Francesco Carlassare

4,5‘‐DIMETHYLANGELICIN: A NEW DNA‐PHOTOBINDING MONOFUNCTIONAL AGENT*

Abstract— 4,5′‐Dimethylangelicin is a new angular furocoumarin showing interesting photochemical and photosensitizing properties. In the dark it forms a complex with native DNA having higher values of the binding parameters than angelicin; by irradiation at 365 nm it is able to photobind with DNA several times faster than angelicin and in about the same degree as psoralen, without forming crosslinkages. It therefore behaves as a pure monofunctional reagent. The same high photobinding capacity with DNA is shown also in vivo in Ehrlich ascites tumor cells and bacterial E. coli cells.

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.

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.

6-Methylangelicins: new monofunctional photochemotherapeutic agents for psoriasis

The monofunctional furocoumarins, the 6‐methylangelicins, were tested for their antiproliferative activity with various animal models and for genotoxicity in micro‐organisms and in mammalian cells. The most active compound was 6,4,4′‐trimethylangelicin, which showed a high antiproliferative effect and reduced genotoxicity in comparison with 8‐methoxypsoralen (8‐MOP). Some of these compounds were also tested clinically by topical application on 17 patients with psoriasis. They appeared to be more active than 8‐MOP in clearing psoriasis without inducing skin phototoxicity. The methylangelicins also caused skin pigmentation.

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

Abstract 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.

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

Abstract 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.

4, 4′, 5′‐TRIMETHYL‐8‐AZAPSORALEN, A NEW‐PHOTOREACTIVE AND NON‐SKIN‐PHOTOTOXIC BIFUNCTIONAL BIOISOSTER OF PSORALEN

Abstract— 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.

5-Methylangelicin: a new highly photosensitizing angular furocoumarin.

5-Methylangelicin, a new highly photosensitizing angular furocoumarin, was studied in 2 different biological systems, the T2 phage and Ehrlich ascites tumor cells; in comparison with angelicin, the parent compound, it was several times more active.

Synthesis and biological properties of a new series of N-pyrido substituted tetrahydrocarbazoles.

A series of methyl and ethyl quaternary pyridiniumtetrahydrocarbazoles was synthesized and studied in comparison with ellipticine, chosen as a reference. In general, their antiproliferative activity, tested in different biological substrates, appeared to be higher than that of the corresponding non-quaternarized compounds. This fact could be attributed to the introduction of a positive charge in the molecule, which can stabilize the molecular complex they form with DNA. In a prokaryotic system, the T2 bacteriophage, both quaternarized and non-quaternarized compounds inhibited its infectivity moderately, in a similar way to ellipticine. This effect seemed to be connected to a direct activity on the virions rather than on the indicator bacteria. In mammalian cells, the pyridiniumtetrahydrocarbazoles were more effective. In particular, they appeared to be very active in inhibiting DNA synthesis in Ehrlich ascites cells; some of them were as effective as ellipticine. However, pyridiniumtetrahydrocarbazoles were less active in comparison with ellipticine when their capacity for inhibiting the clonal growth in Chinese hamster ovary (CHO) cells was tested. A similar picture was obtained studying the formation of chromosome aberrations and of sister chromatid exchanges in the same cells. These different responses can be explained considering that the data on DNA synthesis reflect effects only on DNA replication within a short time, without considering any later consequences; on the contrary, in the long-term tests, other events, which lead to cell killing or genotoxicity, can take place. Pyridiniumtetrahydrocarbazoles damage DNA, inducing double-strand breaks efficiently. These observations, together with the data already obtained on unsubstituted derivatives, suggest the pyridiniumtetrahydrocarbazoles induce antiproliferative and genotoxic effects, very probably by inhibiting topoisomerase II.

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.