Evaluation of photodegradation, phototoxicity and photogenotoxicity of ofloxacin in ointments with sunscreens and in solutions.

Fluoroquinolones are widely used anti-bacterial agents that are known to exhibit moderate to severe phototoxicity. Furthermore some of them reveal photogenotoxicity under UV irradiation. Incidence of side effects due to light exposure may be augmented, if the medicament is used topically. The main goal of this work was to compare the extent of photodegradation of ofloxacin in ointments with various excipients: hydrated or non-hydrated base and the addition of sunscreens: bisoctrizole (Tinosorb M) and bemotrizinol (Tinosorb S). The next goal of present work was the analysis of phototoxicity and photogenotoxicity of ofloxacin photodegradation products in tested ointments and in solutions with the umu-test, the test of mitotic gene conversion with Saccharomyces cerevisiae D7 and the micronucleus assay with V79 Chinese hamster cell line. At the same time an attempt was made to determinate the photodegradation products of ofloxacin in different unguents variants. We observed a significant photoprotective effect in ointment with Tinosorb M. We did not evaluated relevant differences regarding the genotoxicity and toxicity of unguents. However, the pre-irradiated ofloxacin solutions in comparison to samples stored in the dark were significantly more genotoxic to bacteria, slightly increased the number of micronuclei in V79 cell line and were toxic to the yeast strain.

The role of Exo1p exonuclease in DNA end resection to generate gene conversion tracts in Saccharomyces cerevisiae.

The yeast Exo1p nuclease functions in multiple cellular roles: resection of DNA ends generated during recombination, telomere stability, DNA mismatch repair, and expansion of gaps formed during the repair of UV-induced DNA damage. In this study, we performed high-resolution mapping of spontaneous and UV-induced recombination events between homologs in exo1 strains, comparing the results with spontaneous and UV-induced recombination events in wild-type strains. One important comparison was the lengths of gene conversion tracts. Gene conversion events are usually interpreted as reflecting heteroduplex formation between interacting DNA molecules, followed by repair of mismatches within the heteroduplex. In most models of recombination, the length of the gene conversion tract is a function of the length of single-stranded DNA generated by end resection. Since the Exo1p has an important role in end resection, a reduction in the lengths of gene conversion tracts in exo1 strains was expected. In accordance with this expectation, gene conversion tract lengths associated with spontaneous crossovers in exo1 strains were reduced about twofold relative to wild type. For UV-induced events, conversion tract lengths associated with crossovers were also shorter for the exo1 strain than for the wild-type strain (3.2 and 7.6 kb, respectively). Unexpectedly, however, the lengths of conversion tracts that were unassociated with crossovers were longer in the exo1 strain than in the wild-type strain (6.2 and 4.8 kb, respectively). Alternative models of recombination in which the lengths of conversion tracts are determined by break-induced replication or oversynthesis during strand invasion are proposed to account for these observations.

The epistatic relationship between BRCA2 and the other RAD51 mediators in homologous recombination.

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA-damage site.

AKT1 represses gene conversion induced by different genotoxic stresses and induces supernumerary centrosomes and aneuploidy in hamster ovary cells.

The oncogenic kinase AKT1 is frequently overexpressed or activated in sporadic breast and ovarian cancers. In human breast tumors, we have previously shown that AKT1 represses homologous recombination (HR) induced by one double-strand break (DSB). To further analyze the impact of AKT1 on HR, we ectopically expressed wild-type or mutant forms of AKT1 in a hamster ovary cell line containing an intrachromosomal substrate for monitoring HR. In this cell line, AKT1 repressed HR induced by different genotoxic stresses including ionizing radiation, UV-C and one single DSB introduced into the intrachromosomal substrate. Consistently, AKT1 disrupted RAD51 foci formation, showing that AKT1 specifically affects gene conversion. Concomitantly, AKT1 represses both BRCA1 foci formation and HR stimulation resulting from BRCA1 overexpression, showing that AKT1 affects BRCA1-mediated HR functions, also in another species (hamster) and in another type of cell tissue (ovary cells). Finally, consistent with the HR defects, active AKT1 expression induces supernumerary centrosomes and aneuploidy. In addition to its impact on cell proliferation and apoptosis, the present data propose a novel oncogenic function for AKT1, by producing genomic instability as a consequence of HR repression.

Characterization of the mycobacterial NER system reveals novel functions of the uvrD1 helicase.

In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB or the helicase uvrD1 gene and a double knockout lacking both genes were constructed, and their sensitivities to a series of DNA-damaging agents were analyzed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and interstrand cross-links. In addition, it could be shown to exert a protective effect against oxidizing and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria (which lack the postreplicative mismatch repair system) NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches.

Role of the Saccharomyces cerevisiae Rad51 paralogs in sister chromatid recombination.

Rad51 requires a number of other proteins, including the Rad51 paralogs, for efficient recombination in vivo. Current evidence suggests that the yeast Rad51 paralogs, Rad55 and Rad57, are important in formation or stabilization of the Rad51 nucleoprotein filament. To gain further insights into the function of the Rad51 paralogs, reporters were designed to measure spontaneous or double-strand break (DSB)-induced sister or nonsister recombination. Spontaneous sister chromatid recombination (SCR) was reduced 6000-fold in the rad57 mutant, significantly more than in the rad51 mutant. Although the DSB-induced recombination defect of rad57 was suppressed by overexpression of Rad51, elevated temperature, or expression of both mating-type alleles, the rad57 defect in spontaneous SCR was not strongly suppressed by these same factors. In addition, the UV sensitivity of the rad57 mutant was not strongly suppressed by MAT heterozygosity, even though Rad51 foci were restored under these conditions. This lack of suppression suggests that Rad55 and Rad57 have different roles in the recombinational repair of stalled replication forks compared with DSB repair. Furthermore, these data suggest that most spontaneous SCR initiates from single-stranded gaps formed at stalled replication forks rather than DSBs.

Intermediate frequency magnetic fields do not have mutagenic, co-mutagenic or gene conversion potentials in microbial genotoxicity tests.

We used bacterial mutation and yeast genotoxicity tests to evaluate the effects of intermediate frequency (IF; 2 kHz, 20 kHz and 60 kHz) magnetic fields (MFs) on mutagenicity, co-mutagenicity and gene conversion. We constructed a Helmholtz type exposure system that generated vertical and sinusoidal IF MFs, such as 0.91 mT at 2 kHz, 1.1 mT at 20 kHz and 0.11 mT at 60 kHz. Mutagenicity, co-mutagenicity and gene conversion assays were performed for each of the three MF exposure conditions. Mutagenicity testing was performed in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and two strains of Escherichia coli (WP2 uvrA and WP2 uvrA/pKM101) to cover a wide spectrum of point mutations. For co-mutagenicity tests, we used four sensitive test strains (TA98, TA100, WP2 uvrA and WP2 uvrA/pKM101) with five chemical mutagens (t-butyl hydroperoxide (BH, a hydroxyl free radical precursor), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, DNA reactive reagents), benz[a]pyrene (BaP) and 2-aminoanthracene (2AA, DNA reactive promutagens). Gene conversion testing was performed in the yeast test strain, Saccharomyces cerevisiae XD83. We also examined the effects on the repair process of DNA damage by UV irradiation. No statistically significant effects were observed between exposed and control groups in any of the genotoxicity tests, indicating that the IF MFs (0.91 mT at 2 kHz, 1.1 mT at 20 kHz or 0.11 mT at 60 kHz) do not have mutagenic or co-mutagenic potentials for the chemical mutagens tested under these experimental conditions. Our findings also indicate that these IF MFs do not induce gene conversion or affect the repair process of DNA damage in eukaryotic cells.

Antigenotoxic effects of three essential oils in diploid yeast (Saccharomyces cerevisiae) after treatments with UVC radiation, 8-MOP plus UVA and MMS.

Essential oils (EOs) extracted from medicinal plants such as Origanum compactum, Artemisia herba alba and Cinnamomum camphora are known for their beneficial effects in humans. The present study was undertaken to investigate their possible antigenotoxic effects in an eukaryotic cell system, the yeast Saccharomyces cerevisiae. The EOs alone showed some cytotoxicity and cytoplasmic petite mutations, i.e. mitochondrial damage, but they were unable to induce nuclear genetic events. In combination with exposures to nuclear mutagens such as 254-nm UVC radiation, 8-methoxypsoralen (8-MOP) plus UVA radiation and methylmethane sulfonate (MMS), treatments with these EOs produced a striking increase in the amount of cytoplasmic petite mutations but caused a significant reduction in revertants and mitotic gene convertants induced among survivors of the diploid tester strain D7. In a corresponding rho0 strain, the level of nuclear genetic events induced by the nuclear mutagens UVC and 8-MOP plus UVA resulted in the same reduced level as the combined treatments with the EOs. This clearly suggests a close relationship between the enhancement of cytoplasmic petites (mitochondrial damage) in the presence of the EOs and the reduction of nuclear genetic events induced by UVC or 8-MOP plus UVA. After MMS plus EO treatment, induction of these latter events was comparable at least per surviving fraction in wildtype and rho0 cells, and apparently less dependent on cytoplasmic petite induction. Combined treatments with MMS and EOs clearly triggered switching towards late apoptosis/necrosis indicating an involvement of this phenomenon in EO-induced cell killing and concomitant decreases in nuclear genetic events. After UVC and 8-MOP plus UVA plus EO treatments, little apoptosis and necrosis were observed. The antigenotoxic effects of the EOs appeared to be predominantly linked to the induction of mitochondrial dysfunction.

Conversión y reversión génica en Saccharomyces cerevisiae. Un modelo para el estudio del daño producido por radiación gamma / Gen conversion and reversion events in Saccharomyces cerevisiae. A model for the study of gamma radiation damage

Se estudió la radiosensibilidad y la cinética de inducción de eventos de conversión y reversión génica en la cepa D7 de Sacharomyces cerevisiae frente a radiación gamma, en rangos de dosis entre 100-800 Gy y entre 50-300 Gy, respectivamente. Se utilizó una fuente de 60Co PX-g-30 con una tasa de dosis 49,43 Gy/min. La curva de supervivencia celular mostró un DL50 de 150 Gy. La cinética de muerte celular fue lineal con un ajuste superior a 98 por ciento. La inducción de eventos de conversión génica fue significativa respecto al control a partir de 50 Gy. Por el contrario, la reversión génica fue significativa solo a partir de 200 Gy. En general, las frecuencias de eventos de conversión génica fueron superiores a las de reversión, esto sugiere que la radiación gamma induce preferentemente eventos recombinogénicos. Tanto para los eventos de conversión como de reversión génica se obtuvo una dependencia exponencial de la dosis de radiación gamma. Se discutió sobre la utilidad relativa del ensayo para estudios de mutagénesis y antimutagénesis

Conversión y reversión génica en Saccharomyces cerevisiae. Un modelo para el estudio del daño producido por radiación gamma / Gen conversion and reversion events in Saccharomyces cerevisiae. A model for the study of gamma radiation damage

Se estudió la radiosensibilidad y la cinética de inducción de eventos de conversión y reversión génica en la cepa D7 de Sacharomyces cerevisiae frente a radiación gamma, en rangos de dosis entre 100-800 Gy y entre 50-300 Gy, respectivamente. Se utilizó una fuente de 60Co PX-g-30 con una tasa de dosis 49,43 Gy/min. La curva de supervivencia celular mostró un DL50 de 150 Gy. La cinética de muerte celular fue lineal con un ajuste superior a 98 por ciento. La inducción de eventos de conversión génica fue significativa respecto al control a partir de 50 Gy. Por el contrario, la reversión génica fue significativa solo a partir de 200 Gy. En general, las frecuencias de eventos de conversión génica fueron superiores a las de reversión, esto sugiere que la radiación gamma induce preferentemente eventos recombinogénicos. Tanto para los eventos de conversión como de reversión génica se obtuvo una dependencia exponencial de la dosis de radiación gamma. Se discutió sobre la utilidad relativa del ensayo para estudios de mutagénesis y antimutagénesis(AU)

Spontaneous and ultraviolet light-induced direct repeat recombination in mammalian cells frequently results in repeat deletion.

Recombination is enhanced by transcription and by DNA damage caused by ultraviolet light (UV). Recombination between direct repeats can occur by gene conversion without an associated crossover, which maintains the gross repeat structure. There are several possible mechanisms that delete one repeat and the intervening sequences (gene conversion associated with a crossover, unequal sister chromatid exchange, and single-strand annealing). We examined transcription-enhanced spontaneous recombination, and UV-induced recombination between neomycin (neo) direct repeats. One neo gene was driven by the inducible MMTV promoter. Multiple (silent) markers in the second neo gene were used to map conversion tracts. These markers are thought to inhibit spontaneous recombination, and our data suggest that this inhibition is partially overcome by high level transcription. Recombination was stimulated by transcription and by UV doses of 6-12J/m(2), but not by 18J/m(2). About 70% of spontaneous and UV-induced products were deletions. In contrast, only 3% of DSB-induced products were deletions. We propose that these product spectra differ because spontaneous and UV-induced recombination is replication-dependent, whereas DSB-induced recombination is replication-independent.

Characterization of the role played by the RAD59 gene of Saccharomyces cerevisiae in ectopic recombination.

The RAD52 group of genes in the yeast Saccharomyces cerevisiae controls the repair of DNA damage by a recombinational mechanism. Despite the growing evidence for physical and biochemical interactions between the proteins of this repair group, mutations in individual genes show very different effects on various types of recombination. The RAD59 gene encodes a protein with similarity to Rad52p which plays a role in the repair of damage caused by ionizing radiation. In the present study we have examined the role played by the Rad59 protein in mitotic ectopic recombination and analyzed the genetic interactions with other members of the repair group. We found that Rad59p plays a role in ectopic gene conversion that depends on the presence of Rad52p but is independent of the function of the RecA homologue Rad51p and of the Rad57 protein. The RAD59 gene product also participates in the RAD1-dependent pathway of recombination between direct repeats. We propose that Rad59p may act in a salvage mechanism that operates when the Rad51 filament is not functional.

DNA damage caused by etoposide and gamma-irradiation induces gene conversion of the MHC in a mouse non-germline testis cell line.

We have explored the effects of gamma-irradiation and etoposide on the gene conversion frequency between the endogenous major histocompatibility complex class II genes Abk and Ebd in a mouse testis cell line of non-germline origin with a polymerase chain reaction assay. Both gamma-rays and etoposide were shown to increase the gene conversion frequency with up to 15-fold compared to untreated cells. Etoposide, which is an agent that stabilise a cleavable complex between DNA and DNA topoisomerase II, shows an increased induction of gene conversion events with increased dose of etoposide. Cells treated with gamma-rays, which induce strand breaks, had an increased gene conversion frequency when they were subjected to low doses of irradiation, but increasing doses of irradiation did not lead to an increase of gene conversion events, which might reflect differences in the repair process depending on the extent and nature of the DNA damage. These results where DNA damage was shown to be able to induce gene conversion of endogenous genes in mouse testis cells suggests that the DNA repair system could be involved in the molecular genetic mechanism that results in gene conversion in higher eukaryotes like mammals.

Ionizing radiation-induced mutation of human cells with different DNA repair capacities.

We have observed significant differences in the response to ionizing radiation of two closely related human cells lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by alpha-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and alpha-particles. WTK1 is also more mutable by alpha-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while alpha-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-induced tk- mutants of TK6, were not induced significantly by alpha-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.

Induction of gene conversion in yeast cells continuously cultured at high radiation background.

The induction of genetic damage was investigated by culturing diploid yeast Saccharomyces cerevisiae D7 cells continuously at radiation levels ranging from 0.383 microSv/h to 1.275 mSv/h by selecting appropriate concentrations of tritiated water in the growth medium. These radiation levels correspond to 3-10,000 times the natural background. Parameters such as growth kinetics, gene conversion frequency at background radiation and after a challenging dose of acute gamma-radiation or alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were assessed. The gene conversion frequency in most of the assays was in the range of 5-10 convertants per 10(6) cells, as in the case of controls. However, a number of the cultures showed conversion frequencies above 20 per 10(6) viable cells. This stochastic phenomenon occurred more frequently in cells which were incubated at higher radiation levels and for longer durations. This suggests that radiation is responsible for the phenomenon. When subculturing continued beyond 900 h, gene conversion frequencies reverted back to normal values in all cultures in spite of elevated background radiation levels, thus suggesting an adaptive response. The generation time of the cells was 78 min in all cultures irrespective of the radiation level. The response of the cells cultured at elevated background radiation levels to subsequent challenging treatment with gamma-radiation or MNNG was identical to that of the control cultures. Our results suggest that in eukaryotic yeast, low-level radiation may induce an adaptive response to chronic radiation, whereas no such response could be detected when the cells were challenged with acute high-dose exposure or with MNNG.

Radiation-induced mitotic gene conversion frequency in yeast is modulated by the conditions allowing DNA double-strand break repair.

Repair of DNA double-strand breaks (DSB) involves recombinational processes which may lead to gene conversion (intragenic recombination). Using the diploid yeast mutant rad54-3 heteroallelic for his1 (his1-7/his1-1) and temperature conditional for DSB rejoining, radiation induced gene conversion was investigated as dependent on DSB repair under different postirradiation conditions. Gene conversion is negligible under conditions preventing DSB repair (36 degrees C). In contrast, gene conversion is observed when cells are incubated at the permissive temperature (23 degrees C) both under growth and nongrowth conditions. However, there is a much higher yield of convertants for cells incubated under growth as opposed to nongrowth conditions. These results can most plausibly be explained by the cell cycle regulated enhancement of the expression of genes such as PMS and POL3 known to be involved in gene conversion processes and/or the enhanced recombination in transcriptionally active genes. 'Nutrient stress' inducible responses and/or cell cycle specific recombination pathways leading to gene conversion events preferentially in S-phase cells seem to be less likely.

XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination.

The XRS2 gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that XRS2 also encodes an essential meiotic function. Spore inviability of xrs2 strains is rescued by a spo13 mutation, but meiotic recombination (both gene conversion and crossing over) is highly depressed in spo13 xrs2 diploids. The xrs2 mutation suppresses spore inviability of a spo13 rad52 strain suggesting that XRS2 acts prior to RAD52 in the meiotic recombination pathway. In agreement with the genetic data, meiosis-specific double-strand breaks at the ARG4 meiotic recombination hotspot are not detected in xrs2 strains. Despite its effects on meiotic recombination, the xrs2 mutation does not prevent mitotic recombination events, including homologous integration of linear DNA, mating-type switching and radiation-induced gene conversion. Moreover, xrs2 strains display a mitotic hyper-rec phenotype. Haploid xrs2 cells fail to carry out G2-repair of gamma-induced lesions, whereas xrs2 diploids are able to perform some diploid-specific repair of these lesions. Meiotic and mitotic phenotypes of xrs2 cells are very similar to those of rad50 cells suggesting that XRS2 is involved in homologous recombination in a way analogous to that of RAD50.

Effects of K-shell X-ray absorption of intracellular phosphorus on yeast cells.

The effects of K-shell absorption of phosphorus atoms on yeast cells were investigated using synchrotron X-rays that were tuned to the resonance absorption peak (2153 eV). Three types of cellular effect (cell inactivation, induction of gene conversion at the trp-5 locus, and cell membrane impairment (changes in the permeability] were measured. It was demonstrated that the enhancement factor was 1.4 at the resonance peak regarding both lethality and the induction of gene conversion in reference of off-peak irradiation (2146 and 2160 eV). No difference was found between the two off-peak irradiation energies. No cell membrane impairment was detected, irrespective of the X-ray photon energies employed within the fluence range tested. These results strongly suggest that K-shell X-ray absorption in the resonance mode by cellular phosphorus atoms causes significantly more cellular effects than the off-resonance mode of absorption, probably via some specific changes induced in the phosphates of the DNA strand. Calculations using the number of phosphorus atoms in a defined size of the trp locus (2127 base pairs) on the DNA and the absorption cross-section of the resonance mode of phosphorus showed that gene conversion is inducible at a rate of 0.13 per X-ray photon absorption per locus. These results are discussed regarding the modes of K-shell photoabsorption.