Antitumour effects of artesunate via cell cycle checkpoint controls in human oesophageal squamous carcinoma cells.

Artesunate (ART), an effective antimalarial semisynthetic derivative of artemisinin, exhibits antitumour properties, but the mechanism(s) involved remain elusive. In this study, we investigated the antitumour effects of ART on human oesophageal squamous cell carcinoma (ESCC) cell lines. Treatment of ESCC cell lines with ART resulted in the production of excessive reactive oxygen species (ROS) that induced DNA damage, reduced cell proliferation and inhibited clonogenicity via G1-S cell cycle arrest and/or apoptosis in vitro. The administration of ART to nude mice with ESCC cell xenografts inhibited tumour formation in vivo. However, the cytotoxicity of ART strongly differed among the ESCC cell lines tested. Transcriptomic profiling revealed that although the expression of large numbers of genes in ESCC cell lines was affected by ART treatment, these genes could be functionally clustered into pathways involved in regulating cell cycle progression, DNA metabolism and apoptosis. We revealed that p53 and Cdk4/6-p16-Rb cell cycle checkpoint controls were critical determinants required for mediating ART cytotoxicity in ESCC cell lines. Specifically, KYSE30 cells with p53Mut/p16Mut were the most sensitive to ART, KYSE150 and KYSE180 cells with p53Mut/p16Nor exhibited intermediate responses to ART, and Eca109 cells with p53Nor/p16Nor exhibited the most resistance to ATR. Consistently, perturbation of p53 expression using RNA interference (RNAi) and/or Cdk4/6 activity using the inhibitor palbociclib altered ART cytotoxicity in KYSE30 cells. Given that the p53 and Cdk4/6-cyclin D1-p16-Rb genes are commonly mutated in ESCC, our results potentially shed new light on neoadjuvant chemotherapy strategies for ESCC.

Genome-wide identification of replication fork stalling/pausing sites and the interplay between RNA Pol II transcription and DNA replication progression.

BACKGROUND: DNA replication progression can be affected by the presence of physical barriers like the RNA polymerases, leading to replication stress and DNA damage. Nonetheless, we do not know how transcription influences overall DNA replication progression. RESULTS: To characterize sites where DNA replication forks stall and pause, we establish a genome-wide approach to identify them. This approach uses multiple timepoints during S-phase to identify replication fork/stalling hotspots as replication progresses through the genome. These sites are typically associated with increased DNA damage, overlapped with fragile sites and with breakpoints of rearrangements identified in cancers but do not overlap with replication origins. Overlaying these sites with a genome-wide analysis of RNA polymerase II transcription, we find that replication fork stalling/pausing sites inside genes are directly related to transcription progression and activity. Indeed, we find that slowing down transcription elongation slows down directly replication progression through genes. This indicates that transcription and replication can coexist over the same regions. Importantly, rearrangements found in cancers overlapping transcription-replication collision sites are detected in non-transformed cells and increase following treatment with ATM and ATR inhibitors. At the same time, we find instances where transcription activity favors replication progression because it reduces histone density. CONCLUSIONS: Altogether, our findings highlight how transcription and replication overlap during S-phase, with both positive and negative consequences for replication fork progression and genome stability by the coexistence of these two processes.

Comparison of the genotoxicity of propofol and desflurane using the comet assay in the lymphocytes of patients who underwent lumbar discectomy: A randomized trial.

OBJECTIVES: To compare the genotoxic effects of desflurane and propofol using comet assay in patients undergoing elective discectomy surgery. METHODS: This was a randomized controlled study. Patients who underwent elective lumbar discectomy under general anesthesia with propofol or desflurane were included in the study. Venous blood samples were obtained at 4 different time points: 5 minutes before anesthesia induction (T1), 2 hours after the start of anesthesia (T2), the first day after surgery (T3), and the fifth day following surgery (T4). Deoxyribonucleic acid damage in lymphocytes was assessed via the comet assay. RESULTS: A total of 30 patients, 15 in each group, were included in the analysis. The groups were similar in terms of age and gender distribution. There were no significant differences in demographics, duration of surgery, total remifentanil consumption, and total rocuronium bromide consumption. The comet assay revealed that head length, head intensity, tail intensity, tail moment at T1 were similar in the desflurane and propofol groups. Head length, tail length and tail moment measured in the desflurane group at T4 were significantly higher compared to the propofol group. Tail lengths of the desflurane group at T1, T2 and T3 were significantly higher than the corresponding values in the propofol group. CONCLUSION: Propofol and desflurane do not appear to induce DNA damage in lymphocytes. However, when the quantitative data were compared, it was determined that propofol had relatively lower genotoxic potential than desflurane.ClinicalTrials.gov Reg. No.: NCT05185167.

Spatial transcriptomic analysis reveals local effects of intratumoral fusobacterial infection on DNA damage and immune signaling in rectal cancer.

Mucinous colorectal cancer (CRC) is a common histological subtype of colorectal adenocarcinoma, associated with a poor response to chemoradiotherapy. The commensal facultative anaerobes fusobacteria, have been associated with poor prognosis specifically in mesenchymal CRC. Interestingly, fusobacterial infection is especially prevalent in mucinous CRC. The objective of this study was therefore to increase our understanding of beneficial and detrimental effects of fusobacterial infection, by contrasting host cell signaling and immune responses in areas of high vs. low infection, using mucinous rectal cancer as a clinically relevant example. We employed spatial transcriptomic profiling of 106 regions of interest from 8 mucinous rectal cancer samples to study gene expression in the epithelial and immune segments across regions of high versus low fusobacterial infection. Fusobacteria high regions were associated with increased oxidative stress, DNA damage, and P53 signaling. Meanwhile regions of low fusobacterial prevalence were characterized by elevated JAK-STAT, Il-17, Il-1, chemokine and TNF signaling. Immune masks within fusobacterial high regions were characterized by elevated proportions of cytotoxic (CD8+) T cells (p = 0.037), natural killer (NK) cells (p < 0.001), B-cells (p < 0.001), and gamma delta T cells (p = 0.003). Meanwhile, fusobacteria low regions were associated with significantly greater M2 macrophage (p < 0.001), fibroblast (p < 0.001), pericyte (p = 0.002), and endothelial (p < 0.001) counts.

Extracellular vesicles from senescent mesenchymal stromal cells are defective and cannot prevent osteoarthritis.

Age is the most important risk factor in degenerative diseases such as osteoarthritis (OA), which is associated with the accumulation of senescent cells in the joints. Here, we aimed to assess the impact of senescence on the therapeutic properties of extracellular vesicles (EVs) from human fat mesenchymal stromal cells (ASCs) in OA. We generated a model of DNA damage-induced senescence in ASCs using etoposide and characterized EVs isolated from their conditioned medium (CM). Senescent ASCs (S-ASCs) produced 3-fold more EVs (S-EVs) with a slightly bigger size and that contain 2-fold less total RNA. Coculture experiments showed that S-ASCs were as efficient as healthy ASCs (H-ASCs) in improving the phenotype of OA chondrocytes cultured in resting conditions but were defective when chondrocytes were proliferating. S-EVs were also impaired in their capacity to polarize synovial macrophages towards an anti-inflammatory phenotype. A differential protein cargo mainly related to inflammation and senescence was detected in S-EVs and H-EVs. Using the collagenase-induced OA model, we found that contrary to H-EVs, S-EVs could not protect mice from cartilage damage and joint calcifications, and were less efficient in protecting subchondral bone degradation. In addition, S-EVs induced a pro-catabolic and pro-inflammatory gene signature in the joints of mice shortly after injection, while H-EVs decreased hypertrophic, catabolic and inflammatory pathways. In conclusion, S-EVs are functionally impaired and cannot protect mice from developing OA.

Biochemical and molecular evaluation of oxidative stress and mitochondrial damage in fruit fly exposed to carmoisine.

BACKGROUND: In today's world, appearance is an important factor in almost all areas of our lives. Therefore, it has become common to use dyes to color foods to make them look appetizing and visually appealing. However, food additives have negative effects on biochemical processes in cells at both high and low doses. METHODS AND RESULTS: This study investigated the effect of carmoisine, a commonly used food coloring, on oxidative stress and damage parameters in Drosophila melanogaster in terms of both enzymatic and gene expression. The change in mitochondrial DNA copy number (mtDNA-CN), a marker of oxidative stress, was also examined. When the data obtained were analyzed, it was observed that carmoisine caused a significant decrease in GSH levels depending on the increase in dose. SOD, CAT, GPx, and AChE enzyme activities and gene expression levels were also found to be significantly decreased. All groups also showed a significant decrease in mtDNA-CN. The effect of carmoisine on Drosophila melanogaster morphology was also investigated in our study. However, no significant change was observed in terms of morphological development in any group. CONCLUSIONS: When all the findings were evaluated together, it was observed that carmoisin triggered oxidative stress and these effects became more risky at high doses. Therefore, we believe that the consumer should be made more aware of the side effects of azo dyes in food and that the type and concentration of each substance added to food should be specified.

TCAF1 promotes TRPV2-mediated Ca2+ release in response to cytosolic DNA to protect stressed replication forks.

The protection of the replication fork structure under stress conditions is essential for genome maintenance and cancer prevention. A key signaling pathway for fork protection involves TRPV2-mediated Ca2+ release from the ER, which is triggered after the generation of cytosolic DNA and the activation of cGAS/STING. This results in CaMKK2/AMPK activation and subsequent Exo1 phosphorylation, which prevent aberrant fork processing, thereby ensuring genome stability. However, it remains poorly understood how the TRPV2 channel is activated by the presence of cytosolic DNA. Here, through a genome-wide CRISPR-based screen, we identify TRPM8 channel-associated factor 1 (TCAF1) as a key factor promoting TRPV2-mediated Ca2+ release under replication stress or other conditions that activate cGAS/STING. Mechanistically, TCAF1 assists Ca2+ release by facilitating the dissociation of STING from TRPV2, thereby relieving TRPV2 repression. Consistent with this function, TCAF1 is required for fork protection, chromosomal stability, and cell survival after replication stress.

The novel SMYD3 inhibitor EM127 impairs DNA repair response to chemotherapy-induced DNA damage and reverses cancer chemoresistance.

BACKGROUND: SMYD3 has been found implicated in cancer progression. Its overexpression correlates with cancer growth and invasion, especially in gastrointestinal tumors. SMYD3 transactivates multiple oncogenic mechanisms, favoring cancer development. Moreover, it was recently shown that SMYD3 is required for DNA restoration by promoting homologous recombination (HR) repair. METHODS: In cellulo and in vivo models were employed to investigate the role of SMYD3 in cancer chemoresistance. Analyses of SMYD3-KO cells, drug-resistant cancer cell lines, patients' residual gastric or rectal tumors that were resected after neoadjuvant therapy and mice models were performed. In addition, the novel SMYD3 covalent inhibitor EM127 was used to evaluate the impact of manipulating SMYD3 activity on the sensitization of cancer cell lines, tumorspheres and cancer murine models to chemotherapeutics (CHTs). RESULTS: Here we report that SMYD3 mediates cancer cell sensitivity to CHTs. Indeed, cancer cells lacking SMYD3 functions showed increased responsiveness to CHTs, while restoring its expression promoted chemoresistance. Specifically, SMYD3 is essential for the repair of CHT-induced double-strand breaks as it methylates the upstream sensor ATM and allows HR cascade propagation through CHK2 and p53 phosphorylation, thereby promoting cancer cell survival. SMYD3 inhibition with the novel compound EM127 showed a synergistic effect with CHTs in colorectal, gastric, and breast cancer cells, tumorspheres, and preclinical colorectal cancer models. CONCLUSIONS: Overall, our results show that targeting SMYD3 may be an effective therapeutic strategy to overcome chemoresistance.

miR-92b-3p protects retinal tissues against DNA damage and apoptosis by targeting BTG2 in experimental myopia.

BACKGROUND: Myopia is one of the eye diseases that can damage the vision of young people. This study aimed to explore the protective role of miR-92b-3p against DNA damage and apoptosis in retinal tissues of negative lens-induced myopic (LIM) guinea pigs by targeting BTG2. METHODS: Biometric measurements of ocular parameters, flash electroretinogram (FERG), and retinal thickness (RT) were performed after miR-92b-3p intravitreal injection in LIM guinea pigs. The apoptotic rate was detected by Annexin V-FITC/PI double staining, and the change in mitochondrial membrane potential was measured by JC-1 staining. Retinal apoptosis and expression of p53, BTG2, and CDK2 were explored by TdT-mediated dUTP-biotin nick labeling (TUNEL) and immunofluorescence staining assays, respectively. BTG2 and its upstream and downstream molecules at gene and protein levels in retinal tissues were measured by real-time quantitative PCR (qPCR) and Western blotting. RESULTS: Compared with normal controls (NC), the ocular axial length of LIM guinea pig significantly increased, whereas refraction decreased. Meanwhile, dMax-a and -b wave amplitudes of ERG declined, retinal thickness was decreased, the number of apoptotic cells and apoptotic rate in LIM eyes was exaggerated, and the mitochondrial membrane potential significantly decreased. In addition, results of qPCR and Western blot assays showed that the expression levels of p53, BTG2, CDK2, and BAX in LIM guinea pigs were higher than the levels of the NC group, whereas the BCL-2 expression level was decreased. By contrast, the miR-92b-3p intravitreal injection in LIM guinea pigs could significantly inhibit axial elongation, alleviate DNA damage and apoptosis, and thus protect guinea pigs against myopia. CONCLUSION: In conclusion, p53 and BTG2 were activated in the retinal tissue of myopic guinea pigs, and the activated BTG2 could elevate the expression of CDK2 and BAX, and attenuate the expression of BCL-2, which in turn promote apoptosis and eventually lead to retinal thinning and impaired visual function in myopic guinea pigs. The miR-92b-3p intravitreal injection can attenuate the elongation of ocular length and retinal thickness, and inhibit the CDK2, BAX, and p53 expression by targeting BTG2, thereby ameliorating DNA damage and apoptosis in LIM guinea pigs and protecting ocular tissues.

Oxidative stress and DNA alteration on the earthworm Eisenia fetida exposed to four commercial pesticides.

Modern agriculture is mainly based on the use of pesticides to protect crops but their efficiency is very low, in fact, most of them reach water or soil ecosystems causing pollution and health hazards to non-target organisms. Fungicide triazoles and strobilurins based are the most widely used and require a specific effort to investigate toxicological effects on non-target species. This study evaluates the toxic effects of four commercial fungicides Prosaro® (tebuconazole and prothioconazole), Amistar®Xtra (azoxystrobin and cyproconazole), Mirador® (azoxystrobin) and Icarus® (Tebuconazole) on Eisenia fetida using several biomarkers: lipid peroxidation (LPO), catalase activity (CAT), glutathione S-transferase (GST), total glutathione (GSHt), DNA fragmentation (comet assay) and lysozyme activity tested for the first time in E. fetida. The exposure to Mirador® and AmistarXtra® caused an imbalance of ROS species, leading to the inhibition of the immune system. AmistarXtra® and Prosaro®, composed of two active ingredients, induced significant DNA alteration, indicating genotoxic effects. This study broadened our knowledge of the effects of pesticide product formulations on earthworms and showed the need for improvement in the evaluation of toxicological risk deriving from the changing of physicochemical and toxicological properties that occur when a commercial formulation contains more than one active ingredient and several unknown co-formulants.

A unified mechanism for PARP inhibitor-induced PARP1 chromatin retention at DNA damage sites in living cells.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) not only suppress PARP1 catalytic activity but also prolong its association to damaged chromatin. Here, through live-cell imaging, we quantify the alterations in PARP1 dynamics and activity elicited by seven PARPis over a wide range of concentrations to deliver a unified mechanism of PARPi-induced PARP1 chromatin retention. We find that gross PARP1 retention at DNA damage sites is jointly governed by catalytic inhibition and allosteric trapping, albeit in a strictly independent manner-catalytic inhibition causes multiple unproductive binding-dissociation cycles of PARP1, while allosteric trapping prolongs the lesion-bound state of PARP1 to greatly increase overall retention. Importantly, stronger PARP1 retention produces greater temporal shifts in downstream DNA repair events and superior cytotoxicity, highlighting PARP1 retention, a complex but precisely quantifiable characteristic of PARPis, as a valuable biomarker for PARPi efficacy. Our approach can be promptly repurposed for interrogating the properties of DNA-repair-targeting compounds beyond PARPis.

Mapping the tumor stress network reveals dynamic shifts in the stromal oxidative stress response.

The tumor microenvironment (TME) presents cells with challenges such as variable pH, hypoxia, and free radicals, triggering stress responses that affect cancer progression. In this study, we examine the stress response landscape in four carcinomas-breast, pancreas, ovary, and prostate-across five pathways: heat shock, oxidative stress, hypoxia, DNA damage, and unfolded protein stress. Using a combination of experimental and computational methods, we create an atlas of stress responses across various types of carcinomas. We find that stress responses vary within the TME and are especially active near cancer cells. Focusing on the non-immune stroma we find, across tumor types, that NRF2 and the oxidative stress response are distinctly activated in immune-regulatory cancer-associated fibroblasts and in a unique subset of cancer-associated pericytes. Our study thus provides an interactome of stress responses in cancer, offering ways to intersect survival pathways within the tumor, and advance cancer therapy.

Transient caspase-mediated activation of caspase-activated DNase causes DNA damage required for phagocytic macrophage differentiation.

Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every organ before birth to lifelong self-renew. However, the mechanisms for versatile macrophage differentiation remain unknown. Here, we use in vivo genetic and cell biological analysis of the Drosophila larval hematopoietic organ, the lymph gland that produces macrophages. We show that the developmentally regulated transient activation of caspase-activated DNase (CAD)-mediated DNA strand breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling regulates the apoptosis signal-regulating kinase 1 (Ask1)/c-Jun kinase (JNK) axis to control sublethal levels of caspase activation, causing DNA strand breaks during macrophage development. Furthermore, caspase activity is also required for embryonic-origin macrophage development and efficient phagocytosis. Our study provides insights into developmental signaling and CAD-mediated DNA strand breaks associated with multifunctional and heterogeneous macrophage differentiation.

CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity.

DNA replication is a vulnerable cellular process, and its deregulation leads to genomic instability. Here, we demonstrate that chromobox protein homolog 3 (CBX3) binds replication protein A 32-kDa subunit (RPA2) and regulates RPA2 retention at stalled replication forks. CBX3 is recruited to stalled replication forks by RPA2 and inhibits ring finger and WD repeat domain 3 (RFWD3)-facilitated replication restart. Phosphorylation of CBX3 at serine-95 by casein kinase 2 (CK2) kinase augments cadherin 1 (CDH1)-mediated CBX3 degradation and RPA2 dynamics at stalled replication forks, which permits replication fork restart. Increased expression of CBX3 due to gene amplification or CK2 inhibitor treatment sensitizes prostate cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors while inducing replication stress and DNA damage. Our work reveals CBX3 as a key regulator of RPA2 function and DNA replication, suggesting that CBX3 could serve as an indicator for targeted therapy of cancer using PARP inhibitors.

p37 regulates VCP/p97 shuttling and functions in the nucleus and cytosol.

The AAA+-ATPase valosin-containing protein (VCP; also called p97 or Cdc48), a major protein unfolding machinery with a variety of essential functions, localizes to different subcellular compartments where it has different functions. However, the processes regulating the distribution of VCP between the cytosol and nucleus are not understood. Here, we identified p37 (also called UBXN2B) as a major factor regulating VCP nucleocytoplasmic shuttling. p37-dependent VCP localization was crucial for local cytosolic VCP functions, such as autophagy, and nuclear functions in DNA damage repair. Mutations in VCP causing multisystem proteinopathy enhanced its association with p37, leading to decreased nuclear localization of VCP, which enhanced susceptibility to DNA damage accumulation. Both VCP localization and DNA damage susceptibility in cells with such mutations were normalized by lowering p37 levels. Thus, we uncovered a mechanism by which VCP nucleocytoplasmic distribution is fine-tuned, providing a means for VCP to respond appropriately to local needs.

Development of Aptamer-DNAzyme based metal-nucleic acid frameworks for gastric cancer therapy.

The metal-nucleic acid nanocomposites, first termed metal-nucleic acid frameworks (MNFs) in this work, show extraordinary potential as functional nanomaterials. However, thus far, realized MNFs face limitations including harsh synthesis conditions, instability, and non-targeting. Herein, we discover that longer oligonucleotides can enhance the synthesis efficiency and stability of MNFs by increasing oligonucleotide folding and entanglement probabilities during the reaction. Besides, longer oligonucleotides provide upgraded metal ions binding conditions, facilitating MNFs to load macromolecular protein drugs at room temperature. Furthermore, longer oligonucleotides facilitate functional expansion of nucleotide sequences, enabling disease-targeted MNFs. As a proof-of-concept, we build an interferon regulatory factor-1(IRF-1) loaded Ca2+/(aptamer-deoxyribozyme) MNF to target regulate glucose transporter (GLUT-1) expression in human epidermal growth factor receptor-2 (HER-2) positive gastric cancer cells. This MNF nanodevice disrupts GSH/ROS homeostasis, suppresses DNA repair, and augments ROS-mediated DNA damage therapy, with tumor inhibition rate up to 90%. Our work signifies a significant advancement towards an era of universal MNF application.

Iron (II)-based metal-organic framework nanozyme for boosting tumor ferroptosis through inhibiting DNA damage repair and system Xc.

Development of ferroptosis-inducible nanoplatforms with high efficiency and specificity is highly needed and challenging in tumor ferrotherapy. Here, we demonstrate highly effective tumor ferrotherapy using iron (II)-based metal-organic framework (FessMOF) nanoparticles, assembled from disulfide bonds and ferrous ions. The as-prepared FessMOF nanoparticles exhibit peroxidase-like activity and pH/glutathione-dependent degradability, which enables tumor-responsive catalytic therapy and glutathione depletion by the thiol/disulfide exchange to suppress glutathione peroxidase 4, respectively. Upon PEGylation and Actinomycin D (ActD) loading, the resulting FessMOF/ActD-PEG nanoplatform induces marked DNA damage and lipid peroxidation. Concurrently, we found that ActD can inhibit Xc- system and elicit ferritinophagy, which further boosts the ferrotherapeutic efficacy of the FessMOF/ActD-PEG. In vivo experiments demonstrate that our fabricated nanoplatform presents excellent biocompatibility and a high tumor inhibition rate of 91.89%.

Glycyrrhizic acid inhibits DNA damage repair and enhances cisplatin-induced apoptosis of melanoma cells.

This research was designed to prospect the mechanism and impact of glycyrrhizic acid (GA) on DNA damage repair and cisplatin (CP)-induced apoptosis of melanoma cells. First, human melanoma cell SK-MEL-28 was stimulated using GA for 24, 48, and 72 h. Then, the optimal treatment time and dosage were selected. After that, cell counting kit-8 (CCK-8) was employed for testing the cell viability, flow cytometry for the apoptosis, comet assay for the DNA damage of cells, and western blot for the cleaved-Caspase3, Caspase3, Bcl-2, and γH2AX protein expression levels. The experimental outcomes exhibited that as the GA concentration climbed up, the SK-MEL-28 cell viability dropped largely, while the apoptosis level raised significantly, especially at the concentration of 100 µm. In addition, compared with GA or CPtreatment only, CP combined with GA notably suppressed the viability of melanoma cells and promoted cell apoptosis at the cytological level. At the protein level, the combined treatment notably downregulated the Bcl-2 and Caspase3 expression levels, while significantly upregulated the cleaved-Caspase3 and γH2AX expression levels. Besides, CP + GA treatment promoted DNA damage at the DNA molecular level. Collectively, both GA and CP can inhibit DNA damage repair and enhance the apoptosis of SK-MEL-28 cells, and the synergistic treatment of both exhibits better efficacy.

Dose and DNA damage modelling of diffusing alpha-emitters radiation therapy using Geant4.

PURPOSE: Diffusing alpha-emitters radiation therapy (DaRT) is a brachytherapy technique using α-particles to treat solid tumours. The high linear energy transfer (LET) and short range of α-particles make them good candidates for the targeted treatment of cancer. Treatment planning of DaRT requires a good understanding of the dose from α-particles and the other particles released in the 224Ra decay chain. METHODS: The Geant4 Monte Carlo toolkit has been used to simulate a DaRT seed to better understand the dose contribution from all particles and simulate the DNA damage due to this treatment. RESULTS: Close to the seed α-particles deliver the majority of dose, however at radial distances greater than 4 mm, the contribution of ß-particles is greater. The RBE has been estimated as a function of number of double strand breaks (DSBs) and complex DSBs. A maximum seed spacing of 5.5 mm and 6.5 mm was found to deliver at least 20 Gy RBE weighted dose between the seeds for RBEDSB and RBEcDSB respectively. CONCLUSIONS: The DNA damage changes with radial distance from the seed and has been found to become less complex with distance, which is potentially easier for the cell to repair. Close to the seed α-particles contribute the majority of dose, however the contribution from other particles cannot be neglected and may influence the choice of seed spacing.