Radionuclides in radiation-induced bystander effect; may it share in radionuclide therapy?

For many years in radiobiology and radiotherapy predominated the conviction that cellular DNA is the main target for ionizing radiation, however, the view has changed in the past 20 years. Nowadays, it is assumed that not only directed (targeted) radiation effect, but also an indirect (non-targeted) effect may contribute to the result of radiation treatment. Non-targeted effect is relatively well recognized after external beam irradiation in vitro and in vivo, and comprises such phenomena like radiation-induced bystander effect (RIBE), genomic instability, adaptive response and abscopal (out of field) effect. These stress-induced and molecular signaling mediated phenomena appear in non-targeted cells as variety responses resembling that observed in directly hit cells. Bystander effects can be both detrimental and beneficial in dependence on dose, dose-rate, cell type, genetic status and experimental condition. Less is known about radionuclide-induced non-targeted effects in radionuclide therapy, although, based on characteristics of the radionuclide radiation, on experiments in vitro utilizing classical and 3-D cell cultures, and preclinical study on animals it seems obvious that exposure to radionuclide is accompanied by various bystander effects, mostly damaging, less often protective. This review summarizes existing data on radionuclide induced bystander effects comprising radionuclides emitting beta- and alpha-particles and Auger electrons used in tumor radiotherapy and diagnostics. So far, separation of the direct effect of radionuclide decay from crossfire and bystander effects in clinical targeted radionuclide therapy is impossible because of the lack of methods to assess whether, and to what extent bystander effect is involved in human organism. Considerations on this topic are also included.

Influence of local peripheral temporary ischaemia on biochemical and histological effects in small intestine and serum of rats following abdominal irradiation.

The local temporary ischaemia effect on radiation-induced lipid peroxidation, superoxide dismutase isoenzyme activities, and intestinal crypt number was estimated in male WAG-strain rats in vivo. The animals were irradiated in the abdomen area with doses of 2 Gy for ten consecutive days using a Philips 60Co source. The calculated dose rate was 0.595 Gy/min. Local temporary ischaemia was induced by clamping the tail base before each irradiation. The parameters evaluated were: TBA-RS level and enzymatic activities of CuZnSOD, MnSOD in serum and jejunum. The number of jejunum crypts was assigned as a histopathologic parameter. The results showed a clear protection by ischaemic preconditioning for crypt survival. The difference in the number of crypts in irradiated animals with and without local temporary ischaemia was statistically significant (Student's t-test P < 0.05). Also, significant enhancement of TBA-RS was observed in the serum of irradiated animals. Local temporary ischaemia application diminished the concentration of radiation- induced TBA-RS. The differences in the levels of TBA-RS in the serum were statistically significant (ANOVA P < 0.002). In contrast, there was no evident effect on the level of TBA-RS in tissue homogenates in any investigated groups. Some fluctuation of CuZnSOD isoenzyme activity in intestinal tissue was noted; however, the differences were not significant. Local temporary ischaemia had no influence on Mn- SOD activity in serum, and in both irradiated groups the behaviour of this isoenzyme was similar. Also, there were no differences in MnSOD activity measured in tissue homogenates. These findings support results of our previous in vivo studies, suggesting that local temporary ischaemia can prevent oxidative effects of fractionated radiotherapy.

Long-term effects of pioglitazone and metformin on insulin sensitivity in patients with Type 2 diabetes mellitus.

AIM: Despite their comparable glycaemic effects in patients with Type 2 diabetes mellitus (T2DM), pioglitazone and metformin may have different effects on insulin sensitivity because they have different mechanisms of action. We studied the changes in insulin sensitivity, as assessed by the Quantitative Insulin Sensitivity Check Index (QUICKI), in patients with T2DM who used metformin or pioglitazone as monotherapy or in combination therapy with sulphonylurea. METHODS: Data in this report are from two multicentre, randomized, double-blind, double-dummy studies conducted in Europe (monotherapy) or in Europe and Canada (combination therapy study). Patients were randomized to 52 weeks of treatment consisting of a 12-week forced titration period and a 40-week maintenance period. HbA(1c), fasting plasma glucose (FPG) and fasting serum insulin (FSI) were quantified from a single blood sample at weeks 0, 8, 16, 24, 32, 42 and 52. Insulin sensitivity was assessed with QUICKI, which is calculated from FSI and fasting blood glucose (FBG) concentrations using the formula 1/(log(10) FSI + log(10) FBG). Time course effects of the treatments were compared by repeated measures analysis of covariance. RESULTS: As monotherapy, pioglitazone and metformin increased QUICKI compared with baseline (baseline vs. end point [mean +/- sem]; pioglitazone [0.303 +/- 0.001 vs. 0.321 +/- 0.001; P < 0.001] and metformin [0.304 +/- 0.001 vs. 0.315 +/- 0.001; P < 0.001]). Pioglitazone increased insulin sensitivity more than metformin from week 4 through week 52. There were significant increases in QUICKI from baseline in both combination therapy groups (baseline vs. end point; pioglitazone + sulphonylurea [0.305 +/- 0.001 vs. 0.319 +/- 0.001; P < 0.001] and metformin + sulphonylurea [0.306 +/- 0.001 vs. 0.317 +/- 0.001; P < 0.001]). Overall, pioglitazone + sulphonylurea significantly increased insulin sensitivity more than metformin + sulphonylurea. CONCLUSION: Pioglitazone differed from metformin in its effects on insulin sensitivity despite both drugs having comparable glycaemic effects.

Pioglitazone elicits long-term improvements in insulin sensitivity in patients with type 2 diabetes: comparisons with gliclazide-based regimens.

AIMS/HYPOTHESIS: Recent studies have demonstrated that pioglitazone (PIO) has beneficial effects on insulin sensitivity compared with placebo in patients with type 2 diabetes. The effects of PIO and gliclazide (GLIC)-based therapy on insulin sensitivity have not previously been directly compared. This analysis aimed to compare the effects of 52 weeks of treatment with PIO (30-45 mg/day) and GLIC (80-320 mg/day), both titrated to maximum tolerable doses, as monotherapy or in combination with metformin (MET), on insulin sensitivity and lipid parameters known to be related to insulin sensitivity in patients with type 2 diabetes. METHODS: We performed an analysis of 1,880 patients with inadequately controlled type 2 diabetes (HbA1c 7.5-11.0%) who were participants in two parallel-group, double-blind, double-dummy, randomised, multicentre, clinical trials. Measures of insulin sensitivity and lipids were assessed. RESULTS: The PIO- and GLIC-based regimens produced similar levels of glycaemic control (HbA1c). In both trials, insulin sensitivity as assessed using the homeostasis model assessment was improved in patients receiving PIO, but decreased in those receiving GLIC (mean change, baseline to endpoint: PIO 15.5, GLIC -15.6; p<0.001 and PIO+MET 18.9, GLIC+MET -5.3; p<0.001). Improvements in the atherogenic index of plasma (mean change: PIO -0.17, GLIC -0.08; p<0.001 and PIO+MET -0.17, GLIC+MET -0.02; p<0.001), triglycerides (mean change, mmol/l: PIO+MET -0.62, GLIC+MET -0.22; p<0.001) and NEFA (mean change, mmol/l: PIO+MET -0.12, GLIC+MET-0.05; p<0.001) were greater in PIO-treated patients than in patients receiving GLIC. CONCLUSIONS/INTERPRETATION: The PIO-based regimens resulted in improved insulin sensitivity and more favourable insulin sensitivity-related lipid profiles compared with the GLIC-based regimens. These benefits may be important in the management of cardiovascular risk in patients with type 2 diabetes.

Inhibitory effect of local ischemic preconditioning on gamma ray-induced lipid peroxidation in rats: a preliminary study.

We examined the effect of local ischemic preconditioning on postradiation lipid peroxidation in the serum of total body irradiated rats. Markers of peroxidative damage provoked by radiation alone or radiation preceeded by ischemic preconditioning were thiobarbituric acid reactive substances, triglycerides and uric acid concentrations in serum. These data indicated that local ischemic preconditioning modifies the peroxidizing effects of radiation through inhibition of free radical-dependent lipid peroxidation. Other unrecognized mechanisms are probably also involved. Uric acid could act as an antioxidant against radiation alone and local preconditioned ischemia together with radiation.

Micronucleus assay in vivo provides significant prognostic information in human cervical carcinoma; the updated analysis.

PURPOSE: Reanalysis after a 5-year follow-up previously presented relationships between spontaneous and radiation-induced micronucleus frequencies in tumour cells and the clinical outcome of patients with advanced stages (II B-IV B) of cervix carcinomas treated with radiotherapy. MATERIALS AND METHODS: Spontaneous and induced in vitro and in vivo micronucleus frequencies were determined and related to clinical parameters. Data were analysed by the univariate Kaplan-Meier method and multivariate Cox proportional hazards model. RESULTS: In univariate analysis stage, spontaneous micronucleus frequency before radiotherapy (MNSP) and per cent increment of micronucleus level in vivo after 20 Gy in relation to spontaneous pretreatment level were statistically significant predictors of 5-year recurrence-free, disease-free and overall survival. Neither micronucleus frequency (MN/BNC at 2 Gy) nor proliferating fraction (%BNC at 0 Gy) estimated in vitro (in primary culture) were related to radiotherapy outcome. The age of patients was not associated with clinical results. Multivariate analysis demonstrated that the clinical stage of disease, the high frequency of spontaneous micronuclei and low-induced micronucleus frequency were independent and significantly unfavourable predictive factors for disease-free and overall survival. But for local control, only high MNSP and low-induced MN frequency were significant negative predictive variables. CONCLUSIONS: A high frequency of micronuclei before radiotherapy and a slight increase of micronucleus frequency during radiotherapy measured after 10 fractions of 2 Gy were independent on stage, statistically significant adverse predictors of clinical outcome in cervical carcinoma patients treated with radiotherapy.

The increment of micronucleus frequency in cervical carcinoma during irradiation in vivo and its prognostic value for tumour radiocurability.

A potential usefulness of micronucleus assay for prediction of tumour radiosensitivity has been tested in 64 patients with advanced stage (II B-IV B) cervical carcinoma treated by radiotherapy. The study of cellular radiosensitivity in vitro was conducted in parallel with the study of cellular damage after tumour irradiation in vivo. Radiosensitivity of in vitro cultured primary cells isolated from tumour biopsies taken before radiotherapy was evaluated using cytokinesis-block micronucleus assay. Frequency of micronuclei per binucleated cell (MN/BNC) at 2 Gy was used as a measure of radiosensitivity. Radiation sensitivity in vivo was expressed as per cent increment of micronucleus frequency in cells isolated from biopsy taken after 20 Gy (external irradiation, 10 x 2 Gy) over the pre-treatment spontaneous micronucleus level and was called MN20. Very low correlation (r = 0.324) was observed between micronucleus frequency in vitro and in vivo. Although micronucleus frequency at 2 Gy differed widely between tumours evaluated (mean MN/BNC was 0.224; range 0.08-0.416), no significant correlation was observed between this parameter and clinical outcome. The average increment of micronucleus frequency after 20 Gy amounted to 193% of spontaneous level (range 60-610%) and was independent of spontaneous micronucleation before radiotherapy. In contrast to in vitro results, these from in vivo assay seem to have a predictive value for radiotherapy of cervix cancer. The micronucleus increment in vivo that reached at least 117.5% of pretreatment value (first quartile for MN20 data set) correlated significantly with better tumour local control (P < 0.008) and overall survival (P < 0.045). Our results suggest that evaluation of increment of micronucleus frequency during radiotherapy (after fixed tested dose of 20 Gy) offers a potentially valuable approach to predicting individual radioresponsiveness and may be helpful for individualization of treatment strategy in advanced stage cervical cancer.

Modifying effect of vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in mouse cells.

The modifying effect of treatment with vitamins C, E and beta-carotene on the clastogenic activity of gamma rays was investigated in mice. Damage in vivo was measured by the micronucleus assay in bone marrow polychromatic erythrocytes and exfoliated bladder cells. The vitamins were administered orally, either for five consecutive days before or immediately after irradiation with 2 Gy of gamma rays. The results show that pretreatment with vitamin E (100-200 mg/kg/day) and beta-carotene (3-12 mg/kg/day) were effective in protecting against micronucleus induction by gamma rays. Vitamin C depending on its concentration enhanced the radiation effect (400 mg/kg/day), or reduced the number of micronucleated polychromatic erythrocytes (50-100 mg/kg/day). Such effect was weekly observed in exfoliated bladder cells. The most effective protection in both tissues was noted when a mixture of these vitamins was used as a pretreatment. Administration of the all antioxidant vitamins to mice immediately after irradiation was also effective in reducing the radiation-induced micronucleus frequency. The data from the in vitro experiments based on the comet assay show that the presence of the vitamins in culture medium influences the kinetic of repair of radiation-induced DNA damage in mouse leukocytes.

Inverse dose-rate effect for the induction of micronuclei in Lewis lung carcinoma after exposure to cobalt-60 gamma rays.

Induction of micronuclei was used as a measure of the dose-rate effect in Lewis lung carcinoma in vivo. Tumors transplanted on the hind legs of male C57BL mice were irradiated at dose rates of 1 and 0.34 Gy/min, cells were isolated and cultured in vitro, and micronuclei were scored at 24-h intervals. Maximum expression of micronuclei was observed 72 h after plating. The frequency of cells containing micronuclei and the number of micronuclei per single cell were linearly dependent on dose in the range 0-6 Gy. However, a marked inverse dependence on dose rate was observed. The inverse dose-rate effectiveness factor, calculated as the ratio of damage per gray at the lower dose rate to that at the higher dose rate, was 3.25 for frequency of micronuclei and was even higher (4.57) for micronuclei per cell (P < 0.05). Since the differences in exposure time for the different dose rates are not large, our results cannot be explained by the differential effect on cell kinetics during tumor irradiation. However, it cannot be excluded that the differential effect of radiation on division delay and redistribution of cells in the phases of the cell cycle may be expressed during incubation of cells in vitro for micronucleus expression. Furthermore, it can be hypothesized that more cells may die in culture because of interphase death and apoptosis in the higher dose-rate group than in the lower dose-rate group and that these cells were not accessible for the micronucleus assay. The actual explanation for the phenomenon observed requires further experimentation.

The radiosensitivity of human malignant melanomas evaluated by cytokinesis-block micronucleus assay.

Cytokinesis-block micronucleus assay (CB-MNA) was applied for comparison of radiation sensitivity of 25 human malignant melanomas in primary culture. Cells obtained from tumor specimens were irradiated (0-4.Gy) on dishes, incubated with cytochalasin B (2 micrograms/ml) to block cytokinesis, stained in situ and micronuclei (MN) scored in binucleate cells (BNC). Proportions of BNC in nonirradiated controls after fixed time of incubation (96 h) ranged from 2.3 to 38% indicating great differences (C.V. = 74%) in proliferative activity among tumors evaluated. No correlation was observed between proliferative activity and susceptibility of cells to induction of MN by radiation. The great inter-tumor heterogeneity was observed in respect of radiation sensitivity expressed either as normalized (Net) frequency (Fq) of BNC with MN or as number of MN per BNC. Both endpoints differed widely at 2 Gy and 4 Gy as well (Net FqBNC with MN = 0.28-25.4% or 1.5-45% and MN/BNC = 0.004-0.309 or 0.013-0.593 respectively at 2 Gy and 4 Gy) with coefficients of variation ranging from 44 to 57%. Extreme difference in MN frequency was also observed between one primary tumor and its metastasis indicating intra-tumor heterogeneity. Our results suggest that CB-MNA may contribute some clinically useful information for discriminating tumors that will eventually respond to radiotherapy and those that will probably not. However, studies aimed at comparison of MN induction in vitro with clinical radioresponsiveness of malignant melanomas are urgently required.

Activity of creatine kinase MB-isoenzyme in rat serum after heart irradiation and/or farmorubicin (4'-epidoxorubicin) treatment.

Activity of creatine kinase (CK, EC 2.7.3.2) and its CK-MB isoenzyme were evaluated in rat serum after external irradiation of the heart with 20 Gy given as A single dose or 4 x 5 Gy and also after treating the animals with farmorubicin 10 mg/kg in a single dose of 4 x 2.5 mg/kg. Fractionated irradiation or repeated injection of farmorubicin induced high five-fold transient increment of CK-MB isoenzyme activity in rat serum. Combined treatment (4 x 5 Gy heart irradiation, week 1, after 2 days pause 4 x 2.5 mg/kg farmorubicin, week 2) showed increase of MB isoenzyme activity in serum roughly comparable with that measured in animals treated with farmorubicin alone. The behaviour of MB isoenzyme activity after single doses of radiation 1 x 20 Gy or farmorubicin 1 x 10 mg/kg was dissimilar to that after repeated doses and was generally lower. Specially, a high single dose of farmorubicin led at first to suppression of CK-MB isoenzyme activity and then to an increase, however, slower than after fractionated treatment. Generally, elevation of CK-MB isoenzyme activity in rat serum after heart irradiation and farmorubicin injection (scheme independent) was many times higher than the total CK activity increment. Results suggest that estimation of CK-MB isoenzyme activity in serum is more sensitive and definitive evidence of early cardiac damage exerted by gamma-rays and/or farmorubicin than total CK activity.

Early peroxidizing effects of myocardial damage in rats after gamma-irradiation and farmorubicin (4'-epidoxorubicin) treatment.

Changes in lipid peroxide levels (TBA-RS) in rat serum and heart tissue as well as creatine kinase enzyme (CPK) activity in serum were used as early indicators of peroxidizing effects of heart damage after fractionated gamma-irradiation (4 x 5 Gy) and/or farmorubicin (4 x 2.5 mg/kg) treatment. An increase in the TBA-RS and enzyme activity was observed after the action of both agents given separately or in combination. The maximal expression of biochemical effects appeared a few days after irradiation or farmorubicin treatment. The application of the antioxidant, vitamin E, diminished the level of TBA-RS in serum and in heart homogenates plus CPK activity in serum, indicating the involvement of peroxidizing mechanisms in myocardial damage by both agents.

Nitroimidazoles, XIV: Synthesis of 4-nitroimidazoles with 1-substituents containing acid, ester or phenol functions, and radiosensitizing efficiency of some of these compounds.

1,4-Dinitroimidazole and 1,4-dinitro-2-methylimidazole were reacted with aminocarboxylic acids and their esters, aminosulfonic acids, and aminophenol to obtain the corresponding 1-substituted-4-nitroimidazoles. The radiosensitizing efficiency of some esters of 2-(4-nitro-1-imidazolyl)alkanecarboxylic acids was tested.

Mutagenic and clastogenic activity of the chloro-nitroimidazole radiosensitizer P40 in vitro and in vivo.

The hypoxic radiosensitizer 1-(2-hydroxy-3-methoxypropyl)-2-chloro-4-nitroimidazole [P40] was investigated for its mutagenic activity in bacterial Ames test as well as for genotoxic activity in micronucleus assay in vivo. This nitroimidazole showed the weak mutagenicity towards TA100 strain (base pair substitution) and towards TA98 strain (frameshift) only in the highest concentration. P40 induced also a significant increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) at the doses of 0.6 mg/g and 1.2 mg/g. The maximum time response was at 48 h. The decrease of percentage of PCEs suggested the possible cytotoxicity on bone marrow cells after treatment with P40. Positive results in this battery short-term tests provide evidence of clastogenic activity of P40.

Radiosensitization of the rats' rhabdomyosarcoma R 1 by chloronitroimidazole compound--P40 dependent on radiation dose in fractionated radiotherapy.

The radiosensitizing effectiveness of 1-(2-hydroxy-3-methoxy-propyl)-2-chloro-4-nitroimidazole (P40) dependent on various fractionated schedules was tested in the rat solid tumor, rhabdomyosarcoma R 1. P40 combined conventional fractionation of gamma rays exerted no radiosensitizing effect measured as local control and growth delay as well. In contrary, the significant sensitization has been noticed when nontoxic doses of the nitroimidazole were combined with higher (3.7 Gy) doses of radiation. Low toxicity of P40 is encouraging for further experimental studies.

Chloronitroimidazoles as radiosensitizers of hypoxic cells in vitro.

Some results of the first more complex studies in vitro on radio-sensitizing efficiency, cytotoxicity and reactivity with blood-thiols of a series of 4- or 5-nitroimidazoles substituted in the 5, 4 or 2 position with chlorine are presented. The derivatives of 4-nitroimidazole substituted in 5 position ("ortho" position) with Cl show higher radiosensitizing efficiency than one may expect from their reduction potential, E1/2. At the same time they are extremely toxic, especially for aerobic cells. It is considered that high biological activity of ortho-substituted 4-nitroimidazoles is connected with their considerable chemical reactivity towards thiols and suppression of those natural protective compounds in the cells. The corresponding 5-nitro isomers are about tenfold weaker sensitizers, and simultaneously much less cytotoxic, either in aerobic or in hypoxic conditions. The chloro-4(5)-nitroimidazoles nonsubstituted at N-1 and ionizable in aqueous solution are relatively weaker at the same time less toxic radiosensitizers. It is evaluated that potential application in radiotherapy may have those chloronitroimidazoles which show low aerobic cytotoxicity, moderate radiosensitizing ability and no reactivity towards thiols. On the basis of the study in vitro, we have selected such a compound: 1-methyl-2-chloro-4-nitroimidazole (P13) for screening in vivo.

2-Chloro-4-nitroimidazole radiosensitizers of hypoxic tumor cells in vivo.

The transplantable rhabdomyosarcoma in WAG/Rij rats was used to test the in vivo effectiveness of 1-methyl-2-chloro-4-nitroimidazole (P13) and its analog 1-(2-hydroxy-3-methoxy-propyl)-2-chloro-4-nitroimidazole (P40) as tumor-cell radiosensitizers after their i.p. administration at low doses. The results indicate that both compounds administered repeatedly at nontoxic concentrations (70-150 mg/kg body wt.) in combination with moderate fractional doses of irradiation (3.7 Gy) enhance the radiation effect on tumors. The local control of tumors on the 210th day in all experimental groups has been higher than in the control ones. In the case of P40 administered in the maximal dose, the increment in local control is statistically significant (p less than 0.05). The regrowth delay has also been longer after treatment with radiosensitizers. In the course of treatment we did not observe any symptoms of neurotoxicity of the compounds. The mean body weight of rats during the administration of compounds remained on the level of control rats whose tumors were irradiated only.

Radiosensitizing ability and cytotoxicity of some 5(4)-substituted 2-methyl-4(5)-nitroimidazoles.

Radiosensitizing efficiency and cytotoxicity of three 2-methyl-4(5)-nitroimidazoles substituted with electron-withdrawal groups -NO2, -Cl and -OCH3 in 5(4)-position ("ortho" position) have been evaluated in vitro on V 79-379 A cells under aerobic as well as under hypoxic conditions. It has been established that 2-h incubation of cells at 37 degrees C in the presence of increasing (up to 2 mM) concentration of the compounds causes a moderate cytotoxicity for P7(2-methyl-5(4)-chloro-4(5)-nitroimidazole) and P5(2-methyl-4,5-dinitroimidazole). Cytotoxicities of these compounds are much stronger under hypoxic than under aerobic conditions. The compound P9(2-methyl-5(4)-methoxy-4(5)-nitroimidazole) is practically completely nontoxic. The radiosensitizing efficiency of P5, P7, and P9 at 2 mM concentration expressed by ER (enhancement ratio) equals 1.75, 1.57 and 1.14 respectively. A discussion regarding the features of electron-affinity, cytotoxicity and radiosensitizing ability of the compounds studied is presented.

Response of the solid Guerin epitheliomas of rats to fractionated irradiation and a new 4-nitroimidazole.

The transplantable Guerin epithelioma in Wistar rats was used to test the in vivo effectiveness of 1-(2-hydroxy-3-methoxypropyl)-2-methyl-4-nitroimidazole (P1) as a tumour-cell radiosensitizer after its oral administration at relatively low doses. The radiosensitizing ability of P1 was compared with that of metronidazole. The results indicate that P1 is less toxic than metronidazole, and greater concentrations of P1 in blood and tumour tissues are obtained for the same administered dose of the compounds. The radiosensitizing ability of P1, determined from tumour-regression rates and local-control percentage at 130 days, was higher than that of metronidazole.