Lethal and mutagenic photodynamic effect of acridine orange on bacteriophage cd]

Both acridine-sensitized inactivation and mutagenesis in phage sd have been studied and compared with the effect of other mutagens. Inactivation curve was not stictly exponential, with a small shoulder at short light doses and deviation of survival lower than 10(-5). The lethal effect was not reactivated by multiplicity reactivation. Photodynamic damage in phage sd was accompanied by the increase of the rise (but not of the latent) period in the one-step curve of phage multiplication and increase of the burst size. Experiments were carried out at dye concentration of 1-10(-5) M or lower; a strong dark effect of the dye being observed under 2-fold increase of the dye concentration. Plaque-type mutants were formed up to the maximum approximately 1% at the survival approximately 2--10(-5); in comparison with other mutagens photo-sensitized mutagenesis in phage sd was lower. The significant increase in the number of plaque mutants was observed after the illumination of phage fraction surviving the pre-treatment with higher acridine orange concentration (greater than or equal to 2-10(-5)M).     

Chromatographic determination of the association constant between 8-methoxypsoralen and modified beta-cyclodextrin: protective effect of hydroxypropyl-beta-cyclodextrin on 8-methoxypsoralen toxicity in human keratinocytes

The retention of 8-methoxypsoralen (8-MOP) on an immobilised hydroxypropyl-beta-cyclodextrin (HP-beta-CD) column was analysed in HPLC by the determination of its Langmuir distribution isotherm. A such method was used to confirm the potential drug complexing role of this cyclodextrin. The 8-MOP/HP-beta-CD association constant (K) was equal to 29.5 and 18.7 M-1, respectively, at a temperature equal to 5 and 25 degrees C, respectively. These association constant values were used to determine the cytotoxicity profile of human keratinocyte cell line (HaCaT) in relation to the complex concentration. It was showed through these data that HP-beta-CD had a cytoprotective since a reverse effect of HP-beta-CD on 8-MOP cytotoxicity was observed.     

Thiopyronine and 8-methoxypsoralen sensitized photodynamic effect on DNA synthesis in yeast

Summary The synthesis of DNA in growing yeast cells was investigated after photodynamic treatment of the cells with thiopyronine (TP) and visible light or with 8-methoxypsoralen (8-MOP) and UVA light.DNA synthesis was inhibited after photodynamic treatment with 8-MOP but not after photodynamic treatment with TP. This result is further evidence that the photodynamic effect with TP does not attack nuclear DNA in eucaryotic cells.     

Pulse-radiolysis studies of 8-methoxypsoralen.

Both eaq- and .OH have been found to react with 8-methoxypsoralen (8-MOP), giving rate-constants of 1.1 X 10(10) M-1 s-1. Transient spectra of products from the reactions of eaq-, .OH with 8-MOP have been characterized. Rate-constants for the oxidation by 8-MOP of reduced and oxidized DNA bases have also been measured and found to lie in the range 3-6 X 10(9) M-1 s-1. Oxidation of reduced bases occurs by electron transfer with 100 per cent efficiency in all cases. However, for oxidized bases, only approximately 25 per cent of the intermediate yield produced by OH attack undergoes electron transfer; the balance of the oxidized base appears to form adducts with 8-MOP.     

Evidence for uptake of 8-methoxypsoralen and 5-methoxypsoralen by cellular nuclei

The fluorescent appearance of oral mucosa cells treated with 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP) was observed by means of fluorescence microscopy. Fluorescence at the nuclei was weakened in 8-MOP-treated cells, while it was intensified in 5-MOP-treated cells. These findings were consistent with changes in the fluorescence intensities on association of the psoralen derivatives with DNA in aqueous solution. This intensity change of fluorescence and also the blue shift of the fluorescence maximum of the derivatives on association suggested that the environment around the psoralen molecules is as little polar as methanol. From the results of these fluorescence microscopic observations and spectroscopic analysis of fluorescence of derivatives interacting with DNA during equilibrium dialysis, we concluded that 8-MOP, as well as 5-MOP, is incorporated by nuclei of human cells.     

Bacterial radiosensitization by 8-methoxypsoralen.

8-Methoxypsoralen has been shown to act as a radiosensitizer of hypoxic bacterial cells with uvrA, recA and uvrB and/or lexA mutations. No effect of the drug on the radiosensitivity of oxic bacteria with these mutations was observed. This drug differs from O2 and electron-affinic radiosensitizers in that its effect is not purely dose-modifying and can exceed the oxygen effect in certain mutants.     

Repair of 8-methoxypsoralen photoinduced cross-links in yeast

Summary The repair of interstrand cross-links induced by 8-methoxypsoralen plus UVA (365 nm) radiation DNA was analyzed in diploid strains of the yeast Saccharomyces cerevisiae. The strains employed were the wild-type D₇ and derivatives homozygous for the rad18-1 or the rad3-12 mutation. Alkaline step-elution and electron microscopy were performed to follow the process of induction and removal of photoinduced crosslinks. In accordance with previous reports, the D₇ rad3-12 strain failed to remove the induced lesions and could not incise cross-links. The strain D₇ rad18-1 was nearly as efficient in the removal of 8-MOP photoadducts after 2 h of post-treatment incubation as the D₇ RAD⁺ wild-type strain. However, as demonstrated by alkaline step-elution and electron microscopic analysis, the first incision step at DNA cross-links was three times more effective in D₇ rad18-1 than in D₇ RAD⁺. This is consistent with the hypothesis that the RAD18 gene product is involved in the filling of gaps resulting from persistent non-informational DNA lesions generated by the endonucleolytic processing of DNA cross-links. Absence of this gene product may lead to extensive strand breakage and decreased recognition of such lesions by structural repair systems.     

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

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

8-Mop选育肉桂地链霉菌(StreptomycesCinnamonensis)的研究

作者首次报导8－甲氧基补骨（8－Mop)单一选育肉桂地链霉菌（Streptomyces cinnamonensis)的研究结果,曾有摇瓶发酵产量增幅达7．5％的稳产高产株获得,高产株已在国内应用。     

An estimate of genetic risk from 8-methoxypsoralen photochemotherapy

Summary A method based on a combination of cell culture and pharmacokinetic data is explored as a way of estimating the possible genetic risk to man from a mutagenic chemical. 8-methoxypsoralen, which is given to psoriasis patients as part of a photochemotherapy regime, is used, since it represents a real-life situation and possesses properties that make it particularly amenable to this approach. It is concluded that treatment of males for a mean period of 10 years before their spermatozoa were involved in concenption would increase the gene mutation rate in the male germ line by a factor of 0.00125. The implications of this for the incidence of sex-linked and dominant disease in man are elaborated and the assumptions and limitations are set out. The fragility of the estimates is emphasized.     

Repair of 8-methoxypsoralen monoadducts in mouse lymphoma cells

Studies of the repair of DNA lesions at biologically important doses is extremely difficult for most mutagens. With 8-methoxypsoralen (8-MOP) plus longwave ultraviolet light (UVA) as the lesion-inducing agent, however, it is easy to manipulate the relative frequency of different DNA adducts by means of a special experimental protocol (the tap-and-test protocol) and this can be used to measure repair of DNA adducts. Three classes of photoadducts are produced by 8-MOP plus UVA treatment: 3,4-cyclobutane monoadducts, 4',5'-cyclobutane monoadducts, and 8-MOP-DNA interstrand crosslinks. A monoadduct is formed when a photoactivated 8-MOP molecule reacts with a pyrimidine base. An 8-MOP-DNA interstrand crosslink is formed when an existing monoadduct is photoactivated to react with another pyrimidine base on the opposite DNA strand. Thus monoadducts are formed by absorption of one photon of light and crosslinks by absorption of two. In the tap-and-test experiments, cells were exposed to UVA in the presence of 8-MOP and then re-exposed to UVA in the absence of free 8-MOP so that only crosslinks can be produced by the second UVA treatment. By means of this technique we have previously shown that DNA crosslinks are much more effective than monoadducts at producing chromosomal damage (sister-chromatid exchanges and micronuclei) but not mutations (Liu-Lee et al., 1984). If L5178Y mouse lymphoma cells were able to remove monoadducts, incubation prior to the second UVA treatment should lead to decreases in the effect of re-irradiation, because fewer monoadducts would be available for crosslink formation. In this way, we have found that psoralen monoadducts are repaired in these cells and that about 70% of those capable of crosslink formation are removed or otherwise made unavailable for crosslink formation in 6 h.     

Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments

The photoreaction of 8-methoxypsoralen (8-MOP) with DNA fragments of defined sequence was studied. We took advantage of the blockage by bulky adducts of the 3'-5'-exonuclease activity associated with the T4 DNA polymerase. The action of the exonuclease is stopped by biadducts as well as by monoadducts. The termination products were analyzed on sequencing gels. A strong sequence specificity was observed in the DNA photobinding of 8-MOP. The exonuclease terminates its digestion near thymine residues, mainly at potentially cross-linkable sites. There is an increasing reactivity of thymine residues in the order T less than TT much less than TTT in a GC environment. For thymine residues in cross-linkable sites, the reactivity follows the order AT much less than TA approximately TAT much less than ATA less than ATAT less than ATATAA. Repeated A-T sequences are hot spots for the photochemical reaction of 8-MOP with DNA. Both monoadducts and interstrand cross-links are formed preferentially in 5'-TpA sites. Our results highlight the role of the sequence and consequently of the conformation around a potential site in the photobinding of 8-MOP to DNA.     

Photosensitized inactivation of plasmid and bacteriophage lambda DNA: relative contribution to the lethal effect of monoadducts and diadducts of 8-methoxypsoralen and their repair in SOS-induced Escherichia coli cells

The curves of UV (254 nm)-inactivation and inactivation by furocoumarin derivatives + UVA radiation (PUVA) of bacteriophage lambda and biologically active plasmid pBR322 were measured using Escherichia coli K12 bacteria with different defects of DNA repair system as a ghost. The ratio of mono- and diadducts (interstrand cross-links) of 8-methoxypsoralen was determined that are formed after treating the DNA of pBR322 and bacteriophage lambda with PUVA. It is shown that, on the average, about five monoadducts per one diadduct are formed in DNA of pBR322, and about 0.9 monoadducts per one diadduct are formed in lambda phage DNA. An increased (up to 50%) efficiency of SOS-repair of monoadducts of 8-methoxypsoralen in DNA of pBR322 and lambda in the presence of plasmid pKM101 muc+ (incN) was found.     

8-Mop选育天兰淡红链霉菌(S. Coeruleorubidus)高产株

本文首次报导天兰淡红链霉菌（S.coeruleorubidus)经8－甲氧基补骨脂素选育后,获得一株摇瓶产量稳增7．47％的高产株,已在国内应用。     

Photosensitizing effect of protoporphyrin IX in pigmented melanoma of mice

No fluorescence of protoporphyrin IX (PpIX) was measured using a fiber optic probe in pigmented B16F10 melanoma in mice after topical application of 5-aminolevulinic acid methylester (ALA-Me). However, chemical extraction of tissues excised from mice after intratumoral administration of ALA-Me or its parent compound ALA revealed that this tumor had the capability to produce PpIX. Small amounts of endogenous porphyrins, mainly PpIX, were found in the melanoma not treated with these drugs. Topical application of ALA-Me followed by exposure with laser light (633nm) delayed the growth of the tumors slightly. Light alone also had a significant effect on the tumor growth.