Photochemotherapy: identification of a metabolite of 4, 5', 8-trimethylpsoralen.

A compound, 4, 8-dimethyl, 5'-carboxypsoralen (DMeCP), has been identified in mouse urine as a major metabolite of the photoactive drug, 4, 5', 8-trimethylpsoralen (TMeP). This drug is widely used in the treatment of vitiligo and psoriasis. DMeCP is fluorescent, and nonphotosensitizing when tested on guinea pig skin. DMeCP also occurs in the urine of human patients receiving TMeP orally.

Effect of mimosine on DNA synthesis in mammalian cells.

We have designed a general protocol to assess the rate of replicon initiation in mammalian cells in the presence of inhibitors of DNA synthesis. It is based on cross-linking DNA in vivo with trioxsalen, which effectively blocks the movement of the replication forks along DNA, while having little effect on initiation of replication. We applied this protocol to study the effect of the plant amino acid mimosine on the rate of replicon initiation in exponentially growing murine erythroleukemia F4N cells. We found out that during the first 2 h after application of 25-400 microM mimosine, the initiation step was inhibited more efficiently than the overall DNA synthesis. In this respect, the effect of mimosine was similar to that of gamma-ray irradiation and differed from that of hydroxyurea and aphidicolin. The results suggest that in addition to inhibiting the elongation step of DNA synthesis, mimosine inhibits the initiation of DNA replication as well.

DNA interstrand cross-links induced by psoralen are not repaired in mammalian mitochondria.

Although it is generally known that mitochondria are defective in DNA damage processing, little is known about the DNA repair pathways and mechanisms that exist in these vital organelles. Certain lesions that are removed by base excision repair are efficiently removed in mitochondria, whereas some bulky lesions that are removed by nucleotide excision repair are not repaired in these organelles. There has been much interest in whether mitochondria possess activities for recombination repair, and some previous studies have reported such activities, whereas others have not. We have taken the approach of studying the formation and removal of interstrand cross-links (ICLs) in DNA. These lesions are thought to be repaired by a repair mechanism that involves nucleotide excision and recombinational repair. The formation and repair of DNA ICLs by 4'-hydroxymethyl-4,5',8-trimethylpsoralen was investigated in both the nuclear and mitochondrial genomes in hamster cells. Seven-fold-higher levels of ICLs were generated in mtDNA than in the dihydrofolate reductase gene, clearly indicating that the mitochondrial genome is a preferential target of 4'-hydroxymethyl-4,5',8-trimethylpsoralen damage. ICLs were removed efficiently from the dihydrofolate reductase gene, but no repair was observed in mtDNA. Our observations support previous work showing efficient gene-specific repair of these lesions in the nucleus but suggest that repair of this type of ICL does not exist in the mitochondria. The preferential damage of mtDNA and the absence of cross-link repair further suggests that mtDNA may be a biologically important target for psoralen.

Binding of 4'-aminomethyl 4,5',8-trimethyl psoralen to DNA, RNA and protein in HeLa cells and Drosophila cells.

In Drosophila cells and HeLa cells treated with 4'-aminomethyl trioxsalen and ultraviolet light, this compound binds covalently to DNA and RNA. The maximum number of molecules bound to 10(3) base pairs in DNA is 60 and in RNA it is 20. In nuclei treated likewise the number of molecules bound to 10(3) base pairs in DNA can be as high as 376. When cells are irradiated in the frozen state the number of 4'-aminomethyl trioxsalen molecules bound per 10(3) base pairs in DNA is about 40 and in RNA about 20. DNA molecules from cells or nuclei treated with 4'-aminomethyl trioxsalen and ultraviolet light are highly crosslinked and appear as loops interspersed by double stranded regions when analyzed in the electron microscope under denaturing conditions. The loop sizes are heterogeneous and the fraction of double stranded regions increases to almost complete double-strandedness at high degrees of reaction. No secondary structures could be found in ribosomal RNA from Drosophila cells or HeLa cells after treatment with 4'-aminomethyl trioxsalen and ultraviolet light. In cells treated with 4'-aminomethyl trioxsalen and ultraviolet light the RNAase activity is increased considerably suggesting a release of lysosomal enzymes. 4'-aminomethyl trioxsalen and its photodecomposition products bind strongly to cellular proteins.

Penetration and localization of furocoumarins in single living cells studied by microspectrofluorometry.

The microspectrofluorometric technique has been used to study the penetration and the localization of psoralen, 4,5',8-trimethylpsoralen and 4'-aminomethyltrioxsalen in single living L-cells. The concentration of the different compounds inside the cell reached a plateau in 2 min with psoralen and aminomethyltrioxsalen and in 20 min with trioxsalen. Washing of the cells with culture medium produced only a partial removal of the three furocoumarins, distributed apparently in equivalent amount in the nucleus and cytoplasm.

Hyperreactivity of B-Z junctions to 4,5',8-trimethylpsoralen photobinding assayed by an exonuclease III/photoreversal mapping procedure.

We have developed an exonuclease III/photoreversal procedure to map, with base-pair resolution, the bases that have photoreacted with 4,5',8-trimethylpsoralen (Me3-psoralen) forming either monoadducts or interstrand crosslinks in DNA. This assay allows quantification of relative rates of Me3-psoralen photobinding to bases in DNA at levels less than one crosslink per 8000 base-pairs. We demonstrate the applicability of the Me3-psoralen mapping procedure on the Z-forming sequence GAATT(CG)6-TA(CG)6AATTC. The results confirm our previous findings that Me3-psoralen forms crosslinks in the 5'TA within the (CG)6TA(CG)6 sequence when it exists in the B conformation but not when it exists in the Z conformation. In addition, with increasing superhelical density we observe at least a hundred-fold increased Me3-psoralen presumably represent B-Z junctions. The two presumed junctions respond differently with increasing negative superhelical tension, however, suggesting that the structures of these negative superhelical tension, however, suggesting that the structures of these junctions differ. This increased Me3-psoralen photoreactivity provides a positive signal for the presence of Z-DNA. The sequence and assay described here provide a "torsionally tuned probe" for determining the effective superhelical density of DNA in vivo.

The crystal structures of psoralen cross-linked DNAs: drug-dependent formation of Holliday junctions.

The single-crystal structures are presented for two DNA sequences with the thymine bases covalently cross-linked across the complementary strands by 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT). The HMT-adduct of d(CCGCTAGCGG) forms a psoralen-induced Holliday junction, showing for the first time the effect of this important class of chemotheraputics on the structure of the recombination intermediate. In contrast, HMT-d(CCGGTACCGG) forms a sequence-dependent junction. In both structures, the DNA duplex is highly distorted at the thymine base linked to the six-member pyrone ring of the drug. The psoralen cross-link defines the intramolecular interactions of the drug-induced junction, while the sequence-dependent structure is nearly identical to the native Holliday junction of d(CCGGTACCGG) alone. The two structures contrast the effects of drug- and sequence-dependent interactions on the structure of a Holliday junction, suggesting a role for psoralen in the mechanism to initiate repair of psoralen-lesions in mammalian DNA.

A study of the relationship between photosensitizing and therapeutic activity of 4,5',8-trimethylpsoralen, and its major metabolite 4,8-dimethyl, 5'-carboxypsoralen.

The molecular basis for the clinically observed differences in the skin photosensitizing activity and therapeutic effectiveness of the topically applied and orally administered drug trimethylpsoralen (TMP) was investigated. TMP, when tested topically, is a very potent photosensitizing and therapeutically effective furocoumarin in the treatment of psoriasis. When administered orally, however, it is significantly less photosensitizing and therapeutically a less effective drug than the commonly used furocoumarin 8-methoxypsoralen. This decreased reactivity of oral TMP is attributable to its poor solubility and rapid in vivo metabolic transformation to several inactive (nonphotosensitizing) metabolites, one of which is referred to as 4,8-dimethyl,5'-carboxypsoralen (DMeCP). The supporting evidence has been obtained by: (a) isolation of the urinary metabolite DMeCP and subsequent comparison of its properties with the synthetically prepared DMeCP and its methyl ester; (b) examining the dark and photochemical interactions of TMP, DMeCP, and DMeCP methyl ester with DNA and determining their ability to form interstrand cross-links with DNA; and (c) studying the inhibition of DNA and RNA synthesis in Ehrlich ascites tumor cells and the killing of bacteria and T2 bacteriophages. The structure-activity relationship of TMP and DMeCP also has been examined in normal human subjects and in patients with psoriasis. The order of topical therapeutic effectiveness in terms of ability to clear psoriasis plaques appeared to be: TMP greater than 8-MOP greater than DMeCP methyl ester greater than DMeCP. The data also suggest the methyl ester of DMeCP to be an interesting nonphotosensitizing furocoumarin that photoconjugates to DNA better than 8-MOP and is therapeutically effective in psoriasis.

Psoralen photofootprinting of protein-binding sites on DNA.

Using a BAL31 exonuclease assay to determine the sites of 4,5',8-trimethylpsoralen photocrosslinking in DNA we have shown that 5'-TA sites which are accessible to psoralen DNA interstrand photocrosslinking in naked DNA become inaccessible when protein, in casu, lambda-repressor E. coli or RNA polymerase are bound at their recognition DNA sequences (OR1 operator or deo1 promoter, respectively). These results show that psoralens can be used as photofootprinting reagents to study specific protein-DNA interactions.

Skin concentration of 8-methoxypsoralen, 5-methoxypsoralen and 4,5,8-trimethylpsoralen in guinea pigs.

The skin concentrations of 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and 4,5,8-trimethylpsoralen (TMP) were studied in the albino guinea pig following oral administration and intraperitoneal injection. The skin concentration of phototoxic drugs after oral administration peaked at 1.5 h, and the concentration of 8-MOP was 3.5 times greater than that of 5-MOP. The skin concentration of TMP was not detected in our study (limit of sensitivity 5 ng/ml). The highest skin concentrations of 8-MOP and 5-MOP after intraperitoneal injection were achieved for both drugs at 0.5 h, with the level of 8-MOP approximately 1.3 times higher than that of 5-MOP. The level of TMP could not be measured even in the case of intraperitoneal injection.

8-Methoxypsoralen DNA interstrand cross-linking of the ribosomal RNA genes in Tetrahymena thermophila. Distribution, repair and effect on rRNA synthesis.

The distribution and repair of 8-methoxypsoralen-DNA interstrand cross-links in the ribosomal RNA genes (rDNA) in Tetrahymena thermophila have been studied in vivo by Southern blot analysis. It is found that the cross-links at a density of < or = 1/2 x 10(4) base pairs (bp) are distributed equally between three domains (terminal spacer, transcribed region and central spacer) as defined by restriction enzyme analysis (BamHI and ClaI). It is furthermore shown that a dosage resulting in approximately one cross-link per rDNA molecule (21 kbp, two genes) is sufficient to block RNA synthesis. Finally, it is shown that the cross-links in the rDNA molecules are repaired at equal rate in all three domains within 24 h and that RNA synthesis is partly restored during this repair period. The majority of the cells also go through one to two cell divisions in this period but do not survive.

Dark and photoreactivity of 4'-aminomethyl-4,5',8-trimethylpsoralen with T7 phage.

The dark and photoreactions of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with T7 phage were investigated from biological and structural points of view. The dark reaction leads to the structural destabilization of the double helix of the DNA as is shown by optical melting measurements. The genotoxicity of AMT in the dark is comparable with that of known genotoxic drugs as determined by phage inactivation. The photoreaction with UVA light leads to the formation of mono- and di-adducts depending on the wavelength and dose used. Mono- and di-adducts influence DNA stability differently; biologically both types of adducts are genotoxic as measured by action spectra.

Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation.

DNA interstrand cross-links are formed by chemotherapy drugs as well as by products of normal oxidative metabolism. Despite their importance, the pathways of cross-link metabolism are poorly understood. Laser confocal microscopy has become a powerful tool for studying the repair of DNA lesions that can be detected by immunofluorescent reagents. In order to apply this approach to cross-link repair, we have synthesized conjugates of 4,5',8-trimethylpsoralen (TMP) and easily detected compounds such as Lissamine rhodamine B sulfonyl chloride (LRB-SC), biotin, and digoxigenin. These conjugates are activated by UVA, and we have analyzed the intracellular localization of DNA damage and DNA reactivity by confocal and immunofluorescence microscopy. The LRB-SC-TMP conjugate 2 appeared mainly in the mitochondria, while the biotin-TMP conjugate 4 preferentially localized in the cytoplasm. Adducts formed by UVA and digoxigenin conjugates of TMP 7a and 4,5'-dimethylangelicin (DMA) 7b, which forms only monoadducts, were largely localized to the nucleus. Exposure of cells incubated with 7a and 7b to a 364 nm UV laser directed toward defined nuclear regions of interest resulted in localized adduct formation which could be visualized by immunofluorescence. Repair-proficient cells were able to remove the photoadducts, while repair-deficient cells were unable to repair the damage. The results indicated that the digoxigenin-TMP conjugate 7a and digoxigenin-DMA conjugate 7b can be used for studying the repair of laser localized DNA monoadducts and cross-links.

Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA.

Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation, and benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA), at these lesions. RPA, shown previously to bind tightly to these lesions, also binds in the presence of HMGB1, without displacing HMGB1. A discrete ternary complex is formed, containing HMGB1, RPA, and psoralen-damaged DNA. Thus, HMGB1 has the ability to recognize ICLs, can cooperate with RPA in doing so, and likely modulates their repair by the NER machinery. The abundance of HMGB1 suggests that it may play an important role in determining the sensitivity of cells to DNA damage under physiological, experimental, and therapeutic conditions.

Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells.

The rate of covalent photobinding of trimethylpsoralen to DNA is greater when the DNA is wound with negative superhelical tension than when it is relaxed. In vitro the rate of photobinding is directly proportional to the negative superhelical density of the DNA. Thus measurement of the rate of photobinding provides an assay for probing in vivo unrestrained tension in the winding of the DNA double helix. This approach has been applied to measure torsional tension in DNA as it is packaged in living E. coli. Drosophila and HeLa cells. A method is described for measuring the rate of photobinding to intracellular DNA and rRNA, and for using the latter measurement as an internal control of the rate of me3-psoralen photobinding in vivo. This permits more accurate and reproducible measurement of changes in the DNA-psoralen photobinding reaction. The me3-psoralen probe interacts with intracellular bacterial DNA as expected for a purified DNA duplex wound with superhelical density sigma = -0.05 +/- 0.01. This superhelical tension is relaxed in cells when multiple single-strand breaks are introduced into the chromosomal DNA by gamma-irradiation. Similar relaxation occurs when cells are treated with the DNA gyrase inhibitor coumermycin. The results suggest that the DNA double helix is wound with torsional tension in vivo and that DNA supercoils which are equilibrated with this tension are not completely restrained in nucleosome-like structures. Torsional tension in the DNA of eucaryotic cells is not detectable in analogous measurements of the packaged DNA of HeLa and Drosophila cells. The simplest interpretation of this finding is that, within the limits of detection, all superhelical turns in the DNA are restrained in nucleosomes or nucleosome-like structures in these eucaryotic cells.

Base-catalyzed reversal of a psoralen-DNA cross-link.

Base-catalyzed reversal of a psoralen-DNA cross-link has been observed under denaturing alkaline conditions at elevated temperatures. The cross-link was formed between 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen and the two thymidine residues (T) on opposite strands of the double-stranded DNA formed from the self-complementary oligonucleotide 5'-GGGTACCC-3'. In contrast to the photoreversal of the cross-link, which yields mostly the furan-side monoadducted oligonucleotide [Cimino, G. D., Shi, Y., & Hearst, J. E. (1986) Biochemistry 25, 3013-3020], base-catalyzed reversal of the cross-link yields only pyrone-side monoadducted oligonucleotides as identified on the basis of their mobilities on a 20% polyacrylamide-7 M urea gel and their chemical and photochemical properties. A mechanism has been proposed to explain the base-catalyzed reversal reaction. This observation suggests a way to make pyrone-side monoadducted DNA. It also suggests that caution must be taken when psoralen-adducted DNA is treated under denaturing alkaline conditions.

Concentration of trimethylpsoralen in blood and skin after oral administration.

Trimethylpsoralen (TMP) concentrations were determined in blood and skin of 21 patients given oral TMP therapy and were compared to data obtained from 5 patients treated with TMP baths. The quantitative determination was performed by gas chromatography with selected ion monitoring. Concentrations of up to 5.6 ng/ml were detected in whole blood from orally TMP treated patients, with about the same concentrations in patients given TMP baths. Whole skin biopsies from patients ingesting TMP showed concentrations in 14 patients that ranged from 30 to 1250 ng/g skin, median value 85 ng/g. Large interindividual variations were observed. Stripped skin from 5 patients after TMP baths showed a somewhat higher median value of 160 ng/g and their entire skin had fairly high concentrations, with a median value of 390 ng/g, probably mostly bound to stratum corneum. Different concentrations in different parts of the skin may explain the difference in phototoxic capacity when the drug is given locally.