Mutagenic interaction between near-(365 nm) and far-(254 nm)ultraviolet radiation in repair-proficient and excision-deficient strains of Escherichia coli.

The mutational interaction between radiation at 365 and 254 nm was studied in various strains of E. coli by a mutant assay based on reversion to amino-acid independence in full nutrient conditions. In the two repair-proficient strains (K12 AB 1157 and B/r), pre-treatment with radiation at 365 nm strongly suppressed the induction of mutations by far-UV, a phenomenon accompanied by a strong lethal interaction. The frequency of mutations induced by far-UV progressively declined with increasing dose of near-UV. Far-UV-induced mutagenesis to T5 resistance was almost unaltered by pre-treatment with near-UV. In AB 1886 uvrA there was no lethal interaction between the two wavelengths but the mutagenic interaction was synergistic. This synergism was maximal at a 365-nm dose of 8 X 10(5) J m-2. It is proposed that in the wild-type strain, cells containing potentially mutagenic lesions are selectively eliminated from the population because of abortive excision of an error-prone repair-inducing signal. In excisionless strains, 365-nm radiation may be less damaging to the error-prone than to the error-free post-replication repair system. Alternatively, mutation may be enhanced because of the occurrence of error-prone repair of 365-nm lesions by a system that is not induced in the absence of 254-nm radiation.     

Artificial and solar UV radiation induces strand breaks and cyclobutane pyrimidine dimers in Bacillus subtilis spore DNA

The loss of stratospheric ozone and the accompanying increase in solar UV flux have led to concerns regarding decreases in global microbial productivity. Central to understanding this process is determining the types and amounts of DNA damage in microbes caused by solar UV irradiation. While UV irradiation of dormant Bacillus subtilis endospores results mainly in formation of the "spore photoproduct" 5-thyminyl-5,6-dihydrothymine, genetic evidence indicates that an additional DNA photoproduct(s) may be formed in spores exposed to solar UV-B and UV-A radiation (Y. Xue and W. L. Nicholson, Appl. Environ. Microbiol. 62:2221-2227, 1996). We examined the occurrence of double-strand breaks, single-strand breaks, cyclobutane pyrimidine dimers, and apurinic-apyrimidinic sites in spore DNA under several UV irradiation conditions by using enzymatic probes and neutral or alkaline agarose gel electrophoresis. DNA from spores irradiated with artificial 254-nm UV-C radiation accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, while DNA from spores exposed to artificial UV-B radiation (wavelengths, 290 to 310 nm) accumulated only cyclobutane pyrimidine dimers. DNA from spores exposed to full-spectrum sunlight (UV-B and UV-A radiation) accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, whereas DNA from spores exposed to sunlight from which the UV-B component had been removed with a filter ("UV-A sunlight") accumulated only single-strand breaks and double-strand breaks. Apurinic-apyrimidinic sites were not detected in spore DNA under any of the irradiation conditions used. Our data indicate that there is a complex spectrum of UV photoproducts in DNA of bacterial spores exposed to solar UV irradiation in the environment.     

Effects of XeCl UV308 nm laser radiation on survival and mutability of recA-proficient and recA-defective Escherichia coli strains

recA1, recA13 and recA56 are considered null alleles of the Escherichia coli recA gene because they were shown to have essentially no activity in vivo. In this study, we used strains harboring the recA null alleles and their recA-proficient congenic counterpart to assess the lethal and the mutagenic effects elicited by near-UV(308 nm) coherent radiation generated by a XeCl excimer laser. We compared these effects with those produced by a conventional far-UV(254 nm) germicidal lamp. Compared to the germicidal lamp, the excimer laser was able to better discriminate the different recA-defective strains on the basis of their UV-radiation sensitivity, which was progressively higher in the strains with the alleles in the order recA1, recA56 and recA13. This finding was consistent with previous data on residual biochemical activities of the respective mutated RecA proteins in vitro. The discrepancy between the results obtained with the lamp and laser irradiation suggested that the biological response to the two radiations involves distinct mechanisms. This hypothesis was supported by the evidence that exposure to near-UV(308 nm) radiation induced mutagenesis in recA-defective strains at an extent considerably greater than in recA-proficient strains. In contrast, far-UV(254 nm)-radiation-induced mutagenesis was reported to be largely dependent on a functional recA allele.     

Near-UV mutagenesis: photoreactivation of 365-nm-induced mutational lesions in Escherichia coli WP2s.

Reversion to tryptophan independence induced by 365-nm and 254-nm radiation was studied in Escherichia coli WP2s (B/r trp uvrA). Under aerobic conditions, the mutant frequency responses was of the fluence-square or "two-hit" type at both 365 and 254 nm when revertants were assayed on minimal agar supplemented with 2% nutrient broth (SEM plates). In contrast, when mutants were assayed on minimal agar supplemented with tryptophan only, the revertant yield was reduced to very low values at 365 nm, whereas values substantially greater than with SEM plates were obtained at 254 nm. Premutational lesions induced by both 365-nm and 254-nm radiation were photoreactivated more than 10-fold when assayed on SEM plates, implicating pyrimidine dimers as premutational lesions at both wavelengths. The strong photoreactivation of 365-nm-induced mutagenesis contrasted strikingly with the complete absence of photoreactivation of 365-nm-induced lethality in this strain.     

Evaluation of acute corneal damage induced by 222-nm and 254-nm ultraviolet light in Sprague–Dawley rats

Two hundred twenty-two nanometres ultraviolet (UV) light produced by a krypton–chlorine excimer lamp is harmful to bacterial cells but not skin. However, the effects of 222-nm UV light exposure to the eye are not fully known. We evaluated acute corneal damage induced by 222- and 254-nm UV light in albino rats. Under deep anaesthesia, 6-week-old Sprague–Dawley albino rats were exposed to UV light. The exposure levels of corneal radiation were 30, 150, and 600?mJ/cm². Epithelial defects were detected by staining with fluorescein. Superficial punctate keratitis developed in corneas exposed to more than 150?mJ/cm² of UV light, and erosion was observed in corneas exposed to 600?mJ/cm² of UV light. Haematoxylin and eosin staining also showed corneal epithelial defects in eyes exposed to 254-nm UV light. However, no damage developed in corneas exposed to 222-nm UV light. Cyclobutane pyrimidine dimer-positive cells were observed only in normal corneas and those exposed to 254-nm UV light. Although some epithelial cells were stained weakly in normal corneas, squamous epithelial cells were stained moderately, and the epithelial layer that was detached from the cornea exposed to 600?mJ/cm² of light was stained intensely in corneas exposed to 254-nm UV light. In the current study, no corneal damage was induced by 222-nm UV light, which suggested that 222-nm UV light may not harm rat eyes within the energy range and may be useful for sterilising or preventing infection in the future.     

Artificial and Solar UV Radiation Induces Strand Breaks and Cyclobutane Pyrimidine Dimers in Bacillus subtilis Spore DNA

The loss of stratospheric ozone and the accompanying increase in solar UV flux have led to concerns regarding decreases in global microbial productivity. Central to understanding this process is determining the types and amounts of DNA damage in microbes caused by solar UV irradiation. While UV irradiation of dormant Bacillus subtilis endospores results mainly in formation of the “spore photoproduct” 5-thyminyl-5,6-dihydrothymine, genetic evidence indicates that an additional DNA photoproduct(s) may be formed in spores exposed to solar UV-B and UV-A radiation (Y. Xue and W. L. Nicholson, Appl. Environ. Microbiol. 62:2221–2227, 1996). We examined the occurrence of double-strand breaks, single-strand breaks, cyclobutane pyrimidine dimers, and apurinic-apyrimidinic sites in spore DNA under several UV irradiation conditions by using enzymatic probes and neutral or alkaline agarose gel electrophoresis. DNA from spores irradiated with artificial 254-nm UV-C radiation accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, while DNA from spores exposed to artificial UV-B radiation (wavelengths, 290 to 310 nm) accumulated only cyclobutane pyrimidine dimers. DNA from spores exposed to full-spectrum sunlight (UV-B and UV-A radiation) accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, whereas DNA from spores exposed to sunlight from which the UV-B component had been removed with a filter (“UV-A sunlight”) accumulated only single-strand breaks and double-strand breaks. Apurinic-apyrimidinic sites were not detected in spore DNA under any of the irradiation conditions used. Our data indicate that there is a complex spectrum of UV photoproducts in DNA of bacterial spores exposed to solar UV irradiation in the environment.     

Photoreactivation of Escherichia coli after low- or medium-pressure UV disinfection determined by an endonuclease sensitive site assay

Photoreactivation of Escherichia coli after inactivation by a low-pressure (LP) UV lamp (254 nm), by a medium-pressure (MP) UV lamp (220 to 580 nm), or by a filtered medium-pressure (MPF) UV lamp (300 to 580 nm) was investigated. An endonuclease sensitive site (ESS) assay was used to determine the number of UV-induced pyrimidine dimers in the genomic DNA of E. coli, while a conventional cultivation assay was used to investigate the colony-forming ability (CFA) of E. coli. In photoreactivation experiments, more than 80% of the pyrimidine dimers induced by LP or MPF UV irradiation were repaired, while almost no repair of dimers was observed after MP UV exposure. The CFA ratios of E. coli recovered so that they were equivalent to 0.9-, 2.3-, and 1.7-log inactivation after 3-log inactivation by LP, MP, and MPF UV irradiation, respectively. Photorepair treatment of DNA in vitro suggested that among the MP UV emissions, wavelengths of 220 to 300 nm reduced the subsequent photorepair of ESS, possibly by causing a disorder in endogenous photolyase, an enzyme specific for photoreactivation. On the other hand, the MP UV irradiation at wavelengths between 300 and 580 nm was observed to play an important role in reducing the subsequent recovery of CFA by inducing damage other than damage to pyrimidine dimers. Therefore, it was found that inactivating light at a broad range of wavelengths effectively reduced subsequent photoreactivation, which could be an advantage that MP UV irradiation has over conventional LP UV irradiation.     

Comparative studies on photoreactivation of ultraviolet light-induced T4 endonuclease susceptible sites and sister-chromatid exchanges in Potorus cells

Photoreactivation (PR) of T4 endonuclease-susceptible sites (ESS) and sister-chromatid exchanges induced by ultraviolet light was investigated in Potorous tridactylis Pt K2 cells, using monochromatic light from a grating monochromator. Both ESS and SCE showed maximum PR at 350 nm and the action spectra of PR essentially overlapped between ESS and SCE at 350, 400 and 450 nm. Exposure to 325-nm light after UV irradiation induced additional ESS and SCE, but reduction of ESS was shown by increasing exposure to 325-nm light, and further induction of SCE was observed by the same treatment. A possible difference in mechanisms between induction of ESS and SCE is suggested at 325 nm, while similar causes for ESS and SCE, presumably pyrimidine dimers, are suggested by UV (254-nm) irradiation.     

Endonuclease III and endonuclease IV protect Escherichia coli from the lethal and mutagenic effects of near-UV irradiation.

In contrast to the DNA damage caused by far-UV (lambda < 290 nm), near-UV (290 < lambda < 400 nm) induced DNA damage is partially oxygen dependent, suggesting the involvement of reactive oxygen species. To test the hypothesis that enzymes that protect cells from oxidative DNA damage are also involved in preventing near-UV mediated DNA damage, isogenic strains deficient in one or more of exonuclease III (xthA), endonuclease IV (nfo), and endonuclease III (nth) were exposed to increasing levels of far-UV and near-UV. All strains, with the exception of the nth single mutant, were found to be hypersensitive to the lethal effects of near-UV relative to a wild-type strain. A triple mutant strain (nth nfo xthA) exhibited the greatest sensitivity to near-UV-mediated lethality. The triple mutant was more sensitive than the nfo xthA double mutant to the lethal effects of near-UV, but not far-UV. A forward mutation assay also revealed a significantly increased sensitivity for the triple mutant compared to the nfo xthA deficient strain in the presence of near-UV. However, the triple mutant was no more sensitive to the mutagenic effects of far-UV than a nfo xthA double mutant. These data suggest that exonuclease III, endonuclease IV, and endonuclease III are important in protection against near-UV-induced DNA damage.     

207-nm UV Light - A Promising Tool for Safe Low-Cost Reduction of Surgical Site Infections. I: In Vitro Studies

Background

0.5% to 10% of clean surgeries result in surgical-site infections, and attempts to reduce this rate have had limited success. Germicidal UV lamps, with a broad wavelength spectrum from 200 to 400 nm are an effective bactericidal option against drug-resistant and drug-sensitive bacteria, but represent a health hazard to patient and staff. By contrast, because of its limited penetration, ∼200 nm far-UVC light is predicted to be effective in killing bacteria, but without the human health hazards to skin and eyes associated with conventional germicidal UV exposure.

Aims

The aim of this work was to test the biophysically-based hypothesis that ∼200 nm UV light is significantly cytotoxic to bacteria, but minimally cytotoxic or mutagenic to human cells either isolated or within tissues.

Methods

A Kr-Br excimer lamp was used, which produces 207-nm UV light, with a filter to remove higher-wavelength components. Comparisons were made with results from a conventional broad spectrum 254-nm UV germicidal lamp. First, cell inactivation vs. UV fluence data were generated for methicillin-resistant S. aureus (MRSA) bacteria and also for normal human fibroblasts. Second, yields of the main UV-associated pre-mutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) were measured, for both UV radiations incident on 3-D human skin tissue.

Results

We found that 207-nm UV light kills MRSA efficiently but, unlike conventional germicidal UV lamps, produces little cell killing in human cells. In a 3-D human skin model, 207-nm UV light produced almost no pre-mutagenic UV-associated DNA lesions, in contrast to significant yields induced by a conventional germicidal UV lamp.

Conclusions

As predicted based on biophysical considerations, 207-nm light kills bacteria efficiently but does not appear to be significantly cytotoxic or mutagenic to human cells. Used appropriately, 207-nm light may have the potential for safely and inexpensively reducing surgical-site infection rates, including those of drug-resistant origin.     

Efficiency of pyrimidine dimer formation in Escherichia coli across UV wavelengths

AIMS: Inactivation of Escherichia coli as a function of ultraviolet (UV) wavelength was investigated by using the endonuclease-sensitive site (ESS) assay to quantify pyrimidine dimer formation. METHODS AND RESULTS: Ultraviolet dose-response curves were determined based on both log reduction in colony-forming units (CFU) and endonuclease-sensitive sites per kb DNA (ESS/kb) for monochromatic 254-nm low-pressure (LP) UV, polychromatic medium-pressure (MP) UV, 228 and 289-nm UV irradiation. UV irradiation from LP and MP UV sources were approx. equal in both CFU reduction and pyrimidine dimer formation at all UV doses studied; 228-nm irradiation was less effective than LP or MP, and 289-nm irradiation was the least effective in both CFU reduction and pyrimidine dimer formation. These results are in qualitative agreement with the absorption spectrum of pyrimidine bases in DNA. Results indicated an approx. linear relationship between ESS/kb and log CFU reduction. CONCLUSIONS: Formation of pyrimidine dimers in genomic DNA is primarily responsible for UV inactivation of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributed to fundamental understanding of UV disinfection and aids in UV reactor design.     

Detection and repair of a UV-induced photosensitive lesion in the DNA of human cells

Irradiation with UV light results in damage to the DNA of human cells. The most numerous lesions are pyrimidine dimers; however, other lesions are known to occur and may contribute to the overall deleterious effect of UV irradiation. We have observed evidence of a UV-induced lesion other than pyrimidine dimers in the DNA of human cells by measuring DNA strand breaks induced by irradiating with 313-nm light following UV (254-nm) irradiation. These breaks, measured by alkaline sucrose sedimentation, increased linearly with the dose of UV light over the range tested (10-40 J/m2). The breaks cannot be photolytically induced 5 h after a UV dose of 20 J/m2 in normal cells; however, in xeroderma pigmentosum variant cells, the breaks are inducible for up to 24 h after UV irradiation. Xeroderma pigmentosum group A cells in the same 5-h period show an increase in the number of strand breaks seen with 313-nm light photolysis from about 2 to 4 breaks/10(9) dalton DNA. These breaks can then be induced for up to 24 h. These data suggest that, in normal cells, the lesion responsible for this effect is rapidly repaired or altered; whereas, in xeroderma pigmentosum variant cells it seems to remain unchanged. Some change apparently occurs in the DNA of xeroderma pigmentosum group A cells which results in an increase in photolability. These data indicate a deficiency in DNA repair of xeroderma pigmentosum variant cells as well as in xeroderma pigmentosum group A cells.     

Preferential excision repair and non-preferential photoreactivation of pyrimidine dimers in the c-ras sequence of cultured goldfish cells

The time courses of excision repair and photoreactivation of pyrimidine dimers induced by 254-nm UV were examined in the genome overall and in the c-ras sequence of RBCF-1 cells derived from a goldfish, by the use of UV endonuclease of Micrococcus luteus and alkaline agarose gel electrophoresis. Excision repair was more efficient in the ras sequence than in the genome overall, whereas no differences in efficiency of photoreactivation were detected. These results suggest that excision repair is affected by the accessibility of chromatin, while photoreactivation is not.     

Inhibition of semiconservative DNA synthesis in ICR 2A frog cells by pyrimidine dimers and nondimer photoproducts induced by ultraviolet radiation

DNA synthesis was examined in ultraviolet (uv)-irradiated ICR 2A frog cells in which either pyrimidine dimers or nondimer photoproducts represented the major class of DNA lesions. Dimers were induced by exposure of cells to 254 nm uv, while nondimer photoproducts were induced by irradiation of cells with uv produced by a fluorescent sunlamp (FSL) that was filtered through 48A Mylar (removes wavelengths less than 310 nm). The FSL-irradiated cultures were also treated with photoreactivating light (PRL) which removed most of the small number of dimers induced by the irradiation, leaving a relatively pure population of nondimer photoproducts. In addition, cells were exposed to 60Co gamma rays. The cultures were pulse-labeled and the size distribution of the DNA synthesized was estimated using both sucrose gradient sedimentation and alkaline step elution. Using either of these techniques, it was found that the presence of dimers resulted in a reduction principally in the synthesis of high molecular weight (MW) DNA. In contrast, nondimer photoproducts caused a strong inhibition in the synthesis of low MW DNA, as was also observed in gamma-irradiated cells. Hence the induction of pyrimidine dimers in DNA mainly affected the elongation of replicons, whereas nondimer lesions primarily caused an inhibition of replicon initiation.     

Escherichia coli xthA mutants are sensitive to inactivation by broad-spectrum near-UV (300- to 400-nm) radiation.

Logarithmically growing and stationary-phase cells of Escherichia coli mutants lacking exonuclease III (xthA) were sensitive to inactivation by broad-spectrum near-UV (300- to 400-nm) radiation. The same xthA mutants were no more sensitive to far-UV wavelengths (200- to 300-nm) than was a strain bearing a functional xthA allele.     

Is DNA damage the signal for induction of thermal resistance? induction by radiation in yeast

Yeast, as well as higher eukaryotes, are induced to increase thermal resistance (thermotolerance) by prior exposure to a heat stress. Prior exposure to an acute dose of either 60Co gamma or 254-nm ultraviolet radiation, at sublethal or fractionally lethal doses, is shown to cause a marked increase in the resistance of Saccharomyces cerevisiae to killing by heat. Following a radiation exposure, thermal resistance increased with time during incubation in nutrient medium, and the degree of resistance reached was proportional to the dose received. Partial induction by radiation followed by maximum induction by heat did not produce an additive response when compared to a maximum induction by heat alone, suggesting that the same process was induced by both heat and radiation. Irradiation with 254-nm uv light followed by an immediate, partial photoreversal of the pyrimidine dimers with long-wavelength uv light resulted in a reduced level of resistance compared to cells not exposed to the photoreversal light, indicating that the cells specifically recognized pyrimidine dimers as a signal to increase their thermal resistance. Exposure to 254-nm uv or ionizing radiation induced thermal resistance in mutants defective in either excision repair (rad3, uv-sensitive) or recombinational repair (rad52, gamma-sensitive), suggesting that recognition and repair of DNA damage by these systems are not a part of the signal which initiates an increase in resistance to heat. The amount of induction, per unit dose, was greater in the DNA repair-deficient mutants than in the wild-type cells, suggesting that an increase in the length of time during which damage remains in the DNA results in an increase in the effectiveness of the induction. These data indicate that types of DNA damage as diverse as those produced by ionizing radiation and by ultraviolet light are recognized as a signal by the yeast cell to increase its thermal resistance. It is therefore suggested that heat-induced alterations in DNA or in DNA-dependent chromosomal organization may be the signal for heat induction of thermotolerance in this and other eukaryotes.     

Bistranded oxidized purine damage clusters: induced in DNA by long-wavelength ultraviolet (290-400 nm) radiation?

Bistranded clustered DNA damages involving oxidized bases, abasic sites, and strand breaks are produced by ionizing radiation and radiomimetic drugs, but it was not known whether they can be formed by other agents, e.g., nonionizing radiation. UV radiation produces clusters of cyclobutyl pyrimidine dimers, photoproducts that occur individually in high yield. Since long-wavelength UV (290-400 nm) radiation induces oxidized bases, abasic sites, and strand breaks at low yields, we tested whether it also produces clusters containing these lesions. We exposed supercoiled pUC18 DNA to UV radiation with wavelengths of >290 nm (UVB plus UVA radiation), and assessed the induction of bistranded clustered oxidized purine and abasic clusters, as recognized by Escherichia coli Fpg protein and E. coli Nfo protein (endonuclease IV), respectively, as well as double-strand breaks. These three classes of bistranded clusters were detected, albeit at very low yields (37 Fpg-OxyPurine clusters Gbp(-1) kJ(-1) m(2), 8.1 double-strand breaks Gbp(-1) kJ(-1) m(2), and 3.4 Nfo-abasic clusters Gbp(-1) kJ(-1) m(2)). Thus, these bistranded OxyPurine clusters, abasic clusters, and double-strand breaks are not uniquely induced by ionizing radiation and radiomimetic drugs, but their level of production by UVB and UVA radiation is negligible compared to the levels of frequent photoproducts such as pyrimidine dimers.     

Photoreactivation of cells and phages injuried by ultraviolet radiation in the ecological long-wave band]

Photoreactivation (PR) was measured after inactivation by far (254 nm), middle (300-315 nm) and near (315-400 nm) UV radiation of Paramecium caudatum and 8 strains of Escherichia coli differing in PR and dark repair capability. PR volume was high and practically the same after irradiation by far and middle UV, but PR was not observed in near UV-inactivated cells of all the strains. It is proposed that pyrimidine dimers are not significant in near UV lethal lesions in cells, as near UV-irradiated phages (T7 and lambdacI 857) are not photoreactivated in undamaged host bacterial cells.     

Evidence for excision repair in promitochondrial DNA of anaerobic cells of Saccharomyces cerevisiae.

The respiratory adaptation (i.e., essentially mitochondrial biogenesis) in the excision repair-defective rad3-type mutants of Saccharomyces cerevisiae undergoing transition from the anaerobic to the aerobic state is found to be far more sensitive to 254-nm ultraviolet radiation (UV) than that of the RAD wild-type strain. We confirm that mitochondria of aerobic cells of a RAD strain lack the excision repair capacity of UV-induced pyrimidine dimers at all doses tested (1-15 J/m2). In contrast, in promitochondria of anaerobic cells of the wild-type strain excision repair appears to take place. This process is very efficient at low doses (at 0.5-5 J/m2 100% of the UV endonuclease-sensitive sites disappear), whereas at high doses its efficiency is reduced by about 50%. The promitochondrial excision repair of pyrimidine dimers appears to be under nuclear control since it is blocked in the rad2 mutant. Finally photoreactivation is found to be operating in nuclei, mitochondria and promitochondria.