Radiation therapy combined with intracerebral convection-enhanced delivery of cisplatin or carboplatin for treatment of the F98 rat glioma.

BACKGROUND: The purpose of this review is to summarize our own experimental studies carried out over a 13-year period of time using the F98 rat glioma as model for high grade gliomas. We evaluated a binary chemo-radiotherapeutic modality that combines either cisplatin (CDDP) or carboplatin, administered intracerebrally (i.c.) by means of convection-enhanced delivery (CED) or osmotic pumps, in combination with either synchrotron or conventional X-irradiation. METHODS: F98 glioma cells were implanted stereotactically into the brains of syngeneic Fischer rats. Approximately 14 days later, either CDDP or carboplatin was administered i.c. by CED, followed 24 h later by radiotherapy using either a synchrotron or, subsequently, megavoltage linear accelerators (LINAC). RESULTS: CDDP was administered at a dose of 3 µg in 5 µL, followed 24 h later with an irradiation dose of 15 Gy or carboplatin at a dose of 20 µg in 10 µL, followed 24 h later with 3 fractions of 8 Gy each, at the source at the European Synchrotron Radiation Facility (ESRF). This resulted in a median survival time (MeST) > 180 days with 33% long term survivors (LTS) for CDDP and a MeST > 60 days with 8 to 22% LTS, for carboplatin. Subsequently it became apparent that comparable survival data could be obtained with megavoltage X-irradiation using a LINAC source. The best survival data were obtained with a dose of 72 µg of carboplatin administered by means of Alzet® osmotic pumps over 7 days. This resulted in a MeST of > 180 days, with 55% LTS. Histopathologic examination of all the brains of the surviving rats revealed no residual tumor cells or evidence of significant radiation related effects. CONCLUSIONS: The results obtained using this combination therapy has, to the best of our knowledge, yielded the most promising survival data ever reported using the F98 glioma model.

DNA-polyamine cross-links generated upon one electron oxidation of DNA.

The possibility to induce the formation of covalent cross-links between polyamines and guanine following one electron oxidation of double stranded DNA has been evaluated. For such a purpose, a strategy has been developed to chemically synthesize the polyamine-C8-guanine adducts, and efforts have been made to characterize them. Then, an analytical method, based on HPLC separation coupled through electrospray ionization to tandem mass spectrometry, has been setup for their detection and quantification. Using such a sensitive approach, we have demonstrated that polyamine-C8-guanine adducts could be produced with significant yields in double stranded DNA following a one-electron oxidation reaction induced by photosensitization. These adducts, involving either putrescine, spermine, or spermidine, are generated by the nucleophilic addition of primary amino groups of polyamines onto the C8 position of the guanine radical cation. Our data demonstrate that such a nucleophilic addition of polyamines is much more efficient than the addition of a water molecule that leads to 8-oxo-7,8-dihydroguanine formation.

Validation of genes for H-ARS severity prediction in leukemia patients - interspecies comparison, challenges, and promises.

PURPOSE: In a previous baboon-study, a total of 29 genes were identified for clinical outcome prediction of the hematologic, acute, radiation, syndrome (H-ARS) severity. Among them, four genes (FDXR, DDB2, POU2AF1, WNT3) appeared promising and were validated in five leukemia patients. Within this study, we sought further in-vivo validation in a larger number of whole-body irradiated patients. MATERIAL AND METHODS: Peripheral blood was drawn from 10 leukemia patients before and up to 3 days during a fractionated (2 Gy/day) total-body irradiation (TBI) with 2-12Gy. After RNA-isolation, gene expression (GE) was evaluated on 31 genes widely used in biodosimetry and H-ARS prediction employing qRT-PCR. A customized low-density-array (LDA) allowed simultanously analyzing all genes, the 96-well format further examined the four most promising genes. Fold-changes (FC) in GE relative to pre-irradiation were calculated. RESULTS: Five patients suffering from acute-lymphoblastic-leukemia (ALL) respectively non-Hodgkin-lymphoma (NHL) revealed sufficient RNA-amounts and corresponding lymphocyte and neutrophile counts for running qRT-PCR, while acute-myeloid-leukemia (AML) and one myelofibrosis patient could not supply enough RNA. Generally, 1-2µg total RNA was isolated, whereas up to 10-fold differences in RNA-quantities (associated suppressed GE-changes) were identified among pre-exposure and exposure samples. From 31 genes, 23 were expressed in at least one of the pre-exposure samples. Relative to pre-exposure, the number of expressed genes could halve at 48 and 72h after irradiation. Using the LDA, 13 genes were validated in human samples. The four most promising genes (vid. sup.) were either undetermined or too close to pre-exposure. However, they were measured using the more sensitive 96-well format, except WNT3, which wasn´t detectable. As in previous studies, an opposite regulation in GE for FDXR in leukemia patients (up-regulated) relative to baboons (down-regulated) was reconfirmed. Radiation-induced GE-changes of DDB2 (up-regulated) and POU2AF1 (down-regulated) behaved similarly in both species. Hence, 16 out of 23 genes of two species showed GE-changes in the same direction, and up-regulated FDXR as in human studies were revalidated. CONCLUSION: Identified genes for H-ARS severity prediction, previously detected in baboons, were validated in ALL but not in AML patients. Limitations related to leukemia type, associated reduced RNA amounts, suppressed GE changes, and methodological challenges must be considered as factors negatively affecting the total number of validated genes. Based on that, we propose additional controls including blood cell counts and preferably fluorescence-based RNA quantity measurements for selecting promising samples and using a more sensitive 96-well format for candidate genes with low baseline copy numbers.

Photoactivation of gold nanoparticles for glioma treatment.

Radiosensitization efficacy of gold nanoparticles (AuNPs) with low energy radiations (88 keV) was evaluated in vitro and in vivo on rats bearing glioma. In vitro, a significant dose-enhancement factor was measured by clonogenic assays after irradiation with synchrotron radiation of F98 glioma cells in presence of AuNPs (1.9 and 15 nm in diameter). In vivo, 1.9 nm nanoparticles were found to be toxic following intracerebral delivery in rats bearing glioma, whether no toxicity was observed using 15 nm nanoparticles at the same concentration (50 mg/mL). The therapeutic efficacy of gold photoactivation was determined by irradiating the animals after intracerebral infusion of AuNPs. Survival of rats that had received the combination of treatments (AuNPs: 50 mg/mL, 15 Gy) was significantly increased in comparison with the survival of rats that had received irradiation alone. In conclusion, this experimental approach is promising and further studies are foreseen for improving its therapeutic efficacy. FROM THE CLINICAL EDITOR: These investigators report that gold nanoparticles of the correct size can be used to enhance the effects of irradiation in the context of a glioma model. Since many of the glioma varieties are currently incurable, this or similar approaches may find their way to clinical trials in the near future.

Differential Recruitment of DNA Repair Proteins KU70/80 and RAD51 upon Microbeam Irradiation with α-Particles.

In addition to representing a significant part of the natural background radiation exposure, α-particles are thought to be a powerful tool for targeted radiotherapy treatments. Understanding the molecular mechanisms of recognition, signaling, and repair of α-particle-induced DNA damage is not only important in assessing the risk associated with human exposure, but can also potentially help in identifying ways of improving the efficacy of radiation treatment. α-particles (He2+ ions), as well as other types of ionizing radiation, and can cause a wide variety of DNA lesions, including DNA double-strand breaks (DSBs). In mammalian cells, DNA DSBs can be repaired by two major pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). Here, we investigated their dynamics in mouse NIH-3T3 cells through the recruitment of key proteins, such as the KU heterodimer for NHEJ and RAD51 for HR upon localized α-particle irradiation. To deliver α-particles, we used the MIRCOM microbeam, which allows targeting of subnuclear structures with submicron accuracy. Using mouse NIH-3T3 cells, we found that the KU heterodimer is recruited much earlier at DNA damage sites marked by H2AX phosphorylation than RAD51. We also observed that the difference in the response of the KU complex and RAD51 is not only in terms of time, but also in function of the chromatin nature. The use of a microbeam such as MIRCOM, represents a powerful tool to study more precisely the cellular response to ionizing irradiation in a spatiotemporal fashion at the molecular level.

Safe-by-design strategies for lowering the genotoxicity and pulmonary inflammation of multiwalled carbon nanotubes: Reduction of length and the introduction of COOH groups.

Potentially, the toxicity of multiwalled carbon nanotubes (MWCNTs) can be reduced in a safe-by-design strategy. We investigated if genotoxicity and pulmonary inflammation of MWCNTs from the same batch were lowered by a) reducing length and b) introducing COOH-groups into the structure. Mice were administered: 1) long and pristine MWCNT (CNT-long) (3.9 µm); 2) short and pristine CNT (CNT-short) (1 µm); 3) CNT modified with high ratio COOH-groups (CNT-COOH-high); 4) CNT modified with low ratio COOH-groups (CNT-COOH-low). MWCNTs were dosed by intratracheal instillation at 18 or 54 µg/mouse (∼0.9 and 2.7 mg/kg bw). Neutrophils numbers were highest after CNT-long exposure, and both shortening the MWCNT and addition of COOH-groups lowered pulmonary inflammation (day 1 and 28). Likewise, CNT-long induced genotoxicity, which was absent with CNT-short and after introduction of COOH groups. In conclusion, genotoxicity and pulmonary inflammation of MWCNTs were lowered, but not eliminated, by shortening the fibres or introducing COOH-groups.

RENEB/EURADOS field exercise 2019: robust dose estimation under outdoor conditions based on the dicentric chromosome assay.

PURPOSE: Biological and/or physical assays for retrospective dosimetry are valuable tools to recover the exposure situation and to aid medical decision making. To further validate and improve such biological and physical assays, in 2019, EURADOS Working Group 10 and RENEB performed a field exercise in Lund, Sweden, to simulate various real-life exposure scenarios. MATERIALS AND METHODS: For the dicentric chromosome assay (DCA), blood tubes were located at anthropomorphic phantoms positioned in different geometries and were irradiated with a 1.36 TBq 192Ir-source. For each exposure condition, dose estimates were provided by at least one laboratory and for four conditions by 17 participating RENEB laboratories. Three radio-photoluminescence glass dosimeters were placed at each tube to assess reference doses. RESULTS: The DCA results were homogeneous between participants and matched well with the reference doses (≥95% of estimates within ±0.5 Gy of the reference). For samples close to the source systematic underestimation could be corrected by accounting for exposure time. Heterogeneity within and between tubes was detected for reference doses as well as for DCA doses estimates. CONCLUSIONS: The participants were able to successfully estimate the doses and to provide important information on the exposure scenarios under conditions closely resembling a real-life situation.

Long-term exposure of A549 cells to titanium dioxide nanoparticles induces DNA damage and sensitizes cells towards genotoxic agents.

Titanium dioxide nanoparticles (TiO2-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1-50 µg/mL TiO2-NPs, 86% anatase/14% rutile, 24 ± 6 nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO2-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO2-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO2-NPs.

Molecular responses of alveolar epithelial A549 cells to chronic exposure to titanium dioxide nanoparticles: A proteomic view.

Although the biological effects of titanium dioxide nanoparticles (TiO2-NPs) have been studied for more than two decades, the mechanisms governing their toxicity are still unclear. We applied 2D-gel proteomics analysis on A549 epithelial alveolar cells chronically exposed for 2months to 2.5 or 50µg/mL of deeply characterized TiO2-NPs, in order to obtain comprehensive molecular responses that may reflect functional outcomes. We show that exposure to TiO2-NPs impacts the abundance of 30 protein species, corresponding to 22 gene products. These proteins are involved in glucose metabolism, trafficking, gene expression, mitochondrial function, proteasome activity and DNA damage response. Besides, our results suggest that p53 pathway is activated, slowing down cell cycle progression and reducing cell proliferation rate. Moreover, we report increased content of chaperones-related proteins, which suggests homeostasis re-establishment. Finally, our results highlight that chronic exposure to TiO2-NPs affects the same cellular functions as acute exposure to TiO2-NPs, although lower exposure concentrations and longer exposure times induce more intense cellular response. BIOLOGICAL SIGNIFICANCE: Our results make possible the identification of new mechanisms that explain TiO2-NP toxicity upon long-term, in vitro exposure of A549 cells. It is the first article describing -omics results obtained with this experimental strategy. We show that this long-term exposure modifies the cellular content of proteins involved in functions including mitochondrial activity, intra- and extracellular trafficking, proteasome activity, glucose metabolism, and gene expression. Moreover we observe modification of content of proteins that activate the p53 pathway, which suggest the induction of a DNA damage response. Technically, our results show that exposure of A549 cells to a high concentration of TiO2-NPs leads to the identification of modulations of the same functional categories than exposure to low, more realistic concentrations. Still the intensity differs between these two exposure scenarios. We also show that chronic exposure to TiO2-NPs induces the modulation of cellular functions that have already been reported in the literature as being impacted in acute exposure scenarios. This proves that the exposure protocol in in vitro experiments related to nanoparticle toxicology might be cautiously chosen since inappropriate scenario may lead to inappropriate and/or incomplete conclusions.

Comparison between internalizing anti-HER2 mAbs and non-internalizing anti-CEA mAbs in alpha-radioimmunotherapy of small volume peritoneal carcinomatosis using 212Pb.

BACKGROUND AND PURPOSE: We assessed the contribution of antibody internalization in the efficacy and toxicity of intraperitoneal α-radioimmunotherapy (RIT) of small volume carcinomatosis using (212)Pb-labeled monoclonal antibodies (mAbs) that target HER2 (internalizing) or CEA (non-internalizing) receptors. MATERIALS AND METHODS: Athymic nude mice bearing 2-3 mm intraperitoneal tumor xenografts were intraperitoneally injected with similar activities (370, 740 and 1480 kBq; 37 MBq/mg) of (212)Pb-labeled 35A7 (anti-CEA), trastuzumab (anti-HER2) or PX (non-specific) mAbs, or with equivalent amounts of unlabeled mAbs, or with NaCl. Tumor volume was monitored by bioluminescence and survival was reported. Hematologic toxicity and body weight were assessed. Biodistribution of (212)Pb-labeled mAbs and absorbed dose-effect relationships using MIRD formalism were established. RESULTS: Transient hematological toxicity, as revealed by white blood cells and platelets numbering, was reported in mice treated with the highest activities of (212)Pb-labeled mAbs. The median survival (MS) was significantly higher in mice injected with 1.48 MBq of (212)Pb-35A7 (non-internalizing mAbs) (MS = 94 days) than in animals treated with the same activity of (212)Pb-PX mAbs or with NaCl (MS = 18 days). MS was even not reached after 130 days when follow-up was discontinued in mice treated with 1.48 MBq of (212)Pb-trastuzumab. The later efficacy was unexpected since final absorbed dose resulting from injection of 1.48 MBq, was higher for (212)Pb-35A7 (35.5 Gy) than for (212)Pb-trastuzumab (27.6 Gy). These results also highlight the lack of absorbed dose-effect relationship when mean absorbed dose was calculated using MIRD formalism and the requirement to perform small-scale dosimetry. CONCLUSIONS: These data indicate that it might be an advantage of using internalizing anti-HER2 compared with non-internalizing anti-CEA (212)Pb-labeled mAbs in the therapy of small volume xenograft tumors. They support clinical investigations of (212)Pb-mAbs RIT as an adjuvant treatment after cytoreductive surgery in patients with peritoneal carcinomatosis.

Monochromatic minibeams radiotherapy: from healthy tissue-sparing effect studies toward first experimental glioma bearing rats therapy.

PURPOSE: The purpose of this study was to evaluate high-dose single fraction delivered with monochromatic X-rays minibeams for the radiotherapy of primary brain tumors in rats. METHODS AND MATERIALS: Two groups of healthy rats were irradiated with one anteroposterior minibeam incidence (four minibeams, 123 Gy prescribed dose at 1 cm depth in the brain) or two interleaved incidences (54 Gy prescribed dose in a 5 × 5 × 4.8 mm(3) volume centered in the right hemisphere), respectively. Magnetic resonance imaging (MRI) follow-up was performed over 1 year. T2-weighted (T2w) images, apparent diffusion coefficient (ADC), and blood vessel permeability maps were acquired. F98 tumor bearing rats were also irradiated with interleaved minibeams to achieve a homogeneous dose of 54 Gy delivered to an 8 × 8 × 7.8 mm(3) volume centered on the tumor. Anatomic and functional MRI follow-up was performed every 10 days after irradiation. T2w images, ADC, and perfusion maps were acquired. RESULTS: All healthy rats were euthanized 1 year after irradiation without any clinical alteration visible by simple examination. T2w and ADC measurements remain stable for the single incidence irradiation group. Localized Gd-DOTA permeability, however, was observed 9 months after irradiation for the interleaved incidences group. The survival time of irradiated glioma bearing rats was significantly longer than that of untreated animals (49 ± 12.5 days versus 23.3 ± 2 days, p < 0.001). The tumoral cerebral blood flow and blood volume tend to decrease after irradiation. CONCLUSIONS: This study demonstrates the sparing effect of minibeams on healthy tissue. The increased life span achieved for irradiated glioma bearing rats was similar to the one obtained with other radiotherapy techniques. This experimental tumor therapy study shows the feasibility of using X-ray minibeams with high doses in brain tumor radiotherapy.

Intracerebral delivery of carboplatin in combination with either 6 MV photons or monoenergetic synchrotron X-rays are equally efficacious for treatment of the F98 rat glioma.

BACKGROUND: The purpose of the present study was to compare side-by-side the therapeutic efficacy of a 6-day infusion of carboplatin, followed by X-irradiation with either 6 MV photons or synchrotron X-rays, tuned above the K-edge of Pt, for treatment of F98 glioma bearing rats. METHODS: Carboplatin was administered intracerebrally (i.c.) to F98 glioma bearing rats over 6 days using AlzetTM osmotic pumps starting 7 days after tumor implantation. Radiotherapy was delivered in a single 15 Gy fraction on day 14 using a conventional 6 MV linear accelerator (LINAC) or 78.8 keV synchrotron X-rays. RESULTS: Untreated control animals had a median survival time (MeST) of 33 days. Animals that received either carboplatin alone or irradiation alone with either 78.8 keV or 6 MV had a MeSTs 38 and 33 days, respectively. Animals that received carboplatin in combination with X-irradiation had a MeST of > 180 days with a 55% cure rate, irrespective of whether they were irradiated with either 78.8 KeV synchrotron X-rays or 6MV photons. CONCLUSIONS: These studies have conclusively demonstrated the equivalency of i.c. delivery of carboplatin in combination with X-irradiation with either 6 MV photons or synchrotron X-rays.