Micronuclei in Cytokinesis-blocked Lymphocytes of Cancer Patients Following Fractionated Partial-body Radiotherapy

Summary

We applied the cytokinesis-block micronucleus assay to measure chromosome damage in lymphocytes of 11 cancer patients undergoing fractionated partial-body irradiation. Measurements performed before, during and after cessation of radiotherapy showed a dose-related increase in micronucleus frequency in each of the patients studied. When the results for micronucleus frequency (Y) were plotted against the estimated equivalent whole-body dose (X) the dose-response relationship obtained was Y = 75·8X + 49·5 (r = 0·783, P < 0·0001). A general decline in MN frequency was observed during the post-treatment period down to 57 per cent (± 10) after 12 months but there was considerable variation between individuals. The advantages and disadvantages of the application of the cytokinesis-block micronucleus assay as a biological dosemeter for lymphocytes irradiated in vivo are discussed.     

Comparison of the protective effects of melatonin and amifostine on radiation-induced epiphyseal injury

Purpose: We compared the effects of amifostine and melatonin in preventing radiation-induced epiphyseal growth plate injury in rats.

Materials and methods: Four-week-old (65–85 g), growing male Sprague-Dawley rats were randomly assigned to receive radiation alone, at 25 Gy in three fractions (group R), or this dose of fractionated radiation proceeded by prophylactic amifostine 200 mg/kg i.p. (group A), melatonin 15 mg/kg i.p. (group M), or amifostine + melatonin (group AM). The right rear extremity of each animal was irradiated while the contralateral leg was shielded from radiation, as a control. Bone growth based on the length of the tibia, femur, and overall limb was calculated 6 weeks after the treatment.

Results: In groups R, A, M, and AM, the mean growth loss (GL) for the overall limb was 56.9 ± 8.1%, 46.8 ± 7.7%, 36.6 ± 4.3%, and 38.5 ± 5.1%, respectively. The limb length discrepancies (LLD) in groups R, A, M, and AM were 13.8 ± 1.4%, 10.5 ± 0.3%, 7.4 ± 0.7%, and 8.8 ± 1.1%, respectively. Differences in LLD were significant between each treatment group and group R (range: p = 0.0001–0.001). Differences in either of mean GL and LLD were not significant between groups M and AM; however both of these groups had significantly less GL and LLD than group A.

Conclusions: We observed a superior radioprotective function of melatonin over amifostine in preventing radiation-induced epiphyseal growth plate injury, without any increase in radioprotective effect by adding amifostine to melatonin.     

Radioprotective effects of mistletoe extract in zebrafish embryos in vivo

Abstract

Introduction: Radioprotectors can enhance the efficacy of cancer radiotherapy, but their clinical use remains uncommon. The present study aimed to assess the radioprotective potential of mistletoe extract (commercial name: Abnoba Viscum), a well-known complementary cancer medicine, in zebrafish larvae.

Materials and methods: Wild-type AB zebrafish embryos at 4?h-post-fertilization were exposed to 5?Gy 9-MeV electron beam irradiation after being treated for 1?h with 4?mMl/L amifostine or 0.2?mg/ml Abnoba Viscum A, F, M, or Q. Primary endpoints were abnormality-free survival and abnormality-free rates among survivors at 5 days-post-fertilization.

Results: The crude abnormality-free survival rates were 33.7%, 49.0%, 38.8%, 43.9%, 38.1%, and 52.6%, whereas abnormality-free rates among survivors were 36.4%, 49.6%, 37.8%, 45.6%, 52.0%, and 62.8% for the control (with no pharmacologic treatment), amifostine, Abnoba Viscum A, F, M, and Q groups, respectively. Abnormality-free survival rates in the amifostine and Abnoba Viscum Q groups were significantly different from those in the control (p?=?.040 and .012, respectively), with an odds ratio (OR) of 1.90 [95% confidence interval (CI): 1.03–3.51] and 2.20 (95% CI: 1.19–4.08), respectively. Abnormality-free rates among survivors in the amifostine and Abnoba Viscum M and Q groups were significantly different from those in the control group (p?=?.048, .042, and <.001, respectively), with an OR of 1.79 (95% CI: 1.00–3.20), 1.82 (95% CI: 1.02–3.26), and 2.98 (1.67–5.33), respectively.

Conclusion: Abnoba Viscum Q has at least a similar radioprotective effect to that of amifostine. Mistletoe extracts have been clinically applied for a long time and their effectiveness and feasibility have been verified. Abnoba Viscum Q might be a new candidate radioprotectant to enhance cancer radiotherapy efficacy.     

硒对鼻咽癌患者放射治疗后细胞免疫功能的影响

本文作者报道了硒对鼻咽癌患者放疗后细胞免疫功能影响的体外实验，结果表明：适宜浓度的硒（４．３４×１０－７ｍｏｌ／Ｌ）显著提高患者经ＰＨＡ诱导的淋巴细胞转化作用：３Ｈ－ＴｄＲ掺入量由（３．１７±２．４２）×１０４·ｍｉｎ－１·１０－６淋巴细胞增至（５．６７±５．６３）×１０４·ｍｉｎ－１·１０－６淋巴细胞，较对照组提高了（７８．８６±７７．６０）％，Ｐ＜０．０１。同时，硒还显著提高ＮＫ细胞杀伤活性：３Ｈ－ＴｄＲ释放率由（２７．９０±９．２２）％增加至（３４．５６±１３．４５）％，Ｐ＜０．０５，提示：硒在恢复放疗后鼻咽癌患者的免疫功能的治疗中，可能具有一定的价值。     

gamma-H2AX foci formation in peripheral blood lymphocytes of tumor patients after local radiotherapy to different sites of the body: Dependence on the dose-distribution,irradiated site and time from start of treatment

Purpose: To evaluate the relationship between an estimated integral total body radiation dose delivered and phosphorylated histone H2AX protein (γ-H2AX) foci formation in peripheral blood lymphocytes of cancer patients.

Material and methods: γ-H2AX formation was quantified as the mean number of foci per lymphocyte (N_meanH2AX) and the percentage of lymphocytes with ≥n foci. The integrated total body radiation dose was estimated from the dose volume histogram of patient's body corrected for the proportion of the body scanned by computed tomography for 3D treatment planning.

Results: There was a strong linear correlation between the mean number of γ-H2AX foci per lymphocyte in the peripheral blood sample and integrated total body radiation dose (r = 0.83, p < 0.0001). The slope of the relationship was dependent on the site of body irradiated. In comparison to chest irradiation with a slope of 8.7 ± 0.8 foci Gy^?1, the slopes for brain, upper leg and pelvic sites were significantly shallower by ?4.7, ?4.3, and ?3.8 Gy^?1, respectively (p < 0.0001), while the slope for upper abdomen irradiation was significantly larger by 9.1 ± 2.6 Gy^?1 (p = 0.0007). There was a slight time effect since the start of radiotherapy on the slopes of the in vivo dose responses leading to shallower slopes (?1.5 ± 0.7 Gy^?1, p = 0.03) later (≥10 day) during radiotherapy. After in vitro irradiation, lymphocytes showed 10.41 ± 0.12 foci per Gy with no evidence of inter-individual heterogeneity.

Conclusions: γ-H2AX measurements in peripheral lymphocytes after local radiotherapy allow the estimation of the applied integral body dose. The site and time dependence have to be considered.     

Radioprotective effect of amifostine on cells from cancer prone patients and healthy individuals studied by the G2 and PCC assays

Purpose: To investigate whether amifostine is effective at reducing the yield of chromatid breaks when present during G₂‐phase irradiation of human normal cells and cells from cancer prone patients, as well as to study the mechanisms underlying the radioprotective effect of amifostine.

Materials and methods: G₂ chromosomal radiosensitivity in the presence or absence of amifostine was studied in healthy donors, cancer patients, ataxia‐telangietasia (A‐T) patients and five human lymphoblastoid cell lines with genes predisposing to cancer. The yield of chromatid breaks following γ‐irradiation in G₂ phase was obtained at the subsequent metaphase using the G₂ assay. For scoring chromatid damage directly in G₂ or G₀ phase, premature chromosome condensation was used.

Results: When amifostine was present during irradiation, the mean yield of radiation‐induced chromatid breaks as visualized by the G₂ assay was significantly reduced in healthy donors (t‐test, p=0.001), in cells from cancer patients (p=0.001) and in cell lines from patients with genes predisposing to cancer (p=0.01) except ATM^?/? (0.1<p<0.2). However, when chromatid breaks were scored directly in G₂ or G₀ phase using premature chromosome condensation, the presence of amifostine did not affect the yields obtained.

Conclusion: Amifostine reduces the mean yield of chromatid breaks in normal cells and in cells from cancer prone patients when present during G₂ irradiation. Although the precise mechanisms of radioprotection caused by amifostine remain unclear, the results obtained using premature chromosome condensation reveal that amifostine does not act on cells only as a free radical scavenger and as a repair enhancer of DNA damage.     

Randomized Phase III Trial of Postoperative Radiochemotherapy ± Amifostine in Head and Neck Cancer

PURPOSE: Experimental and clinical data suggest a reduction of radiation-induced acute toxicity by amifostine (A). We investigated this issue in a randomized trial comparing radiochemotherapy (RT + CT) versus radiochemotherapy plus amifostine (RC + CT + A) in patients with head and neck cancer. PATIENTS AND METHODS: 56 patients with oro-/hypopharynx or larynx cancer (T1-2 N1-2 G3, T3-4 N0-2 G1-3) were randomized to receive RC + CT alone or RC + CT + A. Patients were irradiated up to 60 Gy (R0) or 70 Gy (R1/2) and received chemotherapy (70 mg/m(2) carboplatin, day 1-5 in week 1 and 5 of radiotherapy). 250 mg amifostine were applied daily before each radiotherapy session. Acute toxicity was evaluated according to the Common Toxicity Criteria (CTC). As for acute xerostomia, patients with laryngeal cancer were excluded from evaluation. RESULTS: 50 patients were evaluable (25 patients in the RC + CT, 25 patients in the RC + CT + A group). Clinical characteristics were well balanced in both treatment groups. Amifostine provided reduction in acute xerostomia and mucositis but had no obvious influence on Karnofsky performance status, body weight, cutaneous side effects, and alopecia. The differences between both groups were statistically significant for acute xerostomia and nonsignificant, but with a trend for mucositis. CONCLUSIONS: According to our results, there is a radioprotective effect on salivary glands and a potential effect on oral mucosa by amifostine in postoperative radiotherapy combined with carboplatin. To improve the radio- and chemoprotective effects of amifostine in clinical practice, the application of a higher dose (> 250 mg) seems to be necessary.     

分割照射对肝癌移植瘤小鼠脾脏免疫细胞的影响

目的探讨肝癌皮下移植瘤小鼠的分割照射对远端脾脏组织的影响。方法构建荷瘤C57BL/6 J小鼠模型,取对数期生长的小鼠肝癌细胞Hepa 1-6接种于小鼠右侧皮下(1×10^7/只),荷瘤小鼠按随机数表法分为荷瘤对照组和照射组,每组20只。另设10只健康小鼠作为健康对照组。照射组皮下肿瘤给予8 Gy×3次X射线局部照射,剂量率为0.883 Gy/min。照射后第7、14天检测小鼠肿瘤脏器指数、脾脏脏器指数、脾脏病理学改变,以及脾脏T淋巴细胞亚群、B淋巴细胞亚群、NK细胞的变化。结果与荷瘤对照组相比,照后7、14 d,照射组小鼠肿瘤脏器指数显著降低(t=4.649、26.34,P<0.05),脾脏中NK细胞显著升高(t=3.952、3.633,P<0.05),CD3+、CD4+、CD3+CD4+淋巴细胞以及CD4+/CD8+比值在照后7 d显著降低(t=3.193、3.656、3.219、2.641,P<0.05),CD3+淋巴细胞在照后14 d显著降低(t=3.031,P<0.05),其余指标脾脏脏器指数、B淋巴细胞、CD3+CD4+淋巴细胞、CD8+淋巴细胞变化不显著。结论肿瘤局部照射可引起远隔器官内淋巴细胞比例失衡,导致机体免疫力下降,为放疗免疫损伤提供了新的诠释。     

Association of genetic variants in apoptosis genes FAS and FASL with radiation-induced late toxicity after prostate cancer radiotherapy

Background and purpose

Fas ligand (FASL) triggers apoptotic cell death by cross-linking with its receptor FAS, and after irradiation, expression of FAS and FASL is increased. In the present study, we investigated the association between common polymorphisms in the genes for FAS and FASL and the risk of late side effects after radiotherapy for prostate cancer.

Patients and methods

The role of FAS (??1377G > A, rs2234767 and ??670A > G, rs1800682) and FASL (??844C > T, rs763110) gene polymorphisms in the development of high-grade late rectal and/or urinary toxicity (defined as late toxicity EORTC/RTOG grade ≥?2) was analyzed in 607 prostate cancer patients treated with radiotherapy. DNA was isolated and the selected polymorphisms were determined by 5’-nuclease (TaqMan) assays.

Results

After a median follow-up time of 82 months, high-grade late rectal and/or urinary toxicity was observed in 175 patients (29.7?%). Univariate analysis revealed a significantly decreased risk of high-grade late toxicity in carriers of the FASL ??844T allele. After adjusting for covariates, patients harboring at least one ??844T allele (CT or TT genotype) remained at decreased risk of high-grade late toxicity compared with patients harboring the CC genotype [hazard ratio (HR) 0.585, 95?%CI 0.39–0.878; p?=?0.010]. For patients with the ??844TT genotype, the HR was 0.404 (95?%CI 0.171–0.956; p?=?0.039) in multivariate analysis. No significant associations were found for the remaining polymorphisms analyzed.

Conclusions

These results provide the first evidence that the presence of the FASL ??844T variant allele may have a protective effect against the development of high-grade late rectal and/or urinary side effects after prostate cancer radiotherapy.     

B-type natriuretic peptide plasma level in 5-year breast cancer survivors after radiotherapy

Abstract

Background: Left-sided breast cancer patients treated with radiotherapy (RT) are at risk for late radiation-induced cardiovascular complications.

Aim: The aim of this study was to investigate the BNP plasma levels in long-term breast cancer survivors who received only RT as well to assess whether cardiac dose was associated with BNP values.

Methods: Plasma samples for BNP measurement were repeated in 29 patients (63?±?11 years) who were alive at 5 years after radiotherapy, free of heart disease and available to provide new blood sample. All patients had BNP measurements at baseline. The ΔBNP was measured to analyze the role of marker variations. No patients received chemotherapy.

Results: The mean cardiac and ventricle dose were 2.1?±?1.0 (range 0.02–4.5) Gy and 3.0?±?1.7 (range 0.02–7.6), respectively. Median value of BNP was 47?pg/mL (interquartile ranges, 26–58.2?pg/mL) at baseline, and 34?pg/mL (interquartile ranges, 17.5–54?pg/mL) at 5 years after radiotherapy. There was no significantly different between two measurements (p?=?ns). Fifteen (52%) reported an improvement in BNP levels, 1 (3%) no changes and 13 (45%) reported a worsening. There was no correlation between ΔBNP and age (p?=?ns).

When patients were stratified according to the median value of dose–volume data, ΔBNP was significantly higher in patients with increased cardiac D_mean (p?=?.02) and left ventricle D_mean (p?=?.009).

Conclusion: At 5 years after radiotherapy, median plasma BNP levels remained within the normal range, but the delta-BNP levels are directly related to the heart and ventricular dose received.     

Hypoxia inducible factor (HIF1α and HIF2α) and carbonic anhydrase 9 (CA9) expression and response of head-neck cancer to hypofractionated and accelerated radiotherapy

Purpose: Tumor hypoxia and low intrinsic radiosensitivity may counteract the efficacy of standard radiotherapy for locally advanced head and neck cancer (HNC). We investigated the involvement of hypoxia-regulated proteins (Hypoxia inducible factors HIF1α, HIF2α and carbonic anhydrase CA9) in HNC resistance to accelerated and hypofractionated radiotherapy.

Materials and methods: Thirty-nine patients with locally advanced HNC received 15 daily fractions of 3.4 Gy amounting to a total tumor dose of 51 Gy (equivalent to 63 Gy in four weeks – one week split); this was combined with platinum chemotherapy and amifostine cytoprotection administered subcutaneously. Immunohistochemical analysis of hypoxia-regulated proteins, namely HIF1α, HIF2α and CA9, was performed in formalin-fixed paraffin-embedded tissues obtained prior to radio-chemotherapy.

Results: HIF1α and HIF2α were expressed in the nuclei and cytoplasm of cancer cells, while CA9 had a membrane reactivity. A high expression of HIF1α, HIF2α and CA9 was noted in 21/39 (53.8%), 20/39 (51.3%) and 23/39 (58.9%) cases, respectively. Complete response was obtained in 85.2% of patients and HIF1α was marginally related with persistent disease after RT (p = 0.05). HIF1α was significantly associated with poor local relapse free survival (LRFS) (p = 0.006) and overall survival (p = 0.008), whilst HIF2α was not. A significant association of CA9 expression with poor LRFS was noted (p = 0.01).

Conclusion: In accord with previously reported studies, high levels of the hypoxia regulated proteins HIF1α and CA9 in HNC predict resistance to platinum based radio-chemotherapy. Whether HIF2α expressing tumors are more sensitive to larger radiotherapy fractions, compared to standard radiotherapy fractionation, is an issue that deserves further investigation.     

Genome damage in testicular seminoma patients seven years after radiotherapy

Abstract

Purpose: Testicular seminoma cancer incidence has significantly increased over the last few decades, and although it is successfully treated by radiotherapy, long-term health risks are still unclear. The aim of the study was to show long-term genome damage in patients with seminoma after radiotherapy.

Materials and methods: Chromosome aberration (CA) and micronucleus (MN) assays seven years after radiotherapy with a total dose of 25 Gy were conducted in 10 testicular seminoma patients aged 23–49 years and results were compared with 10 healthy control subjects matched for age and smoking status.

Results: Although mean CA frequency did not deviate from control values, significantly increased frequencies of dicentrics, double minutes, and ring chromosomes were detected in seminoma patients. MN frequency in binuclear lymphocytes of patients was similar to controls (4.60/1000 vs. 5.82/1000, respectively). Significantly higher MN frequency was detected in mononuclear lymphocytes of patients than in controls (2.55/1000 vs. 0.73/1000, respectively). Average percentage of centromere-positive MN was 62.6% in seminoma patients.

Conclusion: This study shows the persistence of unstable CA in seminoma patients seven years after radiotherapy and the relevance of long-term follow up. MN frequency in mononuclear lymphocytes was shown to be relevant biomarker of long-term genome damage.     

Delayed chromosomal instability in lymphocytes of cancer patients after radiotherapy

Purpose:?To assess possible delayed chromosomal instability (DCI) expressed as elevated chromatid breakage in cells containing previously formed chromosome type aberrations in cultured blood lymphocytes of cancer patients after radiotherapy (RT).

Materials and methods:?Twenty patients treated for uterine cancer with external Co⁶⁰ RT, without chemotherapy, were selected. Blood was taken before, 1–2 days after RT and one year later. Lymphocytes were cultured for 50 and 100?h. Metaphases were stained with fluorescence-plus-Giemsa and analysed for chromosome and chromatid aberrations in 1st (M1) and 3rd plus later (M3+) mitoses.

Results:?RT caused a significant increase of radiation-specific chromosome aberrations in patients' lymphocytes together with DCI, which was observed as an excessive yield of cells containing both chromosome and chromatid aberrations (defined as C_acs&act). This DCI passed successfully through mitoses in?vitro, and at the end of RT a mean yield of ‘extra’ C_acs&act was 3?×?10^?3?×?cell^?1 amongst either M1 or M3+ cells. At the end of RT and one year later DCI in M1 lymphocytes appeared at random amongst patients, but some inter-individual variation was found for DCI presence in M3+ cells at both post-irradiation samplings. As time passed, the mean yield of lymphocytes exhibiting DCI decreased in?vivo and one year after RT reached the pre-treatment level of 1?×?10^?3?×?cell^?1.

Conclusions:?DCI was demonstrated in descendants of human lymphocytes after therapeutic irradiation. The effect diminished one year later, suggesting that the progeny of patients' irradiated stem cells did not produce new daughter lymphocytes exhibiting DCI during the studied post-irradiation period.     

In vitro cellular radiosensitivity in relationship to late normal tissue reactions in breast cancer patients: a multi-endpoint case-control study

Purpose: A minority of patients exhibits severe late normal tissue toxicity after radiotherapy (RT), possibly related to their inherent individual radiation sensitivity. This study aimed to evaluate four different candidate in vitro cellular radiosensitivity assays for prediction of late normal tissue reactions, in a retrospective matched case-control set-up of breast cancer patients.

Methods: The study population consists of breast cancer patients expressing severe radiation toxicity (12 cases) and no or minimal reactions (12 controls), with a follow-up for at least 3 years. Late adverse reactions were evaluated by comparing standardized photographs pre- and post-RT resulting in an overall cosmetic score and by clinical examination using the LENT-SOMA scale. Four cellular assays on peripheral blood lymphocytes reported to be associated with normal tissue reactions were performed after in vitro irradiation of patient blood samples to compare case and control radiation responses: radiation-induced CD8+ late apoptosis, residual DNA double-strand breaks, G0 and G2 micronucleus assay.

Results: A significant difference was observed for all cellular endpoints when matched cases and controls were compared both pairwise and grouped. However, it is important to point out that most case-control pairs showed a substantial overlap in standard deviations, which questions the predictive value of the individual assays. The apoptosis assay performed best, with less apoptosis seen in CD8+ lymphocytes of the cases (average: 14.45%) than in their matched controls (average: 30.64%) for 11 out of 12 patient pairs (p?p?p?Conclusion: This matched case-control study in breast cancer patients, using different endpoints for in vitro cellular radiosensitivity related to DNA repair and apoptosis, suggests that patients’ intrinsic radiosensitivity is involved in the development of late normal tissue reactions after RT. Larger prospective studies are warranted to validate the retrospective findings and to use in vitro cellular assays in the future to predict late normal tissue radiosensitivity and discriminate individuals with marked RT responses.     

Differences in the cellular and humoral immune system between sedentary and endurance-trained elderly males

Aim. – This study aimed to examine the association between active vs. inactive lifestyle and cellular and humoral immune system in the elderly.Methods. – Eleven elderly male athletes (mean age ± S.D.: 67.1 ± 6.0 year) performing regular aerobic exercise for about 38.8 ± 18.5 yeasr (4.4 ± 1.4 d week^–1; 10.0 ± 8.1 h week^–1) and eleven male individuals at similar ages (mean age ± S.D.: 64.9 ± 4.6 year) leading a sedentary life were taken as control group. Immunological assessments were total leukocyte count, lymphocyte subpopulation, natural killer percentages, and IgA, IgG, and IgM concentrations.Results. – Baseline NK cell percentage, and serum IgA and IgM concentrations of master athletes were significantly higher than those of control group (29.3 ± 12.8% vs. 20.0 ± 7.5%; 2.4 ± 0.66 g/l vs. 1.6 ± 0.63 g/l; 1.0 ± 0.47 g/l vs. 0.58 ± 0.31 g/l, respectively; P < 0.05). No statistical differences were obtained in the total leukocyte counts and neutrophils, eosinophils, lymphocytes, monocytes, and the percentages of total T cells, B cells and T cell subsets (CD4+ T cells, CD8+ T cells), and IgG levels of the two groups.Conclusion. – Habitual exercise training may cause a slow down in the age-related decline in NK cell percentages and serum IgA and IgM levels of the elderly.     

Histamine prevents functional and morphological alterations of submandibular glands induced by ionising radiation

Purpose:?Xerostomia is a common, disturbing side-effect among patients treated with radiotherapy for head-and-neck cancer. The aim of the present work was to investigate whether histamine could prevent salivary gland dysfunction and histological alterations exerted by ionising radiation.

Materials and methods:?Forty-eight rats were divided into four groups. Histamine and histamine-5 Gy groups received a daily subcutaneous histamine injection (0.1 mg/kg) starting 24?h before irradiation. Histamine-5 Gy and untreated-5 Gy groups were irradiated with a single dose of whole-body Cesium-137 irradiation. Control and untreated-5 Gy groups were given daily saline injections. Three days post irradiation metacholine-induced salivary secretion was measured or animals were sacrificed and submandibular gland (SMG) removed, stained and histological characteristics were evaluated. Proliferation and apoptosis markers were studied by immunohistochemistry.

Results:?Radiation decreased salivary secretion by 40% in comparison to untreated rats, which was associated with loss of SMG mass, alteration of epithelial architecture, partial loss of secretor granular material, diminished proliferation and a remarkable apoptotic response. In contrast, histamine completely reversed the reduced salivation induced by radiation, conserved glandular mass with normal appearance and preserved the structural organisation of secretor granules. Radiation-induced toxicity is prevented by histamine essentially by suppressing apoptosis of ductal and acinar cells, reducing the number of apoptotic cells per field (19.0?±?3.8 vs. 106.0?±?12.0 in untreated animals, P?<?0.001), and also by preventing the radiation-induced decrease in cell proliferation.

Conclusions:?Histamine prevents morphological and functional radiation-induced damage on SMG, representing a potential radioprotector for treatment of patients undergoing radiotherapy for head and neck malignancies.     

DNA repair foci and late apoptosis/necrosis in peripheral blood lymphocytes of breast cancer patients undergoing radiotherapy

Abstract

Purpose: Double-strand breaks (DSB) repair and apoptosis are assumed to be key factors in the determination of individual variability in response to radiation treatment. In this study we investigated tumor protein p53 (TP53) binding protein 1 (53BP1) and phosphorylated histone 2A family member X (γH2AX) foci, γH2AX pan-staining and late apoptosis/necrosis (LAN) in lymphocytes from breast cancer (BC) patients undergoing radiotherapy.

Materials and methods: BC patients were subjected to local radiotherapy with fractionated doses using linear accelerator. Adverse reactions of patients were classified according to the Radiation Therapy Oncology Group (RTOG)/European Organization for Research and Treatment of Cancer (EORTC) criteria. Blood samples were collected before treatment, at various time-points during and after radiotherapy. Residual 53BP1 and γH2AX foci, γH2AX pan-staining were analyzed in peripheral blood lymphocytes (PBL) using the Metafer system and confocal laser scanning microscopy. LAN cells were counted by the trypan blue (TB) exclusion assay. Statistical analysis was performed using Mann–Whitney test, Spearman rank correlation test and analysis of covariance (ANCOVA).

Results: No statistically significant changes were observed in the levels of γH2AX foci during radiotherapy. In contrast, radiation-induced residual 53BP1 were detected already after the first fraction. Increased individual variability in the 53BP1 focus formation was observed during treatment. The background level of DNA repair foci and its individual variability in response to radiotherapy decreased after the end of radiotherapy indicating successful removal of DNA-damaging effects. A correlation between stage of cancer and 53BP1 focus formation was established which suggests the prognostic value of this test. We show that the fraction of LAN cells negatively correlates with the level of 53BP1 and positively correlates with individual radiosensitivity. Only weak correlation was observed between γH2AX pan-staining and LAN cells. Due to large interindividual variability, both in vivo assays, LAN and focus formation, have shown relatively low predictive power at the individual level.

Conclusions: It is likely that radiosensitive patients have less efficient mechanisms of elimination of apoptotic cells with DNA damage resulting in accumulation of LAN cells and facilitating adverse reactions. Our data suggested that the grade of adverse reaction may positively correlate with LAN cells in PBL before and during radiotherapy.     

Radioprotection in mice following oral delivery of amifostine nanoparticles

Purpose: Amifostine (Ethyol^®) is an approved cytoprotective agent prescribed to reduce certain side-effects in the chemotherapy of ovarian or non-small cell lung cancer, or in radiation treatment of head-and-neck cancer. The usefulness of this drug is further hampered, because it is not effective when given orally. The objective of this part of the project was to evaluate the radioprotective efficacy of orally active amifostine nanoparticles.

Materials and methods: Radioprotective efficacy was evaluated by measuring the ability of the amifostine nanoparticles (equivalent to 500?mg/Kg) to inhibit whole-body gamma irradiation -induced injury in mice. All mice received acute whole-body gamma irradiation from a Cesium-137 source and the radioprotective efficacy of the formulation was determined by measuring 30-day survival at 9?Gy, bone marrow hemopoeitic progenitor cell survival at 9?Gy and 8?Gy, and intestinal crypt cell survival at 11?Gy.

Results: Thirty-day survival, hemopoietic progenitor cell survival, as well as the jejunal crypt cell survival were all significantly enhanced when the mice were treated orally with the amifostine nanoparticles 1?h prior to irradiation.

Conclusions: These results clearly and unequivocally demonstrate that the amifostine nanoparticles developed in our laboratory provides significant protection from acute whole-body gamma irradiation injury in mice.     

Caffeine ingestion and antigen‐stimulated human lymphocyte activation after prolonged cycling

This study investigated the effect of caffeine ingestion on antigen‐stimulated T‐ (CD4⁺ and CD8⁺) and natural killer (NK)‐ (CD3^?CD56⁺) cell activation after prolonged, strenuous cycling. In a randomized cross‐over design, nine male endurance cyclists (age: 22 ± 3 years, V?O_2peak: 62 ± 4 mL/kg/min, mean ± SD) cycled for 90 min at 70% V?O_2peak 60 min after ingesting 6 mg/kg body mass of caffeine (CAF) or placebo (PLA). Venous blood samples were obtained before supplementation, pre‐exercise, immediately post‐exercise and 1 h post‐exercise. Whole blood was stimulated with Pediacel (five in one) vaccine. At 1 h post‐exercise the number of antigen‐stimulated CD4⁺ cells expressing CD69 decreased on CAF compared with PLA [15 (17) × 10⁶ vs 23 (22) × 10⁶ cells/L, P<0.05]. In addition, the geometric mean fluorescence intensity (GMFI) of CD69 expression on antigen‐stimulated CD8⁺ cells decreased on CAF compared with PLA 1 h post‐exercise [78 (10)% vs 102 (24)%, P<0.05]. At the same time‐point GMFI of CD69 expression on antigen‐stimulated CD3^?CD56⁺ cells was increased on CAF compared with PLA [103 (9)% vs 87 (8)%, P<0.05]. These findings suggest that caffeine reduces antigen‐stimulated CD69 expression on T cells while at the same time increases NK‐cell activation 1 h after intensive cycling.     

Increase in efficacy of cancer radiotherapy by combination with whole-body low dose irradiation

Purpose:Design of cancer radiotherapy protocol to reduce radiation dose and increase treatment efficacy in Lewis lung cancer (LLC) model.

Methods: C57BL/6J mice subcutaneously implanted with LLC were treated by conventional radiotherapy (2Gy × 6) combined with LDWBI (low dose whole-body irradiation; the second, third, fifth and sixth local doses of 2Gy each substituted by LDWBI with 0.075Gy) and/or gene therapy (intratumor injection of pEgr-IL-18-B7.1 plasmid 24 h before the first and fourth local doses). Immunologic mechanisms were explored.

Results: Cancer control was most significantly improved in the group receiving local radiotherapy combined with LDWBI and gene therapy as shown by prolongation of mean survival time by 60.4%, reduction in average tumor weight by 70.8%, decrease in pulmonary metastasis by 66.9% and decrease in intratumor angiogenesis by 64.8% as compared to local radiotherapy alone (p < 0.05). These changes in tumor growth and progression were accompanied with up-regulation of host immunity manifested by stimulated NK (natural killer) and CTL (cytotoxic T lymphocyte) activity, IFN (interferon)-gamma and TNF (tumor necrosis factor)-alpha secretion, PKC (protein kinase C)-theta activation and LAMP (lysosomal associated membrane protein)-1 expression.

Conclusion: Combination of conventional radiotherapy with LDWBI and gene transfer could reduce total radiation dose by 2/3 and at the same time improve treatment efficacy of cancer accompanied with up-regulated host anticancer immunity.