Original Article 337
Antimutagenic Effects of Vitamin E on Oncology and Non Oncology Hospital Nurses by Comet Assay
Affiliations
Key words
▶ vitamin E ● ▶ comet assay ● ▶ nurses ● ▶ urine extracts ●
M. Rezaei-Basiri1, H. Rezazadeh1, I. Asvadi-Kermani2, A. Davoodi3, M. A. Eghbal1, M. Golchin2, M. Sarmad2 1
Department of Pharmacology and Toxicology, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz- Iran Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz- Iran 3 Department of Medical Surgical, School of Nursing and Midwifery, Tabriz University of Medical Sciences, Tabriz- Iran 2
Abstract
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Vitamin E is a natural antioxidant and its most common biologically active form is α-tocopherol. The antiproliferative effects of α-tocopherol have been previously demonstrated. In this study the antimutagenic effects of vitamin E on oncology and non oncology hospital nurses was investigated. A total of 138 female nurses from oncology and non oncology hospitals participated in the study. They received 200 mg/day vitamin E for 2 weeks. The urine samples before and after intake of vitamin E were collected and the nucleus of urothelial cells were evaluated with comet assay. The length of epithelial cells nuclei correlated with increased fracture rate of DNA. Nucleolus length of urine epithelial cells of all nursing staff
Introduction received 28.08.2013 accepted 05.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1361163 Published online: December 19, 2013 Drug Res 2014; 64: 337–342 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Dr. H. Rezazadeh Department of Pharmacology and Toxicology School of Pharmacy Tabriz University of Medical Sciences Daneshgah avenue – Tabriz/Iran Tel.: + 98/411/3341 315 Fax: + 98/411/3344 798 [email protected]
▼
Several studies have performed on biological samples to detect genotoxic effects in humans in connection to jobs or environment. It has been reported that multiple professional workers from clinical, pathology laboratories, farmers and welders are exposing to carcinogenic compounds [1–3]. In addition, oncology nursing personnel usually expose to antineoplastic drugs during preparation and administration [4]. However, investigations on the genotoxicity of these drugs are inconsistent. Thereby, in vitro ames, comet assays, chromosomal aberrations and micronucleus tests have essential roles for the evaluation of mutagenicity and carcinogenicity [5]. So far a range of applications of the alkaline comet assay is covered, from investigations of the physicochemical behavior of DNA, through studies of cellular responses to DNA damage, to biomonitoring of human populations [6]. The purpose of present study was to determine the antimutagenic effects of vitamin E on oncology and non oncology nurses by comet assay.
before and after vitamin E treatment were measured and the data were evaluated by student t-test and SPSS. Our study showed that 20 % of nursing staff have apoptosis and DNA fracture in the nucleolus of their urine epithelial cells and DNA damage in the urothelial cells of exposed nurses was significantly higher than the control group (P < 0.05). The antimutagenic activity of vitamin E had significant effects on oncology hospital nurses effectively in repairing DNA damage and decreasing their nucleus length in urine epithelial cells. We propose that the higher therapeutic doses of vitamin E and increasing the length of treatment period will be effective against DNA strand breakage and may have more effect on oncology nurses.
Thus, in the constant quest for new therapeutic, the natural products and phytocomplexes have been tested for their ability to prevent oxidation and mutagenesis [7, 8]. In animal model, vitamin E has been shown to inhibit completely peroxidative injury by restoring renal tissue antioxidants and glutathione redox balance of hyperoxaluriainduced renal injury [9], also the suppressive effects of vitamins on No2-induced DNA single strand breaks has been reported [10]. In the course of this study we investigated the genototoxicity in urothelial cells, in addition the antimutagenic effects of vitamin E in oncology nurses in connection to antineoplastic drugs exposure was evaluated.
Materials and Methods
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Study population The study included 138 female (non pregnant) nurses working in oncology and non-oncology hospitals located at Tabriz/Iran, between September 2011 and May 2012 and their duration of
Rezaei-Basiri M et al. Role of Vitamin E on Urothelial Cell … Drug Res 2014; 64: 337–342
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Authors
338 Original Article
Chemicals and reagents All chemicals used in this study were of analytical grade and obtained from (Merck co, Germany). Vitamin E (dL-alpha-tocopheryl acetate) pearl 200IU purchased from (Zahravi pharmaceutical company, Tabriz, Iran).
Collection of urinary samples Primary samples of all volunteers obtained from morning urine (100 ml) were transferred to laboratory in capped sterile beakers. Then they received orally 200 mg of vitamin E daily for 2 weeks and at the end of 14th day second urine sample (100 ml) were also delivered to laboratory. The samples were maintained in refrigerator for further investigation.
urothelial cells of one nurse volunteer. 100 μl NMA (normal melting agaros) was added to every pit and was coated with 100 μl LMA (low melting agaros) at 37 °C. Then 20 μl urothelial cells were spreaded, the cells finally were coated with 100 μl NMA at 37 °C. Then the prepared slides were kept in refrigerator at 4 °C for 10 min. For lysing the cytoplasm of cells, the slides were kept in the ice cold container with soluble cold, freshly made lysing solution (DMSO and Triton X) for 40 min and kept in dark to avoid from false positive results. The slides were put in electrophoresis tank and electrophoresed in alkaline solution (pH = 13), an ambient temperature of 4 °C for 20 min at an electrical field strength of 25 V. The slides were taken out and surface layer were neutralized by alkaline Tris buffer (pH = 7.4) repeated thrice for 6 min [15], then the slides were coated with 100 μl etidium bromide and washed with distilled water and covered with cover slips by using cytology glue and lamel. The comets were studied with 50X fluorescence Olympus microscope equipped with digital camera.
Positive control The positive control evaluation of the urine epithelial cell suspension of selected healthy students were exposed to hydrogen peroxide 10 % v/v and incubated for 4 h at 37 °C [16]. The nuclear of these cells were processed to comet assay. Finally the photography of all nuclear were prepared by Olympus 50X fluorescence microscopes equipped with digital camera and the nuclear tail length of images were calculated by using the CASP software analysis. The microscope was adjusted as: Objective: 100X, sensitivity: ISO 1 600, Resolution: Live/movie: 1 360 × 1 024, snap: 4 140 × 3 096 (Pixel shift), exposure time: 2.5–15, dark room. Then slides were studied and photographed zigzag in the margins under microscope, and ideal nuclear of urothelial were selected in slides. Red cells nuclear were often visible in the core were diagonal form [17].
Image analysis Calculation of urinary epithelial cells nuclear elongation Minimum of 50 images of urinary tract epithelial cells for each participant of nurses were prepared. Each image adjusted and saved with picture manager software then these images were loaded in CASP software. Using graph pad prism software mean of 50 stretched urinary epithelial cells nuclear was calculated for ▶ Fig. 1 each participant of nurses [18, 19]. Accordingly the ● shows DNA breakage in epithelial cells and calculation of epithelial cells nucleus elongation with CASP software in oncology hospital nurse.
Preparation of urothelial cell suspension The urine samples from the nurse volunteers were centrifuged at 2 000 g for 5 min at 4 °C in a refrigerated centrifuge. The upper sections of urines were taken for mutagenic compounds extraction and were kept in refrigerator. To obtain pure and healthy urothelial cells suspension, the urine sediments were washed and centrifuged with phosphate buffer solution 3 times. 100 μl TRED (Trypsin 0.005 % + EDTA) and 100 μl FFP (Fresh Frozen Plasma) were added to urothelial cells suspension then they were incubated at 37 °C for overnight [12].
Comet assay
Statistical analysis Graph Pad Prism 5 Software and SPSS were used for statistical analysis. The data for comet assay was tail length which calculated to oncology and non oncology hospital nurses then statistical analysis were camparisoned between the 2 subjects. Mean and standard deviation (SD) were analyzed for each parameter. The significance of the differences between control and exposed nurses’ end point means were analyzed using Student’s t-test (mean DNA tail length). The antimutagenic effects of vitamin E (exposure and age) were assessed by Student’s t-test. A p-value < 0.05 was considered as significant.
The comet assay was performed under alkaline conditions [13, 14]. The transparent plastic slides pitted were initially prepared. Every slide pit was belonging to comet assay evaluation of
Rezaei-Basiri M et al. Role of Vitamin E on Urothelial Cell … Drug Res 2014; 64: 337–342
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employment was (range 1–28 years). The first group consisted of 69 nurses working in a specialized oncology hospital, who had regular contact with antineoplastic drugs daily (preparation of solutions and syringes for infusion, administering of antineoplastic drugs and handling of body fluids of patients undergoing chemotherapy). The most frequently used chemotherapeutic drugs were doxorubicin, bleomycin, vinblastine, dacarbazine, methotrexate, fluorouracil, prednisone, epirubicin, irinotecan, leucoverin, prednisone, 6-mercaptopurine, procarbazine, lomustine, cisplatin, etoposide, 6-thioguanine and cyclophosphamide. The handling time varied from 1 to 6 h/day. The second control group 69 nurses were selected from non oncology general hospital with no history of occupational exposure to antineoplastic agents. The exclusion and inclusion criteria of research population and selection criteria of study persons were based on a questionnaire. All subjects were asked to complete a face-to-face questionnaire, which included standard demographic data (age, gender) as well as medical (exposure to X-rays, vaccinations, medication), lifestyle (smoking, coffee, alcohol, diet) and occupational questions (working hours per day, years of exposure, use of protective measures, etc.). It was assured that the exposed nurses and the controls did not statistically differ from each other except for antineoplastic drugs exposure. It was ensured that the exposed and the control subjects had not been taking any medicines, nor had they been exposed to any kind of radiation for 12 months before sample collection. According to the ethical committee of Tabriz university of medical sciences, all subjects involved in the study received detailed information concerning the aims of the research study, available online: http://www.irct.ir [11].
Original Article 339
Parameters
Exposed nurses (n = 69)
Non Exposed nurses (n = 69)
Statistical analysis
Age (years) (mean ± SD) Years of exposure (mean ± SD) Per day exposure (hours) (mean ± SD)
36 ± 7 10.46 ± 7 7.18 ± 0.76
36 ± 7.3 11.13 ± 7 7.78 ± 1.77
P = 0.28 P = 0.33 P = 0.14
Table 1 Demographic characteristics of non-exposed (control) and exposed nurses involved in the study.
t-test, P < 0.05 significant for between groups
Results
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The effect of occupational exposure to antineoplastic drugs on the levels of genetic damage in exposed nurses and non exposed ▶ Table 1 control subjects were assessed by the comet assay. ● represents the distribution of subjects with respect to age, years of exposure and duration of handling chemotherapeutic agents per day. Both groups had similar demographic characteristics. The mean age of the exposed group was 36 ± 7, ranging from 21 to 60 years, and that of controls was 36 ± 7.3, ranging from 21 to 60 years. The extent of DNA damage evaluated by comet assay in urothelial cells of all the study subjects as measured by mean ▶ Table 2, 3. ● ▶ Table 2 shows the comet tail length are listed in ● comparison of DNA mean tail (in μm) by age and years of exposure among oncology hospital nurses and non exposed control group of nurses before vitamin E treatment. The comet tail length significantly increased in exposed nurses. Subjects who were ≥ 36 years of age showed a statistically significant increase in mean DNA damage values than those who were < 36 years in exposed group (21.90 ± 5.71 vs. 19.89 ± 2.24 μm; P < 0.05). However, the result did not show any significant changes in control group (17.62 ± 3.6 vs. 17.93 ± 2.37 μm; P < 0.05). Similarly the comet tail length did not show any significantly increased in DNA damage of exposed nurses with ≥ 10.5 or < 10.5 years of employment (22.87 ± 6.18 vs. 22.32 ± 4.65 μm). Also nurses who handled antineoplastics for ≥ 7.5 h/day did not show any significant increase in DNA damage compared to those who handled them for < 7.5 h/day (21.68 ± 5.34 vs. 22.98 ± 4.82 μm). Similar result was seen in control group (17.97 ± 3.85 vs. 17.11 ± 2.88 μm). ▶ Table 3 shows the comparison of DNA mean tail (in μm) by age ● and years of exposure of oncology hospital nurses and non exposed nurses after vitamin E treatment.
Table 2 Comet assay tail length between exposed nurses and non exposed nurses before E treatment. Parameters
Exposed nurses tail length
Non Exposed nurses tail length
N
Mean ± SD
N
Mean ± SD
Age (years) ≥ 36
N = 37
21.9 ± 5.71
N = 37
< 36
N = 32
19.89 ± 2.24
N = 32
17.62 ± 3.6 P = 0.09 17.93 ± 2.37 P = 0.12
Years of exposure ≥ 10.5
N = 31
22.87 ± 6.18
N = 31
< 10.5
N = 38
22.32 ± 4.65
N = 38
Per day exposure (hours) exposure ≥ 7.5 N = 31 21.68 ± 5.34
N = 31
< 7.5
N = 38
N = 38
22.98 ± 4.82
17.99 ± 3.66 P = 0.12 17.25 ± 3.60 P = 0.17 17.97 ± 3.85 P = 0.17 17.11 ± 2.88 P = 0.12
P < 0.05 significant for between groups, t-test
The comet tail length significantly increased in exposed nurses. Subjects who were ≥ 36 years of age showed a statistically significant increase in mean DNA damage values than those who were < 36 years in exposed group (20.54 ± 5.92 vs. 18.24 ± 3.21 μm; P < 0.05). Similar result was seen in control group (14.73 ± 3.43 vs. 14.33 ± 2.74 μm; P < 0.05). The comet tail length did not show any significantly increased in DNA damage of exposed nurses with ≥ 10.5 or < 10.5 years of employment (19.36 ± 4.56 vs. 20.42 ± 5.86 μm).
Rezaei-Basiri M et al. Role of Vitamin E on Urothelial Cell … Drug Res 2014; 64: 337–342
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Fig. 1 Shows DNA breakage in urine epithelial cells nucleus and the nucleus elongation was calculated with CASP.
340 Original Article
Parameters
Exposed nurses tail
Non exposed nurses tail
length
length
N
Mean ± SD
N
Mean ± SD
Age (years) ≥ 36
N = 37
20.54 ± 5.92
N = 37
< 36
N = 32
18.24 ± 3.21
N = 32
14.73 ± 3.43 P = 0.57 14.33 ± 2.74 P = 0.47
Years of exposure ≥ 10.5
N = 31
20.42 ± 5.86
N = 31
< 10.5
N = 38
19.36 ± 4.56
N = 38
Per day exposure (hours) exposure ≥ 7.5 N = 31 17.94 ± 4.68
N = 31
< 7.5
N = 35
N = 35
20.66 ± 4.97
14.68 ± 3.25 P = 0.54 13.83 ± 3.58 P = 0.57 14.17 ± 3.57 P = 0.64 14.27 ± 3.18 P = 0.53
P < 0.05 (Difference between exposed and non exposed nurses; student’s t-test
Also nurses who handled antineoplastics for ≥ 7.5 h/day did not show any significant increase in DNA damage compared to those who handled for < 7.5 h/day (17.94 ± 4.68 vs. 20.66 ± 4.97 μm). Also same result was seen in control group (14.17 ± 3.57 vs. 14.27 ± 3.18 μm). ▶ Fig. 1 shows DNA breakage in epithelial cells and calculation ● of epithelial cells nucleus elongation with CASP (Comet assay software) in oncology hospital nurses. ▶ Fig. 2 shows DNA breakage in epithelial cells nucleus in an ● oncology hospital nurse before and after 200 mg/day vitamin E ▶ Fig. 2a. large ‘comets’ were treatment. As it is shown in ● observed in cells before vitamin E. However, vitamin E treatment ▶ Fig. 2b. In order to causes decrease in the size of the ‘comet’ ● quantify these effects and examine whether vitamin E itself prevent DNA damage, the mean tail moments of all individual cells from each experimental and control group were calculated. ▶ Table 4 states antimutagenic effects of vitamin E Although ● between all exposed nurses before and after vitamin E treatment as it is clear 200 mg of vitamin E per day had been completely significant effects in diminishing of tail moments in
Fig. 2 a Before vitamin E treatment, tail length = 119. b After vitamin E treatment, tail length = 81.
Rezaei-Basiri M et al. Role of Vitamin E on Urothelial Cell … Drug Res 2014; 64: 337–342
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Table 3 Comet assay tail length between exposed nurses and non exposed nurses after E treatment.
Table 4 Antimutagenicity effects of vitamin E (Comet assay tail length) on (N = 69) women oncology hospital nurses. Parameters
Before vitamin E
2 week after vitamin E
Treatment Tail length
Treatment Tail length
N
Mean ± SD
N
Mean ± SD
Age (years) ≥ 36
N = 37
21.90 ± 5.71
N = 37
< 36
N = 32
19.89 ± 2.24
N = 32
20.54 ± 5.92 P = 0.000 18.24 ± 3.21 P = 0.000
Years of exposure ≥ 10.5
N = 31
22.87 ± 6.18
N = 31
< 10.5
N = 38
22.32 ± 4.65
N = 38
Per day exposure (hours) exposure ≥ 7.5 N = 31 21.68 ± 5.34
N = 31
< 7.5
N = 35
N = 35
22.98 ± 4.82
20.42 ± 5.86 P = 0.000 19.36 ± 4.56 P = 0.000 17.94 ± 4.68 P = 0.000 20.66 ± 4.97 P = 0.000
P < 0.05 (Difference between before and after vitamin-E treatment; student’s t-test
Fig. 3 Antimutagenic effects of vitamin E had been significant effects (the p < 0.05, P = 0.000) in diminishing the tail moments in oncology exposed nurses (n = 31). Daily work: ≥ 7.5 h.
oncology exposed nurses in their urothelial nucleus. Also the ▶ Fig. 3. same result is shown in ●
Discussion
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The present study provides first evidence that vitamin E supplementation has diminished the mutagenic effects of antineoplastic drugs among nursing personnel. Chemotherapy agents with low therapeutic index having major role for chemical control of cancer and on this light, they induce genotoxic effects on normal cells. Most studies published about professionals working with antineoplastic drugs assessed some degree of increasing genotoxicity [4, 14, 20, 21]. A study by Kosgeroglu et al. claimed that with regard to nursing self-protection, when the information and administration scores decrease and increase in the working period (per week), may incur risk exposure to chemotherapeutic drugs [22]. So nursing and healthcare personnel, who admix, administer or dispose of antineoplastic drugs or body waste products from patients being treated with such drugs are at great risk of exposure and they need to be monitored for risks. Several studies have performed on biological samples to detect genotoxic effects in humans in connection to their jobs or envi-
ronment and in this line because of ease to sampling of urine, we assessed the antimutagenicity effects of vitamin E in uroepithelial cells of nursing urine samples by using comet assay. Studies have investigated the genotoxic potential of antineoplastic agents among the medical personnel chronically handling these drugs and it has been found that the DNA damage to be significantly higher than the control [4, 21, 23, 24]. Our results are in line with these studies. Contrary to these findings, some studies by evaluating genotoxicity in health care workers handling antineoplastic drugs showed no statistically significant increase in DNA damage in nurses with respect to controls [25]. The purpose of our study was to evaluate the antimutagenic effect of vitamin E on possible genotoxic risk associated with antineoplastic drugs. Some studies have suggested that, antioxidants especially vitamin E, a chain-breaking lipophilic antioxidant found within biological membranes, is also physiological membrane-bund free radical scavenger [26] and our earlier observation indicates that vitamin E as an effective chemopreventive agent is associated with diminution of oxidative stress [27]. Vitamin E has also shown to have a potent role on unsaturated fatty acids of cell membranes and stabilizes/preserves cell membranes of urothelial tract [28]. In addition, it has been shown that vitamin E in both the cases, by pretreatment/treatment has essential role on epithelial cells counts [29]. In this study, we have evaluated the role of vitamin E on urothelial cells nucleus. It was appeared that antimutagenic activity of vitamin E had significant effects on oncology hospital nurses effectively in repairing DNA damage and decreasing their nucleus length in urine epithelial cells. Accordingly on comparing to non oncology nurses (control), the urothelial cell’s nucleus tail lengths among oncology nurses were larger. Hence, since nurses are subjected to several antineoplastics drugs in job setting, which has been shown to have potent carcinogenic activities and the nursing resistance may decrease and they may encounter with oxidative stress of cellular components [30].
Conclusion
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In summary the damage in urothelial cell nucleus in nurses could not be in a fixed range and depends on several factors likes protective equipments, the working period, the information and administration scores with regard to self-protection, and physiological states of a person, age and nutrition. Additionally, a proper natural diet rich in antioxidants and antimutagenics such as vitamin E should be considered. Also based on experimental evidence we propose that use of vitamin E at higher therapeutic doses would significantly be effective against DNA strand breakage and may have more effect on nucleus of uroepthelial cells of oncology hospital nursing personnel and it might be an alternative to reduce genotoxic risk in exposure to antineoplastic drugs. Also because of ease to sampling of urine, it can be inferred that uroepithelial cells may have essential diagnostic role among oncology nursing personnel.
Rezaei-Basiri M et al. Role of Vitamin E on Urothelial Cell … Drug Res 2014; 64: 337–342
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Original Article 341
Acknowledgement
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The authors acknowledge hematology and oncology research center, Tabriz University of Medical Sciences, Iran, for funding the project.
Conflict of Interest
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“The authors state no conflict of interest”.
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342 Original Article
Antimutagenic Effects of Vitamin E on Oncology and Non Oncology Hospital Nurses by Comet Assay
Affiliations
Key words
▶ vitamin E ● ▶ comet assay ● ▶ nurses ● ▶ urine extracts ●
M. Rezaei-Basiri1, H. Rezazadeh1, I. Asvadi-Kermani2, A. Davoodi3, M. A. Eghbal1, M. Golchin2, M. Sarmad2 1
Department of Pharmacology and Toxicology, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz- Iran Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz- Iran 3 Department of Medical Surgical, School of Nursing and Midwifery, Tabriz University of Medical Sciences, Tabriz- Iran 2
Abstract
▼
Vitamin E is a natural antioxidant and its most common biologically active form is α-tocopherol. The antiproliferative effects of α-tocopherol have been previously demonstrated. In this study the antimutagenic effects of vitamin E on oncology and non oncology hospital nurses was investigated. A total of 138 female nurses from oncology and non oncology hospitals participated in the study. They received 200 mg/day vitamin E for 2 weeks. The urine samples before and after intake of vitamin E were collected and the nucleus of urothelial cells were evaluated with comet assay. The length of epithelial cells nuclei correlated with increased fracture rate of DNA. Nucleolus length of urine epithelial cells of all nursing staff
Introduction received 28.08.2013 accepted 05.10.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1361163 Published online: December 19, 2013 Drug Res 2014; 64: 337–342 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Dr. H. Rezazadeh Department of Pharmacology and Toxicology School of Pharmacy Tabriz University of Medical Sciences Daneshgah avenue – Tabriz/Iran Tel.: + 98/411/3341 315 Fax: + 98/411/3344 798 [email protected]
▼
Several studies have performed on biological samples to detect genotoxic effects in humans in connection to jobs or environment. It has been reported that multiple professional workers from clinical, pathology laboratories, farmers and welders are exposing to carcinogenic compounds [1–3]. In addition, oncology nursing personnel usually expose to antineoplastic drugs during preparation and administration [4]. However, investigations on the genotoxicity of these drugs are inconsistent. Thereby, in vitro ames, comet assays, chromosomal aberrations and micronucleus tests have essential roles for the evaluation of mutagenicity and carcinogenicity [5]. So far a range of applications of the alkaline comet assay is covered, from investigations of the physicochemical behavior of DNA, through studies of cellular responses to DNA damage, to biomonitoring of human populations [6]. The purpose of present study was to determine the antimutagenic effects of vitamin E on oncology and non oncology nurses by comet assay.
before and after vitamin E treatment were measured and the data were evaluated by student t-test and SPSS. Our study showed that 20 % of nursing staff have apoptosis and DNA fracture in the nucleolus of their urine epithelial cells and DNA damage in the urothelial cells of exposed nurses was significantly higher than the control group (P < 0.05). The antimutagenic activity of vitamin E had significant effects on oncology hospital nurses effectively in repairing DNA damage and decreasing their nucleus length in urine epithelial cells. We propose that the higher therapeutic doses of vitamin E and increasing the length of treatment period will be effective against DNA strand breakage and may have more effect on oncology nurses.
Thus, in the constant quest for new therapeutic, the natural products and phytocomplexes have been tested for their ability to prevent oxidation and mutagenesis [7, 8]. In animal model, vitamin E has been shown to inhibit completely peroxidative injury by restoring renal tissue antioxidants and glutathione redox balance of hyperoxaluriainduced renal injury [9], also the suppressive effects of vitamins on No2-induced DNA single strand breaks has been reported [10]. In the course of this study we investigated the genototoxicity in urothelial cells, in addition the antimutagenic effects of vitamin E in oncology nurses in connection to antineoplastic drugs exposure was evaluated.
Materials and Methods
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Study population The study included 138 female (non pregnant) nurses working in oncology and non-oncology hospitals located at Tabriz/Iran, between September 2011 and May 2012 and their duration of
Rezaei-Basiri M et al. Role of Vitamin E on Urothelial Cell … Drug Res 2014; 64: 337–342
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338 Original Article
Chemicals and reagents All chemicals used in this study were of analytical grade and obtained from (Merck co, Germany). Vitamin E (dL-alpha-tocopheryl acetate) pearl 200IU purchased from (Zahravi pharmaceutical company, Tabriz, Iran).
Collection of urinary samples Primary samples of all volunteers obtained from morning urine (100 ml) were transferred to laboratory in capped sterile beakers. Then they received orally 200 mg of vitamin E daily for 2 weeks and at the end of 14th day second urine sample (100 ml) were also delivered to laboratory. The samples were maintained in refrigerator for further investigation.
urothelial cells of one nurse volunteer. 100 μl NMA (normal melting agaros) was added to every pit and was coated with 100 μl LMA (low melting agaros) at 37 °C. Then 20 μl urothelial cells were spreaded, the cells finally were coated with 100 μl NMA at 37 °C. Then the prepared slides were kept in refrigerator at 4 °C for 10 min. For lysing the cytoplasm of cells, the slides were kept in the ice cold container with soluble cold, freshly made lysing solution (DMSO and Triton X) for 40 min and kept in dark to avoid from false positive results. The slides were put in electrophoresis tank and electrophoresed in alkaline solution (pH = 13), an ambient temperature of 4 °C for 20 min at an electrical field strength of 25 V. The slides were taken out and surface layer were neutralized by alkaline Tris buffer (pH = 7.4) repeated thrice for 6 min [15], then the slides were coated with 100 μl etidium bromide and washed with distilled water and covered with cover slips by using cytology glue and lamel. The comets were studied with 50X fluorescence Olympus microscope equipped with digital camera.
Positive control The positive control evaluation of the urine epithelial cell suspension of selected healthy students were exposed to hydrogen peroxide 10 % v/v and incubated for 4 h at 37 °C [16]. The nuclear of these cells were processed to comet assay. Finally the photography of all nuclear were prepared by Olympus 50X fluorescence microscopes equipped with digital camera and the nuclear tail length of images were calculated by using the CASP software analysis. The microscope was adjusted as: Objective: 100X, sensitivity: ISO 1 600, Resolution: Live/movie: 1 360 × 1 024, snap: 4 140 × 3 096 (Pixel shift), exposure time: 2.5–15, dark room. Then slides were studied and photographed zigzag in the margins under microscope, and ideal nuclear of urothelial were selected in slides. Red cells nuclear were often visible in the core were diagonal form [17].
Image analysis Calculation of urinary epithelial cells nuclear elongation Minimum of 50 images of urinary tract epithelial cells for each participant of nurses were prepared. Each image adjusted and saved with picture manager software then these images were loaded in CASP software. Using graph pad prism software mean of 50 stretched urinary epithelial cells nuclear was calculated for ▶ Fig. 1 each participant of nurses [18, 19]. Accordingly the ● shows DNA breakage in epithelial cells and calculation of epithelial cells nucleus elongation with CASP software in oncology hospital nurse.
Preparation of urothelial cell suspension The urine samples from the nurse volunteers were centrifuged at 2 000 g for 5 min at 4 °C in a refrigerated centrifuge. The upper sections of urines were taken for mutagenic compounds extraction and were kept in refrigerator. To obtain pure and healthy urothelial cells suspension, the urine sediments were washed and centrifuged with phosphate buffer solution 3 times. 100 μl TRED (Trypsin 0.005 % + EDTA) and 100 μl FFP (Fresh Frozen Plasma) were added to urothelial cells suspension then they were incubated at 37 °C for overnight [12].
Comet assay
Statistical analysis Graph Pad Prism 5 Software and SPSS were used for statistical analysis. The data for comet assay was tail length which calculated to oncology and non oncology hospital nurses then statistical analysis were camparisoned between the 2 subjects. Mean and standard deviation (SD) were analyzed for each parameter. The significance of the differences between control and exposed nurses’ end point means were analyzed using Student’s t-test (mean DNA tail length). The antimutagenic effects of vitamin E (exposure and age) were assessed by Student’s t-test. A p-value < 0.05 was considered as significant.
The comet assay was performed under alkaline conditions [13, 14]. The transparent plastic slides pitted were initially prepared. Every slide pit was belonging to comet assay evaluation of
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employment was (range 1–28 years). The first group consisted of 69 nurses working in a specialized oncology hospital, who had regular contact with antineoplastic drugs daily (preparation of solutions and syringes for infusion, administering of antineoplastic drugs and handling of body fluids of patients undergoing chemotherapy). The most frequently used chemotherapeutic drugs were doxorubicin, bleomycin, vinblastine, dacarbazine, methotrexate, fluorouracil, prednisone, epirubicin, irinotecan, leucoverin, prednisone, 6-mercaptopurine, procarbazine, lomustine, cisplatin, etoposide, 6-thioguanine and cyclophosphamide. The handling time varied from 1 to 6 h/day. The second control group 69 nurses were selected from non oncology general hospital with no history of occupational exposure to antineoplastic agents. The exclusion and inclusion criteria of research population and selection criteria of study persons were based on a questionnaire. All subjects were asked to complete a face-to-face questionnaire, which included standard demographic data (age, gender) as well as medical (exposure to X-rays, vaccinations, medication), lifestyle (smoking, coffee, alcohol, diet) and occupational questions (working hours per day, years of exposure, use of protective measures, etc.). It was assured that the exposed nurses and the controls did not statistically differ from each other except for antineoplastic drugs exposure. It was ensured that the exposed and the control subjects had not been taking any medicines, nor had they been exposed to any kind of radiation for 12 months before sample collection. According to the ethical committee of Tabriz university of medical sciences, all subjects involved in the study received detailed information concerning the aims of the research study, available online: http://www.irct.ir [11].
Original Article 339
Parameters
Exposed nurses (n = 69)
Non Exposed nurses (n = 69)
Statistical analysis
Age (years) (mean ± SD) Years of exposure (mean ± SD) Per day exposure (hours) (mean ± SD)
36 ± 7 10.46 ± 7 7.18 ± 0.76
36 ± 7.3 11.13 ± 7 7.78 ± 1.77
P = 0.28 P = 0.33 P = 0.14
Table 1 Demographic characteristics of non-exposed (control) and exposed nurses involved in the study.
t-test, P < 0.05 significant for between groups
Results
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The effect of occupational exposure to antineoplastic drugs on the levels of genetic damage in exposed nurses and non exposed ▶ Table 1 control subjects were assessed by the comet assay. ● represents the distribution of subjects with respect to age, years of exposure and duration of handling chemotherapeutic agents per day. Both groups had similar demographic characteristics. The mean age of the exposed group was 36 ± 7, ranging from 21 to 60 years, and that of controls was 36 ± 7.3, ranging from 21 to 60 years. The extent of DNA damage evaluated by comet assay in urothelial cells of all the study subjects as measured by mean ▶ Table 2, 3. ● ▶ Table 2 shows the comet tail length are listed in ● comparison of DNA mean tail (in μm) by age and years of exposure among oncology hospital nurses and non exposed control group of nurses before vitamin E treatment. The comet tail length significantly increased in exposed nurses. Subjects who were ≥ 36 years of age showed a statistically significant increase in mean DNA damage values than those who were < 36 years in exposed group (21.90 ± 5.71 vs. 19.89 ± 2.24 μm; P < 0.05). However, the result did not show any significant changes in control group (17.62 ± 3.6 vs. 17.93 ± 2.37 μm; P < 0.05). Similarly the comet tail length did not show any significantly increased in DNA damage of exposed nurses with ≥ 10.5 or < 10.5 years of employment (22.87 ± 6.18 vs. 22.32 ± 4.65 μm). Also nurses who handled antineoplastics for ≥ 7.5 h/day did not show any significant increase in DNA damage compared to those who handled them for < 7.5 h/day (21.68 ± 5.34 vs. 22.98 ± 4.82 μm). Similar result was seen in control group (17.97 ± 3.85 vs. 17.11 ± 2.88 μm). ▶ Table 3 shows the comparison of DNA mean tail (in μm) by age ● and years of exposure of oncology hospital nurses and non exposed nurses after vitamin E treatment.
Table 2 Comet assay tail length between exposed nurses and non exposed nurses before E treatment. Parameters
Exposed nurses tail length
Non Exposed nurses tail length
N
Mean ± SD
N
Mean ± SD
Age (years) ≥ 36
N = 37
21.9 ± 5.71
N = 37
< 36
N = 32
19.89 ± 2.24
N = 32
17.62 ± 3.6 P = 0.09 17.93 ± 2.37 P = 0.12
Years of exposure ≥ 10.5
N = 31
22.87 ± 6.18
N = 31
< 10.5
N = 38
22.32 ± 4.65
N = 38
Per day exposure (hours) exposure ≥ 7.5 N = 31 21.68 ± 5.34
N = 31
< 7.5
N = 38
N = 38
22.98 ± 4.82
17.99 ± 3.66 P = 0.12 17.25 ± 3.60 P = 0.17 17.97 ± 3.85 P = 0.17 17.11 ± 2.88 P = 0.12
P < 0.05 significant for between groups, t-test
The comet tail length significantly increased in exposed nurses. Subjects who were ≥ 36 years of age showed a statistically significant increase in mean DNA damage values than those who were < 36 years in exposed group (20.54 ± 5.92 vs. 18.24 ± 3.21 μm; P < 0.05). Similar result was seen in control group (14.73 ± 3.43 vs. 14.33 ± 2.74 μm; P < 0.05). The comet tail length did not show any significantly increased in DNA damage of exposed nurses with ≥ 10.5 or < 10.5 years of employment (19.36 ± 4.56 vs. 20.42 ± 5.86 μm).
Rezaei-Basiri M et al. Role of Vitamin E on Urothelial Cell … Drug Res 2014; 64: 337–342
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Fig. 1 Shows DNA breakage in urine epithelial cells nucleus and the nucleus elongation was calculated with CASP.
340 Original Article
Parameters
Exposed nurses tail
Non exposed nurses tail
length
length
N
Mean ± SD
N
Mean ± SD
Age (years) ≥ 36
N = 37
20.54 ± 5.92
N = 37
< 36
N = 32
18.24 ± 3.21
N = 32
14.73 ± 3.43 P = 0.57 14.33 ± 2.74 P = 0.47
Years of exposure ≥ 10.5
N = 31
20.42 ± 5.86
N = 31
< 10.5
N = 38
19.36 ± 4.56
N = 38
Per day exposure (hours) exposure ≥ 7.5 N = 31 17.94 ± 4.68
N = 31
< 7.5
N = 35
N = 35
20.66 ± 4.97
14.68 ± 3.25 P = 0.54 13.83 ± 3.58 P = 0.57 14.17 ± 3.57 P = 0.64 14.27 ± 3.18 P = 0.53
P < 0.05 (Difference between exposed and non exposed nurses; student’s t-test
Also nurses who handled antineoplastics for ≥ 7.5 h/day did not show any significant increase in DNA damage compared to those who handled for < 7.5 h/day (17.94 ± 4.68 vs. 20.66 ± 4.97 μm). Also same result was seen in control group (14.17 ± 3.57 vs. 14.27 ± 3.18 μm). ▶ Fig. 1 shows DNA breakage in epithelial cells and calculation ● of epithelial cells nucleus elongation with CASP (Comet assay software) in oncology hospital nurses. ▶ Fig. 2 shows DNA breakage in epithelial cells nucleus in an ● oncology hospital nurse before and after 200 mg/day vitamin E ▶ Fig. 2a. large ‘comets’ were treatment. As it is shown in ● observed in cells before vitamin E. However, vitamin E treatment ▶ Fig. 2b. In order to causes decrease in the size of the ‘comet’ ● quantify these effects and examine whether vitamin E itself prevent DNA damage, the mean tail moments of all individual cells from each experimental and control group were calculated. ▶ Table 4 states antimutagenic effects of vitamin E Although ● between all exposed nurses before and after vitamin E treatment as it is clear 200 mg of vitamin E per day had been completely significant effects in diminishing of tail moments in
Fig. 2 a Before vitamin E treatment, tail length = 119. b After vitamin E treatment, tail length = 81.
Rezaei-Basiri M et al. Role of Vitamin E on Urothelial Cell … Drug Res 2014; 64: 337–342
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Table 3 Comet assay tail length between exposed nurses and non exposed nurses after E treatment.
Table 4 Antimutagenicity effects of vitamin E (Comet assay tail length) on (N = 69) women oncology hospital nurses. Parameters
Before vitamin E
2 week after vitamin E
Treatment Tail length
Treatment Tail length
N
Mean ± SD
N
Mean ± SD
Age (years) ≥ 36
N = 37
21.90 ± 5.71
N = 37
< 36
N = 32
19.89 ± 2.24
N = 32
20.54 ± 5.92 P = 0.000 18.24 ± 3.21 P = 0.000
Years of exposure ≥ 10.5
N = 31
22.87 ± 6.18
N = 31
< 10.5
N = 38
22.32 ± 4.65
N = 38
Per day exposure (hours) exposure ≥ 7.5 N = 31 21.68 ± 5.34
N = 31
< 7.5
N = 35
N = 35
22.98 ± 4.82
20.42 ± 5.86 P = 0.000 19.36 ± 4.56 P = 0.000 17.94 ± 4.68 P = 0.000 20.66 ± 4.97 P = 0.000
P < 0.05 (Difference between before and after vitamin-E treatment; student’s t-test
Fig. 3 Antimutagenic effects of vitamin E had been significant effects (the p < 0.05, P = 0.000) in diminishing the tail moments in oncology exposed nurses (n = 31). Daily work: ≥ 7.5 h.
oncology exposed nurses in their urothelial nucleus. Also the ▶ Fig. 3. same result is shown in ●
Discussion
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The present study provides first evidence that vitamin E supplementation has diminished the mutagenic effects of antineoplastic drugs among nursing personnel. Chemotherapy agents with low therapeutic index having major role for chemical control of cancer and on this light, they induce genotoxic effects on normal cells. Most studies published about professionals working with antineoplastic drugs assessed some degree of increasing genotoxicity [4, 14, 20, 21]. A study by Kosgeroglu et al. claimed that with regard to nursing self-protection, when the information and administration scores decrease and increase in the working period (per week), may incur risk exposure to chemotherapeutic drugs [22]. So nursing and healthcare personnel, who admix, administer or dispose of antineoplastic drugs or body waste products from patients being treated with such drugs are at great risk of exposure and they need to be monitored for risks. Several studies have performed on biological samples to detect genotoxic effects in humans in connection to their jobs or envi-
ronment and in this line because of ease to sampling of urine, we assessed the antimutagenicity effects of vitamin E in uroepithelial cells of nursing urine samples by using comet assay. Studies have investigated the genotoxic potential of antineoplastic agents among the medical personnel chronically handling these drugs and it has been found that the DNA damage to be significantly higher than the control [4, 21, 23, 24]. Our results are in line with these studies. Contrary to these findings, some studies by evaluating genotoxicity in health care workers handling antineoplastic drugs showed no statistically significant increase in DNA damage in nurses with respect to controls [25]. The purpose of our study was to evaluate the antimutagenic effect of vitamin E on possible genotoxic risk associated with antineoplastic drugs. Some studies have suggested that, antioxidants especially vitamin E, a chain-breaking lipophilic antioxidant found within biological membranes, is also physiological membrane-bund free radical scavenger [26] and our earlier observation indicates that vitamin E as an effective chemopreventive agent is associated with diminution of oxidative stress [27]. Vitamin E has also shown to have a potent role on unsaturated fatty acids of cell membranes and stabilizes/preserves cell membranes of urothelial tract [28]. In addition, it has been shown that vitamin E in both the cases, by pretreatment/treatment has essential role on epithelial cells counts [29]. In this study, we have evaluated the role of vitamin E on urothelial cells nucleus. It was appeared that antimutagenic activity of vitamin E had significant effects on oncology hospital nurses effectively in repairing DNA damage and decreasing their nucleus length in urine epithelial cells. Accordingly on comparing to non oncology nurses (control), the urothelial cell’s nucleus tail lengths among oncology nurses were larger. Hence, since nurses are subjected to several antineoplastics drugs in job setting, which has been shown to have potent carcinogenic activities and the nursing resistance may decrease and they may encounter with oxidative stress of cellular components [30].
Conclusion
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In summary the damage in urothelial cell nucleus in nurses could not be in a fixed range and depends on several factors likes protective equipments, the working period, the information and administration scores with regard to self-protection, and physiological states of a person, age and nutrition. Additionally, a proper natural diet rich in antioxidants and antimutagenics such as vitamin E should be considered. Also based on experimental evidence we propose that use of vitamin E at higher therapeutic doses would significantly be effective against DNA strand breakage and may have more effect on nucleus of uroepthelial cells of oncology hospital nursing personnel and it might be an alternative to reduce genotoxic risk in exposure to antineoplastic drugs. Also because of ease to sampling of urine, it can be inferred that uroepithelial cells may have essential diagnostic role among oncology nursing personnel.
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Original Article 341
Acknowledgement
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The authors acknowledge hematology and oncology research center, Tabriz University of Medical Sciences, Iran, for funding the project.
Conflict of Interest
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“The authors state no conflict of interest”.
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342 Original Article
