High Resolution and Automatable Cytogenetic Biodosimetry Using In Situ Telomere and Centromere Hybridization for the Accurate Detection of DNA Damage: An Overview.

In the event of a radiological or nuclear accident, or when physical dosimetry is not available, the scoring of radiation-induced chromosomal aberrations in lymphocytes constitutes an essential tool for the estimation of the absorbed dose of the exposed individual and for effective triage. Cytogenetic biodosimetry employs different cytogenetic assays including the scoring of dicentrics, micronuclei, and translocations as well as analyses of induced premature chromosome condensation to define the frequency of chromosome aberrations. However, inherent challenges using these techniques include the considerable time span from sampling to result, the sensitivity and specificity of the various techniques, and the requirement of highly skilled personnel. Thus, techniques that obviate these challenges are needed. The introduction of telomere and centromere (TC) staining have successfully met these challenges and, in addition, greatly improved the efficiency of cytogenetic biodosimetry through the development of automated approaches, thus reducing the need for specialized personnel. Here, we review the role of the various cytogenetic dosimeters and their recent improvements in the management of populations exposed to genotoxic agents such as ionizing radiation. Finally, we discuss the emerging potentials to exploit these techniques in a wider spectrum of medical and biological applications, e.g., in cancer biology to identify prognostic biomarkers for the optimal triage and treatment of patients.

Is Response to Genotoxic Stress Similar in Populations of African and European Ancestry? A Study of Dose-Response After in vitro Irradiation.

Background: Exposure to genotoxic stress such as radiation is an important public health issue affecting a large population. The necessity of analyzing cytogenetic effects of such exposure is related to the need to estimate the associated risk. Cytogenetic biological dosimetry is based on the relationship between the absorbed dose and the frequency of scored chromosomal aberrations. The influence of confounding factors on radiation response is a topical issue. The role of ethnicity is unclear. Here, we compared the dose-response curves obtained after irradiation of circulating lymphocytes from healthy donors of African and European ancestry. Materials and Methods: Blood samples from six Africans living in Africa, five Africans living in Europe, and five Caucasians living in Europe were exposed to various doses (0-4 Gy) of X-rays at a dose-rate of 0.1 Gy/min using an X-RAD320 irradiator. A validated cohort composed of 14 healthy Africans living in three African countries was included and blood samples were irradiated using the same protocols. Blood lymphocytes were cultured for 48 h and chromosomal aberrations scored during the first mitosis by telomere and centromere staining. The distribution of dicentric chromosomes was determined and the Kruskal-Wallis test was used to compare the dose-response curves of the two populations. Results: No spontaneous dicentric chromosomes were detected in African donors, thus establishing a very low background of unstable chromosomal aberrations relative to the European population. There was a significant difference in the dose response curves between native African and European donors. At 4 Gy, African donors showed a significantly lower frequency of dicentric chromosomes (p = 8.65 10-17), centric rings (p = 4.0310-14), and resulting double-strand-breaks (DSB) (p = 1.32 10-18) than European donors. In addition, a significant difference was found between African donors living in Europe and Africans living in Africa. Conclusion: This is the first study to demonstrate the important role of ethnic and environmental factors that may epigenetically influence the response to irradiation. It will be necessary to establish country-of-origen-specific dose response curves to practice precise and adequate biological dosimetry. This work opens new perspective for the comparison of treatments based on genotoxic agents, such as irradiation.

Transmission of Induced Chromosomal Aberrations through Successive Mitotic Divisions in Human Lymphocytes after In Vitro and In Vivo Radiation.

The mechanisms behind the transmission of chromosomal aberrations (CA) remain unclear, despite a large body of work and major technological advances in chromosome identification. We reevaluated the transmission of CA to second- and third-division cells by telomere and centromere (TC) staining followed by M-FISH. We scored CA in lymphocytes of healthy donors after in vitro irradiation and those of cancer patients treated by radiation therapy more than 12 years before. Our data demonstrate, for the first time, that dicentric chromosomes (DCs) decreased by approximately 50% per division. DCs with two centromeres in close proximity were more efficiently transmitted, representing 70% of persistent DCs in ≥M3 cells. Only 1/3 of acentric chromosomes (ACs), ACs with four telomeres, and interstitial ACs, were paired in M2 cells and associated with specific DCs configurations. In lymphocytes of cancer patients, 82% of detected DCs were characterized by these specific configurations. Our findings demonstrate the high stability of DCs with two centromeres in close proximity during cell division. The frequency of telomere deletion increased during cell cycle progression playing an important role in chromosomal instability. These findings could be exploited in the follow-up of exposed populations.

Detection and automated scoring of dicentric chromosomes in nonstimulated lymphocyte prematurely condensed chromosomes after telomere and centromere staining.

PURPOSE: To combine telomere and centromere (TC) staining of premature chromosome condensation (PCC) fusions to identify dicentrics, centric rings, and acentric chromosomes, making possible the realization of a dose-response curve and automation of the process. METHODS AND MATERIALS: Blood samples from healthy donors were exposed to (60)Co irradiation at varying doses up to 8 Gy, followed by a repair period of 8 hours. Premature chromosome condensation fusions were carried out, and TC staining using peptide nucleic acid probes was performed. Chromosomal aberration (CA) scoring was carried out manually and automatically using PCC-TCScore software, developed in our laboratory. RESULTS: We successfully optimized the hybridization conditions and image capture parameters, to increase the sensitivity and effectiveness of CA scoring. Dicentrics, centric rings, and acentric chromosomes were rapidly and accurately detected, leading to a linear-quadratic dose-response curve by manual scoring at up to 8 Gy. Using PCC-TCScore software for automatic scoring, we were able to detect 95% of dicentrics and centric rings. CONCLUSION: The introduction of TC staining to the PCC fusion technique has made possible the rapid scoring of unstable CAs, including dicentrics, with a level of accuracy and ease not previously possible. This new approach can be used for biological dosimetry in radiation emergency medicine, where the rapid and accurate detection of dicentrics is a high priority using automated scoring. Because there is no culture time, this new approach can also be used for the follow-up of patients treated by genotoxic therapy, creating the possibility to perform the estimation of induced chromosomal aberrations immediately after the blood draw.