Cytogenetic status of healthy children assessed with the alkaline comet assay and the cytokinesis-block micronucleus cytome assay

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Abstract

In the present study the alkaline comet assay and the cytokinesis-block micronucleus cytome (CBMN Cyt) assay were used to evaluate the baseline frequency of cytogenetic damage in peripheral blood lymphocytes (PBLs) of 50 healthy children from the general population in Croatia (age, 11.62 ± 1.81 years). Mean values of tail length, tail intensity and tail moment, as comet assay parameters, were 12.92 ± 0.10, 0.73 ± 0.06 and 0.08 ± 0.01, respectively. The mean frequency of micronuclei (MN) for all subjects was 2.32 ± 0.28 per 1000 bi-nucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 1.72 ± 0.24 and of nuclear buds (NBUDs) 1.44 ± 0.19. The mean nuclear division index (NDI) was 1.70 ± 0.05. When comet-assay parameters were considered, higher mean values for all three were found for the female population. According to the Mann–Whitney U test applied on the results of the comet assay, the only statistically significant difference between the male and female populations was found for tail length. Similar to the results obtained by the comet assay, girls showed higher mean values of all three measured parameters of the CBMN Cyt assay. This difference was statistically significant for total number of NPBs only. In the case of the NDI, a higher mean value was also obtained in girls, but this difference was not statistically significant. The results obtained present background data that could be considered as normal values for healthy children living in urban areas, and can later on serve as baseline values for further toxicological monitoring. Additionally, the usefulness of both techniques in measuring cytogenetic damage during bio-monitoring of children is confirmed.

Highlights

► Baseline frequency of cytogenetic damage in healthy children was assessed. ► This was done with the alkaline comet and the cytokinesis-block micronucleus assay. ► Results present background data that could be considered as normal values. ► Usefulness of both techniques in child biomonitoring is confirmed.

Introduction

Human bio-monitoring is an essential tool for providing a detection system for a variety of health hazards. Cytogenetic biomarkers provide useful information regarding the DNA-damage status of the population under study. One of the first reliable methods for assessment of the cytogenetic status of populations was the analysis of sister chromatid exchange in the 1960s. Over next decades, several other methods have been developed in order to improve the sensitivity of detecting biomarkers of genotoxicity. In that context, the use of methods like chromosomal aberration analysis and the micronucleus assay became more popular [1], [2], [3], [4]. Recently, the evolution of methods in human bio-monitoring can be traced as we witness the changes in approach and methods used, for instance the introduction of gels and electric current in the comet assay, or specific probes in fluorescence in situ hybridization, and also the automatization of methods to reduce the time needed to analyze results [5], [6], [7], [8].

The number of studies evaluating the effects of environmental exposure in children has rapidly grown in the last years [9], [10], [11], [12]. The reason is that children are more sensitive than adults to the effects of the environmental exposure and medical treatment, and genome damage that occurs at young age may influence the lifetime risk for delayed adverse health effects [13], [14]. This risk is determined by a child's anatomical features and by the longer life expectancy during which it can express that risk [10], [15], [16], [17]. Genetic susceptibility and environmental exposures during vulnerable periods of development are also important contributors to the etiology of many childhood diseases [18], [19]. Among the adverse health effects that could be studied in children exposed to various environmental hazards, cytogenetic damage received special attention after it had been shown that a high frequency of DNA/chromosome damage is predictive of cancer in healthy adults [20], [21].

Since limited information is available about the baseline cytogenetic damage-frequency in children especially with regards to their age, sex and health status, this study presents the results obtained by use of the alkaline comet assay and the cytokinesis-block micronucleus cytome (CBMN Cyt) assay on peripheral blood lymphocytes (PBLs) collected from healthy children selected from the general Croatian population. The aim of the present study was to evaluate the baseline cytogenetic damage in PBLs of healthy children and to investigate if there is a relation with age and gender by measuring the tail length, tail intensity and tail moment as comet assay parameters, in addition to measuring the frequency of micronuclei (MN), nucleoplasmic bridges (NPBs), nuclear buds (NBUDs) and the nuclear division index (NDI). These baseline records may contribute to future genotoxicological monitoring.

Section snippets

Chemicals and media

Chromosome kit P was from Euroclone, Milano, Italy; cytochalasin B, ethidium bromide, low melting-point (LMP) and normal melting-point (NMP) agarose were from Sigma, St. Louis, MO, USA; heparinised vacutainer tubes were from Becton Dickinson, Franklin Lakes, NJ, USA; Giemsa dye was from Merck, Darmstadt, Germany. All other reagents used were laboratory-grade chemicals from Kemika, Zagreb, Croatia.

Population characteristics

The present study comprised a group of 50 healthy children (25 girls and 25 boys), between the ages

Results

This paper presents the results of the alkaline comet assay and the CBMN Cyt assay parameters on PBLs obtained form healthy children in the general Croatian population. Using these two methods we evaluated parameters for measuring baseline frequency of cytogenetic damage.

Discussion

There is increasing interest in the bio-monitoring of children, who are considered as a specific subgroup in public health which could be more sensitive than the average adult [12], [18]. The main reasons why children are studied is the fact that they are still in the phase of active development, which may have influence on a different response to environmental hazards than adults [10], [13]. Although children could be more sensitive, there is another important fact that should be highlighted.

Conclusions

The present study is the first to report the results of assessment of the baseline frequency of cytogenetic damage in PBLs of a healthy child population in Croatia. In addition to an automated image-analysis system that was used to score the comets, scoring of the CBMN Cyt assay parameters was also performed according to the criteria proposed by HUMN project [4], [37]. The proposed baseline values obtained in the present study mainly refer to the children in the age group from 11 to 13 years

Conflict of interests

The authors declare that there have been no conflicts of interests in this research.

Acknowledgements

Authors would like to thank all the volunteers that participated in this study. This work was supported by the Croatian Ministry of Science, Education and Sports (Grant no. 022-0222148-2125).

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