The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB)

Purpose: Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. Materials and methods: Whole blood and separated lymphocyte samples were homogenously irradiated with 60Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. Results: Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. Conclusions: This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories

A functional analysis to differentiate pathogenic from benign variants identified in clinical diagnostic panels for breast cancer

Genetic testing for susceptibility genes through next‑generation sequencing (NGS) has become a widely used technique. Using this, a number of genetic variants have been identified, several of which are variants of unknown significance (VUS). These VUS can either be pathogenic or benign. However, since their biological effect remains unclear, functional assays are required to classify their functional nature. As the use of NGS becomes more mainstream as a diagnostic tool in clinical practice, the number of VUS is expected to increase. This necessitates their biological and functional classification. In the present study, a VUS was identified in the BRCA1 gene (NM_007294.3:c.1067A>G) in two women at risk for breast cancer, for which no functional data has been reported. Therefore, peripheral lymphocytes were isolated from the two women and also from two women without the VUS. DNA from all samples were sequenced by NGS of a breast cancer clinical panel. Since the BRCA1 gene is involved in DNA repair and apoptosis, the functional assays chromosomal aberrations, cytokinesis‑blocked micronucleus, comet, γH2AX, caspase and TUNEL assays were then conducted on these lymphocytes after a genotoxic challenge by ionizing radiation or doxorubicin to assess the functional role of this VUS. The micronucleus and TUNEL assays revealed a lower degree of DNA induced‑damage in the VUS group compared with those without the VUS. The other assays showed no significant differences between the groups. These results suggested that this BRCA1 VUS is likely benign, since the VUS carriers were apparently protected from deleterious chromosomal rearrangements, subsequent genomic instability and activation of apoptosis.publishersversionpublishe

RENEB accident simulation exercise

Purpose: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. Materials and methods: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. Results: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). Conclusions: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested

RENEB intercomparisons applying the conventional Dicentric Chromosome Assay (DCA)

Purpose: Two quality controlled inter-laboratory exercises were organized within the EU project ‘Realizing the European Network of Biodosimetry (RENEB)’ to further optimize the dicentric chromosome assay (DCA) and to identify needs for training and harmonization activities within the RENEB network. Materials and methods: The general study design included blood shipment, sample processing, analysis of chromosome aberrations and radiation dose assessment. After manual scoring of dicentric chromosomes in different cell numbers dose estimations and corresponding 95% confidence intervals were submitted by the participants. Results: The shipment of blood samples to the partners in the European Community (EU) were performed successfully. Outside the EU unacceptable delays occurred. The results of the dose estimation demonstrate a very successful classification of the blood samples in medically relevant groups. In comparison to the 1st exercise the 2nd intercomparison showed an improvement in the accuracy of dose estimations especially for the high dose point. Conclusions: In case of a large-scale radiological incident, the pooling of ressources by networks can enhance the rapid classification of individuals in medically relevant treatment groups based on the DCA. The performance of the RENEB network as a whole has clearly benefited from harmonization processes and specific training activities for the network partners

Integration of new biological and physical retrospective dosimetry methods into EU emergency response plans : joint RENEB and EURADOS inter-laboratory comparisons

Purpose: RENEB, 'Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,' is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation. Materials and methods: The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation induced thermoluminescent signals in glass screens taken from mobile phones. Results: In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques. Conclusions: Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios

Carcinoma da tiróide: caracterização genética e citogenética de uma população de doentes

Tese de doutoramento em Biologia (Genética), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2008As causas do cancro da tiróide não são totalmente conhecidas, exceptuando-se a exposição à radiação ionizante. Este trabalho incidiu sobre o estudo dos carcinomas papilar (CP) e folicular da tiróide (CF) sob duas perspectivas complementares, caracterizando-se genética e citogeneticamente doentes com estas patologias, por comparação com uma população controlo, e avaliando-se o efeito da terapêutica com iodo-131 em termos dos seus efeitos biológicos. A susceptibilidade genética associada ao carcinoma da tiróide foi estudada através da análise de polimorfismos de genes envolvidos na biotransformação (GSTT1, GSTM1, GSTP1) e na reparação de lesões do DNA (ERCC2). A combinação genotípica GSTM1*0, GSTT1*0, GSTP1 Ile/Ile revelou um aumento de risco para CP, mas não para CF da tiróide. Relativamente ao gene ERCC2, e para os polimorfismos Asp312Asn e Lys751Gln, foi evidenciado que os indivíduos simultaneamente homozigóticos para ambas as variantes do gene apresentam um maior risco de desenvolver CP da tiróide. O estudo de âmbito citogenético sobre a possível instabilidade cromossómica presente nestes doentes (aberrações cromossómicas e micronúcleos), efectuado por comparação com um grupo controlo, não revelou diferenças significativas entre ambos os grupos. Os doentes com CP e CF da tiróide são tratados com iodo-131 após tiroidectomia total, uma terapêutica com grande sucesso clínico. É, no entanto, necessário avaliar potenciais efeitos deletérios induzidos por esta radiação ionizante. Foi avaliada, nos linfócitos do sangue periférico destes doentes, a indução de aberrações cromossómicas e micronúcleos em diferentes períodos de tempo (1, 6 e 24 meses) após terapêutica com iodo-131 (2590 MBq). Observou-se que estes parâmetros encontram-se ligeiramente aumentados de um modo persistente e significativo até aos 24 meses. Não foram observadas alterações relevantes em parâmetros relacionados com o stresse oxidativo. Dado que as doses corporais totais de exposição dos doentes tratados com iodo-131 são relativamente baixas, avaliou-se a existência de uma eventual resposta adaptativa (RA), utilizando o ensaio do micronúcleo. Os resultados sugerem a existência de uma RA de carácter transitório, observada apenas um mês após a terapêutica face à genotoxicidade induzida in vitro pela mitomicina C. No seu conjunto, este trabalho realça a importância do papel de determinados polimorfismos na etiologia dos carcinomas papilar e folicular da tiróide e a necessidade de uma monitorização biológica dos doentes para que se compreenda melhor os efeitos induzidos pela terapêutica com iodo-131.The causes of thyroid cancer are not known except for exposure to ionizing radiation. This work focused on the study of papillary (PC) and follicular (FC) thyroid carcinoma from two complementary perspectives, aiming to characterize genetic and cytogenetically patients with these diseases, by comparison with a control population, and evaluating the biological effects resulting from treatment with iodine-131. The individual genetic susceptibility associated with thyroid carcinoma was investigated by assessing the polymorphisms of genes involved in biotransformation (GSTT1, GSTM1,GSTP1) and genes involved in the repair of DNA damage (ERCC2). The combined genotypes GSTM1*0, GSTT1*0, GSTP1 Ile/Ile result in a significant increased risk for PC, but not for FC. For the ERCC2 gene namely for polymorphisms Asp312Asn andLys751Gln, we observed that individuals homozygous for both genotype variants have greater risk for developing PC. The cytogenetic study on the possible chromosomal instability present in these patients (chromosomal aberrations and micronuclei), by comparison to a control group, showed no significant differences between both groups. Patients with thyroid PC and FC are treated with iodine-131 after total thyroidectomy, a therapy with great clinical success. In order to evaluate the potential deleterious effects induced by this ionizing radiation we studied, in lymphocytes from peripheral blood of those patients, the induction of chromosomal aberrations and micronuclei in different periods of time (1, 6 and 24 months) after iodine-131 therapy (2590 MBq). We observed that these parameters were slightly increased, in a persistent and significant way up to 24 months. No relevant changes in the parameters related to oxidative stress were found. Since whole body doses in thyroid cancer patients treated with iodine-131 are relatively low, we have evaluated the possible induction of an adaptive response, using the micronucleus test. Our results suggest the existence of a transient adaptive response, observed only one month after iodine-131 therapy, against the genotoxicity induced in vitro by mitomycin C. This work highlights the importance of the evaluation of genetic polymorphisms in the aetiology of papillary and follicular thyroid carcinoma and the need for a biological monitoring of the patients in order to better understand the effects induced by treatment with iodine-131

Evalution of a challenge assay as na effect biomarker in environmental or occupational biomonitoring studies

Background: Human long-term exposure to environmental or occupational stressors may produce adverse effects, e.g., genotoxic effects that underlie the onset of diseases, among which cancer is the most severe. Besides the instability that a wide variety of genotoxic and carcinogenic agents directly exert on the cell genome, also indirect effects, e.g., interference with the DNA repair capacity deserve to be studied. Such effects have been described for heavy metals, low doses of ionising radiation or complex mixtures, among others. Aim: This work aimed to evaluate the usefulness of the ex vivo challenge assay as a functional biomarker of early biological effects, based on data previously obtained in molecular epidemiology studies. Methods: The first study (S1) comprised a group of individuals with environmental exposure to ionising radiation and heavy metals (IR Group) and a non-exposed group (NIR Group), whereas the second study (S2) included individuals occupationally exposed to environmental tobacco smoke (ETS Group) and the respective control group (NETS Group). In S1, whole blood samples were subjected to 2Gy of ionising radiation before culture set up for translocation analysis through FISH. Blood samples from S2 participants were exposed to an alkylating agent (EMS) and then processed for the comet assay; the level of DNA damage was quantified by the percentage of DNA in tail. The level of chromosome or DNA damage measured after the ex vivo challenge with the genotoxic agents was compared to the basal level obtained for the same individuals in S1 and S2, respectively. Results: The results from both studies showed that peripheral blood lymphocytes (PBLs) exposure to a medium/high dose of a genotoxicant (ionising radiation or EMS), followed by an assessment of chromosome or DNA damage, respectively, allowed to distinguish exposed and control groups. The distinct response consisted of a lower frequency of ionising radiation-induced translocations in the IR comparatively to the NIR group (S1) or a lower level of EMS-induced DNA breaks in the ETS comparatively to the NETS Group (S2). Discussion and Conclusions: The results from both studies agreed in that the use of an ex vivo challenge of PBLs with a genotoxicant allowed the detection of a differential response between the exposed and non-exposed groups. Interestingly, PBLs from exposed individuals were less affected by the ex vivo exposure to a genotoxicant suggesting an adaptive response instead of a reduced DNA repair capacity. These findings are expected to contribute to the development of new biomarkers of early biological effects for human biomonitoring studies.Portuguese Ministry of Health, Calouste Gulbenkian Foundation and Foundation for Science and Technology - ToxOmics (UID/BIM/00009/2013)N/

Exposição Humana a radiação Ionizante: Efeitos Biológicos e Impacto na Saúde

O homem encontra-se exposto a radiação ionizante de origem natural (raios cósmicos e radiação emanada dos solos) mas, alguns grupos populacionais, poderão ainda estar sujeitos a uma exposição em contexto ocupacional (e.g., médicos e trabalhadores de minas de urânio) ou ambiental (e.g., residentes em áreas de influência de minas de urânio). Estão bem documentados os efeitos nefastos da exposição a radiação ionizante na saúde humana incluindo, a longo prazo, um aumento da incidência de várias formas de cancro. Esses processos cancerígenos relacionam-se com a capacidade deste agente físico interagir com a molécula de DNA, levando à indução de mutações génicas e de quebras cromossómicas e, envolvendo também, alterações da expressão ou função de genes implicados na resposta celular ao stress. Através da dosimetria biológica, baseada na análise de anomalias cromossómicas estruturais em linfócitos, é possível quantificar a dose de radiação a que determinado indivíduo foi exposto e detetar precocemente efeitos biológicos, que precedem o aparecimento de sinais de doença. Serão apresentados os resultados mais relevantes dos trabalhos que têm vindo a ser desenvolvidos no Departamento de Genética Humana do INSA no âmbito da investigação dos efeitos genotóxicos da radiação ionizante. Esses trabalhos abrangem quer estudos laboratoriais in vitro e in vivo, quer um estudo de base populacional que pretendeu avaliar o impacto na saúde da exposição ambiental a resíduos radioativos de uma antiga exploração de urânio. Este último estudo revestiu-se de particular interesse pois, conjuntamente com os resultados obtidos noutras componentes relacionadas com o estado de saúde dos indivíduos e com a caracterização da radioatividade ambiental, veio contribuir para a implementação de um processo de remediação ambiental visando proteger a saúde humana e o ambiente