Beam collimation and bolusing material optimizations for 10boron neutron capture enhancement of fast neutron (BNCEFN): definition of the optimum irradiation technique.

PURPOSE: In boron-10 neutron capture enhancement of fast neutron irradiation (BNCEFN), the dose enhancement is correlated to the 10B concentration and thermal neutron flux. A new irradiation technique is presented to optimize the thermal neutron flux. METHODS AND MATERIALS: The coupled FLUKA and MCNP-4A Monte Carlo codes were used to simulate the neutron production and transport for the Nice and Orleans facilities. RESULTS: The new irradiation technique consists of a 20-cm lead blocks additional collimator, placed close to the patient's head, which is embedded in a pure graphite cube. A 24-fold thermal neutron flux increase is calculated between a 5 x 5 cm2 primary collimated field, with the patient's head in the air, and the same field size irradiated with the optimum irradiation technique. This increase is more important for the p(60)+Be Nice beam than for the p(34)+Be Orleans one. The thermal neutron flux is 2.1 x 10(10) n(th)/Gy for each facility. Assuming a 100 microg/g 10B concentration, a physical dose enhancement of 22% is calculated. Moreover, the thermal neutron flux becomes independent of the field size and the phantom head size. CONCLUSION: This technique allows conformal irradiation of the tumor bed, while the thermal neutron flux is enhanced, and spreads far around the tumor.

Combined use of FLUKA and MCNP-4A for the Monte Carlo simulation of the dosimetry of 10B neutron capture enhancement of fast neutron irradiations.

Boron neutron capture enhancement (BNCE) of the fast neutron irradiations use thermal neutrons produced in depth of the tissues to generate neutron capture reactions on 10B within tumor cells. The dose enhancement is correlated to the 10B concentration and to thermal neutron flux measured in the depth of the tissues, and in this paper we demonstrate the feasibility of Monte Carlo simulation to study the dosimetry of BNCE. The charged particle FLUKA code has been used to calculate the primary neutron yield from the beryllium target, while MCNP-4A has been used for the transport of these neutrons in the geometry of the Biomedical Cyclotron of Nice. The fast neutron spectrum and dose deposition, the thermal flux and thermal neutron spectrum in depth of a Plexiglas phantom has been calculated. The thermal neutron flux has been compared with experimental results determined with calibrated thermoluminescent dosimeters (TLD-600 and TLD-700, respectively, doped with 6Li or 7Li). The theoretical results were in good agreement with the experimental results: the thermal neutron flux was calculated at 10.3 X 10(6) n/cm2 s1 and measured at 9.42 X 10(6) n/cm2 s1 at 4 cm depth of the phantom and with a 10 cm X 10 cm irradiation field. For fast neutron dose deposition the calculated and experimental curves have the same slope but different shape: only the experimental curve shows a maximum at 2.27 cm depth corresponding to the build-up. The difference is due to the Monte Carlo simulation which does not follow the secondary particles. Finally, a dose enhancement of, respectively, 4.6% and 10.4% are found for 10 cm X 10 cm or 20 cm X 20 cm fields, provided that 100 micrograms/g of 10B is loaded in the tissues. It is anticipated that this calculation method may be used to improve BNCE of fast neutron irradiations through collimation modifications.

Thiol WR-1065 and disulphide WR-33278, two metabolites of the drug ethyol (WR-2721), protect DNA against fast neutron-induced strand breakage.

The main metabolites of the cytoprotective drug Ethyol (Amifostine, WR-2721) are the thiol WR-1065 and the disulphide WR-33278 (formed by the oxidation of WR-1065). Both metabolites are well-known protectors against DNA damage induced by gamma-rays. Using supercoiled plasmid DNA and restriction fragments we show that they protect efficiently also in the case of fast neutrons. In anoxic conditions WR-1065 (Z = +2) protects by scavenging of OH. and by 'chemical repair' (by H donation from its SH function). WR-33278 (Z = +4) protects by scavenging of OH. and, in the case of the supercoiled plasmid DNA, by reducing the accessibility of radiolytic attack sites via the induction of packaging of DNA in liquid-crystalline condensates (observed by circular dichroism). Because of this second mechanism, the plasmid DNA is more efficiently protected by WR-33278 than by WR-1065, at concentration ratios > 1 drug/4 nucleotides. Moreover, using sequencing gel electrophoresis of irradiated fragments of known sequence, we show that the protection by the two metabolites is non-homogeneously distributed along the DNA sequence, with 'hot spots' of protection and with unprotected regions. Based on presented molecular modelling results we explain the sequence dependence of radioprotection by structural variations induced by the binding of the drugs.

[Computed tomography option on simulator: a 14-month use at the Orléans hospital radiotherapy department]. / L'option scanneur sur simulateur: 14 mois d'utilisation dans le service de radiothérapie du CHR d'Orléans.

The purpose was to evaluate the use of the CT option for simulator during a 14-month period in the radiotherapy department. The CT option has been adapted on the Philips simulator SL23. The virtual tunnel diameter is 92 cm which allows slice acquisition regardless of the contention device used. This system is connected to the treatment planning system through an Ethernet link. Three fields of view are available which cover the standard radiotherapy use. Four hundred and twenty-seven patients benefited from this system over a 14-month period of use. The number of slices acquired per patient regularly increased. The use of the system was rapidly extended to all the standard treatments. Those slices were also used as additional information for these high technology treatments. The good quality of the images and the reliability of this system involved a rapid integration in the treatment preparation procedure. It will not replace the use of the scanner but will significantly improve the treatment quality.

Sequence-modulated radiosensitization of DNA by copper ions.

Plasmid DNA and restriction fragments of 80 and 120 base pairs were irradiated with fast neutrons in the presence of CuCl2. The number of single and double strand breaks is higher in the presence than in the absence of Cu2+ ions. The radiosensitizing effect was lower for solutions of high compared with low ionic strength, and also lower for deoxygenated than for aerated solutions. This effect was inhibited by EDTA, catalase and Tris, but not by ethanol. Superoxide dismutase partially inhibited the effect of low copper concentrations (< 1 Cu2+/nucleotide). Saturation of the solutions with N2O removed the effect for these concentrations of copper. The sensitization occurred preferentially at pyrimidines (thymines > cytosines) situated 5' to one or several purines (guanine > adenine) or located between two purines, at runs of purines (guanine > adenine), and at combinations of such sequences. The results can be only partially explained by a Fenton-like mechanism involving radiation induced hydrated electrons and hydrogen peroxide, which produces OH. radicals at the sites of binding of copper on DNA. The regions around these binding sites may undergo conformational changes. A second path for sensitization could be the enhancement of the efficiency of cleavage by the radiolytically produced OH. radicals in these conformationally modified regions.

Comparative study of DNA radiolysis by fast neutrons and gamma-rays.

The effect of fast neutrons on cells is different from that of gamma-rays: the relative (to gamma) biological effect (RBE) is higher than one and the oxygen enhancement ratio (OER) is lower than that of gamma-rays. We searched for differences between the effects of the two radiations on DNA, the critical target of radiations. Using a model plasmid DNA we observed that for the same absorbed dose, fast neutrons induce twice fewer single strand breaks (SSB) and 1.5 more double strand breaks (DSB) than gamma-rays. A transition metal ion, the Cu++, is a better sensitizer of DNA breakage with fast neutrons than with gamma-rays. In anoxia, cysteamine, a positively charged thiol, is a better radioprotector against neutrons than against gamma-rays. In presence of cysteamine, the OER is lower for neutrons than for gamma-rays. These results are discussed in terms of different physical properties of the two types of radiation.

RBE variation between fast neutron beams as a function of energy. Intercomparison involving 7 neutrontherapy facilities.

In fast neutron therapy, the relative biological effectiveness (RBE) of a given beam varies to a large extent with the neutron energy spectrum. This spectrum depends primarily on the energy of the incident particles and on the nuclear reaction used for neutron production. However, it also depends on other factors which are specific to the local facility, eg, target, collimation system, etc. Therefore direct radiobiological intercomparisons are justified. The present paper reports the results of an intercomparison performed at seven neutrontherapy centres: Orléans, France (p(34)+Be), Riyadh, Saudi Arabia (p(26)+Be), Ghent, Belgium (d(14.5)+Be), Faure, South Africa (p(66)+Be), Detroit, USA (d(48)+Be), Nice, France (p(65)+Be) and Louvain-la-Neuve, Belgium (p(65)+Be). The selected radiobiological system was intestinal crypt regeneration in mice after single fraction irradiation. The observed RBE values (ref cobalt-60 gamma-rays) were 1.79 +/- 0.10, 1.84 +/- 0.07, 2.24 +/- 0.11, 1.55 +/- 0.04, 1.51 +/- 0.03, 1.50 +/- 0.04 and 1.52 +/- 0.04, respectively. When machine availability permitted, additional factors were studied: two vs one fraction (Ghent, Louvain-la-Neuve), dose rate (Detroit), influence of depth in phantom (Faure, Detroit, Nice, Louvain-la-Neuve). In addition, at Orléans and Ghent, RBEs were also determined for LD50 at 6 days after selective abdominal irradiation and were found to be equal to the RBEs for crypt regeneration. The radiobiological intercomparisons were always combined with direct dosimetric intercomparisons and, when possible in some centres, with microdosimetric investigations.

Fast neutron therapy for inoperable or recurrent sacrococcygeal chordomas.

Between 1981 and 1994, 13 patients were referred to the Orleans neutrontherapy department with inoperable or recurrent pelvic chordomas. One patient who had already been irradiated refused the treatment, fearing complications. Among the 12 patients suitable for evaluation, ten had undergone one to five previous surgical operations. The time lapse between the last surgical operation and neutrontherapy was 13 months. Neutrons were used alone or as a boost depending on the tumor volume or treatment purpose. At four years, crude survival and local control probability (Kaplan-Meier) were 61 and 54% respectively. This small series suggests that fast neutrontherapy can provide a good alternative for the treatment of inoperable sacral chordomas.

Radioprotection of DNA by polyamines.

Putrescine, spermidine and spermine are natural polyamines bearing at neutral pH the net electrical charges +2, +3 and +4 respectively. We report here the radioprotective effect of these polyamines on the radiolysis of pBR322 plasmid DNA. We observe a very efficient protection against fast neutron-induced single and double-strand breakage in the presence of spermine and spermidine, and a significantly less efficient protection in the presence of putrescine. An ionic strength dependence is observed for spermidine and spermine, but not for putrescine. Circular dichroism measurements show spermidine- and spermine-induced structural modifications of DNA, i.e. the formation of tightly packaged condensates in the concentration range corresponding to radioprotection. No structural change is observed for concentrations of putrescine affording radioprotection. We explain the radioprotection by: (1) the scavenging of OH radicals in the bulk, essentially observed in the case of putrescine; (2) a local scavenging at the sites of binding of polyamines; and (3) the reduced accessibility of the attack sites in the condensed structures induced by spermine or spermidine.

Radiation-induced damages in single- and double-stranded DNA.

In the present study, we searched for possible effects of DNA strandedness (single and double), on two types of damages, frank strand breaks (FSB, observed at neutral pH) and alkali labile sites (ALS, leading to breaks at alkaline pH) induced by irradiation with gamma-rays (60Co) or fast neutrons (p34,Be). Sequencing gel electrophoresis allowed us to follow the occurrence of these damages at each nucleotide site in single (ss-ss), double (ds-ds), and half single-half double (ss-ds and ds-ss) stranded oligonucleotides. Globally, in DNA with random sequences of bases, no differences in FSB and ALS yield between the single and the double-stranded conformations were observed. One observes, however, an increased alkaline lability at some guanine sites belonging to single-stranded region of ss-ds or ds-ss. Nevertheless, strandedness influences the radiosensitivity of some particular sequences, i.e. the 5'-AATT sequences. This region is less radiosensitive than the rest of DNA in the double helical, but not in the single-stranded conformation. The results are discussed in terms of DNA conformation.

Radiolytic signature of Z-DNA.

Ionizing radiations induce various damages in DNA via the hydroxyl radical OH. generated by the radiolysis of water. We compare here the radiosensitivity of B- and Z-DNA, by using a Z-prone stretch included in a plasmid. In the supercoiled plasmid, the stretch is in the Z-form, whereas it is in the B-form when the plasmid is relaxed. Frank strand breaks (FSB) and alkali-revealed breaks (ARB) were located and quantified using sequencing gel electrophoresis. We show that B- and Z-DNA have the same mean sensitivity towards radiolytic attack, for both FSB and ARB. Nevertheless, the guanine sites are more sensitive, and the cytosine sites less sensitive in Z- than in B-DNA, leading to a characteristic signature of the Z-form. The comparison of experiments with the outcome of a Monte Carlo simulation of OH. radical attack suggests that transfer of initial damage from a guanine base to its attached sugar or the adjacent 3' cytosine is more important in Z-DNA than in B-DNA.

Radiosensitivity of DNA minicircles.

DNA minicircles of 207 bp were constructed by the ligation of linear restriction fragments in the presence of various concentrations of ethidium bromide. Three topoisomers characterized by linking numbers (Lk) of 20, 19 and 18, and with helical repeats of 10.35, 10.9 and 11.5 bp/turn respectively, were obtained. They are called, respectively, relaxed minicircle or topoisomer 0, topoisomer -1 and topoisomer -2. Owing to the limited flexibility of such small circles, the stress created by the lack of 1 or 2 turns cannot be eliminated by a spatial circle-axis writhing (supercoiling) of the circular molecules. These two undertwisted, stressed topoisomers have to adopt a flat, non-crossed shape, similar to that of the relaxed minicircle. The three minicircles were irradiated with gamma-rays or fast neutrons. The same yields of single-strand breaks, double-strand breaks and alkali-induced single-strand breaks were observed for the three topoisomers showing that their base and sugar moieties are attacked equally by gamma photon- or fast neutron-induced radicals. We conclude that untwisting of a B helix does not modify the radiosensitivity of DNA.

Sites of strand breakage in DNA irradiated by fast neutrons.

Therapeutic fast neutrons are densely ionizing particles, with a high relative biological effectiveness relative to 60Co gamma rays (RBE) and a low oxygen enhancement ratio (OER). The molecular basis of their properties is not yet entirely understood. In a previous work, we have shown that neutrons induce a different number of DNA frank strand breaks as compared to gamma photons, and we have revealed the presence of breaks due to the direct effects of neutrons. In the present work, we searched for eventual differences in the chemical nature of the attacked sites in DNA irradiated in oxygenated diluted solution. We compare our results with neutrons to those previously reported by other authors using gamma- or X-rays. Using sequencing gel electrophoresis of short natural DNA restriction fragments, or synthetic oligonucleotides, we have shown that, in the case of neutrons, the attack occurs with almost the same probability, at each nucleotide, as reported for gamma- and X-rays. The doubling of bands in the bottom of gels shows the presence of two types of termini, the 3'-phosphate and the 3'-phosphoglycolate. Upon neutron irradiation, the 3'-phosphate end appears with a higher yield than the 3'-phosphoglycolate, whereas equal amounts were obtained with gamma- or X-rays.

Radioprotection of DNA by a DNA-binding protein: MC1 chromosomal protein from the archaebacterium Methanosarcina sp. CHTI55.

The archaebacterial chromosomal protein MC1 binds tightly and unspecifically to DNA; binding protects DNA against radiolysis by fast neutrons. At low covering of pBR322 plasmid DNA, one bound protein protects some 50 attack sites (phosphate-sugar moieties) against both single (ssb) and double strand breaks (dsb). At high covering of plasmid, protection against dsb becomes almost complete, although about half of the attack sites remain accessible to ssb. DNA restriction fragments were used to investigate the organization of the complexes, and its consequences on DNA radiolysis. Sequencing gel electrophoresis of the radiolytically-broken fragments are almost regular in the absence of protein, showing that breakage occurs at every base. In the presence of the protein, a periodic protection pattern is observed. The period of 11 base pairs is interpreted as the minimum distance between two adjacent MC1 proteins.

Radiolytic footprinting. Beta rays, gamma photons, and fast neutrons probe DNA-protein interactions.

Ionizing radiations induce numerous damages in DNA, especially strand breaks. The hydroxyl radical OH., produced by the radiolysis of water, is mainly responsible for this effect. The fact that strand breakage occurs at all nucleotides and that bound proteins may locally radioprotect DNA at the binding site lead us to develop a radiolytic footprinting method to study DNA-protein interactions. Three different radiations were used: beta rays, gamma photons, and fast neutrons. In order to validate this technique, three well-known interaction systems were tested: the lac repressor-lac operator of Escherichia coli, the cyclic AMP receptor protein (CRP) of E. coli and its specific site in the lac regulation region, and the core nucleosome. Radiolytic footprinting gives results similar to those obtained by more classical probes: DNase I, complexes of orthophenanthroline (OP) and copper, complexes of ethylenediaminetetraacetate ion (EDTA) and iron, and UV light. For the same system (lac repressor), irradiation with either gamma photons or fast neutrons gives identical results.

N-acetylcysteine and captopril protect DNA and cells against radiolysis by fast neutrons.

N-Acetylcysteine and captopril, respectively mucolytic and antihypertensive drugs, contain free sulfhydryl groups. Since in general thiols have well-established radioprotective abilities, we sought putative radioprotective effects of these drugs against therapeutic fast neutrons. We show that pBR322 plasmid DNA is indeed protected against radiolytic strand breakage by both drugs. The oxygen independent protection is consistent with a hydroxyl radical scavenging mechanism. A clonogenicity assay reveals an increase of the survival of SCL-1 cultured keratinocytes irradiated in the presence of the drugs compared with cells irradiated without drugs. Our results suggest possible interferences between treatment with drugs bearing-SH groups and radiotherapy.

[Radiation protection of operators during endoscopic retrograde cholangiopancreatography]. / Radioprotection des opérateurs lors des cholangio-pancréatographies rétrogrades endoscopiques.

Radiation hazards to operators performing retrograde endoscopic cholangio-pancreatographies (RECP) have been investigated. The principles and central elements of French medical radioprotection legislation are recalled. Doses received by the operators, over a one month period representative of their usual work, were measured in two ways: by using a ionisation chamber and with thermoluminescent detectors worn by the staff. Extrapolated to 12 months, the results were inferior to the annual reglementary dose limit: whole body < 1.2 mSv (against 50 mSv), eye lens = 38.4 mSv (against 150 mSv). Further means for reducing dose levels are proposed.

Metal ions protect DNA against strand breakage induced by fast neutrons.

Single and double strand breaks (SSB and DSB) are induced by fast neutrons in plasmid (pBR322) DNA in 1 mM potassium phosphate buffer (pH 7.25). Increasing the concentration of monovalent (Na+, Cs+, Li+), divalent (Mg2+, Ca2+) and trivalent (Al3+, Co3+ (NH3)6) metal cations strongly decreases the yield of DSB. The extent of the observed protection depends on the valence of the cation. The production of SSB is only slightly decreased, except for Al3+ and Co3+ (NH3)6, whose effects are particularly large (complete protection at 1 and 0.1 mM respectively). Circular dichroism spectra show that Al3+ induces an important structural change of DNA at the ion concentration where the protection becomes total. This change is probably a condensation (collapse), as in the well-known case of Co3+ (NH3)6. Our results suggest two mechanisms of protection by metal ions: (i) the induction of structural changes of DNA, that render less accessible the critical sites of attack by OH. radicals; and (ii) the stabilization of the double helical regions between two close-set nicks on opposite strands, that hinders the effective double strand breakage of DNA.

DNA radiolysis by fast neutrons. II. Oxygen, thiols and ionic strength effects.

Plasmid DNA was irradiated with fast neutrons, and the protection by cysteamine against strand breakage (ssb and dsb) was evaluated in the presence and absence of oxygen. In the absence of cysteamine no radiosensitizing effect of oxygen was observed. In anoxia the protection factors, PF(ssb) and PF(dsb) of 1 mM cysteamine (in 50 mM potassium phosphate solution) were lower than the PFs observed with gamma-irradiation. The results agree with the radical repair model, in which the thiol competes with the oxygen produced inside the anoxic neutron-irradiated system, according to the 'oxygen-in-the-track' hypothesis. At low ionic strength in air-saturated solutions, positively charged cysteamine protects more efficiently than negatively charged thiolactate. The dependence of the PFs on the charge of the thiol can be explained by the condensation of counter-ions and depletion of co-ions around DNA, predicted by Manning's theory. Based on the same theory, we propose here an explanation of the ionic strength or by thiolactate at low ionic strength is largely due to scavenging of OH. radicals in the bulk solution. At low ionic strength the PF(ssb) of cysteamine is higher for neutrons than for gamma-irradiation. This might suggest different primary lesions for the two types of irradiation.

DNA radiolysis by fast neutrons.

The effects of fast neutron irradiation on DNA were studied using DNA of the pBR322 plasmid (4362 base pairs), and the results compared to those obtained with 60Co gamma rays. Irradiation of the plasmid DNA in solution with a neutrons beam (p34+Be) of the CERI (CNRS Orléans) cyclotron (with a flat energy spectrum from 34 MeV to low energies) results in half the yield of single-strand breaks (ssb), and 1.5 times higher yield of double-strand breaks (dsb) for neutrons as compared to gamma-rays. Possible specificity of the neutron-induced breaks was examined: the scavenging of OH. radicals by 0.1 mol dm-3 ethanol inhibits all neutron-induced ssb, but only 85 per cent of the dsb. For gamma-irradiation, both ssb and dsb are completely inhibited in these conditions. These results suggest at least three different origins for neutron-induced dsb. The occurrence of around 30 per cent of dsb can be explained by a radical transfer mechanism (proposed by Siddiqi and Bothe (1987) for gamma-irradiation). Around 55 per cent of dsb may be due to the non-random distribution of radicals in high-density tracks of the secondary particles of neutrons, which results in a simultaneous attack of the two strands by OH. radicals. These first two processes are both OH.-mediated and thus are sensitive to ethanol. The direct effect of fast neutrons and their secondaries (recoil protons, alpha-particles and recoil nuclei) can account for the remaining 15 per cent of dsb, not inhibited by 0.1 mol dm-3 ethanol.