Induction in human skin fibroblasts of sister-chromatid exchanges (SCEs) by photoaddition of two new monofunctional pyridopsoralens in comparison to 3-carbethoxypsoralen and 8-methoxypsoralen

Mutation Research,

138 (1984) 63-70

63

Elsevier MTR 0906

Induction in human skin fibroblasts of sister-chromatid exchanges (SCEs) by photoaddition of two new monofunctional pyridopsoralens in comparison to 3-carbethoxypsoralen and 8-methoxypsoralen C. B i l l a r d o n , S. L e v y a n d E. M o u s t a c c h i

*

Institut Curie-Biologie, 26 rue d'Ulrn, 75231 Paris Cedex 05 (France)

(Received 21 October 1983) (Revision received9 May 1984) (Accepted 16 May 1984) Summary The induction of SCEs in human fibroblasts by photoaddition of a pyrido[3,4-c]psoralen (PyPs) and its 7-methyl derivative (MePyPs), two newly synthesized monofunctional agents proposed for photochemothcrapcutic use, was compared to that of another monofunctional agent, 3-carbethoxypsoralen (3-CPs) and to the bifunctional compound, 8-methoxypsoralcn (8-MOP). The yield of SCEs/cell and of S C E s / c h r o m o s o m e was determined at equimolar concentrations (10 -6 M) of all the drugs with increasing doses of 365 nm radiation (UVA). In the dark, the drugs alone had either no effect (8-MOP, PyPs) or a very slight effect (3-CPs, MePyPs). Nor did UVA alone demonstrate at inducing action (14.4 kJ/m2). With all the agents the average frequencies of SCE increased with increasing UVA doses, reaching a plateau level for the monofunctional compounds. The order of effectiveness for the linear part of the induction curves was MePyPs > PyPs > 8-MOP >> 3-CPs, whereas at the maximal levcl the order was 8-MOP > PyPs > MePyPs > 3-CPs. Determination of the frequencies of 2nd generation mitosis indicates that MePyPs is the most cytotoxic. The results focus the attention on the importance of the structure of psoralen monoadducts which, for certain genetic endpoints, might be as efficient as cross-links.

Exposure of cells to bifunctional psoralen derivatives such as 8-methoxypsoralen (8-MOP) and 365 nm radiation (UVA) gives rise to psoralcn monoadducts as well as to cross-links in D N A (for reviews see Song and Tapley, 1979; Parsons, 1980). Derivatives such as angelicin (Dall'Acqua et al., 1971; Ashwood-Smith and Grant, 1977; Kaye et al., 1980) or 3-carbethoxypsoralen (3-CPs) (Avcrbeck et al., 1978; Maga~aa-Schwencke et al., 1980)

* Please send all corrcspondence to: Dr. E. Moustacchi, Institut Curie-Biologie, Pavilion Pasteur, 26, rue d'UIm, 75231 Paris Cedex 05, France. 0165-1218/84/$03.00 © 1984 Elsevier Science Publishers B.V.

are considered to be monofunctional, since following UVA irradiation they form only monoadditions in. DNA. The genotoxity of psoralens has been investigated in a variety of biological systems (for revicw see Scott et al., 1976). This is of particular importance in view of the use of certain furocournarins in dermatology and cosmetology. Testing included induction of mutations in procaryotes and eucaryotes (Alderson and Scott, 1970; lgali et al., 1970; Seki et al., 1978; Bridges et al., 1979; Averbeck and Moustacchi, 1979, 1980; Burger and Simons, 1979; Schimmer et ah, 1980; Venturini et al., 1980; Abel and Schimmer, 1981a, b; Pani et

64

al., 1981; Scott and Maley, 1981), of genetic recombination in phage and in yeast (Cassuto et al., 1977; Averbeck and Moustacchi, 1979), of sisterchromatid exchanges in mammalian cells (Carter et al., 1976; Waksvik et al., 1977; Gaynor and Carter, 1978; Latt and Loveday, 1978; Carrano et al., 1979; Ashwood-Smith et al., 1980; Natarajan et al., 1981; Sahar et al., 1981; Bredberg and Lambert, 1983), and of carcinogenicity in mice (Griffin et al., 1958; Fry et al., 1978; Zajdela and Bisagni, 1981; for review see Grekin and Epstein, 1981). In general, a correlation between genotoxicity and the ability to induce cross-links with DNA was found, monofunctional furocoumarins appearing to be less active than bifunctional ones (Averbeck and Moustacchi, 1979, 1980; Dubertret et al., 1979; Grant et al., 1979; Venturini ct al., 1980; Abel and Schimmer, 1981a, b; Bredberg and Lambert, 1983). On account of the potential carcinogenic risk associated with the actual use of photochemotherapy, in particular for the treatment of psoriasis (Stern et al., 1979) with bifunctional psoralens, two newly synthesized (Moron et al., 1983) monofunctional (Moustacchi et al., 1983; Blais et al., 1984) derivatives have been proposed as an alternative for photochemotherapeutic use (Averbeck et al., 1983). In the present paper, we investigate the photoinduction of sister-chromatid exchanges (SCEs), one of the indicators of genotoxicity, in human skin fibroblasts by two new monofunctional derivatives of psoralen belonging to the family of pyridopsoralens (Moron et al., 1983). These are the pyrido[3,4-c]psoralen (PyPs or compound I) and its 7-methyl derivative (MePyPs or compound II) (Fig. 1). As reference compounds the bifunc-

tional agent 8-MOP and the monofunctional agent 3-CPs were used in parallel experiments. Material and methods Human cell fines Most of the experiments were performed with the GMI603 cell line derived from 3-month-old male embryo skin fibroblasts (Institute for Medical Research, Camden, N J, U.S.A.). For comparison, the FH97 cell line, also derived from a 3month-old male embryo (Seromed, Munich, F.R.G.) was used. Cells were maintained in Eagle's MEM supplemented with 10% fetal calf serum (Gibco, U.S.A.) and 20 /tg/ml gentamycin. Both lines were used between the 10th and 13th passage. Furocoumarins Stock solutions at 1 m g / m l were prepared in ethanol and kept in the dark at 4°C. 8-MOP ( M W = 216) was purchased from Sigma, 3-CPs (MW = 258) and the two pyridopsoralens (compounds I, MW = 2 3 7 and II, MW = 251) were synthesized by J. Moron and E. Bisagni (Institut Curie, Biologie, Orsay). All the products were chromatographically pure. The limits of solubility are 23 /~g/ml for 8-MOP, 13 F g / m l for 3-CPs (Dall'Acqua et al., 1981) and 1 F g / m l for compounds I (PyPs) and II (MePyPs) in aqueous solution (Blais et al., 1984) or culture medium. Treatment of cells 24 h before treatment, the cells were seeded at a 1 : 6 ratio in 9-cm diameter plastic petri dishes (Falcon). Thus treatments were performed on exponentially growing ceils.

N COOC2H

N

s

O

CH~

OCH~ 8-METHOXYPSORALEN

:3_ CARBETHOXYPSORALEN

PYRIDO C3,4_c )- FURO (3,2-9) c o u M A R I N

(.1)

?.METHYL,PYPJDO (.3,4.c) FURO (3,2 -g)CCUMARIN (]])

Fig. 1. Chemical structures of 8-methoxypsoralen (8-MOP), 3-carbethoxypsoralen (3-CPs), pyrido[3,4-c]furo{3,2-g]coumarin (PyPs) and 7-methyl pyrido[3,4-c]furo[3,2-g}coumarin (MePyPs).

65 The growth medium was discarded and replaced by 3 ml of Eagle's MEM without serum containing the furocoumarins at equimolar concentration (]0 -6 M) unless specified otherwise in the text. The concentration used is well below the limits of solubility. The absence of absorption of the medium without serum in the range of emission of the irradiation device (340-380 nm) was spectrophotometrically verified. The cells were incubated for 20 min in the dark in the presence of the drugs at 37 ° C and were subsequently irradiated.

Irradiation The irradiations were performed with a HPWI25 Philips lamp (320-380 nm) mounted horizontally. Cells in open petri dishes placed in the centre of the light beam were irradiated from above. The wavelengths below 340 nm were excluded by a glass filter as already described (Averbeck et ai., 1978). The dose.rate was 10 j / m 2 / s e c as determined by a Black-Ray UV meter J221 (Ultraviolet Products, San Gabriel, CA 91778, U.S.A.).

Chromosome preparations Following irradiation, the cells were washed 3 times in medium without serum and then incubated for 48-72 h in complete growth medium complemented with serum and containing 5/~g/ml 5-bromodeoxyuridine. The cells were then treated with colcemid (Gibco) at a concentration of 0.01 # g / m l medium. After 1 h, the cells were trypsinized and spun down at 800 rpm. The pellet was suspended in 0.075 M KC1; the cells were maintained at room temperature for 20 min, thereafter fixed twice in methanol:glacial acetic acid ( 3 : 1 ) and the slides prepared using the conventional air-drying method. They were then stained with a modification of the Hoechst-plus-Giemsa technique according to Perry and Wolff (1976). The slides were treated for 20 min with a Hoechst solution (5 m g / 1 0 0 ml distilled water), then briefly rinsed in distilled water, immersed in pH 6.8 S0rensen buffer and then exposed for 1 -3 h to 254-nm UV irraditation from a Philips germicidal lamp. The slides were subsequently stained in a

buffered Giemsa solution containing 10 ml of 2.0 M N a H 2 P O 4 and 5 ml of Giemsa (RALR) per 100 ml solution. The frequency of metaphases was determined on samples of at least 1000 cells.

SCE analysis SCE were scored on a minimum of 20 cells for each dose in each experiment. Counting was carried out by projecting a film negative of the slides on a microfilm reader. The experiments were repeated 3 times for each compound. Data have been expressed both in SCEs per cell and in SCEs per chromosome. The former conventional parameter allows comparison with published results and the latter which does not take into account variations in number of chromosomes per cell (spontaneous or induced heteroploidy) gives a more direct indication on the induced effect. Results

In the dark, 8-MOP at the concentration used has a negligible effect on the yield of SCEs which is in accordance with data published by others (Carter et al., 1976; Latt and Loveday, 1978; Sahar et al., 1981). The same is true for PyPs. 3-CPs and MePyPs tend to lead to a very slight increase of the SCE frequency above the control level (Table 1). It may be worth mentioning at this point that 8-MOP, PyPs and MePyPs, but not 3-CPs, were found to induce frameshift mutations in the dark in the Salmonella typhimurium Ames test (Quinto et al., 1984). This indicates that these furocoumarins interact with D N A in the dark in a manner which may depend on the structure of the chromatin. Irradiation at 365 nm in the absence of the drugs did not produce SCEs above the control level even with the highest dose used (14400 J / m 2) (Table 1). Using an equimolar concentration (10 6 M) of all the compounds tested. The yield of SCEs increased with increasing dose of UVA (Table 1). It should be noted that in yeast (Averbeck et al., 1983; Moustacchi et al., 1983) as well as in Chinese hamster V79 cells (Papadopoulo, personal c o m -

66 TABLE 1 INI)UCTION OF SCE BY DIFFERENT FUROCOUMARINS AS A FUNCTION OF UVA DOSE IN GM1603 tlUMAN SKIN I"IBROBI,ASTS The 4 compounds were used at the same concentration of 10 6 M. * refers to the absolute values of SCE frequencies observed whereas other values refer to the increments, i.e. the drug-alone control values were subtracted from values observed in the drug-treated and irradiated samples. The UVA doses given to the UVA-alone control correspond to the highest dose given to the drug-phis-UVA-treated sample (for example 14400 J / m 2 in the experiment with 3-CPs). Standard deviations are given in parentheses. Treatment

8-MOP SCEs/cell

3-CPS

PyPs (compound I)

SCEs/chrom. SCEs/cell SCEs/chrom. SCF,s/cell

MePyPs (compound II)

SCEs/chrom. SCEs/cell SCLs/chrom.

Control *

6.17 (2.9) 0.14 (0.06)

5.27 (3.00) 0.12 (0.07)

6.17 (2.9) 0.14(0.06)

5.69 (3.01) 0.13 (0.01)

UVA alone*

4.17 (2.8) 0.17 (0.08)

4.54(1.3)

0.14(0.03)

7.2 (3.7) 0.17 (0.08)

4.50 (1.04 0.12 (0.04)

Drug alone*

6.46 (3.1) 0.15 (0.07)

7.33 (3.2) 0.18 (0.08)

5.36 (2.0) 0.13 (0.05)

8.85 (3.2) 0.20 (0.07)

Drug+ 200J/m 2 + 400 J / m 2 + 600 J / m 2 + 1 200 J / m 2 + 1800J/m 2 + 2400J/m 2 + 4800 J / m 2 + 7200 J / m 2 + 14400 J / m 2

0.70 (1.24) 4.5 (1.05) 6.75(1.00) 12.54(1.27)

0.01 (0.02) 0.08 (0.02) 0.15(0.02) 0.32(0.04) -

0.88 (1.02) 0.01 (0.02) 0.99(0.96) 0.02(0.02) 3.28 (1.07) 0.08 (0.02) 4.17 (1.30) 0.104 (0.03) 4.67 (1.36) 0.145 (0.03)

munication), following U V A exposure, lethality, m e a s u r e d by c o l o n y - f o r m i n g ability, is greatest with MePyPs, followed by PyPs then by 8 - M O P and is least with 3-CPs. F r o m the delay in h u m a n fibroblast replication, measured by the decrease in frequencies of cells in second division (Table 2), it a ppe a rs that these cells d e m o n s t r a t e the same relative sensitivity according to agents except for PyPs; at a c o n c e n t r a t i o n of 10 -6 M, PyPs does not display a high p h o t o c y t o t o x i c i t y c o m p a r e d to 8M O P or MePyPs. As seen in T a b l e 2, the p r o p o r tion of cells in mitosis decreases as a function of the U V A dose again with the exception of PyPs. With M e P y P s the cytotoxicity is so d r a m a t i c that high U V A doses could not be used. Indeed a b o v e 1200 J / m 2, mitoses are almost absent. F r o m the data in T a b l e 1 it can be seen that the frequency of SCEs after 3-CPs p h o t o a d d i t i o n increases linearly up to a dose of 7200 J / m 2 reaching a plateau at higher doses. F o r 8 - M O P the shape of the dose- response curve is similar to that reported for o t h er m a m m a l i a n cell lines ( N a t a r a j a n et al., 1981); after a slow increase at low doses, the e n h a n c e m e n t in S C E frequency b e c o m e s linear. F o r PyPs and MePyPs, the increase in yield of

5.29 (0.89) 0.11 (0.02) 9.97 (1.09) 0.23 (0.02) 10.44(1.27) 0.24(0.02) 10.69(1.44) 0.32(0.03)

3.65 (1.18) 3.59 (1.30) 4.68 (1.38) 5.14 (2.36)

0.09 (0.03) 0.09 (0.03) 0.11 (0.02) 0.12 (0.03)

-

SCEs appears to be linear up to doses of 1200 and 600 J / m 2 respectively, before reaching a plateau. W h e n c o m p a r i n g the relative efficiency of the 4 f u r o c o u m a r i n s in p r o d u c i n g SCEs at equal U V A doses c o r r e s p o n d i n g to the linear part of the d o s e - e f f e c t relationship, PyPs and M e P y P s are m o r e efficient than 8-MOP, 3-CPs being the least efficient c o m p o u n d . It follows that the ascending p o t e n c y of the agents for S C E i n d u ct i o n in h u m a n fibroblasts in M e P y P s > PyPs > 8 - M O P >> 3-CPs. If the U V A dose required to induce a certain f r eq u en cy of SCEs by M c P y P s is arbitrarily considered to equal 1, doses 1.8, 5.5 and 24 times higher are necessary to p r o d u c e the same yield of SCEs for PyPs, 8 - M O P and 3-CPs respectively. I lowever, when the m a x i m a l levels of i n d u ct i o n are c o m p a r e d , the relative efficiencies follow the o r d er 8 - M O P >/PyPs > M e P y P s / > 3-CPs, whether t h e d a t a ar e e x p r e s s e d in S C E s / c e l l or SCEs/chromosome. Differences in sensitivity to S C E i n d u ct i o n dep e n d i n g on the cell line were noted. I n d e e d when c o m p a r i n g the frequency of SCEs induced in cell lines F H 9 7 and G M 1 6 0 3 (Table 3) after p h o t o a d dition using a 50-fold higher c o n c e n t r a t i o n of 8-

67 TABLE 2 F R E Q U E N C Y OF MITOSIS (2nd G I - N E R A T I O N ) AS A F U N C T I O N OF UVA DOSE FOR T H E D I F F E R E N T F U R O C O U M A R I N S IN GM1603 FIBROBLASTS Numbers in parentheses represent cells in 1st generation mitosis. The frequency of cells in mitosis from the samples treated with the medium alone constitute the controls. This frequency was ver~' close to that of other controls (drug alone, UVA alone) and varied between 2% and 5% of the total population. All drugs were used at a concentration of 10 -6 M. Treatment and UVA dose ( J / m 2 )

Number of cells scored

Number of mitoses

Percentage of control

8-MOP+ 600 + 1 200 + 1800 + 2400

2000 1 000 2000 2000

52 54 28 23 (1)

43 67 35 30

3-CPs + 1200 + 2400 + 4800 + 7200 +14400

2000 3000 2000 2000 2000

28 27 12 13 3

77 50 33 36 16

PyPs+ 600" + 1 200 + 1800 + 2400

2000 2000 2000 2000

18 21 26 30(2)

MePyPs + 200 + 400 + 600 + 900 + 1800 + 2400

5 000 4000 5829 6613 5203 3000

24 9 8 (1) 3 (3) (9) (12)

64 75 92 114 48 22.5 17.1 10.4 5.9 10

TABLE 3 I N D U C T I O N OF SCE BY 8-MOP P H O T O A D D I T I O N IN FH97 H U M A N SKIN FIBROBLASTS * refers to the absolute values of SCE frequencies observed, whereas other v',dues refer to the increment, i.e. the wdnes observed with 8-MOP alone * were subtracted from the values obtained in the 8-MOP-treated and irradiated samples. A concentration of 5 × 1 0 -5 M of 8-MOP was used. Numbers in parentheses are standard deviations. Treatment

SCEs/cell

SCEs/chromosome

Control *

6.40 (3.10)

0.16 (0.07)

UVA alone (1200 J / m 2 ) *

6.39 (2.59)

0.14 (0.06)

8-MOP ',done *

6.20 (2.57)

0.14 (0.06)

8-MOP + 200 J / m 2 8-MOP + 600 J i m z 8-MOP + 1 200 J / m z

1.49 (0.94) 2.40 (1.27) 6.60 (1.23)

0.18 (0.02) 0.24 (0.02) 0.31 (0.03)

MOP, far fewer SCEs are induced in FH97 cells than expected in GM1603 cells. If induction of SCEs is as generally hypothesized, a manifestation of repair of induced lesions, such differences may reflect variations in repair capacity between socalled ' n o r m a l ' cell lines. In any case, in comparing data on SCE induction in human fibroblasts obtained in different laboratories, this factor should be taken into account. Discussion PyPs and MePyPs do not induce cross-links in D N A either in vitro (Blais et al., 1984) or in vivo (Moustacchi et al., 1983). They demonstrate a photoaffinity for D N A in the same range as that of 8-MOP (Moustacchi et al., 1983) and only 2 times higher than that of 3-CPs. At the concentrations used which are well below the limits of solubility, in the low dose range of UVA the two new monofunctional pyridopsoralens turned out to be at least as efficient as the bifunctional psoralen derivative 8-MOP in inducing SCEs. They are by far more efficient than the other monofunctional agent 3-CPs. In connection with our observations, it may be worth recalling that a single laser pulse which should produce only D N A monoadducts with 8-MOP is capable of inducing SCEs in C H O cells (Sahar et al., 1981). Moreover, D N A cross-links did not appear to be necessary for induction of SCEs, even with a potentially bifunctional agent. The 3 monofunctional agents tested here reach a plateau level of SCE induction after doses which differ from one compound to the other. This may imply that the sites for addition by these agents are limited. When such maximal levels are compared, MePyPs which is the most cytotoxic, does not differ greatly from 3-CPs in efficiency, whereas PyPs demonstrates an efficiency close to that of 8-MOP. This finding, taken together with the high efficiency of PyPs and MePyPs, in the low dose range, appears to contradict the general observation that monofunctional psoralen derivatives are less genotoxic than bifunctional ones. It is likely that the amount of total D N A lesions as well as the molecular nature of the photoinduced monoadducts, in particular their steric configuration,

68 m a y play an i m p o r t a n t role. Similarly to 3-CPs, angelicin, a n o t h e r m o n o f u n c t i o n a l psoralen derivative, was found to be a relatively poor SCE-ind u c i n g agent compared to 8 - M O P ( L i n n a i n m a a a n d Wolff, 1982). Indeed with the same U V A exposure, a c o n c e n t r a t i o n of 10 -4 M angelicin was required to induce as m a n y SCEs as 10 -6 M 8-MOP. The fixation of the pyrido group on the 3 - 4 double b o n d position of the f u r o c o u m a r i n (Fig. 1) clearly enhances both p h o t o b i n d i n g to cellular D N A (Magaha-Schwencke, personal comm u n i c a t i o n ; Nocentini, personal c o m m u n i c a t i o n ) , a n d phototoxicity and SCE induction. In diploid yeast (Averbeck, personal c o m m u n i cation) a n d in C h i n e s e h a m s t e r V79 cells (Papadopoulo, personal c o m m u n i c a t i o n ) , PyPs and MePyPs were shown to be more mutagenic than 8 - M O P and far more mutagenic than 3-CPs per unit incident U V A dose. It consequently appears that there is a good correlation between SCE ind u c t i o n a n d mutagenicity in diploid eucaryotic cells in terms of the relative sensitivities to the different agents for the two genetic end-points. The same is true for other c o m p o u n d s in m a m malian cells (Perry and Wolff, 1976). The relationship between SCE i n d u c t i o n and carcinogenicity is still u n d e r debate. It has been proposed that c h r o m o s o m e a n d chromatid exchanges may constitute an i m p o r t a n t step in m a l i g n a n t transformation. Of 40 c o m p o u n d s which were compared for both carcinogenic and SCE i n d u c t i o n activities, 25 clearly displayed both activities, 1 was negative for both end-points, and for 2 c o m p o u n d s SCE and carcinogenicity data were directly contradictory. Of the r e m a i n i n g 12 c o m p o u n d s , the c o m p a r i s o n was inconclusive (Latt et al., 1981). In other words the predictive value for carcinogenic activity of i n d u c t i o n of SCEs is somewhat c o m p a r a b l e to that of other tests. It is clear, however, that testing of the carcinogenic activity of PyPs a n d MePyPs must be performed before definitive conclusions can be drawn. Since i n d u c t i o n of SCEs reflects an interaction of a given chemical with D N A and is likely to result from a break and rejoining repair process which may be error-prone, our results indicate that the two new pyridopsoralens must be used with the same caution as 8 - M O P for photochcmotherapeutic purpose. MePyPs which is extremely

cytotoxic a n d leads to a clearly lower maximal yield of SCE compared to 8 - M O P seems the most promising. These results also focus attention on the importance of the nature of psoralen m o n o a d d u c t s , which could be in certain conditions and for certain e n d - p o i n t s as efficient as cross-links.

Acknowledgements The authors thank Drs. D. Averbeck and R. C h a n e t for critical reading of the m a n u s c r i p t and Dr. J. M o r o n for the supply of pyridopsoralens. This work was supported by the C o m m i s s a r i a t l'Energic A t o m i q u e (Saclay, France), the Centre N a t i o n a l de la Recherche Scientifique and the C o m m i s s i o n des C o m m u n a u t e s Europ6ennes, C o n t r a c t No. BIO-E-397F (SI).

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