CURRENT AWARENESS DOCUMENT
LINEAR AND ANGULAR FUROMARINS PSORALENS. ANGELICINS,
AND RELATED COMPOUNDS
CARCINOGENICITY AND STRUCTURE ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
METABOLISM. ENVIRONMENTAL SIGNIFICANCE.
Prepared by
Yin-Tak Woo, Ph.D., D.A.B.T.
David Y. Lai, Ph.D.
Science Applications Internation Corporation
8400 Westpark Drive
McLean, Virginia 22102
EPA Contract No. 68-02-3948
SAIC Project No. 2-813-07-409
EPA Project Officer and Scientific Editor
Joseph C. Arcos, D.Sc.
Extradivi sional Scientific Editor
Mary F. Argus, Ph.D.
June 1986
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5325 Linear and Angular Furocouraarins Psoralens, AngeLicins and Related
Compounds
53251 Introduction
The linear and angular furocouraarins, mostly derivatives of psoralen and
angelicin, constitute a special class of chemical carcinogens known as photo-
carcinogens Furocoumarins are naturally occurring substances found in five
major plant families — parsley, rue, legume, fig and orchid In certain
plants (e g , celery, parsnips), they are produced in very high amounts,
perhaps as phytoalexins, in response to fungal infection (see Section
5.3 2552) During the last decade, furocoumarins have captured the atten-
tion of many clinicians, moi-eculjir biologists and health protection special-
ists because of their promise as photochemotherapeutic agents for a variety of
skin diseases, as molecular probes for the study of nucleic acid structure and
DNA repair mechanisms, and the use of 5-methoxypsoralen (5-MOP or bergapten)
as a cosmetics ingredient
The historical development of the use of furocoumarins as photochemo-
therapeutic agents has been extensively described by Scott et al (1) and
Fitzpatrick and Pathak (2) Furocoumarin-containing plants have been used as
herbal medicine since 1200-2000 B C The ancient Indian sacred book, "Atharva
Veda" and the Buddhist Bower manuscripts both mention the treatment of
vitiligo (a color imperfection of the skin, also known as leukoderma or "white
leprosy") with a combination of sunlight and extracts from the seeds of a
plant now classified as Psoralea corylifolia L Another important plant, Ammi
majus, a weed found in the Nile Valley, has similarly been used in Egypt for
centuries as a cure for this socially unacceptable skin disease. It was not
until the 1940's that El Mofty (3) showed that the active photochemothera-
400
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peutic principle in seeds of Ammi majus is 8-methoxypsoralen (8-MOP, also
known as Methoxsalen, Xanthotoxin) In 1974, a new milestone was set when
Pamsh j^t_ _al_ (4) reported that a combination of oral administration of 8-MOP
and dermal exposure to UVA* is highly effective in the treatment of a vir-
tually incurable skin disease, psoriasis This new PUVA (acronym for
psoralens plus UVA) therapy has stirred widespread interest throughout the
world Since 1974, several thousand psoriasis patients have received PUVA
treatment in clinical trials in Europe and the United States On May 20,
1982, the U S Food and Drug Administration approved the use of PUVA for the
treatment of psoriasis The effective use of psoralens in the treatment of
vitiligo has also led to the controversial use of 5-MOP as a melanogenic agent
in several suntan lotions (5) Oil of bergamot, a citrus extract which con-
tains 5-MOP, was also used in perfumery in Eau de Cologne (6, 7). Owing to
its unique capacity to intercalate, photoreact and form both monoadducts and
crosslinks with DNA, psoralens have also proved to be a very useful probe for
the study of nucleic acid structure (8-11) and DNA repair mechanism (8, 12)
The potential phototoxicity of furocoumarins and furocoumarin-containing
plant substances has been known for centuries The phototoxicity of furocou-
marins was even mentioned in a 18th century German fairy tale in which the
central character was described to have suffered from photodermatitis after
ingesting figs and exposing himself to sunlight (see 1) Many incidents of
phototoxicity in livestock and in humans exposed to furocoumarin-containing
*0wing to their differential biological activities, the UV spectrum is
generally divided into three regions UVA, which occupies the 320-400 nm
region and is also known as long wave (wavelength) UV, near UV or black light
UVB, which occupies the 280-320 nm region and is sometimes called intermediate
UV or "sunburn" UV UVC, which occupies the region below 280 nm and is also
called short wave UV, far UV or germicidal UV
401
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plant products have been reported in the literature (see Section
532521) The photocarcinogenicity of 8-MOP was discovered by O'Neal and
Griffin (13) in 1957 in a study originally designed to explore the possible
protective effect of the compound The interest in the study of photocarcino-
genicity of furocoumarins has been rekindled recently as the concern over
potential long-term health hazard of human use of furocoumarins as drugs and
cosmetics ingredients grows To date, at least 11 furocoumarins have been
tested for in vivo or in vitro carcinogenic activity The intense interest in
these compounds have generated many recent publications as reflected by the
large number of recent reviews (1, 8, 9, 12, 14, 15) on the subject Two
recent symposia were devoted to review studies of the photobiologic, toxico-
logic and pharmacologic aspects of psoralens (16, 17)
53252 Physicochemical Properties and Biological Effects
532521 PHYSICAL AND CHEMICAL PROPERTIES
The physical and chemical properties of psoralens and related furocou-
marin compounds have been extensively discussed in a number of reviews (1, 8,
15, 18) Synthetically, there are 12 different ways a furan ring can be fused
to a suitably substituted ooumarin derivative to form different types of furo-
coumarins Naturally occurring furocoumarins of plant origin consist of two
types, psoralens and angelicins In psoralens the furan ring is condensed
linearly to the coumarin molecule (the 3,2-position of furan to the 6,7-
position of coumarin), whereas in angelicins the furan ring is condensed
angularly (the 3,2-position of furan to the 7,8-position of coumarin) The
systematic, IUPAC name for the parent compound, psoralen, is 7H-furo[3,2-
g]benzopyran-7-one Two different systems are in use for numbering the carbon
and oxygen atoms in the psoralen molecule In the conventional system the
original numbering sequence of both the coumarin and the furan moieties are
402
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I
(Conventional) (IUPAC)
retained, but the furan positions are distinguisned by the prime sign, such as
1", 2', In the IUPAC system, the atoms on the periphery of the mole-
cule are numbered in a clockwise direction starting from the oxygen atom in
the furan ring The conventional system is still widely used in the litera-
ture on psoralens and is the one adopted in this section
Close to 30 different derivatives of psoralen and angelicin have been
isolated from natural sources (1, 9) The chemical structures of the eleven
compounds which have been tested for carcinogenic activity are depicted in
Table LXVIII Some physical and chemical properties of eight of these com-
pounds are summarized in Table LXIX Most naturally occuring psoralens and
angelicins are white to cream-colored, odorless, needle-like crystals at room
temperature They are practically insoluble in cold water, sparingly soluble
in ether or boiling water, and soluble in hot ethanol, acetone, vegetable
oils, propylene glycol, benzene and chloroform The UV absorption spectra of
most psoralens and angelicins show one or more absorption maxima in three
regions less than 225 nm, 230-270 nm and 290-330 nm (18, 23) The lactone
ring in furocoumarins is susceptible to alkaline hydrolysis. However, if the
alkaline treatment is mild, acidification will reverse the reaction to reform
furocoumarins. Furan-2,3-dicarboxylic acid is obtained when either psoralen
or angelicin derivatives are oxidized with peroxide under alkaline condi-
tions. Treatment of an alkoxy furocouraarin, other than a methoxy derivative,
with an acetic acid-sulfuric acid mixture leads to cleavage of the ether bond
(18).
403
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Table
Structural Formulas of Psoralen and Related Compounds
R,
cr^o
Psoralen (R,=R2=-H)
5-MOP or Bergapten (R^-OCHj, R2=-H)
8-MOP or Methoxsaien (R, =-H, R2=-OCH3)
CH
Imperatorin (R,=-H, R2=-OCH2CH=C' 3
Marmesm (R=-H)
CH3
I
Chalepin(R=-C-CH=CH2)
3-Carbethoxypsoralen*(R^-COOC2H5,R^R3^R^-H)
4,5',8-Tnmethylpsoralen or Tnoxsalen
(R,=-H,R2=R3=R4=-CH3)
u
Synthetic compound
Angelic in (R,=R2=R3=-H)
4,5'-Dimethylangel!cm(R,=R^-CH3,
Rr-H)
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Table LXIX
Physicochemical and Photophysicochemical Properties of Psoralen and Related Compounds'
UV-absorpt ion Fluores
Compound (synonyms m p Solubility
and abbreviations) ( C) (/Jg/tnl H20) ^max £max £365 ^ ^exclt
Psoralen 163-164 37
8-Methoxypsoralen 148 23
(8-MOP, methox-
salen, Xanthotoxin)
5-Methoxypsoralen -- 5
(5-MOP, Bergapten)
4,5' ,8-Trimethyl- 235 1
psoralen (Tri-
oxsalen)
3-Carbethoxy- 153-154 13
psoralen
Angel ic in -- 20
5-Me thy I angel i<_ in 189-190
4,5'-Dimethyl- 185 8
angel it in
295
302 12,010 1,150 360
312 14,200 950 350
298 7,950 1,680 360
317 10,900 6,230 365
299 9,870
307 10,900
298 9,350 125 330
Quantum yield
;cencec Super-
Triplet Singlet oxide
Aemis stated oxygen6 anionf
0 45, 0 12 3 3, 7 6, +++ +++
460 0 06, 0 14 1, + +
460 <0 001 04-05,+ +
430 — 53,+ ++
460 0 32, 0 35 26, ++++ +
0 33 0, +++ ++
•n- -n-
425 — * +
aSummarized from data compiled by Grossweiner (19), Rodighiero and Dal1'Ac qua (20), Joshi and Pathak (21), Averbeck et
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Tne photochemistry of psoraiens has been extensively studied, the readers
are referred to reviews by Song and Tapley (8), Pathak (25), Rodighiero and
DaLl'Acqua (20) and Grossweiner (19) for details The essential features of
the photochemical changes in psoralen brought about by photoactivation are
schematically summarized in Fig 16 Psoralen in ground state (Pgo) may be
photoactivated to an excited singlet state (see Section 322, Vol I) by two
possible types of electronic excitation (a) excitation of the nonbonding
electron in the carbonyl group to antibonding ~JT molecular orbital ( ft*) to
produce the n,Tf* state and (b) excitation of a IT-electron to the antibond-
ing TT* state to produce the 7T,7T* state The resulting excited singlet
1 — ft
state ( PS) is extremely reactive, short-lived (•*• 10 sec) and readily under-
goes spontaneous deactivation by radiative decay resulting in fluorescence, or
undergoes intersystem crossing (electronic spin flip) to the excited triplet
o o o
state ( Pg) The exicted triplet state ( n,lT* or i7 , Tf*) > although still
highly reactive and short-lived, has a considerably longer lifetime ( — 10
sec) and may allow three competing deactivating processes to take place
These are (a) spontaneous radiative decay resulting in phosphorescence (see
Section 322 and Suppletory Note 3, Vol I), (b) type I photochemical reac-
tions, and (c) type II photochemical reactions The type I (anoxic) reactions
involve nonradiative transfer of energy from P to substrates resulting in
s
direct reaction between P0 and substrates In biological systems, these
b
reactions include C- cycloaddition of P to pyrimidines, interstrand cross-
linking, and conjugation to ammo acid moieties of proteins These reactions
are believed to play a major role in contributing to the phototoxic, photo-
mutagenic and photocarcmogenic activities of psoraiens (see Section
53254) The type II (photodynamic) reactions involve indirect, sensitized
o
transfer of energy from P to molecular oxygen (in triplet state) to produce
404
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Nonradiative
transfer of energy
photoactivation (hl>)
Fluorescence decay (V
intersystem crossing (electronic spin flip)
Phosphorescence decay m PJO +• hi/)
Sensitized transfer
of energy
TYPE I
Direct photochemical reactions
a C^ cycloaddition of P to
pyrimidmes
b Coniugation to proteins
TYPE II
Indirect photochemical reactions
a Production of singlet oxygen
33 1
s 2 so 2
b Production of super oxide anion
Fig. 16. Photoexcited states of psoralen and their photophysical radia-
tive decays (fluorescence and phosphorescence) and photochemical reactions.
TWo types of photochemical reactions are shown: type I (direct photochemical
reaction), anoxic and involving nonradiative transfer of energy, and type II
(indirect photochemical reaction), oxygen-dependent and involving sensitized
transfer of energy to molecular oxygen. Abbreviations: Pgo, psoralen in
ground state; 1P8, psoralen in singlet state; 3PS, psoralen in triplet state,
^02, molecular oxygen in triplet state; 102, molecular oxygen in singlet
state; Oo' superoxide anion. [Adapted from M.A. Pathak: NCI Monog. 66, 41
(1984).]
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reactive oxygen species such as singlet oxygen ( C^) , superoxide anion (02*)
and hydroxy radicals ( • OH) Compared to type I reactions, type II reactions
are relatively unimportant in contributing to the photobiological effects of
psoralens Psoralens have high tendency to intercalate into DNA, once inter-
calated, psoralens are less efficient in producing Qj Furthermore,
psoralens themselves can react with CK to form oxidation products thus effec-
tively removing C^ from the system (8) The relative abilities of several
psoralens and angelicins to yield triplet state, singlet oxygen and superoxide
anions are summarized in Table LXIX
5 3 2.5 2 2 BIOLOGICAL EFFECTS OTHER THAN CARCINOGEN 1C ITY
Toxicity and Photobiological Effects Most of the toxicity studies of
furocoumar in compounds focus on their phototoxicity Relatively little data
are available on their systemic toxicity. The acute LDcQ of 8-MOP in rats by
i p injection is 470 mg/kg body weight (26) Lower i p LD5Q values of 189
and 158 mg/kg for male and female Sprague-Dawley rats, and about 250 mg/kg for
CD-I mice, were found using micronized suspension of 8-MOP (27). The corre-
sponding oral LDcQ values of micronized 8-MOP suspension were 791, 699 and 566
mg/kg body weight (27) In a comparative study (route not specified) by
Herold _et_ _al_ (28), 5-MOP was substantially less toxic than 8-MOP, with
reported LD5Q values of 8,100, >30,000 and 9,000 mg/kg body weight for 5-MOP
and 875, 4,400 and 505 mg/kg body weight for 8-MOP in I
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The phototoxicity of psoralen and related compounds has been extensively
studied and was the subject of several recent reviews (l, 30, 31) In mam-
malian skin the most striking features of acute phototoxicity due to psoralens
are delayed erythema and pigmentation Under some conditions, edema and
blistering is also produced The dose-response curve of the phototoxicity of
psoralens is usually very steep The phototoxic effects of psoralens are
manifested by either oral or parenteral administration or by topical applica-
tion The action spectrum* of psoralens lies in the 340-380 nm region, the
higher end of their absorption spectrum Unlike for many photosensitizers,
the photosensitizing effect of psoralens is essentially independent of oxygen
(1, see also Section 532521)
Sporadic outbreaks of psoralen-induced photosensitization in livestock
have been noted after grazing or feeding on plants (e g , Cymopterus watsonn,
Ammi yisnaga, Ammi rnajus) of the parsley family (32) Incidents of dermatitis
in celery workers, lime pickers, and consumers using the perfume "Eau de
Cologne" have also been reported In celery workers' dermatitis, it was esta-
blished that the very high amount of 8-MOP and 4,5',8-trimethylpsoralen
produced in celery in response to fungal disease ("pink rot") is responsible
for the phototoxicity (33, 34) The presence of 5-MOP in lime oil and
bergamot oil (used as an ingredient in Eau de Cologne) may account for the
occasional dermatitis in the workers and consumers (1, 6)
*The action spectrum of a photosensitizing compound is the region of wave-
lengths capable of eliciting photobiological response from sensitized sub-
jects The action spectrum of a compound is generally expected to parallel
its absorption spectrum However, for furocoumarm compounds, the two spectra
are not in good agreement.
406
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The strueture-phototoxicity relationships of furocoumann compounds have
been extensively studied More than 150 furocoumarins and related compounds
have been tested for photosensitizing activity on mammalian skin (rev in
1) Some of these results are summarized in Table LXX and compared to their
ability to photoreact with DNA In general, linear furocoumarins are much
more active than angular furocoraarins. Saturation of the 4',5'-double bond
completely abolishes activity The introduction of a bulky substituent (e g ,
carbethoxy) at one of the two critical double bond (3,4- and 4',5'-) of
psoralen molecule yields a compound (eg , 3-carbethoxypsoraien) with mono-
functional character, having little or no phototoxicity. Compounds with a
large number of substitutents having ionic character tend to ionize in water
and are less stable and less phototoxic Substrtutents that alter the
resonance of the furocoumarm molecule greatly affect its activity The
introduction of electron-donating substituents such as a methyl group in the
5'- or 8-position enhances the phototoxicity of psoralen, whereas methyl sub-
stitution at 3-, 4-, and 4'-position decreases the activity Introduction of
electron-withdrawing substituents (e g , -OH, -CN, -NH2) at the same position
exhibits an opposite effect
Comparison of the phototoxicity of furocoumarins with their photobinding
activity to DNA (see Table LXX) indicates a very good correlation between
phototoxicity and ability to form interstrand cross-links Apparently, the
formation of monoadduct is insufficient to cause significant phototoxicity *
This hypothesis is supported by the findings of Gange and Parrish (31) that
blue light (>380 nm) photoactivates bifunctional 8-MOP to form monoadducts
*Some exceptions to this general rule exist Certain mono functional psoralens
such as 4,6,4'-tnmethylangelicin are phototoxic (25)
407
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Table LXX
Phototoxicity of Furocoumann Compounds and Comparison with
Their Ability to Photobind to DNAa
Phototoxicity
Compound in mice
Psoralen ++++
5 -MOP ++
8-MOP +++
4,5' ,8-Trimethylpsoralen (TMP) ++++
4'-Aminomethyl-TMP +•»•++
3,4'-Dimethyl-8-MOP ++
5-Diethylaminobutoxypsoralen +
3-Carbethoxypsoralen -c
Angel ic in
5-Methylangel icin
4,5 '-Dimethyl angel ic in
Ability
to form
monoadduct s
strong
strong
strong
strong
strong
strong
strong
strong
weak
strong
moderate
Ab 1 1 1 1 y
to form
interstrand
crosslinks
very strong
strong
strong
very strong
very strong
low
moderate
absent
absent
absent
absent
Adapted from P C. Joshi and M.A Pathak [Biochera. Biophys. Res Commun 112,
638 (1983)]
Tested by skin photosensitization in mice in response to topical application
of the compound followed bv UVA exposure
cData from M.P Mullen, M.A Pathak, J D. West, T.J. Harrist, and F.
Dall'Acqua [NCI Monogr. 66, 205 (1984)].
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with DNA, but without forming any crosslink Mice treated with 8-MOP plus
blue light showed no sign of phototoxicity However, if these mice were
treated with UVA 48 hours later to convert monoadducts into crosslinks, photo-
toxicity became evident
Mutagenicity The rautagenicity of psoralen and related compounds has
been extensively studied The readers are referred to an excellent review by
Scott et al (1) for studies published prior to 1976 Numerous studies have
been reported since that time, mostly in vitro assays using a variety of test
organisms such as prophages, bacteria, algae and yeasts The following dis-
cussion focuses mainly on studies using the Ames test and on comparative
studies. It should be emphasized that the mutagenicity of furocoumarin and
related compounds may be affected by a number of unusual factors as discussed
below
a) Light As may be expected from the photochemical reactivity of
furocoumarin compounds, the mutagenicity of these compounds may
differ significantly when assayed in the dark (or under subdued
light) or when assayed with exposure to light (particularly long
wave UV or UVA) Their mechanism of action is believed to involve
noncovalent complexing (intercalation) with DNA for "dark" muta-
genicity and covalent photobinding to DNA for photomutagenicity
(see Section 53254)
b) Phototoxicity A number of furocoumarin compounds cause cell
death after photoactivation The bifunctional furocoumarins are
particularly phototoxic in this respect because of their cross-
linking activity The mutagenicity testing of these compounds is
often complicated by their cytotoxicity (35, 36)
408
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c) Induction of iysogeaic virus Mutagenicity assays using
bacteria as the test organism may be affected by the induction
(activation) of Lysogenic virus by furocoumann compounds leading
to lysis of bacteria (37, 38) Interpretation of bacterial muta-
genicity data should take this factor into consideration
d) DNA repair proficiency of test organisms The proficiency of
DNA repair capability of test organisms plays a critical role in
determining the mutagenicity of furocoumarins in Salmonella
typhimurium (39), Escherichia coli (12, 40), Aspergillus nidulans
(41) and Saccharomyces cerevisiae (42) It has been suggested that
the repair contributes to mutation by preventing cell lethality and
by introducing error during the unscheduled DNA repair synthesis
Over 20 furocoumann compounds have been tested for mutagenic activity
using the Ames test The results of these studies are summarized in Table
LXXI When assayed in the absence of UV-photoactivation, most furocoumarins
are either inactive or are weak mutagens toward frameshift mutants With a
few exceptions, most mutagenic compounds do not require metabolic activa-
tion The weak, direct-acting frameshift mutagenic activity of these
compounds is consistent with the hypothesis that, by virtue of their favorable
molecular size, they produce mutations by noncovalently complexing with
(intercalating into) DNA Among the compounds tested for "dark" mutagenicity,
8-MOP has been by far the most extensively studied, it was found inactive by
most investigators and weakly rautagenic in two studies (44, 50) Psoralen-8-
glycidol ether is an extremely potent muLagen (probably one of the most potent
mutagen ever synthesized), a concentration of 0 03 uraole per plate was suffi-
cient to produce positive results It is probably the presence of a reactive
functional group (epoxide) coupled with the molecular shape and size of the
409
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Table LXXI
"Dark" and Photoactivated Mutagenicity of Psoralen
and Related Compounds in the Ames Test
I of 2
Without UV-photoact ivat ion
Compound3
Psoralen
5-MOP (Bergapten)
8-MOP (Methoxsalen)
Imperatorin
Heraclenin
Oxypeucedaninc
Psoralen-8-glycidol ether
3-Carbethoxypsoralen
4,5" ,8-Trimethylpsoralen
(Trioxsalen)
Pyndo(3,4-c)furo(3,2-g)-
coumarin
7-Methylpyr ido( 3 , 4-c ) f uro-
( 3,2-g)coumann
Marmesin
Chalepin
Angel ic in
5-Methylangelicin
4'- or 5 '-Methylangelicin
- S-9
- (43)
- (44)
- (37, 43,
46-49)
w+ (44, 50)
+ (46)
- (46)
w* (51)
- (46)
w+ (51)
+ (51)
- (44)
- (47, 48)
w+ (44)
w+ (44)
w+ (44)
+ (46)
- (46)
- (43)
w+ (43)
- (43)
With UVA-
+ S-9 photoact ivation
- (43) + (39)
+ (39,45)
- (37, 43, - (37, 47-48)
46-48)
+ (39)
+ (46)
- (46)
- (46)
- (47, 48) - (47, 48)
+ (46)
v+ (46)
- (43) f (39)
w* (43)
- (43)
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Table LXXI (continued)
P 2 of 2
Without UV-photoactivation
Compound3
4,4'- or 5,5'- or 4,5'- or
4' ,5-Dimethylangel icin
4,5 '-Dimethyl-4 '-hydroxy-
methylangelicin
4, 5 '-Dime thy 1-4 '-ace toxy-
angelicin
4, 5 '-Dune thy 1-4 '-dime thy 1-
- S-9
- (43)
- (43)
- (43)
- (43)
With UVA-
+ S-9 photoactwation
- (43)
w+ (43) + (43)
w+ (43)
w+ (43)
aminoethoxymethyl-
angelicin
Esculm (6,7-Dimethoxy-
coumarin)
- (46)
- (46)
aExcept where otherwise indicated, see Table LXVIII for structural formula.
See below for structural formula.
C0xypeucadanin is an isomer of heraclenin with the substituent in the
5-position instead of the 8-position.
Tested in the dark or under subdued light. w+ = weakly mutagenic.
0
0-CH2-CH-CX
Heraclenin (R=-CH3)
Psoralen-8-glycidol ether (R=-H)
Pyndo(3,4-c) furo(3,2-g)-
coumarm (R=-H)
7-Methylpyndo(3,4-c)furo-
(3,2-g) coumarm (R=-CH3)
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psoralen molecule, favorable for intercalation, which makes it highly muta-
genic This is shown by the fact that replacement of the psoralen moiety with
a phenyl ring yields phenyl glycidol ether, which is 30 times less rauta-
genic Introduction of two sterically hindering methyl groups at the terminal
epoxide group yields heraclenm, a naturally occurring compound, which is
1,000 times less mutagenic Interestingly, imperatorin, the unepoxized parent
compound of heraclenm, is a moderately active mutagen with or without meta-
bolic activation
The presence of both photoreactive double bonds (3,4- and 4',5'-) in
psoralen molecules does not seem to be an absolute requirement for "dark"
rautagenicity Blockade of the 3,4-double bond by a pyridine ring yields com-
pounds (pyr idofurocoumar ins) which s-till retain some mutagenic activity
Likewise, saturation of 4',5'-double bond does not necessarily abolish "dark"
mutagenic activity In fact, marmesin is a moderately active "dark" rauta-
gen Uwaifo (46) postulated that the introduction of a tertiary hydroxypropyl
group at the 5'-position may activate the 3,4-double bond, however, the
mechanism is not clear. Among the angular furocoumarins, only 5-methylangeli-
cin is mutagenic in the dark Three derivatives of 4,5'-diraethylangelicin,
bearing a hydrophilic group at 4'-position, are all mutagenic after metabolic
activation (see Table LXXI)
Only six furocoumarin compounds have been tested for photomutagenic
activity in the Ames test A number of studies (see Table LXXI) found both
8-MOP and 4,5',8-trimethylpsoralen nonmutagenic in the commonly used tester
strains (TA98 and TA100) However using a new tester strain (TA102), which is
highly sensitive to oxidative and cross-linking agents and is proficient in
UV-excision repair, Levin et al (39) found several furocoumarins mutagenic
after photoactivation by UVA Quantitatively, the bifunctional furocoumarms
410
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(8-MOP, 5-MOP and psoraien) are considerably more rautagenic than the raonofunc-
tional furocoumarins (angelicins) 4,5'-Diraethyl-4'-hydroxyangelicin appears
to be an unusual compound, it is mutagenic toward TA98 after metabolic activa-
tion in the dark but becomes mutagenic toward TA100 and nonmutagenic toward
TA98 after UVA-photoactivation (43)
Besides the Ames test, furocoumarin compounds have been assayed in a
variety of other test systems With few exceptions, remarkably similar
results have been obtained from these studies When assayed in the dark,
psoraien, 5-MOP, 8-MOP and angelicin are all weak, direct-acting frameshift
mutagens in various strains of Eschenchia coil, whereas 4,5' ,8-tnmethyl-
psoralerj appears to be nonmutagenic (5, 50, 52-54). There is some evidence
that 8-MOP and 4,5',8-trimethylpsoralen can be metabolically activated to a
base-pair substitution mutagen in the dark (47) When assayed with UV-photo-
activation using UV-excision repair-proficient test organisms, bifunctional
furocoumarin compounds are consistently more mutagenic than monofunctional
furocouraarin compounds In E coli, the reported relative order of photo-
mutagneic potency order is psoraien > 8-MOP > 5-MOP > angelicin in one study
(54), and 8-MOP > psoraien > angelicin > 4,5'-dimethylangelicin in another
(40) In the algal system Chlamydomonas reinhardii, both 5-MOP and 8-MOP are
equally strong photomutagens, imperatorin is also strongly photomutagenic
after prolonged UV irradiation, whereas 5'-methylangeiicin is a weak photo-
mutagen (55, 56) In the yeast (Saccharomyces cerevisiae), 4,5' ,8-tnmethyl-
psoralen is highly photomutagenic, the relative photorautagenic potency is
4,5' ,8-tnmethylpsoralen > 5-MOP > 8-MOP > 4,5 '-dimethylangel icin > 3-carbeth-
oxypsoralen in the induction of nuclear mutation, and 4,5'8-trimethylpsoralen
> 5-MOP > 3-carbethoxypsoralen > 8-MOP = 4,5'-dimethylangelicin in the induc-
tion of cytoplasmic petite mutation (57)
411
-------
Teratogemc ity In contrast to the extensive mutagenicity data, there is
little or no information available on the teratogenicity of furocoumarin com-
pounds Herold _et_ _a_l_ (28) reported that 5-MOP exhibits no teratogenic
effects in rabbits and rats, the details of the study are not available
Colombo et_ al_ (58, 59) treated sea urchin sperms with psoralen and long wave-
length UV, just before fertilizing untreated eggs, and observed abnormal
effects (formation of giant cells, polynucleated cells, impairment of
cleavage, shrinkage of cytoplasm, irregular blastomeres, and blastulas and
cytolysis of embryo) in various subsequent developmental stages Neither
psoralen nor long wavelength UV alone exhibited any significant effect on the
development of sea urchin eggs
53253 Carcinogenicity and Structure-Activity Relationships
The careinogenicity of psoralen and related furocoumarin compounds was
first studied by O'Neal and Griffin (13) in 1957 using 8-MOP The interest in
furocoumarin compounds was renewed recently because of their therapeutic value
in the treatment of psoriasis and other skin disorders and the commercial use
of 5-MOP in suntan lotion To date, 11 furocoumarin compounds have been
tested for in vivo or in vitro carcinogenic activity Early studies led to
the conclusion that bifunctional furocoumarins (such as 8-MOP, 5-MOP) are more
carcinogenic than mono functional furocoumarins (such as angelicin, 3-carbo-
ethoxypsoralen). However, more recent studies using derivatives of angelicin
indicate that the above conclusion is not necessarily true for all cases, some
raonofunctlonal furocoumarins can be more carcinogenic than 8-MOP Like their
photomutagenicity, the careinogenicity of furocoumarin compounds can be
affected by a variety of factors as exemplified by the results of studies on
8-MOP.
412
-------
532531 CARCINOGENICITY OF 8-METHOXYPSORALEN (8-MOP)
8-Methoxypsoralen (Methoxsalen, Xanthotoxin) was first found to be photo-
carcinogenic in 1957 by O'Neal and Griffin (13) in mice by i p injection, in
a study intended to explore the potential of the compound as a protective
agent against ultraviolet 1ight-induced skin careinogenesis Subsequently,
the compound was extensively studied in various strains of mice, particularly
in the past several years, because of its use in combination with UVA in the
"PUVA" treatment of psoriasis The careinogenicity studies of 8-MOP reported
prior to 1980 were reviewed by the International Agency for Research on Cancer
(15) The results of these and a number of more recent studies are summarized
in Table LXXII At the time of this writing, 8-MOP was also being tested
under the U.S National Toxicology Program The (.arc inogenicity of 8-MOP is
affected by a variety of factors such as (a) the route of administration,
(b) the type, the intensity and the timing of irradiation used to activate the
compounds, and (c) the strain of the animals used.
The route of administration has an important modulating effect on the
photocaremogenicity of 8-MOP Topical application and i p injection are
substantially more effective for the induction of skin tumors than oral
administration By oral administration, 8-MOP has no significant carcinogenic
effect in mice of various strains as observed in several independent studies
(13, 60-64). In fact, in one of these studies (13), 8-MOP appeared actually
to protect against far-UV-induced skin carcinogenesis (25% incidence in 8-MOP
plus far-UV-exposed mice vs. 62% in control mice exposed to far-UV alone)
The doses administered in the above-cited studies ranged from 0 6 to 40 rag/kg
body weight by gavage or 200 to 1,000 ppm in the diet Various forms of
irradiation (far-UV, near-UV, fluorescent light) were used to photoactivate
8-MOP, usually one hour after dosing The duration of these studies ranged
413
-------
Table LXXII
Photocarcinogenicity of 8-Methoxypsoralen (8-MOP) in Mice of Various Strains3
Strain
Various strains
Swiss albino
Swiss albino
ICR Swiss
Swiss
C3H"/HeN
ICR Swiss
SKH-hairless-1
SKH-hairless-1
or HRS/J/Anl
Albino hairless
XVIInc/z
Swiss albino
Route
oral
oral
i p.
1 P
i.p.
i.p.
topical to ear
skin painting
skin painting
skin painting
topical to ear
skin painting
Princ ipal
organs
Irradiation affected
UV (250-400 run)
UVA
UV (250-320 mn,
320-400 mn)
UV (280-360 nm)
UV (365 nm)
UVA
UV (280-360 nm)
SSRd O290 nm)
UVA
UV (various
sources)
SSRd
UV (365 nm)
UV (300-400 nm)
Noneb
Skinc
Skinc
Skinc
Skin
Skin
Skinc
Skin
Skin
Skin
Skin
Skinc
Skin
References
(13, 60-64)
(60, 65)
(13, 60)
(66)
(67)
(68)
(66)
(69-71)
(72)
(73, 74)
(75)
(67, 76)
(77, 78)
aln all these studies, 8-MOP was found noncarc inogenic without photoactwa-
tion. The tumors were primarily squamous cell or basal cell carcinomas
The duration of these studies ranged from 4 to 12 months.
cEar and/or eye region.
Simulated solar radiation, spectrum includes UVA, UVB, visible and IR light
-------
from 4 to 12 months, although the duration was relatively short, it should be
noted that in studies of similar length, but using topical or i p route,
8-MOP did prove to be carcinogenic A weak photocarcinogenic effect (skin
tumor incidences of 20-35% vs 0% for control) was observed in one study (60,
65) in which Swiss albino mice were given 500 ppm 8-MOP in the diet and
exposed to UVA 10 minutes to 2 hours a day, 5 days a week for 6 weeks to 3
months In contrast to its weak careinogenicity or lack thereof by oral
administration, 8-MOP was consistently found to be photocarcinogenic by i p
or topical administration Daily i p injection of 0 4 mg 8-MOP to 20 Swiss
albino mice 1 hour before 10-minute exposure to UVA for 6 weeks (total UVA
2
exposure 43 joules/cm ) induced skin tumors (mainj.y in ear and eye regions) in
virtually all the animals, none of the irradiated control, mice bore such
tumors (60) Prolonged UVA exposure was less effective because of severe
phototoxicity. Significant increase in the incidence of skin tumors was also
observed in mice of several other strains given i p injection of 0 4 mg 8-MOP
and irradiation with near or intermediate UV (see Table LXXII) Although no
direct comparative studies have been conducted, topical, application of 8-MOP
appears to be equally effective as (if not more than) i p injection for
inducing photocarcinogenesis For example, Nagayo et al (72) induced skin
tumors in 20 of 20 albino SKH-hairless-1 mice after 40 weeks of daily skin
painting with 40 «g 8-MOP, 30-60 minutes prior to UVA irradiation For com-
parison, none of the 36 control mice that received 8-MOP alone or UVA exposure
alone developed tumors Dubertret et ai (67) found skin tumors in 92% of 40
XVIInc/Z mice given 115 topical applications of 15 ug/un2 8-MOP on each ear
and irradiated with UV (365 nm) About 15% of these mice also had secondary
tumors Significant increase in the incidence of skin tumors (43% treated vs
15% control) was observed in Swiss albino mice skin-painted with as low as 5
ug 8-MOP 15-60 minutes before UV (300-400 nm) exposure (77, 78).
-------
The importance of the nature, dose and timing of irradiation in the acti-
vation of 8-MOP to its photocarcinogenic form was first demonstrated by
Griffin and associates. In contrast to the induction of skin tumors in nearly
100% of mice by a combination of i p injections of 8-MOP and 10-minute expo-
sures to UVA, a combination of 8-MOP and short wavelength UV (254 run) induced
fewer tumors than exposure to UV-254 nm alone (60) Prolonging the time of
UVA exposure or reducing the distance between the light source and skin
decreased the tumor incidence because of excessive phototoxicity causing early
death and severe erythemal responses (60) Delaying the onset of UVA exposure
from 1 hour to 20-22 hours after 8-MOP injection completely abolished the
photocarcinogenic effect of the treatment (13). The spectral dependence of
8-MOP photocarcinogenicity has been recently further explored Grube et al
(73) topically applied 250 ug 8-MOP onto the skin of SKH-hairless-1 mice and
exposed them to UV of three different wavelengths (300-400 nm, 320-400 nm, 365
nm) The respective skin tumor (squamous or basal cell carcinoma) incidences
were 17/20, 15/19 and 8/19 and the latent period was considerably shorter in
the first two groups indicating that UV of broader spectrum is more effective
in eliciting a photocarcinogenic response Additional exposure to filtered
fluorescent light (280-400 nm) either before or after combined treatment of
8-MOP and UV-365 nm increased the skin tumor incidence to 100% and shortened
•«
the latent period, suggesting a possible interaction involving the 365 nm-
induced 8-MOP photoadducts and other molecular Lesions induced by emissions
between 280-400 nm (see Section 532533).
In addition to the demonstration of spectral dependence of 8-MOP photo-
care inogenic ity, Grube, Fry and Ley (73, 74) also noted strain difference in
carcinogenic response to 8-MOP In marked contrast to the high incidence of
skin tumors in SHK-hairless-1 mice, 8-MOP plus UV (300-400 nm) produced no
415
-------
skin carcinomas and 8-MOP plus UVA induced only a low incidence (4/19) of skin
carcinomas (and after a prolonged latent period) in HRS/J/Anl hairless mice
The HRS/J/Anl hairless mice were also less susceptible than SKH-hairless-1
mice to the carcinogenic effect of a combination treatment of 8-MOP, UV-365 nm
and filtered fluorescent light (280-400 nm) However, no significant strain
difference was observed in mice receiving 8-MOP plus UV-365 nm The strain
difference in response to 8-MOP plus UVA is annulled by treating the HRS/J/Anl
mice with 12-0-tetradecanoylphorbol-13-acetate (TPA), a typical tumorigenesis
promoter, suggesting that the strain difference is associated with a differ-
ence in expression of dormant tumor cells rather than with tumor initiation
532532 CARCINOGENICITY OF PSORALEN AND RELATED COMPOUNDS
STRUCTURE-ACTIVITY RELATIONSHIPS
In Vivo Can mogenicity The search for a safer drug for treatment of
psoriasis, the concern over human exposure to naturally occurring furocoumarin
compounds used in suntan lotion or folk herbal medicine have led to extensive
studies on derivatives and structural analogs of 8-MOP At least six deriva-
tives of psoralen and its isomer angelic in have been tested for photocarcino-
genic activity in mice The results of these studies are summarized in Table
LXXIII
The carcinogenicity of five furocoumarins has been studied by Zajdela,
Bisagni and associates (67, 76, 80) using inbred XVIInc/Z albino mice The
compounds (dissolved in acetone) were topically applied on the ears of each
*? 9
animal over an approximately 1 cm surface at the dose of 10 or 15 ug/cm , and
irradiated 15 minutes later with UVA or UV-365 nm at the radiation dose of
2
1 68 joules/cm High incidence of skin tumors were observed in mice given
the three bifunctional furocoumarins — psoralen, 8-MOP and 5-MOP (76) The
416
-------
P 1 of 2
Table LXXI1I
Comparative Photocarcinogenicity of Psoralen and Related Compounds in Mice After Skin Painting'
Irrad lat ion
Compound Mouse strain Dosing regimen
Psoralenb XVIInc/z 115 x 10 jig/cm2
8-MOPc XVIInc/z 115 x 10|Jg/cm2
Albino hairless 34 x 0 01% solution
34 x 0 03% solution
SKH-hairless-1 156-195 x 5 ^ig/<-m2
156-195 x 50 jig/cm2
156-195 x 50 jig/cm2
200 x 40 ug
5-MOP XVIInc/z 115 x 10 /ig/un2
Albino hairless 34 x 0 01% solution
34 x 0 03% solution
3-Carbethoxy- XVIInc/z 106 or 196 x 15 ^ig/cm2
psoralen
SKH-hairless-1 156-195 x 50 wg/cm2
Type
UV-365 nm
UV-365 nm
SSRe
SSRe
UVA
UVA
UVA
UVA
UV-365 nm
SSRe
SSRe
UVA
UVA
Dose
(J/cm2)
1 68
1 68
1 5
1.5
1 0
1 0
2 5
0.72
1.68
1.5
1 5
1 68
1.0
, Skin tumor
Int. idence
97%
92%
100%
100%
65%
70%
54%
100%
85%
100%
100%
0%
0%
Latency
av =
av =
T50
T50
T50
T50
T50
T50
av =
T50
T50
40 wk
58 wk
= ~ 22 wkg
= - 18 wk8
= 56 wk
= 60 wk
= 60 wk
= 24 wk
69 wk
= ~ 25 wk8
= ~ 21 wkg
--
Re ference
(76)
(76)
(75)
(75)
(79)
(79)
(79)
(72)
(76)
(75)
(75)
(66)
(79)
-------
Table LXXIII (continued)
of 2
Irradiat ion
Compound Mouse strain
Angel ic in XVIInc/z
SKH-hairless-1
5-Methyl- SKH-hairless-1
angel ic in
4,5'-Dimethyl- SKH-hairless-1
angel icin
Aflatoxin Bj SKH-hairless-1
Dosing regimen
106 or
156-195
156-195
156-195
156-195
156-195
196 x 15 fig/cm2
O
x 50 ug/cm
r\
x 5 ^ig/cm
0
x 50 ug/cm
x 50 ug/cm
o
x 50 fig/ cm
200 x 40 ug
Type
UVA
UVA
UVA
UVA
UVA
UVA
UVA
Skin tumor
Dose
2 f
(J/cm ) Incidence Latency
1 68
2 5-5
1 0
1 0
2 5
7 5
0 72
"moderately carcinogenic"
0 20% T5Q = >60 wk
78% T5Q = 53 wk
97% T5Q = 33 wk
97% T50 = 40 wk
100% T5Q = 42 wk
0%
Reference
(cited
_in_ 80)
(79)
(79)
(79)
(79)
(79)
(72)
aAll compounds listed in this table are noncarcinogenic without photoactivation. At the radiation doses applied
(expressed in joules/cm ), irradiation alone is also without carcinogenic effects
Psoralen is not photocarcinogenic after oral administration (61)
cSee Table LXXII for other careinogenicity studies of 8-MOP.
3-Carbethoxypsoralen is also not photocarcinogenic after i p. injection (67)
eSSR simulated solar radiation Wavelength spectrum includes UVAj UVB, visible and 1R
Latent period for tumor induction TCQ = time for appearance of tumor in 50% of the surviving mice
^Estimated from graph published in the reference
-------
two raonofunctlonal furocoumarins — angelicin and 3-carbethoxypsoralen (3-CP)
— were found to be "moderately carcinogenic" (cited in 80) and inactive (67),
respectively Based on comparison of tumor incidence and latent period, the
relative photocarcinogenic potency of the five compounds follows the order
psoralen > 8-MOP > 5-MOP > angelic in > 3-carbethoxypsoralen (inactive) This
relative order is generally in good agreement with the relative photomutagenic
potency of these compounds and led to the suggestion that mono functional
furocoumarins are less carcinogenic than bifunctional furocoumarins, and may
therefore be potentially safer therapeutic agents for treatment of
psoriasis The finding of photocarcinogenicity of 5-MOP is also of great
importance because the compound was, at one time, used an active ingredient,
to promote pigmentation, in many widely used suntan preparations at concentra-
tions of 12 to 50 ug/ml (5) Based on photomutagenicity data, 5-MOP was
earlier predicted to be photocarcinogenic (5) Like 8-MOP, the photocar-
cinogenicity of 5-MOP can be promoted by the classical tumorigenesis promoter
TPA (see Section 532533) suggesting that photosensitized 5-MOP can
initiate a population of "dormant" tumor cells, which are normally not
expressed but can persist over a long period of time and can progress towards
macroscopic tumors if and when promoted
The photocarcinogenicity of 5-MOP has been confirmed by Young et al (75)
using albino hairless mice The compound was skin-painted on the flanks of
the mice (applied as a 0 01 or 0 03% solution in a mixture of arachis oil and
isopropyl myristate, the volume applied was not specified), and photoactivated
by exposure to simulated solar radiation Compared to 8-MOP, 5-MOP induced
skin tumors with a slightly longer latent period, but the difference was not
considered significant and the two compounds were considered to be equally
potent photocarcinogens
417
-------
The conclusion that bifunctional furocoumarins are more carcinogenic than
their monofunctlonal counterparts has recently been challenged by Mullen et
al (79) using SKH-hairless-1 mice Compounds were topically applied (0 05
ml) on the backs of the mice in 0 01 or 0 1% ethanol solutions and photoac-
tivated 45 minutes later with UVA These investigators did confirm the non-
carcinogenicity of 3-carbethoxypsoraien and the weak carcinogenicity of
angelicin However, two methyl derivatives of angelicin have been found to be
even more carcinogenic than 8-MOP, inducing more skin tumors with a much
shorter latent period under similar conditions (see Table LXXIII) The rela-
tive photocarcinogenic potency of the five furocouraarins tested by Mullen et
al (79) follows the order 5-methylangelicin > 4,5'-dimethylangelicin >
8-MOP > angelicin > 3-carbethoxypsoralen (inactive) The photocarcinogenicity
of 5-methylangelicin appears to be directly dependent on the dose of the com-
pound applied, whereas that of 8-MOP is not Increasing the irradiation dose
has no effect on the photocarcinogenicity of 4,5'-dimethylangelicin but
decreases the 8-MOP-induced tumor incidence due to higher phototoxicity Mice
receiving 0 01 and 0 1% solutions of 8-MOP exhibit severe skin irritation,
scabbing, and epidermal necrosis No sign of phototoxicity was observed in
mice receiving either of the other four mono functional furocoumarins These
findings have led the investigators to hypothesize that the DNA photoadducts
formed with both the mono functional furocoumarins (monoadducts) and the
bifunctional furocoumarins (crosslinks as well as monoadducts), undergo an
error-prone excision-repair process which is potentially carcinogenic The
increased lethality associated with the formation of cross-links by bifunc-
tional furocoumarins may actually reduce the carcinogenic potential of the
affected cells by killing the cells before somatic mutation can take place
The lack of careinogenicity of 3-carbethoxypsoralen was attributed to its
418
-------
photolability According to some unpublished observations (P C Joshi and
M A Pathak, cited in 79), 3-carbethoxypsoralen undergoes rapid transformation
to a less reactive molecule upon irradiation It should, therefore, not be
considered as a typical mono functional furocoumarin compound
In addition to the compounds listed in Table LXXIII, 4, 5' ,8-tnmethyl-
psoralen (trioxsalen) was reported to be carcinogenic in albino mice (M A
Pathak, D M Kramer and T B Fitzpatrick, cited in 79), the details of the
study were not available at the time of this writing Aflatoxin B^, a difuro-
coumarocyclopentenone compound (see Section 5311) structurally related to
furocoumarins, is not carcinogenic after topical application to SKH-hairless-1
mice followed by UVA irradiation (72) Actually, aflatoxin B^ can be photo-
activated to bind to DNA However, the nature of the photobinding of AFBj to
DNA differs from that of metabolically activated DNA binding (see Section
5 3 1.1 4) and is apparently unrelated to the steps leading to carcinogenesis
In Vitro Carcinogenicity Four furocoumann compounds have been tested
for in vitro carcinogenic activity by the cell transformation assay Evans
and Morrow (81) showed that a 8-MOP plus UV treatment of primary mouse, rat
and hamster embryo cultures and several established cell lines (BHl^i/Cl^-},
NIH/3T3 and Balb/c 3T3) caused a high percentage of cell transformation as
judged by altered cell morphology and growth characteristics Subcutaneous
inoculation of 8-MOP-plus-UV-transforraed hamster embryo fibroblasts into 2-
day-old newborn Syrian hamsters produced palpable tumors after 3 days The
same transformed cells grew into spindle cell sarcomas 10-14 days after inocu-
lation into the cheek pouch of adult hamsters The capability of 8-MOP plus
UV to induce in vitro cell transformation has been confirmed by Uwaifo et al
(82) using mouse fibroblast CSH/IOT^ cells With UVA activation, 8-MOP
induced "type II foci" at concentrations of 1 0, 15 and 2 5 ug/ml No such
( 419
-------
transformation by 8-MOP was observed without UVA exposure Among the other
three naturally occurring furocoumarins investigated in the same study,
imperatorin (8-isoamylenoxypsoralen) induced "type III foci" at a concentra-
tion of 10 .ug/ml but was inactive at lower concentrations Marmesin (which is
highly cytotoxic) and chalepin did not induce any foci when tested at concen-
trations of up to 1 0 ug/ml and 5 0 ug/ml, respectively 8-Methoxypsoralen,
imperatorin, marmesin and chalepin have all been isolated from two Nigerian
medicinal plants and are suspected to contribute to the high incidence of skin
cancer in Nigeria and other tropical countries (82)
532533 MODIFICATION OF CARCINOGENESIS BY FUROCOUMARIN COMPOUNDS
There is a scarcity of information on the modification of carcinogenesis
by furocoumarins and the interaction of furocoumarins with other carcinogens
in animals Fry et_ ^1_ (74) reported that SKH-hairless-1 mice are consider-
ably more susceptible than HRS/J/Anl hairless mice to skin carcinogenesis
induced by 8-MOP plus UVA However, treatment of HRS/J/Anl mice with 12-0-
tetradecanoylphorbol-13-acetate (TPA), a typical tumorigenesis promoter,
eliminates this strain difference rendering the two strains equally suscep-
tible The promoting effect of TPA on the photocarcinogenicity of 5-MOP was
also observed by Zajdela and Bisagni (76) using XVIInc/Z mice Treatment witn
TPA 8 months after the termination of 5-MOP plus UVA treatment raised the
tumor incidence from 85% to 100% and increased the percentage of animals with
multiple tumors from 25% to 61% An analysis of the distribution of latent
periods indicated that the additional tumors observed in the promotion experi-
ment arose from accelerating the emergence of tumors which would have had very
long latent periods without promotion. These results indicate that both 8-MOP
and 5-MOP can, after photoactivation, initiate "dormant" tumor cells which can
persist over a long period of time and evolve towards macroscopic tumors if
and when promoted
420
-------
As mentioned in Section 532531, Grube et al (73) demonstrated that
additional exposure to simulated solar radiation either before or after 8-MOP
plus UV-365 run (PUVA) treatment substantially potentiated the photocarcino-
genic effects Mice treated with the simulated solar radiation plus UV-365 run
alone did not develop any tumors The simulated solar radiation (280-400 nm)
contains the UVB (280-320 nm) portion which is known to be photocarcinogenic
(see 83) The results suggest a possible interaction (e g , synergism or
promotion) between PUVA and UVB in photocarcinogenesis In this respect, it
is interesting to note that Roberts et al (84) found that mice treated with
subcarcinogenic doses of PUVA were tumor-susceptible when challenged with UVB-
induced "regressor" tumors cells * The PUVA treatment appeared to generate a
suppressor cell response in mice and apparently allowed the growth of "regres-
sor" tumor cells through immunosuppression Besides animal data, there are
some epidemiologic evidence and a variety of case reports to indicate that
PUVA therapy may potentiate or promote tumor induction in patients who had a
history of prior exposure to substantial amounts of known carcinogens (see^
Section 53255 1). Apparent suppression of cell-mediated immune response
in human skin by PUVA treatment also has been observed (85)
53254 Metabolism and Mechanism of Action
5 3.2.5 4 1 METABOLISM
The pharmacokinetic properties of psoralens have been exensively dis-
cussed in a recent symposium (17) with emphasis on development of dosing
*Tumor cells induced by UVB in mice are of two immunologic types — the "pro-
gressor" cells which grow rapidly when transplanted into normal syngenic
hosts, and the "regressor" cells which are highly immunogenic and are usually
rejected unless transplanted into immunosuppressed syngenic hosts
421
-------
regimens to achieve optimal therapeutic efficacy The metabolism of the two
most widely used psoralen drugs, 8-MOP (methoxsalen) and 4,5' ,8-tnmethyl-
psoralen (trioxsalen) , has been studied by various investigators (25, 86-
90) Seven metabolites have been isolatd from the urine of SKH-hairless-1
mice or of human volunteers given an oral dose of 8-MOP (25, 87) Three major
metabolites have been tentatively identified as (a) 8-hydroxypsoralen (only in
human urine), (b) methoxyfurocoumar i<_ acid, the hydrolyzed open-chain form of
8-MOP, which can be cyclized to the lactone form of 8-MOP, and (c) a glucuro-
nide which, upon hydrolysis, regenerates 8-MOP. Both 8-hydroxypsoralen and
the methoxyf urocourraanc acid are not phototoxic. A fourth major metabolite
may be either (a) the 4',5'-epoxide or 4',5'-dihydrodiol derivative of
8-MOP, or (b) the 3,4-epoxide or 3,4-dihydrodiol derivative of 8-MOP, or
(c) 6-carboxymethyl-7-hydroxy-8-methoxycoumarin The identity of the other
three minor metabolites remains to be elucidated The metabolites identified
in the urine of dogs given an i v dose of 8-MOP include the methoxyfurocou—
marie acid, and 6-carboxymethyl-7-hydroxy-8-methoxycoumarin and its conjugate
(88) In vivo studies showed little or no metabolism of 8-MOP by mouse Liver
homogenates or by microsomes (25) However, rat liver preparations effec-
tively metabolize 8-MOP in v^tro, and the metabolic rate is enhanced by pre-
treatment of the animals with the typical enzyme inducers, phenobarbital or
^ -naphthoflavone (90) The metabolism of 4,5',8-trimethylpsoralen in human
volunteers and mice mainly involves oxidation of the 5'-methyl group (86,
89). The major urinary metabolite is 4,8-dimethyl-5'-carboxypsoralen which is
not phototoxic Using in vitro systems, it has been established that the
5'-methyl group is first hydroxylated and then further oxidized to carboxy
group. Both the S'-hydroxy and 5'-carboxy derivatives of 4,5',8-trimethyl-
psoralen may be further hydroxylated With the possible exception of epoxida-
422
-------
tion, all the above metabolic pathways appear to represent detoxification
For both 8-MOP and 4,5',8-trimethylpsoralen, there is no evidence that
metabolism would enhance the mutagenicity of the compound in the Ames test
(see Table LXXI) Photoactivation appears to be the major, if not the sole,
pathway in the activation of most psoralens to toxic, mutagenic or carcino-
genic intermediates
532542 MECHANISM OF ACTION
Despite extensive studies (rev in 8, 12, 14, 19, 20, 25), the mechanism
of carcinogenesis and mutagenesis by psoralens is not well understood It
appears that, for most psoralens, at least three types of mechanisms (a)
intercalation complexrng with DNA, (b) photoreac£ion with DNA, and (c) epi-
genetic mechanisms, either alone or in combination, may contribute to their
carcinogenic or mutagenic action
It is now generally believed that intercalation complexing with DNA is
probably responsible for the "dark" mutagenicity of most psoralens, and repre-
sents an important initial step in the photobinding of these compounds to DNA
(see discussion below) The tricyclic structure of psoralens, and to a lesser
extent of angelicins, provides a favorable molecular size and shape for inter-
calation complexing with DNA Polynuclear hydrocarbons, as well as poly-
nuclear heteroaromatics structurally related to furocoumarins, are known to
intercalate into DNA, resulting in increased solubility, DNA viscosity^and
melting temperature (Tm) and spectral shifts (see Section 5 1 1 6 2 3 in Vol
IIA and Sections 5 1 5 1 and 5 1.5 2 in Vol IIB) .
The evidence supporting the existence of furocoumarin-DNA intercalation
complexes has been reviewed (1, 20, 91) The usually low solubilities of
furocoumarins in water (see Table LXIX) is significantly enhanced by the addi-
423
-------
tion of DNA Likewise, the solubility of DNA in aqueous solution can be
increased by inclusion of furocoumarin compounds such as 5-MOP Paralleling
this increased solubility, both the viscosity and melting temperature (T ) of
DNA solution are elevated A spectral shift in the absorption maxima and a
decrease in extinction coefficient are observed after furocoumarin-DNA com-
plexing (1) There is also evidence that complexing causes slight unwinding
of the DNA helix (91) The furocoumarin-DNA noncovalent binding constants of
several furocoumanns have been determined (20, 91) There appears to be an
inverse relationship between the binding (association) constants of furocou-
marins and their water solubilities, suggesting that mainly hydrophobic forces
are involved in the complexes (20) just as with the polynuclear hydrocar-
bons The stability of the furocoumarin-DNA intercalation complexes is low
and this is consistent with the observation that most furocoumanns are either
inactive or weak, direct-acting, frameshift mutagens when assayed in the dark
(see Section 532522) Despite the weak intercalation complexing of the
psoralen molecule with DNA, such intercalation plays an important role if the
molecule is coupled with a reactive functional group For example, psoralen-
8-glycidol ether is one of the most potent synthetic mutagens known, substitu-
tion of the psoralen nucleus with a phenyl moiety reduces the activity by more
than 30 times (see Section 5 3.2 522) Furocoumanns show a much lo^er
capacity to form noncovalent complexes with RNA, and photobind to RNA less
effectively than to DNA Moreover, there is some evidence that furocoumarins
do not intercalate randomly but do so preferentially in regions having a high
density of repetitive alternations of purines and pyrimidines (20) A
sequence-specific noncovalent complexing has also been shown to occur before
the covalent binding of the potent carcinogen, aflatoxin B^, to DNA (see
Section 5 3 1.1 4 2)
424
-------
The mechanism of photobinding of psoralens and angelic ins to DNA has been
extensively investigated (rev in 1, 8, 9, 19, 20, 25) Figure 17 summarizes
the proposed mechanism for bifunctional linear furocoumarins Once the furo-
coumann-DNA complex is established, covalent bonds between the furocoumarin
molecule and DNA are formed upon irradiation Firstly, a CA-cycloaddition
reaction involving the 5,6-double bond of pyrimidines and the exicted (triplet
state) 3,4- or 4',5'-double bond of the psoralen molecule occurs, forming 3,4-
or 4',5'-monoadducts Then, the 3,4- or 4',5'-monoadducts may be further
photoactivated to react with a second pyrimidine base to give a diadduct
When a diadduct is formed with the two strands of DNA, a cross-link is
formed Photoreaction of angelicins with DNA yields mainly 4',5'-monoadduct
(20, 92) because the angular structure of angeiicins forecloses the possi-
bility of photoreaction of the 3,4-double bond with a second pyrimidine base
in the sister DNA strand The photoreaction of 3-carbethoxypsoralen is also
expected to yield 4',5'-monoadduct because of the presence of bulky substi-
tuent in the 3-position, however, the identity of this photoadduct has not
been clearly defined (20) The relative ability of psoralens and angelicins
to photoreact with DNA, in terms of total photobinding (mostly monoadducts
plus some crosslinking), ranks as follows 4,5',8-trimethylpsoralen » 4,5'-
dimethylangelicin > psoralen > 8-MOP > 5-MOP > angelicin > 3-carbethoxy-
psoralen (19, 20). For crosslinking, the ranking is 4,5'8-trimethylpsoralen
» psoralen > 8-MOP > 5-MOP, whereas 3-carbethoxypsoralen and angelicin show
little or no crosslinking activity (19) For 4,5',8-trimethylpsoralen, 5-MOP
and 8-MOP, the ratio of raonoadduct to crosslink formation is about 9 1 (20)
The role of monoadduct formation and crosslinking in photocarcmogenesis
and photomutagenesis by furocoumarin compounds is not clearly understood
Crosslinks are expected to be more effective than raonoadducts for inducing
425
-------
0
Thymme
Intercalation
Psora Ien
complexmg
0
^0
0' -^ -0
Weak complex
nr T ""tCHs
^o-^V^o
Weak complex
H
Diadduct (Crosslink)
Fig. 17. Proposed mechanism for photobinding of psoralen to DNA. The
first step is believed to involve weak, intercalation complex ing between
psoralen and pyrimid me (e.g., thymine) residues in DMA. Upon UVA-
irradiation, a CA-cycloaddition reaction takes place, forming 3,4- or
4* ,5'-monoadduct. Further addition of a second molecule of py raid me to
either monoadduct gives rise to diadduct (crosslink). [Modified from B R
Scott, M.A. Pathak, and G.R. Mohn Mutat.,Res. 39. 29 (1976).]
-------
potentially more genotoxic large-scale changes in the genome (93) However,
crosslinks are also more difficult to repair (94) and, consequently, are more
likely to lead to cell lethality than monoadducts Photorautagenicity studies
using various microbial test organisms have consistently shown that bifunc-
tional furocoumarins are more mutagenic than mono functional furocoumarins.
Furthermore, the presence of one or more proficient DNA repair pathways
appears to be a requirement for the expression of photomutagenicity of some
furocoumarins in various microbial test organisms (see Section 532522)
These results suggest that at least one probable mechanism of photomutagenesis
is through "fixation" of the furocoumarin-induced DNA lesions by DNA repair
mechanisms The usually error-prone .SOS repair mechanisms, triggered by furo-
coumarin-DNA photoadducts (both crosslinks and monoadducts), contribute to
photomutagenesis by promoting cell lethality and by introducing errors during
the repair synthesis Photocarcinogenicity studies of 8-MOP in mice indicate
that DNA crosslinking does not play a role in carcinogenesis (73) and actually
appears to reduce the carcinogenic potential of 8-MOP by causing cell death
(see Section 532531) Certain mono functional furocoumanns (e g ,
5-methylangelicin, 4,5'-dimethylangelicin) are in fact more photocarcinogenic
than bifunctional furocoumanns (see Section 532532) The ranking of
photocarcinogenic potency follows the order 4,5'-dimethylangelicin >
psoralen > 8-MOP > 5-MOP > angelicin > 3-carbethoxypsoralen, which is poorly
related to the relative crosslinking activities of the compounds, but corre-
lates well with their relative ability to form monoadducts The lack of cor-
relation between mammalian photocarcinogenicity and microbial photomuta-
genicity is intriguing, the possibility that mammalian and microbial cells may
differ in their ability to repair furocoumarin-DNA photoadducts should be
investigated
426
-------
The mechanisraCs) by which photoadducts of furocoumarins initiate the pro-
cess of carcinogenesis is not known It has been shown that both the struc-
ture and the template functions of DNA are altered by photoreaction with furo-
coumanns (rev in 1, 9, 10), and such changes are expected to be potentially
genotoxic The contributory role of DNA repair pathways to photocarcino-
genesis is not clear The repair of photoadducts is error-prone and it is
possible that it would introduce errors during repair synthesis and lead to
somatic cell mutation However, there is evidence that the excision repair of
both monoadducts (94, 95) and especially of crosslinks (94) of furocoumarins
is relatively inefficient in mammalian cells Certain portions of the
mammalian genome (e g», a specific region of DNA ui monkey cells called
-------
gens, by inducing antigen-specific T-suppressor cells Roberts et al (84)
have shown that transplanted tumor cells, which normally do not grow in
immune-proficient mice, grow rapidly in PUVA-treated mice Thus, it appears
that the immunosuppression brought about by PUVA treatment promotes dormant
tumor cells or allows their growth and expression Consistent with the promo-
tion of carcinogenesis by PUVA treatment, Gange (100) showed that PUVA treat-
ment of hairless albino mice induces epidermal ornithine decarboxylase, the
enzyme which is believed to play a role in tumor promotion by the typical
promoter, 12-0-tetradecanoylphorbol-13-acetate (101). Another possible
indirect mechanism through which PUVA treatment may induce carcinogenesis is
the induction (activation) of, latent tumor virus(es) Coohill and Moore (102)
reported that treatment of Simian virus 40-transformed hamster kidney cells
with PUVA leads to the induction of the SV40 virus, an oncogenic DNA tumor
virus (103) In addition to the specific indirect mechanisms exemplified
above, psoralens can photoconjugate with proteins leading to the inactivation
of cellular enzymes (19) It is possible that random inhibition of some key
enzymes can lead to disturbance of gene expression and loss of control of
nuclear function. Granger et al (104, 105) have shown recently that PUVA
treatment inactivates two of the three enzymic functions of DNA polyraerase of
Escherichia coll, whether this finding has any relevance to mammalian carcino-
genesis remains to be studied The reactive oxygen species ( C>2, Oo'j'OH,
etc ) produced by the interaction of triplet state psoralens with molecular
oxygen (see Section 532521) are also potentially carcinogenic They can
cause direct DNA damage or can initiate membrane lipid peroxidation (19) lead-
ing to the generation of other potentially carcinogenic hydroperoxides and
unsaturated aldehydes (see Vol. IIIA, Section 5.2 1.1 4 1.1) There is also
some evidence that the singlet oxygen may be responsible for at least a part
of the photomutagenic effect of 8-MOP (21, 106).
-------
5_ 3 2 5 5 Environmental Significance
5 3 2 5.5.1 EPIDEMIOLOGIC EVIDENCE
Psoralen-containing herbal medicine, in combination with sunlight, have
been used in the treatment of vitiligo in Egypt and in India since 1200-2000
B C (2) This form of photochemotherapy has gained widespread interest in
modern medicine in the last decade, as oral psoralens plus UVA (PUVA) therapy
has been found to be highly effective in the treatment of psoriasis, and
mycosis fungoides as well as vitiligo Since 1974 several thousand patients
have received PUVA treatment in clinical trials in Europe and the United
States The safety of such therapy has been a subject of great concern as
animal carcinogenicity data indicate increased incidence of skin cancer in
mice given 8-MOP (topically or intraperitoneally) plus UVA Several prospec-
tive epidemiologic studies are currently being undertaken to investigate the
potential excessive risk of skin cancer in patients undergoing PUVA therapy
Considering the long latent period required for the induction of most human
cancer, all of these studies can only be considered to be at the beginning
stage at the time of this writing. The interim results of these studies are
summarized in Table LXXIV It should be noted that some of the patients
selected for PUVA therapy had prior exposure to other treatments (eg , thera-
peutic doses of X-ray, UVB, arsenical compounds, nitrogen mustard) that are
themselves potentially carcinogenic These patients should be considered as a
separate subset in the assessment of carcinogenic risk of PUVA therapy
Moreover, the susceptibility of individual patients to the potential cancer
risk of PUVA therapy is expected to be affected by a variety of host factors,
such as degree of pigmentation, skin type and DNA-repair efficiency
429
-------
1 of 2
Table LXXIV
Summary of Epidemiologic Studies on Induction of Skin Cancer in Patients Receiving PUVA Therapy3
Authors
Study group
Cumulat ive
dose
Follow-up
Results
Honigsmann et al. (107)
Wilson (108)
Lassus et al. (109)
Roenigh and Caro (110)
318 psoriasis patients
in Innsbruck, Austria
300 psoriasis patients
in Utah, USA
568 psoriasis patients
in Helsinki, Finland
631 psoriasis patients
in various treatment
centers in the USA
660 J/cm2
<500 J/cra2
27 months
Not specified 4 years
2 1 years
(range,
1-3.6 years)
1,000 J/cm2 4 years
None of 246 patients with no prior
exposure to other carcinogens
developed cancer (cancer incidence
of 0 08 expected) Four of 72
patients with prior exposure to
arsenic developed skin cancer (an
incidence higher than that of the
general population).
Two patients had skin cancer, one
of the patients had a previous
history of skin cancer
No significant increase in cancer
risk in patients with or without
prior exposure to other
careinogens
Ten patients (3 with prior expo-
sure to ionizing radiation)
developed skin tumors The inci-
dence of skin cancer in patients
with no prior carcinogen exposure
was not higher than that of the
general population
-------
Table LXXIV (continued)
2 of 2
K.
Authors
Wolff, unpublished
Study group
4,600 psoriasis patients
Cumulative
dose
Not specified
Follow-up
6 years
Results
Skin tumor incidence
in 3,450
results (cited in
ref 111)
Stern et al. (112),
Stern (113)
Harrist et al (114)
in Europe
1,380 psoriasis patients
in 16 treatment centers
in the U S A
1,600 J/cm2 5 years
230 vitiligo patients
in India
Not specified 55 months
Henseler and Christopher 381 psoriasis patients Not specified Since 1976
(115) in the Federal Republic
of Germany
patients with no prior exposure to
other carcinogens apparently lower
than expected (0 03 vs 0 08)
Incidence in 1,150 patients with
prior exposure to carcinogen 5
times higher than the general
population
No significant increase in cancer
risk in patients who had no prior
carcinogen exposure and who
received fewer than 80 PUVA treat-
ments Significant increase (9
times higher) in risk of develop-
ing squamous cell carcinomas in
patients with prior carcinogen
exposure
No evidence of increase in skin
cancer risk
No evidence of increase in skin
cancer risk
With one exception, all the studies involved the use of
psoralen, and 4,5" ,8-trimethylpsoralen were used as the
sunlight
8-MOP plus UVA In the study of Harrist £t__al_ (115), 8-MOP,
photosensitizer, and the patients were exposed to natural
'Cumulative PUVA expressed in terms of Joules/cm2 The data shown were those calculated by Stern (113)
-------
As Che results summarized in Table LXXIV indicate, there appears to be no
evidence, thus far, that PUVA therapy Leads to increased incidence of skin
cancers in patients who had no history of prior exposure to other carcinogenic
treatment In fact, in some of these studies, the incidence of skin cancer
was apparently slightly lower than those of the control or general popula-
tion However, considering the short follow-up time in all these studies and
the low cumulative dose in some of these studies, it would be premature to
draw any conclusion at this point Even if PUVA therapy is carcinogenic, a
rise in the skin cancer rates in these patients would not be expected until
the 1990s
In contrast to patients with no prior carcinogen exposure, patients who
had received treatments with X-ray, UVB or arsenicals for their skin disease,
before being subjected to PUVA treatment, developed significantly higher inci-
dences of skin cancer than the control or general population It is not clear
to what extent the increase may be attributed to PUVA therapy. Reviewing the
data from five different studies, Halprin et al (111) calculated that the
maximum incidence of skin cancer in these patients was 2% per year This rate
was not considered to be any higher than that expected from these patients
without PUVA therapy (116) Stern (113), however, is of the opinion that pro-
longed exposure to high doses of PUVA can lead to substantial increase in the
risk of developing skin cancer, particularly in patients previously treated
with ionizing radiation In some patients the tumors appeared within 2 years
after the start of PUVA therapy (112) After 4 years of follow-up, the
patients were nine times more likely to develop squamous cell carcinomas than
the control group (p < 0 001) and two times more likely to develop basal cell
carcinomas He cautioned that the negative results seen in other studies
could be due to shorter follow-up time and lower cumulative dose.
430
-------
In addition to the above studies, there are a number of individual case
reports of rapid induction of skin cancer in patients after receiving PUVA
therapy In all these cases, the patients either had previous carcinogen
exposure or were believed to be "intrinsically susceptible " Holier and
Howitz (117) reported that a 43-year-old vitiligo patient, who had been pre-
viously treated with arsenic trioxide 8 years before, developed multiple basal
cell carcinomas 2 months after receiving 8-MOP plus sunlight treatment for 91
days Verdich (118) detected multiple squamous cell carcinomas in mycosis
fungoides patients who received PUVA therapy after exposure to electron beam
or nitrogen mustard Stern et al (119) observed an increase in skin cancer
rate in patients, with previous histories of yery high skin exposure to tar
and far-UV, within 2-7 years after the start of PUVA therapy. Reed and asso-
ciates (97, 98) found that xeroderma pigmentosum patients (deficient in DNA-
repair enzymes) could develop skin tumors within 1 month after starting PUVA
therapy The extremely short induction time in these cases, together with
animal data, led Bridges and Strauss (120) to hypothesize that PUVA may act as
a "pseudopromotor," allowing the expression of preexistent neoplastic foci
that would have otherwise remained dormant This hypothesis was supported by
the observation by Strauss et al. (85) that PUVA therapy can reduce or abolish
cell-mediated immunity in human skin The totality of the above epidemiologic
evidence and case reports clearly indicate that, at least in certain types of
patients, PUVA therapy can present a substantial increase in cancer risk
Thus, it is imperative to conduct a risk-benefit analysis for each individual
patient selected for PUVA therapy
5 3 2.5 5 2 ENVIRONMENTAL SOURCES, OCCURRENCE AND EXPOSURE
Furocoumarin compounds have a wide distribution in the plant kingdom
(rev. in 1, 9, 32, 121, 122) Over twenty-eight naturally occurring deriva-
431
-------
tives of psoralen or angelicin have been isolated from five major botanical
families (a) Umbeliferae (parsley family) which includes parsley, parsnip,
celery, Ammi ma jus (bishop's weed), Angelica ar change lie, a, (b) Rutaceae (rue
family) which includes bergamot fruits, lime, gas plants, cloves, common rue,
(c) Leguminosae (legume family) which includes Psoralea corylifolia,
Xanthoxylum flavum, (d) Moraceae (fig family) which includes Ficus caria
(fig), and (e) Orchidacea (orchid family) They are present in the fruits,
seeds, leaves or roots of the plants The most frequently found furocoumarins
include 8-MOP (xanthotoxin, methoxsalen), 5-MOP (bergapten) , isopimpinellin
(5,8-dimethoxypsoralen), 4,5',8-triraethylpsoralen (trioxsalen), psoralen,
Vmperatorin (8-isoamylenoxypsoralen) and angelicin (1, 121). The quantitative
data on concentration are relatively scant Ivie et al (123) reported that
parsnip (Past inaca sativ^a) root contains an average of 26 ppm 8-MOP, 10 5 ppm
psoralen and 3 2 ppm 5-MOP These compounds are not destroyed by usual cook-
ing procedures (boiling or microwave) Carrot (Daucus carota) root contains
negligible amounts «0 3 ppm) of the above three compounds Trace amounts of
psoralen, 5-MOP, 8-MOP and isopimpinellin were detected in several varieties
of healthy celery (Apium graveolens), their total concentration did not exceed
1 3 ppm (124) In contrast to healthy celery, celery infected with fungus
(e g , Sclerotinia sclerotxorum) produces high concentrations of psoralens,
perhaps as phytoalexins, in response to disease infection Levels of
psoralens (8-MOP, 4,5',8-trimethylpsoralen) in such cases can reach hundreds
or perhaps thousands of parts per million (9, 33, 125)
Human exposure to furocoumann compounds may originate from a variety of
sources which include (a) photochemotherapeutic or medicinal uses, (b) appli-
cation of cosmetics, (c) occupational exposure and (d) ingestion of psoralen-
containing foodstuffs The use of psoralen-containing plant extracts in
432
-------
photochemotherapy dates back to 1200-2000 B C when boiled extracts of the
fruit of Annni majus and Psoraiea corylifolia was used ia Egypt aad India,
respectively, along with sunlight, to treat vitiligo (2) The interest in
this photochemotherapeutic method has surged since 1974 when Parnsh et al
(4) discovered that oral 8-MOP plus UVA (PUVA) therapy is highly effective in
the treatment of psoriasis Therapy with PUVA has subsequently been found
effective in the treatment of a number of other skin diseases which include
vitiligo, mycosis fungoides (a form of malignant lymphoma which arises in the
skin), polymorphous light eruption, palmar/plantar vesicular dermatitis and
eczema (2) In some cases, PUVA therapy is the only effective treatment
available Currently, over 7,500 patients in the United States and Europe are
receiving PUVA therapy in clinical trials In addition to photochemotherapy,
psoralen-containing plants are also used as folk medicine in some tropical
countries Concoctions of roots of Clausena anista (wild Rutaceae), Ruta
chalepensis, and Afraegle paniculata (Rutaceae) are used in the treatment of
various diseases ranging from hemorrhoids to measles, scarlet fever, headaches
and heart ailment in Guatemala, Nigeria and other tropical countries (126).
These preparations contain 8-MOP, imperatorin, chalepin and marmesin (see 82)
Human exposure to furocoumarin compounds may occur through the use of
some cosmetics products which contain oil of bergamot , a citrus extract with
5-MOP or bergapten content (6, 7). Oil of bergamot was and may still be used
in perfumery in Eau de Cologne which is responsible for the so-called "Eau de
Cologne dermatitis" (erythema and blistering of the skin) in some consumers
(6) 5-Methoxypsoralen was or is perhaps still used as an active ingredient
in many widely used suntan lotions, at concentrations of 12 to 50 -ug/ml, to
promote skin pigmentation (5) With the demonstration of photocarcinogenicity
of 5-MOP in animals (see Section 532532), it is obvious that such
unnecessary exposure should be avoided
-------
Besides cosmetics, dermal exposure to psoralens may also occur to profes-
sional fruit or vegetable pickers Celery workers in Massachusetts (USA) were
reported to develop severe dermatitis after handling rotten celery It was
subsequently established that extracts of celery rot ("pink rot") contained
high amounts of 8-MOP and 4,5' ,8-tnmethylpsoralen (33) which, in combination
with sunlight, produced the characteristic phototoxic effects (see 34)
Occasional dermatitis has also been reported in lime pickers and in workers in
contact with fig leaves, both of which contain psoralens (1, 6)
The extent of human exposure to psoralens from food sources is not known
and is dependent on the individual's diet Human foodstuffs known to contain
psoralerts include figs, parsley, parsnip roots and celery (I, 123, 124) Ivie
et al (123) estimated that consumption of 0 1 kg of either raw or cooked
parsnip root, which contains about 40 ppm psoralens, could expose an indivi-
dual to 4 to 5 mg of psoralens Such exposure might be expected to cause
pharmacological effects in some individuals (the prescribed dosage of
4,5',8-trimethylpsoralen in PUVA therapy for vitiligo is 5 mg) It is impor-
tant to note that individuals consuming food which contain rotten celery or
parsnip could have substantially higher exposure, because the concentration of
psoralens in such products can reach hundreds and perhaps thousands of parts
per million Persons in the high risk group (e g , xeroderma pigmentosum
patients, persons with history of prior exposure to carcinogen) should mini-
mize or avoid the consumption of such food products, particularly if they have
a high exposure to sunlight.
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-------
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SOURCE BOOKS AND MAJOR REVIEWS FOR SECTION 5325
1 Pathak, M A , and Dunnick, J K (eds ) "Photobiologic, Toxicologic,
and Pharmacologic Aspects of Psoralens," Nat Cancer Inst Monograph
No 66, NIH publ No 84-2692, U S Department of Health and Human
Services, Washington, D.C , 1984, 261 pp.
2 Scientific International Research (ed ) "Psoralens in Cosmetics and
Dermatology," Proceeding of the International Symposium, Paris, April
1981, Pergamon, New York, 1982, 447 pp.
3 Scott, B R , Pathak, M A., and Mohn, G R Mutat. Res 39, 29-74
(1976).
4 Song, P -S , and Tapley, K J Jr Photochem Photobiol 29_, 1177-1197
(1979)
5 Parsons, B J Photochem Photobiol 32_, 813-821 (1980)
6 International Agency for Research on Cancer IARC Monogr 24, 101-124
(1980)
7. Busby, W F , and Wogan, G N. Psoralens In "Mycotoxins and N-Nitroso
Compounds Environmental Risks" (R.C Shank, ed ), Vol. II, CRC Press,
Boca Raton, Florida, 1981, pp. 105-119.
8. Bridges, B A Environ Mutagen. _5_, 329-339 (1983)
9. Cimino, G.D , Camper, H B. , Isaac, S T , and Hearst, J E Ann Rev
Biochem. 54 (1985, in press).
443
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