DNA repair and recovery in Escherichia coli after psoralen and angelicin photosensitization.

The correlation between DNA repair and recovery of biological functions was studied using three wild type strains of Escherichia coli and two skinphotosensitizing furocoumarins, psoralen and angelicin, which are well known specific reagents of the pyrimidine bases of DNA. In addition to mono-adducts psoralen is able to form a high number of inter-strand cross-links, while angelicin forms only mono-adducts. Both of these damages were repaired, in a short time, in the following way: at first DNA was cut into small pieces that were then rejoined into molecules of normal size, free from cross-links, while the furocoumarin residue was split from DNA almost quantitatively. Recovery of biological functions was studied performing photosensitization experiments in such a manner that the same amounts of psoralen or of angelicin were linked to bacterial DNA. DNA synthesis, tested just after the damage, was inhibited in a similar extent by both drugs. The same bacteria, however, showed a very different colony-forming capacity; angelicin was much less effective than psoralen with a D37 dose about 2.7 times higher. A similar picture was obtained studying DNA synthesis at different times after photosensitization: in the bacteria damaged by angelicin it was restored while no recovery was observed in cells photosensitized by psoralen. These results suggest that both mono-adducts and cross-links can be chemically repaired more or less in a quantitative measure, but that repair of cross-links in much less effective on cell recovery; this behaviour is very probably connected with the different repair mechanisms of mono-adducts and of cross-links.

Methylangelicins: new potential agents for the photochemotherapy of psoriasis. Structure-activity study on the dark and photochemical interactions with DNA.

The interactions both in the ground and in the excited state between various methylangelicins, previously prepared with the aim to increase the low photobiological activity of the parent angelicin 1, and DNA have been studied. In general, the new methylangelicins show an increased capacity to photobind monofunctionally to DNA and a parallel increment of photobiological activity in comparison with the parent 1. This increase appears to be connected with various factors, such as the augmented affinity toward DNA for the dark complex formation and the electronic effect connected with the introduction into 1 of one or two methyl groups. The new compounds, on the basis of their photobiological activity and their lack of skin phototoxicity, appear as possible agents for the photochemistry of skin diseases characterized by cell hyperproliferation.

Synthesis and biological activity of (hydroxymethyl)- and (diethylaminomethyl)benzopsoralens.

Some benzopsoralens, carrying a hydroxymethyl or a diethylaminomethyl group at the 3, 5, 8, and 11 positions, were prepared, and their biological activity was compared with that of 4-(hydroxymethyl)benzopsoralen (BP). 5-(Hydroxymethyl)benzopsoralen (7b), 11-(hydroxymethyl)benzopsoralen (7c), and 11-(diethylaminomethyl)benzopsoralen (8c) induced marked antiproliferative effects in mammalian cells by simple incubation in the dark; this activity appeared to be related to their ability to inhibit topoisomerase II. Benzopsoralens appeared to be more active, especially BP and 7c, upon UVA activation. Compounds carrying a methyl group at the 4 position together with a hydroxymethyl or diethylaminomethyl at the 8 position (7d and 8d, respectively) were also effective, although to a lower extent; instead, a substituent at the 3 position canceled all activity. Benzopsoralens did not induce interstrand cross-links in DNA in vitro, as seen in the induction of cytoplasmic <> mutations and double-strand breaks in yeast. This behavior is also compatible with their low mutagenic activity in E. coli WP2 and with the absence of any phototoxicity on the skin. For these features, benzopsoralens seem to be interesting potential drugs for PUVA photochemotherapy and photopheresis. The activity shown in the dark is not sufficient for their possible use as antitumor drugs, but it does offer a new model for the study of topoisomerase inhibitors.

Angular furoquinolinones, psoralen analogs: novel antiproliferative agents for skin diseases. Synthesis, biological activity, mechanism of action, and computer-aided studies.

With the aim of obtaining new potential photochemotherapeutic agents, having increased antiproliferative activity and decreased undesired effects, we have prepared some new furoquinolinones. Two of them have been studied in detail: 1,4,6,8-tetramethyl-2H-furo[2,3-h]-quinolin-2-one (8), and 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one (10). These compounds form a molecular complex with DNA, undergoing intercalation inside the duplex macromolecule, as shown by linear flow dichroism. The complexed ligands, by subsequent irradiation with UV-A light, photobind with the macromolecule forming only monocycloadducts with thymine with cis-syn configuration. In order to evaluate the electronic effects induced by the nitrogen atom in position 1 of 8, semiempirical calculations have been performed on both 4,6,4'-trimethylangelicin (TMA) and 8. The results obtained do not clearly differentiate between the two molecules which, at this level of approximation, show the possibility of photoreaction with both the 3,4- and 8,9-olefinic bonds for 8 and the 3,4- and 4',5'-bonds for TMA. In the lower energy conformation of intercalated 8, the furan ring is turned toward the minor groove of the polynucleotide, in such a way that photoreaction of this ring with thymine is favored. These compounds unexpectedly inhibit DNA and RNA synthesis in Ehrlich cells, in the dark. They also show a strong photoantiproliferative activity, 2 orders of magnitude higher than 8-methoxypsoralen (8-MOP), the most used drug for photochemotherapy. Their mutagenic activity on Escherichia coli is similar to that of TMA and 8-MOP. On the basis of these results, the compounds should deserve evaluation of their activity in the treatment of hyperproliferative skin diseases.

4'-Methylangelicins: new potential agents for the photochemotherapy of psoriasis.

Three derivatives of angelicin (1) [4'-methyl-, 4,4'-dimethyl-, and 4',5-dimethylangelicin (2a-c)] have been prepared with the aim of obtaining new agents for the photochemotherapy of psoriasis. These compounds form a complex in the dark with DNA that shows an affinity for the macromolecule higher than that of the parent angelicin (1). A correlation between their octanol/water partition coefficients and the association constants of the complexes has been observed. Compounds 2a-c photobind to DNA to a much higher extent than 1 and also more effectively than 8-methoxypsoralen (8-MOP), taken as reference compound. When activated with UV-A, the three compounds strongly inactivate T2 phage and inhibit epidermal DNA synthesis in mice. Moreover, they show a mutagenic activity markedly lower than that of 8-methoxypsoralen on Escherichia coli wild-type strain. Due to its lack of skin phototoxicity, its low mutagenic activity, and its antiproliferative activity, 2c was chosen for clinical evaluation. It proved to be effective in clearing psoriasis in two patients.

New monofunctional reagents for DNA as possible agents for the photochemotherapy of psoriasis: derivatives of 4,5'-dimethylangelicin.

With the aim of obtaining new agents for the photochemotherapy of psoriasis, we have prepared monofunctional reagents for DNA by starting from 4,5'-dimethylangelicin (2), an angular furocoumarin, and introducing in a 4'-(hydroxymethyl) (3), 4'-(methoxymethyl) (4), or 4'-(aminomethyl) group (5), in way analogous to what other authors have done previously on trioxsalen, a DNA bifunctional reagent. These new compounds form complexes with DNA in the ground state and by successive irradiation (UV-A) undergo monofunctional photoaddition to the macromolecule. Photobinding to DNA was highest for 3 and gradually lower for 4 and 5, respectively. These compounds do not form interstrand photocross-linkages in DNA and do not show any skin phototoxicity. Fluorimetric studies show that their 4',5' double bond is involved in the photoaddition to DNA. Their photobiological activity evaluated on Ehrlich ascites tumor cells and on T2 phages was strictly connected with their photobinding to DNA. The effect of the introduction of hydroxymethyl and methoxymethyl groups in angular 2 is somewhat similar to that previously described for trioxsalen: the introduction of an aminomethyl group in 2 markedly increases the affinity in the dark for DNA but under UV-A irradiation strongly inhibits photobinding to the macromolecule. By contrast, in the analogous derivative of trioxsalen both the affinity for DNA in the dark and the photobinding to DNA increased.

6-Methylangelicins: a new series of potential photochemotherapeutic agents for the treatment of psoriasis.

The possible presence of methylpsoralens as undesired inquinants in synthetic methylangelicins has been avoided through a synthetic pathway starting from umbelliferones carrying a methyl group in the 6-position. The new 6-methylangelicins show a high affinity toward DNA, forming in the dark a molecular complex; the complexed angelicins under UV-A irradiation photobind effectively to the macromolecule, forming only monoadducts. The new compounds show an evident antiproliferative activity by inhibiting DNA synthesis on Ehrlich cells; great differences, however, can be seen between the various compounds. All the compounds are lacking of skin erythemogenic activity. Some of the new 6-methylangelicins, evaluated in terms of mutagenic activity, demonstrate to be less effective than 8-methoxypsoralen (8-MOP), used for a comparison. On the basis of antiproliferative activity, lack of skin phototoxicity, and low mutagenicity, two compounds have been chosen for clinical evaluation. The compounds tested on seven psoriatic patients by topical application and UV-A irradiation proved to be more effective than 8-MOP, used in the same conditions.

Furocoumarin sensitization induces DNA-protein cross-links.

The capacity of some linear and angular furocoumarins to induce DNA-protein cross-links by UVA (320-400 nm) irradiation has been evaluated in Chinese hamster ovary cells. Two linear furocoumarins, psoralen and 8-methoxypsoralen appeared to be capable of inducing DNA-protein cross-links to a noticeable extent. 4'-Methylangelicin and 4,4'-dimethylangelicin formed only reduced amounts of DNA-protein cross-links, while angelicin and 4,6,4'-trimethylangelicin seemed to be unable to induce significant levels of this lesion. The biological significance of this damage remains to be elucidated, but it might have an important role in furocoumarin sensitization. In the examined compounds, the capacity for inducing DNA-protein cross-links appears to be a property of the skin phototoxic furocoumarins. This result suggests the hypothesis of a connection between this damage and the formation of skin erythemas.

Biological properties of some benzopsoralen derivatives.

The biological activity of some benzopsoralen derivatives, prepared with the aim of obtaining new drugs for photochemotherapy, has been studied. The more interesting compounds are 4-hydroxymethyl-4',5'-benzopsoralen and 4-hydroxymethyl-4',5'-tetrahydro-benzopsoralen, which were found to be active in the dark also: DNA and RNA synthesis were both inhibited in Ehrlich cells, even if in a partially reversible fashion, while protein synthesis remained unaffected. In Chinese hamster ovary cells cultured in vitro, the clonal growth was strongly inhibited by incubation in the dark with both drugs, while a number of chromosomal aberrations was observed in the fraction of growing cells. Using alkaline elution, DNA strand breaks were detected. In addition, in the presence of aphidicolin, a specific inhibitor of DNA polymerase, the clonal growing capacity was completely restored; in contrast, the number of DNA strand breaks remained unchanged. All these results suggest that DNA topoisomerases are probably the target of these two benzopsoralens. These compounds are also good sensitizers; by UV-A irradiation they have a good capacity to produce singlet oxygen, but they appeared to be unable to induce erythemas on guinea-pig skin. Under UV-A light, they induced a strong inhibition of DNA synthesis in Ehrlich cells. Thus, benzopsoralens appear to be capable of inducing strong antiproliferative effects by two different mechanisms, by UV-A irradiation and in the dark.

DNA damage induced by 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one, a new furocoumarin analog: photochemical mechanisms.

Some photochemical and photobiological properties of 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one (HFQ) were studied in comparison with its isomer 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one (FQ) and 8-methoxypsoralen (8-MOP). The HFQ photobinds to DNA forming furan-side monoadducts (MAHFQ) that have molecular structure very similar to those of FQ (MAFQ). Unlike MA8-MOP and MAFQ, MAHFQ no longer photoreact. The HFQ, like FQ, produces moderate amounts of singlet oxygen but no superoxide anions. The HFQ and FQ induce numbers of DNA-protein cross-links (DPC), much more plentiful than those of 8-MOP (about two and seven times, respectively) but no interstrand cross-links. The mechanism of DPC formation was studied in vivo in mammalian cells by alkaline elution and in vitro using a new test mixing histones and DNA from calf thymus. The latter is a very useful technique for the double irradiation protocol. The DNA (or histones) are separately exposed to a first UVA dose in the presence of the sensitizer; then, after its unbound molecules have been removed, histones (or DNA) are added to assemble the chromatin-like complex that is irradiated again. According to in vitro and in vivo methods, DPC appear to be formed by FQ and 8-MOP by a biphotonic process that starts with monoadduct induction in DNA, followed by their conversion into DPC. In the resulting lesions, the sensitizer molecule forms a covalent bridge between the two macromolecules (DPC at length greater than zero). Instead, HFQ induces DPC by a monophotonic process; thus, HFQ is probably not a physical part of the bridge between DNA and proteins, which may be linked together directly, like DPC at zero length induced by UVC.

DNA damage induced by 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one, a new furocoumarin analog: biological consequences.

4,6,8,9-Tetramethyl-2H-furo[2,3-h]quinolin-2-one (HFQ) and its isomer FQ (1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one) showed very strong antiproliferative activity in mammalian cells, about two times greater than 8-methoxypsoralen (8-MOP). Both compounds induced DNA-protein cross-links (DPC) but not interstrand cross-links. The FQ generated DPC in a biphotonic process, yielding a new kind of diadduct, whereas HFQ induced DPC by a monophotonic one, probably without its physical participation in the covalent bridge. These lesions gave different toxic responses. Sensitization of FQ led to extensive DNA fragmentation and to a number of chromosomal aberrations. Conversely, HFQ seemed to be completely inactive and 8-MOP gave intermediate results. A strict relationship between DPC formation and induction of chromosomal aberrations was observed. The HFQ did not induce light skin erythemas, whereas FQ was more phototoxic than 8-MOP, thus suggesting that FQ lesions, DPC in particular, may be implicated in skin phototoxicity. Ehrlich ascites cells, a transplantable mouse tumor, inactivated by furoquinolinone sensitization and injected into healthy mice, protected them from a successive challenge by viable tumor cells. This response appeared to be based on an immune mechanism. Comparable amounts of base substitution revertants were scored when testing furoquinolinones and 8-MOP in bacteria but no DPC were detected. This suggests that classic mutagenesis tests on bacteria are insufficient to give adequate information on furocoumarin genotoxicity. Given its features, HFQ can be regarded as an interesting new agent for psoralen plus UVA photochemotherapy and photopheresis.

Photobiological properties of 1'-thieno-4,6,4'-trimethylangelicin.

Some photobiological properties of 1'-thieno-4,6,4'-trimethylangelicin (TTMA), a new isoster of 4,6,4'-trimethylangelicin (TMA) were studied in comparison with the parent compound. The TTMA absorbs UVA light and photobinds in vitro to DNA more efficiently than TMA; however, in Ehrlich cells in vivo TTMA linked to DNA to a lesser extent than the parent compound. In general, the formation of damage into DNA is in line with this last result: In fact, TTMA and TMA form equivalent amounts of interstrand cross-links (ISC) both in vitro in linearized PM2 DNA and in vivo in HeLa cells. In this system TTMA induces DNA-protein cross-links (DPC) more efficiently than TMA; on the contrary, no significant amounts of single-strand breaks were detected with both compounds. The antiproliferative activity of TTMA is consistent with these results, being only slightly more pronounced than that of TMA. Experiments carried out using double irradiation demonstrated that these drugs are capable of inducing antiproliferative effects by biphotonic reactions, including the formation of both ISC and DPC. Thus, replacement of the oxygen atom by a sulfur increases the UV absorption of the drug and its capacity to photobind to DNA in vitro but does not yield a comparable enhancement of its photosensitizing properties in vivo; this might be due to various reasons, for instance to an increase in the lipophilic character that could modify the behavior in vivo.

4,4',5'-trimethyl-8-azapsoralen, a new-photoreactive and non-skin-phototoxic bifunctional bioisoster of psoralen.

Photochemical and photobiological properties of a new isoster of psoralen, 4,4',5'-trimethyl-8-azapsoralen (4,4',5'-TMAP), have been studied. This compound shows a high DNA-photobinding rate, higher than that of 8-methoxypsoralen (8-MOP), forming both monoadducts and inter-strand cross-links. The yield of cross-links, however, is markedly lower than that of 8-MOP. Antiproliferative activity of 4,4',5'-TMAP, in terms of DNA synthesis inhibition in Ehrlich ascites tumor cells, is higher than that of 8-MOP. Mutagenic activity on E. coli WP2 R46+ cells appeared similar to or even lower than that of 8-MOP. This new compound applied on depilated guinea pig skin and irradiated with UVA did not show any skin-phototoxicity. On the basis of these properties 4,4',5'-TMAP appears to be a potential photochemotherapeutic agent.

Comparative mutagenicity of linear and angular furocoumarins in Escherichia coli strains deficient in known repair functions.

Four furocoumarins, two having a linear molecule, psoralen and 8-methyl-psoralen and two having an angular molecule, angelicin and 4,5'-dimethyl-angelicin were tested for mutagenesis in Escherichia coli B wild type and in various strains deficient in known repair systems. The results indicate that both monoadducts and crosslinks are mutagenic. The mutagenic efficiency of the furocourmarins ranks in the following order 8-methylpsoralen psoralen greater than angelicin greater than 4,5'-dimethylangelicin.

Mutagenicity in Salmonella typhimurium of some angelicin derivatives proposed as new monofunctional agents for the photochemotherapy of psoriasis.

A series of angelicin derivatives were tested for their mutagenic activity with and without near-ultraviolet irradiation (NUV) in Salmonella typhimurium strains. After irradiation with NUV, the tested compounds induced different numbers of revertants in strain TA100, indicating that the mutational events involved are base substitutions. In the dark, 3 chemicals behaved as frame-shift mutagens causing reversion in strain TA98.

DNA damage and topoisomerase II inhibition induced by a benzopsoralen derivative.

The ability of 4-hydroxymethyl-4',5'-benzopsoralen (HMBP) to damage DNA of Chinese hamster ovary cells (CHO) and to inhibit the activity of topoisomerase II in vitro has been studied. This compound is characterized by a fourth ring condensed at the furan-side in the psoralen molecule. Contrary to other known furocoumarin derivatives, HMBP induces chromosomal aberrations in mammalian cells without UVA activation. The lesions induced in the dark by HMBP in DNA were studied by alkaline and neutral elution in CHO cells; comparable amounts of single-strand breaks and DNA-protein cross-links as well as the formation of double-strand breaks were detected. Moreover, HMBP appeared to inhibit the activity of mammalian topoisomerase II in vitro, in both the catenation and the decatenation assay. In these experiments the drug was effective only when it was pre-incubated with DNA substrate. These results are also consistent with the cytotoxic and mutagenic activity of HMBP in the dark, as tested on V79 Chinese hamster cells (V79/HGPRT system).

RNA-protein cross-links induced by sensitization with a pyrroloquinolinone derivative, a furocoumarin analogue.

The capacity of 2,6-dimethyl-9-methoxy-4H-pyrrolo [3,2,1-ij] quinolin-4-one (PQ), a furocoumarin analogue, of inhibiting protein synthesis in Ehrlich cells upon UVA irradiation was investigated. Using 8-methoxypsoralen (8-MOP) as a reference, we observed that in our short-term test the block of RNA synthesis do not affect protein synthesis, which is driven by pre-synthesised molecules of m-RNA; actually 8-MOP, studied at 100 microM concentration, practically abolished RNA synthesis without affecting significantly protein synthesis. Studying PQ sensitization in HL60 cells by alkaline elution and protein precipitation, the formation of covalent RNA-protein cross-links was observed. 8-MOP, assayed in severe experimental conditions, induced only moderate amounts of such lesion. On the basis of the data obtained in experiments carried out using various scavengers or exposing cells to UVA light in a nitrogen atmosphere, this damage appeared to be due to singlet oxygen formation, which is generated by PQ to a large extent. These results are consistent with the data obtained by H. Singh and J.A. Vadasz (Singlet oxygen: a major reactive species in the furocoumarin photosensitized inactivation of E.coli ribosomes, Photochem. Photobiol., 28 (1978) 539-545) on E.coli ribosomes. The lower activity we observed with 8-MOP might be attributed to a different sensitivity of whole mammalian cells in comparison with isolated ribosomes.

Synthesis and photobiological properties of acetylpsoralen derivatives.

In an investigation to find monofunctional reactants for DNA which can act as new agents in the photochemotherapy of psoriasis, we have synthesized and studied some methylpsoralen derivatives which contain an acetyl group at one of the two reactive sites of the furocoumarin skeleton (at the 3 or 5' positions). The compounds do not react easily with DNA; their photobiological properties (e.g. the lack of an ability to inhibit DNA synthesis in Ehrlich ascites tumour cells, to induce T2 phage sensitization and to induce erythema in guinea-pig skin) are exactly in line with this behaviour. Some interesting features are shown by 4,8-dimethyl-5'-acetylpsoralen: it is capable of producing a very large amount of singlet oxygen--an order of magnitude higher than the other compounds and 8-methoxypsoralen (used as reference). In spite of this property, 4,8-dimethyl-5'-acetylpsoralen is non-phototoxic to the skin, and its other photobiological properties appear to be in line with its lack of interaction with DNA rather than its enhanced singlet oxygen production.

4,6,4'-Trimethylangelicin induces interstrand cross-links in mammalian cell DNA.

4,6,4'-Trimethylangelicin, a well-known effective photosensitizer described as a pure monofunctional reactant with DNA, can induce interstrand cross-links in mammalian cell DNA in vivo (about 15% relative to 8-methoxypsoralen), as observed using alkaline elution and Chinese hamster ovary cells. Experiments performed using the two-step irradiation method and HeLa cells support these data. In contrast with 4,6,4'-trimethylangelicin, 4'-methylangelicin and 4,4'-dimethylangelicin do not form interstrand cross-links. These results are consistent with those recently reported by Chen et al. (X. Chen, J. Kagan, F. Dall'Acqua, D. Averbeck and E. Bisagni, J. Photochem. Photobiol. B: Biol, 22 (1994) 51-57) using pBR322 and M13 DNA. The cross-linking ability of 4,6,4'-trimethylangelicin does not seem to be related to a particular feature of these DNAs but to the compound itself.

2,6-Dimethyl-9-methoxy-4H-pyrrolo[3,2,1-ij]quinolin-4-one, a new compound with unusual photosensitizing properties.

Some photobiological properties of 2,6-dimethyl-9-methoxy-4H-pyrrolo[3,2,1-ij]quinolin-4-one (PQ) have been studied in comparison with 8-methoxypsoralen (8-MOP). In Ehrlich cells, PQ induced a moderate inhibition in DNA and RNA syntheses in the dark, which appeared to be more pronounced upon UVA irradiation. In contrast to 8-MOP, in the presence of UVA, PQ also affected protein synthesis. Likewise marked antiproliferative effects were also observed in the study of the clonal growth of CHO cells cultivated in vitro. Using alkaline elution and CHO cells, a moderate formation of single-strand breaks (SSBs) and of DNA-protein cross-links (DPCs) was observed by incubation in the dark; upon UVA irradiation the amount of both lesions increased greatly, whereas no inter-strand cross-links (ISCs) were formed. As expected, 8-MOP did not damage DNA in the dark, but induced SSBs, ISCs and DPCs in the presence of UVA. The induction of SSBs by both compounds seems to be directly related to a photochemical event rather than to incisions during DNA repair. As the induction of ISCs, and also the formation of DPCs by 8-MOP and UVA, appears to be based on a two-step reaction involving photo-bound 8-MOP-DNA moieties. In contrast, the formation of DPCs by PQ and UVA seems to involve photosensitization by free PQ molecules connected with SSB and DPC formation rather than with a DNA photo-binding activity. The PQ activity observed in the dark could probably be ascribed to a moderate inhibition of topoisomerases.