Journal of Photochemistry

and Photobiology,

B: Biology,

5 (1990)

167 - 178




Institute of Biophysics, Semmelweis H-l 444, Budapest (Hungary)

Medical University,

RONTb P.O.B. 263.

(Received April 4, 1989; accepted July 11, 1989)

Keywords. Phage photosensitization, dark reaction, optical melting, action spectra.

Summary The dark and photoreactions of 4’-aminomethyl-4,5’,&trimethylpsoralen (AMT) with T7 phage were investigated from biological and structural points of view. The dark reaction leads to the structural destabilization of the double helix of the DNA as is shown by optical melting measurements. The genotoxicity of AMT in the dark is comparable with that of known genotoxic drugs as determined by phage inactivation. The photoreaction with UVA light leads to the formation of mono- and di-adducts depending on the wavelength and dose used. Mono- and di-adducts influence DNA stability differently; biologically both types of adducts are genotoxic as measured by action spectra.

1. Introduction The dark- and light-mediated action of psoralen derivatives on living material results in wide ranging consequences observed as therapeutic and side effects. The primary dark- and photoprocesses in living matter have been studied in important biomolecules such as isolated nucleic acids [ 1, 21, proteins [ 31 and membranes [ 41. These studies identified and characterized interactions from the structural point of view: rate and geometry of intercalation between the base-pairs, mono- and di-adduct formation with nucleic acids, photobinding to proteins or singlet oxygen-mediated action on the membranes. Biological effects observed on simple targets such as viruses [ 51, bacteria [6], yeast [7] and mammalian cells [8] proved that photoreaction leads to lethality and mutagenicity. Biological consequences of the dark reaction have been published only recently, revealing mutagenicity in bacteria [9], phage inactivation [ 10,111 and membrane lysis [ 121. loll-1344/90/$3.50

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Assuming that the main therapeutic effect (i.e. the inhibition of proliferation) is based on the photoreaction with nucleic acids, all dark reactions, even those with photobreakdown products [ll], lead to unwanted side effects as well as photoreaction with any other cell constituents. The therapeutic role of the mono- and di-adducts with nucleic acids is not well established yet but attempts are being undertaken to test their mutual importance using either monofunctional derivatives (e.g. ref. 13) or irradiation with UVA light of various wavelengths [ 141. However, it is difficult to relate biological events observed in vitro to structural changes because of the fundamentally different experimental conditions, i.e. solvent composition, drug-target concentration ratios and light doses used. In vitro studies concerning the solvent composition are mostly performed at low concentration of monovalent ions to ensure a detectable ratio of products. Increasing the ionic strength or using divalent ions (approaching physiological conditions) causes the rate of dark complexing to DNA or chromatin to decrease significantly also weakening the photoreactivity [ 151. Photoreactivity is influenced less by the salt concentration in highly organized nucleosomes than in isolated DNA [ 161. The role of the drug-target ratio can be illustrated by the results presented by Averbeck et al. [17] through the comparison of two psoralen derivatives acting both on DNA and RNA. The degree and the relative order of their dark- and photoreactivities differ in the reactions with isolated and intracellular nucleic acids. This is probably due to the heterogeneous intracellular drug distribution. The third important parameter is the light dose. Using even the same irradiation set-up for a transparent solvent and a cell culture, quantum yields can not be compared because of complex dissipation phenomena. Here we present the results of parallel investigations on -biological and structural effects of dark- and photoreactions between the 4’aminomethyL 4,5’&trimethyl psoralen and T7 bacteriophage. The T7 phage contains double stranded DNA in highly stacked form [ 181, which makes its interactions with psoralens sterically similar to those of chromatins. The genetic structure of the phage is simple. About 90% of T7 DNA is essential for phage development [ 191; it thus offers a high sensitivity for the biological detection of damage [ 201. Our aim was to detect those structural damages which may be responsible for phage inactivation. For the detection of very few damages in the nucleoprotein the use of an indirect method -via optical melting measurements - appeared to be useful. The reliability of the optical melting method was shown earlier, in the case of UVB irradiation [21]. To study the role of mono- and di-adducts in the phage inactivation, the functional and structural action spectra were compared in the 340 - 380 nm wavelength range. 2. Experimental details 2.1. Bacteriophage T7 T7 was grown on Escherichia coli B (wild type) host cells. The cultivation and purification were carried out as described in refs. 22 and 23. Phages


were concentrated on a CsCl gradient and dialyzed against a buffer containing 20 mm01 1-l TRIS, 50 mmol l-l NaCl and 1 mmoll-l EDTA, adjusted to pH 7 with HCl. The buffer was chosen as an optimal compromise allowing the lowest ionic strength preferred for the binding of AMT and supporting phages without significant inactivation [24]. Phage concentration was 1012 ml-’ (150 E.tmolbase 1-l) in the optical investigations, determined by the extinction coefficient [25] corrected for light scattering [26]: e260 = 7300 1 (mol base)-’ cm-‘. 2.2. 4’-aminomethyl-4,5’,8-trimethylpsoralen (AMT) AMT was purchased from H.R.I. Associates (Emeryville, CA). AMT was dissolved in the same buffer as the phages, its concentration was determined also spectrophotometrically (ezso = 2.5 X lo4 1 mol-’ cm-‘; [27]). AMT concentration for the dark reaction varied between 1 and 100 E.tmol 1-l and it was 1.5 pmol 1-l in the photoreaction. 2.3. Treatments 2.3.1. Dark incubation

Phages were incubated with various concentrations (c) of AMT at room temperature in the dark. Samples for biological investigations were taken at different times of incubation which varied between 1 and 24 hours. For optical measurements 2 h of dark incubation was performed. 2.3.2.


AMT and phage solutions (100 nucleotides to 1 AMT molecule) were mixed just before irradiation. Control samples were prepared at the same time and incubated in the dark for the duration of the irradiation. A xenon lamp (2.5 kW) served as a source of UVA, completed with a Jobin Yvon type grating monochromator (1400 grooves mm-‘). The half-width of the irradiating band was 4 nm, the wavelength varied between 340 and 380 nm. The fluence rate (I) incident on the silica cell was determined by a thermopile and was in the order of 10 - 20 W rn-?. Samples were stirred during irradiation for time (t). Incident dose (D) is given as: D = I X t in kJ rne2. Absorbed dose (DA) is calculated taking into account the optical density (OD) of the samples at the wavelength used : DA = D (1 - lOmoD), and is expressed in number of photons absorbed by one phage. In the absence of furocoumarins the UVA irradiation did not induce more than 5% of phage inactivation compared with the non-irradiated cases.

2.4. Phage inactivation

Phage inactivation was calculated either from the decrease in plaqueforming ability on Petri dishes or by an automatic test method based on the light-scattering changes of bacteria caused by the lysis [28]. In both cases survival rates were evaluated as ln(N/N,) where N,-,and N correspond to the number of active phages before and after the treatment respectively.


In the case of incubation in the dark, inactivation indices (MI values) were calculated from the slopes of the kinetic curves according to ref. 28. The MI value gives quantitatively the product of AMT concentration and incubation time leading to the inactivation down to 37% of survival. In photoreaction a similar parameter was defined, the absorbed dose of 340 nm light leading to 37% of survival (DA3,). The ratio of MI and DA3, gives the quality factor (F) as defined previously [ 111. The wavelength dependence of the photoreaction was characterized by the relative quantum yield (a&, related to the quantum yield at 360 nm. 2.5. Optical melting measurements Optical melting measurements were done by measuring the OD at 260 nm on a Per-kin-Elmer 200 UV-vis spectrophotometer coupled on-line to a computer for data processing. The heating rate was 0.5 “C mix-‘, set by an automatic temperature controller (Perkin-Elmer type C 5700710). First derivatives of the melting curves were plotted and analysed by the computer. Melting temperatures of different regions (Ti) were calculated as first momenta, and the half-width (Wi) of the derivative peaks were defined as shown in Fig. 1. This figure presents the derivative melting curve of the non-treated phage in the TRW buffer used. The three transitions were identified previously [ 291. The Ti values of the second and third transitions, as well as their halfwidths, as indicated in Fig. 1 are actually used to characterize the structural changes introduced by AMT interaction.




T (C-1

Fig. 1. Derivative melting curve of T7 phage nucleoprotein, measured at 260 nm. The identification of the three transitions is given in ref. 29. W is the half-width and T is the melting temperature of a given transition.

3. Results and discussion 3.1. Dark reaction 3.1.1. Biological


Figure 2 presents the inactivation of phage T7 in the dark reaction with AMT in two different buffer media and in comparison with the inactivation of two highly genotoxic drugs: nitrofurantoin and methylmethane-thiosulfonate. The points of the experimental curve correspond to an average of three independent measurements, whereas the full line represents the theoretical kinetics. The inactivating effect of AMT in the dark is stronger in the






min mol/l


-06 -

Fig. 2. Inactivation of T7 phage in dark reaction with AMT in comparison with other genotoxic drugs. -, AMT in the TRIS buffer used (MI = 0.02(+0.005) min mol 1-l); --, AMT in phosphate buffer M9 (MI = 0.6(fO.l) min mol l-l) [lo]; - * - . -, nitrofurantoin (MI = 0.5(+0.5) min mol 1-i) [28]; - * . -, methyl-methane-thiosulfate (MI = 0.022(*0.005) min mol 1-l) [28].

case of the TRIS buffer used by us (solid line) than in the more complex phosphate buffer M9 (dashed line) which was used in an earlier investigation [lo], but both are comparable with the two reference compounds [28]. At the studied concentrations and incubation times the inactivation kinetics can be fitted by straight lines in the semilogarithmic representation characteristic for one-hit type genotoxic actions. The inactivation index of AMT in the TRIS buffer for the dark reaction proved to be 0.02(+0.005) min mol 1-l whereas in the M9 buffer its value was found to be 0.6(?0.1) min mol l-i, this latter value similar for a large variety of mono- and bi-functional furocoumarin derivatives. The smaller index obtained in TRIS buffer representing the higher genotoxicity is in accordance with the higher intercalation ability of the AMT in the buffer of low ionic strength, which contains only monovalent cations [ 151. This observed genotoxicity in the dark is not a unique feature of the AMT-T7 phage. reaction. The genotoxicity of AMT and of some other psoralen derivatives was detected in RNA containing MS2 phage [ll]. The relation of the applied T7 test system to some other similar ones concerning different drugs is presented and discussed in ref. 30. 3.1.2. Structural modifications The luminescence intensity emitted by AMT decreases in its dark reaction with the phage, indicating intercalation between the nucleotide base pairs 1311. The parameters of the intercalation have been calculated from spectroscopic measurements. The association constant K proved to be 3.5 X lo3 1 mol-’ while the n value was about 50 using the model described in [32]. These parameters were found to be very sensitive to the ionic strength. On the other hand, in our buffer the dark binding activity was much higher for isolated than for intraphage DNA. The absorption spectrum of T7 phage shows no significant change at the dark interaction with AMT, but changes are detected in its thermal denaturation which was measured by optical melting. A narrowing was



Fig. 3. Thermal stability changes of T7 phage in dark reaction with AMT. IJ = [AMT intercalated ]/[base pairs], determined from fluorescence measurement. (a) Narrowing of the hydrogen bond’s breaking transition (W,) related to the non-treated case; (b) decrease of the strand separation temperature (Ta) related to the non-treated case. The points represent the average of three values and the experimental errors are indicated.

observed in the second transition which belongs to the breaking of the hydrogen bonds between the bases, and a decrease in the temperature of the third transition belonging to the separation and coiling of the two DNA strands. Figure 3 presents these changes in the function of the intercalated AMT ratio. The half-width of the second transition peak is expressed as the percent of the non-treated case W2,rel(Fig. 3a). The change in the third transition temperature is expressed as the difference from the non-treated case ATj (Fig. 3b). In a first approximation, both sets of data can be fitted by straight lines with a significance level P < 0.01. The fraction of the intercalated AMT (u) was determined from the fluorescence intensities [ 311. The narrowing of the hydrogen bond’s breaking transition, i.e. the increased cooperativity of the melting DNA regions, may be explained either by an increased stacking interaction or by the variation in the cooperativity length. The decrease of the strand separation temperature suggests that probably the intercalation is not geometrically symmetric. These changes of the stability are until now the only structural changes observed at the dark reaction of DNA with psoralens. This destabilization in the presence of AMT must be the result of the weakening of some stabilizing interactions - on the level of the double-stranded DNA structure. These modifications may be precursors of the biological inactivation as well. No significant change in the parameters of the first transition were detected, meaning that the DNA-protein capsid interaction is not affected significantly by the dark interaction. Similar decrease of the thermal stability was reported by us earlier in the dark reaction of TMP (4,5’&trimethylpsoralen) with T7 phage and with its isolated DNA [ 331. The dark complex formation is totally reversible. Eliminating the AMT (by dialyzing for 6 h) caused no inactivation or structural changes to be observed proving the non-covalent character of the intercalation. Preliminary room temperature Raman studies on dark complexes suggest that strong stacking interaction exists between the thymin bases and AMT.


3.2. Pho toreaction 3.2.1. Biological effect Figure 4 presents the phage inactivation under UVA irradiation using light of 340 and 370 nm wavelengths. Survivals are plotted against the incident dose (Fig. 4a) as well as against the absorbed dose (Fig. 4b). The apparently large difference between the inactivations by the two wavelengths observed in the incident dose plot vanishes. The initial slopes are similar when the effects of the absorbed photons are compared. Another difference can be seen in this representation: the inactivation at 370 nm follows simple exponential kinetics in this dose range, whereas it shows a break at higher doses of 340 nm light. At doses above this breaking point the absorbed photons seem to act more strongly than those absorbed initially. Assuming that photolesion follows a Poisson distribution in the phages, the observed breaking point coincides with the overall damage concentration of one lesion per phage. The effect of different reparability of the mono- and di-adducts cannot be excluded from these results but it should be examined further. However, the photobinding of AMT to isolated DNA (with no reparation) also was not found to be strictly linear-dependent on the dose [ 141.


-51 (b) Fig. 4. Inactivation of the AMT-sensitized T7 phage in function of the incident (a) and absorbed (b) UVA dose using different wavelength light irradiation. The points represent the averages of three values and the experimental errors are indicated.

3.2.2. Stability changes Figure 5 presents the changes in the temperature of the third thermal transition of the T7 phage in the function of the absorbed dose. At the shorter wavelength irradiation (340 nm) a biphasic change was detected (see Fig. 5a). At lower doses a destabilization takes place (AT3 is negative),

1.5 x 104abd70bs phase

AT3 (C-l 4


3 2

(b) Fig. 5. Changes of the strand-separation temperature (AT3) of the AMT sensitized phage DNA as a function of the absorbed UVA dose. (a); Biphasic change at short wavelength irradiation at lower doses; (b), comparison of different wavelength irradiations in a larger dose-range. The points represent the averages of three values and the experimental errors are indicated.

whereas stabilization occurs at higher doses. The normal stability (section of the abscissa: ATg = 0) is reached at about the same number of absorbed photons, where the breaking point in the inactivation kinetics is observed (see Fig. 4b). As this phase transition is related to the separation of the two DNA strands, the increased T3 may be explained by the formation of crosslinks, and the biphasic character can be due either to the presence of initially formed mono-adducts or to a further intercalation in the case of the free drug -intercalated drug equilibrium is influenced by the cross-link formation. With longer wavelength irradiation no significant change in the T3 temperature was detected even at doses ten times higher than in the biological experiments, showing the absence of cross-links. It also means that the mono-.adducts have biological significance, although to a lesser degree, than the di-adducts. No clear dependence in the parameters of the first and second transition were observed at the applied doses. The above detailed wavelength dependence of the photoreaction led us to measure the action spectra both from biological and structural points of view. 3.2.3. Action spectra Inactivation and structural stability changes of T7 phage were measured after AMT treatment in the function of the irradiating wavelength in the


region of 340 - 380 nm using a narrow (4 nm half-width) light beam. Relative quantum yields Qrel of the phage inactivation were calculated from the survival ratios at lo4 absorbed photons per phage and are presented in Fig. 6a. The quantum yield at 360 nm was taken as reference. Two regions can be separated in Fig. 6 at wavelengths longer than 360 nm. About 20% decrease in the quantum yield is observed. At shorter wavelengths, the value of arei does not change significantly. The structural action spectrum was determined from the AT, values measured at lo5 absorbed photons per phage (Fig. 6b). It was necessary to apply a dose ten times higher than in the biological experiments because of the lower sensitivity of the structural measurement. At wavelengths longer than 365 nm no significant cross-link formation is detected at the doses applied. A small number of cross-links are formed by absorption of photons at 360 nm, and their ratio is increased at the shorter wavelength.






360 nm







Fig, 6. Action spectra of AMT in T7 phage from functional (a) and structural (b) measurements. Qrrel is the relative quantum yield calculated from the survival ratios at 10” absorbed photons per phage and ATE is the change in the strand-separation temperature of the phage DNA determined at lo5 absorbed photons per phage.

The two action spectra are roughly parallel to each other. The slight difference in their breaking point may be due to the ten-fold difference in the doses. On the basis of this parallelism we can conclude that different structural changes (depending on the wavelength) are responsible for the different inactivation yield. On the other hand these action spectra demonstrate that lamps emitting around 365 nm produce an undefined situation concerning the photoproducts. No biological action spectra of AMT have been reported until now. Our preliminary results on MS2 phage (containing RNA in about 80% base-paired form) show no change in the quantum yield in the 340 - 380 nm region. Action spectra of other psoralen derivatives in T7 phage [34] show that in the case of angelicin the quantum yield is roughly constant between 340 and 380 nm whereas for 8-MOP it changes similarly to our results. 5-MOP action spectra in X phage are published only for incident doses [35] presenting the wavelength dependence of the cross-section. Of the structural action spectra published, we compare our results with those described in refs. 14 and 36, where the wavelength dependence of cross-link formation between AMT and isolated DNA is presented. In ref. 14


only incident doses are given, but after recalculation of these data, we obtained a stepwise action spectrum with the decrease between 360 and 370 nm. Using data from ref. 36 a qualitative agreement was observed in the 340 - 360 nm region, with a gradual decrease of the cross-linking at longer wavelengths, which was not observed by us. We have to note that besides the difference in the targets, experimental details including the concentration ratios and the buffers were quite different in refs. 14 and 36, thus impeding the quantitative comparison with our study. Structural action spectra of &MOP in isolated DNA has also been published in refs. 37 and 38. In Fujita et al. [38] the formation of cross-links shows a similar feature as in our case. 3.3. Comparison of the dark and photoreactivities To approach the problem of how important the unwanted dark effects in relation to photoeffect are, we compared the quantitative characteristics leading to the same degree of inactivation in the cases of dark incubation and photoreaction. The F factor, defined in ref. 11, proved to be 50 + 0.5, about four times lower dark-vs.-light effectivity than for &MOP [ 341.

4. Conclusion The parallel investigation of the functional and structural consequences of the AMT-T7 phage dark and photoreactions led us to the following conclusions : Dark complexation induces the destabilization of the double stranded DNA structure leading to biological inactivation, comparable with that of covalently binding known genotoxic substances. The photoreaction is wavelength and dose dependent: at wavelengths longer than 360 nm or at low doses (less than 5 X lo3 absorbed photons per phage) of shorter wavelength irradiation, mostly mono-adducts are formed, while at short wavelength irradiation at higher doses, di-adducts are formed, decreasing and increasing respectively the DNA strand-separation temperature. Both types of photoreactions lead to inactivation, not differing more than 20% in their quantum yields. The biological role of the dark interaction is comparable with that of the UVA irradiation. The observed large variability of the light vs. dark reactivity depending on the psoralen derivative and target is important in the choice of drugs to be applied.

Acknowledgment The authors thank Judit Fidy for calling attention to the problems discussed, and for her continuous interest.


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Dark and photoreactivity of 4'-aminomethyl-4,5',8-trimethylpsoralen with T7 phage.

The dark and photoreactions of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with T7 phage were investigated from biological and structural points of ...
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