Cancer is the second leading cause of death worldwide and is an important public health problem. According to the World Health Organization (WHO), in the coming decades, the impact of cancer in the population will be 80% of the more than 20 million new cases estimated for 2025. In Brazil, in the biennium 2016-2017, about 600,000 new cases of cancer were estimated (INCA, 2015). Among the risk factors, nowadays, radiation and its impacts on human health is a major concern to the etiology of cancer (Samet, 2011). On this occasion, extensive researches are needed in order to diagnose the problems to avoid or at least minimize the deleterious effects of ionizing radiation (IR) (Pernot et al., 2012).

Notably, ionizing radiation (IR), even at low dose may be harmful and can cause dangerous events in biological systems (Kadhim et al., 2013). An increased dose is always harmful. Sometimes, a particular radiation may act by different mechanistic pathways (Yang et al., 2012). Low doses (0.05 to 0.5 Gy) of IR can cause genomic instability, such as chromosomal alterations and cell death (Kadhim et al., 2013), and can lead to a double failure of DNA and epigenetic alterations in histones (Sasakil et al., 2014). Studies with mass spectrometry based on proteome analysis in human skin model, indicated that after 48 h exposure to 3, 10, and 200 cGy x-rays alter and disrupt 135 proteins, where carboxipeptidases and ubiquitin carboxyl terminus isoenzyme hydrolase were the most sensitive, indicating that radiations at any dose can alter enzymatic proteins (Zhang et al., 2014).

IR have a potential for induced genetic instability in germ and somatic cells, characterized by the production of chromosomal groupings, aneuploidy, micronuclei (MN), gene amplification, mutations in derived and other cells having effects of radiation (Camats et al., 2008). Such changes can be highlighted and monitored by the use of any tests, among them the micronucleus (MN) test is one. It is an easy, economical and reproducible test procedure (Maluf and Riegel, 2011). The MN are formed from the chromosomal fragments or entire chromosomes that are not incorporated into the nucleus of the daughter cell during cell division, they are corpuscles containing DNA without structural connection to the core. The presence of MN and nuclear abnormalities can be considered as a preliminary indication for assessing the mutagenic and/or carcinogenic agents, such as ionizing radiation (Sari-Minodier et al., 2002).

To contribute to the health and prevention of cancer in health professionals occupationally exposed to IR, the present study evaluated the toxicogenetic risks of exposure to the IR using hematological and biochemical parameters in peripheral blood; and the application of cytogenetic MN test in the evaluation of the frequency of MN, nuclear-type abnormalities: binucleate cells, buds, bridges nucleo-plasmatics and apoptosis stages (karyorrhexis and karyolysis) in oral epithelial cells. Secondly, hematological parameters and toxicogenetic biomarkers were correlated to the lifestyle (vegetable consumption, smoking, alcohol consumption, protection of use, age, place of work and working time), age and gender of the participants. 

Ethics and legal aspects

All studies were performed in accordance with Brazilian research guidelines (Law 466/2012, National Council of Health, Brazil) and with the Declaration of Helsinki and all procedures were approved by the Ethics Committee on Human Research, based at the Lutheran University of Brazil  (ULBRA, Rio Grande do Sul - CAAE: 38570914.8.0000.5349).

Research subjects

This study was conducted with 45 professionals occupationally exposed to  IR including radiology technicians, technologists in radiology and radiologists from  two clinics: Diagnostic Unit Imaging (UDI 24 hours, Teresina, Brazil) Lucidio Portela Hospital (Teresina, Brazil) and 45 professionals who do not work in sectors related to diagnostic radiology. All participants answered the health questionnaire recommended by the International Commission for Protection against Environmental Mutagens and Carcinogens (ICPEMC) (Carrano and Natarajan, 1998) in order to select the unexposed group as well as for obtaining data on health and lifestyle of the participants to correlate with toxicogenetic parameters.

Haematological and biochemical parameters analysis

Blood samples were collected for hematological and biochemical tests to quantify liver and kidney enzymes such as AST (glutamic-oxalacetic transaminase) and ALT (glutamic pyruvic transaminase), creatinine, urea and alkaline phosphatase (ALP). The tests were performed according to the protocol established by LabtestTM.

Micronucleus (MN) test in oral epithelial cells

Buccal cell samples were obtained by gently rubbing the inside of the cheeks (right and left side) with a cytobrush, which was immersed in 5 ml cold saline (0.9% (w/v) aqueous NaCl) in a conical tube and transported under refrigeration to the Laboratory of Genetic Toxicology, Federal University Piauí (Teresina, Brazil). The samples in saline were centrifuged at 1500 rpm for 10 min and the sedimented cells were then washed with saline (twice) and fixative solution (methanol and glacial acetic acid 3:1) (once) under the same centrifugation conditions. The cell suspension was spread onto a slide with fixative solution and dried at room temperature. The slides were stained with 2% Giemsa solution for 10 min, rinsed in distilled water, and air-dried. Biomarkers of DNA damage (MN), cytokinetic defects (binucleated cells) and cell death (karyorrhexis, pyknotic and karyolytic cells) were scored according to the criteria set by Thomas et al. (2009). For each volunteer, 2000 buccal cells (1000 from each of the duplicate slides) were scored using bright-field optical microscopy at a magnification of 1000. MN were identified taking into consideration in the following conditions: cells with intact main nuclei and cytoplasm; diameter of one-third of the main nucleus; same staining and texture as the main nucleus, and MN was in the same focal plane as the main nucleus. Other anomalies in cells were measured as binucleated, karyolysis, karyorrhexis, and pyknotic cells.

Statistical analysis

Personal characteristics and lifestyle of the groups obtained from the personal health questionnaire were statistically compared by t-student's test using the statistical program SPSS 16.0. The relationship between lifestyle characteristics and cytogenetic data were performed by correlation of Spearman's rho. Cellular data on the frequency of micronuclei and nuclear abnormalities between the groups were assessed by the multiple t-test with significance in Holm-Sidak method, using the statistical program Graphpad Prism 5.0 (Graphpad Inc., San Diego, CA). Values of P < 0.05 were considered statistically significant.

Evaluation of haematological an biochemical parameters in health professionals occupational exposure to IR

Hematological (red and,white series and platelets) and biochemical (AST, ALT, creatinine, urea and ALP) were seen within the normal reference standards. Then, no signs of hematological changes and liver and kidney toxicity were detected (Table 2).

Genetic instabilities induced by occupational exposure to IR in oral epithelium of health professionals

The risk assessment for occupational exposure to hazards has social importance and for worker health (Montano, 2014). Epidemiological studies show an association between IR and cancer, like brain cancers (Smoll et al., 2016). High and low doses of IR induced circulatory diseases are indicative of risks following occupational exposure to mortality risk, as well as with the cancer-inducing ability (Little et al., 2012). The MN test is well founded to establish the risk of DNA damage and studies show a strong association between IR, increased frequency of MN and susceptibility to cancer (Bolognesi et al., 2014).

Workers exposed occupationally at IR in Radiological Diagnostics Clinics in Teresina- -Piauí, showed genetic instability in oral epithelium cells (P <0.001) since it occurred increase in MN frequencies, nuclear bud (NB), binucleate cells (BN) and nucleoplasmatic bridges (NP), when compared individuals not exposed to IR (Figure 1). Previously cytogenetic studies reported impacts in mammalian cells exposed to 0.5-3 Gy with presence of DNA damage confirmed by chromosomal aberrations and MN, which are coming from breaks and loss of chromosomes, suggesting a genetic instability (Plamadeala et al., 2015).

Oral epithelium is important tool for toxicological studies, especially due to its rapid division. MN and other nuclear abnormalities such as sprouts and bridges are considered biomarkers for genotoxic damage and chromosomal instability. MN are fragments of chromosomes or loss of chromosomes during anaphase of cell division due to the poor chromosome segregation as a result of hipometilations in repeated sequences of the centromeric regions (Fenech et al., 2011).

MN, extra nuclear bodies, may be observed in oral exfoliated mucosa cells, as a biomarker for genotoxicity and carcinogenicity. The formation mechanism of NBs may be related to the elimination and amplification or DNA repair. The BNs may be indicative of cytokinesis failure at the end of cell division (Sabharwal et al., 2015). The MN formation may be due to the breakage, indicating clastogenic and chromosomes aneugenic agents (Suzuki et al., 2003; Fenech et al., 2011).

Nuclear changes induced by occupational exposure to IR in oral epithelium of health professionals

Exposure to occupational and environmental radiation sources can cause damage and genetic instability due to its effects as carcinogens. Epidemiological studies show an association between IR at low doses (1 to 5 mGy) and development of leukemia. Leukemia is a complex disease that may have etiologies related to the lifestyle and occupational and environmental exposure, but there are also associations between leukemia (Polychronakis et al., 2013) due to genetic instability related to increased single and double strand DNA breaks that can lead to the chromosomal alterations and formation of MN (Saha et al., 2014), as noted in mutagenicity analysis of professionals occupationally exposed to IR in this study. There are also reports that IR can lead to apoptosis, as observed in embryonic neuronscells (Leuraud et al., 2015).

Nuclear abnormalities such as BNs and apoptosis, karyorrhexis (nuclear fragmentation), and karyolysis (nuclear dissolution) are indicative of cytotoxicity were observed in health professionals exposed to IR. Data were statistically significant (P <0.05) when compared with the control group (Figure 2). BNs may be indicative of cytokinesis failure at the end of cell division and cells with condensed chromatin are indicative of apoptosis (karyorrhexis and karyolysis), as well as picnoses (Sabharwal et al., 2015). Exposure to IR can lead to oxidative events, one of the mechanisms of cytotoxicity and apoptosis, due to the fact that these have direct action on the macromolecules via radiolysis product and intracellular modulation of the communication mechanisms of redox system, which induce stress in cells, tissues and mitochondria, led to the toxic effects of radiation depending on the doses (Einor et al., 2016).

Numerous effects of radiation have been identified in association with mutations and genomic instability (breaks and actions in repair genes), angiogenesis (vascularisation and hypoxia), apoptosis (changes in p53), proliferative signaling (EGCR and TGF-α), suppression of  cell cycle (ATM, p53 lock), energy dysregulation (HIF, c-MyC, glycolytic pathways inhibitors), tumor promotion and inflammation (p53 and ROS) and activation and inactivation of metastases (hypoxia, lactate) (Boss et al., 2014). Given the risks of cancer, studies suggest biological dosimeters guided by genetic instability mechanisms, is noted in summary in Table 3, especially for chromo-somal aberrations that may be markers for evaluation of dose and biological effects induced by IR (Higueras et al., 2015). Numerous effects of radiation (Table 3) have been identified in association with mutations and genomic instability (breaks and actions in repair genes), angiogenesis (vascularization and hypoxia), apoptosis (changes in p53), proliferative signaling (EGCR and TGF-α), suppression of cell cycle (ATM, locking p53), energy deregulation (HIF, c-MyC, glycolytic pathways inhibitors), tumor promotion and inflammation (p53 and ROS) and activation and inactivation of metastases (hypoxia, lactate) (Boss et al., 2014; Lee et al., 2014). Relation among age, gender, DNA damage and cytotoxicity. 

Populations occupationally exposed to chemical and physical genotoxic agents are being carried out with the application of toxicogenetic tests. However, the understanding of the influence of factors related is necessary to age, gender and lifestyle and disease in relation to MN rates (Bonassi et al., 2011), as well as other nuclear abnormalities (Holland et al., 2008).

To evaluate the influence of age for MN induction, cells with karyorrhexis (indicative of apoptosis) and the mean ages were compared with those toxicogenetic biomarkers. The mean of MN of different age intervals of exposed workers was significantly higher compared to the unexposed group. Workers exposed that age range of 18 to 27 years had more production of the MN when compared to other age ranges (Figure 3A). For karyorrhexis, the data were significant only for the group unexposed to X-rays (Figure 3B).

MN test in relation to the age of the exposed workers showed that in all ages, the exposed workers are more susceptible to IR than the unexposed group. These data are in agreement with Maluf and Erdtmann (2001), who evaluated the presence of cellular with Maluf and Erdtmann (2001), who evaluated abnormalities in workers exposed to radiation than the consumption of oxide ethylene and cytostatic agents. In comparison to the age range, the age of professionals between 18 and 27 years showed more significant levels than the other groups with respect to the mutagenicity, and the range of 38 to 45 showed more significant indices with respect to apoptotic changes. These data differ from the data presented by Ladeira et al. (2011), where the evaluation of abnormalities found in the MN test showed no statistical difference in age and sex. However, Jha and Sharma (1991)  revelaed an apoptosis and nuclear abnormalities in workers age between 35 to 50 years exposured to IR.

Regarding gender, it was observed that only karyorrhexis level is statistically significant with a greater predisposition to nuclear fragmentation in males. On the other hand, Fenech and Bonassi (2011) showed that the changes in MN test are always higher in women than the men in all age groups. This occurs due to the hormonal cycle in women and especially the low intake of some micronutrients such as vitamins, folic acid and ions. Although, there were no statistically significant differences (P >0.05) for cytogenetic biomarkers tested in relation to gender differences in groups exposed to IR in the unexposed group. In this study, we observed differences only karyorrhexis when assessed alone (P <0.05, Figure 4).

Correlation between lifestyle factors with cytogenetic biomarkers

Genomic damage has great important in the etiological analysis for the development of degenerative diseases. Radiation and the use of chemicals are needed in different therapies, but also micronutrient deficiencies, lifestyle and other genetic factors are needed in biomonitoring, diagnosis and treatment of diseases in the evaluation of genetic damage (Tolbert et al., 1992).

Herein, after statistical Spearman's Herein, after statistical, positive correlations were observed between smoking and alcohol consumption with MN, age bridges and karyolysis and working time with karyolysis and binucleate cells in oral epithelium (Table 4).

Cytogenetic damage shown in this study did not show positive correlations with age, smoking, alcohol consumption, and genetic diseases, with exposure to IR. Hartmann et al. (1998) also demonstrated an insignificant relationship between smokers/nonsmokers and DNA damage. However, epidemiological studies show that smoking induces DNA damage, and may cause lung cancer (Sram, 1998). To be noted that age-related increased in the risk of aneuploidy and non-disjunction mitotic, changes in chromosomes with a reduced DNA repair are suggested by Migliore et al. (1991), despite of a controversial talk by Betti et al. (1994). These different responses can be related to the size of the samples, as well as varied and individual susceptibility of the population under study.

Occupational exposure of ionizing radiation to the workers caused genetic instability in a independent way of age and gender, induce micronuclei, bridges, buds and binucleate cells. However, an unchanged haematological parameters and hepatic as well as nephritic function were observed. A correlation with risk factors related to lifestyle (smoking and drinking) and IR was established. Our study demonstrates biomonitoring of genetic risks, applying mutagenicity biomarkers and nuclear abnormalities can be alternative tools for the diagnosis and prevention radiation-induced health hazards.

The authors have not declared any conflict of interests.