Relationship between lesions photoinduced by mono- and bi-functional furocoumarins in DNA and genotoxic effects in diploid yeast

The induction of genetic effects was studied in a diploid strain of Saccharomyces cerevisiae (D7) after treatments with the monofunctional furocoumarins 7-methylpyrido[3,4-c]psoralen (MePyPs), pyrido[3,4-c]psoralen (PyPs) and 3-carbethoxypsoralen (3-CPs) and the bifunctional furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) in the presence of 365-nm radiation. The DNA photobinding of radioactively labelled MePyPs, 3-CPs, 5-MOP and 8-MOP was determined in parallel. The DNA-photobinding capacity was highest for MePyPs followed in decreasing order by 5-MOP, 3-CPs and 8-MOP. At a concentration of 5 microM and 4.2 kJ/m2 of 365-nm radiation approximately 160, 66, 60 and 16 adducts per 10(6) base pairs were formed by MePyPs, 5-MOP, 3-CPs and 8-MOP, respectively. The activity of MePyPs and PyPs for the induction of lethal effects lay in the same range as that of 5-MOP whereas 8-MOP was 3 times less active and 3-CPs showed very little activity. For the induction of mitotic gene conversion and genetically altered colonies including mitotic crossing-over the order of activity was about the same as that observed for the induction of lethal effects: MePyPs greater than 5-MOP greater than PyPs greater than 8-MOP much greater than 3-CPs. Nuclear reversions were induced most effectively by 5-MOP, 8-MOP being about 3 times less effective. Up to 4 and 6 kJ/m2 of 365-nm radiation, MePyPs and PyPs, respectively, were less mutagenic than 8-MOP but became more mutagenic at higher doses. At equal survival, the pyridopsoralens were, however, clearly less mutagenic than the bifunctional furocoumarins 8-MOP and 5-MOP. By plotting the genetic data versus the number of lesions induced in DNA, it was shown that the monoadducts induced by the monofunctional furocoumarins MePyPs and 3-CPs exert a relatively low potential for the induction of lethal and nuclear genetic events as compared to photoadditions induced by the bifunctional furocoumarins 8-MOP and 5-MOP. However, at a very high density, the monoadducts induced by MePyPs became as lethal and as mutagenic as the mixture of mono- and biadducts induced by 8-MOP and 5-MOP probably due to overloading of cellular repair capacities.(ABSTRACT TRUNCATED AT 400 WORDS)     

Permeability of psoralen derivatives in lipid membranes

Molecular dynamics simulations have been performed to explore the distribution and translocation of a set of furocoumarins (psoralen derivatives) inside saturated and partially unsaturated lipid membranes. Within the simulations, strong accumulation of the photodynamic drugs is observed near the polar headgroup region, although the populations also extend out into the membrane/water interface as well as to the membrane center. The computed transverse (Dz) diffusion coefficients are in the range 0.01-0.03 x 10(-5) cm2 s(-1)-significantly slower than those reported for small molecules like water, ethane, and ammonia-and are related to the low mobility inside the polar headgroup region. Trimethylpsoralen (TMP) has a very low free energy barrier to transversion, only approximately 10 kJ/mol, whereas 5- and 8-methoxy psoralens (5-MOP, 8-MOP) have the largest barriers of the compounds studied-between 25 and 40 kJ/mol. Upper bounds to the permeation coefficients, obtained by integrating the resistance profiles across the bilayers, range from 5.2 x 10(-8) cm s(-1) for TMP to 4.1 x 10(-12) cm s(-1) for 5-MOP. The current simulations explain the high level of furocoumarin-lipid membrane complexes found in experimental studies of albino Wistar rats exposed to topical application of 8-MOP, and points to the possibility of membrane photodamage as a viable mechanism in psoralen ultraviolet-A treatment.     

Can a mixed damage interfere with DNA‐prottein cross‐links repair?

Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo , by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.     

Chromosome damage induced in human lymphocytes by 5-methoxypsoralen and 8-methoxypsoralen plus UV-A

The induction of structural chromosome aberrations and sister chromatid exchanges (SCE) was studied in human lymphocytes in vitro after treatment with the two bifunctional furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) in the presence of UV-A. The results show that both psoralens induce a dose-dependent increase in the SCE rate as well as in structural chromosome aberrations. 5-MOP was 2.0-2.5 times more effective for the induction of chromosome breaks and had a slightly stronger effect with respect to SCE induction. A significant influence on proliferation kinetics could be observed only with 5-MOP plus UV-A.     

Genotoxic effects and DNA photoadducts induced in Chinese hamster V79 cells by 5-methoxypsoralen and 8-methoxypsoralen

The induction of lethal effects and 6-thioguanine-resistant (6-TGr) mutants were studied in Chinese hamster V79 cells after treatment with the two bifunctional furocoumarins 5- and 8-methoxypsoralens (5-MOP, 8-MOP) in the presence of 365-nm radiation (UVA). The in vivo DNA-photobinding capacity of these two compounds was measured and in parallel the cross-linking capacities of 5-MOP and 8-MOP were determined using the alkaline elution technique. The results show that 5-MOP plus UVA was about 2.5 times more effective than 8-MOP plus UVA for inhibiting cell survival and for inducing the same frequency of 6-TGr mutants (10(-4]. The total number of photoinduced lesions by 5-MOP plus UVA was about 6 times higher than that induced by 8-MOP plus UVA. However, the cross-linking capacities of 5-MOP and 8-MOP were found to be within the same range at equal doses of UVA. At equal number of DNA photoadducts produced, the lesions induced by 5-MOP appeared to be less genetically active than those induced by 8-MOP. The apparently weaker genotoxicity of 5-MOP-induced lesions is likely to be due to the induction of a lower proportion of cross-links by 5-MOP at a given number of photoadducts.     

4-Hydroxymethyl- and 4-methoxymethylfuro[2,3-h]quinolin-2(1H)-ones: synthesis and biological properties

4-Hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ) was prepared by a new profitable way, which allowed to synthesize also 4-methoxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (MOFQ), and 4-hydroxymethyl-6,8-dimethylfuro[2,3-h]quinolin-2(1H)-one (HOHFQ). Some biological activities of the three compounds were studied in comparison with 8-MOP. In the dark, they inhibited topoisomerase II, leading to a moderate antiproliferative activity in mammalian cells. The antiproliferative activity was also tested upon UVA irradiation in mammalian cells: all compounds showed higher activity than 8-MOP, without mutagenicity and skin phototoxicity, with the best results for HOFQ. Photobinding to DNA was investigated, demonstrating a different sequence specificity for these furoquinolinones in comparison with furocoumarins. For all these features, HOFQ and the other analogues appeared very promising photochemotherapeutic agents, whose mechanism of action will be further investigated.     

Two DNA endonuclease activities from normal human and xeroderma pigmentosum chromatin active on psoralen plus ultraviolet light treated DNA

DNA endonuclease activities from the chromatin of normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells were examined on DNA treated with 8-methoxypsoralen (8-MOP) or 4,5',8-trimethylpsoralen (TMP) plus long wavelength ultraviolet (UVA) light, which produce monoadducts and DNA interstrand cross-links, and angelicin plus UVA light, which produces mainly monoadducts. 9 chromatin-associated DNA endonuclease activities were isolated from normal and XPA cells and assayed for activity on PM2 bacteriophage DNA that had been treated with 8-MOP or TMP in the dark and then exposed to UVA light. Unbound psoralen was removed by dialysis and a second dose of UVA light was given. Cross-linking of DNA molecules was confirmed by alkaline gel electrophoresis. In both normal and XPA cells, two DNA endonuclease activities were found which were active on 8-MOP and TMP plus UVA light treated DNA. One of these endonuclease activities, pI 4.6, is also active on intercalated DNA and a second one, pI 7.6, is also active on UVC (254 nm) light irradiated DNA. The major activity against angelicin plus UVA light treated DNA in both normal and XPA cells was found in the fraction, pI 7.6. The levels of activity of both of these fractions on all 3 psoralen-damaged DNAs were similar between normal and XPA cells. These results indicate that in both normal and XPA cells there are at least two different DNA endonucleases which act on both 8-MOP and TMP plus UVA light treated DNA.     

8—MOP对体外培养人癌细胞的光敏灭活作用

本文根据MTT只能被活的增殖细胞中线粒体切断形成紫色甲(?)的原理,测定了8—甲氧基补骨脂素(8—MOP)对体外培养人癌细胞系HCT、KB和BEL细胞的光敏灭活作用。结果表明,8—MOP和UVA光照对这几种人癌细胞有肯定的灭活作用,该作用与8—MOP剂量和光照时间以及细胞种类有关;MTT法可以作为光敏剂活性检测的一种快捷方法。     

Synthesis, photobiological activity and photoreactivity of methyl-thieno-8-azacoumarins, novel bioisosters of psoralen

4,4'-dimethyl-thieno-8-azacoumarin (6) and 4,4',5'-trimethyl-thieno-8-azacoumarin (8) were synthesised. Their photobiological activity was tested on human tumour cell lines. Interestingly, for 6, a photocytotoxic ability higher in HL-60, comparable in HeLa cells, with respect to that of the well-known drug 8-methoxypsoralen (8-MOP), was demonstrated. The covalent photoaddition to DNA occurs by means of the molecular mechanism already demonstrated for furocoumarins. However, it is noteworthy that no skin phototoxicity appears.     

Genetic effect of 3-carbethoxypsoralen, angelicin, psoralen and 8-methoxypsoralen plus 365-nm irradiation in Saccharomyces cerevisiae: induction of reversions, mitotic crossing-over, gene conversion and cytoplasmic "petite" mutations.

The genetic effects of two mono-functional photosensitizing furocoumarins, 3-carbethoxypsoralen (3-CPs) and angelicin, were compared with those of two bi-functional furocoumarins, 8-methoxypsoralen and psoralen in Saccharomyces cerevisiae. A drug concentration of 5 X 10(-5) M plus various doses of 365-nm irradiation at a dose rate of 1.2 kJ m-2 min-1 were used. Per dose of 365-nm irradiation, the frequency of induced nuclear events such as gene mutation and mitotic recombination (conversion and crossing-over) is higher for the bi-functional than for the mono-functional compounds. The higher efficiency of the bi-functional furocoumarins is also evident when the frequency of mutants is expressed as a function of survival. However, the photo-addition of the 4 furocoumarins studied leads to the same response for the induction of recombinational events per viable cell. Amongst genetically altered colonies induced in the diploid strains D5 and D7, the colonies corresponding to the induction of crossing-over are effectively produced by bi-functional furocoumarins, but are rare (D7) or even absent (D5) after treatment with monofunctional furocoumarins. This suggests a certain specificity of genetic alterations produced by the bi-functional agents. 3-CPs is the most effective inducer on the cytoplasmic "petite" mutation in stationary phase cells per unit irradiation dose or per viable cell.     

The repair of psoralen monoadducts by the Escherichia coli UvrABC endonuclease.

We have examined the interactions of UvrABC endonuclease with DNA containing the monoadducts of 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (TMP). The UvrA and UvrB proteins were found to form a stable complex on DNA that contains the psoralen monoadducts. Subsequent binding of UvrC protein to this complex activates the UvrABC endonuclease activity. As in the case of incision at pyrimidine dimers, a stable protein-DNA complex was observed after the incision events. For both 8-MOP and TMP, the UvrABC endonuclease incised the monoadduct-containing strand of DNA on the two sides of the monoadduct with 12 bases included between the two cuts. One incision was at the 8th phosphodiester bond on the 5' side of the modified base. The other incision was at the 5th phosphodiester bond 3' to the modified base. The UvrABC endonuclease incision data revealed that the reactivity of psoralens is 5'TpA greater than 5'ApT greater than 5'TpG.     

Oxidative DNA damage photo-induced by 3-carbethoxypsoralen and other furocoumarins. Mechanisms of photo-oxidation and recognition by repair enzymes

DNA photosensitization by several furocoumarins (including 3-carbethoxypsoralen (3-CPs), 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and angelicin was investigated by using DNA sequencing methodology. 3-CPs induces photo-oxidation of guanine residues leading to alkali-labile sites in DNA (revealed by hot piperidine), whereas 8-MOP, 5-MOP and angelicin do not. There is a preferential photo-oxidation of G when located on the 5' side of GG doublets, likely to reflect a better accessibility of the G moiety in such a context. Mechanisms operating via both radicals (type I) and singlet oxygen (type II) are involved in the photo-oxidation of G residues by 3-CPs. Photo-oxidized G residues are produced independently of the formation of photoadducts, and scavengers of singlet oxygen or radicals do not inhibit photobinding of 3-CPs to DNA. This leads us to propose that covalent photoadducts arise from the intercalated excited sensitizer molecules, whereas G photo-oxidations are produced either by electron transfer reactions involving bound 3-CPs or by energy transfer to molecular oxygen, thereby producing singlet oxygen that subsequently reacts with guanine bases. Quantification of both types of DNA lesions indicated that in vitro photo-oxidized G residues are produced in DNA by 3-CPs plus ultraviolet light at least to the same extent as photoadducts, under our conditions. A calf thymus redoxyendonuclease, equivalent to the endonuclease III of Escherichia coli, specific for oxidative DNA damages, recognizes and cleaves DNA at sites of photo-oxidized G residues. The extent of the cleavage by this enzyme was close to that observed by hot piperidine and followed the amount of photo-oxidized G residues produced when the lifetime of excited oxygen species is modified. The redoxyendonuclease did not incise DNA treated with 8-MOP, 5-MOP or angelicin plus ultraviolet light. The exonuclease III and endonuclease IV of E. coli also involved in the repair of oxidative DNA damage, convert the replicative form I of 3-CPs-treated DNA to replicative form II. This suggests that the lesions recognized by these enzymes are apurinic-like lesions. In view of the low toxicity and mutagenicity of 3-CPs, DNA photo-oxidation products induced by the photodynamic effect of 3-CPs are likely to be efficiently taken care of by the DNA repair system(s). It is clear that 3-CPs photo-induces several classes of DNA damage, including oxidative damage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Investigation of the mutagenic activity in Salmonella typhimurium of the furochromone khellin, proposed as a therapeutic agent for skin diseases.

The photomutagenicity of the furochromone khellin was tested in Ames Salmonella strains using 8-methoxypsoralen (8-MOP) and 4,5', 8-trimethylpsoralen (TMP) as positive controls. When khellin was assayed with strain TA1537, mutation induction was not detectable; in the same strain, an equitoxic dose (52-56% level of survival) of TMP (used at a concentration 12-fold lower than khellin and with a UVA dose 83-fold lower than that used with khellin) yielded an increase in revertants/plate 3-fold above the spontaneous background. In strain TA102, khellin plus UVA treatment yielded a 2-fold increase in revertants/plate above the spontaneous background (79% survival). 8-MOP, however, used at a concentration 8-fold lower than khellin with a UVA dose 13-fold lower than khellin, yielded an increase in revertants/plate about 14-fold above background (66% survival) in the same strain. These data show that khellin has a weak photomutagenic potential and, along with the previously reported low photogenotoxic potential in eukaryotic cell systems, support the notion that khellin may be safer than bifunctional psoralens for clinical use.     

On the mechanism of adaptive response in human cells

There was investigated one of the mechanisms of adaptive response, related to chromosome aberrations induced by gamma-rays, in lymphocytes of healthy donors and donors with hereditary diseases (Marfan's syndrome and homocystinurea) whose cells are repair-deficient. 3H-thymidine treatment was used as an adaptive dose in G1-period of cell cycle and 8-methoxypsoralen (8-MOP), activated with UV-light, was used as a challenge agents. Cells of healthy donors and cells of patients with Marfan's syndrome had normal adaptive response in relation to gamma-irradiation and photomutagenic action of 8-MOP. There was no induction of adaptive response in realation to gamma-irradiation and 8-MOP photomutagenic action in cells of patients with homocystinurea. The cells from donors characterised with normal repair system and lack of adaptive response 8-MOP photomutagenic action wasn't modified by 3H-thymidine. We have found parallelism of adaptive response protective effect against chromosome aberrations, induced by UV activated 8-MOP and gamma-rays in repair proficient cells of healthy donors and repair deficient cells of patients with Marfan's syndrome. These data lead us to conclusion that mechanism of adaption, at least in some cases has no connection with repair process modification.     

Frameshift mutagenicity in Salmonella typhimurium of furocoumarins in the dark

The dark mutagenicity of 4,5',8-trimethylpsoralen (4,5',8-TMP), 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 3-carbethoxypsoralen (3-CPs) and two new pyridopsoralens (PyPs and MePyPs) was tested using the Ames Salmonella plating assay in the absence of metabolic activation. 4,5',8-TMP, 8-MOP and the two pyridopsoralens were found to be weak frameshift mutagens in strain TA1537 whereas 5-MOP and 3-CPs did not demonstrate any significant mutagenic activity. These findings support the notion that the genetic risks of these psoralens in the dark may be considered to be negligible.     

Determination of 8-methoxypsoralen in human plasma, and microdialysates using liquid chromatography-tandem mass spectrometry

The validation of a LC/MS/MS method for the determination of 8-methoxypsoralen (8-MOP) in human plasma and microdialysates after topical application is described. Plasma samples were extracted by liquid-liquid extraction with diisopropylether using 4,5',8-trimethylpsoralen (TMP) as internal standard. Chromatographic separation of plasma sample extracts was carried out using a short narrow-bore Nucleosil C18 column (30 mm x 2.0 mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (80:20, v/v). For mass spectrometric analysis an API 3000 triple quadrupole mass spectrometer was employed. The mass transitions used were m/z 217.2-->174.0 for 8-MOP and m/z 229.1-->142.1 for TMP. Microdialysis samples diluted with an equal amount of acetonitrile did not require any extraction and were analyzed directly on a narrow-bore Nucleosil C18 column (70 mm x 2.0mm i.d.) with acetonitrile/(2 mM ammonium acetate buffer, 2 mM acetic acid) (50:50, v/v) with the mass transition m/z 217.2-->174.0. The assays were validated over the concentration ranges of 0.5-50 ng/ml for plasma samples and 0.25-50 ng/ml for microdialysates, respectively.     

Production of singlet oxygen and superoxide radicals by psoralens and their biological significance

We have investigated a series of linear and angular furocoumarins, capable of forming either the monofunctional adducts (single strand) or bifunctional adducts (interstrand cross-links) with DNA with a view to examine the relationship of their skin photosensitizing potency, their ability to produce singlet oxygen (1O2) or superoxide radicals (O-.2 or HO.2), and their carcinogenic activity. The significance of photochemical interactions of psoralens and DNA is well known in skin photosensitization and skin carcinogenesis. Our data suggest that both monofunctional and bifunctional psoralens produce 1O2 and O-.2, and these reactive forms of oxygen may contribute to the development of skin cancer and membrane-damaging effects of these furocoumarins.     

Effects of three alkoxypsoralens on voltage gated ion channels in Ranvier nodes.

The effects of the phototoxic K+- channel blockers 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP) on Ranvier nodes were compared to those of 5,8-diethoxypsoralen (5,8-EOP) by means of the Hodgkin-Huxley formalism. When these test substances were added individually to the bathing solution (8-MOP: 100 micromol/l; 5-MOP: 50 micromol/l; 5,8-EOP: 10 micromol/l) the following completely reversible effects were observed: 1. 8-MOP, caused a nearly potential-independent decrease of the sodium permeability, P'Na, by ca. 17%. 5-MOP and 5,8-EOP merely decreased the maximal value of P'Na, by ca. 12 and 8% respectively, whereas with weak depolarisations P'Na was unchanged. 2. In the tested potential range the potassium permeability, P'K, was caused to decrease by ca. 9% by 8-MOP, ca. 21% by 5-MOP and ca. 19% by 5,8-EOP. 3. The potassium currents acquired a phasic time course previously described for 8-MOP and 5-MOP. They reached a relative maximum and approached a lower steady-state value, kinfinity, with a time constant tauk at V = 120 mV of about 16 ms (8-MOP), 20 ms (5-MOP) and 94 ms (5,8-EOP). To obtain dose-response relations the drug-induced effects on peak P'K and on the steady state value, kinfinity, were measured. The corresponding apparent dissociation constants (in micromol/l) were 66.6 and 80.1 (for 8-MOP), 87.6 and 25.8 (for 5-MOP), and 13.5 and 6.5 (for 5,8-EOP). In view of the similarity of the actions of 5-MOP and 5,8-EOP as well as the fact that 5,8-EOP is not phototoxic, in future 5,8-EOP may well prove to be a particularly suitable K+-channel blocker for the symptomatic therapy of multiple sclerosis and other demyelinating diseases.     

Inhibitory Potency of 8-Methoxypsoralen on Cytochrome P450 2A6 (CYP2A6) Allelic Variants CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22: Differential Susceptibility Due to Different Sequence Locations of the Mutations

Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC₅₀ values. In general, a similar trend of change in the IC₅₀ and K_m values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6*16, differences in IC₅₀ values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6*16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.