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.     

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.     

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.     

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.     

Kinetic Analysis of Apoptosis Induction in Human Cell Lines by UVA and 8-MOP

Whereas previous studies have indicated that DNA damage as a result of 8-methoxypsoralen (8-MOP) and UVA treatment leads to cell death, this study establishes the minimum concentrations of 8-MOP and UVA necessary to induce apoptosis in human T-lymphocytic and mono-cytic cell lines. In order to assess apoptosis, we used fluorescent microscopy to examine changes in light scattering as well as internucleosomal DNA fragmentation. Generation of a dose response curve showed that the minimum combination of UVA and 8-MOP that was necessary to induce greater than background levels of apoptosis within 24 h of treatment was 0.5 J/cm² UVA and 12.5 ng/mL of 8-MOP. A striking observation was that UVA alone at doses 1.0 J/cm², but not 8-MOP alone (6300 ng/mL), induced significant apoptosis in the Sup-T1 cell line within 24 h. Although the percentage of apoptotic Sup-T1 cells induced by UVA alone was not as great as that of 8-MOP and UVA in combination, a highly significant correlation between the product of the concentration of 8-MOP (ng/mL) times the dose of UVA (J/ cm²) and the percentage of apoptotic cells was observed. This correlation provides an important tool for studying the relationship of UVA-induced DNA damage to apoptosis induction. Moreover, it will provide a means by which early events in the apoptotic pathway can be dissected.     

DNA Photodamage, Repair, Gene Induction and Genotoxicity Following Exposures to 254 nm UV and 8-Methoxypsoralen Plus UVA in a Eukaryotic Cell System

The induction and repair of different types of photodamage and photogenotoxicity in eukaryotic cells have been the subject of many studies. Little is known about possible links between these phenomena and the induction of DNA damage-inducible genes. We explored this relationship using the yeast Saccharomyces cerevisiae, a pertinent eukaryotic model. Previous results showed that the photogenotoxic potential of 8-methoxypsoralen (8-MOP) plus UVA is higher than that of UV (254 nm). Moreover, the induction of the ribonucleotide reductase gene RNR2 by UV and 8-MOP plus UVA in an RNR2-LACZ fusion strain and the formation of DNA double-strand breaks (dsb) as repair intermediates after such treatments suggest that the latter process could involve a signal for gene induction. To further substantiate this, we measured the induction of the DNA repair gene RAD51 in RAD51-LACZ fusion strains using the dsb repair and recombination deficient mutant rad52 and the corresponding wild type, and we determined the formation of dsb by pulsed-field gel electrophoresis. After treatments, the resealing of dsb formed as repair intermediates was impaired in the rad52 mutant. At equal doses, i.e. the same number of lesions, the induction of the RAD51 gene by UV or 8-MOP plus UVA was significantly reduced in the rad52 mutant as compared with the wild type. The same was true when equitoxic doses were used. Thus, the RAD52 repair pathway appears to play an important role not only in dsb repair but also in gene induction. Furthermore, the signaling pathways initiated by DNA damage and its processing are somewhat linked to the photogenotoxic response.     

Genotoxicity of bergapten and bergamot oil in Saccharomyces cerevisiae

In order to determine the genotoxic potential of bergapten (5-methoxypsoralen (5-MOP] and bergamot oil (BO), the genetic effects of 5-MOP and BO (containing equivalent amounts of 5-MOP) were studied in haploid and diploid yeast (Saccharomyces cerevisiae) using solar simulated radiation (SSR). At equal doses of SSR, equal concentrations of 5-MOP alone or 5-MOP in BO have a similar influence on survival and on the induction of cytoplasmic "petite" mutations, reverse and forward mutations, mitotic gene conversion and genetically aberrant colonies including mitotic crossing over. No reciprocity is found between SSR dose and 5-MOP concentration for cytotoxic, mutagenic and recombinogenic effects. In the presence of chemical filters (Parsol 1789, a UVA filter, and Parsol MCX, a cinnamate derivative acting as a UVB filter) considerable protection is observed against the induction of genetic effects by 5-MOP and BO containing 5-MOP in haploid and diploid cells. As indicated by the lower induction kinetics, the protection is higher than expected from the light-absorbing properties, suggesting photochemical interaction. The protection is slightly higher for BO than for 5-MOP. The induction of genetic effects by 5-MOP alone or BO containing 5-MOP is independent of oxygen. Experiments on suction blister fluids taken from patients after topical treatment with BO containing 5-MOP indicate that in comparison with water the bioavailability and thus the genotoxic effects of the compounds are decreased. Moreover, in addition to the filtering effect against the photoinduced genotoxic effects of BO, the presence of chemical filters apparently reduces the penetration of BO containing 5-MOP and provides a reduction in biological effectiveness.     

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.     

The Lack of Efficacy of 4,6,4'-Trimethylangelicin to Induce Immune Suppression in an Animal Model for Photopheresis: a Comparison with 8-MOP

Photopheresis is an extracorporeal form of photochemo-therapy with 8-methoxypsoralen (8-MOP) and UVA (PUVA). Patients ingest 8-MOP and then a psoralen-rich buffy coat is obtained by centrifugation and mixed with saline. This mixture is recirculated through a UVA radiation field and then reinfused. Photopheresis appears to be effective for several T cell-mediated disorders, because the treatment results in a specific immune response against the pathogenic clone of T cells involved. With PUVA therapy, the whole body of the patient is exposed to UVA, after ingestion of 8-MOP. Upon UVA exposure 8-MOP binds to, amongst others, DNA and induces DNA monoadducts and interstrand cross-links. As a result of these photoadducts photocarcinogenicity is a risk in PUVA. In PUVA for psoriasis, it proved that angular furocoumarins, although almost incapable of inducing DNA cross-links (less carcinogenic), are still effective. In order to determine if monoadducts induced by photopheresis could also be effective we used, specifically, 4,6,4'-trimethylangelicin (TMA). In this report, we compare the photodegradation of both TMA and 8-MOP under conditions relevant to the in vivo situation, as well as the effect both compounds have on the viability of rat lymphocytes as measured with the 3–(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. We show that TMA did not induce immunosuppression in vivo , even after extensive irradiation. In addition a dose dependency of 8-MOPNVA versus the induced immune suppression was carried out. It was shown that there is a log doselresponse correlation of r = 0.9205.     

8-METHOXYPSORALEN PLUS UVA INDUCES THE 72 kDa HEAT SHOCK PROTEIN IN ORGAN-CULTURED NORMAL HUMAN SKIN

Abstract The proteins induced by heat and other stressors, called heat shock proteins (HSP) or stress proteins, are considered to play a general role in protection from cellular injury. Exposure to UVA (320400 nm) following application of 8-methoxypsoralen (8-MOP), termed PUVA is commonly used in the field of dermatology. In order to understand the induction of HSP in PUVA-treated human skin, indirect immunofluorescence using a monoclonal antibody specific for the 72 kDa HSP (HSP 72) was carried out in organ-cultured normal human skin that was treated with PUVA. When the organ-cultured skin was treated at 37°C for 1 h with 8-MOP at a final concentration of 10 or 100 μg/mL and exposed to UVA (51.3 kJ/m²), nuclear immunofluorcscence of HSP 72 was detected in the epidermal cells 12 h after UVA irradiation. In contrast, the induction of HSP 72 was not detected either by UVA irradiation or 8-MOP treatment. These results suggest that PUVA treatment is one of the stressors for human skin, and DNA damage caused by PUVA induces HSP 72.     

Turnover of phospholipids in HUT 102 lymphoblasts and chromatographic characterization of purified lecithins after their exposure to long-wave UV light, psoralen, and UV light and psoralen.

The turnover of 32P-labeled phospholipids in HUT 102 lymphoblasts was determined after a 2 h interaction of lymphoblasts with 8-methoxypsoralen (8-MOP) (15 micrograms ml-1), longwave UV light (UVA) irradiation and PUVA (8-MOP and UVA). In parallel experiments, micellar suspensions of lyso-phosphatidylcholine (PtdC), dipalmitoyl-PtdC and dilinoleoyl-PtdC, treated in a similar manner, served for the correlative assessments of cellular lipid changes. The dark reaction, UVA irradiation and PUVA all depressed total phospholipid levels in HUT 102 cells, although only PUVA induced a statistically significant decline. Thin layer chromatography (TLC) analysis revealed that neither UVA nor 8-MOP alone triggered any significant changes in the cellular content of phosphatidylinositol (PtdI), phosphatidylinositol 4-monophosphate (PtdIP) and phosphatidylinositol 4,5-bisphosphate (PtdIP2), whereas the lyso-PtdC and PtdI content of lymphoblasts showed a two-fold increase after PUVA. The TLC analysis of lyso-PtdC and micelles of dipalmitoyl-PtdC did not reveal any detectable changes after the dark reaction with 8-MOP, UVA irradiation and PUVA. In contrast, the derivatives of dark and UVA mediated reactions of 8-MOP with dilinoleoyl-PtdC were detected by TLC. These results suggest that the formation of 8-MOP derivatives of cellular phospholipids effected by PUVA, modulates the turnover of phosphoinositides and the rate of cellular proliferation.     

SV40 INDUCTION FROM A MAMMALIAN CELL LINE BY ULTRAVIOLET RADIATION and THE PHOTOSENSITIZERS 8-METHOXYPSORALEN and ANGELICIN

Abstract SV40-transformed weanling Syrian hamster kidney cells were exposed to long wavelength ultraviolet radiation in the presence of 8-methoxypsoralen or angelicin. The number of viruses induced as a result of this treatment was quantitated on CV-1P cells. The results indicate that angelicin, which is generally believed to form only monoadducts, is a potent viral inducing agent even when compared with 8-methoxypsoralen (8-MOP), which forms both monoadducts and crosslinks. At low radiation exposures 8-MOP is more effective than angelicin; as the radiation exposure is increased angelicin is capable of inducing the same peak level of virus expression as was 8-MOP. It appears that the induction of SV40 is a phenomenon possibly related to the repair of monoadducts.     

Decreased survival of UV-irradiated Staphylococcus aureus in the presence of 8-methoxypsoralen in the post-irradiation plating medium

For Staphylococcus aureus, the presence of 8-methoxypsoralen (8-MOP) in the post-irradiation plating medium increased the lethal effect of far-UV light (FUV; approximately 254 nm) and of 8-MOP plus near-UV light (8-MOP+NUV; approximately 365 nm), an effect similar to that caused by acriflavine which inhibits DNA repair. In the repair-proficient strain, the presence of 8-MOP in the plating medium was almost as effective in inhibiting the repair of damage caused by FUV as that caused by 8-MOP photoadditions. Survival data obtained with Rec(-)-like and Uvr(-)-like strains suggest that 8-MOP in the plating medium, although possibly inhibiting recombination repair, was much more effective in inhibiting excision repair of FUV damage. Regarding 8-MOP+NUV treatment, 8-MOP in the plating medium had a lesser effect in the repair-deficient strains, differing from that observed after FUV treatment, which is consistent with the notion that different types of damage are caused by the two treatments.     

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.     

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.     

PROPHAGE INDUCTION, MUTAGENESIS AND CELL SURVIVAL OF AMES' MUTAGEN TESTER STRAINS AFTER 8-METHOXYPSORALEN PLUS ULTRAVIOLET LIGHT-A

Abstract— The furocoumarin 8-methoxypsoralen (8-MOP) is not detected as a mutagen in the standard Ames test either in the presence or absence of S9-mix and/or ultraviolet light-A (320–400 nm). The Ames strains have recently been shown to harbor bacteriophages that are inducible by carcinogens and mutagens. Psoralen (8-MOP) plus UVA (PUVA) was found to be a potent prophage inducing treatment. Induction was observed in TA1535, TA1538, TA98, TA100, TA1978 and TA1975 with 0.1 μg/ml of 8-MOP and 2.5 kJ/m² of UVA. PUVA is a potent bactericidal treatment at concentrations of 8-MOP above 0.5 μg/ml and 2.5 kJ/m² in tester strains TA1535, TA1538, TA98, and TA100. PUVA is known to be bactericidal, but the cytotoxicity observed in this study was unique in that the frameshift tester strains (TA1538 and TA98) were more sensitive to the lethal effects of PUVA than the base pair tester strains (TA1535 and TA100). The differential cytotoxicity in such closely related strains led to the examination of some of the strains from which the Ames strains were derived. The data suggest mutations introduced into the Ames strains to make them more sensitive to carcinogens and mutagens (Λ uvrB ) have resulted in an altered response to PUVA. It is postulated that TA1535 retains a DNA repair function that is lost by TA1538 during the selection for uvrB deficient strains.
PUVA is also unique in that plasmid pKM101 does not confer enhanced survival of TA100 compared to TA1535 in contrast to many other carcinogens and mutagens. In conclusion, these data show that PUVA induces a pathway leading to prophage induction and demonstrates the potential use of prophage induction assays to detect mutagens and carcinogens that may otherwise be lethal to the Ames tester strains.     

Duck Hepatitis B Virus Inactivation and 8-Methoxypsoralen Photoadduct Formation in Human Platelet Concentrates*

Photochemical inactivation (PCI) of virus and bacteria in platelet concentrates (PC) has been demonstrated using 8-methoxypsoralen (8-MOP) and long-wavelength UV light (UVA). To study inactivation of blood-borne virus, we have employed duck hepatitis B virus (DHBV), a model for human hepatitis B virus. A specific hepatocyte culture infectivity assay, with PCR detection, could measure 5–6 log₁₀ virus kill. The DHBV inactivation in PC was dependent on UVA dose, was enhanced when plasma was reduced from 100% to 20% and was limited by 8-MOP solubility in the reduced-plasma medium. Optimum conditions for PCI were 100 μg/mL 8-MOP in 20% plasma and 80% synthetic platelet storage medium. A radiolabeling assay for 8-MOP photoadducts in hepatocytes seeded into PC confirmed that DHBV inactivation reflected DNA modification and indicated that adduct formation was insensitive to minor variations in conditions. Kinetic modeling indicated that optimum adduct formation was a compromise between 8-MOP dark binding and optical transmittance and that plasma proteins competed for 8-MOP binding. The PCI results in various media correlated with corresponding DNA modification densities and were compared to statistical models incorporating DHBV characteristics and predictions of 8-MOP crosslink formation between DNA strands. Behavior was consistent with one or a small number of lethal modifications per DNA strand, including monoadducts, but probably not crosslinks alone. A minor subpopulation of DHBV was found to be, somewhat more difficult to inactivate, consistent with three-fold lower modification, due possibly to single-stranded DNA character or host repair of photoadducts.     

THE TREATMENT OF MASTOCYTOMA CELLS WITH 8-METHOXYPSORALEN AND LONG-WAVELENGTH ULTRAVIOLET RADIATION ENHANCES CELLULAR IMMUNOGENICITY: PRELIMINARY RESULTS

Evidence for the increased immunogenicity of mastocytoma cells (P815) treated with 8-methoxypsoralen (8-MOP) and long-wavelength ultraviolet radiation (UVA) is presented. A highly tumorigenic clone (P1) became much less tumorigenic (tum^-) after repetitive phototreatments with 8-MOP (16 ng/mL) and UVA (1 J/cm²). The yield of tum^- clones was proportional to the number of phototreatments. In a pilot study in which P1 cells were treated with three successive rounds of 8-MOP/UVA, one clone out of 73 was tum^-. In a second series of experiments, the P1 cells were treated 10 times and 4 out of 100 clones were much less tumorigenic. When some of the tum^-clones were administered intraperitoneally to DBA/2 mice, significant protection against challenge with the original P1 clone was observed. In addition, the transfer of immune cells from tum^--treated mice allowed the transfer of resistance to other tum^- clones to immunosuppressed mice (650 rad). These results are consistent with earlier literature showing the potent mutagen, N -methyl- N' -nitrosoguanidine, led to mutations in P1 that altered the expression of new surface antigens, which stimulated the murine immune system such that there was also cross recognition of shared antigens on untreated P1 cells used to challenge the immunized mice. The increased immunogenicity that resulted from the less mutagenic 8-MOP/UVA treatment may arise by a similar mechanism and may be responsible in part for the efficacy of 8-MOP/UVA photochemotherapy for the treatment of cutaneous T cell lymphoma.     

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)