Exposure to radiation and genetic damage Industrial hygiene
This research project aims to ascertain whether chronic exposure to X rays during clinical diagnoses carried out by dentistry students as part of their academic training might produce any genetic damage. To this end two biotests were carried out: firstly, the micronucleus assay on cells of the buccal mucous to evaluate both clastogenic and aneugenic effects and other abnormalities of the nucleus that might be used as markers of cell damage; and secondly the single cell gel electrophoresis test or comet assay in peripheral blood samples to determine any damage to the single or double chain of the DNA. Two cohorts of dentistry students were analysed, one exposed group and another control group. The exposed cohort involved final-year students who were taking patient X rays during a chronic 3-year exposure period. The control cohort involved first-year students who were not X ray exposed. Results show a significant increase in micronuclei (p< 0.05) and of cells in apoptosis and other abnormalities of the nucleus in chronically radiation-exposed students but there were no differences in comet-assay DNA damage readings as compared with the control group. The conclusion that can be drawn from these results is that the dose received and the X ray exposure time have a cytotoxic effect on cells of the buccal mucous and produce minimum DNA damage in peripheral blood cells.
By D. FRANCO DE DIANA. Biology graduate. Director of the Basic Sciences Department of the Universidad Católica «Nuestra Señora de la Asunción» (email@example.com).
J. SEGOVIA. graduate. Senior research fellow of the Universidad Católica «Nuestra Señora de la Asunción»
D. CASTIGLIONI. Biology graduate. Senior research fellow of the Universidad Católica «Nuestra Señora de la Asunción».
C. VEGA. Doctor in microbiology. Biology graduate. Senior research fellow of the Universidad Católica «Nuestra Señora de la Asunción». Director of the Centro para el Desarrollo de las Ciencias (CEDIC).
N. LÓPEZ ACOSTA. Statistical mathematics graduate. Research fellow of the Facultad de Ciencias Exactas y Naturales of the Universidad Nacional de Asunción.
A. GÓMEZ. Master in Biological Sciences from the Centro para el Desarrollo de las Ciencias (CEDIC).
The main effect of ionising radiation occurs in the DNA molecule and is tied up with the dose and exposure time. Any dose, however slight, implies some risk, so the exposure should be reduced to the minimum possible. Important factors to bear in mind here, however, are the chronic exposure time to which workers and persons in occupational training are exposed, such as healthcare degree students, and also the variation in radiosensitivity from one person to another, depending on age, gender and exposure time.
X ray exposure during panoramic radiographs in adult and child patients produces genotoxic effects in gingival cells, increasing chromosome damage and cell death. These panoramic dental X rays should therefore be sought only in very necessary cases, since this procedure cannot be considered as low risk or risk-free.
Tolbert et al (1992) put forward the use of the micronucleus assay to evaluate the genotoxic effect, whether caused by chromosomal breakage or abnormal mitotic cell divisions, and also to detect other atypical nuclear abnormalities such as pyknosis, karyorrhexis or karyolysis, which are indicators of cell damage.
Another useful test for detecting damage to the DNA chain is single cell gel electrophoresis or the comet assay, which assesses the damage to single and double DNA chains in cell populations without the need of cell proliferation studies. It is a potentially sensitive tool for showing up genotoxic damage induced by various toxins within their radius of action.
In Paraguay both teachers and students in the dentistry degree course are frequently exposed to radiation during image-based diagnoses. Furthermore the exposure is often without any type of protection because the radioprotection biosecurity habit is not strictly practised at this level, despite the existence of standards and legislation regulating this type of activity. Bearing this factor in mind we have carried out micronucleus assays on buccal smears and peripheral blood samples to gauge DNA damage by means of the comet assay. The smears and blood samples came from dentistry students exposed chronically to low X ray doses.
Material and Methods
The research was designed as a cross sectional analytical observational study on exposed and non-exposed cohorts, who were first asked to fill out a survey to find out their state of health, their smoking, drinking and drug-taking habits, exposure time and radioprotection methods while taking X rays.
Students of both sexes aged 24 to 29 carrying out clinical practices in their dentistry degree and who were exposed to ionising radiation while taking dental patient X rays for a period of at least 3 years.
Non exposed population
Dentistry students still in the basic part of the course who have not yet been taking dental radiographs and have therefore suffered no X-ray exposure.
Data collection instruments and techniques
The genotoxic effect was evaluated by means of two biotests: the micronucleus assay on buccal smears and the comet assay.
The buccal smears and peripheral bloods samples of each individual, both of the control and exposed population, were taken simultaneously from June to October.
Exfoliated buccal cell assay
An evaluation was made of whether chronic X ray exposure during the clinical practice of dentistry students causes genetic damage
A sample of buccal cells was obtained by scraping the inside of the cheek with a spatula. The smear was then spread on the microscope slide. The samples were fixed with a 3:1 mixture of methanol/acetic acid and coloured with the Feulgen technique. Microscope observation was carried out by using special filters to bring out the cytoplasm. Two thousand cells were counted per individual and a determination was made of the frequency of micronuclei in both populations, the control and exposed, according to Tolbert (1992) and Fenech (1999).
Comet assay to measure DNA damage
The comet assay was carried out according to Singh et al. (1988) with small modifications and optimising tweaks. Two μl of peripheral blood was taken from the ring finger of each participant. The samples were suspended in 0.5% of low melting point (LMP) agarose and pipetted onto a slide previously covered with a layer of normal melting point (NMP) agarose at 1% and kept at 4°C for 10 minutes; it was then submerged in a lysis buffer (2.5M Nacl, 100 μm Na2EDTA, 10 μm Tris- Hcl Ph 10,1, Triton – X – 100 and 10% DMSO) for one hour at 4°C in the dark to provoke lysis and DNA breakdown. The slides were then exposed to alkaline buffer (1 mM Na2EDTA, 300mM NAOH buffer) Ph >13 for 20 minutes to degrade the DNA.
For the purpose in hand two biotests were conducted: micronucleus assay of buccal smears and single cell gel electrophoresis assay or comet assay of peripheral blood samples
Each slide was submitted to electrophoresis for 20 minutes at 25 V and 300 m A in the same buffer and then rinsed in 0.4 Try-Hcl buffer (Ph 7.5) to eliminate excess alkali and remove detergents. The samples were fixed with alcohol p.a and the slides were finally tinged with ethidium bromide (10 μg/ml) before being examined under a 400 x fluorescence microscope.
100 cells per agarose plate were evaluated by visual check and by means of the software Casp Lab Comet Assay free.
Evaluation by visual check
This was carried out according to the criteria of Speit (1995)  and Collins (2004)  , wherein cells are classified on the assumption that gentoxic damage varies directly with the number of DNA breaks outside the nucleus. To quantify DNA damage a cell damage category was established on the basis of the comet length:
- Category 0: undamaged cells (5 μm).
- Category I: cells with low damage level (5-20 μm).
- Category II: cells with medium damage level (between 20 and 40 μm).
- Category III: cells with high damage level (between 40 and 95 μm).
- Category IV: maximally damaged cells (higher than 95 μm).
Once the percentage of cells in each category has been established, a calculation is then made of the damage index (N) for each sample of the individual:
N= number of cells in category I + 2 X number of cells in category II + 3 X number of cells in category III + 4 X number of cells in category IV
Evaluation by the software Casp Lab Comet Assay IV
Tail moment values were obtained for measuring the DNA percentage in the comet tail and the tail moment olive describing the heterogeneity of the response within a cell population, since it calculates the DNA distribution variations within the tail, measured in μm.
The SPSS statistical package was used for the exploratory analysis of micronucleus data in buccal smears for the control group (n=31) and the exposed group (n=30). The results were analysed statistically by means of the student’s T test, applying thereafter the non parametric Mann-Whitney U test to cross check the hypothesis.
The analysis of DNA damage with the comet assay was carried out by comparing Mann-Whitney U readings for two samples regardless of DNA damage data and tail moment and tail moment olive values.
Demographic variables of the studied populations
Control or non-exposed population
Dentistry students aged 18 to 20 taking the first year of their degree course. 84% of the population is female (25/31) and 16% (5/31) is male.
43.3% (13/30) of the population have some family cancer precedent, be it lung, prostate, uterus or, predominantly, breast cancer. 40% of the analysed population drinks on a casual basis.
The exposed population consists of 30 dentistry students currently taking the 6th year of their degree course. Their ages range from 23 to 29; 83% of this population is female (25/30) and 17% (5/30) is male. 56.7% of the studied population drinks moderately.
267% (8/30) of the population has some type of family cancer precedent, be it bone, prostate, breast or, predominantly, colon. All surveyed students report that they have been working for at least three years in the X ray room; none of the respondents uses any type of protection against the X rays.
Table 1 shows the most important demographic factors of the studied population.
|Demographic and clinical variables||Exposed population||Non-exposed population|
|Gender||80% (24/30) female
20% (6 /30) male
|84% (25/31) female
16% (5/31) male
|Age||23 – 29 años||17 – 20 años|
|A family cancer precedent||26,7%||43,3%|
|Time of X ray exposure for dental radiographs||3 years||0 exposure|
Evaluation of the genotoxic effect by means of the buccal smear micronucleus assay
Table 2 shows the micronuclei frequency found in the non-exposed population and some nuclear abnormalities. Table 3 shows the readings for micronuclei frequency and nuclear abnormalities in the exposed population. Table 4 compares the mean micronuclei frequencies in both populations: 1.68 ± 1.99 for the control population and 6.67 ± 5.2 for the exposed population and nuclear abnormalities. Application of the student’s T test and non parametric Mann-Whitney U test for cross checking of the hypothesis throws up the value of p= 0.006, where significant values are considered to be those below 0.05. The difference in the micronuclei frequency between both populations is therefore significant, ruling out the null hypothesis and confirming the alternative. Nuclear abnormalities like pyknosis, karyorrhexis and condensed chromatin are taken as parameters of genotoxic and cytotoxic evaluation.
|Studen number||Age||Sex||MN/2000 cel||Nuclear abnormalities|
|BN||Cariolisis||Cariorrexis||Broken eggs||Condensed chromatin||Picnosis|
|Studen number||Age||Sex||MN/2000 cel||Nuclear abnormalities|
|BN||Cariolisis||Cariorrexis||Broken eggs||Condensed chromatin||Picnosis|
|Non exposed population||Exposed population|
Figures 1 and 2 show the mean micronuclei frequency in the exposed and non exposed populations, respectively, broken down by age; the readings are higher in the exposed population.
The exposed population was made up by students in the final year of their degree course, who have been taking patient X rays for a chronic exposure period of three years. The control group is made up by students in the first years of their degree course, not exposed to X rays
Figure 3 compares the mean nuclear abnormality rate between the exposed and non-exposed population. This shows an increase in the mean figures of the exposed population, taking pyknosis, karyorrhexis and condensed chromatin to be characteristic indicators of cells in apoptosis, and binucleate cells and broken eggs to be characteristic of the genotoxic effect. Figures 4 and 5 show Feulgen-stained buccal mucous at 1000 x magnification, with micronucleus and without, respectively. Figures 6 and 7 show nuclear abnormalities such as binucleate cells and apoptotic cells with karyorrhexis.
Comet assay evaluation of the genotoxic effect
By visual check
The results show a significant increase in the frequency of micronuclei (p< 0.05) and cells in apoptosis and other abnormalities of the nucleus like karyorrhexis, condensed chromatin, broken eggs and pyknosis in students chronically exposed to X rays
Table 5 shows percentage readings with different levels of cell damage, according to the criterion of Speit (1995) and Collins (2004). This table shows a level I increase in the exposed population as compared with the non exposed population. The readings for levels II, III and IV, however, are not significant, according to the comparison of student’s T readings and the non parametric Mann Whitney U test for independent samples, throwing up a value of p =0.006 less than 0.05 for level I, and ipso facto considered to be statistically significant, and values higher than 0.05 in the other categories. Figure 11 shows the values for levels I, II, III and IV in both populations, with an obvious increase in level I damage in the exposed population. The damage index (N= number of cells in category I + 2 X number of cells in category II + 3 X number of cells in category III + 4 X number of cells in category IV) was 37.8 for the exposed population and 3.4 for the non-exposed population, with a significant difference (p< 0.05) according to the Mann Whitney U test. These values show that the cell damage index in the exposed population is low, given the predominance of cells with level I damage. Figures 8, 9 and 10 show undamaged cells and cells with damage levels I and II, respectively.
|Groups||N||Mean||Standard deviation||Total damage||control group|
|Level 0||control group|
|Level I||control group|
|Level II||control group|
|Level III||grupo control
|Level IV||Control group|
The comet assay assesses percentage cell viability, ascertaining the rate of cells in apoptosis (figure 12). Table 6 shows the mean values obtained of cells in apoptosis for non exposed populations (22.6%) and exposed (47.9%) by means of a visual determination. Figure 13 reflect the values of cells in apoptosis for both populations.
|Groups||N||Mean||Standard deviation||Typical mean error|
Evaluation by the software Casp Lab Comet Assay
No significant differences were found in DNA damage as measured by the comet assay and in comparison with the control population, indicating a cytotoxic rather than genotoxic effect
Table 7 shows the mean comet assay values for tail moment and tail moment olive of the exposed and control populations. Note the slight increase in readings for the exposed population; according to the Student’s T test, however, these differences are not statistically significant . Figures 14 and 15, for their part, show the mean boxplot values of both populations for the tail moment and tail moment olive, respectively.
|Tail moment||1,34± 2,12||0,73±0,15|
|Tail moment olive||0,93±0,49||0,51±0,24|
The results show the need for biomonitoring in other populations exposed chronically to subtoxic X ray doses
Study results show that the X ray dose and exposure time to which X-ray taking dentistry students are exposed produces a low level of damage (level I) to the DNA molecule in peripheral blood cells; the effects are rather cytotoxic than genotoxic. This finding chimes in with the opinion of other authors, who report an increase in cytotoxicity in buccal mucous in adult patients and children after panoramic X rays[14, 15,16], but not a significant increase in the frequency of micronuclei. The significant micronuclei-frequency values found could be due to exposure time (over three years) and the dose received, since the frequency of micronuclei increases with the dose and exposure to X rays (Ribeiro, 2008). These values might stem from failures in the cell division mechanism rather than chromosome breakage.
These findings suggest that exposure should be kept down to the strictly necessary and always with due radioprotection precautions.
BY WAY OF A GLOSSARY
Broken eggs. Nuclear abnormality characterised by the presence of a protuberance of variable size in the cell nucleus. It is related to DNA damage.
Pyknosis. Nuclear abnormality involving a nucleus of greatly reduced size, generally in response to a cellular lesion. It is characteristic of cells in apoptosis or necrosis.
Karyorrhexis. Disintegration of the nucleus and nuclear membrane. The chromatin is condensed in small groups, in apoptotic cells.
Karyolysis. A colourless look to the nucleus due to the dissolution of the chromatin. It occurs as a result of necrosis.
Condensed chromatin. The chromatin appears greatly condensed within the nuclear membrane, in response to high levels of cellular lesion.
To FUNDACIÓN MAPFRE, through its research aid programme (2010).
To the Centro para el Desarrollo de la Ciencia (CEDIC), for allowing us to use their labs.
To Dr. Enrique Zamorano Ponce, of the Universidad del Bío Bío de Chillán (Chile), for his constant assistance and advice and the training received.
To Dr. José Corvalán Amigo, Dean of the Facultad de Ciencias de la Salud of the Universidad Católica, for his help in carrying out the project and offering lab facilities.
To the managers of the Facultad de Odontología Pierre Fauchard, for authorising the participation of the students in this research.
To the dentistry students who took part in the research.
To the medical students of the Universidad Católica «Nuestra Señora de la Asunción» Juan Segovia, Luis Gatti and Daniela Solís for their help in carrying out the micronuclei bioassay.
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