Delayed effects of low level acute irradiation and chronic environmental radioactive contamination on DNA lymphocytes of people living in Dolon, a settlement located in the vicinity of the Semipalatinsk nuclear test site (Kazakhstan).

During 42 years several hundred nuclear tests were performed by the former USSR at the Semipalatinsk Test Site (STS, Kazakhstan), of which more than 100 were done in the atmosphere. We report here the late genetic damage of external exposure to radiation and environmental radioactive contamination in people living in Dolon, a small settlement situated in the vicinity of the STS. The comet assay was applied on DNA lymphocytes of 20 exposed women and 32 non-exposed women living at 500 km from the STS. We observed a statistically significant difference between the exposed and control groups for mean tail moment (MTM) and DNA% in the tail. The mean values of all comet assay parameters (MTM, DNA% in the tail and score) were higher in the group of women born before 1949 as compared to those born after 1950, which could reflect an effect of external irradiation in 1949 due to the most contaminating explosion. These results suggest that people exposed 50 years ago to relatively small doses of external irradiation and/or still living in an environment contaminated by small amounts of long life radionuclides, still present DNA damage which is in agreement with other cytogenetical studies performed at the same site, on the same population.

Effects of low doses of short-term gamma irradiation on growth and development through two generations of Pisum sativum.

The effects of short-term gamma radiation on pea plants were investigated by exposing 5-day-old seedlings with doses ranging from 0 to 60 Gy, and studying plant growth and development over two generations after irradiation. Doses higher than 6 Gy significantly inhibited the G1 plant growth and productivity, and no seedling survived irradiation with 40 Gy and above. These effects were transmitted and were even more severe in the next generation, G2. Irradiated G1 (> or =10 Gy) and G2 (> or = 0.4 Gy) plants were significantly smaller than controls. The mean number of pods produced per plant was reduced by at least 20% at all doses in both G1 and G2. In parallel, the mean numbers of ovules and normally developed seeds per pod were significantly reduced after 10 Gy in G1 and after 0.4 Gy in G2, leading to a significant drop in seed production. This effect was correlated with a linear decrease in male fertility linked to abnormal meiosis (tetrads with micronuclei) as a function of doses from 0 to 10 Gy, in G1 and G2 plants. These long-term changes in plant development demonstrate a genomic instability induced by irradiation. However, there were neither quantitative nor qualitative changes in storage proteins in G1 seeds at any of the irradiation doses tested from 0 to 10 Gy.

Alkaline single-cell gel electrophoresis (comet assay): a simple technique to show genomic instability in sporadic breast cancer.

The alkaline comet assay was used to study the genomic instability of lymphocytes derived from untreated sporadic breast cancer patients (50 cases), and also following their in vitro irradiation up to 5 Gy. We compared the results (mean tail moment (MTM)) with a control population of 25 patients and with breast cancer patients who had been 'cured' of their disease, with a follow-up of 10 years or more (25 cases). At the basal level, 77.5% (P<0.01) of the untreated patients and 73.7% (P<0.05) of the 'cured' women had values higher than the basal cut-off level of 5.3, compared with only 44% of the controls. After in vitro irradiation, 83% of the untreated patients were above the cut-off value of 10.8 at the 5-Gy dose compared with only 48% of the controls (P<0.01). These results support the hypothesis that women affected by sporadic breast cancer have a constitutional genomic instability. The assessment of the prognostic value of this test could be of interest, particularly in women without axillary nodal involvement.

Study of external low irradiation dose effects on induction of chromosome aberrations in Pisum sativum root tip meristem.

The effects of low doses of ionizing radiation have been a matter of important debate over the last few years. The point of discussion concerns the validity of the linear dose-response extrapolation for low doses, used by international organizations, to establish radio-protection norms. Here, we contributed to this discussion by investigating the induction of chromosome aberrations by low to moderate doses ranging from 0 to 10 Gy in root meristem cells of 6-day-old Pisum plantlets. After acute irradiation of plantlets by a (60)Co source, the percentage of root tip meristem cells displaying chromosome aberrations was estimated immediately after irradiation and after 20 h recovery time. The dose-effect curves show non-linear responses, especially in the low dose range (0- 1 Gy), which is of particular interest. After 20 h of recovery, a steep increase of aberrations was observed for cells exposed to 0.4 Gy, followed by a plateau for doses until 1 Gy. There was an irradiation effect on plant growth during the first and second generations, showing the persistence of cell division anomalies as a long term effect of acute irradiation. This result suggests the induction of a genomic instability. Our results, in agreement with some obtained in animals, show rather non-linear dose-effect responses, with notably higher biological effects of low doses than expected.

Genomic instability and breast cancer.

The loss of genomic stability is accepted as being one of the most important aspects of cancer. The correlation between genomic instability and cancer proneness in cases of known genetic syndromes (e.g. ataxia telengectasia, Fanconi anemia) is well established. This study was conducted to assess genomic instability in 19 patients with sporadic breast cancer. We used the comet assay on the lymphocytes of patients before radiotherapy and/or chemotherapy. The alkaline comet assay (single-cell gel electrophoresis) is a sensitive and rapid method for detecting DNA damage (single strand breaks and alkali-labile sites) in G(0) cells, at a single-cell level. This assay was achieved in vitro without irradiation and after exposure (dose ranging from 50 cGy to 5 Gy). The results show that the patients have higher baseline values than controls. At 2 Gy, the mean tail moment, score and the percentage of DNA in the tail increase for both groups but these values are much higher for patients. Our results show that the lymphocyte DNA of cancer patients is more damaged than that of controls with or without irradiation. Our hypothesis is that this baseline DNA damage reflect a genomic instability in sporadic breast cancer. This instability seems to increase after in vitro irradiation.

IL-6 a key cytokine in in vitro and in vivo response of Sertoli cells to external gamma irradiation.

Interleukin 1(IL-1) and IL-6 are cytokines involved in the response to radiation and are known for their radioprotective properties with respect to total-body irradiation. We previously showed that after gamma irradiation of Sertoli cells (SC), we observed an increase in the activity of IL-6 but not of IL-1. The aim of this study was to see whether this response is a function of the differentiation of SC, to analyse the mechanisms responsible for this induction, and to test whether this cytokine has a radioprotective role on germ cells. Unlike IL-1, a dose-dependent increase of IL-6 activity in SC following gamma irradiation at high doses was observed at all ages studied. On the other hand, radio-induction observed at low doses (<1Gy) was dose-independent. IL-6 up-regulation resulted from transcriptional activation as shown by the use of specific inhibitors. The injection of IL-1 and IL-6 in mice prior to whole-body irradiation resulted in an increased survival rate. Moreover, cytokines protected DNA from remaining cells following irradiation as shown by comet assay on germ cells. In conclusion, IL-6 seems to constitute a good marker of exposure to gamma irradiation, both at low and high doses. In addition, we showed that IL-1 and IL-6 have a radioprotective effect at testicular level.

Exposition of humans to low doses and low dose rate irradiation: an urgent need for new markers and new models.

In human radiation protection, the shape of the dose effects curve for low doses irradiation (LDI) is assumed to be linear, extrapolated from the clinical consequences of Hiroshima and Nagasaki nuclear explosions. This extrapolation probably overestimates the risk below 200 mSv. In many circumstances, the living species and cells can develop some mechanisms of adaptation. Classical epidemiological studies will not be able to answer the question and there is a need to assess more sensitive biological markers of the effects of LDI. The researches should be focused on DNA effects (strand breaks), radioinduced expression of new genes and proteins involved in the response to oxidative stress and DNA repair mechanisms. New experimental biomolecular techniques should be developed in parallel with more conventional ones. Such studies would permit to assess new biological markers of radiosensitivity, which could be of great interest in radiation protection and radio-oncology.

Regulatory influence of germ cells on sertoli cell function in the pre-pubertal rat after acute irradiation of the testis.

While germ cell regulation of Sertoli cells has been extensively explored in adult rats in vivo, in contrast, very little is known about germ cell influence on Sertoli cell function at the time when spermatogenesis begins and develops. In the present study various Sertoli cell parameters (number, testicular androgen binding protein (ABP) and testin, serum inhibin-B and, indirectly, follicle-stimulating hormone (FSH)) were investigated after the exposure of 19-day-old rats to a low dose of 3 Grays of gamma-rays. Differentiated spermatogonia were the primary testicular targets of the gamma-rays, which resulted in progressive maturation depletion, sequentially and reversibly affecting all germ cell classes. Testicular weight declined to a nadir when pachytene spermatocytes and spermatids were depleted from the seminiferous epithelium and complete or near complete recovery of spermatogenesis and testicular weight was observed at the end of the experiment. Blood levels of FSH and ABP were normal during the first 11 days after irradiation, when spermatogonia and early spermatocytes were depleted. While the number of Sertoli cells was not significantly affected by the irradiation, from days 11-66 after gamma-irradiation, ABP production declined and FSH levels increased when pachytene spermatocytes and spermatids were depleted and the recovery of these parameters was only observed when spermatogenesis was fully restored. Comparison of the pattern of change in serum levels of inhibin-B and testicular levels of testin and of germ cell numbers strongly suggest a relationship between the disappearance of spermatocytes and spermatids from the seminiferous epithelium and the decrease in levels of inhibin-B and increase in levels of testin from 7 to 36 days post-irradiation. Levels of testin and inhibin-B were restored before spermatogenesis had totally returned to normal. In conclusion, this in vivo study shows that pre-pubertal Sertoli cell function is under the complex control of various germ cell classes. This control presents clear differences when compared with that previously observed in adult animals and depends on the Sertoli cell parameter of interest, as well as on the germ cell type.

Radiation effect on rat Sertoli cell function in vitro and in vivo.

PURPOSE: To investigate the response of Sertoli cell function to 60Co gamma-rays. MATERIALS AND METHODS: Rat Sertoli cells were exposed in vitro and in vivo to 60Co gamma-rays in the dose range 3 Gy to 48 Gy and at 3 Gy and 6 Gy respectively. Cell viability and transferrin and IL-6 production were measured at different times following irradiation. RESULTS: This study confirms the resistance of in vitro irradiated rat Sertoli cells in the dose range 3 Gy to 48 Gy in terms of cell number. Radiation had no effect on the IL-1 activity of Sertoli cells. However, the experiments show that despite the absence of a macroscopic effect, Sertoli cells respond to ionizing radiation by increasing transferrin secretion, transferrin response to (Bu)2cAMP stimulation and IL-6 activity. CONCLUSIONS: Transferrin is involved in the transport of iron into germ cells and in cell differentiation. IL-6 is a potent inhibitor of meiotic DNA synthesis. Radio-induced transferrin and IL-6 could play a role in the protection of germ cells and could explain, in part, the resistance of Sertoli cells to radiation.

[Radiotherapy of stage T1-T2 M0 prostatic adenocarcinoma. Analysis of the carcinologic results of a multicenter study of 610 patients. Groupe Radiothérapie de la Commission de Coopération Médicale Intercentres (CCMI)]. / Radiothérapie de l'adénocarcinome prostatique de stade T1-T2 M0. Analyse des résultats carcinologiques d'une étude multicentrique de 610 patients. Groupe Radiothérapie de la Commission de Coopération Médicale Intercentres (CCMI).

PURPOSE: Retrospective analysis of the results of radiotherapy in localized prostatic adenocarcinoma. Complications were excluded. PATIENTS AND METHODS: Six-hundred-and-ten T1-T2 adenocarcinomas of the prostate were treated with continuous courses of external beam radiation therapy in 19 participating Institutes between January 1983 and January 1988. The mean follow-up was 10.4 years; the mean age of patients at the beginning of radiotherapy was 68.5 years. RESULTS: A 10-year, local control had been achieved in 86% of T1-T2 (81.4% for T2). The 5- and 10-year metastatic relapse rates were 25.3% and 30% (29% and 38.1% for T2), respectively. At 10 years, 62.4% of T1-T2 were recurrence-free; overall survival rate was 45.8% and cause-specific survival rate was 70.5%; 29.9% of T1-T2 patients were alive and disease-free. T category (TNM), pathologic grade, pelvic lymph node status, local tumor control, and obstructive ureteral symptoms were correlated with survival. The influence of pelvic nodes radiation, dose, overall treatment time, previous endocrine treatment, and transuretral resection was not significant for disease-free survival (alive and disease-free) and other endpoints. CONCLUSION: There was no difference between the French series (1975-1982 and 1983-1988). The results of the literature are comparable to ours. As far as prognostic factors are concerned, this report provides evidence that the explainable variables which influence survival depend on the tumor and patient status.

Increased levels of interleukin 5 are associated with the generation of eosinophilia in drug-induced hypersensitivity syndrome.

Hypersensitivity syndrome (HSS) usually refers to severe drug eruption associated with systemic symptoms and eosinophilia. Interleukin (IL)-5 regulates eosinophil counts with the help of IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Blood IL-5 levels have been reported to be increased in patients with eosinophilia secondary to parasitic infections or idiopathic eosinophilia, but have never been evaluated in drug-induced eosinophilia. The aim of our study was to determine whether IL-5, IL-3 and GM-CSF are involved in eosinophilia in patients with drug-induced HSS. Plasma levels of IL-3, IL-5 and GM-CSF were assayed by ELISA in seven patients with drug-induced HSS, in eight patients with cutaneous adverse drug reactions not associated with eosinophilia, and in five patients with eosinophilia unrelated to drug treatment. IL-5 levels were normal in all eight patients with drug eruptions without eosinophilia, and increased in five of the seven patients with HSS. In the latter patients, IL-5 levels peaked several days before highest eosinophil counts were noted, and returned to normal within a few days, even when eosinophilia persisted. In patients with eosinophilia unrelated to drug treatment, IL-5 levels, although significantly increased remained lower than in HSS patients. IL-3 and GM-CSF could not be detected in any group, at any time. Our results show that IL-5 is involved in drug-related eosinophilia. As IL-5 production was only involved in the early stages of the reaction, it is suggested that IL-5 mainly derives from activated lymphocytes rather than eosinophils. Our results support the clinical relevance of previous in vitro findings. Further studies are needed to test whether assays of IL-5 production by lymphocytes of patients stimulated by the suspected drug and/or its metabolites, are useful in establishing causality in drug-induced reactions associated with eosinophilia.

Experimental colonic carcinogenesis: a new animal model.

The aim of this work was to develop an animal model of accessible colonic tumours simulating human carcinogenesis. A segment of the colon was exteriorized to the skin in 60 rats and swabbed with N-methyl-N-nitrosourea. All the animals developed tumoral abnormalities or frank dysplasic tissue 7 months after the beginning of the experiment. This animal model which closely mimics human colonic carcinogenesis is particularly interesting because tumour development can be monitored in live animals without interrupting normal intestinal functions.

[Cancers of the base of the tongue and hypopharynx: results of a multicenter randomized trial of chemotherapy prior to locoregional treatment]. / Cancers de la base de langue et de l'hypopharynx: résultats d'un essai multicentrique randomisé de chimiothérapie avant traitement locorégional.

The authors report the results of a multicentric randomised trial assessing the effects on survival of neoadjuvant chemotherapy with cisplatin (100 mg/m2, D1) and fluorouracil (1 g/m2, D2-4) delivered before regional treatment in patients with squamous cell carcinoma of hypopharynx and base of tongue. 133 patients were enrolled in the study, and 121 were included in the analysis, 64 in group A (regional treatment alone) and 27 in group B (chemotherapy followed by regional treatment). Despite a high objective response rate to chemotherapy (primary tumour: 85%, 24% complete; nodes: 63%), overall survival was not significantly higher in group B than in group A.

[Radiotherapy and curietherapy of squamous cell carcinoma of the posterior pharyngeal wall (excluding the nasopharynx)]. / Radiothérapie et curiethérapie des épithéliomas de la paroi postérieure du pharynx (à l'exception du cavum).

From 1986 to 1992, 55 cases of PPWC were treated with a conservative intent at the Regional Cancer Center (Rennes, France) and Saint-Yves Center (Vannes, France): 16 oropharyngeal posterior wall carcinoma (OP) and 39 hypopharyngeal posterior wall (HP); the mean age of the population was 60.3 years (31-81 years). A previous and simultaneous head and neck cancer was noted in 15 and 13% of cases respectively. Half of the cases (55%) were T1 T2 tumors and 82% were N0 N1. Except for three patients treated by curietherapy (5%), all patients were treated by radiotherapy (RT) alone (75%) or associated with curietherapy (7%) or partial pharyngectomy (13%). 15% received neoadjuvant chemotherapy, mainly for T3 tumors. With a followup of 4-88 months (mean: 23 months) 38% of patients are still alive; 8% of loco-regionally controlled patients died of second cancer or intercurrent disease. The tumor control was 67%. The nodes control was 90%. During the course of the disease, 19% of patients had metastases. The complete response at the end of treatment was 78%. Among these patients, 54% remained definitively free of disease. There is no difference between OP and HP. The analysis of survival curves showed the following points: significant difference between T1 T2, and T2 T3 (P < 0.05), N0 N1 and N2 N3 (P < 0.03), well differentiated histology or not (P < 0.02), RT alone or associated with curietherapy or surgery (P < 0.03) even for limited tumors T1 T2 N0 N1 (P < 0.03). There was no significant difference between group treated or not by chemotherapy even for T3 tumors. These findings do not differ if we consider either OP or HP. We conclude that OP and HP have the same prognostic factors and must be considered as the same clinical entity. For limited tumors T1 T2 N0 N1, patients managed by radiotherapy associated with complementary local treatment (conservative surgery or curietherapy) do better than patients treated by RT alone (plateau 80% at 18 months+vs plateau 25% at 12 months +). For these limited tumors, our recommendation is to treat patients by external RT (50 Gy) and curietherapy boost (20 Gy) rather than by conservative surgery and external RT (70 Gy). These two treatments have the same efficacity but the first one is expected to diminish late complications of RT. Neo adjuvant chemotherapy does not seen to improve survival even for advanced tumors. Generally speaking these results remain poor for locally advanced desease and for undifferentiated tumors. These patients need a new therapeutic approach (concomittant radio-chemotherapy, hyper or hypofonctionnated RT).

Hyperfractionation versus conventional fractionation in oropharyngeal carcinoma: final analysis of a randomized trial of the EORTC cooperative group of radiotherapy.

EORTC protocol 22791 compared once daily fractionation (CF) of 70 Gy in 35-40 fractions in 7-8 weeks, to pure hyperfractionation (HF) of 80.5 Gy in 70 fractions in 7 weeks using 2 fractions of 1.15 Gy per day, in T2-T3 oropharyngeal carcinoma (excluding base of tongue), N0,N1 of less than 3 cm. From 1980 to 1987, 356 patients were entered. In the final analysis (June 1990), the local control was significantly higher (p = 0.02 log-rank) after HF compared with CF. At 5 years, 59% of patients are local disease-free in the HF arm compared to 40% in the CF arm. The superiority of HF was demonstrated in patients staged T3N0,T3N1 but not in T2. The Cox model confirmed that the treatment regimen was an independent significant prognostic factor for locoregional control (p = 0.007 log-rank). This improvement of locoregional control was responsible for a trend to an improved survival (p = 0.08 log-rank). There was no difference in late normal tissue damage between the two treatment modalities.

Unusual combination of Cis platinum and radiotherapy followed by a three fractions per day irradiation in splitcourse: a phase I-II study in brain glioma patients.

An unusual protocol based on a preliminary clinical study on cylindromas metastasized to the lung was proposed to brain glioma patients: Day 2 100 mg/m2 i.v. Cis platinum (Cis PII) followed at days 3 and 5 by 6 Gy irradiation (RT) in two fractions and three days. Five cycles were scheduled at 21 days interval. On disease progression a three fractions per day radiotherapy regimen (3 FRT) in split-course (two series of 22.50 Gy in 15 fractions and five days separated by a two weeks period of rest) was then delivered to the patients. All patients had a measurable mass on the CT scan. 19 were entered into the study: 13 as first line therapy (group A) and six for salvage treatment (group B). Tolerance was globally good. Eight patients were considered responders at the end of five cycles of Cis PII-RT. They were all group A patients. Median symptom-free interval was six months for the whole population. Survival was twelve months. The 3 FRT was well tolerated but does not seem to have improved the therapeutic gain of the chemoradiotherapy combination. The present study concerns patients whose prognosis was poor on inclusion: surgery inadvisable or unsatisfactory and diagnosis mainly based on biopsy only. The number and the duration of responses justify further study into Cis PII as first line therapy as either an effective cytotoxic drug or a potential radio enhancer.

[Randomized trial of initial chemotherapy in 151 locally advanced carcinoma of the cervix (T2b-N1, T3b, MO)]. / Essai randomisé de chimiothérapie initiale dans 151 carcinomes du col utérin localement étendus (T2b-N1, T3b, M0).

From 1982 to 1987, a randomized phase III trial was performed in order to determine the long-term effect of induction chemotherapy before standard pelvic irradiation in stage IIb-N1, III squamous cell carcinomas of the cervix. Patients were randomized to either chemotherapy and radiotherapy (C + R group) vs radiotherapy alone (R group). Radiotherapy for all patients consisted of 50 Gy in the pelvis with a boost by external irradiation or by brachytherapy (cumulative dose of 68 Gy). The chemotherapy regimen was an association of methotrexate (10 mg/m2, D2-4), chlorambucil (4 mg/m2, D1-5), vincristine (0,7 mg/m2, D1), cisplatin (80 mg/m2, D5), given every 3 wks; at least 2 courses were to be given before assessing efficacy and 2 more courses were given to patients who responded. One hundred and fifty-one patients were fully evaluable, after a mean follow-up of 38 mths (range 2-7 years), 76 in the R arm and 75 in the C + R arm. The response rate (greater than 50%) to chemotherapy was 42.5%. After completion of treatment, the complete response rate was 86.8% in the R arm and 86.3% in the C + R arm. The 3 year disease-free survival was 58.7% in the C + R group and 54.5% in the R group, and the median survival was 39.5% and 47 months respectively (NS). The survival of patients with a complete response at the end of radiotherapy was significantly better in the C + R group (when chemotherapy had been active) than in the R group (p = 0.04). Although radiotherapy was not modified whether patients had initial chemotherapy or not, tolerance was not significantly different between the 2 groups. The data collected in this study indicate that: 1) efficacy of induction chemotherapy is the only available predictive test for long-term results, 2) tolerance to treatment is crucial for optimal chemotherapy delivery, 3) higher dose intensity of chemotherapy in cervical carcinoma is associated with a better tumor reduction, and probably a better survival.

Proton NMR relaxation times of experimental Lewis lung carcinoma after irradiation.

NMR proton spin-lattice (T1) and spin-spin (T2) relaxation times were measured ex vivo on Lewis lung carcinoma after in vivo single irradiation with an absorbed dose of 4 Gy. The results were compared to tumoural volume evolution, pathological examinations, and cell kinetic measurements. Tumour growth decreased between the third and the sixth day after irradiation while relaxation times, especially T1, is increased 2 days before the clinical recurrence of the tumour is observed. Pathological morphometric measurements tempted to show that necrosis is less extended after irradiation. Cell cycle analysis demonstrated the G2/M phase blockade by radiation after one day, and its release 4 days later. These phenomena could be important for in vivo radiotherapy follow-up using determination of relaxation times by magnetic resonance imaging (MRI).