Induction Of Photoproducts Research Articles

Fluoroquinolone Photodegradation Influences Specific Basophil Activation

Fluoroquinolones (FQs) are photoreactive drugs, but it is not known whether laboratory light exposure can influence the induction of photoproducts and modify in vitro test results. The basophil activation test (BAT) has proven to be useful for evaluating immunoglobulin E (IgE)-mediated hypersensitivity to FQs, with a higher percentage of positive responders with ciprofloxacin (CIP) than with moxifloxacin (MOX). We studied the effect of laboratory light on CIP and MOX degradation, and drug-protein conjugate formation, and its influence on the BAT for evaluating IgE-mediated hypersensitivity to FQs. The results showed an important decrease in fluorescence emission intensity under light compared to dark conditions for MOX, and that BAT positivity was lower in light (17.9%) than in dark (35.7%) conditions. No changes were found for CIP in either fluorescence emission intensity or BAT results (46.4% in both conditions). We can conclude that light exposure is a critical factor in BAT results when photolabile drugs like MOX are used. Therefore, light is important when interpreting in vitro results.

Immediate Hypersensitivity To Quinolones: Drug Photodegradation Influences The Specific Basophil Activation

Basophil activation test (BAT) can be useful for evaluating immediate hypersensitivity to fluoroquinolones with higher percentage of positive responders with ciprofloxacin than with moxifloxacin. As fluoroquinolones are photoreactive, whether laboratory light exposure can influence in the induction of photoproducts and therefore modify BAT results is unknown. To analyze the effect of laboratory light in ciprofloxacin and moxifloxacin degradation, drug-protein conjugate formation and therefore in basophil activation in patients with immediate hypersensitivity reactions to fluoroquinolones.

P53 Mutant Human Glioma Cells Are Sensitive to UV-C-Induced Apoptosis Due to Impaired Cyclobutane Pyrimidine Dimer Removal

The p53 protein is a key regulator of cell responses to DNA damage, and it has been shown that it sensitizes glioma cells to the alkylating agent temozolomide by up-regulating the extrinsic apoptotic pathway, whereas it increases the resistance to chloroethylating agents, such as ACNU and BCNU, probably by enhancing the efficiency of DNA repair. However, because these agents induce a wide variety of distinct DNA lesions, the direct importance of DNA repair is hard to access. Here, it is shown that the induction of photoproducts by UV light (UV-C) significantly induces apoptosis in a p53-mutated glioma background. This is caused by a reduced level of photoproduct repair, resulting in the persistence of DNA lesions in p53-mutated glioma cells. UV-C-induced apoptosis in p53 mutant glioma cells is preceded by strong transcription and replication inhibition due to blockage by unrepaired photolesions. Moreover, the results indicate that UV-C-induced apoptosis of p53 mutant glioma cells is executed through the intrinsic apoptotic pathway, with Bcl-2 degradation and sustained Bax and Bak up-regulation. Collectively, the data indicate that unrepaired DNA lesions induce apoptosis in p53 mutant gliomas despite the resistance of these gliomas to temozolomide, suggesting that efficiency of treatment of p53 mutant gliomas might be higher with agents that induce the formation of DNA lesions whose global genomic repair is dependent on p53.

Validation of Phage T7 Biological Dosimeter by Quantitative Polymerase Chain Reaction Using Short and Long Segments of Phage T7 DNA ¶

Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.

Validation of Phage T7 Biological Dosimeter by Quantitative Polymerase Chain Reaction Using Short and Long Segments of Phage T7 DNA¶

Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.

Ultraviolet Photoproduct Levels in Melanocytic Nevi and Surrounding Epidermis in Human Skin In Situ

Melanocytic nevi are localized benign proliferations of melanocytes. The number of nevi has been shown to be the major risk marker for the development of cutaneous melanoma. This study compares the induction of photoproducts in nevi and in surrounding skin after exposure to solar-simulating radiation. Cyclobutane pyrimidine dimers (TT=T and TT=C) and 6-4 photoproducts (TT-T and TT-C) were measured in 20 nevi and 20 surrounding skin samples obtained from 14 subjects, using a 32P-postlabeling method. The amount of all four types of photoproducts in nevi was found to be 3-5-fold lower than that in surrounding skin, and the difference was statistically significant (paired t test, p < 0.01). In nevi, the photoproduct level was significantly associated with the color of nevi (the lowest level in the darkest color of nevi; r = -0.86, p < 0.01 for TT=T; r = -0.68, p < 0.01 for TT=C). Our findings suggest that the magnitude of the DNA damage is not a sole risk marker for the development of cutaneous melanoma.

Protective Effects of Tanning on Cutaneous DNA Damage in situ

Background: The incidence of skin cancer, the most common type of cancer in the Western world, has been shown to be associated with the degree of exposure to solar radiation. However, little is known on how human skin can be protected against UV-induced DNA damage by constitutive and induced pigmentation. Objective: To study the effect of skin pigmentation induced by a sunbed-type of treatment on the formation of UV-induced DNA damage in human skin in situ. Methods: A photoproduct assay was performed in untanned and tanned skin of healthy volunteers. Results: There is no significant difference in the induction of photoproducts between untanned and tanned skin. Conclusion: Our data demonstrate that constitutive skin pigmentation is more efficient than the induced one in protection against formation of photoproducts.

Bound transcription factor suppresses photoproduct formation in the NF-kappa B promoter.

The relationship between purified transcription factor p50 binding and ultraviolet light-induced DNA damage formation in the NF-kappa B promoter element was investigated. The effect of bound transcription factor on cyclobutane dimer formation was quantified using Maxam-Gilbert analysis of irradiated substrate digested with T4 phage endonuclease V. Two methods were employed for cleaving (6-4) photoproducts. Sites of (6-4) photoproducts cleaved by piperidine showed a general suppression in the presence of bound p50 protein similar to that observed for cyclobutane dimers. In contrast to piperidine, digestion with ultraviolet damage endonuclease (UVDE) from Saccharomyces pombe subsequent to cyclobutane dimer reversal by photolyase displayed a broader spectrum of damaged sites. Whereas some of these sites were suppressed by bound p50 protein, some remained unaffected and one site showed increased (6-4) photoproduct induction. These data illustrate the advantage of UVDE over piperidine for studying (6-4) photoproducts at the sequence level and suggest that this approach may be useful for footprinting transcription factor binding in other promoters.

Effect of age on the formation and repair of UV photoproducts in human skin in situ

Ultraviolet radiation (UVR)-induced photoproducts can be measured by a number of methods. The newly developed 32P-postlabelling method is feasible in molecular epidemiological studies due to its sensitivity, specificity and little amount DNA needed. We applied the 32P-postlabelling method to investigate the induction and repair of photoproducts (cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts) after UVR in human skin in situ and studied the effects of age, skin type and gender. The study included 30 subjects aged 32–78 years. The photoproduct induction levels varied 7- to 15-fold between the individuals tested. All four types of photoproducts were induced at a higher frequency in the older population (≥50 years) than in the younger population (<50 years). Individuals with skin type I and II had a higher CPD induction frequency than individuals with skin type III and IV. In both cases, the differences in thymidylyl (3′–5′) thymidylyl (3′–5′)-2′-deoxycytidine induction reached statistical significant levels (p<0.05). Photoproduct repair rates 24 h and 48 h after UV irradiation showed a large inter-individual variation. No clear effects of age, skin type or gender on DNA repair could be detected. Our data suggest that UV-induced DNA photoproduct levels increase with age.

Effect of Constitutional Pigmentation on Ultraviolet B-Induced DNA Damage in Fair-Skinned People

Ultraviolet light has been implicated as a dominant factor in skin cancer development. Skin pigmentation is traditionally regarded as an important protection against skin cancer. Yet, little is known about how skin pigmentation is modulating induction of DNA damage, which is the primary event in carcinogenesis. We applied a recently developed 32P-postlabeling technique to measure the effect of constitutional pigmentation on the formation of major ultraviolet-induced DNA damage in human skin in vivo. The induction of photoproducts showed a statistically significant negative correlation with erythemal response and skin pigmentation. Our results demonstrated that the constitutional pigmentation is efficiently guarding DNA against the formation of photoproducts. The difference in melanin content is likely to be one of the reasons for the observed interindividual variation in levels of DNA damage after the uniform exposure to ultraviolet B.

Assessment of the Effects of Various UV Sources on Inactivation and Photoproduct Induction in Phage T7 Dosimeter

The correlation between the biologically effective dose (BED) of a phage T7 biological dosimeter and the induction of cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts ((6-4)PD) in the phage DNA was determined using seven various UV sources. The BED is the inactivation rate of phage T7 expressed in HT7 units. The CPD and (6-4)PD were determined by lesion-specific monoclonal antibodies in an immunodot-blot assay. The various lamps induced these lesions at different rates; the relative induction ratios of CPD to (6-4)PD increased with increasing effective wavelength of irradiation source. The amount of total adducts per phage was compared to the BED of phage T7 dosimeter, representing the average number of UV lesions in phage. For UVC (200-280 nm radiation) and unfiltered TL01 the number of total adducts approximates the reading; however, UV sources having longer effective wavelengths produced fewer CPD and (6-4)PD. A possible explanation is that although the most relevant lesions by UVC are the CPD and (6-4)PD, at longer wavelengths other photoproducts can contribute to the lethal damage of phages. The results emphasize the need to study the biological effects of solar radiation because the lesions responsible for the lethal effect may be different from those produced by various UV sources.

Assessment of the Effects of Various UV Sources on Inactivation and Photoproduct Induction in Phage T7 Dosimeter

Assessment of the Effects of Various UV Sources on Inactivation and Photoproduct Induction in Phage T7 Dosimeter

Establishment and characterization of a monoclonal antibody recognizing the Dewar isomers of (6-4)photoproducts.

We established a monoclonal antibody (DEM-1) that recognizes UV-induced DNA damage other than cyclobutane pyrimidine dimers or (6-4)photoproducts. The binding of DEM-1 antibody to 254 nm UV-irradiated DNA increased with subsequent exposure to UV wavelengths longer than 310 nm, whereas that of the 64M-2 antibody specific for the (6-4)photoproduct decreased with this treatment. Furthermore, the increase in DEM-1 binding was inhibited by the presence of the 64M-2 antibody during the exposure. We concluded that the DEM-1 antibody specifically recognized the Dewar photoproduct, which is the isomeric form of the (6-4)photoproduct. The DEM-1 antibody, however, also bound to DNA irradiated with high fluences of 254 nm UV, suggesting that 254 nm UV could induce Dewar photoproducts without subsequent exposure to longer wavelengths of UV. Furthermore, an action spectral study demonstrated that 254 nm was the most efficient wavelength for Dewar photoproduct induction in the region from 254 to 365 nm, as well as cyclobutane dimers and (6-4)photoproducts, although the action spectrum values in the UV-B region were significantly higher compared with those for cyclobutane dimer and (6-4)photoproduct induction.

WAVELENGTH DEPENDENT FORMATION OF THYMINE DIMERS AND (6‐4)PHOTOPRODUCTS IN DNA BY MONOCHROMATIC ULTRAVIOLET LIGHT RANGING FROM 150 TO 365 nm

We investigated the wavelength dependence of cyclobutane thymine dimer and (6-4)photoproduct induction by monochromatic UV in the region extending from 150 to 365 nm, using an enzyme-linked immunosorbent assay with two monoclonal antibodies. Calf thymus DNA solution was irradiated with 254-365 nm monochromatic UV from a spectrograph, or with 220-300 nm monochromatic UV from synchrotron radiation. Thymine dimers and (6-4)photoproducts were fluence-dependently induced by every UV below 220 nm extending to 150 nm under dry condition. We detected the efficient formation of both types of damage in the shorter UV region, as well as at 260 nm, which had been believed to be the most efficient wavelength for the formation of UV lesions. The action spectra for the induction of thymine dimers and (6-4)photoproducts were similar from 180 to 300 nm, whereas the action spectrum values for thymine dimer induction were about 9- and 1.4-fold or more higher than the values for (6-4)photoproduct induction below 160 nm and above 313 nm, respectively.

CELL SURVIVAL CHARACTERISTICS AND MOLECULAR RESPONSES OF ANTARCTIC PHYTOPLANKTON TO ULTRAVIOLET‐B RADIATION1

ABSTRACTTwelve species of Antarctic diatoms were studied to assess UV sensitivity in relation to cellular and molecular aspects of DNA damage and repair. Responses of cell survival, induction of DNA damage, and DNA repair capacity were determined. There was a wide range of interspecific UV‐sensitivity among diatoms. D37 values (average fluence to kill one cell) ranged from 681 J · m−2 (most sensitive) to 25,338 J · m−2 (most resistant). Molecular analysis (by radioimmunoassay) of UV‐induced DNA damage [induction of cys‐syn cyclobutane dimers and pyrimidine (6‐4) pyrimidone photoproducts] also revealed considerable variability among species [0.98–84 lesions · (108 daltons DNA)−1 induced by exposure to 2500 J · m−2]. Repair of DNA damage ranged from 0.18 to 2.72 lesions removed · (108 daltons DNA)−1 in 6 h; removal represented 0.72–73.5% of initial damage. Comparison of cellular responses associated with photoenhanced repair and nucleotide excision (“dark”) repair indicated that light‐mediated correction of UV damage was an important factor in cell survival. There was a relationship between the number of photoproducts induced and cell survival, but not between repair efficiency and survival. The data also indicate a general dependence of photoproduct induction and D37 values on cell size and shape (expressed as the surface area: volume ratio which ranged from 0.07 to 0.66 between species) and suggest that these factors are indicators of UV sensitivity. Smaller cells with greater surface area: volume ratios sustained more damage per unit of DNA, had lower D37 values, and were more sensitive to UV exposure. The wide species variations observed in molecular and cellular responses to UV exposure emphasize the ecological implications of changes in natural UV regimes. These changes can act as determinants of cell size and taxonomic structure within phytoplankton communities and have as yet unknown effects on trophic interactions within the Antarctic ecosystem.

In situ (6-4)photoproduct determination by laser cytometry and autoradiography

The UV-induced (6-4)photoproducts and their repair in individual human cells were quantitatively determined by argon-laser imaging microspectrofluorometry or autoradiography with a well-characterized monoclonal antibody against (6-4)photoproducts. (6-4)Photoproduct induction curves were linear as a function of UV dose, using both methods. The formation of (6-4)photoproducts was detected in the cells irradiated with as low as 10 and 25 J/m2 of UV by autoradiography and laser cytometry, respectively. Normal cells repaired more than 80% of the initial damage within 4 h post irradiation. In contrast, almost no repair was observed in xeroderma pigmentosum cells (complementation group A) within 8 h.

Cyclobuta-dithymidine induction by solar-simulating UV radiation in human skin: II. Individual responses

The purpose of this study was to examine cyclobuta-dithymidine (TOT) photoproduct induction and persistence in human skin exposed in situ to simulated solar UV radiation. Small areas of untanned skin in nineteen individuals were exposed using a solar UV apparatus that simulates both the spectrum and intensity of the UV portion of summer midday sunlight at 39°N latitude. The equivalent of approximately 60 min of sunlight exposure (72KJ/m2) was administered, and TOT photoproducts were quantitated by enzyme-linked immunosorbent assay (ELISA) of DNA extracted from skin punch biopsies. Net yields of TOT photoproducts were determined in 10 individuals, with the majority (7 of 10) between 0.02 and 0.12 TOT per kilobase (Kb). The three remaining individuals had higher levels of photoproducts but were not unusually sensitive to solar UV as determined by minimal erythema dose (MED). Percentage loss of TOT photoproducts 4 h after exposure was determined in nine individuals to be 80.2 ±14.0%.

Sequence effect on alkali-sensitive sites in UV-irradiated SV40 DNA.

Ultraviolet light at 254 nm induces various kinds of DNA damage. We have located and quantified the pyrimidine (6-4) pyrimidone photoproducts along three hundred and forty two nucleotides of SV40 DNA. The level of photoproduct induction varies greatly according to the position on the DNA, but unlike what happens with pyrimidine dimers, the very adjacent nucleotides do not play a major role in the frequency of formation. A new alkali-sensitive site has been found on the ACA sequence after UV irradiation. This complex lesion is insensitive to the T4 endonuclease V and the E. coli photolyase, and may be involved with mutagenesis.

Induction of photoproducts in synthetic polynucleotides by far and near ultraviolet radiation.

AbstractSingle and double‐stranded polynucleotides of thymine and cytosine have been used to analyse the photoproducts produced by irradiation with far or near UV light. Reversed‐phase high performance liquid chromatography was used to detect and quantify cyclobutane dimers and Pyr(6–4)Pyo adducts produced by 254 nm. At 320 nm 10‐times less Thy(5–6)Thy dimers and one half the number of Cyt(5–6)Cyt dimers were induced; no Pyr(6–4)Pyo adducts were evident.HPLC has recently been applied to the isolation and characterization of various nucleic acid substituents and their UV‐photoproducts. The relative retention times of pyrimidines and their UV photoproducts on HPLC reflect differences in the hydrophobicity of the compounds being separated. Hence, the more hydrophobic (less polar) a compound is, the greater its capacity to bind to the column and the greater its retention time; for T o T, C&lt;&gt;T and C&lt;&gt;C dimers this difference may result from variations in the number of methyl moieties (Cadet et al., 1983). The retention time of the compound also depends on its stereochemistry. Separation of the four stereoisomers of T&lt;&gt;T by HPLC shows that compounds which are molecular equivalents can be more or less accessible to the non‐polar stationary phase depending on their conformation (Cadet, 1980). The relatively long retention times of the bipyrimidine photoadducts suggest a structural configuration which allows greater access to the hydrophobic moieties of the molecule.It is intriguing to consider that this difference in conformation may also be reflected in DNA–protein interactions such as binding by UV‐endonucleases or antibodies directed against UV photodamage. Proteins can be used as sensitive probes for photoproduct induction and repair but an accurate evaluation of their specificity is required. It was the intent of this paper not only to compare the induction parameters for various dimers in pyrimidine homo‐polymers but to provide controlled substrates which can be used to define the relative binding efficiencies of repair enzymes and antibodies for different types of photoproducts.