J.F. Whitfield

THE EFFECTS OF X-RADIATION ON THE CHROMATIN STRUCTURE AND HISTONE COMPONENTS OF RAT THYMOCYTE NUCLEI.

Abstract Irradiation caused the condensed, reticular chromatin structures of the nuclei of rat thymocytes to disaggregate and the nuclei to become structurally homogeneous. This was accompanied by the appearance of histones in the cytoplasm while the histones which remained in the nucleus could no longer be stained with fast green although they could be stained by the Sakaguchi reaction (which stains protein-bound arginine). The development of these changes was prevented or strongly retarded by increasing the NaCl concentration of the medium after irradiation. It was also retarded by the addition of spermidine to the cultures immediately after irradiation. It is suggested that these procedures owed their effectiveness to their ability to cause chromatin condensation. The rate of appearance of radiation-induced nuclear changes was strongly influenced by the inorganic phosphate concentration of the medium. It is proposed that chromatin disaggregation is primarily caused by an increase in the inorganic phosphate concentration in the nucleus since phosphate is known to competitively remove the chromatin-condensing histones from their salt-like linkages with DNA. High salt concentrations would retard the radiation effects by condensing the chromatin and thus rendering it less accessible to the phosphate and they might assist a damaged sodium-dependent transportation of metabolic intermediates into the nucleus. Spermidine can react with phosphate and thereby reduce the effective intranuclear phosphate concentration as well as rendering the chromatin less accessible to phosphate attack by aggregation.

The effect of X-irradiation on the nicotinamide adenine dinucleotides (NAD-NADH) content of rat thymocytes

Abstract Isolated rat thymocytes, treated with X-rays (1500 r) or subjected to sham irradiation, were incubated at 37 °C for 4 1 2 hr. Samples were taken at different time intervals for cytological examination and nicotinamide adenine dinucleotide (NAD and NADH) measurements. In normal thymocytes NAD decreased slightly after 1 1 2 hr of incubation and then remained constant, while NADH declined slowly throughout the experimental period. The findings may be the result of the isolation procedure and of the natural ageing of cultures. X-rays caused a severe drop of NAD and NADH in the cells, the decrease being much greater than in normal cultures. However, in both cases (irradiated and unirradiated thymocytes) the change occurred at the same time and to approximately the same degree as the disappearance of nuclear structure (nuclear homogenisation) which is considered the symptom of cell death. The losses of pyridine nucleotides by X-ray treated thymocytes accompanied rather than caused the death of cells. Therefore they should not be regarded as early and prevailing events in the process of radiation injury, but as unspecific and late effects of cellular damage. In fact at all the phases of experiment, the irradiated thymocytes with regularly stained nuclei seemed to contain a normal amount of pyridine nucleotides. The cytological and the NAD-NADH changes in irradiated cultures are not necessarily associated or closely linked together. In fact, the proper addition of nicotinamide or spermidine to the incubation medium allowed them to occur independently.

THE EFFECTS OF X-RADIATION ON LACTATE METABOLISM OF MAMMALIAN CELLS.

Abstract There were two distinct phases of lactate production in suspension cultures of irradiated (1500 r) rat thymocytes. Lactate was produced during the first hour after irradiation. Production then stopped only to resume after 3 hr. Unirradiated cells did not produce lactate after an initial production during the isolation procedure. The first phase of lactate production in irradiated cultures was probably a combined result of the isolation trauma and exaggeration of this trauma by radiation. The second phase followed the disappearance of the characteristic reticulo-granular chromatin structures of the nucleus and the disappearance of stainable DNA-associated histone. These cytological symptoms accompany cell death. In a medium without phosphate, the postirradiation development of cytological damage was inhibited and the cells consumed the lactate produced during isolation instead of making more. In the presence of phosphate, there was a strong stimulation of lactate production and development of cytological damage by irradiation. Exposure of cells to spermidine (0.10 M) or a high Na+ concentration (260 mEq/l) immediately after irradiation strongly retarded the development of cytological damage, but they did not retard lactate production. These agents strongly stimulated lactate production in unirradiated thymocyte cultures. Exposure of thymocytes to 0.10 M nicotinamide from 0 to 3 hr after irradiation prevented further development of cytological damage and further production of lactate. Irradiation (1500 and 1700 r) of suspension cultures of the more radio-resistant L mouse cells which rapidly consume lactate during the later phases of growth resulted in a transient inability to consume lactate. The cells fully regained their ability to consume lactate 8 to 16 hours after irradiation. Possible mechanisms underlying these initial metabolic effects of irradiation are outlined and discussed.

Mitotic stimulation in normal and irradiated suspension cultures of rat bone marrow by an elevated salt concentration

Abstract Increasing the sodium concentration of the medium of suspension cultures of rat bone marrow accelerated the entry of normal cells into mitosis. A similar acceleration was obtained in irradiated (75 and 100 r) suspensions and the duration of postirradiation mitotic delay was strongly reduced.

THE EFFECTS OF X-RADIATION AND NICOTINAMIDE ON THE RESPIRATION OF RAT THYMOCYTES

SummaryThe respiration rate of thymocyte suspension cultures irradiated with 1·5 kr remains normal until the process of nuclear structural homogenization is well under way. Reduction of respiration rate by cyanide, nicotinamide and a very high dose of x-radiation (30 kr) is associated with a strong retardation of nuclear changes.It is proposed that the inhibition of respiration is, in fact, responsible for the inhibition of nuclear structural changes by these agents. A possible mechanism for this effect of respiratory inhibition is discussed.

Prevention of nuclear changes in irradiated rat thymocytes by nicotinamide

Abstract Nicotinamide added to suspension cultures of thymocytes immediately to 2 hr after X-irradiation retards or prevents the development of the cytological symptoms of nuclear damage and cell death.

Reduction of mitotic delay in irradiated suspension cultures of rat thymocytes by an elevated salt concentration

Abstract Increasing the concentration of NaCl in the medium of thymocyte cultures from 0.12 M to 0.15 M after irradiation with 225 and 300 r reduces the duration of the initial mitotic delay. This procedure had no effect on cultures irradiated with 150 r.

SYNCHRONISATION OF CELL DIVISION IN SUSPENSION CULTURES OF L STRAIN MOUSE CELLS.

Abstract Synchronous cell division can be induced in randomly growing populations of strain L mouse fibroblasts by suspending the cells in a medium without serum for 54 to 58 hr followed by addition of serum and halving the cell concentration.

FACTORS INFLUENCING NUCLEAR DAMAGE IN RAT THYMOCYTES IRRADIATED WITH 30 KR OF X-RAYS.

Abstract The rate of appearance of nuclear damage in rat thymocytes irradiated with 30 Kr was unexpectedly slow in a medium containing 15 m M phosphate. However, increasing the phosphate concentration to 30 m M doubled the rate of development of pycnosis. These results were obtained using preparations made with cells mixed with serum prior to fixation. When serum was not used the cells with damaged nuclei were largely destroyed by the cytological manipulations and the observed rate of development of nuclear damage was much lower.

The demonstration of cytoplasmic basic proteins in strain L mouse fibroblasts

Abstract Cytoplasmic basic proteins stained with alkaline fast green could be seen in L strain mouse cells provided the cells were not dehydrated with alcohol and xylol prior to mounting. These basic proteins were located in fibrillar structures which also contained RNA. If the stained cells were dehydrated with alcohol and xylol and mounted in Canada balsam, only the nuclei remained stained while the stain was completely extracted from the cytoplasm.