Method validation to assess in vivo cellular and subcellular changes in buccal mucosa cells and saliva following CBCT examinations

Abstract

Objectives: Cone-beam CT (CBCT) is a medical imaging technique used in dental medicine. However, there are no conclusive data available indicating that exposure to X-ray doses used by CBCT are harmless. We aim, for the first time, to characterize the potential age-dependent cellular and subcellular effects related to exposure to CBCT imaging. Current objective is to describe and validate the protocol for characterization of cellular and subcellular changes after diagnostic CBCT. Methods: Development and validation of a dedicated two-part protocol: 1) assessing DNA double strand breaks (DSBs) in buccal mucosal (BM) cells and 2) oxidative stress measurements in saliva samples. BM cells and saliva samples are collected prior to and 0.5 h after CBCT examination. BM cells are also collected 24 h after CBCT examination. DNA DSBs are monitored in BM cells via immunocytochemical staining for γH2AX and 53BP1. 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG) and total antioxidant capacity are measured in saliva to assess oxidative damage. Results: Validation experiments show that sufficient BM cells are collected (97.1 ± 1.4 %) and that γH2AX/53BP1 foci can be detected before and after CBCT examination. Collection and analysis of saliva samples, either sham exposed or exposed to IR, show that changes in 8-oxo-dG and total antioxidant capacity can be detected in saliva samples after CBCT examination. Conclusion: The DIMITRA Research Group presents a two-part protocol to analyze potential age-related biological differences following CBCT examinations. This protocol was validated for collecting BM cells and saliva and for analyzing these samples for DNA DSBs and oxidative stress markers, respectively.

Quantification of DNA Double Strand Breaks and Oxidation Response in Children and Adults Undergoing Dental CBCT Scan

“…This study aimed to characterize the short-term radiation-induced effects associated with CBCT examinations, specifically in children. To this end, the number of DNA DSBs was monitored in BMCs and 8-oxo-dG levels as well as total antioxidant capacity were monitored in saliva samples using previously optimized protocols 55 .…”

“…The DIMITRA study is an non-interventional, prospective study that focusses on radiation-induced effects related to diagnostic CBCT exposure in children. It is a multicentre study carried out in three European centres: the Oral and MaxilloFacial Surgery -Imaging & Pathology department (Katholieke Universiteit Leuven, Leuven, Belgium), the Dental Medicine Department of the Bretonneau Hospital (Paris, France) and the Iuliu Hatieganu University of Medicine and Pharmacy (Cluj-Napoca, Romania) 55 . All experiments and methods were performed in accordance with relevant guidelines and regulations.…”

“…Ethical approval was obtained at the participating sites (Commissie Medische Ethiek KU Leuven, B322201525196, Belgium; Comité d'Evaluation de l'Ethique des projets de Reserche Biomédicale Paris Nord, N°16-021, France; Comisia de Etica UMF Iuliu Hatieganu Cluj-Napoca, 208/21.04.2015, Romania). In case of underage children, both parents needed to consent unless one parent has explicit permission from the other parent 55 .…”

“…were described in detail by Belmans et al 55 . Briefly, synthetic swabs were used to collect BMCs just before, 30 minutes and 24 hours after CBCT examination using a protocol modified from Thomas et al 45 .…”

“…Power analysis. Power analysis was based on validations experiments that were performed prior to this study 55 . The number of participants (N) are the numbers described in this manuscript.…”