photochemical interaction of furocoumarins with bromodeoxyuridine and polydeoxy-nucleotides...

Photochemistry and Phorobiology, 1978, Vol. 27, pp. 551-557 Pergamon Press. Printed in Great Britain

PHOTOCHEMICAL INTERACTION OF FUROCOUMARINS

NUCLEOTIDES CONTAINING BROMODEOXYURIDINE: ITS BIOLOGICAL IMPLICATIONS

E. BEN-HUR and E. RIKLIS Israel Atomic Energy Commission, Nuclear Research Centre-Negev, P.O. Box 9001,

Beer Sheva, Israel

(Received 25 April 1977; accepted 7 September 1977)

Abstract-The photoreaction of 5-bromodeoxyuridine (BUdR) exposed to 360 nm light in the presence of the furocoumarins, 4,5’,8-trimethylpsoralen (TMP) and 8-methoxypsoralen (&MOP), was studied and compared to those of thymidine. BUdR reacted with furocoumarins, producing cyclobutane- containing adducts, as does thymidine. Furocoumarins reacted also with BUdR-containing polymer, poly(dA-BUdR) in the double stranded form, at a rate similar to that of thymidine-containing polymer, poly(dA4T). Polyamines, which slow the photoreactions of TMP with DNA, had no effect on its binding to the two former polynucleotides. It is suggested that because of the similar photoreactions of BUdR and thymidine with furocoumarins, this combination could be used to elucidate the mechanism by which BUdR sensitizes biological systems. In Escherichia coli some sensitization by BUdR of TMP plus 360nm light killing was observed. It is therefore concluded that at least part of the sensitization of bacteria by BUdR to UV and ionizing radiation is caused by interference with the repair processes. Since no such sensitization was observed in a uur B mutant, BUdR apparently impairs the efficiency of the excision resynthesis pathway of repair.

WITH BROMODEOXYURIDINE AND POLYDEOXY-

INTRODUCTION

Thymidine may be replaced by its analog 5-bromodeoxyuridine (BUdR) in the DNA of various biological systems. This substitution increases the sensitivity to light of wavelengths below 320nm as well as to ionizing radiation (Greer, 1960). The molecular basis for this sensitization is still unknown. The explanation is sought in terms of increased cross-section for pho- toprocesses in DNA containing BUdR, the difference in lesions produced or differences in the capability of repair (Hutchinson, 1973). Photochemically, one of the main differences between thymidine and BUdR is that the latter does not produce cyclobutane containing photoproducts, in contrast to the naturally occurring pyrimidines (Peter and Drewer, 1970).

Furocoumarins (psoralens) sensitize many biological systems to near-UV light (300-400 nm). The effects produced are lethal (Oginsky et a/. , 1959; Ben-Hur and Elkind, 1973a), mutagenic (Igali e t al., 1970) and carcinogenic (Griffin et al., 1958). The photochemical reactions are with pyrimidine bases in DNA. Psoralen (Fig. 1) and its derivatives intercalate with DNA and upon exposure to near UV light its 3,4- or 4‘,5‘ double

Figure 1. The chemical structure of psoralen.

bonds form cyclobutane rings with the 5,6-double bond of the pyrimidine (Krauch et a/ . , 1967; Musajo et a/., 1967). The psoralen monoadducts in DNA can further react with a pyrimidine base in the comple- mentary DNA strand to produce DNA crosslinks (Cole, 1970; Dall’Acqua e t a/., 1970; Ben-Hur and Elkind, 1973b). The latter products appear to be the lethal lesions in bacteria (Cole, 1971) and in mammalian cells (Ben-Hur and Elkind, 1973a).

This work deals with the interaction of psoralen derivatives with BUdR and with a polynucleotide containing BUdR under the action of 360 nm light. The biological implications for the mechanism of BUdR sensitization were investigated in Escherichia coli, using this photochemical system, and preliminary results have been presented (Ben-Hur and Riklis, 1976).

MATERIALS AND METHODS

Near U V irradiation. A sample of 0.5 mL frozen aqueous solution, containing 1 p M 4,5’,8-trimethylpsoralen (TMP) (Paul B. Elder Co., Bryan, Ohio), 0.01 M phosphate buffer and nucleosides (2mM) was exposed in a 15mm dish covered with 1 cm thick solution of CoCI2 in acetone (2 g/t) which completely cuts off all wavelengths below 325 nm, so that no light was directly absorbed by BUdR (Ben-Hur and Elkind, 1972). The light source was two tubular fluor- escent black light lamps in a reflector (UV Products Inc., lamp No. 50058) with maximum emission at about 360 nm. The incident fluence was 20Jm-’s-’ as measured by a calibrated black light meter (model 5-221, UV Products Inc., CA). Poly[dA-(S-BUdR)].poly[dA-(5-BUdR)] (P-L Biochemical Inc.), poly (dA-dT) . poly(dA-dT), calf thymus DNA (Sigma Chemical Co.) and E. coli DNA (obtained by phenol extraction) were irradiated as above, at 25°C

,551

552 E. BEN-HUR and E. RIKLIS

and concentration of 500mg/(. Samples of 0.1 mY were taken for analysis. All irradiated solutions contained 0.1 pCi/m/ of C3H]-TMP, 3.9 Ci/mM (custom tritiated by New England Nuclear, MA), in addition to the unlabeled TMP.

Spectrophotometric analysis. Samples of 10 mY frozen aqueous-dioxane (1 : 1) solution containing 5 mM 8-methoxypsoralen (8-MOP) (Sigma) and 5 mM BUdR or thymidine were exposed in 100mm petri dishes as above. Absorption spectra were recorded after various exposure times, on a hundred fold dilution in water of the thawed samples, using a Cary model 17 spectro photometer.

Binding of T M P to polymers. After near UV-irradiation in the presence of labeled TMP, samples of the polynucleotides and of native DNA were gel filtered through a Sephadex B-25 column (1.4 x 15 cm), polynucleotides were eluted with 0.01 M phosphate buffer and 2mY fractions were collected. Absorbance at 260 nm and radioactivity were determined. The latter was measured in a dioxane- based cocktail in a liquid scintillation spectrometer. The polymers containing bound TMP appeared in the fourth fraction, while free TMP eluted in fraction 15-20.

Photoproduct analysis. Following irradiation of nucleosides in the presence of labeled TMP, the solution was thawed and spotted on paper, and developed for radio- chromatographic separation and analysis of photoproducts (Riklis, 1965). Descending chromatography for about 16 h

was carried out in n-butano1:acetic acid:water = 80:12:30 (v/v/v). The chromatogram was scanned by a radiochromatogram scanner (Packard, Inc.) and the regions containing photoproducts were eluted for further analysis.

Bacterial cell growth and suruioal. Bacteria ( E . coli KMBL 1056 thy- end I- and its uur B mutant KMBL 1054) were grown to a density of about 1 x 109/mC in mini- mal medium containing either thymidine or BUdR (5 mg/t), harvested by centrifugation and resuspended in saline containing 1 p M TMP. After about 10 min at room temperature the cells were irradiated with near UV light at 4"C, serially diluted and plated on nutrient agar, under subdued light. Viability counts were made after 16 h incu- bation at 37°C.

RESULTS

Photoproducts of B U d R and thymidine with trimethylpsoralen

BUdR and thymidine were exposed t o near UV light for 1 h in frozen solution in the presence of 3H-TMP, as described in Materials and Methods. After irradiation the thawed solution was chromato- graphed and the radiochromatograms were scanned.

b 0.63 0.9 0.

C 0.94

n 0 0.2 0.4 0.6 0.8 1.0

R t

Figure 2. Radiochromatogram scans of: (a) C3H]-TMP; (b) labeled TMP, irradiated for 1 h in frozen solution with thymidine as described in Materials and Methods; (c) labeled psoralen irradiated with

BUdR as in (b).

Mechanism of bromouracil sensitization :553

The results are shown in Fig. 2. Evidently in both cases, under our conditions, about half the TMP reacted with the nucleosides. In order to characterize the products of the photoreaction, the spots were eluted from the paper, purified by rechromatography and then irradiated with 10 kJ m-’ at 254 nm in solution at room temperature. Under these conditions, cyclobutane containing photo-adducts of furocoumarins with pyrimidines are split, producing free furocoumarin and the corresponding pyrimidine (Krauch et al., 1967; Musajo et al., 1967). Indeed, paper chromatography after irradiation at 254 nm revealed that the major part of both thymidine and BUdR derived photoproducts disappeared while TMP reappeared (Fig. 3). In the case of BUdR an ad- ditional peak appeared at R, 0.49 (Fig. 3b). This is probably the result of debromination of the BUdR-TMP adduct by far UV. The results shown in Fig. 3 indicate that, like all the other pyrimidines studied, BUdR produces cyclobutane containing adducts with furocoumarins.

Spectrophotometric analysis of the photoreaction

Since the above results were unexpected, we did

not wish to base our conclusions solely on chromatographic behavior of the photoproducts. Therefore, spectrophotometric analysis of the photoreactions was undertaken. Figure 4 shows the absorption spectra of BUdR exposed to near UV light for various times in the presence of %MOP, as described in Materials and Methods. 8-MOP was used instead of TMP because of the low solubility of the latter. Evi- dently, there is a progressive disappearance of 8-MOP (absorption above 320 nm is contributed solely by 8-MOP) and BUdR (peak at 285 nm) with increasing exposure time. Reirradiation at 254 nm of the thawed solution at room temperature caused a red shift of the absorption spectrum and the reappearance of the BUdR peak. Similar results were obtained upon exposure of thymidine and uridine with furocoumarins (unpublished data; see also Krauch et al., 1967). These results are taken as evidence that the photoproducts of BUdR and furocoumarins are of the cyclobutane type. Furthermore, they indicate that the bromine atom remains in the photoproduct. Control experiments in which BUdR was exposed to 254nm light indicate that under these conditions only about 1-2% of it was debrominated to yield uridine.

a

b

0.91

0.49

I I I I 0 0.2 0.4 0.6 0.8 1.0

R I

Figure 3. Radiochromatogram scans of: (a) C3H]-TMP adduct with thymidine after irradiation with lOkJrn-’ of 254nm light in solution; (b) labeled psoralen adduct with BUdR irradiated as in (a).


( n m )

Figure 4. Absorption spectra of BUdR and 8-methoxypsoralen exposed to 360 nm light as 'described in Materials and Methods. Exposure times are 0, 1 and 4h, as indicated. The dotted line is the spectrum obtained from the 4 h exposure sample upon reirradiation with 10 kJ m-' of 254 nm light.

Binding of trimethylpsoralen to polymers In order to characterize the photoreactivity of

BUdR with furocoumarins, its behavior within a polynucleotide was studied. The double stranded alternating copolymer poly(dA4BU). poly(dA4BU) was used and compared to a similar polymer in which BUdR is replaced by thymidine, namely poly(dA4T) poly(dA4T). Calf thymus DNA was also included for comparison. The results of photo-induced binding of C3H]-TMP to these polymers are shown in Fig. 5 . As reported previously (Chandra et al., 1973) we see that the rate of binding to poly(dA4T) is much higher than to native calf thymus DNA. There is no marked difference between the binding rate of psoralen to the polynucleotide containing BUdR, compared to thymidine containing polynucleotides. This is consistent with the observations made with the nucleosides.

Another aspect under study of furocoumarins photochemistry with polynucleotides was the effect of polyamines. Polyamines slow the rate of the photoreactions of T M P with DNA (Ben-Hur, 1975) and it was of interest to see if polydeoxynucleotides differ in this respect. A priori it can be assumed that because

of the higher thymidine content the effect will be greater in poly(dA4T) than in DNA (Mahler and Mehrotra, 1963). Figure 5 shows that this is not so. While SmM cadaverine reduced the rate of TMP binding to DNA by a factor of 1.7, in agreement with the previous report (Ben-Hur, 1975), there was no effect on either poly(dA-dT) or its BUdR-containing analog.

D N A crosslinking by trimethylpsoralen

The results of T M P binding to polynucleotides (Fig. 5 ) do not discriminate between monofunctional adducts and crosslinks. To obtain information about the latter we have measured the crosslinking of BUdR-substituted E . coli DNA following exposure to TMP-plus near UV light. The method employed was to follow the denaturation-renaturation behavior of the DNA (Ben-Hur, 1975). Exposure times of 1 and 2 min reduced the percentage of nonrenaturing DNA (i.e. DNA that d o not contain interstrand crosslinks) to 65% and 40%, respectively, in the case of BUdR- substituted DNA. The rate of crosslinking of nonsub- stituted DNA was similar; 55% and 30% for 1 and 2 min exposure, respectively. Thus, there appears to

Mechanism of bromouracil sensitization 555

IRRADIATION TIME ( Min )

Figure 5. Rate of TMP binding to polymers under exposure to near UV light. Polymers used were: DNA, squares; poly(dA-dT) . poly(dA-dT), triangles; poly(dA-BUdR) . poly(dA-BUdR), circles. Filled symbols: irradiation in the presence of cadaverine at a concentration of 5 mM (DNA) or 20 mM (polynucleotides). The higher cadaverine concentration was because the higher ionic strength (0.1 M phosphate buffer instead of 0.01 M in the case of DNA), required to maintain the double-stranded

structure of the polynucleotides.

be no qualitative dimerence in the effect of BUdR- substitution on the formation of T M P monoadducts versus crosslinks. Both products are induced at a similar rate in normal and BUdR substituted DNA.

Survival of E. coli exposed to TMP-plus-near U V light

Neither near UV light alone (in the dose range studied) nor TMP alone affect the survival of E. coli KMBL (wild type) significantly. However, simul- taneous exposure to both these agents drastically reduces survival. The survival curve obtained as a function of near UV light exposure is shown in Fig. 6. After an initial shoulder the bacteria are killed exponentially. Also shown in Fig. 6 is the response of cells in which thymidine was replaced by BUdR. It is apparent that BUdR substitution affects the survival response of the cells to TMP-plus-near UV light. The effect involves elimination of the shoulder on the survival curve while the final slope remains un-

changed. This sensitization effect of BUdR is absent in the mutant E. coli uur B (Fig. 6). This mutant differs from the wild type only in being deficient in UV endonuclease activity and is, therefore, unable to per- form excision repair of UV damage in DNA. This result strongly indicates that BUdR sensitization is the result of interference with cellular capability of excision repair.

DISCUSSION

An observation described in this paper which was somewhat unexpected is that BUdR reacts with furocoumarins under near UV light exposure just as thymidine does. This is true for the nucleosides as well as their polymers. It was unexpected because the photochemistry of BUdR is drastically different from that of thymidine and the former was not shown to pro: duce cyclobutane-containing photoproducts up to


11

0.1

z 0 I- U

P v

0 z ? a

0.01 YI

0.001 I . 0 20 4 0 60

IRRADIATION T I M E (min)

Figure 6. Survival of colony-forming ability of E. coli KMBL exposed to near UV light in the presence of 1 pA4 TMP. Cells were grown with either thymidine or BUdR (filled symbols) at a concentration of 5 mg/f during two cell divisions prior to exposure. Circles: wild type cells. Triangles: E. coli KMBL

1054 uur B cells.

now. Halogen substitution in position 5 of pyrimidines decreases the electron density at this position, and since polarization of the 5,6-double bond is im- portant for the dimerization reaction, compounds containing halogens should have a smaller tendency to dimerize by forming a cyclobutane ring (Wacker, 1963). In spite of this it appears that when the photoreaction does not proceed by direct excitation of the pyrimidine moiety but via excitation of a furocoumarin (Song et al., 1971), BUdR can produce cyclobutane-containing photoproducts just as readily as thymidine.

Another unexpected observation is that polyamines do not affect the rate of the photoinduced binding of TMP to polydeoxynucleotides, unlike in the case of DNA, where polyamines slow the rate of its photoreactions with TMP (Ben-Hur, 1975). In the latter case it was suggested that polyamines affected the photochemical reactions due to their stabilizing effect on the DNA double-helix structure, making the dis- tortion required for the photoadduct formation more

difficult (Ben-Hur, 1975). It is not clear why this does not apply in the case of the polydeoxynucleotides.

It is suggested that because of the quantitatively and qualitatively similar photoreactions of BUdR and thymidine with furocoumarins, this combination could be used in biological systems to elucidate the mechansim of BUdR sensitization. Specifically, if the sensitization is due to BUdR lesions produced by UV light or ionizing radiation, no sensitization should be observed in the BUdR-furocoumarin system, where only BUdR photoproducts involving furocoumarin should occur. If sensitization is found in this system, it will strongly suggest that the mere presence of BUdR in DNA makes the cell more sensitive to lesions in its DNA, probably by interfering with the repair of these lesions. The results obtained with E. coli cells, showing sensitization by BUdR of the response to furocoumarins-plus-near UV light (Fig. 6) indicate that BUdR exerts in bacteria part of its sensitizing action by interference with repair mechan- isms. Furthermore, since no sensitization was

Mechanism of bromouracil sensitization 557

observed in E . coli uur B the interference of BUdR must be exclusively with the excision repair pathway.

A similar effect, i.e. elimination of the shoulder of the survival curve by low BUdR concentration, was also observed for X-irradiated bacteria (Lett e t al., 1970) and mammalian cells (Shipley e t al., 1971). It thus appears that the presence of BUdR in the DNA inhibits the repair of sublethal damage, probably by interfering at low dose range with the efficiency of repair by excision-resynthesis. The failure to observe such an effect of BUdR on the excision of UV- induced pyrimidine dimers in bacteria (Lion, 1968)

may reflect the fact that excision was measured following UV fluences which kill over 99% of the cells. Alternatively, BUdR may impair a latter step in the repair process. O u r data with another photochemical system, however, support the idea that BUdR inhibits the excision, rather than a latter step (Ben-Hur et al., 1978) of the repair system.

Acknowledgements-We are grateful to Mrs. A. Prager for her contribution and to Miss R. Volinsky for help in some of the biological experiments. We also thank Dr. I. Rosen- thal for obtaining the absorption spectra and for critical reading of the manuscript.

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Cole, R. S. (1970) Biochim. Biophys. Acta 217, 3CL39. Cole, R. S . (1971) J . Bacteriol. 107, 846-852. Dall'Acqua, F., S. Marciani and G. Rodighiero (1970) Griffin, A. C., R. E. Hakim and J. Knox Greer, S . (1960) J . Gen. Microbiol. 22, 618-634. Hutchinson, F. (1973) Quart. Rev. Biophys. 6 , 201-246. Igali, S., B. A. Bridges, M. J. Ashwood-Smith and B. R. Scott (1970) Mutat. Res. 9, 21-30. Krauch, C. H., D. M. Kramer and A. Wacker (1967) Photochem. Photobiol. 6, 341-354. Lett, J. T., I. Caldwell and J. G. Little (1970) J . Mol . Biol. 48, 395-408. Lion, M. B. (1968) Biochim. Biophys. Acta 155, 505-520. Mahler, H. R. and B. D. Mehrotra (1963) Biochim. Biophys. Acta 68, 211-233. Musajo, L., F. Bordin and R. Bevilacqua (1967) Photochem. Photobiol. 6, 927-931. Oginsky, E., G. Green, D. Griffith and W. Fowlks (1959) J . Bacteriol. 78, 821-833. Peter, H. and R. Drewer (1970) Photochem. Photobiol. 12, 269-282. Riklis, E. (1965) Can. J . Biochem. 43, 1207-1219. Shipley, W. A,, M. M. Elkind and W. B. Prather (1971) Radiat. Res. 47, 4 3 H 4 9 . Song, P-S., M. L. Harter, T. A. Moore and W. C. Herndon (1971) Photochem. Photobiol. 14, 521-530. Wacker, A. (1963) Prog. Nucleic Acid. Res. 1, 369-399.

(1976) Trans. Isr. Nucl. SOC. 4, 135-137. Photochem. Photobiol. 27, 55S563.

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FEBS Lett . 9, 121-123. (1958) J . Inoest. Dermatol. 31, 289-295.

photochemical interaction of furocoumarins with bromodeoxyuridine and polydeoxy-nucleotides...

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