Effect of 2-deoxy-D-glucose on the liquid-holding reactivation of yeast cells photodamaged by psoralen.
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Coumarins↗
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Structural specificity in the lethal and mutagenic activity of furocoumarins in yeast cells.
Using monofunctional (Angelicin) and bifunctional furocoumarins (Psoralen and 8 Methoxypsoralen) plus 365 nm light it is shown that both damages, the induced monoadducts and/or crosslinks in DNA, provoke lethal and mutgenic effects in haploid and diploid cells of Saccharomyces cerevisiae. Bifunctional furocoumarins are about 20 times more effective in cell killing than Angelicin. Diploid cells are always more resistant than haploid cells. Dark repair (agar haolding) increases survival. This effect can be at least in part correlated to the release of bound material from DNA in dark repair conditions. Bifunctional psoralens (10 mug/ml) are at least 10-fold more effective in inducing nuclear gene black mutations (his- to HIS+) than Angelicin (10 mug/ml) plus 365 nm light or 254 nm ultraviolet light. In contrast cytoplasmic "petite" (delta-) mutations are about as frequently induced by Angelicin plus 365 nm light as by 254 nm UV light. Bifunctional furocoumarins are less effective. The frequency of cytoplasmic "petite" mutations per survivors decreases during dark repair conditions more efficiently after Angelicin than after Psoralen plus 365 nm light treatment.
Cell Survival↗
Inverse relationship between melanogenesis and endogenous hydroquinone.
Inhibition or stimulation of melanogenesis have been found to occur as a result of the alteration of hydroquinone levels in the body. Substances which stimulate melanogenesis are found to lower the level of hydroquinone in amphibia, and evidence for the relationship is also given by mammalian experiments.
Animals↗
Conversion of psoralen DNA monoadducts in E. coli to interstrand DNA cross links by near UV light (320-360 nm): inability of angelicin to form cross links, in vivo.
Both angelicin and psoralen monoadducts formed in vivo in E. coli by near UV light produce lethal and mutagenic effects. However psoralen monoadducts are converted to cross links by higher doses of UV; angelicin monoadducts are not. The relevance of these results to psoralen photosensitization is discussed.
Binding Sites↗
Quantitative determination of psoralen and angelicin from some medicinal herbs by high performance liquid chromatography.
A reversed-phase high performance liquid chromatographic method was developed to determine the contents of psoralen and angelicin from some medicinal herbs. The optimum eluent for chromatography was 20 v/v% acetonitrile in water on a Zorbax 300SB C18 column. The identification was carried out by comparing the retention time and mass spectra of the relevant peaks with their standards. The variation of the concentration of psoralen and angelicin was wide between different species. The seeds of Psoralea corylifolia showed the highest contents of psoralen (7.8 mg/g) and angelicin (2.3 mg/g) among the tested herbs.
Chromatography, High Pressure Liquid↗
Effects of psoralen on the structural integrity of cultured osteoblasts. Phase contrast, immunofluorescent, and electronmicroscopic evaluation.
The 72-h dark interaction of cultured osteoblasts with 0.5-1.0 microgram/ml 8-methoxypsoralen (8-MOP) resulted in the accumulation of cytoplasmic lipid droplets (steatosis) in target cells. These methanol-extractable lipid droplets and 8-MOP, however, had no microscopically detectable effect on the organization of alpha-actin and beta-tubulin-containing filaments. On the ultrastructural level, psoralen effects ranged from negligible to unambiguous structural alterations of target cells. The latter consisted of blebbing, segmental deletions of the nuclear envelope, and structural unraveling of nucleoli. Moreover, the dilatation of the rough endoplasmic reticulum, the decrease in the number of ribosomes, and the extensive vacuolation of cytoplasm constituted yet another hallmark of the 8-MOP effect in the absence of light. Whereas psoralen has induced a number of structural alterations in some, but not in all osteoblasts, more than 95% of the target cells have, nonetheless, remained viable. Taken together, our results suggest that the dark reaction of psoralen with osteoblasts results mainly in transient structural alterations of affected cells.
Animals↗
Mutant allele pso7-1, that sensitizes Saccharomyces cerevisiae to photoactivated psoralen, is allelic with COX11, encoding a protein indispensable for a functional cytochrome c oxidase.
The yeast gene PSO7 was cloned from a genomic library by complementation of the pso7-1 mutant's sensitivity phenotype to 4-nitroquinoline-1-oxide (4NQO). Sequence analysis revealed that PSO7 is allelic to the 1.1-kb ORF of the yeast gene COX11 which is located on chromosome XVI and encodes a protein of 28-kDa localized in the inner mitochondrial membrane. Allelism of PSO7/COX11 was verified by non-complementation of 4NQO-sensitivity in diploids homo- and hetero-allelic for the pso7-1 and cox11::TRP1 mutant alleles. Sensitivity to 4NQO was the same in exponentially growing cells of the pso7-1 mutant and the cox11::TRP1 disruptant. Allelism of COX11 and PSO7 indicates that the pso7 mutant's sensitivity to photoactivated 3-carbethoxypsoralen and to 4NQO is not caused by defective DNA repair, but rather is due to an altered metabolism of the pro-mutagen 4NQO in the absence of cytochrome oxidase (Cox) in pso7-1/cox11::TRP1 mutants/disruptants. Lack of Cox might also lead to a higher reactivity of the active oxygen species produced by photoactivated 3-carbethoxypsoralen. The metabolic state of the cells is important for their sensitivity phenotype since the largest enhancement of sensitivity to 4NQO between wild-type (WT) and the pso7 mutant occurs in exponentially growing cells, while cells in stationary phase or growing cells in phosphate buffer have the same 4NQO resistance, irrespective of their WT/mutant status. Strains containing the pso7-1 or cox11::TRP1 mutant allele were also sensitive to the oxidative stress-generating agents H(2)O(2) and paraquat. Mutant pso7-1, as well as disruptant cox11::TRP1, harboured mitochondria that in comparison to WT contained less than 5% and no detectable Cox activity, respectively.
Alleles↗
Therapy for mycosis fungoides.
Treatment of mycosis fungoides (MF) is indicated to reduce symptoms, improve clinical appearance, prevent secondary complications, and prevent progression of disease, all of which may have an impact on survival. Treatment of MF includes topical and systemic therapies, which can be administered alone or in combination. Psoralen and ultraviolet A radiation is effective in early-stage MF, inducing complete remissions in most patients. Psoralen and ultraviolet A radiation may also be combined with low doses of interferon (IFN)-alpha to treat stage I/II disease. However, early aggressive therapy with radiation and chemotherapy does not improve the prognosis. Local radiotherapy or total skin electron beam irradiation has been used with success to control advanced skin disease. Extracorporeal photopheresis may also be used successfully, but it is not generally available. Once the disease becomes refractory to topical therapy, IFN-alpha single-agent or combination chemotherapy may be administered, but the duration of response is often less than 1 year and ultimately all patients will relapse and become refractory. Among chemotherapeutic agents, pentostatin, gemcitabine, and liposomal doxorubicin seem to be particularly effective. Response rates after combined modality therapy with total skin electron beam irradiation and chemotherapy/IFN-alpha appear similar to response rates of chemotherapy alone. Therefore, there is a great need for the further development of novel emerging treatment modalities, such as retinoids (ie, bexarotene) and immunotherapeutic agents (ie, cytokines, tumor vaccines, and monoclonal antibodies), all of which appear to have significant therapeutic potential in patients with MF. Biologically based therapies may reduce the need for genotoxic therapies, such as cytostatics and radiotherapy.
Combined Modality Therapy↗
Multiple end labeling of oligonucleotides with terbium chelate-substituted psoralen for time-resolved fluorescence detection.
A new procedure for the photochemical functionalization and the subsequent nonradioactive labeling of synthetic oligonucleotides with psoralen derivatives was developed where a double-stranded poly(A-T) tail is attached to the 5'- or 3'-end of the oligonucleotide to be labeled. The double-stranded poly(A-T) tail is covalently crosslinked by psoralen molecules which carry reactive thiol or amino groups for the attachment of labels. A NH2-specific terbium chelate exhibiting long-lived fluorescence was attached to the functional groups of the intercalated psoralen molecules. Oligonucleotides substituted in this way hybridize readily and can be sensitively detected by time-resolved fluorescence measurements.
Base Sequence↗
Accurate measurement of psoralen-crosslinked DNA: direct biochemical measurements and indirect measurement by hybridization.
This paper evaluates methods to measure crosslinkage due to psoralen plus light in total DNA and in specific sequences. DNA exposed in cells or in vitro to a bifunctional psoralen and near ultraviolet light accumulates interstrand crosslinks. Crosslinkage is the DNA mass fraction that is attached in both strands to a crosslink. We show here biochemical methods to measure psoralen photocrosslinkage accurately in total DNA. We also describe methods to measure photocrosslinkage indirectly, in specific sequences, by nucleic acid hybridization. We show that a single 4,5',8-trimethylpsoralen (TMP) crosslink causes at least 50 kbp of alkali-denatured DNA contiguous in both strands with it to snap back into the duplex form when the denatured preparation is returned to neutral pH. This process was so efficient that the DNA was not nicked by the single-strand nuclease S1 at 100-fold excess after snapping back. Uncrosslinked DNA was digested to acid-soluble material by the enzyme. Crosslinkage therefore equals the fraction of S1-resistant nucleotide in this kind of experiment. We alkali-denatured DNA samples crosslinked to varying degrees by varying TMP concentration at constant light exposure. We then measured crosslinkage by ethidium bromide (EtBr) fluorometry at pH 11.8; by EtBr fluorometry at neutral pH of S1 digests of the DNA; and by the fraction of radioactivity remaining acid insoluble in S1-digests of DNA labeled uniformly with [3H]deoxythymidine. These assays measure distinct physical properties of crosslinked DNA. Numerical agreement is expected only when all three measurements are accurate. Under optimum conditions, the three methods yielded identical results over the range of measurement. Using alkaline EtBr fluorescence in crude cell lysates, we detected crosslinks at frequencies in the range of 1.6 X 10(-7) per base pair. These levels were compatible with cell survival, attesting to the sensitivity of the measurement system. Crosslinkage affected hybridization as well. One crosslink prevented all alkali-denatured DNA contiguous in both strands with it from hybridizing to complementary DNA either on solid supports or in solution. Strand-length effects on crosslinkage and on reassociation caused solution hybridization levels to exceed those predicted by simple theory. In a quantitative, dot-blotting assay hybridization was linear up to membrane saturation by denatured, uncrosslinked DNA of any strand length.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
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.
Coumarins↗
Biological effects and repair of damage photoinduced by a derivative of psoralen substituted at the 3,4 reaction site: photoreactivity of this compound and lethal effect in yeast.
A newly synthesized linear psoralen derivative, 3-carbethoxypsoralen is shown to bind to yeast nucleic acids after 365 nm light treatment. As compared to 8-methoxypsoralen, a well-known bifunctional furocoumarin, 3-carbethoxypsoralen exhibits a high photoaffinity for DNA in vivo. Both compounds bind and photoreact more efficiently in vivo than in vitro. In contrast to 8-methoxypsoralen, 3-carbethoxypsoralen does not form cross-links in yeast DNA as demonstrated by heat denaturation-reassociation studies at least in the range of doses used. Thus 3-carbethoxypsoralen reacts as a monofunctional compound. Wild-type cells of Saccharomyces cerevisiae are 6 times more resistant to 3-carbethoxypsoralen than to 8-methoxypsoralen plus 365 nm light treatment in terms of lethal effect. In comparison to angelicin, another monofunctional (but angular) furocoumarin, 3-carbethoxypsoralen is more photoreactive. When the photoaffinity for DNA of 8-methoxypsoralen and 3-carbethoxypsoralen are considered in relation to photoinduced cell killing, it is clear that monoadducts are very efficiently repaired in wild-type cells. In contrast to the additivity obtained with 8-methoxypsoralen, a synergistic interaction of the two different repair pathways blocked by the rad2 and the rad9 mutation is observed after 3-carbethoxypsoralen plus 365 nm light. Dark holding experiments show that the excision repair function which is present in wild-type and rad9-4 cells is important for dark recovery.
Coumarins↗
The reactivities of furocoumarin excited states with DNA in solution. A laser flash photolysis and fluorescence study.
The effect of DNA on both the fluorescence emission spectra and yields and lifetimes of the triplet stae of psoralen and 8-methoxypsoralen in aqueous solution has been determined. The changes in the fluorescence spectra are similar in nature for both of these furocoumarins and are attributed to binding of the drug to DNA. The yield of the 8-methoxypsoralen triplet state when bound to DNA was found to be similar, if not identical, to that measured in the absence of DNA. This contrasts sharply with data obtained for psoralen from which it is concluded that either the yield of bound psoralen triplet states is very low, if not zero, or that the lifetime of such species is less than 50 ns. The relevance of this data to the molecular basis of skin photosensitisation by furocoumarins is discussed.
Ficusin↗
8-Methoxypsoralen: its isolation and gas chromatographic determination from aqueous solutions and serum.
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Chromatography, Gas↗
Inactivation of human liver cytochrome P-450 by the drug methoxsalen and other psoralen derivatives.
The effects of psoralen derivatives on cytochrome P-450 have been studied in human liver microsomes. CO-binding cytochrome P-450 was decreased by 33% after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 microM methoxsalen (8-methoxypsoralen). No destruction of cytochrome P-450 was observed when either NADPH or methoxsalen was omitted. A similar (27%) decrease in CO-binding required a 100-times higher concentration of allylisopropylacetamide (2 mM). The activities of 7-ethoxycoumarin deethylase and benzo(a)pyrene hydroxylase were decreased by about 50% in the presence of 12.5 microM methoxsalen. At this low concentration, neither cimetidine nor SKF 525-A or piperonyl butoxide had any significant inhibitory effect. Monooxygenase activities were also decreased in the presence of 12.5 microM bergapten (5-methoxypsoralen) or 12.5 microM psoralen, but not with 12.5 microM trioxsalen (trimethylpsoralen). CO-binding cytochrome P-450 was not decreased after 10 min of incubation with 1.5 mM EDTA, an NADPH-regenerating system and 20 microM trioxsalen. We conclude that methoxsalen is an extremely potent suicide inhibitor of cytochrome P-450 in human liver microsomes. Bergapten and psoralen are also inhibitory whereas trioxsalen has little effects. In the latter derivative, a methyl group is attached on the furan ring and may hinder its metabolic activation and the inactivation of cytochrome P-450.
5-Methoxypsoralen↗
The action of a mammalian endonuclease on psoralen-bound DNA.
The sequential actions of two enzymes believed to be involved in DNA repair, namely a mammalian endonuclease and the bacterial DNA polymerase I on psoralen bound 32P-labeled DNA, was studied. When ultraviolet-irradiated DNA is exposed to the sequential action of the endonuclease, the formation of single-strand breaks prepares the DNA for the exonucleolytic excision of thymine dimers. The mammalian endonuclease purified from rat liver to electrophoretic homogeneity is inactive on normal DNA, DNA irradiated at 360 nm or DNA mixed with psoralen without irradiation. Incubation of psoralen-bound DNA labeled with 32P with the endonuclease releases the isotope in the acid soluble indicating that psoralen-bound DNA is susceptible to the endonucleolytic attack. Sedimentation of DNA on sucrose gradients indicates that there is no collapse of the DNA molecule after treatment with the endonuclease. Moreover, there is no release of the adduct in the acid soluble after treatment with DNA polymerase, indicating that the 5--3 min exonucleolytic activity of that enzyme is impaired by the remaining crosslinks. The crosslinks also inhibit the incorporation of [3H] dATP in presence of DNA polymerase I.
2-Acetylaminofluorene↗
Effect of psoralen-induced photodermatitis on tryptophan metabolism in rats.
Tryptophan metabolism 'via kynurenine' has been studied in rats before and after induction of experimental light-conditioned dermatitis with psoralen. Tryptophan load in animals during the acute phase of dermatitis (one day after induction) causes a markedly increased urinary excretion of total metabolites in comparison with that obtained before dermatitis. During this phase of the skin disease tryptophan pyrrolase activity is significantly increased and kynureninase activity significantly decreased in liver in respect to the control animals. Kynurenine aminotransferase activity shows no significant variations in both liver and kidneys. After 6 days of dermatitis, when the skin damage is in repair, both the excretory values of the urinary metabolites after L-tryptophan load and the enzymic activities are similar to those before dermatitis.
Animals↗
Psoralene and melanogenesis in relation to vitiligo.
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Animals↗