Influence of anoxia on the induction of mutations by phenylalanine radicals during gamma-irradiation of plasmid DNA in aqueous solution

When DNA is irradiated in aqueous solution, most of the damage is inflicted by water-derived radicals. This is called the indirect effect of ionizing radiation. However in whole cells not only the primary formed water radicals play a role, because some cellular compounds form secondary radicals which can also damage DNA. It is known that the amino acid phenylalanine is able to react with water radicals, resulting in the production of secondary phenylalanine radicals which can damage and inactivate DNA. In a previous study the influence of the presence of phenylalanine during gamma-irradiation of DNA in aqueous solution under oxic conditions was studied. Under anoxic irradiation conditions different amounts and types of reactive water-derived radicals are formed compared to oxic conditions and also different phenylalanine radicals are formed. Therefore, this study examines the influence of the presence of phenylalanine under anoxic conditions on the gamma-radiation-induced mutation spectrum. The results indicate that phenylalanine radicals are damaging to DNA, but less effective compared to primary water radicals. On the mutational level, in the presence of phenylalanine radicals under anoxic conditions, the amount of mutations on G:C base pairs was significantly decreased as compared to oxic conditions. Furthermore, the results of this study indicate that nucleotide excision repair is involved in repair of both inactivating and mutagenic damage induced by phenylalanine radicals under anoxic conditions.     

Radiosensitization in multifraction schedules. II. Greater sensitization by 2-nitroimidazoles than by oxygen

The objective of this study was to characterize the extent of and mechanisms involved in radiosensitization by 2-nitroimidazoles in multifraction schedules using low doses per fraction. For this purpose, contact-inhibited monolayers of C3H 10T1/2 cells were given 1.7 Gy every 12 h and plated 12 h after the last dose received to allow full repair of potentially lethal damage (PLD). Severe hypoxia was obtained by a 1-h gassing procedure at room temperature immediately before each irradiation. No toxicity occurred as a consequence of multiple exposures to 5 mM misonidazole (MISO) or SR 2508 (2508) during the deoxygenation procedure. Experimental conditions during the pregassing and irradiation (presence of drug and gas mixture) were appropriately manipulated to test for the different mechanisms of radiosensitization demonstrated by nitroimidazoles. A very low oxygen enhancement ratio (OER) results under these conditions (1.34). Exposure to 5 mM MISO or 2508 during the deoxygenation and irradiation of hypoxic cells resulted in greater radiosensitization than could be accounted for by oxygen-mimetic sensitization alone (MISO and 2508 enhancement ratios were greater than the OER). Pregassing cells with N2 in the presence of 5 mM drug sensitized cells which were subsequently irradiated under aerobic conditions (drug free), indicating the occurrence of the "preincubation effect" (which does not require hypoxia or the drug's presence during the irradiation). Thus, for the hypoxic irradiations, the preincubation effect could account for the greater sensitization by nitroimidazoles than by oxygen. The presence of 5 mM drug only during the irradiation of aerobic cells produced radiosensitization in both multifraction and single-dose experiments with delayed plating. This sensitization has been previously shown to involve reduced PLD repair. Finally, maximum radiosensitization occurred in the multifraction schedule when a transient period of hypoxia with drug preceded an aerobic irradiation with drug present, thus combining the benefits of both the preincubation effect and PLD repair inhibition. This work demonstrates the possibility that effects other than oxygen-mimetic radiosensitization could be largely responsible for the sensitization seen in multifraction schedules, particularly when the OER is already low and only transient periods of hypoxia occur.     

Different oxygen enhancement ratios for induced and unrejoined DNA double-strand breaks in eukaryotic cells

DNA double-strand breaks (DSBs) are 2.9 times more frequently induced in yeast cells exposed to sparsely ionizing 30-MeV electrons under oxic compared to anoxic conditions. The rejoining of DSBs induced under anoxic conditions was investigated under conditions allowing repair of potentially lethal damage and compared to the rejoining of DSBs induced in oxic cells. In contrast to the biphasic rejoining kinetics of DSBs induced in oxic cells, the rejoining kinetics of DSBs induced in anoxic cells is complicated by the formation of secondary DSBs. These arise during postirradiation incubation of cells, presumably as a consequence of repair processes acting on radiation-induced lesions other than DSBs. These secondary DSBs may at least partially explain the finding that a greater fraction of unrejoinable DSBs is present in cells irradiated under anoxic compared to oxic conditions. As a consequence, the oxygen enhancement ratio of the yield of the remaining DSBs is decreasing in the course of DSB rejoining.     

Radiation sensitization by thiol-binding agents of radio-resistant and radiosensitive Escherichia coli and the oxygen effect

Dose-survival curves of four strains of E. coli-B/r her- try-, Bs-1, B/r CSH, and 15T-were obtained in the presence and in the absence of non-toxic levels of several thiol-binding agents-N-ethylmaleimide (NEM), iodoacetamide (IA), and hydroxymercuribenzoate (HMB). The degree of radiosensitization by these agents was estimated from the increase in slope of the dose-survival curve, under anoxic and aerobic conditions. Chemical sensitization of both radioresistant and radiosensitive strains of bacteria with the thiol-binding enzyme poisons NEM, IA, and HMB has been observed. NEM sensitized only under anoxic conditions, IA sensitized under both anoxic and aerobic conditions, although to a much greater extent aerobically, and HMB sensitized only under aerobic conditions. The formation of long-lived radiolytic products toxic to bacteria is observed in sensitization by IA. When sensitization occurs, the magnitude of the dose-modifying factor is equal to or greater than the oxygen enhancement ratio. Inhibition of an energy-requiring enzymic repair process does not seem to be involved as a primary mechanism of sensitization. Interference by thiol-binding agents with a "rapid repair" process which is different from the usual "enzymic repair," and which operates in radio-sensitive as well as in radioresistant bacteria, is suggested.     

Effects of the differentiating agents sodium butyrate and N-methylformamide on the oxygen enhancement ratio of human colon tumor cells

We have previously shown that chronic adaptation of human tumor cells to the differentiation-inducing agents N-methylformamide (NMF) and sodium butyrate (NAB) increases the sensitivity of oxic cells to graded single doses of X rays. These studies were carried out to define the sensitivity of hypoxic cells after adaptation. Clone A colon tumor cells were grown for three passages in medium containing 170 mM NMF or 2 mM NAB and irradiated in suspension culture, after gassing with either oxygen (60 min) or ultrapure nitrogen (90 min), and complete survival curves were generated. Using the linear-quadratic equation to describe the data, it was found that NMF and NAB produced increased X-ray killing of hypoxic cells. At the 10% level of survival, the dose-modifying factors were about 1.20 and 1.25 for NMF- and NAB-adapted hypoxic cells, respectively, as compared to hypoxic control cells. However, since both oxic and hypoxic cells exhibited increased sensitivity after NMF and NAB adaptation, there was no major change in the oxygen enhancement ratio.     

Growth of the phototrophic purple sulfur bacterium Thiocapsaroseopersicina under oxic/anoxic regimens in the light

Abstract The phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in sulfide-limited continuous cultures exposed to oxic/anoxic regimens in continuous light. Synthesis of bacteriochlorophyll a (BChl a ) did not occur during the oxic periods, but started immediately upon the creation of anoxic conditions. In contrast, protein synthesis continued during both oxic and anoxic periods. Consequently, the specific content of BChla fluctuated. Despite the presence of oxygen and the fluctuating BChl a content, growth occurred predominantly in a phototrophic mode and respiration was virtually zero.
BChl a synthesis continued at high rates during anoxic periods, thus compensating for the lack of synthesis during oxic periods. When cultivated under regimens with oxic periods shorter than 12 h the highest specific BCh a content was 27 μg·mg⁻ protein. In contrast, when cultivated under regimens with oxic periods longer than 12 h the specific BChl a content was always lower than 27μg·mg⁻ length of the oxic periods. During the anoxic periods, BChl a synthesis occurred at the maximal velocity of 1.2 μg·mg⁻¹ protein·h⁻, but the length of the anoxic periods was not sufficient to allow the BChl a content to reach the maximum level.
Cultivation under continuously oxic conditions eventually resulted in pigmentless cells growing chemolithotrophically. The BChl a synthesizing ability was not lost during prolonged exposure to oxygen.
It was concluded that T. roseopersicina is very well adapted to oxic/anoxic cycles.     

Suppression of delayed-type hypersensitivity to oxazolone in whole-body-irradiated mice and protection by WR-2721

The effect of whole-body irradiation on cellular immunity, as measured in vivo by delayed-type hypersensitivity (DTH) to oxazolone (4- ethoxymethylene -2-phenyl- oxazol -5-one), was determined in CD2F1 mice. DTH, determined by changes in ear swelling after challenge with oxazolone, was significantly depressed in irradiated mice (500-900 rad of 60Co) in a dose-dependent fashion when animals were irradiated after sensitization and before challenge with oxazolone. Administration of WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] 30 min before irradiation (2 days after sensitization) resulted in protection against suppression of DTH, which was dependent on drug and radiation dose. An effective dose of WR-2721 (200 mg/kg body wt) provided an approximate dose-modifying factor of 1.3. The data suggest that WR-2721 interacts with cells involved in that DTH response (lymphocytes and/or macrophages) and that WR-2721 may be useful in protecting against radiation-induced decrements in cell-mediated immunity.     

Changes in the survival curve shape of E. coli cells following irradiation in the presence of uncouplers of oxidative phosphorylation

Four uncouplers of oxidative phosphorylation (UOP) (carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, 4-hydroxybenzylidenemalonitrile and N-phenylanthranilic acid) have been found to alter the shape of the radiation survival curves of several cell lines of E. coli when present during irradiation in oxia. Incubation of cells with high concentrations of UOP for 30 min before irradiation induced an increase in extrapolation number (n) in cell lines AB 1157 (wild-type), AB 1886(uvrA-) and KMBL(polA-) but not GR 501(lig-)ts, AB 2463(recA-) and AB 2480(uvrA-recA-). In addition the UOP all effect a decrease in mean lethal dose (D0) even when tested at low concentrations or short contact times. Studies with wild-type cells correlate the increase in n with measured increased levels of ATP (above oxic control cells) produced upon incubation with UOP. The increased levels of ATP most likely arise from the UOP overstimulating glycolysis. The decrease in D0 cannot be associated with any of the repair pathways investigated and it is concluded that the highly lipophilic UOP directly or indirectly potentiate other target(s) to radiation damage as well as DNA under oxic conditions. Treatment of the cells with UOP did not result in the deleterious depletion of energy substrates, loss of non-protein thiols or the production of cytotoxins upon irradiation.     

The relative biological effectiveness of 14.5-MeV neutrons for the induction of gene conversion and mutation in yeast

The relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) were determined in the yeast Saccharomyces cerevisiae for the induction of gene conversion (the product of recombinational repair) and mutation (the product of error prone repair) by 14.5-MeV neutrons in comparison with 60Co gamma rays and 150 KVp X rays. Neutron irradiation in oxic or anoxic conditions induced significantly higher yields of convertants and mutants than sparsely ionizing radiations under the same conditions. RBEs for both gene conversion and mutation under anoxia were significantly higher than under oxic conditions. RBEs for mutant induction under anoxia were lower than the RBEs for gene conversion under the same conditions. The data support the hypothesis that the production of lesions leading to the genetic consequences of gene conversion and mutation differ in their dependence upon LET and the presence of oxygen during irradiation, and therefore the two DNA repair processes which produce these end points recognize, at least in part, different classes of damage.     

Radiation-induced DNA single-strand scission and its rejoining in spermatogonia and spermatozoa of mouse

Gamma-ray-induced DNA single-strand scissions and the ability to repair the scissions in spermatogonia from young mice and in spermatozoa from adult mice were studied quantitatively by an alkaline sucrose density-gradient centrifugation method. The average size of DNAs in non-irradiated spermatogonia was 2.6–3.0 × 10⁸ daltons, similar to those of a spermatid-rich population, and the size of DNA in non-irradiated spermatozoa was 1.2 × 10⁸ daltons.In spermatogonia, the radiosensitivity of DNA was 0.42 single-strand breaks/ 10¹² daltons of DNA/rad in oxic conditions and only 0.24 under anoxic conditions. In spermatozoa the break efficiency of DNA was 0.22 single-strand breaks/10¹² daltons of DNA/rad under oxic conditions and altered little under anoxic irradiation. The DNA scissions were efficiently repaired in spermatogonia within 10 min, whereas the breaks in spermatozoa were not rejoined at all even after two days of post-irradiation time.The radiosensitivities of DNA, repair capability and non- and/or slow-reparable DNA scissions were compared in spermatogonium-rich, spermatid-rich and spermatozoan-rich populations.     

Differential regulation of periplasmic nitrate reductase gene (napKEFDABC) expression between aerobiosis and anaerobiosis with nitrate in a denitrifying phototroph Rhodobacter sphaeroides f. sp. denitrificans

A denitrifying phototroph, Rhodobacter sphaeroides f. sp. denitrificans, has the ability to denitrify by respiring nitrate. The periplasmic respiratory nitrate reductase (Nap) catalyses the first step in denitrification and is encoded by the genes, napKEFDABC. By assaying the ss-galactosidase activity of napKEFD-lacZ fusions in wild type and nap mutant cells grown under various growth conditions, the environmental signal for inducing nap expression was examined. Under anoxic conditions with nitrate, nap genes expression in the wild-type strain was highest in the dark, and somewhat lowered by incident light, but that of the napA, napB, and napC mutant strains was low, showing that nap expression is dependent on nitrate respiration. Under oxic conditions, both the wild type and nap mutant cells showed high ss-galactosidase activities, comparable to the wild-type grown under anoxic conditions with nitrate. Myxothiazol, a specific inhibitor of the cytochrome bc (1) complex, did not affect the beta-galactosidase activity in the wild-type cells grown aerobically, suggesting that the redox state of the quinone pool was not a candidate for the activation signal for aerobic nap expression. These results suggested that the trans-acting regulatory signals for nap expression differ between anoxic and oxic conditions. Deletion analysis showed that the nucleotide sequence from -135 to -88 with respect to the translational start point is essential for nap expression either under anoxic or oxic conditions, suggesting that the same cis-acting element is involved in regulating nap expression under either anoxic with nitrate or oxic conditions.     

Evidence against the “oxygen-in-the-track” hypothesis as an explanation for the radiobiological low oxygen enhancement ratio at high linear energy transfer radiation

Oxygen sensitizes cells toward the effect of ionizing radiation. This sensitization, quantified by the oxygen enhancement ratio (OER), decreases with increasing ionization density or linear energy transfer (LET) of the radiation applied. One explanation for the decreased OER at high LET offers the oxygen-in-the-track hypothesis. It claims that oxygen is produced in the track of densely ionizing particles providing an oxic microenvironment around the relevant cellular target molecules, even if cells are exposed under anoxic atmospheric conditions. Experimental evidence is presented against this hypothesis. It is based on the different kinetic pattern of DNA double-strand-break rejoining observed in yeast cells exposed under oxic or anoxic conditions to 3.5 MeV -particles.     

Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions.     

Degradation of and sensitivity to cholate in Pseudomonas sp. strain Chol1

A facultative anaerobic bacterium, Pseudomonas sp. strain Chol1, degrading cholate and other bile acids was isolated from soil. We investigated how strain Chol1 grew with cholate and whether growth was affected by the toxicity of this compound. Under anoxic conditions with nitrate as electron acceptor, strain Chol1 grew by transformation of cholate to 7,12-dihydroxy-1,4-androstadiene-3,17-dione (DHADD) as end product. Under oxic conditions, strain Chol1 grew by transformation of cholate to 3,7,12-trihydroxy-9,10-seco-1,3,5(10)-androstatriene-9,17-dione (THSATD), which accumulated in the culture supernatant before its further oxidation to CO₂. Strain Chol1 converted DHADD into THSATD by an oxygenase-dependent reaction. Addition of cholate (≥10 mM) to cell suspensions of strain Chol1 caused a decrease of optical density and viable counts but aerobic growth with these toxic cholate concentrations was possible. Addition of CCCP or EDTA strongly increased the sensitivity of the cells to 10 mM cholate. EDTA also increased the sensitivity of the cells to DHADD and THSATD (≤1.7 mM). The toxicity of cholate and its degradation intermediates with a steroid structure indicates that strain Chol1 requires a strategy to minimize these toxic effects during growth with cholate. Apparently, the proton motive force and the outer membrane are necessary for protection against these toxic effects.     

Effect of oxygen on the germination and culturability of Bacillus atrophaeus spores

International Microbiology - The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various...     

Vibrio coralliilyticus Search Patterns across an Oxygen Gradient

The coral pathogen, Vibrio coralliilyticus shows specific chemotactic search pattern preference for oxic and anoxic conditions, with the newly identified 3-step flick search pattern dominating the patterns used in oxic conditions. We analyzed motile V. coralliilyticus cells for behavioral changes with varying oxygen concentrations to mimic the natural coral environment exhibited during light and dark conditions. Results showed that 3-step flicks were 1.4× (P = 0.006) more likely to occur in oxic conditions than anoxic conditions with mean values of 18 flicks (95% CI = 0.4, n = 53) identified in oxic regions compared to 13 (95% CI = 0.5, n = 38) at anoxic areas. In contrast, run and reverse search patterns were more frequent in anoxic regions with a mean value of 15 (95% CI = 0.7, n = 46), compared to a mean value of 10 (95% CI = 0.8, n = 29) at oxic regions. Straight swimming search patterns remained similar across oxic and anoxic regions with a mean value of 13 (95% CI = 0.7, n = oxic: 13, anoxic: 14). V. coralliilyticus remained motile in oxic and anoxic conditions, however, the 3-step flick search pattern occurred in oxic conditions. This result provides an approach to further investigate the 3-step flick.     

Effect of cellular glutathione content on the induction of DNA double strand breaks by 25 MeV electrons

The effect of endogenous glutathione (GSH) on the induction of DNA double strand breaks (dsb) by 25 MeV electrons was investigated using stationary haploid yeast cells defective in gamma-glutamyl-cysteine-synthetase (gsh 1) containing less than 5 per cent of the normal GSH content. In gsh 1 cells the induction of dsb is increased by a factor of 1.5 under oxic and 1.8 under anoxic irradiation conditions: whereas the oxygen enhancement ratio was only slightly decreased (1.9) compared to wild-type cells (2.4).     

Growth and final product formation by <Emphasis Type="Italic">Bifidobacterium infantis</Emphasis> in aerated fermentations

Fermentation conditions were developed to allow Bifidobacterium infantis to grow in the presence of air. Batch fermentations in TPYG medium, starting from anoxic conditions followed by the application of low airflow rates [0.02–0.1 air volume, per liquid media volume, per minute (vvm)], were analyzed for growth, oxygen uptake, and product formation by the bacterium. Under all aerated fermentations, B. infantis showed high aerotolerance, with a maximum oxygen-specific consumption rate of 0.34 mmol oxygen per gram dry cell weight per hour in the presence of 0.06 vvm. Similar growth yields were obtained under oxic and anoxic conditions (0.11–0.13 and 0.11 g dry cell weight per mmol glucose, respectively). Oxygen also influenced metabolite formation since lactate production and its molar relation to acetate increased and formate decreased with aeration rate. Under anoxic conditions, a maximum concentration of 8.1 mM lactate and an acetate/lactate ratio of 3.5:1 were obtained, while under oxic conditions the lactate concentration increased more than two-fold and the acetate/lactate molar ratio decreased to 1.5:1. The possibility of balancing acetate/lactate molar ratios for organoleptic purposes as well as for obtaining good growth under microaerated conditions was demonstrated.