Proliferative changes in two murine tumours in response to single or fractionated doses of X-rays

Abstract. Studies were carried out to investigate proliferative changes in two murine experimental tumours in response to radiation. Results were generated using bro-modeoxyuridine labelling and flow cytometry. This study demonstrates the possible ambiguity of previous studies using tritiated thymidine in which inability to discriminate normal and tumour cell components in murine tumours may lead to different values for cell kinetic parameters. In particular, the sarcoma F appeared to have a growth fraction of 0.62 when all cells were considered; in reality the growth fraction of the tumour cells only (based on DNA content discrimination) was close to unity. Radiation, administered either as single or fractionated doses, caused little change in the proliferative characteristics of the sarcoma F tumour but had profound effects on the adenocarcinoma Rhodesia tumour. Major changes were the accumulation of cells in G₂ for several days after the end of the radiation treatment in both tumours and a dramatic drop in labelling index of the Rhodesia tumour. In neither tumour was there any evidence to suggest an increase in tumour cell proliferation during or after the irradiations. The diploid cells within the sarcoma F tumour showed an initial depression of labelling index followed by a rapid increase overshooting the control labelling index at higher radiation doses. Much of the effects could be attributed to cell cycle delays.     

The response of the skin of swine to increasing single exposures of 250-KVP X-rays

When the skin of the shoulder ("A" field) and lower back ("C" field) is irradiated through 10-cm-diameter fields with 250-kVp x-rays, having a HVL of 0.87 mm copper, a dose range is reached between approximately 1600 rads and 3000 rads in which a moist reaction is or is not formed. If a moist reaction is formed, it either heals completely, partially, or not at all. The evolution, time course, and dose dependence of the moist reaction that occurs following irradiation has been determined. The moist reaction is found at 17.5 +/- 0.6 days in the "A" field, and 20.8 +/-0.8 days in the "C" field. The reaction evolves to involve from 5 % to 100% of the field by 24.9 +/- 0.5 days in the "A" field and by 28.5 +/- 1.0 days in the "C" field. Healing is complete by 36.0 +/-1.0 days in the "A" field and by 38.0 +/- 1.3 days in the "C" field. The area of the field involved with a moist reaction at the time of maximal involvement is dose-dependent. The area of the field involved with a moist reaction at the time of complete healing is also dose-dependent. The dose at which 50 % of the fields were not healed was 2273 +/-103 rads in the "A" field, 2578 +/-141 rads in the "C" fields, and 2437 +/- 89 rads in the "A" and "C" fields. The values in the "C" field are significantly different from those in the "A" field except for the dose at which 50 % of the fields were not healed and the time at which the field was maximally healed.     

The response of mouse skin to hyperthermia combined with fast neutrons or X-rays

The effects of hyperthermia combined with fast neutrons (mean energy approximately 7.5 MeV) or X-rays (250 kVp) were studied in the skin of the mouse ear and foot. Hyperthermia was achieved by immersion in water at temperatures of 41.5-43.0 degrees C for 1 hour. The heat treatments used caused no observable tissue injury other than transient erythema but they enhanced the response to both neutrons and X-rays. The enhancement of neutron damage increased as the heating temperature was increased, as is well known for X-rays. When heat was given after irradiation the thermal enhancement ratio (t.e.r.) for neutrons was similar to that for X-rays. When heat was given before irradiation the neutron t.e.r. was less than that for X-rays. Consequently, the relative biological effectiveness of fast neutrons compared with X-rays was not altered by giving heat after irradiation but it was reduced by giving heat before irradiation.     

The response of mouse skin to combined hyperthermia and X-rays.

The effects of combined hyperthermia and X-irradiation were studied in the skin of the mouse ear. Ears were heated for 1 hour by immersion in a waterbath at temperatures ranging from 37 degrees C--43 degrees C. These heat treatments had little visible effect alone, but when combined with X-rays, enhanced the radiation response. Enhancement depended on the degree of heating. When heat was given immediately after X-rays, the radiation dose to cause a given skin reaction had to be reduced by about 10 per cent for 37 degrees C and about 40 per cent of 43 degrees C. The timing and sequence of the two treatments were important. Heat after X-rays was less effective than heat before X-rays. When heat followed X-rays, the enhancing effect was lost completely if the interval exceeded 4 hours. When heat preceded X-rays, the effect was lost more slowly, depending on temperature. The implications of this for the treatment of cancer by combined therapy are discussed.     

DNA damage in Chinese hamster cells repeatedly exposed to low doses of X-rays

In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90-95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.     

Adaptive response to 2 low doses of X-rays in human blood lymphocytes

Human peripheral blood lymphocytes exposed to a single adaptive dose of 1 cGy X-rays or 2 adaptive doses, each of 1 cGy, were found to be equally resistant to the induction of chromosome damage by subsequent challenge with a high dose of 1 Gy X-rays, as compared to cells that were not pre-exposed. They responded with a significantly reduced incidence of chromatid and isochromatid breaks. These results indicate the presence of an inducible chromosomal repair mechanism in human blood lymphocytes and confirm the observations made by earlier investigators. The incidence of chromosome damage was found to be similar in the lymphocytes pre-exposed to a single or 2 adaptive doses, suggesting that, under the conditions tested, the second adaptive dose did not offer any additional protection against the chromosome damage induced by the challenge dose.     

Effects of m-aminobenzamide on the response of Chinese hamster cells to hyperthermia and/or radiation

The modifying effects of m-aminobenzamide (m-ABA), an inhibitor of poly(ADP-ribose) synthesis, on 42 degrees C hyperthermia- and/or radiation-induced cell killing were examined in Chinese hamster V-79 cells. When cells were exposed to 42 degrees C hyperthermia in combination with m-ABA (10 mM), cell survival decreased compared with that for 42 degrees C hyperthermia alone. Thermosensitizing effects of m-ABA changed with treatments in a decreasing order of during and after heating greater than during heating greater than after heating. Treatments with m-ABA during and/or after X irradiation enhanced radiation-induced cell killing. When cells were exposed to combined treatment with X irradiation, 42 degrees C hyperthermia (60 min), and m-ABA (24 hr), cell survival decreased markedly compared with that for X irradiation alone. However, with both X----42 degrees C and X----42 degrees C----m-ABA, the enhancement ratios (ER), designated as D0 ratio, were similar. These results suggest that the mechanisms of radiosensitization by m-ABA may be similar to those of 42 degrees C hyperthermia.     

Late occurring lesions in the skin of rats after repeated doses of X-rays

Late radiation damage, characterized by atrophy and necrosis in the skin and subcutaneous tissues, has been demonstrated in both the tail and feet of rats. The incidence of necrosis increased with total dose. These total doses, in the range 72-144 Gy, were given as 4-8 treatments of 18 Gy, each dose separated from the next by an interval of 28 days. This treatment protocol minimized acute epithelial skin reactions. The same regime applied to the skin on the back of rats resulted in a very severe acute reaction occurring after the second to fifth dose of 18 Gy. This was surprising since back skin, like tail skin, is less sensitive to large single doses of radiation than that of the foot. The late radiation reaction in the foot and tail of rats are compared and contrasted with other attempts to assess late effects in rodent skin and with late changes seen in pig skin.     

The induction by X-rays of chromosome aberrations in male Guinea-pigs, rabbits and golden hamsters. III. Dose-response relationship after single doses of X-rays to spermatogonia.

The induction by X-rays of translocations in spermatogonia was studied by cytological means in spermatocytes derived from them. In the rabbit and guinea-pig hump shaped dose-response curves were obtained, with a linear relationship at the low doses. The shapes of the curves were similar to those reported for the mouse, except that the maximum occurred at 600-700 rad in the mouse as opposed to 300 rad in the guinea-pig and rabbit. Unlike the guinea-pig and rabbit, the golden hamster showed a hump dose-response curve without a definite peak value and with little decrease in yield at high radiation doses. Over the low dose range 100-300 rad, the slopes of the curves of translocation yield were in the order:mouse (highest), rabbit, guinea-pig and hamster. Data on sterile periods suggested that the amount of spermatogonial killing in the rabbit and guinea-pig was as great or greater than in the mouse, and that in the golden hamster it was most severe. It is suggested that the differing shapes of the dose-response curves can be explained by a lower sensitivity to translocation induction in the test species and, also especially in the golden hamster, a greater sensitivity to cell killing. The possibility of extrapolating from these data to other species is discussed.     

The differential response of the skin in young and old rats to a combination of X-rays and 'wet' or 'dry' hyperthermia

The left hind feet of groups of female rats aged 7, 14 and 52 weeks were irradiated at three dose levels of X-rays (20, 25 or 30 Gy). Hyperthermia (42.5 degrees C for 1 h) was carried out immediately following irradiation using either 'wet' or 'dry' heat, achieved by immersion in either water or fluorocarbon liquid. The results demonstrated that 'wet' heat produced a consistently greater enhancement of the irradiation damage than 'dry' heat. The thermal enhancement ratio for irradiation plus 'wet' heat was approximately 1.5 and for irradiation plus 'dry' heat it was in the range 1.17 to 1.39. Immersion of the feet in fluorocarbon liquid at 37 degrees C did not significantly modify the irradiation response of the skin. The lower thermal enhancement ratios obtained using immersion in fluorocarbon liquid at 42.5 degrees C are close to those obtained in large animal studies and also similar to the limited amount of data from clinical studies where microwave or ultrasound heating techniques were used. It has been demonstrated that there are large age-related differences in the response of the rat foot skin to irradiation alone. It has also been shown in the present study, using rats of the same age, that the response to irradiation plus hyperthermia was less age dependent. This finding may reflect the differing methods by which damage occurs in tissue after irradiation or hyperthermia.     

Effects of single or repeated administrations of methamphetamine on immune response in mice.

The present study aimed to clarify the connection between immune responses and the administration frequency of methamphetamine (MAP) in male and female mice. Male and female ddY mice were given single or multiple (repeated for 10 days) intraperitoneal injections of MAP (5.0 mg/kg/day). The following immune parameters were examined; the number of leukocytes in peripheral blood and the proliferative activity (phytohemagglutinin;PHA, lipopolysaccharide; LPS response) and natural killer (NK) cell activity in splenic lymphocytes. Further, the differences in metabolic function in the spleen in response to MAP (and its metabolite amphetamine) in male and female mice were measured by gas chromatography. The results of the present study were that; 1) single and repeated MAP injections reduced leukocytes; 2) single MAP injection increased the proliferative response of splenic lymphocytes to PHA stimulation in only male mice, but the response to LPS stimulation was slightly increased in both male and female mice; 3) single and repeated MAP injections reduced NK cell activity of splenic lymphocytes, and especially in female mice with 5 injections of MAP; 4) with 10 MAP injections the NK cell activity and leukocytes recovered to the level of controls; and 5) the metabolic activity of MAP was reduced in female mice treated acutely with MAP in comparison to male mice. These results appear to indicate that immune responses to MAP were involved in the different results shown for administration frequency, sex difference and metabolic process of MAP.     

Adaptive response to chromosome damage in cultured human lymphocytes primed with low doses of X-rays

Human lymphocytes exposed to 0.02 Gy of X-rays in the G1 but not the G0 phase became less susceptible to the induction of chromosome aberrations of the chromosome type by subsequent exposure to 3 Gy of X-rays. The induction of chromatid-type aberrations was not affected by the pretreatment with the priming dose. The expression of this adaptive-type response was transitory, being maximum at 5 h, and disappeared at 9 h after the initial low-dose exposure. Cell-cycle analysis excluded the possibility of a spurious consequence of differential cell-cycle progression.     

Single or multiple daily doses of aminoglycosides: a meta-analysis.

OBJECTIVE--To assess relative efficacy and toxicity of aminoglycosides given by single daily dose compared with multiple daily doses. DESIGN--Meta-analysis of 21 randomised trials identified through MEDLARS (1966 to January 1995). Data were overviewed with fixed effects and random effects models and with meta-regression analysis. SUBJECTS--Total of 3091 patients with bacterial infection, most without pre-existing renal disease. INTERVENTIONS--Patients were randomized to receive aminoglycosides once daily or multiple times daily with similar total daily dose. MAIN OUTCOME MEASURES--Clinical failure of treatment, nephrotoxicity, ototoxicity, and mortality. RESULTS--Single daily dose regimen produced a non-significant decrease in risk of antibiotic failures (random effects risk ratio 0.83 (95% confidence interval 0.57 to 1.21)). Benefit of once daily dosing was greater when the percentage of pseudomonas isolates in a trial was larger. Once daily administration reduced risk of nephrotoxicity (fixed effects risk ratio 0.74 (0.54 to 1.00)). Similar trends were noted for patients with febrile neutropenia and for children. There was no significant difference in ototoxicity between the two dosing regimens, but the power of the pooled trials to detect a meaningful difference was low. There was no significant difference in mortality. CONCLUSIONS--Once daily administration of aminoglycosides in patients without pre-existing renal impairment is as effective as multiple daily dosing, has a lower risk of nephrotoxicity, and no greater risk of ototoxicity. Given the additional convenience and reduced cost, once daily dosing should be the preferred mode of administration.     

Effects of hyperthermia on the mouse testis and its response to X-rays, as assayed by weight loss.

The effects of both hyperthermia alone and X-rays combined with hyperthermia on mouse testis have been investigated. Testis weight on heating time was observed for temperatures in the range 39.5 to 43.75 degrees C. The relationship between the reaction rate and the reciprocal of absolute temperature indicated that, over the temperature range considered, the activation energy associated with such thermal damage was (646 +/- 45) x 10(3) J mol-1. No evidence was obtained to indicate a change in slope of the Arrhenius plot over this temperature range. Finally, despite the high sensitivity of the testis to heat and X-rays, no thermal enhancement of the weight loss after irradiation was observed when thermal treatments which, if given alone would produce some observable damage, were administered immediately after irradiation.     

A cell kinetic model to explain the time of appearance of skin reaction after X-rays or ultraviolet light irradiation.

Skin reactions to various doses of X-rays (300 and 10 kV) and ultraviolet light (u.v.) have been compared using hairless mice. Two regions of epidermis with widely differing cell kinetics and gross structure have been compared. Little evidence could be found to support the idea that the early phases of the reaction are dependent on cell cycle time. The data can be explained by a model based on the assumption that epidermis contains only a small fraction of clonogenic (stem) cells and this fraction may vary in different epidermal regions. X-rays appear to exert their greatest destructive action on these clonogenic cells while u.v. is more indiscriminate in its action, killing both clonogenic and non-clonogenic cells.     

Protein binding of indobufen enantiomers: pharmacokinetics of free fraction-studies after single or multiple doses of rac-indobufen

The binding of the enantiomers of indobufen (INDB) to human serum proteins was investigated using the racemic mixture or the pure (+)-S-enantiomer in a concentration range of 2.5-100.0 mg/L. In addition, the pharmacokinetics of free (unbound) and total INDB enantiomers were studied 1) following administration of a single 200 mg rac-INDB tablet to healthy volunteers, and 2) in obliterative atherosclerosis patients at steady state. The free fraction of INDB was obtained by ultrafiltration. Using the racemic mixture, the binding parameters of the two enantiomers were different, showing enantioselectivity in protein binding. The (-)-R-enantiomer was bound more strongly to human serum albumin, with association constant K = 11.95 +/- 0.98 x 10(5) M(-1) and n = 0.72 +/- 0.02 binding sites. The comparable data for the (+)-S-enantiomer were K = 4.65 +/- 0.02 x 10(5) M(-1), n = 0.92 +/- 0.01. When the binding of (+)-S-enantiomer was studied alone, the association constant K (2.10 +/- 0.18 x 10(5) M(-1)) was lower and the number of binding sites was increased, to n = 1.87 +/- 0.17. Competition occurred between the enantiomers, with the (-)-R-enantiomer displacing its antipode. The fraction of both enantiomers bound to serum proteins was 99.0%, which increased with decreasing initial concentration of the enantiomers. In healthy volunteers the (+)-S-enantiomer was eliminated faster than its (-)-R antipode, resulting in a lower AUC for the (+)-S-enantiomer. Significant differences were observed in the total INDB enantiomer concentrations. The mean unbound fraction of (-)-R- and (+)-S-INDB was 0.45% and 0.43%, respectively. Levels of the free (+)-S-enantiomer were higher than its (-)-R-antipode at steady state in patients with obliterative atherosclerosis who also took other drugs. The free enantiomer fraction increased to around 1% upon repeated administration. We conclude that the more rapid elimination of the (+)-S enantiomer is associated with its weaker binding to serum proteins.     

Effects of diets high in corn oil or in extra virgin olive oil on oxidative stress in an experimental model of breast cancer

Molecular Biology Reports - Experimental evidence highlights the importance of dietetic factors on breast cancer. In this work we aimed to analyze the effects two oils, corn oil (rich in n-6...