Mutagenicity in Salmonella typhimurium of some angelicin derivatives proposed as new monofunctional agents for the photochemotherapy of psoriasis

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Mutation Research, 88 (1981) 17--22 © Elsevier/North-Holland Biomedical Press

MUTAGENICITY IN Salmonella typhimurium OF SOME ANGELICIN DERIVATIVES PROPOSED AS NEW MONOFUNCTIONAL AGENTS FOR THE PHOTOCHEMOTHERAPY OF PSORIASIS

S. VENTURINI, M. TAMARO, C. MONTI-BRAGADIN and F. CARLASSARE 1 Institute of Microbiology, University of Trieste, Trieste; and 1 Institute of Pharmaceutical Chemistry, University of Padua, Padua (Italy) (Received 14 April 1980) (Revision received 28 July 1980) (Accepted 4 August 1980)

Summary A series of angelicin derivatives were tested for their mutagenic activity with and without near-ultraviolet irradiation (NUV) in Salmonella typhimurium strains. After irradiation with NUV, the tested compounds induced different numbers of revertants in strain TA100, indicating that the mutational events involved are base substitutions. In the dark, 3 chemicals behaved as frame-shift mutagens causing reversion in strain TA98.

Some linear furocoumarins are often used in the photochemotherapy of several human skin disorders, such as psoriasis and vit'.fligo. These chemicals are administered orally or topically and then the skin of the patient is irradiated with 320--380 nm near-ultraviolet irradiation (NUV). Furocoumarins intercalate with DNA and, upon exposure to NUV, bind covalently to pyrimidine bases of DNA forming mono-adducts and crosslinks (Dall'Acqua et al., 1971; Musajo and Rodighiero, 1972; Cole and Sinden, 1974; Scott et al., 1976). These photo-adducts inhibit nucleic acid synthesis and cell division, and it is to this action that the therapeutic activity is ascribed. Because furocoumarin therapy produces some undesirable effects, such as skin phototoxicity and the risk of skin cancer (Urbach 1959; Stern et al., 1979), which are connected with crosslink formation, some new angular furocoumarins, namely angelicin and its derivatives, which form only mono-adducts, have been studied. Their capacity to inhibit cell division and their absence of skin phototoxicity are known (Bordin et al., 1978; Bordin et al., 1979), which leads to the suggestion that these compounds be used in the photochemotherapy of skin diseases. We have tested the mutagenic activity of a series of angelicin derivatives in

18 Ames's Salmonella typhimurium tester strains both with and without NUV, with the aim of finding a compound with high therapeutic efficiency and low genotoxic effect. Materials and methods Chemicals tested Angelicin, 4-methylangelicin, 5-methylangelicin, 5'-methylangelicin, 5,5'-dimethylangelicin, 4,5'
R1

R2 Fig. 1. T h e m o l e c u l a r s t r u c t u r e o£ angelicin and its derivatives.

Compound

R1

R2

R3

R4

( A ) Angelicin (B) 4 - M e t h y l a n g e l i c i n

H --CH 3

H H

H H

H H

(C) 5 - M e t h y l a n g e l i c i n (D) 5'-Methylangelicin

H H

H H

H --CH 3

--CH 3 H

(E) 5 , 5 ' - D i m e t h y l a n g e l i c i n (F) 4,5'-Dimethylangelicin

H --CH 3

H H

----CH3 --CH 3

----CH3 H

4'-Hydroxymethyl-4,5'-dimethylangelicin

--CH 3

--CH2 OH

---CH 3

H

(H) 4'-Methoxymethyl-4,5'-dimethylangelicin

---CH 3

---CH20CH 3

--CH 3

H

(I) H y d r o x y c h l o r i d e o f 4 ' - a m i n o m e t h y l - 4 , 5 ' dimethylangelicin

--CH 3

- - C H 2 N H 2 • HCI

---CH 3

H

(G)

19 cent lamp F 15 T 8 BLB 15 W, at 30 cm above the cultures. Irradiated cells, 0.1 ml, were added to 2 ml of molten 0.6% top agar (containing 0.5 mM L-histidine and 0.5 mM biotin), kept at 42°C and poured onto plates of Vogel-Bonnet agar VBMA (Vogel and Bonner, 1956). After 48 h of incubation at 37°C in the dark, revertant colonies were counted. Survivors were counted by plating appropriate dilutions of irradiated cells on the same medium. The doses of NUV used had no significant mutagenic effect in the strains used. For dark mutagenesis, various amounts of chemicals dissolved in ethanol and 0.1 ml of overnight bacterial culture were added to molten top agar and poured onto plates of VBMA. Plates were incubated in the dark at 37°C for 48 h. Control plates received ethanol alone.

o x m

e~ m IZ I-

a m D a z

I U f / ~ /

* H

F i g . 2. M u t a g e n i c a c t i o n o f a n g e l i c i n a n d i t s d e r i v a t i v e s p l u s N U V o n Salmonella typhimurium T A 1 0 0 . E a c h p o i n t is t h e m e a n o f at l e a s t 3 E x p t s . S t a n d a r d e r r o r s o f t h e m e a n s are n o t r e p o r t e d f o r g r a p h i c a l reasons; they ranged between 1 and 8% of each mean.

20

Results and discussion The mutagenic action of angelicin derivatives plus NUV on Salmonella typhimurium strain TA100 is shown in Fig. 2. These substances behave as monofunctional reagents, forming for geometrical reasons adducts with only a single DNA strand ( A s h w o o d ~ m i t h and Grant, 1977; Seki et al., 1978; Bordin et al., 1978). The mutation frequency (ratio of m u t a n t colonies to survivors) depends on the nature of substituents in the angelicin molecule. The hydrochloride of 4'-aminomethyl-4,5' 4-r~ethylangelicin (B) > 5'-methylangelicin (D) ~- 4'hydroxymethyl-4,5'-dimethylangelicin (G) ~- angelicin (A) > 4'-methoxymethyl-4,5'
14

lO.i" Fu q(

,=. o z

>_

l o -~o~

16 3

F i g . 3 . S u r v i v a l o f c o l o n y - f o r m i n g ability o f Salmonella t y p h i m u r i u m T A 1 0 0 p r e s e n c e o f angelicin derivatives.

e x p o s e d to N U V in the

21 20(1-

j B

15o_ <

a. b. z

.C

1oo_

IE

I 5o_

.ADEFGH I I 25 50

Fig. 4. M u t a g e n i c

i 100

I 200

I 300

[ 400

I 500

jug//plat •

a c t i o n o f a n g e l i c i n a n d its derivatives in t h e d a r k i n

Salmonella t y p h i m u r i u m T A 9 8 .

the dose of 500 pg per plate are shown in Fig. 4. There was no evidence of mutagenic activity in the dark up to the maximal dose of 1000 pg per plate for all other angelicin derivatives (Fig. 4). Dall'Acqua et al. (personal communication) have recently found that the photo-binding capacity towards DNA was highest for 4'-hydroxymethyl-4,5'dimethylangelicin (G) and lower for the 4'-methoxymethyl-4,5'-dimethylangelicin (H) and the hydrochloride of 4'-aminomethyl-4,5'
22 because of its strong photobiological activity with no mutagenic effect in the dark and low mutagenicity after N U V irradiation. Acknowledgements We thank Mrs. S. Saincich, Mrs. A. Presti and Mr. C. Gamboz for their skilful technical assistance. References A m e s , B.N., J. M c 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 s f o r 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 t h e 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 t e s t , M u t a t i o n Res., 3 1 , 3 4 7 - - 3 6 4 . A s h w o o d - S m i t h , M.J., a n d E. G r a n t ( 1 9 7 7 ) C o n v e r s i o n o f p s o r a l e n D N A m o n o a d d u c t s in E. coli t o inters t r a n d D N A c r o s s - l i n k s b y n e a r U V l i g h t ( 3 2 0 - - 3 6 0 n m ) : I n a b i l i t y o f a n g e l i c i n t o f o r m c r o s s - l i n k s in vivo, E x p e r i e n t i a , 3 3 , 3 8 4 - - 3 8 6 . B o r d i n , F., F. B a c c i c h e t t i a n d F. Carlassaxe ( 1 9 7 8 ) 4 , 5 ' - D i m e t h y l a n g e l l c i n , a n e w v e r y active f u n c t i o n a l f u r o c o u m a r i n , Z. N a t u r f o r s c h . , 3 3 c , 6 1 8 . B o r d i n , F., F. C a r l a s s a r e , F. B a c c i c h e t t i , F. G u i o t t o , P. R o d i g h i e r o , D. V e d a l d i a n d F. D a l l ' A c q u a ( 1 9 7 9 ) 4,5'-Dimethylangelicin: a new DNA-photobinding monofunctional agent, Photochem. Photobiol., 29, 1063--1070. C o l e , R . S . , m i d R . R . S i n d e n ( 1 9 7 4 ) P s o r a l e n crnss-links in D N A : B i o l o g i c a l c o n s e q u e n c e a n d c e l l u l a r repair, in: O.S. Nygaard, H.I. Adler and W.K. Sinclair (Eds.), Radiation Research Proceedings of the Vth International Congress of Radiation Research, Seattle, W A , Academic Press, N e w York, pp. 582-589. D a l l ' A c q u a , F . , S. M a r c i a n i , L. C i a v a t t a a n d G. R o d i g h i e r o ( 1 9 7 1 ) F o r m a t i o n o f i n t e r - s t r a n d c r o s s - l i n k i n g s in t h e p h o t o r e a c t i o n s b e t w e e n f u r o c o u m a r i n s a n d D N A , Z. N a t u r f o r s c h . , 2 6 , 5 6 1 - - 5 6 9 . M u s a j o , L., a n d G. R o d i g h i e r o ( 1 9 7 2 ) M o d e o f p h o t o s e n s i t i z i n g a c t i o n o f f u r o c o u m a r i n s , P h o t o p h y s i o l ogy, 7,115--147. Scott, B.R., M.A. Pathak and G.R. Mohn (1976) Moleeular and genetic basis of furoeoumarin reactions, Mutation Res., 39, 29--74. S e k i , T., K. N o z u a n d S. K o n d o ( 1 9 7 8 ) D i f f e r e n t i a l c a u s e s o f m u t a t i o n a n d killing i n E. coli a f t e r p s o r a l e n plus light treatment: monoadduets and cross-links, Photochem. Photobiol., 27, 19--24. S t e r n , R . S . , L . A . T h i b o d e a u , R . A . K 1 e i n c r m a n , J . A . P a r r i s h a n d T.B. F i t z p a t r i c k ( 1 9 7 9 ) R i s k o f c u t a n e o u s c a r c i n o m a in p a t i e n t s t r e a t e d w i t h o r a l m e t h o x y p s o r a l e n p h o t o c h e m o t h e r a p y f o r p s o r i a s i s , N. Engi. J. Med., 300, 809--813. U r h a e h , F. ( 1 9 5 9 ) M o d i f i c a t i o n o f u l t r a v i o l e t c a r c i n o g e n e s i s b y p h o t o a e t i v e a g e n t s : p r e l i m i n a r y r e p o r t , J. Invest. D e r m a t o l . , 3 2 , 3 7 3 - - 3 7 8 . V o g e l , H . J . , a n d D.M. B o n n e t ( 1 9 5 6 ) A c e t y l o r n i t h i n a s e o f E. coli p a r t i a l p u r i f i c a t i o n a n d s o m e p r o p e r t i e s , J. Biol. C h e m . , 2 1 8 , 9 7 .