Psoralen Derivatives Research Articles

Photoaddition of 8-methoxypsoralen to E. coli DNA polymerase I. Role of psoralen photoadducts in the photosensitized alterations of pol I enzymatic activities.

Abstract— We have previously demonstrated that 8‐methoxypsoralen (8‐MOP)‡ plus UVA is able to inactivate the three enzymatic activities of E. coli DNA polymerase I and that oxygen is required for these reactions (M. Granger et al., (1982) Photochem. Photobiol., 36, 175–180). We now show that UV‐A irradiation produces a covalent incorporation of the psoralen derivative into the enzyme either in the presence or in the absence of oxygen. The excited psoralen binds directly to the protein in an oxygen‐independent reaction; no complex was detected in the absence of irradiation. Fluorescence measurements reveal that at least two photoadducts are formed.The 8‐MOP‐photomodified enzyme is still fully active but further irradiation leads to an inhibition of the 5′→ 3′ polymerase activity whereas the 5′→ 3′ exonuclease activity is not affected. A major part of the inhibition reaction is shown to be oxygen‐dependent but singlet oxygen quenchers have no effect on the kinetics. This oxygen‐dependent reaction is attributed to a photosensitization, due to covalently bound 8‐MOP, of neighbouring amino acids through an intermediate reactive oxygen species which is not singlet oxygen. The oxygen‐independent reaction is attributed to a direct photosensitization through, for example, a radical mechanism.

252Cf-plasma desorption mass spectrometry of covalently bound nucleic acid adducts: Psoralen-nucleosides photoadducts

Psoralen-nucleosides photocycloadducts have been studied by 252Cf-Plasma Desorption Mass Spectrometry. The present study involves isolation, high performance liquid chromatography purification and mass spectrometry analysis of the two major monoaddition products formed in the photoreaction of the naturally occurring 8-methoxypsoralen with thymidine. Structural informations have also been obtained concerning photocycloadducts of a new linear psoralen derivative 3-carbethoxypsoralen with thymidine and cytidine. A photobinding of 3-carbethoxypsoralen with adenosine has also been studied. The mass spectra of the adducts have been obtained directly without any derivatization. In all cases they showed molecular and quasimolecular ions in the positive and/or negative mode. It is concluded that 252Cf-PDMS is a powerful and easy technique for studying xenobiotic-modified nucleic acids.

Induction in Saccharomyces cerevisiae of mitotic recombination by mono and bifunctional agents: Comparison of the pso 2-1 and rad52 repair deficient mutants to the wild-type

The induction in Saccharomyces cerevisiae of mitotic gene conversion and crossing-over by photoaddition of mono and bifunctional psoralen derivatives as well as by mono (HN1) and bifunctional (HN2) nitrogen mustards or 254 nm ultraviolet (UV) radiation was compared in wild type and in a mutant, pso2-1, initially selected for sensitivity to DNA cross-linking agents. The induction of the same recombinational events by photoaddition of one mono and one bifunctional derivative of psoralen was also examined in the rad52-1 diploid strain. The non-reciprocal (gene conversion) and the reciprocal (crossing-over) events follow the same patterns for each agent tested regarding the relative sensitivities of the wild type and repair-deficient strains. The pso2-1 strain retains the same or even a greater ability to perform recombination than the wild type following exposure to monofunctional agents, as well as to HN2 and UV. In contrast, this mutant is not able to perform mitotic recombination as efficiently as the wild type specifically after photoaddition of bifunctional furocoumarins. Such a difference is not simply due to the relative toxicity of the inducing agents. The rad52-1 mutant is blocked in induced recombination by the photoaddition of both the mono and bifunctional furocoumarins tested and the same was already established for UV and γ-rays. The rad52-1 and pso2-1 mutant strains have in common a high sensitivity in the G2 phase of the cell cycle and a loss of the ability to restitute high molecular weight DNA after the appearance of DNA double strand breaks related to the incision of inter-strand cross-links. This leads to a generalised block in recombination ability in rad52 whereas in the pso2-1 mutant the nature of the deficiency in recombination is much more specific.

The Reaction of Psoralens and Related Benzofurans with Singlet Oxygen An Account of Current Research

AbstractRecent work dealing with the role of singlet oxygen in the biological activity of the psoralens has been reviewed. The reaction of singlet oxygen with benzofurans gives products which result from initially formed dioxetanes. Evidence for dioxetane intermediates was obtained from experiments using diphenyl sulfide as a trapping agent. Psoralen derivatives which contain alkyl or aryl groups on the furan double bond react readily with singlet oxygen to give dioxetane cleavage‐type products. Psoralens which lack furan substituents but possess powerful electrondonating groups in the 5‐ and 8‐positions give rise to peroxidic intermediates which are stable only at low temperature.

Reactions of furocoumarins. II. Synthetic aminomethyl psoralens via chloromethylation or benzylic bromination

AbstractSeveral psoralen derivatives have been synthesized in order to evaluate their efficacy as photochemotherapeutic (PUVA) agents, including a variety of 4′‐substituted‐4,5′,8‐trimethypsoralen compounds (1d‐j). Improved synthesis of the very potent photosensitizers 8‐methylpsoralen (6a) and 4,8‐dimethylpsoralen (6b) are described and 6a has been shown to undergo formylation in the 4′‐position. Free radical bromination of 6a and 6b with NBS affords primarily 8‐bromomethyl derivatives (8a and d), which are readily converted into the 8‐aminomethyl derivatives (8c and f) by the Gabriel method. If the 4′‐position is blocked, electrophilic substitution apparently occurs primarily in the 5′‐position of the psoralen system. At least, chloromethylation of 4′‐methylpsoralen (9) affords mainly 5′‐chloromethyl derivatives (10a and d), which also lead to aminomethylpsoralens (10c and f).

Site-directed psoralen crosslinking of DNA.

A technique has been developed for placing site-specific crosslinks into DNA by using a psoralen derivative containing a sulfhydryl group. Plasmid pBR322 was nicked at the unique BamHI restriction site, and mercurated pyrimidines were introduced by nick-translation. Then the psoralen was specifically directed to this site in the dark through a Hg-S linkage prior to irradiation to form a crosslink. The location of interstrand cross-links was examined by electron microscopy of Pst I-cut molecules and found to be at or near the BamHI site.

Small nuclear RNA U2 is base-paired to heterogeneous nuclear RNA.

Eukaryotic cells contain a set of low molecular weight nuclear RNA's. One of the more abundant of these is termed U2 RNA. The possibility that U2 RNA is hydrogen-bonded to complementary sequences in other nuclear RNA's was investigated. Cultured human (HeLa) cells were treated with a psoralen derivative that cross-links RNA chains that are base-paired with one another. High molecular weight heterogeneous nuclear RNA was isolated under denaturing conditions, and the psoralen cross-links were reversed. Electrophoresis of the released RNA and hybridization with a human cloned U2 DNA probe revealed that U2 is hydrogen-bonded to complementary sequences in heterogeneous nuclear RNA in vivo. In contrast, U2 RNA is not base-paired with nucleolar RNA, which contains the precursors of ribosomal RNA. The results suggest that U2 RNA participates in messenger RNA processing in the nucleus.

Photopromotion of sister chromatid exchanges by psoralen derivatives.

Several derivatives of 8-methoxypsoralen and 4,5'8-trimethylpsoralen were compared with respect to their photopromotion of sister chromatid exchanges. Peripheral blood leukocytes from heparinized blood of human volunteers were cultivated for 24 h. Selected flasks received psoralens (6.5 X 10(-8) M) and/or were irradiated (UV-A, 365 nm, 0.9 J/cm2). Bromodeoxyuridine (20 micrograms/ml) was added and flasks were further incubated for 48 h when colchicine was added. Spreads of mitotic chromosomes were prepared, stained with Hoechst 33258 and Giemsa, and scored for the number of sister chromatid exchanges (SCEs) per 100 chromosomes. Results were: Control (16.5); 8-methoxypsoralen (23.9); 5'-methyl-8-methoxypsoralen (25.7); 4-methyl-8-methoxypsoralen (27.8); 4,5'-dimethyl-8-methoxypsoralen (31.9); 4'-methoxymethyl, 4,5'8-trimethylpsoralen (42.9); 4'aminomethyl, 4,5'8-trimethylpsoralen-hydrochloride (59.1); 4'hydroxymethyl,4,5'8-trimethylpsoralen (75). In general, the psoralens with strong affinity for DNA as determined by KD values promoted more SCEs than did those with lower affinity. Trimethylpsoralen and its derivatives promoted more SCEs than did 8-methoxypsoralen and its derivatives, which may be indicative of relative DNA cross-linking potential.

Mapping the in vivo arrangement of nucleosomes on simian virus 40 chromatin by the photoaddition of radioactive hydroxymethyltrimethylpsoralen.

Intracellular simian virus 40 (SV40) chromatin was photoreacted with a 3H-labeled psoralen derivative, hydroxymethyltrimethylpsoralen (HMT), at 48 h postinfection. Psoralen compounds have been shown to readily penetrate intact cells and, in the presence of long-wavelength UV light, form covalent adducts to DNA, preferentially at regions unprotected by nucleosomes. The average distribution pattern of [3H]HMT on the SV40 genome was determined by specific activity measurements of the DNA fragments generated by HindIII plus HpaII or by AtuI restriction enzyme digestion. At levels of 1 to 10 [3H]HMT photoadducts per SV40 molecule, the radiolabel was found to be distributed nonrandomly. Comparison of the labeling pattern in vivo with that of purified SV40 DNA labeled in vitro revealed one major difference. A region of approximately 400 base pairs, located between 0.65 and 0.73 on the physical map, was preferentially labeled under in vivo conditions. This finding strongly suggests that the highly accessible region near the origin of replication, previously observed on isolated SV40 "minichromosomes," exists on SV40 chromatin in vivo during a lytic infection.

Photochemotherapy: a new promising chemical derivative.

A new synthetic psoralen (5'aminomethyl-4,4',8-trimethylpsoralen) is presented which possesses potential superior photochemotherapeutic efficacy. Results of studies conducted on the guinea pig and hairless mouse in our laboratories reveal that following oral administration there is a more rapid onset of photosensitization, a more rapid loss of photosensitization indicating apparent chemical inactivation or excretion, and a lower systemic toxicity on a milligram to milligram basis when compared to conventional psoralen derivatives. These results indicate that the use of this new derivative will minimize potential side effects such as ocular complications and inadvertent additional photosensitization that might lead to premature aging and carcinogenesis due to the slow onset and prolonged photosensitization following administration that are characteristic of conventional psoralen derivatives.

New procedure using a psoralen derivative for analysis of nucleosome associated DNA sequences in chromatin of living cells.

Sites for restriction endonuclease cleavage in double helical DNA are blocked from cleavage when the photoaffinity drug trimethylpsoralen is photobound at or near the site. In general, Hind III sites are about 15 fold more sensitive to inactivation than the other restriction sites which were tested, although sensitivity of different Hind III sites seems to vary somewhat depending on base sequences adjacent to the site. Hind III sites can be inactivated in two ways; one which completely blocks action of the specific restriction endonuclease and one permitting the introduction of a swivel which relaxes DNA supercoiling without producing a double strand break. Nucleosomes and perhaps other protein-DNA complexes can protect the underlying DNA sequence from trimethylpsoralen photobinding and thus protect restriction sites from inactivation. This property can be exploited to determine if specific sites are accessible to the psoralon probe in vivo and thus to establish if specific nucleotide sequences are nucleosome associated. Using this procedure evidence is obtained that nucleosomes on SV40 DNA in living infected cells are either distributed randomly or at many discrete alternate sites that approach a random distribution.

Spectroscopic study of the dark interaction and of the photoreaction between a new monofunctional psoralen : 3-carbethoxy psoralen, and DNA

3-carbethoxypsoralen (3-CPs) is a new linear psoralen derivative. Its dark interaction and photoreaction with DNA has been studied and compared with that of a well known bifunctional psoralen : 8-methoxypsoralen (8-MOP). 3-CPs is able to form in the dark a non covalent complex with native DNA. After irradiation of this complex with UV-A light (365 nm) 3-CPs is able to link covalently to DNA. Heat denaturation and renaturation patterns of treated DNA clearly show that, in contrast to 8-MOP, 3-CPs does not form DNA interstrand cross links. Fluorescence studies show that the photobinding of 3-CPs gives rise to the formation of monoadducts involving the 4',5' double bond of this molecule.

Base-pairing interactions between small nuclear RNAs and nuclear RNA precursors as revealed by psoralen cross-linking in vivo

The psoralen derivative 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) reacts with base-paired regions of RNA and forms interstrand covalent cross-links. Since psoralens permeate living cells, they can be used to probe RNA-RNA interactions in vivo. We used AMT to investigate whether small nuclear RNAs are base-paired with high molecular weight nuclear RNA in the cell. Intact HeLa cells were treated with AMT, and high molecular weight RNA was isolated under denaturing conditions from nuclei or from subnuclear fractions. The presence of base-paired snRNAs in the high molecular weight nuclear RNA was examined by electrophoresis after photochemical reversal of the cross-links. We found snRNA U3 and 5.8S rRNA to be cross-linked to nucleolar RNA. In contrast, snRNA U1 was cross-linked to high molecular weight RNA in ribonucleo-protein particles containing hnRNA. The U1 base-paired to hnRNA was identified by its hybridization with a cloned U1 DNA sequence after reversal of the cross-links. These results demonstrate that U1 is base-paired with hnRNA in vivo, suggesting a role in mRNA processing.

Mutagenesis induced by mono- and bi-functional alkylating agents in yeast mutants sensitive to photo-addition of furocoumarins (pso).

The inactivation and the induction of forward and reverse mutations by a mono- and a bifunctional nitrogen mustard in 3 pso mutants of Saccharomyces cerevisiae, initially selected for their sensitivity to psoralen photo-addition, were compared with that of the wild-type. The pso1-1 mutant was very sensitive to both alkylating agents, and the mutagenicity was abolished. This correlates with the defect in the error-prone repair capacity for lesions induced by psoralen photo-addition and radiations already observed for this mutant. Therefore it appears that the PSO1+ gene product acts on a spectrum of DNA lesions. The pso2-1 mutant was highly sensitive to the lethal effect of the bifunctional nitrogen mustard and was only slightly sensitive to the monofunctional one. For both agents a reduction in induced mutagenesis was seen. The same was true for mono- and bifunctional psoralen derivatives. The pso2-1 mutant having the same sensitivity as the wild-type to UV and ionizing radiations, it is suggested that the PSO2+ gene product is predominantly necessary for the repair of cross-links irrespective of their molecular nature. In contrast with psoralen photo-induced inactivation the pso3-1 mutant had the same sensitivity as the wild-type to alkylating agents. However, a reduction in induced mutagenesis was seen in both cases. This response was modulated according to dose and type of mutation. Consequently, it appeared that the PSO3+ gene product acts specifically on psoralen photo-induced sub-lethal lesions and on a fraction of premutagenic lesions independently of their structure.

Interaction of psoralen derivatives with the RNA genome of Rous sarcoma virus

We have examined the interaction of several psoralen derivatives with the RNA genome of Rous sarcoma virus (RSV). In the presence of long-wavelength uv light, 4,5′,8-trimethylpsoralen (trioxsalen) and its 4′-hydroxymethyl (HMT) and 4′-aminomethyl (AMT) derivatives all efficiently crosslinked the subunits of the RSV 70 S RNA complex into a nondissociable state. These psoralen derivatives also were able to penetrate virions and crosslink the RNA complex in situ. The ability of psoralen to crosslink the subunits of viral RNA in situ is consistent with the presumption that the RSV genome exists as a complex within the virion and suggests an approach for a detailed examination of regions of secondary structure. The addition of psoralen adducts to viral RNA correlated with a loss of virus infectivity and a decrease in the amount and length of viral DNA synthesized in vitro and in vivo. Our results indicate that the RSV genome contains a significant amount of secondary structure that is reactive with psoralen compounds and that the addition of HMT to viral RNA causes premature chain termination during viral DNA synthesis.

Sister-chromatid exchange induction in Chinese hamster ovary cells by 8-methoxypsoralen and brief pulses of laser light assessment of the relative importance of 8-methoxypsoralen—DNA monoadducts and crosslinks

Brief (⩽20 nsec) 5–15 mJ pulses of near-ultraviolet laser light were employed to promote, in a controlled manner, the reaction between 8-methoxypsoralen and the DNA of Chinese hamster ovary (CHO) cells. According to Johnston et al. [21], a single brief laser pulse should induce only monoadducts between 8-methoxypsoralen and DNA, while multiple laser pulses are needed to induce 8-methoxypsoralen—DNA crosslinks. In the present study, a single laser pulse was found sufficient to cause a significant elevation in the sisterchromatid exchange (SCE) frequency in CHO cells exposed to 0.75–1.2× 10 −5 m 8-methoxypsoralen. Additional laser pulses caused less than proportional further increments in SCEs, although the total yield of 8-methoxy-psoralen adducts in similarly exposed solutions of isolated DNA increased linearly. Higher numbers of laser pulses induced detectable amounts of 8-methoxypsoralen-DNA crosslinks and distorted the DNA flow histogram of CHO cells in a manner consistent with retarded cell cycle traverse. It is concluded that 8-methoxypsoralen—DNA monoadducts are sufficient to induce SCEs in CHO cells, while 8-methoxypsoralen—DNA crosslinks might have relatively little overall effect on SCE frequencies. These results, together with previous data correlating SCE induction and mutagenesis [10] are discussed in the context of strategies for evaluating psoralen derivatives for their suitability in the phototherapy of skin disorders.

Psoralen Photochemistry and Nucleic Acid Structure

Many new psoralen derivatives have been synthesized in an effort to enhance their water solubility and their binding to nucleic acids. Availability of the very soluble strongly binding compounds has improved our abilities to follow the optical changes associated with the photochemistry of psoralens with DNA. Changes in both absorbance and fluorescence are presented in this review. A kinetic model for the photochemistry concludes that the detailed kinetics is dominated by the equilibrium constant for intercalation of the psoralen in the DNA, the quantum yield for photoaddition to DNA once intercalated and the quantum yield for photodestruction of the drug in water. With these 3 parameters the kinetics of photochemistry is predictable. The values of these parameters for numerous derivatives of 8-methoxypsoralen and 4,5',8 trimethylpsoralen are presented. Application of this photochemistry to a study of nucleic acid secondary structure in chromatin, fd bacteriophage, and in ribosomes is reviewed.

Laser Microirradiation of Chinese Hamster Cells at Wavelength 365 nm: Effects of Psoralen and Caffeine

Cells of a V79 subline of the Chinese hamster were microirradiated at wavelength 365 nm in the presence of the psoralen derivative, trioxsalen. Microirradiation was accomplished by a pulsed argon laser microbeam either in anaphase or in interphase 3 hr after mitosis. Inhibition of clonal growth and formation of micronuclei at the first postirradiation mitosis were observed after microirradiation of anaphase chromosomes and of small parts of the interphase nucleus. Microirradiation of the cytoplasm beside the interphase nucleus or between the sets of chromosomes moving apart from each other in anaphase did not produce these effects. Anaphase experiments showed that only the daughter cell which received microirradiated chromatin exhibited an abnormal growth pattern. Most interestingly, shattering of the whole chromosome complement could be induced by microirradiation of small parts of the interphase nucleus and post-treatment with caffeine. Since microirradiation of chromatin in the absence of psoralen was not effective, we consider formation of psoralen photoadducts to nucleic acids in microirradiated chromatin to be the specific cause of the effects. We suggest that DNA photolesions in chromosome segments present in the microirradiated part of the nucleus can induce shattering of all the chromosomes in the microirradiated nucleus. Several possibilities are discussed to explain this unexpected finding.

In situ photochemical crosslinking of HeLa cell mitochondrial DNA by a psoralen derivative reveals a protected region near the origin of replication.

The in vivo association with proteins of HeLa cell mitochondrial DNA (mtDNA) has been investigated by analyzing the pattern of in situ crosslinking of the DNA by 4'-hydroxymethyl-4, 5',8-trimethylpsoralen (HMT). Either isolated mitochondria or whole cells were irradiated with long wavelength UV light in the presence of ths psoralen derivative, and the mtDNA was then isolated and analyzed in the electron microscope under totally denaturing conditions. No evidence of nucleosomal structure was found. The great majority of the molecules (approximately 90%) had a double-stranded DNA appearance over most of their contour length, with one to several bubbles occupying the rest of the contour, while the remaining 10% of the molecules appeared to be double-stranded over their entire length. Analysis of restriction fragments indicated the presence, in approximately 80% of the molecules, of a protected segment (300 to 1500 bp long) in a region which was centered asymmetrically around the origin of replication so as to overlap extensively the D-loop. Control experiments showed that at most 30% of the bubbles found near the origin could represent D-loops or expanded D-loops: furthermore, it could be excluded that some sequence peculiarity would account for the preferential location of bubbles near the origin of replication. The data have been interpreted to indicate that, in at least 55% of HeLa cell mtDNA molecules, the region around the origin is protected from in situ psoralen crosslinking by proteins or protein complexes which are associated in vivo with the DNA.

Isolation and characterization of pso mutants sensitive to photo-addition of psoralen derivatives in Saccharomyces cerevisiae.

We have isolated mutants sensitive to photo-addition of bi-functional and mono-functional derivatives of psoralen in Saccharomyces cerevisiae. Three of these pso mutants were analyzed in detail. They segregate in meiosis like Mendelian genes and complement each other, as well as existing radiation-sensitive (rad and rev) mutants. The study of heterozygous diploid strains (PSO+/pso) indicates that the three pso genes are recessive. The mutant pso1--1 demonstrates a cross-sensitivity to UV and gamma-rays, whereas mutants pso2--1 and pso3--1 are specifically sensitive to photo-addition of psoralen derivatives. The comparison of exponentially growing cells to stationary-phase cells demonstrates that for the three mutants the defect in repair capacity of DNA cross-links and monoadducts concerns G1 and early S-phase cells. The pso2--1 mutant is, however, also defective in G2 repair and loses diploid resistance when it is in the homozygous state.--The block in repair capacity in these novel mutants is discussed in relation to the three other repair pathways known to be involved in the repair of furocoumarins photo-induced lesions in yeast DNA.