoralen along with the structural for- mulae of 8-methoxypsoralen, bergapten, xanthotoxol , 8-methylpsoralen and trimethylpsoralen are shown in Fig. 3. Another numbering system (see ref. [185]) has also been proposed the ring index system, but this has not been commonly used. Similarly, the 1963 Chemical Abstracts indexing of psoralen as a lactone of 6-hydroxy-5-benzofuranacrylic acid or the 1967 indexing as 7H- furo (3,2-g)(1)benzopyran-7-one is also not in common usage. To avoid con- fusion in the following account the compounds will be referred to by their common names.

Furocoumarins and radiant energy

Non-ionizing radiation

General considerations Some basic notions on how chemical energy is related to simple molecular

reactions and how radiant energy can initiate these reactions is necessary for an understanding of the photoreactions of furocoumarins. In a given population of molecules, at a particular temperature, each molecule possesses a certain amount of energy. The energy of the individual molecules is governed by the spatial ar- rangement of the component parts of the molece and is affected by collision with other molecules. Thus, molecules in a given population exhibit a range of energies which are distributed about a mean energy value. The majority of chemical reactions only occur when the energy of the reactants is equal to or exceeds a certain amount of energy (the energy of activation Eact) and collision occurs. In the absence of radiant energy the Eae t of the molecules is attained by collision. Reactions of this type are usually accelerated by increases in tempera- ture which results in increased molecular movements, as well as collision; and thus, additional molecules are activated. Molecules can also acquire Eae t by ab- sorbing electromagnetic energy in the form of visible or UV light. The absorp- tion of radiation also excites valency electrons to a higher orbital level resulting in a very reactive molecule.

A general account of the electronic transitions that take place when photo- sensitizers are excited by radiation has been reviewed by Lamola [162]. Brief- ly, the electronic orbitals involved are the bonding orbitals (~ or p electronic orbitals) corresponding to the s and p orbitals of the atom. In the unexcited state of the furocoumarins these valency electrons are paired resulting in stabil-

36

ity of the molecules. This might explain why the furocoumarins are virtually chemically and biologically inactive in the absence of radiation. When absorp- tion of radiation occurs these electrons are excited from the ground state to one of the unoccupied orbitals of the molecule with unchanged electron spin (the singlet excited state). Electrons in this state are short lived, less than 10 -s of a second for some furocoumarins [168], and either decay to the ground state (fluorescence) or are converted to the triplet state by a change in spin of an electron. (The triplet state may occur wi thout passage through the singlet state.) The lifetime of the triplet state of the furocoumarins ranges from 0.34 to 1.22 sec [168]. Electrons decay from this excited state when no reaction takes place and phosphorescence occurs. In biological systems, the triplet state of furocoumarins is usually long-lived (10 -3 of a second and longer); and there- fore, can play an important role in the transfer of energy and initiation of bio- logical changes responsible for photosensitization. The long duration of the reactive state increases the probabili ty that a molecule in the triplet state can react chemically with the target molecules.

From luminescent spectroscopy and molecular orbital calculations, the photoreactivi ty of furocoumarins towards pyrimidine bases of DNA was inter- preted in terms of the triplet excited states of these molecules and the ability to localize excitation energy in the region of the 3,4-double bond [252,253]. It was also predicted that 3,4-double bond of psoralen in the triplet state is more reactive than the 4' ,5 ' ,-double bond [252] (see section p. 60).

Some factors that affect furocoumarin photosensitization The degree of photoreact ion elicited by the various furocoumarins in biolog-

ical material are affected by a large number of factors. Most of these have been studied with respect to cutaneous sensitization, however, they also appear to apply to other biological photoreact ions of these compounds.

Absorption spectra. According to the Grothus and Draper's basic law of photochemist ry , a photochemical reaction can only take place when radiation of a specific wavelength is absorbed by the system. Furocoumarins exhibit maxima's in absorption spectrum at regions below 320 nm [101,206,287]. One might, therefore, expect that wavelengths in the region of maximal absorption (i.e., below 320 nm) would have greatest photosensitizing effect. However, wavelengths above 320 nm have the greatest photosensitizing bffect. Buck et al. [40], Pathak et al. [208] and Nakayama et al. [197] reported the action spec- t rum of 8-methoxypsoralen for erythemal induction in human skin and guinea pig skin. Their data indicated that human skin and the skin of other mammals can be optimally photosensit ized by longwave UV light after topical applica- tion or oral administration of 8-methoxypsoralen. The maximum photosensi- tivity reaction occured at 340--360 nm. In the absence of 8-methoxypsoralen, these wavelengths did not induce any reaction. Wavelengths which were maxi- mally absorbed by furocoumarins (e.g. 2 1 7 , 2 4 8 , 2 9 5 , 3 0 3 nm, etc.) did not elic- it any augmented erythemal response in the presence of photosensitizing agent like 8-methoxypsoralen or trimethylpsoralen [40,206,208]. Thus, it appears that the absorption spectrum m,d the sensitizing action spectrum are not in agreement as would be expected from the above law, suggesting that the photo- chemical behavior of these compounds was rather unusual.

37

Several a t tempts to resolve this apparent conflict have been made. For in- stance, Yeargers and Augenstein found that when appropriate corrections for inherent errors in instrumentation were made in the absorption spectrum, there was reasonable agreement in the absorption and action spectrum of 8-methoxy- psoralen [287,288] . Then experimentally, other workers using Candida albi- cans spore inactivation and minimal erythemal dose in guinea pig skin, found that these action spectra exhibited maxima below 320 nm indicating that the 8- methoxypsoralen molecule was probably not abnormal in its photochemical behavior [202]. However, this group of experimenters seemed to have over- looked the possibility that the absorption spectrum of the compound in the biological material may be different from that of the test tube situation. A study of the wavelengths that are absorbed by the stratum layers of animal skin or corneum and transmitted to the viable cells of epidermis reveals, however, that wavelengths below 290 nm do not penetrate appreciably to the viable cells of epidermis where the important photochemical reaction occurs. The non- viable, dead horny cells of the stratum corneum absorb most of the radiation of wavelengths shorter than 290 nm. One cannot, therefore, expect a very close relationship between this absorption spectrum and the photosensitizing action spectrum of psoralen in the region below 290 nm. The transition of photoac- tivated psoralens to triplet state appears to be optimal in the longwave ultra- violet region (320--360 nm).

Relationship between structure and photosensitivity. The relationship be- tween the chemical structure of furocoumarins as well as related compounds and their photosensitizing capacity has been studied extensively by laboratories in Italy [44,186,189,238] and America [214,217,219] . More than one hun- dred and fifty different compounds have been tested for their photosensitizing potency on the skin, including several compounds structurally related to furo- coumarins, such as coumarins, benzofurans and pseudopsoralens. A consider- able difference exists in the photoreactivity of the various furocoumarins in producing their photosensitizing effects in skin; some compounds are very ac- tive (e.g. psoralen and certain methyl substi tuted psoralen derivates); others are only weekly active (e.g. 5-methoxypsoralen, 3-butylpsoralen); and many others are completely inactive (isopsoralen, isobergapten, and dihydropso- ralens). In general, furocoumarins with linear structures are more active than those with angular structures. Thus, the reactivity of psoralen is greater than angelicin and the corresponding linear form of bergapten is more active than isobergapten. Molecules containing bonds with a large amount of ionic char- acter tend to ionize when dissolved in polar solvents, such as water, whereas, those that increase the stability of the molecule or increase its insolubility in a polar solvent also increase the capacity of the molecules to cause photosensiti- zation and vice versa.

Any substi tution that alters the resonance of the furocoumarins molecule alters the activity of the photosensitizer. Therefore , introduction of electron donating moieties such as methyl groups in the 5' or 8 position enhances the ac- tivity of psoralen, whereas, addition of methyl groups at the 4 or 4' position decreases the activity and at the 3 position methyl substitution severely reduces the reactivity. Alternatively, introduction of electron withdrawing groups such as hydroxyl , cyano, nitro, amino or acetylamino at the same position exhibits

38

A c t i v e s t P u c t u r e s

PsoraLen CH 3

4 methyLpsor'alen

~ 0

CH 3 CH s

4~4" d imethyLpsoPalen

-%oyyko CH 3

4,5' d ime tny tpsoca !en

I n a c t i v e stPuctdPeS

O C,

I s o p s o r a l e n s Pseudopsor a le r t

\ '

o/[-.<.jA~S - [.\.//

Pseudopso ra [e n S

>0

CH3

CH3

4~8 d i m e t Byl psoca len

OCH 3

O O O

B me t h o x y p s o p a l e n

CH 3 HBC O ~ O O

CHs

4,5" , 8 - tP imet hyl psoPalen

OCH s

5 m e t hoxypsor -a ten

3 - m e t hyLpsoPalen 4~5£ dihydr-opsocaLens

COU mar' ins F-uPans Ben zod fuPal,s

0 FuPanochPO m o d e s O x a z o l o c o u m a r i n s

Naph tb /o fu ran Naph tbo alpha pyroNe

Fig. 4. Structural character is t ics o f act ive (capable o f induc ing p h o t o s e n s i t i z a t i o n ) and inact ive (non- p h o t o s e n s i t i z i n g ) furocoumv.r ins and re lated c o m p o u n d s .

the opposite effect. Thus, addition of hydroxyl groups into the parent mole- cule psoralen as in xanthotoxo l (8-hydroxypsoralen) or bergaptol (5-hydroxy- psoralen) completely removes the photobiological potency. This decreased ac- tivity is partially restored when the hydrogen of the hydroxyl group is replaced by a methyl group as in xanthotox in (8-methoxypsoralen) and bergapten (5- methoxypsoralen) . However, alkylating groups of longer chain length reduces the activity to zero. Hydrogenation of the 4',5' position in the furan ring (di- hydro furocoumarins) resulted in complete loss of the photosensitizing potency of psoralen molecule (see Fig. 4).

Dose and concentration of photosensitizer. One of the prime conditions governing the response of photobiologically active furocoumarins is the con- centration of the agent at the biological site of action at the time of irradiation. In monocellular systems, such as microorganisms, maximal response is usually attained after 1 or 2 h when an equilibrium in the concentration of the furo-

39

coumarin outside and inside the cell is established [4]. In most of these sys- tems, too, there is a critical point beyond which increases in concentration may either fail to augment or even reduce the response to radiation [199]. This change in furocoumarin sensitivity may result from the limited solubility of the compound in water (most of the compounds are dissolved initially in ethanol or acetone for experimental purposes; see [117] for other solvents), a shielding or screening effect--reducing the dose of the impinging radiation, a quenching effect of the furocoumarin, or a photopolymerizat ion of the sensitizer.

With complex multicellular organisms, such as man, the route of administra- tion (topical or oral) of the compound is very important. Each route has a series of factors which affects the concentration of the furocoumarin at the bio- logical important substrate. For orally administered compounds these include [204]: the amount of the drug that can be administered safely wi thout any systemic effects (e.g. nausea, vomiting, etc.); the rate of absorption, the rate of excretion, the ability of the organism to detoxify or modify the administered drug to an inactive form, as well as the time interval between administration and exposure to radiation. Topical applications are strongly influenced by the following factors [123,144] : the amount of the sensitizer that is applied, con- centration of the photosensitizer in the vehicle, partical size of the sensitizer, partit ion coefficient between sensitizer and vehicle, change in permeability of the skin caused by the vehicle, and finally the release of the compound from the vehicle. In a clinical s tudy of the photosensitivity of 50 mg of 8-methoxy- psoralen on healthy adult male subjects, maximal response was obtained when the carrier vehicles were a hydrophilic ointment or lanolin. These carriers were significantly more effective than petroleum and much better than Car- bowax, from which little of the 8-methoxypsoralen was released [141].

Temperature. In contrast to thermal reactions, which are approximately doubled by a rise in temperature of 10°C (Q~0 = 2--3), photochemical reaction rates are unaffected or reduced by increases in temperature (Q~0 ~< 1). Orginsky et al. [199] found that step-wise increases in the temperature of irradiation from 0 to 45 ° C resulted in a proportional diminuation of the inactivation of E. coli by 8-methoxypsoralen plus blacklight. In contrast photosensitization pro- duced by photodynamical ly active dyes was strongly increased by elevation of the temperature of the irradiation [199].

Similarly, increased inactivation of T4 phage, as well as increased inactiva- tion of E. coli strain B and mutat ion to t ryptophan independence, were ob- served as the temperature of 8-methoxypsoralen photosensitization was lowered to --50°C. These changes were suggested by Ashwood-Smith and Grant to be associated with increased cross-linking in the DNA [13]. At even lower temperatures (--13°C to --196°C) much larger amounts of energy were required to produce changes in DNA, lethality and mutat ion induction. At --196°C bacteria are very resistant to the biological effects of photosensitiza- tion and no cross-linking of DNA was observed. However, a new pattern of DNA damage is apparent. Irradiation temperature thus affects both the nature and quanti ty of induced photoproducts as well as the biological consequences of such changes [13]. However, the formation of molecular complexes with isolated DNA is not influenced by the temperature [72]. Whereas, the rate of photoreactions between furocoumarins and single-stranded nucleic acids (de-

40

natured DNA and RNA) are influenced by the temperature [74]. This may suggest that in vivo additional factors, as yet unspecified, may be involved in furocoumarin photoreactivation. One obvious candidate might be repair, how- ever, this requires further investigation.

Hydrogen ion concentration (pH). E. coli B irradiated in the presence of 8- methoxypsoralen exhibited essentially the same inactivation effect at all pH values whereas inactivation of B/r gradually increased as the pH increased from pH 4.3 to pH 8.7 [201]. Two possible explanations of this phenomenon are that an increased absorption of 8-methoxypsoralen occurred at alkaline pH by E. coli B/r or an increase in sensitivity of the targets occurs with this strain as the pH approaches 8.7. The furocoumarins are known to undergo opening of the lac- tone ring at alkaline pH. It is not known whether this open chain furocouma- ric acid moiety of 8-methoxypsoralen is more reactive than linearly annulated tricyclic 8-methoxypsoralen.

Activity is oxygen independent. Various oxidation-reduction reactions are unaffected by photosensit ization with furocoumarins. These include the oxida- tion of a-terpinene to ascaridol, haemolysis of red blood cells, both in the pres- ence and absence of oxygen, the oxidation of serum protein or unsaturated fat ty acids [189,191] , giant cell formation in mammalian tissue culture [67], and the inhibition of enzymes in vitro and in vivo [213,218]. The killing of bacteria in the presence of 8-methoxypsoralen and subsequent irradiation with 365 nm was also found to be independent of the presence of oxygen [199].

In a parallel s tudy with the same biological end point, the inactivation of Sarcina lutea, Mathews [171] demonstrated that photosensitization by 8- methoxypsoralen was curtailed by the presence of oxygen; whereas, the photo- dynamic action of toluidene blue required molecular oxygen for reaction. This difference in oxygen requirement is typical of that between photosensitizers and dyes exhibiting a photodynamic effect (see reviews [33,108,250,258]) .

Reduct ion of the photosensitizing effect of furocoumarins by oxygen can be explained in terms of triplet energy transfer to oxygen, generating singlet oxy- gen and quenching the photoreactive triplet state [208,227]. Other paramag- netic agents, such as Mn 2÷, Ni 2÷, and Co 2÷ also reduce the photoreactivity of psoralen and pyrimidines by this process [31]. The singlet state excitation of the furocoumarins is unaffected by molecular oxygen, however, in this state of excitation the furocoumarin molecules are less reactive than in their triplet state [12].

Radiation variables. The amount of photic energy absorbed (intensity of im- pinging radiation and duration of radiation exposure) and the wavelength (see section p. 36) have a considerable effect on the degree of furocoumarin photo- reaction. Each of these parameters of sunlight vary according to the latitude, time of the day, season, elevation above sea level and the local atmospheric conditions. Thus, in the natural environment estimates of the dose of radiation received at the reactive site, are highly variable and extremely difficult to deter- mine. Longwave ultraviolet radiation {320--400 nm) and particularly 320--360 nm wavelengths augment furocoumarin reaction. Presence of shorter wave- lengths 290--320 nm, will certainly be additive to the effects of 320--360 nm radiation. These wavelengths (290--320 nm) alone will cause erythemal reac- tion wi thout the presence of furocoumarin.

41

In the laboratory situation, an estimate of the rate of photoreaction, includ- ing some radiation variables, has been given by Ecker [83]. This worker rep- resented the 8-methoxypsoralen/E. coli DNA photoreact ion by:

kl 8-MOP + DNA ~ photo product (1)

k2 2(8-MOP) ~ photodimer (2)

React ion (1) was not a first order reaction and an estimate of the rate con- stant k~ was given as 7 × 10 -9 min -~ D -~ C -~ (at 365 nm); whereas, k2 had a value of 3 × 10 -s mo1-1 min- ' D -1 (D is the incident dose rate in ergs/mm2/sec, C, is the concentrat ion of the nucleotide pairs in the DNA). According to Ecker these estimates do not take into account the absorption cross-section of the near ultraviolet excited molecule or complex; or the quantum yield of the photoreact ion [83]. Incidently, there is also no consideration of heterogeneity the DNA base arrangement along the DNA chain. Experimentally, this has been demonstrated to have a marked effect on the degree of cross-linking of psoralen in synthetic polynucleotides. A very high amount of cross-linking of psoralen occured in poly d(A-T) after irradiation, whereas, under the same conditions of irradiation of the polymer poly dA • dT no cross-linking occurred with psoralen [52].

Erythemal threshold of human subjects. Erythemal threshold of the subject greatly influences the photoreactions. The reactivity of normal human skin to UV radiation (290--320 nm) is variable and is principally affected by (a) the thickness of the stratum corneum, (b) the tanning ability or genetic capacity of the individual to produce melanin. The melanin content of the melanocytic and the Malpighian cells is genetically pre<letermined. There are individuals whose skin will reveal minimal or poor tanning ability, moderate tanning ability or profuse tanning ability. The tolerance of these peoples' skin to UV radiation (290--320 nm) is highly variable and is principally influenced by the melanin content of the skin. Psoralen induced photosensitization reactions (so called photo toxic reactions which are manifested by erythema, edema and blistering reactions) are also influenced by tl~e thickness of the stratum corneum and the melanin content of the epidermis.

Drug induced photoreact ion occurs frequently in subjects with fair skin and less frequently in pigmented individuals. Fair skinned individuals (who burn readily and tan poorly) receiving 40 mg of oral 8-methoxypsoralen require as little as 2 J /cm 2 of ultraviolet-A (320--400 nm) energy to produce minimal photo toxic (photosensitization) reaction. Contrary to this, pigmented individ- uals (who rarely sunburn but tan profusely) receiving 40 mg oral 8-MOP will require as much as 6--8 J /cm 2 of ultraviolet-A energy to produce photosensiti- zation reaction. Increment of exposure dose in fair-skinned individuals can result in blistering reaction, whereas in pigmented individuals, it may only reveal minimal or moderate sunburn reaction.

Various methods have been evolved to test photosensitizing agents on human and guinea pig skin (see reviews [121,175]) . These basically fall into four types. In the first, 2.5--5 pg/cm 2 of the compound in solution to be tested is applied to the backs and arms of human volunteers and after evaporation the area is irradiated. In the case of the furocoumarins, a Phillips MPW 125 lamp or

42

an Osram HWA 500W lamp emitting mostly in the 365 nm region is placed at a constant distance [184,189] and the minimum exposure time required to in- duce an erythema is evaluated at 24 and 48 h after exposure is determined. Arbitrarily, compounds not producing any effect after 60 rain irradiation were considered to be inactive.

The second and third systems involved depilated guinea pigs. These experi- ments are of two types: one uses a fixed dose of the compounds to be tested combined with variable radiation exposures and the other a fixed dose of ir- radiation combined with varying doses of the compound to be tested. The first situation parallels that of the previously mentioned human clinical experi- ments. Caporale et al. [44] used this method to determine the minimal ery- thema dose required to produce an effect using 2.5 pg/cm 2 of various methyl derivatives of psoralen. In the second situation various concentrations of sub- stances have been applied to guinea pig skin and irradiated for a constant pe- riod of time [214,216,217]. In topical testing, approximately 0.5--2 J/cm 2 of ultraviolet-A (320--400 nm) energy is required to produce distinct photosensi- tization reaction when the concentration of the sensitizing agent is in the range of 5--25 pg/2.5 cm 2 (Pathak, unpublished observation). The fourth testing procedure involves the determination of minimum phototoxic dose or MPD after oral administration of the test compound. The photosensitizing com- pound like 8-methoxypsoralen is administered orally (0.6 mg/kg, about 42 mg/ 70 kg). The back is exposed to ultraviolet-A {320--400 nm) radiation between 2 and 3 h after oral ingestion. Approximately 6 exposure sites (2 × 2 inch) are prepared and each site is sequentially exposed to 1, 2, 3, 4, 6, 8 J/cm 2 of ultra- violet-A radiation. The sites are observed at 24 and 48 h after exposure. The ex- posure dose which produces minimally perceptible erythema reaction is scored as MPD. MPD for a fair skinned person who sunburns easily may range between 1--2 J/cm 2 where as MPD for a pigmented person who tans wells but does not burn readily, may range from 4--8 J/cm 2. Although guinea pig skin is less sen- sitive than human skin [44] the results obtained with the different methods are strictly analoguous.

Vitiligo Vitiligo is an acquired progressive achromia of the skin, due to a functional

abnormality of the melanocytes [ 108 ], with common familial incidence [ 165 ]. It occurs in about 2% of the world's population [ 165 ], and has been treated for the last 3000 years with plant substances and sunlight. This folk remedy was clinically tested using isolates of the plant substances with vitiligous patients by E1-Mofty [87]. The positive result of repigmentation in these studies was con- firmed in trials using oral or topical applications of furocoumarins combined with sunlight or near-UV radiation [94,99,106,224,251].

The augmentation of melanin pigmentation in normal skin by topical appli- cation or oral administration of psoralens like 4,5',8-trimethylpsoralen or 8- methoxypsoralen and subsequent exposure to UV light (320--400 nm) involves the interaction of melanocytes and kerationcytes. During this process the fol- lowing events have been observed [221].

(a) Mitotic activity in melanocytes within 48 to 72 h. (b) An increase in the number of functional melanocytes as the result of proliferation and/or activa-

43

tion of melanocytes. The number of functional melanocytes is doubled, and at times tripled, within 3 to 6 days after exposure and remains elevated for nearly 30 to 60 days. (c) Hyper t rophy of melanocytes and increased arborization of melanocytic dendrites. (d) The number of melanosomes in melanocytes is in- creased as the result of increased synthesis of melanosomes in the melanocytes. (e) Tyrosinase activity increases as new tyrosinase is synthesized in the prolif- eratory melanocytes. (f) After topical application of trimethylpsoralen, or oral administration of 8-methoxypsoralen and subsequent exposure to UV light it has been observed that the pattern of melanosome distribution in caucasian skin changes, possibly as a result of increase in the size of melanosomes. It should be emphasized that this switch in the distribution pattern of melano- somes from an aggregated to a non-aggregated form is not total.

In the unexposed skin of caucasians, the melanosomes in kerat inocyte are predominantly in an aggregated (complex) form. Melanosomes in the keratino- cyte after furocoumarin plus UV exposure were predominantly in discrete non- aggregated form.

The mechanism of the photosensitizing effect resulting in repigmentation of pigmentless areas of vitiligo patients by furocoumarins is obscure, However, persons with vitiligo which were treated with psoralen, 8-methoxypsoralen or 5-methoxypsoralen and exposed to sunshine or to 365 nm UV light exhib- ited certain skin reactions [147]. After a latent period of 6 h, erythema reaches its maximum intensity within 36--48 h. This is followed by melanin production. In severe cases, edema, vesication, desquamation and ulceration of the skin have been observed. The effect depends on the concentrat ion of the furocoumarin and the susceptibility of the individual (see section p. 36). Humidi ty increased the effectiveness of topically administered furocoumarin in humans [119], bu t patients with a low level of a-2-globulin react more slowly to oral t rea tment [86]. A combinat ion of psoralen and corticosteriods increases the efficacy of "curing" leukoderma [86].

The stimulation of melanin pigmentation in vitilegenous skin probably oc- curs as a result of proliferation of follicular melanocytes and their subsequent migration over the outer hair root sheet to the dermo-epidermal junction. In repigmenting vitiligenous macule one often sees perifollicular repigmentation indicating the migration of melanocytes from the hair bulb region.

A histological s tudy of psoralen therapy by Becker [25] found that the stra- tum corneum was thickened; pigment product ion was correlated directly with the amount of inflammation developed and psoralen tanning, reported by other workers [80,135] , was produced by large numbers of melanin granules retained in the stratum corneum and basal cell layer [25]. These observations were con- firmed by Zimmerman [291].

8-Methoxypsoralen sensitization by ionizing radiation E1-Mofty and his co-workers reported in 1964 [90] that administration of

100 mg of 8-methoxypsoralen in the diet of albino rats two hours before ir- radiation, or 0.33 mg daily for 15 da:~s sensitizes the animals against 900 R of 230 kV X-rays. Survival was 8.6 and 6 days respectively depending on the route of administration in irradiated rats against 12.6 days for the irradiated controls only. On the other hand, the exposure of the skin to graded doses of beta ir-

44

radiation up to 1200 reps resulted in a radioprotective effect. In this case, the appearance of the skin reaction was delayed, the degree of reaction diminished, as well as an enhanced rate of recovery, was observed. These studies have not been confirmed by other investigators. Another interesting experiment by Musajo showed that a 5,8<lihydroxypsoralen, a non-skin photosensitizing furo- coumarin, exhibited a radiosensitizing action when added to ascites cells ir- radiated with X-rays. Contrary to these findings no affect of protection or sen- sitization for either the induction of inactivation or mutat ion by ionizing radia- tion has been observed for E. coli [ 133] or Aspergillus nidulans (Scott, unpub- lished observations).

Biochemical effects

Pharmacological action and metabolic alteration Despite the increasingly widespread ingestion of furocoumarins in the treat-

ment of psoriases the vitiligo and an increasing tolerance of skin to UV ra- diation, little is known of their metabolism in man and animals. Some prelim- inary observations gathered by Pathak and his associates (unpublished) con- cerning the absorption and detoxification of therapeutically useful furocou- marins can be summarized as follows. (1) Penetration and fate of topically applied furocoumarins. Penetration and resorption of 8-methoxypsoralen into various skin layers of human subjects was investigated by Kammerau and his associates [142]. Tritium labeled 8-meth- oxypsoralen (12 pCi) was mixed in various ointment bases to yield a 1% 8- methoxypsoralen concentration in white vaseline aqueous wool wax alcohol (a hydrophilic solvent without emulsion) and in an aqueous hydrophilic ointment (a hydrophilic sohient with emulsion plus a hydrophilic polyethylene glycol mixture) were used as an ointment base. It was observed that nearly 70 to 95% of the applied 8-methoxypsoralen does not enter the horny layer. The aqueous wool wax alcohol ointment gave maximum pentration (4--6 X 10 -S M in epi- dermis) and penetration could be detected within 10 minutes and highest con- centration in the epidermis and dermis was achieved within 100 min (see also section p. 38). Four hours after application, 8-methoxypsoralen could be de- tected in urine and regardless of the ointment base employed, 8-methoxy- psoralen could not be detected in urine 40 h after application, although skin treated topically with 8-methoxypsoralen can remain photosensitive for as long as 48--72 h if the applied material is not washed off. Topical application of tri- methylpsoralen to guinea pig skin and subsequent irradiation results in (a) photodecomposi t ion of trimethypsoralen and (b) photoconjugation of tri- methylpsoralen to proteins of stratum corneum. The photoproduct is soluble in chloroform, alcohol and shows very little absorption in 290--340 nm region. The photoproduct is incapable of inducing cutaneous photosensitization.

(2) The kinetics of absorption, metabolism and excretion of furocoumarins (psoralen, trimethylpsoralen and 8-methoxypsoralen) have been examined in mice and human volunteers [209]. Groups of mice received 0.25 ml [3H]- psoralen (2780 mM/ml; specific activity 4.33 X 109 DPM/mM and [3H]tri- methylpsoralen (3920 Mm/ml; specific activity 10.5 X 10 ~° DPM/mM) either orally of intraperitoneally, and their urine, blood, faeces, skin and viscera were

45

analyzed at 2, 4, 6, 8, 12, 24 and 48 h after administration. Highest levels of psoralen in blood, skin and liver tissues are observed between 2 and 4 h. Over 70% of the orally or intraperitoneally administered dose of either psoralen or trimethylpsoralen is excreted in the urine within 8 h. Within 12 h nearly 90% of the administered dose is excreted. The excretion through faeces is not more than 5% of the administered dose. Likewise the excretion of 8-methoxy- psoralen or trimethylpsoralen in human subjects also occurs quite rapidly after oral ingest~ion. Over 80% of the administered dose of furocoumarins can be accounted for in the urine within the first 8 h and over 90% within 12 h. At 24 h, furocoumarin cannot be detected in urine specimens.

These observations are in close agreement with the clinical and experimental data about the manifestation of photosensitization reaction gathered in pa- tients and laboratory animals. It is known that human skin as well as guinea pig skin remains maximally photosensitive between 2 and 4 h after oral administra- tion of 8-methoxypsoralen. Six hours after an oral dose the skin cannot be photosensitized.

Attempts to identify the metabolities of psoralen have been also partially successful. Psoralen and 8-methoxypsoralen appear to be detoxified and ex- creted as glucuronides and hydroxylated moieties (hydroxylat ion either at 3 or 8 position). It is also apparent that 8-methoxypsoralen is converted to a furo- coumaric acid moiety. Trimethylpsoralen on the other hand is metabolized to carboxylated moiety; 4,8<limethyl-5-carboxypsoralen (DMECP) has been identified in mouse urine as a major fluorescent metabolite. A DMECP-like metabolite is also present in the urine of patients receiving oral trimethylpso- ralen. There are other fluorescent and non-fluorescent metabolites (at least two) that are formed and remained to be identified. It is of interest to know that the liver is the major site of metabolism. Hepatic microsomal action exhib- iting mixed function oxidase activity is involved in oxidation, hydroxylat ion and glucuronide formation of psoralens. The metabolic DMECP is non-photo- sensitizing when it is tested on mammalian skin. However, no apparent work has been undertaken to determine ~vhether or not furocoumarins are metabol- ically converted to deleterious genetic agents not requiring excitation by ra- diation, but several workers have examined the affect of furocoumarin on the biochemistry of the cell.

Ali and Agarwala [6,7] examined the effect of unirradiated and irradiated 8- methoxypsoralen on the glucose oxidation of brain and liver homogenates after Griffin [111] reported measureable amounts of 3H-labeled 8-methoxypsoralen in these organs. In the absence of irradiation no effect was observed while vari- ous doses of radiation in combination with 8-methoxypsoralen produced in- creasing inhibition of the glucose oxidation in both brain and liver tissue. [88, 89]. Such an observation was accounted for by the inactivation of - -SH groups by irradiation products of 8-methoxypsoralen. In contrast to these in vitro studies the in vivo studies in which male albino rats were fed 8-methoxypso- ralen mixed in with food for one week revealed inhibition in the glucose oxi- dation only in the livers by 23%, brain being almost unaffected [8].

Depression of macromolecular synthesis has also been observed for Ehrlich ascites tumor cells irradiated in the presence of psoralen [30]. However, oxy- gen consumption by these cells or by rat liver mitochondria was unaffected,

46

whereas the brain endogenous oxygen uptake was increased by low levels of furocoumarin derivatives and depressed by high levels [30,140,150,173]. Other studies on copper and glutathione levels in the blood and liver of male albino rats have shown that oral administration of 8-methoxypsoralen produced an in- crease in the copper level of the blood and a decrease in the liver. Such a pro- cess appears to be under the control of the pituitary gland, since changes in the copper levels were not observed when this organ was removed (El Auty 1959 quoted in ref. [243]). Finally pigment formation involving dopa oxidation is stimulated in the presence of 8-methoxypsoralen and near-UV radiation [5,39] as well as phenylalanine lyase and pisatin synthesis [114].

Inh ib i t i on o f D N A syn thes i s - - psoriasis t r e a t m e n t Many investigators have demonstrated that scheduled DNA synthesis is in-

hibited by photosensitization with furocoumarins (see Table II for summary). Furocoumarins not exhibiting skin-photosensitizing activity, such as xantho- toxol, are not able to inhibit incorporation of labelled precursors, the index used to measure DNA synthesising ability. For the cases in which the inhibition occurs, it appears that the degree of inhibition depends on the concentration of the active furocoumarin, the dose of radiation exposure, the type of furocou- marins, and the type of biological material used. This latter point, however, re- quires clarification because even though different strains of normal mouse spleen cells exhibit a difference in the dose of radiation required to produce the same effect with psoralen [34], human fibrobtasts treated with 8-methoxypso- ralen exhibited no difference over the range of doses studied [181,203]. The studies by Walter and Voorhees [280] also suggested a direct effect on DNA synthesis rather than a late GI-~ S inhibition or a block of the epidermal cell cycle occurs. All these reports suggested that a therapeutic use for psoralens might be in the t reatment of proliferating epidermal cell diseases such as psori- asis [280].

Psoriasis [24] is a common, chronic intractable skin disease that affects 1 to 3% of the worlds population [129]. It is a disorder of keratinization of the normal turn-over rate from basic to mature squamous cells to the epidermis; in normal epidermis the turnover rate of tissue is approximately once every 28 days whereas, in psoriatic epidermis, the division of the cells occurs every 3 to 4 days [281]. This dramatic increase in psoriatic epidermal turnover results in hyperplasia of epidermis and an increased population of cells in the S phase (DNA synthesis phase) at any one time [281].

Photosensitizing agents other than furocoumarins have been previously em- ployed in the t reatment of psoriasis. This includes topical coal tar preparations followed by frequent exposures to ultraviolet light (UV-B 290--320 nm radi- ation or UV-A 320--400 nm radiation) as is recommended in the Goecher- man regimen [221]. Recently topical therapy of psoriasis with psoralen and blacklight (UV-A or 320--400 nm) has been proposed by several investigators [ 268,269,277,280,283 ].

A pilot clinical study with topically applied trioxsalen to 11 patients with chronic plaque type psoratic lesions who were then exposed to blacklight, found improvement in 8 cases and a complete recovery in two [277]. Control sites subjected to exposure to blacklight or psoralen alone showed no visible

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48

change. A similar result was obtained with a large number of patients who had psoriasis vulgaris [280]. In this s tudy total recovery occurred in 34 patients, 23 and 11 patients exhibited improved and moderate recovery respectively, while the remaining six patients ' t reatment were discontinued after no improvement was observed. Lesions in the shaded areas of the scalp and ano-genital region al- so did not heal. It should be pointed out that topical t reatment with 8-meth- oxypsoralen or trimethylpsoralen invariably results in intense irregular, often long lasting hyperpigmentat ion and a high incidence of blistering reaction in the psoratic plaques as well as in the normal skin adjacent to psoratic plaques. Topical therapy in laboratories, is time consuming and often impractical. Re- cently, successful t reatment of psoriasis with orally administered 8-methoxy- psoralen and blacklight has been reported [204,284].

The interaction of an orally administered drug like 8-methoxypsoralen and exposure of skin to UV light produces therapeutic effects in patients with pso- riasis. This beneficial approach referred to as photochemotherapy involves the ingestion of 8-methoxypsoralen (0.6 mg/kg, body weight; average dose 40 mg) and delivery of UV-A (320--360 nm) to the entire body surface without signif- icant UV-B (290--320 nm) or infra-red. The patient is exposed to UV-A 2 h after an oral dose of 8-methoxypsoralen. The UV-A energy is delivered through a specially designed high intensity light system [284]. The initial exposure doses are determined primarily on the basis of complexion, degree of pigmenta- tion and patients ' sunburn and tanning histories, and usually range from 1--5 J/ cm 2. The initial report [204] on 21 patients showed complete clearing of ex- tensive psoriasis in 21 of 21 patients. A recent series of 91 patients indicated that this photochemotherapeut ic approach is significantly bet ter than the topical approach [284]. Over 360 psoriatic patients have been cleared success- fully by the Fitzpatrick and Wolff group and this t reatment is now institution- ally carried by 16 other University Centers throughout the United States with similar good results.

Inhibition of template activity Photobinding of furocoumarin to DNA induces loss of template activity for

RNA synthesis [35,54,91,238]. Several derivatives of furocoumarin (e.g. pso- ralen, 8-methoxypsoralen, 4,5' ,8-trimethylpsoralin, etc.) were used in this s tudy of the template activity of DNA. The inhibitory effect on template activ- ity has been correlated with the skin photosensitizing ability of various furo- coumarins. Aqueous solutions of 0.1% DNA were irradiated in presence of 10 g/ml of furocoumarins. The incorporation of [14C]AMP into RNA was mea- sured in the presence of irradiated and non-irradiated DNA solutions. Highest inhibition was observed with trimethylpsoralen, followed by psoralen, 8-meth- oxypsoralen and 5-methoxypsoralen. Xanthotoxol (8-hydroxypsoralen) re- vealed practically no inhibition of RNA polymerase reaction.

Irradiation of ribosomes in the presence of various furocoumarins and as- saying their template efficiency for protein synthesis in a cell free system is less conclusive. A partial inhibition was found for 8-methoxypsoralen, psoralen and bergapten which by contrast exhibit different skin photosensit ization proper- ties and also exhibit different capacities to photobind to RNA. Further inves- tigations in this area are desirable to understand the manner in which the nu-

49

cleo protein particles composed of protein and ribosomal RNA influence the functional activity of ribosomes and how furocoumarin photosensitization af- fects template activity.

Inactivation and toxicity

The first demonstrat ion of the photosensitizing effect of furocoumarins on bacterial inactivation was reported in 1958 by Fowlk et al. [102]. Using a filter paper-disc diffusion assay method they found 13 out of 25 furocoumarins tested to be active in inducing photosensitized lethal effects. The gram positive bacteria were more sensitive than the gram negative bacteria. Since that time other workers have demonstrated that a combined treatment of 8-methoxy- psoralen or psoralen and near ultraviolet light are also effective in inactivating a wide variety of other microorganisms and tissue culture cells (see sections A and B of Table III). In the case of inactivated viruses and tumor cells when the treated material was introduced into a host organism, such as a rabbit, the inac- tivated cells induced a protection against successive infection of the same viable infective material [67,136,188,193,195,198]. Recently, it has been demon- strated that the photosensiting inactivation of biological material is correlated with the formation of cross-links in the DNA by furocoumarin molecules (see section p. 62). This may also account for the absence of an inactivating effect of psoralen and near ultraviolet light on RNA viruses causing Teschen disease [67,193]. In a comparative study of the inactivating effect of equimolar con- centrations of different furocoumarins [67] on tissue culture cells it was ob- served that the degree of inactivation paralleled their skin photosensitizing abil- ity. Unreactive furocoumarins, from the point of view of skin sensitization, were also inactive in inactivating biological material.

At the multicellular level in the absence of light, furocoumarins at concen- trations of approx. 500 mg/kg body weight of rats and mice (see section C, Table III) have a toxic effect resulting in death. Similarly, plant growth is in- hibited; perhaps this might be the natural function of these compounds. With fish at a concentration of 1 : 93,000 parts of water these compounds narcotise or kill fish [257]. The narcotic effect is prevalent amongst cold blooded ani- mals and may be due to the action these compounds exert on the heart. Khad- zhai and others [148,149,153] have demonstrated that at about 10 ppm, im- peritorin, 8-isomylene-oxypsoralen, 8-methoxypsoralen, 5-methoxypsoralen and 9<limethylaminoethoxypsoralen, added to isolated rabbit heart, lowers the arterial blood pressure and tonus of the smooth muscle. The rhythm of the heart is stimulated and the amplitude of the heart contractions decreases.

In human beings in the latter part of the 19th century a large number of plants were reported to induce dermatitic action in people who unwittingly came into contact with them. (see section D, Table III for some examples.) In 1938 Kuske [158] provided the major unifying concept to these observations when he demonstrated experimentally that furocoumarins could be isolated from these plants and that when combined with sunlight on the surface of skin a phototoxic reaction resulted. However, under controlled clinical studies where the doses of the skin active furocoumarins were limited to an oral t reatment of 30--40 rag, only tanning and occasionally minor side effects were observed

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(nausea, itching, vomiting). Above this concentration various deleterious effects were noted, ranging from severe erythema to bullous eruption (see section E, Table III). Some subjects in these trials reported epigastric disturbances, nausea and sickness with orally administered medication; no such side effects were reported when the furocoumarins were administered topically. In all these studies and others [105,166,259] tests of liver function, blood counts and urine analysis were all normal. The inital concern that furocoumarin may be hepatotoxic (based on uncontrolled studies) has been dispelled by subsequent well-controlled studies [98,275,284] . Also, the 25 years of world-wide expe- rience of hundreds of physicians in India, Egypt and other countries including the USA who have treated thousands of vitiligo patients has demonstrated that 8-methoxypsoralen is a non-toxic drug when used at a concentrat ion in the therapeutic dose range. This is probably due to several factors such as: (a) oral- ly ingested furocoumarins and rapidly metabolized in the liver and excreted in the urine [ 209,210,222]; (b) orally ingested furocoumarins are rapidly excreted; over 90% of the administered dose is excreted within 8 - 1 2 h in the urine; (c) furocoumarins occur naturally in common edible plants such as celery, parsley, carrots, figs, etc.; (d) the human body has presumably evolved certain enzyme pathways to rapidly metabolize these furocoumarins.

Cataracts and morphologic changes in eyes have been described in certain experimental animals (that received extreme doses of 8-methoxypsoralen and UV-A for excessive periods) [103]. In patients receiving therapeutic doses of psoralen, no such adverse findings have been reported [221].

Carcinogenicity

The first investigation of the effect of 8-methoxypsoralen on tumor induc- tion was reported by O'Neal and Griffin in 1957 [200]. They indicated that in female Swiss albino mice fed dietary 8-methoxypsoralen the incidence of ear tumors induced by ultraviolet radiation of wavelengths below 320 nm (15 min daffy exposure for 110 days giving a total exposure of 15.2 to 17.5 × 108 ergs/ cm 2) was considerably less than that of the control receiving irradiation only. The degree of protect ion provided by the dietary 8-methoxypsoralen was pro- portional to concentrat ion up to an optimal dose of 0.5 gm/kg of ground Purina Laboratory Chow. (25% incidence compared with 6S% tumor induction in the control mice examined at 210 days.) However, in the same experimental series they found that intraperitoneal administration of 8-methoxypsoralen (0.4 mg/mouse/day for 110 days) appeared to increase tumor incidence (57% incidence compared with 68% tumor induction in control mice examined at 210 days). In the latter case, the incidence of tumor formation was found to be both concentrat ion and time dependent . No increase in tumor induction com- pared with the control was detected if the time interval between injection and exposure was greater than 20 h. Throughout this series of experiments the mice were kept in a room lighted by overhead fluorescent fixtures.

Fluorescent lamps are normally harmless and ineffective in inducing tumors in albino Swiss mice [116]. Dietary amounts of 0.5 g of 8-methoxypsoralen per kg of body weight combined with radiation exposure are also ineffective bu t interperitoneal injections of 0.4 mg 1 h before exposure results in 40%

53

tumor incidence in albino mice [112,116]. Large dietary doses (1 g 8-methoxy- psoralen per kg of diet} also led to tumor incidence after exposure to fluores- cent light. These experiments might explain why the cancer incidence in the mice receiving interperitoneal injections of 8-methoxypsoralen and treated with UV light (of wave-lengths less than 320 nm) exhibit a higher incidence of cancer than those mice receiving only irradiation. The additional exposure to overhead lighting coupled with high concentrations of the photosensitizer is probably responsible for the increased tumor incidences in the intraperitoneally injected 8-methoxypsoralen compared with the irradiated control [200].

The above studies indicated that different wavelengths of ultraviolet irradia- tion combined with 8-methoxypsoralen resulted in both protect ion against and induction of ultraviolet induced skin tumors in Swiss mice. Compounding these studies Griffin et al [112] in 1958 demonstrated that either interperitoneal in- jections (0.4 mg daily) or dietary 8-methoxypsoralen (0.5 gm/kg of chow) and subsequent exposure to germicidal radiation (254 nm) resulted in no tumor in- duction in Swiss mice. However in combinat ion with germicidal UV light (254 nm) exposures (total energy 8.3 × 108 ergs/cm 2) given over a period of 10 months the same tumor incidence was observed as in the irradiation mice re- ceiving 8-methoxypsoralen by an interperitoneal route. Similarly, exposed dietary treated mice exhibited a reduced tumor formation. When radiation from a Woods-Horn lamp (366) was employed (total dose of 108--10 l° ergs/ cm 2 over a period of 3 months) tumor formation was noted for both routes of administration of the 8-methoxypsoralen. No tumors were detected in mice receiving only 366 nm UV irradiation and the greater tumor induction was ob- served in the intraperitoneally injected 8-methoxypsoralen. Urbach [275] has also confirmed these findings of Griffin et al. [112].

Contrary to the foregoing findings, Pathak et al. [212] could detect no pro- tective effect of 8-methoxypsoralen or psoralen against ultraviolet tumor in- duction in mice. These workers administrated by oral intubation doses similar to those which had been given in the diet in the initial studies [200]. Recently Langner et al. [162] also reported the lack of tumor induction in hairless mice treated with oral 8-methoxypsoralen. Mice received either 1, 10, 20 or 40 mg per kg body weight of oral 8-methoxypsoralen. In mice treated with 30 and 40 mg/kg of 8-methoxypsoralen, severe progressive skin changes suggestive of ac- tinic damage (ulceration, scarring, mutilation) were observed. However, there was no evidence of any tumor induction and malignancies. Even though these findings possibly require further investigation supporting evidence that furo- coumarins at different concentrations variously protect organisms against far- UV light (265 nm, sensitize them to near-UV radiations (365 mm) as well as inhibition of dark repair of the photoproducts , has been repoted by Bridges [37,38]. He found that when 8-methoxypsoralen was presented at 100 pg/ml for 15 min before irradiation of E. coli WP2 with far-UV light (265 mm), even if washed out prior to exposure, there was a 3--4 fold reduction in mutant yield, whereas t reatment immediately after exposure was wi thout effect. In this case, 8-methoxypsoralen was thought to act by lowering the number of pyri- midine dimer products produced. When 8-methoxypsoralen was presented in broth enriched plates (40 pg/ml) increased numbers of mutants, by a factor of 5, in the UV treated WP2 but no increase in WP2 Hcr bacteria were observed

54

compared with the UV-treated bacteria plated on broth-enriched plates. These results have been confirmed by Igali and Gazso [134]. Previously, irradiation of these same strains in the presence of 8-methoxypsoralen with near ultraviolet light resulted in inactivated and induction of mutations [ 132].

The use of psoralen compounds as possible prophylactic agents against can- cer of the skin has been tested clinically [167]. In a double blind trial using single 20 mg daily oral doses per patient of either 8-methoxypsoralen or a pla- cebo no significant differences were observed between the drugs after a total trial period of 24 months. This suggested that the use of 8-methoxypsoralen as a prophylactic agent for skin cancer is ineffective. However, as pointed out above the various reactions exhibited by 8-methoxypsoralen in mice appear to depend on the concentration of the drug and the dose of radiation received, thus it is possible that in this particular trial the effective prophylactic dose was not reached. Finally, it must be pointed out that no significant increases in skin cancer induction was reported, although, like the absence of a prophylactic re- sponse of the compound this may be due to the trials monitoring for cancer being too short. This is particularly important if skin cancer like other forms of cancers appears to have a latency period of 20 years or so.

Teratogenicity

Apparently the only investigation that might be regarded as a teratogenic study is that of Colombo et al. [67] with sea urchin eggs. Neither psoralen nor near-UV light separately exhibited any effect on the development of sea urchin eggs. Combined treatment, however, of sperm used to fertilize untreated eggs variously produced giant cell [67] and polynucleate cell [66] formation in the developing embryos. Other effects were also noted which were dose dependent. With 100 pM (18 pg/ml) of psoralen and radiation doses greater than 100 ergs/cm 2 impairment of cleavage shrinkage of cytoplasm after fertilization with the formation of many asters was observed. Embryos which progressed to the 16-32 cell blastomere stage were irregular and those that managed to reach the blastula stage were also irregular as well as the embryo being cytolysed. At radiation doses of less than 100 ergs/cm 2 cleavage was regular until the blastula stage was reached at the same time as the untreated Paraccntrotus lividus em- bryo. However, at the primary mesenchyme formation stage further develop- ment was halted. The stopped blastulae or early gastrulae remained intact ap- proximately until the time when the control embryos reached the prism stage when they degenerated.

Mutagenicity

The mutagenic effect of furocoumarins was initially indicated by Musajo [178] and recently the subject of mutagenic induction by photosensitization has been generally reviewed by Zelle [290]. Musajo found that 5-methoxypso- ralen and psoralen were almost as effective as the very potent agent trypaflavin in the induction of chromosome aberrations. When tested on onion root tips, a 4-h incubation at 5 × 10 -s M concentration of these compounds induced aber- rations in 40% of the mitoses. At higher concentrations there was a total inhibi-

55

tion of mitosis [178]. It was not mentioned whether these experiments were performed in the presence or absence of light. However, recently, chromatid and chromosome aberrations have been reported to be induced by 8-methoxypsoralen [58] photosensitization in normal and Fanconi's anemic leukocytes [240].

With the exception of the report by Clarke and Wade [58] in which 8-meth- oxypsoralen is described as a mutagen inducing in the dark frame-shift muta- tions in a sensitive strain of E. coli K 12, furocoumarins do not exhibit any detectable mutagenic activity in the absence of radiation (see Table IV). With other bacterial strains used to routinely detect frame-shift mutagens no detect- able effect was observed in the absence of radiation [246]. The type of mu- tagenic alteration produced in the dark by 8-methoxypsoralen in E. coli K12/ ND 160 described by Clarke and Wade [58] may thus be different from those produced by other frame-shift mutagens (and thus only detectable in this par- ticular strain of bacteria), or may be the result of unknown cellular processes. Alternatively, the differing results may reflect differences in the test systems, i.e. count of mutan t papillae on wild-type colonies versus counts of mutant colonies with a background lawn of prototrophic bacteria.

The first decisive report on the mutagenic activity of furocoumarins came from Altenburge in 1956 [9]. He stated that neither 8-methoxypsoralen nor near-UV radiation alone are mutagenic when applied to the polar cap cells of Drosophilia eggs, but 8-methoxypsoralen in combination with very weak doses of near ultraviolet light gave mutat ion frequency of about 2.7% sex-linked recessive lethals compared to a control frequency of 0.3%. Similar results with Drosophila melanogaster have also been obtained by other workers [198].

Mathews [171] demonstrated the induction of mutat ion in Sarcina lutea. She observed that the induction of penicillin resistant mutants was not miti- gated by the presence of normal carotenoid pigment, as was found to be the case for the photodynamic action by visible light and toluidine blue [146]. On the basis of her finding, she concluded that photosensitization by 8-methoxy- psoralen was due to damage of cellular DNA and not cellular enzyme proteins.

The mutagenic effect of 8-methoxypsoralen was further characterized by Drake and McGuire with T4 bacteriophage [82]. They showed that about 4 X 10 -4 mutants were induced per lethal hit; a frequency characteristic of a weak mutagen. On the basis of the revertibility of mutants (induced by 8- methoxypsoralen plus near-UV radiation) by specific chemical mutagens, the majority of the induced mutants were concluded to be base-pair transitions (the substitution in DNA of one pyrimidine by another, or one purine by an- other), with a substantial minority of transversions (substitutions of a pyrimi- dine for a purine or vice versa). Transitions were also thought to be the type of mutat ion induced by the combined action of 8-methoxypsoralen and near-UV radiation [ 132,134].

Igali et al. [132] found that both types of t ryptophan revertants were in- duced in roughly equal numbers by 8-methoxypsoralen photosensitization. Similarly, equality of induced mutat ion frequency at different loci have also been obtained with T4 bacteriophage and Aspergillus nidulans [4,247]. With the bacteriophage T4 although there were small differences in the frequencies of rI and rII mutants, each was induced to approximately the same extent. Furthermore, the 91 rII mutants induced by the treament when mapped were

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found to be essentially randomly distributed throughout the rII locus. By con- trast, about half the spontaneously occuring rII mutants are found to be lo- cated in two highly mutable sites [21].

An equal number of mutants was also induced at the suppressor loci of methionine and the 2-thioxanthine resistant loci of Aspergillus nidulans [4, 247]. In the former forward mutational system each of three phenotypic clas- ses, comprising of two genetic loci each, exhibited equal frequencies of muta- tion after t reatment with 100 pg/ml of 8-methoxypsoralen and a variety of near ultraviolet radiation doses, whereas, the mutant distribution assayed by the 2-thioxanthine system which can detect mutat ion at more than 13 gene loci, was not statistically different from that which would be expected if the individual loci responded equally to the same extent [247]. The mutagenic in- duction thus appears to be radically different from other mutagens in that it acts randomly throughout the DNA.

At the chromosomal level of organization, peripheral blood lymphocytes from patients with and without Fanconi's anemia have been tested for their susceptibility to chromosome breakage after photosensitization in vitro. Again neither the t reatment with 8-methoxypsoralen (0.1 pg/ml) alone nor irradiation with 355 nm (2 × 10 s ergs/nm 2) UV alone were effective in increasing the yield of chromosome aberrations in either type of cell. However, simultaneous treat- ment with 8-methoxypsoralen and black light produced an extensive amount of chromatid aberrations in both cell types, compared to the untreated controls [240]. The Fanconi 's lymphocytes were 43.6 times more susceptible to chromosome breaks than the "normal" lymphocytes, and associated with the breaks, in both cases, a large number of chromatid exchanges were also ob- served [240]. Other types of nuclear events involving the nucleolus and spindle apparatus have also been reported for combined t reatment of other types of cells in vitro [95,172].

Cytoplasmic mutat ion has also been reported to be inducible by furocou- marin photosensitization. Trimethylpsoralen or 8-methoxypsoralen plus long wave UV light were found by Swanbeck and Thesso to be potent inducers of respiration deficient mutants in yeast [262]. (These mutations are believed to induce changes occurring in mitochondrial genes.) Trimethylpsoralen was about 100 times as potent as 8-methoxypsoralen and a significant increase in the mutat ion rate was obtained at a concentration of 10-7--10 .8 M.

Recent observations by Averbeck et al. [14] on photo-induced mutations by psoralens in yeast cells are also interesting. These investigators examined the mutagenic effects of psoralen and 8-methoxypsoralen which in presence of UV radiation form mono-functional (single strand linkage) and bifunctional (cross- links) adducts with nucleic acids. They also compared the mutagenic effects of angelicin (isopsoralen) that forms mono-functional adducts only. It was ob- served that: (a) psoralen and 8-methoxypsoralen were more effective in killing yeast cells than isopsoralen; (b) psoralen and 8-methoxypsoralen that are able to form crosslinks with DNA in presence of UV-A irradiations were more ef- ficient in the induction of nuclear mutations than irradiation of yeast cells with 254 nm UV light; (c) isopsoralen which forms mono-functional adducts only induced nuclear mutations at a frequency comparable to that obtained with 254 nm UV light; (d) cytoplasma "pet i te" mutations which indicate damage of

59

mitochondrial DNA were about as frequently induced by angelicin plus 365 nm light as with 254 nm UV light. Bifunctional adduct forming furocoumarins were significantly less effective in inducing petite mutations.

Repair of damage

An interaction of psoralen molecules with DNA under the influence of light at 365 nmJeads to the formation of mono-functional and bifunctional (cross- links) adducts with pyrimidines. The consequence of such cross-links of the DNA structure can be drastic as the genetic information encoded in the DNA structure would no longer be decipherable at the point of linkage of psoralen with pyrimidines. Thus, the important cell functions depending on translational and transcriptional processes would be inhibited or modified as long as there is alteration or reversion of nucleic acid sequence. In most cases, this is expressed in the form of cessation of cell division or death. Experimental evidence gathered in guinea pig skin and cultured human fibroblasts treated with tri- methylpsoralen or psoralen and UV light indicates that the damage due to the photobinding of furocoumarins is efficiently repaired by an efficient dark re- pair process [20,50].

Using bacterial and yeast strains defective in different DNA repair processes, evidence has been gathered suggesting that the excision as well as the recom- bination repair functions are needed to repair psoralen plus 365 nm light in- duced damage in DNA [16,17].

Apart from the fact that it is not photoreversible [20,51,53,132] damage produced by 8-methoxypsoralen plus near-UV light (366 nm) affects cellular material in essentially the same way as that produced by far-UV radiation (265 nm), in the process of excision repair, post-replication repair and mutagens.

Initially repair of damage induced by 8-methoxypsoralen plus ultraviolet radiation was studied using various repair deficient strains of E. coli WP2. It was found that both premutational and potentially lethal damage induced by photosensitization was excisable as judged by the comparative sensitivities of strains possessing and lacking excision repair capacity. A "reck-less" Rec- strain was sensitive to the lethal effect of 8-methoxypsoralen plus near UV radiation and was not detectably mutable; mutabili ty was also lacking in bacteria of an exr - mutant . Similar results were also obtained with various 8-methoxypsoralens plus near-UV radiation treated T phages assayed for survival with a variety of E. coli host strains differing in known dark-repair functions [107]. In these studies it was shown that in the case of phages T3 and T7 the photosensitized damage was repaired by excision repair and to a lesser extent by the host cell rec gene system. No influences of exr or p o I A gene function were detected. With phage T5 B the induced potentially lethal damage was found to be re- paired by the HCR (excision repair) and poIA system, but not by the exr or rec host system. In haploid cells of Saccharomyces carrying the single genes rad- 2-20 (defective in excision repair) or radg_4 (presumably deficient in a recom- binational repair) and the double mutan t rad2_2o radg_4 were more sensitive than the wild type to an exposure to 8-methoxypsoralen and near-UV. The double mutants ' greater sensitivity than either component indicates that at least two pathways are involved in the repair of this induced damage [15]. This was

60

also indicated by the induction of repressed phage ~ in various repair deficient E. coli K 12 strains after t reatment with trimethylpsoralen plus 360 nm light [128]. Greater sensitivity to inactivation in repair deficient strains compared with the corresponding wild-type strains was also observed with Bacillus sub- tilus [96] and T4 bacteriophage [230]. Repair deficient E. coli strains have also been shown to exhibit a greater 8-methoxypsoralen photobiologically in- duced lethal and mutagenic effect in the stationary phase than in the logarith- mic phase compared to those possessing repair capacity.

Biochemically, the biological consequences of light-induced psoralen deoxy- ribonucelic adducts and several E. coli functions (inactivation, and transfer of F lac ÷ episome) are correlated [59--62,64,65] . Wild-type E. coli cells survive treatments producing about 55--70 crosslinks per genome, while those defec- tive in excision repair (uv f ) and genetic recombination (recA-) can only sur- vive with 5--20 crosslinks per genome [62]. The double mutant (uvr- recA-) is killed by treatments producing not more than one crosslink per genome. Further, investigation by Cole demonstrated that during a 30-min incubation after psoralen plus irradiation, crosslinks were excised and cellular DNA cut in- to discrete pieces and Karu et al. [143] in their similar s tudy suggested that the crosslinking did not grossly distort the duplex structure of DNA. Additional incubation resulted in covalent joining of these fragments into high molecular weight DNA [59]. This rejoining did not occur in cells carrying a mutat ion at recA. Cole proposed that a mechanism involving sequential excision and genetic recombination may also account for the skin decrease in label bound to DNA to guinea pig after irradiation [20] and the 75% loss of 3H observed 72 h after irradiation of E. coli in the presence of furocoumarin [49]. Only 25% of this lost label was accounted for by crosslinks.

Proposed mechanism of action

The molecular mechanism of photosensit ization by the active furocoumarins is generally accepted to consist of a series of steps: (1) molecular complexing, (2) photobinding of furocoumarins to DNA and bases, and (3) cross-linking between strands of DNA.

This series of steps is summarized in Fig. 5 and the experimental evidence supporting each is discussed in the following paragraphs.

Molecular complexing Furocoumarins are able to form molecular complexes when added to an

aqueous solution of nucleic acids. Since these complexes of nucleic acids-furo- coumarins involve weak bonding forces, such as Van der Waal's, hydrogen bonding and hydrophilic forces, their formation is easily reversed.

Evidence supporting the existence of such complexes is that the solubility of furocoumarins in water is increased in the presence of nucleic acids [191]. With bergapten the solubility in a DNA solution was greater than in an RNA solution [76]. Accompanying this increased solubility, the viscosity of the DNA in- creases by 3.3 to 6.5% [237] and the melting point temperature of the DNA is increased by 1 to 1.5°C [77]. Spectroscopic properties of a DNA--furocou- marin complex also have an absorption maximum shift of 9--12 nm compared

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with the absorption maximum of the component parts examined separately [76] and the molar extinction coefficient is sharply decreased [76] . From these various observations and those on the photoadducts it is inferred that the furocoumarins intercalate between two pyrimidine bases of the DNA. Because both biologically active and inactive furocoumarins with DNA exhibit these effects this feature does not explain the photobiological property of the active furocoumarins. However, complexing is an important preliminary condition for the subsequent photobiological reaction.

Photobinding of furocoumarins to DNA and bases Fluorescence spectra of mixtures of furocoumarins and DNA exhibit only

slight modifications from the spectra of the components examined separately. However, after irradiation not only does the fluorescence intensity increase, but new maxima at shorter wavelengths appear, indicating that new species are formed. With inactive furocoumarins this spectral modification does not occur [193] . Associated with this the Tm value of the DNA changes markedly [_77]. Confirmation that the furocoumarins link to DNA was finally obtained when [~4C]bergapten was irradiated in the presence of DNA and one bergapten

62

molecule per 154 nucleotides of the DNA was found to be linked [72,196]. Further studies indicated that 1 : 1 adducts of [14C]psoralen and 3H nucleo- tides are formed when mixtures of polyU, polyC and polyT are irradiated in the presence of psoralen [95].

Only pyrimidine (thymine, cytosine or uracil) bases, nucleotides and nucleo- sides react with active furocoumarins [155,190,193,221,220]. No evidence has been obtained from either chromatographic studies or labelled radioactive tracer studies that furocoumarin react with purine (adenine or guanine) bases, nucleosides [190,193,194]. The new complexes formed between pyrimidines and furocoumarins were elucidated after isolation by column chromatography by elementary analysis plus UV, infra-red and nuclear magnetic resonance spec- troscopy [183,181,220,221]. The spectral studies indicated that active furo- coumarins combined with pyrimidine bases by C 4-cyclo addition. The pyrimi- dine bases react with their 5,6 double bond (in a way analogous to the forma- tion of pyrimidine dimers by UV-B alone), while the bonds involved from the furocoumarin component were either the 3,4 double bond or the 4',5, double bond. With the 4',5' adduct the coumarin nucleus remains intact [163,166]. These adducts, therefore, unlike those involving the 3,4 double bond have an absorption spectrum similar to the 7-hydroxycoumarin derivatives. Because the 3,4 photo adduct radically alters the molecule involved the UV absorption spectra is correspondingly altered. These two isomers are formed at different extents depending on the irradiation conditions; mainly, that of temperature [68].

Cross-linking between opposite strands of DNA If bonding of the active furocoumarin to the pyrimidine bases involves its 3,

4 double bond, no further absorption of radiation occurs with wavelengths greater than 320 nm, however, when the 4',5, double bond is involved, the complex can absorb additional quanta of radiant energy at 365 nm and react with another pyrimidine moiety in the opposite strand of the DNA. When such a reaction occurs the furocoumarin molecule crosslinks across the complemen- tary strands of the DNA. This concept is supported mainly by observations in- volving measurements of denatured and renatured DNA.

For instance, the absorbance of an irradiated mixture of psoralen and native DNA increases with increasing temperature (25--100 ° C) and does not decrease when the temperature is lowered [70,71]. Flow dichromism at 250 nm also in- dicates that only with the irradiated mixture of the components is the charac- teristic order of DNA restored after renaturing [170]. Direct evidence that cross-linking occurs between two strands of DNA with irradiation in the pres- ence of tripsoralen or psoralen is that after heat denaturation separation into single stranded DNA as observed by chromatography on a hydroxyapat i te column and alkaline sucrose gradient centrifugation decreases with radiation dose [63]. These results are similar to those observed with light scattering mea- surements which indicated that the molecular weight of irradiated mixtures was twice that of unirradiated mixtures after denaturation and renaturation [170]. Incidentally, these studies also indicate that no fission of the DNA backbone oc- curs. Furthermore, because the light scattering measurements [78] and flow dichromism measurements [170] of the irradiated DNA furocoumarin mixture

63

exhibit no differences from the unirradiated DNA furocoumarin mixtures, it is unlikely that cross-linking of the DNA furocoumarin mixture distorts the DNA helix to any extent.

The efficiency of forming cross-links varies from furocoumarin to furocou- marin. Cross-linking ability was greatest with 8-methoxypsoralen and com- pletely absent with angelicin [74]. This behavior of the furocoumarins has been explained by the efficiency of each compound in complying with the steric requirements for stacking between two pyrimidines in the complemen- tary strands [70]. Thus, the angular structure of angelicin does not exhibit cross-linking because the angular form of the molecule does not have the cor- rect steric relationship for reacting with the two pyrimidines. Also linear com- pounds with a methyl group in the 4 position sterically interfere with the methyl group of a second thymine reducing the ability of the substituted linear furocoumarins to cross-link DNA. No such interference is obtained for 5 or 8 substituted compounds.

Overall, there appears to be a correlation between the ability of furocou- marins to form cross-links and the degree of skin sensitization [69,70,73,74]. Similarly, the inactivation of chinese hamster cells by tripsoralen sensitization [27,28] and the inactivation of bacteria, phage [75] or the loss of transfer of intact F l a c ÷ episomes [62] by furocoumarin sensitization are mainly correlated with the formation of cross-links because the monomeric damage is removed by repair systems of these cells. However, as yet no one seems to have obtained a corresponding correlation between cross-linking or the monomeric damage and the induction of mutat ion.

Genetic hazards to man

Skin-sensitizing furocoumarins in combination with long-wavelength ultra- violet radiation (UV-A) induce point/gene mutations and chromosome aberra- tions in a variety of organisms. From these results, especially the chromosome aberrations induced in human cells in culture, it may be expected that genetic alterations may also be induced in man. Because unmetabolized furocoumarins do not seem to exhibit mutagenic properties without concomitant radiation it is not likely that genetic alterations in adult human germinal cells will occur due to the inability of the radiation to penetrate to these cells. It is, however, still possible that somatic cells, especially those of the epidermis, may undergo genetic alteration after combined treatment of furocoumarins and radiation, a process which may theoretically lead to cancer of the skin. The risk of such an outcome is dependent on a number of factors; the most important ones are concentration of the compound, the dose of radiation at the biologically im- portant site, and the duration of the treatment.

In the natural environment radiation exposure is highly variable and usually restricted to the areas of the skin having the highest pigment density, hence the maximum protected areas. The maximal possible radiation exposure is also slight in many areas of the world and in areas with possible high intensities of radiation. Exposure from the sun is usually restricted by clothing, etc. Added to this in areas of high intensities of radiation the majority of the population have the capacity to stimulate melinin pigment production. This biochrome is the

64

unique absorber of UV radiation (UV-A and UV-B). Ingestion of the active furocoumarins from food has not been accurately estimated although it is sug- gested that a maximal level of 1 mg per 24 h in the summer can be ingested [235]. Also the presence of these compounds in food, such as figs, exhibits a seasonal variation which reduces this level. Further reduction in the .ingested amount is also achieved when these food substances are boiled, because these compounds can be steam distilled. It should be pointed out that certain spices (cloves, coriander, fennel, etc.) which are rich sources of furocoumarins are in- gested daily by people living in tropical countries (India, Malaysia, Indonesia, Mexico, etc.). Perhaps an epidemiological study between these different races would indicate whether or not a hazard exists for the population.

With medical and cosmetic uses of these compounds the exposure is greater. Pathak, Dall 'Acqua and Rodighiero (unpublished observation quoted in ref. [221]) examined the rate of absorption, excretion and time at which they elicit maximum photosensitivity after single oral doses of psoralen or trimethylpso- ralen (40--250 mg/kg). With man, mice and guinea pigs the maximum concen- tration of the drug in the blood was found 2 to 3 h after oral ingestion; this corresponded to the time of maximal photosensitivity of the skin. Analysis of the excreta revealed that over 90% of the administered psoralens is excreted within 12 h and the remainder in the next 12 h. Repeated exposure to mice and guinea pigs also showed that these agents are primarily detoxified in the liver to either the hydroxylated products (hydroxylat ion either at the 3 or 8 position) or in the form of a glucuronate; no accumulation in the skin, liver or other organs was observed. This study, thus, establishes when and for how long the human to whom the drug is administered is possibly at risk. However, the corresponding data on the possible induction of cancer by various combined doses of radiation plus furocoumarins (oral as well as topical) is fragmentary and needs to be established. Although extensive clinical experience during the last 25 years has established that the non-toxic therapeutic dose (20--50 mg/70 kg ) [161] has so far not given rise to the de- velopment of skin carcinomas in patients with vitiligo who have been treated continuously with psoralens and deliberately exposed to radiation. Also, there are indications that at certain concentrations furocoumarins may be an effec- tive prophylactic against skin cancer by promoting melanogenesis and thicken- ing of the epidermis. However, like the induction of the skin cancer, the period of investigation in humans might as yet not been long enough, particularly if a latent period is involved in the induction of skin cancer.

Therefore, at the moment the evidence would tend to indicate that a thera- peutic t reatment regime of furocoumarins combined with radiation is not hazardous to man nor is the natural exposure from environmental sources. However, further examination especially into the relationship between radia- tion dose and concentration of these compounds versus the induction of dele- terious genetic effects plus a genetic investigation of the metabolic breakdown products is required to establish long term safety and effectiveness of these in- teresting therapeutic agents.

6 5

Ac knowledgements

The references cited in this review were obtained from various computerized sources, for these we acknowledge the expertise of Miss Barker of the University of Nottingham England (for the Biological Abstracts Search), Mr. John Wassom (for the EMIC search) and Mr. Ralph Hester (for the Chemcon, Index Medline and Science Citation Index searches). Special thanks is also due to Mrs. Jean Gordner and Mrs. Barbara Nichols for obtaining most of the information con- tained within this review, and Miss Amy Keeter for the unenviable task of typing it. Drs. Sparrow and Setlow are also thanked for their helpful criticims and suggestions in the latter stage of the preparation of the review. Dr. Madhu A. Pathak was supported by USPHS Grant No. CA 05003.

References

1 A b o u S h a d y , M.A.A. , A m m i majus Linn, Thesis for Master of P h a r m a c y , Facu l ty of Medicine, Cairo Univers i ty , 1 9 4 8 .

2 A h m a d , V.U. and A. Basha, I so la t ion of psora lene a nd angel ic in f r o m Psoralea plicata Pak., J. Sci. Ind . Res. , 15 ( 1 9 7 2 ) 1 5 8 - - 1 5 9 .

3 Akac i , B. a n d A. Damjan ic , D e t e r m i n a t i o n of t he ra t io of psora len to b e r g a p t e n in the leaves of Dal- m a l t i a n var ie t ies o f Ficus cariea, Acta . P h a r m . Jugos law. , 22 ( 1 9 7 2 ) 55- -58 .

4 Alde r son , T. a n d B.R. Scot t , The pho tosens i t i za t ing e f fec t of 8 - m e t h o x y p s o r a l e n on the inac t iva t ion an d m u t a t i o n o f Aspergi l lus con ida by near u l t ra-viole t l ight , Mu ta t i on Res. , 9 ( 1 9 7 0 ) 5 6 9 - -5 7 8 .

5 Ali, R. and C. Agarwa la , Mode of ac t ion of psora len in p i g m e n t p r o d u c t i o n . I I I . Pho to -ox ide of di- h y d r o x y p h e n y l a m i n e in p resence of psora len , India J. B iochem. , 2 ( 1 9 6 5 ) 2 7 1 - -2 7 4 .

6 Ali, R. a n d C. Agarwa la , E f f ec t of feed ing psora len on c o p p e r c o n t e n t of d i f f e ren t o rgans in a lb ino ra ts , Exper i en t i a . , 25 ( 1 9 6 9 ) 24 - -25 .

7 Ali, R. a n d C. Agarwa la , Mode of ac t i on of psora len in p i g m e n t p r o d u c t i o n . II . I nac t iva t ion of - -SH gaps by i r r ad ia ted psora len , J. Scient . Ind . Res. , 21C ( 1 9 6 2 ) 3 2 1 - - 3 3 3 .

8 All, R. a n d C. Agarwa la , In v i t ro and in v ivo e f fec t of n o r m a l an d i r rad ia ted psora len on glucose oxi- da t ion of b ra in and liver, E n z y m e , 18 ( 1 9 7 4 ) 3 2 1 - - 3 2 6 .

9 A l t enbu rge , E., S tudies of the e n h a n c e m e n t of m u t a t i o n ra te by carc inogens , Texas Rep. Biol. Med. , 14 (1956) 481 .

10 An tone l l a , G., Sintesi di me t i l i xan to toss ine , Gazz . Chim. Ital. , 88 (1958) 4 1 5 - - 4 2 9 . 11 Ar le t t , C.F. , S o m e obse rva t ions on UV and g a m m a - i n d u c e d m u t a t i o n s to 8 -azaguanine res is tance in

c u l t u r e d Chinese h a m s t e r cells, In t . J. Radia t . Biol., 20 (1971) 182. 12 Ar le t t , C.F., Mutagenesis in cu l t u r ed m a m m a l i a n cells, Studia Biophysica , 36 - -37 ( 1 9 7 3 ) 1 3 9 - -1 4 7 . 13 A s h w o o d - S m i t h , M.J. a nd E. Gran t , E f fec t of t e m p e r a t u r e on d o s e - d e p e n d e n t changes in the se-

d i m e n t a t i o n charac te r i s t i cs of bac te r ia l D N A p r o d u c e d , in vivo, by near -u l t rav io le t i r rad ia t ion and 8- m e t h o x y p s o r a l e n , C ryob io logy , 2 ( 1 9 7 4 ) 1 6 8 - - 1 6 9 .

14 A ve rbeck , D., S t ruc tu ra l specif ic i ty of p h o t o a c t i v e f u r o c o u m a r i n s induc ing genet ic d a m a g e in yeas t , 7 th In t . Congr. Pho tob io l . , R o m e , 1976 , pp. 137.

15 A v e r b e c k , D. a n d E. Mous tacch i , 8 -Me thoxypso ra l en plus 365 n m light e f fec ts and repa i r in yeast , Biochim. Biophys . Acta . , 395 (1975) 3 9 3 - - 4 0 4 .

16 A v e r b c c k , D. a n d E. Mous tacch i , 8 - M e t h o x y p s o r a l e n plus 365 n m by ef fec ts an d repai r in yeast , Bio- ch im. Biophys . Acta . , ( 1 9 7 6 ) in press.

17 A v e r b e c k , D.E. , R.K. Biswas and P. Chandra , P h o t o - i n d u c e d m u t a t i o n s by Psoralens in yeas t cells in P h o t o c h e m o t h e r a p y , in E. J u n g (ed.) F .K. S c h a t t a u e r Verlag, S tu t tga r t , G e r m a n y , (1976) 9 7 - - 1 0 4 .

18 A v r a m e n k o , L .G. , G.K. N i k o n o v o and G.P. Sharova , Acyl der iva t ives in a series of d i h y d r o f u r o - and f u r o e o u m a r i n s , and the co r re l a t ion b e t w e e n the i r s t ruc tu re and spasmoly t i c ac t iv i ty , Sb. Naueh . Rab . vses. Nauch-issled. Inst . Lek. Rast . , 1 ( 1 9 7 0 ) 1 5 3 - - 1 5 7 .

19 B a n d o p a d h y a y , M., S.B. Malik a nd T.R. Seshardr i , Coumar ins f r o m roo t s and seeds of Heracleum

canidicans, Ind i an J. Chem. , 11 (1973) 4 1 0 - - 4 1 2 . 20 Baden, H.P., J.M. Par r ing ton , J .D .A. De lhan ty and M.A. Pa thak , DNA synthes is in n o r m a l and

X e r o d e r m a p i g m e n t o s u m f ibroblas ts fo l lowing t r e a t m e n t wi th 8 - m e t h o x y p s o r a l e n and long wave l ight , Biochim. Biophys. Acta . , 262 (1972) 2 4 7 - - 2 5 5 .

21 Baskin, J .M., G.J . L u d l o w , T.M. Harr is and F.T. Wolf, Psoralen inh ib i to r seeds of Psoralea subacaulis

( L e g u m i n o s e a ) . , 6 ( 1 9 6 7 ) 1 2 0 9 - - 1 2 1 3 .

66

22 Baskin, J .M., F.T. Murrel l a nd F.T. Wolf, Psoralen in the seeds of Psoralea psoraloidea, Phy to . Rev. Inst . Bot. Exp. , 24 ( 1 9 6 7 ) 1 2 0 9 - - 1 2 1 3 .

23 Baskin, J.M. and J .T . Murrel l , Presence of psora len in the roots , leaves and f lowers of Psoralea sub- acaulis, J. Tenn . Acad. Sci., 43 ( 1 9 6 8 ) 25- -26 .

24 Becke , R.F . , Psoriasis: its na tu re and t r e a t m e n t , Drugs, 4 (1972) 85- -89 . 25 Becker , S.W., Psoralen p h o t o t h e r a p e u t i c agents , J. A m e r . Med. Assoc. , 202 ( 1 9 6 7 ) 4 2 2 - - 4 2 4 . 26 Be iderbeck , R., 8 - M e t h o x y p s o r a l e n m a c m t A g r o b a c t e r i u m tume fae iens , Ka lanchoe da igremont iana

und die T u m o r i n d u k t i o n e m p f i n d l i c h fur langwell iges UV, Z. Na tu r fo r sch . , 29c (1974) 5 7 2 - -5 7 6 . 27 Ben-Hut , E. and M.M. Elkind, DNA cross-l inking in Chinese h a m s t e r cells exposed to near -u l t rav io le t

l ight in the p resence of 4 ,SP,8- t r imethylpsora len , Biochim. Biophys. Acta . , 331 ( 1 9 7 3 ) 1 8 1 - -1 9 3 . 28 Ben-Hur , E. and M.M. Elkind, Psoralen plus near -u l t rav io le t l ight inac t iva t ion of cu l tu red Chinese

h a m s t e r cells a n d its r e la t ion to DNA cross-links, Mu ta t i on Res., 18 (1973) 3 1 5 - - 3 2 4 . 29 Berlin, C., F e i g e n b a u m d e r m a t i t i s , D e r m a t o l . Wochsch , 90 ( 1 9 3 0 ) 52 - -73 . 30 Bevi lacqua, R., F. Baccichet t i a nd L. Masajo, Ef fec t of i r rad ia t ion a t 365 n m in the presence of pso-

ra len on the o x y g e n c o n s u m p t i o n in ra t liver m i t r o c h o n d r i a an d in Ehrl ich ascites t u m o r cells. I tal ian J. B iochem. , 22 ( 1 9 7 3 ) 1 6 0 - - 1 6 6 .

31 Bevi lacqua, R. and F. Bordin, Photo-C 4 -cyc loadd i t ion of psora len and p y r i m i d i n e bases: effects of o x y g e n and p a r a m a g n e t i c ions, P h o t o c h e m . Pho tob io l . , 17 (1973) 1 9 1 - - 1 9 4 .

32 Bloomf ie ld , M., Sacred b o o k s of the east: h y m n s of A t h a r a Venda , Vol. XLI I , C la rendon Press, Oxfo rd , 1897 .

33 Blum, H.F. , P h o t o d y n a m i c ac t ion a nd disease caused by l ight (Re inho ld , New York , 1941) . This b o o k , w i th an up -da t ed a p p e n d i x , was r e p r in t e d in 1964 , by the Hafne r Publishing Co., N.Y.

34 Bordin, F., F. Bacc iche t t i a nd L. Musajo, Nucleic acids an d p ro t e in synthes is in mice t u m o r an d nor- ma l cells a f t e r i r rad ia t ion a t 365 n m in the p resence of psora len , Exper ien t ia , 30 (1974) 29- -30 .

35 Bordin , F., F. Baccichet t i , R. Bevi lacqua a nd L. Musajo, Inh ib i t ion of p ro t e in synthes is in Ehrl ich ascites t u r n o u t cells by i r r ad ia t ion (365 n m ) in the presence of sk in-photosens i t iz ing f u r o c o u m a r i n s , Exper i en t i a , 29 (1973) 2 7 2 - - 2 7 3 .

36 Bordin , F., F. Bacc iche t t i a nd L. Musa]o, Inh ib i t ion of nucle ic acid synthes is in Eher i ich ascites t u m o u r cells by i r r ad ia t ion in vi t ro in the presence of sk in-photosens i t iz ing f u r o c o u m a r i n s , Ex- per ient ia , 28 ( 1 9 7 2 ) 148.

37 Bridges, B.A., Gene t i c d a m a g e i nduc e d by 254 n m ul tra-violet light in Escherichia coll. 8-Methoxy- psora len as a p ro t ec t ive agen t and repa i r inh ib i tor , P h o t o c h e m . Pho tob io l . , 14 (1971) 6 5 9 - - 6 6 2 .

38 Bridges, B.A., 8 - M e t h o x y p s o r a l e n a nd rad ia t ion d a m a g e in bac te r ia , I n t e rna t . J. Radia t . Biol., 20

( 1 9 7 1 ) 185. 39 Brown, S.A., Role of d i h y d r o p s o r a l e n in f u r o c o u m a r i n m e t a b o l i s m , Can. J. Biochem. , 51 (1973)

9 6 5 - - 9 6 8 . 40 Buck, H.N. , I .A. Magnus a nd A.D. Por te r , The ac t ion s p e c t r u m of 8 - m e t h o x y p s o r a l e n for e r y t h e m a

on h u m a n skin. P re l imina ry s tudies wi th a m o n o c h r o m a t o r , British J. Derma to l . , 72 (1960) 2 4 9 - - 2 5 5 .

41 Capora le , G. and G. Antone l l a , Sintesi di a lcuni der ivat i f u r o c o u m a r i n i a , F a r m a c o ' Ed. Sci., 13 (1958) 3 6 3 - - 3 6 7 .

42 Capora le , G., F. Da l l 'Acqua , S. Magno, S. Maxciani an d A. Capozzi , Biosynthesis of psoralen, b e r g a P t e n and x a n t h o t o x i n in R u t a graveolans, Herb. Hung. , 10 (1971) 71- -77 .

43 Capora le , G., F. Da l l 'Acqua , A. Capozzi a nd S, Marciani° Biosynthesis of f u r o c o u m a r i n s in the leaves of Ficus carica, Ann. Chim. ( R o m e ) , 62 (1972) 536 - -545 .

44 Capora le , G., L. Musajo, G. Rod igh ie ro and F. Baccichet t i , Sk in-photosens i t iz ing ac t iv i ty of some me thy l -pso ra l ens , Exper ien t i a , 23 (1967) 9 6 5 - - 9 8 6 .

45 Car ter , D.M., D.V. McMacken a nd E.S. Condi t , Defense of cu t an eo u s cells against UV i r radia t ion . P h o t o m e d i a t e d b ind ing of t r i m e t h y l p s o r a l e n to D N A of m e l a n o m a cells in cu l tu re , J. Invest . De rma-

tol . , 60 ( 1 9 7 3 ) 2 7 0 - - 2 7 3 . 46 Car ter , D.M., B.V. J e ga s o thy a nd E.S. Condi t , Defense of cu t an eo u s cells against UV i r radia t ion . II.

Res t r i c t ed p h o t o m e d i a t e d b ind ing of t r i m e t h y l p s o r a l e n to p i g m e n t e d skin in vivo, J. Inves t . Derma- tol . , 60 ( 1 9 7 3 ) 2 7 4 - - 2 7 7 .

47 Ce t akon , M.M. and I .N. V i r o k u r o n , Psoralen t r e a t m e n t of Alcopec ia arccia in chi ldren, Pediatr ia , 4 6 ( 1 9 6 7 ) 1968 .

48 Chandra , P., P h o t o d y n a m i c ac t ion: a valuable too l in mo lecu l a r b io logy , in U. Gallo and L. Santa- mar i a (eds.) Resea rch Progress in Organic , Biological Medicinal Chemis t ry , Vol. 3, 1972, PP. 2 3 2 - - 2 5 8 .

49 Chandra , P., R .K. Biswas, F. Da l l 'Acqua , S. Marciani , F. Baccichet t i , D. Vedald i and C. Rodighiero , Pos t - i r rad ia t ion da rk r e c o v e r y of p h o t o d a m a g e to DNA in d u ced by fu rocournar ins , Biophysik , 9 ( 1 9 7 3 ) 1 1 3 - - 1 1 9 .

50 Chandra , P., F. Da l l 'Acqua , S. Mareiani and J. Rodigh ie ro , Studies on the repai r of D N A pho to - d a m a g e d by fu roeoumar ins° in M.A. Pa thak et al. (eds.) Sunl ight and man , Univers i ty of T o k y o Press, 1974 , pp. 4 1 1 - - 4 1 7 .

67

51 C h a n d r a , P. , S. K r a f t , A. Wake r , S. R o d i g h i e r o , F. D a l l ' A c q u a a n d S. Marc i an i , S tud i e s o n the r eac - t i v a t i o n o f b a c t e r i a p h o t o d a m a g e b y p s o r a l e n , B i o p h y s i k , 7 ( 1 9 7 1 ) 2 5 1 - - 2 5 8 .

52 C h a n d r a , P., S. Marc i an i , F. D a l l ' A c q u a , D. V e d a l d i , G. R o d i g h i e r o a n d R . K . Biswas , S t r u c t u r a l spe- c i f i c i ty o f p o l y d e o x y r i b o n u c l e o t i d e s f o r p h o t o r e a e t i o n w i t h p s o r a l e n , F E B S Le t t . , 35 ( 1 9 7 3 ) 2 4 3 - - 2 4 6 .

5 3 C h a n d r a , P. , G. R o d i g h i e r o , F. D a l l ' A e q u a , S. Marc i an i , S. K r a f t a n d A. Wacke r , R e a c t i v a t i o n o f b a c t e r i a p h o t o d a m a g e d b y f u r o c o u m a r i n s . S t u d . B i o p h y s . , 29 ( 1 9 7 1 ) 5 3 - - 6 1 .

5 4 C h a n d r a , P. a n d A. W a c k e r , P h o t o d y n a m i e e f f ec t s o n t h e t e m p l a t e a c t i v i t y o f nuc le i c ac ids , Z. N a t u r f o r s c h g . , 2 1 b ( 1 9 6 6 ) 6 6 3 - - 6 6 6 .

55 C h a k r a b o r t y , D.P. , A. Das G u p t a a n d P .K. Bose , O n the a n t i f u n g a l a c t i o n of s o m e n a t u r a l c o u m a r i n s , A n n a l s o f B i o c h e m i s t r y a n d E x p e r i m e n t a l Med ic ine , XVI I ( 1 9 5 7 ) 5 9 - - 6 2 .

5 6 Cla rk , J .H . , P h o t o s e n s i t i z a t i o n b y 8 - m e t h o x y p s o r a l e n , J . Inves t . D e r m a t o l . 37 ( 1 9 6 2 ) 1 7 1 - - 1 7 4 . 57 C la rke , C .H. a n d M.J . Wade , E v i d e n c e t h a t ca f f e ine , 8 - m e t h o x y p s o r a l e n a n d s t e ro ida l d i a m e r s a re

f r a m e s h i f t m u t a g e n s f o r E. col i K - 1 2 , M u t a t i o n Res . , 26 ( 1 9 7 5 ) 1 2 3 - - 1 2 5 . 58 C l o u d , T.M. , R . H a k i n a n d A.C. Gr i f f in , P h o t o s e n s i t i z a t i o n of the eye w i t h m e t h o x y s a l e n . II.

C h r o n i c e f f ec t s , A r c h . O p h t h a l . , 66 ( 1 9 6 1 ) 6 8 0 - - 6 9 4 . 59 Cole , R.S . , R e p a i r o f D N A c o n t a i n i n g i n t e r s t r a n d c ross l inks in Escherichia coli: s e q u e n t i a l e x c i s i o n

a n d r e c o m b i n a t i o n , P roc . Nat l . A c a d . Sci. U S A , 7 0 ( 1 9 7 3 ) 1 0 6 4 - - 1 0 6 8 . 6 0 Cole , R .S . P s o r a l e n m o n o a d d u c t s a n d i n t e r s t r a n d cross- l lngs in D N A , B i o c h i m . B i o p h y s . A c t a , 2 5 4

( 1 9 7 1 ) 3 0 - - 3 9 . 61 Cole , R .S . , Pso ra Ien - - a n e w D N A c ross - l i nk ing r e a g e n t a n d s o m e b io log i ca l a n d b i o c h e m i c a l conse-

q u e n c e s o f p s o r a l e n c ross l inks , B i o p h y s . Soc . A n n . Meet . , 1 5 ( 1 9 7 1 ) 1 8 0 A . 6 2 Cole , R.S . , I n a c t i v a t i o n o f Escherichia cole, F' e p i s o m e s a t t r a n s f e r , a n d b a c t e r i o p h a g e l a m b d a b y

p s o r a l e n p lus 3 6 0 n m l ight : s i gn i f i c ance of d e o x y r i b o n u c l e i c ac id c ross- l inks , J. Bae te r io l . , 1 0 7 ( 1 9 7 1 ) 8 4 6 - - 8 5 2 .

6 3 Cole , R .S . , L i g h t i n d u c e d c ross l i nk ing o f D N A in t h e p r e s e n c e o f f u r o c o u m a r i n p s o r a l e n , S tud i e s w i t h p h a g e l a m b d a , Escher ichia coil , a n d m o u s e l e u k e m i c cells, B i o c h i m . B i o p h y s . Ac t a . , 2 1 7 ( 1 9 7 0 ) 3 0 - - 3 9 .

6 4 Cole~ R.S . a n d R . R . S i n d e n , P s o r a l e n cross- l inks in D N A - b i o l o g i c a l c o n s e q u e n c e s a n d ce l lu lar r epa i r , R a d i a t i o n R e s e a r c h , 59 ( 1 9 7 4 ) 2 4 3 - - 2 5 3 .

6 5 Cole , R .S . a n d D. Z u s m a n , S e d i m e n t a t i o n p r o p e r t i e s o f p h a g e D N A m o l e c u l e s c o n t a i n i n g l igh t - in - d u c e d p s o r a l e n c ross l inks , B i o c h i m . B i o p h y s . A c t a , 2 2 4 ( 1 9 7 0 ) 6 6 0 - - 6 6 2 .

66 C o l o m b o , G. , P h o t o s e n s i t i z a t i o n o n sea u r c h i n s p e r m to long wave u l t r av io l e t l igh t b y p s o r a l e n , Cell Res , , 4 8 ( 1 9 6 7 ) 1 6 7 - - 1 6 9 .

6 7 C o l o m b o , G. , A .C . Levis a n d V. T o r l o n e , P h o t o s e n s i t i z a t i o n o f m a m m a l i a n cells a n d a n i m a l v i ruses b y f u r o c o u m a r i n s , P rog r . b i o c h e m . P h a r m a c o l . , 1 ( 1 9 6 5 ) 3 9 2 - - 3 3 9 .

6 8 D a U ' A c q u a , F . , S. Marc i an i , F . B o r d i n a n d R . Bev i l aqua , S tud ie s o n the p h o t o r e a c t i o n ( 3 6 5 n m ) b e t w e e n p s o r a l e n a n d t h y m i n e , Ric . Sci. ( R o m a ) , 38 ( 1 9 6 8 ) 1 0 9 4 - - 1 0 9 9 .

69 D a l i ' A c q u a , F . , S. Marc i an i , L. C i a v a t t a a n d G. R o d i g h i e r o , F o r m a t i o n of i n t e r s t r a n d c ross - l ink ings in t h e p h o t o r e a c t i o n s b e t w e e n f u r o c o u m a r i n s a n d D N A , S t u d . B i o p h y s . , 29 ( 1 9 7 1 ) 6 3 - - 7 0 .

7 0 D a U ' A c u q a , F. , S. Marc i an i , L. C i a v a t t a a n d G. R o d i g h i e r o , F o r m a t i o n of i n t e r s t r a n d c ross - l ink ings in t h e p h o t o r e a c t i o n s b e t w e e n f u r o c o u m a r i n s a n d D N A , Z. N a t u r f o r s c h . , 2 6 b ( 1 9 7 1 ) 5 6 1 - - 5 6 9 .

71 D a l l ' A c q u a , F., S. M a r c i a n i a n d G. R o d i g h i e r o , I n t e r - s t r a n d c ross - l inkages o c c u r i n g in t he p h o t o r e a c - t i v a t i o n b e t w e e n p s o r a l e n a n d D N A , F E B S Le t t . , 9 ( 1 9 7 0 ) 1 2 1 - - 1 2 3 .

72 D a l l ' A c q u a , F . , S. M a r c i a n i a n d G. R o d i g h i e r o , P h o t o r e a c t i v i t y ( 3 6 5 5 A) of va r ious s k i n - p h o t o s e n s i - t i z i ng f u r o c o u m a r i n s w i t h nuc le i c ac ids , L. N a t u r f o r s c h . , 2 4 b ( 1 9 6 9 ) 3 0 7 - - 3 1 4 .

73 D a l i ' A c q u a , F . , S. Marc i an i , D. V e d a l d i a n d H. R o d i g h i e r o , F o r m a t i o n o f i n t e r - s t r a n d c ro s s l i nk ings o n D N A o f gu inea pig sk in a f t e r a p p l i c a t i o n of p s o r a l e n a n d i r r a d i a t i o n a t 3 6 5 n m , FEBS Lef t . , 27 ( 1 9 7 2 ) 1 9 2 - - 1 9 4 .

74 D a l l ' A c q u a , F . , S. Marc ian i , D. V e d a l d i a n d D. R o d i g h i c r o , Sk in p h o t o s e n s i t i z a t i o n a n d c ros s l ink ings f o r m a t i o n in na t ive D N A b y f u r o c o u m a r i n s , Z. N a t u r f o r s c h . , 2 9 c ( 1 9 7 4 ) 6 3 5 - - 6 3 6 .

7 5 D a l l ' A c q u a , F . , S, Marc ian i , D. V e d a l d i a n d G. R o d i g h i e r o , F o r m a t i o n o f i n t e r - s t r a n d c ro s s l i nk ings of D N A in r e p r e s s e d p h a g e l a m b d a , Gene t i c s , 7 3 (Supp l . 1) ( 1 9 7 3 ) 8 5 - - 9 0 .

7 6 D a l l ' A c q u a , F. a n d G. R o d i g h i e r o , The d a r k i n t e r a c t i o n b e t w e e n f u r o c o u m a r i n s nuc l e i c ac ids , A t t i . A c a d , Naz. L ince i . C1. Sci. Fis. Mat . N a t u r . R e n d . , 4 0 ( 1 9 6 6 ) 4 1 2 - - 4 2 2 .

77 D a l l ' A c q u a , F. a n d G. R o d i g h i e r o , C h a n g e s in t he m e l t i n g cu rve o f D N A a f t e r the p h o t o r e a c t i o n w i t h sk in - sens i t i z ing f u r o c o u m a r i n s , A t t i . A e a d . Naz . L ince i . CI. Sci. Fis. Mat . N a t u r . R e n d . , 4 0 ( 1 9 6 6 ) 5 9 5 - - 6 0 0 .

78 D a l l ' A c q u a , F . , M. T e r h o j e v i c k a n d F. B e n v e n u t o , L i g h t - s c a t t e r i n g a n d v i scos ime t r i c s tud ie s o n D N A a f t e r t he p h o t o r e a c t i o n w i t h f u r o c o u m a r i n s , Z. N a t u r f o r s c h . h , 23 ( 1 9 6 8 ) 9 4 3 .

79 D a m j a n i e , A. a n d B. Akac i , F u r o c o u m a r i n s in Fieus caricia, P l a n t a Med. , 26 ( 1 9 7 4 ) 1 1 9 - - 1 2 3 . 8 0 Danie l s , F . J r . , C .E . H o p k i n s , D.J . I m b r i e , L. Be rge r son , O. Miller, F . C r o w e a n d T.B. F i t z P a t r i c k ,

F ie ld t r ia ls o f t h e s u n t a n p r o m o t i n g e f f ec t o f m e t h o x y s a l e n , J . Inves t . D e r m a t o l . , 32 ( 1 9 5 9 ) 3 2 1 - - 3 2 9 .

68

81 Del Vivo, G., A p ropos i t o de l iaz ione di a lcuni olei essenzial, silla p i g m e n t o z i o n e , Giorn. Ital. Derma- tol . Sif., 71 ( 1 9 3 0 ) 4 6 7 - - 4 8 0 .

82 Drake , J.W. and J. McGui re , Proper t i es of r m u t a n t s of b ac t e r i o p h ag e T4 p h o t o d y u a m i c a l l y i n d u ced in the p resence of t h i o p y r o n i n and psora len , J . Vi ro logy , 1 ( 1 9 6 7 ) 2 6 0 - - 2 6 7 .

83 Ecker , R.E. , P h o t o r e a c t i o n of 8 - m e t h o x y p s o r a l e n a n d DNA in vivo, R e p o r t an l -7970 (1972) 65~--66. 84 Eidler , Y.A. , G .L . Ge nk ina a n d T.T. Shaki rov , Gas-liquid c h r o m a t o g r a p h i c d e t e r m i n a t i o n of fu rocou-

m a t i n s in the p r e p a r a t i o n Psorberan , Kh im. Prir. Soedin. , 10 ( 1 9 7 4 ) 86- -97 . 85 Eidler, Y.A. , G.L. G e n k i n a a nd T.T. Shaki rov , S p e c t r o p h o t o m e t r i c analysis of a p sobe ran prepara-

t ion , K h i m . Prir. Soedin. , 10 (1974) 1 5 5 - - 1 5 6 . 86 E1-Hefrawi, M., M.F.S. E1-Haway a nd A. Rasheed , E l ec t ro p h o re t i c s tudies of se rum pro te ins in

vi t i l igenous pa t i en t s t r e a t e d w i t h oral psora len a n d UV i r rad ia t ion , J. Inves t . De rma to l . , 40 (1963) 1 1 1 - - 1 1 9 .

87 El Mof ty , A.M., A p r e l im ina ry clinical r e p o r t on the t r e a t m e n t of l e u k o d e r m a w i t h A m m i majus, Linn. J. R o y . Egy t i an MA., 31 ( 1 9 4 8 ) 6 5 1 - - 6 6 5 .

88 E1 Mof ty , A., A.M. El Mof ty , M. Abdu la l andM.F.S . E1-Hawary, Studies of the m o d e of ac t ion of psora len der iva t ives I the i r e f fec t on coppe r , g lu t a th ione levels in b lood and liver, J. Inves t . Derma- tol . , 32 ( 1 9 5 9 ) 6 4 5 - - 6 4 9 .

89 El Mof ty , A., A.M. El Mof ty , M. Abdula l and M.F.S. E1-Hawary, Studies of the m o d e of ac t ion of psora len derivat ives. I I . The p i tu i t a ry adrena l c on t ro l of c o p p e r m e t a b o l i s m and its r esponse to psora len , J. Invest . D e r m a t o l . , 32 ( 1 9 5 9 ) 6 5 0 - - 6 5 9 .

90 E1 Mof ty , A.M., A.M. N o t a w a i a nd S.H. T o p o u d z a d a , A p re l imina ry r e p o r t on the r ad io p ro t ec t i v e a nd radiosens i t iz ing e f fec t of 8 - m e t h o x y p s o r a l e n on a lb ino rats , J. Invest . Derma to l . , 43 (1964) 1 4 9 - - 1 5 0 .

91 Epste in , J .H . and K. F u k u y a m a , A s t u d y of 8 - m e t h o x y p s o r a l e n (8 MOP) induced p h o t o x i c ef fec t on m a m m a l i a n ep i de rma l m a c r o m o l e c u l a r synthes is in vivo, J. Inves t . Derma to l . , 54 (1970) 3 5 0 - - 3 6 1 .

92 Eps te in , W.L., K. F u k u y a m a and J .H. Eps te in , UV l ight DNA repa i r and skin cancers in man-sun ex- posure X e r d e r m a p i g m e n t o s u m , Fed. Proc. , 30 (1971) 1 7 6 6 - - 1 7 7 1 .

93 F a h m y , F .R. and P. A d u - S h a d y , A m r n i ma jus l inea: p h a r m a c o g n a s t i c a l s tudy and isolat ion of crysta l - line c o n s t i t u t a n t a m m o i d i n , Quar t . J. P h a r m . a nd Pha rmaco l . , 20 (1947) 2 8 1 - -2 9 1 .

94 Fa rah , F.S., A .K. K u r b a n and M.T. Chaglassin, T r e a t m e n t of virti l igo pa t ien ts wi th psora len and tri- macio l ine by m o u t h , Brit. J . D e r m a t o l . , 79 ( 1 9 6 7 ) 89- -91 .

95 Far id , S.C. a n d H. Kra uc h , P h o t o c h e m i c a l a nd biological r eac t ions of the f u r o c o u m a r i n s , Rad ia t i on Res. , 31 ( 1 9 6 7 ) 8 6 9 - - 8 8 6 .

96 Fe lkner , I.C., M.L. H a r t e r a nd A.D. L a u m b a c h , 4 -Ni t roqu ino l ine - l -ox ide and psora len lesions in Bacil lus subt i l l is , Ann . Meet . A m e r . Soc. Microbiol . , 1973 , ( 1 9 7 3 ) 70.

97 F i t zpa t r i ck , T.B., C.E. Hopk ins , D.D. Bl ickers taff a nd S. Swif t , A u g m e n t p i g m e n t a t i o n and s tero re- sponses of n o r m a l h u m a n skin to suns r ad i a t i on fo l lowing oral admin i s t r a t i on of 8 - m e t h o x y p s o r a l e n , J. Invest . De rma to l . , 25 ( 1 9 5 5 ) 1 8 7 - - 1 9 5 .

98 F i t zpa t r i ck , T.B., J .E. Imbr i e , D.H. L a b b y , Studies on l iver f u n c t i o n in subjects rece iv ing 8 - m e t h o x y - psora len , J. A m e r i c a n Med. Assoc ia t ion , 167 (1958) 1 5 8 6 - - 1 5 8 9 .

99 F i t zpa t r i ck , T.B. a nd M.A. Pa thak , His tor ica l aspects of m e t h o s a l e n and f u r o c o u m a r i n , J. Invest . De rma to l . , 32 (1965) 2 2 5 - - 2 3 8 .

100 Fowlks , W.L., T he m e c h a n i s m of the p h o t o d y n a m i c ef fec t , J. Inves t . Derma to l . , 32 (1959) 2 3 3 - - 247 .

101 Fowlks , W.L., The c h e m i s t r y of psora len , J. Inves t . De rma to l . , 32 (1959) 2 4 9 - - 2 5 4 . 102 Fowlks , W.L., D.G. Gr i f f i th a nd E.L. Oginsky, Pho tosens i t i za t ion of b a c t e r i a ' b y f u r o c o u m a r i n s and

re la ted c o m p o u n d s , Na tu re , 181 ( 1 9 5 8 ) 5 7 1 - - 5 7 2 . 103 F r e e m a n , R .G. , Morpholog ic changes resul t ing f r o m pho tosens i t i za t ion of the eye wi th 8 - m e t h o x y -

psora len . A c o m p a r a t i v e s tudy wi th c o n v e n t i o n a l UV in jury , Tex. Rep. Biol. Med. , 24 ( 1 9 6 6 ) 558- -

596. 104 F r e u n d , E., " U b e r b isher n o c b n icht besch r i ebene k/ inst l iche hautvcrf~/rbungen, De rma to l . Wosh-

schr . , 63 ( 1 9 1 6 ) 931 . 105 Fukush i , S.I. and M. T a n a k a , Studies on the c o m p o n e n t s of Fieus capica. VIII . On the m e c h a n i s m of

in jury of in jur ious ac t ion of psora len on Orcel p lan t g r o w t h r e sp i r a to ry e n z y m e s , Agric. Biol. Chem. , 28 ( 1 9 6 4 ) A7 (abs t rac t f r o m the Japanese original) .

106 Gla t t , O.S., T r e a m e n t of viti l igo, Medical T imes , 89 ( 1 9 6 1 ) 1 2 0 3 - - 1 2 9 7 . 107 Godsel l , A., T. Collis a nd C. Clarke, In f luence of hos t cell repa i r on survival of phages T3, T7 and

T5B af te r t r e a t m e n t wi th 8 - m e t h o x y p s o r a l e n plus long wave length UV light, S tudia Biophys . , 36 - -37 ( 1 9 7 3 ) 8 1 - - 9 6 .

108 Goldberge , L.C., The clinical use ol 8 - m e t h o x y p s o r a l e n , AntiBiol . Med. Clin. Ther . , 6 ( 1 9 5 9 ) 4 0 7 - - 411 .

109 Gonza lez , A.G. , J .T . Barroso, J .R. Luis a nd L.F. Rodr iguez , C o m p o n e n t s of Umbel i i fe rae . III . Struc- tu re of a n e w c o u m a r i n of H e r a c l e u m granatense , Ann . Quire. , 70 (1974) 3 6 9 - -3 7 0 .

69

110 Gonza lez , G.A. , R .R . Es tevez a n d E. Diaz Chicho, Ne w sources of na tu ra l coumar ins . XXVII . Pre- l iminary s t udy of c o u m a r i n s a nd alkaloids of R u t a bracteosa. Ann. Qn im, 70 (1974) 2 8 1 - - 2 8 2 .

111 Gri f f in , A.C., Me t hoxs a l e n in UV carc inogenes is in m o u s e , J. Invest . De rma to l . , 32 ( 1 9 5 9 ) 3 6 7 - - 3 7 2 . 112 Grif f in , A.C., R.E. Ha k in a n d J. K n o x , The wave leng ths ef fec ts u p o n e r y t h e m a l and carc inogenic re-

sponse in psora len t r e a t e d mice , J. Inves t . D e r m a t o l . , 31 ( 1 9 5 8 ) 2 8 9 - - 2 9 5 . 113 Gross, R. and L.B. Rob in s on , Belock d e r m a t i t u s ' unusua l de rma t i t i s an d p i g m e n t fo l lowing use of

p e r f u m e , Arch . D e r m a t o l . S y m p . , 21 ( 1 9 3 0 ) 6 3 7 - - 6 4 1 . 114 Hadwige r , L .A. , I n d u c t i o n of p h e n y l a lanine lyase a n d pisat in by pho tosens i t ive psora len com-

p o u n d s , P lant phys io l . , 49 (1972) 7 7 9 - - 7 8 2 . 115 H a k i m , R.E. , R .G. F r e e m a n , A.C. Grif f in , a n d J.M. K n o x , E x p e r i m e n t a l t ox ico logy studies on 8-

m e t h o x y p s o r a l e n in an imals e xpos e d to long UV, J. P h a r m . Expt l . The rap . , 131 ( 1 9 6 0 ) 3 9 4 - - 3 9 9 . 116 H a k i m , R.E. , A.C. Griff in a nd M. K n o x , E r y t h e m a a n d t u m o r f o r m a t i o n in m e t h o x s a l e n t r e a t ed

mice exposed to f luo rescen t l ight, Arch. De rma to l . , 82 ( 1 9 6 0 ) 5 2 2 - - 5 7 7 . 117 H a m m o n d , G.S., C.A. S t rou t a n d A.A. L a m o l a , Mechan i sms of p h o t o c h e m i c a l r eac t ions in so lu t ion .

XXV. T he p h o t o d i m e r i z a t i o n of c o u m a r i n , J. A m e r . C h e m . Soc., 86 ( 1 9 6 4 ) 3 1 0 3 - - 3 1 0 6 . 118 Harbe r , L.C., P h o t o c h e m i s t r y of psoriasis, New Eng land J. Med. , 291 (1974) 1 2 5 1 - - 1 2 5 2 . 119 Ha rbe r , L.C. a n d R.L. Baer, Ef fec t of h u m i d i t y on the pho tosens i t ive response of 8 - m c t h o x y p s o -

ralen, J. Inves t . D e r m a t o l . , 44 ( 1 9 6 5 ) 61 - -65 . 120 Haiiffs Tales t r ans l a t ed b y Margare t H. GaUia, The S to ry of Li t t le Muck, William Brooks & Co. L td .

pp. 1 3 1 - - 1 6 6 . 121 Ha rbc r , L.C. , R .L . Baer a n d D.R. Bickers, Techn iques of eva lua t ion of p h o t o t o x i c i t y an d pho toa l -

lergy in biologic sys t ems , inc luding m a n wi th pa r t i cu la r emphas i s on i m m u n o l o g i c aspects , in M.A. P a t h a k et al. (eds.) Sunl igh t and Man, Proceed ings of I n t e rn a t i o n a l Confe rence on Pho tosens i t i za t ion and P ro t ec t ion , T o k y o (1972) , Univers i ty of T o k y o Press, Chap te r 33, 1974 , pp . 515---528.

122 Her i chove , A., Cyto log ica l a nd e l ec t ron mic roscop ic s tudy of the inf luence of i sopropy l -n -pheny l - c a r b a m a t e on m e r i s t e m a t i c cells of ba r l ey ( H o r d e u m sa t iv ium) , Acta . Fac. R e r u m . Nat . Univ. Com- enianae Physiol . Plant , 4 ( 1 9 7 1 ) 29 - -62 .

123 Higuchi , T., Phys ica l chemica l analysis of pe r i c u t a ne ou s abso rp t i on process f r o m c r e a m s and oint- m e n t s , J. Soc. Cosmet . Chem. , 97 ( 1 9 6 0 ) 85 - -97 .

124 Hi rsch lberger , A. and H. Fuchs , 0 h e r die Ursache u n d die V o r a n s e t z u n g e n zur E n t s t e h u n g der Wiesendermat i t i s , im besonde re s in der die d u r e h pas t inak h e r v o r g e r u f e n e h a u t e n Zi indung, Muech. M e d . Wochschr . 83 ( 1 9 3 6 ) 1 9 6 5 - - 1 9 .

125 Hoern le , A .F .R . , Bower m a n u s c r i p t G o v e r n m e n t Pr in t ing Calcu t ta , India , pp. 1 8 9 3 - - 1 9 1 2 . 126 Hol l ander , A., Neus Aus der A m e r i k a n i s c h e n Derma to log ie (What ' s n e w in A m e r i c a n d e r m a t o l o g y ) ,

H a u t a r z t , 25 ( 1 9 7 4 ) 3 6 3 - - 3 6 7 . 127 Hopk ins , C.E., J .C. Belsario a nd E.J. MacDona ld , Psoralens p rophy lax i s vs skin cancer : r e p o r t on

clinical trial . II . , J . Inves t . De rma to l . , 41 ( 1 9 6 3 ) 2 1 9 - - 2 2 3 . 128 H o w a r d - F l a n d e r s , P. and P .F . Lin, Gene t i c r e c o m b i n a t i o n i n d u c e d by DNA cross-links in repress

phage l a m b d a , Genet ics , 73 (Suppl . ) (173) 85 - -90 . 129 H u r s t h o u s e , M.W., P h o t o d e r m a t o s e s : causes a nd m a n a g e m e n t , Drugs, 6 (1973) 2 5 5 - - 2 6 0 . 1 3 0 Ibn El Bitar, Mof rada EI -Adwiya , q u o t e d f r o m refs. 99 , 221. 131 Ison, A. a n d H. Blank, Tes t ing d rug p h o t o x i c i t y in mice , J. Inves t . Derma to l . , 49 ( 1 9 6 7 ) 5 0 8 - -5 1 1 . 132 Igali, S., B.A. Bridges, M.J. A s h w o o d - S m i t h a nd B.R. Scot t , Mutagenesis in E. coli. IV. Photosensi t i -

za t ion to near u l t ravio le t l ight by 8 - m e t h o x y p s o r a l c n , Muta t ion Res., 9 (1970) 21- -30 . 133 Igali, S. and L. Gazso , Photosens i t i z ing , le thal and m u t a g e n i c ac t ion of 8 - m e t h o x y p s o r a l e n an d near

UV l ight in Escherichia eoli, Stud. Biophys . , 29 ( 1 9 7 1 ) 45 - -52 . 134 Igali, S., and L .G. Gazso, Pro tec t ive ef fec ts of 8 - m e t h o x y p s o r a l e n on the le tha l and m u t a g e n i c ac-

t ion of u l t rav io le t i r rad ia t ion a t 254 n m in Escherichia coli strains w i th d i f fe ren t repai r capaci t ies , Mu ta t i on Res. , 21 ( 1 9 7 3 ) 37.

135 Im br i e , D.J . , F. Daniels , Jr . L. Bergeson, C.E. Hopk ins and T.B. F i t zpa t r i ck , Inc reased e r y t h e m a l t h r e sho l d six weeks a f t e r a single exposu re to sunl ight , J. Invest . De rma to l . , 32 ( 1 9 5 9 ) 3 3 1 - - 3 3 4 .

136 Jahnes , L.C.P~W.G., E.C. Mat ino a nd J .H. Lodge , Ant ivi ra l ac t iv i ty of 8 - m e t h o x y p s o r a l e n , Fed. Proc . , 21 ( 1 9 6 2 ) 457 .

137 Ja in , A.C., G.K. G u p t a and P.R. Rao , I so la t ion a nd cons t i t u t ion of coryl in , a new isoflavin f r o m the frui ts of Psoralea coryli fol ia, Ind i an J. Chem. , 12 ( 1 9 7 4 ) 6 5 9 - - 6 6 0 .

138 Jensen , T. and K.A. Mansen, Arch. D e r m a t o l . , 40 ( 1 9 3 9 ) 5 6 6 - - 5 6 8 . 139 Joseph , R., M.S. S h a n t h a m m a , F. R e h a n a a nd K. Nand , I n d u c e d m u t a t i o n s in Serratia marcescens by

nea r -UV light in the p resence of psora len , Exper ien t i a , 30 ( 1 9 7 4 ) 3 6 0 - - 3 6 1 . 140 Judis , J . , T he p h o t o - o x i d a t i o n of d i h y d r o x y p h e n y l a l i n c in the p resence of 8 - m e t h o x y p s o r a l e n , J. A m .

P h a r m . Assoc. Sci. Ed. , 49 ( 1 9 6 0 ) 4 4 7 - - 4 5 0 . 141 K a i d b e y , K .H . and A.M. K l i n g m a n , Top ica l photosens i t i ze rs . In f luence of vehicles on pen i t r a t ion ,

Arch . D e r m a t o l . , 110 ( 1 9 7 4 ) 8 6 8 - - 8 7 0 . 142 K a m m e r a u , B., U. Kleba , A. Zesch, H. Schaefer , Pene t r a t ion , p e r m e a t i o n and resp i ra t ion of 8-

m e t h o x y p s o r a l e n , Arch . D e r m . Forsch. , ( 1976) in press.

70

1 4 3 K a r u , A .E . a n d L. S t u a r t , U n c o u p l i n g o f t he r e c B C a t A T P a s e f r o m D N A a s e b y D N A c ros s - l i nked w i t h p s o r a l e n , P roc . Na t l . A c a d . Sci. U S A , 69 ( 1 9 7 2 ) 2 8 5 5 - - 2 8 5 9 .

1 4 4 K a t z , M. a n d B.J . P o u l s e n , A b s o r p t i o n o f d r u g s t h r o u g h the sk in , in B,B. B r o d i c a n d J . Gi le t te (eds.) H a n d b o o k o f E x p e r i m e n t a l p h a r m a c o l o g y , S p r i n g e r Ver lag , Vol . 28 , 1 9 7 1 , PP. 1 1 4 - - 1 7 4 .

1 4 5 K a u f m a n , K.D. , S y n t h e t i c f u r o c o u m a r i n s . I. A n e w s y n t h e s i s o f m e t h y l - s u b s t i t u t e d p s o r a l e n s a n d i sopso ra l ens , J . Org. C h e m . , 2 6 ( 1 9 6 0 ) 1 1 7 - - 1 2 1 .

I 4 6 K a u f m a n , K.D. , F .D . Gaiser , T .D . L e t h a n d L .R . W o r d e n , S y n t h e t i c f u r o c o u m a r i n s . II. S y n t h e s i s of severa l a l k l a t e d p s o r a l e n a n d o f d i h y d r o i s o p s o r a l e n , J . Org. C h e m . , 26 ( 1 9 6 0 ) 2 4 4 2 - - 2 4 4 6 .

147 K e l l y , E .W. , J r . a n d M. P inkes , L o c a l a p p l i c a t i o n o f 8 - m e t h o x y p s o r a l e n in vi t i l igo, J . Invest . D e r m a - to l . , 2 5 ( 1 9 5 5 ) 4 5 3 .

1 4 8 K h a d z h a i , Ya . I . a n d V .F . K u z n e t s o v a , F a r m a k o l o g i c h e s k e i ss ledoveneie f o t o s e n s i b i l r z i u i n s k c h e g o p r e p a r a t o b o r o b s a n a , F a r m a k o i . i. T o k o s i k o l 26 ( 1 9 6 3 ) 2 1 9 - - 2 2 4 .

1 4 9 K h a d z h a i , Ya . I . a n d V .F . K u z n e t s o v a , De i ak i f a r m a k o l o g i c h n i d a n i p r o biil l i m p e r a t o r i n a f a r m a t - sevt , Zn . , 1 7 ( 1 9 6 2 ) 5 7 - - 6 3 .

1 5 0 K i t a g a w a , M., C o u m a r i n der iva t ives fo r m e d i c a l p u r p o s e s . VII . E f f e c t o f c o u m a r i n der iva t ives o n the r e s p i r a t i o n o f r a t b r a i n t i s sue , J . P h a r m . Soc . J a p a n , 7 4 ( 1 9 5 4 ) 2 7 1 - - 2 7 8 .

1 5 1 K i t c h e v a t z , M., Gale c r o d t e u s e ( lds ions des ong les avec p r g s s e n t a t i o n de m o n l a g e ) , Bull. Soc. F r a n c , D e r m a t o l . , Sympi l i s . , 4 3 ( 1 9 3 6 ) 7 8 7 - - 7 9 1 .

1 5 2 K n i p l , Y.S. a n d O.N. Ku laeva , E f f e c t o f c o u m a r i n s a n d s y n t h e t i c g r o w t h r e t a r d a n t s o n R N A a n d p r o t e i n s y n t h e s i s in d e t a c h e d Bar ley M leaves in t he d a r k a n d l ight , F iz io l R a s t . , 17 ( 1 9 7 0 ) 5 4 9 - - 5 5 7 .

1 5 3 K o m i s s a r e n k o , N .F . a n d E .A . V a s i l ' C h e n k o , 9 - D i e t h y l a m i n o e t h o x y p s o r a l e n a n d i ts s p a s m o l y t i c ac- t i v i t y , K h i m i y a Pri~. Soedin.~ 1 0 ( 1 9 7 4 ) 8 0 - - 8 8 ( in Russ i an ) .

1 5 4 K r a u c h , C . H . S . F a r i d , S. D r a f t a n d A. Wacke r , Z u m W i x k u n g s m e c h i s m u s p h o t o d y m i s c h e r F u r o c o u - m a r i n e , B i o p h y s i k , 2 ( 1 9 6 5 ) 3 0 1 - - 3 0 2 .

1 5 5 K r a u c h , C .H. , D.M. K r ~ m e r a n d A. W a c k e r , Z u m W i r k u n g s m e c h a . n i s m u s p h o t o d y a m i s c h e r F u r o - c u m a r i n e , p h o t o r e a k t i o n y o n [ 4 - 1 4 C ] p s o r a l e n m i t DNS, R N S , h o m o p o l y n u c l e o t i d e a n d n u c l e o -

s iden , P h o t o c h e m . P h o t o b i o l . , 6 ( 1 9 6 7 ) 3 4 1 - - 3 5 4 . 1 5 6 K r y z h a r e k o v , A .N. a n d N .A . K a m b u l i n , I n f o r m a t i o n o n the p h a r m a c o l o g y of the p s o r a l e n p r e p a r a -

t i o n i s o l a t e d f r o m Psoralcn drupacea, T a s h k e n t i s k i i Med. Ins t . , 3 ( 1 9 6 3 ) 2 2 - - 2 8 . 1 5 7 K u s k e , H. , P e r c u t a n e o u s p h o t o s e n s i t i z a t i o n b y ac t ive s u b s t a n c e s a n d e x t r a c t s f r o m p lan t s , D e r m a -

to l . , 8 2 ( 1 9 4 0 ) 2 7 3 - - 2 7 5 . 1 5 8 Kuske~ H. , E x p e r i m e n t e l l e U n d e r s u c h u n g e n zu r P h o t o s e n s i b i l i s i e r u n g des H a u t d u t c h p f l a n z l i c h e

W i r k s t o f f s , A r c h v . F. , D e r m a t o l . Syph i l . , 1 7 8 ( 1 9 3 8 ) 1 1 2 - - 1 2 3 . 1 5 9 K u z o k i n a , I .N. , G .A . D u z n e t s o v a a n d A.M. Smixnov , S y n t h e s i s o f c o u m a r i n s in t he r o o t t issue of cul-

t u r e s o f Ruta graveolens, H e r b a H u n g . , 1 0 ( 1 9 7 1 ) 3 9 - - 4 2 . 1 6 0 K u z o k i n a , I .N. , G .A . K u z n e t s o v a a n d A.M. S m i r n o v , C o u m a r i n s y n t h e s i s in t he r o o t t i ssue of Ruta

graveolens g r o w n u n d e r s ter i le c o n d i t i o n s , Izv. A k a d . N a u k . U S S R , se t Biol . , 6 ( 1 9 7 1 ) 9 2 9 - 9 3 2 . 1 6 1 L a b b y , D.H. , J .D . I m b r i e a n d T.B. F i t z p a t r i c k , S tud ie s of l iver f u n c t i o n in sub jec t s r ece iv ing m e t h -

o x s a l e n , J . Inves t . D e r m . , 3 2 ( 1 9 5 9 ) 2 7 3 - - 2 7 6 . 1 6 2 L a m o l a , A . A . , F u n d a m e n t a l A s p e c t s o f the s p e c t r o s c o p y a n d p h o t o c h e m i s t r y o f o rgan i c c o m p o u n d s ;

e l e c t r o n i c e n e r g y t r a n s f e r in b io log ica l s y s t e m s ; a n d p h o t o s e n s i t i z a t i o n , in T.B. F r i t z p a t r i c k et al . (eds . ) S u n l i g h t a n d Man , P r o c e e d i n g s o f I n t e r n a t i o n a l C o n f e r e n c e o n P h o t o s e n s i t i z a t i o n a n d P ro t ec - t i o n , T o k y o ( 1 9 7 2 ) , Un ive r s i t y o f T o k y o Press . , C h a p t e r 2, 1 9 7 4 , PP. 1 7 - - 5 5 .

1 6 3 L a n g n e r , A. , H. Wolska , M.A. C h o r l z e l s k a a n d M. P a w i n s k a , D e r m a l t o x i c i t y o f 8 - m e t h o x y p s o r a l e n in ha i r less m i c e i r r a d i a t e d w i t h l o n g w a v e UV, J. Inves t . D e r m a t . , 6 4 ( 1 9 7 5 ) 2 7 9 - - 2 8 0 .

1 6 4 Legge , R .T . , O c c u p a t i o n a l d isease o f sk in a n d h a n d s in C a l i f o r n i a i n d u s t r y , Cal i f . S t a t e J . Med . ,

( 1 9 2 1 ) 4 6 1 - - 4 6 3 . 1 6 5 L e r n e r , A .B. , Vi t i l igo. J . Inves t . D e r m a t o l . , 32 ( 1 9 5 9 ) 2 8 5 . 1 6 6 L o n d o n , I .D. , E v a l u a t i o n o f m e t h o x s a l e n in t h e t r e a t m e n t o f vi t i l igo, J. Inves t . D e r m a t o l . , 32 ( 1 9 5 9 )

3 1 5 - - 3 1 7 . 1 6 7 M a c D o n a l d , E .J . , A .C . Gr i f f in , C .E . H o p k i n s , L. S m i t h , H. G a r r e t t a n d G.I . Black , Pso ra l en p r o p h y -

lax is vs sk in c a n c e r : r e p o r t o f c l in ica l t r ia ls . I. J . Inves t . D e r m a t o l . , 41 ( 1 9 6 3 ) 2 1 3 - - 2 1 7 . 1 6 8 M a n t u l i n , W,W. a n d P. S o n g , E x c i t e d s ta te o f sk in -sens i t i z ing c o u m a r i n s a n d p so ra l ens , J. A m e r .

C h e m . Soc . , 9 5 ( 1 9 7 3 ) 5 1 2 2 - - 5 1 2 9 . 1 6 9 Marc l an i , S., F. D a l l ' A c q u a , L. Gue l f i a n d D. Veda ld i , P h o t o r e a c t i v i t y ( 3 6 5 n m ) o f s o m e c o u m a r i n s

a n d 4 t ,5 t d i h y d r o f u r o c o u m a r i n s w i t h nuc le i c ac ids , Z. N a t u r f o r s c h . , 26 ( 1 9 7 1 ) 1 1 2 9 - - 1 1 3 6 . 1 7 0 Marc i an i , S., M. Te rbo jev ic a n d F. D a l l ' A c q u a , L i g h t s c a t t e r i n g a n d f l ow d i c h r o i s m s tud ie s o n D N A

a f t e r t he p h o t o r e a c t i o n w i t h p s o r a l e n , Z. N a t u r f o r s c h . , 27 ( 1 9 7 2 ) 1 9 6 - - 2 0 0 . 171 M a t h e w s , M.M., C o m p a r a t i v e s t u d y of t he l e tha l p h o t o s e n s i t i z a t i o n of Sarcina lutea b y 8 - m e t h o x y -

p s o r a l e n a n d t o l u i d e n e b l u e , J . Bac . , 8 5 ( 1 9 6 3 ) 3 2 2 - - 3 2 8 . 1 7 2 M a t s u h a s h i , S., E l e c t r o n m i c r o s c o p t c s t ud i e s o n the m o r p h o l o g i c c h a n g e s w i t h t ime of the n u c l e o l u s

b y 8 - m e t h o x y p s o r a l e n p h o t o x i c r e a c t i o n , N a g o y a S h i r i t s u D a i g a k u I g a k k a l Zasshi , 21 ( 1 9 7 1 ) 7 2 7 - -

7 4 6 .

7 1

1 7 3 M o n t a n a r i , R . a n d P. R o v e s t i , E n d g e n o u s a n d e x o g e n o u s p i g m e n t a t i v e a c t i o n o f t r i m e t h y l p s o r a l e n e - b a s e d s u b s t a n c e s , Riv . I ta l , Essenze . P r o f u m i . P i an t e Off ic . A r o m i . Sapon i . G o s m e t . Ae roso l . , 53 ( 1 9 7 1 ) 4 2 4 - - 4 2 6 .

1 7 4 M o o r e , T .A . , W.W. M a n t y l i n a n d P. S o n g , E x c i t e d s t a t e s a n d r e a c t i v i t y of c a r c i n o g e n i c b e n z p y r e n e : a c o m p a r i s o n w i t h sk in - sens i t i z ing e o u m a r i n s , P h o t o c h e m . P h o t o b i o l . , 18 ( 1 9 7 3 ) 1 8 5 - - 1 9 4 .

1 7 5 M o r i k a w a , F . , Y. N a k a y a m a , M. F u k u d a , M. H u m a n o , Y. Y o k o y a m a , T. N a g u r a , M. I sh iha r i a n d K. T o d a , T e c h n i q u e s f o r e v a l u a t i o n o f p h o t o x i e i t y a n d p h o t o a U e r g y in l a b o r a t o r y a n i m a l s a n d m a n , in T.B. F r i t z p a t r i c k et al . (eds . ) S u n l i g h t a n d Man , P r o c e e d i n g s o f I n t e r n a t i o n a l C o n f e r e n c e o n P h o t o - s e n s i t i z a t i o n a n d P r o t e c t i o n , T o k y o ( 1 9 7 2 ) , Un ive r s i t y o f T o k y o Press , C h a p t e r 34 , 1 9 7 4 , pp . 5 2 9 - - 5 5 9 .

1 7 6 Muker i i , A. , T o x i c i t y s t ud i e s o n p s o r a l e n , i s o p s o r a l e n a n d i m p e r a t o n i n in a l b i n o ra t s , J . Sci. Ind . Res . , 19 ( 1 9 6 0 ) 2 2 3 - - 2 2 5 .

1 7 7 Musa jo , L. , P h o t o c h e m i c a l i n t e r a c t i o n s b e t w e e n s k i n - p h o t o s e n s i t i z i n g f u r o c o u m a r i n s a n d D N A , in G. Silini (ed . ) R a d i a t i o n R e s e a r c h , P r o c e e d i n g s o f t h e 3 r d In t . C o n g r e s s o f R a d i a t i o n R e s e a r c h he ld a t C o r t i n a d ' A m p e z z o , I t a ly , J u n e - - J u l y 1 9 6 6 , 8 0 3 - - 8 1 2 .

1 7 8 Musa jo , L. , I n t e r c e s s a n t i p r o p r i e t a del le f u r o c o u m a r i n e , n a t u r a l i . II. F a m a c o , E. Sci . , 10 ( 1 9 5 5 ) 2 - - 7 . 1 7 9 Musa jo , L. a n d F. B a c c i c h e t t i , P r o t e c t i o n aga in s t Gra f f i s l e u k e m i a in mice t r e a t e d w i t h l e u k e m i c cells

p h o t o i n a c t i v a t e d b y p s o r a l e n , Eur . J . C a n c e r . , 8 ( 1 9 7 2 ) 3 9 7 - - 3 9 8 . 1 8 0 Musa jo , L. , F . B o r d i n a n d R . Bev i l acqua , P h o t o r e a c t i o n s a t 3 6 5 5 A, l i nk ing the 3-4 d o u b l e b o n d of

f u r o c o u m a r i n s w i t h p y r i m i d i n e bases , P h o t o c h e m . P h o t o b i o l . , 6 ( 1 9 6 7 ) 9 2 7 - - 9 3 1 . 181 Musa jo , L. , F. B o r d i n , L. Busu l inn i , F. B a c c i c h e t t i , R. Revi lac , 5 0 C o e f f ec t on m o u s e E h r l i c h ase i tes

t u m o r cel l in t h e p r e s e n c e o f s o m e c o u m a r i n der iva t ives , 1 9 6 2 S e c o n d In t . S y m p . R a d i o s e n s - p r o t e c - rive Drugs N o . 69 , R o m a , I n s t i t u t o , S u p e r i o r e de San i t a .

1 8 2 Musa jo , L., G. R o d i g h i e r o , G. C a p o r a l e , F. D a l l ' A c q u a , S. Marc ian i , F. B o r d i n , F . B a c c i c h e t t i a n d R. Bev i l acqua , P h o t o r e a c t i o n s b e t w e e n s k i n - p h o t o s e n s i t i z i n g f u r o c o u m a r i n s a n d nuc le i c Acids , In M.A. P a t h a k e t al (eds . ) S u n l i g h t a n d Man , P r o c e e d i n g s o f I n t e r n a t i o n a l C o n f e r e n c e in P h o t o s e n s i t i z a t i o n a n d P r o t e c t i o n , T o k y o ( 1 9 7 2 ) , Un ive r s i t y o f T o k y o Press , C h a p t e r 23 , 1 9 7 4 , p p . 3 6 9 - - 3 8 8 .

1 8 3 Musa jo , L. , F. B o r d i n , G. C a p o r a l , S. M a r c i a n i a n d C. R a g a t t i , P h o t o r e a c t i o n s a t 3 6 5 5 A b e t w e e n p y r i m i d i n e bases a n d sk in p h o t o s e n s i t i z i n g f u r o c o u m a r i n s , P h o t o c h e m . P h o t o b i o l . , 6 ( 1 9 6 7 ) 7 1 1 - - 7 1 9 .

1 8 4 Musa jo , L. , G. C a p o r a l e a n d G. R o d i g h i e r o , I s o l a m e t o Del B e r g a p t e n e da l s e d a n o e da l P r e z z e m o l o , G a z z . C h i m . I ta l , 8 4 ( 1 9 5 4 ) 8 7 0 - - 8 7 3 .

1 8 5 Musa]o , L. a n d G. R o d i g h i e r o , P h o t o - C 4 c y c l o - a d d i t i o n r e a c t i o n s t o nuc le ic ac ids in U. Gai lo a n d L. S a n t a m a r i a (eds.) R e s e a r c h P rog re s s in o r g a n i c , b io log i ca l a n d m e d i c a l c h e m i s t r y , Vol . 3, N o r t h - H o l - l a n d , A m s t e r d a m , 1 9 7 2 , p p . 1 5 5 - - 1 8 1 .

1 8 6 Musa jo , L. a n d G. R o d i g h i e r o , M o d e of p h o t o s e n s i t i z i n g a c t i o n o f f u r o c o u m a r i n s , P h o t o p h y s i o l o g y , 7 ( 1 9 7 2 ) 1 1 5 - - 1 4 7 .

1 8 7 Musa jo , L. a n d G. R o d i g h i e r o , P h o t o s e n s i t i z i n g p r o p e r t i e s o f f u x o c o u m a r i n s , H e r b a H u n g . , 1 0 ( 1 9 7 1 ) 7 9 - - 9 4 .

1 8 8 Musa jo , L. a n d G. R o d i g h i e r o , S tud i e s o n the C-4 c y c l o a d d i t i o n r e a c t i o n s b e t w e e n s k i n - p h o t o s e n - s i t i z ing f u r o c o u m a r i n s a n d nuc l e i c ac ids , P h o t o c h e m . P h o t o b i o l , 11 ( 1 9 7 0 ) 2 7 - - 2 5 .

1 8 9 Musa jo , L. a n d G. R o d i g h i e r o , The s k i n - p h o t o s e n s i t i z i n g f u r o c o u m a r i n s , E x p e r i e n t i a , 1 8 ( 1 9 6 2 ) 1 5 3 - - 1 6 1 .

1 9 0 Musa]o , L. a n d G. R o d i g h i e r o , Sul m e c c a n i s m o d ' a z i o n e de l la f u r o c o u m a r i n e Po tosens ib i l i z za t r i c i , At t i . A c a d . N a x . L ince i . Cl. Sci . Fis. Mat . N a t u r . R e n d . , 3 8 ( 1 9 6 5 ) 5 9 1 - - 5 9 9 .

1 9 1 Musa jo , L. , G. R o d i g h i e r o a n d L. S a n t a m a r i a , Le sos taze f o t o d i n a m i c h e c o n p a r t i c o l a r e r i g u a r d o alle f u r o c o u m a r i n e , A t t i . Del la Soc . I ta l . Pa to l . , 5 ( 1 9 5 7 ) 1 - - 7 0 .

1 9 2 Musa jo , L. , G. R o d i g h i e r o , G. C a p o r a l e a n d C. A n t o n e l l o , Ul te r io r i r i c h e r c h e sui r a p p o r t i f r a cons t i - t u z i o n e e p r o p r i e t a ' f o t o d i n a m i c h e nel c a m p o del la f u r o c o u m a r i n e , F a r m . (Pavia) EC. Sci., 13 ( 1 9 5 8 ) 3 5 5 .

1 9 3 Musa jo , L. , G. R o d i g h i e r o , G. C o l o m b o , V. T o r o l o n e a n d F. D a l l ' A c q u a , P h o t o s e n s i t i z i n g f u r o c o u - m a r i n s : i n t e r a c t i o n s w i t h D N A a n d p h o t o - i n a c t i v a t i o n of D N A c o n t a l n g viruses , E x p e r i e n t i a , 21 ( 1 9 6 5 ) 2 2 - - 2 4 .

1 9 4 Musa jo , L. , G. R o d i g h i e r o , A . E recc i a , F . D a l l ' A c q u a a n d G. Malesani , Sk in p h o t o s e n s i t i z i n g f u r o c o u - m a t i n s : p h o t o c h e m i c a l i n t e r a c t i o n vs D N A a n d - - 0 1 4 C H 3 b e r g a p t e n (5 m e t h o x y p s o r a l e n ) , P h o t o - c h e m . P h o t o b i o l . , 5 ( 1 9 6 6 ) 7 3 9 - - 7 4 5 .

1 9 5 Musa jo , L. , P. V i sen t in i , F . B a c c i c h e t t i a n d M.A. Razz i , P h o t o i n a c t i v a t i o n o f Eh r l i ch asc i tes t u m o r cells in v i t ro o b t a i n e d w i t h s k i n - p h o t o s e n s i t i z i n g f u r o c o u m a r i n s , E x p e r i e n t i a , 23 ( 1 9 6 7 ) 3 3 5 - - 3 3 6 .

1 9 6 M u s t a f a , A. , F u r o p y r a n s a n d f u r o p y r o n e s , I n t e r s c i ence ( L o n d o n ) , ( 1 9 6 7 ) 1 4 - - 9 0 . 1 9 7 N a k a y a m a , Y., F. M o r i k a w a , M. F u k u d a , M. M a m a n o , K. T o d a a n d M.A. P a t h a k , M o n o c h r o m a t i c

r a d i a t i o n a n d i ts a p p l i c a t i o n - - l a b o r a t o r y s t ud i e s o n the m e c h a n i s m o f e r y t h e m a a m d p i g m e n t a t i o n i n d u c e d b y p s o r a l e n s in P a t h a k , M.A. Marbes , L .C. , Seji , M. a n d K u k i t a , A. (eds . ) S u n l i g h t a n d Man , Un ive r s i t y o f T o k y o Press , 1 9 7 4 , pp . 5 9 1 - - 6 1 2 .

1 9 8 N ico l e t t i , N. a n d G. T r i p p a , S u l l ' a z i o n e m u t a g e n a del p s o r a l e n e i r r a d i a t e s c o n luce u l t r a v i o l e t t a in Drosophila melanogaster, R e n d . A c c a d . Naz . L ince i . ( R o m a ) , 43 ( 1 9 6 7 ) 2 5 9 - - 2 6 3 .

72

199 Oginsky , E.K. , G.S. Green , D.G. Gr i f f i th a nd W.L. Fowlks , Le tha l pho tosens i t i za t i on of bac te r ia w i th 8 - m e t h o x y p s o r a l e n to long wave l eng th UV rad ia t ion , J. Bac ter io logy , 78 (1959) 8 2 1 - - 8 3 3 .

200 O 'Neal , M.A. a n d A.C. Griff in , The e f fec t of oxyps o ra l en u p o n u l t ravio le t carc inogenes is in albino m i ce , Cance r Research , 17 ( 1 9 5 7 ) 9 1 1 - - 9 1 6 .

201 O p p e n h e i n , M. and A. Fessler, t~ber eine St re i fenf i / rnuge bull6se d e r m a t i t u s ( F r e i b a d u n d wiesender- mi t i t i s ) , D e r m a t o l . Wochschen . , 86 ( 1 9 2 8 ) 1 3 8 - - 1 8 7 .

202 Owens , D.W., J.M. Gi lksman , R .G. F r e e m a n a nd R. Carnes, Biologica ac t ion s p e c t r u m of 8 - m e t h o x y - Psora len d e t e r m i n e d by m o n o c h r o m a t i c l ight , J. Invest . Derma to l . , 52 ( 1 9 6 8 ) 4 3 5 - - 4 4 0 .

203 Pa r r ing ton , J .M., J .D.A. De lhan ty a nd H.P. Baden, Q u o t e d in ref . 16, Ann . H u m a n . Gene t . L o n d o n , 35 ( 1 9 7 1 ) 149 .

204 Parrish, J .A . , T.B. F i tpa t r i ck , L. T a n e n b a u m and M.A. Pa thak , P h o t o c h e m i s t r y of psorisis wi th oral m e t h o x y p s o r a l e n and l ongwave UV light, Ne w England J. Med., 291 ( 1 9 7 4 ) 1 2 0 7 - - 1 2 1 1 .

205 Pa thak , M.A., P h y t o p h o t o d e r m a t i t i s , in T.B. F i t z pa t r i c k et al. (eds.) Sunl igh t and Man, Proceed ings o f I n t e r n a t i o n a l Confe rence on Pho tosens i t i za t ion a nd P ro tec t ion , T o k y o (1 9 7 2 ) , Univers i ty of T o k y o Press, C hap t e r 32~ 1974 , pp. 4 9 5 - - 5 1 4 .

206 Pa thak , M.A., Mechan i sm of spora len pho tosens i t i za t i on an d in vivo biological ac t i on s p e c t r u m of 8- m e t h o x y p s o r a l e n , J. Inves t . De rma to l . , 37 ( 1 9 6 1 ) 3 9 1 - - 4 0 6 .

207 Pa thak , M.A., Basic aspects of c u t a n e o u s pho tosens i t i za t i on , in F. Urbach , (cd.) The biological ef- fects o f UV Rad ia t i on wi th Emphas i s on the Skin, P e r g a m o n , L o n d o n , 1 9 6 9 , pp. 4 8 9 - -5 1 1 .

208 Pa thak , M.A., B. Allen, D. Ingra in a nd J .H. Fe l lman , Pho tosens i t i za t ion and e f fec t of u l t ravio le t r a d i a t i on on the p r o d u c t i o n of u n p a i r e d e l ec t rons in the p resence of f u r o c o u m a r i n s , Biochim. Bio- phys . Acta , 54 ( 1 9 6 1 ) 5 0 6 - - 5 1 5 .

209 Pa thak , M.A., F. D a l l ' Ac qua , G. Rod igh ie ro and J .A. Parrish, Clinical Resea rch XXI I , 5 7 8 A, 1974 . 210 Pa t hak , M.A., F. Da l l 'Acqua , G. Rod igh ie ro and J .A. Parrish, Metabo l i sm of psoralens , J. Invest .

D e r m a t o l o g y , 62 ( 1 9 7 4 ) 347 . 211 Pa thak , M.A., F. Daniels and T.B. F i t zpa t r i ck , The p resen t ly k n o w n d i s t r ibu t ion of f u r o c o u m a r i n s

(psora len) in plants , J. Inves t . De rma to l . , 39 (1962) 2 2 5 - - 2 4 9 . 212 Pa thak , M.A. , F. Daniels, C.E. H o p k i n s a n d T.B. F i t zpa t r i ck , Ul t rav io le t carc inogenesis in a lbino and

p i g m e n t e d mice rece iv ing f u r o c o u m a r i n s , psora len and 8 - m e t h o x y p s o r a l e n , Na tu re , 183 ( 1 9 5 9 ) 7 2 8 - - 7 3 0 .

213 Pa thak , M.A. and J .H. F e n m a n , Pho tosens i t i za t ion by f u r o c o u m a r i n s : psoralens , 3rd In t e rna t iona l Congress on Pho t ob io logy , C o p e n h a g e n ( 1 9 6 0 ) in B. Chr is tensen and B. Buchlaaann, (Eds.) Progress in p h o t o b i o l o g y , Elsevier Publ ishing Co., A m s t e r d a m .

214 Pa thak , M.A., J .H. Fe l lman a nd D. K a u f m a n , The ef fec ts of s t ruc tu ra l a l t e ra t ions on the e r y t h e m a l f u r o c o u m a r i n s : Psoralens , J. Inves t . D e r m a t o l . , 35 (1960) 1 6 5 - - 1 8 3 .

215 Pa thak , M.A. a n d J .H. Fe l lman , Act iva t ing and f lou rescen t wave- leng ths of f u r o c o u m a r i n s , psoralens , Na tu re . 185 (1960) 3 8 2 - - 3 8 3 .

216 Pa thak , M.A. and T.B. F i t zpa t r i ck , Bioassay of na tu ra l and syn the t i c f u r o c o u m a r i n s (psoralen) , J. Inves t . D e r m a t o l . , 32 ( 1 9 5 9 ) 5 0 9 - - 5 1 8 .

217 Pa thak , M.A. and T.B. F i t zpa t r i ck , Re la t ionsh ip of mo lecu l a r con f igu ra t ion to the ac t iv i ty of furo- c o u m a r i n s w h i ch increase the c u t a n e o u s r e sponses fo l lowing long wave u l t ravio le t r ad ia t ion , J. In- vest . Dc rma to l . , 32 ( 1 9 5 9 ) 255.

218 Pa thak , M. A. a n d W.L. Fowlks , The in vivo de s t ruc t i on of succinic d e h y d r o g e n a s e ac t iv i ty of guinea- pig skin by Pho tosens i t i za t ion wi th psora len or m e t h o x y s a l c n , J. Inves t . De rma to l . , 37 (1961) 1 8 3 - - 187.

219 Pa thak , M.A., K.D. K a u f m a n a nd L .R. Worden , Ef fec t of s t ruc tu ra l a l t e ra t ions on the photosens i t i - za t ion p o t e n c y of f u r o c o u m a r i n s (psora lens) a nd re la ted c o m p o u n d s , J. Inves t . Derma to l . , 58 ( 1 9 6 7 ) 1 0 3 - - 1 1 8 .

220 Pa thak , M.A. and D.M. K r a m e r , Pho tosens i t i za t ion of skin in vivo by f u r o c o u m a r i n s (psoralcns) ,

Biochim. Biophys . Acta . , 195 ( 1 9 6 9 ) 1 9 7 - - 2 0 6 . 221 Pa thak , M.A. , D.M. Kr~/mer and T.B. F i t zpa t r i ck , P h o t o b i o l o g y and p h o t o c h e m i s t r y of f u rocou -

m a t i n s (Psoralens) , in M.A. P a t h a k et al. (eds.) Sunl ight and Man, Proceedings Univers i ty of T o k y o Press. C hap t e r 22, 1974 , pp. 3 3 5 - - 3 6 8 .

222 Pa thak , M.A., B. Mandula , Y. N a k a y a m a and J .A. Parrish, Cu taneous pho tosens i t i za t ion an d in vivo m e t a b o l i s m of psora len , J. Inves t . D e r m a t o l o g y , 64 ( 1 9 7 5 ) 279.

223 Perone , V.B., The na tu ra l o c c u r r e n c e and uses of tox ic c o u m a r i n s , Microbial Toxins , 8 ( 1 9 7 2 ) 67 - -92 . 224 Per ry , H.O. , C.W. S o d e r s t r o m and R.W. Schulze , Arch . Derma to l . , 98 ( 1 9 6 8 ) 1 7 8 - -1 8 2 .

Pe tzo ld t , D. and A.L. Reich , Oral 8 - m e t h o x y p s o r a l e n and t r imac in o lo n e in virti l igo, Hau t a r z t , 25 ( 1 9 7 4 ) 1 9 1 - - 1 9 4 .

225 P h y l a d e l p h y , A., De rma to l . , Wochschr . , 92 (1931) 713, q u o t e d in ref. [211 , 2 2 1 ] . 226 Pinkus, H., Viti l igo - - w h a t is i t? J. Invest . Derma to l . , 32 (1959) 2 8 1 - - 2 8 9 . 227 Poppe , W. and Grossweiner , Photodyna~'nic sensi t iza t ion by 8 - m e t h o x y p s o r a l e n via the singlet o x y g e n

m e c h a n i s m , P h o t o c h e m i s t r y a nd P ho tob io logy , 22 (1975) 2 1 7 - - 2 1 9 .

73

228 Porcaxd, O.R. and P. Shubiak , Liquid c h r o m a t o g r a p h i c d e t e r m i n a t i o n of b e r g a p t e n c o n t e n t in t r e a t e d or na t u r a l b e r g a m o t oil, J. of the Assoc ia t ion of Official Ana ly t i ca l Chemis ts , 57 ( 1 9 7 4 ) 1 4 5 - - 1 4 7 .

229 Queval , P. and E. Basagni, Ne w synthes is of psora len an d re la ted c o m p o u n d s , Eur . J. Med. Chem. Ch im Ther . , 9 ( 1 9 7 4 ) 3 3 5 - - 3 4 0 .

230 R a k h n a n k u l o v , U., S.A. K h a m i d k h o d z a e v a nd E.E. K o r o t k o v a , Ficus carica leaves as a source of psora len and b e r g a p t e n , Uzb . Biol. Zh. 18 ( 1 9 7 4 ) 36 - -37 .

231 Re inha rd , E., G. C o r d u a n and W. V a n Brocke , Essential oil an d c o u m a r i n s in tissue cu l tu res of R u t a graveolens, H e r b a Hung. , 10 ( 1 9 7 1 ) 9 - -26 .

232 Rip ley , L.S., I n d u c t i o n of t ransvers ions at b a c t e r i o p h a g e T4 A :T base-pai r sites by chemica l m u t a - gens and by m u t a n t DNA po lymerase s , Genet ics , 74 ( s u p p l e m e n t ) ( 1 9 7 3 ) $ 2 3 0 - - 2 3 1 .

233 Richards , D.E. , The isola t ion a n d iden t i f i ca t ion of toxic c o u m a r i n , Microbial Toxins , 8 (1972) 3 - - 44.

234 Rod igh ie ro , G., I n f luence of na tu ra l f u r o c o u m a r i n s on the g e r m i n a t i o n of seeds and on the g r o w t h of l e t tuce sp rou t s and roo ts , Giorn . Biochim, 3 ( 1 9 5 4 ) 1 3 8 - - 1 4 6 .

235 Rod igh ie ro , G. and G. Allegri, Quan t i t a t ive d e t e r m i n a t i o n of b e r g a p t e n in celery and pars ley, F a r m a c o Ed. Sci., 14 ( 1 9 5 9 ) 7 2 7 - - 7 3 5 .

236 Rod igh ie ro , G. and C. An tone l l o , Psoralen and b e r g a p t e n c o n t e n t in leaves o f Ficus carica, F a r m a c o Ed. Sci., 14 ( 1 9 5 9 ) 679---685.

237 Rod igh ie ro , G., G. Capora le a nd T. Dolcher , Osservazioni sul la t t iv i ta c i tologica di a lcune fu rocou- mar ine e sul loro c o m p a r t a m e n t o di f ron t e a l l ' ac ido desoss i robonuc le in ico , Rend . Accad . Naz. Lincei ( R o m a ) , 30 ( 1 9 6 1 ) 8 4 - - 8 9 .

238 Rod igh ie ro , G., P. Cha nd ra a n d A. Wacker , S t ruc tu ra l speci f ic i ty for the pho to - inac t iva t ion of nucle i acids by f u r o c o u m a r i n s , FEBS Le t t . , 10 ( 1 9 7 0 ) 29 - -32 .

239 Sams , W.M., P h o t o d y n a m i c ac t ion of l ime oil, (Ci trus auran t i fdena) , A.M.A. Arch . D e r m a t o l . , 44 ( 1 9 4 1 ) 5 7 1 - - 5 8 7 .

240 Sasaki , M.S. a n d A. T o n o m u r a , A high suscept ib i l i ty of Fancon i ' s a n e m i a to c h r o m o s o m e b reakage by D N A cross-l inking agents , Cancer Res., 33 ( 1 9 7 3 ) 1 8 2 9 - - 1 8 3 6 .

241 Scaife, J .F . , D N A repa i r in t h y m u s l y m p h o c y t e s a f t e r i r rad ia t ion wi th psoralen, In t . J. Rad ia t i on Biol., 21 (1972) 1 9 7 - - 2 0 0 .

242 Scheel , L.D. , T ox i ca n t s occur r ing in na tu ra l foods . 2nd ed., Nat . Acad . Sci. Wash ing ton D.C., 1973 , pp. 5 5 8 - - 5 7 2 .

243 Scheel , L .D. , Biological ac t i on of the coumar in s , Microbial Toxins , 8 ( 1 9 7 2 ) 4 7 - -6 6 . 244 Scheel , L .D. , The f u r o c o u m a r i n s - - a fami ly of p h o t o t o x i c c o m p o u n d s in b i o c h e m i s t r y of s o m e food

b o r n e mic rob ia l toxins , Matches a n d Wagen (eds.) The MIT Press, 1 9 6 6 , pp. 1 0 9 - - 1 1 8 . 245 Scheel , L.D. , V.P. Perone , R.L. La rk in a n d R.E. Kupel , The isolat ion an d cha rac t e r i za t i on of two

p h o t o t r o p i c f u r o c o u m a r i n s (psora lens) f r o m diseased ce lery , B iochem. , 2 ( 1 9 6 3 ) 1 1 2 7 - - 1 1 3 1 . 246 Scot t , B.R. and G.H. Mohn, Fai lure to de t ec t a m u t a g e n i c ac t iv i ty of 8 - m e t h o x y p s o r a l e n (in the

dark) in s t rains of Salmone l la t y m p h i m u r i u m and Escher ichia coli , M u t a t i o n Res. (in p repa ra t i on ) . 247 Scot t , B.R. and T. A lde r son , The r a n d o m (non-specif ic) f o rwa rd m u t a t i o n of gene loci in Asperg i l lus

n idulans conid ia a f t e r pho tosens i t i za t i on to near -u l t rav io le t light (365 n m ) by 8 - m e t h o x y p s o r a l e n , Muta t i on Res. , 12 ( 1 9 7 1 ) 29 - -34 .

248 S e m p u k u , K. , P repa ra t ion of s o m e p h o s p h a t e esters of 5 - m e t h o x y - 8 - h y d r o x y p s o r a l e n , Chem. Pha rm. Bull., 21 ( 1 9 7 3 ) 4 3 7 - - 4 3 9 .

249 Sepulveda , A.J . , L.J . Viguera a nd P.J. Sanchez , R u t a mon tana . VI. Iso la t ion an d cha rac te r i sa t ion of b e r g a p t e n a n d psora len f r o m the leaves of R. m o n t a n a , Ann . Quire. , 69 (1973) 3 6 5 - - 3 6 8 .

250 Shel ley, W., Photosens i t i ze rs , in Rees (Ed. ) D e r m a t o s e s due to e n v i r o n m e n t a l and phys ica l factors , Charles T h o m a s Publishers , 1 9 6 2 , pp. 8 8 - - 1 0 3 .

251 Sidi, E. and J . Bourged is -Gavard in , The t r e a t m e n t of vitiligo, J. Inves t . De rma to l . , 18 ( 1 9 5 2 ) 3 9 1 - - 394 .

252 Song, P., M.L. H a t t e r , T .A. Moore a nd W.C. H e r n d o n , L u m i n e s c e n c e spec t ra an d p h o t o c y c l o a d d i t i o n of the exc i t ed c o u m a r i n s to DNA bases, P h o t o c h e m . Photob io l . , 14 ( 1 9 7 1 ) 5 2 1 - -5 3 0 .

253 Song, P. and W.H. G o r d o n , Spec t roscop ic s tudy of exc i t ed states of c o u m a r i n , J. Phys. Chem. , 74 ( 1 9 7 0 ) 4 2 3 4 - - 4 2 4 0 .

254 Sp//th, E. and H. Ho lzen , Che m. Bet., 66 ( 1 9 3 3 ) 1137 , q u o t e d in ref. 199. 255 Sp~/th, E. and O. Pesta, l~ber Nat / i r l iche C u m a r i n e XI Die K o n s t i t u t i o n des Angel ic ins (aus Angel ica

Archange l i ca 1), Bet. Deut . Chem. Ges. , 67 B (1934) 8 5 3 - - 8 6 0 . 256 Sp~/th, E., Die na t u r l i c he n Cuma r ine , Che m. Ber, 70 ( 1 9 3 7 ) 8 3 - - 1 1 7 . 257 Sp~/th, E. a n d F. K u f f n e r , Na tu ra l c o u m a r i n s a nd the i r toxic ac t ion of fish, Monas t sch Chem. , 69

( 1 9 3 6 ) 7 5 - - 1 1 4 . 258 Spikes, J .D. and R. St ra ight , Sensi t ized p h o t o c h e m i c a l prosesses in biological sys tems , Ann . Rev.

Phys. Chem. , 18 ( 1 9 6 7 ) 4 0 9 - - 4 3 6 . 259 S tegmaie r , O.C., The use of m e t h o x y s a l e n in sun tann ing , J. Invest . De rma to l . , 32 ( 1 9 5 9 ) 345. 260 S t e m p l e , N.R. and W.H. Watson, Crys ta l a nd mo lecu la r s t ruc tu re of x a n t h o t o x i n , Ac ta CristaUogr.

Sect . , B 28 ( 1 9 7 2 ) 2 4 8 5 - - 2 4 8 9 . 261 S towers , J .H. , Syphil is , Brit. J . D e r m a t o l . 9 ( 1 8 9 7 ) 285 , q u o t e d in reI. 188. 262 S w a n b e c k , G. and M. Thesso, I n d u c t i o n of r e sp i r a to ry def ic ien t m u t a n t s in yeast by psora len and

light, J. Inves t . D e r m a t o l . , 63 (1974) 2 4 2 - - 2 4 4 .

74

263 S w a n b e c k , G., Thyres son , M. MSk, A. Bredberg a nd B. L a m b e r t , T r e a t m e n t of Psoriasis wi th oral psora lens a n d longwave u l t rav io le t l ight , Ac ta D e r m a t o v e n e r ( S t o c k h o l m ) , 55 (1975) 3 6 7 - - 3 7 6 .

264 Szybatski , W., Special mic rob io log ica l sys t em. II. Observa t ion on chemica l mutagenes i s in mic roor - ganisms, Ann . NY Acad . Sci., 76 ( 1 9 5 8 ) 4 7 5 - - 4 8 9 .

265 Thorns , H., The cons t i t u t i on of x a n t h o t o x i n a nd its re la t ionship to be rgap t en , Ber. Deut . Chem. Gesellsch. , 44 ( 1 9 1 1 ) 3 3 2 5 - - 3 3 3 2 .

266 T o w n s e n d , M.E., D.A. H o p w o o d and H.M. Wright , Eff ic ient mutagenes i s by near UV in thb presence of 8 - m e t h o x y p s o r a l e n in S t r e p t o m y c e s , J o h n Innes Inst . Ann . Rep. , 61 (1970) 56---57.

267 T o w n s e n d , M.E., H.M. Wright a nd D.A. H o p w o o d , Eff ic ient mutagenes i s by near u l t rav io le t light in the presence of 8 - m e t h o x y p s o r a l e n in S t r e p t o m y c e s , J. Appl . Bacteriol . , 34 (1971) 7 9 9 - -8 0 1 .

268 Tronn ie r , M. and D. Schtile, First resul ts of t h e r a p y wi th longwave u l t ravio le t a f t e r photosens i t iza- t ion of skin, in G.O. Schenck (ed.) Progress in Pho tob io logy Proceed ing of the VI In t e rna t iona l Con- gress of P h o t o b i o l o g y Biochem. (1972) , Den t sche Gesselschef t for L ich fo r schung , F rank fu r t , 1974 , pp . 340.

269 Tronn ie r , M. and D. Sch/ile, Zur D e r m a t o g i s c h e n therap ie von d e r m a t o s e n m i t longwel l ingen UV nach photosens ib i l i z ie rung der H a u t mi t m e t h o x s a l e n Erste Ergcbisse bei der Psoriasis vulgaris, Z. H a u t Geschl . Krk , 48 ( 1 9 7 3 ) 3 8 5 - - 3 9 3 .

270 Trosko , J .E . and M. I soun , S tudies on the pho tosens i t i z ing e f fec t of t r i sora len on h u m a n cells in vi t ro , Radia t . Res., 43 (1970) 266 .

271 Trosko , J .E. and M. I soun , Photosens i t i z ing e f fec t of t r ipsora len on DNA synthes is in h u m a n cells g r o w n in v i t ro , 19 (1971) 87 - -02 .

272 Trosko , J .E. and M. I soun , Synergis t ic e f fec ts of sunl ight and the drug t r i sora len and caffe ine on h u m a n cells in vi t ro , EMS Newsle t t e r , 3 (1970) 39.

273 Tucke r , H.A. , Celery a nd c o n t a c t de rmat i t i s , J. Am. Med. Associa t ion , 169 (1959) 1968 . 274 Tucke r , H.H. , Clinical and l a b o r a t o r y t o l e rnace s tudies in vo lun tee r s given oral m e t h o x s a l e n , J. In-

ves t igat ive D e r m a t . , 32 (1959) 2 7 7 - - 2 8 0 . 275 Urbach , F., Modi f ica t ion of u l t rav io le t carc inogenesis by p h o t o a c t i v e agents , J. Invest . Derma to l . , 32

( 1 9 5 9 ) 3 7 3 - - 3 7 8 . 276 V i n o k u r o v , I .N., F u n c t i o n a l cond i t ion of the adrena l co r t ex in pa t i en t s wi th diffuse fo rms of al-

copec ia w h e n psora lene and cor t i co id s teroids arc used s imul t aneous ly , Ves ta De rma to l . Vcnerol . , 41 ( 1 9 6 7 ) 26- -32 .

277 Walter , J .F. and J . J . Voorhees , Psoriasis i m p o r v e d by psora lcn plus b lack light, Ac ta D c r m a t o v e n e r ( S t o c k h o l m ) , 53 (1973) 4 6 9 - - 4 7 2 .

278 Wacker , A., H. Del lweg, L. Trager , A. Korhause r , E. L o d e m a n n , G. T/ irck, H. Selzer, P. Chandra and H. I s h i m o t o , Organic P h o t o c h e m i s t r y of Nucleic acids, P h o t o c h e m . Pho tob io l , 3 (1964) 3 6 9 - - 3 9 4 .

279 Walter, J .F . , J .J . Voorhees , W.H. Kelsey a nd E.A. DueU, Psoralen plus b lack light inhibi ts ep id e rma l DN A synthes is , Arch. of D e r m a t o l . 107 (1973) 8 6 1 - - 8 6 5 .

280 Weber , G., C o m b i n e d 8 - m c t h o x y p s o r a l e n a nd b lack l ight t h e r a p y of psor ias is - technique and results , British J . De rma to l . , 90 (1974) 3 1 7 - - 3 2 3 .

281 Weinstein, G.D. , J. Walesco and P. Fros t , Biological and pha rmaco log ica l re la t ionships of psoriasis and m e t h o t r e x a t e , in E.M. Farber and A.J . Cox (eds.) Psoriasis, Proceedings of the In t e rna t iona l S y m p o s i u m , S t an fo rd , Cal ifornia , S t a n f o r d Univers i ty Press, 1971 , pp. 3 9 3 - - 4 0 7 .

282 White, J.C., Notes on de rmat i t i s ve ne na t a , Boston Med. Surg. J., 28 (1897) . 283 Willis, I and D.E.R. Harris , Res is tant psoriasis c o m b i n e d m e t h o x s a l e n an th ra l in t h e r a p y , Arch Der-

ma to l . , 107 (1973) 3 5 8 - - 3 6 2 . 284 Wolff , K., T.B. F i t zpa t r i ck and J .A. Parrish, P h o t o c h e m o t h e r a p y of Psoriasis wi th oral 8 - m e t h y o x y p s o -

ralen, A r c h Derma to l . , 112 ( 1 9 7 6 ) 9 4 3 - - 9 5 0 . 285 Worden , L .R. , K.D. K a u f m a n , J .A. Weis and T .K. Schaof , Syn the t i c f u r o c o u m a r i n s . IX. A new syn-

the t i c rou te to psora len , J. Org. Chem. , 34 (1969) 2 3 1 1 - - 2 3 1 3 . 286 Wu, C.M., P.E. Koehler , J.C. Ayres , I so la t ion and iden t i f i ca t ion of x a n t h o t o x i n ( 8 - m c t h o x y p s o r a l e n )

an d Bergap ten ( 5 - m e t h o c y p s o r a l e n ) f r o m celery in fec ted wi th Sclerot in ia sc l e ro t io rum, Appl . Micro- biol . , 23 (1972) 8 5 2 - - 8 5 6 .

287 Yeargers , E. and L. Augens te in , Abso rp t i on and emiss ion s p e c t r u m of psora len and emiss ion spec- t r u m of psora len and 8 - m e t h o x y p s o r a l e n in p o w d e r s and in sol, J. Invest . Derma to l . , 44 (1965) 1 8 1 - - 1 8 7 .

288 Yeargers , E. and L. Augens te in , Molecular orb i ta l ca lcula t ions on photoscns i t i z ing psora len deriva- tives, Na tu re ( L o n d o n ) , 212 ( 1 9 6 6 ) 2 5 1 - - 2 5 3 .

289 Yarosh , E.A. and G.K. N ikonov , F u r o c o u m a r i n s of the f rui t clusters of the fig, Tr. Vses. Semin, Biol. Akt iv . (Lech) V e s h c h e s t v a m Plodov Yagod 4 (1972) 3 9 0 - - 3 9 4 .

290 Zelle, M.R., Gene t i c effects of p h o t o d y n a m i c ac t ion , in F. Urbach (ed.) The Biological Effec ts of u l t ravio le t Rad ia t i on (wi th emphas i s on the skin), P e r g a m o n Press (Proceedings of the first In ter- na t iona l Confe rence ) , 1969 , 2 8 0 - - 2 9 6 .

291 Z i m m c r m a n , M.C., His tological changes in i r rad ia ted skin, a f te r in jec t ion of 8 - m e t h o x y p s o r a l e n , J. Inves t . Derm. , 32 ( 1 9 5 9 ) 2 6 9 - - 2 7 1 .

292 Jo u rna l of P h a r m a c o l o g y and E x p e r i m e n t a l The rapeu t i c s (Ba l t imore) , 131 (1961) 394. 293 Chemica l Biological Coord ina t i on Center , S u m m a r y Tests, 5 (1953) 62. 294 Chemica l Biological C o o r d i n a t i o n Center , S u m m a r y Tests, 7 ( 1 9 5 5 ) 689. 295 The Merck Index : An E n c y c l o p e d i a of Chemica l and Drugs, Merck Co. Inc. R a h w a y , New Jersey 8

(1968) 677 .