Mutagenesis vol. 22 no. 6 pp. 395–401, 2007 Advance Access Publication 13 September 2007
doi:10.1093/mutage/gem032
Genotoxicity assessment in oncology nurses handling anti-neoplastic drugs
P. V. Rekhadevi, N. Sailaja, M. Chandrasekhar, M. Mahboob, M. F. Rahman and Paramjit Grover* Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, Andhra Pradesh, India
Many anti-neoplastic drugs are used globally during chemotherapy in the treatment of cancer. However, occupational exposure to anti-cancer drugs can represent a potential health risk to humans. Investigations on the genotoxicity of these drugs are inconsistent. Further, information on the genotoxic potential of anti-neoplastic drugs in medical personnel from India is not available. Hence, the aim of this study was to carry out genotoxicity monitoring of nurses from the oncology department of a hospital in South India, occupationally exposed to antineoplastic drugs under routine working conditions. The level of genome damage was determined in whole blood with the comet assay as well as micronucleus test (MNT) and in buccal epithelial cells with MNT alone of 60 nurses handling anti-neoplastic drugs and 60 referents matched for age and sex. Urinary cyclophosphamide (CP), used as a marker for drug absorption, was also measured in the urine of the nurses. The DNA damage observed in the lymphocytes of exposed nurses was significantly higher than the controls. Similarly, a significant increase in micronuclei (MN) frequency with peripheral blood lymphocytes and buccal cells was observed in the exposed nurses compared to controls (P < 0.05). Multiple regression analysis showed that occupational exposure and age had a significant effect on mean comet tail length as well as on frequency of MN. The mean value of CP in urine of the nurses handling antineoplastic drugs was (mean standard deviation; 0.44 0.26 mg/ml). Our study has shown that increased genetic damage was evident in nurses due to occupational exposure to anti-neoplastics. This data corroborate the need to maintain safety measures to avoid exposure and the necessity of intervention in the case of exposure when using and handling anti-neoplastic drugs.
Introduction Worldwide, anti-neoplastic drugs are used in the treatment of cancer. The drugs, which are administered as infusions or bolus injections, are usually prepared individually for each patient. The health hazard for medical personnel administering these drugs is a major concern as cytostatic drugs are classified as potentially carcinogenic, mutagenic or teratogenic (1). Hospital personnel are exposed to anti-neoplastic drugs during performance of their duties. Exposure can occur mainly to hands and sporadically to other body parts as well. Contact with patients
treated with anti-neoplastic drugs via their excreta and clothing also leads to significant exposure. In order to ascertain occupational exposure to anti-neoplastic drugs, measurement of biological markers can be considered suitable. Different methods for monitoring biological effects are available. Biomonitoring studies of human populations exposed to potential mutagens assess the risk of genetic disease or cancer by analyzing the relationship between internal exposure and biological effects in target cells under consideration of confounding factors. Biomarkers of effect that indicate exposure to a causative agent and that reflect the individual risk of disease are numerous (2). The most commonly used biomarkers in cancer epidemiology include measurements of DNA damage, such as DNA breaks, micronuclei (MN), chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) (3). To monitor internal exposure to anti-neoplastic drugs, various analytical methods have been used by the researchers for the determination of few classes of substances in urine or blood from which extrapolations to other anti-neoplastic drugs can be made. The genotoxicity of anti-neoplastic drugs has been evaluated in numerous short-term tests using a variety of end points during the last three decades. Several anti-neoplastic drugs have shown positive genotoxicity with in vitro test systems (4–6). A significant genotoxic effect for many anti-cancer drugs has been observed in animal studies (7–9) as well as in cancer patients treated with anti-neoplastic drugs (10–12). Results of studies on the genotoxic effects of anti-neoplastic drugs in health care workers occupationally exposed to these drugs are inconsistent. Some investigations reveal a significant increase in SCE, CA, MN and comet tail length of personnel exposed to anti-neoplastic drugs (13–21), although negative results have also been reported (22–26). In some studies on subjects occupationally exposed to anti-neoplastic drugs, an induction of MN was either found in peripheral blood lymphocytes (PBLs) (27,28) or in exfoliated buccal cells (29,30). Whereas, some investigations showed an enhancement in DNA migration (31,32). The contrasting results observed in the cytogenetic studies in populations exposed to anti-neoplastic drugs could be because of the variation in exposure (e.g. pharmacy technicians, nurses and cleaning personnel). The contradicting results may also be due to differences in the anti-neoplastic agents used, in the protective measures employed or in the genotoxic biomarker evaluated. However, data from one study in one particular occupational setting cannot be used to judge the genetic risk in another occupational setting. This justifies this study (and other studies as well), despite the availability in the literature of investigations of this kind (but on different populations, with different exposures). Among the hospital personnel, nurses in oncology departments frequently prepare and administer the drugs. Moreover, they may come in contact with the contaminated excrement of the treated patients. A survey of literature on the genotoxic
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effects of occupational exposure to anti-neoplastic drugs revealed no studies from India. Hence, the present study was initiated to assess the genome damage associated with antineoplastic drug exposure in oncology department nurses employed in a hospital in India. The genotoxicity was evaluated by using the comet assay. This assay has become an important tool in the area of human biomonitoring studies to assess genetic damage in exposed populations. The comet assay enables the assessment of genetic damage in a great variety of cells. In traditional human biomonitoring studies, white blood cells are readily available and lymphocytes are preferred for the measurement of genotoxic effects. However, even though longterm diseases are not expected to affect blood cells, lymphocytes are regarded as sentinel cell types being early warning signals for adverse health effects (2). To substantiate our results, the micronucleus test (MNT) in buccal cells and in PBL was also carried out. The buccal cell MNT, first proposed by Stich et al. (33), is useful as a biomarker of genetic damage caused by lifestyle habits, exposures to environmental pollutants and medical procedures. The non-invasive nature of this technique makes it an attractive candidate for the biomonitoring of human populations (34). In human population studies, the frequency of MN is most commonly determined in cultured PBL after being stimulated to proliferate by phytohemagglutinin (PHA). The most frequently applied methodology uses the cytokinesis-block MN technique in which scoring is limited to cells that have divided once since mitogen stimulation (35). To quantify the biological uptake of anti-neoplastic drugs or their metabolites in the urine of exposed subjects, methods for their detection have been developed (19,22,25,26,28,29,32). Hence, another goal of the present study was to investigate whether internal exposure of the anti-neoplastic drugs being handled by the oncology nurses of this study had occurred, especially cyclophosphamide (CP) by measuring the urinary concentrations of CP. Besides, the influence of confounding factors like age and duration of exposure on the differences in genotoxicity in the study subjects with comet assay and MNT were also analyzed. Materials and methods Study population The study involved 120 subjects divided into two groups. The first group consisted of 60 nurses exposed to anti-neoplastic drugs, employed in a hospital located at Hyderabad, India. All the study subjects were females and their average duration of employment was 13.61 years (range 6–23 years). They worked for 8 h/day for 6 days a week. All study nurses during their service in the oncology department were in contact with anti-neoplastic drugs daily (preparation of solutions and syringes for infusion, administering of antineoplastic drugs and handling of body fluids of patients undergoing chemotherapy). The exposed subjects handled a diversity of anti-neoplastic drugs. Many of them reported handling more than five anti-neoplastic agents. The most frequently used anti-cancer drugs were cisplatin, carboplatin, adriamycin, bleomycin and endoxane. The handling time varied from 1 to 6 h/day with an average of 4.06 h/day. The control group (60 subjects) was selected from the general population with no history of occupational exposure to anti-neoplastic agents or any particular environmental agent. The 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, etc.) as well as medical (exposure to X-rays, vaccinations, medication, etc.), lifestyle (smoking, coffee, alcohol, diet, etc.) and occupational questions (working hours per day, years of exposure, use of protective measures, etc.). Details on individual work place exposure (drugs handled, therapy involved, methodologies utilized for drug administration, precautionary measures taken, waste disposal methods, etc.) were also taken. Only those subjects who had worked for at least 5 years in the oncology department were considered eligible. It was assured that the exposed nurses and the controls did not statistically differ from each other except for occupational exposure. It was also ensured that the exposed and the control subjects had not been taking any
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medicines, nor had they been exposed to any kind of radiation for 12 months before sample collection. All the study subjects were non-smokers and did not consume alcohol. Table I shows the main characteristics of both groups. The local ethical committee approved the study. Informed consent was obtained from each individual prior to the beginning of the study. All subjects involved in the study received detailed information concerning the aims of the research study. Blood, buccal smear and urine samples were collected from all the exposed personnel on the last day of their 6-day work shift in the morning hours before the beginning of their work shift. Six controls and six exposed samples were taken every 7 days. Sampling was carried out over a period of 3 months. Samples were coded to avoid possible bias. Collection of blood samples Venous blood (1.5 ml) was collected once from all the study and control group subjects using heparinized syringes. The samples were transported on ice to the laboratory and were processed within 2 h. DNA damage analysis using the comet assay Blood (40 ll) was taken from the collected samples for the comet assay, which was carried out according to Singh et al. (36) with slight modifications. Cell viability determined by the trypan blue exclusion technique ranged from 92 to 96% (data not shown). Slides were prepared in duplicate per subject. The method followed has been described earlier (37). Analysis was performed using a 400 objective with Olympus BX 51 fluorescent microscope equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. Slides were randomized and coded to blind the scorer. All slides were scored by one person to avoid inter-scorer variability. A total of 100 individual cells were screened per subject (50 cells from each slide). Undamaged cells resemble an intact nucleus without a tail and damaged cell has the appearance of a comet. The length of the DNA migrated in the comet tail, which is an estimate of DNA damage, was measured using an ocular meter and calculated as follows: comet tail length (lm) 5 (maximum total length) (head diameter). MN assay (buccal epithelial cells) The MNT was carried out on the buccal epithelial cells of 60 nurses and 60 controls. Buccal cell samples were obtained by rubbing the inside of the cheeks of study subjects with a toothbrush. The cells were collected in sample bottles containing 20 ml of buffer solution (0.1 M ethylenediaminetetraacetic acid, 0.01 Tris–HCl and 0.02 M NaCl, pH 7) and transported to our institute for processing. After three washes in the buffer solution by centrifugation at 400 g for 10 min, 50 ll of cell suspension was dropped onto pre-heated (55°C) slides and allowed to air-dry for 15 min on a slide warmer. The slides were made in triplicates for each subject. The slides were fixed in 80% cold methanol for 30 min, air-dried overnight at room temperature and stored at 20°C until use. The slides were stained with a DNA-specific dye, 4#,6-di-amidino-2phenylindole dihydrochloride (1 lg/ml). A total of 5000 buccal epithelial cells were screened per subject and the frequency of MN per 1000 cells was calculated (38). Scoring was done with Olympus BX 51 fluorescent microscope. MN assay (PBL) The MNT was conducted according to the method of Fenech and Morley (35). Briefly, 0.5 ml of whole blood was mixed with 4.5 ml of RPMI-1640 medium supplemented with 20% fetal calf serum, 100 U/ml penicillin and 100 lg/ml streptomycin. PHA-M (0.2 mg/ml) was added to stimulate the culture. Cultures were incubated in duplicates at 37°C for 72 h. Cytochalasin-B was added at the 44th hour of culture growth at a final concentration of 5 lg/ml to arrest the cells at cytokinesis. The cultures were harvested by centrifugation after 72 h. The lymphocytes were subjected to mild hypotonic treatment with 0.075 M KCl for 5 min, and then fixed in fresh fixative solution (3:1, methanol:acetic acid). This fixation was repeated twice. Few drops of cell suspension were smeared on precooled microscopic slides and air-dried. The slides were stained using 10% Giemsa of pH 6.8 for 10 min. One thousand binucleated lymphocytes (500 cells per culture) were scored at 400 magnification.
Table I. Demographic characteristics of controls and exposed nurses Parameters
Control (n 5 60)
Exposed group (n 5 60)
Age (years) 37.95 5.64 38.21 5.61 (mean SD) Years of exposure 13.61 4.81 (mean SD) Per day exposure (hours) 4.06 0.73 (mean SD)
t 5 0.25 P 5 0.79
Genotoxicity assessment in oncology nurses Urine collection Approximately 15 ml of urine was collected from each subject in polypropylene sample vials, aliquot and stored at 20°C until required for analysis. A total of 52 samples were obtained from exposed subjects (n 5 60). The other subjects could not provide the samples for various reasons. No samples were taken from control personnel. Standard preparation and calibration Calibration curves were constructed from the analysis of standard CP samples which were freshly prepared by adding 100 ll of the CP (5 lg/ml) using ethyl acetate as solvent. The CP concentrations of the standard samples ranged from 0 to 5 lg/ml. A linear graph for area versus concentration of CP (lg/ml) was obtained. Sample preparation To 5 ml of urine sample 500 ll of 1 M Tris buffer was added. The pH was adjusted to 9.0. The samples were extracted thrice with 20 ml of tertiary butyl methyl ether. The ether layer was washed with 5 ml of 10% sodium bicarbonate and evaporated under nitrogen at 30°C until a residue of 2 ml was obtained. The solution was then transferred to conical tubes with screw caps and further evaporated to dryness. The organic phase was then redissolved in 100 ll ethyl acetate and derivatized with tri-fluoro acetic anhydride for 30 min at 70°C. The dried samples were then dissolved in 100 ll toluene, mixed and sonicated for 5– 10 min and analyzed by gas chromatography–mass spectrometry (GC–MS) (39). Determination of CP by GC–MS The GC–MS analysis of urine for CP was performed using an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a model 5973 N mass selective detector and a CP Si 8 CB capillary column (Varian Inc., Middleburg, The Netherlands; length 30 m, 250 lm internal diameter, 0.25 lm film thickness). The column oven was programmed initially from 50°C (held for 2 min) at 10°C/min ramp to a final temperature of 280°C. The final temperature hold time was 5 min. Helium (99.99%) was used as a carrier gas at a flow rate of 1 ml/min. The inlet and GC–MS interface temperature were maintained at 280°C. The samples were introduced in 10:1 split injection ratio. The mass spectrometer was scanned from m/z 29 to 600 for all GC–MS experiments. Samples were injected by an auto sampler (Agilent 7683 injector). Quantification was based on internal standard with concentrations ranging from 0 to 5 lg/ml. Limit of detection and quantification (signal/ noise ratio 10:1) for CP was found to be 0.04 and 0.13 lg/l, respectively. Retention time for CP was 10.2 min.
significantly increased in PBL of exposed nurses (mean value 13.66 versus 6.21 lm; P , 0.05). Subjects who were 35 years of age showed a statistically significant increase in mean DNA damage values than those who were ,35 years in exposed group (14.65 versus 11.95 lm; P , 0.05). Similar result was seen in control group also (6.39 versus 5.90 lm; P , 0.05). The comet tail length significantly increased in PBL of exposed nurses with .10 years of employment (14.57 versus 11.85 lm; P , 0.05). Nurses who handled antineoplastics for 4 h/day did not show any significant increase in DNA damage compared to those who handled them for ,4 h/day (13.70 versus 13.65 lm; P , 0.05) as seen in Table III. Multiple linear regression analysis for mean DNA tail length showed a statistically significant positive association for age and duration of exposure, whereas a negative association was seen for years of exposure. A positive association of urinary CP concentrations and mean DNA tail was observed in exposed subjects. However, the association was not statistically significant (Table IV). MN frequency in buccal cells The exposed nurses revealed a significant induction of MN when compared with controls (2.66 versus 1.86; P , 0.05) as seen from Table II. The exposed as well as the control subjects showed a significant difference in MN frequency between subjects 35 and ,35 years of age. Further, the MN frequency was significantly higher in subjects with a longer duration of exposure (2.82 versus 2.35; P , 0.05). Duration of handling anti-neoplastics for 4 h/day did not have any significant effect
Table II. Inter-group comparison of mean DNA damage (comet tail length in lm) and MN frequency in controls and exposed nurses Group
Statistical analysis The samples were coded at the time of preparation and scoring. They were decoded before statistical analysis for comparison. Mean and standard deviation (SD) was calculated for each biomarker. As the distribution of DNA mean tail length and MN did not significantly differ from the normal distribution, untransformed data were used. The significance of the differences between controls and exposed nurses’ end point means were analyzed using Student’s ttest (mean DNA tail length and MN frequency). All calculations were performed using Graph Pad Prism 4 Software package for windows. Mean values and SDs were computed for the scores and the statistical significance (P , 0.05) of effects (exposure and age) was determined using Student’s t-test. Multiple linear regression analysis was performed to assess the association between end points and the independent variables.
Control Exposed
n
60 60
DNA damage (lm) (mean SD) 6.21 0.92 13.66 2.37*
Lymphocytes
1.86 0.62 2.66 0.83*
3.11 1.09 6.53 1.15*
Table III. DNA mean tail (in lm) by age and years of exposure in controls and exposed nurses Control
Exposed group
n
Mean SD
n
Mean SD
22 38
5.90 0.92 6.39 0.88* P 5 0.04 t 5 2.01
22 38
11.95 2.10 14.65 1.93* P , 0.0001 t 5 5.05
Years of exposure ,10 10
20 40
11.85 2.08 14.57 1.97* P , 0.0001 t 5 4.95
Per day exposure (hours) ,4 4
17 43
13.70 1.99 13.65 2.53 P 5 0.93 t 5 0.07
Results
Comet assay The extent of DNA damage evaluated by comet assay in leukocytes of all the study subjects as measured by mean comet tail length is presented in Table II. The comet tail length
Buccal cells
*P , 0.05.
Parameters
The effect of occupational exposure to anti-neoplastic drugs on the levels of genetic damage in exposed nurses and control subjects was assessed by the comet assay and MNT. Table I represents the distribution of subjects with respect to age, years of exposure and duration of handling anti-neoplastics per day. The two groups studied had similar demographic characteristics. The mean age of the exposed group was 38.21 5.61, ranging from 28 to 49 years, and that of controls was 37.95 5.64, ranging from 28 to 45 years.
MN frequency (%) (mean SD)
Age (years) ,35 35
*P,0.05.
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Table IV. Multiple linear regression analysis of end points in exposed nurses End point
Independent variables
Frequency ratio (95% CI)
DNA mean tail length
Age Years of exposure Per day exposure
19.50 (12.48–26.53) 0.50 (6.84–5.84) 4.77 (3.65–5.88)
MN frequency (Buccal cells)
Age Years of exposure Per day exposure
31.93 (27.32–36.55) 8.62 (4.63–12.61) 4.43 (379–5.07)
MN frequency (lymphocytes)
Age Years of exposure Per day exposure
34.30 (16.69–31.91) 2.91 (3.75–9.58) 5.19 (4.12–6.26)
Urinary CP concentrations
Age Years of exposure Per day exposure
35.44 (32.62–38.26) 12.14 (9.55–14.74) 4.10 (3.17–4.49)
DNA mean tail length MN frequency (buccal cells) MN frequency (lymphocytes)
12.77 (11.56–13.99) 2.75 (2.28–3.23) 6.25 (5.58–6.92)
R2 (%)
P
F
,0.0001* ,0.0001* 0.20
29.28 20.41 1.63
0.006* 0.01* 0.23
8.14 6.87 1.43
0.0005* 0.0019* 0.03*
13.79 10.63 4.57
0.80
0.78
0.78 0.02* 0.17 0.61
5.25 1.89 0.25
0.05 0.61 0.26
3.78 0.25 1.28
0.77
0.007 The relative influence of independent variables (age and years of exposure) on the frequencies of mean DNA damage and MN frequencies was tested by multiple linear regression analysis. The significance for each model level was 0.05 R2 (%) 5 multiple determination coefficient. Frequency ratio is expressed with exponentiated regression coefficients and their 95% confidence interval (CI). *P , 0.05.
Table V. MN frequency (‰) in buccal epithelial cells with respect to age and years of exposure in controls and exposed nurses Parameters
Age (years) ,35 35
Control
Exposed group
n
Mean SD
n
Mean SD
22 38
1.36 0.49 2.15 0.49* P , 0.0001 t 5 6.00
22 38
2.36 0.95 2.84 0.71* P 5 0.03 t 5 2.20
20 40
2.35 0.98 2.82 0.71* P 5 0.03 t 5 2.13
Years of exposure ,10 10 Per day exposure (hours) 4 ,4
17 43
2.82 0.72 2.60 0.87 P 5 0.36 t 5 0.19
*P , 0.05.
on MN frequency in buccal cells (2.82 versus 2.60; P . 0.05) as seen in Table V. MN frequency showed a statistically significant positive association with age and years of exposure by multiple linear regression analysis. The results were found to be statistically significant except for daily exposure. Urinary CP concentrations showed a positive association with MN frequency but was not statistically significant. MN frequency in lymphocytes The results of MN frequency in PBL of oncology nurses increased significantly, as compared with controls (6.53 versus 3.11; P , 0.05). The difference in MN frequency between 35-year-old subgroup and ,35-year-old subgroup was significant (6.92 versus 5.86; P , 0.05). Similar result was seen in control group also (3.52 versus 2.40; P , 0.05). Duration of 398
exposure to anti-neoplastics had a significant effect on MN frequency in lymphocytes of nurses who had been working in oncology departments for 10 years over those who worked for ,10 years (6.82 versus 5.95; P , 0.05). Handling antineoplastic drugs for 4 h/day caused a rise in MN frequency than handling them for ,4 h (7.00 versus 6.34; P , 0.05) as seen in Table VI. Multiple linear regression analysis showed a statistically significant positive association of MN frequency in lymphocytes with age and duration of exposure. MN frequency in lymphocytes showed a positive association with urinary CP concentrations. However, the results were not statistically significant. Urinary CP concentrations The analyses of urine samples from exposed personnel for the presence of any anti-neoplastic drugs were performed by GC–MS. Samples were analyzed for the presence of CP. Varying concentrations of CP in the range of 0.08–0.9 lg/ml of urine were found in 42 subjects only. The mean CP concentration in 42 subjects was 0.44 0.26 lg/ml. The rest of the samples had CP below the limit of detection. Age, years of exposure and duration handling anti-neoplastic drugs per day showed a positive association with urinary CP concentrations. The correlation was found to be statistically significant only for age. Discussion During the past decades, cancer chemotherapeutic agents have begun to offer major hope for chemical control of cancer. Although ideal anti-neoplastic drugs should only destroy cancer cells and not do harm to normal tissues, most of these drugs have genotoxic effects on normal cells (40). Because these drugs directly or indirectly affect DNA, not only the cancer patients but also the medical personnel chronically handling these drugs are at a higher risk for acquiring DNA damage (41). The aim of this study was to assess the genotoxic damage in nurses occupationally exposed to anti-neoplastic
Genotoxicity assessment in oncology nurses
Table VI. MN frequency (‰) in lymphocytes with respect to age and years of exposure in controls and exposed nurses Parameters
Control
Exposed group
n
Mean SD
n
Mean SD
22 38
2.40 0.79 3.52 1.03* P , 0.0001 t 5 4.31
22 38
5.86 0.71 6.92 1.19* P , 0.0004 t 5 3.37
Years of exposure ,10 10
20 40
5.95 0.82 6.82 1.19* P , 0.0004 t 5 2.93
Daily exposure (hours) 4 ,4
17 43
7.00 1.27 6.34 1.06* P 5 0.45 t 5 2.01
Age (years) ,35 35
drugs in a hospital in India. Besides, the internal exposure of the nurses to anti-neoplastic drugs (CP) was determined. The health concerns raised by the nurses handling anti-neoplastic drugs were irritation of skin, eyes, nausea, vomiting, infertility, low birth weight and abortions. None of the exposed personnel took any protective measures like use of gloves and worked without safety cabinets. In the present study, nurses handling anti-neoplastic drugs and a control group were monitored by the comet assay in PBL and the MNT in buccal epithelial cells and PBL. Comet assay and MNT are the most promising short-term genotoxicity assays for human risk assessment and their combination is recommended to monitor populations chronically exposed to genotoxic agents (18). A significant increase in comet tail length was detected in the nurses as compared to the controls using the comet assay in the current study. Many studies that have investigated the genotoxic potential of individuals handling anti-neoplastic agents using comet assay have found DNA damage to be significantly higher than the controls (16,18,20,31,32,42). Our results are in line with these studies. Contrary to these findings, researchers evaluating DNA damage in health care workers handling anti-neoplastic drugs showed no statistically significant increase in DNA damage in nurses with respect to controls with comet assay (26). MNT has emerged as one of the methods preferred by researchers for assessing chromosome damage because it enables both chromosome loss and chromosome breakage to be measured reliably. The method is now applied to various cell types for monitoring populations for genetic damage. In the current study, the MNT in exfoliated buccal cells and PBL was conducted in nurses exposed to anti-neoplastic drugs. In the MNT with exfoliated buccal cells, significant differences between nurses and control groups were observed. Similarly, the analysis of MN frequencies in PBL showed significant induction in exposed group in comparison to controls. Previous investigations reporting genotoxic effects using MNT with buccal smears in nurses occupationally exposed to anti-neoplastic drugs are scanty, but consistent with our results (14,28,29,30). Investigations reporting the genotoxic effect of occupational exposure to anti-neoplastic drugs with MNT using PBL are also available. The results, however, were controversial. Several investigations have revealed positive results (13,15,21). How-
ever, negative results were also found (22–25,29–32). Genotoxic effects with SCE in individuals handling anti-neoplastic drugs are also reported which are in accordance with our results (15,17). Similarly, genotoxic damage in nurses occupationally exposed to anti-cancer drugs by the analysis of CA has been found and supports our findings (13,15,17,19). Our results of multiple regression analysis indicated that years of exposure to anti-neoplastic drugs was a factor affecting the increase in DNA mean tail length and in MN frequency. Similarly, the older nurses had significantly larger mean tail length as well as increased MN frequency than the younger nurses. Other investigations on genotoxic effect of occupational exposure to anti-neoplastic drugs have shown similar increase in genetic damage with years of exposure (13,16,42). In contrast, a study revealed no effect of DNA damage of duration of occupational exposure (18). However, contrary to our results, some studies found that age had no influence on the genotoxicity parameters in populations exposed to antineoplastic drugs (18,28). The positive genotoxicity observed in the exposed subjects of the present study may be due to lack of protective measures. Likewise, a significant rise in genetic damage in the nurses handling anti-neoplastic drugs without adequate protective equipment was observed (17,19,28,43,44). Since the genotoxicity may be due to combined effects of all or some of the anti-neoplastic drugs, it is not possible to attribute damage to any particular agent. Results of this study as well as investigations performed on subjects occupationally exposed to anti-neoplastic drugs using different genotoxicity end points suggest that mixtures of anti-neoplastic drugs in long-term occupational exposure may act as clastogens on the DNA molecule of somatic cells. An evaluation of exposure obtained by biological monitoring of the exposed subjects to at least one anti-neoplastic agent was also assessed from the levels of urinary CP in our study. The data confirm the genotoxic results suggesting a relationship between the amount of anti-neoplastic drugs handled and genotoxic effects. Some studies on biological monitoring of oncology nurses are in accordance with our results (19,28,23). On the other hand, some studies have found no association between biological monitoring and genotoxicity in anti-neoplastic drug-exposed subjects (22,25,23). The contrasting results could be due to protection measures employed. A crucial early event in carcinogenesis is the induction of the genomic instability phenotype, which enables an initiated cell to evolve into a cancer cell by achieving a greater proliferative capacity. It is well known that cancer results from an accumulation of multiple genetic changes that can be mediated through chromosomal changes and that have the potential to be cytogenetically detectable. It can be hypothesized that the level of genetic damage in PBL and buccal cells reflects the amount of damage in the precursor cells that lead to the carcinogenic process in target tissues (45). Evidence that cytogenetic biomarkers are positively correlated with cancer risk has been strongly validated in recent results from both cohort and nested case–control studies showing that CAs are a marker of cancer risk (46,47), reflecting both the genotoxic effects of carcinogens and individual cancer susceptibility. The results obtained in our investigation as well as studies by other authors suggest that there is possible genotoxic damage of oncology nurses related to occupational exposure. As a whole, there is concern that the present handling practices of anti-neoplastic drugs used in hospitals in India are not 399
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sufficient to prevent exposure. Our results emphasize the importance of sufficient safety devices, their proper use and training of personnel prior to employment in order to avoid possible health hazards caused by anti-cancer drugs. Funding Sidhu Singh Foundation, USA. Acknowledgements The authors express their sincere thanks to Dr J. S. Yadav, Director, Indian Institute of Chemical Technology, Hyderabad, for providing facilities and his encouragement during the study.
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