Non-targeted effects of radiation: bystander responses in cell and tissue models.

The standard paradigm for radiation effects in cellular systems has involved direct damage to DNA and in particular, DNA double strand breaks as the triggering lesions leading to mutation, cell death and transformation. Recently, however, a growing body of evidence has reported non-targeted effects, which are not a direct consequence of the initial lesions produced in cellular DNA. These have included bystander responses, genomic instability, gene induction, adaptive responses and low dose hypersensitivity. A common observation of these responses is that they dominate at low doses and saturate with increasing dose. Non-targeted effects may therefore have consequences for extrapolation of risk estimates to low doses if these are important in vivo. A range of experimental techniques is being used to study non-targeted responses, including microbeam approaches. Microbeams have considerable advantages in that they allow individual cells and subcellular targets to be selected within populations with precise low doses and, if required, exact dose rates. Recent advances also allow targeting of 3-D cell systems. The mechanisms underlying non-targeted responses appear to involve production of reactive oxygen species and direct cell-to-cell signalling via gap junctional intercellular communication although significant differences exist in different cell types. The triggering lesions for these responses remain unclear however. Some non-targeted responses may be inter-related, for example in the case of bystander responses and instability and may be part of a general stress response system in irradiated populations. Some non-targeted effects may also act as protective mechanisms; if they lead to the removal of potentially damaged cells from the population.

A review of studies of ionizing radiation-induced double-strand break clustering.

Underpinning current models of the mechanisms of the action of radiation is a central role for DNA damage and in particular double-strand breaks (DSBs). For radiations of different LET, there is a need to know the exact yields and distributions of DSBs in human cells. Most measurements of DSB yields within cells now rely on pulsed-field gel electrophoresis as the technique of choice. Previous measurements of DSB yields have suggested that the yields are remarkably similar for different types of radiation with RBE values < or = 1.0. More recent studies in mammalian cells, however, have suggested that both the yield and the spatial distribution of DSBs are influenced by radiation quality. RBE values for DSBs induced by high-LET radiations are greater than 1.0, and the distributions are nonrandom. Underlying this is the interaction of particle tracks with the higher-order chromosomal structures within cell nuclei. Further studies are needed to relate nonrandom distributions of DSBs to their rejoining kinetics. At the molecular level, we need to determine the involvement of clustering of damaged bases with strand breakage, and the relationship between higher-order clustering over sizes of kilobase pairs and above to localized clustering at the DNA level. Overall, these studies will allow us to elucidate whether the nonrandom distributions of breaks produced by high-LET particle tracks have any consequences for their repair and biological effectiveness.

The relationship between the RBE of alpha particles and the radiosensitivity of different mutations of Chinese hamster cells.

The RBE of alpha-particles in different mutations of Chinese hamster cells was determined with the aim of identifying differences in the sensitivity to x-ray and alpha-particle-induced DNA damage. Two parental lines of Chinese hamster cells and four radiosensitive mutants were irradiated with different single doses of x-rays and alpha-particles and clonogenic cell survival was determined. Radiosensitivity to x-rays varied by a factor of 5 between the cell strains whereas sensitivity to alpha-particle irradiation was almost identical among all strains. The RBE is only determined by the sensitivity of the cells towards x-rays. Since cells with different defects of repair or cell cycle control have different radiosensitivities, we conclude that the effects of x-ray irradiation and the RBE are mostly determined by the activity of repair processes.

The role of higher-order chromatin structure in the yield and distribution of DNA double-strand breaks in cells irradiated with X-rays or alpha-particles.

PURPOSE: To determine whether the non-random distributions of DNA double-strand breaks in cells observed after alpha-particle irradiation are related to the higher-order structure of the chromatin within the nucleus. MATERIALS AND METHODS: Chinese hamster V79 cells were irradiated as either cellular monolayers, nuclear monolayers with condensed chromatin or nuclear monolayers with relaxed chromatin, and the yields and distribution of DSB measured using two pulsed-field gel electrophoresis protocols capable of separating fragments of 10kbp to 5.7 Mbp. RESULTS: Using conventional FAR analysis, the effect of isolating nuclear monolayers and changing the chromatin condensation state was less for alpha-particle irradiated substrates than for X-irradiated ones. When the total number of breaks was measured by separating and quantifying all the fragments produced in the 10kbp to 5.7Mbp region, the difference between the observed yields of breaks in X-irradiated cells (7.3 x 10(-9) DSB/Gy/bp) relative to alpha-particles (12.1 x 10(-9) DSB/Gy/bp) was largely removed when nuclear monolayers with decondensed chromatin were exposed. The yields, although similar, increased to 44.4 x 10(-9) DSB/Gy/bp for X-irradiated decondensed nuclear monolayers and 46.6 x 10(-9) DSB/Gy/bp for alpha-particle irradiated monolayers. However, the alpha-particle DSB distributions remained non-random. CONCLUSIONS: Our results suggest that the non-random distribution of breaks observed in cells with alpha-particle irradiation, which leads to a high probability for the production of regionally multiply damaged sites, is not related to the underlying chromatin condensation state present in the nucleus.

Quantification of DNA damage by PFGE: development of an analytical approach to correct for the background distribution.

PURPOSE: To analyse the currently existing methods to infer the extent of cellular DNA damage induced by ionizing radiation when the pulsed field gel electrophoresis (PFGE) technique is used. RESULTS AND CONCLUSIONS: PFGE is currently the method of choice for the measurement of radiation-induced double-strand breaks (dsb). For accurate determination of both the yields and distributions of breaks, separation of a large range of fragment sizes is required. In the conventional analysis of PFGE experiments, the background distribution of fractionated molecules is, normally, simply subtracted from the irradiated measured distribution, for each molecular weight region available. This work shows that this approach may lead to incorrect estimation of the breakage frequencies. An alternative approach based on correcting the fitting functions for the actual nonrandom damage present in the control unirradiated samples has been developed.

A study of DNA fragmentation patterns in cells irradiated with charged particles: evidence for non-random distributions.

Many studies have shown that the effectiveness of radiations of varying LET is similar when yields of dsb have been measured, despite large differences in biological response. Recent evidence has suggested however, that current techniques underestimate the yields of dsb. By monitoring the fragmentation of DNA over a wide range of fragment sizes (< 10 kbp > 6 Mbp) by pulsed field electrophoresis, RBE values greater than 1.0 for radiations of around 100 keV/mm have been determined. The data provide evidence for the production of correlated breaks produced within cells as particle tracks traverse the nucleus. The highly ordered structure of DNA within mammalian cells may lead to clustering of breaks over distances related to the repeating unit structures of the chromatin. As well as these regionally damaged sites, a major contributor to radiation effectiveness will be the localised clustering of damage in the 1-20 bp region. A major effort is required to elucidate the relative importance of these levels of clustering and their importance in biological response.

DNA double-strand break distributions in X-ray and alpha-particle irradiated V79 cells: evidence for non-random breakage.

Many studies have shown that with increasing LET of ionizing radiation the RBE (relative biological effectiveness) for dsb (double strand breaks) induction remains around 1.0 despite the increase in the RBE for cell killing. This has been attributed to an increase in the complexity of lesions, classified as dsb with current techniques, at multiply damaged sites. This study determines the molecular weight distributions of DNA from Chinese hamster V79 cells irradiated with X-rays or 110 keV/micron alpha-particles. Two running conditions for pulsed-field gel-electrophoresis were chosen to give optimal separation of fragments either in the 225 kbp-5.7 Mbp range or the 0.3 kbp to 225 kbp range. Taking the total fraction of DNA migrating into the gel as a measure of fragmentation, the RBE for dsb induction was less than 1.0 for both molecular weight regions studied. The total yields of dsb were 8.2 x 10(-9) dsb/Gy/bp for X-rays and 7.8 x 10(-9) dsb/Gy/bp for alpha-particles, measured using a random breakage model. Analysis of the RBE of alpha-particles versus molecular weight gave a different response. In the 0.4 Mbp-5.7 Mbp region the RBE was less than 1.0; however, below 0.4 Mbp the RBE increased above 1.0. The frequency distributions of fragment sizes were found to differ from those predicted by a model assuming random breakage along the length of the DNA and the differences were greater for alpha-particles than for X-rays. An excess of fragments induced by a single-hit mechanism was found in the 8-300 kbp region and for X-rays and alpha-particles these corresponded to an extra 0.8 x 10(-9) and 3.4 x 10(-9) dsb/bp/Gy, respectively. Thus for every alpha-particle track that induces a dsb there is a 44% probability of inducing a second break within 300 kbp and for electron tracks the probability is 10%. This study shows that the distribution of damage from a high LET alpha-particle track is significantly different from that observed with low LET X-rays. In particular, it suggests that the fragmentation patterns of irradiated DNA may be related to the higher-order chromatin repeating structures found in intact cells.

Inactivation of V79 cells by low-energy protons, deuterons and helium-3 ions.

Previous work by ourselves and by others has demonstrated that protons with a linear energy transfer (LET) about 30 keVmum(-1)are more effective at killing cells than doubly charged particles of the same LET. In this work we show that by using deuterons, which have about twice the range of protons with the same LET, it is possible to extend measurements of the RBE of singly charged particles to higher LET (up to 50 keVmum(-1). We report the design and use of a new arrangement for irradiating V79 mammalian cells. Cell survival measurements have been made using protons in the energy range 1.0-3.7 MeV, deuterons in the energy range 0.9-3.4MeV and 3He2+ ions in the energy range 3.4-6.9 MeV. This corresponds to volume-averaged LET (within the cell nucleus) between 10 and 28 keVmum(-1) for protons, 18-50 keVmum(-1) for deuterons, and 59-106 keVmum(-1) for helium ions. Our results show no difference in the effectiveness of protons and deuterons matched for LET. However, for LET above about 30 keVmum(-1) singly charged particles are more effective at inactivating cells than doubly-charged particles of the same LET and that this difference can be understood in terms of the radial dose distribution around the primary ion track.

Effect of radiation quality on lesion complexity in cellular DNA.

Understanding the critical lesions induced by ionizing radiation in DNA and their relationship to cellular effects is an important challenge in radiation biology. Much evidence has suggested that DNA double-strand breaks (dsb) are important lesions. Establishing a cause and effect relationship between initial levels of DNA dsb, their repair rate or the level of residual unrepaired breaks, and cellular effects has proved difficult in mammalian cells. Several studies have measured yields of DNA dsb after irradiation with radiations of differing linear energy transfer (LET). In general the RBEs for dsb induction (20-100 keV/microns) have been lower than the RBEs measured for cell survival and in many cases are around 1.0. Several studies have shown differences in the rejoining of dsb with less dsb rejoined after high-LET irradiation in comparison with low-LET radiation. These results suggest that there may be differences in the types of lesions induced by different radiations and scored as DNA dsb using current techniques. Track structure modelling studies have suggested that some lesions induced will be clustered at the sites of energy depositions and that uniquely large energy deposition events are produced by high-LET radiations. Assays need to be developed to measure complex lesions in both model DNA and cellular systems. Different levels of complexity need to be considered such as clustering of radicals close to DNA, localized areas of DNA damage (1-20 bp) and lesions which may be induced over larger distances. Studies using new and existing assays of DNA damage, coupled with irradiation at various LETs, are directed at understanding the role of lesion complexity in relation to cellular effects.

Transendoscopic Nd:YAG laser surgery for treatment of epiglottal entrapment and dorsal displacement of the soft palate in the horse.

Transendoscopic neodymium:yttrium-aluminum-garnet (Nd:YAG) laser was used to treat 12 standing horses with epiglottic entrapment (EE) or dorsal displacement of the soft palate (DDSP), or both. In four horses, transendoscopic laser staphylectomy was performed. The most common presenting complaints were respiratory stridor, cough, and exercise intolerance. Ten horses with EE healed without epiglottic complications; in one horse, partial adhesion of the aryepiglottic fold to one side of the epiglottis was corrected surgically through a laryngotomy incision. One horse with DDSP had no further signs, one continued to have continual DDSP, and two had induced DDSP. Transendoscopic Nd:YAG laser proved to be a feasible means of correcting EE and selected cases of DDSP.

Transendoscopic neodymium:yttrium aluminum garnet laser irradiation in horses.

A neodymium:yttrium aluminum garnet (Nd:YAG) laser was used to study effects of applying laser irradiation transendoscopically to the corniculate process of the arytenoid cartilage in horses. Dosimetry was established initially in vitro in 10 corniculate cartilages that were irradiated and examined histologically to determine penetration depths at selected power settings. Eleven horses were given xylazine IV and butorphoral tartrate IV, and their left ventricle and corniculate process were irradiated. Six horses had left laryngeal hemiplegia and were euthanatized and necropsied 14 weeks after laser application and evaluation for upper airway stridor. Endoscopy was performed in the 5 other horses; they were euthanatized and necropsied at selected intervals to characterize the healing process. Healing was by second intention and was complete at 14 weeks. Two horses developed buds of granulation tissue along the laser incision, which resolved after a second laser application. Scar tissue formation resulted in left of midline displacement of the dorsal portion of the right corniculate process. The left ventricle healed without complications and was totally ablated. All horses had inspiratory stridor when exercised 14 weeks after laser irradiation.

Neodymium:yttrium, aluminum, and garnet laser energy delivered transsclerally to the ciliary body of dogs.

Using a hand-held optical fiber, a surgical neodymium:yttrium, aluminum, and garnet laser was used transsclerally on the left ciliary body of 25 dogs. Dogs were assigned to groups and were given low (100 J) and high (238 J) energy levels. In 12 dogs given 100-J energy, the intraocular pressure decreased a mean value of 6 mm of Hg below that in the untreated right eye. However, intraocular pressure returned to the pretreatment value 1 week after treatment. Intraocular lesions were not found by histologic examination 28 days later. In the second group of 13 dogs treated with 238-J energy, 6 were euthanatized 7 days after treatment and 7 were euthanatized 28 days after treatment. A mean decrease in intraocular pressure of 10 mm of Hg in the treated left eye, relative to the untreated right eye, persisted throughout the evaluation period. In eyes examined histologically 7 days after treatment, ciliary hemorrhage and necrosis were prominent. Other histologic changes were minimal and consisted principally of iris stromal hemorrhage. Of 7 dogs examined for 28 days after treatment, 6 remained sighted; in 1 dog, extensive intraocular hemorrhage occurred, eventually resulting in phthisis bulbi. Ciliary atrophy and fibrosis were the important histologic lesions observed 28 days after treatment. Laser energy delivered transsclerally by this optical system effectively induced ciliary necrosis and may be an effective treatment for canine glaucoma.

Mechanism of uptake of sulfonated metallophthalocyanines by cultured mammalian cells.

The uptake of several metallophthalocyanine tetrasulfonates by cultured Chinese hamster cells was studied. Uranyl- and chloraluminium were taken up at the highest rate followed by Ni-, Zn-, Cu-, Co- and dichlorosilicon-phthalocyanine tetrasulfonate. The uptake from the growth medium containing 10% serum in which only 15% of the dye is not bound to serum proteins was 5--18-fold slower than in the absence of serum, suggesting that most of the uptake is of free dye. Using gel filtration to separate tightly protein-bound dye from the free dye, binding curves were constructed that varied in slope and saturation values for the different compounds. At saturation, the number of dye molecules bound per serum albumin molecule varied from 1 for vanadyl- and cobalt-, to 4 for uranyl-phthalocyanine tetrsulfonate. Absorption spectra of the various phthalocyanines indicated that under physiological conditions, all dyes, with the exception of chloroaluminum- and dichlorosilicon-phthalocyanine tetrasulfonate, were aggregated. The rate of uptake was unrelated to the state of aggregation. The rate of uptake was temperature dependent at intervals longer than 1 h. At shorter times, very little temperature dependence was observed. These results suggest that the uptake process takes place in two steps. The first step is passive, involving binding of metallophthalocyanine tetrasulfonate to a receptor on the cell membrane, while the second one is active and involves internalization of the bound dye.

Neodymium (Nd):YAG laser surgery in the equine larynx: a pilot study.

Laryngeal surgery in the equine is customarily and routinely performed by means of a ventral laryngotomy incision. Such procedures are usually performed under deep general anesthesia with the horse in dorsal recumbency. The objective of this work was to determine the efficacy of an endoscopic approach coupled to a Nd:YAG laser fiber in performing arytenoidectomy. Arytenoidectomy is commonly indicated in the treatment of arytenoid chondritis and in the failure of prosthetic implantation for left laryngeal hemiplegia. This preliminary study was undertaken to determine the feasibility of the endoscopic laser approach to ablate the most difficult of the structures of the larynx, considering tissue structure and density. This procedure was accomplished by using heavy sedation and local anesthesia and was performed in the standing position. This approach appears to be feasible and makes other adjacent structures accessible via the endoscope coupled to a surgical laser. Indications for future pilot applications of laser surgery in the equine larynx are entrapment of the epiglottis, dorsal soft palate displacement, and the ablation of laryngeal cysts and polyps. This pilot study appears to offer a new modality for discrete surgical ablation of this structure, with minimal postoperative complications and a reduced or equivalent convalescent period in this valuable species of animal.

Glycemic index of foods: a physiological basis for carbohydrate exchange.

The determine the effect of different foods on the blood glucose, 62 commonly eaten foods and sugars were fed individually to groups of 5 to 10 healthy fasting volunteers. Blood glucose levels were measured over 2 h, and expressed as a percentage of the area under the glucose response curve when the same amount of carbohydrate was taken as glucose. The largest rises were seen with vegetables (70 +/- 5%), followed by breakfast cereals (65 +/- 5%), cereals and biscuits (60 +/- 3%), fruit (50 +/- 5%), dairy products (35 +/- 1%), and dried legumes (31 +/- 3%). A significant negative relationship was seen between fat (p less than 0.01) and protein (p less than 0.001) and postprandial glucose rise but not with fiber or sugar content.

Reversible bone marrow granulomas-adverse effect of oxyphenbutazone therapy.

A 48-year-old woman treated with oxyphenbutazone developed fever, gastrointestinal disturbances, mucocutaneous reactions, leukopenia, eosinophilia and thrombocytopenia. Bone marrow biopsy showed granulomatous lesions. Following withdrawal of the drug, all signs and symptoms subsided and the blood changes and the bone marrow biopsy normalized. The granulomatous reaction in the bone marrow is considered to be a hypersensitivity manifestation of oxyphenbutazone.

The final F.F.A.R.A.C.S. examination.

The development of the Final Examination of Fellowship of the Faculty of Anaesthetists of the Royal Australasian College of Surgeons is described from its inception in 1956 to the present. A statistical analysis was made of the examinations in 1969 and 1970. The correlations between marks for essay questions, within the multiple choice examination and between clinical examinations were low, suggesting that the reliability of these tests was unsatisfactory. At this same time, applied anatomy was added to the subjects in this examination. The examination was restructured with more emphasis on the oral and multiple choice examination and the marking system was revised. Continuing analysis has shown higher correlations between and within most parts of the examination. The correlations for the essay marks have remained lower, but essays have been retained in an attempt to assess and encourage the skills involved. Feedback of teaching and learning information obtained from analysis of the examination is provided to Regional Education Officers and Supervisors of Training.