THE PHOTOTOXICITY OF 8-M-ETHOXYTHIONEPSORALEN AND 6-M-ETHYLTHIONECOUMARIN

The phototoxicity of 8-methoxythionepsoralen (8-MOTP) and 6-methylthione coumarin (6-MTC) when activated by UV-A has been investigated using a variety of Escherichia coli strains, Haemophilus influenzae transforming DNA and Escherichia coli pBR322 plasmid DNA. The results demonstrate that 8-MOTP is a strictly oxygen independent photosensitizer that is about 500-fold less efficient in forming lesions leading to equivalent lethality than is the parent compound from which it is derived (8-MOP). As is true for 8-MOP, 8-MOTP is capable of inducing histidine independent mutations in E. coli and inactivating transforming DNA consistent with DNA being a target for lesions induced by this molecule in the presence of UV-A. 6-MTC is a strongly oxygen dependent photosensitizer activated by UV-A when tested with either E. coli cells or transforming DNA in contrast to the parent compound (6-methylcoumarin; 6-MC) which is not phototoxic when treated with UV-A. These results imply that the membrane may be an important target leading to lethality. 6-MTC in the presence of UV-A can inactivate pBR322 plasmid and Haemophilus influenzae transforming DNA activity in vitro suggesting that DNA is a potential target for this molecule when activated by UV-A.     

Oxygen-independent inactivation of Haemophilus influenzae transforming DNA by monochromatic radiation: action spectrum, effect of histitine and repair.

Abstract— The action spectrum for the oxygen-independent inactivation of native transforming DNA from Haemophilus influenzae with near-UV radiation revealed a shoulder beginning at 334 and extending to 460 nm. The presence of 0.2 M histidine during irradiation produced a small increase in inactivation at 254, 290 and 313 nm, a large increase at 334 nm and a decrease in inactivation at 365, 405 and 460 nm. Photoreactivation did not reverse the DNA damage produced at pH 7.0 at 334, 365, 405 and 460 nm, but did reactivate the DNA after irradiation at 254, 290 and 313 nm. The inactivation of DNA irradiated at 254, 290 and 313 nm was considerably greater when the transforming ability was assayed in an excision-defective mutant compared with the wild type, although DNA irradiated at 334, 365, 405 and 460 nm showed smaller differences. These results suggest that the oxygen-independent inactivation of H. influenzae DNA at pH 7 by irradiation at 334, 365, 405 and 460 nm is caused by lesions other than pyrimidine dimers.     

THE EFFECTS OF HOST-CELL REACTIVATION ON ASSAY OF U.V.-IRRADIATED HAEMOPHILUS INFLUENZAE TRANSFORMING DNA

Abstract— The host cell reactivation (HCR) mechanism in Haemophilus influenzae cells is inhibited by sub-microgram concentrations of acriflavine (as is already known to be true for Escherichia coli ). Exposure of these cells to similar concentrations of the drug during bacterial transformation increases the apparent ultraviolet light (u.v.) sensitivity of previously irradiated transforming DNA, indicating a repair of this DNA after uptake by the cells under normal conditions. Repair is inhibited by applying acriflavine at any time up to one hour after competent cells contact the irradiated transforming DNA. The fraction of the u.v. damage repaired by HCR is very different for different genetic markers. Those markers which are most u.v. sensitive when assayed in the absence of acriflavine are most poorly repaired, suggesting that this is the reason for their higher sensitivity. For all markers the fraction of the damage repairable by in vitro photoreactivation is approximately constant, and strongly overlaps the damage repairable by HCR. The degree of HCR achieved can be altered by experimental treatment of the H. influenzae DNA prior to transformation. Thus treatment of irradiated DNA with an enzyme from Micrococcus lysodeikticus –known to attack u.v. damaged, but not undamaged DNA–prevents subsequent intracellular repair of the same u.v. lesions whose repair is inhibited by acriflavine. Similarly, partial replacement of the thymine in transforming DNA by 5-bromouracil (BU) strongly inhibits repair of subsequent u.v. damage. As in bacteriophage, the BU effect is relieved if the u.v. exposure occurs in the presence of cysteamine. It is clear that intracellular repair must be considered in interpreting experiments with u.v.-irradiated transforming DNA.     

CU(II) SENSITIZES pBR322 PLASMID DNA TO INACTIVATION BY UV-B(280–315 nm)

Abstract— Copper(II), in the presence of UV-B radiation(280–315 nm), can generate single-strand breaks in the sugar-phosphate backbone of pBR322 plasmid DNA. A low level of single-strand backbone breaks occurs in the presence of Cu(II) alone, but UV-B irradiation increases the rate by the more than 100-fold. Concomitant with the damage to the DNA backbone is a loss of transforming activity. Oxygen is required for generation of the single-strand breaks but not for the loss of transforming activity. A DNA glycosylase (Fpg), which participates in the repair of certain DNA nitrogenous base damage, does not repair plasmid DNA damaged by Cu(II). The hydroxyl radical scavenging compound DMSO is only somewhat effective at protecting the physical and biological properties of the DNA. These results with Cu(II) are compared to those obtained previously with pBR322 plasmid DNA in the presence of Fe(III) and UV-A.     

PHOTOSENSITIZED INACTIVATION OF DNA BY MONOCHROMATIC 334-nm RADIATION IN THE PRESENCE OF 2-THIOURACIL: GENETIC ACTIVITY AND BACKBONE BREAKS

Abstract Monochromatic 334-nm radiation delivered under aerobic conditions inactivates the genetic activity (ability to transform auxotrophic recipient cells to nutritional prototrophy) of isolated transforming Bacillus subtilis DNA. The presence of superoxide dismutase (SOD), catalase, and mannitol reduces the 334-nm inactivation. The rate of inactivation of the genetic activity by 334-nm radiation is enhanced fivefold by the sensitizer 2-thiouracil (s²Ura). This enhancement is substantially reversed when the irradiations are performed in the presence of mannitol, and, to a lesser extent, SOD. Catalase slightly reduces the s²Ura enhancement of 334-nm inactivation of transforming activity. Backbone breaks induced in the same DNA by aerobic 334-nm radiation were also enhanced markedly by the presence of s²Ura; this enhancement was reversed by the presence of mannitol and, to a lesser extent, SOD during irradiation. Catalase had no effect upon s²Ura-enhanced, 334-nm-induced SSBs. Whereas DNA breakage may be responsible for a portion of the inactivation of the DNA by the photosensitized reaction between s²-Ura and 334-nm radiation, it is not the only inactivating lesion, because the yield of SSBs per lethal hit per unit length of DNA is not constant for all the irradiation conditions studied. The results support a complex role for active oxygen species in inactivation of transforming activity and DNA breakage by s²Ura-enhanced 334-nm radiation. They are also consistent with the formation of superoxide anion, hydroxyl radical, and possibly also singlet molecular oxygen, generated from ground-state molecular oxygen by reactive s²Ura in both Type I and II reactions.     

Never Cared for What They Do: High Structural Stability of Guanine-Quadruplexes in the Presence of Strand-Break Damage

DNA integrity is an important factor that assures genome stability and, more generally, the viability of cells and organisms. In the presence of DNA damage, the normal cell cycle is perturbed when cells activate their repair processes. Although efficient, the repair system is not always able to ensure complete restoration of gene integrity. In these cases, mutations not only may occur, but the accumulation of lesions can either lead to carcinogenesis or reach a threshold that induces apoptosis and programmed cell death. Among the different types of DNA lesions, strand breaks produced by ionizing radiation are the most toxic due to the inherent difficultly of repair, which may lead to genomic instability. In this article we show, by using classical molecular simulation techniques, that compared to canonical double-helical B-DNA, guanine-quadruplex (G4) arrangements show remarkable structural stability, even in the presence of two strand breaks. Since G4-DNA is recognized for its regulatory roles in cell senescence and gene expression, including oncogenes, this stability may be related to an evolutionary cellular response aimed at minimizing the effects of ionizing radiation.     

Biological consequences of cyclobutane pyrimidine dimers.

In the skin many molecules may absorb ultraviolet (UV) radiation upon exposure. In particular, cellular DNA strongly absorbs shorter wavelength solar UV radiation, resulting in various types of DNA damage. Among the DNA photoproducts produced the cyclobutane pyrimidine dimers (CPDs) are predominant. Although these lesions are efficiently repaired in the skin, this CPD formation results in various acute effects (erythema, inflammatory responses), transient effects (suppression of immune function), and chronic effects (mutation induction and skin cancer). The relationships between the presence of CPD in skin cells and the subsequent biological consequences are the subject of the present review.     

Repair of furocoumarin-plus-UVA-induced damage and mutagenic consequences in eukaryotic cells

In the presence of near-UV radiation (UVA) furocoumarins (psoralens) photoinduce defined lesions in DNA, i.e. monoadducts and interstrand crosslinks. Their use in photochemotherapy (psoralen plus UVA (PUVA) treatment) and cosmetics raises questions concerning the repairability of these lesions and their genotoxic consequences. We have analysed the repair of psoralen photoadducts in cultured eukaryotic cells, such as yeast and mammalian cells, for furocoumarins of photochemotherapeutic interest. In yeast, the interaction of repair pathways differs in exogenous (plasmid) and endogenous (chromosomal) DNA. The order of mutagenic activity is 4,5',8-trimethylpsoralen greater than 5-methoxypsoralen greater than 8-methoxypsoralen greater than 7-methylpyrido[3,4-c]psoralen greater than 3-carbethoxypsoralen. The mutagenicity is dependent on psoralen functionality, concentration and bioavailability, maximal UVA dose, wavelength, dose (fluence) rate and presence or absence of chemical filters. It probably involves an inducible component. Chromosome breakage occurs during the repair period after PUVA treatment. It appears that the genotoxic effects of psoralens are produced by a specific arrangement of induced photolesions and the interaction of different repair systems.     

MECHANISMS OF CITRAL PHOTOTOXICITY

Citral, a monoterpene aldehyde synthesized by several plant genera, has been reported to exhibit antimicrobial activity. For the first time, we report that critral exhibits UV-A (315-400 nm) light enhanced oxygen-dependent toxicity against a series of Escherichia coli strains differing in DNA repair and catalase proficiency. Those E. coli strains carrying a gene leading to catalase deficiency (katF) are particularly sensitized to inactivation by citral and UV-A treatment when compared to catalase proficient strains (katF+). Consistent with these in vivo observations, citral when treated with UV-A in vitro produces H2O2. When tested against Fusarium oxysporum and F. solani, fungal root pathogens of Citrus, enhanced toxicity by citral in the presence of UV-A was demonstrated, while dark toxicity was negligible. When the plasmid pBR322 was treated with citral in the presence of UV-A, a change in conformation from the covalently closed circular to the open circular and, ultimately, the linear form was observed. The change in plasmid conformation corresponded to a reduction in transforming activity. Holding plasmid DNA which had been treated with UV-A light in the presence of citral at 4 degrees C for 22 h in the dark resulted in continued degradation of the DNA and loss of transforming activity. Holding plasmid DNA treated with UV-A or citral alone under identical conditions had no detectable effect on either plasmid conformation or transforming activity.     

Nanopore detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine in immobilized single-stranded DNA via adduct formation to the DNA damage site

The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine, which occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo- and heteropolymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also for detecting DNA damage, which will be an important component to any sequencing efforts.     

DNA damage induced via independent generation of the radical resulting from formal hydrogen atom abstraction from the C1′-position of a nucleotide

Background: Deoxyribonucleotide radicals resulting from formal C1′-hydrogen atom abstraction are important reactive intermediates in a variety of DNA-damage processes. The reactivity of these radicals can be affected by the agents that generate them and the environment in which they are produced. As an initial step in determining the factors that control the reactivity of these important radical species, we developed a mild method for their generation at a defined site within a biopolymer.Results: Irradiation of oligonucleotides containing a photolabile nucleotide produced C1'-DNA radicals. In the absence of potential reactants other than O₂, approximately 90% of the damage events involve formation of alkaline-labile lesions, with the remainder resulting in direct strand breaks. The ratio of alkaline-labile lesions to direct strand breaks (∼ 9:1) is independent of whether the radical is generated in single-stranded DNA or double-stranded DNA. Strand damage is almost completely quenched under anaerobic conditions in the presence of low thiol concentrations. Competition studies with 02 indicate that the trapping rate of C1′-DNA radicals by β-mercaptoethanol is ∼ 1.1 x 10⁷ M⁻¹s⁻¹Conclusions: The mild generation of the C1'-DNA radical in the absence of exogenous oxidants makes it possible to examine their intrinsic reactivity. In the absence of other reactants, the formation of direct strand breaks from C1′-radicals is, at most, a minor pathway. Competition studies between β-mercaptoethanol and 02 indicate that significantly higher thiol concentrations than those in vivo or some means of increasing the effective thiol concentration near DNA are needed for these reagents to prevent the formation of DNA lesions arising from the C1'-radical under aerobic conditions.     

Analysis of a photosensitive lesion induced by sunlamp UV greater than 315 nm exposure of 254-nm-irradiated human cells

Normal human skin fibroblasts were exposed to 0-10 J m-2 of 254 nm UV, incubated 0-16 h and then treated with 0-150 kJ m-2 of sunlamp UV greater than 315 nm. For each treatment, the cells were subjected to alkaline elution in order to measure the yield of single strand breaks (ssb) produced. It was found that treatment of 254-nm-irradiated cells with sunlamp UV greater than 315 nm resulted in the production of a higher level of ssb than that produced by separate exposures. Hence, lesions are produced by the 254 nm irradiation that are photolyzed through exposure to sunlamp UV greater than 315 nm. Approximately 50% of these lesions are removed following a 2-4 h incubation of the 254-nm-irradiated cells and nearly complete removal is achieved by 16 h. In addition, the profiles for elutions performed at pH 12.8 with cells exposed to the combined treatment were indicative of the presence of alkali labile sites. The repair kinetics of this lesion and alkaline lability of the photolysis product suggest that this photosensitive lesion may represent pyrimidine(6-4)pyrimidone photoproducts. Hence, this approach may represent a relatively simple and sensitive assay for the measurement of this DNA damage.     

Radiosensitization by a modified nucleotide that produces DNA interstrand cross-links under hypoxic conditions

This paper describes the reactivity of a molecule that combines two desirable chemical processes into one molecule for the first time. Interstrand cross-links (ISCs) are an effective family of lesions produced by cytotoxic agents that target DNA. For instance, ISCs are the source of mitomycin C's cytotoxicity. Radiosensitizing agents are molecules that enhance DNA damage produced by ionizing radiation, especially under O2-deficient conditions. Phenyl selenide 1 is the first example of a modified nucleotide that can be incorporated in DNA by polymerases, which produces ISCs when DNA containing it is exposed to gamma-radiolysis under O2-deficient conditions. These experiments suggest that 1 could be useful as a novel type of radiosensitizing agent.     

DNA SYNTHESIS-KINETICS, CELL DIVISION DELAY, AND POST-REPLICATION REPAIR AFTER UV IRRADIATION OF FROZEN CELLS OF E. COLI B/r

Abstract— Previous studies have shown that the relative yields of photoproducts produced in the DNA of Escherichia coli cells UV irradiated at -79°C differ from those produced at +21°C; the yield of DNA-protein cross-links was markedly enhanced at -79°C while the yield of thymine dimers was reduced. In the present studies, cells of E. coli B/r thy were frozen at -79°C, and then UV irradiated (254nm) while frozen(4.7 J m^-2), or after thawing (22 Jm^-2). Essentially the same survival, cell division delay, and DNA synthesis kinetics were observed for these two samples after irradiation, even though the UV fluence differed by a factor of ˜5. This supports previous observations that a correlation exists between the magnitude of the effects of UV radiation upon DNA synthesis kinetics and on cell survival. The weight average molecular weight of the pulse labeled DNA in the sample irradiated at +21°C was one-half that of the sample irradiated at -79°C, and complete repair of daughter-strand gaps was observed in both cases. Thus, UV-induced lesions produced in cells at -79°C (i.e. DNA-protein cross-links) appear to be amenable to post-replicational repair. While the overall DNA synthesis kinetics were the same for the two irradiation procedures, the apparent number of lesions produced per unit length of DNA was not. This suggests that each of the lesions produced in frozen cells, although apparently fewer in number, must cause a longer local delay in DNA synthesis than those lesions produced at +21°C.     

Evidence for glycosidic bond rotation in a nucleobase peroxyl radical and its effect on tandem lesion formation

Nucleobase peroxyl radicals are the major reactive intermediates formed in DNA when the biopolymer is exposed to gamma-radiolysis under aerobic conditions. The major reaction pathways for the peroxyl radical (1) derived from 5,6-dihydro-2'-deoxyuridin-6-yl involve pi-bond addition to or hydrogen atom abstraction from the adjacent nucleotides to produce tandem lesions. The ability to independently generate 1 at a defined site in DNA enabled us to probe its reactivity by varying the local DNA structure. The effect of DNA structure variation reveals that 1 reacts from its syn- and anti-conformations in competition with trapping by thiol. These experiments also reveal that tandem lesions will be produced as a mixture of diastereomers, which could impact their biological effects.     

Photophysics of Acetophenone Interacting with DNA: Why the Road to Photosensitization is Open

Deoxyribonucleic acid photosensitization, i.e. the photoinduced electron‐ or energy‐transfer of chromophores interacting with DNA, is a crucial phenomenon that triggers important DNA lesions such as pyrimidine dimerization, even upon absorption of relatively low‐energy radiation. Oxidative lesions may also be produced via the photoinduced production of reactive oxygen species. Aromatic ketones, and acetophenone in particular, are well known for their sensitization effects. In this contribution we model the structural and dynamical properties of the acetophenone/DNA aggregates as well as their spectroscopic and photophysical properties using high‐level hybrid quantum mechanics/molecular mechanics methods. We show that the key steps of the photochemistry of acetophenone in gas phase are conserved in the macromolecular environment and thus an ultrafast singlet–triplet conversion of acetophenone is expected prior to the transfer to DNA.     

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.     

RECOVERY OF HAEMOPHILUS INFLUENZAE FROM ULTRAVIOLET AND X-RAY DAMAGE

Abstract— Results of experiments on reactivation of ultraviolet (u.v.)-irradiated Haemophilus influenzae and cellular reactivation of u.v.-damaged transforming deoxyribonucleic acid (DNA) and bacteriophage are reported. Liquid-holding recovery (LHR) is small for the u.v.-sensitive mutant BC100 which, relative to the wild type, also has greatly reduced host-cell reactivation (HCR) of u.v.-inactivated phage, and competent cultures show reduced competent cell reactivation (CCR) of u.v.-inactivated transforming DNA. BC100 cells can be transformed with DNA isolated from the wild type strain Rd to a u.v. resistance similar to that of Rd, and irradiation of the DNA reduces the transformation frequency for this marker (uvr). The u.v.-resistant mutant BC200 displays very little LHR under the usual conditions where reactivation occurs after plating. The colony-forming ability (cfa) of irradiated BC200 is greater than that of Rd, but HCR and CCR are the same on this mutant as on the wild type. The major difference between Rd and BC200 is the enhanced u.v. survival of cfa of the latter. It was determined that this difference reflects cell lysis of irradiated Rd and lack of lysis in BC200 cultures. That lysis is closely correlated with damage to the bacterial chromosome is suggested by the finding that the lytic response of Rd (as determined turbidimetrically) can be negated by the liquid-holding procedure, but lysis of BC100 (which lacks comparable DNA-repair ability) can be only partially inhibited by this procedure. LHR occurs when post-plating dark recovery is incomplete, is temperature-sensitive, and occurs unimpeded when post-u.v. protein synthesis is inhibited by chloramphenicol. It is suggested that enzymatically catalyzed reactivation of DNA occurs or is initiated during liquid-holding of u.v.-irradiated H. influenzae Rd and that the necessary enzyme(s) exists prior to appearance of u.v. lesions in the DNA. Results are reported for X-ray inactivation of transforming DNA as assayed on BC100, Rd and BC200 and of the cfa of the three strains.