105

Mutation Research, 249 (1991) 105-110 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100131G

MUT 04983

Furoquinoline alkaloids as photosensitizers in Chlamydomonas reinhardtii Oskar Schimmer and Irmgard Kiihne Institat fib" Botanik und PharmazetaischeBiologic der Universitdt Erlangen-Niirnberg, D-8520 Erlangen (F.R. G.)

(Received 30 January 1990) (Revision received 13 December 1990) (Accepted 17 December 1990)

Keywords: Dictamnine; Furoquinoline alkaloids; Structure-activity relationships; UV-A; Photomutagenicity; Phototoxicity

Summary Seven naturally occurring furoquinoline alkaloids were investigated for their photobiological activity using arg-1 cells of Chlamydomonas reinhardtii. UV-A-mediated toxicity of the compounds was calculated from the colony-forming ability of the treated cells. The UV-A-mediated mutagenicity was measured by counting the number of Arg + revertants induced by the treatment. Dictamnine was found to be the strongest mutagen as well as the most toxic compound of the group. The mutagenic activities were measured as mutation frequencies at equal substance concentration and ranked in the following order: dictamnine > ~-fagarine > maculine > evolitrine > kokusaginine > skimmianine = flindersiamine. An increase in the number of substituents on the lateral aromatic nucleus greatly decreased the photomutagenicity. Except for evolitrine, a similar ranking order was found as reported for the dark mutagenicity of these compounds in Salmonella typhimurium strain TA98. Based on the result that furoquinolines are able to intercalate into DNA, we assume that the different mutagenic potencies m a y reflect differences in the geometry of the intercalation complex, which is important for the subsequent photochemical reaction.

In a recent paper we reported on the UV-Amediated phototoxicity and photomutagenicity of 2 furoquinoline alkaloids, dictamnine and ~,fagarine, in the unicellular green alga Chlamydomonas reinhardtii (Schimmer and Kiihne, 1990). Both compounds exhibited a photobiological behavior analogous to that of linear furocoumarins which were reported earlier to be photosensitizers

Correspondence: Dr. O. Schimrner, Institut for Botanik und Pharmazeutische Biologic der Universit~it Erlangen-Niirnberg, Staudtstr. 5, D-8520 Erlangen (F.R.G.).

in Chlamydomonas reinhardtii (Schimmer et al., 1980; Schimmer, 1981, 1983). The chemical structure of furoquinoline alkaloids is very similar to that of linear furocoumarins. This similarity prompted us to test further compounds for their photobiological activities. The aim of this study was to elucidate the structure-mutagenicity relationships in furoquinoline alkaloids as well as to obtain basic data on the photomutagenicity of these natural substances. The investigation was performed with a mutant strain of Chlamydomonas reinhardtii which has been shown to be suitable for detecting com-

106 OCH 3

R tH

DICTAMNINE R t

EVOLITRINE y-FAGARINE

-H

-H

SKIMMIANINE KOKUSAGININE MACULINE FLINDERSIAMINE

-H

-H

-OCH 3

-O\

-O\ CH?

R" R ttt

-H

-OCH

-H

-I-4

3

CH~

-H

-OCH 3

-OCH 3

-O /

_O /

-OCH 3

-OCH 3

-H

-H

-OCH 3

Fig. 1. Chemical structures of the furoquinoline alkaloids.

p o u n d s reacting via U V - A with D N A , as d e m o n strated b y the papers m e n t i o n e d above.

sured o n the surface of the petri dishes (UVX digital s p e c t r o r a d i o m e t e r with sensor UVX-36, U V P Inc., San Gabriel, CA, U.S.A.).

Materials and methods

Mutant strain Test compounds

The a r g i n i n e - r e q u i r i n g arg-1, m a t i n g type ( - ) of

T h e sources a n d purities of the f u r o q u i n o l i n e alkaloids have b e e n p u b l i s h e d b y Paul±n± et al. (1989). The chemical structures of the c o m p o u n d s are shown in Fig. 1.

Irradiation conditions U V - A was o b t a i n e d from two Philips black light blue fluorescent l a m p s (TL 4 0 W / 0 8 ) . The i r r a d i a t i o n i n t e n s i t y was a b o u t 2 W / m 2 as mea-

Chlamydomonas reinhardtii was o b t a i n e d from Dr. R. Loppes ( U n i v e r s i t y of Li+ge, Belgium). The m u t a t i o n is located o n linkage group I a n d is characterized b y a deficiency in acetylglutamyl p h o s p h a t e reductase. T h e arg-1 strain displays a stringent r e q u i r e m e n t for a r g i n i n e a n d is easily suppressed. Most Arg + revertants are caused by suppressor m u t a t i o n s , as evidenced b y backcrosses with the wild type. M o r e i n f o r m a t i o n a b o u t

TABLE 1 UV-A-MEDIATED PHOTOTOXICITY OF DICTAMNINE IN arg-1 CELLS OF Chlamydomonas reinhardtii Concentration (#g/ml)

UV-A

0 1 1 10 10 10

+ + + + + -

Treatment time (min) 60 30 60 30 60 60

Survival (%)± SD, n = 10 Expt. 1 1~.0±5.8 a 95.8±8.2 52.8±8.1 50.8±7.7 0.8±0.6 98.9±7.9

Expt. 2

Expt. 3

Expt. 4

1~.0±6.5 87.6±6.7 74.7±5.8 39.8±5.5 19.2±5.6 113.9±3.2

1~.0±6.7 n.t. 67.9±5.9 n.t. 12.8±3.1 n.t.

1~.0±8.5 n.t. n.t. 34.4±2.1 0.2±0.3 92.2±9.2

a The number of colonies/plate corresponding to the 100% values were 168.9_+9.1 (Expt. 1), 224.3+ 14.6 (Expt. 2), 226.2_+15.1 (Expt. 3) and 114.1 +9.7 (Expt. 4). n.t., not tested.

107 TABLE 2 SURVIVAL AND MUTATION INDUCTION BY FUROQUINOLINE ALKALOIDS PLUS UV-A (30 min) IN arg-1 CELLS OF

Chlamydomonas reinhardtii Compound

Concentration

Expt. 1

(#g/ml)

Survival(%) 5: SD (n = 6)

Arg +/plate -+SD (n = 10)

Expt. 2 Survival(%) _+SD (n = 6)

Arg +/plate 5: SD (n = 10)

Dictamnine

0 2 5 10 20

100 + 8.9 a 100 + 7.6 85 +11.3 64 + 8.7 < 0.4