Furoquinoline alkaloids as photosensitizers in Chlamydomonas reinhardtii

Furoquinoline alkaloids as photosensitizers in Chlamydomonas reinhardtii

105 Mutation Research, 249 (1991) 105-110 © 1991 Elsevier Science Publishers B.V. 0027-5107/91/$03.50 ADONIS 002751079100131G MUT 04983 Furoquinoli...

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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








-OCH 3


-O\ CH?

R" R ttt








-OCH 3

-OCH 3

-O /

_O /

-OCH 3

-OCH 3



-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)


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.


Chlamydomonas reinhardtii Compound


Expt. 1


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

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

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

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


0 2 5 10 20

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

<0.1 5.0+1.76 22.2+4.81 19.3+3.80 0.75:0.95

100 +11.0 104 +11,0 84 + 8.0 58 ___ 5,2 1.4+ 0,9

0.1+0.32 2.8+1.14 10.7+3.56 14.9:t:5.00 < 0.1


0 2 5 10 20

100 85 102 69 72


0 2 5 10 20

100 -+ 91 _+ 93 + 83 _+ 1.1+


0 2 5 10 20


5:7.7 5:4.3 4-10.7 5:6.5 _+ 9.8

0.35:0.48 1.2_+1.03 7.3-+4.45 15.0-+3.23 31.8_+5.18

100 101 88 74 54

_+ 7.4 _+ 4.3 5:6.4 _+ 6.0 _+ 4.0

9.7 9.8 8.1 5.2 1.1

<0.1 1.1+0.74 5.6-+ 2.95 11.6_+3.84 5.95:2.73

100 _+ 89 + 80 + 70 + 1.9+

100 97 86 95 102

+ 3.3 5:6.5 + 4.5 5:3.2 + 4.3

< 0.1 1.55:1.43 4.7+2.16 14.4_+4.86 29.9_+5.42

0 2 5 10 20 40

100 85 96 109 96 -

5:10.2 5:10.2 5:12.2 + 7.8 5:6.1


0 10 20 40

100 105 98 102


0 10 20

0.2+0.42 1.05:0.66 3.3+3.47 12.7-+1.83 14.9+2.88

5.6 7,0 5,6 4,2 0.7

0.4+0.70 0.75:0.67 3.6 5:1,90 15.65:4.09 1.7+1.06

100 84 95 76 85

_+10.3 + 8.4 +12.1 + 5.4 _+ 6.5

< 0.1 1.8_+1.13 5.1+1.37 8.9-+2.88 17.9+4.23

0.1+0.32 0.2_+0.42 0.55:0.71 1.2_+1.03 2.8-+1.13 -

100 100 86 89 78 50

+12.0 + 5.4 5:4.1 + 6.6 + 8.9 _+ 4.1

<0.1 0.2+0.42 0.7_+0.95 3.1+1.59 5.9+1.37 9.7-+3.10

+ 9.8 + 7.1 + 6.8 +11.9

<0.1 0.3-+0.48 0.85:0.92 0.85:0.79

100 100 98 94

+ 4.7 5:7.8 + 6.4 -+ 6.0

0.2+0.42 0.4+0.7 1.1+1.1 0.9-+0.87

100 + 8.4 90 + 4.5 93 + 7.2

0.15:0.32 1.35:1.06 1.35:1.64

100 -+ 6.3 89 + 6.9 87 + 7.2

<0.1 0.25:0.42 0.8-t-0.77

a Corresponding colonies/plate ( × 105) of the controls were dictamnine 47+4.2 (1) and 50+ 5.5 (2); "t-fagarine 99+ 7.7 (1) and 72 + 5.3 (2); evolitrine 107 + 10.4 (1) and 158 + 8.8 (2); maculine 96 + 3.2 (1) and 80 + 8.2 (2); kokusaginine 46 + 4.7 (1) and 80 + 9.6 (2); skimmianine 41 +4.0 (1) and 58 + 2.7 (2); flindersiamine 94+ 7.9 (1) and 54+ 3.4 (2). t h e m u t a n t s t r a i n a n d its s u i t a b i l i t y f o r m u t a t i o n e x p e r i m e n t s c a n b e f o u n d in t h e C h l a m y d o m o n a s S o u r c e B o o k ( H a r r i s , 1988).

treated. The furoquinoline alkaloids were dissolved in D M S O a n d a d d e d to the algal s u s p e n sion (100-ml samples) immediately before irradiation.

Mutation assay T h e a s s a y is d e s c r i b e d in d e t a i l i n t h e p a p e r p u b l i s h e d b y S c h i m m e r a n d K i i h n e (1990). I n all


e x p e r i m e n t s a s y n c h r o n o u s cell p o p u l a t i o n s w e r e

to elucidate the U V - A - m e d i a t e d

Separate experiments were performed in order toxicity of dic-


tamnine. 20-ml samples of the algal suspension (cell density about 1-3 × 106/ml) were treated in falcon flasks. After irradiation the suspension was diluted 1:1000 with phosphate buffer (0.02 M, p H 6.9) and spread onto agar plates. Toxicity was estimated by counting the number of colonies after a growth period of 7 days in continuous light (5000 lux). The data in Table 1 are the mean of 10 plates _+ SD of 4 individual experiments.

densities than in those with lower cell densities. No significant reduction in colony-forming cells was detected after treatment of the algal cells with 1 /~g d i c t a m n i n e / m l plus UV-A for a 30-min period. Treatment for 60 min, however, reduced the survival to 50-75%. UV-A light alone did not change the survival and was therefore kept as a control. The toxicity of the remaining compounds was studied only in connection with the mutagenicity experiments, in which 100-ml samples of algal suspensions were treated. Except for evolitrine, which exhibited as high a toxicity as dictamnine at the highest concentration, only few or no toxic effects were observed (Table 2). As some compounds (evolitrine, skimmianine, flindersiamine) were available only in very small amounts, further experiments could not be performed. Dictamnine (10 t~g/ml) did not induce Arg + revertants, when the arg-1 cells were treated in the dark (data not shown). Irradiation with UV-A alone did not change the spontaneous mutation frequency either (data not shown). But treatment with dictamnine plus UV-A at a constant fluence of 3.6 k J / m 2 resulted in a dose-dependent mutagenicity (Table 2). A dose of 2 /~g d i c t a m n i n e / m l enhanced the mutation frequency significantly (Table 3). The other compounds also enhanced the number of Arg ÷ revertants per plate with increasing doses (Table 2), but the mutation frequencies as well as the numbers of revertants varied considerably at a given dose (Tables 2 and 3).


Mutation frequency. The interexperimental variation was checked using the F-test ( a = 0.05) which established homogeneity of variance. The data of 3 experiments were then pooled and analyzed with Student's t-test. Spontaneous mutation frequencies < 1 × 10 8 were rounded off. Results Dictamnine (10 t~g/ml) did not affect the survival of the arg-1 cells when the cells were treated in the dark (Table 1). However, when the cells were irradiated simultaneously with UV-A, a significant decrease in colony-forming cells was observed after 30 min of exposure. Irradiation for 60 min resulted in survival rates ranging from 0.2 to 19.2% (Table 1). Under equal treatment conditions the percentage of survival partly depended on the cell density, as demonstrated in Table 1. Especially when we used a high, toxic dose the survival was higher in Experiments with higher cell TABLE 3 MUTATION




P L U S U V - A (30 min) I N arg-1 C E L L S O F


reinhardtii Compound

Dictamnine y-Fagarine Maculine Evolitrine Kokusaginine Skimmianine Flindersiamine

A r g ÷ r e v e r t a n t s p e r 108 s u r v i v i n g cells _+ SE (n = 3) at a c o n c e n t r a t i o n ( / ~ g / m l ) 0 a





1.3 + 0.3 2.3 _+0.7 1.0+0 1.3_+0.3 2.0_+0.6 2.3_+0.7 1.0 _+0

108 + 4 * * * 16 + 1 * * * 24+4"* 8_+2 * 4_+2 4_+1 2 _+ 1

483 + 42 * * * 73 -+ 13 * * 81+19" 43_+ 8 * 12_+ 1 * * 3_+ 0 4 _+ 1

648 -+ 79 * * 239 + 12 * * * 160_+ 9 " * * 137_+ 4 * * * 45_+12 * 8_+ 1 * 8+ 3

n.c. 435 + 29 * * * 295_+17 * * * n.c. 86_+11 * * 19_+ 2 * * 18 _+ 2 * *

a Control, D M S O + UV-A. n.c., n o t calculated, see T a b l e 2. Values significantly d i f f e r e n t f r o m control: * P < 0.05; * * P <

0.01; * * * P < 0.001.

109 Dictamnine exhibited the strongest effect. Moderate activities were observed when 3,-fagarine, maculine or evolitrine were investigated. The mutagenic potential of maculine was higher than that of 7-fagarine compared on equal survival. A smaller but significant and dose-dependent mutagenicity was detected with kokusaginine. The activity of this compound was further enhanced using a dose of 40 # g / m l . Skimmianine and flindersiamine revealed only marginal activities. Both compounds were capable of enhancing the number of revertants but the activity of skimmianine could not be further enhanced by a higher dose of 40 /tg/ml (Table 2). Nevertheless we cannot exclude a considerable mutagenic response at toxic doses of skimmianine or flindersiamine. Discussion

The chemical structure of furoquinoline alkaloids is similar in many ways to that of linear furocoumarins with which they co-occur in many members of the Rutaceae family, e.g., Rum graveolens (Schimmer and Kiihne, 1990). The most important common structure is the unsubstituted furane ring, which is linearly condensed to a planar bicyclic aromatic system. Because of the planarity of their molecules, both groups of compounds are able to intercalate into DNA and to form reversible complexes with DNA in the dark (Pfyffer et al., 1982a; for furocoumarins cf. Song and Tapley, 1979). This dark intercalation complex with DNA is responsible for frameshift mutations induced by furoquinoline alkaloids in E. coli (Ashwood-Smith et al., 1982) and Salmonella typhimurium (HMele and Schimmer, 1988). In contrast, furoquinoline alkaloids are biologically inactive in the dark in Chlamydomonas reinhardtii, as are furocoumarins. But when the cells are irradiated with UV-A both types of compound act as photosensitizers. The phototoxicity of furoquinolines observed in this study is generally lower than that caused by bifunctional furocoumarins. The most toxic compounds, dictamnine and evolitrine, reveal toxic effects comparable to that of monofunctional furocoumarins, e.g., angelicin in Chlamydomonas reinhardtii (Schimmer et al., 1981). Monofunctional furocoumarins have also been reported to

be less toxic in yeast than bifunctional furocoumarins (Averbeck et al., 1981). It is also well established that dictamrfine behaves as a monofunctional photoreagent and binds only through its furyl double bond to D N A (Pfyffer et al., 1982a,b). It is therefore reasonable to assume that the low phototoxicity of furoquinoline alkaloids in Chlamydomonas depends on their monofunctional nature. Compounds which are substituted on the lateral aromatic nucleus show a reduced activity when compared to the unsubstituted dictamnine. This agrees well with an earlier report on the toxicity of furoquinolines in bacteria (Towers et al., 1981). The mutagenic potency of the furoquinoline alkaloids studied varies by a factor of about 80 (Table 3). Dictamnine is the strongest mutagen of the group, but it is less active than 5-MOP, the strongest mutagen of the furocoumarin group in Chlamydomonas reinhardtii (Schimmer et al., 1980; Schimmer and Kiihne, 1990). Generally compounds with 1 substituent on the lateral aromatic nucleus are more active than those with 2 methoxyl groups. However, maculine, bearing a methylene dioxo bridge (Fig. 1), shows that the activity did not depend solely on the number and position of substituents, but also on their influence upon the planarity of the basic molecule. Considering the planar structure of furoquinoline alkaloids and assuming that all the compounds are capable of intercalating into DNA, substituents and their influence upon the planarity may determine the geometry of the intercalation complex formed in the dark. The efficiency of the subsequent photochemical reaction may then depend on the position of the reactive furyl double bond, i.e., on its distance from the DNA bases. The smaller this distance is, the more adducts are formed at a given UV-A fluence. Certainly other properties of the compounds tested might be important, e.g., their photobinding capacity. However, the importance of the dark interaction for the subsequent photochemical reaction is also indicated by the fact that the ranking order in photomutagenicity in Chlamydomonas resembles that of dark mutagenicity in Salmonella TA98 (Paulini et al., 1989), although the variability between the compounds is considerably

110 h i g h e r t h a n in S a l m o n e l l a . E v o l i t r i n e is the o n l y c o m p o u n d t h a t d o e s n o t fit i n t o this r a n k i n g o r d e r . But t h e r e a r e at least 2 p o s s i b i l i t i e s to e x p l a i n this exception. As evidenced by thin-layer chromatogr a p h y , e v o l i t r i n e is p a r t l y d e c o m p o s e d u n d e r the treatment conditions. Another possible explanat i o n d e p e n d s o n t h e p r o m u t a g e n c h a r a c t e r o f furo q u i n o l i n e s in S a l m o n e l l a . D i f f e r e n c e s in the m e t a b o l i s m of e v o l i t r i n e c o u l d b e r e s p o n s i b l e for its s t r o n g e r m u t a g e n i c i t y in S a l m o n e l l a , w h i c h is s o m e w h a t u n e x p e c t e d c o n s i d e r i n g the fact t h a t u n s u b s t i t u t e d c o m p o u n d s like d i c t a m n i n e are g e n erally more active than substituted derivatives.

References Ashwood-Smith, M.J., G.H.N. Towers, Z. Abramowski, G.A. Poulton and M. Liu (1982) Photobiological studies with dictamnine, a furoquinoline alkaloid, Mutation Res., 102, 401-412. Averbeck, D., S. Averbeck and F. Dall'Acqua (1981) Mutagenic activity of three monofunctional and three bifunctional furocoumarins in yeast (Saccharomyces cerevisiae), Farmaco Ed. Sci., 36, 492-505. Hiifele, F., and O. Schimmer (1988) Mutagenicity of furoquinoline alkaloids in the Salmonella/microsome assay. Mutagenicity of dictamnine is modified by various enzyme inducers and inhibitors, Mutagenesis, 3, 349-353. Harris, E.H. (1989) The Chlamydomonas Source Book, Academic, San Diego, CA, Paulini, H., R. Waibel and O. Schimmer (1989) Mutagenicity

and structure-mutagenicity relationships of furoquinolines, naturally occurring alkaloids of the Rutaceae, Mutation Res., 227, 179-186. Pfyffer, G.E., I. Panfil and G.H.N. Towers (1982a) Monofunctional covalent photobinding of dictamnine, a furoquinoline alkaloid, to DNA as target in vitro, Photochem. Photobiol., 35, 63-68. Pfyffer, G.E., B.U. Pfyffer and G.H.N. Towers (1982b) Monoaddition of dictamnine to synthetic double-stranded polydeoxyribonucleotides in UVA and the effect of photomodified DNA on template activity, Photochem. Photobiol., 35, 793-797. Schimmer, O. (1981) Vergleich der photomutagenen Wirkungen von 5-MOP (Bergapten) und 8-MOP (Xanthotoxin) in Chlamydomonas reinhardtii, Mutation Res., 89, 283-296. Schimmer, O. (1983) Effect of re-irradiation with UV-A on inactivation and mutation induction in arg- cells of Chlamydomonas reinhardtii pretreated with furocoumarins plus UV-A, Mutation Res., 109, 195-205. Schimmer, O., and I. Kiihne (1990) Mutagenic compounds in an extract from Rutae Herba (Ruta graveolens L.). II. UV-A-mediated mutagenicity in the green alga Chlamydomonas reinhardtii by furoquinoline alkaloids and furocoumarins present in a commercial tincture from Rutae Herba, Mutation Res., 243, 57-62. Schimmer, O., R. Beck and U. Dietz (1980) Phototoxizitat und Photomutagenit~it von Furocumarinen bei Chlamydomonas reinhardtii, Planta Med. 40, 68-76. Song, P.S., and K.J. Tapley (1979) Photochemistry and photobiology of psoralens, Photochem. Photobiol., 29, 1177-1197. Towers, G.H.N., E.A. Graham, I.D. Spenser and Z. Abramowski (1981) Phototoxic furanoquinolines of the Rutaceae, Planta Med., 41, 136-142.