73
Mutation Research, 79 (1980) 73--77
© Elsevier/North-Holland Biomedical Press
Short Communication
MUTAGENICITY OF PSORALEN EPOXIDES
G. WAYNE IVIE a, JAMES T. MACGREGOR b and BRUCE D. HAMMOCKc
a Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research,
Science and Education Administration, U.S. Department o f Agriculture, College Station, T X
77840, b Western Regional Research Laboratory, Agricultural Research, Science and
Education Administration, U.S. Department o f Agriculture, Berkeley, CA 94710, and
c Department o f Entomology, University o f California, Riverside, CA 92502 (U.S.A.)
(Received 14 January 1980)
(Revision received 31 March 1980)
(Accepted 2 May 1980)
Linear furocoumarins (psoralens) occur widely in nature as constituents of
hundreds of plant species from several families (Ivie, 1978b; Pathak et al.,
1962). Many psoralens are potent photosensitizers, and some are used medicinally as light-activated drugs for the treatment of certain skin disorders, including skin depigmentation (leukoderma) and psoriasis (Pathak et al., 1962; Scott
et al., 1976). Humans are also exposed to psoralens by the consumption of certain foods, and dermally through perfumes and fragrances, skin-tanning preparations, and the exposure of field workers and food processors (Pathak et al.,
1962; Scott et al., 1976). Many cases of psoralen p h o t o t o x i c i t y in humans have
been d o c u m e n t e d as a result of these interactions (Pathak et al., 1962), and
livestock and poultry are sometimes likewise affected (Ivie, 1978b).
Psoralens crosslink with DNA in the presence of activating ultraviolet light
and are mutagenic, but have little (Bridges and Mottershead, 1977) or no (Scott
et al., 1976) mutagenicity in the absence of light. For this reason, psoralens are
n o t generally considered to pose a significant genetic, mutagenic, or carcinogenic hazard to man (Scott et al., 1976). However, some plant psoralens have
a-epoxy ether substituents that could dramatically affect their light-independent mutagenic potential. Many epoxides are mutagenic (Wade et al., 1978,
1979), and because psoralens readily intercalate between pyrimidine bases of
DNA strands (Scott et al., 1976), the potential for subsequent light-independent alkylation reactions by the epoxide moiety of psoralen epoxides may be
greatly increased. We report here that 2 naturally occurring psoralen epoxides
are only weakly mutagenic in the dark, but that a closely related synthetic psoralen epoxide is a p o t e n t mutagen, and in fact may be the most mutagenic alkyl
epoxide yet studied.
Mutagenicity studies were conducted with histidine-dependent strains
(TA100, TA1535, TA98 and TA1537) of the bacterium S a l m o n e l l a t y p h i m u -
74
rium, using the plate-incorporation assay developed by Ames et al. (1975). Mutagenicity of the test c o m p o u n d s was evaluated both with and without the
addition of rat liver enzyme (S-9) preparations (Ames et al., 1975) and in some
cases with added mouse-liver epoxide hydrase enzymes ( H a m m o c k et al.,
1979).
The natural psoralen epoxides studied were heraclenin and oxypeucedanin
(Fig. 1), which are the major psoralen epoxides occuring in most plants. Oxypeucedanin was isolated from the ripened seed of A m m i rnajus (Ivie, 1978a),
and herclenin was synthesized by reported procedures (Ivie, 1978a). Psoralen-8glycidyl ether (PSGE, Fig. 1) was prepared by the reaction of epibromohydrin
with x a n t h o t o x o l (8-hydroxypsoralen) (Ivie, 1978a). Glycidol and phenyl glycidyl ether (PGE) were obtained commercially, and the dimethyl analog of glycidol (glycidol-DiMe, Fig. 1) was prepared by epoxidation of commercially obtained 3-methyl-2-buten-l-ol. Structures of all c o m p o u n d s were confirmed by
nuclear magnetic resonance (NMR) and mass spectral analysis, and each
appeared to be pure on the basis of NMR and/or thin-layer chromatography.
Appropriate levels of these c o m p o u n d s were added to the test plates via 100 pl
of DMSO (Ames et al., 1975). Control plates received DMSO only, and ariatoxin B1, MNNG, or 9-aminoacridine were used as positive controls to confirm
proper functioning of the asSay system (Table 1). To minimize the possibility
of light-induced mutations by the test compounds, all phases of the assays were
c o n d u c t e d either in the dark or under low intensity artificial lighting that
emitted essentially no long- or short-wave ultraviolet radiation.
Heraclenin is a weak mutagen in S. typhimuriurn strains TA100, TA98 and
>200(] o
,
>1700 o
7
~- 1000
/
oi
800
o ,.
CH CH ~ '
/
~
/
/
/
600
i
~400~z
I
......
f o ~1~o
i.o
I
~]
~..../....
o ....
.o. iH
HO--CH CH--c
,o-~
,.
G ycidol
//
~'/
~:"
J
CH
POE//
c~°Xc~H
i'
/
'
0001
k~
/
/I
,,. / /
/ ,6
Oxypeucedanin
'CH,
J
~ + o
I
OOl
'
I
I
' 'ilil
I
I ~
O1
/ ,'
'
1
"
10
I
.....
I
I00
~UMOL PER PLATE
F i g . 1. M u t a g e n i e i t y o f p s o r a l e n e p o x i d e s a n d r e l a t e d c o m p o u n d s i n a h i s t i d i n e - d e p e n d e n t s t r a i n o f Salmonella typhirnurium (TA100). The numbers of revertant colonies occurring in untreated (control) plates
(X- -+ S . D . = 1 1 9 -+ 1 3 ) h a v e b e e n s u b t r a c t e d . A f l a t o x i n B 1 w a s u s e d as a p o s i t i v e c o n t r o l (see T a b l e 1, f o o t n o t e b ) . D a t a o b t a i n e d o n a s s a y s p e r f o r m e d w i t h a d d e d r a t - l l v e r e n z y m e s are n o t i n c l u d e d i n t h e f i g u r e .
Addition of these enzymes had no effect on the mutagenicity of any of the compounds studied, with the
e x c e p t i o n o f P S G E a n d P G E w h e r e m u t a g e n i c a c t i v i t y w a s d e c r e a s e d b y as m u c h as 1 o r d e r o f m a g n i t u d e .
75
TABLE 1
MUTAGENICITY OF PSORALEN EPOXIDES AND
D E P E N D E N T S T R A I N S OF Salmonella t y p h i m u r i u m
Compound a
H e r a c l e nin
RELATED
COMPOUNDS
IN
3 HISTIDINE-
R e v e r t a n t c o l o n i e s p e r p l a t e b,c
pmol per
plate
TA1535
TA98
TA1537
0.35
1.05
3.50
10.50
0
0
0
TOX d
10
23
63
211
1
13
51
TOX d
Oxypeucedanin
0,35
3.50
ND e
ND e
10
14
7
9
PSGE
0,002
0.008
0.031
0,105
0
29
206
660
8
32
64
192
0
4
34
187
PGE
0,033
0,33
1.00
3.33
32
215
588
1216
8
0
5
13
1
2
5
5
Glycidol
0.40
1.35
4.05
13.51
261
721
:>1500
~2600
12
0
0
13
0
0
0
4
Glycidol-DiMe
0.98
2.94
9.80
29.40
0
0
0
0
0
0
0
0
0
3
1
1
a S t r u c t u r e s o f t h e c o m p o u n d s are s h o w n in Fig. 1.
b A v e r a g e o f 2--3 d e t e r m i n a t i o n s . T h e n u m b e r s o f r e v e r t a n t c o l o n i e s o c c u r r i n g in u n t r e a t e d ( c o n t r o l )
p l a t e s h a v e b e e n s u b t r a c t e d . T h e s e w e r e (X-+ S.D.) 16 ± 3 ( T A 1 5 3 5 ) , 25-+ 7 ( T A 9 8 ) , a n d 8-+ 2
( T A 1 5 3 7 ) . D a t a o b t a i n e d f r o m assays p e r f o r m e d w i t h a d d e d rat-liver e n z y m e s are n o t i n c l u d e d in t h e
t a b l e , b u t w i t h e v e r y c o m p o u n d a n d b a c t e r i a l strain t e s t e d , m u t a g e n i c a c t i v i t y was e i t h e r n o t a f f e c t e d
or w a s s i g n i f i c a n t l y d e c r e a s e d .
c T h e n u m b e r s o f r e v e r t a n t c o l o n i e s o c c u r r i n g in positive c o n t r o l p l a t e s w e r e as follows: T A 1 0 0 a n d
T A 9 8 ( a f l a t o x i n B I , 0.5 # g / p l a t e , ~ 1 0 0 0 ) ; T A 1 5 3 5 ( M N N G , 5.0 p g / p l a t e , :>2500); T A 1 5 3 7 ( 9 - a m i n o a c r i d i n e , 1 0 0 p g / p l a t e , :>2000).
d G r o w t h inhibition of the b a c k g r o u n d lawn.
e Not determined.
TA1537, and is not mutagenic in strain TA1535 (Fig. 1, Table 1). Its 5-substit u t e d isomer, oxypeucedanin, was available in only very small quantities, b u t
the limited studies c o n d u c t e d with oxypeucedanin suggested that it is no more
active, and possibly even less so, than heraclenin. On the other hand, the synthetic psoralen epoxide PSGE, which differs from the natural psoralens only in
that it lacks dimethyl substitution at the epoxide moiety, is a potent mutagen
in strains TA100 and TA1535 (Fig. 1, Table 1) and, considering the low levels
of spontaneous reversions in strains TA98 and TA1537 (Table 1), is quite
mutagenic in these strains as well. These data suggest that psoralen epoxides,
in the dark, act both as base-pair substitution and frameshift mutagens, whereas
the absence of appreciable activity of PGE and glycidol in strains TA98 and
76
TA1537 suggests that these epoxides have base-pair mutagen specificity (Ames
et al., 1975). Our data on PGE and glycidol agree with those previously
reported by others (Greene et al., 1979; Wade et al., 1979).
Studies in which the epoxide moiety of PSGE was converted to the diol synthetically or by the incorporation of mouse-liver epoxide hydrase (Hammock et
al., 1979) revealed that the diol has greatly reduced mutagenicity and thus confirms that the epoxide is largely if not totally responsible for the mutagenicity
observed in these tests. Dimethyl substitution at the epoxide moiety dramatically diminishes mutagenicity, as evidenced by comparisons of the data obtained
with PSGE and its naturally occurring dimethyl analogs and of glycidol with its
dimethyl analog (Fig. 1). These dimethyl-substituted epoxides are as much as 3
orders of magnitude less mutagenic than their unsubstituted analogs. Electrondonating effects of the methyl substituents, leading to lower reactivity of the
epoxide (Wade et al., 1978), and perhaps steric effects probably account for
the reduced activity observed.
While these studies indicate that naturally occurring psoralen epoxides have
low mutagenic potential and are thus unlikely to be of significant toxicological
significance in man, they further show that a synthetic psoralen epoxide,
PSGE, is highly mutagenic in certain strains of S. typhimuriurn (Fig. 1, Table
1). It appears, in fact, that PSGE may be the most mutagenic alkyl epoxide yet
studied, and its mutagenicity is clearly enhanced by the presence of the psoralen nucleus. The related epoxide PGE differs from PSGE only in the nature
of the aromatic substituent (phenyl versus psoralen), y e t PSGE is as much as
30-fold more mutagenic than PGE (Fig. 1). The most likely explanation for the
enhanced activity of PSGE is that the psoralen moiety, which readily forms
molecular complexes with DNA (Scott et al., 1976), greatly facilitates the
intercalation of PSGE into DNA strands, where the epoxide reacts at some as
y e t undetermined site. Because the psoralen nucleus complexes with and, in the
presence of light, forms mono- or di-adducts with pyrimidine bases (Scott et
al., 1976), PSGE and its analogs represent unique trivalent alkylating agents
that should be very useful models for mechanistic studies of both light and
dark reactions of psoralens and epoxides with DNA.
Acknowledgment
We thank R o b e r t E. Wilson for technical assistance in performing the mutagenicity assays.
References
A m e s , B.N., J. M e C a n n a n d E. Y a m a s a k i ( 1 9 7 5 ) M e t h o d for d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h the
S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31, 3 4 7 - - 3 6 4 .
Bridges, B.A., a n d R.P. M o t t e r s h e a d ( 1 9 7 7 ) F r a m e s h i f t m u t a g e n e s i s in b a c t e r i a b y 8 - m e t h o x y p s o r a l e n
( m e t h o x a l e n ) in t h e d a r k , M u t a t i o n Res., 44, 3 0 5 - - 3 1 2 .
G r e e n e , E.J., M.A. F r i e d m a n , J . A . S h e r r o d a n d A.J. S a l e r n o ( 1 9 7 9 ) I n v i t r o m u t a g e n i c i t y a n d cell transf o r m a t i o n s c r e e n i n g of p h e n y l g l y c i d y l e t h e r , M u t a t i o n Res., 67, 9 - - 1 9 .
H a m m o c k , B.D., S.S. Gill, S.M. M u m b y a n d K. O t a ( 1 9 7 9 ) C o m p a r i s o n o f e p o x i d e h y d r a s e s in t h e soluble
a n d m i c r o s o m a l f r a c t i o n s of m a m m a l i a n liver, in: R.S. B b a t n a g a r (Ed.), M o l e c u l a r Basis of E n v i r o n m e n t a l T o x i c i t y , A n n A r b o r Science Pub., A n n A r b o r , pp. 2 2 9 - - 2 7 2 .
Ivie, G.W. ( 1 9 7 8 a ) L i n e a r f u r o c o u m a r i n s ( p s o r a l e n s ) f r o m t h e seed of T e x a s A m m i m a j u s L. ( b i s h o p ' s
w e e d ) , J. Agric. F o o d . C h e m . , 26, 1 3 9 4 - - 1 4 0 3 .
77
Ivie, G.W. ( 1 9 7 8 b ) T o x i c o l o g i c a l significance of p l a n t f u r o c o u m a r i n s , in: R.F. K e e l e r , K.R. v a n K a m p e n
a n d L.R. J a m e s (Eds.), E f f e c t s of P o i s o n o u s Plants on L i v e s t o c k , A c a d e m i c Press, N e w Y o r k , pp.
475--485.
P a t h a k , M.A., F. Daniels a n d T.B. F i t z p a t r i c k ( 1 9 6 2 ) T h e p r e s e n t l y k n o w n d i s t r i b u t i o n of f u r o c o u m a r i n s
( p s o r a l e n s ) in plants. J. I n v e s t . D e r m a t o l . , 39, 2 2 5 - - 2 3 9 .
S c o t t , B.R., M.A. P a t h a k a n d G.R. M o h n ( 1 9 7 6 ) M o l e c u l a r a n d g e n e t i c basis of f u r o c o u m a r i n r e a c t i o n s ,
M u t a t i o n Res., 39, 2 9 - - 7 4 .
W a d e , D.R., S.C. A i r y a n d J.E. S i n s h e i m e r ( 1 9 7 8 ) M u t a g e n i c i t y of aliphatic e p o x i d e s , M u t a t i o n Res., 58,
217--223.
Wade, M.J., J.W. M o y e r a n d C.H. Hine ( 1 9 7 9 ) M u t a g e n i c a c t i o n of a series of e p o x i d e s , M u t a t i o n Res., 66,
367--371.