Mutagenesis, Vol. 16, No. 1, 71-78,
January 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Application of the alkaline comet assay in human biomonitoring for genotoxicity: a study on Croatian medical personnel handling antineoplastic drugs
Laboratory of Mutagenesis, Institute for Medical Research and Occupational Health, HR-10 000 Zagreb, Croatia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The alkaline comet assay was used to evaluate the genotoxicity towards peripheral blood lymphocytes of medical personnel regularly handling various antineoplastic drugs with different safety precautions. The study population consisted of 50 exposed subjects working in the oncology, pulmology, gynaecology and haematology units of nine Croatian hospitals and 20 unexposed control subjects. Peripheral blood lymphocytes from the subjects were embedded in agarose on a microscope slide and lysed; the DNA was unwound and subjected to electrophoresis at pH 13. Staining with a fluorescent dye was used to identify cells with DNA damage, as judged by increased migration of genetic material from the cell nucleus. DNA damage was quantified by measuring the displacement between the genetic material of the nucleus and the resulting tail using an image analysis system. Three parameters were used as indicators of DNA damage: i.e. tail length, percentage of DNA in the tail and tail moment. Statistically significant differences in all three parameters were observed between the exposed and control groups. Within the exposed group, there were marked differences between individuals in the comet tail parameters. In the majority of exposed subjects an effect on DNA damage of age or duration of occupational exposure could be excluded. In the exposed group, the highest level of DNA damage was recorded in subjects who used only latex gloves in their work with antineoplastic drugs. The observed DNA damage was lower in exposed subjects who used more than one type of protective equipment and who worked in a well-ventilated safety cabinet. No statistically significant differences were found between the mean values of comet tail parameters for smoking and non-smoking subpopulations from the exposed group. In view of the results obtained, the alkaline comet assay, as a simple, rapid and sensitive method, appears to be a promising additional test for biomonitoring purposes in human populations.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Antineoplastic drugs include chemically unrelated classes of agents capable of inhibiting tumour growth by disrupting cell division and killing actively growing cells (McDevitt et al., 1993
). Clinical and laboratory studies have shown that many of these drugs are mutagens, carcinogens or teratogens in humans and animals, and these drugs can have many different acute and chronic adverse effects in patients receiving therapeutic doses, including cancer (Baker and Connor, 1996).
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 to these toxic agents. Primary routes for exposure in unprotected individuals include dermal absorption, ingestion and inhalation resulting from aerosolization of powder or liquid during reconstitution and spillage during drug preparation or administration to patients. In addition, patients may excrete these drugs and their metabolic by-products, so personnel who handle waste products from these patients can be exposed to drugs in this way (Connor, 1993; Valanis et al., 1993).
Evidence about the health hazards of exposure of medical personnel to antineoplastic drugs in hospitals are conflicting and inconclusive. Various effects have been reported, including acute toxicity (McDiarmid et al., 1988), nausea, headache, dizziness and hair loss (Valanis et al., 1993), liver damage (Sotaniemi et al., 1983), spontaneous abortions (Stücker et al., 1990), urine mutagenicity (Kolmodin-Hedman et al., 1983; Pohlová et al., 1986; Thiringer et al., 1991), chromosomal damage (Nikula et al., 1984; Oesterreicher et al., 1990; Grummt et al., 1993; Anwar et al., 1994), induction of micronuclei (Anwar et al., 1994; Machado-Santelli et al., 1994; Garaj-Vrhovac and Kopjar, 1998a; Kevekordes et al., 1998) and sister chromatid exchange in peripheral lymphocytes (Norppa et al., 1980; Milkovi
-Kraus and Horvat, 1991; 
arda
et al., 1991; Thiringer et al., 1991), as well as DNA damage (Fuchs et al., 1995; Garaj-Vrhovac and Kopjar, 1998b; Undeger et al., 1999).
To minimize the risk of occupational exposure, several guidelines for the handling of antineoplastic drugs and safety recommendations have been issued (OSHA, 1986; Valanis et al., 1992; Baker and Connor, 1996). The most commonly recommended safety precautions include the use of the proper type of biological safety cabinet, with vertical air-flow, and protective equipment such as latex gloves, gowns, surgical masks and hair covers for employees handling antineoplastic agents (Valanis et al., 1992; Connor, 1993, 1995; Fuchs et al., 1995). However, the guidelines differ from country to country, but in their practical application from hospital to hospital (Grummt et al., 1993; Baker and Connor, 1996).
A variety of cytogenetic methods and mutagenicity tests of urine have been used to monitor populations exposed to antineoplastic drugs. Although they provide information about damage in individual cells, cytogenetic techniques are of limited value, because of the need for proliferating cell populations and because DNA damage must be processed into microscopically visible lesions.
During the last 10 years, a rapid and sensitive technique, the comet assay [single-cell gel electrophoresis (SCGE)] has gained widespread acceptance for genotoxicity testing. It has recently been used for various in vitro and in vivo studies to monitor exposure to mutagens and carcinogens that induce DNA damage (Tice, 1990; Fairbairn, 1995; Olive, 1999). In molecular epidemiology studies, DNA damage evaluated by the comet assay is used as a biomarker of exposure (Betti et al., 1994, 1995; Collins et al., 1997; Garaj-Vrhovac and Kopjar, 1998b, 
rám et al., 1998; Piperakis et al., 1999).
The comet assay permits the detection of primary DNA damage and the study of repair kinetics at the level of single cells (Singh et al., 1988; Olive et al., 1990; Hellman et al., 1995; Olive, 1999). There is a variety of possible modifications of the assay, facilitating the detection of single-strand DNA breaks, alkali-labile sites, double-strand DNA breaks, incomplete excision repair sites and interstrand cross-links. The comet assay can also be used to assess DNA fragmentation associated with cell death or related to apoptosis (Olive, 1999; Piperakis et al., 1999).
Compared with other genotoxicity tests, the comet assay is a cheap and simple method. However, it is advisable to use a computer-assisted image-analysis system to measure the comet parameters. Although the comet assay requires isolated cells of the same type, there has been increasing interest in this test in the past years, mainly because of its advantages, sensitivity and rapidity (Cotelle and Ferard, 1999).
In the present study, the alkaline comet assay was used to evaluate the genotoxic effects of antineoplastic drugs on the peripheral lymphocytes of medical personnel handling various antineoplastic drugs with different safety precautions.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Subjects of study
The population studied comprised 70 volunteer blood donors, 50 of whom had been occupationally exposed to antineoplastic drugs and 20 of whom were unexposed control subjects. Each person completed a standardized questionnaire which included items concerning age, health status, occupational exposure to antineoplastic drugs at the time of the study and the drug-handling safety precautions taken. The questionnaire also included items concerning exposure to other potential mutagenic hazards unconnected with work, such as smoking, alcohol and drug consumption, viral diseases, recent vaccinations and radiodiagnostic examinations.
The exposed group consisted of 25 female smokers and 25 female non-smokers aged 20–55 years (mean age 37 years) working in the oncology, pulmology, gynaecology and haematology units of nine Croatian hospitals. They were in daily or almost daily contact with antineoplastic drugs (preparing solutions and syringes for infusion, administering of antineoplastic drugs, handling of body fluids of patients undergoing chemotherapy). The exposed subjects handled a diversity of antineoplastic drugs. Many of them reported working with more than five antineoplastic agents. The most frequently used drugs were cyclophosphamide, vincristine, vinblastine, cis-platinum, 5-fluorouracil, bleomycin, methotrexate and adriamycin. The handling time varied from 1–6 h/day. Mean exposure time for the exposed group was 12.9 years (range 1–30 years). With regard to the type of safety precautions used, the exposed group in our study was divided into four subgroups. The first subgroup comprised 20 subjects who used only latex gloves in their daily work with antineoplastic drugs. The second subgroup comprised eight subjects who used latex gloves and surgical masks as individual protective equipment. In both of these subgroups, antineoplastic drugs were prepared in a separate corner of a room occasionally used for smoking and eating. The third subgroup comprised 19 subjects who used latex gloves and a safety cabinet with vertical air-flow. The subjects in the fourth subgroup (three nurses) used latex gloves, surgical masks and a safety cabinet with vertical air-flow when handling antineoplastic drugs. The safety cabinets used by subjects in the third and fourth subgroups for the preparation of antineoplastic drugs were located in a separate room set aside for this purpose only.
Exposed subjects had not been subjected to diagnostic X-ray examinations during the year before the beginning of this study. They were not on birth control pills during the study and were not taking other medications. In the exposed group, seven nurses reported cases of one spontaneous abortion, while one nurse reported a case of two spontaneous abortions in the first trimester.
Control subjects were healthy students and office employees (10 females and 10 males), chosen from the general Croatian population. All were non-smokers aged 20–50 years (mean age 30 years). None of the them had ever had any contact with antineoplastic drugs. They had not been occupationally exposed to known genotoxic agents. None of the control subjects reported alcohol consumption. During the year before blood sample collection, the controls had not been subjected to diagnostic examinations using X-rays or non-ionizing radiation. None had received any therapeutic irradiation. They were not taking any medications or oral contraceptives.
Blood sampling
Peripheral blood samples of the exposed and control subjects were collected by venipuncture into heparinized tubes during the period September 1998 to September 1999. All samples were coded, cooled and processed within 24 h after collection. The alkaline comet assay was performed immediately after blood transportation.
The comet assay
The comet assay was carried out under alkaline conditions, basically as described by Singh et al. (1988). Fully frosted slides were covered with 1% normal melting point (NMP) agarose (Sigma). After solidification, the gel was scraped off the slide. The slides were then coated with 0.6% NMP agarose. When this layer had solidified a second layer containing the whole blood sample mixed with 0.5% low melting point (LMP) agarose (Sigma) was placed on the slides. After 10 min of solidification on ice, slides were covered with 0.5% LMP agarose. Slides were then immersed for 1 h in ice-cold freshly prepared lysis solution [2.5 M NaCl, 100 mM disodium EDTA, 10 mM Tris–HCl, 1% sodium sarcosinate (Sigma), pH 10] with 1% Triton X-100 (Sigma) and 10% dimethyl sulfoxide (Kemika) added fresh to lyse cells and allow DNA unfolding. The slides were then placed on a horizontal gel electrophoresis tank, facing the anode. The unit was filled with fresh electrophoresis buffer (300 mM NaOH, 1 mM disodium EDTA, pH 13.0) and the slides were placed in this alkaline buffer for 20 min to allow DNA unwinding and expression of alkali-labile sites. Denaturation and electrophoresis were performed at 4°C under dim light. Electrophoresis was carried out for 20 min at 25 V (300 mA). After electrophoresis the slides were rinsed gently three times with neutralization buffer (0.4 M Tris–HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained with ethidium bromide (20 µg/ml) and covered with a coverslip. Slides were stored at 4°C in sealed boxes until analysis.
Comet capture and analysis
A total of 50 randomly captured comets from each slide were examined at 400x magnification using an epifluorescence microscope (Zeiss) connected through a black and white camera to an image analysis system (Comet Assay II; Perceptive Instruments Ltd, UK). A computerized image analysis system was used to acquire images, compute the integrated intensity profile for each cell, estimate the comet cell components and evaluate the range of derived parameters. To quantify DNA damage, the following comet parameters were evaluated: tail length, percentage of DNA in tail and tail moment. Tail length (i.e. the length of DNA migration) is related directly to the DNA fragment size and presented in micrometres. It was calculated from the centre of the cell. Tail moment was calculated as (tail length x % of DNA in tail)/100.
Statistical analysis
The various parameters measured in the exposed and control groups (tail length, percentage of DNA in the tail and tail moment) were evaluated using the non-parametric Mann–Whitney U-test. The level of significance was set at 5%.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The present paper illustrates the results of an alkaline comet assay on peripheral blood lymphocytes obtained from 50 subjects handling antineoplastic drugs and 20 control subjects. Using Comet Assay II software, we evaluated parameters for measuring DNA damage. To describe the comet global parameters, tail length, tail DNA % and tail moment are used.
Results of the alkaline comet assay are summarized in Tables I–IV. Figure 1 gives the mean values of tail length (a), percentage of DNA in tail (b) and tail moment (c) for the exposed and control group. Figure 2 shows the distribution of tail length, percentage of DNA in tail and tail moment in the exposed and control groups.
|
|
|
|
|
|
Statistically significant differences in all comet assay parameters were observed between the exposed and control groups (Figure 1
). The distribution of comet tail parameters in control subjects was characterized by nuclei with small comets (Table IV). Within the exposed group there were marked differences between individuals in terms of comet tail parameters (Table II). In the majority of exposed subjects the influence of age or duration of occupational exposure on the DNA damage was excluded (Table I). Less DNA damage was observed in the exposed subjects using more than one type of individual protective equipment and working in a well-ventilated safety cabinet (Tables I and II). In the exposed group, the highest level of DNA damage was recorded in subjects in the first subgroup (i.e. those who only used latex gloves while working with antineoplastic drugs). No statistically significant differences were found between the mean tail parameters for smoking and non-smoking subpopulations in the exposed group (Table III).
Tail length
The lengths of comet tails in exposed subjects were in the range 14.52 ± 1.12 µm to 23.44 ± 5.21 µm (mean 17.46 ± 1.99 µm) (Table I, Figure 1a). The observed values were significantly different (P < 0.05) from those in control subjects, which were in the range 11.68 ± 0.76 µm and 14.42 ± 1.11 µm (mean 12.55 ± 0.82 µm) (Table IV, Figure 1a).
With regard to the type of safety precautions used, some differences in mean tail lengths were observed between the four exposed subgroups (Tables I and II). Mean tail length was greatest in subjects in the first subgroup (who only used latex gloves in daily work with antineoplastic drugs) and smallest in subjects in the fourth subgroup (who used latex gloves, surgical masks and a safety cabinet). There were statistically significant (P < 0.05) differences in the mean values of tail length between the first and the third exposed subgroups and between the first and fourth exposed subgroups (Table II). No other significant differences were found between the four exposed subgroups.
With regard to smoking habits, there were minor, although not statistically significant, differences (P > 0.05) in mean tail length between exposed smokers and non-smokers (Table III).
Percentage of DNA in the comet tail
The percentage of DNA in the the tail ranged between 70.67 ± 10.19% and 85.34 ± 3.59% (mean 81.49 ± 4.31%) in the exposed group (Table I, Figure 1b) while that in the control group was 70.01 ± 10.35% to 80.99 ± 1.48% (mean 76.01 ± 3.70%) (Table IV, Figure 1b). Differences observed between the exposed and control group were statistically significant (P < 0.05).
With regard to the safety precautions used, some differences in the mean percentage of DNA in the tail were observed between the four exposed subgroups (Table II). The mean percentage of tail DNA was greatest in subjects in the first subgroup who handled antineoplastic drugs using only latex gloves, while it was smallest in subjects in the fourth subgroup who used latex gloves, surgical masks and a safety cabinet as protective equipment. There were statistically significant (P < 0.05) differences in the mean percentage of tail DNA between the first and the third exposed subgroups and between the first and the fourth exposed subgroups. No other significant differences were recorded between the four exposed subgroups.
There were no significant differences (P > 0.05) between the mean percentages of DNA in the comet tail determined in lymphocytes taken from exposed smokers and exposed non-smokers (Table III).
Tail moment
The tail moments for the exposed group ranged between 9.98 ± 1.98 and 19.96 ± 4.82 (mean 14.31 ± 2.16) (Table I, Figure 1c). The observed values differed significantly (P < 0.05) from those in control subjects, which were in the range 8.41 ± 1.83 to 11.66 ± 1.20 (mean 9.78 ± 0.91) (Table IV, Figure 1c).
As in the case of the mean tail length and mean percentage of tail DNA, mean tail moments varied between the four subgroups of exposed subjects (Table II). The mean tail moment was greatest in subjects in the first subgroup, who used only latex gloves as protective equipment, and smallest in subjects in the fourth subgroup, who used latex gloves, surgical masks and a safety cabinet. There were statistically significant (P < 0.05) differences in the mean tail moments between the first and the third exposed subgroups and between the first and the fourth exposed subgroups. No other significant differences were noticed between the four exposed subgroups.
There were no statistically significant differences (P > 0.05) between the mean tail moments recorded in exposed smokers and exposed non-smokers (Table III).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Among the individuals occupationally exposed to mutagenic and carcinogenic agents, the population exposed to antineoplastic drugs deserves special attention. The results of the present study using the alkaline comet assay demonstrate that handling antineoplastic drugs without appropriate protection is associated with a significant increase in DNA damage in the peripheral blood lymphocytes of exposed subjects. These results are in agreement with our previous findings (Garaj-Vrhovac and Kopjar, 1998b
) as well as with the findings of Undeger et al. (1999) obtained using the same method.
In the lymphocytes taken from healthy volunteers, there was a low background level of DNA damage. Control subjects were predominantly characterized by nuclei with small comets. Only minor variation in tail length, percentage of DNA in tail and tail moments were observed between individuals in control subjects. All the measures of DNA damage in peripheral lymphocytes taken from the exposed subjects were significantly greater than in controls. In the majority of lymphocyte nuclei taken from exposed subjects, tail length, percentage of DNA in tail and tail moment increased and the range was broader. This indicates an accumulation of DNA damage as a consequence of chronic occupational exposure to small amounts of various antineoplastic drugs. It should be emphasized that, in the majority of exposed subjects, no relationship was found between DNA migration and age. This lack of age in modulating DNA migration confirms previous observations (Betti et al., 1995; Gutiérrez et al., 1998). In contrast, Singh et al. (1991) reported higher levels of DNA damage in subjects aged > 60 years, but in this case the electrophoresis time was increased to 40 min to achieve higher resolution in DNA migration. However, it is noteworthy that none of the subjects in our study was aged >55 years.
The observation that the extent of DNA damage was not strictly dependent on the exposure period reflects differences in individual exposure, sensitivity and efficiency of DNA repair mechanisms. Similar results in populations exposed to antineoplastic drugs have been reported by Oestereicher et al. (1990) and Grummt et al. (1993) using cytogenetic tests in their studies. It is well known that DNA damage (especially chromosome aberrations) in peripheral lymphocytes of exposed subjects accumulate over a long period when antineoplastic drugs are handled without protection. The accumulation may be related to the populations of long-lived peripheral lymphocytes, whose half-life is estimated to be 3–4 years (Nikula et al., 1984).
Many antineoplastic drugs induce single-strand breaks and alkali-labile sites, which are predominantly measured in the comet assay. Because both types of DNA damage are continuously and efficiently repaired, the measured damage level is a result of equilibrium between the amount of DNA damage inflicted and the speed of repair. Generally, the type, level and persistence of DNA damage in lymphocytes of exposed populations depend on the kind of antineoplastic drugs used as well as on the concentrations of drugs producing the mutagenic response. On the other hand, the relationship between the onset of DNA damage and the time after exposure depend on the mechanism of DNA damage.
For the treatment of most drug-sensitive tumours, a drug cocktail is used, so nurses may be simultaneously exposed to several drugs. The spectrum of antineoplastic drugs, the quantities used and the amount of time per day for which antineoplastic drugs were handled could not be determined retrospectively.
Among the most commonly used antineoplastic drugs are clastogenic and aneugenic agents, which require protective equipment to avoid genotoxic risks to the individuals managing them. Some of the subjects studied here did not follow recommendations for the management of such drugs, so biomonitoring is of great value. The development of occupational hygiene standards in our country has been relatively slow compared with that in other European countries. It was, therefore, possible to find medical personnel with relatively high exposure. Although the practical application of safety precautions in the handling of antineoplastic drugs varies from hospital to hospital, it has increased over the last 10 years. According to information given by the medical personnel, antineoplastic agents were often handled without any special protective measures until 1991. Today, latex gloves usually are worn for parenteral administration of these drugs to patients, while surgical masks and a vertical laminar air-flow cabinet are used where available for making dilutions. In the present study it was observed that older nurses were less likely to use personal protective equipment than younger ones.
Our study has clearly shown that the extent of DNA damage, determined by use of the alkaline comet assay, was significantly lower in nurses administering antineoplastic drugs carefully and taking precautions to protect themselves during their work. The best protection was found to be the simultaneous use of gloves, surgical mask and a safety cabinet. This is in agreement with other observations (Kolmodin-Hedman et al., 1983; Thiringer et al., 1991; Valanis et al., 1993).
Although safety precautions minimize exposure to antineoplastic drugs, medical personnel are at risk of absorbing very small amounts of these drugs which are sufficient to cause mutations in their genome. To ensure maximum occupational safety, the measurement of biological markers is deemed appropriate for monitoring such exposure. Various methods have been established for monitoring biological effects which should be considered as an internal dosimeter in detection of increased genotoxic and presumably also carcinogenic risks (Sorsa and Anderson, 1996; Kevekordes et al., 1998). Among these approaches are the detection of chromosomal aberrations, sister chromatid exchanges, micronuclei, alkaline elution technique, measurement of excretion of urine thioethers and urine mutagenicity. By using various tests, several groups have studied mutagenicity among nurses and pharmacists handling antineoplastic drugs; some of these studies found evidence of mutations whereas others did not (Waksvik et al., 1981; Barale et al., 1985; Stücker et al., 1986; Sarto et al., 1990; Milkovi-Kraus and Horvat, 1991; arda et al., 1991; Sessink et al., 1994; DeMéo et al., 1995; Ensslin et al., 1997). The heterogeneity of the results is probably due to differences in the sensitivities of the test systems, the extent and type of exposure and different safety precautions applied.
In the present study the alkaline comet assay of peripheral blood lymphocytes was evaluated for use in the biomonitoring of subjects exposed to antineoplastic drugs. Results obtained confirmed that this method is a very promising tool for estimation of DNA damage at the individual cell level in in vivo studies, as reported by other authors (Betti et al., 1994, 1995; Ashby et al., 1995; Hellman et al., 1997, 1999; Olive, 1999; Piperakis et al., 1999).
Although some authors have observed a connection between cigarette smoking and increased DNA migration in human lymphocytes during the comet assay (Betti et al., 1994, 1995), in the present study no significant differences were seen between the mean comet tail parameters for smoking and non-smoking subpopulations in the exposed group (P > 0.05, see Table III). Our results indicate that cigarette smoking is not a very potent confounding factor when peripheral lymphocytes are used in the comet assay for biomonitoring purposes. These observations are in agreement with findings by Hellman et al. (1997, 1999), Undeger et al. (1999) and Wojewódzka et al. (1999).
There may be several reasons for the lack of difference between the smoking and non-smoking subgroups of exposed subjects. Possibly due to an adaptive response in smokers, the effect of additional occupational exposure was more pronounced in non-smokers. Smokers probably showed a tendency towards a lower mean DNA strand break level than non-smokers. Similar results were obtained by using the alkaline elution method (Fuchs et al., 1995).
As previously mentioned, a low level of damage assessed experimentally in an individual may be the result of an actual low number of lesions or of a high efficiency of repair (Wojevodzka et al., 1999). A slightly increased level of DNA single strand breaks is therefore not automatically associated with an increased incidence of gene mutations, chromosomal aberrations or other permanent genetic alterations.
However, because DNA damage is an inevitable event which, if left unrepaired, can lead to tumorigenesis through mutational activation of protooncogenes and inactivation of tumour supressor genes, we are of the opinion that employees with persistently elevated levels of DNA damage, estimated using the comet assay and other cytogenetic tests, should be protected by avoiding exposure or by transfer to an alternative workplace in the hospital.
In conclusion, the results of this study confirm that the alkaline comet assay is applicable for detection of genotoxic effects induced in vivo by occupational exposure to various mutagens. The relative simplicity and rapidity of the method, combined with the important practical factor that few cells are required for the analysis, makes it attractive for biomonitoring purposes in human populations.
|
Acknowledgments |
|---|
This investigation was supported in part by the Croatian Ministry of Science and Technology (grant no. 00220107).
|
Notes |
|---|
1 To whom correspondence should be addressed. Email: nkopjar{at}imi.hr
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Anwar,W.A., Salama,S.I., Elserafy,M.M., Hemida,S.A. and Hafez,A.S. (1994) Chromosomal aberrations and micronucleus frequency in nurses occupationally exposed to cytotoxic drugs. Mutagenesis, 9, 315–317.
Ashby,J., Tinwell,H., Lefevre,P.A. and Browne,M.A. (1995) The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment. Mutagenesis, 10, 85–90.
Baker,E.S. and Connor,T.H. (1996) Monitoring occupational exposure to cancer chemotherapy drugs. Am. J. Health-Syst. Pharm., 53, 2713–2723.
Barale,R., Sozzi,G., Toniolo,P., Borghi,O., Reali,D., Loprieno,N. and Della Porta,G. (1985) Sister-chromatid exchanges in lymphocytes and mutagenicity in urine of nurses handling cytostatic drugs. Mutat. Res., 157, 235–240.[Web of Science][Medline]
Betti,C., Davini,T., Gianessi,L., Loprieno,N. and Barale,R. (1994) Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects. Mutat. Res., 307, 323–333.[Web of Science][Medline]
Betti,C., Davini,T., Giannessi,L., Loprieno,N. and Barale,R. (1995) Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects. Mutat. Res., 343, 201–207.[Web of Science][Medline]
Collins,A., Dusinska,M., Franklin,M., Somorovska,M., Petrovska,H., Duthie,S., Fillion,L., Panayiotidis,M., Raslova,K. and Vaughan,N. (1997) Comet assay in human biomonitoring studies—reliability, validation, and applications. Environ. Mol. Mutagen., 30, 139–146.[Web of Science][Medline]
Connor,T.H. (1993) An evaluation of the permeability of disposable propylene-based protective gowns to a baterry of cancer chemotherapy drugs. Appl. Occup. Environ. Hyg., 8, 785–789.
Connor,T.H. (1995) Permeability testing of glove materials for use with cancer chemotherapy drugs. Oncology, 52, 256–259.[Web of Science][Medline]
Cotelle,S. and Ferrard,J.F. (1999) Comet assay in genetic ecotoxicology: a review. Environ. Mol. Mutagen., 34, 246–255.[Web of Science][Medline]
DeMéo,M.P., Mérono,S., DeBaille,A.D., Botta,A., Laget,M., Guiraud,H. and Duménil,G. (1995) Monitoring exposure of hospital personnel handling cytostatic drugs and contaminated materials. Int. Arch. Occup. Environ. Health, 66, 363–368.[Web of Science][Medline]
Ensslin,A.S., Huber,R., Pethran,A., Römmelt,H., Schierl,R., Kulka,U. and Fruhmann,G. (1997) Biological monitoring of hospital pharmacy personnel occupationally exposed to cytostatic drugs: urinary excretion and cytogenetic studies. Int. Arch. Occup. Environ. Health, 70, 205–208.[Web of Science][Medline]
Fairbairn,D.W., Olive,P.L. and O'Neill,K.L. (1995) The comet assay: a comprehensive review. Mutat. Res., 33, 37–59.
Fuchs,J., Hengstler,J.G., Jung,D., Hiltl,G., Konietzko,J. and Oesch,F. (1995) DNA damage in nurses handling antineoplastic agents. Mutat. Res., 342, 17–23.[Web of Science][Medline]
Garaj-Vrhovac,V. and Kopjar,N. (1998a) Micronuclei in cytokinesis-blocked lymphocytes as an index of occupational exposure to antineoplastic drugs. Radiol. Oncol., 32, 385–392.
Garaj-Vrhovac,V. and Kopjar,N. (1998b) The comet assay—a new technique for detection of DNA damage in genetic toxicology studies and human biomonitoring. Period. Biol., 100, 361–366.
Grummt,T., Grummt,H.J. and Schott,G. (1993) Chromosomal aberrations in peripheral lymphocytes of nurses and physicians handling antineoplastic drugs. Mutat. Res., 302, 19–24.[Web of Science][Medline]
Gutiérrez,S., Carbonell,E., Galofre,P., Creus,A. and Marcos,R. (1998) Application of the single cell gel electrophoresis (SCGE) assay to the detection of DNA damage induced by 131I treatment in hyperthyroidism patients. Mutagenesis, 13, 95–98.
Hellman,B., Vaghef,H. and Boström,B. (1995) The concepts of tail moment and tail inertia in the single cell gel electrophoresis assay. Mutat. Res., 336, 123–131.[Web of Science][Medline]
Hellman,B., Vaghef,H., Friis,L. and Edling,C. (1997) Alkaline single cell gel electrophoresis of DNA fragments in biomonitoring for genotoxicity: an introductory study on healthy human volunteers. Int. Arch. Occup. Environ. Health, 69, 185–192.[Web of Science][Medline]
Hellman,B., Friis,L., Vaghef,H. and Edling,C. (1999) Alkaline single cell gel electrophoresis and human biomonitoring for genotoxicity: a study on subjects with residental exposure to radon. Mutat. Res., 442, 121–132.[Web of Science][Medline]
Kevekordes,S., Gebel,T.W., Hellwig,M., Dames and Dunkelberg,H. (1998) Human effect monitoring in cases of occupational exposure to antineoplastic drugs: a method comparison. Occup. Environ. Med., 55, 145–149.
Kolmodin-Hedman,B., Hartvig,P., Sorsa,M. and Falck,K. (1983) Occupational handling of cytostatic drugs. Arch. Toxicol., 54, 25–33.[Web of Science][Medline]
Machado-Santelli,G.M., Marcilio Carqueira,E., Tosello Oliviera,C. and de Bragenca Pereira,C.A.A. (1994) Biomonitoring of nurses handling antineoplastic drugs. Mutat. Res., 322, 203–208.[Web of Science][Medline]
McDevitt,J.J., Lees,P.S.J. and McDiarmid,M.A. (1993) Exposure of hospital pharmacists and nurses to antineoplastic agents. J. Occup. Med., 35, 57–60.[Web of Science][Medline]
McDiarmid,M., Egan,T. and Pharm,D. (1988) Acute occupational exposure to antineoplastic agents. J. Occup. Med., 30, 984–987.[Web of Science][Medline]
Milkovi-Kraus,S. and Horvat. (1991) Chromosomal abnormalities among nurses occupationally exposed to antineoplastic drugs. Am. J. Ind. Med., 19, 771–774.[Web of Science][Medline]
Nikula,E., Kiviniitty,K., Leisti,J. and Taskinen,P.J. (1984) Chromosome aberrations in lymphocytes of nurses handling cytostatic agents. Scand. J. Work Environ. Health, 10, 71–74.[Web of Science][Medline]
Norppa,H., Sorsa,M., Vainio,H., Gröhn,P., Heinonen,E., Holsti,L. and Nordman,E. (1980) Increased sister chromatid exchange frequencies in lymphocytes of nurses handling cytostatic drugs. Scand. J. Work Environ. Health, 6, 299–301.[Web of Science][Medline]
Oestereicher,U., Stephan,G. and Glatzel,M. (1990) Chromosome and SCE analysis in peripheral lymphocytes of persons occupationally exposed to cytostatic drugs handled with and without use of safety covers. Mutat. Res., 242, 271–277.[Web of Science][Medline]
Olive,P.L. (1999) DNA damage and repair in individual cells: applications of the comet assay in radiobiology. Int. J. Radiat. Biol., 75, 395–405.[Web of Science][Medline]
Olive,P., Banáth,J.P. and Durand,R.E. (1990) Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells using the `comet' assay. Radiat. Res., 122, 86–94.[Web of Science][Medline]
OSHA (1986) Work Practice Guidelines for Personnel Dealing with Cytotoxic (Antineoplastic) Drugs. Publication 8–1.1. Occupational Safety and Health Administration, US Department of Labor, Washington DC.
Piperakis,S.M., Visvardis,E.E. and Tassiou,A.M. (1999) Comet assay for nuclear DNA damage. Methods Enzymol., 300, 184–194.[Web of Science][Medline]
Pohlová,H., 
erná,M. and Rössner,P. (1986) Chromosomal aberrations, SCE and urine mutagenicity in workers occupationally exposed to cytostatic drugs. Mutat. Res., 174, 213–217[Web of Science][Medline]
arda,S., Gök,S. and Karakaya,A.E. (1991) Sister chromatid exchanges in lymphocytes of nurses handling antineoplastic drugs. Toxicol. Lett., 55, 311–315.[Web of Science][Medline]
Sarto,F., Trevisan,A., Tomanin,R., Canova,A. and Fiorentino,M. (1990) Chromosomal aberrations, sister chromatid exchanges, and urinary thioethers in nurses handling antineoplastic drugs. Am. J. Ind. Med., 18, 689–695.[Web of Science][Medline]
Sessink,P.J.M., Cerna,M., Rössner,P., Pastorkova,A., Bavarova,H., Frankova,K., Anizon,R.B.M. and Bos,R.P. (1994) Urinary cyclophosphamide excretion and chromosomal aberrations in peripheral blood lymphocytes after occupational exposure to antineoplastic agents. Mutat. Res., 309, 193–199.[Web of Science][Medline]
Singh,N.P., McCoy,M.T., Tice,R.R. and Schneider,L.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell. Res., 175, 184–191.[Web of Science][Medline]
Singh,N.P., Danner,D.B., Tice,R.R., Brant,L., Morrel,C.H. and Schneider,E.L. (1991) Basal DNA damage in individual human lymphocytes with age. Mutat. Res., 256, 1–6.[Web of Science][Medline]
Sorsa,M. and Anderson,D. (1996) Monitoring of occupational exposure to cytostatic anticancer agents. Mutat. Res., 355, 253–261.[Web of Science][Medline]
Sotaniemi,E., Sutinen,S. and Arranto,A. (1983) Liver damage in nurses handling cytostatic agents. Acta. Med. Scand., 214, 181–189.[Web of Science][Medline]
Stücker,I., Caillard,J.F., Collin,R., Gout,M., Poyen,D. and Hémon,D. (1990) Risk of spontaneous abortion among nurses handling antineoplastic drugs. Scand. J. Work Environ. Health, 16, 102–107.[Web of Science][Medline]
Stücker,I., Hirsch,A., Doloy,T., Bastie-Sigeac,I.B. and Hemon,D. (1986) Urine mutagenicity, chromosomal abnormalities and sister chromatid exchanges in lymphocytes of nurses handling cytostatic drugs. Int. Arch. Occup. Environ. Health, 57, 195–205.[Web of Science][Medline]
rám,R.J., Podrazilová,K., Dejmek,J., Mra
ková,G. and Pilík,T. (1998) Single cell gel electrophoresis assay: sensitivity of peripheral white blood cells in human population studies. Mutagenesis, 13, 99–103.
Thiringer,G., Granung,G., Holmén,A., Högstedt,B., Järvholm,B., Jönsson,D., Persson,L., Wahlström,J. and Westin,J. (1991) Comparison of methods for the biomonitoring of nurses handling antitumor drugs. Scand. J. Work Environ. Health, 17, 133–138.[Web of Science][Medline]
Tice,R.R., Andrews,P.W., Hirai,O. and Singh,N.P. (1990) The single cell gel (SCG) assay: an electrophoretic technique for the detection of DNA damage in individual cells. In: Witmer,C.M., Snyder,R.R., Hollow,D.J., Kalf,G.F., Kocsis,J.J. and Sipes,J.G. (eds), Biological Reactive Intermediates, Vol. IV: Molecular and Cellular Effects and their Impact on Human Health. Plenum Press, New York, pp. 157–164.
Undeger,U., Basaran,N., Kars,A. and Guc,D. (1999) Assessment of DNA damage in nurses handling antineoplastic drugs by alkaline comet assay. Mutat. Res., 439, 277–285.[Web of Science][Medline]
Valanis,B., Vollmer,W.M., Labuhn,K., Glass,A. and Corelle,C. (1992) Antineoplastic drug handling protection after OSHA guidelines. J. Occup. Med., 2, 149–155.
Valanis,B., Vollmer,W.M., Labuhn,K.T. and Glass,A.G. (1993) Acute symptoms associated with antineoplastic drug handling among nurses. Cancer Nursing, 16, 288–295.[Web of Science][Medline]
Waksvik,H., Klepp,O. and Brogger,A. (1981) Chromosome analyses of nurses handling cytostatic agents. Cancer Treat. Rep., 65, 607–610.[Web of Science][Medline]
Wojewódzka,M., Kruszewski,M., Iwanenko,T., Collins,A.R. and Szumiel,I. (1999) Lack of adverse effect of smoking habit on DNA strand breakage and base damage, as revealed by the alkaline comet assay. Mutat. Res., 440, 19–25.[Web of Science][Medline]
Received on June 28, 2000; accepted on September 7, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  F. Rombaldi, C. Cassini, M. Salvador, J. Saffi, and B. Erdtmann Occupational risk assessment of genotoxicity and oxidative stress in workers handling anti-neoplastic drugs during a working week Mutagenesis, March 1, 2009; 24(2): 143 - 148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. V. Rekhadevi, N. Sailaja, M. Chandrasekhar, M. Mahboob, M. F. Rahman, and P. Grover Genotoxicity assessment in oncology nurses handling anti-neoplastic drugs Mutagenesis, November 1, 2007; 22(6): 395 - 401. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Hoffmann, J. Hogel, and G. Speit The effect of smoking on DNA effects in the comet assay: a meta-analysis Mutagenesis, November 1, 2005; 20(6): 455 - 466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Deng, M. Zhang, J. He, W. Wu, L. Jin, W. Zheng, J. Lou, and B. Wang Investigating genetic damage in workers occupationally exposed to methotrexate using three genetic end-points Mutagenesis, September 1, 2005; 20(5): 351 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Garaj-Vrhovac and N. Kopjar The alkaline Comet assay as biomarker in assessment of DNA damage in medical personnel occupationally exposed to ionizing radiation Mutagenesis, May 1, 2003; 18(3): 265 - 271. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||