Fast neutron r.b.e. for lethality and genotoxicity in a wild-type and a repair-deficient strain of yeast

The relative biological effectiveness (r.b.e.) of cyclotron-produced fast neutrons (11 MeV) in relation to 60Co gamma-rays, was studied in a wild-type and a DNA repair-deficient yeast strain for cell killing and genotoxicity. In the wild-type (D7) strain the r.b.e. varied from 2.7 to 4.1 for lethality, 2.8 to 7.1 for reverse mutation and 3.5 to 7.8 for mitotic gene conversion. At different survival levels, the repair deficient strain (D7 rad 52/rad 52) generally showed a lower r.b.e. for both cell killing and genotoxicity (25.2 to 37.2 per cent reduction for the cell death and 24.8 to 70.6 per cent for mutation and gene conversion) compared to the wild type. Except at very low dose levels, the r.b.e. values for cell killing and genotoxicity were similar within a given strain. At similar survival levels, neutrons were no more genotoxic than gamma-rays.     

DNA strand break and rejoining in cultured human fibroblasts exposed to fast neutrons or gamma rays

The production and rejoining of DNA single-strand and double-strand breaks have been monitored in monolayer cultures of proliferating human skin fibroblasts by means of sensitive techniques. Cells were irradiated with low doses of either 60Co gamma-rays or 14.6 MeV neutrons at 0 degrees C (0-5 Gy for measurement of single-strand breaks by alkaline elution and 0-50 Gy for double-strand breaks measured by neutral elution). The yield of single-strand breaks induced by neutrons was 30 per cent of that produced by the same dose of gamma-rays; whilst in the induction of double-strand breaks neutrons were 1.6 times as effective as gamma-rays. Upon post-irradiation incubation of cells at 37 degrees C, neutron-induced single-strand and double-strand breaks were rejoined with a similar time-course to gamma-induced breaks. Rejoining followed biphasic kinetics; of the single-strand breaks, 50 per cent disappeared within 2 min after gamma-rays and 6-10 min after neutrons. Fifty per cent of the double-strand breaks disappeared within 10 min, after gamma-rays and neutrons. Cells derived from patients suffering from ataxia-telangiectasia showed the same capacity for repair of single- and double-strand breaks induced by 14.6 MeV neutrons, as cells established from normal donors. The comparison of neutrons and gamma-rays in the induction of DNA breaks did not explain the elevated r.b.e. on high LET radiation. However, a study of the variation in the spectrum of lesions induced by different radiation sources will probably contribute to the clarification of the relative importance of other radio products.     

Modification of high LET radiation-induced damage and its repair in yeast by hypoxia.

The lethal response of a diploid yeast strain BZ34 to densely ionizing radiations from the reaction 10B(n, alpha)7 Li was studied. The values for relative biological effectiveness (r.b.e.) and oxygen enhancement ratio (o.e.r.) for this radiation compare favourably with the data obtained with charged particles on the same strain of yeast. Recovery from potentially lethal damage was also studied by post-irradiation holding under non-nutrient conditions. In order to understand the role of oxygen in the recovery process, the investigation covered the following treatment regimens: (a) aerobic irradiation and aerobic holding (A-A), (b) aerobic irradiation and hypoxic holding (A-H), (c) hypoxic irradiation and hypoxic holding (H-H) and (d) hypoxic irradiation and aerobic holding (H-A). It has been found that the presence of oxygen is essential for recovery from the damage induced by both gamma rays and high linear energy transfer (LET) radiations. The extent of recovery was larger for gamma-induced damage than for damage induced by high LET radiation (alpha + 7Li) for the A-A condition. In the H-H condition, while only a slight recovery was seen for gamma-induced damage, it was totally absent for high LET damage. For the modality A-H, it was found that there is not recovery from the sparsely ionising gamma radiation-induced damage. The implications of these results for the treatment of malignant tumours by radiotherapy are briefly discussed.     

Response of mouse intestine to 14 MeV neutrons.

At the Hamburg-Eppendorf Hospital neutron facilities the relative biological effectiveness (r.b.e.) of d,T-neutrons was determined with respect to survival of mouse intestinal crypts. (CBA/Rij x C57BL/Rij)F1 mice were irradiated to the whole body at different depths inside a tissue-equivalent phantom. Irradiations were carried out with a collimated neutron beam at about 6 rad/min given in single doses ranging from 450 to 1000 rad. For reference, gamma-rays from a 60Co therapy unit were used. The number of surviving intestinal crypts per circumference of the jejunum was determined 3 1/2 days after irradiation according to the method of Withers and Elkind. The number of surviving stem cells was calculated on the basis of Poisson statistics. The doses necessary to reduce survival to ten crypt stem cells per circumference amounted to 689 +/- 19 rad for neutrons and 1449 +/- 29 rad for 60Co gamma-rays. From these figures an r.b.e. of 2 . 1 +/- 0 . 1 is obtained. Measurements at different depths in the phantom did not show any variation of r.b.e. with depth along the axis of the neutron beam.     

Induction and repair of DNA strand breaks in cultured mammalian cells following fast neutron irradiation.

Induction and repair of DNA breaks following irradiation with NIRS cyclotron neutrons were studied in cultured mammalian cells (L5178Y) in comparison to those following gamma-rays. The yield of the total single-strand breaks, 3'OH terminals and sites susceptible to S1 endonuclease following fast neutrons was found to be approximately 50 per cent of that following gamma-irradiation. On the other hand, the yield of double-strand breaks was slightly higher after fast neutrons than after gamma-rays. The percentage of the total single-strand breaks remaining unrejoined at 3 hours after post-irradiation incubation was found to be distinctly higher after the fast neutrons than after gamma-rays. The neutron-induced damage appears to carry a higher proportion of alkali-labile lesions compared to gamma-rays. It was concluded that the increase in the yield of double-strand breaks and of unrejoinable breaks is responsible for a high r.b.e. of the cyclotron neutrons.     

The relationship between o.e.r., r.b.e. and number of fractions for X-ray- or neutron-induced skin damage.

The skin reactions in aerated and hypoxic mouse tails after single or fractionated doses of 250 kV X-rays or fast neutrons (6 MeV deuterons on beryllium) have been measured. The o.e.r. for one to sixteen fractions of X-rays remains constant, while that for one to ten fractions of neutrons decreases with increasing neutron fractionation and decreasing neutron dose/fraction. The o.e.r. for X-rays was 1.7, for single-neutron doses 1.4, and for ten fractions of neutrons 1.25. It was anticipated that the o.e.r. for neutron-induced damage would decrease further as neutron fractionation is increased because the contribution to damage from the highest LET components of dose, the alpha and heavy recoil particles, would increase relative to the lowe LET components. The r.b.e. values obtained for skin damage were higher at all neutron doses/fraction examined in this study on tails than all those previously obtained in studies on skin at other sites on four species. This may be due to the influence of hypoxia on the r.b.e. measurements in the mouse tail.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

Effect of neon ions on synchronized Chinese hamster cells

The variation in radiosensitivity across the cell cycle after exposure to neon ions and 60Co gamma-rays is reported for cultured hamster cells. The cells were first synchronized by mitotic selection, then resynchronized in the region of the G1/S boundary by treatment with 10(-3)M hydroxyurea. Although the use of hydroxyurea improves the synchrony, it does sensitize cells at the G1/S boundary to some degree. The cells were exposed at the plateau and the distal peak position of a neon ion beam modified by a 10 cm wide ridge filter. The results indicate that the variation (ratio of maximum to minimum survival after fixed doses of radiation that are approximately matched to produce similar cell killing) was approximately 80 to 100-fold for 60Co gamma-rays and neon ions at the plateau, and 25-fold for distal peak neon ions. While the r.b.e. of distal peak neon ions decreased rapidly with increasing dose for cells in late S-phase, the r.b.e. is independent of dose for cells at the G1/S boundary.     

Release of iron from ferritin by xanthine oxidase. Role of the superoxide radical.

Mobilization of iron from ferritin by xanthine oxidase was studied under aerobic and anaerobic conditions. Aerobic iron release amounted to approx. 3.7 nmol/ml in 10 min. This amount was decreased by approx. 30% under anaerobic conditions. Aerobic iron mobilization involved two mechanisms. About 70% was released by O2.- generated by xanthine oxidase. The rest was released by O2(.-)-independent mechanisms, which also accounted for the total iron release when O2 was absent. A possible transfer of reducing equivalents directly from xanthine oxidase to ferritin is discussed. The results imply that, in pathological conditions with increased formation of O2.-, iron may be released from ferritin. Furthermore, in hypoxic tissues xanthine oxidase can release iron from ferritin by an O2(.-)-independent process. Free iron is liable to catalyse the formation of the extremely reactive and damaging OH. radical.     

Survival of V79 cells following simultaneous irradiation with X-rays and neutrons in air or hypoxia

V79 Chinese hamster cells have been irradiated with X-rays and neutrons given simultaneously. The oxygen enhancement ratio and r.b.e. were measured as a function of the proportion of the dose due to the neutrons, which varied from 0 to 100 per cent. These were compared with the values calculated assuming the two types of radiation act independently, following an approach suggested by Curtis. The o.e.r. was less than the predicted value when the neutrons contributed less than about 40 per cent of the total dose. The r.b.e. also did not vary as predicted on the basis of independent action. The 'oxygen gain factor' reached half its maximum value when the proportion of the dose due to neutrons was only about 27 per cent. The results imply that there may be interaction between the damage caused by X-rays and neutrons and that beams having only 20 to 30 per cent of their dose due to high l.e.t. radiation, could be of therapeutic benefit.     

The influence of oxygen on the induction of radiation damage in DNA in mammalian cells after sensitization by intracellular glutathione depletion

Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and non-protein-bound SH (NPSH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10-20 per cent for the three types of radiation damage. Also under severely hypoxic conditions (0.01 microM oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0-30 per cent). At somewhat higher concentrations of oxygen (0.5-10 microM) however, the sensitization amounted to about 90 per cent for the induction of ssb and dsb and about 50 per cent for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen.     

Some mechanisms involved in the radiosensitization of E. coli B/r by paracetamol.

Paracetamol, a widely-used analgestic and antipyretic drug, sensitized E. coli B/r to 60Co gamma-rays under hypoxic conditions. Part of the sensitizing effect has been shown to be due to an electron adduct of the drug. Paracetamol inhibited both post-irradiation DNA and protein syntheses. The targets involved in the inhibition of post-irradiation DNA synthesis have been shown to be different in the presence of the sensitizer. Increased DNA degradation after irradiation was also observed when E. coli B/r were irradiated in the presence of the drug. The presence of paracetamol during hypoxic irradiation of E. coli B/r resulted in the enhancement of DNA single-strand scissions with no apparent effect on their rejoining.     

Sensitization of cultured Chinese hamster cells to 42 degrees C hyperthermia by pentalenolactone, an inhibitor of glycolytic ATP synthesis

The antibiotic pentalenolactone, a specific inhibitor of glyceraldehydephosphate dehydrogenase, was used to investigate the effect of glycolytic adenosine triphosphate (ATP) synthesis on the survival response of aerobic and hypoxic Chinese hamster cells treated with 42 degrees C hyperthermia. Data obtained with aerobic cells, incubated in balanced salt solutions supplemented with different substrates for ATP production, showed that 50 microM pentalenolactone blocked ATP synthesis via glycolysis but not by oxidative phosphorylation. The glycolytic inhibition was reversed upon transfer of the cells to antibiotic-free medium, and minimal cytotoxicity (less than 20 per cent) was observed. Hypoxic cultures were obtained by incubating dense cell suspensions (2 X 10(6)/ml) to produce metabolic oxygen depletion. Concomitant with the development of hypoxia, pentalenolactone-treated cells became ATP-depleted; cellular ATP levels were reduced by about 70-fold as compared to hypoxic cells in the antibiotic-free medium. The ATP-depleted cells were more sensitive to killing by hyperthermia. Comparison of the 42 degrees C survival curves for control and the antibiotic-treated hypoxic cells yielded a dose-modifying factor of 4 (5 per cent survival level). The results indicate that inhibition of glycolytic ATP synthesis, for example by pentalenolactone, can selectively sensitize hypoxic cells to the lethal effects of mild hyperthermia.     

Aerobic and anaerobic metabolism of the freshwater oligochaete Tubifex sp.

The freshwater oligochaete Tubifex shows several mechanisms of metabolic adaptations, enabling the worms to occupy saprobial habitats of extremely variable oxygen content. Under normoxic conditions the metabolism of the worms is mainly aerobic with a respiratory ratio of 0.7. Under hypoxic conditions, metabolism of energy sources via aerobic and anaerobic pathways is observed. During complete anoxia acetate and propionate are the main products of glycogen degradation and they are excreted in constant rates into the water. A retransfer of the worms to aerobic conditions enables them to regain aerobic metabolic state within about 60 min.In two Tubifex habitats, which we have characterized, concentrations of dissolved organic material (DOM) were low in the surface water, but high in the interstitial water from sediments. The short-chain fatty acids acetate and propionate reached concentrations up to 1 mmole/liter. Employing radioisotope techniques, we demonstrated that Tubifex can achieve an integumentary uptake of acetate and propionate from artificial tap water at naturally occurring concentrations of 5 to 1000 M. Levels of uptake (600 to 800 nmoles/g wwt.hr) and transport characteristics are very similar to those of marine invertebrates associated with detritus rich sediments. The uptake is susceptible to inhibition by structurally analogous compounds and to metabolic inhibition. Furthermore, DOM uptake in Tubifex is susceptible to inhibition by oxygen depletion, ouabain and Na⁺-depletion. The results may suggest that a carrier system for DOM transport exists in the integument of the worms. The uptake system is highly specific for aliphatic C2 and C3 carboxylic acids. The absorbed volatile fatty acids are rapidly metabolized. Only 15 min after absorption, a considerable amount of radioactivity is present in the glycogen storage of the animals. Depending on the substrate concentration assumed to be available for uptake, up to 40 per cent of the oxidative requirement of the worms may be attained by using dissolved organic material from the interstitial water of their habitat.Supported by the Deutsche Forschungsgemeinschaft (Ho 631/9-9).     

Comparative effects of 60Co gamma-rays and neon and helium ions on cycle duration and division probability of EMT 6 cells. A time-lapse cinematography study

Exponentially growing cultures of EMT 6 cells were irradiated in vitro with neon ions, helium ions or 60Co gamma-rays. Time-lapse cinematography allowed the determination, for individual cells, of cycle duration, success of the mitotic division and the age of the cell at the moment of irradiation. Irradiation induced a significant mitotic delay increasing proportionally with the delivered dose. Using mitotic delay as an endpoint, the r.b.e. for neon ions with respect to 60Co gamma-rays was 3.3 +/- 0.2 while for helium ions it was 1.2 +/- 0.1. Mitotic delay was greatest in those cells that had progressed furthest in their cycle at the time of irradiation. No significant mitotic delay was observed in the post-irradiation generation. Division probability was significantly reduced by irradiation both in the irradiated and in the post-irradiated generation. The reduction in division probability obtained with 3 Gy of neon ions was similar to that obtained after irradiation with 6 Gy of helium ions or 60Co gamma-rays.     

High-speed running performance is largely unaffected by hypoxic reductions in aerobic power.

We tested the importance of aerobic metabolism to human running speed directly by altering inspired oxygen concentrations and comparing the maximal speeds attained at different rates of oxygen uptake. Under both normoxic (20.93% O2) and hypoxic (13.00% O2) conditions, four fit adult men completed 15 all-out sprints lasting from 15 to 180 s as well as progressive, discontinuous treadmill tests to determine maximal oxygen uptake and the metabolic cost of steady-state running. Maximal aerobic power was lower by 30% (1.00 +/- 0.15 vs. 0.77 +/- 0.12 ml O2. kg-1. s-1) and sprinting rates of oxygen uptake by 12-25% under hypoxic vs. normoxic conditions while the metabolic cost of submaximal running was the same. Despite reductions in the aerobic energy available for sprinting under hypoxic conditions, our subjects were able to run just as fast for sprints of up to 60 s and nearly as fast for sprints of up to 120 s. This was possible because rates of anaerobic energy release, estimated from oxygen deficits, increased by as much as 18%, and thus compensated for the reductions in aerobic power. We conclude that maximal metabolic power outputs during sprinting are not limited by rates of anaerobic metabolism and that human speed is largely independent of aerobic power during all-out runs of 60 s or less.     

The r.b.e. of fast fission neutrons (2 MeV) for chronic radiation damage of the large bowel of rats after single dose and fractionated irradiation

Late damage in the rectum of rats was studied after local irradiation with 2 MeV fast fission neutrons and the results were compared with those for 300 kV X-rays. A similarity between latency times, and between clinical, gross pathological and histological appearances of the rectal obstruction after the two types of radiation was observed. The r.b.e. evaluated from ED 50/200 days values was 1.8 for single doses, 2.1 for two fractions and 3 for five fractions and these are similar to r.b.e.s from other investigations of chronic intestinal radiation damage. Using linear quadratic analysis, a limiting r.b.e. of 5 was obtained. The beta-values are similar for both radiation qualities. Because of the high gamma-contamination (25 per cent) in the RENT I beam a possible correction of the r.b.e. for neutrons only, assuming interaction, is discussed.     

Influence of cellular environment on toxicity of nitroheterocycles.

The preferential sensitivity of hypoxic cells to nitroheteroxycles is thought to result from the actions of toxic intermediates of drug reduction produced under hypoxic conditions. However, a lack of oxygen also alters the biochemical state of the cell and may indirectly enhance the sensitivity, of hypoxic cells to these drugs. This hypothesis was tested by 'conditioning' mouse L-929 cells in oxygen-free buffer, then exposing the cells to nitrofurazone under both aerobic and anaerobic conditions. After conditioning, the rate of cell inactivation by nitrofurazone was equal in air or nitrogen-equilibrated buffer. Pretreatment of cells in 1 muM rotenone or 0.5 mM 2,4-dinitrophenol for one hour under aerobic conditions increased the sensitivity of the cells to nitrofurazone under aerobic conditions. Similar rates of cell killing were obtained when mouse L-cells were heated in buffer for 30 min at 43 degrees before incubation with nitrofurazone in either air or nitrogen. Also, incubation of cells with nitrofurazone in the presence of 0.1% glucose, or at a cell density less than 10(5) cells/ml significantly enhanced cell killing, especially under aerobic conditions. Thus, the intracellular state of the cell, manipulated by altering the cellular environment, influenced the cellular sensitivity to nitrofurazone. Similar results were not, however, obtained with the nitroimidazoles, dimetronidazole and misonidazole; pretreatment for 2 h in buffer under anaerobic conditions did not increase the sensitivity of L cells to subsequent drug treatment in air-equilibrated buffer.     

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.     

Negative pion irradiation of mammalian cells. 1. Survival characteristics of monolayers and spheroids of Chinese hamster lung cells.

Monolayers and spheroids of Chinese hamster cells (V79) were subjected to negative pion irradiation under aerobic conditions. R.b.e. values in the pion peak of 1-8 and 1-5 were obtained for monolayers and spheroids, respectively, whereas the r.b.e. for the plateau was found to be slightly higher than 1. In addition, it was observed that the higher resistance of the V79 spheroid cells than the monolayers to gamma-irradiation is not diminished in the pion peak, suggesting that the underlying phenomenon of intercellular communication influences cell survival even after high-LET irradiation.