The effect of low level exposure to diagnostic and therapeutic radiation sources at workplace is a concern to a large number of health care workers (UNSCEAR, 1982). Effects of ionizing radiation at chronic low doses have been considered as mutagenic and carcinogenic in humans. Cytogenetic studies have demonstrated that even low levels of chronic radiation exposure can potentially increase the frequency of chromosomal aberrations (Jha AN and Sharma, 1991, Cardio RS et al., 2001) and aneuploidy (Thierens H et al., 2000). Importantly, the epidemiological studies have shown that health workers occupationally exposed to ionizing radiation may show increased risk of hematological malignancies (Smith and Doll, 1981; Mvirhead et al., 1999).
The monitoring of radiation exposure in health workers is primarily done by estimation of absorbed dose using film badges at regular intervals. Although, the assessment of genetic integrity in lymphocytes has been developed as a tool for bio monitoring of the radiation exposure (Touil N et al., 2002; Thierens H et al., 2000; Maffei F et al., 2002), these techniques are restricted to only circulating lymphocytes and there is no reports available using germ cells.
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Reproductive function has been shown to be sensitive to changes in the physical and chemical environments (Younglai EF et al., 2005). However, the association between occupational radiation exposure and risk to human fertility is inconclusive (Schull WJ, 1984; Baranski B et al., 1993; Bonde JP, 1999, sjweh). Evidences from laboratory studies indicate that testicular irradiation in mouse can lead to DNA fragmentation in sperm which eventually results in variety of checkpoint responses in early embryos (Adiga et al., 2007) and transgenerational genomic instability in somatic and germ cell compartments in the offspring (Adiga et al., 2010). It has been demonstrated that sperm DNA fragmentation in human is correlated with poor semen quality, decreased fertilization rate and impaired embryonic development (Jayaraman et al., 2011; Borini A et al., 2006HR, Ji B.T. et al., 1997). Hence, there is a widespread fear of damage to the reproductive system from occupational exposure with implication for fertility disorders and adverse reproductive outcome (Fischbeina et al., 1997).
In light of these considerations, there is a need to investigate the possible adverse effects of occupational radiation exposure on semen characteristics including sperm genetic integrity in a chronically exposed population.
Materials and Methods
This study comprised 134 male volunteers out of which 83 were occupationally exposed to ionizing radiation and 51 non-exposed control subjects. The occupationally exposed volunteers were selected from various hospitals having diagnostic or therapeutic radiation facility (x/Î²/Î³-rays). The non-exposed volunteers were employees of the same hospitals but not exposed to radiation sources. The volunteers who fulfilled the criteria were given a set of questionnaire to get the information about the duration of stay at their work place, type of radiation source they handle, life style, habits, history of diseases in addition to problems related to reproduction such as incidence of infertility and miscarriage in their partners. The questionnaire also included other confounding factors influencing semen quality and sperm DNA integrity such as, smoking, alcohol, previous exposure to mutagenic agents etc. Processing and evaluation of the samples of the two groups were performed in the university infertility research laboratory. The study was approved by the Institutional Ethical Committee and the written consent was taken from all the volunteers.
The occupational exposure level of the subjects was routinely monitored by personal exposure measurement devices. The cumulative exposure level of each subject was collected from the radiation safety officers of the respective hospitals where subjects were enrolled.
Semen samples were obtained between 3-7 days of sexual abstinence by masturbation in sterile containers. Semen analysis was performed within one hour of collection under sterile condition. Upon completion of liquefaction, the sample was mixed well and evaluated for physical and microscopic characteristics according to WHO criteria (1999).
Single cell gel electrophoresis (alkaline comet) assay
Single cell gel electrophoresis (alkaline comet) assay was performed as described earlier (Kalthur et al., 2008) with minor modifications. Briefly, the spermatozoa were suspended in sterile phosphate buffered saline (PBS) and the sperm density was kept constant by appropriate dilution in order to maintain the uniform distribution of the spermatozoa during electrophoresis. The sperm suspension was mixed with equal volume of 0.75% low melting agarose (Cat No. A 9414, Sigma Chemical Co, USA) and layered on a slide pre-coated with 1% normal agarose (Cat No. 9539, Sigma Chemical Co, USA). A third coat of agarose was layered over the second layer followed by overnight incubation in lysing solution (2.5M NaCl, 100mM disodium EDTA, 10mM Trizma base, pH 10, 1% Triton X-100, 5% DMSO) under alkaline conditions (pH 10) at 4Â°C. Subsequently, 10 mM dithiothreitol (DTT) was added to the lysing solution to ensure decondensation of sperm DNA. Sperm DNA unwinding was carried out by immersing the slides in electrophoresis buffer (10N NaOH, 200mM EDTA, pH >13), 25V (VcM= 0.74V/cm, 300 mA) for 30 minutes followed by neutralization of slides in 0.4M Tris HCl buffer for 15 min. The slides were dehydrated in chilled absolute alcohol for 30 min and then stained with ethidium bromide (2mg/mL). The cells were observed under a fluorescent microscope (Imager-A1, Zeiss, Germany) and image was captured using 40X objective. Each slide was coded to avoid observer s bias and a minimum of 50 images were captured from each slide randomly avoiding the anode end and edges of the slides. The damaged sperm attain a shape of comet with tail region consisting of fragmented DNA and head region with intact DNA. The comet evaluation of the captured images was done using Kinetic Imaging software (Komet 5.5, UK).
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Terminal deoxynucleotidyl transferase dUTP nick end lebelling (TUNEL) assay
The TUNEL assay was performed as described earlier (Jayaraman et al., 2011). Briefly, one drop of semen was placed on a poly-L-lysine coated cover slip and allowed to dry at room temperature. The cells were fixed in 4% paraformaldehyde solution for 30 min followed by permeabilization using 0.2% Triton X-100 for 30 min. The spermatozoa were incubated in terminal deoxynucleotidyl transferase and nucleotide mix (Apoalert DNA fragmentation assay kit, Cat No. 630108; Clontech, Mountain View, CA) for 1 hour at 37Â°C in a humidified chamber. The cells were washed, counterstained with propidium iodide (10 mg/mL), and mounted on a glass slide. The TUNEL positive cells which exhibited a strong nuclear green fluorescence under a fluorescence microscope (Imager-A1; Zeiss, Gottingen, Germany) were assessed. A total of 2,000 spermatozoa were assessed from each subjects and expressed as percentage of TUNEL positive spermatozoa.
Sperm Chromatin Structure Assay (SCSA)
The sperm density in the ejaculate was adjusted to a concentration of approximately 1-2 millions/mL. After removing seminal plasma by repeated washing, the spermatozoa were fixed in 70% ethanol and stored until analysis. Prior to analysis, the spermatozoa were treated with a low-pH (pHÂ 1.2) detergent solution containing 0.1% Triton X-100, 0.15Â mol/l NaCl and 0.08Â mol/l HCl for 30Â seconds followed by staining with 6Â mg/l purified Acridine Orange (AO, Cat No 74395, Sigma Aldrich Co, USA) in a phosphate-citrate buffer, pHÂ 6.0. Cells were analyzed using a FAC Sort flow cytometer (Becton Dickinson, San Jose, CA), equipped with an air-cooled argon ion laser. A minimum of 5000 events were accumulated for each measurement. Under these experimental conditions, when excited with a 488Â nm light source, AO intercalated with double-stranded DNA emits green fluorescence and AO associated with single-stranded DNA emits red fluorescence. Sperm chromatin damage was quantified by the metachromatic shift from green (native, double-stranded DNA) to red (denatured, single-stranded DNA) fluorescence and displayed as red (fragmented DNA) versus green (DNA stainability) fluorescence intensity cytogram patterns. The data was entered in Excel and analyzed to determine the DNA fragmentation index (DFI= Red fluorescence/ [Red + Green fluorescence]).
Fluorescence in situ hybridization (FISH)
The in situ hybridization was performed as described by Sarrate and Anton (2009) with minor modifications. The slides containing spermatozoa were air dried followed by fixation in freshly prepared Carnoy's fixative (methanol: acetic acid, 3:1). Sperm decondensation was achieved using 25 mM dithiothreitol dissolved in lysis solution for 5 min at room temperature followed by washing with 2 X saline-sodium citrate (SSC) buffer. Slides were dried for 10-15 min and then immersed in ageing solution (2 X SSC, pH 7.4) at 73oC for 2 minutes followed by washing in water. The cells were treated with protease solution (Pepsin, Cat. No. P7012; Sigma Aldrich Inc. USA) dissolved in 10 mM HCl for 15 min, washed in PBS and then dehydrated by serial graded ethanol solutions. About 12.5 ÂµL of FISH probes (AneuVysion Multicolor DNA Probe Kit, Vysis CEP 18, X, Y-alpha satellite, LSI 13 and 21, Abbott Molecular Inc.USA) were added onto the cells and denatured at 73Â°C for 5 min followed by hybridization for 16 h at 37Â°C in a hybridization chamber (Thermobrite, Abbott molecular, USA). The slides were placed in 2 X SSC/ 0.1 % NP-40 at room temperature for 1 min and agitated. The slides were washed in 0.4 X SSC/ 0.3% NP-40 at 73Â°C for 2 min followed by washing in 2 X SSC/ 0.1 % NP-40 at room temperature for 1 min and then counter stained with DAPI. The slides were observed using appropriate filter under fluorescence microscope (Imager-A1, Zeiss, Germany) at 100 X magnification. All the slides were coded to avoid observer's bias before microscopic evaluation.
The data represent mean and standard error (Mean Â± SEM) of the values. The statistical significance level was calculated by unpaired 't'test with wetch correction using GraphPAD Instat software, San Diego, CA, USA. Linear regression analysis was applied to assess the correlation between radiation exposure level and sperm characteristics in exposed subject. The graphs were plotted using origin 6.0 (Northampton, MA, USA).
The mean age of the exposed and non exposed subjects included in this study was 27.74 Â± 0.75 and 28.03 Â± 0.83 years and the difference was not statistically significant. The exposed group had an average work experience of 6.51 Â± 0.67 years. The cumulative radiation exposure level of sixty two volunteers were <0.50mSv whereas twenty one volunteers were exposed to radiation dose of â‰¥0.50mSv as per their personal exposure measurement devices record (Table 1).
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A direct comparison of the semen characteristics between the exposed and non exposed populations are given in table-2. Although, the semen volume and sperm concentration were not significantly different between exposed and non-exposed groups, the motility characteristics, especially total and rapid progressive motility were significantly declined in the exposed group (P<0.01). Further, the sperm viability was also significantly compromised in the exposed group (P<0.05). A significant decline in morphologically normal sperm was observed in the exposed group (P<0.0001). The defects are more localized in the head when compared to rest of the structural abnormalities. An attempt to determine the sperm head vacuoles between two groups revealed a significantly higher incidence of vacuoles in the exposed group (P<0.01).
Sperm DNA integrity
The alkaline comet assay was performed to quantify the amount of single and double strand breaks in the spermatozoa occupationally exposed subjects. The mean head DNA in the exposed group was 84.99 Â± 1.24, which was significantly lower than non-exposed (89.57 Â± 0.87; P<0.001) group. The olive tail moment (OTM, product of the tail length and the fraction of total DNA in the tail) in the exposed group was approximately 1.8 fold higher than the non exposed subjects (P<0.05) (Fig 1A). Although, the TUNEL results are in agreement with comet data, the number of TUNEL positive cells are not statistically significant between the groups (Fig 1B). Since flow cytometry based sperm chromatin assay provides independent measurement of sperm DNA integrity, it is considered as a useful tool for epidemiological studies (Spano et al, 1998, HR), Therefore, we used this technique to validate the comet and TUNEL results. A significantly higher DFI was observed in the exposed group when compared to non-exposed group (P<0.0001) (Fig 1C).
Incidence of sperm aneuploidy
Sperm aneuploidy assessment was performed in 23 exposed subjects out of which 12 subjects had the mean cumulative absorbed dose of 5.43 Â± 1.01 mSv. The mean absorbed dose of 11 subjects was <0.05 mSv hence this subgroup was considered as internal control. Except the exposure level, the other factors like age, and smoking habits were not significantly different between the two groups. From each subject, a minimum of 5500 spermatozoa evaluated for each chromosome to determine the incidence of disomies/trisomies. Incidence of 13, 18, 21, X and Y disomies was not significantly different between the groups studied, although, the overall incidence of aneuploidy was moderately high in the test group (Table-3).
While earlier studies have clearly demonstrated the detrimental effect of occupational radiation exposures on the genetic integrity of somatic cells, the impact on semen characteristics and genetic integrity of male gamete is not elucidated. Here for the first time, this study has demonstrated that exposure to ionizing radiation at workplace can significantly impair the semen characteristics and sperm DNA integrity. These findings have profound implication for the fertility of health workers and importantly on the health of children born to exposed fathers as DNA damaged sperm can still fertilize the oocyte (Ahmadi et al...) which eventually carries substantial risk of transgenerational genomic instability in the offspring (Adiga et al., 2010).
Semen parameters and occupational influence
Nevertheless, the fact that effects on genetic integrity of peripheral lymphocytes have previously been observed in various epidemiological studies (ref.............), which emphasizes the biological significance of these findings.
Notwithstandingly, the specialized nature of human spermatozoa, the results observed in this study may also apply to......radiation induced damage to other cell types.
Occupational exposure have been associated with sperm DNA damage and the damage has been related to the.....
Well designed epidemiological and toxicological studies can inform clinicians about risks to sperm DNA integrity posed by environmental contaminations and help them to advise their parents about considering suh exposures (Barratt CLR et al., HR, 2010). Earlier studies have indicated that environmental toxicants can potentially induce sperm DNA fragmentation (Evenson & Wixon, 2005; Barratt CLR, 2010 HR).
Cross sectional studies of semen quality suffer often of low participation rates which may bias the internal validity of a study. Further, selection bias is unlikely since the age of the subjects did not differ between two groups.
We found increased incidence of semen abnormalities and sperm DNA fragmentation in the individuals exposed to radiation at workplace.
The design of this study provided distinct strengths in the evaluation of risks....The acquisition of dosimeter measurements of exposure was done with no direct contact with study subjects.
The present study is unique in its examination of the possible influence of radiation exposure. Although, the workers handling the radiation sources receive higher exposure of ionizing radiation than environmental exposure, the existence of a significantly increased gamete abnormalities pose a potential public health risk.
Significantly elevated number of centromere positive micronuclei in medical workers exposed to ionizing radiation suggest possible aneugenic effects of chronic exposure to IR (Thierens H et al., 2000). However, evaluation of aneuploidy in sperm did not reveal any association in the present study.
The cytogenetic studies of hospital workers also showed a significant increase in structural chromosomal abnormalities (Bigatti et al., 1988; Barquinero et al., 1993).
For a better understanding of the potential effects of ionizing radiation and genetic integrity of spermatozoa, this study presents the results ofâ€¦
Our results indicated that overall semen abnormalities are higher in exposed subjects than unexposed controls.
Moreover the influence of confounding factors such as smoking and age on sperm abnormalities was investigated by multiple regression analysis. Smoking can influence the genetic damage induced in humans by ionizing radiation (Maffei F et al., 2002) possibly by increasing the radiosensitivity of the cells (Wang LE, 2000, RadRes).
The strengths of the present study are 1. use of large number of exposed subjects and comparison with age matched controls and 2. simultaneous use of three biomarkers to determine the sperm DNA integrity. The comet assay which has been widely acceptable to assess the radiation induced damage in radiation health workers (Touil Â© N et al.,2002).
The most striking observation wasâ€¦
This work was supported by Indian Council of Medical Research (ICMR) Grant # 2010-00020 and DST-SERC Grant # SR/SO/HS-0080/2007.
Declaration of Author's roles
DK has performed the experiments and analyzed the data, SR, SU and NJ have performed the experiment, GPK and HK have assisted in designing the study, in evaluating the data and in writing the manuscript, CS, Srinidhi have assisted in data analysis and writing manuscript, PK has given advice concerning study design, SKA has designed the study, analyzed the data and written the manuscript.