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.     

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.     

Design, synthesis and photobiological properties of 3,4-cyclopentenepsoralens

The QSAR directed synthesis of tetracyclic psoralen derivatives (3-5) characterised by the condensation of a cyclopentane ring at the level of the 3,4 double bond of the tricyclic psoralen moiety is reported. The new compounds present a methoxy (3), a hydroxy (4) or a dimethylaminopropoxy (5) side chain inserted in position 8 of the lead chromophore. The evaluation of photoantiproliferative activity on human tumour cell lines reveals for 5 an ability to inhibit cell growth significantly higher with respect to that of the reference drug, 8-MOP. Interestingly, the enhancement in antiproliferative activity is accompanied by the disappearance of skin phototoxicity. On the other hand, no significant photobiological activity was scored for 3 and 4. The ability to photoreact with DNA, evaluated by isolating the 4',5' monoadduct and by estimating the ability to form interstrand cross-links, appeared to be significant for 5, practically negligible for 3 and 4. Furthermore, a back-projection of the more active compound identifies structural features suitable for further synthetic modifications.     

Synthesis and photocytotoxic activity of new alpha-methylene-gamma-butyrolactone-psoralen heterodimers

The synthesis of a new alpha-methylene-gamma-butyrolactone-psoralen heterodimer 2 is reported. Its photoantiproliferative activity and skin phototoxicity were compared with that of 5-methoxypsoralen (5-MOP) and another heterodimer 1. Both derivatives show a significant phototoxicity toward malignant cell lines including melanoma cells A375 compared to their intrinsic cytotoxicity in the dark. Both compounds were found to be nonphototoxic on mice skin and therefore could be active potential drugs in photochemotherapy.     

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.     

Lethal and mutagenic effects photoinduced in haploid yeast (Saccharomyces cerevisiae) by two new monofunctional pyridopsoralens compared to 3-carbethoxypsoralen and 8-methoxypsoralen

The photobiological effects of two monofunctional pyridopsoralens (PPs), pyrido[3,4-c]psoralen and pyrido[3,4-c]-7-methylpsoralen were studied and compared to those of 3-carbethoxypsoralen (3-CPs) and 8-methoxypsoralen (8-MOP) in a haploid wild-type strain of yeast (Saccharomyces cerevisiae). The capacity of PPs to photoinduce lethal effects in the presence of 365-nm radiation was not only higher than that of the monofunctional compound 3-CPs, but also higher than that of the bifunctional compound 8-MOP. This activity was apparently independent of oxygen, and it was found that it was probably due to the induction of monoadducts in DNA. A high effectiveness of PPs on the induction of cytoplasmic 'petite' mutations was observed suggesting a high photoaffinity towards mitochondrial DNA. In contrast to 8-MOP, the strong cell killing activity of PPs was not accompanied by a strong inducing effect on nuclear mutations (HIS+ reversions or canR forward mutations). For these endpoints, PPs were less effective per unit dose of 365-nm radiation and also less efficient per viable cell than 8-MOP. From this, it appears that the lesions photoinduced by the former compounds show a more lethal than (nuclear) mutagenic potential. Furthermore, the fact that PPs were even less mutagenic (nuclear) per viable cell than the monofunctional compound 3-CPs suggests that the activity of these agents may differ in frequency and nature of lesions induced. The photobiological activity of PPs in haploid yeast appears to be in line with the recent proposition for their use in photochemotherapy.     

Novel angular furo and thieno-quinolinones: synthesis and preliminary photobiological studies

A number of new furo and thienoquinolinones carrying an electron-withdrawing function or unsubstituted at the position 3 were synthesized in order to obtain new potential photochemotherapeutic agents with increased antiproliferative activity and decreased toxic side effects. Our interest in studying the SAR of these derivatives also prompted us to investigate the influence of N-methylation on biological activity, by preparing N-methyl derivatives. The antiproliferative activity of all the newly synthesized compounds was evaluated and compared to 8-methoxypsoralen (8-MOP), the drug widely used in PUVA-therapy. The 3-unsubstituted thienoquinolinones were generally the most potent derivatives, followed by the furo-analogues. In particular, the unsubstituted thieno[2,3-h]quinoline-2(1H)one showed the highest activity in T2 bacteriophage, HeLa cells and Ehrlich cells tests. All the compounds, assayed on Escherichia coli WP2 TM9, showed a similar mutagenic activity, very close to that of 8-MOP. Except for 2-oxo-1,2-dihydrothieno[2,3-h]quinoline-3-carboxylic acid, which appeared to be very effective, all compounds generated singlet oxygen to slightly larger amounts when compared to 8-MOP. The N-methyl analogues only induced moderate skin erythemas on albino guinea pigs, while all other derivatives appeared to be entirely inactive. On the basis of these results, the unsubstituted thieno[2,3h]quinoline 2(1H)one seems to be the most interesting potential drug for PUVA photochemotherapy and photopheresis.     

Psoralen activity and binding sites in melanotic and amelanotic human melanoma cells

The biological activity and specific binding sites of 8-methoxypsoralen (8-MOP) are assayed using two human melanoma cell lines, melanotic SK-Mel 28 and amelanotic C32TG. Long-term (72 hr) treatment with 8-MOP at a concentration of 10(-4)M results in an increase in melanogenesis and a decrease in proliferation, similar in both cell lines. Daily exposure of these cells to ultraviolet A (UVA) irradiation (1.28 mJ/cm(2)) does not enhance the response to the compound. Daily pulse application (30 min daily) of 8-MOP does not promote any response. However, in combination with UVA, 8-MOP pulse treatment becomes as effective as the long-term treatment. A decrease in cell proliferation in the constant presence of 8-MOP is not coupled with apoptosis, since no increase in the number of apoptotic nuclei was observed after the treatment. The flow cytometry indicates that 8-MOP arrests the cells at the G0/G1 phase, irrespective of the presence or absence of UVA light. In view of the lack of epidermal growth factor (EGF) receptors in both cell lines, it is not likely that such an arrest is associated with the down-regulation of EGF receptors by 8-MOP. It is noted that this compound elicits a biphasic cell response, since cell proliferation increases after the first 24-hr treatment, whereas it decreases in the subsequent 48 hr and thereafter. Competition binding assays using 3H-8-MOP disclosed: 1) the specific binding of the compound in both cell lines occurs in the presence or absence of UVA light, and 2) a higher binding rate at low concentrations of the compound is in SK-Mel 28 (72%) rather than C32TG (58%) cells. The competition assays in the presence of UVA suggest a possible occurrence of covalent bindings between psoralen and receptor, as DNA covalent binding accounted to only 3-5% of the total binding in both cell lines.     

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.     

Effects of 8-methoxypsoralen and ultraviolet light A on EGF receptor (HER-1) expression

Activation of psoralens by ultraviolet light irradiation at 308-400 nm (UVA) is used in the photochemical treatment of psoriasis. While the major effect of this activation is the formation of DNA adducts, it was recently demonstrated that psoralens can also bind to specific saturable high affinity cellular receptors, and that this is associated with inhibition of epidermal growth factor (EGF)-receptor binding. In view of these findings, we have examined whether 8-methoxy-psoralen (8-MOP) itself, or in combination with UVA, influences expression of the human EGF-receptor gene ("HER-1") in a human keratinocyte cell line. We have found that 8 MOP alone, and to a lesser extent UVA, induce a striking increase in cellular levels of HER-1 RNA. The combination of 8-MOP with UVA produces less induction of HER-1 RNA than that obtained with 8-MOP alone. We suggest, therefore, that this effect of 8-MOP is not due to DNA damage, but may reflect a separate effect of this compound on receptor-mediated signal transduction.     

DNA photobinding of 7-methylpyrido[3,4-c]psoralen and 8-methoxypsoralen. Effects on macromolecular synthesis, repair and survival in cultured human cells

The photobinding to DNA of tritiated 7-methylpyrido[3,4-c]psoralen (MPP), a recently synthesized monofunctional compound of therapeutical interest, and of 8-methoxypsoralen (8-MOP) was determined in cultured normal human fibroblasts. Employing compounds at 10(-6) M, MPP photobinds approximately 11 times more efficiently than 8-MOP: one molecule is fixed respectively per 7.5 X 10(4) or 8.1 X 10(5) base pairs/kJ . m-2 of 365-nm radiation (UVA). Removal of bound material from DNA is slow and limited in 48-72 h of post-treatment incubation to 30-40% of initial adducts formed by MPP and to 50-60% of those of 8-MOP. For equivalent photobinding MPP and 8-MOP induce similar inhibitions of DNA synthesis. However, the recovery of DNA synthesis during post-treatment incubation is lower after photoaddition of MPP than after that of 8-MOP. MPP also exerts a much higher lethal effect than 8-MOP: one lethal hit corresponds to about 4400 and to 19,900 adducts per cell respectively. Alkaline elution experiments confirmed the monofunctional nature of MPP and indicated that in MPP-damaged cells DNA breaks accumulate with time of post-treatment incubation. In contrast, after photoaddition of 3-carbethoxypsoralen (3-CPs), another monofunctional furocoumarin, or irradiation with 254-nm UV, DNA breaks are induced only transiently. In 8-MOP-treated cells, DNA cross-links appear to be partially repaired. In conclusion, MPP monoadducts turn out to constitute more cytotoxic lesions than 8-MOP mono- and bi-adducts.     

Photochemotherapy using pyridopsoralens

Aiming to decrease the acute side effects and genotoxic hazards of PUVA, pyrido (3,4-C) psoralen (PP) and 7-methyl pyrido (3,4-C) psoralen (MPP) were synthesized and studied. Their UVA maximum absorption lies at 325 and 330 nm, respectively. Their photostability is comparable to that of 8-MOP. They complex to DNA in the dark, and, in the presence of UVA, produce only monoadditions to DNA, as shown by fluorescence and DNA denaturation-renaturation studies. In diploid eukaryotic yeast they are more effective than 8-MOP for the induction of lethal effects and mitochondrial damage. Their mutagenic activity per unit dose of UVA is in the same range as that of 8-MOP. However, per viable cell they are clearly less mutagenic than 8-MOP. This difference is also observed for recombinogenic activity. No oxygen effect is observed. In mammalian cells the following ranges of effectiveness are found: inhibition of DNA synthesis in human fibroblasts: MPP greater than PP greater than 8-MOP; mutagenic activity in V79 Chinese hamster cells: MPP greater than PP greater than 8-MOP; cell transforming ability in C3H embryonic mouse cells: MPP greater than 8-MOP greater than PP as a function of UVA dose, and: 8-MOP greater than MPP greater than PP as a function of survival; induction of sister chromatic exchanges (SCE) per unit dose: MPP greater than PP greater than 8-MOP in the linear part of the induction curve, and : 8-MOP greater than PP greater than MPP at the maximum level of SCE obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

PUVA (8-methoxy-psoralen plus ultraviolet A) induces the formation of 8-hydroxy-2'-deoxyguanosine and DNA fragmentation in calf thymus DNA and human epidermoid carcinoma cells.

The objective of this study is to investigate if 8-methoxy-psoralen (8-MOP) plus ultraviolet A (UVA) radiation (PUVA) induces oxidative DNA damage. When calf thymus DNA was incubated with 8-MOP and irradiated with UVA (335-400 nm), the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was substantially increased by approximately 6-fold. Formation of 8-OHdG proportionally correlated with both UVA fluence and 8-MOP concentrations. Human epidermoid carcinoma cells were incubated with 10 microg 8-MOP per milliliter, followed by irradiation of 25 kJ/m2 UVA. The level of 8-OHdG increased by nearly 3-fold in PUVA-treated cells compared to 8-MOP and UVA controls. The formation of 8-OHdG correlated with DNA fragmentation as determined by spectrofluorometry. To investigate the reactive oxygen species (ROS) involved in PUVA-induced oxidative DNA damage, less or more specific ROS quenchers were added to DNA solution prior to PUVA treatment. The results showed that only sodium azide and genistein significantly quenched PUVA-induced 8-OHdG, whereas catalase, superoxide dismutase, and mannitol exhibited no effect. The quencher study with cultured cells indicated that N-acetyl-cysteine and genistein protected oxidative DNA damage as well as DNA fragmentation by PUVA treatment. Our studies show that PUVA treatment is able to induce the formation of 8-OHdG in purified DNA and cultured cells and suggest that singlet oxygen is the principle reactive oxygen species involved in oxidative DNA damage by PUVA treatment.     

Synthesis,cellular internalization and photodynamic activity of glucoconjugated derivatives of tri and tetra(meta-hydroxyphenyl)chlorins

Glucoconjugated tri and tetra(meta-hydroxyphenyl)chlorins have been synthesized in order to explore how glucoconjugation of the macrocycle affects the photoactivity of the molecule. Internalization processes, photosensitizing efficacy of TPC(m-O-GluOH)(3) and TPC(m-O-GluOH)(4), in HT29 human adenocarcinoma cells have been compared to those of tetra(meta-hydroxyphenyl) chlorin (m-THPC, Foscan). The tetra glucoconjugated chlorin, TPC(m-O-GluOH)(4), was found to be poorly internalized and weakly photoactive. In contrast, the asymmetric and more amphiphilic compound TPC(m-O-GluOH)(3), exhibited superior phototoxicity compared to m-THPC. Drug concentration, temperature and sodium azide effects indicated that TPC(m-O-GluOH)(3) internalization partly proceeds via an active receptor-mediated endocytosis mechanism. Cellular uptake appeared as a saturable process and remained 30% lower than for mTHPC. However, a maximum phototoxicity in HT29 cells (survival fraction of 2+/-0.6%) were observed for concentration as low as 2 microM. A 4-fold higher concentration of m-THPC was necessary to observe the same level of photoactivity. This higher phototoxicity has been correlated to a greater mitochondrial affinity. On the basis of these results, work is in progress to further evaluate the potential of glycosylated chlorins in photodynamic therapy (PDT).     

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)     

DNA crosslinking and cell survival in human lymphoid cells treated with 8-methoxypsoralen and long wavelength ultraviolet radiation

8-Methoxypsoralen (8-MOP) when irradiated with long wavelength ultra-violet radiation (UV-A) inhibits DNA synthesis in lymphocytes in vitro and in vivo. 8-MOP binds reversibly to DNA in the dark; when exposed to UV-A, covalent monoadducts and cross-links are formed with the DNA. The present study correlates the cytotoxic effects of 8-MOP plus UV-A with DNA crosslinking. E-B virus transformed human lymphoblastoid cells were suspended in a colorless salt solution containing 8-MOP and exposed to UV-A from fluorescent lamps filtered to remove radiation below 320 nm (22.5 J/m²-sec). Cells were then returned to complete medium and assayed for survival (by daily counts of viable cells and by cloning in microtiter wells) and for DNA crosslinking by alkaline elution. 8-MOP alone or UV-A alone resulted in minimal to no alterations in survivial or in DNA crosslinking. DNA crosslinking was found to be linearly dependent on 8-MOP concentration (in the range of 0.01–1.0 μg/ml) for 3 different UV-A doses (3000–15000 J/m²). The surviving fraction declined exponentially as a function of the relative number of DNA crosslinks. These results suggest that the cytotoxic effects of photoactivated 8-MOP in human lymphoblastoid cells may depend on DNA interstrand crosslinks.     

Repair of exogenous (plasmid) DNA damaged by photoaddition of 8-methoxypsoralen in the yeast Saccharomyces cerevisiae

The contribution of different repair pathways to the repair of 8-methoxypsoralen (8-MOP) plus UVA induced lesions on a centromeric plasmid (YCp50) was investigated in the yeast Saccharomyces cerevisiae using the lithium acetate transformation method. The pathways of excision-resynthesis (RAD1) and recombination (RAD52) were found to be involved in the repair of exogenous as well as of genomic DNA. Mutants in RAD6 and PSO2 genes showed the same transformation efficiency with 8-MOP plus UVA treated plasmid as wild-type cells suggesting that these latter pathways involved in mutagenesis are not operating on plasmid DNA although required for the repair of 8-MOP photoadducts induced in genomic DNA. These results indicate that DNA-repair gene products may be differently involved in the repair of exogenous and endogenous DNA depending on the repair system and the nature of the DNA damage considered.     

Analysis of mutagenic activity and ability to induce replication of polyoma DNA sequences by different model metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) requires metabolic activation to express its carcinogenic activity. This activation leads to the formation of methylating and pyridyloxobutylating agents. To determine the possible biological effects mediated by each of these metabolic pathways we have studied the activities of model compounds that are metabolized to either a methylating or pyridyloxobutylating species. Each model compound was evaluated for its mutagenic activity in both prokaryotic and eukaryotic cell systems. The model compounds were also tested for their ability to induce asynchronous replication of viral DNA sequences. We demonstrate here that both the methylating model compound acetoxymethylmethylnitrosamine (AMMN) and the pyridyloxobutylating model compound 4-(acetoxymethyl)-1-(3-pyridyl)-1-butanone (NNKOAc) were mutagenic in strains TA98, TA100, and TA1535 but not TA102. While NNKOAc appeared to be 10 times more potent than AMMN in Salmonella, AMMN was found to be a more potent mutagen in mammalian G12 cells. Both chemicals could induce asynchronous replication of polyoma DNA sequences in rat fibroblast cells carrying an integrated copy of the polyoma virus with AMMN appearing to be more active. Measurement of DNA adduct levels suggest that the damage produced by NNKOAc was at least as active as that produced by AMMN when viewed on a per adduct basis. The possible implications of the biological activities exhibited by methylating and pyridyloxobutylating model compounds to NNK induced carcinogenesis are discussed.     

Comparative bacterial mutagenicity studies with 8-methoxypsoralen and 4,5',8-trimethylpsoralen in the presence of near-ultraviolet light and in the dark

2 strains of S. typhimurium, TA98 and TA100, and 2 strains of E. coli, WP2(pKM101) and WP2uvrA-(pKM101) were used to study mutagenesis by 8-methoxypsoralen (8-MOP) and 4,5',8-trimethylpsoralen (4,5',8-TMP) in the dark and in the presence of near-ultraviolet (NUV) light both without metabolic activation and with rat-liver S9 at 3 levels (4, 10 and 30% in standard cofactors). The S9-independent base substitution mutagenic activity of 8-MOP plus NUV light was confirmed in WP2(pKM101), and a similar activity was seen for 4,5',8-TMP, although neither substance was active in TA100. The frameshift mutagenic activity of 8-MOP in the dark in TA98 was not confirmed despite histidine levels which would ensure DNA replication, but this may be due to the lower concentrations of 8-MOP achieved in the common solvent system adopted. Both 8-MOP and 4,5',8-TMP were mutagenic in WP2uvrA-(pKM101) after microsomal activation, and the responses were similar whether experiments were conducted in the dark or in NUV light. In view of the oral administration of 8-MOP to psoriasis patients, this finding may be of relevance in risk assessment, and tends to suggest that topical application of 4,5',8-TMP to psoriatic patients may present reduced risk of malignant disease.     

W-reactivation and W-mutagenesis in lambda and T7 bacteriophages: a comparative study of the action of ultraviolet radiation (254 nm) and of the photosensitizing agents, 8-methoxypsoralen and angelicin

Monoadducts and interstrand cross-links are formed in DNA after psoralen plus light treatment of bacteriophage lambda . Survival and clear plaque mutation frequency of lambda after photosensitization with 8-methoxypsoralen (8-MOP) are increased when the wild type host is slightly UV-irradiated (W-reactivation and W-mutagenesis). The recA13, lexA1 and uvrA6 mutations block W-reactivation and W-mutagenesis of lambda treated with 8-MOP plus light. Using the technique of "repeated irradiation" we showed that the mutagenic effect of 8-MOP plus light treatment on phage is due mainly to formation of cross-links in DNA. The mutagenic activity of monoadducts had been studied by using angular furocoumarin, angelicin which forms mainly monoadducts in DNA. Upon W-mutagenesis of phage lambda treated with angelicin plus light a high mutagenic effect is observed. The results indicate that the mutagenic activity of monoadducts is 15-20 fold slower as compared to that of cross-links. W-reactivation and W-mutagenesis of UV-irradiated (254 nm) bacteriophage lambda are also observed after 8-MOP plus light treatment of Escherichia coli uvrA and wild type hosts. It is possible that the difference in mutagenic activity of psoralen adducts could depend on the repair mechanism of adducts: cross-links repair in bacterial and lambda DNA is controlled by lexA gene (error-prone SOS-repair mechanism), while monoadducts can be efficiently repaired by error-free excision and recombination.