COMPARATIVE STUDIES ON THE LETHAL, MUTAGENIC, AND RECOMBINOGENIC EFFECTS OF ULTRAVIOLET-A,-B,-C, AND VISIBLE LIGHT WITH AND WITHOUT 8-METHOXYPSORALEN IN Saccharomyces cerevisiae

Genetic effects of UV-A, UV-B, UV-C, and the combination of 8-methoxypsoralen (8-MOP) with UV-A or visible light were studied in the haploid strain XV185-14C and diploid strain D5 of Saccharomyces cerevisiae. The induction of his+, lys+, and hom+ reverse mutations was measured in strain XV185-14C. In strain D5 we measured the induction of genetically altered colonies, particularly twin spot colonies arising from a mitotic crossing-over. UV-C and UV-B induced point mutations at the three loci in the haploid strain and mitotic crossing-over and other genetic alterations in the diploid strain. UV-C was more mutagenic and recombinogenic than UV-B. UV-A or visible light alone did not induce genotoxic effects at the doses tested. However, UV-A plus 8-MOP produced lethal and mutagenic effects in the haploid strain XV185-14C, although mutagenic activity was less than that of UV-B. Visible light plus 8-MOP also induced genotoxic effects in strain XV185-14C. In the diploid strain D5, UV-A plus 8-MOP induced a higher frequency of genetic alterations than UV-B at comparative doses. Visible light plus 8-MOP was also genetically active in strain D5. The haploid strain was more sensitive to the lethal effects of UV-C, UV-B, UV-A, and impure visible light plus 8-MOP than the diploid strain.     

COMPARATIVE STUDIES ON THE LETHAL, MUTAGENIC, AND RECOMBINOGENIC EFFECTS OF ULTRAVIOLET-A,-B,-C, AND VISIBLE LIGHT WITH AND WITHOUT 8-METHOXYPSORALEN IN Saccharomyces cerevisiae

Abstract
Genetic effects of UV-A, UV-B, UV-C, and the combination of 8-methoxypsoralen (8MOP) with UV-A or visible light were studied in the haploid strain XV185–14C and diploid strain D5 of Saccharomyces cerevisiae. The induction of his+, lys+, and horn+ reverse mutations was measured in strain XV185–14C. In strain D5 we measured the induction of genetically altered colonies, particularly twin spot colonies arising from a mitotic crossing-over. UV-C and UV-B induced point mutations at the three loci in the haploid strain and mitotic crossing-over and other genetic alterations in the diploid strain. UV-C was more mutagenic and recombinogenic than UV-B. UV-A or visible light alone did not induce genotoxic effects at the doses tested. However, UV-A plus 8-MOP produced lethal and mutagenic effects in the haploid strain XV185–14C, although mutagenic activity was less than that of UV-B. Visible light plus 8-MOP also induced genotoxic effects in strain XV185–14C. In the diploid strain D5, UV-A plus 8-MOP induced a higher frequency of genetic alterations than UV-B at comparative doses. Visible light plus 8-MOP was also genetically active in strain D5. The haploid strain was more sensitive to the lethal effects of UV-C, UV-B, UV-A, and impure visible light plus 8-MOP than the diploid strain.     

MUTAGENIC AND RECOMBINOGENIC ACTION OF DNA MONOADDUCTS PHOTOINDUCED BY THE BIFUNCTIONAL FUROCOUMARIN 8-METHOXYPSORALEN IN YEAST (Saccharomyces cerevisiae)

Abstract— For the same furocoumarin 8-MOP and the same total number of photoadditions, the genetic activity of DNA monoadducts and a mixture of mono- and biadducts photoinduced by the bifunctional furocoumarin 8-methoxypsoralen (8-MOP) is compared in the yeast Saccharomyces cerevisiae. In the presence of 8-MOP, 405 nm irradiation induces only monoadducts, whereas 365 nm irradiation induces mono- and biadducts (interstrand cross-links) in DNA. This is shown by heat denaturation-renaturation experiments on calf thymus DNA treated in vitro and by alkaline step elution analysis of DNA from treated yeast cells. For the same photobinding of tritiated 8-MOP to DNA in diploid yeast, about 20 times higher doses are needed with 405 nm than with 365 nm irradiation. Re-irradiation experiments reveal that part of the monoadducts induced by 8-MOP and 405 nm irradiation can be effectively converted into DNA interstrand cross-links by exposures to 365 nm radiation after washing-out of unbound 8-MOP molecules. 8-MOP and 405 nm irradiation induce per lethal hit cytoplasmic "petite" mutations in yeast as efficiently as the monofunctional furocoumarin 3-carbethoxypsoralen (3-CPs) and 365 nm irradiation, both treatments being much more efficient than 8-MOP and 365 nm irradiation. At equal survival, treatments with 8-MOP and 405 nm radiation are clearly less efficient than treatments with 8-MOP and 365 nm radiation for the induction of forward ( CAN *) and reverse ( HIS +) mutations in haploïd yeast and for the induction of mutations ( ILV +) and genetically aberrant colonies including mitotic crossing-over in diploid yeast. The two treatments are equally efficient for the induction of mitotic gene conversion. At equal photobinding of 8-MOP, the monoadducts induced by 405 nm irradiation are found less effective than the mixture of mono-and biadducts induced by 365 nm irradiation for the induction of cell killing, mutations and mitotic recombination.     

Genetic effects and repair of DNA photo-adducts induced by 8-methoxypsoralen and homopsoralen (pyranocoumarin) in diploid yeast

The relationship between DNA mono- and di-adducts and genetic effects induced by the pyranocoumarin 8,8-desmethylxanthyletine (homopsoralen) HP and 365 nm radiation (UVA) was investigated in the diploid yeast strain D7 (Saccharomyces cerevisiae) taking 8-methoxypsoralen (8-MOP) as a reference compound. The number of DNA cross-links (CLs) induced was determined using alkaline step elution analysis. The induction and removal of total photo-adducts was followed using radioactively labelled compounds. HP showed the same photobinding capacity as 8-MOP. As a function of UVA dose, it was less effective than 8-MOP for the induction of CLs and genetic effects. However, as a function of CLs induced, HP was shown to be more effective for the induction of lethal effects and mitotic recombination than 8-MOP but equally effective for the induction of mutations. The results suggest that, although CLs are recognized as genetically effective lesions, at a given number of CLs, HP induced mono-adducts efficiently contribute to the induction of lethal effects and mitotic recombination but less to the induction of mutations. Using a re-irradiation protocol, HP was brought to yield the same relative amounts of CLs at the same number of total adducts as single UVA exposures with 8-MOP. In these conditions, mutation induction and the kinetics for the removal of photo-adducts were the same for both agents indicating that not only the removal of adducts but also mutation induction are highly dependent on the relative level of CLs induced.     

8-METHOXYPSORALEN-PHOTOINDUCED DNA CROSSLINKS AS DETERMINED IN YEAST BY ALKALINE STEP ELUTION UNDER DIFFERENT REIRRADIATION CONDITIONS. RELATION WITH GENETIC EFFECTS

Abstract— DNA damage induced by 8-methoxypsoralen (8-MOP) plus near UV light (UVA) was analyzed in diploid yeast using the alkaline step elution technique. The presence of 8-MOP and UVA induced DNA interstrand cross-links was revealed by the increase of DNA retained on elution filters as compared to untreated controls. The fraction of DNA retained on filters increased linearly with UVA dose. The amount of cross-links was estimated from the fraction of DNA retained on filters using a dose of -radiation leading to a number of DNA strand breaks at least equivalent to the number of 8-MOP induced photoadducts.
When 8-MOP treated cells were exposed to monochromatic light, 365 nm light induced monoadducts and cross-links whereas 405 nm light induced only monoadducts. When submitting 8-MOP plus 405 nm light treated cells to 365 nm irradiation, after removal of unbound 8-MOP by washing, a portion of 8-MOP plus 405 nm light induced monoadducts was converted into cross-links. The amount of monoadducts transformed into cross-links was dependent on the dose of 365 nm irradiation up to a maximum likely to correspond to the number of suitably positioned furan-side monoadducts that could be converted into biadducts. When 8-MOP plus 365 nm light treated cells were reirradiated with 365 nm light, following the same protocol, the maximum level of cross-linking obtainable in yeast was lower than that obtained with 8-MOP in a 405 nm plus 365 nm reirradiation protocol.
In the presence of 8-MOP single exposures to 405 nm light were found to be only slightly genotoxic. However, when followed by second exposures to 365 nm light, a dose-dependent increase in genetic effects, i.e. mutation and gene conversion, was observed in parallel to the induction of DNA crosslinks. These results stress again the prominent role of DNA cross-links in the genotoxicity of 8-MOP.     

METABOLISM OF 5-METHOXYPSORALEN BY Saccharomyces cerevisiae

Incubation of methoxypsoralen (5-MOP) in the presence of diploid yeast cells (Saccharomyces cerevisiae) before UV-A exposure leads to an incubation-time dependent decrease of photoinduced genotoxic effects. The reduction in photoinduced genotoxicity is stronger in cells grown in the presence of 20% glucose and containing high levels of cytochrome P-450 than in cells grown in the presence of 0.5% glucose and containing undetectable levels of cytochrome P-450. Inhibition of P-450 activity by specific inhibitors, such as tetrahydrofuran and metyrapone, strongly affects the observed decrease in 5-MOP genotoxicity, indicating the involvement of P-450 in 5-MOP metabolism. As demonstrated by spectrophotometric and chromatographic (HPLC) analysis during incubation of 5-MOP with P-450 containing yeast cells, 5-MOP gradually disappears from the cell supernatant of the incubation mixture. The reduction in the chromatographic peak corresponding to 5-MOP is accompanied by the appearance of a new peak that probably corresponds to a metabolite. As shown by the use of P-450 specific inhibitors, the metabolite appears to be due to P-450 mediated 5-MOP metabolisation. Its UV absorption spectrum suggests an alteration of the pyrone moiety of the 5-MOP molecule.     

3-CARBETHOXYPYRANOCOUMARIN, A PHOTOREACTIVE DERIVATIVE OF XANTHYLETIN WITH INTERESTING PHOTOBIOLOGICAL PROPERTIES

Abstract— The photobiological activity of the newly synthesized pyranocoumarin derivative 3-carbethoxypyranocoumarin, so-called 3-carbethoxyhomopsoralen (3-CHPs) was studied in comparison to the known bifunctional furocoumarin 8-methoxypsoralen {8-MOP) and to the monofunctional furocoumarin 3-carbethoxypsoralen (3-CPs) in the presence of 365 nm irradiation using two eukaryotic cell systems, the yeast Saccharomyces cerevisiae and cultured normal human skin fibroblasts. 3-Carbethoxyhomopsoralen is shown to be a photobiologically active compound capable of effectively photoinducing cytoplasmic "petite" mutants (mitochondrial damage), nuclear reversions and mitotic gene conversion in the diploid yeast strain D₇. Per unit dose it is more effective than 8-MOP and 3-CPs for the induction of cytoplasmic "petite" mutants but less effective than 8-MOP for the induction of nuclear reversions and mitotic gene conversion. A very moderate effect on cell survival is accompanied by a relatively strong genetic activity per viable cell. In human fibroblasts 3-CHPs produces a stronger inhibition of DNA synthesis than 8-MOP and 3-CPs at low doses of 365 nm radiation. During post-treatment incubation human fibroblasts recovered more easily from DNA synthesis and growth inhibitions photoinduced by 3-CHPs than from those photoinduced by 8-MOP. The results are in accord with the notion that 3-CHPs is a highly photoreactive monofunctional compound inducing easily repairable lesions with a low lethal but significant mutagenic potential.     

Phototumorigenesis studies of 5-methoxypsoralen in bergamot oil: evaluation and modification of risk of human use in an albino mouse skin model

The skin of the female hairless albino mouse (Skh 1) was used to study the enhancement of solar simulated radiation (SSR) tumorigenesis by 5-methoxypsoralen (5-MOP) in model perfumes that contain bergamot oil. This work was done in association with yeast mutagenicity studies and human skin phototoxicity studies. Analyses of time-to-onset of tumour observation with 5-MOP at 0, 5, 15 and 50 ppm show a highly significant 5-MOP dose effect and the data indicate that 5-MOP has phototumorigenic potential even at 5 ppm. The addition of 0.5% UVB and 0.5% UVA sunscreens significantly reduces the tumorigenicity associated with the vehicle (i.e. 5-MOP at 0 ppm) and 5-MOP at all concentrations. Pairwise comparisons of 5-MOP (at 5 or 15 ppm) plus sunscreens with vehicle plus sunscreens show that the sunscreens afford total protection at the lower 5-MOP concentrations. Additional studies show that a 5-6 h delay between 5-MOP application and SSR exposure defers the time-to-onset of tumours as does intermittent 5-MOP and SSR treatment. A comparison of 5-MOP at 50 ppm in bergamot oil with 5-MOP at 50 ppm prepared from pure 5-MOP crystals shows identical results, indicating that the active phototumorigenic agent in bergamot oil is 5-MOP and not other related compounds, which may be present at greater concentrations. Analyses of tumour histology at death show, in general, similar patterns of malignancy for all groups. Thus although it is possible to delay tumorigenesis by various strategies, the tumours that eventually develop are just as likely to be malignant, if not more so, when compared with non-delayed groups.     

FAILURE OF UVR DOSE RECIPROCITY FOR SKIN TUMORIGENESIS IN HAIRLESS MICE TREATED WITH 8-METHOXYPSORALEN

Abstract— 8-Methoxypsoralen (8-MOP) phototumorigenesis was studied in hairless albino mice irradiated with solar simulated radiation (SSR). Animals were exposed to three different daily doses of SSR after application of an oily vehicle with or without 8-MOP. Tumor production was dependent on daily SSR dose irrespective of topical treatment. The phototumorigenic potency of SSR was increased by the vehicle and to a much greater extent by inclusion of 8-MOP. Irrespective of topical treatment, a failure in dose reciprocity for tumorigenesis was demonstrated; in terms of cumulative SSR dose, the lower daily doses were more tumorigenic than the higher doses. This reciprocity failure in 8-MOP photosensitized mice is similar to that previously observed for UV-B phototumorigenesis. The relevance of these findings to therapeutic use of psoralens is discussed.     

New aspects of the repair and genotoxicity of psoralen photoinduced lesions in DNA.

Several approaches are described aiming at a better understanding of the genotoxicity of psoralen photoinduced lesions in DNA. Psoralens can photoinduce different types of photolesions including 3,4- and 4',5'-monoadducts and interstrand cross-links, oxidative damage (in the case of 3-carbethoxypsoralen (3-CPs)) and even pyrimidine dimers (in the case of 7-methylpyrido(3,4-c)psoralen (MePyPs)). The characterization and detection of different types of lesions has been essential for the analysis of their possible contributions to genotoxicity. For example, oxidative damage photoinduced by 3-CPs can be detected by the formamidopyrimidine glycosylase (FPG) protein. Furthermore, it is shown how the presence of MePyPs induced monoadducts may interfere with the photoreactivation of concomitantly induced pyrimidine dimers, how the ratio of monoadducts and interstrand cross-links (CL) affects the occurrence of double-strand breaks during the repair of photolesions and genotoxicity. In vitro treatment of yeast plasmids, followed by transformation, also indicates that the repair of photoadducts on exogenous DNA differs for 8-methoxy-psoralen (8-MOP) induced mono- and diadducts and for monoadducts alone. The recombinational rad52 dependent pathway is not needed for the repair of 8-MOP induced monoadducts. The results obtained suggest that the genotoxic effects of psoralens are conditioned by the nature, number, ratio and sequence distribution of the photolesions induced in DNA.     

Molecular dosimetry of 8-MOP + UVA-induced DNA photoadducts in Saccharomyces cerevisiae: correlation of lesions number with genotoxic potential

Acid hydrolysis of purified DNA extracted from cells of a haploid repair-proficient (RAD) yeast strain that had been treated with 8-MOP + UVA revealed the existence of two major and one minor thymine photoproduct. At survival levels of the RAD strain between 100% and 1% furanside monoadducts constituted the major DNA lesion, followed by diadducts that, at the lowest survival level, nearly reached 50% of the thymine photoproducts; pyrone-side monoadducts were only detectable at the highest UVA exposure dose applied and clearly constitute a minority photoproduct. The number of induced diadducts was verified by determination of interstrand cross-links via denaturation and renaturation of 8-MOP + UVA-treated DNA from RAD and rad2 yeast strain. 8-MOP + UVA was shown to induce two types of locus-specific mutations: reversion of the lys1-1 ochre allele was between 20- to 50-fold higher than that of the his4-38 frameshift allele. Mutant yield for the lys 1-1 reversion was the same in RAD and excision repair-deficient rad2-20 strains whereas frameshift mutagenesis was about eightfold higher in the rad2-20 background.     

Effect on topical 5-methoxypsoralen on tumorigenesis induced in albino and pigmented hairless mouse skin by UV irradiation

A new line of the Skh:HRII hairless pigmented mouse (black juvenile coat) is described which has been selectively bred for the capacity to respond consistently to simulated solar UV radiation with a continuous and strong tan. This mouse demonstrates a degree of protection from chronic UV-induced tumorigenesis when compared with the Skh:HRI hairless albino mouse, and has been used here to study the effect of induced melanogenesis on phototumorigenesis. Mice were irradiated for 10 weeks with incremental doses of simulated solar UV radiation (UVA + B) from a fluorescent tube source which induced tumours in 100% of albino mice and 93% of black mice by 200 days (minimally oedemal), or with 60% of this dose (sub-oedemal) which induced tumours in 85% of albino mice and 65% of black mice. Mice were also exposed to the UVA component of these radiation sources, obtained by window glass filtration. The effect of topical 5-methoxypsoralen (5-MOP) was examined, at either 0.003% with minimally oedemal UVA + B or its UVA component alone, or at 0.01% with sub-oedemal UVA + B or its UVA component alone, in both albino and black mice. The 5-MOP concentrations were selected as the maximum concentration which did not increase the erythema and oedema responses after a single exposure to minimally oedemal or sub-oedemal UVA + B. At 200 days, the tumorigenic response to sub-oedemal UVA + B was significantly increased by topical 5-MOP, to 100% in albinos and 93% in black mice. In contrast, tumorigenesis in response to minimally oedemal UVA + B was unaffected by topical 5-MOP. The UVA component alone of either irradiation regime was not tumorigenic under these conditions. When combined with topical 5-MOP, the UVA of minimally oedemal UVA + B became moderately tumorigenic, and resulted in a tumour incidence of 23% in albinos and 14.5% in black mice. However, the UVA component of sub-oedemal UVA + B, when combined with topical 5-MOP, was highly tumorigenic specifically in albino mice, inducing tumours in 93% of albino mice but in only 27% of black mice. Tan intensity resulting from minimally oedemal UVA + B was not enhanced by topical 5-MOP, and its UVA component combined with 5-MOP resulted in only a minimal tan. However, the tan intensity resulting from sub-oedemal UVA + B with topical 5-MOP was strongly increased, although its UVA component combined with 5-MOP did not produce a perceptible tan.(ABSTRACT TRUNCATED AT 400 WORDS)     

ACTIVATION OF THE HUMAN IMMUNODEFICIENCY VIRUS PROMOTER BY UVA RADIATION IN COMBINATION WITH PSORALENS OR ANGELICINS

Abstract— The effects of mono- and bifunctional furocoumarins plus UVA radiation (PUVA and related treatments) on the human immunodeficiency virus-1 (HIV-1) promoter were studied using HeLa cells stably transfected with the chloramphenicol acetyl transferase gene under the control of the HIV-1 promoter. The experiments were performed with three psoralens (5-methoxypsoralen, 5-MOP; 8-methoxypsoralen, 8-MOP; and 4′-aminomethyl-4,8,5′-trimethyl-psoralen, AMT) and four angelicins (angelicin; 4,5′-diniethylangclicin, 4,5′-DMA; 6,4′-dimethylangelicin, 6,4′-DMA; and 4,6,4′-trimethylangelicin, TMA). The drugs alone and UVA radiation alone showed no erect on the HIV promoter. However, when the cells were incubated with the furocoumarins at 0.1–40 μg/mL and then irradiated. the HIV promoter was activated in distinct fluence ranges, i.e. (1) no promoter activity was discernible at low fluences (e.g. at 0.1 μg/mL of 8-MOP up to 100 kJ/m²), (2) as the fluence was increased, the promoter activity increased to reach a maximum (10–50-fold with respect to the unexposcd samples), and (3) as the fluence was further increased, the promoter activity decreased. Similar (although shifted on the fluence scale) pattcrns were observed with either > 340-nm UVA radiation or with UVA radiation contaminated with a small amount of UVB radiation (typical for PUVA lamps). The effective fluences were inversely related to the drug concentration. Experiments with 5-MOP and 8-MOP indicated reciprocity of the drug concentration and radiation hence. The HIV promoter response patterns were similar for monofunctional angelicins and bifunctional psoralens. This indicated that the furocoumarin-DNA crosslinks are not a prerequisite for the promoter activation and that the monoadducts suffice to elicit the HIV promoter response. The HIV promoter-activating effectiveness of diKcrent drugs correlated with their photosensitizing potential. Thus, among psoralens the effectiveness order was AMT >. 5-MOP >8-MOP, and among angelicins: TMA > 6,4′-DMA > 4,5′-DMA > angelicin. The ektiveness did not vary substantially for 5-MOP, 8-MOP, 4,5′-DMA, and 6,4′-DMA. The combined drug and UVA radiation doses were higher than those that elicit cellular responses or those that may be received by the human white blood cells during cxtracorporeal PUVA therapy (photopheresis).     

CERTAIN SINGLET OXYGEN QUENCHERS AFFECT THE PHOTOREACTION BETWEEN 8-MOP and DNA

The effect of certain chemicals known as singlet oxygen quenchers on the photoreaction between 8-MOP and DNA has been studied in vitro; sodium azide, l,4-diazabicyclo-(2,2,2)-octane, p-carotene and dimethylsolfoxide (used as a solvent) appeared to be capable of reducing significantly the 8-MOP ability to induce both monoadducts and cross-links in DNA. Therefore, these chemicals seem to be not useful in studying the singlet oxygen implication in the induction of biological effects of 8-MOP sensitization.     

Relative affinity of 5-methoxypsoralen and 8-methoxypsoralen towards beta-cyclodextrin: a fluorescence, circular dichroism and chromatographic study

The relative affinity of 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) towards beta-cyclodextrin, a good model for the study of lipophilic interactions in biological systems and a potential drug carrier, has been investigated using spectroscopic and chromatographic methods. The fluorescence emission of 5-MOP in aqueous solution containing beta-cyclodextrin (10(-2) M) is found to be markedly blue shifted and enhanced by a factor of 6 whereas no significant changes are observed for 8-MOP. The existence of an induced circular dichroism is evidence for the formation of a 1:1 inclusion complex (association constant K = 400 +/- 50 M-1). Moreover, chromatographic results obtained with a beta-cyclodextrin linked stationary phase are consistent with our spectroscopic results and might have interesting analytical implications. These results clearly demonstrate that, in contrast to 8-MOP, 5-MOP exhibits a strong affinity for hydrophobic medium. Interesting pharmacological and analytical applications may result from the possible inclusion of psoralen derivatives into beta-cyclodextrin.     

MUTAGENIC EFFECTS PHOTOINDUCED IN MAMMALIAN CELLS IN VITRO BY TWO MONOFUNCTIONAL PYRIDOPSORALENS

Abstract— The photobiological activity of the two monofunctional pyridopsoralens pyrido (3,4-c) psoralen (PyPs) and 7-methyl pyrido (3,4-c) psoralen (MePyPs) was studied in mammalian cells in vitro taking 8-methoxypsoralen (8-MOP) as a reference compound.
In the presence of 365-nm irradiation (UVA) MePyPs was found to be more effective than 8-MOP in terms of DNA photobinding capacity and inhibition of cell cloning ability in Chinese hamsterV–79 cells. As a function of UVA dose and of the number of total photoadducts induced MePyPs produced a higher frequency of 6-thioguanine resistant mutants than 8-MOP. PyPs showed an intermediate response for cell killing and mutation induction. At equal cytotoxic levels both monofunctional pyridopsoralens exhibited the same mutagenic activity as the Afunctional furocoumarin 8-MOP.
The antiproliferative effect being taken as indicative for an efficient photochemotherapeutic activity against psoriasis, the inhibition of cloning capacity induced by MePyPs plus UVA was studied in parallel on human skin fibroblasts. Such cells were more sensitive to 8-MOP photoadditions thanV–79 cells and even more so to MePyPs photoadditions. Data obtained on the rate of DNA semi conservative synthesis on both cell lines following treatments with the two compounds are in line with these observations.     

UVA-induced binding of 8-methoxypsoralen to cells of Saccharomyces cerevisiae: separation and characterization of DNA photoadducts

We present methods for the determination of UVA-induced binding of 8-methoxypsoralen (8-MOP) to nucleic acids and protein and for a quantitative assay of radioactively labelled 8-MOP plus UVA induced DNA photoproducts in the yeast Saccharomyces cerevisiae. For the dose range up to 60 kJ m-2, with a wild-type survival of 1% or higher, binding to DNA is 100-fold and to RNA 10- to 20-fold more efficient than that to protein. Between 20% and 65% of the 8-MOP binds to macromolecules that are neither nucleic acids nor protein. The number of DNA-bound 8-MOP molecules for the haploid genome rises from 14 (unirradiated control) to 349 at the highest UVA exposure dose (60 kJ m-2). Gel chromatography reveals three types of DNA thymine photoproduct, the pyrone-side monoadducts, the furan-side monoadducts and the diadducts. Among these, pyrone-side monoadducts always constitute the smallest fraction, regardless of whether the treatment is with in vitro or in vivo 8-MOP plus UVA.     

Phototoxic properties of perfumes containing bergamot oil on human skin: photoprotective effect of UVA and UVB sunscreens

As part of an international cooperative study of the photophysical, photomutagenic and photocarcinogenic properties of bergamot oil and the effect of UVA and UVB sunscreens, the phototoxic properties of model perfumes containing 5, 15 and 50 ppm 5-methoxypsoralen (5-MOP) in bergamot oil with and without a sunscreen have been investigated on human skin. It has been confirmed that the photosensitivity of human skin is maximal 2 h after perfume application. Interestingly the addition of a UVA sunscreen is more efficient for decreasing the phototoxic properties of bergamot oil than is a UVB sunscreen. The addition of sunscreens in a model perfume containing 50 ppm 5-MOP in bergamot oil can reduce the phototoxic properties of this perfume to a toxicity equivalent to that produced by the application of a model perfume containing 15 ppm 5-MOP without sunscreens. However, despite their promising protective effect in vitro, UVB and UVA sunscreens at low concentration (0.5%-1%) in perfumes cannot suppress the phototoxicity of bergamot oil on human skin.     

PHOTOBINDING OF PSORALENS TO BACTERIAL MACROMOLECULES IN SITU AND INDUCTION OF GENETIC EFFECTS IN A BACTERIAL TEST SYSTEM. EFFECTS OF SINGLET OXYGEN DIAGNOSTIC AIDS D2O AND DABCO

The photobinding of radiolabeled psoralen and 8-methoxypsoralen (8-MOP) to biological macromolecules under conditions that affect the lifetime of singlet oxygen (¹O₂) is reported. These conditions are: increase of ¹O₂ lifetime in D₂O and ¹O₂ quenching with DABCO. The photobinding to calf thymus DNA was studied in vitro and the covalent photobinding to DNA and other biological macromolecules (RNA, proteins) was also studied in intact bacteria. The results of the DNA photobinding experiments have been related to the induction of genetic damage in a bacterial test system. In addition, laser flash photolysis has been used to measure the effect of D₂O and DABCO on the psoralen and 8-MOP triplet lifetimes. In general D₂O increases the triplet lifetimes and DABCO quenches the triplet states with the probable formation of radicals. The results suggest that the covalent photobinding of 8-MOP to various biological macromolecules in situ is a basis for cell damage occurring at various cellular targets. Analysis of the results of the mutagenicity test suggests that in the presence of D₂O the mechanism of induction of genetic lesions is not changed and therefore largely seems to be independent of singlet oxygen.     

Induction of mitotic arrest and aneuploidy by organic arsenic compounds in human lymphocytes

Inorganic arsenic is methylated in the mammalian body to methylarsonic acid (MMA), dimethylarsinic acid (DMA) and trimethylarsine oxide (TMA). To achieve a more precise understanding of arsenic carcinogenicity, we examined the genotoxic effects of organic arsenic compounds on human lymphocytes by assessing induction of mitotic arrest, sister chromatid exchange (SCE) and aneuploidy. MMA, DMA and TMA arrested mitosis, DMA induced hyperdiploid cells, and DMA and TMA induced tetraploid cells. Of the three arsenic metabolites tested, DMA had the strongest effects on cell mitosis and aneuploidy induction. DMA arrested mitosis and induced c-mitosis significantly. These results suggest that DMA arrests mitosis and induces aneuploidy through spindle disruptions similar to those observed with known spindle poisons, such as colchicine or vinblastine. Since aneuploidy has been thought to be associated with tumor induction or neoplastic transformation, induction of aneuploidy by organic metabolites of arsenic may play a major role in arsenic carcinogenesis in humans. © 1997 John Wiley & Sons, Ltd.