Gy Dose Of X-rays Research Articles

The induction of a radiation-induced bystander effect in fish transcends taxonomic group and trophic level

Purpose: To extend the investigations of bystander effect induction in fish of the same species as the irradiated fish, to bystander effect induction between fish species and between trophic levels.Materials and methods: To investigate interspecies bystander effect induction, zebrafish and medaka were irradiated with a 0.5 Gy X-ray dose and then swum with non-irradiated fish of the same and opposite species. To investigate trophic level bystander effect induction, California blackworms were irradiated with the same X-ray dose and then fed to non-irradiated rainbow trout.Results: Reductions in clonogenic survival of the HPV-G (non-transformed human keratinocytes, immortalized with the human papilloma virus) reporter cell line, treated with tissue explant media, revealed that zebrafish and medaka induced a pro-apoptotic bystander effect in the other species and that, in trout, the normally anti-apoptotic effect caused by the consumption of non-irradiated blackworms was significantly reduced or lost if the blackworms had been irradiated.Conclusions: These results are the first to show that a radiation- induced bystander effect can transcend taxonomic group and trophic level in fish. This provides further evidence that bystander signals are widespread and conserved and may be transmitted through an ecosystem, as well as between individuals of the same species.

Genomic characterization of a three-dimensional skin model following exposure to ionizing radiation

This study aimed at characterizing the genomic response to low versus moderate doses of ionizing radiation (LDIR versus MDIR) in a three-dimensional (3D) skin model, which exhibits a closer tissue complexity to human skin than monolayer cell cultures. EpiDermFT skin plugs were exposed to 0, 0.1 and 1 Gy doses of X-rays and harvested at 5 min, 3, 8 and 24 h post-irradiation (post-IR). RNA was interrogated for global gene expression alteration. Our results show that MDIR modulated a larger number of genes over the course of 24 h compared to LDIR. However, immediately and throughout the first 3h post-IR, LDIR modulated a larger number of genes than MDIR, mostly associated with cell–cell signaling and survival promotion. Significant modulation of pathways was detected only at 3 h post-IR in MDIR with induction of genes promoting apoptosis. Collectively, the data show different dynamics in the response to LDIR versus MDIR, especially in cell-cycle distribution. LDIR-exposed tissues showed signs of attempted cell-cycle re-entry as early as 3 h post-IR, but were arrested beyond 8 h at the G1/S checkpoint. At 24 h, cells appeared to accumulate at the G2/M checkpoint. MDIR-exposed tissues did not exhibit a prolonged G1/S arrest but rather a prolonged G2/M arrest, which was sustained at least up to 24 h. By 24 h cells exhibited signs of recovery in both LDIR- and MDIR-exposed tissues. In summary, the most pronounced difference in the initial cellular response to LDIR versus MDIR is the promotion of protection and survival in LDIR versus the promotion of apoptosis in MDIR.

Radiation-induced bystander effect in healthy G 0 human lymphocytes: Biological and clinical significance

To study the bystander effects, G 0 human peripheral blood lymphocytes were X-irradiated with 0.1, 0.5 and 3 Gy. After 24 h, cell-free conditioned media from irradiated cultures were transferred to unexposed lymphocytes. Following 48 h of medium transfer, viability, induction of apoptosis, telomere shortening, reactive oxygen species (ROS) levels and micronuclei (after stimulation) were analyzed. A statistically significant decrement in cell viability, concomitant with the loss of mitochondrial membrane potential, telomere shortening, increases in hydrogen peroxide (H 2O 2) and superoxide anion (O 2 −) with depletion of intracellular glutathione (GSH) level, and higher frequencies of micronuclei, were observed in bystander lymphocytes incubated with medium from 0.5 and 3 Gy irradiated samples, compared to lymphocytes unexposed. Furthermore, no statistically significant difference between the response to 0.5 and 3 Gy of irradiation in bystander lymphocytes, was found. However, when lymphocytes were irradiated with 0.1 Gy, no bystander effect with regard to viability, apoptosis, telomere length, and micronuclei was observed, although a high production of ROS level persisted. Radiation in the presence of the radical scavenger dimethyl sulfoxide (DMSO) suppressed oxidative stress induced by 3 Gy of X-rays with the effective elimination of bystander effects, suggesting a correlation between ROS and bystander signal formation in irradiated cells. The data propose that bystander effect might be mostly due to the reactions of radiation induced free radicals on DNA, with the existence of a threshold at which the bystander signal is not operative (0.1 Gy dose of X-rays). Our results may have clinical implications for health risk associated with radiation exposure.

Frozen human cells can record radiation damage accumulated during space flight: mutation induction and radioadaptation

To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.3-fold) in thymidine kinase deficient (TK(-)) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK(-) mutation frequency upon exposure to a subsequent 2 Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores.

Transcriptional modulation of micro-RNA in human cells differing in radiation sensitivity

Purpose: The molecular basis of gene regulation in cells exposed to ionising radiation is not fully understood. Gene regulation occurs at the transcriptional and post-transcriptional levels. Recent studies have suggested that micro-RNA (miRNA) plays a significant role at the post-transcriptional gene regulation. miRNA are a recently identified class of RNA molecules 18–24 nucleotides in length that have been shown to negatively regulate the stability or translation of target messenger RNA. We hypothesised that ionising radiation induced stress response is controlled in part by miRNA and that a difference in tumour protein 53 (p53) status corresponds with altered expression in miRNA responses to ionising radiation.Materials and methods: To test this hypothesis, we investigated the relative expression of several miRNA by quantitative real-time polymerase chain reaction (QPCR) in human cell lines TK6 and WTK1 that differ in p53 status and radiosensitivity after exposure to high and low doses of X-radiation.Results: The suitability of several endogenous miRNA controls was tested for relative quantification by QPCR. The baseline expression of 21 miRNA targets in TK6 and WTK1 cells indicated a wide range of modulation between the two cell lines without exposure to ionising radiation. Differences in the relative expression of miRNA were observed among the two cell lines after radiation treatment. The expression patterns of many miRNA markedly differed within the same cell line after exposure to either 0.5 Gy or 2 Gy doses of X-rays. The expression of eight miRNA belonging to the lethal-7 (let-7) family, which are negative regulators of the rat sarcoma, RAS oncogene, was upregulated in irradiated TK6 cells but was downregulated in WTK1 cells. Alterations in the myelocytomatosis oncogene, c-MYC induced cluster of miRNA were also observed. The micro RNA, miR-15a and miR-16 were upregulated in 0.5 Gy-irradiated TK6 cells but were downregulated after a 2 Gy dose of X-rays. In contrast miR-15 and miR-16 were repressed in 0.5 Gy-exposed WTK1. The miR-21 was upregulated in 0.5 Gy-treated TK6 cells and its target genes programmed cell death factor 4 (hPDCD4) phosphatase and tensin homolog (hPTEN), and sprouty homolog 2 (hSPRY2) were found to be downregulated in these cells. The miR-21 was downregulated in 2 Gy-irradiated TK6 cells, and all three of its target genes were upregulated in 2 Gy-exposed TK6 cells.Conclusion: Taken together, these results establish the involvement of miRNA in radiation response and may potentially help explain the mechanisms of gene regulation in the cellular response to ionising radiation exposure.

Distribution of spatial photoneutrons inside a 70kg water phantom via neutron activation analysis

This study evaluated spatial Phi(th) inside a 70kg water phantom using the NAA method. Fifty indium foils were placed inside the water phantom and exposed under 15MV LINAC for 2.5min to yield the 10Gy X-ray dose. The Phi(th) value at the isocenter of the water was 1.03x10(6)ncm(-2)/Gy-X, and the maximum quantity of Phi(th) appeared at the water surface along the z-axis, 3.99x10(6)ncm(-2)/Gy-X. The thermal neutron dose at isocenter of the water phantom occupied approximately 0.151% of the whole photo and neutron dose.

Non-irradiated Bystander Fibroblasts Attenuate Damage to Irradiated Cancer Cells

Introduction and aim: Radiation-induced bystander effect is described as a different type of responses, displayed by non-irradiated neighbouring cells, induced by signals transmitted from irradiated cells. These responses in bystander cells include genetic damage (SCE, micronuclei, genomic instability), apoptosis induction and other non-necessarily detrimental effects. Bystander effect might bear some implications for coexisting normal cells non-targeted by cancer radiotherapy. However, it is possible, that bystander effect can act in opposite direction, and non-irradiated cells can in some way influence the response of targeted cells. Our experiments in vitro were aimed at evaluating this concept. Materials and methods: Lung Lewis carcinoma cells (LLC), growing in monolayer in 6-well plates, were irradiated with 2 or 4 Gy dose of X-rays (using a 6 MeV accelerator suited for therapeutic purposes). After irradiation, the cells were co-cultured with non-irradiated NIH3T3 mice fibroblasts, the latter growing (in monolayer) in special inserts. Such system allowed separation of the two kinds of cells, with medium freely circulating through the separation membrane (pore size 0.4 µm). Thus, species released by irradiated cells could be transmitted to non-irradiated neighbours and vice versa. Results and discussion: The bystander effects, caused by irradiated cancer cells, which were observed in non-targeted fibroblasts included dose-dependent elevation of micronucleus and apoptosis frequency indicating that irradiated cancer cells can induce damage in normal fibroblast cells. However, irradiated LLC cancer cells co-incubated with fibroblasts presented lower levels of this type of cytogenetic damage and apoptosis in comparison with LLC cells incubated after irradiation without fibroblasts on inserts or without inserts at all. Our current studies attempt to search for agents and signalling responsible for observed bilateral bystander effects. Supported by the Polish Ministry of Science and High Education, grant no N406 101/31/3870

Radiosensitivity of Prostate Cancer and BPH Patients Studied by DNA Repair Capacity, CA and FISH.

An individual’s genetic constitution and lifestyle, e.g., diet and levels of physical activity, can affect the body’s response to various exogenous agents including therapeutic treatment. There is a strong need to combine studies on variability in a cellular response to genotoxic exposure with predisposition of the patient to diseases development and healing. In this study a variation in responses to challenging dose of X rays in lymphocytes from healthy donors and prostate cancer patients were compared on the molecular and mitotic levels. Blood was collected from healthy donors BPH (Benign Prostate Hyperplasia), and prostate cancer patients. Among cancer patients 33% never smoke 46.7% were former smokers. Immediately after collecting the blood a challenging irradiation was performed followed by culturing procedures for classic cytogenetics and FISH techniques with a probe for the whole chromosome 1. In DNA damage investigations, isolated and cryopreserved lymphocytes were thawed and their viability examined before molecular studies. To evaluate individual susceptibility, defrosted lymphocytes were exposed to 4 Gy dose of X-rays and the extent of DNA damage was studied right after irradiation with the alkaline version of the single cell gel electrophoresis (SCGE) assay. To asses variability in the DNA repair competency the residual (unrepaired) DNA damage was detected again after one hour of incubation, during which irradiated cells had the condition allowing to complete the fast DNA repair process. Visible difference between DNA susceptibilities to the challenging dose was observed between investigated groups. However, variation between individuals in repair efficiency of the DNA damage induced by the challenging treatment was significantly higher in lymphocytes of prostate cancer patients than that of BPH and healthy donors (p<.05). That findings correlate with results from cytogenetic studies. Significantly higher amount of chromosomal damage was detected in irradiated lymphocytes of prostate cancer than that in BPH patients by classic cytogenetics. Results obtained by FISH technique have also shown a statistically significantly higher level of translocations frequency (4.3 ± 0.33 vs 2.9 ± 0.31, p<.005) in chromosome 1 of prostate cancer lymphocytes than that of BPH. Our results, clearly suggest that vulnerability of chromosome 1 and DNA repair competence assays have shown a possible potency as predictive assays in preclinical studies. *the MSC foundation’ fellow from Kazakh National University, Almaty, Kazakhstan

Dose-rate Effects are Dependent on Cell Lines

Background: A number of studies, including our own, have indicated that low dose rate of ionizing radiation might be (within some range) more effective in killing cells than high dose rate. This phenomenon is defined as “inverse dose-rate effect” and is mediated mainly by post radiation oxidative reactions, as radiation peroxidative damage increases with decreased dose rate. Knowledge of biological dose rate-dependent effects might have influence upon the extension of therapeutic maneuvers and limitation of normal tissue reaction. Aim: The aim of the present study was to evaluate the dose-rate effects on lymphoblastic cells since this type of cells is a target in total body irradiation. Materials and methods: Lymphoblastic K562 cells were exposed to 2 Gy X-ray dose (using a 6 MeV accelerator for therapeutic purposes), at high dose rate (2Gy/min), low dose rate (0.07 Gy/min) and at high dose rate, but total dose was given as short separated pulses delivered during the same time as low dose rate (28 min 34 sec). Cells were suspended in RPMI medium supplemented with serum and irradiated in culture flasks. After irradiation cells were incubated for 0.5 – 60 h and micronuclei, apoptotic, necrotic and mitotic cells were scored under fluorescent microscope. Activity of superoxide dismutase isoenzymes (MnSOD and CuZuSOD), and glutathione peroxidise as well as end-product of lipid peroxidation (malondialdehyde) were also measured. Results and discussion: Our results indicated that 2 Gy dose at high dose rate induced higher yield of micronuclei than the same dose at low dose rate or 2 Gy dose given in many pulses. At the same time high dose-rate was less effective in apoptosis induction in K562 cells than low dose-rate or pulsed dose. This effect may differ in different cell lines, since our study on murine carcinoma AT478 cells indicated opposite trend. Furthermore, there was no dose-rate effect in the activation of antioxidant enzymes in lymphoblastic cells, whereas inverse dose rate effect was observed for carcinoma cells. The observed differences may also result from the type of cell growth, K562 grow in suspension, but AT478 are adherent cells. The adherent cells may suffer higher damage from low dose rate during long time of irradiation, in part due to bystander effects. The ongoing study is aimed at evaluating the mechanisms responsible for observed differences and possible signalling activated at different dose rate irradiation. Supported by the Polish Ministry of Science and High Education, grant no N406 101/31/3870

Occupational exposure to mercury vapour on genotoxicity and DNA repair

We have conducted a population study to investigate whether current occupational exposure to mercury can cause genotoxicity and can affect DNA repair efficiency. Blood samples from 25 exposed workers and 50 matched controls were investigated for the expression of genotoxicity. The data indicate that mercury exposure did not cause any significant differences between the workers and controls in the baseline levels of DNA strand breaks (as measured by the alkaline version of the single cell gel electrophoresis [SCGE] assay) or sister chromatid exchanges (SCE). However, the exposure produced elevated average DNA tails length in the SCGE assay and frequency of chromosome aberrations. In the studies, isolated lymphocytes were exposed to 6 J/m 2 UV-C light or 2 Gy dose of X-rays in a challenge assay and repair of the induced DNA damage was evaluated using the SCGE assay. Results from the UV-light challenge assay showed no difference between the workers and controls in the expression of DNA strand breaks after exposure followed by incubation in the absence or presence of the cellular mitogen (phytohemagglutinin, PHA). No difference in DNA strand breaks between the workers and controls was seen immediately after the X-ray challenge, either. However, significant differences were observed in cells that were incubated for 2 h with and without phytohemagglutinin. Data from the X-rays challenge assay were further used to calculate indices that indicate DNA repair efficiency. Results show that the repair efficiencies for the workers (69.7% and 83.9% in un-stimulated and stimulated lymphocytes, respectively) were significantly lower than that of matched controls (85.7% and 90.4%, respectively). In addition, the repair efficiency showed a consistent and significant decrease with the duration of occupational exposure to mercury (from 75.7% for <10 years employment, to 65.1% for 11–20 years and to 64.1% for 21–35 years) associated with increase of cytogenetic damage. Our study suggests that the occupational exposure to mercury did not cause a direct genotoxicity but caused significant deficiency in DNA repair. Our observations are consistent with previous studies using the standard chromosome aberration assay to show that exposure to hazardous environmental agents can cause deficiency in DNA repair. Therefore, these affected individuals may have exposure-related increase of health risk from continued exposure and in combination with exposure to other genotoxic agents.

Use of the radiation-inducible WAF1 promoter to drive iNOS gene therapy as a novel anti-cancer treatment.

Inducible nitric oxide synthase (iNOS) gene therapy has been identified as a potential anti-tumour strategy. A major problem common to most gene therapy strategies is targeting of treatment to the tumour volume. In this study we report on the use of the X-ray-inducible WAF1 promoter to achieve targeting of iNOS expression to the tumour volume. A WAF1/iNOS/liposome complex was injected directly into RIF-1 and HT29 tumours in mice. A 4 Gy dose of X-rays was applied to induce the WAF1 promoter followed, 8 h later, by treatment doses of 10 or 20 Gy. Tumour volume was measured, and growth curves plotted. Intra-tumoural injection of WAF1/iNOS combined with a priming dose of X-rays to induce the WAF1 promoter, followed by a treatment dose, resulted in sensitiser enhancement ratios of 2.0 and 1.3 in RIF-1 and HT29 tumours, respectively, compared with radiation treatment alone. PCR analysis of organ tissue after intra-tumoural injection of WAF1/iNOS showed that vector sequences were detected in all tissue tested; however, Western blot analysis revealed that iNOS protein levels were significantly increased only in tumour and the surrounding dermal tissue that had been exposed to the 4 Gy inducing dose. iNOS gene therapy in combination with an inducible promoter results in significant tumour cell radiosensitisation. The WAF1 promoter may be a good candidate for a gene therapy as it is silent in normal tissue yet can be induced by the tumour environment.

Marked increase in the rate of ocular lens regeneration in the newt, Cynops pyrrhogaster, following partial body exposure to low dose X-rays.

In recent years, concern over the stimulating effects of low-dose X-rays has been growing. Therefore, the effects of low-dose X-irradiation on lens regeneration in the newt were examined. Newts were subjected to sham or whole-body X-ray exposure at a dose of 0.2 Gy or 0.4 Gy, delivered at a rate of 0.43 Gy min(-1). The eyeballs were fixed in formalin solution, embedded in paraffin and assessed histologically. On day 14 after lens removal, unexposed animals showed the formation of a hollow epithelial vesicle of depigmented cells continuous with the laminae of the iris corresponding to the expected regeneration stage (Reyer's regeneration stage II). In contrast, lenses from newts exposed to a 0.2 Gy dose of X-rays showed some formation of the primary lens fibre nucleus corresponding to the fibre differentiation stage (Reyer's regeneration stage III-early). Thus, low-dose X-irradiation induced regeneration compared with the unexposed groups. An acceleration from Reyer's stage II to III-early was also found on day 14 following irradiation of only the upper belly, including the spleen. The effects of low-dose X-irradiation on lens regeneration may be mediated by changes in immune activity.

Cell cycle progression and radiation survival following prolonged hypoxia and re-oxygenation

Purpose : To investigate cell cycle progression and radiation survival following prolonged hypoxia and re-oxygenation. Materials and methods : NHIK 3025 human cervical carcinoma cells were exposed to extremely hypoxic conditions (<4ppm O 2) for 20h and then re-oxygenated. The subsequent cell cycle progression was monitored by analysing cell cycle distribution at different time-points after re-oxygenation using two-dimensional flowcytometry. The clonogenic survival after a 3.6Gy X-ray dose was also measured at each of these time-points. The measured radiation survival was compared with theoretical predictions based on cell cycle distribution and the radiation age-response of the cells. Results : Following re-oxygenation the cells resumed cell cycle progression, completed S-phase, and then accumulated in G 2. Non-clonogenic cells remained permanently arrested in G 2, while the remainder of the cells completed mitosis after a few hours delay. The radiation survival of the hypoxia-pretreated cell population remained lower than for an exponentially growing control population for the investigated 50h of re-oxygenation. However, following 7h of re-oxygenation, the radiation survival of the hypoxia-treated cell population correlated well with theoretically predicted values based on cell cycle distribution and radiation age-response. Conclusions : The work demonstrates that prolonged hypoxia followed by re-oxygenation results in a G 2 delay similar to that observed after DNA damage. Furthermore, chronic hypoxia results in decreased radiation survival for at least 50h following the reintroduction of oxygen. The hypoxia-induced radiosensitization following 7h of re-oxygenation could in large part be explained by the synchronous cell cycle progression that occurred.

A comparison of single-dose and fractionted total-body irradiation on the development of pneumonitis following bone marrow transplantation

Purpose : A review of 132 consecutive patients who received bone marrow transplant for various malignancies was conducted to determine factors associated with increased risk in developing interstitial pneumonitis (IP) as the result of total body irradiation (TBI). Twenty-four patients were excluded because 22 did not receive TBI and two had insufficient records. Methods and Materials : Patients were conditioned with TBI and various drug regimens. Eighteen patients received a single 6.0 Gy dose of x-rays. The remaining 90 were treated with three doses of 3.33 Gy separated by 24 h. All patients were followed for at least 18 months for the purposes of determining the IP incidence. Results : Twenty-seven of these 108 (25%) patients developed IP; 19 (17.6%) died. The 2-year estimated incidence of IP was 24 and 18.6% for fatal IP. The etiology was determined to be idiopathic in 12 patients, the result of cytomegalovirus in 6 patients, and caused by a variety of other infectious organisms in 9 patients. We were unable to demonstrate a statistically significant increase in IP with age (adults vs. children), dose regimen, use of methotrexate for graft-vs.-host disease prophylaxis, the presence of acute graft-vs.-host disease, time from diagnosis to transplant, or transplant type (allogeneic vs. autologous). Conclusions : The incidence of fatal IP reported here is similar to that reported by other institutions utilizing hyperfractionated TBI protocols. Our data do not support the need for hyperfractionation to reduce the risk of IP.

The time-course of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood

The time-course of micronucleated polychromatic erythrocytes (MPCE) in mouse bone marrow and peripheral blood, induced by an acute 0.1 Gy dose of X-rays, was determined using flow cytometric analysis, which made frequent sampling possible and allowed use of a dose low enough not to affect erythroid cell proliferation. The frequency of MPCE (fMPCE) began to increase in the bone marrow at 10 h after irradiation and reached a maximum at 28 h after irradiation. In the peripheral blood fMPCE began to increase at 20 h after irradiation and peaked at about 40 h after irradiation. The time-course found is discussed on the basis of data on the differentiation of erythroid cells. The results indicate that the micronuclei registered in polychromatic erythrocytes may originate from lesions induced not only during the last cell cycle but also during earlier ones. After an acute dose of 1.0 Gy of X-rays the maximum fMPCE was delayed both in bone marrow and peripheral blood reflecting an effect on the cell cycle progression of erythroblasts.

Course of c-mycmRNA Expression in the Regenerating Mouse Testis Determined by Competitive Reverse Transcriptase Polymerase Chain Reaction

The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

THE INFLUENCE OF INTESTINAL METAPLASIA ON DEVELOPMENT OF GASTRIC TUMORS IN WISTAR RATS RECEIVING X-RAYS AND MNNG, MNU OR DMH

Five-week-old male Wistar:Crj rats received two 10 Gy doses of X-rays to the gastric region at a 3-day interval. Two months thereafter the animals were treated with 100 mg/liter of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 4 months in their drinking water, N-methyl-N-nitrosourea (MNU) 2 mg/l ml/weekly/ for a total of 10 times by gastric intubation, or dimethylhydrazine (DMH) 20 mg/kg i.m./weekly/20 times. After 1 year, the animals were sacrificeded. There was thus no direct evidence of a histogenetic link between intestinal metaplasia and gastric carcinogenesis in the present results.

Induction and Repair of DNA Double-Strand Breaks

Induction and repair of DNA double-strand breaks (DSBs) was measured using a pulsed-field gel electrophoresis system. A cell line of methotrexate-resistant EMT-6 cells that contain numerous double-minutes (DMs) 3 million base pairs in size was employed. The electrophoretic mobility of these DMs depends on whether they have zero, one, or more than one DSB. With no DSBs the DMs remain as circles and are trapped in the origin of electrophoresis, but with one DSB the DMs migrate as a discrete band and can be detected easily through hybridization with a gene-specific probe. Using a clamped homogeneous electrical field apparatus, the induction of DSBs in the 1.5 to 12 Gy X-ray dose range is studied and is shown to be linear. Double-strand break repair following 7.5 Gy is studied, and is shown to be exponential. The kinetics of both induction and repair of DSBs induced in DM DNA was compared to the induction and repair of DSBs in chromosomal DNA and is shown to be similar. The kinetics of repair of DSBs following 7.5 Gy for cells embedded in agarose and cells in suspension is shown to be similar.

Radiosensitivity testing of human malignant gliomas

Radiotherapy remains the main treatment modality for patients with malignant gliomas and is the only treatment which significantly prolongs survival. Clonogenic and tetrazolium based colorimetric assays (MTT) of early passage cultures have been performed following 2 Gy doses of x-rays in order to determine if in vitro radiosensitivity is a factor in response to treatment. Of 47 biopsies received, 39 were established in primary culture. A value of surviving fraction to 2 Gy (SF2) was obtained in 85% of growth assays and 64% of clonogenic assays. The mean SF2 value for the MTT was 0.56 which was significantly higher than the 0.42 obtained for the clonogenic assay. There was, however, reasonable qualitative agreement in assessing relative radiosensitivity/radioresistance (r = 0.7). Mean SF2 values for grade 3 tumors were 0.52 (MTT) and 0.35 (clonogenic) as against mean SF2 values of 0.63 (MTT) and 0.47 (clonogenic assay) for grade 4 tumors. In 24 patients with adequate follow-up, no direct correlation was found between SF2 and survival, although mean SF2 values for patients surviving greater than 18 months was significantly less (p = 0.01) than patients surviving less than 18 months as determined by the MTT assay.

Manipulation of oxygenation in a human tumour xenograft with BW12C or hydralazine: effects on responses to radiation and to the bioreductive cytotoxicity of misonidazole or RSU-1069.

The influence of altered tumour oxygenation on the responses to radiation and/or bioreductive 2-nitroimidazole compounds was studied in a well differentiated, human, colon adenocarcinoma (MAWI), grown as a subcutaneous xenograft in nude mice. Tumour growth delays were measured after local, single 5-18 Gy doses of X-rays. BW12C, which inhibits dissociation of oxyhaemoglobin, produced radioprotection similar to that resulting from clamping off the tumour blood supply during irradiation. Hydralazine, a vasoactive agent, also appeared to give radioprotection. BW12C or misonidazole (MISO) alone had no measurable inhibitory effect on xenograft growth. Hydralazine or RSU-1069 slightly increased the time for tumours to reach 6 times their original volumes. When hydralazine was given 40 min after a dose of 800 mg/kg of MISO, without X-rays, growth delays in excess of 5 tumour volume doubling times resulted and fewer tumour cells were present in histological sections. Lower doses of MISO combined with hydralazine were ineffective. Other combinations of bioreductive cytotoxic agents and methods of manipulation of tumour blood flow/oxygenation induced slight and inconsistent growth delays. Hydralazine was injected after irradiation of tumours in mice previously treated with various doses of MISO in an attempt to exploit the bioreductive cytotoxic potential of MISO in conjunction with its radiosensitizing properties; however, tumour growth delays were similar with or without hydralazine after irradiation. Thus, post-irradiation restriction of tumour blood flow appears to be an ineffective therapeutic strategy in this human xenograft tumour model.